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

A study on nosocomial pathogens in ICU with special reference to multiresistant Acinetobacter baumannii harbouring multiple plasmids R.B. Patwardhan, PK. Dhakephalkar*, K.B. Niphadkar" & B.A. Chopade*

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

Received April 19, 2007

Background & objectives: Antibiotic resistant bacteria! nosocomial infections are a leading probiem in intensive care units (ICU). Present investigation was undertaken to know antibiotic resistance in Acinetobacter baumannii and some other pathogens obtained from c端nical samples from ICU causing nosocomial infections. Special emphasis was given on p!asmid mediated transferable antihiotic resistance in Acinetobacter. Methods: The clinical specimens obtained from ICU, were investigated to study distribution of nosocomia! pathogens (272) and their antibiotic resistance profile. Acinetobacter iso!ates were identified by API2ONE system. Antimicrobia! resistance was studied with minimum inhibitory concentration (MIC) by double dilution agar plate method. The plasmid profile of 26 antibiotic resistant iso!ates of Acinetobacter was studied. Curing of R-p!asmids was determined in tbree antibiotic resistant p!asmid containing /4. baumannii Isolates. Plasmid transfer was studied by transformation. ResuUs: Majctr 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 infections (29.4%), pneumonia (10.7%) and broncbitis (7.4%). Acinetobacter iso!ates displayed high !eve! of antibiotic resistance (up to l()24ng/ml) to most of antibiotics. More tban 90 per cent i.so!ates of Acinetobacter were resistant to a minimum of 23 antibiotics. P!asmid profile of Acinetobacter iso!ates showed presence of 1-4 plasmids. Ethidium bromide cured p!asmids pUPI280, pUPI281, pUPI282 with curing efTiciencies 20, 16 and U per cent respectively whi!e acridine orange cured plasmids pUPI280, pUP1281 with curing efficiencies 7 and 18 per cent retrospectively. IVaasformation frequency of E. coti HBlOl with pUPI281 was 4.3xW transfomiants/ng plasmid DNA. Interpretation & conclusions: A. baumannii was found to be associated with urinary tract infections, respiratory tract infections, septicaemia, hacteraemia, meningitis and wound infections. A. baumannii displayed higher resistance to more number of antibiotics tban other nosocomia! pathogens from ICU. Antihiotic sensitivity of A. baumannii cured isolates confirmed p!asmid borne nature of antihiotic resistance markers. Transfer of antibiotic resistant plasmids from Acinetobacter to otber nosocomial patbogens ean create complications in the treatment of the patient. Therefore, it is very important to target Acinetobacter which is associated with nosocomial infections. Key words Acinetobaeter baumannii - antibiotic resistance - ICU - nosocomial infections - plasmid curing 178


PATWARDHAN et al: ACINETOBACTER IN NOSOCOMIAL INFLECTIONS IN ICU

Antimicrobial resistance in nosocomial infections is increasing with both morbidity and mortality greater when infection is caused by drug resistant organisms'. 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'-\ Acinetobacter is most frequently isolated bacterium in clinical specimens. Members of genu^ 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^. Acinetobacter baumannii is the commonest isolate from Gram-negative sepsis in immunocompromized patients, posing risk for high mortality\ Outbreaks of Acinetobacter infections are linked to contaminated respiratory equipment, intravascuiar access devices, bedding materials and transmission via hands of hospital personnel''. During recent years, A. baumannii has become a significant pathogen especially in intensive care units^ It typically colonizes skin and indwelling plastic devices of the hospitalized patients^ 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"". Multidrug resistance of Acinetobacter isolates is a growing problem and has been widely reported'^. Resistance in Acinetobacter to majority of commercially available antimicrobials (aminoglycosides, cephalosporins, quinolones and imipenem) raises an important therapeutic problem'^'-". The presence of resistance plasmids (R-piasmids) is a significant feature of this organism'"'"'. More than 80 per cent of Acinetobacter isolates carry multiple indigenous plasmids of variable molecular size'^. The plasmids

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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'^ In Acinetobacter associated nosocomial infections, the major problem encountered is the readily transferable antimicrobial resistance expressed by this organism'^. 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 CSE (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, P.seudomonas aeruginosa, Staphylococcus aureus and Streptococcus pyogenes were identified on basis of morphological, cultural and biochemical characteristics^'^'. Acinetobacter was identified on basis of five preliminary tests viz.. Gram staining, capsule staining, motility, oxidase and catalase tests. Phenotypic identification was perfonned by biochemical t e s t s - ' - . Chromosomai DNA transformation assay of Juni was used to confirm Genus

Acinetobacter^^. A. baumannii isolates (26) were confirmed by using AP12ONE system-''.


