prevalence of ndm per veb imp acinetobacter...

Research ArticlePrevalence of blaNDM blaPER blaVEB blaIMP andblaVIM Genes among Acinetobacter baumannii Isolatedfrom Two Hospitals of Tehran Iran

Fatemeh Fallah1 Maryam Noori2 Ali Hashemi2 Hossein Goudarzi2

Abdollah Karimi1 Soroor Erfanimanesh2 and Shadi Alimehr1

1 Pediatric Infections Research Center Mofid Children Hospital Shahid Beheshti University of Medical Sciences Tehran Iran2Department of Microbiology Shahid Beheshti University of Medical Sciences Tehran Iran

Correspondence should be addressed to Ali Hashemi hashemi1388yahoocom

Received 28 January 2014 Revised 1 June 2014 Accepted 8 June 2014 Published 15 July 2014

Academic Editor Enrico Tortoli

Copyright copy 2014 Fatemeh Fallah et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Background and Objectives The aim of this study was to determine the frequency of blaNDM blaPER blaVEB blaIMP and blaVIMtype genes among A baumannii isolates from hospitalized patients in two hospitals in Tehran Iran Patients and MethodsAntibiotic susceptibility tests were performed by Kirby-Bauer disc diffusion and Broth microdilution methods The frequencyof MBL (metallo-beta-lactamase) and ESBL (extended-spectrum-beta-lactamase) producers was evaluated by CDDT The 120573-lactamases genes were detected by PCR and sequencing methods Results The resistance of A baumannii isolates against testedantibiotics was as follows 103 (954) to ceftazidime 108 (100) to cefotaxime 105 (957) to cefepime 99 (917) to imipenem99 (917) to meropenem 87 (806) to amikacin 105 (972) to piperacillin 100 (926) to ciprofloxacin 103 (954) topiperacillintazobactam 44 (407) to gentamicin 106 (981) to ampicillinsulbactam 106 (981) to co-trimoxazole 87 (806)to tetracycline and 1 (18) to colistin Using combined disk diffusion test 91 (842) and 86 (8686) were ESBL and MBLproducers respectively The prevalence of blaPER-1 blaVEB-1 blaIMP-1 and blaVIM-1 genes was 71 (7803) 36 (395) 3 (348) and15 (1744) respectively Conclusions The prevalence of ESBLs and MBLs-producing A baumannii strains detected in this study isa major concern and highlights the need of infection control measures

1 Background

Multidrug-resistant bacterial strains have emerged asthe causes of nosocomial infections worldwide Recentlypandrug-resistant (PDR) bacterial strains which areresistant to all antibacterial agents except the polymyxinsand tigecycline and extensively drug-resistant (XDR)bacterial strains which are resistant to all antibacterialagents were isolated from hospital-acquired infectionsThere is a huge risk of these ldquosuperbugsrdquo extending into thecommunity and threatening public health [1] A baumanniiis an important cause of nosocomial infections and aleading cause of mortality and morbidity among hospitalizedpatients and has been associated with a wide variety ofdiseases in hospitalized patients in the intensive care units

(ICU) [2] Nowadays increasing drug resistant rate amongA baumannii strains is a major concern worldwide [3] Themost common mechanism of resistance is the production of120573-lactamases including enzymes of Ambler classes A D andB with their genes being often associated withmobile geneticelements such as plasmids [4] Carbapenem resistance causedby acquiring the MBLs is considered to be more seriousthan other resistance mechanisms because MBLs can almosthydrolyse all beta-lactam antibiotics except monobactams[3] Furthermore the MBL-encoding genes located onintegrons can be disseminated easily from one bacteriumto another Many MBLs have been found in A baumanniiincluding imipenemase (IMP) Sao Paulo metallo (SPM)Verona integron-encoded metallo-beta-lactamases (VIM)Seoul imipenemase (SIM) Japan Kyorin University Hospital

Hindawi Publishing CorporationScientificaVolume 2014 Article ID 245162 6 pageshttpdxdoiorg1011552014245162

2 Scientifica

imipenemase (KHM) German imipenemase (GIM)New-Delhi metallo-beta-lactamase (NDM-1) andAustralianimipenemase (AIM) [5 6] The IMP-type enzymes firstdetected in Japan in the late 1980s have since been reportedworldwide in Enterobacteriaceae and in Gram-negativenonfermenters (mostly in P aeruginosa and Acinetobacterspp) More than 20 different IMP allotypes have beendescribed belonging to various sublineages [4]The differentIMP types often have a defined area in the globe howeversome of which (eg IMP-1 IMP-4 and IMP-7) werediscovered in different areas which shows their potential forintercontinental dissemination The VIM-type 120573-lactamase(Verona integron-encoded metallo-120573-lactamases) was firstdescribed in amultidrug-resistant P aeruginosa strain in Italyduring the 1990s and has since been reported worldwideMore than 33 different VIM allotypes are described[4]

