Indian J Med Res 128, August 2008, pp 178-187

A study on nosocomial pathogens in ICU with special reference tomultiresistant Acinetobacter baumannii harbouring multipleplasmids

R.B. Patwardhan, PK. Dhakephalkar*, K.B. Niphadkar" & B.A. Chopade*

Department of Microbiology, University of Pune, 'Mierobial Sciences Division, Agharkar ResearchInstitute, "Department of Microbiology, KEM hospital & * Institute of Bioinformatics & Biotechnology &Department of Microbiology, University of Pune, Pune, India

Received April 19, 2007

Background & objectives: Antibiotic resistant bacteria! nosocomial infections are a leading probiem in intensivecare units (ICU). Present investigation was undertaken to know antibiotic resistance in Acinetobacterbaumannii and some other pathogens obtained from cünical samples from ICU causing nosocomial infections.Special emphasis was given on p!asmid mediated transferable antihiotic resistance in Acinetobacter.

Methods: The clinical specimens obtained from ICU, were investigated to study distribution of nosocomia!pathogens (272) and their antibiotic resistance profile. Acinetobacter iso!ates were identified by API2ONEsystem. Antimicrobia! resistance was studied with minimum inhibitory concentration (MIC) by doubledilution agar plate method. The plasmid profile of 26 antibiotic resistant iso!ates of Acinetobacter wasstudied. Curing of R-p!asmids was determined in tbree antibiotic resistant p!asmid containing /4. baumanniiIsolates. Plasmid transfer was studied by transformation.

ResuUs: Majctr infections found in ICU were due to Acinetobacter baumannii, Escherichia coli, Klebsiellapneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus pyogenes. The infectionrate was maximum in urinary tract (44.4%) followed by wound infections (29.4%), pneumonia (10.7%) andbroncbitis (7.4%). Acinetobacter iso!ates displayed high !eve! of antibiotic resistance (up to l()24ng/ml) tomost of antibiotics. More tban 90 per cent i.so!ates of Acinetobacter were resistant to a minimum of 23 antibiotics.P!asmid profile of Acinetobacter iso!ates showed presence of 1-4 plasmids. Ethidium bromide cured p!asmidspUPI280, pUPI281, pUPI282 with curing efTiciencies 20, 16 and U per cent respectively whi!e acridineorange cured plasmids pUPI280, pUP1281 with curing efficiencies 7 and 18 per cent retrospectively.IVaasformation frequency of E. coti HBlOl with pUPI281 was 4.3xW transfomiants/ng plasmid DNA.

Interpretation & conclusions: A. baumannii was found to be associated with urinary tract infections,respiratory tract infections, septicaemia, hacteraemia, meningitis and wound infections. A. baumanniidisplayed higher resistance to more number of antibiotics tban other nosocomia! pathogens from ICU.Antihiotic sensitivity of A. baumannii cured isolates confirmed p!asmid borne nature of antihiotic resistancemarkers. Transfer of antibiotic resistant plasmids from Acinetobacter to otber nosocomial patbogens eancreate complications in the treatment of the patient. Therefore, it is very important to target Acinetobacterwhich is associated with nosocomial infections.

Key words Acinetobaeter baumannii - antibiotic resistance - ICU - nosocomial infections - plasmid curing

178

PATWARDHAN et al: ACINETOBACTER IN NOSOCOMIAL INFLECTIONS IN ICU 179

Antimicrobial resistance in nosocomial infectionsis increasing with both morbidity and mortality greaterwhen infection is caused by drug resistant organisms'.This increase is due to overuse and misuse ofantimicrobial agents, immunosuppressed patients andexogenous transmission of bacteria, usually by hospitalpersonnel. Nosocomial infections are typicallyexogenous, the source being any part of the hospitalecosystem, including people, objects, food, water andair in the hospital. These infections are opportunisticand microorganisms of low virulence can cause diseasein hospital patients whose immune mechanisms areimpaired. The outcome is that many antibiotics can nolonger be used for the treatment of infections causedby such organisms and the threat to the usage of otherdrugs increases'-\

