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.     

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 Spy1908–1910 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 2010–2013, 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.     

Analysis of the roles of NrdR and DnaB from Streptococcus pyogenes in response to host defense

Toxic shock syndrome caused by Streptococcus pyogenes (S. pyogenes) is a re‐emerging infectious disease. Many virulence‐associated proteins play important roles in its pathogenesis and the production of these proteins is controlled by many regulatory factors. CovS is one of the most important two‐component sensor proteins in S. pyogenes, and it has been analyzed extensively. Our recent analyses revealed the existence of a transposon between covS and nrdR in several strains, and we speculated that this insertion has some importance. Hence, we examined the significances of the NrdR stand‐alone regulator and DnaB, which is encoded by the gene located immediately downstream of nrdR in S. pyogenes infection. We established an nrdR‐only knockout strain, and both nrdR and partial dnaB knockout strain. These established knockout strains exhibited a deteriorated response to H₂O₂ exposure. nrdR and partial dnaB knockout strain was more easily killed by human polynuclear blood cells, but the nrdR‐only knockout strain had no significant difference compared to wild type in contrast to the combined knockout strain. In addition, the mouse infection model experiment illustrated that nrdR and partial dnaB knockout strain, but not the nrdR‐only knockout strain, was less virulent compared with the parental strain. These results suggest that DnaB is involved in response to host defense.     

Reliable five-minute test strip method for identification ofStreptococcus pyogenes

A novel and rapid method (Strep Strip, Lab M) for identification ofStreptococcus pyogenes was evaluated. The method combines the established test for pyroglutamyl aminopeptidase (PYR) with a rapid chromogenic test for beta-glucosidase on a paper strip. The test was evaluated with 274 clinical isolates and 237 culture collection isolates of beta-haemolytic streptococci.Streptococcus pyogenes was identified with 100% specificity. Six isolates identified as Lancefield group A and which might therefore be assumed to beStreptococcus pyogenes were shown by the test strip method not to be this species. The beta-glucosidase test on the test strip allowed differentiation between enterococci andStreptococcus pyogenes (both PYR positive) in all cases. The test strip method represents an economical and accurate method for identification ofStreptococcus pyogenes in clinical material.     

The nature of innate and adaptive interleukin-17A responses in sham or bacterial inoculation

Streptococcus pyogenes is the causative agent of numerous diseases ranging from benign infections (pharyngitis and impetigo) to severe infections associated with high mortality (necrotizing fasciitis and bacterial sepsis). As with other bacterial infections, there is considerable interest in characterizing the contribution of interleukin‐17A (IL‐17A) responses to protective immunity. We here show significant il17a up‐regulation by quantitative real‐time PCR in secondary lymphoid organs, correlating with increased protein levels in the serum within a short time of S. pyogenes infection. However, our data offer an important caveat to studies of IL‐17A responsiveness following antigen inoculation, because enhanced levels of IL‐17A were also detected in the serum of sham‐infected mice, indicating that inoculation trauma alone can stimulate the production of this cytokine. This highlights the potency and speed of innate IL‐17A immune responses after inoculation and the importance of proper and appropriate controls in comparative analysis of immune responses observed during microbial infection.     

Screening for vancomycin‐resistant enterococci: results of a survey in Stockholm

Fang H, Nord CE, Ullberg M. Screening for vancomycin‐resistant enterococci: results of a survey in Stockholm. APMIS 2010; 118: 413–7. Vancomycin‐resistant enterococci (VRE) are emerging in Stockholm hospitals. To rapidly screen for VRE from stool and rectal swab samples, a detection assay combining broth enrichment and real‐time PCR was set up. From September to December 2008, 6914 samples were screened for VRE using the broth‐PCR combined assay. Of them, 5463 samples were reported as negative the day after sampling. Among the 6914 samples, 47 was screened as vanA probable and 44 of them were confirmed as VRE; 1314 samples was screened as vanB probable and 93 of them were confirmed as VRE. The 44 vanA‐type VRE isolates, detected from 31 patients, were clustered into four genetic types by pulsed‐field gel electrophoresis (PFGE). The same PFGE type was observed in multiple isolates recovered from the same patient with vanA VRE. The 93 vanB‐type VRE isolates were detected from 60 patients, 59 of them harboring VRE with PFGE type 1. One patient with vanB VRE had two isolates of distinct PFGE types (types 1 and 5). PFGE type 1 and PFGE type 2 were predominant clones in vanB and vanA strains, respectively, represented by 98.3% (59/60) of patients with vanB VRE and 77.4% (24/31) of patients with vanA VRE.     

