Characterisation of fluoroquinolone-resistant clinical isolates of Streptococcus pyogenes in Barcelona, Spain

Resistance mechanisms and clonal relationships were determined for six Streptococcus pyogenes isolates with low- or high-level ciprofloxacin resistance. Four isolates displayed reduced susceptibility to ciprofloxacin and levofloxacin and had alterations in ParC: Ser80-->Pro (isolate emm3.1); Ser79-->Ala (two isolates emm6.0); and a double substitution Ser79-->Phe and Ala121-->Val (isolate emm12.27). Two isolates (emm12.26) displayed high-level resistance to ciprofloxacin and levofloxacin, as well as to other quinolones. These isolates had the same double substitution in ParC as isolate emm12.27, and an additional substitution (Ser81-->Tyr) in GyrA. Resistance patterns, emm typing and sequencing of the quinolone resistance-determining regions defined two clusters containing three and two isolates, respectively.     

Biotypes of group A streptococci from patients with pharyngitis and soft tissue infections

This study determined the biotypes of group A streptococci (GAS) isolated from 66 pharyngeal and 62 skin and soft-tissue infections. Among all GAS isolates tested, the most common biotypes were 1 and 3, irrespective of the isolation source and the severity of clinical symptoms. However, compared with the pharyngeal group, a more heterogeneous distribution of biotypes was observed among the cutaneous group of isolates, including seven isolates that were non-typeable but had an identical biotype pattern, suggesting that they may represent a new biotype.     

Activity of telithromycin compared with seven other agents against 1039 Streptococcus pyogenes pediatric isolates from ten centers in central and eastern Europe

In total, 1039 pediatric Streptococcus pyogenes isolates from Bulgaria, Croatia, the Czech Republic, Hungary, Latvia, Lithuania, Poland, Romania, Slovakia and Slovenia were studied. All strains were susceptible to penicillin G, levofloxacin, and quinupristin–dalfopristin, 91–100% to telithromycin, and 82–100% to erythromycin, azithromycin, and clarithromycin, and 90–100% to clindamycin. Macrolide resistance occurred mainly in Slovakia (25%), the Czech Republic (17.3%), and Croatia (15.8%). Overall, 9.7% of S. pyogenes isolates were erythromycin resistant due to erm(B) - or erm(A) -encoded methylases (72.3%) or to a mef(A) -encoded efflux pump (25.7%). One strain had alterations of both 23S rRNA (A2058G Escherichia coli numbering) and ribosomal protein L22 (G95D).     

Colonisation rates of Streptococcus pyogenes and Staphylococcus aureus in the oropharynx of a young adult population

There are very few reports on the rates of oropharyngeal colonisation by Streptococcus pyogenes and Staphylococcus aureus in young adults. The present study found colonisation rates of 9.6% and 26.2%, respectively. These rates are two-fold higher than historical rates, indicating that these organisms may be more prevalent than thought previously. This finding may have important clinical consequences in certain populations, and requires further investigation.     

Prevalence of emm types 1 and 12 from invasive Streptococcus pyogenes disease in Greece—results of enhanced surveillance

Among a total of 101 isolates from the first systematic multicentre surveillance effort concerning invasive Streptococcus pyogenes disease in Greece, conducted between 2003 and 2005 and covering 38% of the population, emm types 1 and 12 were prevalent, being responsible for 27 and nine cases, respectively. The isolates from the remaining 65 cases were assigned to 26 other emm types. Erythromycin resistance (12 isolates) was primarily mef (A)-mediated, although all emm type 1 strains were susceptible. Tetracycline resistance, due mostly to tet (M), was detected in 26 isolates. Subtyping by pulsed-field gel electrophoresis yielded 50 chromosomal fingerprints, thus discriminating further among ten of the 28 observed emm types.     

