Table Of ContentShakibaieetal.AntimicrobialResistanceandInfectionControl2012,1:1
http://www.aricjournal.com/1/1/1
RESEARCH Open Access
Antibiotic resistance patterns and extended-
b
spectrum -lactamase production among
Acinetobacter spp. isolated from an intensive care
Unit of a hospital in Kerman, Iran
Mohammad Reza Shakibaie*, Saied Adeli and Mohammad Hosain Salehi
Abstract
Background: TheglobalincreaseinmultidrugresistanceofAcinetobacterspp.hascreatedwidespreadproblemsinthe
treatmentofpatientsinintensivecareunits(ICUs)ofhospitals.ToassessthesensitivityofAcinetobacterisolatesto
antibioticsroutinelyusedinICUs,weinvestigatedantibioticresistancepatternsandextended-spectrumb-lactamase
(ESBL)productionamongAcinetobacterspp.isolatedfromtheICUofauniversityhospitalinKerman,Iran.
Methods: FifteenisolatesofAcinetobacterspp.wererecoveredfromonehundredclinicalspecimenscollectedfrom
theICUofAfzalipoorHospitalinKerman,Iran,fromOctober2010toJune2011.Preliminaryantibioticsensitivitytesting
wascarriedoutusingthedisk-diffusionbreakpointassay,andMICsofdifferentantibioticsweredeterminedusingthe
E-test.ESBLproductionwasdetectedbyadouble-disksynergytestandconfirmedbyaphenotypicconfirmatorytest.
Substratehydrolysisinthepresenceandabsenceofthefollowinginhibitorswascarriedoutusingtherapidfixed-time
method:para-chloromercuribenzoate(p-CMB),clavulanicacid,sulbactam,andNaCl.
Results: Overall, 73.3% of the isolates were resistant to imipenem (MIC range 240-128 µg/mL) and 66% to
ciprofloxacin (MIC range 240-64 ± 0.08 µg/mL). All of the isolates were fully resistant (MIC 240 µg/mL) to
piperacillin, while 93.3%, 53.3%, and 93.3% were resistant to piperacillin + tazobactam (MIC 240 µg/mL), amikacin
(MIC range 128-16 µg/mL), and cefepime (MIC range 240-60 µg/mL), respectively. The isolates were also resistant
to chloramphenicol and tetracycline: MICs of these two agents were ≥ 240 µg/mL. The test for ESBL production
was positive for only three isolates (nos. 1, 10, and 15). The rate of substrate hydrolysis was highest in the presence
of p-CMB (80.2 ± 0.02) and lowest in the presence of NaCl (2.1 ± 0.01) (P ≤ 0.05).
Conclusions: Many isolates of Acinetobacter spp. are resistant to almost all antibiotics routinely used in the ICU of
our hospital, including imipenem, ciprofloxacin, and piperacillin + tazobactam. Three isolates were ESBL producers.
The other isolates exhibited high resistance to b-lactams, but they did not produce any ESBL enzymes.
Keywords: Acinetobacter spp, antibiotic resistance, MIC, extended-spectrum β-lactamase
Introduction pathogeninthe hospitalenvironment. The contribution
Acinetobacter is a genus of gram-negative bacteria of Acinetobacter spp. to nosocomial infection has
belonging to the Gammaproteobacteria. They are non- increased over the past three decades, and many out-
motile,oxidasenegative,highlypleomorphicandusually breaks ofhospitalinfection involving Acinetobacterspp.
occurinpairs.ThegenusAcinetobacterhasoccupiedan havebeenreportedworldwide[1-3].
increasingly important position as an opportunistic Although generally regarded as commensals ofhuman
skin and the human respiratory tract, Acinetobacter spp.
havealsobeenimplicatedasthecauseofseriousinfectious
*Correspondence:[email protected]/mohammadreza.shakibaie@gmail.
com diseasessuchaspneumonia,urinarytractinfections,endo-
DepartmentofMicrobiology,KermanUniversityofMedicalSciences,Kerman, carditis, wound infections, meningitis, and septicemia,
Iran
©2011Shakibaieetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycited.
Shakibaieetal.AntimicrobialResistanceandInfectionControl2012,1:1 Page2of8
http://www.aricjournal.com/1/1/1
involvingmostlypatientswithimpairedhostdefenses[2]. nonsusceptible Acinetobacter baumannii isolates,
Acinetobacterspp.haveemergedasparticularlyimportant respectively.
organisms late-onset ventilator associated pneumonia in Sinhaetal.[9]recovered150clinicalisolatesofAcineto-
theintensivecareunit(ICU).Thisisprobablyrelated,at bacterandidentifiedthemusingvariousphenotypictests.
