Updated emm-typing protocol for Streptococcus pyogenes.

OBJECTIVES: PCR-based typing of the emm gene Streptococcus pyogenes often results in the amplification of multiple bands. This has resulted in the misclassification of strains into types based on non-emm gene sequences. We aimed to improve the specificity of the emm typing PCR reaction using a primer called CDC3, the sequence for which has been previously used to identify emm genes in silico. METHODS: The proposed primer CDC3 was validated in silico from a global database of 1688 GAS genomes and in vitro with 32 isolates. PCR reactions were performed on genomic DNA from each isolate, using the published CDC1 forward primer with the CDC2 reverse primer or the new CDC3 reverse primer. The products were examined by gel electrophoresis, and representative PCR products were sequenced. RESULTS: In 1688 S. pyogenes genomes, the previous CDC2 reverse primer annealed in silico in 1671 emm genes and also in 2109 non emm genes in close proximity, whereas the new CDC3 primer annealed in 1669 emm genes only. The remaining 19 genes without a CDC3 binding site were chimeric emm genes. The PCR pair CDC1+CDC3 produced a single band at appropriate molecular weight in all 32 isolates tested, while the CDC1+CDC2 pair produced more than one band in 13 of 32 isolates (40%). CONCLUSIONS: The new CDC3 primer is more specific for emm genes than the previous CDC2 primer and represents a simple solution to reduce the potential for mistyping S. pyogenes strains.

Multistate population and whole genome sequence-based strain surveillance of invasive pneumococci recovered in the USA during 2017.

OBJECTIVES: We aimed to provide population-based and whole-genome sequence (WGS) -based characterization of invasive pneumococcal disease isolates collected from multistate surveillance in the USA during 2017. METHODS: We obtained short-read WGS from 2881 isolates with associated bioinformatics pipeline strain feature predictions. For quality control, capsular serotypes and antimicrobial MICs were also obtained conventionally from 442 isolates. Annotated WGS were provided (inclusive of serotypes, MICs, multilocus sequence types, pilus type(s)) from 2723 isolates. For 158 isolates with suboptimal WGS, antimicrobial MICs were obtained conventionally. RESULTS: There were 127 isolates from children <5 years of age and 2754 isolates from those ≥5 years old in 2017. One of 43 different serotypes was predicted for 2877 of the 2881 isolates. Serotypes in the 13-valent conjugate vaccine together with 6C (PCV13+6C) accounted for 816 (28.3%) isolates, with PCV13 serotype 3 being the most common serotype overall. Non-PCV13-6C- serotypes accounted for 2065 (71.7%) isolates, comprising 96 (75.6%) isolates from children < 5 years old and 1969 (61.4%) isolates from those aged ≥5 years. Of 36 different categories of recently emerged serotype-switch variants, three showed marked increases relative to 2015-2016 in that the number from 2017 surpassed the number from 2015-2016 combined. Two of these included antimicrobial-resistant serotype 11A and 35B serotype-switch variants of the ST156 clonal complex. CONCLUSIONS: PCV13+6C strains are still identified in 2017 but non-PCV13-type strains impose a considerable burden. This well-annotated year 2017 WGS/strain data set will prove useful for a broad variety of analyses and improved our understanding of invasive pneumococcal disease-causing strains in the post-PCV13 era.

Prolonged and large outbreak of invasive group A Streptococcus disease within a nursing home: repeated intrafacility transmission of a single strain.