New-Delhi-metallo-beta-lactamase (NDM-1) a new typeof MBL was first detected in two K pneumoniae and Esche-richia coli strains isolated from a Swedish patient who wasadmitted to a hospital in New Delhi India In recent yearsthe emergence and dissemination of NDM-1-producing iso-lates have been reported in several countries includingUSA Canada Sweden UK Austria Belgium France TheNetherlands Germany Japan Africa Oman and Australia[4] NDM-producing bacteria are commonly resistant toalmost all groups of antibiotics including fluoroquinolonesaminoglycosides and 120573-lactams (especially carbapenems)but are susceptible to colistin and sometimes tigecyclineThe 119887119897119886NDM-1 gene has been detected on different largeplasmids which were readily transferable among bacteriamaking NDM-1-producing bacteria a serious clinical andpublic health threat [7] Extended spectrum beta-lactamases(ESBLs) are a rapidly evolving group of 120573-lactamase enzymesproduced by some bacteria These enzymes have the abilityto hydrolyze cephalosporins and aztreonam but are inhibitedby 120573-lactamase inhibitors such as clavulanic acid [8] ESBLgenes are often located on plasmids and many of them arederived frommutations in TEM (Temoneira) genes and SHV(Sulphydryl variable) detected by amino acid substitutionsaround the active site Apart fromTEM and SHVESBL typesisolates may additionally produce CTX-M (Cefotaximase-Munchen) 120573-lactamase [8] Other clinical types include119887119897119886KPC 119887119897119886VEB 119887119897119886PER 119887119897119886BEL-1 119887119897119886BES-1 119887119897119886SFO-1 119887119897119886TLA and119887119897119886BIC [9] But in recent years new families of extended-spectrum-beta-lactamases have also emerged all over theworld including PER (for Pseudomonas extended resis-tance) and VEB (for Vietnamese extended-spectrum-beta-lactamase) families [10]

2 Objectives

The aim of this study was to determine the frequency ofthe 119887119897119886NDM 119887119897119886PER 119887119897119886VEB 119887119897119886IMP and 119887119897119886VIM type genesamong A baumannii strains isolated from patients admittedto Loghman Hakim and Milad hospitals Tehran Iran fromyear 2012 to 2013

3 Patients and Methods

31 Bacterial Identification From June 2012 to May 2013one hundred and eight nonduplicate nonconsecutive isolatesof A baumannii were recovered from blood wound urinesputum and respiratory tract of patients from 2 hospitals(fifty-eight isolates belonged to Loghman Hakim and fiftyisolates to Milad hospitals) in Tehran Iran The isolates wereidentified by conventional biochemical methods [2] and alsoconfirmed for blaOXA-51 gene by PCR

32 Antimicrobial Susceptibility Testing Antimicrobial susce-ptibility test to imipenem (IPM 10120583g) meropenem (MEM10 120583g) ceftazidime (CAZ 30 120583g) cefotaxime (CTX 30 120583g)amikacin (AK 30 120583g) piperacillintazobactam (PTZ10010 120583g) piperacillin (PIP 100 120583g) ampicillin (AMP10 120583g) tetracycline (TE 10 120583g) colistin sulphate (CT10 120583g) ciprofloxacin (CIP 5 120583g) cefepime (FEP 30 120583g)trimethoprim-sulfamethoxazole (TS 25 120583g) and gentamicin(GEN 10 120583g) (Mast UK) was performed by the Kirby-Bauerdisk diffusion method on Mueller Hinton agar (MerckGermany) based on Clinical Laboratory Standards Institute(CLSI) Guidelines 2012 [11] Escherichia coli ATCC 25922was used as the quality control strain

33 Minimum Inhibitory Concentration (MIC) Strains resis-tant to imipenem meropenem ceftazidime cefepime cefo-taxime and colistin using the disk diffusion test wererechecked by the broth microdilution method according tothe guidelines of the CLSI 2012 [11]

34 Phenotypic Detection of MBL Combined disk diffusiontest (CDDT) was performed for identification of MBLsby imipenem and meropenem (Mast Group MerseysideUK) alone and in combination with EDTA An increase inzone diameter of ge7mm around the Imipenem+EDTA andMeropenem+EDTA disks compared to that of Imipenem andMeropenemdisks alone respectively was considered positivefor MBL production [12]

