A study on nosocomial pathogens in ICU with special reference to multiresistant Acinetobacter baumannii harbouring multiple plasmids.
|Article Type:||Clinical report|
Intensive care units
Cross infection (Research)
Nosocomial infections (Research)
Drug resistance in microorganisms (Research)
|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|
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
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.
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.
[FIGURE 1 OMITTED]
[FIGURE 2 OMITTED]
[FIGURE 3 OMITTED]
[FIGURE 4 OMITTED]
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.
[FIGURE 5 OMITTED]
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.
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
(1.) Hosein IK, Hill DW, Jenkins LE, Magee JT. Clinical significance of emergence of bacterial resistance in the hospital environment. Sym Ser Soc J Appl Microbiol, 2002; 31: 90S-7S.
(2.) Courvalin P. Evasion of antibiotic action by bacteria. J Antimicrob Chemother 1996; 37: 855-69.
(3.) Chopra I. Research and development of antibacterial agents. Curr Opin Microbiol 1998; 1 : 495-501.
(4.) Bergogne-Berezin E, Towner KJ. Acinetobacter spp. as nosocomial pathogens: microbiological, clinical and epidemiological features. Clin Microbiol Rev 1996; 9 : 148-65.
(5.) Koprnova J, Svetlansky I, Babel AR, Bilikova E, Hanzen J, Zuscakova IJ. Prospective study of antibacterial susceptibility, risk factors and outcome of 157 episodes of Acinetobacter baumannii bacteremia in 1999 in Slovakia. Scand J Inject Dis 2001; 33 : 891-5.
(6.) Wisplinghoff H, Edmond MB, Pfaller MA, Jones RN, Wenzel RP, Selfert H. Nosocomial blood stream infections caused by Acinetobacter species in United States hospitals: Clinical features, molecular epidemiology and antimicrobial susceptibility. Clin Infect Dis 2000; 31 : 690-7.
(7.) Catalano M, Quelle LS, Jeric PE, Martino AD, Maimone SM. Survival of Acinetobacter baumannii on bed rails during and outbreak and during sporadic cases. J Hosp Inject 1999; 42 : 27-35.
(8.) Larson E. A decade of nosocomial Acinetobacter. Am J Infect Control 1984; 12 : 14-8.
(9.) Jawad A, Seifert H, Snelling AM, Heritage J, Hawkey PM. Survival of Acinetobacter baumannii on dry surfaces: comparison of outbreak and sporadic isolates. J Clin Microbiol 1998; 36 : 1938-41.
(10.) Seifert H, Baginski R, Schulze A, Pulverer G. Antimicrobial susceptibility of Acinetobacter species. J Antimicrob Chemother 1993; 37 : 750-3.
(11.) Gulati S, Kapil A, Dass B, Dwivedi SN, Mahapatra AK. Nosocomial infections due to Acinetobacter baumannii in a neurosurgery ICU. Neurol India 2001; 49 : 134-7.
(12.) Matthew EF, Patra KK, Ioannis AB. The diversity of definitions of multidrug resistant (MDR) and pandrug resistant (PDR) Acinetobacter baumannii and Pseudomonas aeruginosa. J Med Microbiol 2006; 55 : 1619-29.
(13.) Smolyakov R, Borer A, Riesenberg K, Schlaeffer F, Alkan M, Porath A, et al. Nosocomial multidrug resistant Acinetobacter baumannii bloodstream infection: risk factors and outcome with ampicillin-sulbactam treatment. J Hosp Infect 2004; 56 : 165-6.
(14.) Towner KJ. Clinical importance and antibiotic resistance of Acinetobacter spp. Proceedings of a symposium held on 4-5 November 1996 at Eilat. Israel J Med Microbiol. 1997; 46 : 721-46.
(15.) Joshi SG. Assignment of antibiotic resistance to naturally occurring plasmids from clinical isolates of Acinetobacter species. PhD Thesis. University of Pune, Pune, India; 1998.
(16.) Joshi SG, Litake GM, Ghole VS, Niphadkar KB. Plasmid borne extended spectrum [beta] lactamase in a clinical isolate of Acinetobacter baumannii. J Med Microbiol 2003; 52 : 1125-27.
(17.) Gerner-Smidt P. Ferquency of plasmids in strains of Acinetobacter calcoaceticus. J Hosp Infect 1989; 14 : 23-8.