Acinetobacter is most frequently isolated bacteriumin clinical specimens. Members of genu^ Acinetobacterare Gram-negative, non-motile, non-spore formingencapsulated coccobacilli belonging to familyNeisseriaceae. It is an opportunistic pathogen found tobe associated with a wide spectrum of infectionsincluding nosocomial pneumonia, meningitis,endocarditis, skin and soft tissue infections, urinary tractinfections, conjunctivitis, burn wound infections andbacteraemia^. Acinetobacter baumannii is thecommonest isolate from Gram-negative sepsis inimmunocompromized patients, posing risk for highmortality\ Outbreaks of Acinetobacter infections arelinked to contaminated respiratory equipment,intravascuiar access devices, bedding materials andtransmission via hands of hospital personnel''. Duringrecent years, A. baumannii has become a significantpathogen especially in intensive care units^ It typicallycolonizes skin and indwelling plastic devices of thehospitalized patients^ Persistence of endemic A.baumannii isolates in ICU seems to be related to theirability for long-term survival on inanimate surfaces inpatients' immediate environment and their widespreadresistance to the major antimicrobial agents"".

Multidrug resistance of Acinetobacter isolates is agrowing problem and has been widely reported'^.Resistance in Acinetobacter to majority of commerciallyavailable antimicrobials (aminoglycosides,cephalosporins, quinolones and imipenem) raises animportant therapeutic problem'^'-". The presence ofresistance plasmids (R-piasmids) is a significant featureof this organism'"'"'. More than 80 per cent ofAcinetobacter isolates carry multiple indigenousplasmids of variable molecular size'^. The plasmids

present in Acinetobacter can be readily transferredexperimentally to other pathogenic bacteria bytransformation and conjugation. Also Acinetobacteracquires R plasmids from various pathogenic bacteriaas well. Acinetobacter has the capacity to serve as apotential reservoir of transmissible drug resistancegenes especially in nosocomial environment'^ InAcinetobacter associated nosocomial infections, themajor problem encountered is the readily transferableantimicrobial resistance expressed by this organism'^.

The growing number of nosocomial infections andrapid increase in antibiotic resistant Acinetobacterisolates has prompted us to investigate incidence andprevalence of antibiotic resistant Acinetobacter isolatedin 2003 from different clinical samples from ICU ofKEM hospital. Pune, India. Antibiotic resistance pattern,plasmid profile, plasmid curing as well as plasmidtransfer study in A. baumannii isolates were carried outto confirm the plasmid borne nature of antibioticresistant markers.

Material & Methods

Clinical specimens: Bacterial resistance to severalantibiotics was studied in 272 different bacterialpathogens (Gram-positive and Gram-negative) fromclinical samples (urine, pus, sputum, blood, etc.) fromKing Edward Memorial Hospital (KEM Hospital,affiliated to the University of Pune), Pune, fromFebruary 2003 to December 2003. Clinical samplesincluding urine ( 150), sputum (85), pus (64), blood (73),peritoneal fluid (26), Foley's tip (32), abdominalwashing (38), bronchial washings (10), and CSE (5)were collected from variety of patients from intensivecare unit. Clinical samples were investigated to findthe distribution of nosocomial pathogens in causingdifferent opportunistic infections and their antibioticresistance profile.

Identification of Acinetobacter and other nosocomialpathogens: Clinical isolates of Acinetobacter,Escherichia coli. Klebsiella pneumoniae, P.seudomonasaeruginosa, Staphylococcus aureus and Streptococcuspyogenes were identified on basis of morphological,cultural and biochemical characteristics^'^'. Acinetobacterwas identified on basis of five preliminary tests viz..Gram staining, capsule staining, motility, oxidase andcatalase tests. Phenotypic identification was perfonnedby biochemical tes t s - ' - . Chromosomai DNAtransformation assay of Juni was used to confirm GenusAcinetobacter^^. A. baumannii isolates (26) wereconfirmed by using AP12ONE system-''.

180 INDIAN J MED RES, AUGUST 2008

Control strains and culture conditions: Antibioticresistance pattern was studied in 26 isolates of A.baumannii. All the isolates resistant to multipleantibiotics were screened for presence of plasmids.Control strains used for antibiotic resistance includedE.coli (RP4), E.coli (R751), E.coU (HBIOI). A.calcoaceticus MTCC127, A. calcoaceticus MTCC1271and A. calcoaceticus MTCC 1425. Control strains usedfor plasmid profile studies included P. aeruginosa(RIP64 ) , E. coli ( pRK20I3), S. typhi (R136 ), E. coliK12 (pBR322), E. coli K 12 (RP4) and E. coli V517provided by Mierobial Type Culture Collection(MTCC), Institute of Mierobial Technology,Chandigarh, India. Cultures were grown aerobically at37"C, with constant shaking at 150 rpm for 16-18 h.

