A study on nosocomial pathogens in ICU with special ... · Antimicrobial resistance in nosocomial infections is increasing with both morbidity and mortality greater when infection

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

R.B. Patwardhan, P.K. Dhakephalkar*, K.B. Niphadkar ** & B.A. Chopade+

Department of Microbiology, University of Pune, *Microbial 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 bacterial nosocomial infections are a leading problem in intensivecare units (ICU). Present investigation was undertaken to know antibiotic resistance in Acinetobacterbaumannii and some other pathogens obtained from clinical samples from ICU causing nosocomial infections.Special emphasis was given on plasmid mediated transferable antibiotic resistance in Acinetobacter.Methods: The clinical specimens obtained from ICU, were investigated to study distribution of nosocomialpathogens (272) and their antibiotic resistance profile. Acinetobacter isolates were identified by API2ONEsystem. Antimicrobial resistance was studied with minimum inhibitory concentration (MIC) by doubledilution agar plate method. The plasmid profile of 26 antibiotic resistant isolates of Acinetobacter wasstudied. Curing of R-plasmids was determined in three antibiotic resistant plasmid containing A. baumanniiisolates. Plasmid transfer was studied by transformation.Results: Major 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%) andbronchitis (7.4%). Acinetobacter isolates displayed high level of antibiotic resistance (up to 1024µg/ml) tomost of antibiotics. More than 90 per cent isolates of Acinetobacter were resistant to a minimum of 23 antibiotics.Plasmid profile of Acinetobacter isolates showed presence of 1-4 plasmids. Ethidium bromide cured plasmidspUPI280, pUPI281, pUPI282 with curing efficiencies 20, 16 and 11 per cent respectively while acridineorange cured plasmids pUPI280, pUPI281 with curing efficiencies 7 and 18 per cent retrospectively.Transformation frequency of E. coli HB101 with pUPI281 was 4.3×104 transformants/µg plasmid DNA.Interpretation & conclusions: A. baumannii was found to be associated with urinary tract infections,respiratory tract infections, septicaemia, bacteraemia, meningitis and wound infections. A. baumanniidisplayed higher resistance to more number of antibiotics than other nosocomial pathogens from ICU.Antibiotic sensitivity of A. baumannii cured isolates confirmed plasmid borne nature of antibiotic resistancemarkers. Transfer of antibiotic resistant plasmids from Acinetobacter to other nosocomial pathogens cancreate complications in the treatment of the patient. Therefore, it is very important to target Acinetobacterwhich is associated with nosocomial infections.

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

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

178

Antimicrobial resistance in nosocomial infectionsis increasing with both morbidity and mortality greaterwhen infection is caused by drug resistant organisms1.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 increases2, 3.

Acinetobacter is most frequently isolated bacteriumin clinical specimens. Members of genus 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 andbacteraemia4. Acinetobacter baumannii is thecommonest isolate from Gram-negative sepsis inimmunocompromized patients, posing risk for highmortality5. Outbreaks of Acinetobacter infections arelinked to contaminated respiratory equipment,intravascular access devices, bedding materials andtransmission via hands of hospital personnel6. Duringrecent years, A. baumannii has become a significantpathogen especially in intensive care units7. It typicallycolonizes skin and indwelling plastic devices of thehospitalized patients8. 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 agents9-11.

Multidrug resistance of Acinetobacter isolates is agrowing problem and has been widely reported12.Resistance in Acinetobacter to majority of commerciallyavailable antimicrobials (aminoglycosides,cephalosporins, quinolones and imipenem) raises animportant therapeutic problem13,14. The presence ofresistance plasmids (R-plasmids) is a significant featureof this organism15,16. More than 80 per cent ofAcinetobacter isolates carry multiple indigenousplasmids of variable molecular size17. 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 environment18. InAcinetobacter associated nosocomial infections, themajor problem encountered is the readily transferableantimicrobial resistance expressed by this organism19.

