Egyptian Journal of Medical Microbiology, January 2007 Vol. 16, No. 1

Transcription

1 Chromagar Staph aureus Versus Blood Agar and Mannitol Salt Agar for Isolation and Identification of Staphylococcus aureus from Suppurative Skin Lesions Wafaa M.K.Bakr and Heba S.Selim Microbiology Department, High Institute of Public Health, Alexandria University Chromagar Staph aureus (CHROMagar Company, Paris, France) is a chromogenic agar medium proposed for the detection of Staphylococcus aureus by incorporation of a chromatic substrate into a suitable isolation medium and then detection of the activities of a specific bacterial enzymes by color changes thus eliminating the need for time consuming and costly biochemical identification. To evaluate this medium, a of 105 suppurative skin lesion swabs were cultured onto CHROMagar Staph aureus agar, blood agar (gold standard) and mannitol salt agar. After 24 h of incubation a of 83 S. aureus strains were recovered on blood agar. Chromagar Staph aureus succeded to isolate 82 strains with mauve color in the first 24 h with a sensitivity of % and a specificity of %,with no further isolation after extending the incubation time to 48 h. As regards mannitol salt agar, 43 (51.8%) strains were recovered as yellow colonies after 24 h of incubation compared with 61(73.4%) strains after incubation for 48 h. These results were associated with a sensitivity of 50.60% after 24 reaching up to 73.49% after 48 h. We conclude that CHROMagar Staph aureus compared favorably to conventional media for rapid detection of S. aureus in clinical samples and achieved a higher sensitivity and specificity than mannitol salt agar for the isolation and presumptive identification of S. aureus from suppurative skin lesions. Key words: Chromagar Staph aureus, Blood Agar,Mannitol Salt Agar, Staphylococcus aureus INTRODUCTION Staphylococcus aureus (S. aureus ) is an important and frequent cause of of skin and soft tissue infection and is consequently one of the most common pathogens sought in clinical microbiology laboratories. (1) Reliable and rapid methods to identify these organisms are crucial in any clinical laboratory. (2,3) Diagnosis of S. aureus skin infection is generally performed by culture of swabs onto nonspecific media and confirmation of suspected colonies by biochemical and/or serological tests. (4) Most commonly, this involves testing colonies of staphylococci for the coagulation of plasma, the fermentation of mannitol (mannitol salt agar [MSA]), the production of thermostable nuclease (DNase), egg yolk lipase hydrolysis (lipovitellin-salt-mannitol agar [LSM]), and the production of natural pigment. (2,3,5) Colonies of S. aureus may be atypical and difficult to differentiate from coagulase staphylococci,a large number of agglutination tests may be required to rule out the presence of S. aureus. (6) A number of culture media have been developed to increase the specificity of S. aureus detection, including mannitol-salt agar and Baird-Parker medium. (7,8) A more recent approach has been the use of CHROMagar Staph aureus, which employs chromogenic enzyme substrates in a selective agar medium, allowing the detection of S. aureus with a high degree of sensitivity and specificity. (9) The present study examines the use of a chromogenic plate medium, CHROMagar Staph aureus (CAS), and Mannitol Salt Agar (MSA) versus the gold standard Blood agar (BA) for the isolation and identification of S. aureus from pyoderma (suppurative skin lesions).the criteria for medium evaluation included colony growth reaction, color reproducibility for the identification of S. aureu. MATERIAL AND METHODS Culture media. CHROMagar Staph aureus was provided by the CHROMagar Company, Paris, France. The medium contained agar (15 g/liter), peptones (40 g/liter), NaCl (25 g/liter), and a proprietary chromogenic mix (3.5 g/liter). The medium was prepared as instructed by the manufacturer by avoiding heating at over 100 C. Columbia blood agar 63

