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Rapid Identification and Antimicrobial Susceptibility Profiling of Anaerobic Bacteria in Clinical Specimens

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Rapid Identification and Antimicrobial Susceptibility Profiling of Anaerobic Bacteria in Clinical Specimens Egyptian Journal of Medical Microbiology, July 2012 Vol. 21, No. 3 Rapid Identification and Antimicrobial Susceptibility Profiling of Anaerobic Bacteria in Clinical Specimens *Wafaa N. El-tayab, **Fatma Alzhraa M. Gomaa *Associate Professor of Microbiology & Immunology, Faculty of Pharmacy Misr International University, Cairo, Egypt **Associate Professor of Microbiology & Immunology, Faculty of Pharmacy for girls- Al-Azhar University, Egypt ABSTRACT Objective: The aim of the current study was to evaluate the ability of API 20A, and Microscan Rapid- Anaerobe identification system to identify a spectrum of clinically significant anaerobic isolates against the gold standard conventional method. Also to determine the antimicrobial susceptibility of anaerobic isolates using different methods. Materials and Methods: Anaerobes were isolated from the different clinical specimens and all isolates were initially identified by conventional tests. Identification of isolates were made also by using the API 20A and Microscan systems. Antimicrobial susceptibility of all isolates were determined by disc diffusion and broth-disk method and compared to MIC determined by broth dilution method. Results: A total of 51 isolates were recovered from clinical specimens. API 20A and Microscan correctly identified 70.6% and 82.4% of strains to species level respectively. Eight strains were misidentified by API 20A and 9 were misidentified by Microscan. B. fragilis group isolates were the most encountered clinically significant isolates among the gram-negative anaerobes. Penicillin and ampicillin had poor activity against B. fragilis group, Prevotella spp. Eubacterium spp. and Veillonella spp. Members of the B. fragilis group were more resistant to cefoxitin, than other anaerobes (66.6%). Resistance to clindamycin varied among the species range from 11.1% to 50%. Chloramphenicol was effective against all isolates. Fusobacterium spp. were highly susceptible to penicillin and 33.3 % of Fusobacterium isolates were resistant to metronidazole. Clostridium perfringens isolates were susceptible to all agents tested. Clostridium species other than C. perfringens were more resistant than C. perfringens, with 75% of the isolates resistant to penicillin, 12.5% resistant to clindamycin, and 18.7% resistant to metronidazole. Disk diffusion method has correlated well with MIC values but give poor correlation most tested C. perfringens. The overall correlation of the broth-disk results with MIC values was 97.6% in all tests. Conclusions: It was concluded that Microscan RAID was easier to perform and interpret and has the potential to provide rapid identification of anaerobes with an automated reader but more expensive than API. Both system give reliable rapid identification of anaerobes. Our study indicate that there continue to be changes in susceptibility of anaerobes over time and the antimicrobial resistance patterns should be monitored regularly in order to guide empirical therapy. Attention should be paid to standardize broth-disk method as an easy, rapid and most practical to be applicable to routine use for clinical laboratories. INTRODUCTION expensive and is beyond the capabilities of many clinical laboratories. In the past few decades there have been Commercial methods which have gained great advances in the understanding of the role acceptance for identification of anaerobes played by anaerobes in the pathogenesis of include API 20A, and rapid kits detecting severe bacterial infections. The infectious preformed enzymes, such as MicroScan potential of these organisms has been (American Microscan, West Sacramento, CA) clarified(1), and the development of species- and have been developed and are very useful for group-specific differences in antimicrobial anaerobic bacteriology. MicroScan systems susceptibility patterns has been defined(2). mainly detect glycosidases and aminopeptidases Anaerobes isolation and identification are and some other enzymes by means of an important part of the functions of a clinical chromogenic substrates. A heavy inoculum and microbiology laboratory. Conventional short incubation period (4 hours in air) biochemical testing of anaerobes together with minimizes contamination and gives rapid (4) gas-liquid chromatography (GLC) are the most results . The microbiology laboratory without accurate identification method(3), but it has the facilities for conventional anaerobe testing disadvantages of being time consuming and 29 Egyptian Journal of Medical Microbiology, July 2012 Vol. 21, No. 3 requires an accurate and reliable method for information obtained from the primary plates, anaerobe