Background: The emergence and rapid spread of metallo-beta-lactamase (MBL) producing Acinetobacter spp. are of great concern worldwide due to limited treatment options. Epidemiologic studies of the causing genes are important for prevention.
Methods:  In  this  study,  70  imipenem-resistant  Acinetobacter  strains  were isolated from health care associated infections. These isolates were screened for detection of metallo-beta-lactamase (MBL) using inhibitor potentiated disk diffusion tests with thylenediaminetetraacetic acid (EDTA). PCR was designed for detection of bla vim and bla imp-1 using specific primers.
Results: Among these 70 strains, 50 strains appeared to produce metallo-beta-lactamase. Three isolates were detected by PCR to carry metallo-beta-lactamase gene bla vim, but bla imp-1 gene was not detected.
Conclusion: These findings suggest that in our area other genetic elements are responsible for resistance against metallo-beta-lactams

Faeze Sabet

Jalil Vandyosefi

Kiarash Ghazvini

Neda Noori

Sara Ashrafi

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J Med Bacteriol. Vol. 2, No. 3, 4 (2013): pp. 11-16 jmb.tums.ac.irISMB TUMSFrequency of IMP-1 and VIM Genes among Metallo-beta-Lactamase Producing Acinetobacter spp. Isolated from Health CareAssociated Infections in Northeast of IranNeda Noori 1, Jalil Vandyosefi 1, Faeze Sabet 2,Sara Ashrafi 2, Kiarash Ghazvini 2*1 Department of Microbiology, College of Basic Sciences, Karaj Branch, Islamic Azad University, Alborz, IR Iran2 Antimicrobial Resistance Research Center, Buali Research Institute, Department of Microbiology and Virology, MedicalSchool, Mashhad University of Medical Sciences, Mashhad, IR IranARTICLE  INFO ABSTRACTArticle type:Original ArticleBackground: The emergence and rapid spread of metallo-beta-lactamase (MBL)producing Acinetobacter spp. are of great concern worldwide due to limitedtreatment options. Epidemiologic studies of the causing genes are important forprevention.Methods: In this study, 70 imipenem-resistant Acinetobacter strains wereisolated from health care associated infections. These isolates were screened fordetection of metallo-beta-lactamase (MBL) using inhibitor potentiated diskdiffusion tests with ethylenediaminetetraacetic acid (EDTA). PCR was designedfor detection of bla vim and bla imp-1 using specific primers.Results: Among these 70 strains, 50 strains appeared to produce metallo-beta-lactamase. Three isolates were detected by PCR to carry metallo-beta-lactamasegene bla vim, but bla imp-1 gene was not detected.Conclusion: These findings suggest that in our area other genetic elements areresponsible for resistance against metallo-beta-lactams.Article history:Received: 12 Apr 2013Revised: 20 May 2013Accepted: 25 Jun 2013Keywords:AcinetobacterCarbapenemaseIran Please cite this paper as: Noori N, Vandyosefi J, Sabet F, Ashrafi S, Ghazvini K. Frequency of IMP-1 and VIM Genes among Metallo-beta-Lactamase Producing Acinetobacter spp. Isolated from HealthCare Associated Infections in Northeast of Iran. J Med Bacteriol. 2013; 2 (3, 4): pp. 11-16.*Corresponding Author: Kiarash Ghazvini, PhD., Antimicrobial Resistance Research Center, Buali Research Institute, Department ofMicrobiology and Virology, Medical School, Mashhad University of Medical Sciences, Mashhad, IR Iran, Tel: +98 915 1248938, E-mail:kiarash_ghazvini@yahoo.comNoori N et al. Frequency of IMP-1 and VIM Genes among …12 J Med Bacteriol. Vol. 2, No. 3, 4 (2013): pp. 11-16       jmb.tums.ac.irIntroductionAcinetobacter is a Gram-negative, opportunisticpathogen which causes various infectionsspecially nosocomial infections (1-3). Theemergence and rapid spread of multidrug-resistant (MDR) isolates of Acinetobacterspp. are of great concern worldwide (4, 5).In recent years, carbapenems has beenproposed as a last resort for treating seriousinfections attributable to multidrug-resistantisolates of Acinetobacter. The most commonmechanism of carbapenem resistance amongAcinetobacter spp. is the acquisition ofcarbapenemases, including certain class Bmetallo-beta-lactamases (MBLs) (6).Metallo-beta-lactamases can hydrolyze betalactams from all classes except themonobactams. The genes encoding metallo-beta-lactamases are often found on mobilegenetic units, such as transposons andplasmids so can rapidly spread (7, 8).The IMP and VIM-type metallo-beta-lactamase producing Acinetobacter spp.have been increasingly isolated from clinicalspecimens in many part of the world (9).Higher mortality has been reported inpatients infected with the IMP-1- producingstrains. Therefore reliable epidemiologicaldata about the prevalence and thecontributing genes is essential for theoptimal control of the spread of resistance(9).Although several studies have reported thefrequency of MBL-producing Acinetobacterspp. and the contributing genes but there isnot any report about the metallo-beta-lactamase producing Acinetobacter spp. innortheast of Iran (10, 11). The purpose ofthis study was to investigate the frequency ofIMP-1 and VIM genes among metallo-beta-lactamase producing Acinetobacter spp.isolated from health care associatedinfections in Northeast of Iran, Mashhad.Materials and MethodsClinical isolates of Acinetobacter spp. werecollected during a period of 6 months frompatients admitted to the Ghaem andEmamreza university hospital of Mashhad.Acinetobacter spp. was isolated fromdifferent clinical specimens, includingwound (41 specimens) and respiratorysecretions (29 specimens). The isolates wereidentified using standard laboratorymethods. Antimicrobial susceptibility ofisolates was determined using the standardKirby-Bauer disk diffusion methodaccording to Clinical and LaboratoryStandard Institute (CLSI) (12). Colistin,Ceftizoxime, Cefepime, Imipenem,Ceftriaxo, Ciprofloxacin, Sulfamethoxazol,Gentamycin, Piperacilin, Meropenem werethe antibiotics evaluated in this study.The imipenem / imipenem + EDTA diskmethod was performed for detection ofmetallo-beta-lactamase. The isolates with anincrease in inhibition zone diameterproduced by imipenem + EDTA comparedwith imipenem were considered positive formetallo-beta-lactamase production. Allisolates positive for metallo-beta-lactamaseproduction were screened by PCR for blaVIM and bla IMP-1 genes with primersspecific for these genes (Table 1).Frequency of IMP-1 and VIM Genes among … Noori N et al.J Med Bacteriol. Vol. 2, No. 3, 4 (2013): pp. 11-16       jmb.tums.ac.ir 13Table 1. PCR Primers for the detection of genesencoding metallo-beta-lactamases (bla VIM andblaIMP-1)AmpliconSizeNucleotide SequenceGenesIdentified500 bpTTTGGTCGCATATCGCAACGCCATTCAGCCAGATCGGCATbla VIM587 bpCTACCGCAGCAGAGTCTTTGAACCAGTTTTGCCTTACCATbla IMP-1DNA from all imipenem resistant isolateswere extracted by boiling and used astemplate in PCR assay to amplify blaVIM,blaIMP-1 genes. The amplification conditionwas: initial denaturation at 95°C for 5 min,followed by 35 cycle (1min at 94°C, 1min at62°C and 1 min at 72°C with a finalextension for 10 min at 72°C for bla IMP-1gene; and initial denaturation for 5 min at95°C, followed by 35 cycle (1min at 94°C,1min at 56°C and 1 min at 72°C and 10 minat 72°C for blaVIM gene. PCR products wereelectrophoresed and then purified byAccuPrep PCR Purification Kit (Bioneer).DNA Sequencing was performed based onSanger Dideoxynucleotides by Bioneersequencing company.  