(18.) Chopade BA, Wise PJ, Towner KJ. Plasmid transfer and behaviour in Acinetobacter calcoaceticus EBF65/65. J Gen Microbiol 1985; 131 : 2805-11.
(19.) Bergogne-Berezin E. The increasing role of Acinetobacter species as a nosocomial pathogen. Curr Inject Dis Rep 2001; 3 : 440-4.
(20.) Kloos WE, Schleifer KH. Genus IV Staphylococcus Rosenbach. 1884, 18AL. In: Sneath PHA, Mair NS, Sharpe ME, Holt JG, editors. Bergey's Manual of systematic bacteriology. Baltimore: Williams and Wilkins; 1986. p. 1013-35.
(21.) Bouvet PJM, Grimont PAD. Taxonomy of the Genus Acinetobacter with the recognition of Acinetobacter baumannnii sp. nov., Acinetobacter haemolyticus sp. nov., Acinetobacter johnsonnii sp. nov., and Acinetobacter junii sp. nov., and emended descriptions of Acinetobacter calcoacticus and Acinetobacter lwoffii. Int J Syst Bacteriol 1986; 36 : 228-40.
(22.) Bouvet PJM, Grimont PAD. Identification and biotyping of clinical isolates of Acinetobacter. Ann Inst Pasteur Microbiol 1987; 138 : 569-78.
(23.) Juni E. Interspecies transformation of Acinetobacter: genetic evidence for a ubiquitous genus. J Bacteriol 1972; 112 : 917-31.
(24.) Towner KJ, Chopade BA. Biotyping of Acinetobacter calcoaceticus using the API2ONE system. J Hosp Infect 1987; 10 : 145-51.
(25.) Bauer AW, Kirby WMM, Sherris JC, Turck M. Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol 1996; 45 : 493-6.
(26.) National Committee for Clinical Laboratory Standards (now Clinical and Laboratory Standards Institute, CLSI). Performance standards for antimicrobial susceptibility testing; 12th information supplement (M100-S1). Villanova, PA; NCCLS: 2002.
(27.) Jacques FA, Goldstein FW. Disc susceptibility test, In: Lorian V, editor. Antibiotics in laboratory medicine, 2nd ed. Williams & Wilkins, Baltimore, MD, USA; 1986. p. 27-63.
(28.) Dhakephalkar PK, Chopade BA. High levels of multiple metal resistance and its correlation to antibiotic resistance in environmental isolates of Acinetobacter. BioMetals 1994; 7 : 67-74.
(29.) National Committee for Clinical Laboratory Standards (now Clinical and Laboratory Standards Institute, CLSI). Methods for dilution antimicrobial susceptibility testing for bacteria that grows aerobically. Approved Standards M7-A4. Villanova, PA: NCCLS, 1997.
(30.) Kado CI, Liu ST, Rapid procedure for detection and isolation of large and small plasmids. J Bacteriol 1981 ; 145 : 1365-73.
(31.) Sambrook J, Fritsch EF, Maniatis T. Plasmid vectors. In: Molecular cloning: A laboratory manual, 2nd ed. Cold Spring Harbour: CSH Press. 1989; P.1.1-1.109.
(32.) Deshpande NM, Dhakephalkar PK, Kanekar PP. Plasmid mediated dimethoate degradation in Pseudomonas aeruginosa MCMB-427. Lett Appl Microbiol 2001; 33 : 275-9.
(33.) Pimentel JD, Low J, Styles K, Harris OC, Hughes A, Athan E. Control of an outbreak of multidrug resistant Acinetobacter baumannii in an ICU and a surgical ward. J Hosp Infect 2005; 59 : 249-53.
(34.) Jeong SH, Bae IK, Kwon SB, Lee K, Yong D, Woo GJ, et al. Investigation of a nosocomial outbreak of Acinetobacter baumannii producing PER-1 extended spectrum [beta]-lactamase in an ICU. J Hosp Infect 2005; 59 : 242-8.
(35.) Jeon BC, Jeong SH, Bae IK, Kwon SB, Lee K, Yong D, et al. Investigation of a nosocomial outbreak of imipenem resistant Acinetobacter baumannii producing the OXA-23 [beta]-lactamase in Korea. J Clin Microbiol 2005; 43 : 2241-5.
