Linezolid resistance emerged in two isolates of ST36 MRSA colonizing the lungs of two paediatric cystic fibrosis patients. Subtherapeutic levels of linezolid may have facilitated the selection of resistance.

Mutations in mutS and mutL, which encode DNA mismatch repair (MMR) proteins, can confer hypermutator phenotypes and may facilitate the emergence of mutational antibiotic resistance in bacteria. Linezolidresistant enterococci (LRE) rarely emerge during therapy and contain mutations in 23S rRNA genes. As enterococci with defective MMR could be prone to the development of oxazolidinone resistance mutations, we investigated 13 clinical isolates of Enterococcus faecium, including 2 LRE, for mutations in mutSL. A 4,944-bp fragment spanning mutSL was sequenced from two pairs of linezolid-resistant (MICs, 64 g/ml) and linezolidsusceptible (MICs, 2 g/ml) E. faecium isolates (one pair from Austria and one pair from the United Kingdom) identical by pulsed-field gel electrophoresis. The pairs represented distinct strains in which linezolid resistance had emerged during therapy. The MutSL peptides of all four isolates had amino acid substitutions compared with the sequence of E. faecium strain DO (used for genome sequencing). These were Val352Ile (one pair of isolates only) and Met628Leu in MutS and Leu387Pro, Tyr406Phe, Thr415Ser, Phe427Leu, and Phe565Ile in MutL. The significance of these changes remains unknown; these isolates did not show a demonstrable hypermutator phenotype. The same substitutions were found in two of nine geographically diverse linezolidsusceptible enterococcal isolates; the other seven isolates had MutSL sequences identical to that of strain DO. Multilocus sequence typing revealed that all isolates with alternate MutSL peptides belonged to a distinct lineage of a prevalent E. faecium clonal complex, designated CC17. Further studies are needed to investigate the prevalence of these MutSL mutations and their possible roles in the emergence of E. faecium strains resistant to oxazolidinones and other antibiotic classes.

RWJ-416457, a novel oxazolidinone, had modal MICs of 0.5 to 1 g/ml for linezolid-susceptible staphylococci and enterococci, versus linezolid MICs for these organisms of 1 or 2 g/ml. RWJ-416457 MICs for mutants with 23S rRNA mutations were 2 to 32 g/ml, versus linezolid MICs of 8 to 64 g/ml; actual values reflected the proportion of gene copies mutated.Oxazolidinones are among the very few genuinely new antimicrobial classes developed in the past 30 years, with linezolid the sole analogue currently marketed. At launch, linezolid had nearly universal activity against gram-positive bacteria, with MICs for staphylococci, enterococci, and streptococci tightly clustered from 0.5 to 4 g/ml (3). Licensing trials showed equivalence to standard therapies in pneumonia infections and in skin and skin structure infections (10), while pooled subgroup analysis suggested superiority over vancomycin in methicillin-resistant Staphylococcus aureus (MRSA) pneumonia (11), and a recent prospective trial indicated superiority over vancomycin in MRSA skin and skin structure infections (9).Linezolid resistance is sometimes selected in vivo, particularly if treatment is prolonged or if the drug dosage is reduced (3). Most of the clinical mutants have G2576T mutations in domain V of their 23S rRNA genes, giving G2576U in the corresponding rRNA product, although other 23S rRNA mutations are occasionally found. More than one 23S rRNA gene copy must be altered to confer resistance, meaning that internal recombination events must follow modification of the first gene copy (4). This complexity doubtless explains the continued rarity of resistance, which remains essentially undetectable in large-scale epidemiological surveys.Numerous further oxazolidinone analogues have been synthesized in the search for improvements over linezolid, but none has yet progressed beyond phase 1 testing. RWJ-416457 is a novel investigational analogue (1) now being developed by Ortho McNeil (Raritan, NJ). We evaluated its activity against linezolid-susceptible and -resistant staphylococci and enterococci. The MICs of RWJ-416457, linezolid, and other comparators (quinupristin-dalfopristin, ampicillin, oxacillin, gentamicin, vancomycin, and teicoplanin) were determined by CLSI agar dilution methodology (5) for clinical isolates (ca. 100 each) of linezolid-susceptible Staphylococcus aureus, coagulase-negative staphylococci, and enterococci, all collected in the United Kingdom. These collections were designed to include equal proportions of MRSA and methicillin-susceptible S. aureus and of Enterococcus faecium and Enterococcus faecalis. The enterococcal collections were deliberately loaded with vancomycin-resistant organisms, since oxazolidinones are particularly used in infections due to these organisms. Both the staphylococci and enterococci were selected for epidemiological diversity, being sourced from a wide geographic spread of hospitals. Nevertheless, the MRSA collections were dominated by the nationally prevalent epidemic MRSA-15 and -16 lineages, which account ...

The aim of this work was to develop a molecular method using loop-mediated isothermal amplification (LAMP) for detection of extended spectrum β-lactamase (ESBL)-producing Enterobacteriaceae from meat, and to compare it with different isolation agars and microarrays. LAMP assays were developed for CTX-M groups 1, 2, and 9 and OXA-10-like genes. Chicken, lamb, beef, pork, and turkey samples were spiked with 10, 100, and 1,000 cfu/gram using 8 strains of ESBL-producing Enterobacteriaceae (CTX-M sequence types 1, 2, 3, 14, 15, OXA-11, SHV-2, TEM-52) +/- a mix of competitor organisms. Samples were enriched overnight in buffered peptone water (BPW) +/- antibacterials before plating to CHROMagar CTX, OXOID ESBL Brilliance agar, and MacConkey agar with 1 mg/L cefotaxime. Selected BPW broths were also tested using LAMP assays, microarrays and using cefpodoxime discs on agar. For isolation/detection of ESBL producers from beef, pork, lamb, and turkey spiked with 10 or 100 cfu/gram ESBL (natural flora only), all agars and the LAMP assays showed 100% sensitivity and specificity for ESBL spike strains. For chicken samples, both LAMP and chromogenic agars showed improved sensitivity and specificity for isolation of ESBLs compared with MacConkey agar, particularly with competitor bacteria added. In comparison, the cefpodoxime disc method and microarray showed reduced sensitivity.

Abstract. Staphylococcus pseudintermedius is a commensal and opportunistic pathogen of dogs. It is mainly implicated in canine pyoderma, as well as other suppurative conditions of dogs. Although bacterial culture is routinely used for clinical diagnosis, molecular methods are required to accurately identify and differentiate S. pseudintermedius from other members of the Staphylococcus intermedius group. These methods, owing largely to their cost, are not easy to implement in nonspecialized laboratories or veterinary practices. In the current study, loop-mediated isothermal amplification (LAMP), a novel isothermal nucleic acid amplification procedure, was employed to develop a rapid, specific, and sensitive S. pseudintermedius assay. Different detection strategies, including the use of a lateral flow device, were evaluated. The assay was evaluated for crossreactivity against 30 different bacterial species and validated on a panel of 108 S. pseudintermedius isolates, originating from different dog breeds and locations within the United Kingdom. The assay was specific, showing no cross-reactivity during in silico and in vitro testing. When tested using DNA extracts prepared directly from 35 clinical surgical site swabs, the assay could detect S. pseudintermedius in less than 15 min, with a diagnostic sensitivity of 94.6%, superior to that of a polymerase chain reaction method. The LAMP assay also had an analytical sensitivity in the order of 10 1 gene copies, and the amplified products were readily detected using a lateral flow device. The LAMP assay described in the present study is simple and rapid, opening up the possibility of its use as a diagnostic tool within veterinary practices.