The sequence datawere studied using chromas and MEGAsoftwares.ResultsAcinetobacter spp. were screened formetallo-beta-lactamases production amonghospitalized patients. Imipenem resistantclinical isolates were isolated from 36 malepatients and 34 females during 6 monthsfrom Mashhad. Antimicrobial susceptibilityprofiles of these 70 isolates were shown intable 2.Table 2. Antimicrobial susceptibility profilesof the isolatesN (%) of ResistanceAntibiotics23 (32.8)Colistin70 (100)Ceftizoxime64 (91.4)Cefepime70 (100)Imipenem70 (100)Ceftriaxon70 (100)Ciprofloxacin63 (90)Sulfamethoxazol62 (88.5)Gentamycin68 (97.1)Piperacilin70 (100)MeropenemAmong 70 imipenem resistant isolates, 50appeared to produce metallo-beta-lactamasesinhibitor potentiated disk diffusion tests withethylenediaminetetraacetic acid (EDTA). Ofthese 50 metallo-beta-lactamases positivestrains, 3 were found to harbored blaVIMgene, but no blaIMP-1 gene was identifiedamong our strains by PCR. The sequencedata showed close similarity among thesethree blaVIM genes.DiscussionThe aim of this study was to assess theprevalence of blaIMP-1 and blavim metallo-beta-lactamases genes. Among these 70imipenem nonsusceptible strains, 50 strainsappeared to produce metallo-beta-lactamases.Of these 50 strains, 3 were found to carryblaVIM gene, but no blaIMP-1 gene wasidentified among our strains by PCR. Such ahigh prevalence of metallo-beta-lactamaseproducing Acinetobacter in hospitals indicatesthe urgent need for action to prevent furtherspread of these bacteria. Previous experienceswith Methicillin-Resistant Staphylococcusaureus, vancomycin-resistant Enterococcus andNoori N et al. Frequency of IMP-1 and VIM Genes among …14 J Med Bacteriol. Vol. 2, No. 3, 4 (2013): pp. 11-16       jmb.tums.ac.irextended-spectrum β-lactamase-producingbacteria indicate that once resistant bacteria canbecome wide-spread they cannot be controlled(13). So it is strongly recommended thathospitals must promote infection controlpractices.Our results showed that these two genes arenot the major source for resistance againstimipenem. And it can be assumed that innortheast of Iran other metallo-beta-lactamasgenes such as SPM-1, GIM-1, SIM-1 orNDM-1 may contribute in resistance againstmetallo-beta-lactams.Since the first report of blaIMP-1 and blaVIMmetallo-beta-lactamase in Japan and Italyrespectively, metallo-beta-lactamases producingGram-negative bacilli have been increasinglyreported in many countries (14). In a studyduring 2002 to 2004 in France, 46% ofimipenem nonsusceptible P. aeruginosaisolates were metallo-beta-lactamases positiveusing phenotypic methods while 97.2% ofthem were PCR positive for metallo-beta-lactamases genes (15). In another studywhich conducted in India during 2011 byAmudhan et al, among 179 imipenemnonsusceptible isolates, 144 isolates (%80.4)were metallo-beta-lactamases producing. Inthis study PCR detected the metallo-beta-lactamases genes blaVIM/blaIMP in 51.4% ofcases (16). And in Japan during 2012 among54 Acinetobacter isolates 48 isolates werepositive for MBL genes. The blaIMP-1 geneswere detected in four isolates (17). In lee’sstudy during 2003 in Korean, 11 isolates(28.9%) of 38 metallo-beta-lactamasespositive isolates of Acinetobacter spp. carryblaIMP (18). In another study in southernTaiwan, among 123 strains of gram negativebacilli, 116 strains (94.3%) carry the intI1gene and 21 strains contain integron-associated blaVIM-3, blaVIM-11 and blaIMP-8genes. This finding suggests that clinicalspread of this blaVIM-11 gene is a matter ofgreat concern for carbapenem resistance(19).The results of these studies differ from theresults of ours. Although in all abovementioned study blaVIM was much morecommon than blaIMP which is somehowsimilar to our study but in our study thefrequency of these genes were much lower.This aspect of our results is different fromabove mentioned results which confirmeddifferent source and level of resistance