These results suggest that the number of clinical PRGBS isolates with a tendency to multidrug resistance increased rapidly between 2005-06 and 2012-13 in Japan.

Streptococcal toxic shock syndrome (STSS) is a re-emerging infectious disease in Japan and many other developed countries. Epidemiological studies have revealed that the M1 serotype of Streptococcus pyogenes is the most dominant causative isolate of STSS. Recent characterization of M1 isolates revealed that the mutation of covS, one of the two-component regulatory systems, plays an important role in STSS by altering protein expression. We analyzed the M1 S. pyogenes clinical isolates before or after 1990 in Japan, using two-dimensional gel electrophoresis (2-DE) and pulsed-field gel electrophoresis (PFGE). PFGE profiles were different between the isolates before and after 1990. Markedly different profiles among isolates after 1990 from STSS and pharyngitis patients were detected. Sequence analysis of two-component regulatory systems showed that covS mutations were detected not only in STSS but also in three pharyngitis isolates, in which proteins from the culture supernatant displayed the invasive type. The mutated CovS detected in the pharyngitis isolates had impaired function on the production of streptococcal pyrogenic exotoxin B (SpeB) analyzed by 2-DE. These results suggest that several covS mutations that lead to the malfunction of the CovS protein occurred even in pharyngeal infection.

The number of reports concerning Escherichia coli clinical isolates that produce glutathione S-transferases responsible for fosfomycin resistance (FR-GSTs) has been increasing. We have developed a disk-based potentiation test in which FR-GST producers expand the growth inhibition zone around a Kirby-Bauer disk containing fosfomycin in combination with sodium phosphonoformate (PPF). PPF, an analog of fosfomycin, is a transition-state inhibitor of FosA PA , a type of FR-GST from Pseudomonas aeruginosa. Considering its mechanism of action, PPF was expected to inhibit a variety of FR-GSTs. In the presence of PPF, zone enlargement around the disk containing fosfomycin was observed for FosA3-, FosA4-, and FosC2-producing E. coli clinical isolates. Moreover, the growth inhibition zone was remarkably enlarged when the Mueller-Hinton (MH) agar plate contained 25 g/ml glucose-6-phosphate (G6P). When we retrospectively tested 12 fosfomycin-resistant (MIC, >256 g/ml) E. coli clinical isolates from our hospital with the potentiation test, 6 FR-GST producers were positive phenotypically by potentiation disk and were positive for FR-GST genes: 5 harbored fosA3 and 1 harbored fosA4. To identify the production of FR-GSTs, we set the provisional cutoff value, 5-mm enlargement, by adding PPF to a fosfomycin disk on the MH agar plates containing G6P. Our diskbased potentiation test reliably identifies FR-GST producers and can be performed easily; therefore, it will be advantageous in epidemiological surveys and infection control of fosfomycin-resistant bacteria in clinical settings.

Verocytotoxin 1 and 2 (VT1 and 2) produced by verocytotoxin-producing Escherichia coli have been considered to play an important role in the pathogenesis of glomerular and tubular damage in the epidemic form of hemolytic uremic syndrome (HUS). VTs are known to be cytotoxic to culture cells by inhibiting cellular protein synthesis. In this in vitro study, the mechanism(s) of tubular damage in HUS and the ability of VT1 to induce apoptosis in normal human renal proximal tubular epithelial cells (HRPTEC) were examined. VTI markedly reduced cell viability of HRPTEC and rapidly inhibited overall protein synthesis. VT1 directly induced apoptotic cell death in HRPTEC in a dose- and time-dependent fashion, and co-incubation with tumor necrosis factor-alpha enhanced the VT1-induced apoptosis. These results suggest that apoptosis induced by VT1, possibly in concert with host cytokines, in renal tubular cells may contribute to the tubular damage in HUS.

