Biomedical Research

Abstract

Acinetobacter spp. are Gram negative coccobacilli causing various nosocomial infections. They possess different types of Beta-lactamases like SHV, TEM and others which lead to treatment failure in case of infections due to this pathogen. Studying their species distribution in various infections and drug resistance pattern is hence important. Hence our study was aimed to study the different infections caused by Acinetobacter spp, in a tertiary care hospital in Eastern India and its antibiotic susceptibility pattern.

Keywords

Acinetobacter, Nosocomial, Resistance.

Introduction

Acinetobacter spp. are Gram Negative, strictly aerobic, non-fastidious, non-fermenting encapsulated coccobacilli causing mostly nosocomial infections. According to most recent scientific literature, Acinetobacter spp. are the second most common non-fermenting Gram negative pathogen isolated from clinical samples after Pseudomonas aeruginosa [1]. They are oxidative in metabolism and coccoid in stationary phase of culture [2]. The genus comprises several species, important among which are A. baumannii group, A. lwoffii, A. johnsonii, A. junii, A. hemolyticus and at least 25 genomospecies [3]. Acinetobacter spp. are important causes of device-related infections and urinary tract infections, but in recent years have also been isolated from bloodstream and other sites, and are notorious for resistance to Beta-lactam antibiotics. The spread of Multidrug resistant Acinetobacter strains among hospitalized patients has become an increasing cause of concern [4]. Hence it is important to look for novel classes of antibiotics which are effective in treating infection due to Acinetobacter spp.

Materials and Methods

The study was conducted from June 2010 to May 2011 in the Department of Microbiology, Bankura Sammilani Medical College, Bankura, West Bengal, India.

One Hundred (100) isolates of Acinetobacter spp. were recovered from various clinical specimens, namely pus (23 samples), urine (54 samples), CSF (12 samples), throat swab (4 samples), vaginal swab (1 sample) and unspecified swab samples (6 samples). Only the isolates grown from samples of patients developing various symptoms after admission in different wards of the hospital for more than 48 hours (Nosocomial isolates) were selected for the study. The isolates were speciated and their antimicrobial resistance pattern was studied. The samples received in the laboratory were inoculated on 5% Sheep Blood Agar and Mac Conkey Agar and incubated overnight aerobically at both 37◦C (to isolate Acinetobacter spp. other than A. johnsonii) and 25◦C (to isolate A. johnsonii, respectively). Thereafter species identification and in-vitro antibiotic susceptibility tests were performed. In case of urine samples, the isolates were subjected to biochemical tests and antimicrobial susceptibility only if the colony count was significant (> 10⁵ CFU/ml). Acinetobacter spp. were identified by characteristic colonies (Non Lactose-fermenting, glistening, small mucoid colonies), Gram staining pattern and standard biochemical reactions [1] (Catalase, Oxidase, Hugh-Leifson’s Oxidation- Fermentation test, Indole production, Citrate utilization, Motility, Urease activity, Reaction in Triple Sugar Iron medium), as shown in Table 1.

After identification by phenotypic methods, antibiotic susceptibility was performed for each isolate by the Kirby-Bauer disc diffusion method on Mueller-Hinton Agar (4) using 0.5 MacFarland Turbidity standard and comparing zone sizes with Control strain P. aeruginosa ATCC 27853. The following antibiotic discs were used:-

Amikacin (30 μg), Penicillin G(10 μg), Piperacillin- Tazobactum(100/10μg), Cefotaxime(30μg), Ceftazidime (30μg) , Gatifloxacin(10μg) , Levofloxacin(5μg), Ciprofloxacin( 5 μg), Cotrimoxazole(25μg), Tetracycline(30μg) (in case of samples other than urine) and Nitrofurantoin (300 μg) (for urine samples only). Susceptibility results were interpreted by measuring the zone diameters produced and correlating them with the CLSI standards [5]. ESBL production was tested using the Double-Disc approximation method using Ceftazidime and Ceftazidime- Clavulinic Acid discs [6].

Results

Among the 100 isolates, 54% (54 isolates) belonged to the A. baumannii group, 44% (44 isolates) were A. lwoffii and 2% (2 isolates) were identified as A. hemolyticus based on biochemical reactions already mentioned. A. johnsonii was not isolated from any of the samples. All the isolates were of nosocomial origin as mentioned before. Acinetobacter spp. formed about 17% of all nosocomial bacterial isolates from all clinical samples processed during the study period, as observed from a record of 100 bacterial isolates recovered from various samples during the same period, and about 33.3% of all bacterial isolates from the I.C.U.

