Chromagar Staph aureus Versus Blood Agar and Mannitol Salt Agar ...

Egyptian Journal of Medical Microbiology, January 2007 Vol. 16, No. 1Chromagar Staph aureus Versus Blood Agar and Mannitol Salt Agarfor Isolation and Identification of Staphylococcus aureus fromSuppurative Skin LesionsWafaa M.K.Bakr and Heba S.SelimMicrobiology Department, High Institute of Public Health, Alexandria UniversityChromagar Staph aureus (CHROMagar Company, Paris, France) is a chromogenic agar mediumproposed for the detection of Staphylococcus aureus by incorporation of a chromatic substrate intoa suitable isolation medium and then detection of the activities of a specific bacterial enzymes bycolor changes thus eliminating the need for time consuming and costly biochemical identification.To evaluate this medium, a total of 105 suppurative skin lesion swabs were cultured ontoCHROMagar Staph aureus agar, blood agar (gold standard) and mannitol salt agar. After 24 h ofincubation a total of 83 S. aureus strains were recovered on blood agar. Chromagar Staph aureussucceded to isolate 82 strains with mauve color in the first 24 h with a sensitivity of 98.79 % and aspecificity of 95.45 % ,with no further isolation after extending the incubation time to 48 h. Asregards mannitol salt agar, 43 (51.8%) strains were recovered as yellow colonies after 24 h ofincubation compared with 61(73.4%) strains after incubation for 48 h. These results wereassociated with a sensitivity of 50.60% after 24 reaching up to 73.49% after 48 h. We conclude thatCHROMagar Staph aureus compared favorably to conventional media for rapid detection ofS. aureus in clinical samples and achieved a higher sensitivity and specificity than mannitol saltagar for the isolation and presumptive identification of S. aureus from suppurative skin lesions .Key words:Chromagar Staph aureus , Blood Agar ,Mannitol Salt Agar , Staphylococcus aureusINTRODUCTIONStaphylococcus aureus (S. aureus ) is animportant and frequent cause of of skin andsoft tissue infection and is consequently oneof the most common pathogens sought inclinical microbiology laboratories. (1) Reliableand rapid methods to identify these organismsare crucial in any clinical laboratory. (2,3)Diagnosis of S. aureus skin infectionis generally performed by culture of swabsonto nonspecific media and confirmation ofsuspected colonies by biochemical and/orserological tests. (4)Most commonly, this involves testingcolonies of staphylococci for the coagulationof plasma, the fermentation of mannitol(mannitol salt agar [MSA]), the production ofthermostable nuclease (DNase), egg yolklipase hydrolysis (lipovitellin-salt-mannitolagar [LSM]), and the production of naturalpigment. (2,3,5)Colonies of S. aureus may be atypicaland difficult to differentiate from coagulasenegativestaphylococci ,a large number ofagglutination tests may be required to rule outthe presence of S. aureus. (6)A number of culture media have beendeveloped to increase the specificity of S.aureus detection, including mannitol-salt agarand Baird-Parker medium. (7,8)A more recent approach has been theuse of CHROMagar Staph aureus, whichemploys chromogenic enzyme substrates in aselective agar medium, allowing the detectionof S. aureus with a high degree of sensitivityand specificity. (9)The present study examines the use ofa chromogenic plate medium, CHROMagarStaph aureus (CAS), and Mannitol Salt Agar(MSA) versus the gold standard Blood agar(BA) for the isolation and identification ofS. aureus from pyoderma (suppurative skinlesions).The criteria for medium evaluationincluded colony growth reaction, colorreproducibility for the identification ofS. aureu.MATERIAL AND METHODSCulture media.CHROMagar Staph aureus wasprovided by the CHROMagar Company,Paris, France. The medium contained agar(15 g/liter), peptones (40 g/liter), NaCl(25 g/liter), and a proprietary chromogenicmix (3.5 g/liter). The medium was prepared asinstructed by the manufacturer by avoidingheating at over 100°C. Columbia blood agar63