Chemicals and cultute media: Antibiotic powders wereobtained from Parke-Davis, Ltd. Mumbai, India.Antibiotic dtscs, chemicals and media were purchasedfrom Hi-Media, Mumbai. India. EDTA and otherchemicals used in plasmid isolation and purificationstudies were purchased from Qualigens (India). Cultureswere grown in Luria-Bertani (L-B) broth for allexperiments.

Detennination of resistance to antibiotics: Antibioticresistance profile was determined by Kirby Bauer discdiffusion method on Mueller Hinton (MH) agar plates(Hi-media, Mumbai)-^ Discs were consistently testedfor efficacy against standards strains recommended byNational Committee for Clinical Laboratory Standards(NCCLS) - as well as others with known antimicrobialsusceptibility pattern. Results were interpreted as percent sensitive (%S) and per cent resistant (%R) isolatesderived using NCCLS"*' and WHO breakpoints^''-'.

Determination of minimal inhibitory concentration(MIC) of antibiotics: Antibiotic susceptibility testingof 26 A. baumannii isolates to 27 antibiotics belongingto different groups was carried out on MH agar. MICwas determined by double dilution agar plate method **.It was determined according to NCCLS (nowclinical Laboratory Standards Institute, CLSI)guidelines-''-''. Concentration range of each antibioticused was 1 |xg/ml to 1024 |xg/ml.

Isolation and purification of plasmid DNA: Plasmidisolation was done using modified Kado and Liu'" andSambrook method". Standard strains having plasmidsof known molecular weight were run with each set.Cultures were grown aerobically in L-B medium^', at37"C, 150 rpm for 16-18 h. Following modificationswere included in the standard protocol. In Kado and

Liu's method cell pellet was suspended in 100 il E-buffer (20 mM tris-acetate and 2 mM sodium salt ofEDTA, pH 7.9) followed by addition of 200-400 (illysing buffer (3% SDS and 50 mM tris, pH 12.6 adjustedwith 2N NaOH). Heat treatment at 65"C for 90 minensured complete lysis. There were no modificationsin lysis procedure for Sambrook method. Phenol:chloroform extraction (protein precipitation) was donefor both the methods. Nucleic acid précipitation for boththe methods was done with equal volume isopropanol.Plasmid pellet thus obtained was dissolved in 30 ^1 TE(10 mM tris, 1 mM EDTA, pH 8) buffer. Agarose gelelectrophoresis was perfonned on 0.8 per cent (w/v)agarose gels prepared in TAE buffer'" (40mM trisacetate and 2 mM sodium EDTA. pH 7.9 adjusted withglacial acetic acid). Plasmid profiles were documentedunder UV light in Gel Documentation System (AlphaInnotech Corp., USA).

Determination of molecular weight of plasmid:Molecular weights of plasmids from A. baumanniiisolates were determined by comparing with standardplasmids, pBR322 (4.36 kb), pRK2013 (47 kb). RP4(57 kb), RIP64 (135 kb) and R136 (59 kb). Images ofgels were captured on Alpha Imager gel documentationsystem and molecular weight of test plasmids wasdetermined by comparing them with standard plasmidsusing the software provided in gel documentationsystem. For reproducibility testing, comparison ofplasmids with standard plasmids was done thrice andan average of 2 readings obtained for each isolate wasaffirmed as the final molecular weight of plasmid. V517series of plasmids (E. coli V517, MTCC 131) was usedas plasmid molecular weight standard.

Curing of antibiotic resistance: The plasmid curing wasperformed in A. baumannii A23 (pUPI280), A.baumannii A24 (pUPI281). A. baumannii A26(pUPI282) (all three plasmids identified in presentstudy) and standard plasmid containing strains E. coliK 12 (RP4) and E. coli K12 (pBR322) by method asdescribed by Deshpande et aP-. The percentage curingefficiency was expressed as number of colonies witbcured phenotype per 100 colonies tested. The physicalloss of plasmid in the cured derivative was confirmedby agarose gel electrophoresis of the plasmid DNApreparation of respective cultures. Antibiotic sensitivecured colonies were also tested for loss of resistance toantibiotics by disc diffusion assay. The experiment wasperfonned in duplicate.

Plasmid transfer by transformation: HBlOl of E. coliwas used as host for transformation experiments.

