clinical relevance and diagnostics of staphylococcus ... · acknowledgements this work was...

Acknowledgements

This work was supported by project CEB (CZ.1.07/2.3.00/20.0183).

References

[1] Pantůček R., Švec P., Dajcs J.J., Machová I., Černohlávková J., Šedo O., Gelbíčová T., Mašlaňová I., Doškař. I., Zdráhal Z., Růžičková V., Sedláček I. (2013): Staphylococcus petrasii sp. nov. including S. petrasii subsp. petrasii subsp. nov. and S. petrasii subsp. croceilyticus subsp. nov., isolated from human clinical specimens and human ear infections. Systematic and Applied Microbiology 36, p. 90-95.

Results

Introduction and purpose

Coagulase-negative staphylococci are ubiquitous organisms that were recognized as opportunistic human pathogens occurring often in immunocompromised individuals.

Staphylococcus petrasii represents a typical coagulase-negative, novobiocin susceptible staphylococcus. This species is phylogeneticaly related to S. haemolyticus, S. hominis, S. devriesei and S. lugdunensis.

S. petrasii was described in May 2013 and so far two subspecies S.

petrasii subsp. petrasii and S. petrasii subsp. croceilyticus have been recognised [1]. Since than additional strains were retrieved from clinical samples by our group.

In this study we summarize existing data about the occurrence, identification and significance of S. petrasii in human clinical

materials.

Conclusions

Strains of S. petrasii commonly occurs in human clinical materials and may act as an oppurtunistic pathogen resistant to certain antibiotics.

S. petrasii may be incorrectly identified as atypical S. warneri, S. pasteuri or S. aureus.

Reliable identification and monitoring of Staphylococcus petrasii is of clinical importance

Using commercial biochemical identification tests kits, strains of S. petrasii might have been identified as atypical S. warneri, S. pasteuri or S. simulans.

Results of STAPHYtest 24 and API Staph test are given in the table 3. Diferentiation of S. petrasii from other novobiocin sensitive Staphylococcus species is given in tables 4 and 5.

S. petrasii strains were sensitive to most of antibiotic tested. However some strains revealed resistance to benzylpenicilin, erythromycin and tetracyline (Table 6)

SmaI macrorestriction analysis clearly separated both S. petrasii subspecies and proved to be a suitable methodfor typing of these strains (Fig. 1).

MALDI-TOF MS method assigned S. petrasii strains as: Staphylococcus sp[1]. 10w414721 RLH, Staphylococcus sp[1]. 901400083 LBK, or Staphylococcus sp[1] 4.PIM .

Rep-PCR and automated ribotyping revealed subspecies-specific clusters of S. petrasii subsp. petrasii and S. petrasii subsp. croceilyticus and distinguished the strains from other Staphylococcus spp. (data not shown).

Table 6:Antibiotic susceptibility profile of Staphylococcus petrasii

Fig.1: Dendrogram based on SmaI macrorestriction analysis

1,* 2 1 3 1Jitka Černohlávková, Petr Petráš, Pavel Švec, Roman Pantůček, Ivo Sedláček

1Czech Collection of Microorganisms, Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic

2Reference laboratory for Staphylococci, National Institut of Public Health, Prague, Czech Republic3Division of Genetisc and Molecular Biology, Department of Experimental Biology, Masaryk University, Brno, Czech Republic

* Corresponding author: [email protected]

Czech Collection of Microorganismshttp://www.sci.muni.cz/ccm/

Clinical relevance and diagnostics of Staphylococcus petrasii, a recently described coagulase-negative species

Staphylococcus petrasii subsp. petrasii Date LocalityMaterial/

specimenSex

Age

(year)Diagnosis/source

NRL/St 06/029 January 2006Nové Město na

Moravě, CZ

medical

equipment - - -

NRL/St 07/045 January 2007 Strakonice, CZ nasal swab F 31healthy woman screend

for bacterial carriage

NRL/St 07/427 May 2007České

Budějovice, CZblood M 56 fever of unknown origin

NRL/St 08/280 April 2008České

Budějovice, CZwound pus M 6 leg wound infection

NRL/St 08/435 May 2008České

Budějovice, CZblood F 41

malignant neoplasm

of lymph nodes

NRL/St 08/525 July 2008 Šternberk, CZ blood M 68 monitoring of ATB

CCM 8420 ( = NRL/St 08/738) September 2008 Hořovice, CZ ear swab F 0 neonatal bacterial sepsis

