Novel species-specific targets for real-time PCR detection of four common pathogenic Staphylococcus spp.

Although nosocomial infections by Staphylococcus epidermidis have gained much attention, this skin-colonizing bacterium has apparently evolved not to cause disease, but to maintain the commonly benign relationship with its host. Accordingly, S. epidermidis does not produce aggressive virulence determinants. Rather, factors that normally sustain the commensal lifestyle of S. epidermidis seem to give rise to additional benefits during infection. Furthermore, we are beginning to comprehend the roles of S. epidermidis in balancing the epithelial microflora and serving as a reservoir of resistance genes. In this Review, I discuss the molecular basis of the commensal and infectious lifestyles of S. epidermidis.

Synthetic oligonucleotide primers of 21 and 24 bases, respectively, were used in the polymerase chain reaction (PCR) to amplify a sequence of the nuc gene, which encodes the thermostable nuclease of Staphylococcus aureus. A DNA fragment of approximately 270 bp was amplified from lysed S. aureus cells or isolated DNA. The PCR product was detected by agarose gel electrophoresis or Southern blot analysis by using a 33-mer internal nuc gene hybridization probe. With S. aureus cells the lower detection limit was less than 10 CFU, and with the isolated target the lower detection limit was 0.69 pg of DNA. The primers recognized 90 of 90 reference or clinical S. aureus strains. Amplification was not recorded when 80 strains representing 16 other staphylococcal species were tested or when 20 strains representing 9 different nonstaphylococcal species were tested. Some of the non-S. aureus staphylococci produced thermostable nucleases but were PCR negative. The PCR product was generated when in vitro-cultured S. aureus was used to prepare simulated clinical specimens of blood, urine, cerebrospinal fluid, or synovial fluid. No PCR product was generated when the sterile body fluids were tested. However, the sensitivity of the PCR was reduced when S. aureus in blood or urine was tested in comparison with that when bacteria in saline were tested. With the bacteria in blood, the detection limit of the PCR was 10(3) CFU. A positive PCR result was recorded when a limited number of clinical samples from wounds verified to be infected with S. aureus were tested, while the PCR product was not detected in materials from infections caused by other bacteria. Generation of PCR products was not affected by exposure of S. aureus to bactericidal agents, including cloxacillin and gentamicin, prior to testing, but was affected by exposure to UV radiation. The PCR for amplification of the nuc gene has potential for the rapid diagnosis of S. aureus infections by direct testing of clinical specimens, including specimens from patients with ongoing antimicrobial therapy.

Bacterial genomes vary extensively in terms of both gene content and gene sequence. This plasticity hampers the use of traditional SNP-based methods for identifying all genetic associations with phenotypic variation. Here we introduce a computationally scalable and widely applicable statistical method (SEER) for the identification of sequence elements that are significantly enriched in a phenotype of interest. SEER is applicable to tens of thousands of genomes by counting variable-length k-mers using a distributed string-mining algorithm. Robust options are provided for association analysis that also correct for the clonal population structure of bacteria. Using large collections of genomes of the major human pathogens Streptococcus pneumoniae and Streptococcus pyogenes, SEER identifies relevant previously characterized resistance determinants for several antibiotics and discovers potential novel factors related to the invasiveness of S. pyogenes. We thus demonstrate that our method can answer important biologically and medically relevant questions.