mechanism of reduced susceptibility to fosfomycin...
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Research ArticleMechanism of Reduced Susceptibility to Fosfomycin inEscherichia coli Clinical Isolates
Yasuo Ohkoshi12 Toyotaka Sato1 Yuuki Suzuki1 Soh Yamamoto1 Tsukasa Shiraishi1
Noriko Ogasawara1 and Shin-ichi Yokota1
1Department of Microbiology Sapporo Medical University School of Medicine Chuo-ku Sapporo 060-8556 Japan2Department of Clinical Laboratory NTT East Sapporo Hospital Chuo-ku Sapporo 060-0061 Japan
Correspondence should be addressed to Shin-ichi Yokota syokotasapmedacjp
Received 15 July 2016 Revised 22 December 2016 Accepted 27 December 2016 Published 19 January 2017
Academic Editor Yun-Peng Chao
Copyright copy 2017 Yasuo Ohkoshi et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
In recent years multidrug resistance of Escherichia coli has become a serious problem However resistance to fosfomycin (FOM)has been low We screened E coli clinical isolates with reduced susceptibility to FOM and characterized molecular mechanismsof resistance and reduced susceptibility of these strains Ten strains showing reduced FOM susceptibility (MIC ge 8 120583gmL) in 211clinical isolates were found and examined Acquisition of genes encoding FOM-modifying enzyme genes (fos genes) andmutationsin murA that underlie high resistance to FOM were not observed We examined ability of FOM incorporation via glucose-6-phosphate (G6P) transporter and sn-glycerol-3-phosphate transporter In ten strains nine showed lack of growth onM9minimumsalt agar supplementedwithG6P Eight of the ten strains showed fluctuated induction byG6P of uhpT that encodes G6P transporterexpression Nucleotide sequences of the uhpT uhpA glpT ptsI and cyaA shared several deletions and amino acid mutations in thenine strains with lack of growth onG6P-supplementedM9 agar In conclusion reduction of uhpT function is largely responsible forthe reduced sensitivity to FOM in clinical isolates that have not acquired FOM-modifying genes or mutations in murA Howeverthere are a few strains whose mechanisms of reduced susceptibility to FOM are still unclear
1 Introduction
Escherichia coli is a causative agent of uncomplicated urinarytract infections in immunocompetent hosts and oppor-tunistic infections in immunocompromised hosts In recentyears fluoroquinolone-resistant andor extended spectrum120573-lactamase- (ESBL-) producing E coli strains have been fre-quently isolated from such patients [1ndash4] In addition theseE coli resistant strains occasionally show cross-resistance toaminoglycosides [5] Thus these multidrug-resistant E colihave an impact on the selection of therapeutically effectivedrugs
Because of this serious concern the use of fosfomycin(FOM) an antibiotic as a bacterial cell wall synthesis inhibitordeveloped 40 years ago has been reevaluated against drug-resistant bacteria especially E coli [6 7] Since FOM hasa unique mode of action that differed from other antibi-otics it is expected to display little cross-resistance to otherantimicrobial agents E coli is the most frequent causative
pathogenic bacterium of acute cystitis [8 9] For instancetrimethoprimsulfamethoxazole and FOMare recommendedas the first-line treatment in acute uncomplicated urinarytract infections according to the US guideline [10] On theother hand the guideline of the Japanese Association forInfectious Diseases and Japan Society for Chemotherapyrecommends fluoroquinolones as the first-line drug andFOM as the second-line drug [11]
FOM inhibits UDP-N-acetylglucosamine enolpyruvyltransferase (MurA) an enzyme involved in the synthesisof the essential peptidoglycan component N-acetylmuramicacid [12] FOM is incorporated into the bacterial cellsvia glucose-6-phosphate (G6P) transporter UhpT and sn-glycerol-3-phosphate (G3P) transporter GlpT [13 14] UhpTis upregulated by exogenously added G6P [15ndash17] It is thusrecommended that the addition of G6P to a growth mediumis used in the measurement of FOMMIC
High resistance to FOM primarily occurs by the acquisi-tion of glutathione S-transferase genes such as fosA fosA2 to
HindawiBioMed Research InternationalVolume 2017 Article ID 5470241 8 pageshttpsdoiorg10115520175470241
2 BioMed Research International
fosA5 fosC fosC2 fosB fosB2 fosX and fosKP96 found in var-ious bacteria [19 21ndash26] and mutation(s) in murA gene [1824 27 28] Furthermore mutations in the transporter genesuhpT and glpT and genes encoding proteins regulating uhpTexpression such as uhpA reduce the susceptibility to FOM[18 19 27ndash29] In addition expression levels of UhpT andGlpT are positively regulated by cyclic AMP (cAMP) [16 17]The levels of cAMP are controlled by phosphoenolpyruvate-protein phosphotransferase I encoded by ptsI and adenylcyclase encoded by cyaA andmutations in these genes resultin reduced susceptibility to FOM [29ndash31]
Nevertheless several surveillance studies report that therate of emergence of E coli isolates showing FOM resistanceor reduced FOM susceptibility has been markedly low [79 32] Although spontaneously mutational rate to acquireFOM resistance is high FOM resistance confers biologicalcosts such as reduced cell growth rate in Gram-negativebacteria [29 33] This indicates that FOM continues to bean effective agent against E coli infections However theoverall up-to-date status of FOM resistance needs to becontinuously surveyed and its molecular characteristics haveto be understood to prevent future emergence and increaseof multidrug-resistant E coli with FOM resistance in theclinic In this study we screened E coli clinical isolates fromJapan showing resistance or reduced susceptibility to FOMand identified the molecular mechanisms of their reducedsusceptibility
2 Materials and Methods
21 Bacterial Strains E coli clinical isolates (211 strains) werecollected in the years 2008-2009 as described previously [45]Thesewere identified and stocked in SapporoClinical Lab-oratory Inc (Sapporo Japan) These strains were collectedfrom a variety of clinical specimens in almost entire area ofHokkaido Prefecture Japan This study was approved by thereview boards of the relevant institutions The strains wereisolated from the following clinical specimens urine (119899 =87 410) catheter urine (119899 = 76 358) sputum (119899 =15 71) stool (119899 = 7 33) vaginal secretion (119899 = 628) pus (119899 = 3 14) aspiration tube (119899 = 3 14)drainage tube intravenous hyperalimentation catheter tuberhinorrhea (two strains from each type of specimen 09)ascites anal gland fluid decubitus injury site intestinal juicestoma PEG insertion site pharynx fluid and synovial fluid(one strain from each type of specimen 05) Identificationwas performed using the MicroScan WalkAway 96 system(Beckman Coulter Tokyo Japan) E coli strain ATCC25922was obtained from American Type Culture Collection (Man-assas VA)
22 Antibiotic Susceptibility FOM imipenem (IPM) andceftazidime (CAZ) were provided by Meiji Seika Pharma(Tokyo Japan) MSD (Tokyo Japan) and Glaxo SmithKline(Tokyo Japan) respectively Minimum inhibitory concentra-tion (MIC) was determined by broth microdilution methodor agar plate dilution method according to the recommen-dations of the Clinical and Laboratory Standards Institute(CLSI) with breakpoints according to CLSI guidelines [34]
Table 1 PCR and real-time RT-PCR primers used in this study
Primer Sequence (51015840-31015840) Reference
murA-fulla F AAACAGCAGACGGTCTATGG[18]R CCATGAGTTTATCGACAGAACG
glpT-full F GCGAGTCGCGAGTTTTCATTG[18]R GGCAAATATCCACTGGCACC
uhpT-full F TTTTTGAACGCCCAGACACC[18]R AGTCAGGGGCTATTTGATGG
uhpT-partial F ATGCTGGCTTTCTTAAACC[19]R TTATGCCACTGTCAACTGC
uhpA-full F GATCGCGGTGTTTTTTCAG[18]R GATACTCCACAGGCAAAACC
uhpA-partial F ATCACCGTTGCCCTTATAGA[19]R TCACCAGCCATCAAACAT
ptsI-full F GAAAGCGGTTGAACATCTGG[18]R TCCTTCTTGTCGTCGGAAAC
cyaA-full F AACCAGGCGCGAAAAGTGG[18]R ACCTTCTGGGATTTGCTGG
rpoD-qPCR F CAAGCCGTGGTCGGAAAA[20]R GGGCGCGATGCACTTCT
uhpT-qPCR F AAGCCGACCCTGGACCTT[20]R ACGGTTTGAACCACATTTTGC
aPrimers designated ldquofullrdquo were used for direct sequencing and ldquoqPCRrdquowereused for real-time RT-PCR
TheMIC of FOM was measured by the agar dilution methodin the absence of G6P in the presence of 25120583gmL G6P andin the presence of 25120583gmL G6P and 2mM cAMP
23 Measurements of Carbohydrate Phosphate TransporterActivity E coli cells were cultured for 24 h inMullerndashHintonbroth and harvested by centrifugation The cell pellet waswashed twice with saline E coli cell suspension with salinewas then used to inoculate to M9 minimum salt (BectonDickinson Franklin Lakes NJ) agar supplemented with 02G6P or 02 G3P [18] Cell growth was observed afterincubation for 24 h in the case of G6P and for 48 h in the caseof G3P
24 Genetic Analysis DNA was isolated using DNeasy Kit(Qiagen Hilden Germany) according to the manufacturerrsquosinstructions Polymerase chain reaction (PCR) was per-formed using KAPATaq Extra HotStart Ready Mix with dye(NIPPON Genetics Tokyo Japan) Serogroups [35 36] andphylogenetic groups [37] were determined by PCR Multilo-cus sequence typing (MLST) was determined according toTartof et al [38] Genes of fosA fosA3 fosC2 [22] fosB2fosC fosX [26] fosB [21] fosA34 [25] and fosKP96 [24]were detected by PCRusing the primers described previouslyGene of fosA5 was detected by PCR using primer set 51015840-ACTGAATCACCTGACCCTGG-31015840 and 51015840-CGCATAATG-GGTGTAGTCGC-31015840 Full nucleotide sequences of six genes(murA uhpT glpT uhpA ptsI and cyaA) were determinedby a combination of direct sequencing and primer walkingwith the respective PCR products PCR primer sequences aregiven in Table 1 The sequencing was performed with Big
BioMed Research International 3
Table 2 Antibiotic susceptibility and the presence of ESBL genes in Ecoli strains with FOMMIC ge 8120583gmL
StrainsMIC (120583gmL)
ESBLageneFosfomycin(ge128)b
Levofloxacin(ge4)b
Gentamicin(ge8)b
Imipenem(ge2)b
Ceftazidime(ge8)b
SRE257 1024lowast le0125 1 0125 0125 mdashSRE91 128 32lowast 4 025 0125 mdashSRE49 128 16lowast 2 0125 025 mdashSRE54 64 ge64lowast ge64lowast 0125 2 CTX-M14SRE237 64 le0125 4 0125 0125 mdashSRE29 32 16lowast ge64lowast 0125 0125 mdashSRE252 32 05 2 0125 0125 mdashSRE280 32 le0125 8lowast 05 0125 mdashSRE18 16 32lowast 2 0125 2 CTX-M2SRE253 8 le0125 4 05 0125 mdashaESBL extended spectrum 120573-lactamasebBreakpoints (120583gmL) are according to CLSIlowastResistantIntermediate
Dye Terminator Kit version 31 and 3730xI DNA analyzer(Applied Biosystems Carlsbad CA) at Hokkaido SystemScience (Sapporo Japan)
25 Real-Time Reverse-Transcription (RT) PCR E coli cellswere grown for 24 h in Luria-Bertani (LB) broth and thecells were harvested and washed twice with M9 minimumsalt solution The suspended cells were used to inoculateto M9 minimum salt solution with or without 02 G6Psupplementation and incubated for 30min at 37∘C RNAwas isolated from the cells using RNeasy Plus Mini Kit(Qiagen) according to themanufacturerrsquos instructions cDNAwas prepared from the RNA using SuperScript III FirstStrand Synthesis Kit (Invitrogen Carlsbad CA) and randomhexamer oligonucleotide primers mRNA levels of uhpT andrpoD were quantified using QuantiFast SYBR Green PCRMastermix (Qiagen) by LightCycler LC480 (Roche BaselSwitzerland) with the cDNA as a template PCR primers weregiven in Table 1 Levels of uhpT transcript were calculated by2minusΔΔct method and data were normalized to the levels of thehouse-keeping gene rpoDmRNA
3 Results
31 Antibiotic Susceptibility Antibiotic susceptibility was ex-amined for 211 E coli clinical isolates MICs were determinedby broth microdilution method for LVX GEN IPM andCAZ and by agar plate dilution method for FOM (Table 2)The distribution of FOM MIC was shown in Figure 1 Threestrains (14) were not susceptible including resistant andintermediate Furthermore seven strains (3) with elevatedFOM MIC (ge8120583gmL le64 120583gmL) were observed out ofnormal distribution of the susceptible strains FOM MICsof these ten strains were not affected by the presence orabsence of G6P By contrast other susceptible strains (MICle 2 120583gmL) showed an increase of FOM MICs in theabsence of G6P (Table 3) Susceptibility to other antibiotics
012
8289
18
0 1 1 3 2 2 0 0 1 00
25
50
75
100N
umbe
r of s
trai
ns
MIC (120583gmL)
S I R0125
025
05 1 2 4 8
16
32
64
128
256
512
1024
2048
N = 211
Figure 1 Distribution of fosfomycinMICs of the E coli clinical iso-lates The breakpoint is according to CLSI guideline S susceptibleI intermediate and R resistant
was examined for strains with reduced susceptibility toFOM (Table 2) LVX-resistant strains comprised 50 andGEN-resistant strains comprised 30 of these strains IPM-resistant and CAZ-resistant strains were not observed how-ever 20 strains shared CTX-M-type ESBL genesThe occur-rence of antimicrobial nonsusceptibility was not significantlyhigh compared to total strains examined (data not shown)Genotypes (ie phylogenetic groups and MLST) and O-serogroups of these strains were variable (Table 3)
32 Analysis of Genes Associated with FOM Sensitivity Weexamined molecular mechanisms underlying the reductionof FOM sensitivity (MIC ge 8 120583gmL) First genes encodingFOM-modifying enzymes were investigated None of thefollowing were detected in the strains examined fosA fosA2to fosA5 fosC fosC2 fosB fosB2 fosX and fosKP96 (datanot shown) Next nucleotide sequences of murA uhpTuhpA glpT ptsI and cyaA genes were determined (Table 3)No murA coding sequence mutations that would result in
4 BioMed Research International
Table3Ch
aracteris
ticso
fEcolistrains
used
inthes
tudy
Strain
Specim
enSero-
grou
p
Phylo-
genetic
grou
pMLS
T
MIC
a
(120583gmL)
Growth
onM9agar
supp
lemented
with
uhpT
expressio
nindu
cedby
G6P
b
Aminoacid
resid
uealternations
inproteins
encodedby
glpT
ptsI
cyaA
murA
uhpA
and
uhpT
genesc
G6Pminus
G6P
+G6P
+cA
MP+
G3P
G6P
cyaA
glpT
murA
ptsI
uhpA
uhpT
SRE2
57Urin
eO1
B295
1024
1024
1024
+minus
124
mdashmdash
mdashVa
l399Leu
163sim
188
deletio
nmdash
SRE9
1Aspira
tion
tube
O1
D64
8128
128
32minus
minus378
mdash155sim
158
deletio
nPh
e176Leu
mdashmdash
Thr3Ala
mdash
SRE4
9Urin
eO25bH4
B2131
128
128
128
+minus
NT
His7
16Leu
mdashmdash
Lys410Arg
ND
ND
SRE5
4Urin
eO25bH4
B2131
6464
64+
minus15
9His7
16Leu
mdashmdash
Ala44
3Thr
Gly452A
spmdash
mdash
SRE2
37Urin
eO25bH4
B2131
6464
64+
minus15
9His7
16Leu
mdashmdash
mdashmdash
mdashSR
E29
Urin
eO25bH4
B2131
3232
32+
+122622
mdashmdash
mdashmdash
mdashmdash
SRE2
52Urin
eO25a
D73
3232
4minus
minus368
ND
Ile171Th
rmdash
Lys145Asn
mdashmdash
SRE2
80Ascites
O12
D1486
3232
32+
minus056
Ser142As
nmdash
mdashmdash
mdashmdash
SRE18
Urin
eND
D405
1616
16+
minus222
mdashmdash
mdashmdash
Met1Ile
mdashSR
E253
Urin
eO18
B295
88
8minus
minus19809
His7
16Leu
mdashmdash
mdashmdash
mdashSR
E40
Decub
itus
O25a
D501
3205
NT
++
28405
SRE4
1Ca
theter
urine
O1
D64
88
05
NT
++
32856
SRE110
Catheter
urine
O25bH4
B2131
805
NT
++
19002
SRE2
05Urin
eND
A131
805
NT
++
73419
SRE2
27Pu
sO1
B295
805
NT
++
2472
8SR
E30
Urin
eO1
D64
88
025
NT
++
7190
8AT
CC25922
NT
NT
3205
NT
++
30869
a FOM
MICsw
ered
etermined
inthep
resence(+)
orabsence(minus)o
fglucose-6-pho
sphate(G
6P)a
ndorc
AMP
b Ecolicellswereincub
ated
inM9minim
umsaltsolutio
nin
thep
resenceo
rabsence
ofG6PTh
euhpTmRN
Alevelswered
etermined
byreal-timeR
T-PC
Randthed
ataweren
ormalized
torpoD
mRN
Alevels
Indu
ctionof
uhpT
expressio
nby
G6P
was
calculated
bydividing
theu
hpTmRN
Alevelinthep
resenceo
fG6P
bytheu
hpTmRN
Alevelinthea
bsence
ofG6P
c Aminoacid
mutations
foun
don
lyin
strains
with
redu
cedFO
Msusceptib
ility(M
ICge8120583
gmL)
comparedwith
strains
with
FOM
MIClt1120583
gmL
NDnot
detectedN
Tno
ttested
BioMed Research International 5
Growth in the presence of G6P +minus
P lt 0001
01
1
10
100
1000
10000
Fold
indu
ctio
n of
uhpT
mRN
A ex
pres
sion
(a)
Fosfomycin MIC (120583gml)
P lt 0005
ge8 le4
01
1
10
100
1000
10000
Fold
indu
ctio
n of
uhpT
mRN
A ex
pres
sion
(b)
Figure 2 Induced expression levels of uhpT expression by the addition of G6P Nine clinical isolates with reduced susceptibility to FOM sixsusceptible clinical isolates and one standard strain are included Data used to generate this figure are given in Table 2 Statistical significancewas determined byMannndashWhitney test (a)Comparison of strains not grown (minus) or strains grown (+) inM9minimumsalt solution containingG6P (b) Comparison of strains with FOMMIC ge 8 120583gmL or le4 120583gmL
changes of amino acid residues in MurA were observed Aresistant strain (SRE257) and an intermediate-resistant strain(SRE91) had mutations in the genes that would result indeletions of a part of amino acid residues in UhpA andGlpT respectively In another intermediate-resistant strain(SRE49) uhpA and uhpT were not detected by PCR ampli-fication with two distinct primer pairs (ldquofullrdquo and ldquopartialrdquo inTable 1) In one strain with MIC 32 120583gmL (SRE252) cyaAwas not detectable by PCR All strains with reduced FOMMIC (ge8120583gmL) except one (SRE29) had several mutationsin one or more genes leading to amino acid deletion or pointmutation(s) of amino acid residues compared with othersusceptible strains
