Wesley E. Kloos

Update on clinical significance of coagulase-negative staphylococci.

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. Images

Isolation and Characterization of Staphylococci from Human Skin I. Amended Descriptions of Staphylococcus epidermidis and Staphylococcus saprophyticus and Descriptions of Three New Species: Staphylococcus cohnii, Staphylococcus haemolyticus, and Staphylococcus xylosus

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. saprophyticus 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. saprophyticus do not reduce nitrate, but most of them produce acetylmethylcarbinol and ferment xylitol. S. haemolyticus is usually hemolysis positive, like S. aureus, but it does not produce coagulase, does not have strong phosphatase and deoxy-ribonuclease activities, and does not ferment mannose.

Isolation and characterization of staphylococci from human skin. II. Descriptions of four new species: Staphylococcus warneri, Staphylococcus capitis, Staphylococcus hominis, and Staphylococcus simulans.

Staphylococci were isolated from the skins of people living in North Carolina and New Jersey and were studied in an attempt to resolve their natural relationships. As a result of this study, four new species are proposed in this paper: Staphylococcus warneri, S. capitis, S. hominis, and S. simulans. The type strains of these species are ATCC 27836, ATCC 27840, ATCC 27844, and ATCC 27848, respectively. The new species were established on the basis of a variety of morphological, physiological, biochemical, and antibiotic characters. Cell wall composition was particularly useful in resolving species and correlated well with other characters. Characteristic pigment production was useful in distinguishing several of the different species. A summary of the character variation found in the species and a scheme for the classification of human cutaneous staphylococci are included in this paper. The predominant staphylococci found on human skin were S. epidermidis and S. hominis.

Isolation and Characterization of Micrococci From Human Skin, Including Two New Species: Micrococcus lylae and Micrococcus kristinae1

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 to M. sedentarius or M. nishinomiyaensis, species that were previously represented by only single strains. (ZoBells strain 541 [ATCC 14392; CCM 314] 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.

Characterization of Staphylococcus sciuri sp.nov. and Its Subspecies1

Several strains previously classified as group III staphylococci, by the scheme of Schleifer and Kocur, and numerous strains isolated from animal and human skin that appeared to be related to group III strains were subjected to a taxonomic study. As a result of this study, all group III and related strains were placed in the newly proposed species Staphylococcus sciuri. This species can be differentiated from all other staphylococci on the basis of colony morphology, cell wall peptidoglycan, acid production from cellobiose and usually from fucose under aerobic conditions, and a combination of other characteristics. Thirty-five strains that produced large colonies, usually moderate to light anaerobic growth in thioglycolate, and acid from galactose, sucrose, glycerol, and often from melezitose were placed in the type subspecies, S. sciuri subsp. sciuri. Nine strains that produced very small, unpigmented colonies, usually no detectable anaerobic growth in thioglycolate, and acid from sucrose and often from galactose, glycerol, lactose, and raffinose were placed in the subspecies S. sciuri subsp. lentus. The type strains of these subspecies are ATCC 29062 and ATCC 29070, respectively. A group of three strains that produced relatively small, unpigmented colonies, moderate anaerobic growth in thioglycolate, and acid from glycerol but failed to produce acid from sucrose, melezitose, raffinose and usually galactose may also deserve subspecies status. A summary of the character variation found in S. sciuri and in other novobiocin-resistant species and a simplified scheme for distinguishing S. sciuri and its subspecies are included in this paper.

Recommended minimal standards for description of new staphylococcal species

In accordance with Recommendation 30b of the International Code of Nomenclature of Bacteria, minimal standards are proposed for the genus Staphylococcus and the description of newly recognized species in this genus. Assignment of a strain to the genus Staphylococcus requires that it is a Gram-positive coccus that forms clusters, produces catalase, has an appropriate cell wall structure (including peptidoglycan type and teichoic acid presence) and G + C content of DNA in a range of 30-40 mol%. The recommended minimal standards for describing a new Staphylococcus species are based on the results of phenotypic and genomic studies of at least five independently isolated strains. They include colony morphology and the results of the following conventional tests: pigment production, growth requirements, fermentative and oxidative activity on carbohydrates, novobiocin susceptibility, enzymic activities (nitrate reductase, alkaline phosphatase, arginine dihydrolase, ornithine decarboxylase, urease, cytochrome oxidase, staphylocoagulase in rabbit plasma, heat-stable nuclease, amidases, oxidases, clumping factor, and haemolytic activity on sheep or bovine blood agar). DNA-DNA hybridization experiments may distinguish species when the difference between the binding in the homologous reaction and the binding in the heterologous reaction expressed as a percentage is less than 70%. In addition, rRNA signature sequence criteria, ribotyping characterization of the nomenclature type strain and other strains of the species, and reference strains of other species is recommended to describe the strains of the new species with sets of genetic attributes and reveal possible grouping errors. This proposal has been endorsed by the members of the Subcommittee on the taxonomy of staphylococci and streptococci of the international Committee on Systematic Bacteriology.

