Prevalence of metallo-b lactamase (MBL) producingPseudomonas spp. and Acinetobacter spp.in a tertiary care hospital in India

Varsha Gupta*, Priya Datta, Jagdish Chander

Department of Microbiology, Government Medical College and Hospital, 1154 A, Sec-32 B,Chandigarh 160030, India

Accepted 12 August 2005Available online 6 October 2005

01do

KEYWORDSMetallo-b lactamases;Pseudomonas;Acinetobacter

63-4453/$30.00 Q 2005 The Britishi:10.1016/j.jinf.2005.08.013

* Corresponding author. Tel.: C91 1E-mail address: varshagupta_99@y

Summary Objective. Gram-negative bacteria with acquired metallo-b lactamasesproduction have been increasingly reported in some countries, necessitating theirdetection. We determined the occurrence of acquired metallo-b lactamases (MBL)producing bacteria in our teaching hospital.Method. The method employed was a rapid technique using a disk of imipenem plus750 mg of EDTA, on Mueller–Hinton plate which was supplemented with 70 mg/ml ofzinc sulfate, to differentiate MBL producing Pseudomonads and Acinetobacter.Result. Out of 200 isolates tested, 7.5% of our Pseudomonads and Acinetobacterwere shown to be MBL producers.Conclusions. Though MBL prevalence is still low in our hospital set up, screening forMBL among Pseudomonas and Acinetobacter isolates should be routinely done inlaboratory.Q 2005 The British Infection Society. Published by Elsevier Ltd. All rights reserved.

Introduction

In any nosocomial setting carbepenems are used asa last resort for treatment of multidrug resistantGram-negative bacilli infection. These are b-lactamantibiotics with an ultra broad-spectrum anti-bacterial activity and stability to almost allclinically relevant extended spectrum and Amp Cb-lactamases. However, since last 10 years,acquired resistance to this life saving anti-microbial

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has been increasingly reported in Pseudomonas andAcinetobacter spp.1 This resistance is due tospecific enzymes called metallo b-lactamases,belonging to Bush’s functional group 2f and group3 and corresponding to molecular class B (Amblerclassification).2,3 These rapidly hydrolyze mostb-lactams including carbapenem. These metallob-lactamases (MBL) are of IMP, VIM and SPM series.2

The metal ion active site appears to decrease theirsusceptibility to b-lactamase inhibitor and enablethem to hydrolyze carbapenem.1

Initially IMP-1 emerged and spread during early1990s in Japan.1 Later this enzyme and its variants

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were detected among different countries. NextVIM-1 was reported in Pseudomonas aeruginosa inItaly.4 Reports of VIM-2 from France, Greece andKorea isolated from P. aeruginosa came in early1995.5 From then on occurrence of various metallob-lactamases has been reported from U.S.A.,Europe, Canada and Asia.6,7

The genes for MBL are inserted in integrons andsome of these integrons are located on conjugativeplasmids. Because of their ability to spread,carbapenem resistance related to MBL productionhas become a serious concern.8 The rapid detectionof MBL positive isolates is necessary in aid ofinfection control and to prevent their dissemina-tion.9 For MBL detection PCR method is mostreliable. But its limitation being that it is expensivefor daily application in routine laboratory, indeveloping countries like India. Also PCR hasbecome difficult with the increased number oftypes of MBLs that are being reported.10

Chelating agents inhibit MBL activity. However,NCCLS documents do not yet contain a method fordetection of MBL producing isolates.11 In our study, aphenotypic method described by Yong et al. hasbeen used to determine prevalence of MBL inPseudomonas and Acinetobacter spp. isolated fromhospitalized patients. In this rapid detection of MBLis made possible with the use of a disk of imipenemplus 750 mg of EDTA.10 We further modified thismethod by adding zinc sulfate to the Mueller–Hintonplate on which the test organism was streaked asdescribed by Lee et al.12 The advantage of addingzinc sulfate was greater sensitivity because thosestrains, which gave initially equivocal or falsenegative MBL results, became positive when zincsulfate was added to the medium.12

Figure 1 Comparison of inhibition zone diameters forMBL producer (TEST) and MBL non-products (CONTROL)P.aeruginosa by Imipenens and Imipenem plus EDTA.

Material and method

This study was conducted in Government MedicalCollege and Hospital, Sector 32, Chandigarh, a 650bedded tertiary care teaching hospital. BetweenJanuary 2004 and September 2004, 200 non-dupli-cate isolates belonging to Pseudomonas and Acine-tobacter spp. were isolated and speciated based ona battery of biochemical tests. These were takenfrom in-door patients. The various clinical speci-mens included—pus, body fluids, tracheal aspirate,bronchoalveolar lavage and sputum. Antibioticsusceptibility was done by Kirby–Bauer disc diffusionmethod. The panel of antibiotics included: Amikacin(30 mg), gentamicin (10 mg), amoxycillin (10 mg),sparfloxacin (5 mg), cefepime (30 mg), ceftazidime(30 mg), amoxycillin/clavulanate (30/10 mg),

cefoperazone/sulbactam (75/35 mg), tobramycin(10 mg) and piperacillin (100 mg). The source ofthe media, antibiotics and chemical is Hi Media,Mumbai Ltd.