33 Function and Expression of Carbohydrate PhosphateTransporters To determine the activity of UhpT and GlpTwe examined cell growth in M9 minimum salt solutionsupplementedwithG6P orG3PNine of ten strainswith FOMMIC ge 8120583gmL did not grow within 24 h on G6P-containingM9 minimum salt agar On the other hand only three often strains did not grow in the presence of G3P (Table 3)and the two strains (SRE91 and SRE252) showed reducedMIC to FOM by addition of cAMP This suggested that thesetwo strains shared insufficient intracellular concentration ofcAMP for full expression of GlpT andor UhpTThese resultssuggested that FOM incorporation through the UhpT systemis involved to a greater extent in the reduction of E coli FOMcompared to the GlpT system
Expression of UhpT is induced by G6P [15ndash17] Wetherefore determined uhpT gene induction by G6P usingquantitative RT-PCR analysis (Table 3 and Figure 2) In
strains with FOM MIC lt 1 120583gmL uhpT expression wasstrongly induced (190- to 730-fold) by G6P By contrast ineight of ten strains with FOM MIC ge 8120583gmL the uhpTinduction was markedly lower (056- to 38-fold) or no uhpTsignal was amplified by PCR Of the remaining two strainswe observed a high induction (200-fold) of uhpT expressionby G6P in SRE253 strain however the strain did not grow onG6P-containing M9 minimum agar High induction (1200-fold) of uhpT by G6P was observed in SRE29 strain and thestrain grew on G6P-containing M9 minimum agar howeverit showed reduced susceptibility to FOM (MIC 32 120583gmL)These results indicated that G6P-dependent growth lacked inmost strains with reduced susceptibility to FOM because oflack of uhpT or a markedly reduced uhpT induction by G6PHowever the changes in uhpT expression and UhpT activitydid not account for the reduced FOM susceptibility of a fewstrains (Figure 2)
4 Discussion
The frequency of FOM resistance has been recognized to below And it has been expected that the frequency of cross-resistance of FOMand other antibiotics is very low because ofa unique mode of action However several reports examinedFOM resistance in ESBL-producing E coli [19 23 24 28] Areport indicates that FOM-resistant and intermediate E coliare more frequently resistant to other types of antimicrobialsthan FOM-susceptible strains [28] These reports suggestthe importance for surveillance of FOM-resistant E coliparticularly for focus on their cross-resistance of FOM inmultidrug resistance
6 BioMed Research International
In this study only one resistant and two intermediatestrains were found among 211 clinical isolates from Japan(Figure 1) This indicated that FOM was a promising candi-date agent against E coli infections as generally describedHowever we found seven strains susceptible according tothe CLST breakpoint but reduced susceptibility (MIC ge8 120583gmL) Under the selective pressure of FOM usage thesestrains might acquire FOM resistance more easily In thepresent study cross-resistance of strains with reduced FOMsusceptibility to other antimicrobial drugs was not signifi-cantly higher than in other strains and these strains were notconcentrating on specific genotypes
Based on previous reports acquisition of FOM-modifying enzymes (encoded by fos genes) and mutationsin murA gene results in resistance that is surpass ofbreakpoints to FOM [18 19 28] The present study didnot identify such strains Another resistance mechanismis altered FOM incorporation into bacterial cells TheG6P and G3P transporters contribute to incorporationof FOM into cells [13 14] Loss of function or decreasedexpression levels of them leads to a reduction of FOMsusceptibility [18 29 39] We found that loss of UhpT(G6P transporter) activity is more dominant than that ofGlpT (G3P transporter) in strains with decreased FOMsusceptibility In resistant (gt128 120583gmL) and intermediate(128 120583gmL) strains we noted amino acid deletion in therespective encoded proteins and no PCR amplificationof the transporter-related genes (Table 3) In strains withFOM MICs between 8 and 64 120583gmL gene mutation(s)leading to alternations of amino acid residues were foundhowever it was unclear whether these contributed tothe reduced susceptibility Among the ten strains withreduced susceptibility nine strains did not grow on G6P-supplemented M9 minimum salt agar This suggested thatin these nine strains G6P-induced UhpT function wasattenuated We found several mutations in cyaA and ptsI insome of these strains Dysfunction of CyaA and PtsI leadsto decrease in intracellular concentration of cAMP andinsufficient expression of GlpT and UhpT [16 17 31] Onlytwo strains (SRE91 and SRE252) with reduced susceptibilityto FOM showed that MIC was decreased by the exogenousaddition of cAMP and these strains did not grow in M9medium supplement with G3P Certainly SRE252 defectedcyaA however SRE91 did not share any mutations in cyaAand pstI Thus reduced susceptibility to FOM in the otherstrains was not explained by the insufficient intracellularconcentration of cAMP caused by dysfunction of cyaAand pstI The mutations in these genes found in this study(Table 3) seemed to scarcely relate with dysfunction of cAMPsynthesis
In conclusion FOM resistance occurs with low frequencyand is independent of resistance to other antimicrobials inE coli clinical isolates from Japan On the other hand weidentified strains with decreased FOM susceptibility Mostof them displayed fluctuated activity of the G6P transporterUhpT However G6P transporter function was altered eventhough the G6P-induced uhpT expression and amino acidsequence of UhpT were preserved in one strain (FOM MIC8 120583gmL) Another strain (FOM MIC 32 120583gmL) displayed
reduced susceptibility to FOM and no alteration of MICin the presence and absence of G6P even though G6P-induced uhpT expression amino acid sequence of UhpT andgrowth on G6P-supplemented M9 minimum salt agar werepreserved The exact molecular mechanism of the reducedsusceptibility of these strains remains unclear and requiresfurther evaluation
Competing Interests
The authors declare no competing interests
Acknowledgments
The authors thank Osamu Kuwahara (Sapporo Clinical Lab-oratory) for the provision of E coli clinical isolates Thisstudy was partly supported by a Grant-in-Aid for ScientificResearch (15H06521) from Japan Society for the Promotionof Science and a grant from Yuasa Memorial Foundation
References
[1] WHO ldquoAntimicrobial resistance global report on surveil-lance 2014rdquo 2014 httpwwwwhointdrugresistancedocu-mentssurveillancereporten
[2] M E Falagas andD E Karageorgopoulos ldquoExtended-spectrum120573-lactamase-producing organismsrdquo Journal of Hospital Infec-tion vol 73 no 4 pp 345ndash354 2009
[3] J D D Pitout ldquoExtraintestinal pathogenic Escherichia coli anupdate on antimicrobial resistance laboratory diagnosis andtreatmentrdquo Expert Review of Anti-Infective Therapy vol 10 no10 pp 1165ndash1176 2012
[4] S Yokota T Sato T Okubo et al ldquoPrevalence of fluoroquin-olone-resistant Escherichia coli O25H4-ST131 (CTX-M15-nonproducing) strains isolated in JapanrdquoChemotherapy vol 58no 1 pp 52ndash59 2012
[5] N Tsukamoto Y Ohkoshi T Okubo et al ldquoHigh prevalenceof cross-resistance to aminoglycosides in fluoroquinolone-resistantEscherichia coli clinical isolatesrdquoChemotherapy vol 59no 5 pp 379ndash384 2014
[6] H Giamarellou and G Poulakou ldquoMultidrug-resistant gram-negative infections what are the treatment optionsrdquoDrugs vol69 no 14 pp 1879ndash1901 2009
[7] M E Falagas A C Kastoris A M Kapaskelis and D E Kara-georgopoulos ldquoFosfomycin for the treatment of multidrug-resistant including extended-spectrum 120573-lactamase produc-ing Enterobacteriaceae infections a systematic reviewrdquo TheLancet Infectious Diseases vol 10 no 1 pp 43ndash50 2010
[8] A Schaeffer ldquoInfections of the urinary tractrdquo inCampbell-WalshUrology A Wein L Kavoussi A Novick A Partin and CPeters Eds pp 223ndash303 Elsevier Philadelphia Pa USA 9thedition 2007
[9] K Ishikawa R Hamasuna S Uehara et al ldquoJapanese nation-wide surveillance in 2011 of antibacterial susceptibility patternsof clinical isolates from complicated urinary tract infectioncasesrdquo Journal of Infection and Chemotherapy vol 21 no 9 pp623ndash633 2015
[10] D N Gilbert H F Chambers GM Eliopoulos andM S SaagTheSanford Guide to AntimicrobialTherapy 2015 AntimicrobialTherapy Sperryville Va USA 45th edition 2015
BioMed Research International 7
[11] The Japanese Association for Infectious Diseases and JapanSociety of ChemotherapyThe JAIDJSC Guide to Clinical Man-agement of Infectious Diseases 2014 The Japanese Associationfor Infectious DiseasesJapan Society of Chemotherapy TokyoJapan 2014 (Japanese)
[12] E D Brown E I Vivas C T Walsh and R Kolter ldquoMurA(MurZ) the enzyme that catalyzes the first committed stepin peptidoglycan biosynthesis is essential in Escherichia colirdquoJournal of Bacteriology vol 177 no 14 pp 4194ndash4197 1995
[13] F M Kahan J S Kahan P J Cassidy and H Kropp ldquoThemechanismof action of fosfomycin (phosphonomycin)rdquoAnnalsof the NewYork Academy of Sciences vol 235 no 1 pp 364ndash3861974
[14] M J Lemieux Y Huang and D-N Wang ldquoGlycerol-3-phosphate transporter of Escherichia coli structure functionand regulationrdquo Research in Microbiology vol 155 no 8 pp623ndash629 2004
[15] H HWinkler ldquoKinetics of exogenous induction of the hexose-6-phosphate transport system of Escherichia colirdquo Journal ofBacteriology vol 107 no 1 pp 74ndash78 1971
[16] T J Merkel J L Dahl R H Ebright and R J KadnerldquoTranscription activation at the Escherichia coli uhpT promoterby the catabolite gene activator proteinrdquo Journal of Bacteriologyvol 177 no 7 pp 1712ndash1718 1995
[17] D T Verhamme P W Postma W Crielaard and K J Helling-werf ldquoCooperativity in signal transfer through the Uhp systemof Escherichia colirdquo Journal of Bacteriology vol 184 no 15 pp4205ndash4210 2002
[18] S Takahata T Ida T Hiraishi et al ldquoMolecular mechanismsof fosfomycin resistance in clinical isolates of Escherichia colirdquoInternational Journal of Antimicrobial Agents vol 35 no 4 pp333ndash337 2010
[19] J Wachino K Yamane S Suzuki K Kimura and YArakawa ldquoPrevalence of fosfomycin resistance among CTX-M-producing Escherichia coli clinical isolates in Japan and iden-tification of novel plasmid-mediated fosfomycin-modifyingenzymesrdquo Antimicrobial Agents and Chemotherapy vol 54 no7 pp 3061ndash3064 2010
[20] K Kurabayashi Y Hirakawa K Tanimoto H Tomita and HHirakawa ldquoRole of the CpxAR two-component signal trans-duction system in control of fosfomycin resistance and carbonsubstrate uptakerdquo Journal of Bacteriology vol 196 no 2 pp248ndash256 2014
[21] P Arca G Reguera and C Hardisson ldquoPlasmid-encodedfosfomycin resistance in bacteria isolated from the urinary tractin amulticentre surveyrdquo Journal of Antimicrobial Chemotherapyvol 40 no 3 pp 393ndash399 1997
[22] J Hou X Huang Y Deng et al ldquoDissemination of thefosfomycin resistance gene fosA3 with CTX-M 120573-lactamasegenes and rmtB carried on incfII plasmids among Escherichiacoli isolates from pets in Chinardquo Antimicrobial Agents andChemotherapy vol 56 no 4 pp 2135ndash2138 2012
[23] S-Y Lee Y-J Park J K Yu et al ldquoPrevalence of acquiredfosfomycin resistance among extended-spectrum 120573-lactamase-producing Escherichia coli and Klebsiella pneumoniae clinicalisolates in Korea and IS26-composite transposon surroundingfosA3rdquo Journal of Antimicrobial Chemotherapy vol 67 no 12Article ID dks319 pp 2843ndash2847 2012
[24] P L Ho J Chan W U Lo et al ldquoPrevalence and molecularepidemiology of plasmid-mediated fosfomycin resistance genesamong blood and urinary Escherichia coli isolatesrdquo Journal ofMedical Microbiology vol 62 no 11 pp 1707ndash1713 2013
[25] G Nakamura J Wachino N Sato et al ldquoPractical agar-based disk potentiation test for detection of fosfomycin-nonsusceptible Escherichia coli clinical isolates producing glu-tathione S-transferasesrdquo Journal of ClinicalMicrobiology vol 52no 9 pp 3175ndash3179 2014
[26] Y Ma X Xu Q Guo P Wang W Wang and M WangldquoCharacterization of fosA5 a newplasmid-mediated fosfomycinresistance gene in Escherichia colirdquo Letters in Applied Microbiol-ogy vol 60 no 3 pp 259ndash264 2015
[27] Y Li B Zheng Y Li S Zhu F Xue and J Liu ldquoAntimicrobialsusceptibility and molecular mechanisms of fosfomycin resis-tance in clinical Escherichia coli isolates in mainland ChinardquoPLoS ONE vol 10 no 8 Article ID e0135269 2015
[28] S-P Tseng S-F Wang C-Y Kuo et al ldquoCharacteriza-tion of fosfomycin resistant extended-spectrum szlig-lactamase-producing Escherichia coli isolates from human and pig inTaiwanrdquo PLoS ONE vol 10 no 8 Article ID e0135864 2015
[29] A I Nilsson O G Berg O Aspevall G Kahlmeter and DI Andersson ldquoBiological costs and mechanisms of fosfomycinresistance in Escherichia colirdquo Antimicrobial Agents and Chem-otherapy vol 47 no 9 pp 2850ndash2858 2003
[30] J C Cordaro T Melton J P Stratis et al ldquoFosfomycinresistance selectionmethod for internal and extended deletionsof the phosphoenolpyruvatesugar phosphotransferase genes ofSalmonella typhimuriumrdquo Journal of Bacteriology vol 128 no3 pp 785ndash793 1976
[31] Y Sakamoto S Furukawa H Ogihara and M YamasakildquoFosmidomycin resistance in adenylate cyclase deficient (cya)mutants of Escherichia colirdquo Bioscience Biotechnology and Bio-chemistry vol 67 no 9 pp 2030ndash2033 2003
[32] G Kahlmeter and H O Poulsen ldquoAntimicrobial susceptibilityof Escherichia coli from community-acquired urinary tractinfections in Europe the ECOsdotSENS study revisitedrdquo Interna-tional Journal of Antimicrobial Agents vol 39 no 1 pp 45ndash512012
[33] D E Karageorgopoulos R Wang X-H Yu and M E FalagasldquoFosfomycin evaluation of the published evidence on the emer-gence of antimicrobial resistance in gram-negative pathogensrdquoJournal of Antimicrobial Chemotherapy vol 67 no 2 pp 255ndash268 2012
[34] Clinical and Laboratory Standards Institute Performance Stan-dards for Antimicrobial Susceptibility Testing 22nd InformationSupplement M100-S22 CLSI Wayne Pa USA 2012
[35] O Clermont J R Johnson M Menard and E DenamurldquoDetermination of Escherichia coli O types by allele-specificpolymerase chain reaction application to the O types involvedin human septicemiardquo Diagnostic Microbiology and InfectiousDisease vol 57 no 2 pp 129ndash136 2007
[36] D Li B Liu M Chen et al ldquoA multiplex PCR method todetect 14 Escherichia coli serogroups associated with urinarytract infectionsrdquo Journal of Microbiological Methods vol 82 no1 pp 71ndash77 2010
[37] O Clermont S Bonacorsi and E Bingen ldquoRapid and simpledetermination of the Escherichia coli phylogenetic grouprdquoApplied and Environmental Microbiology vol 66 no 10 pp4555ndash4558 2000
[38] S Y Tartof O D Solberg A R Manges and L W RileyldquoAnalysis of a uropathogenic Escherichia coli clonal group bymultilocus sequence typingrdquo Journal of Clinical Microbiologyvol 43 no 12 pp 5860ndash5864 2005
8 BioMed Research International
[39] R J Kadner and H HWinkler ldquoIsolation and characterizationof mutations affecting the transport of hexose phosphates inEscherichia colirdquo Journal of Bacteriology vol 113 no 2 pp 895ndash900 1973
Submit your manuscripts athttpswwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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2 BioMed Research International
fosA5 fosC fosC2 fosB fosB2 fosX and fosKP96 found in var-ious bacteria [19 21ndash26] and mutation(s) in murA gene [1824 27 28] Furthermore mutations in the transporter genesuhpT and glpT and genes encoding proteins regulating uhpTexpression such as uhpA reduce the susceptibility to FOM[18 19 27ndash29] In addition expression levels of UhpT andGlpT are positively regulated by cyclic AMP (cAMP) [16 17]The levels of cAMP are controlled by phosphoenolpyruvate-protein phosphotransferase I encoded by ptsI and adenylcyclase encoded by cyaA andmutations in these genes resultin reduced susceptibility to FOM [29ndash31]
Nevertheless several surveillance studies report that therate of emergence of E coli isolates showing FOM resistanceor reduced FOM susceptibility has been markedly low [79 32] Although spontaneously mutational rate to acquireFOM resistance is high FOM resistance confers biologicalcosts such as reduced cell growth rate in Gram-negativebacteria [29 33] This indicates that FOM continues to bean effective agent against E coli infections However theoverall up-to-date status of FOM resistance needs to becontinuously surveyed and its molecular characteristics haveto be understood to prevent future emergence and increaseof multidrug-resistant E coli with FOM resistance in theclinic In this study we screened E coli clinical isolates fromJapan showing resistance or reduced susceptibility to FOMand identified the molecular mechanisms of their reducedsusceptibility
2 Materials and Methods
21 Bacterial Strains E coli clinical isolates (211 strains) werecollected in the years 2008-2009 as described previously [45]Thesewere identified and stocked in SapporoClinical Lab-oratory Inc (Sapporo Japan) These strains were collectedfrom a variety of clinical specimens in almost entire area ofHokkaido Prefecture Japan This study was approved by thereview boards of the relevant institutions The strains wereisolated from the following clinical specimens urine (119899 =87 410) catheter urine (119899 = 76 358) sputum (119899 =15 71) stool (119899 = 7 33) vaginal secretion (119899 = 628) pus (119899 = 3 14) aspiration tube (119899 = 3 14)drainage tube intravenous hyperalimentation catheter tuberhinorrhea (two strains from each type of specimen 09)ascites anal gland fluid decubitus injury site intestinal juicestoma PEG insertion site pharynx fluid and synovial fluid(one strain from each type of specimen 05) Identificationwas performed using the MicroScan WalkAway 96 system(Beckman Coulter Tokyo Japan) E coli strain ATCC25922was obtained from American Type Culture Collection (Man-assas VA)