Age‐related susceptibility to Streptococcus pyogenes infection in mice: underlying immune dysfunction and strategy to enhance immunity

Epidemiological studies have shown that the elderly are at higher risk of severe Streptococcus pyogenes infections. In this study, we used a mouse model that displays the age‐related loss of resistance to S. pyogenes infection seen in humans to investigate the impaired immune mechanism underlying the age‐associated susceptibility to this pathogen. Young (2–3 months old) and aged (>20 months old) BALB/c mice were subcutaneously or intravenously inoculated with S. pyogenes and their capacity to control infection was compared. Aged mice showed faster progression of disease, earlier morbidity, and increased mortality when compared with young animals. Since macrophages are critical for host defence against S. pyogenes, we investigated whether susceptibility of aged mice may be due to an age‐associated decline in the functionality of these cells. Our results showed that macrophages from aged mice were as capable as those from young animals to uptake and kill S. pyogenes, but the number of resident tissue macrophages was significantly reduced in the aged host. Treatment of aged mice with macrophage colony‐stimulating factor (M‐CSF) significantly increased the number of resident macrophages and improved their response to infection. Our results indicate that treatment with M‐CSF can restore, at least in part, the mechanisms affected by immunosenescence and enhance the natural resistance of aged mice to infection with S. pyogenes. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Characterization of biofilms in different clinical M serotypes of Streptococcus pyogenes

Streptococcus pyogenes is a notorious human pathogen responsible for a wide array of infections. The ability of S. pyogenes to form biofilms is an innate property during the pathogenesis of invasive infections. From the eleven M serotypes tested: M56, M74, M100, M65, M89 and st38 formed dense biofilms in 48 h. The present study is the first of its kind to report about the biofilm formation in the serotypes M56, M65 M74 M100 and st38. XTT reduction assay of the biofilms showed decreased metabolic activity with increase in incubation time. The surface architecture of the biofilms when observed by scanning electron microscopy (SEM) revealed the microcolony formation. Confocal laser scanning microscopy (CLSM) was used to compare the surface topography and thickness of biofilms between the biofilm formers with and without the addition of glucose. Interestingly a non-biofilm former (st2147) was induced to form biofilms with the addition of glucose. On correlating the drug (erythromycin) resistance of the various M serotypes with their biofilm forming ability we noticed that erythromycin sensitive strains were found to be good biofilm formers. We also noticed that biofilm formation in S. pyogenes is independent of sil gene.     

Transmission of Streptococcus pyogenes causing successive infections in a family

The objective of this study was to determine the characteristics of Streptococcus pyogenes isolated during a 10-month period from members of a family with infections and asymptomatic carriage. T-serotyping and pulsed-field gel electrophoresis confirmed that distinct GAS clones were introduced into the family over a short period of time.     

Distribution of mef(A)-containing genetic elements in erythromycin-resistant isolates of Streptococcus pyogenes from Italy

In total, 124 Streptococcus pyogenes isolates were obtained from throat cultures of different symptomatic patients. All isolates showed M-phenotype macrolide resistance and contained the macrolide efflux gene mef(A). The isolates were screened for the presence and insertion site of mef(A)-containing genetic elements. In 25.8% of the isolates, mef(A) was found to be carried by elements belonging to the Tn1207.3/Phi10394.4 family inserted in the comEC gene, while 74.2% contained chimeric elements with a different genetic structure and chromosomal location, probably associated with the recently described 60-kb tet(O)-mef(A) element.     

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.     

Development of macrolide resistance by ribosomal protein L4 mutation in Streptococcus pyogenes during miocamycin treatment of an eight-year-old Greek child with tonsillopharyngitis

Streptococcus pyogenes isolates with the same pulsed-field patterns were recovered from the throat cultures of a child with tonsillopharyngitis before and after treatment with miocamycin, a 16-membered macrolide. The initial isolate was macrolide-susceptible, but the isolates after the treatment were resistant to 14 and 15-membered macrolides and had two amino acid (65WR66) deletions in ribosomal protein L4.     

