The clinical significance of coagulase-negative Staphylococcus species (CNS) continues to increase as strategies in medical practice lead to more invasive procedures. Hospitalized patients that are immunocompromised and/or suffering from chronic diseases are the most vulnerable to infection. Since CNS are widespread on the human body and are capable of producing very large populations, distinguishing the etiologic agent(s) from contaminating flora is a serious challenge. For this reason, culture identification should proceed to the species and strain levels. A much stronger case can be made for the identification of a CNS etiologic agent if the same strain is repeatedly isolated from a series of specimens as opposed to the isolation of different strains of one or more species. Strain identity initially can be based on colony morphology, and then one or more molecular approaches can be used to gain information on the genotype. Many of the CNS species are commonly resistant to antibiotics that are being indicated for staphylococcal infections, with the exception of vancomycin. The widespread use of antibiotics in hospitals has provided a reservoir of antibiotic-resistant genes. The main focus on mechanisms of pathogenesis has been with foreign body infections and the role of specific adhesins and slime produced by Staphylococcus epidermidis. Slime can reduce the immune response and opsonophagocytosis, thereby interfering with host defense mechanisms. As we become more aware of the various strategies used by CNS, we will be in a better position to compromise their defense mechanisms and improve treatment.

Staphylococci were isolated from human skin and subjected to a taxonomic study. As a result of this study, three new species are being proposed in this paper: Staphylococcus cohnii, S. haemolyticus, and S. xylosus. The type strains of these species are DSM (Deutsche Sammlung von Mikroorganismen) 20260, DSM 20263, and DSM 20266, respectively. Amended descriptions of S. epidermidis and S. suprophyticus are also given. The main characters for the distinction of staphylococci and micrococci are mentioned. Staphylococci were classified on the basis of cell wall composition, lactic acid configuration, and a variety of morphological and physiological characters. There are some key differential characters of these species which can be determined by simple laboratory procedures. The failure to ferment trehalose and mannitol is typical for S. epidermidis. The fermentation of xylose and/or arabinose is a characteristic of S. xylosus. The failure to ferment sucrose and turanose is typical for s. cohnii. Strains of s. suprophyticus do not reduce nitrate, but most of them produce acetylmethylcarbinol and ferment xylitol. S. haemolyticus is usually hemolysis positive, like S. uureus, but it does not produce coagulase, does not have strong phosphatase and deoxyribonuclease activities, and does not ferment mannose.Although gram-positive, catalase-positive cocci are very common on human skin, the methods used to classify them are not completely satifactory. Generally, the scheme devised by Baird-Parker (2, 3) is employed to divide the family Micrococcaceae. However, with this scheme it is impossible to separate weakly anaerobic staphylococci from micrococci, and such strains are often misidentified as micrococci. In applying Baird-Parker's system, Noble (12,13) has found that the predominant groups of skin cocci are Staphylococcus type 2 (probably S. epidermidis sensu stricto) and Micrococcus type 2. The taxonomic status of the latter type, which may include some weakly anaerobic staphylococci, is not clear. Deoxyribonucleic acid (DNA) base composition and cell wall composition are the characters most; useful in separating Micrococcus from Stuphylococcus (16, 18, 19). The cell wall peptidoglycan of the staphylococci contains large amounts of glycine and is thus clearly different from that of the micrococci. Moreover, all staphylococci contain teichoic acids or similar 50 compounds in their cell walls. In addition, the cell wall composition and the type of lactic acid produced from glucose under anaerobic conditions are very valuable in differentiating staphylococci (20). This paper reports on the results of the application of these criteria, as well as of morphological and physiological characters, to the classification of staphylococci isolated from human skin. Tables 2 through 5. Isolation and culture techniques. The procedures and media used for isolating staphylococci were identical to those used in previous studies on micrococci from human skin (10). MATERIALS AND METHODS

From a total of 40 characters that were previously used to differentiate species of staphylococci, 13 key characters were selected to make a simplified scheme that could be easily used by the routine clinical laboratory for identifying human staphylococci. These key characters included coagulase activity, hemolysis, nitrate reduction, and aerobic acid production from fructose, xylose, arabinose, ribose, maltose, lactose, sucrose, trehalose, mannitol, and xylitol. In the simplified scheme, 924 strains of staphylococci were placed into 11 positions, each of which contained the major portion (>80%) of strains of one of the recognized species. Several positions contained a rare or few uncommon strains of one or more additional species and these could be resolved on the basis of other key characters.