35 Phenotypic Detection of ESBL Detection of ESBLs wastested for all the isolates by combination disk diffusiontest (CDDT) containing ceftazidime (CAZ) and cefotaxime(CTX) alone and with CAZ 30 120583g + clavulanic CA 10 120583gand CTX 30 120583g + clavulanic CA 10 120583g per disc (Mast GroupMerseyside UK) The zones of inhibition were comparedwith the CTX and CAZ discs alone and compared with theCAZ 30 120583g + clavulanic (CA) 10 120583g and CTX 30 120583g + CA10 discs An increase in zone diameter of ge5mm in thepresence of clavulanic acid indicated the presence of ESBL inthe test organism Escherichia coliATCC 25922 andKlebsiellapneumoniaeATCC700603 were used as negative and positivecontrols for ESBL production respectively [11]

36 DNA Extraction Total DNAs of the different bacterialisolates were extracted by the DNA extraction kit (BioneerCompany Korea Cat number K-3032-2)

Scientifica 3

37 Detection of bla119873119863119872

bla119875119864119877

bla119881119864119861

bla119868119872119875

and bla119881119868119872

Genes by PCR PCR was used for screening of the 119887119897119886NDM119887119897119886PER 119887119897119886IMP 119887119897119886VIM and 119887119897119886VEB genes The primers usedfor 119887119897119886NDM 119887119897119886PER 119887119897119886VEB 119887119897119886IMP and 119887119897119886VIM were asfollows NDM-F (51015840-GGTTTGGCGATCTGGTTTTC-31015840)and NDM-R (51015840-CGGAATGGCTCATCACGATC-31015840) for119887119897119886NDM PER-F (5

1015840-GCAACTGCTGCAATACTCGG-31015840) andPER-R (51015840-ATGTGCGACCACAGTACCAG-31015840) for 119887119897119886PERVEB-F (51015840-CGACTTCCATTTCCCGATGC-31015840) and VEB-R (51015840-GGACTCTGCAACAAATACGC-31015840) for 119887119897119886VEB IMP-F (51015840-GAAGGCGTTTATGTTCATAC-31015840) and IMP-R (51015840-GTAAGTTTCAAGAGTGATGC-31015840) for 119887119897119886IMP VIM-F (51015840-GATGGTGTTTGGTCGCATA-31015840) and VIM-R (51015840-CGA-ATGCGCAGCACCAG-31015840) for 119887119897119886VIM The PCR mixturecontained the DNA template forwardreverse primers andmaster mix (Bioneer Company Korea Cat number K-2016)Amplification was carried out with the following thermalcycling conditions 5 minutes at 94∘C and 36 cycles ofamplification consisting of 1 minute at 94∘C 1 minute at52ndash56∘C and 1 minute at 72∘C with 5 minutes at 72∘C forthe final extension PCR product bands were analyzed afterelectrophoresis on a 1 agarose gel at 95V for 45 minutesin 1X TBE containing ethidium bromide and the result waschecked under UV irradiation

38 Sequencing Method The PCR purification kit (BioneerCo Korea) was used to purify PCR products and sequenc-ing was performed by the Bioneer Company (Korea)The nucleotide sequences were analyzed with the Chro-mas 145 software and the BLAST program from theNational Center for Biotechnology Information website(httpwwwncbinlmnihgovBLAST)

39 Statistical Analysis This research was a descriptive-application study MINITAB16 software was used for statis-tical analyses The 119875 value and confidence of intervals werelt005 and 95 respectively

4 Results

Fifty-eight strains were isolated from Loghman Hakim Hos-pital (537) and fifty from Milad Hospital (4629) Fifty-one strains were isolated from female patients (472) andfifty-seven frommales (528) Of the 108 isolates 29 (269)were isolated from urine 4 (37) from wound 57 (528)from tracheal tube 8 (74) from blood 8 (74) frompleural fluid and 2 (19) from other samples The agerange of the patients was 1 to 90 years The isolates wereobtained from patients in different age groups 2ndash29 years(119899 = 10) 30ndash39 (119899 = 14) 40ndash49 years (119899 = 21) 50ndash59 years(119899 = 16) 60ndash69 (119899 = 24) and 70ndash79 years (119899 = 17) andsix isolates were isolated from patients of more than eightyyears of age The resistance of A baumannii isolates to testedantibiotics was 108 (100) to cefotaxime 103 (954) to cef-tazidime 99 (917) tomeropenem 99 (917) to imipenem44 (407) to gentamicin 87 (806) to amikacin 100(926) to ciprofloxacin 105 (957) to cefepime 105 (972)to piperacillin 103 (954) to piperacillintazobactam 106