Epidemiology of invasive Streptococcus pyogenes infections in Germany, 1996–2002: results from a voluntary laboratory surveillance system

A nationwide voluntary laboratory-based surveillance study of invasive Streptococcus pyogenes (group A streptococcus; GAS) infections was conducted in Germany between 1996 and 2002. Demographical and clinical information concerning the patients was obtained from the medical files. Multiple logistic regression analysis was used to determine risk-factors for fatal outcome. Invasive isolates were obtained from 475 patients, with 251 (52.8%) of the isolates cultured from blood. The most frequent emm types were emm1 (36.4%), emm28 (8.8%) and emm3 (8%). The speA, speC and ssa genes were present at variable frequencies in different emm types. The highest frequencies of speA and speC were found in emm1 (speA, 93.6%) and emm4 (speC, 94.7%), respectively. The estimated annual incidence of invasive GAS disease for 1997-2002 was 0.1 cases/100 000 individuals. This apparently low incidence rate might be explained by the voluntary nature of the surveillance system, resulting in relatively few cases being referred to the laboratory. Complete clinical information was available for 165 cases. The overall case fatality rate was 40.6%, and was highest (65.2%) in the group aged 60-69 years. Shock, an age of >or=30 years and adult respiratory distress syndrome were predictors of a fatal outcome in a multiple logistic regression analysis. Overall, 6.7% of the cases were considered to be nosocomial, and nine cases of puerperal sepsis were observed. The study underscores the importance of invasive S. pyogenes disease in Germany.     

Strain variations among Streptococcus pyogenes T1—a possible explanation for bacteremia epidemics?

Objective: To study the genetic diversity among consecutive blood culture isolates of Streptococcus pyogenes T1, in order to examine the possibility of different bacterial clones being responsible for two epidemics during the period 1986–95.
Methods: DNA-fingerprinting patterns were obtained by use of the following methods: macrorestriction of genomic DNA followed by pulsed-field gel electrophoresis (PFGE), random amplified polymorphic DNA (RAPD) analysis, and restriction fragment length polymorphism of the vir regulon (Vir-typing). Possession of the speA gene was investigated by use of a polymerase chain reaction (PCR) method, and further characterization of the gene was done by sequencing of the PCR products from six strains.
Results: All isolates contained the speA gene. Sequencing data from six isolates revealed the presence of the speA2 allele. Using RAPD analysis. it was possible to distinguish between DNA-fingerprnting patterns of the S. pyogenes T1 strains from the two epidemics.
Conclusions: The present study reveals different DNA-fingerprinting patterns from blood culture isolates of S. pyogenes T1 from two local epidemics, 1988–89 and 1994–95. Since the differences seen do not necessarily reflect different antigenic properties or changed virulence among the isolates, further studies addressing this question should be performed.     

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.     

Detection of genes encoding internalization-associated proteins in Streptococcus pyogenes isolates from patients with invasive diseases and asymptomatic carriers

A total of 161 Streptococcus pyogenes isolates from patients with invasive infections or from asymptomatic carriers were examined for genes (prtF1, prtF2, and fba) coding for fibronectin-binding proteins to evaluate their involvement in the pathogenesis of different streptococcal manifestations. We found no significant differences in the presence of these three genes between the two groups. Overall, the prtF2 gene was present in similar percentages among strains from both sources (61% versus 63%). Strains carrying the gene fba were slightly more common among those isolated from asymptomatic carriers (72.6% versus 65%). Also, the prtF1 gene was present in a higher, but not significant, percentage among strains from throat swabs than among isolates from invasive infections (75% versus 64.9%). However, this more detailed characterization of the genes encoding fibronectin-binding proteins allowed us to identify a strong association of genes of the erm class, coding for macrolide resistance, with prtF1 and prtF2 rather than with prtF1 alone. Since macrolide resistance was significantly associated with throat swab isolates, it may be hypothesized that proteins coded by prtF1 and prtF2 genes may be synergic in providing support for cell invasion and/or colonizing or persistence efficiency.     

Identification of macrolide-resistant clones of Streptococcus pyogenes in Portugal

Although the overall level of macrolide resistance (27%) has remained stable in Portugal, a rapid inversion in the dominant phenotypes has been noted, with a sharp decrease in the MLS(B) phenotype paralleled by an increase in the M phenotype. To gain further insight into these changes, 325 macrolide-resistant isolates were characterised using a combination of pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The use of Cfr9I, an isoschizomer of SmaI, to digest M phenotype isolates that were refractory to SmaI digestion allowed direct comparison of MLS(B) and M isolates. The results from PFGE and MLST were highly concordant and identified eight major clones, accounting for 92% of the isolates, each of which was associated exclusively with a single macrolide resistance phenotype. Two major clones were found among MLS(B) isolates, characterised by sequence types (ST) 46 (T12/emm22) and ST52 (T28/emm28), whereas clones characterised by ST39 (T4/emm4) and ST28 (T1/emm1) dominated among M isolates. The clone defined by ST52 corresponded to a bacitracin-resistant clone circulating in Europe, and a novel variant expressing other surface antigens (T12/emm22) was detected. The presence of the four major clones has been reported previously in other European countries, suggesting Europe-wide dissemination of a few macrolide-resistant lineages.     

The effect of carbon dioxide on susceptibility testing of azithromycin, clarithromycin and roxithromycin against clinical isolates of Streptococcus pneumoniae and Streptococcus pyogenes by broth microdilution and the Etest: Artemis Project—first-phase study

Objective: To evaluate the effect of carbon dioxide on the susceptibility testing, using broth microdilution and the Etest (AB Biodisk, Solna, Sweden), of azithromycin, clarithromycin and roxithromycin against Streptococcus pneumoniae and Streptococcus pyogenes .
Methods: Fresh clinical isolates collected from 36 hospital laboratories in 12 countries were evaluated using the Etest in the presence of carbon dioxide. The isolates were retested under ambient conditions (absence of carbon dioxide) using broth microdilution and/or the Etest.
Results: Carbon dioxide falsely elevated azithromycin, clarithromycin and roxithromycin MIC₉₀S for S. pneumoniae , determined by the Etest, approximately 12-fold. Also, the azithromycin MIC₉₀ for S. pyogenes was increased fourfold; the effect was less marked for clarithromycin and roxithromycin. When isolates were retested in the absence of carbon dioxide, using the Etest or microdilution, susceptibilities to azithroymycin were comparable to those to clarithromycin ( S. pneumoniae , 93.4% versus 91.3%; S. pyogenes , 96.4% versus 95.8%). Both organisms were less susceptible to roxithromycin ( S. pneumoniae , 71.3%; S. pyogenes , 85.7%). An internal standard control, consisting of 50 isolates each of S. pneumoniae, S. pyogenes and Haemophilus influenzae , confirmed that azithromycin susceptibility testing resulted in falsely elevated MICs.
Conclusions: Carbon dioxide falsely elevated azithromycin MICs for S. pneumoniae and S. pyogenes , with an apparent reduction in susceptibility. When the in vitro activity of azithromycin and other macrolides against S. pneumoniae and S. pyogenes is being evaluated, awareness of the pH effect is essential.     

Bacitracin-resistant clone of Streptococcus pyogenes isolated from pharyngitis patients in Belgium

We report 16 bacitracin-resistant Streptococcus pyogenes isolates recovered from pharyngitis patients in Belgium, 14 of which belonged to a particular emm type (emm28). All 16 isolates were constitutively resistant to macrolides and carried erm(B). The emergence of a bacitracin-resistant S. pyogenes clone raises questions about the continued reliability of bacitracin susceptibility testing for S. pyogenes identification.