Egyptian Journal of Medical Microbiology, January 2007 Vol. 16, No. 1base (Acumedia, Baltimore, Md.)supplemented with 5% human blood (bloodagar) and mannitol-salt agar (Oxoid-CM85),were prepared according to the manufacturer'sinstructions.Clinical samples:Suppurative skin lesions of a total of105 pyoderma patients admitted to theoutpatient clinic of the Main UniversityHospital, Alexandria, Egypt, were included inthis study. Swabs were used to collectsuppurative exudates from skin lesions, thenimmediately delivered to the lab forprocessing. Each swab was inoculated andspread onto each of the three types of media(BA, MSA and CAS). All plates wereaerobically incubated at 37°C, BA for 24 h (10), CAS for 48 h (according to manufacturerinstructions) and MSA for 48 h. (10)Identification of S. aureus.Golden yellow hemolytic or nonhemolytic colonies on blood agar, yellowmannitol fermenting colonies on MSA andmauve colonies on CAS were regarded aspresumptive S. aureus and were confirmedwith Gram stain reactions, reactions tocatalase (3% [wt/vol] hydrogen peroxide,reactions to slide and tube coagulase (rabbitplasma), and DNase activity were used foridentification. (10) RESULTSEighty three confirmed strains of S.aureus were isolated (from 105 swabs takenfrom suppurative skin lesions) on one or moreagar culture media within 48 h of incubation.A total of 83 strains were isolated onBA after 24 h of incubation.On CSA, 83 strains were recovered asmauve colonies after 24 h and no furthergrowth was detected after incubation for 48 h.One of these mauve colony was not provedto be S. aureus (false positive). These resultswere associated with sensitivity of 98.79 %and a specificity of 95.45 %. (Table 1)As regards MSA, 43 strains wererecovered as yellow colonies after 24 h ofincubation compared with 62 strains afterincubation for 48 h. One yellow colony wasnot proved to be S. aureus (false positive).These results were associated with asensitivity of 50.60% and 73.49 % after 24and 48 h. respectively. (Table 2, 3)Table 1: • Chromagar Staph aureus versus blood agar for detection of S. aureus after 24incubation hours.Blood agarChromagartotalpositivenegativepositive 82 1 83negative 1 21 22total 83 22 105•After extending incubation to 48 hours mauve color of colonies start to fade away in most ofthe platesSensitivity = 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 24incubation hours.Bl.agarMSAtotalpositivenegativepositive 42 41 83negative 1 21 22total 43 62 105Sensitivity = 42/42+41 = 50.60%Specificity = 21/21+1= 95.45%64

Egyptian Journal of Medical Microbiology, January 2007 Vol. 16, No. 1Table 3: Mannitol salt agar versus blood agar for detection of S. aureus after 48incubation hours.Blood agarMSAtotalpositivenegativepositive 61 22 83negative 1 21 22total 62 43 105Sensitivity = 61/61+22= 73.49%Specificity = 21/21+1= 95.45%DISCUSSIONIsolation of S. aureus is usuallyaccomplished with the use of conventionalmedia such as blood agar. The disadvantageof such media is the need of confirmatorytests to differentiate S. aureus from colonieswith identical colony appearance or whenswarming colonies of Proteus cover those ofS. aureus on such ordinary media. (2)Performing such tests on all coloniesresembling staphylococci can be timeconsumingand labor intensive. (8,11)The use of chromogenic media canpotentially reduce the number of confirmatorytests that are necessary for the detection of S.aureus. (4,8)The ideal characteristics of anycandidate chromogenic medium are thedetection of S. aureus with high specificityand an isolation rate at least comparable toconventional media after 18 to 20 h ofincubation. (4,8)In this study, CAS was highlysensitive (98.79%) for S. aureus, where itsucceeded to detect 82 out of the total 83 S.aureus strains after 24 h incubation. The samesensitivity (98.5%) was obtained by Carricajoet al., (11) 2001; and a near results wereobtained by Gaillot et al., (8) 95.5%, 2000,Perry et al., (4) 96.2% , 2003,and D’Souza etal., (12) 69%, 2005. Slightly higher sensitivity(99.5%) was obtained by Flyhart et al. (13) ,2004.In the present study sensitivity ofCAS did not increase with extending theincubation time to 48 h, in the contrary itdecreased, as the mauve color of somecolonies faded away. The same result wasreported by Kluytmans et al., (14) who reportedthat after 48 h the sensitivity of CAS wasslightly lower.In contrast, D’Souza and Baron., (12)reported that the sensitivity of CAS afterovernight incubation was 82% and increasedto 96% after additional 24 h incubation.Mannitol-salt agar (MSA) is one ofthe most widely used selective media forisolation of S. aureus. It consists of mannitolto indicate the presence of S. aureus and avariable concentration of sodium chloride toinhibit the growth of other microorganisms. (15)In the present study MSA had a lowsensitivity of 73.49%, in contrast to thereports of other authors who reported asensitivity reaching up to 96% and 97.1%respectively. (12,14)The low sensitivity of MSA in this study wasdue to the high false negative rate which wecould not find an explanation to, except thatthese strains might be inhibited by the highsalt component of this medium. (16)MSA showed a sensitivity of 50.60%after 24 h incubation which markedlyincreased to 73.49% after further 24 h. Thisseems to be consistent with the findings ofD’Souza and Baron (12) who mentioned thatthe sensitivity of MSA increased from 71%after 24 h to 96% after extending theincubation to another 24 h.In conclusion, CSA comparedfavorably to conventional media for rapiddetection of S. aureus in clinical samples andachieved a higher sensitivity and specificitythan MSA for the isolation and presumptiveidentification of S. aureus from suppurativeskin lesions .The use of CSA is much less laborintensive than conventional methods andrequires fewer reagents for confirmation ofsuspect colonies of S. aureus. (12,17)Sometimes, strains which wereconsidered positive after 24 h were negativeafter 48 h. This may be due to variability in65

