Clinical isolates of Staphylococcus aureus (a total of 206) and S. epidermidis (a total of 188) from various countries were tested with multiplex PCR assays to detect clinically relevant antibiotic resistance genes associated with staphylococci. The targeted genes are implicated in resistance to oxacillin ( mecA ), gentamicin [ aac (6′)- aph (2")], and erythromycin ( ermA , ermB , ermC , and msrA ). We found a nearly perfect correlation between genotypic and phenotypic analysis for most of these 394 strains, showing the following correlations: 98% for oxacillin resistance, 100% for gentamicin resistance, and 98.5% for erythromycin resistance. The discrepant results were (i) eight strains found to be positive by PCR for mecA or ermC but susceptible to the corresponding antibiotic based on disk diffusion and (ii) six strains of S. aureus found to be negative by PCR for mecA or for the four erythromycin resistance genes targeted but resistant to the corresponding antibiotic. In order to demonstrate in vitro that the eight susceptible strains harboring the resistance gene may become resistant, we subcultured the susceptible strains on media with increasing gradients of the antibiotic. We were able to select cells demonstrating a resistant phenotype for all of these eight strains carrying the resistance gene based on disk diffusion and MIC determinations. The four oxacillin-resistant strains negative for mecA were PCR positive for blaZ and had the phenotype of β-lactamase hyperproducers, which could explain their borderline oxacillin resistance phenotype. The erythromycin resistance for the two strains found to be negative by PCR is probably associated with a novel mechanism. This study reiterates the usefulness of DNA-based assays for the detection of antibiotic resistance genes associated with staphylococcal infections.

Molecular methods for the rapid identification of methicillin-resistantStaphylococcus aureus(MRSA) are generally based on the detection of anS. aureus-specific gene target and themecAgene. However, such methods cannot be applied for the direct detection of MRSA from nonsterile specimens such as nasal samples without the previous isolation, capture, or enrichment of MRSA because these samples often contain both coagulase-negative staphylococci (CoNS) andS. aureus, either of which can carrymecA.In this study, we describe a real-time multiplex PCR assay which allows the detection of MRSA directly from clinical specimens containing a mixture of staphylococci in <1 h. Five primers specific to the different staphylococcal cassette chromosomemec(SCCmec) right extremity sequences, including three new sequences, were used in combination with a primer and three molecular beacon probes specific to theS. aureuschromosomalorfXgene sequences located to the right of the SCCmecintegration site. Of the 1,657 MRSA isolates tested, 1,636 (98.7%) were detected with the PCR assay, whereas 26 of 569 (4.6%) methicillin-susceptibleS. aureus(MSSA) strains were misidentified as MRSA. None of the 62 nonstaphylococcal bacterial species or the 212 methicillin-resistant or 74 methicillin-susceptible CoNS strains (MRCoNS and MSCoNS, respectively) were detected by the assay. The amplification of MRSA was not inhibited in the presence of high copy numbers of MSSA, MRCoNS, or MSCoNS. The analytical sensitivity of the PCR assay, as evaluated with MRSA-negative nasal specimens containing a mixture of MSSA, MRCoNS, and MSCoNS spiked with MRSA, was ∼25 CFU per nasal sample. This real-time PCR assay represents a rapid and powerful method which can be used for the detection of MRSA directly from specimens containing a mixture of staphylococci.

Staphylococcus aureusis the cause of serious infections in humans, including endocarditis, deep-seated abscesses, and bacteremia, which lead to toxic and septic shock syndromes. Rapid and direct identification of this bacterium specifically and ubiquitously directly from clinical specimens would be useful in improving the diagnosis ofS. aureusinfections in the clinical microbiology laboratory. A wide variety of kits based on biochemical characteristics efficiently identifyS. aureus, but the rapidity and the accuracy of each of these methods combined with testing of clinically relevant antibiotic resistance genes need to be improved. On the basis of hybridization assays with randomly selected clones from anS. aureusgenomic library, we have identified a chromosomal DNA fragment which is specific forS. aureusand which detected all 82S. aureusisolates tested. This 442-bp fragment was sequenced and was used to design a set of PCR amplification primers. The PCR assay was also specific and ubiquitous for the identification from bacterial cultures of 195 clinical strains ofS. aureusisolated from a variety of anatomical sites and obtained from hospitals throughout the world. The PCR assay that we have developed is simple and can be performed in about 1 h. This DNA-based test provides a novel diagnostic tool for the diagnosis ofS. aureusinfections.

Enterococci are becoming major nosocomial pathogens, and increasing resistance to vancomycin has been well documented. Conventional identification methods, which are based on culturing, require 2 to 3 days to provide results. PCR has provided a means for the culture-independent detection of enterococci in a variety of clinical specimens and is capable of yielding results in just a few hours. However, all PCR-based assays developed so far are species specific only for clinically important enterococci. We have developed a PCR-based assay which allows the detection of enterococci at the genus level by targeting the tuf gene, which encodes elongation factor EF-Tu. Initially, we compared the nucleotide sequences of the tuf gene from several bacterial species (available in public databases) and designed degenerate PCR primers derived from conserved regions. These primers were used to amplify a target region of 803 bp from four enterococcal species ( Enterococcus avium , E. faecalis , E. faecium , and E. gallinarum ). Subsequently, the complete nucleotide sequences of these amplicons were determined. The analysis of a multiple alignment of these sequences revealed regions conserved among enterococci but distinct from those of other bacteria. PCR primers complementary to these regions allowed amplification of genomic DNAs from 14 of 15 species of enterococci tested ( E. solitarius DNA could not be amplified). There was no amplification with a majority of 79 nonenterococcal bacterial species, except for 2 Abiotrophia species and several Listeria species. Furthermore, this assay efficiently amplified all 159 clinical isolates of enterococci tested (61 E. faecium , 77 E. faecalis , 9 E. gallinarum , and 12 E. casseliflavus isolates). Interestingly, the preliminary sequence comparison of the amplicons for four enterococcal species demonstrated that there were some sequence variations which may be used to generate species-specific internal probes. In conclusion, this rapid PCR-based assay is capable of detecting all clinically important enterococci and has potential for use in clinical microbiology laboratories.

Group B streptococcal infections are an important cause of neonatal morbidity and mortality. A rapid method for the detection of this organism in pregnant women at the time of delivery is needed to allow early treatment of neonates. We studied the efficacy of two polymerase-chain-reaction (PCR) assays for routine screening of pregnant women for group B streptococci at the time of delivery. We obtained anal, vaginal, and combined vaginal and anal specimens from 112 pregnant women; in 57 women, specimens were obtained before and after the rupture of the amniotic membranes. The specimens were tested for group B streptococci by culture in a standard selective broth medium, with a conventional PCR assay, and with a new fluorogenic PCR assay. Among the 112 women, the results of the culture of the combined vaginal and anal specimens were positive for group B streptococci in 33 women (29.5 percent). The two PCR assays detected group B streptococcal colonization in specimens from 32 of these 33 women: the one negative PCR result was in a sample obtained after the rupture of membranes. As compared with the culture results, the sensitivity of both PCR assays was 97.0 percent and the negative predictive value was 98.8 percent. Both the specificity and the positive predictive value of the two PCR assays were 100 percent. The length of time required to obtain results was 30 to 45 minutes for the new PCR assay, 100 minutes for the conventional PCR assay, and at least 36 hours for culture. Colonization with group B streptococci can be identified rapidly and reliably by a PCR assay in pregnant women in labor both before and after the rupture of membranes.