A study on nosocomial pathogens in ICU with special reference to multiresistant Acinetobacter baumannii harbouring multiple plasmids.
Article Type: Clinical report
Subject: Intensive care units (Health aspects)
Cross infection (Research)
Nosocomial infections (Research)
Drug resistance in microorganisms (Research)
Authors: Patwardhan, R.B.
Dhakephalkar, P.K.
Niphadkar, K.B.
Chopade, B.A.
Pub Date: 08/01/2008
Publication: Name: Indian Journal of Medical Research Publisher: Indian Council of Medical Research Audience: Academic Format: Magazine/Journal Subject: Biological sciences; Health Copyright: COPYRIGHT 2008 Indian Council of Medical Research ISSN: 0971-5916
Issue: Date: August, 2008 Source Volume: 128 Source Issue: 2
Topic: Event Code: 310 Science & research
Geographic: Geographic Scope: India Geographic Code: 9INDI India
Accession Number: 188642289
Full Text: Background & objectives: Antibiotic resistant bacterial nosocomial infections are a leading problem in intensive care units (ICU). Present investigation was undertaken to know antibiotic resistance in Acinetobacter baumannii 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 nosocomial pathogens (272) and their antibiotic resistance profile. Acinetobacter isolates were identified by API2ONE system. Antimicrobial resistance was studied with minimum inhibitory concentration (MIC) by double dilution agar plate method. The plasmid profile of 26 antibiotic resistant isolates of Acinetobacter was studied. Curing of R-plasmids was determined in three antibiotic resistant plasmid containing A. baumannii isolates. Plasmid transfer was studied by transformation.

Results: Major infections found in ICU were due to Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus pyogenes. The infection rate was maximum in urinary tract (44.4%) followed by wound infectious (29.4%), pneumonia (10.7%) and bronchitis (7.4%). Acinetobacter isolates displayed high level of antibiotic resistance (up to 1024[micro]g/ml) to most 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 plasmids pUPI280, pUPI281, pUPI282 with curing efficiencies 20, 16 and 11 per cent respectively while acridine orange cured plasmids pUPI280, pUPI281 with curing efficiencies 7 and 18 per cent retrospectively. Transformation frequency of E. coli HB101 with pUPI281 was 4.3x[10.sup.4] transformants/[micro]g plasmid DNA. Interpretation & conclusions: A. baumannii was found to be associated with urinary tract infections, respiratory tract infections, septicaemia, bacteraemia, meningitis and wound infectious. A. baumannii displayed 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 resistance markers. Transfer of antibiotic resistant plasmids from Acinetobacter to other nosocomial pathogens can create complications in the treatment of the patient. Therefore, it is very important to target Acinetobacter which is associated with nosocomial infections.

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

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Antimicrobial resistance in nosocomial infections is increasing with both morbidity and mortality greater when infection is caused by drug resistant organisms (1). This increase is due to overuse and misuse of antimicrobial agents, immunosuppressed patients and exogenous transmission of bacteria, usually by hospital personnel. Nosocomial infections are typically exogenous, the source being any part of the hospital ecosystem, including people, objects, food, water and air in the hospital. These infections are opportunistic and microorganisms of low virulence can cause disease in hospital patients whose immune mechanisms are impaired. The outcome is that many antibiotics can no longer be used for the treatment of infections caused by such organisms and the threat to the usage of other drugs increases (2,3).

Acinetobacter is most frequently isolated bacterium in clinical specimens. Members of genus Acinetobacter are Gram-negative, non-motile, non-spore forming encapsulated coccobacilli belonging to family Neisseriaceae. It is an opportunistic pathogen found to be associated with a wide spectrum of infections including nosocomial pneumonia, meningitis, endocarditis, skin and soft tissue infections, urinary tract infections, conjunctivitis, burn wound infections and bacteraemia (4). Acinetobacter baumannii is the commonest isolate from Gram-negative sepsis in immunocompromized patients, posing risk for high mortality (5). Outbreaks of Acinetobacter infections are linked to contaminated respiratory equipment, intravascular access devices, bedding materials and transmission via hands of hospital personnel (6). During recent years, A. baumannii has become a significant pathogen especially in intensive care units (7). It typically colonizes skin and indwelling plastic devices of the hospitalized patients (8). Persistence of endemic A. baumannii isolates in ICU seems to be related to their ability for long-term survival on inanimate surfaces in patients' immediate environment and their widespread resistance to the major antimicrobial agents (9-11).

