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We report the complete genome sequence of Mycobacterium heckeshornense strain JMUB5695, which was isolated from necrotizing granulomatous lesions in a lung cancer patient. The complete genome consists of a 4,865,109-bp chromosome with a GC content of 65.9% and contains no plasmids.

The association of Panton-Valentine leukocidin (PVL) toxin with necrotizing soft tissue infection (NSTI) caused by Staphylococcus aureus remains controversial. Here, we report the complete genome sequence of the PVL-negative S. aureus strain JMUB1273, isolated from a patient with pervasive NSTI.

Severe community-acquired pneumonia (CAP) caused by methicillin-resistant Staphylococcus aureus (MRSA) is relatively rare and is usually associated with rapid progression to death. Here, we report the complete genome sequence of the MRSA strain JMUB3031, which was isolated from a patient with fatal CAP.

Heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) clinical strain Mu3 spontaneously generates VISA strains at an extremely high frequency (≥1 × 10 −6 ). The generated VISA strains usually grow more slowly than does the parent hVISA strain, but they form colonies on vancomycin-containing agar plates before 48 h of incubation. However, we noticed a curious group of VISA strains, designated “slow VISA” (sVISA), whose colonies appear only after 72 h of incubation. They have extremely prolonged doubling times but have vancomycin MICs of 8 to ∼24 mg/liter when determined after 72 to ∼144 h of incubation. We established strain Mu3-6R-P (6R-P), which has a vancomycin MIC of 16 mg/liter (at 72 h), as a representative sVISA strain. Its cell wall was thickened and autolytic activity was decreased compared to the respective qualities of the parent hVISA strain Mu3. Whole-genome sequencing of 6R-P revealed only one mutation, encoded by rpoB (R512P), which replaced the 512th arginine of the RNA polymerase β-subunit with proline. Its VISA phenotype was unstable, and the strain frequently reverted to hVISA with concomitant losses of pinpoint colony morphology and cell wall thickness and reduced autolytic activity. Sequencing of the rpoB genes of the phenotypic revertant strains revealed mutations affecting the 512th codon, where the proline of 6R-P was replaced with leucine, serine, or histidine. Slow VISA generated in the tissues of an infected patient serves as a temporary shelter for hVISA to survive vancomycin therapy. The sVISA strain spontaneously returns to hVISA when the threat of vancomycin is lifted. The rpoB (R512P) mutation may be regarded as a regulatory mutation that switches the reversible phenotype of sVISA on and off.

Three types of phenotypic expression of β-lactam resistance have been reported in methicillin-resistant Staphylococcus aureus (MRSA): heterogeneous, homogeneous, and Eagle-type resistance. Heterogeneous-to-homogeneous conversion of β-lactam resistance is postulated to be caused by a chromosomal mutation ( chr* ) in addition to the expression of the mecA gene. Eagle-type resistance is a unique phenotype of chr* occurring in pre-MRSA strain N315 whose mecA gene expression is strongly repressed by an intact mecI gene. We here report that certain mutations of the rpoB gene, encoding the RNA polymerase β subunit, belong to chr* . We studied homogeneous MRSA (homo-MRSA) strain N315ΔIP-H5 (abbreviated as ΔIP-H5), which was obtained from hetero-MRSA strain N315ΔIP by selection with 8 mg/liter imipenem. Whole-genome sequencing of ΔIP-H5 revealed the presence of a unique mutation in the rpoB gene, rpoB (N967I), causing the amino acid replacement of Asn by Ile at position 967 of RpoB. The effect of the rpoB (N967I) mutation was confirmed by constructing a revertant H5 rpoB (I967N) strain as well as an N315-derived mutant, N315 rpoB (N967I). H5 rpoB (I967N) regained the hetero-resistance phenotype, and the N315 rpoB (N967I) strain showed an Eagle-type phenotype similar to that of the typical Eagle-type MRSA strain N315h4. Furthermore, subsequent whole-genome sequencing revealed that N315h4 also had a missense mutation of rpoB (R644H). Introduction of the rpoB (N967I) mutation was accompanied by decreased autolysis, prolonged doubling time, and tolerance to bactericidal concentrations of methicillin. We consider that rpoB mutations are the major cause for heterogeneous-to-homogeneous phenotypic conversion of β-lactam resistance in MRSA strain N315 and its derived strains.

