Evaluation of the automicrobic system for the identification of Streptococcus mutans

1102 Notes Eur. J. Clin. Microbiol. Infect. Dis. rather than an extrinsic contamination during the preparation and infusion in hospital. The batch number of the H6pital Erasme strain was not recorded. However, our observation of identical genomic profiles, together with the fact that this particular patient received RATG provided by the same distributor at the same time as the outbreak in our institution, makes a common source of contamination very likely. The bacteraemia in that hospital could not have been prevented by an interhospital alert policy, however, because it occurred at the time as we started our investigation. All patients in this study recovered from the Ochrobactrum anthropi bacteraemia, some even without antimicrobial therapy. This is in agreement with the previously reported low pathogenicity of this organism (1). This outbreak reminds us that despite stringent manufacturing procedures currently in practice, a certain risk of contamination of nonterminally sterilized injectable drugs persists. Since this contamination does not always cause turbidity, prevention is possible only by the early detection of outbreaks and the investigation of the source. Interhospital communication is advisable because these drugs are often distributed to different centres. This report points out the need for a permanent contact between infection control teams. Acknowledgement The authors wish to thank Dr. F. Potvlieghe (La Louvi6re) for providing strains and information. References 1. Cieslak T J, Robb ML, Drabick C J, Fisher GW: Catheter-associated sepsis caused by Ochrobactrum anthropi: report of a case and review of related nonfermentative bacteria. Clinical Infectious Diseases 1992, 14: 902-907. 2. Ezzedine H, Mourad M, Van Ossel C, Logghe C, Squifflet JP, Renault F, Wauters G, Gigi J, Wilmotte L, Haxhe J J: An outbreak of Ochrobactrum anthropi bacteraemia in five organ transplant patients. Journal of Hospital infection 1994, 27: 35-42. 3. Barbut F, Mario N, Delmee M, Gozian T, Petit JC: Genomic fingerprinting of Clostridium difficile isolates by using a random amplified polymorphic DNA (RAPD) assay. FEMS Microbiology Letters 1993, 114: 161-166. 4. Versalovic J, Koeuth T, Lupski JR: Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Research 1991, 19: 6823-6831. 5. Struelens M J, Cariier E, Maes N, Serruys E, Quint WGV, van Belkum A: Nosocomial colonisation and infection with multiresistant Acinetobacter baumanii; outbreak delineation using genomic DNA macrorestriction analysis and PCR genome fingerprinting. Journal of Hospital infection 1993, 25: 15-32. 6. Whyte W, Niven L, Bell NDS: Microbial growth in smallvolume pharmaceuticals. Journal of Parenteral Science & Technology 1989, 43: 208-212. Evaluation of the Automicrobic System for the Identification of Streptococcus mutans A. de la Higuera, J. Lidbana*, J. Guti6rrez, A. Garcfa-Mendoza, A. Castillo The performance of the Automicrobic System with the Vitek gram-positive identification card (bioMerieux, France) in identifying strains of Streptococcus mutans was studied. Of 160 strains assayed, 72.5 % were confirmed to be Streptococcus mutans; the remainder were identified as other species of streptococci (Streptococcus bovis, Streptococcus uberis, Streptococcus anginosus, Streptococcus sanguis I and II, Streptococcus intermedius, and Streptococcus constellatus). Viridans streptococci comprise a large group of microorganisms, some of which form part of the normal flora of the oral cavity and have no known pathogenicity. Others, however, behave as pathogens, and cause a variety of infections in humans and animals. Among the diseases associated with Streptococcus mutans are local infections such as dental caries and, less frequently, periodontal diseases (1-3), bacteremia, endocarditis, and abscesses (4-6). The clinical consequences of these microorganisms make it necessary to develop appropriate, rapid, and reliable systems to identify them. Because most strains cannot be grouped according to serological category on the basis of the Lancefield classification and capsular antigens, Department of Microbiology, Odontology Section, University of Granada, Avda. de Madrid 11, 18012 Granada, Spain. Vol. 14, 1995 Notes 1103 and because cross-reactions are frequent, rapid latex-based tests are unsuitable. In addition, the wide battery of manual biochemical tests required are not routinely available in many laboratories. In response to these problems, several