Pyogenes Isolates Research Articles

HMGB1 in severe soft tissue infections caused by Streptococcus pyogenes

Extracellular High Mobility Group Box 1 (HMGB1) has been associated with acute and chronic inflammatory conditions. However, little is known about HMGB1 in necrotizing bacterial infections. We hypothesized that the local HMGB1 response is excessive in severe soft tissue infections (STIs), which are characterized by necrosis and hyperinflammation. To explore this, tissue biopsies were collected from patients with varying severity of Streptococcus pyogenes skin and STIs, including erysipelas, cellulitis, and necrotizing fasciitis. Tissue sections were immunostained for HMGB1, S. pyogenes, and inflammatory cell infiltrates and results quantified by acquired computerized image analysis (ACIA). HMGB1 expression increased in parallel to disease severity and was significantly higher in necrotizing fasciitis than in erysipelas (p = 0.0023). Confocal microscopy of sections co-stained for HMGB1 and cell markers revealed both extracellular and cytoplasmic HMGB1, the latter of which was found predominantly in macrophages. To further verify macrophages as main source of activation triggered HMGB1 release, human macrophages were infected with clinical S. pyogenes isolates. The results demonstrated infection triggered release of HMGB1. Dual staining's visualized HMGB1 in areas close to, but not overlapping, with neutrophils, indicating a potential chemotactic role. In vitro transmigration experiments showed a chemotactic effect of HMGB1 on neutrophils. The data furthermore provided in vivo support that HGMB1 may form immunostimulatory complexes with IL-1β. Taken together, the findings provide the first in vivo evidence that HMGB1 is abundant at the local site of severe bacterial STIs and its levels correlated to severity of infections; hence, indicating its potential value as a biomarker for tissue pathology.

Distribution of small native plasmids in Streptococcus pyogenes in India

Complete characterization of a Streptococcus pyogenes population from a defined geographic region comprises information on the plasmids that circulate in these bacteria. Therefore, we determined the distribution of small plasmids (<5kb) in a collection of 279 S. pyogenes isolates from India, where diversity of strains and incidence rates of S. pyogenes infections are high. The collection comprised 77 emm-types. For plasmid detection and discrimination, we developed PCRs for different plasmid replication initiation protein genes, the putative repressor gene copG and bacteriocin genes dysA and scnM57. Plasmid distribution was limited to 13 emm-types. Co-detection analysis using aforementioned PCRs revealed four distinct plasmid sub-types, two of which were previously unknown. Representative plasmids pA852 and pA996 of the two uncharacterized plasmid sub-types were sequenced. These two plasmids could be assigned to the pMV158 and the pC194/pUB110 family of rolling-circle plasmids, respectively. The majority of small plasmids found in India belonged to the two newly characterized sub-types, with pA852- and pA996-like plasmids amounting to 42% and 22% of all detected plasmids, respectively. None of the detected plasmids coded for a known antibiotic resistance gene. Instead, all of the four plasmid sub-types carried known or potential bacteriocin genes. These genes may have influence on the evolutionary success of certain S. pyogenes genotypes. Notably, pA852-like plasmids were found in all isolates of the most prevalent emm-type 11.0. Together, a priori fitness of this genotype and increased fitness due to the acquired plasmids may have rendered type emm11.0 successful and caused the prevalence of pA852-like plasmids in India.

Molecular Epidemiological Characteristics of Streptococcus pyogenes Strains Involved in an Outbreak of Scarlet Fever in China, 2011

ObjectiveTo investigate molecular characterization of streptococcus pyogenes isolates involved in an outbreak of scarlet fever in China in 2011. MethodsSeventy-four Streptococcal pyogenes involved in an outbreak of scarlet fever were isolated from pediatric patients in the areas with high incidence in China from May to August of 2011. Emm genotyping, pulsed-field gel electrophoresis (PFGE), superantigen (SAg) genes and antimicrobial susceptibility profiling were analyzed for these isolates. ResultsA total of 4 different emm types were identified. Emm12 was the most prevalent type which contained four predominating PFGE patterns corresponding to four different virulence and superantigen profiles. Emm12 (79.7%) and emm1 (14.9%) accounted for approximately 94% of all the isolates. The speA gene was all negative in emm12 isolates and positive in emm1 isolates. All strains were resistant to erythromycin, and 89.4% of them were resistant to erythromycin, tracycline, and clindamycin simultaneously. ConclusionSeveral highly diversified clones with a high macrolide resistance rate comprise a predominant proportion of circulating strains, though no new emm type was found in this outbreak. The data provide a baseline for further surveillance of scarlet fever, which may contribute to the explanation of the outbreak and development of a GAS vaccine in China.