Multidrug resistance of Acinetobacter isolates is a growing problem and has been widely reported (12). Resistance in Acinetobacter to majority of commercially available antimicrobials (aminoglycosides, cephalosporins, quinolones and imipenem) raises an important therapeutic problem (13,14). The presence of resistance plasmids (R-plasmids) is a significant feature of this organism (15,16). More than 80 per cent of Acinetobacter isolates carry multiple indigenous plasmids of variable molecular size (17). The plasmids present in Acinetobacter can be readily transferred experimentally to other pathogenic bacteria by transformation and conjugation. Also Acinetobacter acquires R plasmids from various pathogenic bacteria as well. Acinetobacter has the capacity to serve as a potential reservoir of transmissible drug resistance genes especially in nosocomial environment (18). In Acinetobacter associated nosocomial infections, the major problem encountered is the readily transferable antimicrobial resistance expressed by this organism (19).

The growing number of nosocomial infections and rapid increase in antibiotic resistant Acinetobacter isolates has prompted us to investigate incidence and prevalence of antibiotic resistant Acinetobacter isolated in 2003 from different clinical samples from ICU of KEM hospital, Pune, India. Antibiotic resistance pattern, plasmid profile, plasmid curing as well as plasmid transfer study in A. baumannii isolates were carried out to confirm the plasmid borne nature of antibiotic resistant markers.

Material & Methods

Clinical specimens: Bacterial resistance to several antibiotics was studied in 272 different bacterial pathogens (Gram-positive and Gram-negative) from clinical samples (urine, pus, sputum, blood, etc.) from King Edward Memorial Hospital (KEM Hospital, affiliated to the University of Pune), Pune, from February 2003 to December 2003. Clinical samples including urine (150), sputum (85), pus (64), blood (73), peritoneal fluid (26), Foley's tip (32), abdominal washing (38), bronchial washings (10), and CSF (5) were collected from variety of patients from intensive care unit. Clinical samples were investigated to find the distribution of nosocomial pathogens in causing different opportunistic infections and their antibiotic resistance profile.

Identification of Acinetobacter and other nosocomial pathogens: Clinical isolates of Acinetobacter, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus pyogenes were identified on basis of morphological, cultural and biochemical characteristics (20). Acinetobacter was identified on basis of five preliminary tests viz., Gram staining, capsule staining, motility, oxidase and catalase tests. Phenotypic identification was performed by biochemical tests (21,22). Chromosomal DNA transformation assay of Juni was used to confirm Genus Acinetobacter (23). A. baumannii isolates (26) were confirmed by using API2ONE system (24).

Control strains and culture conditions: Antibiotic resistance pattern was studied in 26 isolates of A. baumannii. All the isolates resistant to multiple antibiotics were screened for presence of plasmids. Control strains used for antibiotic resistance included E.coli (RP4), E.coli (R751), E.coli (HB101), A. calcoaceticus MTCC 127, A. calcoaceticus MTCC 1271 and A. calcoaceticus MTCC 1425. Control strains used for plasmid profile studies included P. aeruginosa (RIP64), E. coli (pRK2013), S. typhi (R136), E. coil K12 (pBR322), E. coli K 12 (RP4) and E. coli V517 provided by Microbial Type Culture Collection (MTCC), Institute of Microbial Technology, Chandigarh, India. Cultures were grown aerobically at 37[degrees]C, with constant shaking at 150 rpm for 16-18 h.