least in part, to the increasingly invasive diagnostic and Antibiotic susceptibility wasdetermined by the standard
therapeutic procedures used in hospital ICUs in recent disk-diffusionmethod.Mostisolateswereresistanttothe
years[4-6]. antibioticstested,includingthethird-generationcephalos-
Acinetobacter spp. have acquired resistance to almost porins. ESBL production wasdetected in 28% of the iso-
allcurrentlyavailableantimicrobialagents,includingthe lates. In the double-disk approximation test, most of the
aminoglycosides,thequinolones,andbroad-spectrumb- ESBLs in Acinetobacter isolates could be detected with
lactams. The spectrum of antibiotic resistance of these cefepimeandcefotaxime.
organisms, together with their survival capabilities, InonestudyintheUK, theantimicrobialsusceptibility
makes them a threat in hospital environments, as docu- of Acinetobacter obtained from clinical specimens in 54
mentedbyrecurringoutbreaksbothinhighlydeveloped laboratorieswasinvestigated.Themajorityoftheisolates
countries and elsewhere. Most strains are resistant to were found to be more resistant to cefotaxime, ceftazi-
cephalosporins,whileresistancetocarbapenemsisbeing dime, piperacillin, piperacillin+tazobactam,gentamicin,
reported increasingly [7,8]. One particular attribute of and tetracycline than the other gram negative bacteria
these strains is the production of extended-spectrum [12].
beta-lactamase(ESBL)enzymes that conferresistanceto Littleinformationisavailableontheantibioticresistance
b-lactams[9].Guillouetal.[10]screened 100isolatesof ofAcinetobacterspp.isolatedfromhospitalsinIran.Khos-
Acinetobacterspp.andfoundthat81%ofthestrainspro- roshahi andSharifi [13]recovered 400isolates from ICU
ducedtwotypesofb-lactamases(TEMandCARB). patients in four university hospitals in Isfahan, Iran, of
Acinetobacter baumannii hospital isolates produce which 15 (3.75%) belonged to A. baumannii. Antibiotic
mainly cephalosporinase-type enzyme and are inhibited sensitivitytestingshowedfour(26.6%)isolateswereresis-
by 25 mM of clavulanic acidbut not by 1 mM EDTA or tanttoimipenemandmeropenem.Similarly,Farhanietal.
100 mM para-chloromercuribenzoate (p-CMB). The b- [14]recovered60isolatesofAcinetobacterspp.fromSha-
lactamases produced by Acinetobacter lwoffii ULA-501, hid Beheshti Hospital in Kashan, Iran. Among these, 48
A.baumanniiULA-187,andA.baumanniiAC-14strains wereA.baumannii,sixwereA.lwoffi,andsixwereother
have been purified and characterized, and their kinetic Acinetobacterspp.Theywereresistanttoamikacin,tobra-
interactions with several b-lactam molecules, including mycin,ampicillin+sulbactam,andimipenem.
substrates and inhibitors, have been studied in detail Toassessthelevelofsensitivitytoantibioticsroutinely
[11]. Three b-lactamase enzymes were identified and usedintheICUofourhospitalandtodeterminethepro-
appeared tobe cephalosporinase-type b-lactamaseswith ductionofESBLs,weinvestigatedtheantibioticresistance
different acylation efficiencies (kcat/Km ratio values). pattern and ESBL production in Acinetobacter spp. iso-
Their hydrolytic activitieswere inhibitedby benzylpeni- latedfromtheICUoftheAfzalipoorHospitalinKerman,
cillin, piperacillin, and cefotaxime, none of which Iran.
behavedassubstratesfortheenzyme.Carbenicillinwasa
substrate for the b-lactamase from A. lwoffii ULA-501, Methods
althoughitactedasatransientinactivatoroftheenzymes Source of bacteria
produced by the two A. baumannii strains. Clavulanic Morethanonehundredclinicalspecimenswerecollected
acid was unable to inactivate the three b-lactamases, fromtheICUofAfzalipoorHospital(themainuniversity
whereas sulbactam behaved as an inactivator only at a hospital) inthe city ofKerman,Iran, from October 2010
high concentration (1 mM) that was difficult to achieve to June 2011. The majority of the patients were hospita-
duringantibiotictherapy[11]. lized for 4 days, and 73% of them were on ventilator-
Kimetal.[7]studiedtheprevalenceanddiversityofcar- assisted life support. The average age ofthe patientswas
bapenemases among imipenem-nonsusceptible Acineto- 63 ± 0.8 years. Specimens of lung aspirates, blood, or
bacter isolates in Korea. A total of 190 imipenem- urinewerecollectedbyalaboratorytechnicianandtrans-