Emergence of Streptococcus pyogenes emm102 Causing Toxic Shock Syndrome in Southern Taiwan during 2005–2012

BackgroundStreptococcal toxic shock syndrome (STSS) is an uncommon but life-threatening disease caused by Streptococcus pyogenes.MethodsTo understand the clinical and molecular characteristics of STSS, we analyzed clinical data and explored the emm types, superantigen genes, and pulsed-field gel electrophoresis of causative S. pyogenes isolates obtained between 2005 and 2012.ResultsIn total, 53 patients with STSS were included in this study. The median age of the patients was 57 years (range: 9–83 years), and 81.1% were male. The most prevalent underlying disease was diabetes mellitus (45.3%). Skin and soft-tissue infection accounted for 86.8% of STSS. The overall mortality rate was 32.1%. Underlying diseases had no statistical impact on mortality. A total of 19 different emm types were identified. The most prevalent emm type was emm102 (18.9%), followed by emm11 (17%), emm1 (11.3%), emm87 (9.4%), and emm89 (7.5%). There was no statistically significant association between emm type and a fatal outcome. Among the superantigen genes, speB was the most frequently detected one (92.5%), followed by smeZ (90.6%), speG (81.1%), speC (39.6%), and speF (39.6%). The majority of emm102 strains were found to have speB, speC, speG, and smeZ. The presence of speG was negatively associated with a fatal outcome (P = 0.045).ConclusionsOur surveillance revealed the emergence of uncommon emm types, particularly emm102, causing STSS in southern Taiwan. Characterization of clinical, epidemiological, and molecular characteristics of STSS will improve our understanding of this life-threatening disease.

Extensive Diversity of Streptococcus pyogenes in a Remote Human Population Reflects Global-Scale Transmission Rather than Localised Diversification

The Indigenous population of the Northern Territory of Australia (NT) suffers from a very high burden of Streptococcus pyogenes disease, including cardiac and renal sequelae. The aim of this study was to determine if S. pyogenes isolated from this population represent NT endemic strains, or conversely reflect strains with global distribution. emm sequence typing data were used to select 460 S. pyogenes isolates representing NT S. pyogenes diversity from 1987–2008. These isolates were genotyped using either multilocus sequence typing (MLST) or a high resolution melting-based MLST surrogate (Minim typing). These data were combined with MLST data from other studies on NT S. pyogenes to yield a set of 731 MLST or Minim typed isolates for analysis. goeBURST analysis of MLST allelic profiles and neighbour-joining trees of the MLST allele sequences revealed that a large proportion of the known global S. pyogenes MLST-defined diversity has now been found in the NT. Specifically, fully sequence typed NT isolates encompass 19% of known S. pyogenes STs and 43% of known S. pyogenes MLST alleles. These analyses provided no evidence for major NT-endemic strains, with many STs and MLST alleles shared between the NT and the rest of the world. The relationship between the number of known Minim types, and the probability that a Minim type identified in a calendar year would be novel was determined. This revealed that Minim types typically persist in the NT for >1 year, and indicate that the majority of NT Minim types have been identified. This study revealed that many diverse S. pyogenes strains exhibit global scale mobility that extends to isolated populations. The burden of S. pyogenes disease in the NT is unlikely to be due to the nature of NT S. pyogenes strains, but is rather a function of social and living conditions.

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.