(36.) Navon VS, Ben AR, Carmeli Y. Update on Pseudomonas aeruginosa and Acinetobacter baumannii infections in the healthcare setting. Curr Opi Infect Dis 2005; 18 : 306-13.
(37.) Singh AK, Sen MR, Anupurba S, Bhattacharya P. Antibiotic sensitivity pattern of bacteria isolated from nosocomial infections in ICU. J Commun Dis 2002; 34 : 257-63.
(38.) Hsueh PR, Teng LJ, Chen CY, Chen WH, Yu CJ, Ho SW, et al. Pan drug-Resistant Acinetobacter baumannii causing nosocomial infections in a university hospital, Taiwan. Emerg Infect Dis 2002; 8: 827-32.
(39.) Chopade BA, Patwardhan RB. Acinetobacter: A potential pathogen of urinary tract infection: A review. Mutropnishad 2002; 1: 165-81.
(40.) Richards DA, Toop LJ, Chambers ST, Sutherland MG, Harris BH, Ikram RB, et al. Antibiotic resistance in uncomplicated urinary tract infection: problems with interpreting cumulative resistance rates from local community laboratories. N Z Med J 2002; 25115 : 12-4.
(41.) Dy ME, Nord JA, LaBombardi VJ, Kislak JW. The emergence of resistant strains of Acinetobacter baumannii: clinical and infection control implications. Infect Control Hosp Epidemiol 1999; 20 : 565-7.
(42.) Patil JR, Jog NR, Chopade BA. Isolation and characterization of Acinetobacter from upper respiratory tract of healthy humans and demonstration of lectin activity. Indian J Med Microbiol 2001; 19:30-5.
(43.) Kapil A. Antibiotics in gram negative sepsis. Trop Gastroenterol 2000; 21 : 95-102.
(44.) Wroblewska MM, Dijkshoorn L, Marchel H, Van Den Barselaar M, Swoboda-Kopec E, Van Den Broek PJ, et al. Outbreak of nosocomial meningitis caused by Acinetobacter baumannii in neurosurgical patients. J Hosp Infect 2004; 57 : 300-7.
(45.) Prashanth K, Badrinath S. In vitro susceptibility pattern of Acinetobacter species to commonly used cephalosporins, quinolones and aminoglycosides, Indian J Med Microbiol 2004; 22 : 97-103.
(46.) Prashanth K, Badrinath S. Nosocomial infections due to Acinetobacter species: Clinical findings, Risk and Prognostic Factors. Indian J Med Microbiol. 2006; 24 : 39-44.
(47.) Jain R, Danziger LH. Multidrug resistant Acinetobacter infections: An emerging challenge to clinicians. Ann Pharmacother2004; 38 : 1449-59.
(48.) Chopade BA, Patwardhan RB, Dhakephalkar PK. Acinetobacter infections in India: Genetic and molecular biological studies and some approaches to the problem. In: Sushil Kumar AK, Sen GPD, Sharma RN, editors. Tropical diseases: Molecular biology and control strategies. New Delhi: CSIR Publications & Information Directorate, 1994. p. 704-17.
(49.) Deshpande LM, Kapadnis BE Chopade BA. Metal resistance in Acinetobacter and its relation to [beta]-lactamase production. BioMetals 1993; 6 : 55-9.
(50.) Pardesi KR, Yavankar SP, Chopade BA. Plasmid distribution and antimicrobial susceptibility patterns of Acinetobacter genospecies from healthy skin of a tribal population in western India. Indian J Med Res 2007; 125: 79-88.
(51.) Kapil A. The challenge of antibiotic resistance: Need to contemplate. Indian J Med Res 2005; 121: 83-91.
(52.) Heritier C, Dubouix A, Poirel L, Marty N, Nordmann P. A nosocomial outbreak of Acinetobacter baumannii isolates expressing the carbapenem hydrolyzing oxacillinase OXA-58. J Antimicrob Chemother 2005; 55: 115-8.
Reprint requests: Prof. B.A. Chopade, Director, Institute of Bioinformatics & Biotechnology & Department of
University of Pune, Ganeshkhind, Pune 411 007, India
e-mail: firstname.lastname@example.org, email@example.com
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)
|Gale Copyright:||Copyright 2008 Gale, Cengage Learning. All rights reserved.|