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 CSF (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, Pseudomonasaeruginosa, Staphylococcus aureus and Streptococcuspyogenes were identified on basis of morphological,cultural and biochemical characteristics20. Acinetobacterwas identified on basis of five preliminary tests viz.,Gram staining, capsule staining, motility, oxidase andcatalase tests. Phenotypic identification was performedby biochemical tests21,22. Chromosomal DNAtransformation assay of Juni was used to confirm GenusAcinetobacter23. A. baumannii isolates (26) wereconfirmed by using API2ONE system24.

PATWARDHAN et al: ACINETOBACTER IN NOSOCOMIAL INFECTIONS IN ICU 179

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.coli (HB101), A.calcoaceticus MTCC127, A. calcoaceticus MTCC1271and A. calcoaceticus MTCC1425. Control strains usedfor plasmid profile studies included P. aeruginosa(RIP64 ) , E. coli ( pRK2013), S. typhi (R136 ), E. coliK12 (pBR322), E. coli K 12 (RP4) and E. coli V517provided by Microbial Type Culture Collection(MTCC), Institute of Microbial Technology,Chandigarh, India. Cultures were grown aerobically at370C, with constant shaking at 150 rpm for 16-18 h.

Chemicals and culture media: Antibiotic powders wereobtained from Parke-Davis, Ltd. Mumbai, India.Antibiotic discs, 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.

Determination of resistance to antibiotics: Antibioticresistance profile was determined by Kirby Bauer discdiffusion method on Mueller Hinton (MH) agar plates(Hi-media, Mumbai)25. Discs were consistently testedfor efficacy against standards strains recommended byNational Committee for Clinical Laboratory Standards(NCCLS) 26 as well as others with known antimicrobialsusceptibility pattern. Results were interpreted as percent sensitive (%S) and per cent resistant (%R) isolatesderived using NCCLS26 and WHO breakpoints26,27.

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 method28.It was determined according to NCCLS (nowclinical Laboratory Standards Institute, CLSI)guidelines26,29. Concentration range of each antibioticused was 1 µg/ml to 1024 µg/ml.

Isolation and purification of plasmid DNA: Plasmidisolation was done using modified Kado and Liu30 andSambrook method 31. Standard strains having plasmidsof known molecular weight were run with each set.Cultures were grown aerobically in L-B medium31, at370C, 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 µl E-buffer (20 mM tris-acetate and 2 mM sodium salt ofEDTA, pH 7.9) followed by addition of 200-400 µllysing buffer (3% SDS and 50 mM tris, pH 12.6 adjustedwith 2N NaOH). Heat treatment at 650C 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 precipitation for boththe methods was done with equal volume isopropanol.Plasmid pellet thus obtained was dissolved in 30 µl TE(10 mM tris, 1 mM EDTA, pH 8) buffer. Agarose gelelectrophoresis was performed on 0.8 per cent (w/v)agarose gels prepared in TAE buffer30 (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 al32. The percentage curingefficiency was expressed as number of colonies withcured 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 wasperformed in duplicate.

Plasmid transfer by transformation: HB101 of E. coliwas used as host for transformation experiments.

180 INDIAN J MED RES, AUGUST 2008

Competent cells of E. coli HB101 were prepared usingcalcium chloride method31. Transformation experimentswere performed by “heat shock method”31 using plasmidpUPI281 (Apr, Gmr, Kmr) from A. baumannii A24 andcompetent cells of E. coli HB101 as recipient.Transformation efficiency was calculated as number oftransformants per µg 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 8as A. junii by API20NE 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 (18.6%),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 isolatesof Acinetobacter were found resistant to 23 antibioticscompared to Pseudomonas (15 antibiotics), Klebsiella(11antibiotics) 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 Klebsiella and 43 per centStaphylococcus were resistant to these many antibiotics.Streptococcus was least serious in terms of antibioticresistance. Surprisingly E. coli which can acquire ortransmit R-plasmids very 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 µg/ml from different groupsincluding β 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. baumannii isolates with highantibiotic resistance were identified and tested against27 antibiotics from different groups. A wide range ofconcentrations of antibiotics (1-1024 µg/ml) was testedagainst A. baumannii. Majority of isolates toleratedmore than 512 µg/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 β-lactam antibiotics tested except

