During a 13-month period, 11 equine patients visiting a veterinary teaching hospital for various diagnostic and surgical procedures developed postprocedural infections from which methicillin (oxacillin)-resistant Staphylococcus aureus (MRSA) strains were isolated. The S. aureus isolates were identified by conventional methods that included Gram staining, tests for colonial morphology, tests for clumping factor, and tests for coagulase and urease activities and were also tested with the API STAPH IDENT system. Antimicrobial susceptibility tests were performed by the disk diffusion method. The biochemical profile and antibiogram of each isolate suggested that the isolates may have come from a common source. Because MRSA strains are very uncommon animal isolates but are rather common human isolates, a nasal swab specimen for culture was collected voluntarily from five persons associated with equine surgery and recovery in an attempt to identify a possible source of the organisms. MRSA strains were isolated from three of the five people, with one person found to be colonized with two biotypes of MRSA. The MRSA isolates from the people appeared to be identical to the isolates from horses. Further study of the isolates included SmaI andEagI macrorestriction analysis by pulsed-field gel electrophoresis conducted in two different laboratories. The results indicated that both the equine and human isolates were members of a very closely related group which appear to have originated from a common source. On the basis of the pattern associated with the infection, it is speculated that the members of the Veterinary Teaching Hospital staff were the primary source of the infection, although the specific mode of transmission is unclear.

Delimiting the genus. nov., are described on the basis of a phylogenetic analysis comparing 165 rRNA sequences, DNA-DNA liquid hybridization, DNA base composition, normalized ribotype patterns, macrorestriction pattern analysis and estimation of genome size using PFGE, cell wall composition, phenotypic characteristics and plasmid profiles. Compared with their closest relatives, members of the genus Staphylococcus, these organisms demonstrated significantly lower 165 rRNA sequence similarities (934-9503 %), higher DNA G+C content (38-45 mol YO), absence of cell wall teichoic acids (with the possible exception of M. caseolyticus), unique ribotype pattern types and macrorestriction patterns, smaller genome size (approx. 1500-1800 kb) and generally larger Gram-stained cell size (14-205 pm in diameter). Macrococci can be distinguished from most species of staphylococci (except Staphylococcus sciuri, Staphylococcus witulus and Staphylococcus lentus) by their oxidase activity. The four Macrococcus species can be distinguished from one another on the basis of DNA-DNA hybridization, ribotype pattern types, macrorestriction patterns and their phenotypic properties, including colony morphology, cell morphology, haemolysins, Staph Latex agglutination, acid production from a variety of carbohydrates, acetoin production, nitrate reduction, aesculin hydrolysis, and DNase and urease activities. The type species is M. equipercicus. The type strains of M. equipercicus, M. caseo/yticus, M. bovicus and M. carouselicus are ATCC 5183IT ( (Schleifer et al., 1982). These strains were isolated from the milk of cattle, but more recently several strains have been isolated from the abscesses of slaughtered lambs (De La Fuente et al., 1992) and one from the milk of goats (De Buyser et al., 1992).In a preliminary investigation (Ballard et al., 1995), we sampled the skin of 15 cattle, 25 goats, 14 horses, 10 ponies, 4 whales, 25 dolphins and meat products for the presence of Staphylococcus caseolyticus. This species was isolated from only three samples of raw beef and the skin of a pilot whale and so can still be thought of as a relatively uncommon species. Surprisingly, we discovered a group of three new species related to Staphylococcus caseolyticus living on the skin of cattle, horses and ponies. Phenotypic characterization. The following characteristics were determined as described previously Kloos et al., 1976; Webster et al., 1994;: Gram-stained cell morphology and cell arrangement, colony morphology and pigmentation, motility, anaerobic growth in thioglycollate semi-solid medium, catalase activity, acetylmethylcarbinol (acetoin) production, nitrate reduction, oxidase activity, pyrrolidonylarylamidase activity, aesculin hydrolysis, DNase activity, thermonuclease activity, ornithine decarboxylase activity, urease activity, staphylocoagulase activity, lysostaphin susceptibility, haemolysis of sheep, bovine and horse blood, and carbohydrate reactions. The presence of clumping factor and/or protein A was tested using the Staph Latex Kit ...

