|Year : 2011 | Volume
| Issue : 3 | Page : 302-304
Metallo beta lactamase producing Pseudomonas aeruginosa and Acinetobacter baumannii
S John1, R Balagurunathan2
1 Department of Biochemistry, Indian Institute of science, Bangalore - 560 012, India
2 Department of Microbiology, Periyar University, Salem - 636 011, Tamil Nadu, India
|Date of Submission||28-Mar-2011|
|Date of Acceptance||27-Jun-2011|
|Date of Web Publication||17-Aug-2011|
Department of Biochemistry, Indian Institute of science, Bangalore - 560 012
This study aims in identifying MBLs particularly Zn requiring Molecular Class B enzymes produced by Pseudomonas aeruginosa and Acinetobacter baumannii .The resistance by these organisms are in a rise against all antibiotics including carbapenems and no prescribed CLSI guidelines is available for detecting them. Clinical isolates antibiotic susceptibility was determined by number of phenotypic tests by addition of 50mM of 10 μl zinc as cofactor for metallo beta lactamase production along with 0.5M ETDA of 5μl (930 μg per disk) plain disks. Increase in zone size of the meropenem -EDTA disk compared to the meropenem disk without EDTA was recorded positive. For Zn requiring MBLs zone towards both disks of EDTA and Zn along with meropenem is detected by DDST.
Keywords: Metallo-b-lactamases, MIC of meropenem, zinc substitution
|How to cite this article:|
John S, Balagurunathan R. Metallo beta lactamase producing Pseudomonas aeruginosa and Acinetobacter baumannii. Indian J Med Microbiol 2011;29:302-4
|How to cite this URL:|
John S, Balagurunathan R. Metallo beta lactamase producing Pseudomonas aeruginosa and Acinetobacter baumannii. Indian J Med Microbiol [serial online] 2011 [cited 2015 Mar 30];29:302-4. Available from: http://www.ijmm.org/text.asp?2011/29/3/302/83918
| ~ Introduction|| |
The leading cause of resistance to β-lactam antibiotics like penicillin, cephalosporin, cephamycin and /or monobactam among gram negative bacteria is due to extended-spectrum β- lactamase produced by these organisms they inactivate the antibiotics by hydrolysing the β-lactam ring. Most ESBL producing organisms are in the family Enterobacteriaceae commonly in E.coli and Klebsiella pneumonia, but Acinetobacter and Pseudomonas are also reported. The high resistance for antibiotics and the rapid dissemination of metallo beta lactamase producing isolates in hospitals require an effective phenotypic method for identifying them as there are no prescribed CLSI guidelines available for detecting them. Molecular class B enzymes have zinc in their active site  so the catalytic activity of this enzyme can be enhanced for detection by incorporating it for the tests, unlike serine carbapenem resistance they are not inhibited by clavulanic acid, sulbactam and tazobactam.  This study aims to provide an early, rapid and effective phenotypic method for identifying MBLs in hospitals especially in processing ICU samples for carbapenems drug resistant Pseudomonas aeruginosa and Acinetobacter baumannii which is being increasingly reported.
| ~ Materials and Methods|| |
Isolates of MDR gram negative organisms over a period of one year that showed decreased susceptibility to oxyimino-cephalosporins were tested phenotypically as per CLSI guidelines. Non repetitive ceftazidime resistant Enterobacteriaceae (E.coli, Klebsiella spp, Enterobacter spp), Acinetobacter spp, and Pseudomonas spp that were frequently isolated from samples of urine, blood, sputum, pus, endotracheal aspirates, bronchial secretion, wound and vaginal swabs were selected for the study. Antimicrobial susceptibility testing was initially determined with cephalosporins, aminoglycosides, penicillins and carbapenems (Hi-Media). Zone diameter was measured and interpreted as per CLSI guidelines for quality control of disc diffusion tests ATCC E.coli 25922 and an in-house carbapenem resistant Acinetobacter baumannii were used and they showed the expected zone pattern. The test isolates opacity was adjusted to 0.5 McFarland's standard and a lawn was spread on Mueller Hinton agar plates for all the tests.
| ~ Test for ESBLs Production|| |
Disk approximation method
Isolates found resistant or with decreased susceptibility to any one of the third generation cephalosporin antibiotics were selected as ESBLs producers. All ESBL producers showed enhanced zone diameter of 5 mm or greater for ceftazidime -clavulanic acid with respect to ceftazidime alone. The carbapenem MDR resistant Acinetobacter spp and Pseudomonas spp were further screened for MBL production.
| ~ Test for Metallo β-lactamase Detection|| |
Disk potentiation test 
Two 10μg meropenem disks were placed on inoculated plates wide apart, to one of the disk10 μl of 50mM zinc sulphate was added after drying, 0.5M ETDA of 5μl (930 μg per disk) was then dispensed.EDTA (Sigma Chemicals) of pH 8 was prepared with distilled water and sterilized by autoclaving. After overnight incubation, an increase in zone size of ≥ 7mm around the meropenem -EDTA disk compared to the meropenem disk alone was recorded positive.