Major outbreak of toxic shock-like syndrome caused by Streptococcus mitis

Severe illness caused by viridans streptococci rarely occurs in immunocompetent hosts. Between December 1990 and May 1991, thousands of patients in the YangZi River Delta area of Jiangsu Province, China, suffered from scarlet fever-like pharyngitis. Fewer cases occurred in subsequent years with the same seasonality. Approximately half of the cases developed complications characteristic of streptococcal toxic shock-like syndrome (TSLS). Throat cultures yielded predominant growth of alpha-hemolytic streptococci. All cases admitted to Haian People's Hospital were investigated. Clinical specimens were collected, medical records were reviewed, and bacterial isolates were identified phenotypically and analyzed by 16S rRNA gene sequencing and pulsed-field gel electrophoresis (PFGE). Proteins were purified from culture supernatants by extraction, ammonium sulfate precipitation, and fast-protein liquid chromatography. Biological activities of protein components were determined by subcutaneous inoculation into rabbits. A total of 178 cases of non-beta-hemolytic streptococcal scarlet fever-like pharyngitis were studied. In 88 (79.3%) of 111 patients, oropharyngeal swab cultures grew morphologically identical alpha-hemolytic streptococci. A protein in culture supernatants was pyrogenic in rabbits, was mitogenic for splenocytes, and enhanced rabbit susceptibility to endotoxin challenge. The N-terminal amino acid sequence of this 34-kDa protein showed no homology with known Streptococcus pyrogenic exotoxins. The organism was identified as Streptococcus mitis based on biochemical and 16S rRNA sequence analyses. Representative outbreak isolates from 1990 to 1995 displayed identical PFGE patterns. This TSLS outbreak in southeastern China was caused by a toxigenic clone of S. mitis. An apparently novel toxin may explain the unusual virulence of this organism.     

Emergence ofStreptococcus pyogenes strains resistant to erythromycin in Gipuzkoa, Spain

The aim of this study was to determine the evolution of resistance to macrolides and other antibiotics in strains ofStreptococcus pyogenes isolated in the province of Gipuzkoa, Spain. During the period 1984–1996, all 2561 strains ofStreptococcus pyogenes studied showed full susceptibility to penicillin. Until 1990, only 1.2% ofStreptococcus pyogenes isolates were resistant to erythromycin. Since then, resistance to erythromycin increased every year until 1995, when 34.8% (87/250) ofStreptococcus pyogenes strains were found to be resistant. In 1996 the rate of resistance to erythromycin was 17.8% (75/422). During the study period, 96.1% (246/256) of theStreptococcus pyogenes isolates resistant to erythromycin were susceptible to clindamycin. Of the remaining erythromycin-resistantStreptococcus pyogenes strains, resistance to clindamycin was constitutive in seven strains and inducible in three. When investigated by the polymerase chain reaction (PCR), allStreptococcus pyogenes strains resistant to erythromycin and susceptible to clindamycin showed the 1.4 kb fragment of themefA gene, recently described as the novel macrolide-efflux-resistance determinant. The most frequent T-agglutination patterns amongStreptococcus pyogenes resistant to erythromycin were T4 and T8,25. The emergence and rapid spread of erythromycin-resistantStreptococcus pyogenes in Gipuzkoa and its relationship to the presence of themefA gene are described.     

Characterization of levofloxacin non-susceptible clinical Streptococcus pyogenes isolated in the central part of Italy

We investigated the prevalence, genetics, and clonality of fluoroquinolone non-susceptible isolates of Streptococcus pyogenes in the central part of Italy. S. pyogenes strains (n?=?197) were isolated during 2012 from patients with tonsillopharyngitis, skin, wound or invasive infections and screened for fluoroquinolone non-susceptibility (resistance to norfloxacin and levofloxacin minimum inhibitory concentration (MIC) = 2 mg/L) following EUCAST guidelines. First-step topoisomerase parC and gyrA substitutions were investigated using sequencing analysis. Clonality was determined by pulsed field gel electrophoresis (PFGE; SmaI digestion) and by emm typing. The fluoroquinolone non-susceptible phenotype was identified in 18 isolates (9.1 %) and correlated with mutations in parC, but not in gyrA, the most frequent leading to substitution of the serine at position 79 with an alanine. Most of the fluoroquinolone non-susceptible isolates belonged to the emm-type 6, even if other emm-types were also represented (emm75, emm89, and emm2). A significant level of association was measured between PFGE and both emm type and substitutions in parC. The prevalence of fluoroquinolone non-susceptible Streptococcus pyogenes isolates in Italy is of concern and, although the well-known emm type 6 is dominant, other types are appearing and spreading.     