Table 1 Antimicrobial susceptibility test results of 108 isolatedAcinetobacter baumannii

Antibiotic Resistantnumber ()

Intermediatenumber ()

Sensitivenumber ()

Gentamicin 44 (407) 7 (65) 57 (528)Ampicillinsulbactam 106 (981) 0 (00) 2 (18)Amikacin 87 (806) 4 (37) 17 (157)Imipenem 99 (917) 3 (28) 6 (56)Cefotaxime 108 (100) 0 (00) 0 (00)Cefepime 105 (957) 2 (18) 1 (18)Piperacillin 105 (972) 2 (18) 1 (09)Ciprofloxacin 100 (926) 1 (09) 7 (65)Meropenem 99 (917) 0 (00) 9 (83)Piperacillintazobactam 103 (954) 1 (18) 4 (37)Ceftazidime 103 (954) 0 (00) 5 (47)Co-trimoxazole 106 (981) 0 (00) 2 (18)Tetracycline 87 (806) 9 (83) 12 (111)Colistin 2 (18) 0 (00) 106 (982)

Table 2 Minimum inhibitory concentration of different antimicro-bial agents among 108 Acinetobacter baumannii isolates

Antibiotics MIC (120583gmL)Range MIC50 MIC90

Meropenem 1ndash256 32 128Imipenem 2ndash256 128 256Ceftazidime 2ndashgt512 256 512Cefepime 1ndash256 64 128Cefotaxime 2ndashgt512 256 512Colistin 025ndash128 le1 2

(981) to ampicillinsulbactam 106 (981) to cotrimox-azole 87 (806) to tetracycline and 1 (18) to colistin(Table 1) The results of MIC test of different antibioticson A baumannii isolates are shown in Table 2 By usingthe combined disk diffusion test (CDDT) it was foundthat among 99 imipenem nonsusceptible A baumanniistrains 86 (8686) were MBL producers and out of 108cefotaxime-nonsusceptible A baumannii strains 91 (842)were ESBL producers The prevalence of 119887119897119886PER-1 and 119887119897119886VEB-1genes among 91 ESBL-producing A baumannii isolates was71 (7803) and 36 (395) respectively and for IMP-1and VIM-1 genes among metallo-beta-lactamase-producingA baumannii isolates it was 3 of 86 (348) and 15 of86 (1744) respectively 119887119897119886OXA-51 has been investigatedand was detected in all isolates Fortunately 119887119897119886NDM genewas not detected in isolates Sequencing of PCR prod-ucts showed conserved regions for the restriction sequence119887119897119886PER-1 119887119897119886IMP 119887119897119886VIM and 119887119897119886VEB-1 genes which was con-firmed by BLAST in NCBI The nucleotide sequence datareported in this paper have been submitted to the Gen-Bank sequence database and assigned accession numbersKF723587 KF723588 KF723589 and KF723590 for 119887119897119886PER-1gene KF723586 for 119887119897119886VEB-1 gene and KF723585 for 119887119897119886IMP-1

4 Scientifica

Seq1 Seq2 Seq3 Seq4 Consensus


-------GPAALHDYIQSMGIKETGGVANEAQMHADEQVQYQNWTSMKGAAKILKKFEQK

LLEFLVGGPAALHDYIQSMGIKETAVVANEAQMHADDQVQYQNWTSMKGAAEILKKFEQK

---- LVGGPAALHDYIQSMGIKETAVVANEAQMHADE

E

QVQYQNWTSMKGAAEILKKFEQK

--------- AALHDYIQSMGIKETAVVANEAQMHAD QVQYQNWTSMKGAAEILKKFEQK

gpAALHDYIQSMGIKETavVANEAQMHADQVQYQNWTSMKGAAeILKKFEQK

10 20 30 40 50 60

70 90

TQLSETSQALLWKWMVQ

Q

Q

TTTGPERLKGLLP

TQLSETSQALLWKWMVE TTTGPERLKGLLP

TQLSETSQALLWKWMV TTTGPERLKGLLP


TQLSETSQALLWKWMVTTTGPERLKGLLP

80

Figure 1 Multiple sequence alignment (seq1 related to Milad hospital and seq2 seq3 and seq4 related to Loghman Hakim hospital)

The sequences of 119887119897119886PER-1 gene in A baumannii strainsisolated from Loghman Hakim hospital were 100 thesame but were not similar to the sequences of 119887119897119886PER-1gene in A baumannii strains isolated from Milad hospi-tal (httpmultalintoulouseinrafrmultalinmultalinhtml)(Figure 1)