Anti-&lt;i&gt;Streptococcus pyogenes&lt;/i&gt; Activity of Selected Medicinal Plant Extracts Used in Thai Traditional Medicine

Purpose: To evaluate the anti-Streptococcus pyogenes activity of selected medicinal plants used in Thai traditional medicine. Methods: Sixty-nine extracts of 51 selected Thai medicinal plant species were tested for anti-S. pyogenes activity by paper disc agar diffusion and broth microdilution methods. Results: Ten plants including Boesenbergia pandurata (Roxb.) Schltr., Cinnamomum bejolghota (Buch.-Ham.) Sweet, Cinnamomum porrectum (Roxb) Kosterm, Eleutherine americana Merr., Gymnopetalum cochinchinensis (Lour.) Kurz, Piper betle L., Quercus infectoria G. Olivier, Quisqualis indica L, Rhodomyrtus tomentosa (Aiton) Hassk., and Walsura robusta Roxb. demonstrated good antibacterial activity against S. pyogenes NPRC 101. These plants were selected and further evaluated for their anti-S. pyogenes activity against 11 isolates of S. pyogenes from patients with upper respiratory tract infections. Three plants including Boesenbergia pandurata, Eleutherine americana, and Rhodomyrtus tomentosa exhibited good antibacterial activity against all S. pyogenes isolates and produced similar activities against different tested isolates. Boesenbergia pandurata and Rhodomyrtus tomentosa demonstrated antibacterial activity with the same minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) range of 3.91 - 31.25 µg/ml whereas Eleutherine americana displayed MIC and MBC values of 250 and 250-500 µg/ml against all S. pyogenes isolates. Conclusion: Boesenbergia pandurata, Eleutherine americana, and Rhodomyrtus tomentosa have great antibacterial potentials against S. pyogenes.

Comparative analysis of emm types, superantigen gene profiles and antibiotic resistance genes among Streptococcus pyogenes isolates from ocular infections, pharyngitis and asymptomatic children in south India

Group A Streptococcus (Streptococcus pyogenes) is responsible for a wide array of infections and incidence is high in developing countries like India. Although distribution of emm types of S. pyogenes in India has been described, its association with the virulence genes and ocular isolates is less concentrated. In the present study emm type surveillance as well as its association with toxin gene profile was analyzed. Ocular infected cases such as lacrimal abscess, corneal ulcers, mucocoele showed the presence of 20 S. pyogenes isolates. For noninvasive isolates, we screened 370 pharyngitis cases and 400 asymptomatic school children and recovered 33 pharyngitis and 14 carrier isolates respectively. 14 Emm type distributions were observed in ocular isolates, 11 emm types each in pharyngitis and asymptomatic carrier isolates. The two dominant emm types, emm49 and emm63 were accounted for 33% of the total S. pyogenes isolates. Among ocular isolates, slo, smeZ, speB and speG were found in >50% of isolates, in pharyngitis smeZ (48%), speB (45%) and speG (42%) genes were found to be prevalent. Alarmingly, carrier isolates showed more prevalence to virulence genes than the ocular and pharyngitis isolates with speF (79%), speB, speG (64%), slo and sil (64%). Among the three groups, pharyngitis isolates harbored more prtF1 (33%) and prtF2 (94%) than the asymptomatic carriers (28% and 71%) and the ocular isolates (45% and 40%). 450bp Size band in prtF1 and 350bp size band in prtF2 showed dominance. Among the three groups tested, the distribution of ermB and mefA was high in pharyngitis isolates (30%) where 10 isolates showed the presence of both genes. None of the isolates showed the presence of ermA and tetO genes. Dendrogram generated based on the virulence and antibiotic resistance gene profiles revealed that except one cluster, all other clusters showed some correlation with ocular, pharyngitis and asymptomatic carrier isolates, irrespective of their emm types.

Molecular characterization of Group A streptococcal isolates causing scarlet fever and pharyngitis among young children: A retrospective study from a northern Taiwan medical center