22 Antibiotic Susceptibility FOM imipenem (IPM) andceftazidime (CAZ) were provided by Meiji Seika Pharma(Tokyo Japan) MSD (Tokyo Japan) and Glaxo SmithKline(Tokyo Japan) respectively Minimum inhibitory concentra-tion (MIC) was determined by broth microdilution methodor agar plate dilution method according to the recommen-dations of the Clinical and Laboratory Standards Institute(CLSI) with breakpoints according to CLSI guidelines [34]
Table 1 PCR and real-time RT-PCR primers used in this study
Primer Sequence (51015840-31015840) Reference
murA-fulla F AAACAGCAGACGGTCTATGG[18]R CCATGAGTTTATCGACAGAACG
glpT-full F GCGAGTCGCGAGTTTTCATTG[18]R GGCAAATATCCACTGGCACC
uhpT-full F TTTTTGAACGCCCAGACACC[18]R AGTCAGGGGCTATTTGATGG
uhpT-partial F ATGCTGGCTTTCTTAAACC[19]R TTATGCCACTGTCAACTGC
uhpA-full F GATCGCGGTGTTTTTTCAG[18]R GATACTCCACAGGCAAAACC
uhpA-partial F ATCACCGTTGCCCTTATAGA[19]R TCACCAGCCATCAAACAT
ptsI-full F GAAAGCGGTTGAACATCTGG[18]R TCCTTCTTGTCGTCGGAAAC
cyaA-full F AACCAGGCGCGAAAAGTGG[18]R ACCTTCTGGGATTTGCTGG
rpoD-qPCR F CAAGCCGTGGTCGGAAAA[20]R GGGCGCGATGCACTTCT
uhpT-qPCR F AAGCCGACCCTGGACCTT[20]R ACGGTTTGAACCACATTTTGC
aPrimers designated ldquofullrdquo were used for direct sequencing and ldquoqPCRrdquowereused for real-time RT-PCR
TheMIC of FOM was measured by the agar dilution methodin the absence of G6P in the presence of 25120583gmL G6P andin the presence of 25120583gmL G6P and 2mM cAMP
23 Measurements of Carbohydrate Phosphate TransporterActivity E coli cells were cultured for 24 h inMullerndashHintonbroth and harvested by centrifugation The cell pellet waswashed twice with saline E coli cell suspension with salinewas then used to inoculate to M9 minimum salt (BectonDickinson Franklin Lakes NJ) agar supplemented with 02G6P or 02 G3P [18] Cell growth was observed afterincubation for 24 h in the case of G6P and for 48 h in the caseof G3P
24 Genetic Analysis DNA was isolated using DNeasy Kit(Qiagen Hilden Germany) according to the manufacturerrsquosinstructions Polymerase chain reaction (PCR) was per-formed using KAPATaq Extra HotStart Ready Mix with dye(NIPPON Genetics Tokyo Japan) Serogroups [35 36] andphylogenetic groups [37] were determined by PCR Multilo-cus sequence typing (MLST) was determined according toTartof et al [38] Genes of fosA fosA3 fosC2 [22] fosB2fosC fosX [26] fosB [21] fosA34 [25] and fosKP96 [24]were detected by PCRusing the primers described previouslyGene of fosA5 was detected by PCR using primer set 51015840-ACTGAATCACCTGACCCTGG-31015840 and 51015840-CGCATAATG-GGTGTAGTCGC-31015840 Full nucleotide sequences of six genes(murA uhpT glpT uhpA ptsI and cyaA) were determinedby a combination of direct sequencing and primer walkingwith the respective PCR products PCR primer sequences aregiven in Table 1 The sequencing was performed with Big
BioMed Research International 3
Table 2 Antibiotic susceptibility and the presence of ESBL genes in Ecoli strains with FOMMIC ge 8120583gmL
StrainsMIC (120583gmL)
ESBLageneFosfomycin(ge128)b
Levofloxacin(ge4)b
Gentamicin(ge8)b
Imipenem(ge2)b
Ceftazidime(ge8)b
SRE257 1024lowast le0125 1 0125 0125 mdashSRE91 128 32lowast 4 025 0125 mdashSRE49 128 16lowast 2 0125 025 mdashSRE54 64 ge64lowast ge64lowast 0125 2 CTX-M14SRE237 64 le0125 4 0125 0125 mdashSRE29 32 16lowast ge64lowast 0125 0125 mdashSRE252 32 05 2 0125 0125 mdashSRE280 32 le0125 8lowast 05 0125 mdashSRE18 16 32lowast 2 0125 2 CTX-M2SRE253 8 le0125 4 05 0125 mdashaESBL extended spectrum 120573-lactamasebBreakpoints (120583gmL) are according to CLSIlowastResistantIntermediate
Dye Terminator Kit version 31 and 3730xI DNA analyzer(Applied Biosystems Carlsbad CA) at Hokkaido SystemScience (Sapporo Japan)
25 Real-Time Reverse-Transcription (RT) PCR E coli cellswere grown for 24 h in Luria-Bertani (LB) broth and thecells were harvested and washed twice with M9 minimumsalt solution The suspended cells were used to inoculateto M9 minimum salt solution with or without 02 G6Psupplementation and incubated for 30min at 37∘C RNAwas isolated from the cells using RNeasy Plus Mini Kit(Qiagen) according to themanufacturerrsquos instructions cDNAwas prepared from the RNA using SuperScript III FirstStrand Synthesis Kit (Invitrogen Carlsbad CA) and randomhexamer oligonucleotide primers mRNA levels of uhpT andrpoD were quantified using QuantiFast SYBR Green PCRMastermix (Qiagen) by LightCycler LC480 (Roche BaselSwitzerland) with the cDNA as a template PCR primers weregiven in Table 1 Levels of uhpT transcript were calculated by2minusΔΔct method and data were normalized to the levels of thehouse-keeping gene rpoDmRNA
3 Results
31 Antibiotic Susceptibility Antibiotic susceptibility was ex-amined for 211 E coli clinical isolates MICs were determinedby broth microdilution method for LVX GEN IPM andCAZ and by agar plate dilution method for FOM (Table 2)The distribution of FOM MIC was shown in Figure 1 Threestrains (14) were not susceptible including resistant andintermediate Furthermore seven strains (3) with elevatedFOM MIC (ge8120583gmL le64 120583gmL) were observed out ofnormal distribution of the susceptible strains FOM MICsof these ten strains were not affected by the presence orabsence of G6P By contrast other susceptible strains (MICle 2 120583gmL) showed an increase of FOM MICs in theabsence of G6P (Table 3) Susceptibility to other antibiotics
012
8289
18
0 1 1 3 2 2 0 0 1 00
25
50
75
100N
umbe
r of s
trai
ns
MIC (120583gmL)
S I R0125
025
05 1 2 4 8
16
32
64
128
256
512
1024
2048
N = 211
Figure 1 Distribution of fosfomycinMICs of the E coli clinical iso-lates The breakpoint is according to CLSI guideline S susceptibleI intermediate and R resistant
was examined for strains with reduced susceptibility toFOM (Table 2) LVX-resistant strains comprised 50 andGEN-resistant strains comprised 30 of these strains IPM-resistant and CAZ-resistant strains were not observed how-ever 20 strains shared CTX-M-type ESBL genesThe occur-rence of antimicrobial nonsusceptibility was not significantlyhigh compared to total strains examined (data not shown)Genotypes (ie phylogenetic groups and MLST) and O-serogroups of these strains were variable (Table 3)
32 Analysis of Genes Associated with FOM Sensitivity Weexamined molecular mechanisms underlying the reductionof FOM sensitivity (MIC ge 8 120583gmL) First genes encodingFOM-modifying enzymes were investigated None of thefollowing were detected in the strains examined fosA fosA2to fosA5 fosC fosC2 fosB fosB2 fosX and fosKP96 (datanot shown) Next nucleotide sequences of murA uhpTuhpA glpT ptsI and cyaA genes were determined (Table 3)No murA coding sequence mutations that would result in
4 BioMed Research International
Table3Ch
aracteris
ticso
fEcolistrains
used
inthes
tudy
Strain
Specim
enSero-
grou
p
Phylo-
genetic
grou
pMLS
T
MIC
a
(120583gmL)
Growth
onM9agar
supp
lemented
with
uhpT
expressio
nindu
cedby
G6P
b
Aminoacid
resid
uealternations
inproteins
encodedby
glpT
ptsI
cyaA
murA
uhpA
and
uhpT
genesc
G6Pminus
G6P
+G6P
+cA
MP+
G3P
G6P
cyaA
glpT
murA
ptsI
uhpA
uhpT
SRE2
57Urin
eO1
B295
1024
1024
1024
+minus
124
mdashmdash
mdashVa
l399Leu
163sim
188
deletio
nmdash
SRE9
1Aspira
tion
tube
O1
D64
8128
128
32minus
minus378
mdash155sim
158
deletio
nPh
e176Leu
mdashmdash
Thr3Ala
mdash
SRE4
9Urin
eO25bH4
B2131
128
128
128
+minus
NT
His7
16Leu
mdashmdash
Lys410Arg
ND
ND
SRE5
4Urin
eO25bH4
B2131
6464
64+
minus15
9His7
16Leu
mdashmdash
Ala44
3Thr
Gly452A
spmdash
mdash
SRE2
37Urin
eO25bH4
B2131
6464
64+
minus15
9His7
16Leu
mdashmdash
mdashmdash
mdashSR
E29
Urin
eO25bH4
B2131
3232
32+
+122622
mdashmdash
mdashmdash
mdashmdash
SRE2
52Urin
eO25a
D73
3232
4minus
minus368
ND
Ile171Th
rmdash
Lys145Asn
mdashmdash
SRE2
80Ascites
O12
D1486
3232
32+
minus056
Ser142As
nmdash
mdashmdash
mdashmdash
SRE18
Urin
eND
D405
1616
16+
minus222
mdashmdash
mdashmdash
Met1Ile
mdashSR
E253
Urin
eO18
B295
88
8minus
minus19809
His7
16Leu
mdashmdash
mdashmdash
mdashSR
E40
Decub
itus
O25a
D501
3205
NT
++
28405
SRE4
1Ca
theter
urine
O1
D64
88
05
NT
++
32856
SRE110
Catheter
urine
O25bH4
B2131
805
NT
++
19002
SRE2
05Urin
eND
A131
805
NT
++
73419
SRE2
27Pu
sO1
B295
805
NT
++
2472
8SR
E30
Urin
eO1
D64
88
025
NT
++
7190
8AT
CC25922
NT
NT
3205
NT
++
30869
a FOM
MICsw
ered
etermined
inthep
resence(+)
orabsence(minus)o
fglucose-6-pho
sphate(G
6P)a
ndorc
AMP
b Ecolicellswereincub
ated
inM9minim
umsaltsolutio
nin
thep
resenceo
rabsence
ofG6PTh
euhpTmRN
Alevelswered
etermined
byreal-timeR
T-PC
Randthed
ataweren
ormalized
torpoD
mRN
Alevels
Indu
ctionof
uhpT
expressio
nby
G6P
was
calculated
bydividing
theu
hpTmRN
Alevelinthep
resenceo
fG6P
bytheu
hpTmRN
Alevelinthea
bsence
ofG6P
c Aminoacid
mutations
foun
don
lyin
strains
with
redu
cedFO
Msusceptib
ility(M
ICge8120583
gmL)
comparedwith
strains
with
FOM
MIClt1120583
gmL
NDnot
detectedN
Tno
ttested
BioMed Research International 5
Growth in the presence of G6P +minus
P lt 0001
01
1
10
100
1000
10000
Fold
indu
ctio
n of
uhpT
mRN
A ex
pres
sion
(a)
Fosfomycin MIC (120583gml)
P lt 0005
ge8 le4
01
1
10
100
1000
10000
Fold
indu
ctio
n of
uhpT
mRN
A ex
pres
sion
(b)
Figure 2 Induced expression levels of uhpT expression by the addition of G6P Nine clinical isolates with reduced susceptibility to FOM sixsusceptible clinical isolates and one standard strain are included Data used to generate this figure are given in Table 2 Statistical significancewas determined byMannndashWhitney test (a)Comparison of strains not grown (minus) or strains grown (+) inM9minimumsalt solution containingG6P (b) Comparison of strains with FOMMIC ge 8 120583gmL or le4 120583gmL
changes of amino acid residues in MurA were observed Aresistant strain (SRE257) and an intermediate-resistant strain(SRE91) had mutations in the genes that would result indeletions of a part of amino acid residues in UhpA andGlpT respectively In another intermediate-resistant strain(SRE49) uhpA and uhpT were not detected by PCR ampli-fication with two distinct primer pairs (ldquofullrdquo and ldquopartialrdquo inTable 1) In one strain with MIC 32 120583gmL (SRE252) cyaAwas not detectable by PCR All strains with reduced FOMMIC (ge8120583gmL) except one (SRE29) had several mutationsin one or more genes leading to amino acid deletion or pointmutation(s) of amino acid residues compared with othersusceptible strains
33 Function and Expression of Carbohydrate PhosphateTransporters To determine the activity of UhpT and GlpTwe examined cell growth in M9 minimum salt solutionsupplementedwithG6P orG3PNine of ten strainswith FOMMIC ge 8120583gmL did not grow within 24 h on G6P-containingM9 minimum salt agar On the other hand only three often strains did not grow in the presence of G3P (Table 3)and the two strains (SRE91 and SRE252) showed reducedMIC to FOM by addition of cAMP This suggested that thesetwo strains shared insufficient intracellular concentration ofcAMP for full expression of GlpT andor UhpTThese resultssuggested that FOM incorporation through the UhpT systemis involved to a greater extent in the reduction of E coli FOMcompared to the GlpT system
Expression of UhpT is induced by G6P [15ndash17] Wetherefore determined uhpT gene induction by G6P usingquantitative RT-PCR analysis (Table 3 and Figure 2) In
strains with FOM MIC lt 1 120583gmL uhpT expression wasstrongly induced (190- to 730-fold) by G6P By contrast ineight of ten strains with FOM MIC ge 8120583gmL the uhpTinduction was markedly lower (056- to 38-fold) or no uhpTsignal was amplified by PCR Of the remaining two strainswe observed a high induction (200-fold) of uhpT expressionby G6P in SRE253 strain however the strain did not grow onG6P-containing M9 minimum agar High induction (1200-fold) of uhpT by G6P was observed in SRE29 strain and thestrain grew on G6P-containing M9 minimum agar howeverit showed reduced susceptibility to FOM (MIC 32 120583gmL)These results indicated that G6P-dependent growth lacked inmost strains with reduced susceptibility to FOM because oflack of uhpT or a markedly reduced uhpT induction by G6PHowever the changes in uhpT expression and UhpT activitydid not account for the reduced FOM susceptibility of a fewstrains (Figure 2)
4 Discussion
The frequency of FOM resistance has been recognized to below And it has been expected that the frequency of cross-resistance of FOMand other antibiotics is very low because ofa unique mode of action However several reports examinedFOM resistance in ESBL-producing E coli [19 23 24 28] Areport indicates that FOM-resistant and intermediate E coliare more frequently resistant to other types of antimicrobialsthan FOM-susceptible strains [28] These reports suggestthe importance for surveillance of FOM-resistant E coliparticularly for focus on their cross-resistance of FOM inmultidrug resistance
6 BioMed Research International
In this study only one resistant and two intermediatestrains were found among 211 clinical isolates from Japan(Figure 1) This indicated that FOM was a promising candi-date agent against E coli infections as generally describedHowever we found seven strains susceptible according tothe CLST breakpoint but reduced susceptibility (MIC ge8 120583gmL) Under the selective pressure of FOM usage thesestrains might acquire FOM resistance more easily In thepresent study cross-resistance of strains with reduced FOMsusceptibility to other antimicrobial drugs was not signifi-cantly higher than in other strains and these strains were notconcentrating on specific genotypes
Based on previous reports acquisition of FOM-modifying enzymes (encoded by fos genes) and mutationsin murA gene results in resistance that is surpass ofbreakpoints to FOM [18 19 28] The present study didnot identify such strains Another resistance mechanismis altered FOM incorporation into bacterial cells TheG6P and G3P transporters contribute to incorporationof FOM into cells [13 14] Loss of function or decreasedexpression levels of them leads to a reduction of FOMsusceptibility [18 29 39] We found that loss of UhpT(G6P transporter) activity is more dominant than that ofGlpT (G3P transporter) in strains with decreased FOMsusceptibility In resistant (gt128 120583gmL) and intermediate(128 120583gmL) strains we noted amino acid deletion in therespective encoded proteins and no PCR amplificationof the transporter-related genes (Table 3) In strains withFOM MICs between 8 and 64 120583gmL gene mutation(s)leading to alternations of amino acid residues were foundhowever it was unclear whether these contributed tothe reduced susceptibility Among the ten strains withreduced susceptibility nine strains did not grow on G6P-supplemented M9 minimum salt agar This suggested thatin these nine strains G6P-induced UhpT function wasattenuated We found several mutations in cyaA and ptsI insome of these strains Dysfunction of CyaA and PtsI leadsto decrease in intracellular concentration of cAMP andinsufficient expression of GlpT and UhpT [16 17 31] Onlytwo strains (SRE91 and SRE252) with reduced susceptibilityto FOM showed that MIC was decreased by the exogenousaddition of cAMP and these strains did not grow in M9medium supplement with G3P Certainly SRE252 defectedcyaA however SRE91 did not share any mutations in cyaAand pstI Thus reduced susceptibility to FOM in the otherstrains was not explained by the insufficient intracellularconcentration of cAMP caused by dysfunction of cyaAand pstI The mutations in these genes found in this study(Table 3) seemed to scarcely relate with dysfunction of cAMPsynthesis
In conclusion FOM resistance occurs with low frequencyand is independent of resistance to other antimicrobials inE coli clinical isolates from Japan On the other hand weidentified strains with decreased FOM susceptibility Mostof them displayed fluctuated activity of the G6P transporterUhpT However G6P transporter function was altered eventhough the G6P-induced uhpT expression and amino acidsequence of UhpT were preserved in one strain (FOM MIC8 120583gmL) Another strain (FOM MIC 32 120583gmL) displayed
reduced susceptibility to FOM and no alteration of MICin the presence and absence of G6P even though G6P-induced uhpT expression amino acid sequence of UhpT andgrowth on G6P-supplemented M9 minimum salt agar werepreserved The exact molecular mechanism of the reducedsusceptibility of these strains remains unclear and requiresfurther evaluation
Competing Interests
The authors declare no competing interests
Acknowledgments
The authors thank Osamu Kuwahara (Sapporo Clinical Lab-oratory) for the provision of E coli clinical isolates Thisstudy was partly supported by a Grant-in-Aid for ScientificResearch (15H06521) from Japan Society for the Promotionof Science and a grant from Yuasa Memorial Foundation
References
[1] WHO ldquoAntimicrobial resistance global report on surveil-lance 2014rdquo 2014 httpwwwwhointdrugresistancedocu-mentssurveillancereporten
[2] M E Falagas andD E Karageorgopoulos ldquoExtended-spectrum120573-lactamase-producing organismsrdquo Journal of Hospital Infec-tion vol 73 no 4 pp 345ndash354 2009
[3] J D D Pitout ldquoExtraintestinal pathogenic Escherichia coli anupdate on antimicrobial resistance laboratory diagnosis andtreatmentrdquo Expert Review of Anti-Infective Therapy vol 10 no10 pp 1165ndash1176 2012