Chemicals and culture media: Antibiotic powders were obtained from Parke-Davis, Ltd. Mumbai, India. Antibiotic discs, chemicals and media were purchased from Hi-Media, Mumbai, India. EDTA and other chemicals used in plasmid isolation and purification studies were purchased from Qualigens (India). Cultures were grown in Luria-Bertani (L-B) broth for all experiments.

Determination of resistance to antibiotics: Antibiotic resistance profile was determined by Kirby Bauer disc diffusion method on Mueller Hinton (MH) agar plates (Hi-media, Mumbai) (25). Discs were consistently tested for efficacy against standards strains recommended by National Committee for Clinical Laboratory Standards (NCCLS) (26) as well as others with known antimicrobial susceptibility pattern. Results were interpreted as per cent sensitive (%S) and per cent resistant (%R) isolates derived using NCCLS (26) and WHO breakpoints (26,27).

Determination of minimal inhibitory concentration (MIC) of antibiotics: Antibiotic susceptibility testing of 26 A. baumannii isolates to 27 antibiotics belonging to different groups was carried out on MH agar. MIC was determined by double dilution agar plate method (28). It was determined according to NCCLS (now clinical Laboratory Standards Institute, CLSI) guidelines (26,29). Concentration range of each antibiotic used was 1 [micro]g/ml to 1024 [micro]g/ml.

Isolation and purification of plasmid DNA: Plasmid isolation was done using modified Kado and Liu (30) and Sambrook method (31). Standard strains having plasmids of known molecular weight were run with each set. Cultures were grown aerobically in L-B medium (31), at 37[degrees]C, 150 rpm for 16-18 h. Following modifications were included in the standard protocol. In Kado and Liu's method cell pellet was suspended in 100 [micro]l E-buffer (20 mM tris-acetate and 2 mM sodium salt of EDTA, pH 7.9) followed by addition of 200-400 [micro]l lysing buffer (3% SDS and 50 mM tris, pH 12.6 adjusted with 2N NaOH). Heat treatment at 65[degrees]C for 90 min ensured complete lysis. There were no modifications in lysis procedure for Sambrook method. Phenol: chloroform extraction (protein precipitation) was done for both the methods. Nucleic acid precipitation for both the methods was done with equal volume isopropanol. Plasmid pellet thus obtained was dissolved in 30 [micro]l TE (10 mM tris, 1 mM EDTA, pH 8) buffer. Agarose gel electrophoresis was performed on 0.8 per cent (w/v) agarose gels prepared in TAE buffer (30) (40mM tris acetate and 2 mM sodium EDTA, pH 7.9 adjusted with glacial acetic acid). Plasmid profiles were documented under UV light in Gel Documentation System (Alpha Innotech Corp., USA).

Determination of molecular weight of plasmid: Molecular weights of plasmids from A. baumannii isolates were determined by comparing with standard plasmids, pBR322 (4.36 kb), pRK2013 (47 kb), RP4 (57 kb), RIP64 (135 kb) and R136 (59 kb). Images of gels were captured on Alpha Imager gel documentation system and molecular weight of test plasmids was determined by comparing them with standard plasmids using the software provided in gel documentation system. For reproducibility testing, comparison of plasmids with standard plasmids was done thrice and an average of 2 readings obtained for each isolate was affirmed as the final molecular weight of plasmid. V517 series of plasmids (E. coli V517, MTCC 131) was used as plasmid molecular weight standard.

Curing of antibiotic resistance: The plasmid curing was performed in A. baumannii A23 (pUPI280), A. baumannii A24 (pUPI281), A. baumannii A26 (pUPI282) (all three plasmids identified in present study) and standard plasmid containing strains E. coli K 12 (RP4) and E. coli K12 (pBR322) by method as described by Deshpande et al (32). The percentage curing efficiency was expressed as number of colonies with cured phenotype per 100 colonies tested. The physical loss of plasmid in the cured derivative was confirmed by agarose gel electrophoresis of the plasmid DNA preparation of respective cultures. Antibiotic sensitive cured colonies were also tested for loss of resistance to antibiotics by disc diffusion assay. The experiment was performed in duplicate.