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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.coU (HBIOI). A. calcoaceticus MTCC127, A. calcoaceticus MTCC1271 and A. calcoaceticus MTCC 1425. Control strains used for plasmid profile studies included P. aeruginosa (RIP64 ), E. coli ( pRK20I3), S. typhi (R136 ), E. coli K12 (pBR322), E. coli K 12 (RP4) and E. coli V517 provided by Mierobial Type Culture Collection (MTCC), Institute of Mierobial Technology, Chandigarh, India. Cultures were grown aerobically at 37"C, with constant shaking at 150 rpm for 16-18 h. Chemicals and cultute media: Antibiotic powders were obtained from Parke-Davis, Ltd. Mumbai, India. Antibiotic dtscs, 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. Detennination of resistance to antibiotics: Antibiotic resistance profile was determined by Kirby Bauer disc diffusion method on Mueller Hinton (MH) agar plates (Hi-media, Mumbai)-^ Discs were consistently tested for efficacy against standards strains recommended by National Committee for Clinical Laboratory Standards (NCCLS) -^ 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"*' and WHO breakpoints^''-'. 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^**. It was determined according to NCCLS (now clinical Laboratory Standards Institute, CLSI) guidelines-''-''. Concentration range of each antibiotic used was 1 |xg/ml to 1024 |xg/ml. Isolation and purification of plasmid DNA: Plasmid isolation was done using modified Kado and Liu'" and Sambrook method". Standard strains having plasmids of known molecular weight were run with each set. Cultures were grown aerobically in L-B medium^', at 37"C, 150 rpm for 16-18 h. Following modifications were included in the standard protocol. In Kado and

L.i

Liu's method cell pellet was suspended in 100 ^il Ebuffer (20 mM tris-acetate and 2 mM sodium salt of EDTA, pH 7.9) followed by addition of 200-400 (il lysing buffer (3% SDS and 50 mM tris, pH 12.6 adjusted with 2N NaOH). Heat treatment at 65"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 prĂŠcipitation for both the methods was done with equal volume isopropanol. Plasmid pellet thus obtained was dissolved in 30 ^1 TE (10 mM tris, 1 mM EDTA, pH 8) buffer. Agarose gel electrophoresis was perfonned on 0.8 per cent (w/v) agarose gels prepared in TAE buffer'" (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 aP-. The percentage curing efficiency was expressed as number of colonies witb 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 perfonned in duplicate. Plasmid transfer by transformation: HBlOl of E. coli was used as host for transformation experiments.


PATWARDHAN et al: AdNETOBACTER IN NOSOCOMIAL INFECTIONS IN ICU

Competent cells of £. coli HBlOl were prepared using calcium chloride method*'. Transformation experiments were performed by "heat shock method"^' using plasmid pUPI281 (Ap'. Gm'. KnV) from A. baumannii A24 and competent cells of E. coli HB1Ü1 as recipient. Transformation efficiency was calculated as number of transformants per ^tg 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 ICU. Urine was most common source of Acinetobacter. From 36 isolates of Acinetobacter, 28 were identified and confirmed as A. baumannii and 8 'às, A. junii hy AP120NE system. Urinary tract infections (43.38%) were most predominant infections (Table I). Other infections detected were septicemia (1.84%), pneumonia (10.66%), wound infections (29.41%), bronchitis (7.35%), tuberculosis (0.74%), bacteraemia (5.15%) and meningitis (1.5%). The commonest organisms from urinary tract were E. coli (50.8%), followed by Klebsiella (22%). Acinetobacter {\%.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

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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 oí Acinetobacter were found resistant to 23 antibiotics compared to Pseudomonas (15 antibiotics), Klebsiella (1 lantibiotics) 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 Klehsiella and 43 per cent Staphylococcus were resistant to these many antibiotics. Streptococcus was least serious in tenns of antibiotic resistance. Surprisingly E. coli which can acquire or transmit R-plasmids vei'y 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 ^g/ml from different groups including ß 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. batanannii isolates with high antibiotic resistance were identified and tested against 27 antibiotics from different groups. A wide range of concentrations of antibiotics ( I -1024 |Xg/ml) was tested against A. baumannii. Majority of isolates tolerated more than 512 |ig/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 ß-lactam antibiotics tested except

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

Acinetobacter Pseudomonas E. coli Ktebsiella Staphytococcus Streptococcus Total Percentage

Urinary tract infections No. (%)

Septicaemia No. (%) -

22 (!8.6) 10(8.5) 60(50,8) 26(22) — — !!8 43.38

2(40) 2(40) !(20) — — — 5

Respiratory tract infections Pneumonia Tuberculosi,'; Bronchitis No. (%) No, {%) No. (%) —. 2(6.9) 3(!0.3) 8(27.6) 5(17.2) ! I (37.9) 29 !0.66

1(50) — — . — 1(50) — 2 0.74

1(5) 1(5) .— 3(15) 5(25) !0(50) 20 7.35

Wound infections No. (%)

Bacteraemia No. (%)

Meningitis No. (%)

Total number

7(8.7) 19(23.7) 9((ll.2) I3(!6.2) 26(32,5) 6(7.5) 80 29.4!