innortheast of Iran in contrast to other parts ofthe world.In a study in Iran during 2012 by DrShahcheraghi et al, 13 metallo-beta-lactamases resistant Acinetobacter strainswere isolated which carry blaVIM-1 butsimilarly in that study also no blaIMP geneswere detected among the strains (20).Another study in iran was performed bypeymani during 2010 in tabriz among 63carbapenem non-susceptible isolates. In hisstudy 31 (49%) isolates were found to bemetallo-beta-lactamases producers. Amongthese 31 metallo-beta-lactamases producingisolates, 19 (61%) isolates carried the blaIMPgene and 9 (29%) isolates carried the bla VIMgene (21).ConclussionOur finding are different in some aspectfrom the results of the other studies and thisdifference makes our study importantbecause it reveals that in the North East ofIran these genetic elements are not the mainFrequency of IMP-1 and VIM Genes among … Noori N et al.J Med Bacteriol. Vol. 2, No. 3, 4 (2013): pp. 11-16       jmb.tums.ac.ir 15mechanism for metallo-beta-lactamasesproduction. So, further studies are needed toinvestigate the genetic source of metallo-beta-lactamases production.AcknowledgementsThis work was supported by MashhadUniversity of Medical Sciences and alsoFradid institute of biology. We also thank DrMohamad Reza Akbari Eidgahi for hisvaluable assistance.Conflict of interestNone declared conflicts of interest.References1. Kuoa HY, Changc KC, Kuoe JW, etal. Imipenem: a potent inducer ofmultidrug resistance in Acinetobacterbaumannii. Int J AntimicrobAgents2011; 32: 854-9.2. Lee k, Yong D, Chong Y. Multidrug-Resistant Acinetobacter spp.Increasingly Problematic NosocomialPathogens. Yonsei Med 2011; 52:879-91.3. Liu SY, Lin JY, Chu C, et al.Integron-associated imipenemresistance in Acinetobacterbaumannii isolated from a regionalhospital in Taiwan. Int JAntimicrobAgents 2006; 27: 81-4.4. Mezzatesta ML, Migliavacca R,Giani T, et al. Epidemiologicalcharacterization and distribution ofcarbapenem resistant Acinetobacterbaumannii clinical isolates in Italy.Bacteriology 2011; 18: 160-6.5. Shahcheraghi F, Abbasalipour M,Feizabadi MM, et al. Isolation andgenetic characterization of metallo-β-lactamase and carbapenamaseproducing strains of Acinetobacterbaumannii from patients at Tehranhospitals. Iran J Microbiol 2011; 2(3): 68-74.6. Hujer M, Hujer A, Hulten E, et al.Analysis of Antibiotic ResistanceGenes in Multidrug-ResistantAcinetobacter spp. Isolates fromMilitary and Civilian Patients Treatedat the Walter Reed Army MedicalCenter. Antimicrob AgentsChemother 2006; 50: 4114-23.7. Lee YT, Huang L, Chiang D, et al.Differences in phenotypic andgenotypic characteristics amongimipenem non susceptibleAcinetobacter isolates belonging todifferent genomic species in Taiwan.Int J Antimicrob Agents 2009; 34:580-4.8. Brown NG, Horton LB, Huang W,et al. Analysis of the FunctionalContributions of Asn 233 inMetallo-beta-Lactamase IMP-1.Antimicrob Agents Chemother2011; 55: 5696-702.9. Lee K, Yong D, Yum JH, et al.Evaluation of E-test MBL forDetection of blaIMP-1 andblaVIM-2 Allele-Positive ClinicalIsolates of Pseudomonas spp. andAcinetobacter spp. J ClinMicrobiol 2005; 43: 2-944.Noori N et al. Frequency of IMP-1 and VIM Genes among …16 J Med Bacteriol. Vol. 2, No. 3, 4 (2013): pp. 11-16       jmb.tums.ac.ir10. 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Frequency of IMP-1 and VIM Genes among Metallo-beta-Lactamase Producing Acinetobacter spp. Isolated from Health Care Associated Infections inNortheast of Iran

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Frequency of IMP-1 and VIM Genes among Metallo-beta-Lactamase Producing Acinetobacter spp. Isolated from Health Care Associated Infections inNortheast of Iran

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