L . i

PATWARDHAN et al: AdNETOBACTER IN NOSOCOMIAL INFECTIONS IN ICU 181

Competent cells of £. coli HBlOl were prepared usingcalcium chloride method*'. Transformation experimentswere performed by "heat shock method"^' using plasmidpUPI281 (Ap'. Gm'. KnV) from A. baumannii A24 andcompetent cells of E. coli HB1Ü1 as recipient.Transformation efficiency was calculated as number oftransformants per tg of plasmid DNA.

Results

Nosocomial infections in intensive care unit: A total of272 bacterial isolates were obtained from clinicalspecimens like blood, urine, pus, sputum, CSF,peritoneal fluid, abdominal washing and Foley'scatheter tube. These were identified as Acinetobacter(36), A. baumannii (28), A. junii (8) E. coli (74), K.pneumoniae (52), P. aeruginosa (36), S. aureus (47)and S. pyogenes (27) (Table I). Maximum numbers ofpathogens were isolated from urine, pus and sputum.E. coli was found to be most predominant isolate foundfrom ICU. Urine was most common source ofAcinetobacter. From 36 isolates of Acinetobacter, 28were identified and confirmed as A. baumannii and 8'às, A. junii hy AP120NE system. Urinary tract infections(43.38%) were most predominant infections (Table I).Other infections detected were septicemia (1.84%),pneumonia (10.66%), wound infections (29.41%),bronchitis (7.35%), tuberculosis (0.74%), bacteraemia(5.15%) and meningitis (1.5%). The commonestorganisms from urinary tract were E. coli (50.8%),followed by Klebsiella (22%). Acinetobacter {\%.6%),and Pseudomonas (8.5%). Staphylococcus aureus wascommonest organism isolated from blood (71.4%)(Table I). The frequent organisms from respiratory tractwere Streptococcus, Klebsiella, Staphylococcus andAcinetobacter. Pseudomonas and Acinetobacter werefound in equal proportion in causing septicaemia. E. coli,Acinetobacter and Pseudomonas were isolated from

CSF specimens. Acinetobacter was isolated from almostall types of nosocomial infections in KEM hospital.

Prevalence of antibiotic resistance in nosocomialinfections: Antibiotic resistance profile revealed thatmajority of bacterial isolates were resistant to multipleantibiotics (27) (Table II). More than 90 per cent isolatesoí Acinetobacter were found resistant to 23 antibioticscompared to Pseudomonas (15 antibiotics), Klebsiella(1 lantibiotics) or E. coli (7 antibiotics). About 94 percent Acinetobacter isolates were found to be resistantto 20 or more antibiotics tested, while only 68 per centPseudomonas, 49 per cent Klehsiella and 43 per centStaphylococcus were resistant to these many antibiotics.Streptococcus was least serious in tenns of antibioticresistance. Surprisingly E. coli which can acquire ortransmit R-plasmids vei'y effectively did not display ahigh level of resistance to antibiotics. More than 90 percent E. coli isolates were resistant to only 7 antibiotics.Resistance was detected more in A. baumannii than inA. junii. All Acinetobacter isolates were resistant to 12antibiotics at 1024 ^g/ml from different groupsincluding ß lactam, aminoglycosides, quinolones andothers. Resistance to antibiotics in Gram-positivebacteria was less as compared to Gram-negativebacteria.

Antibiotic resistance patterns in clinical isolates of A.baumannii: Twenty six A. batanannii isolates with highantibiotic resistance were identified and tested against27 antibiotics from different groups. A wide range ofconcentrations of antibiotics ( I -1024 |Xg/ml) was testedagainst A. baumannii. Majority of isolates toleratedmore than 512 |ig/ml of antibiotic from all the groupsand most showed high level of resistance to multipleantibiotics.

More than 80 per cent isolates of A. baumannii werehighly resistant to ß-lactam antibiotics tested except

Bacteria

AcinetobacterPseudomonasE. coliKtebsiellaStaphytococcusStreptococcusTotalPercentage

Table I. Percentage of nosocomial infections caused by durèrent pathogens in !CU

Urinarytract

infectionsNo. (%)

22 (!8.6)10(8.5)

60(50,8)26(22)

——

!!843.38

SepticaemiaNo. (%) -

2(40)2(40)!(20)

———5

Respiratory tract infections

PneumoniaNo. (%)

—.2(6.9)

3(!0.3)8(27.6)5(17.2)

! I (37.9)29

!0.66

Tuberculosi,'No, {%)

1(50)—— .—

1(50)—

20.74

; BronchitisNo. (%)

1(5)1(5)

.—3(15)5(25)

!0(50)20

7.35

WoundinfectionsNo. (%)

7(8.7)19(23.7)9((ll.2)I3(!6.2)26(32,5)

6(7.5)80

29.4!

