Detection of Metallo- -lactamase Producing Clinical Gram-Negative Non-Fermenting

Isolates ‡*Darakhshan Guhar, *Seema Irfan, *Afia Zafar, *Tanveer Ahsan, *Rumina Hasan

‡CESAT, Islamabad *Clinical Laboratory, Department of Pathology and Microbiology, Aga Khan University and Hospital, Stadium

Road, Karachi, Pakistan *darakhshan.guhar@yahoo.com

Abstract:-There is a remarkable increase in antibiotic resistance due to metallo- -lactamase-production among gram-negative non-fermenting organisms. Early detection of metallo- -lactamase (MBL) producing isolates is necessary to prevent their dissemination. Imipenem-EDTA disk method was used for the detection of MBL producing strains among multidrug resistant (MDR) Acinetobacter species and Pseudomonasaeruginosa. One hundred MDR Acinetobacter spp. and forty-two Pseudomonas aeruginosa were screened for MBL production, from January to June 2001. MBL was produced by 96.6 % of imipenem-resistant Acinetobacter isolates, whereas 100% imipenem-resistant Pseudomonas aeroginosa isolates were MBL producers. MBLs are responsible for carbapenem resistance in these MDR Acinetobacter spp. and Pseudomonasaeruginosa There is an urgent need to have a simple screening test to take infection control measures on time.

I. INTRODUCTION

MBLs, like all -lactamases, can be divided into those that are normally chromosomally mediated and those that are encoded by transferable genes [1, 2]. Reports of resistance vary, but a general consensus appears to prevail that quinolone and broad-spectrum-lactam resistance is increasing in members of the family Enterobacteriaceae and Acinetobacter spp. and that treatment regimes for the eradication of Pseudomonas aeruginosa infections are becoming increasingly limited. However, Acinetobacter spp. and Pseudomonas aeruginosa majorly constitute for nosocomial infections [3, 4]. Unfortunately, there are no standardized phenotypic methods available and the testingcriteria are likely to depend on whether the gene is carried by P. aeruginosa or a member of the Enterobacteriaceae, i.e., the evincible level of resistance. For example, most Enterobacteriaceae and some Acinetobacter spp. carrying MBL genes will appear sensitive, with imipenem MICs of between 1 and 2 μg/ml. Therefore, the implementation of a

screening plate to detect MBLs, as has been advocated for extended spectrum -lactamases, must take account of the genus of the bacterium, i.e., Pseudomonads intrinsically have higher carbapenem MICs than Enterobacteriaceae. It is plausible that for screening Enterobacteriaceae for the presence of MBLs, a plate could contain ceftazidime with and without EDTA, but this would only be effective if the bacterium did not also produce an extended-spectrum

-lactamase, which cannot be assumed [1].

II. METHODOLOGY This study was conducted in the Clinical

Microbiology Laboratory of The Aga Khan University Hospital, Karachi, Pakistan. One hundred MDR clinical isolates of Acinetobacter species and forty-two Psedomonas aeruginosa were screened for MBL production. These organisms were isolated from respiratory secretions, wound swab, urine and blood culture specimens of intensive care unit patients admitted from January to April 2001. A multidrug resistant isolate defined as an isolate with resistance against two or more drugs or drug classes of therapeutic relevance [5].

The antibiotic susceptibility was performed by Kirby Bauer disk diffusion method against the antibiotics recommended by CLSI (Clinical Laboratory Standards Institute) [5]. This was further confirmed by minimum inhibitory concentration on agar dilution method as recommended by CLSI. E. coli ATCC 25922 and P. aeruginosa ATCC 27853 were used as control strains. The organisms were considered as susceptible to imipenem if MIC was <4μg/mL and resistant if MIC was >16μg/mL [6].

These isolates were screened for MBL production by using IPM-EDTA-disk synergy test introduced by Yong D et al. A solution of 0.5 M of EDTA was prepared and 10-μg-imipenem disks were treated with this solution to obtain a concentration of 750μg. Test strains were adjusted to the McFarland 0.5 standard and were inoculated to Mueller Hinton agar. A 10-μg-imipenem disk and an imipenem plus 750 μg EDTA

Proceedings of 2014 11th International Bhurban Conference on Applied Sciences & Technology (IBCAST)Islamabad, Pakistan, 14th – 18th January, 2014 73

978-1-4799-2319-9/14/$31.00 © 2014 IEEE

were placed on agar. A negative control disk containing only 750 μg EDTA was also placed. After overnight incubation, the established zone diameter difference of > 7 mm between imipenem disk and imipenem plus EDTA was interpreted as EDTA synergy positive [7].

III. RESULTS Impenem-resistance by disc diffusion method was

found in 90% of Acinetobacter spp. and among these 96.6% was appeared as MBL producers. Moreover, 59.5% of Pseudomonas aeruginosa isolates showed resistance against imipenem. By screening with Imipenem-EDTA disk method, 100% of them showed MBL production (Table 1). The average zone diameter difference between imipenem disk and imipemen plus EDTA disk were 16 mm and 14 mm for MBL positive Acinetobacter spp. and Pseudomonas aeruginosa, respectively (Fig. 2 and 3)

Table 1: Representation of resistance and production of MBL production

Figure 1: MDR strains with their average zone diameter difference

Figure 2: Clinical isolates of Acinetobacter species showing MBL presence or absence by imipenem EDTA disk method

IV. DISCUSSION We used the Imipenem-EDTA disk diffusion method as a screening test for metallo- -lactamase production. This study showed a very high percentage of imipenem resistant Acinetobacter spp. (97%) and Pseudomonas aeruginosa (100%) isolates showed MBL production. Our results are reliable as has the characteristic similarities with reports from other hospitals. A majority of our isolates also showed resistance to other important groups of antibiotics including third generation cephalosporin, aminoglycoside, and quinolone, which is the feature of majority of metallo- -lactamase producing isolates. Worldwide, it has been reported that the emergence of MBLs producing Acinetobacter spp. and Pseudomonas aeruginosa in our clinical strains are alarming and reflects excessive use of carbapenem [3]. Therefore, early screening method is crucial need for laboratory. However, the Imipenem-EDTA disk diffusion method as a screening test for metallo- -lactamase production can be used [8].

ACKNOWLEDGMENT We thanks the Department of Clinical

Microbiology of The Aga Khan University Hospital, Karachi for providing the opportunity to do this research based work.

REFERENCES [1] Timothy R and Mark A., “Metallo- -lactamses:the quiet

before the storm,” Clinical Microbiology Reviews, pp. 306–325, April 2005.

[2] Lim H.M., J.J.Pene, and R.W. Shaw, “Cloning nucleotide sequence, and expression of the Bacillus cerus.,” J. Bacteriol, vol. 170,pp2873-2878, 1988

[3] Gales, A.C.,L.C. Menezes, “Diemination in district Brazilian regions of an epdemic cerbapenem-resistant Pseudomonas aeruginosa producing SPM metallo- -lactamase,” J. Antimicrob.Chemother, vol. 52, pp. 699-702, 2003.

[4] Lee, K., “VIM-and IMP-type metallo- -lactamase-producing Acinetobacter spp.and Pseudomonas aeruginosa in Korean Hospial,” Emerg. Infect.Dis. vol.9, pp. 868-871, 2003.

[5] Nolte F.S., Metchock B. Mycobacterium. In: Murray PR, baron EJ, Pfaller MA, Tenover FC, Yolkia RH, editors. Manual of Clincal microbiology laboratory, sixth edition. Washington DC: American Society for Microbiology Press, pp. 400-37, 1999.

[6] Clinical laboratory standard institute. Performance standards for antimicrobial susceptibility testing: sixteenth informational supplement, vol. 26, 2006.

[7] Yong D., Lee K., Yum J.H., Shin HB, RossoliniGM, Chong Y., “Imipoenem EDTA disk method for differentiation of metallo- -lactamase producing clinical isolates of Acinetobacter spp.and Pseudomonas aeruginosa,”J Clin Microbiol, vol.40, pp.3798-801, 2002.

Isolates (n) Imipenem

resistant by disk

diffusion

MBL producer

Average zone

diameter difference

(mm)

Acinetobacter specie 100 90 83

(96.6%) 16

Pseudomonas aeruginosa 42 25 25

(100%) 14

Proceedings of 2014 11th International Bhurban Conference on Applied Sciences & Technology (IBCAST)Islamabad, Pakistan, 14th – 18th January, 2014 74