5 Discussion

A baumannii is responsible for hospital-acquired infectionsand has recently become one of the most importanthealthcare-associated infections in hospitals Infectioncaused by this bacterium often leads to significant mortalityand morbidity [13] Many researchers have reported theoutbreak of PDR A baumannii Although A baumannii is anincreasingly common nosocomial pathogen that can causeserious infections in intensive care units [14] resistancerates of isolates were as follows 103 (954) to ceftazidime99 (917) to meropenem 99 (917) to imipenem 44(407) to gentamicin 87 (806) to amikacin 100 (926)to ciprofloxacin 105 (957) to cefepime 105 (972)to piperacillin 103 (954) to piperacillintazobactam106 (981) to ampicillinsulbactam 106 (981) tocotrimoxazole 87 (806) to tetracycline and 1 (18)to colistin Also no susceptible isolate to cefotaxime wasdetected So the best coverage against the study isolateswas obtained with colistin sulphate and gentamicin Basedon different studies it is clear that emergence of resistantA baumannii strains is increasing worldwide [2] Thesestudies showed that the PDR strains were resistant notonly to beta lactams including the third generation ofcephalosporins and carbapenems but also to other drugcategories including aminoglycosides and fluoroquinolonesAnother concerned problem is related to their multidrugresistance which restricts the treatment procedure [2]Resistance to beta-lactams is related to various enzymes thatare produced including extended spectrum beta-lactamases(ESBLs) and metallo-beta-lactamases (MBLs) which belongto Ambler A and B divisions [6 15] In this study by using

the CDDT method 86 (8686) A baumannii isolates wereidentified as MBL producers Safari et al reported that theresistance rates of A baumannii isolates were 85 9497 84 95 and 98 against imipenem meropenemciprofloxacin amikacin piperacillintazobactam andcefotaxime respectively Results of 119864-test MBL illustratedthat 99 of all isolates were MBL producers [16] Peymaniet al reported that among 63 carbapenem nonsusceptibleA baumannii isolates 31 (49) were found to be MBLproducers [17] Most of the time the MBL producers canhydrolyze a wide range of antibiotics except aztreonam [18]Usually restrictions in phenotypicmethodsmake researchersconfirm phenotypic results by using molecular methods Onthe other hand there are different genes which encode thebeta-lactamases Among MBL genes IMP is more importantespecially in Iran [19] however its first report was fromJapan in 1980 [12]The other gene is VIMwhich was reportedbefore from Ahwaz another city of Iran [12] In our studythe IMP enzyme was identified only in three A baumanniistrains and VIM gene was detected among seventeen Abaumannii strains by using PCR and further sequencingwhich may be related to differences in the time of studiesand consequently changes in antibiotics prescription or usedprimers [20] The MBL coding gene is 119887119897119886NDM-1 which wasidentified recently and reported from New Delhi India forthe first time and after that from other countries includingPakistan The close distance of these countries to Iran andlarge number of trips between the countries on one side andthe ease of resistance transfer among bacteria on the otherhand led us to think that it may be probable for our isolatesto have the same gene These kinds of studies are valuableto prevent distribution of resistant bacteria to other partsof the world Finally by accurate MBL enzyme screeningand further precise supervision of the hospital practitionersit will be possible to control the spread of multidrugresistantA baumannii strains and decrease related infectionsespecially in ICU patients Based on the available dataA baumannii are growing to become the most importantcommon ESBL producing bacteria and consequently makingtheir eradication difficult In this study 91 (842)

Scientifica 5

of A baumannii were identified as ESBL producers byphenotypic tests which was more than Owlia et alrsquos study(21) Farajnia et alrsquos study in Iran (70) and another studyin Poland (20) [15 21] The high rate of ESBL prevalence inIran and its widespread dissemination is cause of worry Inthis study the prevalence of 119887119897119886PER-1 genes among 91 of ESBL-producing A baumannii isolates was 71 (7803) Accordingto the present study PER-1 is the most common ESBLgenotype among A baumannii strains which is inconsistentto other studies that showed different prevalence of PER-1The prevalence of this genotype was reported 51 in Iran46 in Turkey and 546 in South Korea Screening forVEB genotype revealed that 36 (395) of A baumanniiisolates contained VEB-1 gene The prevalence of this genewas reported to be 10 in Iran and 4761 in the USA [10]The prevalence of 120573-lactamase-producing isolates and theirisolation from life-threatening infections is dramaticallyincreasing worldwide Intensity pressure for usage ofantimicrobial drugs by patients resulted in eradication ofnormal flora and situation of MDR isolates substitutionThisstudy showed that 120573-lactamase producing A baumanniistrains are an emerging threat in ICUs and should besupervised by implementation of timely identification andstrict isolation methods that will help to reduce their severeoutcomes and mortality rate of patients

