Wide geographical dissemination of the multiresistant Staphylococcus capitis NRCS-A clone in neonatal intensive-care units.

Nosocomial late-onset sepsis represents a frequent cause of morbidity and mortality in preterm neonates. The Staphylococcus capitis clone NRCS-A has been previously described as an emerging cause of nosocomial bacteraemia in French neonatal intensive-care units (NICUs). In this study, we aimed to explore the possible unrecognized dissemination of this clone on a larger geographical scale. One hundred methicillin-resistant S. capitis strains isolated from neonates (n = 86) and adult patients (n = 14) between 2000 and 2013 in four different countries (France, Belgium, the UK, and Australia) were analysed with SmaI pulsed-field gel electrophoresis (PFGE) and dru typing. The vast majority of NICU strains showed the NRCS-A pulsotype and the dt11c type (96%). We then randomly selected 14 isolates (from neonates, n = 12, three per country; from adult patients, n = 2), considered to be a subset of representative isolates, and performed further molecular typing (SacII PFGE, SCCmec typing, and multilocus sequence typing-like analysis), confirming the clonality of the S. capitis strains isolated from neonates, despite their distant geographical origin. Whole genome single-nucleotide polymorphism-based phylogenetic analysis of five NICU isolates (from the different countries) attested to high genetic relatedness within the NRCS-A clone. Finally, all of the NRCS-A strains showed multidrug resistance (e.g. methicillin and aminoglycoside resistance, and decreased vancomycin susceptibility), with potential therapeutic implications for infected neonates. In conclusion, this study represents the first report of clonal dissemination of methicillin-resistant coagulase-negative Staphylococcus clone on a large geographical scale. Questions remain regarding the origin and means of international spread, and the reasons for this clone's apparent predilection for neonates.

Identification of Streptococcus spp. causing bovine mastitis by PCR-RFLP of 16S-23S ribosomal DNA.

Traditional microbiological methods for identification of Streptococcus spp. causing bovine mastitis have been demonstrated to be less than highly reliable. PCR-RFLP analysis of 16S-23S ribosomal DNA was used to characterise seven reference strains of streptococcal mastitis pathogens as well as four reference strains of other gram-positive, catalase-negative cocci of bovine origin to allow comparative identification of field isolates. RFLP analysis of PCR products, using a combination of two restriction endonucleases in single reactions (HaeIII and AluI, HaeIII and RsaI or AluI and RsaI) generated unique patterns for species of Streptococcus, Enterococcus and Lactococcus. One hundred field isolates of Streptococcus spp. collected from cows with clinical or subclinical mastitis were tested. Fifty-seven isolates, classified by conventional tests as S. uberis, were identified as 47 S. uberis and six S. parauberis by their unique RFLP patterns. The remaining four isolates had RFLP patterns distinct from the reference strains and two of these were identified as closely related to S. iniae and two to Aerococcus viridans by 16S rRNA sequencing. Conventional identification of 17 S. agalactiae and 26 S. dysgalactiae subsp. dysgalactiae was confirmed by RFLP. Use of a combination of restriction enzymes in a single tube enabled the rapid, accurate, cost effective and easily performed identification of all major streptococcal mastitis pathogens.

Molecular epidemiology of coagulase-negative staphylococcal bacteraemia in a newborn intensive care unit.

We isolated 55 coagulase-negative staphylococci (CoNS) over two separate 12-month periods (26 in 1993 and 29 in 1996) from the blood of neonates in a neonatal intensive case unit (NICU) in Melbourne, Australia and compared them by pulse-field gel electrophoresis profile (PFGE), random amplification of polymorphic DNA (RAPD) and antibiogram. The most common species were Staphylococcus epidermidis, S. haemolyticus and S. warneri. The majority of such isolates were resistant to penicillin and to either or both of methicillin and gentamicin. During 1993, there was an increase in the number of CoNS bloodstream infections compared with previous years. S. epidermidis was the most common isolate, with 88% assessed as clinically relevant. Using the three typing systems, we identified one likely epidemic clone of S. epidermidis, the isolates of which were resistant to penicillin, gentamicin and erythromycin and possessed the mecA gene. There was complete correlation between the detection of mecA and the phenotypic expression of resistance when zone diameters in the disc diffusion assay were interpreted according to the latest NCCLS guidelines (1999). Profiles of the remaining 1993 isolates were generally heterogeneous, suggesting independent acquisition with some evidence of cross-infection. The predominant bloodstream isolates in 1996 were heterogeneous multi-resistant strains of S. epidermidis, S. haemolyticus and S. warneri, about half of which were assessed as clinically relevant. These data support the view that CoNS are significant nosocomial pathogens in NICU and that resistant clones may be transmitted between babies. Molecular epidemiological tools are helpful for understanding transmission patterns and sources of infection, and are useful for measuring outcomes of intervention strategies implemented to reduce nosocomial CoNS sepsis. PFGE was found to be more discriminatory than RAPD, but the latter provides results in a more timely manner.