nonsusceptibleAcinetobacterisolatesfrom12Koreanhos- ferredimmediatelytotheMicrobiologyDepartmentofthe
pitalsin2007wereusedtodeterminespecies,prevalence, Kerman University of Medical Sciences in sterile screw-
andantimicrobial susceptibility ofOXA carbapenemase- captubescontaining5mLoftrypticsoybroth(TSB)med-
producing and metallo-b-lactamase-producing isolates. ium.Priortocollectionofthespecimens,criteriasuchas
bla -like and ISAba1-associated bla -like previousantimicrobialtherapy,immunosuppression,and
OXA-23 OXA-51
genes were detected in 80% and 12% of 178 imipenem- presenceofbacteremiaduetootherpathogensbeforeand
Shakibaieetal.AntimicrobialResistanceandInfectionControl2012,1:1 Page3of8
http://www.aricjournal.com/1/1/1
after colonization by Acinetobacter were taken into Substrate hydrolysis in the presence and absence of
consideration. inhibitors
Inordertoeliminatethepossibilityoffalse-positiveESBL
Bacterial identification testsduetointrinsicsusceptibilityofisolatestob-lacta-
Isolates were identified preliminarily by the chromoso- mase inhibitors (which could result in false-positive or
mal transformation assay [15] using an auxothrophic false-negative tests), the rapid fixed-time assay wasused
strain of Acinetobacter calcoaceticus BD143 trpE27, and to measure b-lactamase activity, based on the reduction
species identification was carried out using the biochem- of iodine by hydrolysis of cefotaxime/ceftazidime under
ical and sugar utilization tests as described by Bouvet anultravioletlightspectrophotometerat450nm.Inthis
and Grimont [16]. Isolates were further identified by case, the cultures were centrifuged at 8,000 rpm at 4°C
Gram stain, motility, characteristics on nutrient agar for 15 minutes after overnight growth in Luria-Bertani
and Cysteine-Lactose-Electrolyte-Deficient (CLED) agar, broth medium, and the cell pellet was washed with 0.01
catalase and oxidase tests, acidity or alkalinity in triple Msterilephosphatebuffer(pH8.0).Thesuspensionwas
sugar iron (TSI) agar slants, growth on citrate agar sonicated with a Lab sonic sonicator (Germany) for 15
slants, hemolytic patterns on blood agar, glucose oxida- secondsusinga 50% on/off pulsedcycle.Sonication was
tion in Hugh and Leifson medium containing 1% glu- followedbyfreeze-thawingat-70°Cfor10minutes.The
cose, and ability to grow at 44°C. sonicated cells were centrifuged at 12,000 rpm for 10
minutes and then observed microscopically to see the
Antibiotic susceptibility tests disintegratedbacterialcells.
Antibiotic sensitivity of the isolates was determined The sonicated solutions of the Acinetobacter isolates
using the Kirby-Bauer disk-diffusion breakpoint assay on werethendilutedwith2.5mLof0.01Mphosphatebuffer
Mueller-Hinton agar using Oxoid disks (purchased from (pH8.0).To this preparation, 0.5mL of 200µg/mL sub-
Hi-Media, India) as recommended previously by the strate(cefotaxime andceftazidime)wasaddedseparately
Clinical and Laboratory Standards Institute (CLSI, pre- and incubated at room temperature for 30 minutes. In
viously called NCCLS) (2007 guidelines) [17]. MICs of caseofb-lactamaseinhibitors,0.5mLof0.5mMp-CMB
different antibiotics were determined using the E-test (Fluka, Germany),100mMNaCl,200µg/mLsulbactam/
(Hi-Media). Susceptibility to the following antimicrobial clavulanic acid, and200µg/mLcloxacillinwereaddedto
agents was tested: cefotaxime, cefepime, cefazolin, cipro- thecrudeenzymepreparation15minutesbeforeaddition
floxacin, piperacillin, piperacillin + tazobactam, ceftazi- ofthesubstrates.Thereactionwasstoppedbytheaddition
dime, imipenem, tetracycline, gentamicin, amikacin, and of5mLiodinereagentcontaining0.32NI and1.2MKI
2
chloramphenicol. Isolates were considered susceptible if withrapidstirringatroomtemperature.Absorbancewas
the MIC was ≤ 2 µg/mL and resistant if the MIC was ≥ measuredat540nm,andtheresultswerecomparedwith
8 µg/mL. A standard culture of A. calcoaceticus BD413 preparationcontainingtheinhibitors.Simultaneously,two
was used as sensitive bacterium at an inoculum of 1.5 × blanks, one containing 3 mL phosphate buffer (pH 7.5)
107CFU/mL. and5mLofiodinereagentandtheothercontaining5mL
of phosphate buffer (pH 7.5), were run alongside of the
Detection of ESBL tests.