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors would like to thank the personnel of PediatricInfections Research Center and the Microbiology Depart-ment of Shahid Beheshti University of Medical Sciences fortheir cooperation

References

[1] S Y Tan S L Chua Y Liu N Hoiby L P Andersen andM Givskov ldquoComparative genomic analysis of rapid evolutionof an extreme-drug-resistant Acinetobacter baumannii clonerdquoGenome Biology and Evolution vol 5 no 5 pp 807ndash818 2013

[2] F Shahcheraghi M Abbasalipour M M Feizabadi G HEbrahimipour and N Akbari ldquoIsolation and genetic character-ization of metallo-beta-lactamase and carbapenamase produc-ing strains of acinetobacter baumannii from patients at tehranhospitalsrdquo Iranian Journal of Microbiology vol 3 no 2 pp 68ndash74 2011

[3] M Rahmati Roodsari F Fallah A Taherpour M HakemiVala and A Hashemi ldquoCarbapenem-resistant bacteria andlaboratory detection methodsrdquo Archives of Pediatric InfectiousDiseases vol 1 no 4 pp 188ndash191 2013

[4] F Fallah A Taherpour M H Vala and A Hashemi ldquoGlobalspread of New Delhi metallo-beta-lactamase-1(NDM-1)rdquo Ira-nian Journal of Clinical Infectious Diseases vol 6 no 4 pp 171ndash177 2011

[5] F Fallah M Hakemi Vala H Goudarzi et al ldquoIdentifica-tion of extended-spectrum-betalactamases(ESBLs) metallo-beta-lactamases (MBLs) Amp-C andKPC120573-lactamases amongKlebsiella pneumoniae isolated from adults and pediatricpatients in IranrdquoAfrican JMicrobiol Res vol 7 no 25 pp 3254ndash3261 2013

[6] G Cornaglia H Giamarellou andGM Rossolini ldquoMetallo-120573-lactamases a last frontier for 120573-lactamsrdquoThe Lancet InfectiousDiseases vol 11 no 5 pp 381ndash393 2011

[7] Y Chen Z Zhou Y Jiang and Y Yu ldquoEmergence of NDM-1-producing Acinetobacter baumannii in Chinardquo Journal ofAntimicrobial Chemotherapy vol 66 no 6 pp 1255ndash1259 2011

[8] A Taherpour and A Hashemi ldquoDetection of OqxAB effluxpumps OmpK35 and OmpK36 porins in ex-tended-spectrum-120573-lactamase-producing Klebsiella pneumoniae isolates fromIranrdquo Hippokratia vol 17 no 4 pp 355ndash358 2013

[9] R H Dhillon and J Clark ldquoESBLs a clear and present dangerrdquoCritical Care Research and Practice vol 2012 Article ID 62517011 pages 2012

[10] S Farajnia F Azhari M Y Alikhani M K Hosseini APeymani and N Sohrabi ldquoPrevalence of PER and VEB typeextended spectrum betalactamases among multidrug resistantAcinetobacter baumannii isolates in North-West of Iranrdquo Ira-nian Journal of Basic Medical Sciences vol 16 no 6 pp 751ndash7552013

[11] Clinical and Laboratory Standards Institute (CLSI) ldquoPer-formance standards for antimicrobial susceptibility testingTwenty-second informational supplementrdquo Tech Rep M100-S22 Fort Wayne Ind USA 2012

[12] F Fallah R S Borhan and A Hashemi ldquoDetection ofbla(IMP) and bla(VIM) metallo-beta-lactamases genes amongPseudomonas aeruginosa strainsrdquo International Journal of Burnsand Trauma vol 3 no 2 pp 122ndash124 2013

[13] P E Fournier and H Richet ldquoThe epidemiology and controlof Acinetobacter baumannii in health care facilitiesrdquo ClinicalInfectious Diseases vol 42 no 5 pp 692ndash699 2006

[14] B Y Lee SMMcglone Y Doi R R Bailey and L HHarrisonldquoEconomic value of Acinetobacter baumannii screening in theintensive care unitrdquo Clinical Microbiology and Infection vol 17no 11 pp 1691ndash1697 2011

[15] P Owlia L Azimi A Gholami B Asghari and A R LarildquoESBL- and MBL-mediated resistance in Acinetobacter bau-mannii a global threat to burn patientsrdquo Infezioni in Medicinavol 20 no 3 pp 182ndash187 2012