Ribotype Delineation and Description of Staphylococcus sciuri Subspecies and Their Potential as Reservoirs of Methicillin Resistance and Staphylolytic Enzyme Genes

Three subspecies of Staphylococcus sciuri, S. sciuri subsp. sciuri Kloos, Schleifer, and Smith 1976, 23AL emend. Kloos et al. 1997 [corrected], S. sciuri subsp. carnaticus subsp. nov., and S. sciuri subsp. rodentium subsp. nov., are described on the basis of their ribotype patterns, DNA-DNA liquid hybridization data, and phenotypic characteristics. Normalized ribotyping subdivided the S. sciuri patterns into three blocks of patterns, each corresponding to a subspecies. Each subspecies formed a separate, well-defined DNA similarity group when DNA-DNA hybridizations were conducted under stringent (70 degrees C) reassociation conditions. S. sciuri subsp. sciuri could be distinguished from the other subspecies on the basis of its ability to produce acid from D-cellobiose, alkaline phosphatase activity, and inability to produce either clumping factor or protein A. S. sciuri subsp. carnaticus could be distinguished by its ability to produce acid aerobically from D-xylose and maltose, inability to produce acid from D-melezitose, and smaller colony size on P agar. S. sciuri subsp. rodentium could be distinguished by its positive reaction in the latex agglutination test for clumping factor and/or protein A and generally higher frequencies and levels of oxacillin and methicillin resistance. All 40 strains of S. sciuri tested (including representatives of all three subspecies) hybridized with the mecA gene probe. All strains of S. sciuri subsp. sciuri, 79% of the strains of S. sciuri subsp. carnaticus and 89% of the strains of S. sciuri subsp. rodentium exhibited extracellular, staphylolytic enzyme activity. This activity was associated with an enzyme(s) that immunoblotted with a lysostaphin-specific monoclonal antibody; however, only three strains hybridized with a lysostaphin (end) gene probe. The type strain of S. sciuri subsp. carnaticus is DD 791 (= ATCC 700058), and the type strain of S. sciuri subsp. rodentium is DD 4761 (= ATCC 700061).

Delimiting the genus Staphylococcus through description of Macrococcus caseolyticus gen. nov., comb. nov. and Macrococcus equipercicus sp. nov., Macrococcus bovicus sp. nov. and Macrococcus carouselicus sp. nov.

Four species of the newly proposed genus Macrococcus, namely macrococcus caseolyticus gen. nov., comb. nov. (formerly Staphylococcus caseolyticus Schleifer, Kilpper-Bälz, Fischer, Faller and Endl 1982, 19VP), Macrococcus equipercicus sp. nov., Macrococcus bovicus sp. nov. Macrococcus carouselicus sp. nov., are described on the basis of a phylogenetic analysis comparing 16S 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 closet relatives, members of the genus Staphylococcus, these organisms demonstrated significantly lower 16S rRNA sequence similarities (93.4-95.3%), higher DNA G+C content (38-45 mol%), 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 (1.1-2.5% microns in diameter). Macrococci can be distinguished from most species of staphylococci (except Staphylococcus sciuri, Staphylococcus vitulus and Staphylococcus lentus) by thier 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, Staphy 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. caseolyticus, M. bovicus and M. carouselicus are ATTCC 51831T (= DD 9350T) ATCC 13548T (= TDD 4508T) (Schleifer et al. 1982, ATCC 51825T (= DD 4516T) and ATCC 51828T (= DD 9348), respectively.

Deoxyribonucleotide Sequence Relationships Among Bordetella Species

Deoxyribonucleotide sequence relationships among currently recognized Bordetella species (Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica) were examined by deoxyribonucleic acid (DNA) hybridization involving the hydroxyapatite batch procedures of Brenner and co-workers. The results indicated that strains from all species tested were highly related. At the stringent criterion (80°C), the relative binding of B. pertussis DNA to B. parapertussis DNA was 75 ± 9%, and to B. bronchiseptica DNA it was 73 ± 8%. Intraspecies binding was 93 ± 8%. Under similar conditions, the relative binding of B. parapertussis DNA to B. bronchiseptica DNA was 85 ± 9%. The various so-called Bordetella species may be reconsidered as representing different subspecies belonging to a single species.

Identification of the Staphylococcus sciuri Species Group with EcoRI Fragments Containing rRNA Sequences and Description of Staphylococcus vitulus sp. nov.

Strains of a new species, Staphylococcus vitulus, were isolated from food and a variety of mammals. This species was recognized on the basis of the results of an analysis of genomic EcoRI fragments containing portions of the rRNA operons. The patterns of hybridized fragments obtained from strains belonging to the new taxon were sorted into a distinguishable cluster and were distinct from the Staphylococcus lentus and Staphylococcus sciuri patterns. The results of DNA-DNA hybridization reactions demonstrated that strains in this cluster were more closely related to S. lentus and S. sciuri than to other Staphylococcus species and yet were significantly different. While these strains had some of the phenotypic characteristics of the S. sciuri species group, the newly recognized taxon could be distinguished by its very small colonies on P agar, absence of alkaline phosphatase activity, and lack of acid production from L-arabinose, maltose, N-acetylglucosamine, D-mannose, and raffinose. The type strain of the new species is strain DD 756 (= ATCC 51145).