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ORIGINAL ARTICLE
Year : 2013  |  Volume : 2  |  Issue : 1  |  Page : 18-21

Prevalence of Pseudomonas aeruginosa and Acinetobacter spp producing metallo-β-lactamase in a tertiary care hospital


1 Department of Microbiology, Institute of Post-Graduate Medical Education and Research, Kolkata, India
2 Department of Microbiology, National Medical College, Kolkata, India

Date of Web Publication13-Mar-2013

Correspondence Address:
Kalidas Rit
70B T. C. Mukherjee Street, P.O. Rishra, Dist. Hooghly, West Bengal - 712 248
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2277-8632.108507

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  Abstract 

Background: Hospital acquired infection (HAI) due to multidrug resistant pathogens are increasing day by day. Among which MBL producing Pseudomonas aeruginosa and Acinetobacter spp contribute a major portion of HAI.
Aims: The objective is to determine the antibiotic susceptibility pattern of P. aeruginosa and Acinetobacter spp. showing imipenem resistance and prevalence of MBL producing strains among them in a tertiary care hospital.
Materials and Methods: Over a period of 5 months in a tertiary care hospital, 60 Pseudomonas aeruginosa and 50 Acinetobacter spp. were isolated. These were tested phenotypically for detection of metallo-β-lactamase (MBL) production, by imipenem-ethylenediaminetetraacetic acid (EDTA) combined disc method and by four fold or more reduction of minimum inhibitory concentration (MIC) against imipenem with EDTA.
Result and Conclusion: The prevalence of MBL producing Pseudomonas aeruginosa was found to be 41% and Acinetobacter spp. 22%. Antibiotic susceptibility tests revealed multidrug resistance for most antibiotics but 100% sensitivity for colistin. A big proportion of these positive strains were isolated from patients with multiple risk factors like prolonged hospital stay, indwelling urinary catheter, continuous mechanical ventilation, prolonged antimicrobial therapy, intravenous lines. The overall mortality rate among them was as high as 21%.

Keywords:  Acinetobacter spp, Imipenem - ethylenediaminetetraacetic acid, combined disc method, Metallo-β-lactamase, minimum inhibitory concentration, Psuedomonas aeruginosa


How to cite this article:
Rit K, Chakraborty B, Dey R, Chakrabarty P, Naha A, Saha R. Prevalence of Pseudomonas aeruginosa and Acinetobacter spp producing metallo-β-lactamase in a tertiary care hospital. J NTR Univ Health Sci 2013;2:18-21

How to cite this URL:
Rit K, Chakraborty B, Dey R, Chakrabarty P, Naha A, Saha R. Prevalence of Pseudomonas aeruginosa and Acinetobacter spp producing metallo-β-lactamase in a tertiary care hospital. J NTR Univ Health Sci [serial online] 2013 [cited 2020 Apr 4];2:18-21. Available from: http://www.jdrntruhs.org/text.asp?2013/2/1/18/108507


  Introduction Top


The most common cause of bacterial resistance to beta-lactam antibiotics is the production of beta-lactamases. Among these, metallo-β-lactamases (MBLs) are zinc-dependent enzymes that have been gaining increasing attention because of their ability to hydrolyze nearly all known beta-lactams and of the lack of useful inhibitors.

MBL producing non-fermenters, Pseudomonas aeruginosa and Acinetobacter spp. are important cause of nosocomial infections. The appearance of MBL and their spread among bacterial pathogens is a matter of concern with regard to the future of antimicrobial therapy. The present study was undertaken to determine the incidence of MBL producing P. aeruginosa and Acinetobacter spp. in a tertiary care hospital and to determine their antibiotic susceptibility pattern.

MBLs are enzymes belonging to Amblers Class B that can hydrolyze a wide variety of beta-lactam antibiotics including penicillins, cephalosporins, and carbapenems, and requires divalent cation, usually zinc, as a cofactor for enzyme activity. [1] The first plasmid-mediated MBL was reported in Japan in 1991. They are usually produced by Enterobacteriaceae family and non-fermenters particularly Acinetobacter spp. and Pseudomonas spp. [2],[3],[4] The rapid dissemination of these highly resistant metallo-β-lactamase [3] producing isolates need an effective phenotypic method for their proper identification. Hence, phenotypic detection of P. aeruginosa and Acinetobacter spp. producing MBL was done to study the prevalence of these MBL-producing strains in our tertiary care centre.