Group B Streptococcus (GBS; Streptococcus agalactiae ) is a leading cause of serious neonatal infections. The Centers for Disease Control and Prevention recommends GBS screening for all pregnant women during the 35th to 37th weeks of gestation. Although GBS screening has been performed mainly by the culture-based method, it takes several days to obtain a reliable result. In this study, we developed a rapid immunochromatographic test (ICT) for the detection of GBS-specific surface immunogenic protein in 15 min using an overnight enrichment culture. The ICT was prepared using two anti-Sip monoclonal antibodies. This ICT was able to detect recombinant Sip levels of 0.5 ng/ml, or about 10 6 CFU/ml of GBS cells, in tests with 9 GBS strains of different serotypes. The cross-reactivity test using 26 species of microorganism showed no detectable false-positive result. Reactivity of the ICT with 229 GBS strains showed one false-negative result that was attributable to the production of truncated Sip. Among 260 enrichment cultures of vaginal swabs, 17 produced red to orange pigments in Granada medium, and they were all GBS and Sip positive. Among 219 pigment-negative cultures, 12 were GBS positive and 10 were Sip positive. Two Sip-negative cultures contained GBS cells below the limit of detection by the ICT. Among 207 GBS-negative cultures, only one was Sip positive, which was attributable to GBS cell debris. Thus, the sensitivity and specificity of the ICT appeared to be 93.1% and 99.6%, respectively. The newly developed ICT is readily applicable to clinical use in the detection of GBS.

Acute phlegmonous gastritis is an uncommon endogenous bacterial gastritis presenting with a high mortality rate. Here, we report the complete genome sequence of an emm 89 Streptococcus pyogenes strain, JMUB1235, which is the causative agent of acute phlegmonous gastritis.

We describe here the genetic analysis of a vancomycin-susceptible Staphylococcus aureus (VSSA) strain, Mu50Ω, a strain related to vancomycin-intermediate S. aureus (VISA) strain Mu50. Using a combination of Mu50Ω whole-genome sequencing and genome engineering, we observed a stepwise evolution of vancomycin resistance from VSSA to VISA after the mutated vraS and graR genes of Mu50 were engineered into Mu50Ω.

In order to better understand the mechanism of daptomycin resistance, we generated a daptomycinnonsusceptible derivative strain, strain 103d1 (MIC 3.0 g/ml), by in vitro exposure of methicillinresistant Staphylococcus aureus strain N315IP (MIC 0.5 g/ml) to daptomycin. We also obtained a daptomycin-susceptible phenotypic revertant strain, strain 103d1-10 (MIC 1.0 g/ml), by passaging 103d1 in drug-free medium for 10 days. The resultant triple-isogenic strains were analyzed for their phenotypes and gene expression by microarray analysis. No significant differences in the membrane fluidities of 103d1 and 103d1-10 compared to the membrane fluidity of N315IP were observed. Resistant strain 103d1 had the highest membrane potential, followed by strains 103d1-10 and N315IP. The vancomycin and teicoplanin MICs also increased. Teichoic acid genes (tagA, tagG), mprF encoding lysyl-phosphatidylglycerol, and cls encoding cardiolipin synthase were downregulated in 103d1 and 103d1-10. The vraF and vraG genes, which encode ATP binding cassette transporter proteins, were upregulated in 103d1. The vraSR two-component regulatory system was upregulated, and electron microscopy revealed that the cell wall of 103d1 was significantly thicker than that of the parental strain. Taken together, daptomycin exposure selected a daptomycin-nonsusceptible strain with a phenotype similar to that of heterogeneous vancomycin-intermediate S. aureus and a transcription profile that partially overlapped that of heterogeneous vancomycin-intermediate S. aureus. After the isolation of the first methicillin-resistant Staphylococcus aureus (MRSA) strains with reduced susceptibility to vancomycin in 1997, the need for a drug with a new target increased (20, 21). Due to the multiple-drug resistance profile of most S. aureus strains isolated nowadays, there are few therapeutic options available. Daptomycin is a calcium-dependent cyclic lipopeptide approved by the U.S. Food and Drug Administration in 2003 and, afterwards, by the European Agency for Evaluation of Medicinal Products for the treatment of complicated skin and soft tissue infections caused by susceptible bacteria. The U.S. Food and Drug Administration also

Multistep genetic alteration is required for methicillin-resistant Staphylococcus aureus (MRSA) to achieve the level of vancomycin resistance of vancomycin-intermediate S. aureus (VISA). In the progression of vancomycin resistance, strains with heterogeneous vancomycin resistance, designated hetero-VISA, are observed. In studying the whole-genome sequencing of the representative hetero-VISA strain Mu3 and comparing it with that of closely related MRSA strains Mu50 (VISA) and N315 (vancomycin-susceptible S. aureus [VSSA]), we identified a mutation in the response regulator of the graSR two-component regulatory system. Introduction of mutated graR , designated graR *, but not intact graR , designated graRn , could convert the hetero-VISA phenotype of Mu3 into a VISA phenotype which was comparable to that of Mu50. The same procedure did not appreciably increase the vancomycin resistance of VSSA strain N315, indicating that graR * expression was effective only in the physiological milieu of hetero-VISA cell to achieve a VISA phenotype. Interestingly, the overexpression of graR * increased the daptomycin MICs in both Mu3 and N315 and decreased the oxacillin MIC in N315.

