Potential Impact of Rapid Blood Culture Testing for Gram-Positive Bacteremia in Japan with the Verigene Gram-Positive Blood Culture Test

Background. Early detection of Gram-positive bacteremia and timely appropriate antimicrobial therapy are required for decreasing patient mortality. The purpose of our study was to evaluate the performance of the Verigene Gram-positive blood culture assay (BC-GP) in two special healthcare settings and determine the potential impact of rapid blood culture testing for Gram-positive bacteremia within the Japanese healthcare delivery system. Furthermore, the study included simulated blood cultures, which included a library of well-characterized methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) isolates reflecting different geographical regions in Japan. Methods. A total 347 BC-GP assays were performed on clinical and simulated blood cultures. BC-GP results were compared to results obtained by reference methods for genus/species identification and detection of resistance genes using molecular and MALDI-TOF MS methodologies. Results. For identification and detection of resistance genes at two clinical sites and simulated blood cultures, overall concordance of BC-GP with reference methods was 327/347 (94%). The time for identification and antimicrobial resistance detection by BC-GP was significantly shorter compared to routine testing especially at the cardiology hospital, which does not offer clinical microbiology services on weekends and holidays. Conclusion. BC-GP generated accurate identification and detection of resistance markers compared with routine laboratory methods for Gram-positive organisms in specialized clinical settings providing more rapid results than current routine testing

Rapid Acquisition of Linezolid Resistance in Methicillin-Resistant Staphylococcus aureus: Role of Hypermutation and Homologous Recombination.

BACKGROUND:We previously reported the case of a 64-year-old man with mediastinitis caused by Staphylococcus aureus in which the infecting bacterium acquired linezolid resistance after only 14 days treatment with linezolid. We therefore investigated relevant clinical isolates for possible mechanisms of this rapid acquisition of linezolid resistance. METHODS:Using clinical S. aureus isolates, we assessed the in vitro mutation rate and performed stepwise selection for linezolid resistance. To investigate homologous recombination, sequences were determined for each of the 23S ribosomal RNA (23S rRNA) loci; analyzed sequences spanned the entirety of each 23S rRNA gene, including domain V, as well as the 16S-23S intergenic spacer regions. We additionally performed next-generation sequencing on clinical strains to identify single-nucleotide polymorphisms compared to the N315 genome. RESULTS:Strains isolated from the patient prior to linezolid exposure (M5-M7) showed higher-level linezolid resistance than N315, and the pre-exposure strain (M2) exhibited more rapid acquisition of linezolid resistance than did N315. However, the mutation rates of these and contemporaneous clinical isolates were similar to those of N315, and the isolates did not exhibit any mutations in hypermutation-related genes. Sequences of the 23S rRNA genes and 16S-23S intergenic spacer regions were identical among the pre- and post-exposure clinical strains. Notably, all of the pre-exposure isolates harbored a recQ missense mutation (Glu69Asp) with respect to N315; such a lesion may have affected short sequence recombination (facilitating, for example, recombination among rrn loci). We hypothesize that this mechanism contributed to rapid acquisition of linezolid resistance. CONCLUSIONS:Hypermutation and homologous recombination of the ribosomal RNA genes, including 23S rRNA genes, appear not to have been sources of the accelerated acquisition of linezolid resistance observed in our clinical case. Increased frequency of short sequence recombination may have resulted from a recQ variant in the infecting organism

Challenges in evaluating forearm muscle activity based on the compound muscle action potential of the flexors of the whole forearm

Objective: Muscle strength, which correlates with the compound muscle action potential (CMAP), can also be estimated by measuring the CMAP. Therefore, we evaluated the CMAP of the flexor muscles of the whole forearm to identify their muscle strength. Methods: Fourteen healthy volunteers were enrolled. The elbow was determined to be the stimulation point, and the recording site for the flexor muscles of the whole forearm was set at approximately 8 cm distal to the elbow. We prospectively evaluated the baseline-to-peak amplitude of the CMAP of the whole forearm flexor muscles (WFFM), including that obtained from the median nerve stimulation (WFFMm), ulnar nerve stimulation (WFFMu), and their sum (WFFMsum). Additionally, we analyzed the relationships between WFFMm and WFFMu amplitudes with other quantitative parameters, including grip strength and routine CMAP amplitudes. Results: The CMAP’s test–retest analysis revealed high reliability. Grip power was significantly correlated with WFFMm and WFFMsum and mildly correlated with WFFMu. Tip-pinch strength with WFFMm and flexor pollicis longus (FPL) measurements correlated significantly. Lateral-pinch strength was significantly correlated with the first dorsal interosseous muscle (FDI) measurements but not with WFFM. The abductor digiti minimi (ADM) and abductor pollicis brevis (APB) were not correlated with grip power or pinch strength. Conclusions: By electrophysiology examination, this study demonstrated that WFFMm is involved in grip power and other pinch strengths. This method may serve as a novel tool for measurement of distal muscle strengths. Significance: This is the first study to attempt to evaluate the muscle strength of forearm flexor muscles by measuring the CMAP

List of strains used.

<p>List of strains used.</p

Phylogenetic tree of SNPs in the M2 and ST5 (MLST) reference strains.

<p>Phylogenetic tree of SNPs in the M2 and ST5 (MLST) reference strains.</p

Pattern of 16S-23S IGS regions in parental strains and isogenic linezolid (LZD)-resistant mutants.

<p>Pattern of 16S-23S IGS regions in parental strains and isogenic linezolid (LZD)-resistant mutants.</p

Stepwise selection of LZD-resistant mutants of MRSA strains.

<p>Strains M2 and M3 achieved MICs over 32 μg/ml in the short term, and reached 128 μg/ml by the end of the study. Strains M13 and N315 were used as controls in this experiment; neither strains achieved MICs of 32 μg/ml for two consecutive passages.</p