[4] S Yokota T Sato T Okubo et al ldquoPrevalence of fluoroquin-olone-resistant Escherichia coli O25H4-ST131 (CTX-M15-nonproducing) strains isolated in JapanrdquoChemotherapy vol 58no 1 pp 52ndash59 2012
[5] N Tsukamoto Y Ohkoshi T Okubo et al ldquoHigh prevalenceof cross-resistance to aminoglycosides in fluoroquinolone-resistantEscherichia coli clinical isolatesrdquoChemotherapy vol 59no 5 pp 379ndash384 2014
[6] H Giamarellou and G Poulakou ldquoMultidrug-resistant gram-negative infections what are the treatment optionsrdquoDrugs vol69 no 14 pp 1879ndash1901 2009
[7] M E Falagas A C Kastoris A M Kapaskelis and D E Kara-georgopoulos ldquoFosfomycin for the treatment of multidrug-resistant including extended-spectrum 120573-lactamase produc-ing Enterobacteriaceae infections a systematic reviewrdquo TheLancet Infectious Diseases vol 10 no 1 pp 43ndash50 2010
[8] A Schaeffer ldquoInfections of the urinary tractrdquo inCampbell-WalshUrology A Wein L Kavoussi A Novick A Partin and CPeters Eds pp 223ndash303 Elsevier Philadelphia Pa USA 9thedition 2007
[9] K Ishikawa R Hamasuna S Uehara et al ldquoJapanese nation-wide surveillance in 2011 of antibacterial susceptibility patternsof clinical isolates from complicated urinary tract infectioncasesrdquo Journal of Infection and Chemotherapy vol 21 no 9 pp623ndash633 2015
[10] D N Gilbert H F Chambers GM Eliopoulos andM S SaagTheSanford Guide to AntimicrobialTherapy 2015 AntimicrobialTherapy Sperryville Va USA 45th edition 2015
BioMed Research International 7
[11] The Japanese Association for Infectious Diseases and JapanSociety of ChemotherapyThe JAIDJSC Guide to Clinical Man-agement of Infectious Diseases 2014 The Japanese Associationfor Infectious DiseasesJapan Society of Chemotherapy TokyoJapan 2014 (Japanese)
[12] E D Brown E I Vivas C T Walsh and R Kolter ldquoMurA(MurZ) the enzyme that catalyzes the first committed stepin peptidoglycan biosynthesis is essential in Escherichia colirdquoJournal of Bacteriology vol 177 no 14 pp 4194ndash4197 1995
[13] F M Kahan J S Kahan P J Cassidy and H Kropp ldquoThemechanismof action of fosfomycin (phosphonomycin)rdquoAnnalsof the NewYork Academy of Sciences vol 235 no 1 pp 364ndash3861974
[14] M J Lemieux Y Huang and D-N Wang ldquoGlycerol-3-phosphate transporter of Escherichia coli structure functionand regulationrdquo Research in Microbiology vol 155 no 8 pp623ndash629 2004
[15] H HWinkler ldquoKinetics of exogenous induction of the hexose-6-phosphate transport system of Escherichia colirdquo Journal ofBacteriology vol 107 no 1 pp 74ndash78 1971
[16] T J Merkel J L Dahl R H Ebright and R J KadnerldquoTranscription activation at the Escherichia coli uhpT promoterby the catabolite gene activator proteinrdquo Journal of Bacteriologyvol 177 no 7 pp 1712ndash1718 1995
[17] D T Verhamme P W Postma W Crielaard and K J Helling-werf ldquoCooperativity in signal transfer through the Uhp systemof Escherichia colirdquo Journal of Bacteriology vol 184 no 15 pp4205ndash4210 2002
[18] S Takahata T Ida T Hiraishi et al ldquoMolecular mechanismsof fosfomycin resistance in clinical isolates of Escherichia colirdquoInternational Journal of Antimicrobial Agents vol 35 no 4 pp333ndash337 2010
[19] J Wachino K Yamane S Suzuki K Kimura and YArakawa ldquoPrevalence of fosfomycin resistance among CTX-M-producing Escherichia coli clinical isolates in Japan and iden-tification of novel plasmid-mediated fosfomycin-modifyingenzymesrdquo Antimicrobial Agents and Chemotherapy vol 54 no7 pp 3061ndash3064 2010
[20] K Kurabayashi Y Hirakawa K Tanimoto H Tomita and HHirakawa ldquoRole of the CpxAR two-component signal trans-duction system in control of fosfomycin resistance and carbonsubstrate uptakerdquo Journal of Bacteriology vol 196 no 2 pp248ndash256 2014
[21] P Arca G Reguera and C Hardisson ldquoPlasmid-encodedfosfomycin resistance in bacteria isolated from the urinary tractin amulticentre surveyrdquo Journal of Antimicrobial Chemotherapyvol 40 no 3 pp 393ndash399 1997
[22] J Hou X Huang Y Deng et al ldquoDissemination of thefosfomycin resistance gene fosA3 with CTX-M 120573-lactamasegenes and rmtB carried on incfII plasmids among Escherichiacoli isolates from pets in Chinardquo Antimicrobial Agents andChemotherapy vol 56 no 4 pp 2135ndash2138 2012
[23] S-Y Lee Y-J Park J K Yu et al ldquoPrevalence of acquiredfosfomycin resistance among extended-spectrum 120573-lactamase-producing Escherichia coli and Klebsiella pneumoniae clinicalisolates in Korea and IS26-composite transposon surroundingfosA3rdquo Journal of Antimicrobial Chemotherapy vol 67 no 12Article ID dks319 pp 2843ndash2847 2012
[24] P L Ho J Chan W U Lo et al ldquoPrevalence and molecularepidemiology of plasmid-mediated fosfomycin resistance genesamong blood and urinary Escherichia coli isolatesrdquo Journal ofMedical Microbiology vol 62 no 11 pp 1707ndash1713 2013
[25] G Nakamura J Wachino N Sato et al ldquoPractical agar-based disk potentiation test for detection of fosfomycin-nonsusceptible Escherichia coli clinical isolates producing glu-tathione S-transferasesrdquo Journal of ClinicalMicrobiology vol 52no 9 pp 3175ndash3179 2014
[26] Y Ma X Xu Q Guo P Wang W Wang and M WangldquoCharacterization of fosA5 a newplasmid-mediated fosfomycinresistance gene in Escherichia colirdquo Letters in Applied Microbiol-ogy vol 60 no 3 pp 259ndash264 2015
[27] Y Li B Zheng Y Li S Zhu F Xue and J Liu ldquoAntimicrobialsusceptibility and molecular mechanisms of fosfomycin resis-tance in clinical Escherichia coli isolates in mainland ChinardquoPLoS ONE vol 10 no 8 Article ID e0135269 2015
[28] S-P Tseng S-F Wang C-Y Kuo et al ldquoCharacteriza-tion of fosfomycin resistant extended-spectrum szlig-lactamase-producing Escherichia coli isolates from human and pig inTaiwanrdquo PLoS ONE vol 10 no 8 Article ID e0135864 2015
[29] A I Nilsson O G Berg O Aspevall G Kahlmeter and DI Andersson ldquoBiological costs and mechanisms of fosfomycinresistance in Escherichia colirdquo Antimicrobial Agents and Chem-otherapy vol 47 no 9 pp 2850ndash2858 2003
[30] J C Cordaro T Melton J P Stratis et al ldquoFosfomycinresistance selectionmethod for internal and extended deletionsof the phosphoenolpyruvatesugar phosphotransferase genes ofSalmonella typhimuriumrdquo Journal of Bacteriology vol 128 no3 pp 785ndash793 1976
[31] Y Sakamoto S Furukawa H Ogihara and M YamasakildquoFosmidomycin resistance in adenylate cyclase deficient (cya)mutants of Escherichia colirdquo Bioscience Biotechnology and Bio-chemistry vol 67 no 9 pp 2030ndash2033 2003
[32] G Kahlmeter and H O Poulsen ldquoAntimicrobial susceptibilityof Escherichia coli from community-acquired urinary tractinfections in Europe the ECOsdotSENS study revisitedrdquo Interna-tional Journal of Antimicrobial Agents vol 39 no 1 pp 45ndash512012
[33] D E Karageorgopoulos R Wang X-H Yu and M E FalagasldquoFosfomycin evaluation of the published evidence on the emer-gence of antimicrobial resistance in gram-negative pathogensrdquoJournal of Antimicrobial Chemotherapy vol 67 no 2 pp 255ndash268 2012
[34] Clinical and Laboratory Standards Institute Performance Stan-dards for Antimicrobial Susceptibility Testing 22nd InformationSupplement M100-S22 CLSI Wayne Pa USA 2012
[35] O Clermont J R Johnson M Menard and E DenamurldquoDetermination of Escherichia coli O types by allele-specificpolymerase chain reaction application to the O types involvedin human septicemiardquo Diagnostic Microbiology and InfectiousDisease vol 57 no 2 pp 129ndash136 2007
[36] D Li B Liu M Chen et al ldquoA multiplex PCR method todetect 14 Escherichia coli serogroups associated with urinarytract infectionsrdquo Journal of Microbiological Methods vol 82 no1 pp 71ndash77 2010
[37] O Clermont S Bonacorsi and E Bingen ldquoRapid and simpledetermination of the Escherichia coli phylogenetic grouprdquoApplied and Environmental Microbiology vol 66 no 10 pp4555ndash4558 2000
[38] S Y Tartof O D Solberg A R Manges and L W RileyldquoAnalysis of a uropathogenic Escherichia coli clonal group bymultilocus sequence typingrdquo Journal of Clinical Microbiologyvol 43 no 12 pp 5860ndash5864 2005
8 BioMed Research International
[39] R J Kadner and H HWinkler ldquoIsolation and characterizationof mutations affecting the transport of hexose phosphates inEscherichia colirdquo Journal of Bacteriology vol 113 no 2 pp 895ndash900 1973
Submit your manuscripts athttpswwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
BioMed Research International 3
Table 2 Antibiotic susceptibility and the presence of ESBL genes in Ecoli strains with FOMMIC ge 8120583gmL
StrainsMIC (120583gmL)
ESBLageneFosfomycin(ge128)b
Levofloxacin(ge4)b
Gentamicin(ge8)b
Imipenem(ge2)b
Ceftazidime(ge8)b
SRE257 1024lowast le0125 1 0125 0125 mdashSRE91 128 32lowast 4 025 0125 mdashSRE49 128 16lowast 2 0125 025 mdashSRE54 64 ge64lowast ge64lowast 0125 2 CTX-M14SRE237 64 le0125 4 0125 0125 mdashSRE29 32 16lowast ge64lowast 0125 0125 mdashSRE252 32 05 2 0125 0125 mdashSRE280 32 le0125 8lowast 05 0125 mdashSRE18 16 32lowast 2 0125 2 CTX-M2SRE253 8 le0125 4 05 0125 mdashaESBL extended spectrum 120573-lactamasebBreakpoints (120583gmL) are according to CLSIlowastResistantIntermediate
Dye Terminator Kit version 31 and 3730xI DNA analyzer(Applied Biosystems Carlsbad CA) at Hokkaido SystemScience (Sapporo Japan)
25 Real-Time Reverse-Transcription (RT) PCR E coli cellswere grown for 24 h in Luria-Bertani (LB) broth and thecells were harvested and washed twice with M9 minimumsalt solution The suspended cells were used to inoculateto M9 minimum salt solution with or without 02 G6Psupplementation and incubated for 30min at 37∘C RNAwas isolated from the cells using RNeasy Plus Mini Kit(Qiagen) according to themanufacturerrsquos instructions cDNAwas prepared from the RNA using SuperScript III FirstStrand Synthesis Kit (Invitrogen Carlsbad CA) and randomhexamer oligonucleotide primers mRNA levels of uhpT andrpoD were quantified using QuantiFast SYBR Green PCRMastermix (Qiagen) by LightCycler LC480 (Roche BaselSwitzerland) with the cDNA as a template PCR primers weregiven in Table 1 Levels of uhpT transcript were calculated by2minusΔΔct method and data were normalized to the levels of thehouse-keeping gene rpoDmRNA
3 Results
31 Antibiotic Susceptibility Antibiotic susceptibility was ex-amined for 211 E coli clinical isolates MICs were determinedby broth microdilution method for LVX GEN IPM andCAZ and by agar plate dilution method for FOM (Table 2)The distribution of FOM MIC was shown in Figure 1 Threestrains (14) were not susceptible including resistant andintermediate Furthermore seven strains (3) with elevatedFOM MIC (ge8120583gmL le64 120583gmL) were observed out ofnormal distribution of the susceptible strains FOM MICsof these ten strains were not affected by the presence orabsence of G6P By contrast other susceptible strains (MICle 2 120583gmL) showed an increase of FOM MICs in theabsence of G6P (Table 3) Susceptibility to other antibiotics
012
8289
18
0 1 1 3 2 2 0 0 1 00
25
50
75
100N
umbe
r of s
trai
ns
MIC (120583gmL)
S I R0125
025
05 1 2 4 8
16
32
64
128
256
512
1024
2048
N = 211
Figure 1 Distribution of fosfomycinMICs of the E coli clinical iso-lates The breakpoint is according to CLSI guideline S susceptibleI intermediate and R resistant
was examined for strains with reduced susceptibility toFOM (Table 2) LVX-resistant strains comprised 50 andGEN-resistant strains comprised 30 of these strains IPM-resistant and CAZ-resistant strains were not observed how-ever 20 strains shared CTX-M-type ESBL genesThe occur-rence of antimicrobial nonsusceptibility was not significantlyhigh compared to total strains examined (data not shown)Genotypes (ie phylogenetic groups and MLST) and O-serogroups of these strains were variable (Table 3)
32 Analysis of Genes Associated with FOM Sensitivity Weexamined molecular mechanisms underlying the reductionof FOM sensitivity (MIC ge 8 120583gmL) First genes encodingFOM-modifying enzymes were investigated None of thefollowing were detected in the strains examined fosA fosA2to fosA5 fosC fosC2 fosB fosB2 fosX and fosKP96 (datanot shown) Next nucleotide sequences of murA uhpTuhpA glpT ptsI and cyaA genes were determined (Table 3)No murA coding sequence mutations that would result in
4 BioMed Research International
Table3Ch
aracteris
ticso
fEcolistrains
used
inthes
tudy
Strain
Specim
enSero-
grou
p
Phylo-
genetic
grou
pMLS
T
MIC
a
(120583gmL)
Growth
onM9agar
supp
lemented
with
uhpT
expressio
nindu
cedby
G6P
b
Aminoacid
resid
uealternations
inproteins
encodedby
glpT
ptsI
cyaA
murA
uhpA
and
uhpT
genesc
G6Pminus
G6P
+G6P
+cA
MP+
G3P
G6P
cyaA
glpT
murA
ptsI
uhpA
uhpT
SRE2
57Urin
eO1
B295
1024
1024
1024
+minus
124
mdashmdash
mdashVa
l399Leu
163sim
188
deletio
nmdash
SRE9
1Aspira
tion
tube
O1
D64
8128
128
32minus
minus378
mdash155sim
158
deletio
nPh
e176Leu
mdashmdash
Thr3Ala
mdash
SRE4
9Urin
eO25bH4
B2131
128
128
128
+minus
NT
His7
16Leu
mdashmdash
Lys410Arg
ND
ND
SRE5
4Urin
eO25bH4
B2131
6464
64+
minus15
9His7
16Leu
mdashmdash
Ala44
3Thr
Gly452A
spmdash
mdash
SRE2
37Urin
eO25bH4
B2131
6464
64+
minus15
9His7
16Leu
mdashmdash
mdashmdash
mdashSR
E29
Urin
eO25bH4
B2131
3232
32+
+122622
mdashmdash
mdashmdash
mdashmdash
SRE2
52Urin
eO25a
D73
3232
4minus
minus368
ND
Ile171Th
rmdash
Lys145Asn
mdashmdash
SRE2
80Ascites
O12
D1486
3232
32+
minus056
Ser142As
nmdash
mdashmdash
mdashmdash
SRE18
Urin
eND
D405
1616
16+
minus222
mdashmdash
mdashmdash
Met1Ile
mdashSR
E253
Urin
eO18
B295
88
8minus
minus19809
His7
16Leu
mdashmdash
mdashmdash
mdashSR
E40
Decub
itus
O25a
D501
3205
NT
++
28405
SRE4
1Ca
theter
urine
O1
D64
88
05
NT
++
32856
SRE110
Catheter
urine
O25bH4
B2131
805
NT
++
19002
SRE2
05Urin
eND
A131
805
NT
++
73419
SRE2
27Pu
sO1
B295
805
NT
++
2472
8SR
E30
Urin
eO1
D64
88
025
NT
++
7190
8AT
CC25922
NT
NT
3205
NT
++
30869
a FOM
MICsw
ered
etermined
inthep
resence(+)
orabsence(minus)o
fglucose-6-pho
sphate(G
6P)a
ndorc
AMP
b Ecolicellswereincub
ated
inM9minim
umsaltsolutio
nin
thep
resenceo
rabsence
ofG6PTh
euhpTmRN
Alevelswered
etermined
byreal-timeR
T-PC
Randthed
ataweren
ormalized
torpoD
mRN
Alevels
Indu
ctionof
uhpT
expressio
nby
G6P
was
calculated
bydividing
theu
hpTmRN
Alevelinthep
resenceo
fG6P
bytheu
hpTmRN
Alevelinthea
bsence
ofG6P
c Aminoacid
mutations
foun
don
lyin
strains
with
redu
cedFO
Msusceptib
ility(M
ICge8120583
gmL)
comparedwith
strains
with
FOM
MIClt1120583
gmL
NDnot
detectedN
Tno
ttested
BioMed Research International 5
Growth in the presence of G6P +minus
P lt 0001
01
1
10
100
1000
10000
Fold
indu
ctio
n of
uhpT
mRN
A ex
pres
sion
(a)
Fosfomycin MIC (120583gml)
P lt 0005
ge8 le4
01
1
10
100
1000
10000
Fold
indu
ctio
n of
uhpT
mRN
A ex
pres
sion
(b)
Figure 2 Induced expression levels of uhpT expression by the addition of G6P Nine clinical isolates with reduced susceptibility to FOM sixsusceptible clinical isolates and one standard strain are included Data used to generate this figure are given in Table 2 Statistical significancewas determined byMannndashWhitney test (a)Comparison of strains not grown (minus) or strains grown (+) inM9minimumsalt solution containingG6P (b) Comparison of strains with FOMMIC ge 8 120583gmL or le4 120583gmL
changes of amino acid residues in MurA were observed Aresistant strain (SRE257) and an intermediate-resistant strain(SRE91) had mutations in the genes that would result indeletions of a part of amino acid residues in UhpA andGlpT respectively In another intermediate-resistant strain(SRE49) uhpA and uhpT were not detected by PCR ampli-fication with two distinct primer pairs (ldquofullrdquo and ldquopartialrdquo inTable 1) In one strain with MIC 32 120583gmL (SRE252) cyaAwas not detectable by PCR All strains with reduced FOMMIC (ge8120583gmL) except one (SRE29) had several mutationsin one or more genes leading to amino acid deletion or pointmutation(s) of amino acid residues compared with othersusceptible strains
33 Function and Expression of Carbohydrate PhosphateTransporters To determine the activity of UhpT and GlpTwe examined cell growth in M9 minimum salt solutionsupplementedwithG6P orG3PNine of ten strainswith FOMMIC ge 8120583gmL did not grow within 24 h on G6P-containingM9 minimum salt agar On the other hand only three often strains did not grow in the presence of G3P (Table 3)and the two strains (SRE91 and SRE252) showed reducedMIC to FOM by addition of cAMP This suggested that thesetwo strains shared insufficient intracellular concentration ofcAMP for full expression of GlpT andor UhpTThese resultssuggested that FOM incorporation through the UhpT systemis involved to a greater extent in the reduction of E coli FOMcompared to the GlpT system
Expression of UhpT is induced by G6P [15ndash17] Wetherefore determined uhpT gene induction by G6P usingquantitative RT-PCR analysis (Table 3 and Figure 2) In
strains with FOM MIC lt 1 120583gmL uhpT expression wasstrongly induced (190- to 730-fold) by G6P By contrast ineight of ten strains with FOM MIC ge 8120583gmL the uhpTinduction was markedly lower (056- to 38-fold) or no uhpTsignal was amplified by PCR Of the remaining two strainswe observed a high induction (200-fold) of uhpT expressionby G6P in SRE253 strain however the strain did not grow onG6P-containing M9 minimum agar High induction (1200-fold) of uhpT by G6P was observed in SRE29 strain and thestrain grew on G6P-containing M9 minimum agar howeverit showed reduced susceptibility to FOM (MIC 32 120583gmL)These results indicated that G6P-dependent growth lacked inmost strains with reduced susceptibility to FOM because oflack of uhpT or a markedly reduced uhpT induction by G6PHowever the changes in uhpT expression and UhpT activitydid not account for the reduced FOM susceptibility of a fewstrains (Figure 2)