Double disk synergy test
This test was performed with an overnight broth culture of the test strain inoculated on the MHA plate and allowed to dry. 5μl of the EDTA solution was added to a 6-mm blank filter paper disk (Whatman filter paper) which contained approximately 930 μg of EDTA. A 10 mg meropenem disk was placed in the centre of the plate flanked by EDTA disk and 10 μl of 50mM zinc sulphate disk [Figure 2] at a distance of 15 mm.After overnight incubation, the presence of an enlarged zone of inhibition towards the EDTA disk was interpreted positive for an MBL producer and an extended zone towards Zn for the putative ambler class B MBLs.
Modified hodge test:
Modified Hodge test was done although not recommended for MBL detection (MHT has been originally described by the Centre for Disease Control for Carbapenemases detection in Enterobacteriaceae) with carbapenem sensitive ATCC E.coli 25922 spread on the MHA plate. A 10 μg meropenem disk was placed in the centre of the plate and 10 μl of 50mM zinc sulfate solution was added to the disk along with EDTA. Carbapenem resistant NLF isolates were streaked with good inoculum of 0.5cm around the meropenem starting from the disk to outwards in four different directions. The plates were incubated at 37°C overnight; zone around the meropenem disk with clover leaf indent is taken as positive test.
| ~ MIC of Meropenem by Broth Microdilution Method|| |
Strains found resistant to meropenem by the disk diffusion test were tested by CLSI broth microdilution method using Mueller-Hinton broth. Doubling dilutions for meropenem ranging from 0.25 μg/mL though 64 μg/mL were tested. Meropenem powder for injection (Astra Zeneca, U.K. Ltd.) was used for the test. ATCC Pseudomonas aeruginosa 27853 was used as the control strain. MIC of 16 μg /mL or above was interpreted as resistant to meropenem.
| ~ Results|| |
The ESBL isolates includes E.coli 63.5%, Enterobacter spp 66.3% and Klebsiella spp 56.1% these resistant isolates are treatable with available β -lactam inhibitors. MBL was positive in 36 of 242 (14.8%) of Acinetobacter baumannii 2 of 331 (27.7%) of Pseudomonas aeruginosa.As a result of widespread application of antimicrobial agents the frequency of drug resistance to Pseudomonas is raising dramatically  imipenem resistance was77.5%, but from literatures  the resistance pattern for imipenem was 26%. Acinetobacter spp imipenem resistance 25.6% had been reported  but in this study we find 99% resistance. Acinetobacter spp accounting for 86% of resistance  among all the multidrug resistant isolates as shown in [Figure 1] was sensitive only for tigecycline and colistin. MIC determined by the broth microdilution method for the MBL positive isolates showed low-level resistance or sensitivity to meropenem. Taking an MIC of 16 μg/mL or above as resistant to meropenem, five isolates obtained from twelve patients, was 64 μg/mL and found to have MICs to meropenem in the sensitive zone. The remaining isolates showed MICs of 0.25-0.5 μg/mL. Meropenem disks incorporated with EDTA and Zn in other two tests is not clear for interpretation although an increase in zone size of 3 to 4mm was observed in comparison with meropenem alone may be due to EDTA.Few strains showed synergy with EDTA and Zn, suggesting that putative DDST possibly identifies Ambler ClassB MBLs.
|Figure 1: Resistance percentage of Acinetobacter spp and Pseudomonas spp for individual antibiotics|
Click here to view
| ~ Discussion|| |
The horizontal spread of resistance factors into environmental gram negative bacteria has seen the emergence of multidrug resistant Acinetobacter, Pseudomonas, Serratia, Stenotrophomonas spp.A few tested Klebsiella spp showed improved zone in MHT with zinc sulfate although reports of no significant improvement was shown.  AmpC detection was done on few MBL negative Acinetobacter spp but the number is not substantial to furnish, along with MIC for colistin and efflux mechanism. Among all the tests used for MBL detection DDST at 15mm distance is more reliable to CDT and MHT, sensitivity was also clear when zinc sulfate was dispensed to the disks as cofactor for metallo beta lactamase production. Plain disks with 50mM zinc sulfate and 0.5M EDTA at 15mm from meropenem on either sides (instead of adding to the meropenem disk) showed synergy with both the disks(Zn and EDTA) as in [Figure 2] a putative identification test. Hence compared to all the phenotypic tests for MBL detection DDST is reliable and reproducible, with separate EDTA and Zn disks it is also enabling identification of ambler class B MBLs but freshly prepared EDTA gives good result. Heteroresistant isolates were observed in ESBL double disk synergy test using amoxyclav and ceftazidime as in [Figure 3] in Acinetobacter spp, but had clear zone for MBLs detection.
|Figure 2: Detection of carbapenem resistance by double disk synergy test. Meropenem disks are fl anked by plain disks of EDTA and Zinc sulphate. Molecular Class B MBLs producing positive isolates showing zone towards EDTA and Zn disks|
Click here to view
|Figure 3: ESBL double disk synergy test with amoxyclav and ceftazidime showing heteroresistance.|
Click here to view
Low-level resistance to carbapenems as determined by MIC has been reported in several studies  which were detected resistant by disk diffusion method. All the 21 meropenem-resistant isolates were also resistant to most of the other antimicrobials tested one of the major concerns emerging from this study is the fact that six meropenem-resistant isolates were resistant to cefoperazone-sulbactam also. If carbapenemases spread widely there will be a situation where many nosocomial Gram-negative infections become untreatable so a simple phenotypic test in hospitals for differentiating MBLs is very critical for treating ICU patients. Further studies are underway to confirm the positive molecular class B MBLs producing strains and standardize this putative test.
| ~ Acknowledgement|| |
We thank Dr.Indumathi VA, Gokula Metropolis Clinical Laboratories, Bangalore -560 054, for providing generous gifts of bacterial strains with plasmids.
| ~ References|| |
|1.||Tamilselvi A, Mugesh G, Nethaji M. "Antibiotic Resistance: Mono- and Dinuclear Zinc Complexes as Metallo-ß-Lactamase Mimics". Chemistry 2006;12:7797-806. |
|2.||Walsh TR, Toleman MA, Poirel L, Nordmann P. Metallo beta lactamases: The quiet before storm? Clin Microbiol Rev 2005;18:306-25. |
|3.||Jarlier V, Nicolas MH, Fournier G, Philippon A. Extended broad spectrum beta-lactamases conferring transferable resistance to newer beta-lactam agents in Enterobacteriaceae: Hospital prevalence and susceptibility patterns. Rev Infect Dis 1988;10:867-78. |
|4.||Yong D, Lee K, Yum JH, Shin HB, Rossolini GM, Chong Y. Imipenem - EDTA disk method for differentiation of metallo beta lactamases producing clinical isolates of Pseudomonas spp. and Acinetobacter spp. J Clin Microbiol 2002;40:3798- 801. |
|5.||Lee K, Chong Y, Shin HB, Kim YA, Yong D, Yum JH. Modified Hodge and EDTA disk synergy test to screen metallo beta lactamases producing strains of Pseudomonas spp and Acinetobacter spp. Clin Microbiol Infect 2001;7:88-91. |
|6.||National committee for Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard. 5th ed. NCCLS document M7-A5. Wayne, PA: NCCLS; 2000. |
|7.||Livermore DM. Multiple mechanisms of antimicrobial resistance in Pseudomonas aeruginosa: Our worst nightmare. Clin Infect Dis 2002;34:634-40. |
|8.||Lone R, Shah A, Kadri SM, Lone S, Shah F. Nosocomial Multi-Drug-Resistant Acinetobacter Infections - Clinical Findings, Risk Factors and Demographic Characteristics. Bangladesh J Med Microbiol 2009;3:34-8. |
|9.||Shete VB, Ghadage DP, Muley VA, Bhore AV. Acinetobacter septicemia in neonates admitted to intensive care units. J Lab Physicians 2009;1:73-6. |
|10.||Bergogne-Bérézin E, Towner KJ. Acinetobacter spp as nosocomial pathogens: Microbiological, clinical and epidemiological features. Clin Microbiol Rev 1996;9:148-65. |
|11.||Rizvi M, Fatima N, Rashid M, Shukla I, Malik A, Usman A, et al. Extended spectrum AmpC and metallo beta lactamase in Serratia and Citrobacter spp in a disc approximation assay. J Infect Dev Ctries 2009;3:285-94. |
|12.||Afzal-Shah M, Livermore DM. Worldwide emergence of Carbapenem-resistant Acinetobacter spp. J Antimicrob Chemother 1998;41:576-7. |
[Figure 1], [Figure 2], [Figure 3]