[16] M SafariM SaidijamA Bahador R Jafari andMY AlikhanildquoHigh prevalence of multidrug resistance and metallo-beta-lactamase (MbetaL) producing Acinetobacter baumannii iso-lated from patients in ICU wards Hamadan Iranrdquo Journal ofResearch in Health Sciences vol 13 no 2 pp 162ndash167 2013

[17] A Peymani M Nahaei S Farajnia et al ldquoHigh prevalence ofmetallo-120573-lactamase-producing Acinetobacter baumannii in ateaching hospital in Tabriz Iranrdquo Japanese Journal of InfectiousDiseases vol 64 no 1 pp 69ndash71 2011

[18] Z Liu W Li J Wang et al ldquoIdentification and characterizationof the first Escherichia coli strain carrying NDM-1 gene inChinardquo PLoS ONE vol 8 no 6 Article ID 3677923 2013

[19] S Yousefi S Farajnia M R Nahaei et al ldquoDetection ofmetallo-120573-lactamase-encoding genes among clinical isolatesof Pseudomonas aeruginosa in northwest of Iranrdquo DiagnosticMicrobiology and Infectious Disease vol 68 no 3 pp 322ndash3252010

6 Scientifica

[20] F Fallah A Taherpour R S Borhan A Hashemi M Habibiand N S Sajadi ldquoEvaluation of Zataria MultiFlora Boiss andCarum copticum antibacterial activity on IMP-type metallo-beta-lactamase-producing Pseudomonas aeruginosardquo Annals ofBurns and Fire Disasters vol 26 no 4 pp 193ndash198 2013

[21] P Sacha P Wieczorek D Ojdana et al ldquoSusceptibility pheno-types of resistance and extended-spectrum beta-lactamases inAcinetobacter baumannii strainsrdquo Folia Histochemica et Cytobi-ologica vol 50 no 1 pp 46ndash51 2012

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2 Scientifica

imipenemase (KHM) German imipenemase (GIM)New-Delhi metallo-beta-lactamase (NDM-1) andAustralianimipenemase (AIM) [5 6] The IMP-type enzymes firstdetected in Japan in the late 1980s have since been reportedworldwide in Enterobacteriaceae and in Gram-negativenonfermenters (mostly in P aeruginosa and Acinetobacterspp) More than 20 different IMP allotypes have beendescribed belonging to various sublineages [4]The differentIMP types often have a defined area in the globe howeversome of which (eg IMP-1 IMP-4 and IMP-7) werediscovered in different areas which shows their potential forintercontinental dissemination The VIM-type 120573-lactamase(Verona integron-encoded metallo-120573-lactamases) was firstdescribed in amultidrug-resistant P aeruginosa strain in Italyduring the 1990s and has since been reported worldwideMore than 33 different VIM allotypes are described[4]

New-Delhi-metallo-beta-lactamase (NDM-1) a new typeof MBL was first detected in two K pneumoniae and Esche-richia coli strains isolated from a Swedish patient who wasadmitted to a hospital in New Delhi India In recent yearsthe emergence and dissemination of NDM-1-producing iso-lates have been reported in several countries includingUSA Canada Sweden UK Austria Belgium France TheNetherlands Germany Japan Africa Oman and Australia[4] NDM-producing bacteria are commonly resistant toalmost all groups of antibiotics including fluoroquinolonesaminoglycosides and 120573-lactams (especially carbapenems)but are susceptible to colistin and sometimes tigecyclineThe 119887119897119886NDM-1 gene has been detected on different largeplasmids which were readily transferable among bacteriamaking NDM-1-producing bacteria a serious clinical andpublic health threat [7] Extended spectrum beta-lactamases(ESBLs) are a rapidly evolving group of 120573-lactamase enzymesproduced by some bacteria These enzymes have the abilityto hydrolyze cephalosporins and aztreonam but are inhibitedby 120573-lactamase inhibitors such as clavulanic acid [8] ESBLgenes are often located on plasmids and many of them arederived frommutations in TEM (Temoneira) genes and SHV(Sulphydryl variable) detected by amino acid substitutionsaround the active site Apart fromTEM and SHVESBL typesisolates may additionally produce CTX-M (Cefotaximase-Munchen) 120573-lactamase [8] Other clinical types include119887119897119886KPC 119887119897119886VEB 119887119897119886PER 119887119897119886BEL-1 119887119897119886BES-1 119887119897119886SFO-1 119887119897119886TLA and119887119897119886BIC [9] But in recent years new families of extended-spectrum-beta-lactamases have also emerged all over theworld including PER (for Pseudomonas extended resis-tance) and VEB (for Vietnamese extended-spectrum-beta-lactamase) families [10]