OBJECTIVES: Multiple invasive group A Streptococcus (GAS) infections were reported to public health by a skilled nursing facility (facility A) in Illinois between May 2014 and August 2016. Cases continued despite interventions including antibiotic prophylaxis for all residents and staff. Two other geographically close facilities reported contemporaneous outbreaks of GAS. We investigated potential reasons for ongoing transmission. METHODS: We obtained epidemiologic data from chart review of cases and review of facility and public health records from previous investigations into the outbreak. Infection control practices at facility A were observed and evaluated. Whole genome sequencing followed by phylogenetic analysis was performed on available isolates from the three facilities. RESULTS: From 2014 to 2016, 19 invasive and 60 noninvasive GAS infections were identified at facility A occurring in three clusters. Infection control evaluations during clusters 2 and 3 identified hand hygiene compliance rates of 14% to 25%, appropriate personal protective equipment use in only 33% of observed instances, and deficient wound-care practices. GAS isolates from residents and staff of all three facilities were subtype emm89.0; on phylogenetic analysis, facility A isolates were monophyletic and distinct. CONCLUSIONS: Inadequate infection control and improper wound-care practices likely led to this 28-month-long outbreak of severe infections in a skilled nursing facility. Whole genome sequencing and phylogenetic analysis suggested that intrafacility transmission of a single highly transmissible GAS strain was responsible for the outbreak in facility A. Integration of genomic epidemiology tools with traditional epidemiology and infection control assessments was helpful in investigation of a facility-wide outbreak.

The successional formation and release of domoic acid in a Pseudo-nitzschia bloom in the Juan de Fuca Eddy: A drifter study.

Blooms of Pseudo-nitzschia species are frequent, but presently unpredictable, in the Juan de Fuca Eddy region off the coasts of Washington (US) and British Columbia (Canada). This upwelling eddy region is proposed to be the bloom commencement site, before cells are entrained into the coastal surface currents. During a shipboard study, we characterized the different stages of the Pseudo-nitzschia bloom development from its initiation and intensification, to its eventual sinking and dissipation. Specifically, we followed a water mass using lagrangian ARGOS-tracked drifters released at the eddy water mass and quantified production of dissolved and particulate domoic acid, and the physiological status of the Pseudo-nitzschia cells with regards to photosynthesis, nutrient needs and sinking rates, along with its relationship with competing species - in this case, the marine euglenoid, Eutreptiella spp. The drifter study allows for an interpretation of the presence or absence of Pseudo-nitzschia and domoic acid against active environmental factors - particularly copper and iron.

Short-read whole genome sequencing for determination of antimicrobial resistance mechanisms and capsular serotypes of current invasive Streptococcus agalactiae recovered in the USA.

OBJECTIVES: Our objective was to evaluate and exploit a whole genome sequence (WGS) bioinformatics pipeline for predicting antimicrobial resistance and capsular serotypes from invasive group B streptococci (iGBS). METHODS: For 1975 iGBS recovered during 2015 from CDC's Active Bacterial Core surveillance, we compared pipeline predictions with broth dilution testing. Fifty-six isolates from earlier surveillance were included for testing ß-lactams. Conventional serotyping was compared to WGS-based assignments for 302 isolates. RESULTS: All 28 isolates with reduced susceptibility to ß-lactam antibiotics harboured one of 19 rare PBP2x types. Resistances to erythromycin/clindamycin (808/1975 isolates, 41.0%), erythromycin (235/1975, 11.9%) and lincosamide/streptogramin A/pleuromutilins (56/1975, 2.8%) were predicted by the presence of erm-methylase, mef and lsa determinants, respectively (41 of 56 lsa gene-positive isolates also contained lnu, erm and/or mef genes). Presence of both erm and lsa determinants (25 isolates) predicted non-susceptibility to quinupristin/dalfopristin. Most isolates (1680/1975, 85.1%) were tet gene-positive, although 41/1565 (2.6%) tetM-positive isolates were tetracycline-susceptible. All 53 fluoroquinolone-resistant isolates contained ParC and/or GyrA substitutions. Resistances to rifampin (eight isolates), trimethoprim, chloramphenicol and vancomycin (two isolates each) were predicted by the pipeline. Resistance to macrolides/lincosamides without pipeline prediction was rare and correlated to divergent resistance genes or rRNA A2062G substitution. A selection of 267 isolates assigned WGS-based serotypes were also conventionally serotyped. Of these, 246 (92.1%) were in agreement, with the remaining 21 (7.8%) conventionally non-serotypeable. For 32 of 1975 isolates (1.6%), WGS-based serotypes could not be assigned. CONCLUSION: The WGS-based assignment of iGBS resistance features and serotypes is an accurate substitute for phenotypic testing.