Table I. Percentage of nosocomial infections caused by different pathogens in ICU

Bacteria Urinary Septicaemia Respiratory tract infections Wound Bacteraemia Meningitis Totaltract No. (%) infections No. (%) No. (%) number

infections Pneumonia Tuberculosis Bronchitis No. (%)No. (%) No. (%) No. (%) No. (%)

Acinetobacter 22 (18.6) 2(40) — 1(50) 1(5) 7(8.7) 2(14.2) 1(25) 36Pseudomonas 10(8.5) 2(40) 2(6.9) — 1(5) 19(23.7) — 2(50) 36E. coli 60(50.8) 1(20) 3(10.3) — — 9((11.2) — 1(25) 74Klebsiella 26(22) — 8(27.6) — 3(15) 13(16.2) 2(14.2) — 52Staphylococcus — — 5(17.2) 1(50) 5(25) 26(32.5) 10(71.4) — 47Streptococcus — — 11(37.9) — 10(50) 6(7.5) — — 27Total 118 5 29 2 20 80 14 4 272Percentage 43.38 1.84 10.66 0.74 7.35 29.41 5.15 1.5 100


ceftazidime and ceftriazone whereas 54 and 61.6 percent resistance was observed at MIC more than 512µg/ml. Less than 5 per cent isolates could be inhibitedat128 µg/ml in β-lactam group antibiotics. AllA. baumannii isolates were resistant to penicillin andcefuroxime at 512-1024 µg/ml. More than 90 per centisolates were resistant to ampicillin, amoxicillin, andpiperacillin at 512-1024 µg/ml (Fig. 1). Cefuroximeshowed maximum level of resistance in cephalosporingroup. Resistance of Acinetobacter to quinolones wasless as compared to aminoglycosides and β-lactamantibiotics (Fig. 2). 100 per cent resistance was observedto nalidixic acid at 512-1024 µg/ml. More than 80 percent isolates were resistant to ciprofloxacin andnorfloxacin at 512-1024 µg/ml. 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 512µg/ml. High level of resistance (MIC 512-1024) wasdetected for amikacin and streptomycin. For clindamycin92 per cent isolates were resistant at 512-1024 µg/ml.Resistance to tetracycline was high as compared todoxycycline of same group. At 512-1024 µg/mltetracycline more than 96 per cent isolates of A.baumannii were resistant; 65 per cent isolates wereresistant to chloramphenicol at 512-1024 µg/ml inphenolics group (Fig. 4). For erythromycin, 54 per cent

Table II. Determination of degree of antibiotic resistance in clinical pathogenic bacterial isolates

AntibioticPer cent isolates showing antibiotic resistance

Acinetobacter Pseudomonas E. coli Klebsiella Staphylococcus Streptococcusspp. spp. spp. spp. spp.