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Micrococci were commonly isolated from the skins of people living in various regions of the United States. Not all micrococci isolated in this investigation could be identified with the currently recognized species of Micrococcus, viz., M. luteus, M. varians, or M. roseus, and these micrococci therefore became the subject of further taxonomic study. As a result of this study, two new species are proposed: Micrococcus lylae and M. kristinae. The type strains of these species are ATCC 27566 and ATCC 27570, respectively. Numerous strains were isolated that were similar t o M. sedentarius or M. nishinomiyaensis, species that were previously represented by only single strains. (ZoBell's strain 5 4 1 [ ATCC 14392; CCM 3141 is designated herein as the type strain of M. sedentarius.) A few micrococci were left unclassified. A variety of morphological, physiological, biochemical, and genetic characters were examined for their use as taxonomic criteria, and key characters, many of which can be determined by simple laboratory procedures, were selected for species differentiation. The more sophisticated studies of aliphatic hydrocarbons and cell-wall peptidoglycans were also very useful in the taxonomy of the micrococci. The predominant micrococci found on human skin were M. luteus and M. varians.The medical literature has, for many years, made reference to a group of aerobic, saprophytic bacteria known as Sarcina that have been commonly isolated from human skin ( 14, 18,28,29,33). Recent taxonomic studies have indicated that most aerobic strains maintained in various culture collections under the name of Sarcina belong t o the species Micrococcus luteus (Schroeter 1872) Cohn 1872 or Micrococcus varians Migula 1900 (19, 21, 22, 24, 40). In line with this, Marples et al. (30) recently made reference t o the species M. luteus in studies on the aerobic microflora of the human scalp. M. varians has not yet been reported in studies of human skin. However, it was not until 1973 (22) that a comprehensive report on the taxonomic status of this species was made available. Earlier reports were somewhat confusing, and, with the lack of a clearly defined species status, cutaneous isolates may have been simply placed in Baird-Parker's Micrococcus subgroups together with other species (2). The purpose of this investigation was t o classify species of micrococci found on human skin by using the most current taxonomic critieria. We also became involved with evaluating existing taxonomic criteria, exploring new characters, and estimating species variation in cutaneous populations.Some of the preliminary results of this study have been previously reported (W. E. Kloos and K. H. Schleifer, Abst. Annu. Meet. Amer. SOC. Microbiol., 73rd, Miami Beach, p-116, 1973). MATERIALS AND METHODSBacterial straips. Micrococci were isolated from the healthy skins o f two groups of people. One group was composed of 20 people living in Raleigh, N.C., who were sampled once each month for 6-13 months. Samples from people of the Raleigh longitudinal study w...

