Increases in group A streptococcal infections in the pediatric population in Houston, TX, 2022.

Beginning in October 2022, we observed a substantial increase in the total number of cases of invasive GAS disease (iGAS) in the pediatric population in Houston, TX. Emm12 GAS strains were disproportionately represented but the overall proportion of iGAS infections observed during the current spike was similar to pre-pandemic years.

The Integrative Conjugative Element ICESpyM92 Contributes to Pathogenicity of Emergent Antimicrobial-Resistant emm92 Group A Streptococcus.

Antimicrobial resistance-encoding mobile genetic elements (MGEs) may contribute to the disease potential of bacterial pathogens. We previously described the association of Group A Streptococcus (GAS) derived from invasive disease with increasingly frequent antimicrobial resistance (AMR). We hypothesized that a 65-kb AMR-encoding MGE (ICESpyM92), highly conserved among closely related emergent invasive emm92 GAS, contributes to GAS disease potential. Here, we provide evidence that a combination of ICESpyM92- and core genome-dependent differential gene expression (DGE) contributes to invasive disease phenotypes of emergent emm92 GAS. Using isogenic ICESpyM92 mutants generated in distinct emm92 genomic backgrounds, we determined the presence of ICESpyM92 enhances GAS virulence in a mouse subcutaneous infection model. Measurement of in vitro and ex vivo DGE indicates ICESpyM92 influences GAS global gene expression in a background-dependent manner. Our study links virulence and AMR on a unique MGE via MGE-related DGE and highlights the importance of investigating associations between AMR-encoding MGEs and pathogenicity.

Population Genomics Reveals Distinct Temporal Association with the Emergence of ST1 Serotype V Group B Streptococcus and Macrolide Resistance in North America.

Identified in the 1970s as the leading cause of invasive bacterial disease in neonates and young infants, group B Streptococcus (GBS) is now also recognized as a significant cause of morbidity and mortality among adults with underlying medical conditions and the elderly. Concomitant with the increasing incidence of GBS invasive disease in adults is the rise of resistance among GBS isolates to second line antibiotics. Previous research shows that among serotype V GBS, one of the most common capsular types causing adult invasive disease, sequence type 1 (ST1), accounts for an overwhelming majority of adult invasive disease isolates and frequently harbors macrolide resistance. In this study, using whole-genome sequencing data from strains isolated in the United States and Canada over a 45-year period, we examined the association of antimicrobial resistance with the emergence of invasive serotype V ST1 GBS. Our findings show a strong temporal association between increased macrolide resistance and the emergence of serotype V ST1 GBS subpopulations that currently co-circulate to cause invasive disease in adults and young infants. ST1 GBS subpopulations are defined, in part, by the presence of macrolide resistance genes in mobile genetic elements. Increased frequency of macrolide resistance-encoding mobile genetic elements among invasive GBS ST1 strains suggests the presence of such elements contributes to GBS virulence. Our work provides a foundation for the investigation of genetic features contributing to the increasing prevalence and pathogenesis of serotype V GBS in adult invasive disease.

The LiaFSR Transcriptome Reveals an Interconnected Regulatory Network in Group A Streptococcus.

The mechanisms by which bacteria sense the host environment and alter gene expression are poorly understood. LiaFSR is a gene regulatory system unique to Gram-positive bacteria, including group A Streptococcus (GAS), and responds to cell envelope stress. We previously showed that LiaF acts as an inhibitor to LiaFSR activation in GAS. To better understand gene regulation associated with LiaFSR activation, we performed RNA sequencing on isogenic deletion mutants fixed in a LiaFSR "always on" (ΔliaF) or "always off" (ΔliaR) state. Transcriptome analyses of ΔliaF and ΔliaR in GAS showed near perfect inverse correlation, including the gene encoding the global transcriptional regulator SpxA2. In addition, mutant transcriptomes included genes encoding multiple virulence factors and showed substantial overlap with the CovRS regulon. Chromatin immunoprecipitation quantitative PCR demonstrated direct spxA2 gene regulation following activation of the response regulator, LiaR. High SpxA2 levels as a result of LiaFSR activation were directly correlated with increased CovR-regulated virulence gene transcription. Furthermore, consistent with known virulence gene repression by phosphorylated CovR, elevated SpxA2 levels were inversely correlated with CovR phosphorylation. Despite increased transcription of several virulence factors, ΔliaF (high SpxA2) exhibited a paradoxical virulence phenotype in both in vivo mouse and ex vivo human blood models of disease. Likewise, despite decreased virulence factor transcription with ΔliaR (low SpxA2), increased virulence was observed in an in vivo mouse model of disease-a phenotype attributable, in part, to known SpxA2-associated speB transcription. Our findings provide evidence of a critical role of LiaFSR in sensing the host environment and suggest a potential mechanism for gene regulatory system cross talk shared by many Gram-positive pathogens.