Plasmid transfer by transformation: HB101 of E. coli was used as host for transformation experiments. Competent cells of E. coli HB 101 were prepared using calcium chloride method (31). Transformation experiments were performed by "heat shock method" (31) using plasmid pUPI281 (Apr, Gmr, Kmr) from A. baumannii A24 and competent cells of E. coli HB101 as recipient. Transformation efficiency was calculated as number of transformants per [micro]g of plasmid DNA.

Results

Nosocomial infections in intensive care unit: A total of 272 bacterial isolates were obtained from clinical specimens like blood, urine, pus, sputum, CSF, peritoneal fluid, abdominal washing and Foley's catheter 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 of pathogens were isolated from urine, pus and sputum. E. coli was found to be most predominant isolate found from 1CU. Urine was most common source of Acinetobacter. From 36 isolates of Acinetobacter, 28 were identified and confirmed as A. baumannii and 8 as A. junii by API2ONE 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 commonest organisms from urinary tract were E. coli (50.8%), followed by Klebsiella (22%), Acinetobacter (18.6%), and Pseudomonas (8.5%). Staphylococcus aureus was commonest organism isolated from blood (71.4%) (Table I). The frequent organisms from respiratory tract were Streptococcus, Klebsiella, Staphylococcus and Acinetobacter. Pseudomonas and Acinetobacter were found in equal proportion in causing septicaemia. E. coli, Acinetobacter and Pseudomonas were isolated from CSF specimens. Acinetobacter was isolated from almost all types of nosocomial infections in KEM hospital.

Prevalence of antibiotic resistance in nosocomial infections: Antibiotic resistance profile revealed that majority of bacterial isolates were resistant to multiple antibiotics (27) (Table II). More than 90 per cent isolates of Acinetobacter were found resistant to 23 antibiotics compared to Pseudomonas (15 antibiotics), Klebsiella (11antibiotics) or E. coli (7 antibiotics). About 94 per cent Acinetobacter isolates were found to be resistant to 20 or more antibiotics tested, while only 68 per cent Pseudomonas, 49 per cent Klebsiella and 43 per cent Staphylococcus were resistant to these many antibiotics. Streptococcus was least serious in terms of antibiotic resistance. Surprisingly E. coli which can acquire or transmit R-plasmids very effectively did not display a high level of resistance to antibiotics. More than 90 per cent E. coli isolates were resistant to only 7 antibiotics. Resistance was detected more in A. baumannii than in A. junii. All Acinetobacter isolates were resistant to 12 antibiotics at 1024 [micro]g/ml from different groups including [beta] lactam, aminoglycosides, quinolones and others. Resistance to antibiotics in Gram-positive bacteria was less as compared to Gram-negative bacteria.

Antibiotic resistance patterns in clinical isolates of A. baumannii: Twenty six A. baumannii isolates with high antibiotic resistance were identified and tested against 27 antibiotics from different groups. A wide range of concentrations of antibiotics (1-1024 [micro]g/ml) was tested against A. baumannii. Majority of isolates tolerated more than 512 [micro]g/ml of antibiotic from all the groups and most showed high level of resistance to multiple antibiotics.

More than 80 per cent isolates of A. baumannii were highly resistant to [beta]-lactam antibiotics tested except ceftazidime and ceftriazone whereas 54 and 61.6 per cent resistance was observed at MIC more than 512 [micro]g/ml. Less than 5 per cent isolates could be inhibited at128 [micro]g/ml in [beta]-1actam group antibiotics. All A. baumannii isolates were resistant to penicillin and cefuroxime at 512-1024 [micro]g/ml. More than 90 per cent isolates were resistant to ampicillin, amoxicillin, and piperacillin at 512-1024 [micro]g/ml (Fig. 1). Cefuroxime showed maximum level of resistance in cephalosporin group. Resistance of Acinetobacter to quinolones was less as compared to aminoglycosides and [beta]-1actam antibiotics (Fig. 2). 100 per cent resistance was observed to nalidixic acid at 512-1024 [micro]g/ml. More than 80 per cent isolates were resistant to ciprofloxacin and norfloxacin at 512-1024 [micro]g/ml. Resistance level was low to ofloxacin and sparfloxacin as compared to other antibiotics of this group.