2(14.2)

1(25) 2(50) 1(25) — — — 4 1.5

36 36 74 52 47 27 272 100

— — 2(14.2) 10(71.4) —• 14 5.15


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Table IL Determination (if degree of antibiotic resistance in cUnica! pathogenic bacteria! isolates Per cent iso!ates sliowing antibiotic resistance Antibiotic

Streptococcus

Klebsiella spp.

Staphylococcus spp,

87 80.0 70.0 80.0 70.0 80.0 90.0 90.0

83.3 88.9 83.3 88.9 88.9 88.9 71.0 70.0

82.4 17.7 35.3 47.1 47.1 52.9 47.1 47.1

15.38 00 35,3 38.5 38.5 38.5 38.5 38.5

71.4 !00 92.8 92.8 85.7

30.0 80.0 70.0 80.0 72.0

38.9 88.9

7!.O

41.2 52.9 35.3 58.8 52.0

53,9 77.0 46.2 38.5 38.5

96.2 !00 100 96,2 96.2 96.2

78.6 78.0 100 100 78.5 50.0

80.0 72.0 80.0 80.0 80.0 80.0

94.5 70.0 88.9 88.9 83.4 50.0

47.1 43.0 52.9 52.9 47,! !7.65

46.2 46.2 38.5 38.5 46.2 23. i

88.5 100

75.0 !00

80.0 80.0

7!,0 77.8

17.7 41.2

22.0 53.9

!00

73,0

72.0

77.8

41.2

41.2

100 100 65.4 0 !()0

100 !00 60.0 !00 82.0

90,0 92.0 40.0 90.0 73,0

94.5 90.0 42.0 !00 62.0

58.8 41.2 42.0 70.6 42,2

38.5 38.5 46.2

Pseudomonas spp.

E. coli

96.2 96.3 !00

92.0 100 87.0 71.4 85.7 57,! 92.8 90.0

96.2 96.2 96.2 80.8 100

Acinetobacter

spp.

spp.

ß tactam: Penicillin Ampicillin Amoxicillin Piperacillin Ccfotaxime Ceftazidime Ceftriazone Ccfuroxime

!Ü0

96.2 100 92.3

too

Aminoglyeosides: Amikacin Gentamycin Streptomycin Tobramycin Clindamycin Quinolones: Ciprofloxacin Lometioxacin Naüdixic acid Nortloxacin Ofloxacin Sparfloxacin Tetracyclines: Doxycycline Tetracycline Phenolies: Ch!oramphenico! Others: Erytliromycin Vancomycin Rifampicin Polymyxin B Trimethopriiii

'

ceftazidime and ceftriazone whereas 54 and 61.6 per cent resistance was observed at MIC more than 512 ^g/ml. Less than 5 per cent isolates could be inhibited at 128 |ig/ml in ß-lactam group antibiotics. All A. baumannii isolates were resistant to penicillin and cefuroxime at 512-1024 pig/ml. More than 90 per cent isolates were resistant to ampicillin, amoxicillin, and piperaciliin at 512-1024 ^ig/ml (Fig. 1). Cefuroxime showed maximum level of resistance in cephalosporin group. Resistance of Acinetobacter to quinolones was less as compared to aminoglycosides and ß-lactam antibiotics (Fig. 2). 100 per cent resistance was observed to naiidixic acid at 512-1024 fig/ml. More than 80 per cent isolates were resistant to ciprofloxacin and norfloxacin at 512-1024 |Xg/mI. Resistance level was

88.9 83.3

38,5 4!.2

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 ^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 |J.g/ml. Resistance to tetracycline was high as compared to doxycycline of same group. At 512-1024 [Lg/m\ tetracycline more than 96 per cent isolates of A. haumannii were resistant; 65 per cent isolates were resistant to chloramphenicoi at 512-1024 |ag/ml in phenolies group (Fig. 4). For erythromycin, 54 per cent


PATWARDHAN et al: AdNETOBACTER IN NOSOCOMIAL INFECTIONS IN ICU

CH

ER

Va

Hf

PB

183

ip

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

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

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

W\\ n

^

1= NA NR OuliiDloneiintIblotIn

OF

Fig. 4. Response oí Aeinetobaeter species to different antibiotics Dc- DoxycycÜne; Tc- Tetracycüne; CH- Chloramphenico!; EREryt!iromycin; Va- Vancomycin; Rp- Rifampicin; PB- Po!ymyxin B; Tp- Trimethoprim.