The distribution of Staphylococcus and Micrococcus species and associated coryneform bacteria, Acinetobacter, Klebsiella, Enterobacter, Bacillus, and Streptomyces on skin was determined during October 1971 from samples collected on persons living in North Carolina and New Jersey. Persistence of these organisms on skin was estimated in temporal studies conducted during the period from June 1971 to June 1972 on persons living in North Carolina. Staphylococci and coryneforms were the most predominant and persistent bacteria isolated from the nares and axillae. Staphylococci, coryneforms, micrococci, and Bacillus were the most predominant and persistent bacteria isolated from the head, legs, and arms. Acinetobacters were most frequently isolated during the warmer months of the years. Staphylococcus aureus and S. epidermidis were the most predominant and persistent staphylococci isolated from the nares, whereas S. epidermidis and S. hominis were the most predominant and persistent staphylococci isolated from the axillae, head, legs, and arms. S. capitis was often isolated from the head and arms and S. haemolyticus was often isolated from the head, legs, and arms. S. simulans, S. xylosus, S. cohnii, S. saprophyticus, S. warneri, and an unclassified coagulase-positive species were only occasionally isolated from skin. Micrococcus luteus was the most predominant and persistent Micrococcus isolated from skin and preferred regions of the head, legs, and arms. M. varians was the second most frequent Micrococcus isolated. M. lylae, M. sedentarius, M. roseus, M. kristinae, and M. nishinomiyaensis were only occasionally isolated from skin. M. lylae was most frequently isolated during the colder months of the years. Numerous studies have been reported on the composition of the bacterial flora of human skin (12, 16, 17, 21, 24, 26, 30, 32). Most have resolved cutaneous bacterial populations into major groups or genera, e.g., Micrococcaceae (coagulase-positive and coagulase-negative staphylococci, micrococci, and/or Sarcina), streptococci, Mimae, nonlipophilic and lipophilic diphthe-' Paper no. 4671 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, N.C. 27607. species have been recognized. Recent systematic studies of Staphylococcus and Micrococcus species isolated from human skin have amended descriptions ofStaphylococcus epidermidis, S. saprophyticus, and Micrococcus sedentarius and characterized new species including S. cohnii, S. haemolyticus, S. xylosus, S. warneri, S. capitis, S. hominis, S. simulans, M. kristinae, and M. lylae (13-15, 27). Previously, certain staphylococci that failed to produce or produced only small amounts of acid from glucose under anaerobic conditions were misclassified as micrococci, and aerobic Sarcina were erroneously separated from the genus Micrococcus (2, 3, 10, 20, 21, 32, 33). Also several of the new species of staphylococci, if isolated, would have been misclassified as S. epidermidis (2-4, 6), and M. lylae and M. sedentarius would have been either misclass...

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In an effort to explore the origin and/or reservoirs of the genetic determinant(s) of methicillin resistance in Staphylococcus aureus, we examined over 200 strains representing 13 different species within the genus Staphylococcus for the presence of the mecA gene, using a DNA probe internal to this gene prepared from a methicillin-resistant strain of S. aureus. Occasional mecA- positive isolates were detected among several staphylococcal species. On the other hand, each one of the 134 isolates of Staphylococcus sciuri, a species considered taxonomically the most primitive among staphylococci and found primarily on rodents and primitive mammals, gave positive reaction with the DNA probe when tested under conditions of high stringency. About two thirds (99) of these isolates, all of which belonged to S. sciuri subspecies "sciuri," as well as 9 of the 11 species carnaticum isolates, showed only marginal, if any, resistance to methicillin (minimal inhibitory concentration of 0.75-6.0 micrograms/ml), while most of the remaining isolates that belonged to the subspecies "rodentius" (13 isolates in all) expressed antibiotic resistance with a heterogeneous phenotype similar to those seen in many methicillin-resistance strains of S. aureus In SmaI digests of chromosomal DNA isolated from such "methicillin-resistant S. aureus-like" strains, the mecA probe hybridized with DNA fragments in the range of 145-180 kb, while in subspecies "sciuri" and carnaticum isolates the mecA hybridizing fragment was located in the SmaI fragment with the highest molecular size (> or = 400 kb). A DNA probe comprising an internal sequence to the regulatory gene mecI from Staphylococcus epidermidis identified the presence of sequences with low degree of homology in isolates of the three S. sciuri subspecies. The mecA-reacting sequences in these bacteria differed from mecA of S. aureus in several respects (e.g., by the absence of a ClaI restriction site from mecA of subspecies "sciuri" and carnaticum, and in some isolates of subspecies "rodentius." The uniform presence of mecA in each one of a large number of S. sciuri strains belonging to distinct ribotypes and macrorestriction patterns and recovered over a 20-year period from a wide variety of animal sources and geographic sites suggests that mecA may be a native genetic element with an as yet unidentified physiologic function in this staphylococcal species.