Among aminoglycosides, 5 antibiotics were tested (Fig. 3). More than 80 per cent isolates were resistant to aminoglycoside antibiotics except tobramycin where 65.3 per cent resistance was observed at MIC more than 512 [micro]g/ml. High level of resistance (MIC 512-1024) was detected for amikacin and streptomycin. For clindamycin 92 per cent isolates were resistant at 512-1024 [micro]g/ml. Resistance to tetracycline was high as compared to doxycycline of same group. At 512-1024 [micro]g/ml tetracycline more than 96 per cent isolates of A. baumannii were resistant; 65 per cent isolates were resistant to chloramphenicol at 512-1024 [micro]g/ml in phenolics group (Fig. 4). For erythromycin, 54 per cent isolates were resistant at 512-1024 [micro]g/ml. For polymyxin B A. baumannii isolates were resistant only up to 128[micro]g/ ml. For doxycycline, rifampicin and trimethoprim resistance level was low as compared to other antibiotics.

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Plasmid profile in A. baumannii: Multiple plasmids were found in all isolates of A. baumannii. Plasmid number found in 26 isolates of A. baumannii was in the range from 1 to 5. In 9 isolates sharp plasmids were observed (Fig. 5). They were used for further genetic experiments. Sambrook method was found to be better since it showed sharp plasmid bands than Kado and Liu method. Molecular sizes of all plasmids ranged from 4 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 by ethidium bromide and acridine orange was detected in A. baumannii A23, A24 and A26 (Table III). Ethidium Bromide cured plasmids pUPI280, pUPI281, pUPI282 with curing efficiencies 20, 16 and 11 per cent respectively while acridine orange was able to cure plasmids pUPI280, pUPI281 with curing efficiencies 7 and 18 per cent respectively. Acridine orange was unable to cure plasmid RP4 from E. coli and pUPI282 from A. baumannii A26. The plasmid cured isolates of A. baumannii and reference strains showed absence of plasmid on agarose gel electrophoresis which clearly confirmed their plasmid elimination.

Plasmid transfer by transformation: Plasmid pUPI281 (Apr, Gmr, Kmr) was transferred from A. baumannii A24 to E. coli HB101by transformation. Frequency of transformation of E. coli HB101 with pUPI281 was observed to be 4.3x [10.sup.4] transformants/[micro]g plasmid DNA.

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Discussion

There are several reports on outbreaks of multidrug resistant Acinetobacter baumannii in an ICU (33-35). In ICU critically ill patients are always at higher risks of developing nosocomial infections with antibiotic resistant strains. The emergence and spread of multidrug resistant A. baumannii and its genetic potential to carry and transfer diverse antibiotic resistant determinants pose a major threat in hospitals (36).

In the present study, most common bacterial pathogens in ICU acquired infections were Acinetobacter, Pseudomonas, Klebsiella, E. coli, Staphylococcus and Streptococcus. Infection rate was highest in urinary tract followed by wound infections, pneumonia and bronchitis. Urinary tract infection was higher as compared to other studies which ranged from 13 to 19 per cent (37). The foremost causes of urinary tract infections in hospitals are E. coli, P. aeruginosa, Klebsiella, Proteus, Enterococci and Candida (38). In this study total numbers of organisms isolated from urine were 118. E. coli were most predominant organisms followed by Klebsiella, Acinetobacter and Pseudomonas. Interestingly percentage of Acinetobacter causing UTI in present study was much higher than previous reports (39). Though E. coli was the most predominant organism in causing UTI, it did not display high level of resistance to antibiotics. In a study by Hsueh et al (38), the most frequent isolates from UTI were Candida spp. (23.6%) followed by E. coli (18.6%) and P. aeruginosa (11%). Singh et al (37), showed presence of E. coli, P. aeruginosa, Proteus mirabilis and Enterococcus faecalis in equal proportion in causing UTI. In the present study Enterococcus and Candida were not isolated.