Detection of MBL production

A total of 75 strains of Pseudomonas spp. andAcinetobacter spp. which showed reduced suscep-tibility to ceftazidime (inhibition zone diameter!18 mm) and/or to imipenem (inhibition zonediameter !16 mm) were further screened for MBLproduction by a phenotypic method as described byYong et al.10 In this test, organisms were inoculatedon plates of Mueller–Hinton agar as recommendedby NCCLS.10 50 mM zinc sulfate was added toMueller–Hinton agar to achieve a final concen-tration of 70 mg/ml as mentioned by Lee et al.12 A0.5 M EDTA solution was prepared by dissolving186.1 g of disodium EDTA$2H2O in 1000 ml ofdistilled water, adjusting its pH to 8.0 by usingNaOH. The mixture was sterilized by autoclaving.Two 10 mg-imipenem disks were placed on the testorganism, and 750 mg of EDTA from the preparedsolution is added to one imipenem disk kept onMueller–Hinton agar plate supplemented with zincsulfate. The inhibition zones of the imipenem andimipenem–EDTA disks were compared after 18–24 hof incubation at 37 8C, for detection of MBLproduction in Pseudomonas spp.10 For Acinetobac-ter spp. we measured the zone of inhibition ofimipenem plus EDTA disk12.

Interpretation

For Pseudomonas spp. if the increase in theinhibition zone with the imipenem and EDTA diskwas R7-mm than imipenem disc alone, it was takenas MBL positive (Fig. 1).10

Prevalence of metallo-b lactamase, India 313

For Acinetobacter spp.—the inhibition zone withthe imipenem and EDTA disk was R17-mm for MBLpositive and for MBL negative the inhibition zones ofimipenem and EDTA were %14 mm.10

All the MBL positive isolates were repeatedlychecked for reproducibility. These MBL positivestrains were further tested for susceptibility withaztreonam.

Result

Out of 75 strains showing reduced susceptibility toceftazidime and/or imipenem, 13 (7.5%) were MBLpositive. These included nine P. aeruginosa, twoAcinetobacter cbc and one each Pseudomonasfluorescence and Pseudomonas putida.

All 13 MBL positive strains were resistant toceftazidime, cefepime, piperacillin, augmentin,cefoperazone/sulbactam and imipenem. However,resistance to amikacin amongst the MBL producerswas 72 and 80% of the strains were resistant totobramycin. Also 75% of strains were resistant tosparfloxacin as well. All MBL positive strains were,however, sensitive to aztreonam.

Discussion

The emergence of Gram-negative bacterial specieswith acquired resistance to various broad-spectrumb-lactams is becoming a worldwide clinicalproblem.13 P. aeruginosa and Acinetobacterspecies are commonly isolated as nosocomialpathogen. MBL production by these multidrugresistant bacteria is an emerging threat and acause of concern for treating physicians.13

The resistance to carbapenem in P. aeruginosaoften results from reduced levels of drug accumu-lation or increased expression of pump efflux. Theresistance is due to chromosomally encoded orplasmid mediated enzymer.14 Most of these MBLconfer resistance not only to carbepenems but alsoto other b-lactams and are poorly inhibited by thepresence of b-lactamase inhibitors such as clavu-lanic acid, sulbactam and tazobactam.14

Of the 200 isolates only 13 strains (7.5%) wereMBL positive. Eight P. aeruginosa, all MBL positive,were isolated from endotracheal secretions fromventilated patients from ICU. One P. aeruginosa,two Acinetobacter cbc and one P. putida MBLpositive strains were isolated from pus (pleuralfluid, bronchial washing) of hospitalized patients.Blood culture from an ICU patient yielded MBLpositive P. fluorescence strain.

Lee et al. evaluated the efficiency of screeningfor MBL in relation to susceptibility of isolates. Heshowed that for MBL production screening could bereduced by 59.6% if imipenem non-susceptibilityrather than ceftazidime resistance isolates weretested.12 However, since in our setting resistance toimipenem is low, isolates resistant to ceftazidimewere also screened for MBL production.

The method using a disk with imipenem plus750 mg of EDTA is highly sensitive in differentiatingMBL producing isolates. The specificity is excellentfor Pseudomonads and good for Acinetobacters.10

According to Lee et al. all the strains which gaveinitially equivocal or false negative MBL resultsbecame positive when zinc sulfate was added to themedium.12

In a similar study by Navaneeth et al. fromBangalore, India 12% of P. aeruginosa were MBLproducers.15 Also consistent with their finding, allour MBL producing isolates were resistant tob-lactams (ceftazidime, cefepime, piperacillin),carbapenem (imipenem) and b-lactam/b-lacta-mase inhibitor (cefoperazone/sulbactam).15 AllMBL producers were sensitive to aztreonam.

In conclusion, though MBL prevalence is low inour hospital, screening for MBL among Pseudomo-nas and Acinetobacter isolates should be routinelydone in laboratory. Also antibiotics rotation andhospital infection control as a part of hospitalcurriculum will aid in limiting the spread ofresistant organisms.

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