Little information is available on the differences in frequency of pyrogenic exotoxin genes between strains of group A streptococci that cause scarlet fever and those that cause pharyngotonsillitis in children in Taiwan. This study retrospectively monitored the presence of pyrogenic exotoxin genes, the emm typing, and the susceptibility of macrolide drugs in Streptococcus pyogenes isolated from children diagnosed with scarlet fever and pharyngotonsillitis in northern Taiwan. Isolates of S. pyogenes were recovered from children with scarlet fever (n = 21) and acute pharyngotonsillitis (n = 29) during 2000-2011. The isolates were characterized according to the presence of spe genes and emm typing. Antibiograms were determined by the disk diffusion method and agar dilution test. Polymerase chain reaction was used to detect the presence of erm genes in isolates that showed nonsusceptibility to erythromycin. All isolates underwent additional genotyping by pulsed-field gel electrophoresis. In isolates from patients with scarlet fever, the frequencies of pyrogenic exotoxin genes were 9.5% for speA, 81.0% for speB, 4.8% for speC, and 71.4% for speF. In isolates from patients with pharyngotonsillitis, the frequencies were 17.2% for speA, 72.4% for speB, 13.8% for speC, and 69.0% for speF. There were no significant differences in frequencies of the exotoxin genes between the two groups of isolates. Eight emm sequence types were identified from all group A streptococci isolates. The most common types were emm12 followed by emm1 and emm4. The erythromycin resistant rate was 4/50 (8%). The ermB gene was detected in only one isolate from a patient with pharyngotonsillitis. Pulsed-field gel electrophoresis had a total of three sets of clustered strains, which showed >80% homology and belonged to the same emm type. There were no significant differences in frequencies of the spe genes between S. pyogenes isolates from patients with scarlet fever and patients with pharyngotonsillitis. The most common emm type was emm12. Low erythromycin resistance in S. pyogenes was observed.

Streptococcus pyogenes isolated in a Tunisian pediatric population: Emm types, T types, virulence factors and genes of resistance to macrolide and tetracycline

Aims: The aims of our study were to determine epidemiological markers of S. pyogenes isolates as well as the antimicrobial activities against these strains and we determined the macrolide and tetracycline resistance genes. Methodology and Results: We studied the epidemiological markers of 148 Streptococcus pyogenes isolates collected from children in Tunisia between October 2000 and December 2006. Antimicrobial susceptibility was performed according to the CA-SFM guidelines. T-serotypes were determined by slide agglutination. Virulence factor genes (pyrogenic exotoxin gene and superantigen gene) and macrolide and tetracycline resistance genes were revealed by PCR method. The emm types were determined by sequencing the variable 5’ end of the emm gene. The predominant markers were T3/12/13/B3264, emm22 and speB. All isolates were susceptible to β-lactam antibiotics (penicillin and amoxicillin). Resistance to tetracycline was observed in 65 isolates (43.9%), and strains harboured the tet(M) gene alone in 95.4% (62/65) or both tet(M) and tet(L) genes in 4.6% (3/65). Six strains (4%) were resistant to erythromycin among these; five were also resistant to clindamycin. Five strains of genotypes emm11, emm22, emm28, and emm76 expressed the constitutive MLSB phenotype with the presence of the ermB gene alone (3 strains) or in association with the mefA gene (2 strains). One emm4 strain expressed the M phenotype and harboured the mefA gene alone. Conclusion, Significance and Impact of Study: This work provided the molecular characteristics of paediatric S. pyogenes isolates in Tunisia. Our study affirmed that this micro-organism still susceptible to β-lactam antibiotics but showed an increase in macrolide resistance. We concluded that the epidemiology and the molecular characteristics of S. pyogenes strains were different around the world.

In vitro and in vivo expression of virulence genes in Trueperella pyogenes based on a mouse model

Trueperella pyogenes is an important opportunistic pathogen causing a number of pyogenic infections in ruminants and other animals. This microorganism expresses several extracellular virulence proteins that contribute to its pathogenic potential. To investigate the expression levels of haemolytic exotoxin pyolysin, neuraminidases, collagen-binding protein and fimbriae of T. pyogenes in routine culture and infection process, 10 T. pyogenes isolates which simultaneously harbored ftsY, plo, cbpA, fimA, fimC, nanP, and nanH genes were injected into 10/group Kunming (KM) mice to determine their virulence. In vitro expressions of these genes were determined by quantitative PCR. Subsequently, three typical isolates including an avirulent, a moderately virulent and a strongly virulent isolate were respectively injected into mouse model to determine the in vivo expression of these genes. Finally, significant correlation was observed between collagen-binding protein, neuraminidases, and fimbriae. The expression level of pyolysin was negatively correlated with the survival of injected mice. The time course of virulence gene expression was monitored based on the expression of virulence genes in mouse model. In conclusion, the in vitro and in vivo gene expression study showed a clear difference in virulence gene expression between virulent and non-virulent isolates.