Staphylococcus was predominant in causing wound infections. Other organisms detected were Pseudomonas, Klebsiella, E. coli, Acinetobacter and Streptococcus. These results were comparable with previous findings (40,41). Isolation rate of Staphylococcus was maximum in causing respiratory tract infections (RTIs) in the present study. Other organisms causing RTIs included Streptococcus, Klebsiella, E. coli, Pseudomonas and Acinetobacter. In a previous report A. lwoffii and A. junii were isolated from upper respiratory tract of healthy humans (42), while in this study A. baumannii was found to be associated with tuberculosis and bronchitis. In a study by Singh et al (37), most frequent isolates causing RTIs were Klebsiella (24.48%), followed by Proteus (18.33%) and E. coli (12.24%).

Other nosocomial infections included bacteraemia septicaemia and meningitis. In the present study, isolation of Pseudomonas and Acinetobacter in blood stream infections along with Staphylococcus suggests possibilities of sepsis resulting from nosocomial infections. Kapil (43) has reported outbreak of bacteraemia due to A. baumannii in leukemia patients in a tertiary care hospital in Delhi. In our study organisms causing meningitis were Pseudomonas, followed by equal proportions of Acinetobacter and E. coli. Wroblewska et al (44) reported outbreak of nosocomial meningitis caused by A. baumannii in neurosurgical patients.

Acinetobacter is reported for about 10 per cent of nosocomial infections in ICU patients (45). In this study Acinetobacter was isolated in a significant proportion from clinical samples in ICU infections, and multidrug resistant Acinetobacter isolates were found to be associated with almost all types of nosocomial infections like UTIs, RTIs, septicaemia, bacteraemia, meningitis and wound infections. In a recent study by Prashanth and Badrinath (46) reported multidrug resistant Acinetobacter responsible for majority of infections. Presence of multidrug resistant plasmid harbouring A. baumannii, causing all types of nosocomial infections could lead to therapeutic problems.

All bacterial isolates showed high frequency of resistance to multiple antibiotics but maximum resistance was observed in Acinetobacter isolates. Acinetobacter isolates have a propensity to readily develop resistance to second and third generation antibiotics such as cefotaxime, ciprofloxacin, and giving rise to therapeutic problems (47). As higher generation antibiotics are being developed to overcome problem of resistance against available antibiotics, bacteria are developing mechanisms to resist newer antimicrobials. In this study A. baumannii isolates showed resistance to both old and new generation antibiotics.

Member of genus Acinetobacter have been shown to be resistant to [beta]-lactam and aminoglycoside antibiotics (48,49) and thought to be a reservoir of antibiotic resistant genes in hospital environment. However, Acinetobacter isolated from healthy skin exhibited higher susceptibility to antibiotics as compared to clinical and environmental isolates (50). A correlation between metal and antibiotic resistance has been established among clinical and environmental isolates (28). In the present study all isolates of A. baumannii were found resistant to clinically achievable levels of most commonly used antibiotics. For relatively new antibiotics such as broad spectrum cephalosporins (cephotaxime, cephazidime, ceftriazone) and tobramycin slightly less resistance was observed. Partial susceptibility was observed for quinolones like ofloxacin, sparfloxacin, lomefloxacin and other antibiotics. Maximum susceptibility was detected against polymyxin B. Despite the rising clinical importance of A. baumannii compared to other nosocomial pathogens, this organism has been widely overlooked.

The major problem encountered by ICU clinicians relates to readily transferable antibiotic resistance expressed by Acinetobacter. A. baumannii has the ability to acquire resistance to many major classes of antibiotics (19) Multiple antibiotic resistance in Acinetobacter was reported previously but plasmid borne nature of antibiotic resistance has been reported only in a few cases in India (16). Clinical isolates of Acinetobacter harbour plasmids of different molecular sizes ranging from 15-56kb (48). We found plasmids having molecular sizes 4-50kb.