Two disk-diffusion methods were employed in this
investigation, both described previously [18]. Briefly, the Statistical analysis
first method is based on the original double-disk stan- All analyses were performed using SPSS, version 16.0
dard test (DDST); isolates are examined for the expan- (SPSSInc,Chicago,IL,USA).AllPvaluesweretwo-tailed;
sion of the cefotaxime + clavulanic acid inhibition zone P≤0.05wasconsideredstatisticallysignificant.Meansand
adjacent to disks containing cefotaxime alone and amox- standard deviations (SD) were calculated as required for
icillin + clavulanic acid 30 + 10 mg. In the second numericalvariables.
method, a disk containing 30 mg of ceftazidime is
placed adjacent to a combination disk containing cefta- Results
zidime (30 mg) with clavulanic acid (10 mg) on sterile Bacterial isolates
Mueller-Hinton agar inoculated with ESBL-positive and FromOctober2010toJune2011,morethanonehundred
non-ESBL-producing standard cultures of Acinetobacter specimenswerecollectedfromtheICUofAfzalipoorHos-
isolates. An expansion of > 5 mm or 50% (according to pital inKerman, Iran.Fromthese specimens, fifteeniso-
the manufacturer’s guidelines) indicates ESBL produc- lates were iden tified as Acinetobacter spp. by different
tion. The antibiotic disks described above were obtained biochemical tests and a chromosomal transformation
from Oxoid, Mast, and Beckton Dickinson, UK. assay using an auxothrophic strain of A. calcoaceticus
Shakibaieetal.AntimicrobialResistanceandInfectionControl2012,1:1 Page4of8
http://www.aricjournal.com/1/1/1
BD413trpE27. The study was conducted in the ICU of +tazobactam,andciprofloxacinisofparticularlyconcern
AfzalipoorHospitalinKerman,Iran.Patientswereeither becausetheseantibioticsareusuallyreservedforseverely
admitted directly to the ICU or transferred from other ill patients. The breakpoints obtained by disk-diffusion
wards,namelyinternalmedicine,surgery,obstetrics,neu- testingwerefurtherconfirmedbydeterminationofMICs,
rology, and cardiology wards. Post-operative patients asshowninTable2. Forisolatenumbers1,2, 5,7,9,10,
requiring ventilation were admitted to the critical care and15,theMICsofimipenem,piperacillin,piperacillin+
unit,whilepatientswithmedicalconditionsnecessitating tazobactam,andciprofloxacinwere240µg/mL.
ventilationwereadmittedtotheICU. All of the Acinetobacter isolates were also highly resis-
Overall,71%ofthehospitalizedpatientsweremaleand tant to third-generation cephalosporins (cefotaxime and
29% were female (P ≤ 0.5). The average age of the ceftazidime), with MICs exceeding 240 µg/mL. For iso-
patients was 63 ± 0.8 years. The specimens were col- late number 13, the MICs of imipenem and amikacin
lected from the lung, blood, and urine of the patients were each 4 µg/mL, and the MICs of ceftazidime + cefo-
hospitalized for 4 days in the ICU. A greater proportion taxime and ciprofloxacin were each 5 µg/mL. Figure 1 (a
of Acinetobacter spp. was isolated from lung aspirates and b) shows the MICs of gentamicin and amikacin for
(76%) than from urine samples (4%) from patients with Acinetobacter isolates 1 and 12, respectively, as deter-
urinary sepsis. The lung aspirates were homogenized mined by the E-test. The imipenem-resistant isolates
with5mL0.05mMPIPSbufferandcentrifugedat8,000 were multiresistant, but, most (46%) were sensitive to
rpmbeforebeinginoculatedontothemedium. amikacin using the CLSI breakpoint of 4µg/mL. The
ThecoloniesonCLEDagarwerecircular,smooth,con- important observation was the complete resistance of all
vex, translucent, mucoid, and nonpigmented. They were isolates when tested with the amoxicillin + clavulanic
gram-negative,encapsulated,non-spore-formingcoccoba- acid combination disk (Table 2).
cilli.The lactose utilization testforallisolateswasnega-
tive.Theorganismswerenonmotileandnonhemolytic. ESBL production
TheDDSTmethodwasusedtodetecttheproductionof
Antibiotic susceptibility ESBL, and results were confirmed by the presence of a
Theresultsofantibioticsusceptibilitytestingbythedisk- zone of inhibition around a disk containing a combina-
diffusiontestareshowninTable1. Isolatenumbers1,2, tion of the cephalosporin (ceftazidime) and clavulanic
4, 5, 10, and 15 were completely resistant to antibiotics acid when compared with the zone around a disk con-
routinely used in the ICU, while isolate no. 13 was the tainingthecephalosporin(ceftazidime)alone.Onlythree
only one sensitive to most of the antibiotics. The emer- isolates (nos. 1, 10, and 15) were capable of producing
gence ofresistancetoimipenem,piperacillin,piperacillin ESBL. The remaining isolates exhibited a highdegree of
Table 1Antibiotic susceptibility of Acinetobacter spp.isolated fromthe ICUofAfzalipoor Hospital
Acinetobacter CTX CPM CZ CP PIP CAZ IMP Te Gm AK PIT C
Isolates
1 R R R R R R R R R R R R
2 R R R R R R R R R S R R
3 R R R S R S S R I I R R
4 R R R R R R R R R I R R
5 R R R S R R R R R I R R
6 R R R R R R S R I S R R
7 R R R R R R R R I R R I
8 R R R S R R S S R R R R
9 R R R R I R R R R I R R
10 R R R R R R R R R R R R
11 R R R S R I S R I S R R
12 R R R R R R R I S S R R
13 S R R S I S S R S S S I
14 R R R I R R R R R S R I
15 R R R R R R R R R I R R
Susceptibilitywasdeterminedbythedisk-diffusionbreakpointmethod.