Epidemiological typing of bovine streptococci by pulsed-field gel electrophoresis.

Pulsed-field gel electrophoresis (PFGE) was used to investigate the epidemiology of streptococcal mastitis in dairy cattle. The most prevalent streptococcal species, Streptococcus uberis (60-80% of streptococcal isolates), was highly heterogeneous, with different cows only rarely sharing the same pulsotype. S. agalactiae was rarely encountered, however all eight isolates from one farm generated identical PFGE profiles, which differed from those of all other isolates examined, confirming cow-to-cow transmission. Fifty-two isolates of S. dysgalactiae from 27 cows on 5 farms generated 6 different profiles. However, on individual farms, only one or two pulsotypes usually predominated. This species is generally regarded as an environmental pathogen but our data suggest that cow-to-cow transmission of S. dysgalactiae may occur. In spite of the variation in PFGE profiles of isolates from different cows, persistent infections in individual cows were usually caused by the same pulsotype of S. uberis or S. dysgalactiae.

Virulence of Staphylococcus epidermidis in a mouse model: significance of extracellular slime.

The ability to produce large quantities of biofilm on solid surfaces in vitro is believed to distinguish potentially pathogenic strains of Staphylococcus epidermidis from commensals. Biofilm consists of staphylococcal cells encased in a matrix of extracellular polysaccharide (also referred to as slime), firmly adherent to each other and to the underlying surface structure. The association of slime with colonization of catheter surfaces in vivo has been examined extensively. Less attention has been paid to the contribution of slime to infections that occur in the absence of an inserted device. In a mouse model of subcutaneous infection without an implanted device 10 S. epidermidis strains (5 slime-positive, 5 slime-negative) produced abscesses; thus a foreign body is not essential for the expression of virulence by S. epidermidis. Biofilm-positive strains produced significantly more abscesses, that persisted longer than biofilm-negative strains. In these chronic infections, large numbers of staphylococci were associated with macrophages and viable staphylococci were cultured from specimens of pus collected at autopsy. Thus slime or components of slime appear to delay the clearance of S. epidermidis from host tissues, possibly by interfering with intracellular killing mechanisms. However, differences in the capacity to produce abscesses, within both the slime-positive and slime-negative groups, indicate that other factors also contribute to the virulence of S. epidermidis.

A study of phenotypic variation of Staphylococcus epidermidis using Congo red agar.

This study examines a series of phenotypic variants of Staphylococcus epidermidis that were generated from a pair of parent variants, isolated from valvular tissue of a patient with prosthetic valve endocarditis. The variants were initially classified by examining their colonial morphology on Congo red agar. In addition to differences in Congo red binding and colonial morphology, they differed in the expression of several surface components and enzymes. Despite these phenotypic differences, all variants had the same restriction endonuclease profile of plasmid DNA. Examination of a collection of clinical isolates demonstrated that phenotypic variation is a common property of S. epidermidis. The ability to express different combinations of surface components and enzymes could contribute to the virulence of S. epidermidis strains by enabling these organisms to colonize a range of diverse environments.

Adherence measured by microtiter assay as a virulence marker for Staphylococcus epidermidis infections.

Staphylococcus epidermidis strains isolated from clinical sources showed a wide range of abilities to adhere to glass and plastic materials. The degree of adherence depended on a number of factors, most notably, the composition of the growth medium. Adherence was enhanced by the addition of glucose or oleic acid to the growth medium and inhibited by serum. We have demonstrated a statistically significant association between the quantitative assessment of adherence to polystyrene tissue culture plates and clinical relevance. No such association was found when adherence was assessed by the qualitative adherence assay. Possible new approaches for assessing the clinical relevance of coagulase-negative staphylococcal isolates are discussed.

Species identification, antibiotic sensitivity and slime production of coagulase-negative staphylococci isolated from clinical specimens.

Two hundred and seventy-five consecutive clinical isolates of coagulase-negative staphylococci, including strains associated with disease, contaminants and skin colonizers, were speciated, tested for slime production and for their sensitivity to a range of antibiotics. Staphylococcus epidermidis was the most commonly identified species, comprising 63% of all isolates. Slime production was detected in half the strains of Staph. epidermidis, Staph. haemolyticus and Staph. saprophyticus but was rare in other species. Most Staph. haemolyticus strains and approximately half of the Staph. epidermidis strains were resistant to five or more antibiotics. A significant association was found between slime production and multiple antibiotic resistance. For catheter-associated strains, clinical relevance was predictable by species i.e. Staph. epidermidis. Multi-resistant slime-positive Staph. haemolyticus strains, although infrequently associated with disease, were common skin colonizers, presumably acquired from the hospital environment. We describe a practical and inexpensive scheme for the speciation of human coagulase-negative staphylococcal isolates.