Using whole genome sequencing to identify resistance determinants and predict antimicrobial resistance phenotypes for year 2015 invasive pneumococcal disease isolates recovered in the United States.

Our whole genome sequence (WGS) pipeline was assessed for accurate prediction of antimicrobial phenotypes. For 2316 invasive pneumococcal isolates recovered during 2015 we compared WGS pipeline data to broth dilution testing (BDT) for 18 antimicrobials. For 11 antimicrobials categorical discrepancies were assigned when WGS-predicted MICs and BDT MICs predicted different categorizations for susceptibility, intermediate resistance or resistance, ranging from 0.9% (tetracycline) to 2.9% (amoxicillin). For ß-lactam antibiotics, the occurrence of at least four-fold differences in MIC ranged from 0.2% (meropenem) to 1.0% (penicillin), although phenotypic retesting resolved 25%-78% of these discrepancies. Non-susceptibility to penicillin, predicted by penicillin-binding protein types, was 2.7% (non-meningitis criteria) and 23.8% (meningitis criteria). Other common resistance determinants included mef (475 isolates), ermB (191 isolates), ermB + mef (48 isolates), tetM (261 isolates) and cat (51 isolates). Additional accessory resistance genes (tetS, tet32, aphA-3, sat4) were rarely detected (one to three isolates). Rare core genome mutations conferring erythromycin-resistance included a two-codon rplD insertion (rplD69-KG-70) and the 23S rRNA A2061G substitution (six isolates). Intermediate cotrimoxazole-resistance was associated with one or two codon insertions within folP (238 isolates) or the folA I100L substitution (38 isolates), whereas full cotrimoxazole-resistance was attributed to alterations in both genes (172 isolates). The two levofloxacin-resistant isolates contained parC and/or gyrA mutations. Of 11 remaining isolates with moderately elevated MICs to both ciprofloxacin and levofloxacin, seven contained parC or gyrA mutations. The two rifampin-resistant isolates contained rpoB mutations. WGS-based antimicrobial phenotype prediction was an informative alternative to BDT for invasive pneumococci.

Strain features and distributions in pneumococci from children with invasive disease before and after 13-valent conjugate vaccine implementation in the USA.

The effect of second-generation pneumococcal conjugate vaccines on invasive pneumococcal disease (IPD) strain distributions have not yet been well described. We analysed IPD isolates recovered from children aged <5 years through Active Bacterial Core surveillance before (2008-2009; n = 828) and after (2011-2013; n = 600) 13-valent pneumococcal conjugate vaccine (PCV13) implementation. We employed conventional testing, PCR/electrospray ionization mass spectrometry and whole genome sequence (WGS) analysis to identify serotypes, resistance features, genotypes, and pilus types. PCV13, licensed in February 2010, effectively targeted all major 19A and 7F genotypes, and decreased antimicrobial resistance, primarily owing to removal of the 19A/ST320 complex. The strain complex contributing most to the remaining ß-lactam resistance during 2011-2013 was 35B/ST558. Significant emergence of non-vaccine clonal complexes was not evident. Because of the removal of vaccine serotype strains, positivity for one or both pilus types (PI-1 and PI-2) decreased in the post-PCV13 years 2011-2013 relative to 2008-2009 (decreases of 32-55% for PI-1, and >95% for PI-2 and combined PI-1 + PI-2). ß-Lactam susceptibility phenotypes correlated consistently with transpeptidase region sequence combinations of the three major penicillin-binding proteins (PBPs) determined through WGS analysis. Other major resistance features were predictable by DNA signatures from WGS analysis. Multilocus sequence data combined with PBP combinations identified progeny, serotype donors and recipient strains in serotype switch events. PCV13 decreased the frequency of all PCV13 serotype clones and concurrently decreased the frequency of strain subsets with resistance and/or adherence features conducive to successful carriage. Our results serve as a reference describing key features of current paediatric IPD strains in the USA after PCV13 implementation.

Ice cover extent drives phytoplankton and bacterial community structure in a large north-temperate lake: implications for a warming climate.

Mid-winter limnological surveys of Lake Erie captured extremes in ice extent ranging from expansive ice cover in 2010 and 2011 to nearly ice-free waters in 2012. Consistent with a warming climate, ice cover on the Great Lakes is in decline, thus the ice-free condition encountered may foreshadow the lakes future winter state. Here, we show that pronounced changes in annual ice cover are accompanied by equally important shifts in phytoplankton and bacterial community structure. Expansive ice cover supported phytoplankton blooms of filamentous diatoms. By comparison, ice free conditions promoted the growth of smaller sized cells that attained lower total biomass. We propose that isothermal mixing and elevated turbidity in the absence of ice cover resulted in light limitation of the phytoplankton during winter. Additional insights into microbial community dynamics were gleaned from short 16S rRNA tag (Itag) Illumina sequencing. UniFrac analysis of Itag sequences showed clear separation of microbial communities related to presence or absence of ice cover. Whereas the ecological implications of the changing bacterial community are unclear at this time, it is likely that the observed shift from a phytoplankton community dominated by filamentous diatoms to smaller cells will have far reaching ecosystem effects including food web disruptions.

Diatom assemblages promote ice formation in large lakes.

We present evidence for the directed formation of ice by planktonic communities dominated by filamentous diatoms sampled from the ice-covered Laurentian Great Lakes. We hypothesize that ice formation promotes attachment of these non-motile phytoplankton to overlying ice, thereby maintaining a favorable position for the diatoms in the photic zone. However, it is unclear whether the diatoms themselves are responsible for ice nucleation. Scanning electron microscopy revealed associations of bacterial epiphytes with the dominant diatoms of the phytoplankton assemblage, and bacteria isolated from the phytoplankton showed elevated temperatures of crystallization (T(c)) as high as -3 °C. Ice nucleation-active bacteria were identified as belonging to the genus Pseudomonas, but we could not demonstrate that they were sufficiently abundant to incite the observed freezing. Regardless of the source of ice nucleation activity, the resulting production of frazil ice may provide a means for the diatoms to be recruited to the overlying lake ice, thereby increasing their fitness. Bacterial epiphytes are likewise expected to benefit from their association with the diatoms as recipients of organic carbon excreted by their hosts. This novel mechanism illuminates a previously undescribed stage of the life cycle of the meroplanktonic diatoms that bloom in Lake Erie and other Great Lakes during winter and offers a model relevant to aquatic ecosystems having seasonal ice cover around the world.

Laboratory-based diagnosis of pneumococcal pneumonia: state of the art and unmet needs.

In view of the increasing use of pneumococcal vaccines, especially in the developing world, there is a need for appropriate diagnostics to understand the aetiology of pneumonia, to define the burden of pneumococcal disease, and to monitor vaccine efficacy and effectiveness. This article summarizes a meeting on the diagnosis, detection and serotyping of pneumococcal disease organized by PATH and Fondation Mérieux (18-20 October 2009, Fondation Mérieux Conference Centre, Les Pensières, France). Workers and experts met to discuss the gaps in the microbiology-based diagnosis of Streptococcus pneumoniae disease, with special emphasis on pneumonia. The meeting was designed to evaluate the state of the art of pneumococcal diagnostics and serotyping methodologies, identify research and development needs, and propose new guidelines to public health authorities to support the introduction of vaccines. Regarding detection, the main recommendations were to encourage chest X-rays and antigen detection in urine. Large-scale studies are needed to evaluate the diagnostic utility of test algorithms that associate chest X-rays, antigen detection in urine, S. pneumoniae quantitative PCR in nasopharyngeal aspirates and sputum, and C-reactive protein or procalcitonin measurement in blood. Efforts should be focused on proteomics to identify pneumococcus-specific antigens in urine or host markers in blood expressed during pneumonia. It was recommended to develop S. pneumoniae typing capacities, to understand the epidemiology of pneumococcal disease, and to evaluate vaccine effectiveness. Simple and effective approaches are encouraged, and new technologies based on beads, microarrays or deep sequencing should be developed to determine, in a single test capsular serotype, resistance profile and genotype.

Streptococcus pyogenes emm and T types within a decade, 1996-2005: implications for epidemiology and future vaccines.

Streptococcus pyogenes group A (GAS) is a primary human pathogen. We performed genetic emm sequence and serological T-antigen typing of 819 mostly invasive GAS isolates recovered in Israel during 1996-2005. Of the 72 emm types found, the six most prevalent types (1, 81, 89, 14, 28, 5) comprised 30.2% of all isolates, and emm-type changes were observed over the years. The predicted coverage of the 26-valent S. pyogenes vaccine formulated for usage in the USA was predicted to be only approximately 60%. On the basis of different emm-T antigen type associations, some Israeli strains are probably different clonal types than those found in USA. About 2% of GAS had emm types that were originally associated with S. dysgalactiae subsp. equisimilis emm genes. Therefore, routine emm typing allows meaningful GAS strain surveillance, and provides data relevant to better vaccine coverage.

A rapid pneumococcal serotyping system based on monoclonal antibodies and PCR.

Streptococcus pneumoniae expresses at least 91 different polysaccharide (PS) capsules and the currently available serotyping methods are tedious to perform. We have been developing a rapid pneumococcal serotyping assay (named the 'multibead assay') based on the capacity of pneumococcal lysates to inhibit the ability of 24 different anti-capsule antibodies to bind to latex beads coated with 24 different PSs (serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9N, 9V, 14, 18C, 19A, 19F, 23F, 2, 8, 10A, 11A, 12F, 15B, 17F, 20, 22F and 33F). Because the polyclonal antibodies used for 10 serotypes (2, 8, 10A, 11A, 12F, 15B, 17F, 20, 22F and 33F) had limited serotype specificity, we replaced them with monoclonal antibodies for the 10 serotypes. To extend the serotype coverage beyond the 24 serotypes, we have adapted multiplexed PCR for five additional serotypes (15A, 15C, 16F, 35B and 38) to be useful with the pneumococcal lysates prepared for the multibead assay. We then validated the combined assay with 157 clinical isolates from the Centers for Disease Control and Prevention and found that the new combined assay produced results that are concordant with the quellung reaction. The combined assay is robust and could be used to rapidly identify the serotypes of the majority of pneumococci ( approximately 90 %). In addition, the assay validation study suggests the presence of serological subtypes within serotype 11A.

Evaluation of synthetic, M type-specific peptides as antigens in a multivalent group A streptococcal vaccine.

The recent development of emm gene sequence-based typing methodology has allowed group A streptococci (GAS) M serotype prevalence data to be determined. This information has been used to identify the components of a multivalent M protein peptide vaccine that could theoretically prevent most of the GAS-mediated diseases in the USA. In this study, we have evaluated in mice the immunogenicity and protective ability of multiple synthetic, M type-specific peptides, derived from the N-termini of three prevalent GAS serotypes (three peptides per serotype, total of nine peptides). At least one peptide, representing each of the three M types tested, was immunogenic. Five of the nine synthetic peptides tested, elicited an immune response in mice, and sera raised against four of the peptides, all possessed functional activity as demonstrated in a bactericidal assay. In vivo nasopharyngeal challenge experiments were carried out with peptides from the M1 (peptide M1-3) and M3 (peptide M3-2) proteins induced in vivo immune protection by reducing intranasal carriage. Reduction in colonization for M1-3 and M3-2 was 90% (P=0.02) and 66% (P<0.17), respectively. A reduction in colonization of 67% (P=0.03) was observed for M3-2 immunized mice when M43, a heterologous serotype, was used as the challenge strain. These results show the utility of synthetic, M type-specific peptides as antigens in a multivalent GAS vaccine.

Epidemiologic analysis of invasive and noninvasive group a streptococcal isolates in Hong Kong.

Since the mid-1980s, there has been a resurgence of severe forms of invasive group A streptococcal (GAS) disease in many Western countries. In Hong Kong, a similar increase has also been observed in recent years. One hundred seven GAS isolates collected from 1995 to 1998 from individuals with necrotizing fasciitis, toxic shock syndrome, meningitis, or other type of bacteremic sepsis (invasive group, n = 24) as well as from individuals with minor skin and throat infections (noninvasive group, n = 83) were characterized through serologic and/or emm sequence typing. Thirty-two M protein gene sequence types were identified. Types M1, M4, and M12 were the most prevalent in both the invasive group and the noninvasive group; together they accounted for 70.8 and 37.3% of the isolates, respectively. No clear pattern of skin and throat infection M types was observed. Type M1 was overrepresented in the invasive and pharyngeal isolates. The same pulsed-field gel electrophoresis pattern was shared by most invasive and all pharyngeal M1 isolates. Overall, resistance to erythromycin (32%) and tetracycline (53%) was high, but M1 isolates were significantly less likely to have resistance to either antimicrobial agent than non-M1 isolates. One novel emm sequence type, stHK, was identified in an isolate from a patient with necrotizing fasciitis. Minor emm gene sequence alterations were noted for 31 isolates, and for 13 of these isolates, deletion, insertion, or point mutations were seen in the hypervariable 50 N-terminal residues.

Fesselin, a synaptopodin-like protein, stimulates actin nucleation and polymerization.

Fesselin is a proline-rich actin binding protein that has recently been isolated from smooth muscle [Leinweber, B. D., Fredricksen, R. S., Hoffman, D. R., and Chalovich, J. M. (1999) J. Muscle Res. Cell Motil. 20, 539-545]. Fesselin is similar to synaptopodin [Mundel, P., Heid, H. W., Mundel, T. M., Krüger, M., Reiser, J., and Kriz, W. (1997) J. Cell Biol. 139, 193-204] in terms of its size, isoelectric point, and sequence although synaptopodin is not present in smooth muscle. The function of fesselin is unknown. Evidence is presented here that fesselin accelerates the polymerization of actin. Fesselin was effective on actin isolated from either smooth or skeletal muscle at low ionic strength and in the presence of 100 mM KCl. At low ionic strength, fesselin decreased the time for 50% polymerization to about 1% of that in the absence of fesselin. The lag phase characteristic of the slow nucleation process of polymerization was eliminated as the fesselin concentration was increased from very low levels. Fesselin did not alter the critical concentration for actin but did increase the rate of elongation by approximately 3-fold. The increase in elongation rate constant is insufficient to account for the total increase in polymerization rate. It is likely that fesselin stabilizes the formation of actin nuclei. Time courses of actin polymerization at varied fesselin concentrations and varied actin concentrations were simulated by increasing the rate of nucleation and both the forward and reverse rate constants for elongation.

Necrotizing fasciitis due to penicillin-resistant Streptococcus pneumoniae: case report and review of the literature.

Necrotizing fasciitis (NF) is a life-threatening infection involving rapid necrosis of subcutaneous and fascial tissues. Streptococcus pneumoniae (SPN) soft tissue infection is exceedingly uncommon, reported primarily in patients with immunosuppression or other underlying conditions. We report a case of NF and septic shock in a healthy 32-year-old man, whose only predisposing factor was antecedent blunt trauma. Pathological examination and culture of the extensive tissue debridement were positive only for SPN. The serotype 9V isolate was penicillin (PCN)-resistant (MIC=2.0), and closely-related by pulse field gel electrophoresis and multilocus fingerprinting to clone France 9V-3, an important genetic reservoir for increasing PCN-resistance worldwide. This unique case has implications for our pathogenic under-standing and empiric management of NF.

Genetic and phenotypic features of Streptococcus pyogenes strains isolated in Brazil that harbor new emm sequences.

In the present study, 37 group A Streptococcus (GAS) strains belonging to 13 new emm sequence types identified among GAS strains randomly isolated in Brazil were characterized by using phenotypic and genotypic methods. The new types were designated st204, st211, st213, st809, st833, st854, st2904, st2911, st2917, st2926, st3757, st3765, and st6735. All isolates were susceptible to the antimicrobial agents tested, except to tetracycline. They all carried the speB gene, and 94.6% produced detectable SpeB. Most strains belonging to a given emm type had similar or highly related pulsed-field gel electrophoresis profiles that were distinct from profiles of strains of another type. The other characteristics were variable from isolate to isolate, although some associations were consistently found within some emm types. Unlike the other isolates, all type st213 isolates were speA positive and produced SpeA. Strains belonging to st3765 were T6 and opacity factor (OF) negative. Individual isolates within OF-positive emm types were associated with unique sof gene sequence types, while OF-negative isolates were sof negative by PCR. This report provides information on new emm sequence types first detected in GAS isolates from a geographic area not extensively surveyed. Such data can contribute to a better understanding of the local and global dynamics of GAS populations and of the epidemiological aspects of GAS infections occurring in tropical regions.

Group A streptococcal genotypes from pediatric throat isolates in Rome, Italy.

In a study assessing genetic diversity, 114 group A streptococcus (GAS) isolates were recovered from pediatric pharyngitis patients in Rome, Italy. These isolates comprised 22 different M protein gene (emm) sequence types, 14 of which were associated with a distinct serum opacity factor/fibronectin binding protein gene (sof) sequence type. Isolates with the same emm gene sequence type generally shared a highly conserved chromosomal macrorestriction profile. In three instances, isolates with dissimilar macrorestriction profiles had identical emm types; in each of these cases multilocus sequence typing revealed that isolates with the same emm type were clones having the same allelic profiles. Ninety-eight percent of the pharyngeal isolates had emm types previously found to be highly associated with mga locus gene patterns commonly found in pharyngeal GAS isolates.

Analysis of immunoreactivity to a Streptococcus equi subsp. zooepidemicus M-like protein To confirm an outbreak of poststreptococcal glomerulonephritis, and sequences of M-like proteins from isolates obtained from different host species.

The etiologic agent of a large 1998 outbreak of poststreptococcal acute glomerulonephritis (PSGN) in Nova Serrana, Brazil, was found likely to be a specific strain of Streptococcus equi subsp. zooepidemicus from contaminated cheese (S. Balter et al., Lancet 355:1776-1780, 2000). In the present study, we used a serologic screen for a known surface-exposed virulence factor to confirm the epidemiologic findings. Using primers flanking a previously characterized M-like protein gene (J. F. Timoney et al., Infect. Immun. 63:1440-1445, 1995), we amplified and sequenced the M-like protein (designated Szp5058) gene and found it to be identical among four independent acute-phase PSGN patient isolates. Convalescent-phase sera from 33 of 44 patients in the PSGN outbreak were found to contain antibodies highly reactive to a purified Szp5058 fusion protein, compared with 1 of 17 control sera (P < 0. 0001), suggesting that Szp5058 was expressed during infection and further implicating this strain as the cause of the PSGN outbreak. The predicted signal sequence and cell wall association motif of Szp5058 were highly conserved with the corresponding sequence from S. equi subsp. zooepidemicus SzpW60, while the predicted surface-exposed portions differed markedly between these two proteins. The 5' end of the szp5058 gene, including its variable region, was identical to the szp gene from another strain associated with a previous PSGN outbreak in England (M. Barham et al., Lancet i:945-948, 1983), and the corresponding szp sequence found from the Lancefield group C type strain isolated from a guinea pig. In addition, the hypervariable (HV) portion of szp5058 was identical to a previously published HV sequence from a horse isolate (J. A. Walker and J. F. Timoney, Am. J. Vet. Res. 59:1129-1133, 1998). Three other strains of S. equi subsp. zooepidemicus, including another strain previously associated with a PSGN outbreak, were each found to contain a distinct szp gene. Two of these szp genes had HV regions identical to szp regions from isolates recovered from different host species.