Strain-Dependent Effect of Capsule on Transmission and Persistence in an Infant Mouse Model of Group A Streptococcus Infection.

Streptococcus pyogenes (group A Streptococcus [GAS]) is a human pathogen responsible for a wide range of diseases. Asymptomatic carriage of GAS in the human pharynx is commonplace and a potential reservoir for GAS transmission. Early studies showed that GAS transmission correlated with high bacterial burdens during the acute symptomatic phase of the disease. Human studies and the nonhuman primate model are generally impractical for investigation of the bacterial mechanisms contributing to GAS transmission and persistence. To address this gap, we adapted an infant mouse model of pneumococcal colonization and transmission to investigate factors that influence GAS transmission and persistence. The model recapitulated the direct correlation between GAS burden and transmission during the acute phase of infection observed in humans and nonhuman primates. Furthermore, our results indicate that the ratio of colonized to uncolonized hosts influences the rates of GAS transmission and persistence. We used the model to test the hypothesis that capsule production influences GAS transmission and persistence in a strain-dependent manner. We detected significant differences in rates of transmission and persistence between capsule-positive (emm3) and capsule-negative (emm87) GAS strains. Capsule was associated with higher levels of GAS shedding, independent of the strain background. In contrast to the capsule-positive emm3 strain, restoring capsule production in emm87 GAS did not increase transmissibility, and the absence of capsule enhanced persistence only in the capsule-negative (emm87) strain background. These data suggest that strain background (capsule positive versus capsule negative) influences the effect of capsule in GAS transmission and persistence and that as-yet-undefined factors are required for the transmission of capsule-negative emm types.

Clinical laboratory response to a mock outbreak of invasive bacterial infections: a preparedness study.

Large hospital-based clinical laboratories must be prepared to rapidly investigate potential infectious disease outbreaks. To challenge the ability of our molecular diagnostics laboratory to use whole-genome sequencing in a potential outbreak scenario and identify impediments to these efforts, we studied 84 invasive serotype emm59 group A streptococcus (GAS) strains collected in the United States. We performed a rapid-response exercise to the mock outbreak scenario using whole-genome sequencing, genome-wide transcript analysis, and mouse virulence studies. The protocol changes installed in response to the lessons learned were tested in a second iteration. The initial investigation was completed in 9 days. Whole-genome sequencing showed that the invasive infections were caused by multiple subclones of epidemic emm59 GAS strains likely spread to the United States from Canada. The phylogenetic tree showed a strong temporal-spatial structure with diversity in mobile genetic element content, features that are useful for identifying closely related strains and possible transmission events. The genome data informed the epidemiology, identifying multiple patients who likely acquired the organisms through direct person-to-person transmission. Transcriptome analysis unexpectedly revealed significantly altered expression of genes encoding a two-component regulator and the hyaluronic acid capsule virulence factor. Mouse infection studies confirmed a high-virulence capacity of these emm59 organisms. Whole-genome sequencing, coupled with transcriptome analysis and animal virulence studies, can be rapidly performed in a clinical environment to effectively contribute to patient care decisions and public health maneuvers.

Increasing Incidence of Streptococcus anginosus Group Intracranial Infections Associated With Sinusitis, Otitis Media, and Mastoiditis in Children.