Virulence determinants and biofilm production among Trueperella pyogenes recovered from abscesses of captive forest musk deer

Trueperella pyogenes (formerly Arcanobacterium) is commonly isolated from domesticated or wild ruminants as an opportunistic pathogen. To investigate the role of virulence determinants (VDs) and biofilm production in T. pyogenes isolates, a total of 36 T. pyogenes were collected from abscesses of forest musk deer in Miyaluo Farm (Sichuan Province, China). The prevalence of VDs and associations with clonal types, antibiotic resistance and biofilm production were analyzed by PCR and bioassay. Finally, T. pyogenes isolates were separated into three clonal types based on the DNA fingerprinting of BOX-PCR. Isolates with less VDs obtained from sick forest musk deer were mainly belonged to Type 1, and the isolates with robust VD repertoire obtained from dead forest musk deer were included in Type 3. Accordingly, resistant isolates exhibited significant lower virulence than susceptible ones. Majority of T. pyogenes isolates of this study were capable of producing a biofilm. However, no VDs presence and antibiotic resistance were statistically associated with biofilm production. In conclusion, the current study demonstrated that T. pyogenes was probably the primary pathogen of abscesses in the forest musk deer. Moreover, as an animal origin pathogen, the increasing resistance of T. pyogenes isolates could also associate with a decreased virulence.

Identification and Cluster Analysis of Streptococcus pyogenes by MALDI-TOF Mass Spectrometry

BackgroundWhole-cell matrix–assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) has been successfully applied for bacterial identification and typing of many pathogens. The fast and reliable qualities of MALDI-TOF MS make it suitable for clinical diagnostics. MALDI-TOF MS for the identification and cluster analysis of Streptococcus pyogenes, however, has not been reported. The goal of our study was to evaluate this approach for the rapid identification and typing of S. pyogenes.Methods65 S. pyogenes isolates were obtained from the hospital. The samples were prepared and MALDI-TOF MS measurements were conducted as previously reported. Identification of unknown spectra was performed via a pattern recognition algorithm with a reference spectra and a dendrogram was constructed using the statistical toolbox in Matlab 7.1 integrated in the MALDI Biotyper 2.0 software.ResultsFor identification, 61 of 65 S. pyogenes isolates could be identified correctly by MALDI-TOF MS with BioType 2.0 when compared to biochemical identification (API Strep), with an accuracy of 93.85%. In clustering analysis, 44 of 65 isolates were in accordance with those established by M typing, with a matching rate of 67.69%. When only the M type prevalence in China was considered, 41 of 45 isolates were in agreement with M typing, with a matching rate of 91.1%.ConclusionsIt was here shown that MALDI-TOF MS with Soft Biotype 2.0 and its database could facilitate rapid identification of S. pyogenes. It may present an attractive alternative to traditional biochemical methods of identification. However, for classification, more isolates and advances in the MALDI-TOF MS technology are needed to improve accuracy.

Decline in macrolide-resistant Streptococcus pyogenes isolates from French children

We studied the macrolide resistance and serotypes of 585 group A streptococcus (GAS) isolates collected from French children with pharyngitis. Nineteen isolates (3.2%) were erythromycin-resistant and harbored the following resistance genes: 31.6% mef(A), 15.8% erm(A), and 52.6% erm(B). The 19 isolates included 7 different emm types (4, 1, 11, 2, 28, 12, and 77) and 7 corresponding multilocus sequence types. The current fall in macrolide consumption has led to a very low rate of GAS macrolide resistance.

Is Streptococcus pyogenes resistant or susceptible to trimethoprim-sulfamethoxazole?