e Some important virulence factors have been elucidated in Klebsiella pneumoniae infections. We investigated the relationship between virulence factors and multilocus sequence types (STs) and assessed the risk factors for bacteremia in patients with pneumonia due to K. pneumoniae. From April 2004 through April 2012, a total of 120 K. pneumoniae isolates from patients with pneumonia (23 with bacteremia and 97 without bacteremia) were collected from 10 medical institutions in Japan. Additionally, 10 strains of K. pneumoniae serotype K2 that were isolated >30 years ago were included in this study. These isolates were characterized using multilocus sequence typing (MLST), and the characteristics of their virulence factors, such as hypermucoviscosity phenotype and RmpA and aerobactin production between patients with and without bacteremia, were examined. MLST analysis was performed on the 120 isolates from patients with pneumonia, and some sequence type groups were defined as genetic lineages (GLs). GL65 was more prevalent among patients with bacteremia (21.7%) than in those without bacteremia (7.2%). The majority of the strains with serotype K2 were classified into GL14 or GL65, and rmpA and the gene for aerobactin were present in all GL65-K2 strains but absent in all GL14-K2 strains. In a multivariate analysis, the independent risk factors for bacteremia included GL65 ( K lebsiella pneumoniae is a member of the family Enterobacteriaceae and is one of the most common pathogens causing pneumonia, abscess, bacteremia, and urinary tract infections (1, 2).Over several decades, some virulence factors of K. pneumoniae, including a capsular serotype, the presence of mucoviscosity-associated gene A (magA), and a regulator of mucoid phenotype A (rmpA) gene, have been identified. The strains of serotypes K1 and K2 were found to be virulent in a mouse model (3), and the magA and rmpA genes were found to be associated with hypermucoviscosity (HV), which has an antiphagocytic effect against macrophages and neutrophils (4-6).Clinically, K. pneumoniae was identified as an independent risk factor for mortality in severe community-acquired pneumonia (7). Some studies assessed the relationship between clinical findings and microbiological factors, such as capsular serotype and the presence or absence of rmpA and magA. The site of infections in these studies was mostly intra-abdominal, especially as a liver abscess (5,8). Few studies have focused on respiratory tract infections and investigated the relationship between clinical findings and microbiological factors, including genetic characteristics (9, 10).It is well-known that multilocus sequence typing (MLST), a nucleotide sequence-based genotyping method, is used to characterize genetic relationships among bacterial isolates and to identify and track the global spread of drug-resistant strains (11,12). In order to predict and evaluate bacterial pathogenicity, it is important to first determine the genetic background of the bacteria causing severe infections. Recently, using MLST, s...

The administration of high-dose clindamycin plus benzylpenicillin has been recommended for the treatment of streptococcal toxic shock-like syndrome caused by Streptococcus pyogenes, and clindamycin has been found to be more effective than beta-lactams in retrospective analyses of human cases. Although therapeutic doses of clindamycin have also been shown to be effective against experimental infections and clindamycin has great efficacy against the production of bacterial exoproteins, we recently reported that the level of production of some exoproteins was unchanged or even increased by a subinhibitory dose of clindamycin when it is added upon the initiation of bacterial culture and the treated cultures were analyzed by two-dimensional gel electrophoresis. In this study we further examined the effect of clindamycin on the production of exoproteins by adding it to Streptococcus pyogenes cultures during various growth phases. We found that the levels of production of some proteins, NAD ؉ glycohydrolase, streptolysin O, and streptococcal inhibitor of complement, were increased when clindamycin was added at early-log-phase growth, which was the result that was seen when clindamycin was added at the beginning of culture. However, clindamycin inhibited the production of most types of proteins when it was administered to Streptococcus pyogenes cultures at mid-log-phase growth. In csrS-or mga-knockout bacterial strains, the increase in exoproteins seen in parental strains was considerably inhibited. Our study indicates that the in vitro effect of clindamycin on the production of exoproteins greatly depends on the growth phase of bacteria and some regulatory factors of Streptococcus pyogenes that are involved in this phenomenon.Streptococcus pyogenes is a gram-positive bacterium that is one of the most common agents of upper respiratory tract infections, especially the acute pharyngitis that occurs mainly in children. It is also responsible for poststreptococcal diseases such as rheumatic fever and glomerulonephritis, in addition to increasing numbers of invasive infections, such as streptococcal toxic shock-like syndrome (TSLS), necrotizing fasciitis, bacteremia, and multiple-organ failure (15,20,23).Because S. pyogenes is exquisitely susceptible to ␤-lactam antibiotics, benzylpenicillin (PCG) has been recommended for the treatment of most S. pyogenes infections. However, it has been reported that PCG has reduced efficacy against aggressive infections like TSLS, which is a condition in which large numbers of organisms are present. Inocula with large numbers of organisms reach the stationary phase of growth quickly, and PCG is less effective against slowly growing organisms (22, 24). Moreover, certain penicillin-binding proteins have been shown not to be expressed by S. pyogenes during the stationary phase (24). Conversely, regarding the inhibition of protein synthesis, several investigators have demonstrated that clindamycin (CLI) suppresses the production of a variety of toxins from S. pyogenes (3,13,19,21,22). CLI als...

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Phenotypic and molecular studies have established that cereulide-producing strains of Bacillus cereus are a distinct and probably recently emerged clone within the Bacillus population. We analyzed a set of B. cereus strains, both cereulide producers and nonproducers, by multilocus sequence typing. Consistent with earlier reports, nonproducers demonstrated high heterogeneity. Most cereulide-producing strains and all flagellar antigen type H1 strains were allocated to the known sequence type of exclusively emetic B. cereus strains. Several cereulide-producing strains, however, were recovered at a new phylogenetic location, all of which were serotype H3 or H12. We hypothesize that the group of cereulide producers is diversifying progressively, probably by lateral transfer of the corresponding gene complex.