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Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) is rapidly spreading worldwide. MRSA USA300 is a clone of increasing public health concern among rapidly disseminating CA MRSA strains in the United States (1). [...]

The NADase (Nga) of group A streptococci (GAS) has been implicated in the pathogenesis of diseases such as streptococcal toxic shock-like syndrome (STSS) and necrotizing fasciitis. In this study we found that the proportion of NADase-positive strains among clinical isolates in Japan has increased over time. The GAS strains studied could be divided into three groups: strains lacking NADase activity, strains with low NADase activity, and strains with high NADase activity. The older strains, isolated before 1989, belonged to the 'no activity' group. Analysis using GST-Nga recombinants revealed that nga alleles of representative older strains encode inactive Nga. Mutational analysis of the GST-Nga recombinants suggested that residue 330 could be associated with reduced activity, based upon deduced amino acid sequences. We also investigated NADase activity of streptococcal strains other than GAS. All group G streptococcal isolates from STSS patients possessed nga genes encoding active enzymes.

Streptococcus pyogenes is a causative agent of streptococcal toxic shock syndrome (STSS). The complete genome sequence of a S. pyogenes strain 10–85 isolated from a STSS patient was recently announced. In this study, the genome sequence was dissected and it was found that the genomic region around 200 kbp (region A) and the genomic region around 1600 kbp (region B) were replaced by each other in strain 10–85, when compared with those in reference strains SF370 and A20. In order to address whether this replacement is unique to 10–85, we further analyzed 163 emm1‐type strains. The results indicated that none of the strains isolated before 1990 had the replacement. In contrast, most of the strains isolated at least after 2000 appeared to have the 10–85‐type replacement. [ABSTRACT FROM AUTHOR]

The survival of 58 diarrheagenic strains and of 15 nonpathogenic Escherichia coli strains are determined after drying at 35 degree Celsius for 24 h in paper disks to determine desiccation tolerances of bacterial strains. The effects of pH, food ingredients such as sucrose, storage period, and storage temperature are examined and the survival of these desiccated bacteria in selected dry foods after artificial inoculation are studied.

Streptococcal toxic shock syndrome (STSS) is primarily caused by Streptococcus pyogenes, but it may also be caused by Streptococcus dysgalactiae subsp. equisimilis (SDSE). The analyses of S. pyogenes have revealed the important roles of NAD+‐glycohydrolase (Nga) and CovR/CovS, a two‐component regulatory system. We examined these factors in SDSE by analyzing mainly two isogenic SDSE strains (12‐10‐1 and 12‐10‐3) from the blood of a patient with STSS. The Nga activities were measured and the nucleotide sequences of covR and covS genes were determined. We detected one nucleotide difference between the covR gene of 12‐10‐1 and that of 12‐10‐3, and the Nga activity of 12‐10‐1 was approximately 6.8‐fold more than that of 12‐10‐3. The introduction of covR of 12‐10‐3 into 12‐10‐1 significantly reduced the Nga activity, but the introduction of 12‐10‐1 covR into itself had only a little effect. In addition, the knockout of covR or covS of 12‐10‐3 remarkably increased the Nga activity. We are the first to report that strains with wild‐type and mutated covR were isolated simultaneously from an SDSE STSS patient and that the CovR/CovS two‐component regulatory system is involved in the Nga activity in SDSE as well as in S. pyogenes. [ABSTRACT FROM AUTHOR]

In order to determine desiccation tolerances of bacterial strains, the survival of 58 diarrheagenic strains (18 salmonellae, 35 Shiga toxin-producing Escherichia coli [STEC], and 5 shigellae) and of 15 nonpathogenic E. coli strains was determined after drying at 35°C for 24 h in paper disks. At an inoculum level of 107 CFU/disk, most of the salmonellae (14/18) and the STEC strains (31/35) survived with a population of 10³ to 104 CFU/disk, whereas all of the shigellae (5/5) and the majority of the nonpathogenic E. coli strains (9/15) did not survive (the population was decreased to less than the detection limit of 10² CFU/disk). After 22 to 24 months of subsequent storage at 4°C, all of the selected salmonellae (4/4) and most of the selected STEC strains (12/15) survived, keeping the original populations (10³ to 104 CFU/disk). In contrast to the case for storage at 4°C, all of 15 selected strains (5 strains each of Salmonella spp., STEC O157, and STEC O26) died after 35 to 70 days of storage at 25°C and 35°C. The survival rates of all of these 15 strains in paper disks after the 24 h of drying were substantially increased (10 to 79 times) by the presence of sucrose (12% to 36%). All of these 15 desiccated strains in paper disks survived after exposure to 70°C for 5 h. The populations of these 15 strains inoculated in dried foods containing sucrose and/or fat (e.g., chocolate) were 100 times higher than those in the dried paper disks after drying for 24 h at 25°C. [ABSTRACT FROM AUTHOR]