automatic systems have been developed (e.g., MicroScan, Baxter USA, and ATB and Automicrobic System, bioM6rieux, France), which claim to facilitate the identification of streptococci. We investigated the performance of the Automicrobic System (AMS) in identifying Streptococcus mutans. Material and Methods. A total of 160 strains of Streptococcus mutans was obtained from clinical oral isolates and identified using conventional methods. In addition, three reference strains of Streptococcus mutans (NCT 10832, ATCC 25175, and NCTC 10449) were used. Cultured microorganisms were Gram stained and subj ected to the following tests: arginine hydrolysis; esculin hydrolysis in the presence and in the absence of bile; mannitol, raffinose, and inulin fermentation; and resistance to 2 U bacitracin. The culture media, techniques, and interpretation of results were in accordance with the methods of Cowan and Steel (7). The identification criteria were those of Maiden et al. (8) (arginine hydrolysis negative, esculin hydrolysis in the presence of bile negative and in its absence positive, mannitol, raffinose, and inulin fermentation positive, and resistance to 2 U bacitracin positive). All strains of Streptococcus mutans identified by conventional means were reidentified with the AMS. This system repeats these conventional assays in an automated fashion. We used a Vitek GPI card (V.1305, version 4.2), and followed the manufacturer's recommendations for inoculation and reading. All assays were repeated three times to verify the results. Results and Discussion. The AMS correctly identified 116 of the 160 (72.5 %) strains as Streptococcus mutans. Of the remaining 44 (27.5 %) strains misidentified by the AMS, 45.5 % were identified as Streptococcus boris, 9.1% as Streptococcus intermedius, 9.1% as Streptococcus anginosus, 4.5 % as Streptococcus sanguis 1, 9.1% as Streptococcus sanguis II, 13.6 % as Streptococcus uberis, and 9.1% as Streptococcus constellatus. Of the reference strains, NCTC 10832 and ATCC 25175 were identified by the AMS as Streptococcus mutans, while NCTC 10449 was identified as Streptococcus bovis. The reproducibility of the tests was 100 % - that is, the same results were obtained in each of the three assays. The AMS uses the same biochemical assays as are used in conventional analyses. Tables 1 and 2 summarize the results of these tests, which did not match the conventionally obtained results both for strains identified as Streptococcus mutans and for other strains. The accurate identification of viridans streptococci continues to be an obstacle in studies of this group. The classification of these microorganisms has been modified many times (9-11). Changes in the taxonomic classification of these microorganisms have been frequent and have led to the development of new techniques for their identification. Of these methods, studies of the homology between nucleic acids, genetic fingerprinting (12, 13), and examination of characteristics such as antigenic structure and physiological properties have helped clarify the classification of these microorganisms. Table 1" Results of conventional biochemical assays of strains identified by conventional methods and the Antimicrobic System as Streptococcus mutans. No. of BAC ESC ARG MAN RAF INU strains 6 + - - + + - 2 + - - + + + 2 + + + + + - 2 + + + + - + 28 + + - + + + 76 + + - + + - BAC, bacitracin resistance; ESC, esculin hydrolysis; ARG, arginine hydrolysis; MAN, mannitol fermentation; RAF, raffinose fermentation; INU, inulin fermentation; +, positive;-, negative. Table 2" Results of conventional biochemical assays of strains identified by the Automicrobic System as species other than Streptococcus mutans. Species No. of BAC ESC ARG MAN RAF INU strains S. bovis 20 + + - + + - 2 .... + + 2 + + + + - - 2 + + - + + - 2 + + + + + - 4 .... + - 2 + + .... 6 + + - + - - 4 + + .... S. intermedius S. anginosus S. sanguis ll S. sanguis I S. uberis S. constellatus BAC, bacitracin resistance; ESC, esculin hydrolysis; ARG, arginine hydrolysis; MAN, mannitol fermentation; RAF, raffinose fermentation; INU, inulin fermentation; +, positive;-, negative. 