species identification. i.e. gram stain, colony morphology of a pure Routine isolation, identification and isolate, hemolysis on BAP...etc. (9). susceptibility testing of anaerobic bacteria Level II identification: It includes group of present several difficulties leading to defects in testes for identification of anaerobic organisms the determination of local susceptibility patterns to genus level and in some cases to species level which will guide empirical treatment using simple tests as antibiotic identification protocols(5). Over the past 10 years, there has test, growth in presence of 20% oxygall, indole been a significant problem with increasing production, urease activity, sugar fermentation, resistance to antimicrobial agents among lecithinase & lipase reaction, esculin hydrolysis, anaerobic bacteria (6,7). gelatin liquefaction and catalase production. Also, anaerobic bacteria are no longer Anaerobic bacteria were identified according to entirely predictable in their susceptibility to according to Jousimies-Somer et al.(10) and agents that might be selected for empiric Summanen(3) therapy(8). Much effort has been exerted in API system recent years to standardize the techniques for Suspensions of growth were made in antibiotic susceptibility testing. For the most Lombard-Dowell broth to a density of a part, the success of these efforts has been McFarland standard of 3 or greater. Strips were limited to the aerobic and facultative bacteria. inoculated according to manufacturer’s The development of simple procedures for the instructions and incubated for 24 h at 37°C. anaerobes has been more difficult. Reagents were then added, and results were read To this end, the present study aimed to according to manufacturer's instructions. Where evaluate the ability of API 20A, and Microscan interpretation of sugar fermentation tests was Rapid-Anaerobe identification system to difficult, clear positive (yellow) and negative identify a spectrum of clinically significant (purple) wells from the same strip were used for anaerobic isolates against the gold standard comparison. Sugar reactions were not compared conventional method. Also to determine the among strips. Tests were assigned numerical antimicrobial susceptibility of anaerobic isolates values from which a profile number was using different methods. generated. Identification was made by using the API Analytical Profile Index. Numbers which MATERIALS & METHODS did not appear in the index were referred to the firm's computer facilities. For the purpose of Anaerobic isolates this study, supplemental tests were not The anaerobes were isolated from the performed. following clinical specimens: Identification of bacterial isolates using pus/wound/aspirates, blood, tissue and other Microscan system sites. All strains were tested within Microscan Rapid Anaerobe IDentification approximately 1 month of isolation. All isolates panel (RAID) (DADE BEHRING, West were initially identified by conventional tests (3). Sacramento, USA), is an in-vitro kit for Isolation and identification of anaerobic identification of anaerobes isolated from clinical bacteria by conventional biochemical tests: specimens. It consists of 24 dehydrated Specimens were cultured for anaerobic substrates. It is rehydrated and inoculated with bacteria by inoculation of different selective and the bacterial suspension (3-5 McFarland) and non selective media. Colombia Blood agar, incubated aerobically for 4 hrs. Because of the kanamycin-vancomycin blood agar plates Microscan RAID panel detects preformed supplemented with vitamin k and hemin, enzymes, growth of the organism in the panel is bacteroid bile esculin agar and neomycin not necessary. Colombia blood agar were inoculated for Antimicrobial susceptibility testing: isolation of anaerobic bacteria and incubated in Antibiotics anaerobic Gas-Pak jar (BBL Becton Dickinson Metronidazole 5µg/ disc, Clindamycin 2µg/ Microbiology System, Cockeysville, MD, disc, Piperacillin/Tazobactam 110µg/ disc, USA). Primary plates of various media were Ceftrixone 30 µg/ disc, Amoxicillin/Clavulanic examined for different colonies morphologies acid 30 µg/ disc, Cefoxtine 30 µg/ disc, and gram stained smears prepared from these Penicillin 10µg/disc and Chloramphenicol 30µg/ colonies. disc (oxoid). Disks were stored refrigerated in Identification included two levels: bottles containing desiccant. Level I identification: It is a presumptive Antimicrobial susceptibility testing by disc identification of many anaerobes depending on diffusion method: Agar plate disc diffusion 30 Egyptian Journal of Medical Microbiology, July 2012 Vol. 21, No. 3 technique has been used as general method. Broth-Dilution Method for Determining the Columbia blood agar supplemented with Antibiotic Susceptibility of Anaerobic isolates (11) vitamin K1 and hemin was used . MICs were determined by the broth- Determination of MIC by the broth
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