CTX=cefotaxime,CPM=cefepime,CZ=cefazolin,CP=ciprofloxacin,PIP=piperacillin,PIT=piperacillin+tazobactam,CAZ=ceftazidime,IMP=imipenem,Te
=tetracycline,Gm=gentamicin,AK=amikacin,C=chloramphenicol,R=resistant,I=intermediate,S=sensitive.SterileMuller-Hintonagarwasusedfor
determinationofantibioticsusceptibilityandtheinoculumconcentrationwas1.5×107
Shakibaieetal.AntimicrobialResistanceandInfectionControl2012,1:1 Page5of8
http://www.aricjournal.com/1/1/1
Table 2Minimum inhibitory concentrations (MICs) ofdifferentantibiotics for Acinetobacterspp. isolatedfrom the ICU
ofAfzalipoor Hospital
Acinetobacter MIC*
isolates (µg/mL)
CPM Gm AK Am PIT CAZ C PIP Te IMP CP
1 120 240 128 >240 >240 >240 60 >240 >240 240 >240
2 30 240 4 >240 >240 >240 >240 >240 >240 240 >240
3 10 10 4 240 30 240 >240 >240 >240 4 2
4 240 >240 16 >240 >240 >240 60 >240 >240 128 128
5 >240 >240 16 >240 240 >240 60 >240 >240 240 >240
6 30 5 4 240 120 240 120 128 >240 10 5
7 240 >240 32 >240 >240 >240 60 >240 >240 240 240
8 60 240 64 >240 5 >240 10 128 4 10 4
9 240 >240 16 240 >240 240 >240 16 >240 240 >240
10 240 >240 128 >240 >240 >240 120 >240 >240 240 >240
11 10 5 4 240 60 240 120 128 16 10 4
12 60 5 32 >240 >240 >240 120 >240 >240 128 >240
13 5 5 4 >240 30 5 10 16 >240 4 5
14 60 120 4 >240 60 >240 10 >240 >240 128 64
15 30 240 4 240 30 240 60 128 240 240 240
*MICforeachantibioticwasdeterminedtwicebytheE-testonMueller-Hintonagar,andCFU/mLwaskeptat1.5×107.
CPM=cefepime,Gm=gentamicin,AK=amikacin,Amp=ampicillin,PIT=piperacillin+tazobactam,CAZ=ceftazidime,C=chloramphenicol,PIP=piperacillin,
Te=tetracycline,IMP=imipenem,CP=ciprofloxacin.
resistancetothird-generationcephalosporinsbutdidnot equipmentwasreportedbyCefaietal.[21].Inourinvesti-
produceESBL. gation, specimens were collected from the lung, blood,
and,inone case (isolate12),urine ofseverelyill patients
Substrate hydrolysis hospitalized inthe ICU for 4days. The average ofage of
Theratesofsubstratehydrolysisandtheinhibitorprofiles theinfectedpatientswas63±0.8years.TheMICsofanti-
of b-lactamase produced by the Acinetobacter isolates bioticsroutinelyusedintheICUofourhospitalindicated
fromtheICUofAfzalipoorHospitalareshowninTable3. theemergenceofresistanceinsomeAcinetobacterisolates
Thehighestrateofceftazidimeandcefotaximehydrolysis (numbers1,2,5,7,9,10,and15)toalmostalltheantibio-
wasobservedinthepresenceofp-CMB(80.2±0.02µM/ tics used,including imipenem,ciprofloxacin, andpipera-
mL), while lowest rate was observed when NaCl (2.1 ± cillin+tazobactam.Thesefindingscomplicatethetherapy
0.01µM/mL)wasusedassubstrate(P≤0.05).Therateof of infections caused by Acinetobacter spp. It has been
substrate hydrolysis decreased inthe presence ofsulbac- suggestedthattheoverwhelminguseofantibiotics,parti-
tam(Table3).Toeliminatethepossibilityoffalse-positive cularlyfluoroquinolones(e.g.,ciprofloxacin)andcarbape-
ESBL tests due to intrinsic susceptibility of b-lactamase nems (e.g., imipenem), has resulted in the emergence of
inhibitors or the other mechanisms, the MICofcefotax- more resistant forms of colonizing strains [6]. ESBL was
imeandceftazidimefortheESBL-producingisolateswas produced by three isolates, while the remaining isolates
measuredinthepresenceandabsenceoftheb-lactamase demonstratednoenzymaticactivity.Theseresultssuggest
inhibitor clavulanic acid. The MICs of ceftazidime and thatintrinsicresistancetoantibioticsinnon-ESBL-produ-
cefotaximeweredecreasedfrom>240µg/mLto8µg/mL cing isolates may be due to mutation(s) in the genome
and4µg/mL,respectively. involvedinantibioticsusceptibility,resultinginhighMIC
values.Theimportantobservationwastheinsensitivityof
Discussion theAcinetobacterisolatestoamoxicillin+clavulanicacid,
Acinetobacter spp. are resistant to the most commonly despitesusceptibilitytoceftazidime+clavulanicacid.