As an aggressive pathogen, Staphylococcus aureus poses a significant public health threat and is becoming increasingly resistant to currently available antibiotics, including vancomycin, the drug of last resort for gram-positive bacterial infections. S. aureus with intermediate levels of resistance to vancomycin (vancomycin-intermediate S. aureus [VISA]) was first identified in 1996. The resistance mechanism of VISA, however, has not yet been clarified. We have previously shown that cell wall thickening is a common feature of VISA, and we have proposed that a thickened cell wall is a phenotypic determinant for vancomycin resistance in VISA (L. Cui, X. Ma, K. Sato, et al., J. Clin. Microbiol. 41: 5-14, 2003). Here we show the occurrence of an anomalous diffusion of vancomycin through the VISA cell wall, which is caused by clogging of the cell wall with vancomycin itself. A series of experiments demonstrates that the thickened cell wall of VISA could protect ongoing peptidoglycan biosynthesis in the cytoplasmic membrane from vancomycin inhibition, allowing the cells to continue producing nascent cell wall peptidoglycan and thus making the cells resistant to vancomycin. We conclude that the cooperative effect of the clogging and cell wall thickening enables VISA to prevent vancomycin from reaching its true target in the cytoplasmic membrane, exhibiting a new class of antibiotic resistance in gram-positive pathogens.

The glycopeptide vancomycin has been used successfully for the treatment of serious infections caused by multidrug-resistant Staphylococcus aureus for several decades, and some isolates were reported to be susceptible only to this antibiotic (30, 36). However, with the worldwide emergence of glycopeptide-intermediate S. aureus (GISA [or vancomycin-intermediate S. aureus; VISA]) strains with vancomycin MICs of 8 or 16 mg/liter (5, 17), the likelihood of this resistance reaching global magnitude causes great concern about the possibility of losing the last treatment option for multidrug-resistant staphylococcus infection. Several factors have been reported to be involved in the mechanism of glycopeptide resistance in VISA, including thickened cell wall (7, 11), accumulation of surplus cell wall material (48), reduced peptidoglycan cross-linking (4, 12), inactivation of penicillin-binding protein D (PBP4) (9, 47), a decrease in the level of the muropeptide amidation (4, 7), and/or other cell wall alterations, such as increased glycan chain length (23). The phenomenon of thickened cell wall was discovered in our early study of the first VISA strain, Mu50 (11). Subsequent investigation of the isogenic cells with different cell wall thickness prepared from a single colony of Mu50 cells demonstrated that cell wall thickening is the major factor contributing to glycopeptide resistance in Mu50 (7). Recently we found that 16 VISA clinical isolates collected from seven countries all showed a distinct thickened cell wall compared to glycopeptide-susceptible S. aureus strains (6, 21, 41). An immanent link between cell wall thickness and the level of glycopeptide resistance was also demonstrated in a comparison study using triple sets of 16 VISA strains, their glycopeptide-susceptible derivatives (vancomycin-resistant S. aureus [VRSA]-P) produced by passage on drug-free medium, and phonotically glycopeptide-resistant revertants (VRSA-PR) selected from VRSA-P on vancomycin plates (5, 6). Contrary to the phenotypic mechanism, the genetic mechanism of VISA is far from being clear. Different VISA strains have been studied by many researchers, and altered expression of genes such as pbp2 (46), pbpD (9, 47), sigB (2, 11), ddh (3, 34), tcaA (33), and vraSR (26) were reported. Several sets of up- and down-regulated genes associated with vancomycin resistance, including regulators, were also reported (26, 35, 54). However, until now none of the genes was altered without exception in VISA strains and none could be attributed to VISA strains specifically. Thus there is a need for additional knowledge on the genetic mechanism(s) of glycopeptide resistance acquisition in VISA and for defining genetic alteration(s) resulting in phenotypic change, such as cell wall thickening. Recently, in an effort to efficiently discover genes involved in the mechanism of glycopeptide resistance in S. aureus, a DNA microarray which consists of PCR products covering the whole genome of N315 was used to identify open reading frames (ORFs) that are expressed differently between VISA and VSSA strains. In this report, we describe our findings from transcriptional profiling studies on genes that are differentially expressed as well as genetic studies on genes that are increased in transcription in glycopeptide-resistant strains.