Conc of BIlibinlitBIMg'rntf ÏIZ 1024

DC

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

Fig. 2. Response of Acinetobacter species to quinolone antibiotics RC- Ciprofloxacin; LF- Loniefloxacin; NA- Nalidixic acid: NRNorfloxacin; OP- Ofloxacin; DC- Spartloxacin.

Coilc o t JI1iriUI01ICTil|Ut'HÜ^ '12-1024

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

Plasmid profile in A. baumannii: Multiple plasmids were found in all isolates of A. baumannii. Plasmid numher 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. bautnannii A23, A24 and A26 (Table III). Ethidium Bromide cured plasmids pUPI280, pUPI28l, pUP1282 with curing efficiencies 20, 16 and 11 per cent respectively while acridine orange was able to cure plasmids pUP1280, pUPI28l 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 .4. baumannii and reference strains sbowed absence of plasmid on agarose gel electrophoresis which clearly confirmed their plasmid elimination. Pkhsmid transfer by transformation: Plasmid pUPI281 (Ap^ Gm',, Km') was transferred from A. baumannii A24 to E, coli HBlOlby transformation. Frequency of transformation of E. coli HBlOl with pUPI281 was observed to be 4.3x 10"* transformants/fxg plasmid DNA.


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Table III. Curing of R-plasmids in clinical isolates oí A. baumannii with EtBr and acridine orange Plasmid curing agents Bacteria! Íso!ates

A. baumannii A23 A. baumannii A24 A. haumannii A26 E. coli K !2 E eoli K!2

P!asmid cured Antibiotic resistance cured

pUP1280 pUPI28t pUPI282 RP4 pBR322

Ap', Gm' , Km'. Cm'. Am' Ap'. Gm' .Km^ Ap'. Gm' . Km'. Sf Lf Ap'. TC. Km' Ap'. Tc'

Elhidium Bromide

Acridine Orange

S!C ( |ig/ml)

Per cent curing efficiency

SIC (^g/m!)

Per cent curing efficiency

512 256 256 128 128

20 !6 11 14 23

512 256 256 64 128

18 — — 14

7

SIC. SubinhibitoiT concentration. Total 3(X) c!ones tested, —. Below detection limit (none of the 300 clones tested showed curing of p!asmid)

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

Discussion There are several reports on outbreaks of multidrug resistant Acinetohacter hautmmnii in an ICU""^^ In ICU critically ill patients are always at higber risks of developing nosocomial infections witb antibiotic resistant strains. Tbe emergence and spread of multidmg resistant A. battmannii and its genetic potential to carry and transfer diverse antibiotic resistant determinants pose a major threat in hospitals^**. 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". The foremost causes of urinary tract infections in hospitals are E. coli, P. aeruginosa, Klebsiella, Proteus, Enterococci and Candida^'^. 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^". 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 aP^, the most frequent isolates from UTI were Candida spp. (23.6%) followed by E. coli ( 18.6%) and R aeruginosa ( 11 %). Singh et aP\ showed presence of E. coli, P. aeruginosa, Proteus mirabilis and Enteroeoccus faecalis in equal proportion in causing UTI. In the present study Entetococcus 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 fmdings-^""". 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. Iwoffii and A. junii were isolated from upper respiratory tract of healthy humans'*-, while in this study A- baumannii was found to be associated with tuberculosis and bronchitis. In a study by Singh et aP\ most frequent isolates causing RTIs were Klebsiella (24.48%), followed by Proteus (18.33%) and E. coli (12.24%). Other nosocomial infections included bacteraemia septicaemia and meningitis. In the present study, isolation of Pseudomonas and Acinetobacter in blood


PATWARDHAN et al: AdNETOBACTER IN NOSOCOMIAL INFECTIONS IN ICU

stream infections along with Staphylococcus suggests possibilities of sepsis resulting from nosocomial infections. Kapil*" 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 aP^ 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'*^ 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^^ 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'*''. 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 Ă&#x;-lactam and aminoglycoside antibiotics'^'^-^''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^". A correlation between metal and antibiotic resistance has been established among clinical and environmental isolates'^ 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, cephaxidime, ceftriazone) and tobramycin slightly less resistance was observed. Partial susceptibility was observed for quinolones like

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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 bas the ability to acquire resistance to many major classes of antibiotics'**. 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'^. Clinical isolates of Acinetobacter harbour plasmids of different molecular sizes ranging froml5-56kb^\ 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. i 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 ^'. The clinical A. haumannii isolate as well as unrelated environmental A. baumannii isolate had a similar carbapenem resistance piasmid suggesting spread of this genetic character". 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. batimannii isolates harbouring R-plasmids were found resistant to multiple antibiotics. Transfer of antibiotic resistant plasmids to olher 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 feĂźowship under Faculty improvement programme, X"'p!an (F,No-34-8/2Ăź03 wroj and the financial support. I


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



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