1104 Notes Eur. J. Clin. Microbiol. Infect. Dis. Streptococcus mutans is currently considered a member of the socalled mutans group. Of the seven species in this group (Streptococcus mutans, rattus, cricetus, sobrinus, ferus, downei, and macacae ), only the first four are human pathogens (9, 14). The AMS with the Vitek GPI card was developed for the identification of the major strains of streptococci involved in human disease and of other gram-positive bacteria. This system includes most of the conventional tests for mutans streptococci, and makes it possible to compare the results of the automated system with a battery of conventional tests. The 72.5 % rate of identification by the AMS of the strains we tested was higher than the figure given in an earlier study (18). Of the 116 strains identified by the Vitek card, the results fully matched those obtained with conventional methods only in 28 cases (24.1%) (Table 1). When we compared the results of the conventional tests done manually with those of the same tests done by the AMS, we found that although 100 % of the 116 strains correctly identified as mutans streptococci were resistant to bacitracin and mannitol fermentation according to the automated system, agreement between the two series of assays was lower for other tests. The greatest discrepancy was found for the inulin fermentation test, which, according to the AMS, was positive in only 32 of the 116 (27.6 %) strains. Differences between automatic and manual results were also described in an earlier study of other identification systems (19). The possibility that strains not identified by the AMS as Streptococcus mutans were, in fact, different species was ruled out by the results of the manually performed conventional tests. This was exemplified by streptococci identified by the AMS as Streptococcus bovis, while in the conventional assays, none of the strains grew in bile esculin agar (7, 8, 15). Some authors have reported Streptococcus interrnedius, Streptococcus constellatus, and Streptococcus anginosus to be mannitol negative (8, 15), although one study found that some strains were able to ferment this sugar (20). Although strains may differ in their behavior in this assay, a negative result in the arginine hydrolysis test rules out the presence of these three species (7, 8, 15). Because neither Streptococcus sanguis I nor Streptococcus sanguis II is able to ferment mannitol (7, 8, 15), these species cannot have been among our sample of 160 strains of Streptococcus mutans, all of which are able to metabolize this polyalcohol. Because none of the strains we investigated was able to hydrolyze arginine (7, 8, 15), the presence of Streptococcus uberis was likewise ruled out. In conclusion, identification of strains of Streptococcus mutans by the AMS did not entirely match the results of manual identification with conventional assays. The discrepancies we found are common when modifications of conventional methods are used, including micromethods or commercial identification systems (19, 21, 22), and may be caused by the shorter incubation times used by these systems. Although the shorter incubation times are sufficient for other species, they are inadequate for oral streptococci, and particularly Streptococcus mutans, given their slow growth rates. Acknowledgements This study was partially supported by the Andalusian Regional Government through the research project "Microbiology, Immunology, and Epidemiology of Oral Diseases." We thank Ms. Karen Shashok for translating the original manuscript into English. References 1. Liebana J, Castillo A: Oral ecology and its pathogenic relation with the origin of primary periodontitis associated with plaque. Annales de Biologie Clinique 1991,49: 338344. 2. Liebana J, Castillo A: Physiopathology of primary periodontitis associated with plaque. Microbial and host factors. A review. Part 1. Australian Dental Journal 1994, 39: 228-232. 3. Li~bana J, Castillo A: Physiopathology of primary periodontitis associated with plaque. Microbial and host factors. A review. Part 2. Australian Dental Journal 1994, 39: 310-315. 4. Lu JR, Wu HC: Morphologic and biochemical characteristics of viridans streptococci isolated from dental plaque. Chung-Hua Min Kuo Wei Sheng Wu Chi Mien I Hsueh Tsa Chih 1992, 25: 91-100. 5. UIIman RF, Miller S J, Strampfer M J, Cunha BA: Streptococcus mutans endocarditis: report of three cases and review of the literature. Heart and Lung 1988, 17: 209-- 212. 6. Ullman RF, Strampfer M J, Cunha BA: Streptococcus mutans vertebral osteomyelitis. Heart and Lung 1988, 17: 319-321. 7. Barrow GI, Feltham RKA (ed): Cowan and Steel's manual for identification of medical bacteria. Cambridge University Press, Cambridge, 1993, p. 50-93. 8. Maiden MFJ, Lai CH, Tanner A: Characteristics of oral gram-positive bacteria. In: Slots J, Taubman MA (ed): Contemporary oral microbiology and immunology. Mosby-Year Book, St. Louis, 1992, p. 342-372. Vol. 14, 1995 Notes 1105 9. Douglas CW, Heath J, Hampton KK, Preston FE: Identity of viridans streptococci isolated from cases of infective endocarditis. Journal of Medical Microbiology 1993, 39: 179-182. 