  Materials and Methods Top


The study was conducted over a period of five months from February 1, 2011 to June 2011. From different clinical samples randomly selected like blood, urine, pus, and bronchial aspirates, a total non-duplicative, 60 isolates of P. aeruginosa and 50 isolates of Acinetobacter spp. were included in this study. The isolates were identified by conventional methods. Antimicrobial susceptibility of all isolates was performed by disc diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. [5],[6] Antibiotics tested for non-fermenters by the disc diffusion method were piperacillin(100 ug), piperacillin/tazobactum (100 ug/10ug), ceftazidime (30 ug), cefepime (30 ug), cefoperazone (30 ug), ceftriaxone (30 ug), ampicillin and clavulanic acid (20 ug/10 ug), ofloxacin (30 ug), amikacin (30 ug), levofloxacin (5 ug), ciprofloxacin (5 ug), colistin (10 ug), chloramphenicol (30 ug), polymixin-B (50 Units), [HiMedia Laboratories Mumbai, India], imipenem (10 ug) [BD diagnostics Pvt. Ltd, Gurgaon]. Minimum inhibitory concentration (MIC) of each bacterial isolate against imipenem was determined by agar dilution method according to CLSI guidelines. MBL production was detected in imipenem resistant isolates by imipenem-EDTA combined disc method [Figure 1] and reduction of MIC value by four fold or more after addition of EDTA powder (750 μg) to imipenem.[4],[5],[6],[7],[8] The MIC range of imipenem tested was 0.125 ug/ml - 1024 ug/ml. The pure form of the drug was obtained from Ranbaxy Laboratories, Mumbai. Pseudomonas aeruginosa ATCC 27853 was used as control.
Figure 1: Comparison of inhibition zone diameters produced by disks with imipenem and imipenem plus 750 μg of EDTA by MBL producers

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Case files of these 110 patients were also reviewed for assessment of five common risk factors and followed until discharge or demise for detection of overall mortality rate.


  Results Top


Thirty Pseudomonas aeruginosa out of 60 and 15 Acinetobacter spp. out of 50 were resistant to imipenem. Among the imipenem resistant isolates 25 P. aeruginosa and 11 Acinetobacter spp. were found to be MBL producers [Figure 2] by both MIC assay and imipenem-EDTA combined disc method. All the MBL producing isolates of P. aeruginosa and Acinetobacter spp. were multidrug resistant with sensitivity pattern different from that of the MBL non-producers, [Table 1] but for only colistin, all isolates were found to be 100% susceptible. Out of five risk factors assessed three were found to be significantly (P < 0.05) associated with MBL production like hospital stay for more than 8 days, indwelling urinary catheter, continuous mechanical ventilation for more than 48 hours, but intravenous line or prolonged antimicrobial therapy within 30 days of positive culture were not significantly associated. The overall mortality in MBL positive patients was found to be 21% and incidence of MBL was more in male patients (63%).
Figure 2: Comparison of the number of MBL producing P aeruginosa and Acinetobacter spp. (n = 25 + 11) among the isolates showing imipenem resistance (n = 30 + 15), out of total number of P aeruginosa and Acinetobacter spp (n = 60 + 50) isolated

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Table 1: Antibiotic sensitivity (%) of P. aeruginosa MBL positive (N = 25), MBL negative (N = 5) and Acinetobacter spp. MBL positive (N = 11), MBL negative (N = 4) isolates showing imipenem resistance

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  Discussion and Conclusion Top


This study shows that the prevalence rate of MBL producing Pseudomonas aeruginosa is 41% and Acinetobacter spp. is 22% when isolated from clinical samples in our hospital, which is well-corroborated with similar studies conducted in Indian subcontinent. [9],[10] These MBL producing isolates were multidrug resistant except for colistin (100% sensitivity) and for polymyxin-B (>90% sensitivity), hence, cautious use of these antibiotics is recommended. [11] Also, some important risk factors were studied for association with MBL producing bacterial infections. [9] It was found that prolonged hospital stay for more than 8 days is a significant risk factor for these infections (P < 0.0001). Other parameters studied and found significant were patients with indwelling urinary catheter at time of positive culture (P-0.0007), and on continuous mechanical ventilation for more than 48 hours (P-0.0115). But intravenous line for more than 5 days or any antimicrobial therapy either received or receiving within 30 days of positive culture were not found to be significantly associated with development of MBL infection. Hence, modification of some of these risk factors if possible may have some value in controlling spread of MBL producing organism.

As MBL will hydrolyze virtually all classes of beta-lactam rings, their continued spread will be a clinical catastrophe. So, it is very essential to identify MBL producers and to determine their prevalence rate to assess their clinical burden for a specified area. Though MIC detection is the gold standard, this study shows that imipenem - ethylenediaminetetraacetic acid (EDTA) combined disc method is comparable with the MIC detection (both methods showed same results) and at the same time this combined disc method is simple, reliable, less cumbersome, and cheap.

In general, MBL-producing bacteria have a common multidrug-resistant phenotype that not only includes carbapenems, but also fourth generation cephalosporins, aminoglycosides, and fluoroquinolones. So appropriate therapeutic protocols with judicious use of carbapenems and a regular screening and monitoring system should be established and implemented to prevent wider spread of this worrisome resistance determinant.


  Acknowledgement Top


We acknowledge Dr. Prasanta K Maiti, Professor and Head of the Dept, Department of Microbiology, Institute of Post-Graduate Medical Education and Research. 244 AJC Bose Road. Kolkata - 700 020 for encouraging and supporting in this research work. E-mail: prasantamaiti123@rediffmail.com

 
  References Top

1.Bush K, Jacoby GA, Medeiros AA. A functional classification scheme for beta-lactamases and its correlation with molecular structure. Antimicrob Agents Chemother 1995;39:1211-33.  Back to cited text no. 1
[PUBMED]    
2.Manoharan A, Chatterjee S, Mathai D, SARI Study Group. Detection and characterization of metallo beta lactamases producing Pseudomonas aeruginosa. Indian J Med Microbiol 2010;28:241-4.  Back to cited text no. 2
    
3.Walsh TR, Toleman MA, Poirel L, Nordmann P. Metallo-beta-lactamases: The quiet before the storm? Clin Microbiol Rev 2005;18:306-25.  Back to cited text no. 3
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4.Yong D, Lee K, Yum JH, Shin HB, Rossolini GM, Chong Y. Imipenem-EDTA disk method for differentiation of metallo-beta-lactamase-producing clinical isolates of Pseudomonas spp. and Acinetobacter spp. J Clin Microbiol 2002;40:3798- 801.  Back to cited text no. 4
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5.Wikler MA, Cockerill FR, Craig WA, Low DE, Sheehan DJ, Tenover FC, et al. Clinical Laboratory Standards Institute. Performance Standards for Antimicrobial Disk Susceptibility Tests. 9 th ed. vol. 26., 2006. p. M2-A9.  Back to cited text no. 5
    
6.Bauer AW, Kirby WM, Sherris JC, Turek M. Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Path 1966;45:493-6.  Back to cited text no. 6
    
7.Hemalatha V, Sekar U, Kamat V. Detection of metallo betalactamase producing Pseudomonas aeruginosa in hospitalized patients. Indian J Med Res 2005;122:148-52.  Back to cited text no. 7
    
8.Lee K, Chong Y, Shin HB, Kim YA, Yong D, Yum JH. Modified Hodge and EDTA disk synergy tests to screen metallo-beta-lactamase producing strains of Pseudomonas and Acinetobacter species. Clin Microbiol Infect 2001;7:88-91.  Back to cited text no. 8
    
9.Shanthi M, Sekar U. Multi-drug resistant Pseudomonas aeruginosa and Acinetobacter baumanii infections among hospitalized patients: Risk factors and outcomes. J Assoc Physicians India 2009;57:636, 638-40, 645.  Back to cited text no. 9
    
10.Jayakumar S, Appalaraju B. Prevalence of multi and pan drug resistant Pseudomonas aeruginosa with respect to ESBL and MBL in a tertiary care hospital. Indian J Pathol Microbiol 2007;50:922-5.  Back to cited text no. 10
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11.Deshmukh DG, Damle AS, Bajaj JK, Bhakre JB, Patwardhan NS. Mettalo-beta-lactamase-producing clinical isolates from patients of a tertiary care hospital. J Lab Physicians 2011;3:93-7.  Back to cited text no. 11
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