We have previously shown that a thickened cell wall is responsible for the vancomycin resistance of vancomycin-resistant Staphylococcus aureus (VRSA) (equivalent to vancomycin-intermediate S. aureus and glycopeptide-intermediate S. aureus ) strain Mu50 (L. Cui, H. Murakami, K. Kuwahara-Arai, H. Hanaki, and K. Hiramatsu, Antimicrob. Agents Chemother. 44:2276-2285, 2000). However, the mechanism of vancomycin resistance in other VRSA strains remained unclear. In this study, 16 clinical VRSA strains from seven countries were subjected to serial daily passage in drug-free medium. After 10 to 84 days of passage in the nonselective medium, passage-derived strains with decreased MICs of vancomycin (MIC, −6 to 1.64 × 10 −3 . The data indicated that vancomycin-resistant cells are frequently generated from the passage-derived strains even after vancomycin selective pressure is lifted. Cell wall thicknesses and MICs of glycopeptides (vancomycin and teicoplanin) and beta-lactams (imipenem and oxacillin) were determined for a total of 48 strains, including 15 sets of three strains: the clinical VRSA strain, the passage-derived strain, and the vancomycin-resistant mutant strain obtained from the passage-derived strain. No simple correlation between glycopeptide and beta-lactam MICs was seen, while significant correlations between MICs of vancomycin and teicoplanin ( r = 0.679; P < 0.001) and between MICs of imipenem and oxacillin ( r = 0.787; P < 0.001) were recognized. Moreover, all of the VRSA strains had significantly thickened cell walls, which became thinner with the loss of vancomycin resistance during drug-free passages and again became thick in the resistant mutant strains. The data showed that cell wall thickness had high correlation with the MICs of the two glycopeptides (correlation coefficients, 0.908 for vancomycin and 0.655 for teicoplanin) but not with those of the beta-lactam antibiotics tested. These results together with coupled changes of cell wall thickness and vancomycin MICs in 16 isogenic sets of strains indicate that thickening of the cell wall is a common phenotype of clinical VRSA strains and may be a phenotypic determinant for vancomycin resistance in S. aureus.

Staphylococcus aureus Mu50, which has reduced susceptibility to vancomycin, has a remarkably thickened cell wall with an increased proportion of glutamine nonamidated muropeptides. In addition, Mu50 had enhanced glutamine synthetase and l -glutamine d -fructose-6-phosphate aminotransferase activities, which are involved in the cell-wall peptidoglycan synthesis pathway. Furthermore, significantly increased levels of incorporation of 14 C-labeled d -glucose into the cell wall was observed in Mu50. Unlike a femC mutant S. aureus strain, increased levels of production of nonamidated muropeptides in Mu50 was not caused by lower levels of glutamine synthetase activity but was considered to be due to the glutamine depletion caused by increased glucose utilization by the cell to biosynthesize increased amounts of peptidoglycan. After the cells were allowed to synthesize cell wall in the absence or presence of glucose and glutamine, cells with different cell-wall thicknesses and with cell walls with different levels of cross-linking were prepared, and susceptibility testing of these cells demonstrated a strong correlation between the cell-wall thickness and the degree of vancomycin resistance. Affinity trapping of vancomycin molecules by the cell wall and clogging of the outer layers of peptidoglycan by bound vancomycin molecules were considered to be the mechanism of vancomycin resistance of Mu50. The reduced cross-linking and the increased affinity of binding to vancomycin of the Mu50 cell wall presumably caused by the increased proportion of nonamidated muropeptides may also contribute to the resistance to some extent.