availableantibiotics;hence,theyareabletosurviveinthe In a recent study, a DDST method that combined
hospitalenvironmentunderhostileconditionsandalsoto amoxicllin+clavulanicacidwithcefepimeweresuccess-
colonizesusceptiblepatientstreatedwithbroad-spectrum fully detected the SHV-5 b-lactamase in a Klebsiella
antibiotics. They are able to survive on various surfaces pneumoniaestrainthatproducedaplasmid-borneAmpC
(bothmoistanddry)inthehospitalenvironment[19,20]. enzyme [22]. In another report, the use of cefepime
An outbreak of Acinetobacter respiratory tract infection increased the sensitivity of the DDST with extended-
resulting from incomplete disinfection of ventilator spectrum cephalosporins for the detection of ESBLs in
Shakibaieetal.AntimicrobialResistanceandInfectionControl2012,1:1 Page6of8
http://www.aricjournal.com/1/1/1
Figure 1 MICs of gentamicin [Gm (a)] and amikacin [AK (b)] for Acinetobacter isolates as determined by the E-test. The MIC was
measuredaslowestconcentrationoftheantibioticthatinhibitsthevisiblegrowthoftheorganism.Mueller-HintonagarwithinitialCFU/mL1.5
×107after24hoursofincubationat37°Cwasusedasmedium.
enterobacteria from 16% to 61% when the disks were were applied at a shorter distance (20 mm) [23]. These
appliedatthestandarddistanceof30mmfromamoxicil- results suggested that the inh ibition of the activities of
lin + clavulanic acid and from 71% to 90% when disks the AmpC enzyme andeffluxpumps mightenhance the
Shakibaieetal.AntimicrobialResistanceandInfectionControl2012,1:1 Page7of8
http://www.aricjournal.com/1/1/1
Table 3Rates ofsubstrate(b-lactam) hydrolysis andinhibitor profiles ofAcinetobacter spp.isolated fromthe ICUof
Afzalipoor Hospital.
Substrate MIC(µg/mL) b-lactamase Substrate
inhibitor hydrolysis(µmol)
CTX >240 - 80±0.04
CTX >240 P-CMB(50mM) 80.2±0.02
CTX >240 NaCl(100mM) 2.1±0.01
CTX >240 Sulbactam(200µg/ml) 10.2±0.03
CTX >240 Clavulanic(200µg/ml) 7.5±0.02
CAZ >240 - 80±0.04
CAZ >240 P-CMB(50mM) 80.2±0.02
CAZ >240 NaCl(100mM) 2.1±0.01
CAZ >240 Sulbactam(200µg/ml) 30.2±0.03
CAZ >240 Clavulanic(200µg/ml) 7.5±0.02
b-lactamaseinhibitorswereadded15minbeforeadditionofthesubstrate.
P≤0.05wasconsideredsignificantofassociation.Theaboveresultsrepresentthemeanoftwoindependentexperiments.Meansandstandarddeviations(SD)
werecalculatedasrequiredfornumericalvariables.
CTX=cefotaxime,CAZ=ceftazidime.
abilities of DDST to detect ESBLs in Pseudomonas duringa4-monthperiodfrom12patientshospitalizedat
aeruginosa. theValenciennesHospital.Sevenclonallyrelatedbla
VEB-1
Inourstudy,therateofhydrolysisofceftazidime/cefo- positive A. baumannii strains were identified in the
taximewashighestinthepresenceofp-CMBandlowest immediateenvironmentofthehospitalizedpatients.
whenNaClandclavulanic acid(200µg/ml)were usedas Wefoundsimilarresultsinourstudywithisolateno.1,
substrate (P ≥0.05). The results possibly explain the which might be a carbapenemase-producing, blaVIM-
expandedzoneofinhibitionaroundtheceftazidime/cefo- positive isolate. Further research must be conducted to
taxime+clavulanicaciddiskandconfirmedthattheESBL determine the mechanism of resistance to the above
productionamongtheisolateswasnotduetotheintrinsic antibiotics.