Elimination of plasmid from antibiotic resistant A. baumannii and antibiotic sensitivity of A. baumannii cured isolates confirmed plasmid borne nature of antibiotic resistance markers. In three isolates of A. baumannii, plasmid elimination was observed by using conventional curing agents like acridine orange and ethidium bromide. The cured isolates showed very low MIC values as compared to original isolates. Physical loss of plasmid from cured strains showed plasmid borne nature of antibiotic resistance markers.

Transferable plasmid mediated antibiotic resistances poses a great threat as it can achieve much larger dimension due to wide and rapid dissemination. This transferable resistance is carried on R-plasmids (51). The clinical A. baumannii isolate as well as unrelated environmental A. baumannii isolate had a similar carbapenem resistance plasmid suggesting spread of this genetic character (52). A single plasmid which acts as vector of resistance genes can carry a number of genes coding for multiple drug resistance. In the present study, A. baumannii isolates harbouring R-plasmids were found resistant to multiple antibiotics. Transfer of antibiotic resistant plasmids to other nosocomial pathogens can create complications in the treatment of patients. Thus Acinetobacter needs to be considered as an important pathogen and steps must be taken to contain Acinetobacter nosocomial infections.

Acknowledgment

One of the authors (RBP) acknowledges University Grants Commission, for teacher fellowship under Faculty improvement programme, Xth plan (F.No.34-8/2003 wro) and the financial support.

Received April 19, 2007

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Reprint requests: Prof. B.A. Chopade, Director, Institute of Bioinformatics & Biotechnology & Department of

Microbiology

University of Pune, Ganeshkhind, Pune 411 007, India

e-mail: directoribb@unipune.ernet.in, chopade@unipune.ernet.in

R.B. Patwardhan, RK. Dhakephalkar, * K.B. Niphadkar ** & B.A. Chopade (+)

Department of Microbiology, University of Pune, * Microbial Sciences Division, Agharkar Research Institute, ** Department of Microbiology, KEM hospital & (+) Institute of Bioinformatics & Biotechnology & Department of Microbiology, University of Pune, Pune, India
Table I. Percentage of nosocomial infections caused by different
pathogens in ICU

Bacteria          Urinary     Septicaemia
                   tract        No. (%)
                 infections
                  No. (%)

Acinetobacter      22(18.6)         2(40)
Pseudoinonas        10(8.5)         2(40)
E. coli            60(50.8)        1 (20)
Klebsiella           26(22)            --
Staphylococcus           --            --
Streptococcus            --            --
Total                   118             5
Percentage            43.38          1.84

Bacteria            Respiratory tract infections

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

Acinetobacter           --           1(50)         1(5)
Pseudoinonas        2(6.9)              --         1(5)
E. coli            3(10.3)              --           --
Klebsiella         8(27.6)              --        3(15)
Staphylococcus     5(17.2)           1(50)        5(25)
Streptococcus     11(37.9)              --       10(50)
Total                   29               2           20
Percentage           10.66            0.74         7.35

Bacteria           Wound      Bacteraemia   Meningitis    Total
                 infections     No. (%)      No. (%)     number
                  No. (%)

Acinetobacter       7(8.7)       2(14.2)       1(25)         36
Pseudoinonas      19(23.7)            --       2(50)         36
E. coli            9(11.2)            --       1(25)         74
Klebsiella        13(16.2)       2(14.2)          --         52
Staphylococcus    26(32.5)      10(71.4)          --         47
Streptococcus       6(7.5)            --          --         27
Total                   80            14           4        272
Percentage           29.41          5.15         1.5        100

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

                    Per cent isolates showing antibiotic resistance
Antibiotic
                   Acinetobacter   Pseudornonas   E. coli   Klebsiella
                       spp.            spp.                    spp.
[beta] lactam:
Penicillin               100           92.0          87        83.3
Ampicillin              96.2            100        80.0        88.9
Amoxicillin              100           87.0        70.0        83.3
Piperacillin            92.3           71.4        80.0        88.9
Cefotaxime               100           85.7        70.0        88.9
Ceftazidime             96.2           57.1        80.0        88.9
Ceftriazone             96.3           92.8        90.0        71.0
Cefuroxime               100           90.0        90.0        70.0