The clinical vancomycin-intermediate Staphylococcus aureus (VISA) strain Mu50 carries two mutations in the vraSR and graRS two-component regulatory systems (TCRSs), namely, vraS(I5N ) and graR(N197S ) (hereinafter designated graR *). The clinical heterogeneously vancomycin-intermediate S. aureus (hVISA) strain Mu3 shares with Mu50 the mutation in vraS that encodes the VraS two-component histidine kinase. Previously, we showed that introduction of the plasmid p graR *, carrying the mutated two-component response regulator graR *, converted the hVISA strain Mu3 into VISA (vancomycin MIC = 4 mg/liter). Subsequently, however, we found that the introduction of a single copy of graR * into the Mu3 chromosome by a gene replacement method did not confer on Mu3 the VISA phenotype. The gene-replaced strain Mu3 graR * thus obtained remained hVISA (MIC ≤ 2 mg/liter), although a small increase in vancomycin MIC was observed compared to that of the parent strain Mu3. Reevaluation of the Mu3 and Mu50 genomes revealed the presence of another mutation responsible for the expression of the VISA phenotype in Mu50. Here, we demonstrate that in addition to the two regulator mutations, a third mutation found in the Mu50 rpoB gene, encoding the RNA polymerase β subunit, was required for Mu3 to achieve the level of vancomycin resistance of Mu50. The selection of strain Mu3 graR * with rifampin gave rise to rpoB mutants with various levels of increased vancomycin resistance. Furthermore, 3 (33%) of 10 independently isolated VISA strains established from the heterogeneous subpopulations of Mu3 graR * were found to possess rpoB mutations with or without an accompanying rifampin-resistance phenotype. The data indicate that a sizable proportion of the resistant hVISA cell subpopulations is composed of spontaneous rpoB mutants with various degrees of increased vancomycin resistance.

Vancomycin-intermediate Staphylococcus aureus (VISA) is generated from vancomycin-susceptible Staphylococcus aureus by multiple spontaneous mutations. We previously reported that sequential acquisition of mutations in the two-component regulatory systems vraSR and graRS was responsible for the VISA phenotype of strain Mu50. Here we report on the identification of a novel set of regulator mutations, a deletion mutation in two-component regulatory system walRK (synonyms, vicRK and yycFG ), and a truncating mutation in a proteolytic regulatory gene, clpP , responsible for the raised vancomycin resistance in a laboratory-derived VISA strain, LR5P1-V3. The contributory effect of the two mutations to vancomycin resistance was confirmed by introducing the walK and clpP mutations into the vancomycin-susceptible parent strain N315LR5P1 by a gene replacement procedure. The vancomycin MIC of N315LR5P1 was raised from 1 to 2 mg/liter by the introduction of the walK or clpP mutation, but it was raised to 4 mg/liter by the introduction of both the walK and clpP mutations. The vancomycin MIC value of the double mutant was equivalent to that of strain LR5P1-V3. Like VISA clinical strains, LR5P1-V3 and the double mutant strain LR5P1 walK * clpP * exhibited a thickened cell wall, slow growth, and decreased autolytic activity. Transcriptional profiles of the mutants with gene replacements demonstrated that introduction of both the walK and clpP mutations could alter expression of dozens or hundreds of genes, including those involved in cell envelope and cellular processes, intermediary metabolism, and information pathway. A mutation prevalence study performed on 39 worldwide clinical VISA strains showed that 61.5, 7.7, 10.3, and 20.5% of VISA strains harbored mutations in walRK , clpP , graRS , and vraSR , respectively. The mutation of walRK was most frequently carried by VISA strains. Together, these results suggested that the mutations of walK and clpP identified in LR5P1-V3 constitute a new combination of genetic events causing vancomycin resistance in Staphylococcus aureus .

Methicillin-resistant Staphylococcus aureus (MRSA), regarded as a tenacious pathogen in the hospital, has recently become increasingly prevalent as a community pathogen. We evaluated the prevalence and characteristics of methicillin-resistant staphylococci in the Japanese community by testing nasal samples of 818 children of five day care centers and two kindergartens in three districts. We found that methicillin-resistant staphylococci are already prevalent among healthy children. Among 818 children, 35 children (4.3%) carried MRSA and 231 children (28.2%) carried methicillin-resistant coagulase-negative staphylococci (MRC-NS). The types of staphylococcal cassette chromosome mec (SCC mec ) found among 44 MRSA isolates were as follows: type IIa, 11 isolates; type IIb, 19 isolates; and type IV, 14 isolates. The type IIb SCC mec element was a new SCC mec element found in this study. Eleven (25%) strains which belonged to clonal complex 5 (CC5) carried type IIa SCC mec , and they produced type 2 coagulase and toxic shock syndrome toxin 1. They were indistinguishable from health care-associated MRSA (H-MRSA) strains in Japan, represented by strain N315. On the other hand, 33 (75%) strains, most of which belonged to CC78 or CC91, carried small SCC mec elements, such as type IIb or type IV, and they produced type 1 or type 3 coagulase and exfoliative toxin. The data indicated that MRSA clones distinct from H-MRSA have disseminated in healthy children. The fact that MRC-NS strains were prevalent in the community suggested that they might serve as a reservoir for the SCC mec element carried by MRSA strains disseminated in the community.