2 base (Acumedia, Baltimore, Md.) supplemented with 5% human blood (blood agar) and mannitol-salt agar (Oxoid-CM85), were prepared according to the manufacturer's instructions. Clinical samples: Suppurative skin lesions of a of 105 pyoderma patients admitted to the outpatient clinic of the Main University Hospital, Alexandria, Egypt, were included in this study. Swabs were used to collect suppurative exudates from skin lesions, then immediately delivered to the lab for processing. Each swab was inoculated and spread onto each of the three types of media (BA, MSA and CAS). All plates were aerobically incubated at 37 C, BA for 24 h (10), CAS for 48 h (according to manufacturer instructions) and MSA for 48 h. (10) Identification of S. aureus. Golden yellow hemolytic or non hemolytic colonies on blood agar, yellow mannitol fermenting colonies on MSA and mauve colonies on CAS were regarded as presumptive S. aureus and were confirmed with Gram stain reactions, reactions to catalase (3% [wt/vol] hydrogen peroxide, reactions to slide and tube coagulase (rabbit plasma), and DNase activity were used for identification. (10) RESULTS Eighty three confirmed strains of S. aureus were isolated (from 105 swabs taken from suppurative skin lesions) on one or more agar culture media within 48 h of incubation. A of 83 strains were isolated on BA after 24 h of incubation. On CSA, 83 strains were recovered as mauve colonies after 24 h and no further growth was detected after incubation for 48 h. One of these mauve colony was not proved to be S. aureus (false ). These results were associated with sensitivity of % and a specificity of %. (Table 1) As regards MSA, 43 strains were recovered as yellow colonies after 24 h of incubation compared with 62 strains after incubation for 48 h. One yellow colony was not proved to be S. aureus (false ). These results were associated with a sensitivity of 50.60% and % after 24 and 48 h. respectively. (Table 2, 3) Table 1: Chromagar Staph aureus versus blood agar for detection of S. aureus after 24 Blood agar Chromagar After extending incubation to 48 hours mauve color of colonies start to fade away in most of the plates Sensitivity = T+ve/T+ve+F-ve = 82/82+1= 98.79% Specificity = T-ve / T-ve+F+ve = 21/21+1= 95.45% Table 2: Mannitol salt agar versus blood agar for detection of S. aureus after 24 Bl.agar MSA Sensitivity = 42/42+41 = 50.60% Specificity = 21/21+1= 95.45% 64

3 Table 3: Mannitol salt agar versus blood agar for detection of S. aureus after 48 Blood agar MSA Sensitivity = 61/61+22= 73.49% Specificity = 21/21+1= 95.45% DISCUSSION Isolation of S. aureus is usually accomplished with the use of conventional media such as blood agar. The disadvantage of such media is the need of confirmatory tests to differentiate S. aureus from colonies with identical colony appearance or when swarming colonies of Proteus cover those of S. aureus on such ordinary media. (2) Performing such tests on all colonies resembling staphylococci can be timeconsuming and labor intensive. (8,11) The use of chromogenic media can potentially reduce the number of confirmatory tests that are necessary for the detection of S. aureus. (4,8) The ideal characteristics of any candidate chromogenic medium are the detection of S. aureus with high specificity and an isolation rate at least comparable to conventional media after 18 to 20 h of incubation. (4,8) In this study, CAS was highly sensitive (98.79%) for S. aureus, where it succeeded to detect 82 out of the 83 S. aureus strains after 24 h incubation. The same sensitivity (98.5%) was obtained by Carricajo et al., (11) 2001; and a near results were obtained by Gaillot et al., (8) 95.5%, 2000, Perry et al., (4) 96.2%, 2003,and D Souza et al., (12) 69%, Slightly higher sensitivity (99.5%) was obtained by Flyhart et al. (13), In the present study sensitivity of CAS did not increase with extending the incubation time to 48 h, in the contrary it decreased, as the mauve color of some colonies faded away. The same result was reported by Kluytmans et al., (14) who reported that after 48 h the sensitivity of CAS was slightly lower. In contrast, D Souza and Baron., (12) reported that the sensitivity of CAS after overnight incubation was 82% and increased to 96% after additional 24 h incubation. Mannitol-salt agar (MSA) is one of the most widely used selective media for isolation of S. aureus. It consists of mannitol to indicate the presence of S. aureus and a variable concentration of sodium chloride to inhibit the growth of other microorganisms. (15) In the present study MSA had a low sensitivity of 73.49%, in contrast to the reports of other authors who reported a sensitivity reaching up to 96% and 97.1% respectively. (12,14) The low sensitivity of MSA in this study was due to the high false rate which we could not find an explanation to, except that these strains might be inhibited by the high salt component of this medium. (16) MSA showed a sensitivity of 50.60% after 24 h incubation which markedly increased to 73.49% after further 24 h. This seems to be consistent with the findings of D Souza and Baron (12) who mentioned that the sensitivity of MSA increased from 71% after 24 h to 96% after extending the incubation to another 24 h. In conclusion, CSA compared favorably to conventional media for rapid detection of S. aureus in clinical samples and achieved a higher sensitivity and specificity than MSA for the isolation and presumptive identification of S. aureus from suppurative skin lesions. The use of CSA is much less labor intensive than conventional methods and requires fewer reagents for confirmation of suspect colonies of S. aureus. (12,17) Sometimes, strains which were considered after 24 h were after 48 h. This may be due to variability in 65

