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.

https://cmr.asm.org/content/cmr/7/1/117.full.pdf

Update on clinical significance of coagulase-negative staphylococci.

The name Staphylococcus (staphyle, bunch of grapes) was introduced by Ogston (1883) for the group micrococci causing inflammation and suppuration. He was the first to differentiate two kinds of pyogenic cocci: one arranged in groups or masses was called “Staphylococcus” and another arranged in chains was named “Billroth’s Streptococcus.” A formal description of the genus Staphylococcus was provided by Rosenbach (1884). He divided the genus into the two species Staphylococcus aureus and S. albus. Zopf (1885) placed the mass-forming staphylococci and tetrad-forming micrococci in the genus Micrococcus. In 1886, the genus Staphylococcus was separated from Micrococcus by Flügge (1886). He differentiated the two genera mainly on the basis of their action on gelatin and on relation to their hosts. Staphylococci liquefied gelatin and were parasitic or pathogenic or both whereas micrococci were variable in their action on gelatin and were saprophytic. The genera Staphylococcus, Micrococcus and Planococcus, containing Gram-positive, catalase-positive cocci, were later placed in the family Micrococcaceae. Evans et al. (1955) proposed separating staphylococci from micrococci on the basis of their relation to oxygen. The facultative anaerobic cocci were placed in the genus Staphylococcus and the obligate aerobic cocci in the genus Micrococcus. By the mid-1960s, a clear distinction could be made between staphylococci and micrococci on the basis of their DNA base composition (Silvestri and Hill, 1965). Members of the genus Staphylococcus have a DNA G+C content of 33–40 mol%, whereas members of the genus Micrococcus have a high G+C content of around 70 mol%. Further studies have shown that staphylococci can be distinguished from micrococci and other catalase-positive cocci on the basis of their cell wall composition (Schleifer and Kandler, 1972; Endl et al., 1983), cytochrome profile (Faller et al., 1980) and menaquinone pattern (Collins and Jones, 1981), susceptibility to lysostaphin and erythromycin (Schleifer and Kloos, 1975b), bacitracin (Falk and Guering, 1983), and furazolidone (Baker, 1984). Comparative immunochemical studies of catalases (Schleifer, 1986), DNA-DNA hybridization studies, DNA-rRNA hybridization studies (Schleifer et al., 1979; Kilpper et al., 1980), and comparative oligonucleotide cataloguing of 16S rRNA (Ludwig et al., 1981) clearly demonstrated the epigenetic and genetic difference of staphylococci and micrococci. Members of the genus Staphylococcus form a coherent and well-defined group of related species that is widely divergent from those of the genus Micrococcus. Until the early 1970s, the genus Staphylococcus consisted of three species: the coagulase-positive species S. aureus and the coagulase-negative species S. epidermidis and S. saprophyticus, but a deeper look into the chemotaxonomic and genotypic properties of staphylococci led to the description of many new staphylococcal species. Currently, 36 species and several subspecies are recognized in the genus Staphylococcus (Table 1). The genus Macrococcus has been described on the basis of comparative 16S rRNA analysis, DNA-DNA hybridization studies, ribotype patterns, cell wall composition, and phenotypic characteristics (Kloos et al., 1998a). Macrococci can be separated from staphylococci on the basis of a generally higher DNA G+C content (38– 45 mol%), absence of cell wall teichoic acids (with the possible exception of M. caseolyticus), unique ribotype patterns, and generally larger cells. Members of the genus Macrococcus are also oxidase positive whereas most staphylococci (exceptions: S. lentus, S. sciuri and S. vitulus) are oxidase negative. There are four species in the genus Macrococcus (Table 1).

The Genera Staphylococcus and Macrococcus

The nucleotide sequences of the 16S rRNA gene (rDNA) in 38 taxa of the genus Staphylococcus were compared phylogenetically. Based on phylogenetic tree analysis, staphylococcal species were divided into 12 cluster groups. These cluster groups were in very good agreement with species groups determined by DNA-DNA reassociation studies. These genealogical classifications were consistent with the results of the production of coagulase or oxidase and with resistance to novobiocin. These suggest that the phylogenetic relationship of the genus Staphylococcus is accurately represented by the results obtained from the sequence analysis of 16S rDNA.

Phylogenetic relationships of 38 taxa of the genus Staphylococcus based on 16S rRNA gene sequence analysis.

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.

Recommended minimal standards for description of new staphylococcal species

This review provides a state-of-the-art description of the performance of Sanger cycle sequencing of the 16S rRNA gene for routine identification of bacteria in the clinical microbiology laboratory. A detailed description of the technology and current methodology is outlined with a major focus on proper data analyses and interpretation of sequences. The remainder of the article is focused on a comprehensive evaluation of the application of this method for identification of bacterial pathogens based on analyses of 16S multialignment sequences. In particular, the existing limitations of similarity within 16S for genus- and species-level differentiation of clinically relevant pathogens and the lack of sequence data currently available in public databases is highlighted. A multiyear experience is described of a large regional clinical microbiology service with direct 16S broad-range PCR followed by cycle sequencing for direct detection of pathogens in appropriate clinical samples. The ability of proteomics (matrix-assisted desorption ionization-time of flight) versus 16S sequencing for bacterial identification and genotyping is compared. Finally, the potential for whole-genome analysis by next-generation sequencing (NGS) to replace 16S sequencing for routine diagnostic use is presented for several applications, including the barriers that must be overcome to fully implement newer genomic methods in clinical microbiology. A future challenge for large clinical, reference, and research laboratories, as well as for industry, will be the translation of vast amounts of accrued NGS microbial data into convenient algorithm testing schemes for various applications (i.e., microbial identification, genotyping, and metagenomics and microbiome analyses) so that clinically relevant information can be reported to physicians in a format that is understood and actionable. These challenges will not be faced by clinical microbiologists alone but by every scientist involved in a domain where natural diversity of genes and gene sequences plays a critical role in disease, health, pathogenicity, epidemiology, and other aspects of life-forms. Overcoming these challenges will require global multidisciplinary efforts across fields that do not normally interact with the clinical arena to make vast amounts of sequencing data clinically interpretable and actionable at the bedside.

Performance and Application of 16S rRNA Gene Cycle Sequencing for Routine Identification of Bacteria in the Clinical Microbiology Laboratory

A new coagulase-negative species of the genus Staphylococcus, Staphylococcus muscae, is described on the basis of the results of a study of four strains that were isolated from flies. 16S rRNA sequences of the type strains of S. muscae, Staphylococcus schleiferi, and Staphylococcus sciuri were determined and used, together with the corresponding sequences of Staphylococcus aureus and Staphylococcus epidermidis, for a comparative analysis. The new species is characterized taxonomically; this species is differentiated from the other novobiocin-susceptible staphylococci by its physiological and biochemical activities, cell wall composition, and levels of genetic relatedness. The type strain of this species is strain MB4 (= CCM 4175).

V. Hájek

Papers

Staphylococcus muscae, a new species isolated from flies