sensitivity of Acinetobacter isolates to b-lactamase
inhibitors. Conclusions
The resultsofantibiotic susceptibility testing of gram- The results of this study showed that the majority of
negative bacilli strains isolated fromthe ICUofthe Fun- ICU isolates of Acinetobacter spp. were highly resistant
deni Clinical Institute, Bucharest, Romania [4], showed to the antibiotics most commonly used in the ICU set-
that80%of19strainsofAcinetobacterspp.isolatedfrom ting, including imipenem, ciprofloxacin, cefepime, and
nasal and pharyngeal exudates and bronchial secretions piperacillin + tazobactam, all of which are already used
from immune-deficient patients were highly resistant to extensively in Iranian hospitals. Tests for ESBL produc-
imipenem and were also resistant to the majority of the tion and substrate hydrolysis by resistant strains
antibioticstested. revealed the unique property of the ESBL (sensitivity to
Similarly,inonestudyonAcinetobactersusceptibilityin cefotaxime + clavulanic acid and to ceftazidime + clavu-
Iran [24], it was found that the rates of sensitivity of the lanic acid, and high rate of hydrolysis by p-CMB) pro-
isolatestoimipenem,piperacillin+tazobactam,andAmi- duced by our isolates. These findings have important
kacinwere50.7%,50%,and38.2%,respectively.However, implications for physicians, microbiologists, and hospital
ithasrecently become obvious that increasedexpression administrators involved in the treatment of ICU patients
of chromosomal genes for efflux systems plays a major in Iran.
role in multidrug resistance [25]. Lagatolla et al. [26]
reportedthatMICsofimipenemfortheblaVIM-positive
isolates were always ≥ 64 µg/mL (range 64-512 µg/mL). Acknowledgements
TheauthorsthanktheUniversityCouncilforResearchAffairsofKerman
Most of the blaVIM-positive isolates (49 of 64 [76%]) UniversityofMedicalSciencesforprovidinggrantno.89/77toMR
exhibited a multidrug-resistant phenotype that included Shakibaie.TheMicrobiologyDepartmentandICUstaffofAfzalipoorHospital,
Kerman,Iran,arealsoacknowledgedforhelpprovidedduringthisresearch.
all of the drugs tested (imipenem, meropenem, ceftazi-
dime,piperacillin,aztreonam,amikacin,gentamicin,tobra- Authors’contributions
mycin,andciprofloxacin)[26].InoneoutbreakinFrench MRSandSAhavemadesubstantiveintellectualcontributionstothisstudy.
MHShelpedinthelaboratorypreparationandsetting.Allauthorsreadand
hospital[27],twelveclonallyrelatedandmultidrug-resis-
approvedthefinalmanuscript.
tant Acinetobacter baumannii isolates were recovered
Shakibaieetal.AntimicrobialResistanceandInfectionControl2012,1:1 Page8of8
http://www.aricjournal.com/1/1/1
Competinginterests 22. PerezF,HujerAM,HujerKM,DeckerB,RatherP,BonomoR:Global
Theauthorsdeclarethattheyhavenocompetinginterests. challengeofmultidrug-resistantAcinetobacterbaumannii.Antimicrob
AgentsChemother2007,51:3471-3484.
Received:27August2011 Accepted:13October2012 23. JacobyGA,HanP:Detectionofextended-spectrumbetalactamasesin
Published:23January2012 clinicalisolatesofKlebsiellapneumoniaeandEscherichiacoli.JClin
Microbiol1996,34:908-911.
24. FeizabadiM,FatollahzadehB,RasoolinejadM,AligholiM,SoroushS,
References
MohammadiS:Antimicrobialsusceptibilitypatternsanddistributionof
1. Bergogne-BerezinE,TownerKJ:Acinetobacterspp.asnosocomial
blaOXAgenesamongAcinetobacterspp.isolatedfrompatientsin
pathogens:microbiological,clinical,andepidemiologicalfeatures.Clin
Tehranhospitals.JpnJInfectDis2008,61:274-278.
MicrobiolRev1996,9:148-165.
25. CoyneS,CourvalinP,PérichonB:Efflux-mediatedantibioticresistancein
2. PelegAY,SeifertH,PatersonDL:Acinetobacterbaumannii:emergenceofa
Acinetobacterspp.AntimicrobAgentsChemother2011,55:947-953.
successfulpathogen.ClinMicrobiolRev2008,21:538-582.
26. LagatollaC,ToninEA,Monti-BragadinC,DolzaniL,GombacF,BearziC,
3. LooverenMV,GoossensH,VanMH:Antimicrobialresistanceof
EdalucciE,GionechettiF,RossoliniGM:Endemiccarbapenem-resistant
Acinetobacterspp.inEurope.ClinMicrobiolInfect2004,10:684-704.
4. BorcanE,GhiţăCM,ChifiriucMC,MăruţescuL,IsarC,LazărV:Antibiotic Pseudomonasaeruginosawithacquiredmetallo-β-lactamase
determinantsinEuropeanhospital.EmergInfectDis2004,10:535-538.
resistanceofGramnegativebacillistrainsisolatedfromtheIntensive
27. PirelL,MenuteauO,AgoliN,CattoenC,NordmannP:Outbreakof
CareUnitinFundeniClinicalInstitute,Bucharest,Romania.RoumArch
extended-spectrumbeta-lactamasesVEB-1-producingisolatesof
MicrobiolImmunol2009,68:228-234.