4 Discussion
The frequency of FOM resistance has been recognized to below And it has been expected that the frequency of cross-resistance of FOMand other antibiotics is very low because ofa unique mode of action However several reports examinedFOM resistance in ESBL-producing E coli [19 23 24 28] Areport indicates that FOM-resistant and intermediate E coliare more frequently resistant to other types of antimicrobialsthan FOM-susceptible strains [28] These reports suggestthe importance for surveillance of FOM-resistant E coliparticularly for focus on their cross-resistance of FOM inmultidrug resistance
6 BioMed Research International
In this study only one resistant and two intermediatestrains were found among 211 clinical isolates from Japan(Figure 1) This indicated that FOM was a promising candi-date agent against E coli infections as generally describedHowever we found seven strains susceptible according tothe CLST breakpoint but reduced susceptibility (MIC ge8 120583gmL) Under the selective pressure of FOM usage thesestrains might acquire FOM resistance more easily In thepresent study cross-resistance of strains with reduced FOMsusceptibility to other antimicrobial drugs was not signifi-cantly higher than in other strains and these strains were notconcentrating on specific genotypes
Based on previous reports acquisition of FOM-modifying enzymes (encoded by fos genes) and mutationsin murA gene results in resistance that is surpass ofbreakpoints to FOM [18 19 28] The present study didnot identify such strains Another resistance mechanismis altered FOM incorporation into bacterial cells TheG6P and G3P transporters contribute to incorporationof FOM into cells [13 14] Loss of function or decreasedexpression levels of them leads to a reduction of FOMsusceptibility [18 29 39] We found that loss of UhpT(G6P transporter) activity is more dominant than that ofGlpT (G3P transporter) in strains with decreased FOMsusceptibility In resistant (gt128 120583gmL) and intermediate(128 120583gmL) strains we noted amino acid deletion in therespective encoded proteins and no PCR amplificationof the transporter-related genes (Table 3) In strains withFOM MICs between 8 and 64 120583gmL gene mutation(s)leading to alternations of amino acid residues were foundhowever it was unclear whether these contributed tothe reduced susceptibility Among the ten strains withreduced susceptibility nine strains did not grow on G6P-supplemented M9 minimum salt agar This suggested thatin these nine strains G6P-induced UhpT function wasattenuated We found several mutations in cyaA and ptsI insome of these strains Dysfunction of CyaA and PtsI leadsto decrease in intracellular concentration of cAMP andinsufficient expression of GlpT and UhpT [16 17 31] Onlytwo strains (SRE91 and SRE252) with reduced susceptibilityto FOM showed that MIC was decreased by the exogenousaddition of cAMP and these strains did not grow in M9medium supplement with G3P Certainly SRE252 defectedcyaA however SRE91 did not share any mutations in cyaAand pstI Thus reduced susceptibility to FOM in the otherstrains was not explained by the insufficient intracellularconcentration of cAMP caused by dysfunction of cyaAand pstI The mutations in these genes found in this study(Table 3) seemed to scarcely relate with dysfunction of cAMPsynthesis
In conclusion FOM resistance occurs with low frequencyand is independent of resistance to other antimicrobials inE coli clinical isolates from Japan On the other hand weidentified strains with decreased FOM susceptibility Mostof them displayed fluctuated activity of the G6P transporterUhpT However G6P transporter function was altered eventhough the G6P-induced uhpT expression and amino acidsequence of UhpT were preserved in one strain (FOM MIC8 120583gmL) Another strain (FOM MIC 32 120583gmL) displayed
reduced susceptibility to FOM and no alteration of MICin the presence and absence of G6P even though G6P-induced uhpT expression amino acid sequence of UhpT andgrowth on G6P-supplemented M9 minimum salt agar werepreserved The exact molecular mechanism of the reducedsusceptibility of these strains remains unclear and requiresfurther evaluation
Competing Interests
The authors declare no competing interests
Acknowledgments
The authors thank Osamu Kuwahara (Sapporo Clinical Lab-oratory) for the provision of E coli clinical isolates Thisstudy was partly supported by a Grant-in-Aid for ScientificResearch (15H06521) from Japan Society for the Promotionof Science and a grant from Yuasa Memorial Foundation
References
[1] WHO ldquoAntimicrobial resistance global report on surveil-lance 2014rdquo 2014 httpwwwwhointdrugresistancedocu-mentssurveillancereporten
[2] M E Falagas andD E Karageorgopoulos ldquoExtended-spectrum120573-lactamase-producing organismsrdquo Journal of Hospital Infec-tion vol 73 no 4 pp 345ndash354 2009
[3] J D D Pitout ldquoExtraintestinal pathogenic Escherichia coli anupdate on antimicrobial resistance laboratory diagnosis andtreatmentrdquo Expert Review of Anti-Infective Therapy vol 10 no10 pp 1165ndash1176 2012
[4] S Yokota T Sato T Okubo et al ldquoPrevalence of fluoroquin-olone-resistant Escherichia coli O25H4-ST131 (CTX-M15-nonproducing) strains isolated in JapanrdquoChemotherapy vol 58no 1 pp 52ndash59 2012
[5] N Tsukamoto Y Ohkoshi T Okubo et al ldquoHigh prevalenceof cross-resistance to aminoglycosides in fluoroquinolone-resistantEscherichia coli clinical isolatesrdquoChemotherapy vol 59no 5 pp 379ndash384 2014
[6] H Giamarellou and G Poulakou ldquoMultidrug-resistant gram-negative infections what are the treatment optionsrdquoDrugs vol69 no 14 pp 1879ndash1901 2009
[7] M E Falagas A C Kastoris A M Kapaskelis and D E Kara-georgopoulos ldquoFosfomycin for the treatment of multidrug-resistant including extended-spectrum 120573-lactamase produc-ing Enterobacteriaceae infections a systematic reviewrdquo TheLancet Infectious Diseases vol 10 no 1 pp 43ndash50 2010
[8] A Schaeffer ldquoInfections of the urinary tractrdquo inCampbell-WalshUrology A Wein L Kavoussi A Novick A Partin and CPeters Eds pp 223ndash303 Elsevier Philadelphia Pa USA 9thedition 2007
[9] K Ishikawa R Hamasuna S Uehara et al ldquoJapanese nation-wide surveillance in 2011 of antibacterial susceptibility patternsof clinical isolates from complicated urinary tract infectioncasesrdquo Journal of Infection and Chemotherapy vol 21 no 9 pp623ndash633 2015
[10] D N Gilbert H F Chambers GM Eliopoulos andM S SaagTheSanford Guide to AntimicrobialTherapy 2015 AntimicrobialTherapy Sperryville Va USA 45th edition 2015
BioMed Research International 7
[11] The Japanese Association for Infectious Diseases and JapanSociety of ChemotherapyThe JAIDJSC Guide to Clinical Man-agement of Infectious Diseases 2014 The Japanese Associationfor Infectious DiseasesJapan Society of Chemotherapy TokyoJapan 2014 (Japanese)
[12] E D Brown E I Vivas C T Walsh and R Kolter ldquoMurA(MurZ) the enzyme that catalyzes the first committed stepin peptidoglycan biosynthesis is essential in Escherichia colirdquoJournal of Bacteriology vol 177 no 14 pp 4194ndash4197 1995
[13] F M Kahan J S Kahan P J Cassidy and H Kropp ldquoThemechanismof action of fosfomycin (phosphonomycin)rdquoAnnalsof the NewYork Academy of Sciences vol 235 no 1 pp 364ndash3861974
[14] M J Lemieux Y Huang and D-N Wang ldquoGlycerol-3-phosphate transporter of Escherichia coli structure functionand regulationrdquo Research in Microbiology vol 155 no 8 pp623ndash629 2004
[15] H HWinkler ldquoKinetics of exogenous induction of the hexose-6-phosphate transport system of Escherichia colirdquo Journal ofBacteriology vol 107 no 1 pp 74ndash78 1971
[16] T J Merkel J L Dahl R H Ebright and R J KadnerldquoTranscription activation at the Escherichia coli uhpT promoterby the catabolite gene activator proteinrdquo Journal of Bacteriologyvol 177 no 7 pp 1712ndash1718 1995
[17] D T Verhamme P W Postma W Crielaard and K J Helling-werf ldquoCooperativity in signal transfer through the Uhp systemof Escherichia colirdquo Journal of Bacteriology vol 184 no 15 pp4205ndash4210 2002
[18] S Takahata T Ida T Hiraishi et al ldquoMolecular mechanismsof fosfomycin resistance in clinical isolates of Escherichia colirdquoInternational Journal of Antimicrobial Agents vol 35 no 4 pp333ndash337 2010
[19] J Wachino K Yamane S Suzuki K Kimura and YArakawa ldquoPrevalence of fosfomycin resistance among CTX-M-producing Escherichia coli clinical isolates in Japan and iden-tification of novel plasmid-mediated fosfomycin-modifyingenzymesrdquo Antimicrobial Agents and Chemotherapy vol 54 no7 pp 3061ndash3064 2010
[20] K Kurabayashi Y Hirakawa K Tanimoto H Tomita and HHirakawa ldquoRole of the CpxAR two-component signal trans-duction system in control of fosfomycin resistance and carbonsubstrate uptakerdquo Journal of Bacteriology vol 196 no 2 pp248ndash256 2014
[21] P Arca G Reguera and C Hardisson ldquoPlasmid-encodedfosfomycin resistance in bacteria isolated from the urinary tractin amulticentre surveyrdquo Journal of Antimicrobial Chemotherapyvol 40 no 3 pp 393ndash399 1997
[22] J Hou X Huang Y Deng et al ldquoDissemination of thefosfomycin resistance gene fosA3 with CTX-M 120573-lactamasegenes and rmtB carried on incfII plasmids among Escherichiacoli isolates from pets in Chinardquo Antimicrobial Agents andChemotherapy vol 56 no 4 pp 2135ndash2138 2012
[23] S-Y Lee Y-J Park J K Yu et al ldquoPrevalence of acquiredfosfomycin resistance among extended-spectrum 120573-lactamase-producing Escherichia coli and Klebsiella pneumoniae clinicalisolates in Korea and IS26-composite transposon surroundingfosA3rdquo Journal of Antimicrobial Chemotherapy vol 67 no 12Article ID dks319 pp 2843ndash2847 2012
[24] P L Ho J Chan W U Lo et al ldquoPrevalence and molecularepidemiology of plasmid-mediated fosfomycin resistance genesamong blood and urinary Escherichia coli isolatesrdquo Journal ofMedical Microbiology vol 62 no 11 pp 1707ndash1713 2013
[25] G Nakamura J Wachino N Sato et al ldquoPractical agar-based disk potentiation test for detection of fosfomycin-nonsusceptible Escherichia coli clinical isolates producing glu-tathione S-transferasesrdquo Journal of ClinicalMicrobiology vol 52no 9 pp 3175ndash3179 2014
[26] Y Ma X Xu Q Guo P Wang W Wang and M WangldquoCharacterization of fosA5 a newplasmid-mediated fosfomycinresistance gene in Escherichia colirdquo Letters in Applied Microbiol-ogy vol 60 no 3 pp 259ndash264 2015
[27] Y Li B Zheng Y Li S Zhu F Xue and J Liu ldquoAntimicrobialsusceptibility and molecular mechanisms of fosfomycin resis-tance in clinical Escherichia coli isolates in mainland ChinardquoPLoS ONE vol 10 no 8 Article ID e0135269 2015
[28] S-P Tseng S-F Wang C-Y Kuo et al ldquoCharacteriza-tion of fosfomycin resistant extended-spectrum szlig-lactamase-producing Escherichia coli isolates from human and pig inTaiwanrdquo PLoS ONE vol 10 no 8 Article ID e0135864 2015
[29] A I Nilsson O G Berg O Aspevall G Kahlmeter and DI Andersson ldquoBiological costs and mechanisms of fosfomycinresistance in Escherichia colirdquo Antimicrobial Agents and Chem-otherapy vol 47 no 9 pp 2850ndash2858 2003
[30] J C Cordaro T Melton J P Stratis et al ldquoFosfomycinresistance selectionmethod for internal and extended deletionsof the phosphoenolpyruvatesugar phosphotransferase genes ofSalmonella typhimuriumrdquo Journal of Bacteriology vol 128 no3 pp 785ndash793 1976
[31] Y Sakamoto S Furukawa H Ogihara and M YamasakildquoFosmidomycin resistance in adenylate cyclase deficient (cya)mutants of Escherichia colirdquo Bioscience Biotechnology and Bio-chemistry vol 67 no 9 pp 2030ndash2033 2003
[32] G Kahlmeter and H O Poulsen ldquoAntimicrobial susceptibilityof Escherichia coli from community-acquired urinary tractinfections in Europe the ECOsdotSENS study revisitedrdquo Interna-tional Journal of Antimicrobial Agents vol 39 no 1 pp 45ndash512012
[33] D E Karageorgopoulos R Wang X-H Yu and M E FalagasldquoFosfomycin evaluation of the published evidence on the emer-gence of antimicrobial resistance in gram-negative pathogensrdquoJournal of Antimicrobial Chemotherapy vol 67 no 2 pp 255ndash268 2012
[34] Clinical and Laboratory Standards Institute Performance Stan-dards for Antimicrobial Susceptibility Testing 22nd InformationSupplement M100-S22 CLSI Wayne Pa USA 2012
[35] O Clermont J R Johnson M Menard and E DenamurldquoDetermination of Escherichia coli O types by allele-specificpolymerase chain reaction application to the O types involvedin human septicemiardquo Diagnostic Microbiology and InfectiousDisease vol 57 no 2 pp 129ndash136 2007
[36] D Li B Liu M Chen et al ldquoA multiplex PCR method todetect 14 Escherichia coli serogroups associated with urinarytract infectionsrdquo Journal of Microbiological Methods vol 82 no1 pp 71ndash77 2010
[37] O Clermont S Bonacorsi and E Bingen ldquoRapid and simpledetermination of the Escherichia coli phylogenetic grouprdquoApplied and Environmental Microbiology vol 66 no 10 pp4555ndash4558 2000
[38] S Y Tartof O D Solberg A R Manges and L W RileyldquoAnalysis of a uropathogenic Escherichia coli clonal group bymultilocus sequence typingrdquo Journal of Clinical Microbiologyvol 43 no 12 pp 5860ndash5864 2005
8 BioMed Research International
[39] R J Kadner and H HWinkler ldquoIsolation and characterizationof mutations affecting the transport of hexose phosphates inEscherichia colirdquo Journal of Bacteriology vol 113 no 2 pp 895ndash900 1973
Submit your manuscripts athttpswwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
4 BioMed Research International
Table3Ch
aracteris
ticso
fEcolistrains
used
inthes
tudy
Strain
Specim
enSero-
grou
p
Phylo-
genetic
grou
pMLS
T
MIC
a
(120583gmL)
Growth
onM9agar
supp
lemented
with
uhpT
expressio
nindu
cedby
G6P
b
Aminoacid
resid
uealternations
inproteins
encodedby
glpT
ptsI
cyaA
murA
uhpA
and
uhpT
genesc
G6Pminus
G6P
+G6P
+cA
MP+
G3P
G6P
cyaA
glpT
murA
ptsI
uhpA
uhpT
SRE2
57Urin
eO1
B295
1024
1024
1024
+minus
124
mdashmdash
mdashVa
l399Leu
163sim
188
deletio
nmdash
SRE9
1Aspira
tion
tube
O1
D64
8128
128
32minus
minus378
mdash155sim
158
deletio
nPh
e176Leu
mdashmdash
Thr3Ala
mdash
SRE4
9Urin
eO25bH4
B2131
128
128
128
+minus
NT
His7
16Leu
mdashmdash
Lys410Arg
ND
ND
SRE5
4Urin
eO25bH4
B2131
6464
64+
minus15
9His7
16Leu
mdashmdash
Ala44
3Thr
Gly452A
spmdash
mdash
SRE2
37Urin
eO25bH4
B2131
6464
64+
minus15
9His7
16Leu
mdashmdash
mdashmdash
mdashSR
E29
Urin
eO25bH4
B2131
3232
32+
+122622
mdashmdash
mdashmdash
mdashmdash
SRE2
52Urin
eO25a
D73
3232
4minus
minus368
ND
Ile171Th
rmdash
Lys145Asn
mdashmdash
SRE2
80Ascites
O12
D1486
3232
32+
minus056
Ser142As
nmdash
mdashmdash
mdashmdash
SRE18
Urin
eND
D405
1616
16+
minus222
mdashmdash
mdashmdash
Met1Ile
mdashSR
E253
Urin
eO18
B295
88
8minus
minus19809
His7
16Leu
mdashmdash
mdashmdash
mdashSR
E40
Decub
itus
O25a
D501
3205
NT
++
28405
SRE4
1Ca
theter
urine
O1
D64
88
05
NT
++
32856
SRE110
Catheter
urine
O25bH4
B2131
805
NT
++
19002
SRE2
05Urin
eND
A131
805
NT
++
73419
SRE2
27Pu
sO1
B295
805
NT
++
2472
8SR
E30
Urin
eO1
D64
88
025
NT
++
7190
8AT
CC25922
NT
NT
3205
NT
++
30869
a FOM
MICsw
ered
etermined
inthep
resence(+)
orabsence(minus)o
fglucose-6-pho
sphate(G
6P)a
ndorc
AMP
b Ecolicellswereincub
ated
inM9minim
umsaltsolutio
nin
thep
resenceo
rabsence
ofG6PTh
euhpTmRN
Alevelswered
etermined
byreal-timeR
T-PC
Randthed
ataweren
ormalized
torpoD
mRN
Alevels
Indu
ctionof
uhpT
expressio
nby
G6P
was
calculated
bydividing
theu
hpTmRN
Alevelinthep
resenceo
fG6P
bytheu
hpTmRN
Alevelinthea
bsence
ofG6P
c Aminoacid
mutations
foun
don
lyin
strains
with
redu
cedFO
Msusceptib
ility(M
ICge8120583
gmL)
comparedwith
strains
with
FOM
MIClt1120583
gmL
NDnot
detectedN
Tno
ttested
BioMed Research International 5
Growth in the presence of G6P +minus
P lt 0001
01
1
10
100
1000
10000
Fold
indu
ctio
n of
uhpT
mRN
A ex
pres
sion
(a)
Fosfomycin MIC (120583gml)
P lt 0005
ge8 le4
01
1
10
100
1000
10000
Fold
indu
ctio
n of
uhpT
mRN
A ex
pres
sion
(b)
Figure 2 Induced expression levels of uhpT expression by the addition of G6P Nine clinical isolates with reduced susceptibility to FOM sixsusceptible clinical isolates and one standard strain are included Data used to generate this figure are given in Table 2 Statistical significancewas determined byMannndashWhitney test (a)Comparison of strains not grown (minus) or strains grown (+) inM9minimumsalt solution containingG6P (b) Comparison of strains with FOMMIC ge 8 120583gmL or le4 120583gmL
changes of amino acid residues in MurA were observed Aresistant strain (SRE257) and an intermediate-resistant strain(SRE91) had mutations in the genes that would result indeletions of a part of amino acid residues in UhpA andGlpT respectively In another intermediate-resistant strain(SRE49) uhpA and uhpT were not detected by PCR ampli-fication with two distinct primer pairs (ldquofullrdquo and ldquopartialrdquo inTable 1) In one strain with MIC 32 120583gmL (SRE252) cyaAwas not detectable by PCR All strains with reduced FOMMIC (ge8120583gmL) except one (SRE29) had several mutationsin one or more genes leading to amino acid deletion or pointmutation(s) of amino acid residues compared with othersusceptible strains