BacteraemiaNo. (%)

2(14.2)——

2(14.2)10(71.4)

—•14

5.15

MeningitisNo. (%)

1(25)2(50)1(25)

———4

1.5

Totalnumber

363674524727

272100

!82 INDIAN J MED RES, AUGUST 2008

Antibiotic

ß tactam:PenicillinAmpicillinAmoxicillinPiperacillinCcfotaximeCeftazidimeCeftriazoneCcfuroxime

Aminoglyeosides:AmikacinGentamycinStreptomycinTobramycinClindamycin

Quinolones:CiprofloxacinLometioxacinNaüdixic acidNortloxacinOfloxacinSparfloxacin

Tetracyclines:DoxycyclineTetracycline

Phenolies:Ch!oramphenico!

Others:ErytliromycinVancomycinRifampicinPolymyxin BTrimethopriiii

Table IL Determination

Acinetobacterspp.

!Ü0

96.2100

92.3too

96.296.3!00

96.296.296.280.8100

96.2' !00

10096,296.296.2

88.5100

!00

100100

65.40

!()0

(if degree of antibiotic resistance

Per cent

Pseudomonasspp.

92.0100

87.071.485.757,!92.890.0

71.4!00

92.892.885.7

78.678.0100100

78.550.0

75.0!00

73,0

100!00

60.0!00

82.0

iso!ates sliowing

E. coli

8780.070.080.070.080.090.090.0

30.080.070.080.072.0

80.072.080.080.080.080.0

80.080.0

72.0

90,092.040.090.073,0

in cUnica! pathogenic

antibiotic resistance

Klebsiellaspp.

83.388.983.388.988.988.971.070.0

38.988.988.983.37!.O

94.570.088.988.983.450.0

7!,077.8

77.8

94.590.042.0!00

62.0

bacteria! isolates

Staphylococcusspp,

82.417.735.347.147.152.947.147.1

41.252.935.358.852.0

47.143.052.952.947,!

!7.65

17.741.2

41.2

58.841.242.070.642,2

Streptococcus

spp.

15.3800

35,338.538.538.538.538.5

53,977.046.238.538.5

46.246.238.538.546.223. i

22.053.9

41.2

38.538.546.2

38,54!.2

ceftazidime and ceftriazone whereas 54 and 61.6 percent resistance was observed at MIC more than 512^g/ml. Less than 5 per cent isolates could be inhibitedat 128 |ig/ml in ß-lactam group antibiotics. AllA. baumannii isolates were resistant to penicillin andcefuroxime at 512-1024 pig/ml. More than 90 per centisolates were resistant to ampicillin, amoxicillin, andpiperaciliin at 512-1024 ^ig/ml (Fig. 1). Cefuroximeshowed maximum level of resistance in cephalosporingroup. Resistance of Acinetobacter to quinolones wasless as compared to aminoglycosides and ß-lactamantibiotics (Fig. 2). 100 per cent resistance was observedto naiidixic acid at 512-1024 fig/ml. More than 80 percent isolates were resistant to ciprofloxacin andnorfloxacin at 512-1024 |Xg/mI. Resistance level was

low to ofloxacin and sparfloxacin as compared to otherantibiotics of this group.

Among aminoglycosides, 5 antibiotics were tested(Fig. 3). More than 80 per cent isolates were resistant toaminoglycoside antibiotics except tobramycin where 65.3per cent resistance was observed at MIC more than 512^g/ml. High level of resistance (MIC 512-1024) wasdetected for amikacin and streptomycin. For clindamycin92 per cent isolates were resistant at 512-1024 |J.g/ml.Resistance to tetracycline was high as compared todoxycycline of same group. At 512-1024 [Lg/m\tetracycline more than 96 per cent isolates of A.haumannii were resistant; 65 per cent isolates wereresistant to chloramphenicoi at 512-1024 |ag/ml inphenolies group (Fig. 4). For erythromycin, 54 per cent

PATWARDHAN et al: AdNETOBACTER IN NOSOCOMIAL INFECTIONS IN ICU 183

ipCH ER Va Hf PBOthn andbtoan

O32-128»igirni B128-512ug/ml OS12-1024Mg/ml

Fig. 1. Response of Aeinetobaeter species to ß lactam antibiotics.Antibiotic concentration range used for MIC 0-1024 \ig/m\: PG-Penicillin; AM- Ampicillin; Am- Amoxicillin: PC- Piperacillin; CF-Cefülaxime; Ca- Ceftazidime; Ci- Ceftriazone; CB- Cefuroxime.