Streptococcus pyogenes is commonly believed to be resistant to trimethoprim-sulfamethoxazole (SXT), resulting in reservations about using SXT for skin and soft tissue infections (SSTI) where S. pyogenes is involved. S. pyogenes' in vitro susceptibility to SXT depends on the medium's thymidine content. Thymidine allows S. pyogenes to bypass the sulfur-mediated inhibition of folate metabolism and, historically, has resulted in apparently reduced susceptibility of S. pyogenes to sulfur antibacterials. The low thymidine concentration in Mueller-Hinton agar (MHA) is now regulated. We explored S. pyogenes susceptibility to SXT on various media. Using two sets of 100 clinical S. pyogenes isolates, we tested for susceptibility using SXT Etests on MHA containing defibrinated horse blood and 20 mg/liter β-NAD (MHF), MHA with sheep blood (MHS), MHA alone, MHA with horse blood (MHBA), and MHA with lysed horse blood (MHLHBA). European Committee on Antibacterial Susceptibility Testing (EUCAST) breakpoints defined susceptibility (MIC, ≤ 1 mg/liter) and resistance (MIC, >2 mg/liter). In study 1, 99% of S. pyogenes isolates were susceptible to SXT on MHA, MHBA, and MHLHBA, with geometric mean MICs of 0.04, 0.04, and 0.05 mg/liter, respectively. In study 2, all 100 S. pyogenes isolates were susceptible to SXT on MHF, MHS, MHA, and MHLHBA with geometric mean MICs of 0.07, 0.16, 0.07, and 0.09 mg/liter, respectively. This study confirms the in vitro susceptibility of S. pyogenes to SXT, providing support for the use of SXT for SSTIs. A clinical trial using SXT for impetigo is ongoing.

Molecular epidemiology, antimicrobial susceptibilities and resistance mechanisms of Streptococcus pyogenes isolates resistant to erythromycin and tetracycline in Spain (1994–2006)

BackgroundGroup A Streptococcus (GAS) causes human diseases ranging in severity from uncomplicated pharyngitis to life-threatening necrotizing fasciitis and shows high rates of macrolide resistance in several countries. Our goal is to identify antimicrobial resistance in Spanish GAS isolates collected between 1994 and 2006 and to determine the molecular epidemiology (emm/T typing and PFGE) and resistance mechanisms of those resistant to erythromycin and tetracycline.ResultsTwo hundred ninety-five out of 898 isolates (32.8%) were erythromycin resistant, with the predominance of emm4T4, emm75T25, and emm28T28, accounting the 67.1% of the 21 emm/T types. Spread of emm4T4, emm75T25 and emm28T28 resistant clones caused high rates of macrolide resistance. The distribution of the phenotypes was M (76.9%), cMLSB (20.3%), iMLSB (2.7%) with the involvement of the erythromycin resistance genes mef(A) (89.5%), msr(D) (81.7%), erm(B) (37.3%) and erm(A) (35.9%).Sixty-one isolates were tetracycline resistant, with the main representation of the emm77T28 among 20 emm/T types. To note, the combination of tet(M) and tet(O) tetracycline resistance genes were similar to tet(M) alone reaching values close to 40%. Resistance to both antibiotics was detected in 19 isolates of 7 emm/T types, being emm11T11 and the cMLSB phenotype the most frequent ones. erm(B) and tet(M) were present in almost all the strains, while erm(A), mef(A), msr(D) and tet(O) appeared in less than half of them.ConclusionsSpanish GAS were highly resistant to macrolides meanwhile showed minor resistance rate to tetracycline. A remarkable correlation between antimicrobial resistance and emm/T type was noticed. Clonal spread of emm4T4, emm75T25 and emm28T28 was the main responsable for macrolide resistance where as that emm77T28 clones were it to tetraclycline resistance. A wide variety of macrolide resistance genes were responsible for three macrolide resistance phenotypes.

Genotypic characterization and evaluation of an antibiotic resistance of Trueperella pyogenes (Arcanobacterium pyogenes) isolated from milk of dairy cows with clinical mastitis

Trueperella pyogenes, recently reclassified from the genus Arcanobacterium, is considered the causative agent of acute suppurative mastitis called summer mastitis. T. pyogenes produces a variety of known and putative virulence factors that include pyolysin and factors promoting adhesion to host cells. The objective of this study was to report the presence of virulence genes in T. pyogenes isolates that were identified as etiological agents of clinical mastitis in cows, as well as to determine antimicrobial resistance and distribution of selected determinants that can be associated with phenotypic resistance among these isolates. The presence of genes (plo, nanH, nanP, cbpA, fimA, fimC, fimE, fimG, tet(W), erm(X), erm(B)) was examined by conventional PCRs. Resistance to 10 antimicrobial agents was determined by the broth microdilution method. Among T. pyogenes isolates of bovine mastitis origin the genes encoding all virulence factors occurred. Besides pyolysin gene plo, the fimA was the only gene detected in all isolates, whereas other virulence factor genes were found with different frequencies. Phenotypic antimicrobial resistance was observed to tetracycline (85.5% isolates) and erythromycin (9.1%). Isolates non-susceptible to erythromycin simultaneously exhibited increased MIC of pirlimycin. Beta-lactams were active against isolates. We found the correlation between the presence of tetracycline and macrolide resistance genes and corresponding resistance phenotype. Genotypic characterization of a large number of T. pyogenes isolates from different herds performed in this study may be useful in explanation, which virulence factors play a significant role in the establishment of bovine mammary gland infection.