Aminoglycosides:
Amikacin                96.2           71.4        30.0        38.9
Gentamycin              96.2            100        80.0        88.9
Streptomycin            96.2           92.8        70.0        88.9
Tobramycin              80.8           92.8        80.0        83.3
Clindamycin            100.0           85.7        72.0        71.0

Quinolones:
Ciprofloxacin           96.2           78.6        80.0        94.5
Lomefloxacin             100           78.0        72.0        70.0
Nalidixic acid           100            100        80.0        88.9
Norfloxacin             96.2            100        80.0        88.9
Ofloxacin               96.2           78.5        80.0        83.4
Sparfloxacin            96.2           50.0        80.0        50.0

Tetracychnes:
Doxycycline             88.5           75.0        80.0        71.0
Tetracycline             100            100        80.0        77.8

Phenolics:
Chloramphenicol          100           73.0        72.0        77.8

Others:
Erythromycin             100            100        90.0        94.5
Vancomycin               100            100        92.0        90.0
Rifampicin              65.4           60.0        40.0        42.0
Polymyxin B                0            100        90.0         100
Trimethoprim             100           82.0        73.0        62.0

                    Per cent isolates showing
                      antibiotic resistance
Antibiotic
                   Staphylococcus   Streptococcus
                        spp.            spp.
[beta] lactam:
Penicillin              82.4            15.4
Ampicillin              17.7              00
Amoxicillin             35.3            35.3
Piperacillin            47.1            38.5
Cefotaxime              47.1            38.5
Ceftazidime             52.9            38.5
Ceftriazone             47.1            38.5
Cefuroxime              47.1            38.5

Aminoglycosides:
Amikacin                41.2            53.9
Gentamycin              52.9            77.0
Streptomycin            35.3            46.2
Tobramycin              58.8            38.5
Clindamycin             52.0            38.5

Quinolones:
Ciprofloxacin           47.1            46.2
Lomefloxacin            43.0            46.2
Nalidixic acid          52.9            38.5
Norfloxacin             52.9            38.5
Ofloxacin               47.1            46.2
Sparfloxacin            17.7            23.1

Tetracychnes:
Doxycycline             17.7            22.0
Tetracycline            41.2            53.9

Phenolics:
Chloramphenicol         41.2            41.2

Others:
Erythromycin            58.8            38.5
Vancomycin              41.2            38.5
Rifampicin              42.0            46.2
Polymyxin B             70.6            38.5
Trimethoprim            42.2            41.2

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

                     Plasmid
Bacterial isolates   cured     Antibiotic resistance cured

A. baumannii A23     pUP1280   [Ap.sup.r], [Gm.sup.r], [Km.sup.r],
                                 [Cm.sup.r], [Am.sup.r]
A. baumannii A24     pUP1281   [Ap.sup.r], [Gm.sup.r], [Km.sup.r]
A. baumannii A26     pUP1282   [Ap.sup.r], [Gm.sup.r], [Km.sup.r],
                                 [St.sup.r] [Lf.sup.r]
E. coli K 12         RP4       [Ap.sup.r], [Tc.sup.r], [Km.sup.r]
E. coli K 12         pBR322    [Ap.sup.r], [Tc.sup.r]

                          Plasmid curing agents

                            Ethidium Bromide
Bacterial isolates
                         SIC        Per cent curing
                     ([micro]/ml)     efficiency
A. baumannii A23         512              20

A. baumannii A24         256              16
A. baumannii A26         256              11

E. coli K 12             128              14
E. coli K 12             128              23

                         Plasmid curing agents

                            Acridine Orange
Bacterial isolates
                          sic        Per cent curing
                     ([micro]g/ml)     efficiency
A. baumannii A23          512               7

A. baumannii A24          256              18
A. baumannii A26          256              --

E. coli K 12               64              --
E. coli K 12              128              14

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