AcinetobacterbaumanniiinaFrenchhospital.JClinMicrobiol2003,
5. JosephM,SistlaS,DuttaTK,BadheAS,RasithaD,ParijaS:Ventilator-
41:3542-3547.
associatedpneumoniainatertiarycarehospitalinIndia:roleofmulti-
drugresistantpathogens.JInfectDevCtries2010,4:218-225.
doi:10.1186/2047-2994-1-1
6. BlotS,VandewoudeK,ColardynF:Nosocomialbacteremiainvolving
Citethisarticleas:Shakibaieetal.:Antibioticresistancepatternsand
Acinetobacterbaumanniiincriticallyillpatients:amatchedcohortstudy. extended-spectrumb-lactamaseproductionamongAcinetobacterspp.
IntensiveCareMed2003,29:471-475. isolatedfromanintensivecareUnitofahospitalinKerman,Iran.
7. KimC,LeeY,LeeH,WooJ,SongW,KimM,LeehW,JungSH,LeeK, AntimicrobialResistanceandInfectionControl20121:1.
ChongY:Prevalenceanddiversityofcarbapenemasesamong
imipenem-nonsusceptibleAcinetobacterisolatesinKorea:emergenceof
anovelOXA-182.DiagnMicrobiolInfectDis2010,68:432-438.
8. TakahashiA,YomodAS,KobayashiI,OkuboT,TsunodaM,IyobeS:
Detectionofcarbapenemase-producingAcinetobacterbaumanniiina
hospital.AntimicrobAgentsChemother2000,38:526-529.
9. SinhaM,SrinivasaH,MacadenR:Antibioticresistanceprofile&extended
spectrumbeta-lactamase(ESBL)productioninAcinetobacterspecies.
IndianJMedRes2007,126:63-67.
10. GuillouJ,ValleeE,Bergogne-BerezinE,PhilliponA:Distributionofβ-
lactamaseandphenotypeanalysisinclinicalstrainsofAcinetobacter
calcoaceticus.JAntimicrobAgentsChemother1988,45:22597-22604.
11. PerilliM,FeliciA,OratoreA,CornagliaG,BonfiglioG,RossoliniGM,
AmicosanteG:Characterizationofthechromosomalcephalosporinases
producedbyAcinetobacterlwoffiiandAcinetobacterbaumanniiclinical
isolates.AntimicrobAgentsChemother1996,40:715-719.
12. HenwoodC,GatwardT,WarnerM,JamesD,StockdeleW,SpenceR,
TownerKJ,LivermoreMD,WoodfordN:Antibioticresistanceamong
clinicalisolatesofAcinetobacterintheUKandinvitroevaluationof
tigacycline(GAR-936).JAntimicrobChemother2002,49:479-487.
13. KhosroshahiN,SharifiM:IsolationofA.baumanniiresistantto
carbapenemsfromICUpatients.IranJMedMicrobiol2009,1(3):33-38.
14. FarahaniR,MoniriR,ShajariG,NazemShiraziMH,MusaviG,GhasemiA,Haj
AghazadehS:AntibioticresistanceofAcinetobactersp.isolatedfrom
ShahidBeheshtiHospital,Kashan.Faiz2009,4:60-66[http://feyz.kaums.ac.
ir].
15. JunieE:InterspeciestransformationofAcinetobacter:geneticevidence
foraubiquitousgenus.JBacteriol1972,1112:917-931.
16. BouvetPJ,GrimontPA:Identificationandbiotypingofclinicalisolatesof
Acinetobacter.AnnInstPasteurMicrobiol1987,138:569-578.
17. ClinicalandLaboratoryStandardsInstitute:Performancestandardsfor
antimicrobialsusceptibilitytesting:17 informationalsupplement.
th
Wayne,PA:CLSI2007,M100-S17.
Submit your next manuscript to BioMed Central
18. PatersonDL,BonomoRA:Extended-spectrumB-lactamases:aclinical
update.ClinicalMicrobiolRev2005,1:657-686. and take full advantage of:
19. TzelepiE,GiakkoupiA,SofianouD,LoukovaV,KemeroglouA,TsakrisA:
Detectionofextended-spectrumbeta-lactamasesinclinicalisolatesof • Convenient online submission
EnterobactercloacaeandEnterobacteraerogenes.JClinMicrobiol2000,
38:542-546. • Thorough peer review
20. JiangXF,HanLZ,YuanFY,JiangYF,LiuY,NiY,LuY,HongXH:Double • No space constraints or color figure charges
inhibitorparallelinhibitdisktesttoeffectivelyanalyzeESBLsin
• Immediate publication on acceptance
Enterobacteriaceaeisolates.ChinJLabMed2005,28:1018-1021.
21. CefaiC,RichardsJ,GouldFK,McPeakeP:AnoutbreakofAcinetobacter • Inclusion in PubMed, CAS, Scopus and Google Scholar
respiratorytractinfectionresultingfromincompletedisinfectionof • Research which is freely available for redistribution
ventilatoryequipment.JHospInfect1990,15:177-182.
Submit your manuscript at
www.biomedcentral.com/submit