β lactam:Penicillin 100 92.0 87 83.3 82.4 15.38Ampicillin 96.2 100 80.0 88.9 17.7 00Amoxicillin 100 87.0 70.0 83.3 35.3 35.3Piperacillin 92.3 71.4 80.0 88.9 47.1 38.5Cefotaxime 100 85.7 70.0 88.9 47.1 38.5Ceftazidime 96.2 57.1 80.0 88.9 52.9 38.5Ceftriazone 96.3 92.8 90.0 71.0 47.1 38.5Cefuroxime 100 90.0 90.0 70.0 47.1 38.5Aminoglycosides:Amikacin 96.2 71.4 30.0 38.9 41.2 53.9Gentamycin 96.2 100 80.0 88.9 52.9 77.0Streptomycin 96.2 92.8 70.0 88.9 35.3 46.2Tobramycin 80.8 92.8 80.0 83.3 58.8 38.5Clindamycin 100 85.7 72.0 71.0 52.0 38.5Quinolones:Ciprofloxacin 96.2 78.6 80.0 94.5 47.1 46.2Lomefloxacin 100 78.0 72.0 70.0 43.0 46.2Nalidixic acid 100 100 80.0 88.9 52.9 38.5Norfloxacin 96.2 100 80.0 88.9 52.9 38.5Ofloxacin 96.2 78.5 80.0 83.4 47.1 46.2Sparfloxacin 96.2 50.0 80.0 50.0 17.65 23.1Tetracyclines:Doxycycline 88.5 75.0 80.0 71.0 17.7 22.0Tetracycline 100 100 80.0 77.8 41.2 53.9Phenolics:Chloramphenicol 100 73.0 72.0 77.8 41.2 41.2Others:Erythromycin 100 100 90.0 94.5 58.8 38.5Vancomycin 100 100 92.0 90.0 41.2 38.5Rifampicin 65.4 60.0 40.0 42.0 42.0 46.2Polymyxin B 0 100 90.0 100 70.6 38.5Trimethoprim 100 82.0 73.0 62.0 42.2 41.2


isolates were resistant at 512-1024 µg/ml. For polymyxinB A. baumannii isolates were resistant only up to 128µg/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. Plasmidnumber 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. baumannii A23, A24 and A26 (Table III). EthidiumBromide cured plasmids pUPI280, pUPI281, pUPI282with curing efficiencies 20, 16 and 11 per centrespectively while acridine orange was able to cureplasmids pUPI280, pUPI281 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 A.baumannii and reference strains showed absence ofplasmid on agarose gel electrophoresis which clearlyconfirmed their plasmid elimination.

Plasmid transfer by transformation: Plasmid pUPI281(Apr, Gmr, Kmr) was transferred from A. baumannii A24to E. coli HB101by transformation. Frequency oftransformation of E. coli HB101 with pUPI281 wasobserved to be 4.3×104 transformants/µg plasmid DNA.

Fig. 1. Response of Acinetobacter species to β lactam antibiotics.Antibiotic concentration range used for MIC 0-1024 µg/ml; PG-Penicillin; AM- Ampicillin; Am- Amoxicillin; PC- Piperacillin; CF-Cefotaxime; Ca- Ceftazidime; Ci- Ceftriazone; CB- Cefuroxime.

Fig. 2. Response of Acinetobacter species to quinolone antibioticsRC- Ciprofloxacin; LF- Lomefloxacin; NA- Nalidixic acid; NR-Norfloxacin; OF- Ofloxacin; DC- Sparfloxacin.

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

Fig. 4. Response of Acinetobacter species to different antibioticsDc- Doxycycline; Tc- Tetracycline; CH- Chloramphenicol; ER-Erythromycin; Va- Vancomycin; Rp- Rifampicin; PB- PolymyxinB; Tp- Trimethoprim.


Discussion

There are several reports on outbreaks of multidrugresistant Acinetobacter baumannii in an ICU33-35. In ICUcritically ill patients are always at higher risks ofdeveloping nosocomial infections with antibioticresistant strains. The emergence and spread of multidrugresistant A. baumannii and its genetic potential to carryand transfer diverse antibiotic resistant determinantspose a major threat in hospitals36.

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 cent37. The foremost causes of urinary tractinfections in hospitals are E. coli, P. aeruginosa,Klebsiella, Proteus, Enterococci and Candida38. 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 reports39. Though E. coli was themost predominant organism in causing UTI, it did notdisplay high level of resistance to antibiotics. In a studyby Hsueh et al38, the most frequent isolates from UTIwere Candida spp. (23.6%) followed by E. coli (18.6%)and P. aeruginosa (11%). Singh et al37, showed presenceof E. coli, P. aeruginosa, Proteus mirabilis andEnterococcus faecalis in equal proportion in causingUTI. In the present study Enterococcus and Candidawere not isolated.