33 Function and Expression of Carbohydrate PhosphateTransporters To determine the activity of UhpT and GlpTwe examined cell growth in M9 minimum salt solutionsupplementedwithG6P orG3PNine of ten strainswith FOMMIC ge 8120583gmL did not grow within 24 h on G6P-containingM9 minimum salt agar On the other hand only three often strains did not grow in the presence of G3P (Table 3)and the two strains (SRE91 and SRE252) showed reducedMIC to FOM by addition of cAMP This suggested that thesetwo strains shared insufficient intracellular concentration ofcAMP for full expression of GlpT andor UhpTThese resultssuggested that FOM incorporation through the UhpT systemis involved to a greater extent in the reduction of E coli FOMcompared to the GlpT system
Expression of UhpT is induced by G6P [15ndash17] Wetherefore determined uhpT gene induction by G6P usingquantitative RT-PCR analysis (Table 3 and Figure 2) In
strains with FOM MIC lt 1 120583gmL uhpT expression wasstrongly induced (190- to 730-fold) by G6P By contrast ineight of ten strains with FOM MIC ge 8120583gmL the uhpTinduction was markedly lower (056- to 38-fold) or no uhpTsignal was amplified by PCR Of the remaining two strainswe observed a high induction (200-fold) of uhpT expressionby G6P in SRE253 strain however the strain did not grow onG6P-containing M9 minimum agar High induction (1200-fold) of uhpT by G6P was observed in SRE29 strain and thestrain grew on G6P-containing M9 minimum agar howeverit showed reduced susceptibility to FOM (MIC 32 120583gmL)These results indicated that G6P-dependent growth lacked inmost strains with reduced susceptibility to FOM because oflack of uhpT or a markedly reduced uhpT induction by G6PHowever the changes in uhpT expression and UhpT activitydid not account for the reduced FOM susceptibility of a fewstrains (Figure 2)
4 Discussion
The frequency of FOM resistance has been recognized to below And it has been expected that the frequency of cross-resistance of FOMand other antibiotics is very low because ofa unique mode of action However several reports examinedFOM resistance in ESBL-producing E coli [19 23 24 28] Areport indicates that FOM-resistant and intermediate E coliare more frequently resistant to other types of antimicrobialsthan FOM-susceptible strains [28] These reports suggestthe importance for surveillance of FOM-resistant E coliparticularly for focus on their cross-resistance of FOM inmultidrug resistance
6 BioMed Research International
In this study only one resistant and two intermediatestrains were found among 211 clinical isolates from Japan(Figure 1) This indicated that FOM was a promising candi-date agent against E coli infections as generally describedHowever we found seven strains susceptible according tothe CLST breakpoint but reduced susceptibility (MIC ge8 120583gmL) Under the selective pressure of FOM usage thesestrains might acquire FOM resistance more easily In thepresent study cross-resistance of strains with reduced FOMsusceptibility to other antimicrobial drugs was not signifi-cantly higher than in other strains and these strains were notconcentrating on specific genotypes
Based on previous reports acquisition of FOM-modifying enzymes (encoded by fos genes) and mutationsin murA gene results in resistance that is surpass ofbreakpoints to FOM [18 19 28] The present study didnot identify such strains Another resistance mechanismis altered FOM incorporation into bacterial cells TheG6P and G3P transporters contribute to incorporationof FOM into cells [13 14] Loss of function or decreasedexpression levels of them leads to a reduction of FOMsusceptibility [18 29 39] We found that loss of UhpT(G6P transporter) activity is more dominant than that ofGlpT (G3P transporter) in strains with decreased FOMsusceptibility In resistant (gt128 120583gmL) and intermediate(128 120583gmL) strains we noted amino acid deletion in therespective encoded proteins and no PCR amplificationof the transporter-related genes (Table 3) In strains withFOM MICs between 8 and 64 120583gmL gene mutation(s)leading to alternations of amino acid residues were foundhowever it was unclear whether these contributed tothe reduced susceptibility Among the ten strains withreduced susceptibility nine strains did not grow on G6P-supplemented M9 minimum salt agar This suggested thatin these nine strains G6P-induced UhpT function wasattenuated We found several mutations in cyaA and ptsI insome of these strains Dysfunction of CyaA and PtsI leadsto decrease in intracellular concentration of cAMP andinsufficient expression of GlpT and UhpT [16 17 31] Onlytwo strains (SRE91 and SRE252) with reduced susceptibilityto FOM showed that MIC was decreased by the exogenousaddition of cAMP and these strains did not grow in M9medium supplement with G3P Certainly SRE252 defectedcyaA however SRE91 did not share any mutations in cyaAand pstI Thus reduced susceptibility to FOM in the otherstrains was not explained by the insufficient intracellularconcentration of cAMP caused by dysfunction of cyaAand pstI The mutations in these genes found in this study(Table 3) seemed to scarcely relate with dysfunction of cAMPsynthesis
In conclusion FOM resistance occurs with low frequencyand is independent of resistance to other antimicrobials inE coli clinical isolates from Japan On the other hand weidentified strains with decreased FOM susceptibility Mostof them displayed fluctuated activity of the G6P transporterUhpT However G6P transporter function was altered eventhough the G6P-induced uhpT expression and amino acidsequence of UhpT were preserved in one strain (FOM MIC8 120583gmL) Another strain (FOM MIC 32 120583gmL) displayed
reduced susceptibility to FOM and no alteration of MICin the presence and absence of G6P even though G6P-induced uhpT expression amino acid sequence of UhpT andgrowth on G6P-supplemented M9 minimum salt agar werepreserved The exact molecular mechanism of the reducedsusceptibility of these strains remains unclear and requiresfurther evaluation
Competing Interests
The authors declare no competing interests
Acknowledgments
The authors thank Osamu Kuwahara (Sapporo Clinical Lab-oratory) for the provision of E coli clinical isolates Thisstudy was partly supported by a Grant-in-Aid for ScientificResearch (15H06521) from Japan Society for the Promotionof Science and a grant from Yuasa Memorial Foundation
References
[1] WHO ldquoAntimicrobial resistance global report on surveil-lance 2014rdquo 2014 httpwwwwhointdrugresistancedocu-mentssurveillancereporten
[2] M E Falagas andD E Karageorgopoulos ldquoExtended-spectrum120573-lactamase-producing organismsrdquo Journal of Hospital Infec-tion vol 73 no 4 pp 345ndash354 2009
[3] J D D Pitout ldquoExtraintestinal pathogenic Escherichia coli anupdate on antimicrobial resistance laboratory diagnosis andtreatmentrdquo Expert Review of Anti-Infective Therapy vol 10 no10 pp 1165ndash1176 2012
[4] S Yokota T Sato T Okubo et al ldquoPrevalence of fluoroquin-olone-resistant Escherichia coli O25H4-ST131 (CTX-M15-nonproducing) strains isolated in JapanrdquoChemotherapy vol 58no 1 pp 52ndash59 2012
[5] N Tsukamoto Y Ohkoshi T Okubo et al ldquoHigh prevalenceof cross-resistance to aminoglycosides in fluoroquinolone-resistantEscherichia coli clinical isolatesrdquoChemotherapy vol 59no 5 pp 379ndash384 2014
[6] H Giamarellou and G Poulakou ldquoMultidrug-resistant gram-negative infections what are the treatment optionsrdquoDrugs vol69 no 14 pp 1879ndash1901 2009
[7] M E Falagas A C Kastoris A M Kapaskelis and D E Kara-georgopoulos ldquoFosfomycin for the treatment of multidrug-resistant including extended-spectrum 120573-lactamase produc-ing Enterobacteriaceae infections a systematic reviewrdquo TheLancet Infectious Diseases vol 10 no 1 pp 43ndash50 2010
[8] A Schaeffer ldquoInfections of the urinary tractrdquo inCampbell-WalshUrology A Wein L Kavoussi A Novick A Partin and CPeters Eds pp 223ndash303 Elsevier Philadelphia Pa USA 9thedition 2007
[9] K Ishikawa R Hamasuna S Uehara et al ldquoJapanese nation-wide surveillance in 2011 of antibacterial susceptibility patternsof clinical isolates from complicated urinary tract infectioncasesrdquo Journal of Infection and Chemotherapy vol 21 no 9 pp623ndash633 2015
[10] D N Gilbert H F Chambers GM Eliopoulos andM S SaagTheSanford Guide to AntimicrobialTherapy 2015 AntimicrobialTherapy Sperryville Va USA 45th edition 2015
BioMed Research International 7
[11] The Japanese Association for Infectious Diseases and JapanSociety of ChemotherapyThe JAIDJSC Guide to Clinical Man-agement of Infectious Diseases 2014 The Japanese Associationfor Infectious DiseasesJapan Society of Chemotherapy TokyoJapan 2014 (Japanese)
[12] E D Brown E I Vivas C T Walsh and R Kolter ldquoMurA(MurZ) the enzyme that catalyzes the first committed stepin peptidoglycan biosynthesis is essential in Escherichia colirdquoJournal of Bacteriology vol 177 no 14 pp 4194ndash4197 1995
[13] F M Kahan J S Kahan P J Cassidy and H Kropp ldquoThemechanismof action of fosfomycin (phosphonomycin)rdquoAnnalsof the NewYork Academy of Sciences vol 235 no 1 pp 364ndash3861974
[14] M J Lemieux Y Huang and D-N Wang ldquoGlycerol-3-phosphate transporter of Escherichia coli structure functionand regulationrdquo Research in Microbiology vol 155 no 8 pp623ndash629 2004
[15] H HWinkler ldquoKinetics of exogenous induction of the hexose-6-phosphate transport system of Escherichia colirdquo Journal ofBacteriology vol 107 no 1 pp 74ndash78 1971
[16] T J Merkel J L Dahl R H Ebright and R J KadnerldquoTranscription activation at the Escherichia coli uhpT promoterby the catabolite gene activator proteinrdquo Journal of Bacteriologyvol 177 no 7 pp 1712ndash1718 1995
[17] D T Verhamme P W Postma W Crielaard and K J Helling-werf ldquoCooperativity in signal transfer through the Uhp systemof Escherichia colirdquo Journal of Bacteriology vol 184 no 15 pp4205ndash4210 2002
[18] S Takahata T Ida T Hiraishi et al ldquoMolecular mechanismsof fosfomycin resistance in clinical isolates of Escherichia colirdquoInternational Journal of Antimicrobial Agents vol 35 no 4 pp333ndash337 2010
[19] J Wachino K Yamane S Suzuki K Kimura and YArakawa ldquoPrevalence of fosfomycin resistance among CTX-M-producing Escherichia coli clinical isolates in Japan and iden-tification of novel plasmid-mediated fosfomycin-modifyingenzymesrdquo Antimicrobial Agents and Chemotherapy vol 54 no7 pp 3061ndash3064 2010
[20] K Kurabayashi Y Hirakawa K Tanimoto H Tomita and HHirakawa ldquoRole of the CpxAR two-component signal trans-duction system in control of fosfomycin resistance and carbonsubstrate uptakerdquo Journal of Bacteriology vol 196 no 2 pp248ndash256 2014
[21] P Arca G Reguera and C Hardisson ldquoPlasmid-encodedfosfomycin resistance in bacteria isolated from the urinary tractin amulticentre surveyrdquo Journal of Antimicrobial Chemotherapyvol 40 no 3 pp 393ndash399 1997
[22] J Hou X Huang Y Deng et al ldquoDissemination of thefosfomycin resistance gene fosA3 with CTX-M 120573-lactamasegenes and rmtB carried on incfII plasmids among Escherichiacoli isolates from pets in Chinardquo Antimicrobial Agents andChemotherapy vol 56 no 4 pp 2135ndash2138 2012
[23] S-Y Lee Y-J Park J K Yu et al ldquoPrevalence of acquiredfosfomycin resistance among extended-spectrum 120573-lactamase-producing Escherichia coli and Klebsiella pneumoniae clinicalisolates in Korea and IS26-composite transposon surroundingfosA3rdquo Journal of Antimicrobial Chemotherapy vol 67 no 12Article ID dks319 pp 2843ndash2847 2012
[24] P L Ho J Chan W U Lo et al ldquoPrevalence and molecularepidemiology of plasmid-mediated fosfomycin resistance genesamong blood and urinary Escherichia coli isolatesrdquo Journal ofMedical Microbiology vol 62 no 11 pp 1707ndash1713 2013
[25] G Nakamura J Wachino N Sato et al ldquoPractical agar-based disk potentiation test for detection of fosfomycin-nonsusceptible Escherichia coli clinical isolates producing glu-tathione S-transferasesrdquo Journal of ClinicalMicrobiology vol 52no 9 pp 3175ndash3179 2014
[26] Y Ma X Xu Q Guo P Wang W Wang and M WangldquoCharacterization of fosA5 a newplasmid-mediated fosfomycinresistance gene in Escherichia colirdquo Letters in Applied Microbiol-ogy vol 60 no 3 pp 259ndash264 2015
[27] Y Li B Zheng Y Li S Zhu F Xue and J Liu ldquoAntimicrobialsusceptibility and molecular mechanisms of fosfomycin resis-tance in clinical Escherichia coli isolates in mainland ChinardquoPLoS ONE vol 10 no 8 Article ID e0135269 2015
[28] S-P Tseng S-F Wang C-Y Kuo et al ldquoCharacteriza-tion of fosfomycin resistant extended-spectrum szlig-lactamase-producing Escherichia coli isolates from human and pig inTaiwanrdquo PLoS ONE vol 10 no 8 Article ID e0135864 2015
[29] A I Nilsson O G Berg O Aspevall G Kahlmeter and DI Andersson ldquoBiological costs and mechanisms of fosfomycinresistance in Escherichia colirdquo Antimicrobial Agents and Chem-otherapy vol 47 no 9 pp 2850ndash2858 2003
[30] J C Cordaro T Melton J P Stratis et al ldquoFosfomycinresistance selectionmethod for internal and extended deletionsof the phosphoenolpyruvatesugar phosphotransferase genes ofSalmonella typhimuriumrdquo Journal of Bacteriology vol 128 no3 pp 785ndash793 1976
[31] Y Sakamoto S Furukawa H Ogihara and M YamasakildquoFosmidomycin resistance in adenylate cyclase deficient (cya)mutants of Escherichia colirdquo Bioscience Biotechnology and Bio-chemistry vol 67 no 9 pp 2030ndash2033 2003
[32] G Kahlmeter and H O Poulsen ldquoAntimicrobial susceptibilityof Escherichia coli from community-acquired urinary tractinfections in Europe the ECOsdotSENS study revisitedrdquo Interna-tional Journal of Antimicrobial Agents vol 39 no 1 pp 45ndash512012
[33] D E Karageorgopoulos R Wang X-H Yu and M E FalagasldquoFosfomycin evaluation of the published evidence on the emer-gence of antimicrobial resistance in gram-negative pathogensrdquoJournal of Antimicrobial Chemotherapy vol 67 no 2 pp 255ndash268 2012
[34] Clinical and Laboratory Standards Institute Performance Stan-dards for Antimicrobial Susceptibility Testing 22nd InformationSupplement M100-S22 CLSI Wayne Pa USA 2012
[35] O Clermont J R Johnson M Menard and E DenamurldquoDetermination of Escherichia coli O types by allele-specificpolymerase chain reaction application to the O types involvedin human septicemiardquo Diagnostic Microbiology and InfectiousDisease vol 57 no 2 pp 129ndash136 2007
[36] D Li B Liu M Chen et al ldquoA multiplex PCR method todetect 14 Escherichia coli serogroups associated with urinarytract infectionsrdquo Journal of Microbiological Methods vol 82 no1 pp 71ndash77 2010
[37] O Clermont S Bonacorsi and E Bingen ldquoRapid and simpledetermination of the Escherichia coli phylogenetic grouprdquoApplied and Environmental Microbiology vol 66 no 10 pp4555ndash4558 2000
[38] S Y Tartof O D Solberg A R Manges and L W RileyldquoAnalysis of a uropathogenic Escherichia coli clonal group bymultilocus sequence typingrdquo Journal of Clinical Microbiologyvol 43 no 12 pp 5860ndash5864 2005
8 BioMed Research International
[39] R J Kadner and H HWinkler ldquoIsolation and characterizationof mutations affecting the transport of hexose phosphates inEscherichia colirdquo Journal of Bacteriology vol 113 no 2 pp 895ndash900 1973
Submit your manuscripts athttpswwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
BioMed Research International 5
Growth in the presence of G6P +minus
P lt 0001
01
1
10
100
1000
10000
Fold
indu
ctio
n of
uhpT
mRN
A ex
pres
sion
(a)
Fosfomycin MIC (120583gml)
P lt 0005
ge8 le4
01
1
10
100
1000
10000
Fold
indu
ctio
n of
uhpT
mRN
A ex
pres
sion
(b)
Figure 2 Induced expression levels of uhpT expression by the addition of G6P Nine clinical isolates with reduced susceptibility to FOM sixsusceptible clinical isolates and one standard strain are included Data used to generate this figure are given in Table 2 Statistical significancewas determined byMannndashWhitney test (a)Comparison of strains not grown (minus) or strains grown (+) inM9minimumsalt solution containingG6P (b) Comparison of strains with FOMMIC ge 8 120583gmL or le4 120583gmL
changes of amino acid residues in MurA were observed Aresistant strain (SRE257) and an intermediate-resistant strain(SRE91) had mutations in the genes that would result indeletions of a part of amino acid residues in UhpA andGlpT respectively In another intermediate-resistant strain(SRE49) uhpA and uhpT were not detected by PCR ampli-fication with two distinct primer pairs (ldquofullrdquo and ldquopartialrdquo inTable 1) In one strain with MIC 32 120583gmL (SRE252) cyaAwas not detectable by PCR All strains with reduced FOMMIC (ge8120583gmL) except one (SRE29) had several mutationsin one or more genes leading to amino acid deletion or pointmutation(s) of amino acid residues compared with othersusceptible strains
33 Function and Expression of Carbohydrate PhosphateTransporters To determine the activity of UhpT and GlpTwe examined cell growth in M9 minimum salt solutionsupplementedwithG6P orG3PNine of ten strainswith FOMMIC ge 8120583gmL did not grow within 24 h on G6P-containingM9 minimum salt agar On the other hand only three often strains did not grow in the presence of G3P (Table 3)and the two strains (SRE91 and SRE252) showed reducedMIC to FOM by addition of cAMP This suggested that thesetwo strains shared insufficient intracellular concentration ofcAMP for full expression of GlpT andor UhpTThese resultssuggested that FOM incorporation through the UhpT systemis involved to a greater extent in the reduction of E coli FOMcompared to the GlpT system