A new subspecies, Staphylococcus horninis subsp. novobiosepticus, isolated from human blood cultures, a wound, a breast abscess and a catheter tip, is described on the basis of a study of 26 strains isolated between 1989 and 1996.DNA-DNA reassociation reactions, conducted under stringent conditions, and macrorestriction pattern analysis demonstrated that these strains are closely related to previously characterized 5. horninis strains isolated from human skin and clinical specimens, but are significantly divergent. 5. horninis subsp. novobiosepticus can be distinguished from 5. horninis (now named 5. horninis subsp. horninis) by its combined characteristics of novobiocin resistance and failure to produce acid aerobically from D-trehalose and N-aCety1-Dglucosamine. Furthermore, all 26 strains of the new subspecies are resistant to nalidixic acid, penicillin G, oxacillin, kanamycin and streptomycin, and were either resistant or had intermediate resistance to methicillin and gentamicin. Most strains were also resistant to erythromycin, clindamycin, chloramphenicol, trimethoprim/sulfamethoxazole and ciprof loxacin. Based on a comparison of the sequences of a 1001 bp rned amplification product from reference methicillin-resistant staphylococci, the rned gene present in S-horninis subsp. novobiosepticus was identified as homologue A, commonly found in S. aureus and many coagulase-negative staphylococcal species. The type strain of 5. horninis subsp. novobiosepticus is ATCC 700236l. Descriptions of S. horninis subsp. novobiosepticus subsp. nov. and 5. horninis subsp. horninis are given and the description of S. horninis is emended.

A previous study (S. H. Goh et al., J. Clin. Microbiol. 34:818-823, 1996) demonstrated that a 600-bp region of the chaperonin 60 (Cpn60) genes from various bacterial isolates could be amplified by PCR with a pair of degenerate primers and that the products could be used as species-specific probes for Staphylococcus aureus, S. epidermidis, S. haemolyticus, S. lugdunensis, S. saprophyticus, and S. schleiferi. To further validate the utility of bacterial Cpn60 genes as universal targets for bacterial identification (ID), reverse checkerboard chemiluminescent hybridization experiments were performed with DNA probes from 34 different Staphylococcus species and subspecies. With the exception of probes from the Cpn60 genes of S. intermedius and S. delphini, which cross hybridized, all were species specific. Two subspecies of both S. capitis and S. cohnii were differentiated from one another, while DNAs from the two S. schleiferi subspecies cross hybridized. When 40 known Staphylococcus isolates were tested in a blind experiment by the Cpn60 gene method, 36 strains, representing six species and one subspecies (S. sciuri, S. caseolyticus, S. hominis, S. warneri, S. hyicus, S. haemolyticus, and S. capitis subsp. ureolyticus), were correctly identified. DNA from the four remaining isolates, known to be S. hyicus bovine strains, failed to hybridize to DNA from the S. hyicus target strain or any other Staphylococcus species. However, DNAs from these S. hyicus isolates did cross hybridize with each other. New DNA sequence data and evidence from previous studies suggest some genetic divergence between the two groups of S. hyicus isolates. Our results demonstrate that this Cpn60 gene-based ID method has the potential to be a basic method for bacterial ID. Studies are in progress to further validate the utility of this Cpn60 gene system for ID of Staphylococcus and other genera, including those of slow-growing microorganisms. MATERIALS AND METHODS Bacterial isolates. All staphylococcal isolates used in this study, with the exception of Staphylococcus saccharolyticus, are from the North Carolina State University Department of Genetics. S. saccharolyticus, a clinical isolate, and all nonstaphylococcal reference isolates are from the culture collection of the Provincial Laboratory, British Columbia Centre for Disease Control. The Cpn60 gene-negative control 600-bp DNA targets were derived from Acinetobacter