Abstract: The molecular mechanism of high level tetracycline resistance in T serotypes 4 and 11 group A streptococcal (GAS) isolates was examined in 61 tetracycline-resistant isolates in Japan. PCR and sequencing analyses revealed that the T serotype/emm genotype, T4/4 isolates carried tet(O) genes, which were genetically homogenous. The T11/11 and T11/89 isolates carried different subtypes of tet(M) genes, which were present on transposons Tn916 and Tn1545, respectively. In addition, these T11 isolates may have obtained the tet(M) gene after the 1990s, because resistance to tetracycline in T11 isolates was rarely found before then. These results strongly suggested that the T4 and T11 GAS isolates acquired tetracycline-resistance via different molecular mechanisms. [Copyright &y& Elsevier]

We found two Shiga toxin producing Escherichia coli O157:H7 strains isolated from humans carrying the stx1 gene with an IS1203-like element (designated as IS1203v1). The IS1203v1 was inserted into the coding region of the A subunit 7 bp upstream from the TGA termination codon, resulting in a loss of two amino acid residues (Ser–Ser) from its C terminus. Toxicity of the Stx1 was confirmed by Vero cell assay. IS1203v1 hardly affected the stx1 gene in either its expression or the toxicity of its product. [Copyright &y& Elsevier]

Although mef(A) and its subclass mef(E) genes have long been considered to play a central role in macrolide efflux-based resistance, we have previously demonstrated that the msr(D) gene located immediately downstream of the mef(A) gene plays a predominant role in Streptococcus pyogenes macrolide resistance. The mef(A) and mef(E) genes are carried by different genetic elements and the resistance associated with mef(A) was reported to be higher than that associated with mef(E); therefore, we further investigated the functional relevance of mef(A)/mef(E) and its associated msr(D). We established additional mef(A)-, mef(E)-, and their associated msr(D)-knockout strains and confirmed the predominance of msr(D) over mef(A)/mef(E). In addition, we performed experiments introducing mef(A), mef(E), and their associated msr(D) genes into mef(A)/mef(E)-msr(D) double-knockout and mef(A)/mef(E) negative strains. Neither mef(A) nor mef(E) genes had effects on erythromycin resistance. However, both associated msr(D) showed significant effects, and the mef(A)-associated msr(D) exhibited more effect than the mef(E)-associated one. These results suggest that an overall functional predominance of msr(D) over mef(A)/mef(E) is conceivable in efflux-based macrolide resistance in at least some S. pyogenes strains. Furthermore, the higher resistance of mef(A) system over mef(E) system could be derived at least in part from functional differences of mef(A)- and mef(E)-associated msr(D). [ABSTRACT FROM AUTHOR]

Group A Streptococcus (GAS) is an important human pathogen that causes many types of infections, including pharyngitis and severe invasive diseases. We recently sequenced the genome of a serotype M3 strain and identified a prophage-encoded secreted phospholipase A2 designated SlaA. To study SlaA structure-activity relationships, 20 site-specific mutants were constructed by alanine-replacement mutagenesis and purified to apparent homogeneity. Enzymatic activity was greatly reduced by alanine replacement of amino acid residues previously described as crucial in the catalytic mechanism of secreted phospholipase A2. Similarly, substitution of five residues in an inferred Ca2+binding loop and three residues in the inferred active site region resulted in loss of activity of 76.5% or greater relative to the wild-type enzyme. Analysis of enzyme substrate specificity confirmed SlaA as a phospholipase A2, with activity against multiple phospholipid head groups and acyl chains located at the sn-2 position. PCR analysis of 1,189 GAS strains representing 48 M protein serotypes commonly causing human infections identified the slaA gene in 129 strains of nine serotypes (M1, M2, M3, M4, M6, M22, M28, M75, and st3757). Expression of SlaA by strains of these serotypes was confirmed by Western immunoblot. SlaA production increased rapidly and substantially on co-culture with Detroit 562 human pharyngeal epithelial cells. Together, these data provide new information about a novel extracellular enzyme that participates in GAS-human interactions. [ABSTRACT FROM AUTHOR]