Response of Different Antibiotic Resistant Group of Streptococcus pyogenes to Environmental Stresses

Streptococcus species is considered as an important pathogen for human and animals. The antibiotic resistance mechanism in this species is continuously increased. On the other side, the tolerance of environmental stresses play an effective role in the severity of many streptococcal causative disease. In this study we assayed survey on the causative agents of pharyngitis and tonsillitis patients. The predominant causative strain was Streptococcus pyogenes with 93% isolating ratio frequency. The other pathogenic species were S. agalactia 5.3% and S. pneumonia 1.7%. According to the antibiotic resistant test the S. pyogenes isolates were classified into six different groups. A selected strain from each antibiotic resistant group was tested for tolerance of a restrictive environmental factors. The variations of the environmental niches of isolates were in consistence with their antibiotic resistant variation.

Epidemiological and molecular characteristics of clinical isolates of Streptococcus pyogenes collected between 2005 and 2008 from Chinese children

The aim of this study was to explore the epidemiological and molecular characteristics of Streptococcus pyogenes in children from different cities in mainland China who were diagnosed with scarlet fever, impetigo and pharyngitis, as well as to detect asymptomatic carriers, between 2005 and 2008, and to compare the results with isolates from rural Chinese children with acute glomerulonephritis in 2005 and in the 1990s. Susceptibility tests to determine MICs and analysis of the presence of erythromycin-resistant genes (mefA, ermB and ermA) and emm gene typing were performed on 466 S. pyogenes isolates from Beijing, Shanghai, Chongqing and Shenzhen. Superantigen genes (speA and speC) were examined by performing PCR on isolates with the most prevalent emm genotype. All isolates were sensitive to penicillin, cefradine and ofloxacin. The highest rate of resistance was against clarithromycin (98.1 %), followed by erythromycin (97.6 %), azithromycin and clindamycin (both 97.2 %), and tetracycline (94.0 %). Among the 466 isolates, 421 (90.3 %) harboured the ermB gene, 145 (31.1 %) were speA-positive and 273 (58.6 %) were speC-positive. The speA gene was common in emm1.0 (88.8 %) and emm6.5 (83.3 %) genotypes. The speC gene was frequently observed in emm4.0 (90.0 %), emm12.0 (69.6 %), emm18.0 (66.7 %), emm22.0 (75.9 %) and emm80.0 (80.0 %) genotypes. The most prevalent emm genotypes in mainland China in recent years were emm1.0 and emm12.0. All isolates remained sensitive to β-lactams and quinolone.

Evaluation of novel Streptococcus pyogenes vaccine candidates incorporating multiple conserved sequences from the C-repeat region of the M-protein

A major challenge for Streptococcus pyogenes vaccine development is the identification of epitopes that confer protection from infection by multiple S. pyogenes M-types. Here we have identified and characterised the distribution of common variant sequences from individual repeat units of the C-repeat region (CRR) of M-proteins representing 77 different M-types. Three polyvalent fusion vaccine candidates (SV1, SV2 and SV3) incorporating the most common variants were subsequently expressed and purified, and demonstrated to be alpha-helical by Circular Dichroism (CD), a secondary conformational characteristic of the CRR in the M-protein. Antibodies raised against each of these constructs recognise M-proteins that vary in their CRR, and bind to the surface of multiple S. pyogenes isolates. Antibodies raised against SV1, containing five variant sequences, also kill heterologous S. pyogenes isolates in in vitro bactericidal assays. Further structural characterisation of this construct demonstrated the conformation of SV1 was stable at different pHs, and thermal unfolding of SV1 is a reversible process. Our findings demonstrate that linkage of multiple variant sequences into a single recombinant construct overcomes the need to embed the variant sequences in foreign helix promoting flanking sequences for conformational stability, and demonstrates the viability of the polyvalent candidates as global S. pyogenes vaccine candidates.