10. Kilpper-B&lz R, Wenzig P, Schleifer KH: Molecular relationship classification of some viridans streptococci as Streptococcus oralis and emended description of Streptococcus oralis. International Journal of Systematic Bacteriology 1985, 35: 482-488. 11. Schmidhuber S, Kilpper-B&lz R, Schleifer KH: A taxonomic study of Streptococcus mitis, S. oralis and S. sanguis. Systematic and Applied Microbiology 1987, 10: 74-77. 12. Adnan S, Li N, Miura H, Hashimoto Y, Yamamoto H, Ezaki T: Covalently immobilized DNA plate for luminometric DNA-DNA hybridization to identify viridans streptococci in under 2 hours. FEMS Microbiology Letters 1993, 106: 139-142. 13. Rudney JD, Larson C J: Use of restriction fragment polymorphism analysis of rRNA genes to assign species to unknown clinical isolates of oral viridans streptococci. Journal of Clinical Microbiology 1994, 32: 437. 14. Coykendall AL: Classification and identification of the viridans streptococci. Clinical Microbiology Reviews 1989, 2: 315-318. 15. Holt JG, Krieg NR, Sneath PHA, Staley JT, Williams ST (ed): Bergey's manual of determinative bacteriology. Williams & Wilkins, Baltimore, 1994, p. 527-558. 16. Gold OG, Jordan HV, Van Houte JV: A selective medium for Streptococcus mutans. Archives of Oral Biology 1973, 18: 1357-1364. 17. Hamada S, Slade HD: Biology, immunology and cariogenicity of Streptoccocus mutans. Microbiological Reviews 1980, 44: 331-384. 18. Schlerka G: The Automicrobic System (AMS): a new test method for the classification and susceptibility testing of bovine mastitis streptococci. Deutsche Tier&rzttiche Wochenschrift 1990, 97: 342-346. 19. Fertally SS, Facklam R: Comparison of physiologic test used to identify non A-hemolytic aerococci, enterococci and streptococci. Journal of Clinical Microbiology 1987, 25: 1845-1850. 20. Whiley RA, Fraser H, Hardie JM, Beighton D: Phenotypic differentiation of Streptococcus intermedius, Streptococcus constellatus, and Streptococcus anginosus strains within the "Streptococcus milleri group." Journal of Clinical Microbiology 1990, 28: 1497-1501. 21. Otdershaw MD, Eisemberg AD, Curzon ME J: Abiochemical micromethod to characterize Streptococcus mutans. Caries Research 1982, 16: 96-102. 22. Westergren G, Krasse B: Evaluation of a micromethod for determination of Streptococcus mutans and Lactobacillus infection. Journal of Clinical Microbiology 1978, 7: 82-83. In Vitro Activity of MDL 62,879 against Gram-Positive Bacteria and Bacteroides Species A. Bartoloni 1., A. Mantella 1, B.E Goldstein 2, M. Denaro 2, R Nicoletti 3, E Paradisi 1 The new thiazolyl peptide antibiotic MDL 62,879 (GE2270 A) showed excellent in vitro activity in testing against staphylococci and streptococci, with MIC90s ranging from 0.23 to 0.9 mg/I. It was very active against Clostridium difficile and Propionibacterium acnes (MIC90 0.06 mg/I in each case) and had variable activity against Bacteroides spp. MDL 62,879 had exceptionally good activity against Enterococcus faecalis, including against a collection of high-level aminoglycoside-resistant isolates where it had an MIC90 of 0.047. The antibiotic was bacteriostatic for enterococcal isolates but bactericidal for a methicillin-resistant isolate of Staphylococcus aureus. MDL 62,879 (GE2270 A) is a novel thiazolyl peptide antibiotic isolated from Planobispora rosea strain ATCC 53773 (1, 2). It inhibits bacterial protein biosynthesis by interacting with elongation factor Tu (EF-Tu) (1, 3, 4). MDL 62,879 has been reported to be active against aerobic and anaerobic gram-positive bacteria and some gram-negative anaerobes (1, 5, 6). In this study we evaluated the in vitro activity of MDL 62,879 in comparison with that of teicoplanin, vancomycin, ramoplanin, ampicillin, and clindamycin, and against aerobic and anaerobic gram-positive bacteria and Bacteroides spp., including a large collection of highlevel aminoglycoside-resistant enterococci. Materials and Methods. MDL 62,879, teicoplanin, and ramoplanin were obtained from Lepetit Research Center (Italy). Vancomycin was 1 Clinica di Malattie Infettive, Universith di Firenze, Nuovo Ospedale S. Giovanni di Dio, via di Torregalli 3, 50143 Firenze, Italy. 2 Lepetit Research Center, Marion Merrel Dow Research Institute, Gerenzano (VA), Italy. 3Laboratorio di Microbiologia e Virologia, Azienda Ospedaliera di Careggi, Firenze, Italy.