CCM 8418T (= CCUG 62727T = NRL/St 10/1050T) December 2009 Trutnov, CZ blood M 59 cerebral hemorrhage

CCM 8419 ( = NRL/St 10/670) July 2010 Strakonice, CZ blood F 87 PACU pateint

NRL/St 11/077 February 2011 Klatovy, CZ blood F 60 abdominal pain

NRL/St 12/012 January 2012 Ústí nad Labem, CZ blood F 0 low birth weight

NRL/St 12/015 January 2012 Kladno, CZ blood F 78 cerebrovascular accident

NRL/St 12/584 September 2012 Tábor, CZ blood F 76 fever of unknown origin

NRL/St 13/057 January 2013 Liberec, CZ blood F 50 respiratory failure

Staphylococcus petrasii subsp. croceilyticus Date LocalityMaterial/

specimenSex

Age

(year)Diagnosis/source

CCM 8421T (= NRL/St 12/153T) May 1998 Texas, USA ear swab M 5 acute otitis externa

NRL/St 12/155 October 1998 Texas, USA ear swab M 1 acute otitis externa

NRL/St 12/154 October 1998 Texas, USA ear swab F 7 acute otitis externa

CCM 8422 (= NRL/St 12/151) October 2001 Texas, USA ear swab F 32 healthy ear

NRL/St 13/014 January 2013 Litomyš, CZ urine M 2 urinary infection

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antibiotics: CXT CIP CLI ERY COT FUS GEN RIF TGC MUP TET CMP LNZ PEN VAN TEI

disc symbol and content (g): FOX 30 CIP 5 DA 2 E 15 SXT25 FD10 CN 10 RD 5 TGC15 MUP200 TE 30 C 30 LZD10 P1 VA5 TEC30

zone breakpoints S ≥ (mm): 25 20 22 21 17 24 22 26 18 30 22 18 19 26 12 14

S. petrasii subsp. petrasii

NRL/St 06/029 S S S S S S S S S S S S S R S S


NRL/St 07/427 S S S S S S S S S S R S S R S S



NRL/St 08/525 S S S S S S S S S S S S S S S S

CCM 8420 (= NRL/St 08/738) S S S S S S S S S S S S S R S S

CCM 8418T (= NRL/St 10/1050

T) S S S S S S S S S S S S S R S S

CCM 8419 (= NRL/St 10/670) S S S S S S S S S S S S S R S S


NRL/St 12/012 S S S R S S S S S S S S S R S S


NRL/St 12/584 S S S R S S S S S S S S S R S S

NRL/St 13/057 S S R ind. R S S S S S S S S S R S S

S. petrasii subsp. croceilyticus

CCM 8421T (=NRL/St 12/153

T) S S S S S S S S S S S S S S S S


NRL/St 12/154 S S R ind. R S S S S S S S S S S S S

CCM 8422 (= NRL/St 12/151) S S R ind. R S S S S S S R S S R S S


*) inducible resistance to clindamycin = R ind.

Table 4: Diferentiation of coagulase-negative species susceptible to novobiocin; uease, sucrose, trehalose positive, and mannitol negative

Table 1: Staphylococcus petrasii subsp. petrasii strains specification

Table 2: Staphylococcus petrasii subsp. croceilyticus strains specification

Table 3: Biochemical profile of S. petrasii using commercial test kits:a) STAPHYtest24 and b) API staph

Table 5: Diferentiation of coagulase-negative species susceptible to novobiocin; urease, sucrose, trehalose and mannitol positive

Species/subspecies NAG PYR GLR

S. simulans +

S. warneri - -

S. pasteuri - -

S. petrasii subsp. petrasii - + -

S. petrasii subsp. croceilyticus - +w +

NAG - N-acetyl glucosamine; ARG - arginine dihydrolase; PYR - pyrrolidonyl arylamidase; GLR - beta-glucuronidase