2 Objectives

The aim of this study was to determine the frequency ofthe 119887119897119886NDM 119887119897119886PER 119887119897119886VEB 119887119897119886IMP and 119887119897119886VIM type genesamong A baumannii strains isolated from patients admittedto Loghman Hakim and Milad hospitals Tehran Iran fromyear 2012 to 2013

3 Patients and Methods

31 Bacterial Identification From June 2012 to May 2013one hundred and eight nonduplicate nonconsecutive isolatesof A baumannii were recovered from blood wound urinesputum and respiratory tract of patients from 2 hospitals(fifty-eight isolates belonged to Loghman Hakim and fiftyisolates to Milad hospitals) in Tehran Iran The isolates wereidentified by conventional biochemical methods [2] and alsoconfirmed for blaOXA-51 gene by PCR

32 Antimicrobial Susceptibility Testing Antimicrobial susce-ptibility test to imipenem (IPM 10120583g) meropenem (MEM10 120583g) ceftazidime (CAZ 30 120583g) cefotaxime (CTX 30 120583g)amikacin (AK 30 120583g) piperacillintazobactam (PTZ10010 120583g) piperacillin (PIP 100 120583g) ampicillin (AMP10 120583g) tetracycline (TE 10 120583g) colistin sulphate (CT10 120583g) ciprofloxacin (CIP 5 120583g) cefepime (FEP 30 120583g)trimethoprim-sulfamethoxazole (TS 25 120583g) and gentamicin(GEN 10 120583g) (Mast UK) was performed by the Kirby-Bauerdisk diffusion method on Mueller Hinton agar (MerckGermany) based on Clinical Laboratory Standards Institute(CLSI) Guidelines 2012 [11] Escherichia coli ATCC 25922was used as the quality control strain

33 Minimum Inhibitory Concentration (MIC) Strains resis-tant to imipenem meropenem ceftazidime cefepime cefo-taxime and colistin using the disk diffusion test wererechecked by the broth microdilution method according tothe guidelines of the CLSI 2012 [11]

34 Phenotypic Detection of MBL Combined disk diffusiontest (CDDT) was performed for identification of MBLsby imipenem and meropenem (Mast Group MerseysideUK) alone and in combination with EDTA An increase inzone diameter of ge7mm around the Imipenem+EDTA andMeropenem+EDTA disks compared to that of Imipenem andMeropenemdisks alone respectively was considered positivefor MBL production [12]

35 Phenotypic Detection of ESBL Detection of ESBLs wastested for all the isolates by combination disk diffusiontest (CDDT) containing ceftazidime (CAZ) and cefotaxime(CTX) alone and with CAZ 30 120583g + clavulanic CA 10 120583gand CTX 30 120583g + clavulanic CA 10 120583g per disc (Mast GroupMerseyside UK) The zones of inhibition were comparedwith the CTX and CAZ discs alone and compared with theCAZ 30 120583g + clavulanic (CA) 10 120583g and CTX 30 120583g + CA10 discs An increase in zone diameter of ge5mm in thepresence of clavulanic acid indicated the presence of ESBL inthe test organism Escherichia coliATCC 25922 andKlebsiellapneumoniaeATCC700603 were used as negative and positivecontrols for ESBL production respectively [11]

36 DNA Extraction Total DNAs of the different bacterialisolates were extracted by the DNA extraction kit (BioneerCompany Korea Cat number K-3032-2)

Scientifica 3

37 Detection of bla119873119863119872

bla119875119864119877

bla119881119864119861

bla119868119872119875

and bla119881119868119872





4 Results





Sensitivenumber ()






4 Scientifica






E




10 20 30 40 50 60

70 90

TQLSETSQALLWKWMVQ

Q

Q

TTTGPERLKGLLP





80



5 Discussion



Scientifica 5




Acknowledgments


References




















6 Scientifica










Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Scientifica 3

37 Detection of bla119873119863119872

bla119875119864119877

bla119881119864119861

bla119868119872119875

and bla119881119868119872





4 Results





Sensitivenumber ()






4 Scientifica






E




10 20 30 40 50 60

70 90

TQLSETSQALLWKWMVQ

Q

Q

TTTGPERLKGLLP





80



5 Discussion



Scientifica 5




Acknowledgments


References




















6 Scientifica










Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

4 Scientifica






E




10 20 30 40 50 60

70 90

TQLSETSQALLWKWMVQ

Q

Q

TTTGPERLKGLLP





80



5 Discussion



Scientifica 5




Acknowledgments


References




















6 Scientifica










Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Scientifica 5




Acknowledgments


References




















6 Scientifica










Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

6 Scientifica










Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

prevalence of ndm per veb imp acinetobacter...

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