Beta-lactam failure in treatment of two group G Streptococcus dysgalactiae subsp. equisimilis Pharyngitis patients

We present two cases of exudative pharyngitis due to Streptococcus dysgalactiae subsp. equisimilis, Lancefield group G. While the participation of this organism as an agent of pharyngitis is well documented, we focus on failure of beta-lactam therapy, a phenomenon that is well described for pharyngitis due to Streptococcus pyogenes. Therefore, these case reports add to our knowledge of pharyngitis caused by non-S. pyogenes streptococci.     

Near absence of methicillin-resistance and pronounced genetic diversity among Staphylococcus epidermidis isolated from healthy persons in northern Sweden

Widerström M, Wiström J, Ek E, Edebro H, Monsen T. Near absence of methicillin‐resistance and pronounced genetic diversity among Staphylococcus epidermidis isolated from healthy persons in northern Sweden. APMIS 2011; 119: 505–12. The main aim of this study was to examine if hospital‐associated clones of multidrug‐resistant Staphylococcus epidermidis (MDRSE), commonly identified in hospitals in our region, also are spread among healthy persons in the community. A total of 124 isolates of S. epidermidis sampled from subjects attending a Travel health clinic, Umeå, Sweden during 2008 were examined with antibiotic susceptibility testing and pulsed‐field gel electrophoresis (PFGE) analysis. Resistance to methicillin or any antibiotic was detected in 2 and 26 of the isolates, respectively. PFGE analysis showed an extensive genetic diversity with 86 different PFGE types, 62 of which were singletons. No isolates belonged to the previously described hospital‐associated MDRSE genotypes, indicating that MDRSE by large are confined to the hospital setting in our region. In conclusion, community‐associated isolates of S. epidermidis showed a low level of methicillin‐resistance and were genetically extremely diverse with no predominating genotype.     

Clonal Relationships Among Isolates of Erythromycin-Resistant <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis> of Different Geographical Origin

The clonal relationships among 261 erythromycin-resistant Streptococcus pyogenes isolates collected in 1986–1997 from nine countries in Europe and North and South America were studied by using two molecular typing methods: Vir typing and random amplified polymorphic DNA (RAPD) analysis. A total of 49 different Vir genotypes (VTs) and 33 different RAPD patterns were noted among the 261 isolates. Isolates that shared the same VT and RAPD pattern were considered to belong to the same clone. Although as many as 60 different clones were found among the isolates studied, only seven clones, comprising 157 of the 261 (60%) isolates, were found in more than one country. Five of these seven clones expressed the M phenotype known to be associated with the drug efflux mechanism, and only two clones expressed the macrolide-lincosamide-streptogramin B-resistance phenotype. The results indicate a polyclonal spread of erythromycin-resistant Streptococcus pyogenes. Furthermore, predominance of the seven clones indicates that erythromycin-resistant Streptococcus pyogenes of the same clonal origin may be widely distributed and found in several different countries. Electronic Publication     

Outbreak of CTX‐M‐15‐producing Klebsiella pneumoniae of sequence type 199 in a Latvian teaching hospital

Dumpis U, Iversen A, Balode A, Saule M, Mikla?evi?s E, Giske CG. Outbreak of CTX‐M‐15‐producing Klebsiella pneumoniae of sequence type 199 in a Latvian teaching hospital. APMIS 2010; 118: 713–6. Previous studies on the epidemiology of extended‐spectrum β‐lactamase (ESBL)‐producing Enterobacteriaceae in Latvia are lacking. ESBL‐producing Klebsiella pneumoniae (n = 32) were subjected to pulsed‐field gel electrophoresis (PFGE) and selected isolates to multi‐locus sequence typing (MLST). Species identification and susceptibility testing were performed using VITEK2, and sequencing of bla_CTX‐M was performed in selected isolates. PFGE revealed one major clone (n = 23), with most of the isolates derived from the ICU. The clone harboured bla_CTX‐M‐15, was sequence type 199 and comprised two ertapenem non‐susceptible isolates. This is the first report of an ESBL outbreak in Latvia, and calls for increased epidemiological typing of ESBL‐producing Enterobacteriaceae, as well as improved infection control routines.     

Role of arginine deiminase in thymic atrophy during experimental Streptococcus pyogenes infection