Species/subspecies ARG PYR GLR

S. lugdunensis -

S. hominis subsp. hominis-

+ -

S. warneri + -

S. petrasii subsp. petrasii + + -

S. petrasii subsp. croceilyticus + +w +

b) API Staph

TCCM 8418 9 isolates*TCCM 8421 4 isolates*

Glucose + 100 + 100

Fructose + 100 + 100

Mannose + 100 - 0

Maltose + 100 + 100

Lactose - 20 - 0

Trehalose + 100 + 100

Mannitol +w 70 - 0

Xylitol - 0 - 0

Melibiose - 0 - 0

Nitrate + 100 + 80

Phosphatase alkaline - 0 - 0

Voges-Proskauer test + 100 + 100

Raffinose - 0 - 0

Xylose - 0 - 0

Sucrose + 100 + 100

-methyl-D-glucoside - 0 - 0

N-acetyl β-D-glucosamine - 0 - 0

Arginine + 100 + 100

Urea - 20 - 0



Material and methods

Bacterial strains: A set of 19 strains of Staphylococcus petrasii (S. petrasii subsp. petrasii and S. petrasii subsp. croceilyticus) were isolated from human clinical material in the Czech Republic and USA between 2006 and 2013. Designation, origin and specification of the strain are given in the Tab.1 and Tab. 2.

Phenotyping: Conventional biochemic tests and the commercial test kits API Staph (Biomerieux), STAPHYtest24 (Erba Lachema) were used for identification and phenotypic characterisation of staphylococcal strains.

Antibiotic susceptibility testing: Antibiotic susceptibility was tested for 16 antibiotics using a disc diffusion method. Oxoid antimicrobial testing discs were used. Inhibition zones were evaluated according to the EUCAST breakpoints (v.3.1 2013-02-11).

Strain identification and typing: Macrorestriction profiling by Pulse Field Gel Electophoresis (PFGE) was performed using SmaI-digested chromosomal DNA. The dendrogram was generated using the band-based Dice similarity coefficient and UPGMA clustering method (BioNumerics software v. 6.6, Applied Maths).

MALDI-TOF was performed using Microflex L mass spectrometerT (Bruker Daltonics) and evaluated with Maldi biotyper software (DBUpdate 4111).

Strain identification was confirmed by rep-PCR and automated ribotyping performed according to [1].

100

90

80

70

60

50

40

NRL/St 12/155

CCM 8422TCCM 8421

NRL/St 12/154

NRL/St 13/014

NRL/St 12/012

NRL/St 13/057

NRL/St 08/280

NRL/St 11/077

NRL/St 06/029TCCM 8418

NRL/St 12/015

CCM 8419

NRL/St 12/584

NRL/St 07/045

NRL/St 08/435

CCM 8240

NRL/St 08/525

NRL/St 07/427



marker

(kbp)

Similarity (%)

67

3

361

32

4

262

208

175

135

11

7

76

52 44 36

a) STAPHYtest 24

TCCM 8418 13 isolates* TCCM 8421 4 isolates*

Voges-Proskauer test + 100 + 100

Urease + 100 + 100

Arginine + 100 + 100

Ornithine - 0 - 0

-Galactosidase - 0 - 0

-Glucuronidase - 0 + 100

-Glucosidase + 100 - 40/50

Phosphatase - 0 - 0

Esculin - 0 - 0

N-acetyl β-D-glucosamine - 0 - 0

Galactose - 30 - 0

Sucrose + 100 + 100

Trehalose + 100 + 100

Mannit - 10 - 0

Maltose + 100 + 100

Xylose - 0 - 0

Mannose + 100 - 0

Lactose - 38 - 0

Sorbitol - 0 - 0

Ribose - 0 - 0

Fructose + 100 + 100

Cellobiose - 0 - 0

L-Arabinose - 0 - 0

Raffinose - 0 - 0

Xylitol - 0 - 0

conventional tests

clumping factor

(3rd generation latex kit)

+ 63 - 0

Deoxyribonuclease +w 100 w - 0

Nitrate reduction + 100 + 75

w - week, * - % of positive reaction



w - week, * - % of positive reaction

clinical relevance and diagnostics of staphylococcus ... · acknowledgements this work was...

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