Egyptian Journal of Medical Microbiology, January 2007 Vol. 16, No. 1the interpretation of the observer or to a truediscoloration after prolonged incubation.Whatever the cause may be, we recommendreading CSA plates after 24 h, as this gives amore accurate result. (13)The cost of CSA is higher than that ofnonselective conventional media. However,there is some efficiency gained with the useof this medium. The more rapid visualizationof the specific mauve pigmentation ofS. aureus allows working through culturesmore quickly; therefore, slide coagulase testscan be substantially reduced or eliminated.Additional biochemical tests or identificationmethods that may be needed on occasion forfinal identification can also be reduced oreliminated. Moreover, there is no need tosubculture organisms for antimicrobialsusceptibility testing. (13)The cost of selective media such asMSA is higher than that of nonselectivemedia. The additional costs incurred with theuse of the MSA and required subcultureplates, slide coagulase, and additionalbiochemical tests narrow the cost differentialand make the change from a selectivemedium to CSA closer to cost-neutral. (12,13)So we recommend the use of CSAagar medium for the isolation and the specificidentification of S.aureus from pyodermalsuppurative skin lesions.REFERENCES1. Paule SM, Pasquariello AC, Hacek DM,Adrienne G. Fisher AG, ThomsonRB,Kaul KL, Peterson LR. Directdetection of Staphylococcus aureus fromadult and neonate nasal swab specimensusing real-time polymerase chainreaction. JMD 2004; 6(3):191-203.2. Philppe P, Michele B, Gerand L,Vandenesch F, Brun Y, Etienne J E.Comparative performances of sixagglutination kits assessed by usingtypical and atypical strains ofStaphylococcus aureus. J Clin Microbiol1997; 35:1138-40.3. Griethuysen V, Bes M, Etienne J,Zbinden R, Kluytmans J. Internationalmulticenter evaluation of latexagglutination tests for identification ofStaphylococcus aureus. J Clin Microbiol2001; 39:86-9.4. Perry JD, Rennison C, Butterworth LA,Hopley ALJ, F. Gould K. Evaluation ofS. aureus ID, a New Chromogenic AgarMedium for Detection of Staphylococcusaureus. J Clin Microbiol 2003; 41:5695-8.5. Merlino J, Leroi M, Bradbury R, VealD, Harbour C. New chromogenicidentification and detection ofStaphylococcus aureus and methicillinresistantS. aureus. J Clin Microbiol2000; 38:2378-80.6. Baumert N, Eiff CV, Schaaff F, PetersSG, Proctor RA, Sahl HG. Physiologyand antibiotic susceptibility ofStaphylococcus aureus small colonyvariants. Microb Drug Resist 2002;8:253-60.7. Baird R M, Lee WH. Media used in thedetection and enumeration ofStaphylococcus aureus. Int J FoodMicrobiol 1995; 26:15-24.8. Gaillot O, Wetsch M, Fortineau N,Berche P. Evaluation of CHROMagarStaph. aureus, a new chromogenicmedium, for isolation and presumptiveidentification of Staphylococcus aureusfrom human clinical specimens. J ClinMicrobiol 2000; 38:1587-91.9. Göran H , Fang H. Evaluation of TwoNew Chromogenic Media, CHROMagarMRSA and S. aureus ID, for IdentifyingStaphylococcus aureus and ScreeningMethicillin-Resistant S. aureus. J ClinMicrobiol 2005; 34:4242-4.10. 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: Americansociety for Microbiology; 1995.p.282-98.11. Carricajo A, Treny A, Fonsale N, BesM, Reverdy M, Gille Y,et al,. Aubert,and A. M. Freydiere. Performance of thechromogenic medium CHROMagarStaph aureus and the Staphychromcoagulase test in the detection andidentification of Staphylococcus aureusin clinical specimens. J Clin Microbiol2001; 39:2581-3.12. D’Souza HA,Baron EJ. BBLCHROMaga Staph aureus is superior tomannitol salt for detection ofStaphylococcus aureus in complex66

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