Expression of gene of arginine deiminase (AD) allows adaptation of Streptococcus pyogenes to adverse environmental conditions. AD activity can lead to L‐arginine deficiency in the host cells’ microenvironment. Bioavailability of L‐arginine is an important factor regulating the functions of the immune cells in mammals. By introducing a mutation into S pyogenes M46‐16, we obtained a strain with inactivated arcA/sagp gene (M49‐16 delArcA), deficient in AD. This allowed elucidating the function of AD in pathogenesis of streptococcal infection. The virulence of the parental and mutant strains was examined in a murine model of subcutaneous streptococcal infection. L‐arginine concentration in the plasma of mice infected with S pyogenes M49‐16 delArcA remained unchanged in course of the entire experiment. At the same time mice infected with S pyogenes M49‐16 demonstrated gradual diminution of L‐arginine concentration in the blood plasma, which might be due to the activity of streptococcal AD. Mice infected with S pyogenes M49‐16 delArcA demonstrated less intensive bacterial growth in the primary foci and less pronounced bacterial dissemination as compared with animals infected with the parental strain S pyogenes M46‐16. Similarly, thymus involution, alterations in apoptosis, thymocyte subsets and Treg cells differentiation were less pronounced in mice infected with S pyogenes M49‐16 delArcA than in those infected with the parental strain. The results obtained showed that S pyogenes M49‐16 delArcA, unable to produce AD, had reduced virulence in comparison with the parental S pyogenes M49‐16 strain. AD is an important factor for the realization of the pathogenic potential of streptococci.     

Characteristics of Streptococcus pyogenes serotype M1 and M3 isolates from patients in Japan from 1981 to 1997

Streptococcus pyogenes isolates obtained in 1981 to 1997 from patients and healthy subjects were characterized by pulsed-field gel electrophoresis (PFGE) patterns, biotyping, and the presence of spe genes encoding streptococcal pyrogenic exotoxins. Changes in the profiles were shown in the serotype M1/T1 isolates from pharyngitis over this period, but not in serotype M3/T3 isolates. The characteristics of isolates from patients with toxic shock-like syndrome (TSLS) were comparable to those of the other isolates, including those from healthy subjects. This finding suggests that further phenotypic and molecular characterization, such as investigating the genomic difference represented by the pathogenicity island, of isolates with apparently the same profiles would be necessary to determine the etiology of diseases caused by S. pyogenes, including TSLS.     

Systemic cytokine response in moribund mice of streptococcal toxic shock syndrome model

Streptococcus pyogenes causes severe invasive disease in humans, including streptococcal toxic shock syndrome (STSS). We previously reported a mouse model that is similar to human STSS. When mice were infected intramuscularly with 10⁷ CFU of S. pyogenes, all of them survived acute phase of infection. After 20 or more days of infection, a number of them died suddenly accompanied by S. pyogenes bacteremia. We call this phenomenon “delayed death”. We analyzed the serum cytokine levels of mice with delayed death, and compared them with those of mice who died in the acute phase of intravenous S. pyogenes infection. The serum levels of TNF-α and IFN-γ in mice of delayed death were more than 100 times higher than those in acute death mice. IL-10 and IL-12, which were not detected in acute death, were also significantly higher in mice of delayed death. IL-6 and MCP-1 (CCL-2) were elevated in both groups of mice. It was noteworthy that not only pro-inflammatory cytokines but also anti-inflammatory cytokines were elevated in delayed death. We also found that intravenous TNF-α injection accelerated delayed death, suggesting that an increase of serum TNF-α induced S. pyogenes bacteremia in our mouse model.     

Genotypic characteristics of multidrug‐resistant Escherichia coli isolates associated with urinary tract infections

Escherichia coli is an important pathogen involved in community‐acquired urinary tract infections (CA‐UTIs). In this study, we analyzed the prevalence of frequently occurring genes and the distribution of integrons in 51 multidrug‐resistant (MDR) E. coli isolates associated with CA‐UTIs. The clonality of these strains was investigated by phylogrouping, multi‐locus sequence typing, and pulsed‐field gel electrophoresis (PFGE). All these strains were found to produce two or more resistance determinants, ceftazidime‐hydrolyzing CTX‐M‐type extended‐spectrum β‐lactamases (ESBLs) and plasmid‐mediated quinolone resistance determinants were the most prevalent (92.2% and 51.0%, respectively). A sulfhydryl variable‐61‐producing E. coli strain was identified for the first time in China. The prevalence of class 1 integrons was 54.9%, class 2 integrons were detected in three isolates but no isolate contained a class 3 integron. Phylogenetic group D was the dominant, observed in 70.6% of the isolates. PFGE analysis revealed a high level of diversity. Twenty‐four distinctive sequence types (STs) including four major STs (ST648, ST224, ST38, and ST405) were identified. To our knowledge, this is the first report on the characterization of MDR E. coli isolates associated with CA‐UTIs in China; our results suggest that an MDR D‐ST648 clone producing CTX‐M‐ESBLs has emerged as a major clone in the community setting.     

Identification of an Insertion Sequence Located in a Region Encoding Virulence Factors of Streptococcus pyogenes

An insertion sequence, IS1562, was identified in a Streptococcus pyogenes strain of the clinically important M1 serotype. IS1562 is located in the mga regulon between the genes coding for the M protein and the C5a peptidase, both important virulence factors. The same or similar insertion sequences were found in most S. pyogenes strains, but the chromosomal location differed among isolates.