4 the interpretation of the observer or to a true discoloration after prolonged incubation. Whatever the cause may be, we recommend reading CSA plates after 24 h, as this gives a more accurate result. (13) The cost of CSA is higher than that of nonselective conventional media. However, there is some efficiency gained with the use of this medium. The more rapid visualization of the specific mauve pigmentation of S. aureus allows working through cultures more quickly; therefore, slide coagulase tests can be substantially reduced or eliminated. Additional biochemical tests or identification methods that may be needed on occasion for final identification can also be reduced or eliminated. Moreover, there is no need to subculture organisms for antimicrobial susceptibility testing. (13) The cost of selective media such as MSA is higher than that of nonselective media. The additional costs incurred with the use of the MSA and required subculture plates, slide coagulase, and additional biochemical tests narrow the cost differential and make the change from a selective medium to CSA closer to cost-neutral. (12,13) So we recommend the use of CSA agar medium for the isolation and the specific identification of S.aureus from pyodermal suppurative skin lesions. REFERENCES 1. Paule SM, Pasquariello AC, Hacek DM, Adrienne G. Fisher AG, Thomson RB,Kaul KL, Peterson LR. Direct detection of Staphylococcus aureus from adult and neonate nasal swab specimens using real-time polymerase chain reaction. JMD 2004; 6(3): Philppe P, Michele B, Gerand L, Vandenesch F, Brun Y, Etienne J E. Comparative performances of six agglutination kits assessed by using typical and atypical strains of Staphylococcus aureus. J Clin Microbiol 1997; 35: Griethuysen V, Bes M, Etienne J, Zbinden R, Kluytmans J. International multicenter evaluation of latex agglutination tests for identification of Staphylococcus aureus. J Clin Microbiol 2001; 39: Perry JD, Rennison C, Butterworth LA, Hopley ALJ, F. Gould K. Evaluation of S. aureus ID, a New Chromogenic Agar Medium for Detection of Staphylococcus aureus. J Clin Microbiol 2003; 41: Merlino J, Leroi M, Bradbury R, Veal D, Harbour C. New chromogenic identification and detection of Staphylococcus aureus and methicillinresistant S. aureus. J Clin Microbiol 2000; 38: Baumert N, Eiff CV, Schaaff F, Peters SG, Proctor RA, Sahl HG. Physiology and antibiotic susceptibility of Staphylococcus aureus small colony variants. Microb Drug Resist 2002; 8: Baird R M, Lee WH. Media used in the detection and enumeration of Staphylococcus aureus. Int J Food Microbiol 1995; 26: Gaillot O, Wetsch M, Fortineau N, Berche P. Evaluation of CHROMagar Staph. aureus, a new chromogenic medium, for isolation and presumptive identification of Staphylococcus aureus from human clinical specimens. J Clin Microbiol 2000; 38: Göran H, Fang H. Evaluation of Two New Chromogenic Media, CHROMagar MRSA and S. aureus ID, for Identifying Staphylococcus aureus and Screening Methicillin-Resistant S. aureus. J Clin Microbiol 2005; 34: Kloose WE, Bannerman TL. Staphylococcus and Micrococcus. In: Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yoken RH (editors). Manual of clinical MICROBIOLOGY. 6 th ed. Washington DC: American society for Microbiology; 1995.p Carricajo A, Treny A, Fonsale N, Bes M, Reverdy M, Gille Y,et al,. Aubert, and A. M. Freydiere. Performance of the chromogenic medium CHROMagar Staph aureus and the Staphychrom coagulase test in the detection and identification of Staphylococcus aureus in clinical specimens. J Clin Microbiol 2001; 39: D Souza HA,Baron EJ. BBL CHROMaga Staph aureus is superior to mannitol salt for detection of Staphylococcus aureus in complex 66

5 mixed infections.am J Clin Pathol, 2005; 123(6): Flayhart D, Lema C, Borek A, Carroll KC.Comparison of the BBL Chromagar Staph aureus agar medium to conventional media for detection of Staphylococcus aureus in respiratory samples. J Clin Microbiol 2004; 42: Kluytmans J, Griethuysen VA, Willemse P, Keulen PV. Performance of CHROMagar selective medium and oxacillin resistance screening agar base for identifying Staphylococcus aureus and detecting methicillin resistance. J Clin Microbiol 2002;40: Gorss EB Prospective, focused surveillance for oxacillin-resistant Staphylococcus aureus. In: Isenberg HD (ed.). Clinical microbiology procedures handbook. ASM Press, Washington, D.C p Jones EM, Bowker KE, Cooke R, Marshall RJ, Reeves DS, MacGowan AP. Salt tolerance of EMRSA-16 and its effect on the sensitivity of screening cultures. J Hosp Infect 1997; 35: Apfalter P, Assadian O, Kalczyk A, Lindenmann V, Makristathis A, Mustafa S,et al. Performance of a new chromogenic oxacillin resistance screen medium (Oxoid) in the detection and presumptive identification of methicillinresistant Staphylococcus aureus. Diagn Microbiol Infect Dis 2002; 44: