Prevalence of emm1 Streptococcus pyogenes having a novel type of genomic composition.

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.

Complete Genome Sequence of emm1 Streptococcus pyogenes 10-85, a Strain Isolated from a Patient with Streptococcal Toxic Shock Syndrome in Japan.

Here, we announce the complete genome sequence of Streptococcus pyogenes strain 10-85 (type emm1), isolated from a patient with streptococcal toxic shock syndrome (STSS). The strain lacks the genomic regions encoding SalR-SalK, a two-component regulatory system, and the adjacent type I restriction modification system.

Homologous role of CovRS two-component regulatory system in NAD+ -glycohydrolase activity in Streptococcus dysgalactiae subsp. equisimilis as in Streptococcus pyogenes.

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.

Functional Predominance of msr(D), Which Is More Effective as mef(A)-Associated Than mef(E)-Associated, Over mef(A)/mef(E) in Macrolide Resistance in Streptococcus pyogenes.

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).

Relevance of spontaneous fabT mutations to a streptococcal toxic shock syndrome to non-streptococcal toxic shock syndrome transition in the novel-type Streptococcus pyogenes isolates that lost a salRK.

Streptococcus pyogenes is a causative agent of streptococcal toxic shock syndrome (STSS). Mutations in covR/S or rgg, negative regulators, can reportedly modulate the severity of infection in this pathogen. Recently, we showed that the regions encoding the SalR-SalK, a two-component regulatory system, were deleted in some emm 1-type isolates (named as 'novel-type'). In this study, the two novel 'STSS' isolates 10-85stss and 11-171stss were more virulent than the two novel 'non-STSS' isolates 11O-2non and 11T-3non when examined using a mouse model of invasive infection. Genome-sequencing experiments using the three strains 10-85stss , 11-171stss , and 11O-2non detected only one single nucleotide polymorphism that causes a non-synonymous mutation in fabT encoding a transcriptional regulator in strain 11O-2non . Loss of fabT reduced the high level of virulence observed in the STSS isolates to that in the non-STSS isolates, and introduction of an intact fabT compensated the lower virulence of 11O-2non , suggesting that the mutation in fabT, but not in covR/S or rgg, is involved in the differential virulence among the novel-type clinical isolates. This type of non-synonymous fabT mutation was also identified in 12 non-STSS isolates (including 11O-2non and 11T-3non ), and most of those 12 isolates showed impaired FabT function.

Description of the Pathogenic Features of Streptococcus pyogenes Isolates from Invasive and Non-Invasive Diseases in Aichi, Japan.

We identified hypervirulent Streptococcus pyogenes in 27 and 420 isolates from patients with invasive and non-invasive diseases, respectively, in Aichi Prefecture, Japan, between 2003 and 2012, in an attempt to understand why the prevalence of streptococcal toxic shock syndrome (STSS) suddenly increased in this location during 2011. Hypervirulent strains belong to the emm1 genotype, with a mutation in the covR/S genes that regulate many other genes, encoding virulence determinants and resulting in the absence of the proteinase streptococcal exotoxin B and the production of virulence factors such as the superantigen streptococcal exotoxin A, the nuclease streptococcal DNase, the cytotoxin NAD-glycohydrolase, and the hemolysin streptolysin O. We found 1 strain from invasive disease and 1 from non-invasive disease with traits similar to those of hypervirulent strains, except that the sda1 gene was absent. We also found 1 non-emm1 strain with phenotypic and genetic traits identical to those of the emm1 hypervirulent strains except that it did not belong to emm1 genotype, from non-invasive diseases cases in 2011. These findings suggested that hypervirulent and hypervirulent-like strains from invasive and non-invasive disease cases could have at least partially contributed to the sudden increase in the number of patients with STSS in Aichi during 2011.

Predominant role of msr(D) over mef(A) in macrolide resistance in Streptococcus pyogenes.

In Japan, the number of patients with streptococcal toxic shock syndrome is reported to be increasing. mef(A) gene-positive macrolide-resistant emm1 strains are thought to possibly contribute to the rise in the frequency of STSS. Although analyses of macrolide-resistant mechanisms, including mef(A) resistance, have been performed mainly in Streptococcus pneumoniae, the role of this gene in Streptococcus pyogenes has not been completely investigated. Therefore, to the best of our knowledge, we established the first mef(A)-knockout strain using an emm1-type S. pyogenes strain, and tested its susceptibility to erythromycin, clarithromycin and azithromycin. We found that the antimicrobial susceptibilities were almost identical to those of the parental strain. Hence, we established a knockout strain for another gene, msr(D), that is located immediately downstream of mef(A). The macrolide resistances of the resulting strain significantly decreased, and were further altered when both mef(A) and msr(D) were knocked out. The introduction of the msr(D) gene into a macrolide-sensitive strain conferred more resistance than the introduction of the mef(A) gene. The erythromycin susceptibilities of knockout strains were further dissected using two additional emm4- and emm75-type S. pyogenes strains. We found almost identical results for both strains except for the mef(A) knockout emm4 type, whose susceptibility was altered, although the change was less than that for the msr(D) knockout. These results suggest that both mef(A) and msr(D) are involved in macrolide resistance in S. pyogenes, and that the msr(D) gene plays a more predominant role in macrolide resistance than mef(A).

Designing multiplex PCR system of Campylobacter jejuni for efficient typing by improving monoplex PCR binary typing method.

Campylobacter jejuni is responsible for the majority of Campylobacter infections. As the molecular epidemiological study of outbreaks, pulsed-field gel electrophoresis (PFGE) is performed in general. But PFGE has several problems. PCR binary typing (P-BIT) method is a typing method for Campylobacter spp. that was recently developed, and was reported to have a similar discriminatory power and stability to those of PFGE. We modified the P-BIT method from 18 monoplex PCRs to two multiplex PCR systems (mP-BIT). The same results were obtained from monoplex PCRs using original primers and multiplex PCR in the representative isolates. The mP-BIT can analyze 48 strains at a time by using 96-well PCR systems and can identify C. jejuni because mP-BIT includes C. jejuni marker. The typing of the isolates by the mP-BIT and PFGE demonstrated generally concordant results and the mP-BIT method (D = 0.980) has a similar discriminatory power to that of PFGE with SmaI digest (D = 0.975) or KpnI digest (D = 0.987) as with original article. The mP-BIT method is quick, simple and easy, and comes to be able to perform it at low cost by having become a multiplex PCR system. Therefore, the mP-BIT method with two multiplex PCR systems has high potential for a rapid first-line surveillance typing assay of C. jejuni and can be used for routine surveillance and outbreak investigations of C. jejuni in the future.

Emergence of type I restriction modification system-negative emm1 type Streptococcus pyogenes clinical isolates in Japan.

Streptococcus pyogenes emm1 type is the dominant cause of streptococcal toxic shock syndrome (STSS) in Japan and many other developed countries. Recently, the number of STSS patients in Japan was reported to be increasing. Hence, we analyzed the S. pyogenes clinical isolates detected in Japan after 2005. We found that the regions encoding the Spy1908­1910 two-component regulatory system and the adjacent type I restriction modification system were deleted in some emm1 type isolates. The isolates with the deletion were detected only in the emm1 strains that were isolated between 2010 and 2013, but not before 2010. Twenty-six of 46 (56.5%) emm1 type isolates were isolated in 2010­2013, and among these isolates, five of seven (71.4%) emm1 type STSS isolates were shown to have that deletion. PFGE and PCR analysis for the presence of several pyrogenic exotoxin-related genes suggested that the emm1 isolates with and without the deletion shared the same genetic background. The emm1 isolates with the deletion could incorporate exogenous plasmids by experimental electroporation transformation far more efficiently. These results suggested that the novel emm1 isolates have occupied a fairly large part of total emm1 isolates.

Phenotypic and genetic analyses of Campylobacter jejuni Lior serotype 76 isolated from chicken meat and clinical specimens.

The aim of this study was to examine the link between Campylobacter jejuni isolates obtained from chicken meat (n = 7) and gastroenteritis patients (n = 744). In total, 751 isolates were subjected to Lior serotyping. All the isolates from chicken meats were serotyped as Lior serotype 76 (LIO76). Among 23 of the identified LIO76 strains, 13 strains (6 from chicken meat and 7 from clinical specimens) were indistinguishable by Penner serotyping, antimicrobial susceptibility testing, and pulsed-field gel electrophoresis. These strains were isolated in 2 different Japanese prefectures in 2004-2005, suggesting that chicken meat is an etiological agent of Campylobacter gastroenteritis and that a diffuse outbreak occurred during this time. Therefore, a continuous surveillance program should be established in Japan in order to prevent Campylobacter gastroenteritis, especially large-scale food-borne outbreaks.

An enrichment medium for increasing a very small number of vibrio parahaemolyticus cells to the detection limit of the loop-mediated isothermal amplification (LAMP) assay.

We developed an enrichment medium for use with the loop-mediated isothermal amplification (LAMP) assay (enrichment media + LAMP assay) to quickly increase a very small number of Vibrio parahaemolyticus cells to the detection limit of the assay. Thirty-nine different enrichment media were prepared based on evaluating 12 potential ingredients. From our assessment of the 39 media, enrichment medium #36, which contained 2% sodium chloride, 1% proteose peptone no. 2, 0.1% trehalose, 0.5% α-ketoglutaric acid, 0.25% pyruvic acid, and 0.5% yeast extract (pH 8.6), was found to be most effective at enhancing the proliferation of V. parahaemolyticus during incubation for 3 h at 40ºC. We compared the detection limits of the LAMP assay, the enrichment medium #36 + LAMP assay, and the cultivation method using bacterial cell and spiked shrimp sample tests. The detection limits of the LAMP assay, the medium #36 + LAMP assay, and the cultivation method were 10(3), 10(0)-10(-1), and 10(-1) CFU ml(-1), respectively. Enrichment medium #36 promoted a 10(3)- to 10(4)-fold increase in the bacterial population, and the detection limit of the enrichment media + LAMP assay was the same as that of the cultivation method.

Antimicrobial ointments and methicillin-resistant Staphylococcus aureus USA300.

We tested 259 methicillin-resistant Staphylococcus aureus isolates and 2 USA300 ATCC type strains for susceptibility to bacitracin and neomycin contained in over-the-counter antibacterial ointments. Resistance to both bacitracin and neomycin was found only in USA300. The use of over-the counter antimicrobial drugs may select for the USA300 clone.

Variation in M protein production among Streptococcus pyogenes strains according to emm genotype.

M protein is an important virulence determinant in Streptococcus pyogenes, but the amounts of M protein in various strains of the species remain to be elucidated. To assess the amount of M protein in strains of each emm genotype, dot blot analysis was performed on 141 clinically isolated strains. Among the cell membrane-associated proteins, M protein was present in greater quantities in the emm1, 3, and 6 strains than in the other emm strains. In addition three strains, one each of the emm1, 3, and 6 types, showed prolific M protein production (M protein-high producers). These three emm genotypes are frequently isolated in clinical practice. Sequencing of the csrRS gene, one of the two-component signal transduction systems implicated in virulence, was performed on 25 strains bearing different amounts of M protein. CsrS mutations, in contrast to CsrR protein, were detected in 11 strains. The M protein-high producer strain of emm1 type carried two amino acid substitutions, whereas the other three emm1 strains carried only one substitution each. The M protein-high producer expressed its emm gene more strongly than the corresponding M protein-low producer did according to TaqMan RT-PCR. These observations suggest that the accumulation of amino acid substitutions in CsrS protein may contribute, at least in part, to the large amount of M protein production seen in several emm genotypes.

Prevalence of a streptococcal inhibitor of a complement-mediated cell lysis-like gene (sicG) in Streptococcus dysgalactiae subsp. equisimilis.

Streptococcus dysgalactiae subsp. equisimilis isolates (n = 110) were analyzed by PCR to determine whether the gene encoding SICG, a homolog of Streptococcus pyogenes SIC, was present. Nineteen strains (17%) had this gene of which 11 (55%) were isolated from patients with invasive disease. All 19 strains possessed group G carbohydrate. Molecular characterization of emm type revealed that the majority of emm sequences were stG643 and stG2078. Only the N-terminal sequence of SICG was similar to that of SIC in S. pyogenes. Although we found no significant relationship between pathogenic severity and sicG possession, further investigation into the mechanism of SICG may elucidate the virulence in S. dysgalactiae subsp. equisimilis infection.

Detection of invasive protein profile of Streptococcus pyogenes M1 isolates from pharyngitis patients.

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.

Characterization of Streptococcus pyogenes isolated from balanoposthitis patients presumably transmitted by penile-oral sexual intercourse.

Streptococcus pyogenes is indigenous to the human pharynx and causes acute pharyngitis. Balanoposthitis is an inflammatory disease of the glans and the foreskin. However, balanoposthitis caused by S. pyogenes is not widely recognized as a sexually transmitted disease. In addition, bacteriological features of the isolates causing balanoposthitis are unclear. The four S. pyogenes strains isolated from adult balanoposthitis were examined. We performed emm typing, T antigen typing, RAPD assay, PCR assay for the streptococcal pyrogenic exotoxin-related genes and antibiotic-resistant genes, and antibiotic susceptibility assay. All four strains were suspected to be transmitted by penile-oral sexual intercourse, were found to be different by genetic analysis, and also harbored some antibiotic-resistant factors. We propose that S. pyogenes should be considered as a causative agent of sexually transmitted disease. The drug resistant S. pyogenes must be taken into account when balanoposthitis patients are treated with antibiotic.

Identification and epidemiological description of enterohemorrhagic Escherichia coli O157 strains producing low amounts of Shiga toxin 2 in Aichi Prefecture, Japan.

Out of 68 Escherichia coli O157 field isolates tested in vitro for Shiga toxin (Stx) 2 production, 12 (17.6%) produced no or a limited amount of Stx2 (Stx 2 non- or low-producing strain; TNLP) even though all 68 possessed the stx(2) gene. The remaining 56 were Stx2 high-producing strains. The 12 TNLPs carried the q21 gene allele, which encodes a transcription antiterminator Q protein and is highly homologous to that of phi21 phage. They also carried nucleotide substitutions and insertions in the promoter region of the stx(2) gene compared with that of O157 EDL933, producing a considerable amount of Stx2. In contrast, the Stx2 high-producing strains carried the q933 gene allele, which was first reported on an stx(2) phage (933W), but not the q21 gene allele, and did not have mutations in the promoter region of the stx(2) gene. These 2 genetic characteristics, i.e., replacement of the q gene and mutation in the promoter region of the stx(2) gene, seemed to determine the amount of Stx2 produced by each strain. The TNLPs were more frequently isolated from healthy carriers than from patients (P<0.05), suggesting that TNLPs are less virulent than those with normal Stx2 production.

Genetic features of clinical isolates of Streptococcus dysgalactiae subsp. equisimilis possessing Lancefield's group A antigen.

Thirteen Streptococcus dysgalactiae subsp. equisimilis isolates possessing Lancefield's group A antigen recovered from people in Japan during 2000 to 2004 were genotyped. The results indicate that a conserved clone has persisted and spread within Japan, and two different emm types were observed within members of this clone.