Staphylococcus was predominant in causing woundinfections. Other organisms detected werePseudomonas, Klebsiella, E. coli, Acinetobacter andStreptococcus. These results were comparable withprevious findings40,41. 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. lwoffii and A. junii were isolated from upperrespiratory tract of healthy humans42, while in this studyA. baumannii was found to be associated withtuberculosis and bronchitis. In a study by Singh et al37,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

Table III. Curing of R-plasmids in clinical isolates of A. baumannii with EtBr and acridine orange

Plasmid curing agentsBacterial isolates Plasmid cured Antibiotic resistance cured Ethidium Bromide Acridine Orange

SIC Per cent curing SIC Per cent curing(µg/ml) efficiency (µg/ml) efficiency

A. baumannii A23 pUPI280 Apr, Gmr, Kmr, Cmr, Amr 512 20 512 7A. baumannii A24 pUPI281 Apr, Gmr, Kmr 256 16 256 18A. baumannii A26 pUPI282 Apr, Gmr, Kmr, Str Lfr 256 11 256 —E. coli K 12 RP4 Apr, Tcr, Kmr 128 14 64 —E. coli K12 pBR322 Apr, Tcr 128 23 128 14

SIC, Subinhibitory concentration. Total 300 clones tested. —, Below detection limit (none of the 300 clones tested showed curing of plasmid)

Fig. 5. Plasmid profile of standard strains and clinical isolates ofA. baumannii harbouring R-plasmids. Lane 1, A. baumannii A4;Lane 2, A. baumanniiA7; Lane 3, A. baumannii A5; Lane 4, A.baumannii A23; Lane 5, A. baumanniiA24; Lane 6, E. coli (RP4);Lane 7, E. coli K12 (pBR322); Lane 8, Reference plasmids fromE. coli MTCC 131. ; Lane 9, A. baumannii A15; Lane 10, A.baumannii A17; Lane 11, A. baumannii A26; Lane 12, A. baumanniiA11; Lane 13, , E.coli (pRK2013); Lane 14, P. aeruginosa MTCC1262 (RIP64); Lane 15, S. typhi MTCC 1264(R136); Lane 16,standard plasmids from E. coli MTCC 131.Images of gel were captured on Alpha Imager gel documentation system


stream infections along with Staphylococcus suggestspossibilities of sepsis resulting from nosocomialinfections. Kapil43 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 al44 reported outbreak of nosocomial meningitiscaused by A. baumannii in neurosurgical patients.

Acinetobacter is reported for about 10 per cent ofnosocomial infections in ICU patients45. 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 Badrinath46 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 problems47. 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 β-lactam and aminoglycosideantibiotics48,49 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 isolates50. A correlationbetween metal and antibiotic resistance has beenestablished among clinical and environmental isolates28.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, cephazidime, 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 has the abilityto acquire resistance to many major classes ofantibiotics19. Multiple antibiotic resistance inAcinetobacter was reported previously but plasmidborne nature of antibiotic resistance has been reportedonly in a few cases in India16. Clinical isolates ofAcinetobacter harbour plasmids of different molecularsizes ranging from15-56kb48. 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.

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 51.The clinical A. baumannii isolate as well as unrelatedenvironmental A. baumannii isolate had a similarcarbapenem resistance plasmid suggesting spread of thisgenetic character52. A single plasmid which acts asvector of resistance genes can carry a number of genescoding for multiple drug resistance. In the present study,A. baumannii isolates harbouring R-plasmids werefound resistant to multiple antibiotics. Transfer ofantibiotic resistant plasmids to other 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.

AcknowledgmentOne of the authors (RBP) acknowledges University Grants

Commission, for teacher fellowship under Faculty improvementprogramme, Xth plan (F.No.34-8/2003 wro) and the financialsupport.


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Reprint requests: Prof. B.A. Chopade, Director, Institute of Bioinformatics & Biotechnology & Department of MicrobiologyUniversity of Pune, Ganeshkhind, Pune 411 007, Indiae-mail: [email protected], [email protected]


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