ACINETOBACTER SPECIES : PHENOTYPIC CHARACTERIZATION AND ANTIMICROBIAL RESISTANCE

Background: Acinetobacter species is ubiquitous, aerobic gram-negative coccobacilli that are now emerging as an important nosocomial pathogen. Objectives: The present study was designed to know the prevalence of Acinetobacter in various clinical samples, their characterization and their antibiotic susceptibility pattern in B.P. Koirala Institute of Health and Sciences, Dharan. Methodology: Hundred Acinetobacter isolates obtained from different clinical specimens were taken. Identification to species level was done according to standard microbiological methods. Antimicrobial susceptibility to 10 antimicrobial agents was performed by Kirby Bauer method with special reference to Minimum inhibitory concentration to meropenem. Result : The predominant Acinetobacter isolate was Acinetobacter calcoaceticus (42%) followed by Acinetobacter baumannii (34%) , Acinetobacter lwoffii (18%) and Acinetobacter junii (6%). Resistance pattern to various drugs were Meropenem (19%), Piperacillin (96%), Piperacillintazobactum (43%) , Amikacin (51%), Ceftazidime (84%), Ceftriaxone (66%), Co-trimoxazole (58%), Gentamicin (57%), Ciprofloxacin (55%), Tetracycline (53%) . Eleven isolates of Acinetobacter were resistant to Meropenem as detected by MIC testing whereas resistant Acinetobacter by disc diffusion technique were 19 in number. Conclusion: Occurrence of Acinetobacter in our hospital as an important clinical isolate is a serious matter of concern. Moreover, its involvement in wide spectrum of diseases and development of resistance to commonly used antimicrobials has further worsened the situation. Prudent use of antimicrobials, effective surveillance of antimicrobial resistance and adherence to infection control practices, perhaps are the key factors that may prevent the development and dissemination of resistance among the local isolates.


Introduction
Members of the genus Acinetobacter have been implicated in a wide spectrum of infectious diseases.Although associated primarily with nosocomial infection, it has also been involved in cases of community acquired infection. 1cinetobacter causes mild to severe illness.The number of nosocomial infections caused by Acinetobacter species has increased in recent years and is of increasing concern in critically ill patients and the risk factors for this infection are not well established. 2Its ubiquitous presence, survival ability and rapid development of resistance to the commonly used antimicrobials are responsible for emergence of this organism as a significant nosocomial and opportunistic pathogen. 3cinetobacter infections have been reported for almost all organ system.It is usually an opportunistic pathogen as evidenced by the fact that 14% to 62% of infections are mixed infections.The most common sites involved are the respiratory and urinary tracts and is common among residents of hospitalized patients. 4In the 1990 to 1992 national nosocomial infections surveillance data, 2% of blood stream infections and 4% of nosocomial pneumonia cases were due to Acinetobacter. 4 Knowledge of the distribution of various species in relation to the variety of infection in hospital setup and to their antimicrobial profile is of utmost importance for effective management of infection caused by the pathogen.
Therefore the study was undertaken to characterize the clinical isolates of Acinetobacter upto species level and to study its antimicrobial susceptibility pattern to commonly used antimicrobials in BP Koirala Institute of Health Science hospital.

Materials and Methods
This study was carried out in the Microbiology unit of Clinical laboratory service (CLS) of B.P. Koirala Institute of Health Sciences hospital from June 2008 to May 2009.One hundred Acinetobacter isolates obtained from blood, pus, urine, corneal scrapings, sputum, aspirates, cerebrospinal fluid, high vaginal swab, catheter tip, peritoneal fluid, wound swab, tissues were taken.
All the samples were subjected to gram staining first except blood and urine.Other clinical specimens were inoculated onto blood and MacConkey agar and incubated at 37 0 C for 24-48 hours.Urine was plated onto Cysteine lactose electrolyte deficient medium.For the blood samples, brain heart infusion broth was used as primary culture medium.After inoculation of the broth incubation, subculture was done on blood agar and MacConkey agar.And was incubated at 37 0 C for 24 hours. 5eliminary identification of Acinetobacters was done by the Gram stain findings, testing for motility and the oxidase reaction in all the samples. 5on-fermenting gram-negative bacilli that were oxidase-negative and nonmotile were identified as Acinetobacter spp.All the isolates of Acinetobacter was identified to species level by using standard microbiological methods which included reaction on triple sugar iron agar, reaction on Sulphite indole motility, urease production, citrate utilization, hemolysis of sheep blood agar, gelatin hydrolysis and growth at 41 0 C and 37 0 C. 6 Antimicrobial susceptibility of all isolates was determined by the standard Kirby Bauer disk diffusion method according to norms of Clinical Laboratory Standards Institute (CSLI).Antibiotics included were Amikacin (30 µg), Ceftriaxone (30 µg), Ceftazidime (30 µg), Ciprofloxacin (5µg), Co-trimoxazole (1.25/23.75µg), Gentamicin (10 µg), Tetracycline (30 µg), Meropenem (10 µg), Piperacillin (100 µg), Piperacillin-tazobactam (100\10µg).Further in vitro susceptibility was determined for meropenem by Minimum Inhibitory Concentration (MIC) with agar dilution method and results were interpreted according to CLSI guidelines.Quality control of susceptibility testing was done by using Pseudomonas aeruginosa (ATCC 27853). 7
Out of 10 antimicrobials tested the isolates were most resistant to piperacillin (96%) and least resistant to meropenem (19%).Antimicrobial susceptibility pattern by Kirby Bauer disc diffusion method exhibited by Acinetobacter is depicted in Table 1.

MIC of Meropenem
Of the 42 isolates of Acinetobacter calcoaceticus, 35 were within or below the susceptible breakpoint for meropenem whereas six were above resistance breakpoint.One fell in the intermediate zone.Similarly 28 isolates of Acinetobacter baumannii were within the susceptible breakpoint, one was in the intermediate zone and five were above the resistance breakpoint.For all 18 isolates of Acinetobacter lwoffi and six isolates of Acinetobacter junii, MIC of meropenem was within the susceptible breakpoint.

Discussion
In the last two decades, Acinetobacter has emerged as a worldwide problem as an important pathogen in hospitalized patients.
Acinetobacter species are often multidrug resistant and associated with life-threatening infections especially in patients with factors that impair normal host resistance. 8In the present study, 100 isolates of Acinetobacter were studied for the characterization and antimicrobial susceptibility.Majority (66 %) were obtained from the admitted patients whereas 14 % were from emergency department and 20 % from outdoor patients.
In our study, 54% of the affected patients were male.Similar pattern was observed in a study conducted in a tertiary care hospital in South India in which male patients formed 58%. 2 Acinetobacter species is a heterogeneous group of organisms which is ubiquitous in nature. 9reater exposure of male to the external environment as compared to female perhaps plays a role in male predominance in acquisition of infections.
Maximum number of Acinetobacter isolates were from blood (25%).Most of our blood isolates were from the cases of neonatal sepsis.Low birth weight, previous antimicrobial therapy, mechanical ventilation, parenteral nutrition and prolonged hospitalization are the known risk factors for bacteremia in such patients. 9 attempt was done to identify all the isolates of genus Acinetobacter upto species level in this study.Four species were encountered at varying frequencies.A. calcoaceticus (42%) was the predominant species, followed by A. baumannii (34%), A. lwoffi and A. junii formed 18% and 6% respectively.This finding is similar to the report published by Communicable Disease Report Weekly . 10edersen et al in 1970 isolated Acinetobacter antitratus in 72 cases, Acinetobacter lwofii in 42 cases. 11Smego et al in 1985 found that 16/25 isolates of Acinetobacter anitratus to be hospital acquired and disease associated and Acinetobacter lwofii in only two cases of bacteremia that was also community acquired. 12inetobacters are known to possess a low potential for virulence.It is their resistance to various antimicrobials, that limits the selection of appropriate drugs for the effective management, thus allowing them to establish themselves as a difficult organism to control and treat. 13ltidrug resistance is well documented phenomenon in clinical strains of Acinetobacter. 14In our study Amikacin and Meropenem showed maximum level of activity with susceptibilities of 49% and 71% respectively.This susceptibility pattern conforms to the recent introduction of these antibiotics in the hospital where the present study was carried out.Increasing resistance to Cephalosporins was observed in our study.The rate of resistance to ceftazidime and ceftriaxone were 87% and 91% respectively.The trend towards resistance to expandedspectrum cephalosporins was also demonstrated by Joly-Guillou et al and seemed to be related to the presence of cephalosporinases. 15However despite such resistance , combination therapy using a third generation Cephalosporin and Amikacin could be the best choice for treating Acinetobacter infections in our set up.
In the present study resistance to meropenem was determined both by disc diffusion and agar dilution method.By agar dilution method, MIC of eleven isolates of Acinetobacter were within the resistant breakpoints, MIC of eighty seven were within the susceptible breakpoints and MIC of two were above the susceptible but below the resistant breakpoint.The isolates found to be resistant by disk diffusion method were found to have MIC range within the susceptible and intermediate breakpoints.Out of nineteen isolates resistant to meropenem by disc diffusion method, seven isolates of Acinetobacter had MIC value ranging from 1-4 µg/ml which is below the recommended MIC breakpoint for resistant isolates and one isolate had MIC value 8 µg/ml.A report from France showed that 17% of Acinetobacter spp. was resistant to meropenem by the agar dilution method. 16Our results were similar to their observation.Low-level resistance to carbapenems as determined by MIC has been reported in several studies. 17,18In a study by Weinbren et al, isolates were detected to be resistant to carbapenem by disk diffusion method and revealed MICs of 0.5-2 µg/ml, which is below the recommended MIC breakpoint for resistant isolates. 18In the study done by Sinha and Srinivasa majority of the isolates resistant to meropenem by disk diffusion method were found to have MICs in the sensitive range which is similar to our study. 19

Conclusion
It can be concluded from the study that Acinetobacter calcoaceticus was the species responsible for majority of Acinetobacter infection in the hospital under study.The increasing trends towards antibiotic resistance reflect the extensive usage of antibiotics in hospitals which in turn exerts selective pressure on Acinetobacter in hospital environment.The infections caused by these organisms are becoming difficult to treat day by day.Effective management and control of Acinetobacter infection does not appear to have a simple answer.Perhaps what is essentially required at this point of time is a multifaceted approach comprising of adherence to good infection control practices, prudent use of antimicrobials and continuous monitoring and surveillance of antimicrobial resistance.