^ 1 =

W\\ nConc of

BIlibinlitBIMg'rntfÏIZ 1024

NA NR OF DCOuliiDloneiintIblotIn

'-123|ig<M •128-51 2 M 9 M •512-IO24ugA>il

Fig. 2. Response of Acinetobacter species to quinolone antibioticsRC- Ciprofloxacin; LF- Loniefloxacin; NA- Nalidixic acid: NR-Norfloxacin; OP- Ofloxacin; DC- Spartloxacin.

Coilc o t

JI1iriUI01ICTil|Ut'HÜ^

'12-1024

Fig. 3. Response of Acinetobacter species to aminoglycosideantibiotics. AK- Amikacin; GM- Gentamycin; ST- Streptomycin;TB- Tobramycin; CL- Cündaniycin.

Fig. 4. Response oí Aeinetobaeter species to different antibioticsDc- DoxycycÜne; Tc- Tetracycüne; CH- Chloramphenico!; ER-Eryt!iromycin; Va- Vancomycin; Rp- Rifampicin; PB- Po!ymyxinB; Tp- Trimethoprim.

isolates were resistant at 512-1024 |j.g/ml. For polymyxinB A. baumannii isolates were resistant only up to 128^g/ml. For doxycycline, rifampicin and trimethoprimresistance level was low as compared to other antibiotics.

Plasmid profile in A. baumannii: Multiple plasmidswere found in all isolates of A. baumannii. Plasmidnumher found in 26 isolates of A. baumannii was in therange from 1 to 5. In 9 isolates sharp plasmids wereobserved (Fig. 5). They were used for further geneticexperiments. Sambrook method was found to be bettersince it showed sharp plasmid bands than Kado andLiu method. Molecular sizes of all plasmids ranged from4 to 50kb by comparing with standard plasmids pBR322(4.36 kb), pRK2013 (47 kb), RP4 (57kb), RIP64 ( 135kb)and R136 (59 kb) and E. coli (V517) (Fig. 5).

Curing of antibiotic resistance: Plasmid curing byethidium bromide and acridine orange was detected inA. bautnannii A23, A24 and A26 (Table III). EthidiumBromide cured plasmids pUPI280, pUPI28l, pUP1282with curing efficiencies 20, 16 and 11 per centrespectively while acridine orange was able to cureplasmids pUP1280, pUPI28l with curing efficiencies 7and 18 per cent respectively. Acridine orange was unableto cure plasmid RP4 from E. coli and pUPI282 from A.baumannii A26. The plasmid cured isolates of .4.baumannii and reference strains sbowed absence ofplasmid on agarose gel electrophoresis which clearlyconfirmed their plasmid elimination.

Pkhsmid transfer by transformation: Plasmid pUPI281(Ap^ Gm',, Km') was transferred from A. baumannii A24to E, coli HBlOlby transformation. Frequency oftransformation of E. coli HBlOl with pUPI281 wasobserved to be 4.3x 10"* transformants/fxg plasmid DNA.

184 !ND!AN J MED RES. AUGUST 2008

Bacteria! Íso!ates

A. baumannii A23A. baumannii A24A. haumannii A26E. coli K !2E eoli K!2

Table III. Curing of R-plasmids

P!asmid cured

pUP1280pUPI28tpUPI282RP4pBR322

SIC. SubinhibitoiT concentration. Total

in clinical isolates oí A.

Antibiotic resistance cured

Ap', Gm'Ap'. Gm'Ap'. Gm'Ap'. TC.Ap'. Tc'

(

, Km'. Cm'. Am'.Km^. Km'. Sf LfKm'

3(X) c!ones tested, —. Below detection

baumannii with EtBr and acridine orange

Plasmid curing agents

Elhidium Bromide

S!C|ig/ml)

512256256128128

Per cent curingefficiency

20

!6111423

Acridine

SIC(^g/m!)

512

256256

64128

Orange

Per cent curingefficiency

7

18——14

limit (none of the 300 clones tested showed curing of p!asmid)

Fig. 5. Plasmid profile of standiird strains and dinica! isolates ofA. haumannii harbouring R-plasmids, Lane L A. baumannii A4;Lane 2. A. ImumanniiAl; Lane 3. A. haumannii A5; Lane 4. A.haumannii A23; Lane 5. A. haumanniiA24: Lane 6. E. coli (RP4);Lane 7. E. coti KI2 (pBR322): Lane 8, Reference p!asmids fromE. coli MTCC ! 3 ! . ; Lane 9, A. baumannii A15; Lane 10. A.baumannii A ! 7: l^ne l\,A. baumannii A26; Lane 12, A. haumanniiAll ; Lane 13, ,£.(<>/( (pRK20!3); Lane ]4. P aeruginosa MTCC!262 (RIP64): Lane 15. S, typhi MTCC 1264(R136): Lane 16.standard plasmids from E. eoli MTCC !3I.Images of gel were captured on Alpha Imager gel documentation system

Discussion

There are several reports on outbreaks of multidrugresistant Acinetohacter hautmmnii in an ICU""^^ In ICUcritically ill patients are always at higber risks ofdeveloping nosocomial infections witb antibioticresistant strains. Tbe emergence and spread of multidmgresistant A. battmannii and its genetic potential to carryand transfer diverse antibiotic resistant determinantspose a major threat in hospitals **.

In the present study, most common bacterialpathogens in ICU acquired infections wereAcinetobacter, Pseudomonas, Klebsiella, E. coli,Staphylococcus and Streptococcus. Infection rate washighest in urinary tract followed by wound infections,pneumonia and bronchitis. Urinary tract infection washigher as compared to other studies which ranged from

13 to 19 per cent". The foremost causes of urinary tractinfections in hospitals are E. coli, P. aeruginosa,Klebsiella, Proteus, Enterococci and Candida^'^. In thisstudy total numbers of organisms isolated from urinewere 118. E. coli were most predominant organismsfollowed by Klebsiella, Acinetobacter andPseudomonas. Interestingly percentage ofAcinetobacter causing UTI in present study was muchhigher than previous reports^". Though E. coli was themost predominant organism in causing UTI. it did notdisplay high level of resistance to antibiotics. In a studyby Hsueh et aP^, the most frequent isolates from UTIwere Candida spp. (23.6%) followed by E. coli ( 18.6%)and R aeruginosa ( 11 %). Singh et aP\ showed presenceof E. coli, P. aeruginosa, Proteus mirabilis andEnteroeoccus faecalis in equal proportion in causingUTI. In the present study Entetococcus and Candidawere not isolated.

Staphylococcus was predominant in causing woundinfections. Other organisms detected werePseudomonas, Klebsiella, E. coli, Acinetobacter andStreptococcus. These results were comparable withprevious fmdings- """. Isolation rate of Staphylococcuswas maximum in causing respiratory tract infections(RTIs) in the present study. Other organisms causingRTIs included Streptococcus, Klebsiella, E. coli,Pseudomonas and Acinetobacter. In a previous reportA. Iwoffii and A. junii were isolated from upperrespiratory tract of healthy humans'*-, while in this studyA- baumannii was found to be associated withtuberculosis and bronchitis. In a study by Singh et aP\most frequent isolates causing RTIs were Klebsiella(24.48%), followed by Proteus (18.33%) and E. coli(12.24%).

Other nosocomial infections included bacteraemiasepticaemia and meningitis. In the present study,isolation of Pseudomonas and Acinetobacter in blood

PATWARDHAN et al: AdNETOBACTER IN NOSOCOMIAL INFECTIONS IN ICU 185

stream infections along with Staphylococcus suggestspossibilities of sepsis resulting from nosocomialinfections. Kapil*" has reported outbreak of bacteraemiadue to A. baumannii in leukemia patients in a tertiarycare hospital in Delhi. In our study organisms causingmeningitis were Pseudomonas. followed by equalproportions of Acinetobacter and E. coli. Wroblewskaet aP^ reported outbreak of nosocomial meningitiscaused by A. baumannii in neurosurgical patients.

Acinetobacter is reported for about 10 per cent ofnosocomial infections in ICU patients'*^ In this studyAcinetobacter was isolated in a significant proportionfrom clinical samples in ICU infections, and multidrugresistant Acinetobacter isolates were found to beassociated with almost all types of nosocomialinfections like UTIs, RTIs, .septicaemia, bacteraemia,meningitis and wound infections. In a recent study byPrashanth and Badrinath^^ reported multidrug resistantAcinetobacter responsible for majority of infections.Presence of multidrug resistant plasmid harbouring A.baumannii, causing all types of nosocomial infectionscould lead to therapeutic problems.

All bacterial isolates showed high frequency ofresistance to multiple antibiotics but maximumresistance was observed in Acinetobacter isolates.Acinetobacter isolates have a propensity to readilydevelop resistance to second and third generationantibiotics such as cefotaxime, ciprofloxacin, and givingrise to therapeutic problems'*''. As higher generationantibiotics are being developed to overcome problemof resistance against available antibiotics, bacteria aredeveloping mechanisms to resist newer antimicrobials.In this study A. baumannii isolates showed resistanceto both old and new generation antibiotics.

Member of genus Acinetobacter have been shownto be resistant to ß-lactam and aminoglycosideantibiotics' ' - ''and thought to be a reservoir of antibioticresistant genes in hospital environment. However,Acinetobacter isolated from healthy skin exhibitedhigher susceptibility to antibiotics as compared toclinical and environmental isolates^". A correlationbetween metal and antibiotic resistance has beenestablished among clinical and environmental isolates'^In the present study all isolates of A. baumannii werefound resistant to clinically achievable levels of mostcommonly used antibiotics. For relatively newantibiotics such as broad spectrum cephalosporins(cephotaxime, cephaxidime, ceftriazone) andtobramycin slightly less resistance was observed. Partialsusceptibility was observed for quinolones like

ofloxacin, sparfloxacin, lomefloxacin and otherantibiotics. Maximum susceptibility was detectedagainst polymyxin B. Despite the rising clinicalimportance of A. baumannii compared to othernosocomial pathogens, this organism has been widelyoverlooked.

The major problem encountered by ICU cliniciansrelates to readily transferable antibiotic resistanceexpressed by Acinetobacter. A. baumannii bas the abilityto acquire resistance to many major classes ofantibiotics'**. Multiple antibiotic resistance inAcinetobacter was reported previously but plasmidborne nature of antibiotic resistance has been reportedonly in a few cases in India'^. Clinical isolates ofAcinetobacter harbour plasmids of different molecularsizes ranging froml5-56kb^\ We found plasmids havingmolecular sizes 4-50kb.

Elimination of plasmid from antibiotic resistant A.baumannii and antibiotic sensitivity of A. baumanniicured isolates confirmed plasmid borne nature ofantibiotic resistance markers. In three isolates of A.baumannii, plasmid elimination was observed by usingconventional curing agents like acridine orange andethidium bromide. The cured isolates showed very lowMIC values as compared to original isolates. Physicalloss of plasmid from cured strains showed plasmidborne nature of antibiotic resistance markers. i

Transferable plasmid mediated antibioticresistances poses a great threat as it can achieve muchlarger dimension due to wide and rapid dissemination.This transferable resistance is carried on R-plasmids ^'.The clinical A. haumannii isolate as well as unrelatedenvironmental A. baumannii isolate had a similarcarbapenem resistance piasmid suggesting spread of thisgenetic character". A single plasmid which acts asvector of resistance genes can carry a number of genescoding for multiple drug resistance. In the present study,A. batimannii isolates harbouring R-plasmids werefound resistant to multiple antibiotics. Transfer ofantibiotic resistant plasmids to olher nosocomialpathogens can create complications in the treatment ofpatients. Thus Acinetobacter needs to be considered asan important pathogen and steps must be taken tocontain Acinetobacter nosocomial infections.

Acknowledgment

One of the authors (RBP) acknowledges University GrantsCommission, for teacher feüowship under Faculty improvementprogramme, X"'p!an (F,No-34-8/2ü03 wroj and the financialsupport. I

186 INDIAN J MED RES. AUGUST 2008

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Reprint requests: Prof. B.A. Chopade, Director, !nstitute of Bioinformatics & Biotechnology & Department of MicrobiologyUniversity of Pune, Ganeshkhind, Pune 41 ! 007, Indiae-mai!: directoribb@unipune.ernet.in, chopade@unipune.ernet,in