Expression of UhpT is induced by G6P [15ndash17] Wetherefore determined uhpT gene induction by G6P usingquantitative RT-PCR analysis (Table 3 and Figure 2) In
strains with FOM MIC lt 1 120583gmL uhpT expression wasstrongly induced (190- to 730-fold) by G6P By contrast ineight of ten strains with FOM MIC ge 8120583gmL the uhpTinduction was markedly lower (056- to 38-fold) or no uhpTsignal was amplified by PCR Of the remaining two strainswe observed a high induction (200-fold) of uhpT expressionby G6P in SRE253 strain however the strain did not grow onG6P-containing M9 minimum agar High induction (1200-fold) of uhpT by G6P was observed in SRE29 strain and thestrain grew on G6P-containing M9 minimum agar howeverit showed reduced susceptibility to FOM (MIC 32 120583gmL)These results indicated that G6P-dependent growth lacked inmost strains with reduced susceptibility to FOM because oflack of uhpT or a markedly reduced uhpT induction by G6PHowever the changes in uhpT expression and UhpT activitydid not account for the reduced FOM susceptibility of a fewstrains (Figure 2)
4 Discussion
The frequency of FOM resistance has been recognized to below And it has been expected that the frequency of cross-resistance of FOMand other antibiotics is very low because ofa unique mode of action However several reports examinedFOM resistance in ESBL-producing E coli [19 23 24 28] Areport indicates that FOM-resistant and intermediate E coliare more frequently resistant to other types of antimicrobialsthan FOM-susceptible strains [28] These reports suggestthe importance for surveillance of FOM-resistant E coliparticularly for focus on their cross-resistance of FOM inmultidrug resistance
6 BioMed Research International
In this study only one resistant and two intermediatestrains were found among 211 clinical isolates from Japan(Figure 1) This indicated that FOM was a promising candi-date agent against E coli infections as generally describedHowever we found seven strains susceptible according tothe CLST breakpoint but reduced susceptibility (MIC ge8 120583gmL) Under the selective pressure of FOM usage thesestrains might acquire FOM resistance more easily In thepresent study cross-resistance of strains with reduced FOMsusceptibility to other antimicrobial drugs was not signifi-cantly higher than in other strains and these strains were notconcentrating on specific genotypes
Based on previous reports acquisition of FOM-modifying enzymes (encoded by fos genes) and mutationsin murA gene results in resistance that is surpass ofbreakpoints to FOM [18 19 28] The present study didnot identify such strains Another resistance mechanismis altered FOM incorporation into bacterial cells TheG6P and G3P transporters contribute to incorporationof FOM into cells [13 14] Loss of function or decreasedexpression levels of them leads to a reduction of FOMsusceptibility [18 29 39] We found that loss of UhpT(G6P transporter) activity is more dominant than that ofGlpT (G3P transporter) in strains with decreased FOMsusceptibility In resistant (gt128 120583gmL) and intermediate(128 120583gmL) strains we noted amino acid deletion in therespective encoded proteins and no PCR amplificationof the transporter-related genes (Table 3) In strains withFOM MICs between 8 and 64 120583gmL gene mutation(s)leading to alternations of amino acid residues were foundhowever it was unclear whether these contributed tothe reduced susceptibility Among the ten strains withreduced susceptibility nine strains did not grow on G6P-supplemented M9 minimum salt agar This suggested thatin these nine strains G6P-induced UhpT function wasattenuated We found several mutations in cyaA and ptsI insome of these strains Dysfunction of CyaA and PtsI leadsto decrease in intracellular concentration of cAMP andinsufficient expression of GlpT and UhpT [16 17 31] Onlytwo strains (SRE91 and SRE252) with reduced susceptibilityto FOM showed that MIC was decreased by the exogenousaddition of cAMP and these strains did not grow in M9medium supplement with G3P Certainly SRE252 defectedcyaA however SRE91 did not share any mutations in cyaAand pstI Thus reduced susceptibility to FOM in the otherstrains was not explained by the insufficient intracellularconcentration of cAMP caused by dysfunction of cyaAand pstI The mutations in these genes found in this study(Table 3) seemed to scarcely relate with dysfunction of cAMPsynthesis
In conclusion FOM resistance occurs with low frequencyand is independent of resistance to other antimicrobials inE coli clinical isolates from Japan On the other hand weidentified strains with decreased FOM susceptibility Mostof them displayed fluctuated activity of the G6P transporterUhpT However G6P transporter function was altered eventhough the G6P-induced uhpT expression and amino acidsequence of UhpT were preserved in one strain (FOM MIC8 120583gmL) Another strain (FOM MIC 32 120583gmL) displayed
reduced susceptibility to FOM and no alteration of MICin the presence and absence of G6P even though G6P-induced uhpT expression amino acid sequence of UhpT andgrowth on G6P-supplemented M9 minimum salt agar werepreserved The exact molecular mechanism of the reducedsusceptibility of these strains remains unclear and requiresfurther evaluation
Competing Interests
The authors declare no competing interests
Acknowledgments
The authors thank Osamu Kuwahara (Sapporo Clinical Lab-oratory) for the provision of E coli clinical isolates Thisstudy was partly supported by a Grant-in-Aid for ScientificResearch (15H06521) from Japan Society for the Promotionof Science and a grant from Yuasa Memorial Foundation
References
[1] WHO ldquoAntimicrobial resistance global report on surveil-lance 2014rdquo 2014 httpwwwwhointdrugresistancedocu-mentssurveillancereporten
[2] M E Falagas andD E Karageorgopoulos ldquoExtended-spectrum120573-lactamase-producing organismsrdquo Journal of Hospital Infec-tion vol 73 no 4 pp 345ndash354 2009
[3] J D D Pitout ldquoExtraintestinal pathogenic Escherichia coli anupdate on antimicrobial resistance laboratory diagnosis andtreatmentrdquo Expert Review of Anti-Infective Therapy vol 10 no10 pp 1165ndash1176 2012
[4] S Yokota T Sato T Okubo et al ldquoPrevalence of fluoroquin-olone-resistant Escherichia coli O25H4-ST131 (CTX-M15-nonproducing) strains isolated in JapanrdquoChemotherapy vol 58no 1 pp 52ndash59 2012
[5] N Tsukamoto Y Ohkoshi T Okubo et al ldquoHigh prevalenceof cross-resistance to aminoglycosides in fluoroquinolone-resistantEscherichia coli clinical isolatesrdquoChemotherapy vol 59no 5 pp 379ndash384 2014
[6] H Giamarellou and G Poulakou ldquoMultidrug-resistant gram-negative infections what are the treatment optionsrdquoDrugs vol69 no 14 pp 1879ndash1901 2009
[7] M E Falagas A C Kastoris A M Kapaskelis and D E Kara-georgopoulos ldquoFosfomycin for the treatment of multidrug-resistant including extended-spectrum 120573-lactamase produc-ing Enterobacteriaceae infections a systematic reviewrdquo TheLancet Infectious Diseases vol 10 no 1 pp 43ndash50 2010
[8] A Schaeffer ldquoInfections of the urinary tractrdquo inCampbell-WalshUrology A Wein L Kavoussi A Novick A Partin and CPeters Eds pp 223ndash303 Elsevier Philadelphia Pa USA 9thedition 2007
[9] K Ishikawa R Hamasuna S Uehara et al ldquoJapanese nation-wide surveillance in 2011 of antibacterial susceptibility patternsof clinical isolates from complicated urinary tract infectioncasesrdquo Journal of Infection and Chemotherapy vol 21 no 9 pp623ndash633 2015
[10] D N Gilbert H F Chambers GM Eliopoulos andM S SaagTheSanford Guide to AntimicrobialTherapy 2015 AntimicrobialTherapy Sperryville Va USA 45th edition 2015
BioMed Research International 7
[11] The Japanese Association for Infectious Diseases and JapanSociety of ChemotherapyThe JAIDJSC Guide to Clinical Man-agement of Infectious Diseases 2014 The Japanese Associationfor Infectious DiseasesJapan Society of Chemotherapy TokyoJapan 2014 (Japanese)
[12] E D Brown E I Vivas C T Walsh and R Kolter ldquoMurA(MurZ) the enzyme that catalyzes the first committed stepin peptidoglycan biosynthesis is essential in Escherichia colirdquoJournal of Bacteriology vol 177 no 14 pp 4194ndash4197 1995
[13] F M Kahan J S Kahan P J Cassidy and H Kropp ldquoThemechanismof action of fosfomycin (phosphonomycin)rdquoAnnalsof the NewYork Academy of Sciences vol 235 no 1 pp 364ndash3861974
[14] M J Lemieux Y Huang and D-N Wang ldquoGlycerol-3-phosphate transporter of Escherichia coli structure functionand regulationrdquo Research in Microbiology vol 155 no 8 pp623ndash629 2004
[15] H HWinkler ldquoKinetics of exogenous induction of the hexose-6-phosphate transport system of Escherichia colirdquo Journal ofBacteriology vol 107 no 1 pp 74ndash78 1971
[16] T J Merkel J L Dahl R H Ebright and R J KadnerldquoTranscription activation at the Escherichia coli uhpT promoterby the catabolite gene activator proteinrdquo Journal of Bacteriologyvol 177 no 7 pp 1712ndash1718 1995
[17] D T Verhamme P W Postma W Crielaard and K J Helling-werf ldquoCooperativity in signal transfer through the Uhp systemof Escherichia colirdquo Journal of Bacteriology vol 184 no 15 pp4205ndash4210 2002
[18] S Takahata T Ida T Hiraishi et al ldquoMolecular mechanismsof fosfomycin resistance in clinical isolates of Escherichia colirdquoInternational Journal of Antimicrobial Agents vol 35 no 4 pp333ndash337 2010
[19] J Wachino K Yamane S Suzuki K Kimura and YArakawa ldquoPrevalence of fosfomycin resistance among CTX-M-producing Escherichia coli clinical isolates in Japan and iden-tification of novel plasmid-mediated fosfomycin-modifyingenzymesrdquo Antimicrobial Agents and Chemotherapy vol 54 no7 pp 3061ndash3064 2010
[20] K Kurabayashi Y Hirakawa K Tanimoto H Tomita and HHirakawa ldquoRole of the CpxAR two-component signal trans-duction system in control of fosfomycin resistance and carbonsubstrate uptakerdquo Journal of Bacteriology vol 196 no 2 pp248ndash256 2014
[21] P Arca G Reguera and C Hardisson ldquoPlasmid-encodedfosfomycin resistance in bacteria isolated from the urinary tractin amulticentre surveyrdquo Journal of Antimicrobial Chemotherapyvol 40 no 3 pp 393ndash399 1997
[22] J Hou X Huang Y Deng et al ldquoDissemination of thefosfomycin resistance gene fosA3 with CTX-M 120573-lactamasegenes and rmtB carried on incfII plasmids among Escherichiacoli isolates from pets in Chinardquo Antimicrobial Agents andChemotherapy vol 56 no 4 pp 2135ndash2138 2012
[23] S-Y Lee Y-J Park J K Yu et al ldquoPrevalence of acquiredfosfomycin resistance among extended-spectrum 120573-lactamase-producing Escherichia coli and Klebsiella pneumoniae clinicalisolates in Korea and IS26-composite transposon surroundingfosA3rdquo Journal of Antimicrobial Chemotherapy vol 67 no 12Article ID dks319 pp 2843ndash2847 2012
[24] P L Ho J Chan W U Lo et al ldquoPrevalence and molecularepidemiology of plasmid-mediated fosfomycin resistance genesamong blood and urinary Escherichia coli isolatesrdquo Journal ofMedical Microbiology vol 62 no 11 pp 1707ndash1713 2013
[25] G Nakamura J Wachino N Sato et al ldquoPractical agar-based disk potentiation test for detection of fosfomycin-nonsusceptible Escherichia coli clinical isolates producing glu-tathione S-transferasesrdquo Journal of ClinicalMicrobiology vol 52no 9 pp 3175ndash3179 2014
[26] Y Ma X Xu Q Guo P Wang W Wang and M WangldquoCharacterization of fosA5 a newplasmid-mediated fosfomycinresistance gene in Escherichia colirdquo Letters in Applied Microbiol-ogy vol 60 no 3 pp 259ndash264 2015
[27] Y Li B Zheng Y Li S Zhu F Xue and J Liu ldquoAntimicrobialsusceptibility and molecular mechanisms of fosfomycin resis-tance in clinical Escherichia coli isolates in mainland ChinardquoPLoS ONE vol 10 no 8 Article ID e0135269 2015
[28] S-P Tseng S-F Wang C-Y Kuo et al ldquoCharacteriza-tion of fosfomycin resistant extended-spectrum szlig-lactamase-producing Escherichia coli isolates from human and pig inTaiwanrdquo PLoS ONE vol 10 no 8 Article ID e0135864 2015
[29] A I Nilsson O G Berg O Aspevall G Kahlmeter and DI Andersson ldquoBiological costs and mechanisms of fosfomycinresistance in Escherichia colirdquo Antimicrobial Agents and Chem-otherapy vol 47 no 9 pp 2850ndash2858 2003
[30] J C Cordaro T Melton J P Stratis et al ldquoFosfomycinresistance selectionmethod for internal and extended deletionsof the phosphoenolpyruvatesugar phosphotransferase genes ofSalmonella typhimuriumrdquo Journal of Bacteriology vol 128 no3 pp 785ndash793 1976
[31] Y Sakamoto S Furukawa H Ogihara and M YamasakildquoFosmidomycin resistance in adenylate cyclase deficient (cya)mutants of Escherichia colirdquo Bioscience Biotechnology and Bio-chemistry vol 67 no 9 pp 2030ndash2033 2003
[32] G Kahlmeter and H O Poulsen ldquoAntimicrobial susceptibilityof Escherichia coli from community-acquired urinary tractinfections in Europe the ECOsdotSENS study revisitedrdquo Interna-tional Journal of Antimicrobial Agents vol 39 no 1 pp 45ndash512012
[33] D E Karageorgopoulos R Wang X-H Yu and M E FalagasldquoFosfomycin evaluation of the published evidence on the emer-gence of antimicrobial resistance in gram-negative pathogensrdquoJournal of Antimicrobial Chemotherapy vol 67 no 2 pp 255ndash268 2012
[34] Clinical and Laboratory Standards Institute Performance Stan-dards for Antimicrobial Susceptibility Testing 22nd InformationSupplement M100-S22 CLSI Wayne Pa USA 2012
[35] O Clermont J R Johnson M Menard and E DenamurldquoDetermination of Escherichia coli O types by allele-specificpolymerase chain reaction application to the O types involvedin human septicemiardquo Diagnostic Microbiology and InfectiousDisease vol 57 no 2 pp 129ndash136 2007
[36] D Li B Liu M Chen et al ldquoA multiplex PCR method todetect 14 Escherichia coli serogroups associated with urinarytract infectionsrdquo Journal of Microbiological Methods vol 82 no1 pp 71ndash77 2010
[37] O Clermont S Bonacorsi and E Bingen ldquoRapid and simpledetermination of the Escherichia coli phylogenetic grouprdquoApplied and Environmental Microbiology vol 66 no 10 pp4555ndash4558 2000
[38] S Y Tartof O D Solberg A R Manges and L W RileyldquoAnalysis of a uropathogenic Escherichia coli clonal group bymultilocus sequence typingrdquo Journal of Clinical Microbiologyvol 43 no 12 pp 5860ndash5864 2005
8 BioMed Research International
[39] R J Kadner and H HWinkler ldquoIsolation and characterizationof mutations affecting the transport of hexose phosphates inEscherichia colirdquo Journal of Bacteriology vol 113 no 2 pp 895ndash900 1973
Submit your manuscripts athttpswwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
6 BioMed Research International
In this study only one resistant and two intermediatestrains were found among 211 clinical isolates from Japan(Figure 1) This indicated that FOM was a promising candi-date agent against E coli infections as generally describedHowever we found seven strains susceptible according tothe CLST breakpoint but reduced susceptibility (MIC ge8 120583gmL) Under the selective pressure of FOM usage thesestrains might acquire FOM resistance more easily In thepresent study cross-resistance of strains with reduced FOMsusceptibility to other antimicrobial drugs was not signifi-cantly higher than in other strains and these strains were notconcentrating on specific genotypes
Based on previous reports acquisition of FOM-modifying enzymes (encoded by fos genes) and mutationsin murA gene results in resistance that is surpass ofbreakpoints to FOM [18 19 28] The present study didnot identify such strains Another resistance mechanismis altered FOM incorporation into bacterial cells TheG6P and G3P transporters contribute to incorporationof FOM into cells [13 14] Loss of function or decreasedexpression levels of them leads to a reduction of FOMsusceptibility [18 29 39] We found that loss of UhpT(G6P transporter) activity is more dominant than that ofGlpT (G3P transporter) in strains with decreased FOMsusceptibility In resistant (gt128 120583gmL) and intermediate(128 120583gmL) strains we noted amino acid deletion in therespective encoded proteins and no PCR amplificationof the transporter-related genes (Table 3) In strains withFOM MICs between 8 and 64 120583gmL gene mutation(s)leading to alternations of amino acid residues were foundhowever it was unclear whether these contributed tothe reduced susceptibility Among the ten strains withreduced susceptibility nine strains did not grow on G6P-supplemented M9 minimum salt agar This suggested thatin these nine strains G6P-induced UhpT function wasattenuated We found several mutations in cyaA and ptsI insome of these strains Dysfunction of CyaA and PtsI leadsto decrease in intracellular concentration of cAMP andinsufficient expression of GlpT and UhpT [16 17 31] Onlytwo strains (SRE91 and SRE252) with reduced susceptibilityto FOM showed that MIC was decreased by the exogenousaddition of cAMP and these strains did not grow in M9medium supplement with G3P Certainly SRE252 defectedcyaA however SRE91 did not share any mutations in cyaAand pstI Thus reduced susceptibility to FOM in the otherstrains was not explained by the insufficient intracellularconcentration of cAMP caused by dysfunction of cyaAand pstI The mutations in these genes found in this study(Table 3) seemed to scarcely relate with dysfunction of cAMPsynthesis
In conclusion FOM resistance occurs with low frequencyand is independent of resistance to other antimicrobials inE coli clinical isolates from Japan On the other hand weidentified strains with decreased FOM susceptibility Mostof them displayed fluctuated activity of the G6P transporterUhpT However G6P transporter function was altered eventhough the G6P-induced uhpT expression and amino acidsequence of UhpT were preserved in one strain (FOM MIC8 120583gmL) Another strain (FOM MIC 32 120583gmL) displayed
reduced susceptibility to FOM and no alteration of MICin the presence and absence of G6P even though G6P-induced uhpT expression amino acid sequence of UhpT andgrowth on G6P-supplemented M9 minimum salt agar werepreserved The exact molecular mechanism of the reducedsusceptibility of these strains remains unclear and requiresfurther evaluation
Competing Interests
The authors declare no competing interests
Acknowledgments
The authors thank Osamu Kuwahara (Sapporo Clinical Lab-oratory) for the provision of E coli clinical isolates Thisstudy was partly supported by a Grant-in-Aid for ScientificResearch (15H06521) from Japan Society for the Promotionof Science and a grant from Yuasa Memorial Foundation
References
[1] WHO ldquoAntimicrobial resistance global report on surveil-lance 2014rdquo 2014 httpwwwwhointdrugresistancedocu-mentssurveillancereporten
[2] M E Falagas andD E Karageorgopoulos ldquoExtended-spectrum120573-lactamase-producing organismsrdquo Journal of Hospital Infec-tion vol 73 no 4 pp 345ndash354 2009
[3] J D D Pitout ldquoExtraintestinal pathogenic Escherichia coli anupdate on antimicrobial resistance laboratory diagnosis andtreatmentrdquo Expert Review of Anti-Infective Therapy vol 10 no10 pp 1165ndash1176 2012
[4] S Yokota T Sato T Okubo et al ldquoPrevalence of fluoroquin-olone-resistant Escherichia coli O25H4-ST131 (CTX-M15-nonproducing) strains isolated in JapanrdquoChemotherapy vol 58no 1 pp 52ndash59 2012
[5] N Tsukamoto Y Ohkoshi T Okubo et al ldquoHigh prevalenceof cross-resistance to aminoglycosides in fluoroquinolone-resistantEscherichia coli clinical isolatesrdquoChemotherapy vol 59no 5 pp 379ndash384 2014
[6] H Giamarellou and G Poulakou ldquoMultidrug-resistant gram-negative infections what are the treatment optionsrdquoDrugs vol69 no 14 pp 1879ndash1901 2009
[7] M E Falagas A C Kastoris A M Kapaskelis and D E Kara-georgopoulos ldquoFosfomycin for the treatment of multidrug-resistant including extended-spectrum 120573-lactamase produc-ing Enterobacteriaceae infections a systematic reviewrdquo TheLancet Infectious Diseases vol 10 no 1 pp 43ndash50 2010
[8] A Schaeffer ldquoInfections of the urinary tractrdquo inCampbell-WalshUrology A Wein L Kavoussi A Novick A Partin and CPeters Eds pp 223ndash303 Elsevier Philadelphia Pa USA 9thedition 2007
[9] K Ishikawa R Hamasuna S Uehara et al ldquoJapanese nation-wide surveillance in 2011 of antibacterial susceptibility patternsof clinical isolates from complicated urinary tract infectioncasesrdquo Journal of Infection and Chemotherapy vol 21 no 9 pp623ndash633 2015
[10] D N Gilbert H F Chambers GM Eliopoulos andM S SaagTheSanford Guide to AntimicrobialTherapy 2015 AntimicrobialTherapy Sperryville Va USA 45th edition 2015
BioMed Research International 7
[11] The Japanese Association for Infectious Diseases and JapanSociety of ChemotherapyThe JAIDJSC Guide to Clinical Man-agement of Infectious Diseases 2014 The Japanese Associationfor Infectious DiseasesJapan Society of Chemotherapy TokyoJapan 2014 (Japanese)
[12] E D Brown E I Vivas C T Walsh and R Kolter ldquoMurA(MurZ) the enzyme that catalyzes the first committed stepin peptidoglycan biosynthesis is essential in Escherichia colirdquoJournal of Bacteriology vol 177 no 14 pp 4194ndash4197 1995
[13] F M Kahan J S Kahan P J Cassidy and H Kropp ldquoThemechanismof action of fosfomycin (phosphonomycin)rdquoAnnalsof the NewYork Academy of Sciences vol 235 no 1 pp 364ndash3861974
[14] M J Lemieux Y Huang and D-N Wang ldquoGlycerol-3-phosphate transporter of Escherichia coli structure functionand regulationrdquo Research in Microbiology vol 155 no 8 pp623ndash629 2004
[15] H HWinkler ldquoKinetics of exogenous induction of the hexose-6-phosphate transport system of Escherichia colirdquo Journal ofBacteriology vol 107 no 1 pp 74ndash78 1971
[16] T J Merkel J L Dahl R H Ebright and R J KadnerldquoTranscription activation at the Escherichia coli uhpT promoterby the catabolite gene activator proteinrdquo Journal of Bacteriologyvol 177 no 7 pp 1712ndash1718 1995
[17] D T Verhamme P W Postma W Crielaard and K J Helling-werf ldquoCooperativity in signal transfer through the Uhp systemof Escherichia colirdquo Journal of Bacteriology vol 184 no 15 pp4205ndash4210 2002
[18] S Takahata T Ida T Hiraishi et al ldquoMolecular mechanismsof fosfomycin resistance in clinical isolates of Escherichia colirdquoInternational Journal of Antimicrobial Agents vol 35 no 4 pp333ndash337 2010
[19] J Wachino K Yamane S Suzuki K Kimura and YArakawa ldquoPrevalence of fosfomycin resistance among CTX-M-producing Escherichia coli clinical isolates in Japan and iden-tification of novel plasmid-mediated fosfomycin-modifyingenzymesrdquo Antimicrobial Agents and Chemotherapy vol 54 no7 pp 3061ndash3064 2010
[20] K Kurabayashi Y Hirakawa K Tanimoto H Tomita and HHirakawa ldquoRole of the CpxAR two-component signal trans-duction system in control of fosfomycin resistance and carbonsubstrate uptakerdquo Journal of Bacteriology vol 196 no 2 pp248ndash256 2014
[21] P Arca G Reguera and C Hardisson ldquoPlasmid-encodedfosfomycin resistance in bacteria isolated from the urinary tractin amulticentre surveyrdquo Journal of Antimicrobial Chemotherapyvol 40 no 3 pp 393ndash399 1997
[22] J Hou X Huang Y Deng et al ldquoDissemination of thefosfomycin resistance gene fosA3 with CTX-M 120573-lactamasegenes and rmtB carried on incfII plasmids among Escherichiacoli isolates from pets in Chinardquo Antimicrobial Agents andChemotherapy vol 56 no 4 pp 2135ndash2138 2012
[23] S-Y Lee Y-J Park J K Yu et al ldquoPrevalence of acquiredfosfomycin resistance among extended-spectrum 120573-lactamase-producing Escherichia coli and Klebsiella pneumoniae clinicalisolates in Korea and IS26-composite transposon surroundingfosA3rdquo Journal of Antimicrobial Chemotherapy vol 67 no 12Article ID dks319 pp 2843ndash2847 2012
[24] P L Ho J Chan W U Lo et al ldquoPrevalence and molecularepidemiology of plasmid-mediated fosfomycin resistance genesamong blood and urinary Escherichia coli isolatesrdquo Journal ofMedical Microbiology vol 62 no 11 pp 1707ndash1713 2013
[25] G Nakamura J Wachino N Sato et al ldquoPractical agar-based disk potentiation test for detection of fosfomycin-nonsusceptible Escherichia coli clinical isolates producing glu-tathione S-transferasesrdquo Journal of ClinicalMicrobiology vol 52no 9 pp 3175ndash3179 2014
[26] Y Ma X Xu Q Guo P Wang W Wang and M WangldquoCharacterization of fosA5 a newplasmid-mediated fosfomycinresistance gene in Escherichia colirdquo Letters in Applied Microbiol-ogy vol 60 no 3 pp 259ndash264 2015
[27] Y Li B Zheng Y Li S Zhu F Xue and J Liu ldquoAntimicrobialsusceptibility and molecular mechanisms of fosfomycin resis-tance in clinical Escherichia coli isolates in mainland ChinardquoPLoS ONE vol 10 no 8 Article ID e0135269 2015
[28] S-P Tseng S-F Wang C-Y Kuo et al ldquoCharacteriza-tion of fosfomycin resistant extended-spectrum szlig-lactamase-producing Escherichia coli isolates from human and pig inTaiwanrdquo PLoS ONE vol 10 no 8 Article ID e0135864 2015
[29] A I Nilsson O G Berg O Aspevall G Kahlmeter and DI Andersson ldquoBiological costs and mechanisms of fosfomycinresistance in Escherichia colirdquo Antimicrobial Agents and Chem-otherapy vol 47 no 9 pp 2850ndash2858 2003
[30] J C Cordaro T Melton J P Stratis et al ldquoFosfomycinresistance selectionmethod for internal and extended deletionsof the phosphoenolpyruvatesugar phosphotransferase genes ofSalmonella typhimuriumrdquo Journal of Bacteriology vol 128 no3 pp 785ndash793 1976
[31] Y Sakamoto S Furukawa H Ogihara and M YamasakildquoFosmidomycin resistance in adenylate cyclase deficient (cya)mutants of Escherichia colirdquo Bioscience Biotechnology and Bio-chemistry vol 67 no 9 pp 2030ndash2033 2003
[32] G Kahlmeter and H O Poulsen ldquoAntimicrobial susceptibilityof Escherichia coli from community-acquired urinary tractinfections in Europe the ECOsdotSENS study revisitedrdquo Interna-tional Journal of Antimicrobial Agents vol 39 no 1 pp 45ndash512012
[33] D E Karageorgopoulos R Wang X-H Yu and M E FalagasldquoFosfomycin evaluation of the published evidence on the emer-gence of antimicrobial resistance in gram-negative pathogensrdquoJournal of Antimicrobial Chemotherapy vol 67 no 2 pp 255ndash268 2012
[34] Clinical and Laboratory Standards Institute Performance Stan-dards for Antimicrobial Susceptibility Testing 22nd InformationSupplement M100-S22 CLSI Wayne Pa USA 2012
[35] O Clermont J R Johnson M Menard and E DenamurldquoDetermination of Escherichia coli O types by allele-specificpolymerase chain reaction application to the O types involvedin human septicemiardquo Diagnostic Microbiology and InfectiousDisease vol 57 no 2 pp 129ndash136 2007
[36] D Li B Liu M Chen et al ldquoA multiplex PCR method todetect 14 Escherichia coli serogroups associated with urinarytract infectionsrdquo Journal of Microbiological Methods vol 82 no1 pp 71ndash77 2010
[37] O Clermont S Bonacorsi and E Bingen ldquoRapid and simpledetermination of the Escherichia coli phylogenetic grouprdquoApplied and Environmental Microbiology vol 66 no 10 pp4555ndash4558 2000
[38] S Y Tartof O D Solberg A R Manges and L W RileyldquoAnalysis of a uropathogenic Escherichia coli clonal group bymultilocus sequence typingrdquo Journal of Clinical Microbiologyvol 43 no 12 pp 5860ndash5864 2005
8 BioMed Research International
[39] R J Kadner and H HWinkler ldquoIsolation and characterizationof mutations affecting the transport of hexose phosphates inEscherichia colirdquo Journal of Bacteriology vol 113 no 2 pp 895ndash900 1973
Submit your manuscripts athttpswwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
BioMed Research International 7
[11] The Japanese Association for Infectious Diseases and JapanSociety of ChemotherapyThe JAIDJSC Guide to Clinical Man-agement of Infectious Diseases 2014 The Japanese Associationfor Infectious DiseasesJapan Society of Chemotherapy TokyoJapan 2014 (Japanese)
[12] E D Brown E I Vivas C T Walsh and R Kolter ldquoMurA(MurZ) the enzyme that catalyzes the first committed stepin peptidoglycan biosynthesis is essential in Escherichia colirdquoJournal of Bacteriology vol 177 no 14 pp 4194ndash4197 1995
[13] F M Kahan J S Kahan P J Cassidy and H Kropp ldquoThemechanismof action of fosfomycin (phosphonomycin)rdquoAnnalsof the NewYork Academy of Sciences vol 235 no 1 pp 364ndash3861974
[14] M J Lemieux Y Huang and D-N Wang ldquoGlycerol-3-phosphate transporter of Escherichia coli structure functionand regulationrdquo Research in Microbiology vol 155 no 8 pp623ndash629 2004
[15] H HWinkler ldquoKinetics of exogenous induction of the hexose-6-phosphate transport system of Escherichia colirdquo Journal ofBacteriology vol 107 no 1 pp 74ndash78 1971
[16] T J Merkel J L Dahl R H Ebright and R J KadnerldquoTranscription activation at the Escherichia coli uhpT promoterby the catabolite gene activator proteinrdquo Journal of Bacteriologyvol 177 no 7 pp 1712ndash1718 1995
[17] D T Verhamme P W Postma W Crielaard and K J Helling-werf ldquoCooperativity in signal transfer through the Uhp systemof Escherichia colirdquo Journal of Bacteriology vol 184 no 15 pp4205ndash4210 2002
[18] S Takahata T Ida T Hiraishi et al ldquoMolecular mechanismsof fosfomycin resistance in clinical isolates of Escherichia colirdquoInternational Journal of Antimicrobial Agents vol 35 no 4 pp333ndash337 2010
[19] J Wachino K Yamane S Suzuki K Kimura and YArakawa ldquoPrevalence of fosfomycin resistance among CTX-M-producing Escherichia coli clinical isolates in Japan and iden-tification of novel plasmid-mediated fosfomycin-modifyingenzymesrdquo Antimicrobial Agents and Chemotherapy vol 54 no7 pp 3061ndash3064 2010
[20] K Kurabayashi Y Hirakawa K Tanimoto H Tomita and HHirakawa ldquoRole of the CpxAR two-component signal trans-duction system in control of fosfomycin resistance and carbonsubstrate uptakerdquo Journal of Bacteriology vol 196 no 2 pp248ndash256 2014
[21] P Arca G Reguera and C Hardisson ldquoPlasmid-encodedfosfomycin resistance in bacteria isolated from the urinary tractin amulticentre surveyrdquo Journal of Antimicrobial Chemotherapyvol 40 no 3 pp 393ndash399 1997
[22] J Hou X Huang Y Deng et al ldquoDissemination of thefosfomycin resistance gene fosA3 with CTX-M 120573-lactamasegenes and rmtB carried on incfII plasmids among Escherichiacoli isolates from pets in Chinardquo Antimicrobial Agents andChemotherapy vol 56 no 4 pp 2135ndash2138 2012
[23] S-Y Lee Y-J Park J K Yu et al ldquoPrevalence of acquiredfosfomycin resistance among extended-spectrum 120573-lactamase-producing Escherichia coli and Klebsiella pneumoniae clinicalisolates in Korea and IS26-composite transposon surroundingfosA3rdquo Journal of Antimicrobial Chemotherapy vol 67 no 12Article ID dks319 pp 2843ndash2847 2012
[24] P L Ho J Chan W U Lo et al ldquoPrevalence and molecularepidemiology of plasmid-mediated fosfomycin resistance genesamong blood and urinary Escherichia coli isolatesrdquo Journal ofMedical Microbiology vol 62 no 11 pp 1707ndash1713 2013
[25] G Nakamura J Wachino N Sato et al ldquoPractical agar-based disk potentiation test for detection of fosfomycin-nonsusceptible Escherichia coli clinical isolates producing glu-tathione S-transferasesrdquo Journal of ClinicalMicrobiology vol 52no 9 pp 3175ndash3179 2014
[26] Y Ma X Xu Q Guo P Wang W Wang and M WangldquoCharacterization of fosA5 a newplasmid-mediated fosfomycinresistance gene in Escherichia colirdquo Letters in Applied Microbiol-ogy vol 60 no 3 pp 259ndash264 2015
[27] Y Li B Zheng Y Li S Zhu F Xue and J Liu ldquoAntimicrobialsusceptibility and molecular mechanisms of fosfomycin resis-tance in clinical Escherichia coli isolates in mainland ChinardquoPLoS ONE vol 10 no 8 Article ID e0135269 2015
[28] S-P Tseng S-F Wang C-Y Kuo et al ldquoCharacteriza-tion of fosfomycin resistant extended-spectrum szlig-lactamase-producing Escherichia coli isolates from human and pig inTaiwanrdquo PLoS ONE vol 10 no 8 Article ID e0135864 2015
[29] A I Nilsson O G Berg O Aspevall G Kahlmeter and DI Andersson ldquoBiological costs and mechanisms of fosfomycinresistance in Escherichia colirdquo Antimicrobial Agents and Chem-otherapy vol 47 no 9 pp 2850ndash2858 2003
[30] J C Cordaro T Melton J P Stratis et al ldquoFosfomycinresistance selectionmethod for internal and extended deletionsof the phosphoenolpyruvatesugar phosphotransferase genes ofSalmonella typhimuriumrdquo Journal of Bacteriology vol 128 no3 pp 785ndash793 1976
[31] Y Sakamoto S Furukawa H Ogihara and M YamasakildquoFosmidomycin resistance in adenylate cyclase deficient (cya)mutants of Escherichia colirdquo Bioscience Biotechnology and Bio-chemistry vol 67 no 9 pp 2030ndash2033 2003
[32] G Kahlmeter and H O Poulsen ldquoAntimicrobial susceptibilityof Escherichia coli from community-acquired urinary tractinfections in Europe the ECOsdotSENS study revisitedrdquo Interna-tional Journal of Antimicrobial Agents vol 39 no 1 pp 45ndash512012
[33] D E Karageorgopoulos R Wang X-H Yu and M E FalagasldquoFosfomycin evaluation of the published evidence on the emer-gence of antimicrobial resistance in gram-negative pathogensrdquoJournal of Antimicrobial Chemotherapy vol 67 no 2 pp 255ndash268 2012
[34] Clinical and Laboratory Standards Institute Performance Stan-dards for Antimicrobial Susceptibility Testing 22nd InformationSupplement M100-S22 CLSI Wayne Pa USA 2012
[35] O Clermont J R Johnson M Menard and E DenamurldquoDetermination of Escherichia coli O types by allele-specificpolymerase chain reaction application to the O types involvedin human septicemiardquo Diagnostic Microbiology and InfectiousDisease vol 57 no 2 pp 129ndash136 2007
[36] D Li B Liu M Chen et al ldquoA multiplex PCR method todetect 14 Escherichia coli serogroups associated with urinarytract infectionsrdquo Journal of Microbiological Methods vol 82 no1 pp 71ndash77 2010
[37] O Clermont S Bonacorsi and E Bingen ldquoRapid and simpledetermination of the Escherichia coli phylogenetic grouprdquoApplied and Environmental Microbiology vol 66 no 10 pp4555ndash4558 2000
[38] S Y Tartof O D Solberg A R Manges and L W RileyldquoAnalysis of a uropathogenic Escherichia coli clonal group bymultilocus sequence typingrdquo Journal of Clinical Microbiologyvol 43 no 12 pp 5860ndash5864 2005
8 BioMed Research International
[39] R J Kadner and H HWinkler ldquoIsolation and characterizationof mutations affecting the transport of hexose phosphates inEscherichia colirdquo Journal of Bacteriology vol 113 no 2 pp 895ndash900 1973
Submit your manuscripts athttpswwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
8 BioMed Research International
[39] R J Kadner and H HWinkler ldquoIsolation and characterizationof mutations affecting the transport of hexose phosphates inEscherichia colirdquo Journal of Bacteriology vol 113 no 2 pp 895ndash900 1973
Submit your manuscripts athttpswwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
Submit your manuscripts athttpswwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology