bPoint mutations with unclear molecular mechanisms are often associated with vancomycin resistance in Staphylococcus aureus. Here, we observed that the walK (G223D) mutation caused decreased expression of genes associated with cell wall metabolism, decreased autolytic activity, thickened cell walls, and reduced vancomycin susceptibility. A phosphorylation assay showed that WalK (G223D) exhibited reduced autophosphorylation, which led to reduced phosphorylation of WalR. An electrophoretic mobility shift assay indicated that WalK (G223D)-phosphorylated WalR had a reduced capacity to bind to the atlA promoter. (2), graRS (3, 4), and walKR (5, 6), were shown to contribute to the development of VISA (5, 7). However, the molecular mechanisms have been incompletely defined (8). WalK is a sensor kinase of the WalKR two-component regulatory system (9-11), and walK mutations across the spectrum of the domains that contribute to two-component regulatory function have been found in many clinical VISA strains isolated from various countries and laboratory-derived VISA (5, 6). Nevertheless, the same mutations are not found in both clinical and laboratoryderived VISA strains. Thus, a key question is whether the mechanism of laboratory-derived VISA strains is analogous to that of clinical VISA strains. WThe laboratory-derived VISA strain, designated SV-1, was selected by serial passage of the susceptible S. aureus strain MW2 through progressively increasing concentrations of vancomycin according to a previously described protocol (5). To identify the genetic changes that confer vancomycin resistance, whole-genome sequencing of wild-type MW2 and SV-1 was completed at the National Center for Gene Research using the paired-end sequencing of Solexa. Five mutations were identified (Table 1) and further confirmed by PCR and sequencing (see the supplemental material). Among the mutations, a single-nucleotide polymorphism within walK (conferring the G223D amino acid change) in SV-1 was also found in the clinical VISA strain JKD6008 (6), supporting the validity of our approach in identifying clinically relevant resistance mechanisms and suggesting that this point mutation probably plays an important role in reduced vancomycin susceptibilities in clinical S. aureus isolates.To determine the effect of the walK mutation, allelic replacement was performed to generate the walK mutant using the vector pBTs, which was derived from pBT2 and pKOR1 (12, 13). To construct pBTs, the segment containing antisense secY, which can inhibit colony formation on agar plates, was cloned into pBT2, and the fragment containing the walK point mutation was cloned into pBTs (see the supplemental material). Antibiotic susceptibilities of the walK mutant were evaluated by determining the MICs of vancomycin and daptomycin using
Background: Delays in the diagnosis of pediatric osteoarticular infections (OAIs) can cause associated acute complications or long-term morbidity. This study attempts to develop a multiplex PCR-based assay that can rapidly and accurately detect the main pathogens involved in pediatric OAIs, namely, methicillinsensitive or methicillin-resistant Staphylococcus aureus, Streptococcus pyogenes and Pseudomonas aeruginosa.Methods: A set of four gene-specific primers suitable for use in a one-tube PCR assay was designed to detect four common pathogens involved in pediatric OAIs, namely, nuc for methicillin-sensitive Staphylococcus aureus, nuc and mecA for methicillin-resistant Staphylococcus aureus, spyM for Streptococcus pyogenes and orpI for Pseudomonas aeruginosa. The multiplex PCR was first evaluated with 39 isolated clinical strains and further with 41 specimens collected from patients suspected of having OAIs.Results: Specific primer pairs were successfully designed, and the targeted genes were simultaneously amplified. The product sizes in the assay for nuc, mecA, spyM and oprI were 233, 158, 336 and 109 bp, respectively. Evaluation of the multiplex PCR with 39 isolated clinical strains and 41 specimens revealed 100% sensitivity and 100% specificity. The limit of detection of the multiplex PCR assay was approximately 1×10 3 CFU at the bacterial cell level.Conclusions: This newly developed multiplex PCR assay, without sequencing, enables a rapid and accurate diagnosis of the major bacterial species in children with OAIs and might serve as an additional diagnostic approach for urgent pathogen determination.
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