Mean age of the patients was 27 years. Gender ratio was 1.46:1 (Male:female). Thus, a slight male preponderance was observed in our study.

Almost all the isolates showed in-vitro resistance to one or more of the antibiotics mentioned earlier. Among the isolates, 29% (29 isolates) were MDR or Multi-Drug resistant (resistance to 3 or>3 different classes of antibiotics) , with 79.3% of total MDR strains belonging to A. baumannii group(23 isolates). Among A. baumannii isolates, 44% of were MDR ones.

Amikacin resistance was found in 55.5% isolates, while the figures for Penicillin G, Piperacillin-Tazobactum, Cotrimoxazole, Levofloxacin, Ofloxacin, Gatifloxacin, Ciprofloxacin, Cefotaxime, Ceftazidime, Imipenem and Nitrofurantoin were 100%, 18.2%, 71.8%, 21.42%, 58.82%, 6.75%, 52.17%, 75.2%, 80%, 15% and 64.28% respectively. Thus there was less in-vitro resistance to Levofloxacin compared to other quinolones as also to Imipenem, and very negligible resistance to Gatifloxacin. Beta-lactams showed reduced efficacy while Amikacin and Piperacillin-Tazobactum were slightly effective. Approximately 78.5% of all the isolates were ESBL producers, determined as per the double disc approximation method [6].

Discussion

Acinetobacter spp., fast emerging as agents of opportunistic nosocomial infection with evolving drug resistance, have become a real problem in hospital set-up, particularly in the critical care units. Moreover it often shows variable Gram reaction with pleomorphism and may mimic many common Gram positive cocci [7]. The pathogen was previously conferred various names like Micro coccus calcoaceticus, Mima polymorpha and Herellea vaginicola. The genus Acinetobacter has undergone significant modification in nomenclature over the last 30 years. The current genus designation, Acinetobacter (derived from the Greek “akinetos”, meaning non-motile) was initially proposed in 1954[8]. The genus now comprises about 28 species, which include A. baumannii, A. calcoaceticus, A. johnsonii, A. junii, A. ursingii and A. hemolyticus among others. There are many genomospecies in addition to the species mentioned. A. calcoaceticus has been recovered from soil and water samples only and never implicated in clinical disease. Hence the nomenclature A. calcoaceticus-baumannii group is no longer acceptable [8]. Acinetobacter spp. are mostly implicated in various nosocomial infections like respiratory tract infections, bloodstream infections, wound infections, urinary tract infections, meningitis and rarely keratitis and other infections [8]. According to our findings, the pathogen was mostly isolated from urine(54% , i.e. 54 isolates) followed by pus(23%, i.e. 23 isolates), CSF(12%,. i.e. 12 isolates) and other samples(11%, i.e. 11 isolates). Acinetobacter spp. have been implicated in about 10% of all ICU infections in Europe [9]. In our experience, the pathogen constituted 16.9% of all nosocomial bacterial isolates. Infection is facilitated by the ability of the bacterium to colonise hospital equipment and to persist on inanimate surfaces for prolonged periods of time ranging from 3 days to 5 months, and Acinetobacter spp. can be detected on various equipment including bedrails, curtains, ventilation equipments (e.g. AMBU bags, Ventilation filter) [8]. Colonisation of patients, health care workers and healthy individuals occurs frequently. Several virulence factors like lipases and siderophores have been studied [10]. Management of Acinetobacter infections is a huge challenge because of the broad array of antimicrobial resistance and the pathogen’s ability to develop new resistance rapidly. Different resistance patterns have been found even with proven clonal isolates of Acinetobacter spp. in the nosocomial setting. Antimicrobial agents that are typically active against the pathogen include the Carbapenems( Imipenem and Meropenem), Amikacin, Sulbactum, Colistin, Rifampin and Tetracyclines. Combination therapy can be considered, but is controversial due to no proven improvement in mortality and increased toxicity [8]. Acinetobacter baumannii strains inherently possess chromosomally encoded AmpC cephalosporinases that mediate resistance to Cephalosporins. Aminoglycosidemodifying enzymes are highly prevalent in multi drugresistant A. baumannii strains. Resistance to Quinolones is mediated by modifications in DNA gyrase or Topoisomerase IV while that to Tetracyclines occurs via efflux pumps or Ribosomal protection [7]. Resistance has been associated with an 86 kb chromosomal region or resistance island, that is responsible for production of resistance to a large number of antimicrobial agents [7]. Multidrug resistance(MDR), i.e. resistance to Cefotaxime, Ceftazidime, Amikacin and Ciprofloxacin [11], is an emerging problem with Acinetobacter spp. Pan drug resistant A. baumannii isolates, i.e. isolates resistant to all antimicrobial agents in-vitro, have been reported from Asia and the Middle-east [8]. In our study, 29% of the isolates were Multi-drug resistant. About 44.4% of A. baumannii isolates were multidrug resistant. Studies have quoted isolation of Multi-drug resistant Acinetobacter spp. from Indian and Asian hospitals. In a review comparing hospitals of 10 Asian countries, 1.2-87% of all Acinetobacter isolates from patients with Hospital Acquired Pneumonia( HAP) were MDR, with MDR strains most prevalent in India and Thailand [12].

In a study from Pune, about 48% to 68.6% A. baumannii isolates were MDR[13]. In a report from 48 European hospitals from 2002 to 2004, 32.4%, 34% and 47.6% isolates showed susceptibility to Ceftazidime, Ciprofloxacin and Gentamicin respectively[9]. In Asia and the Middleeast, rates of non-susceptibility are about 40% for Ceftazidime, 35% for Amikacin and 45% for Ciprofloxacin[9]. We found the corresponding figures for these groups of antimicrobials to be 80%, 55.5% and 52.17% respectively. Thus in our study, slightly higher values were recorded for third generation Cephalosporins and Ciprofloxacin respectively. Acinetobacter spp. constituted about 33.3% of all bacterial isolates and 25% of all microbial isolates from I.C.U. samples, according to our study.

Colistin, also referred as Polymyxin E, typically retains activity against Acinetobacter spp. in the face of broadspectrum antimicrobial resistance. However, it can be nephrotoxic and ototoxic which limits its routine use, though the toxicity has been found to be similar to other antimicrobials in the ICU set-up [8].

Notably, our findings show that Levofloxacin is effective against Acinetobacter spp.(21.42% resistance), while 6.25% isolates were resistant to Gatifloxacin. Another study has reported Levofloxacin resistance in Acinetobacter baumanii in the order of 26% [14]. Thus Levofloxacin, a S-enantiomer of Ofloxacin, being safer and better tolerated than other fluoroquiniolones [15], can be a lesser toxic and cost-effective therapeutic option against Acinetobacter isolates, especially in this part of India. Carbapenem resistance is an emerging problem with Acinetobacter spp. Only a few centres like I.M.S., B.H.U., Varanasi and A.I.I.M.S.,New Delhi, have mentioned Meropenem resistance in Acinetobacter spp. to be about 6.4% and 22.16% respectively in their studies[16,17]. One report from the U.S.A has quoted Imipenem resistance in Acinetobacter baumannii in the order of 23.1% in their study[18]. A similar study from India mentions that about 14.2% of A. baumannii isolates in their study were Imipenem resistant [19]. Acinetobacter spp., in general show greater in vitro resistance to Meropenem than Imipenem [16,17]. Our results show that about 15% of all isolates of Acinetobacter spp. were resistant to Imipenem. Another important finding of our study is that ESBL production was found to be of the order of 78.5%, which is in concordance with results from a study in Meerut, North India which has shown this figure to be about 75% [20]. However, studies from Bangalore, South India, have reported a low prevalence of ESBLs in Acinetobacter spp.(28%) [21]. There are only a handful of reports in the literature regarding the prevalence of ESBLs in Acinetobacter spp. from India, especially Eastern India, and our study thus merits mention.

Limitations of the study

The sample size (100)was not very large and hence the species distribution as mentioned here needs to be correlated with studies taking large number of isolates. Also, the in-vitro resistance of Acinetobacter spp. to Colistin was not studied, especially in the I.C.U., which could be epidemiologically important.

Acknowledgements

The authors are indebted to Mr. Soumitra Roy and Mr. S.P.Dhabal for making timely provisions of culture media and helping in interpreting susceptibility results.

References

1. Gautam V, Singhal L, Ray P. Burkholoderia cepacia complex: Beyond Pseudomonas and Acinetobacter. Ind J Med Microbiol 2011; 29: 4-12.
2. Allen DM, Hartman BJ. Acinetobacter species. In: Mandell GL, Bennett JE, Dolin R, editors. Principles and Practice of Infectious Diseases. Churchill Livingstone. 6th ed. .Philadelphia 2005; pp. 2632-2636.
3. Winn W(Junior), Allen S, Janda W, Koneman E, Procop G, Schreckenberger P, Woods G. The nonfermentative Gram Negative Bacilli. In: Koneman’s Colour Atlas and Textbook of Diagnostic Microbiology. Lippincott Williams and Wilkins. 6th ed..Philadelphia 2006; pp. 353-354.
4. Dijkshoorn L, Nemec A, Seifert H. An increasing threat in hospitals: multi-drig resistant Acinetobacter baumannii. Nat Rev Microbiol 2007; 5: 939-951.
5. Bauer AW, Kirby WMM, Sherris JC, Turck M Antibiotic susceptibility testing by a standardized single disc method. Am J Clin Pathol 1966: 45: 493.
6. Bradford PA. Extended spectrum β-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clin Microbiol Rev 2001; 14: 933-951.
7. Murray CK, Hospental DR. Acinetobacter infection in the ICU. Critic Care Clin 2008; 24: 237-248.
8. Wayne PA: Clinical LaboratoryStandards Institute . Performance Standards for Antimicrobial Susceptibility testing; 17th Informational supplement, 2006; CLSI M2-A9.
9. Peleg AY, Seifert H, Paterson DL. Acinetobacter baumannii: emergence of a successful pathogen. Clin Microbiol Rev 2008: 21: 538-582.
10. Govan JRW. Pseudomonas and Non-fermenters. In: Greenwood D, Slack R, Peutherer J, Barer M, editors. Medical Microbiology. A Guide to Microbial infections: Pathogenesis, Immunity, Laboratory Diagnosis and Control. Churchill Livingstone Elsevier: 17th ed.London 2007; pp. 298.
11. Bergogne-Berezin E, Towner KJ. Acinetobacter spp. as nosocomial pathogens: Microbiological, Clinical and Epidemiological Features. Clin Microbiol Rev 1996; 9: 148-163.
12. Lagamayo EN. Antimicrobial resistance in major pathogens of hospital acquired pneumonia in Asian countries. Am J Inf Control 2008: 36: S101-S108.
13. Joshi SG, Litake GM, Satpute MG, Telang NV, Ghole VS, Niphadkar KB. Clinical and demographic features of infection caused by Acinetobacter species. Ind J Med Sc 2006; 60: 351-360.
14. Heinemann B, Wisplinghoff H, Edmond M, Seifert H. Comparative activities of Ciprofloxacin, Clinafloxacin, Gatifloxacin, Gemifloxacin, Levofloxacin, Moxifloxacin and Trovafloxacin against epidemiologically defined Acinetobacter baumannii strains. Antimicrob Agents Chemother 2000; 44: 2211-2213.
15. Stahlmann R. Clinical toxicological aspects of fluoroquinolones. Toxicol Lett 2002; 127: 269-277.
16. Gaur A, Gang A, Prakash P, Anupurba S, Mohapatra TM. Observations on Carbapenem Resistance by Minimum Inhibitory Concentration in Nosocomial Isolates of Acinetobacter species. J Health Popul Nutr 2008; 26: 183-188.
17. Gupta E, Mohanty S, Sood S, Dhavan B, Das BK, Kapil A. Emerging resistance to carbapenems in a tertiary care hospital in north India. Ind J Med Res 2006; 124: 95-98.
18. Lautenbach E, Synnestvedt M, Weiner MG, Bilker WB, Vo L, Schein J, Kim M. Epidemiology and impact of imipenem resistance in Acinetobacter baumannii. Inf C Hosp Epidemiol 2009; 30: 1186-1192.
19. Gladstone P, Rajendran P, Brahmadathan KN. Incidence of carbapenem resistant nonfermenting gram negative bacilli from patients with respiratory infections in the intensive care units. Ind J Med Microbiol 2005; 23: 189-191.
20. Kansal R, Pandey A, Asthana AK. β-lactamase producing Acinetobacter species in hospitalized patients. Ind J Pathol Microbiol 2009; 52: 456-457.
21. Sinha M, Srinivasa H, Macaden R. Antibiotic resistance profile & extended spectrum beta-lactamase (ESBL) production in Acinetobacter species. Ind J Pathol Microbiol 2007; 126: 63-67.