BACKGROUND: The Streptococcus anginosus group (SAG) pathogens have the potential to cause head and neck space infections, including intracranial abscesses. Several centers noted an increase in intracranial abscesses in children during the SARS-CoV-2 pandemic, prompting a Centers for Disease Control and Prevention health alert in May 2022. We examined the epidemiology of pediatric intracranial abscesses at a tertiary care center with a focus on SAG pre- and post-pandemic. METHODS: Cases of intracranial abscesses of any microbiologic etiology admitted from January 2011 to December 2022 were identified using International Classification of Diseases 10 codes. Subjects were cross-referenced with culture results from the microbiology laboratory at Texas Children's Hospital. Cases included were those associated with either otitis media, mastoiditis or sinusitis and medical records were reviewed. RESULTS: A total of 157 cases were identified and 59.9% (n = 94) were caused by SAG. The incidence of all sinogenic/otogenic intracranial infections (P = 0.002), and SAG-specific infections (P = 0.004), increased from 2011 to 2022. SAG infection was more often associated with multiple surgeries, and these subjects were more likely to require craniotomy or craniectomy. Among sinogenic abscesses, S. intermedius was the most common pathogen, while among otogenic cases, S. pyogenes predominated. From March 2020 to Dec 2022, 9/49 cases tested positive for SARS-CoV-2 (18.4%); characteristics of infection were not significantly different among cases with and without SARS-CoV-2. CONCLUSIONS: Over the last decade, intracranial complications of sinusitis/otitis have been increasing, specifically those caused by SAG; this trend, however, predated the SARS-CoV-2 pandemic. SAG was associated with a greater need for surgical intervention, specifically neurosurgery. Further work is necessary to determine the cause for these rising infections.

Use of whole-genome sequencing to detect transmission of group A Streptococcus in Houston, TX.

We used a combination of local, comprehensive strain surveillance and bacterial whole-genome sequencing to identify potential transmission events of group A streptococcus (GAS) in Houston, TX, USA. We identified pharyngeal and skin and soft tissue sources of infection as having important roles in community GAS transmission, including invasive diseases.

Identification of epidemic scarlet fever group A Streptococcus strains in the paediatric population of Houston, TX, USA.

Scarlet fever (SF) has recently been associated with group A streptococcal (GAS) strains possessing multidrug resistance and specific streptococcal exotoxins. We screened a local surveillance collection of GAS emm12 strains in Houston, TX, USA for antimicrobial resistance and identified a single isolate matching the antimicrobial resistance pattern previously reported for SF clones. Using whole-genome sequencing and combining genome sequence data derived from national surveillance databases, we identified additional emm12 GAS clones similar to those associated with prior SF outbreaks, emphasizing the need for continued surveillance for epidemic emergence in the USA.

ExPortal and the LiaFSR Regulatory System Coordinate the Response to Cell Membrane Stress in Streptococcus pyogenes.

LiaFSR is a gene regulatory system important for response to cell membrane stress in Gram-positive bacteria but is minimally studied in the important human pathogen group A Streptococcus (GAS). Using immunofluorescence and immunogold electron microscopy, we discovered that LiaF (a membrane-bound repressor protein) and LiaS (a sensor kinase) reside within the GAS membrane microdomain (ExPortal). Cell envelope stress induced by antimicrobials resulted in ExPortal disruption and activation of the LiaFSR system. The only human antimicrobial peptide whose presence resulted in ExPortal disruption and LiaFSR activation was the alpha-defensin human neutrophil peptide 1 (hNP-1). Elimination of membrane cardiolipin through targeted gene deletion resulted in loss of LiaS colocalization with the GAS ExPortal and activation of LiaFSR, whereas LiaF membrane localization was unaffected. Isogenic mutants lacking either LiaF or LiaS revealed a critical role of LiaF in ExPortal integrity. Thus, LiaF and LiaS colocalize with the GAS ExPortal by distinct mechanisms, further supporting codependence. These are the first data identifying a multicomponent signal system within the ExPortal, thereby providing new insight into bacterial intramembrane signaling in GAS that may serve as a paradigm for Gram-positive bacteria.IMPORTANCE Bacterial two-component systems sense and induce transcriptional changes in response to environmental stressors, including antimicrobials and human antimicrobial peptides. Since the stresses imposed by the host's defensive responses may act as markers of specific temporal stages of disease progression or host compartments, pathogens often coordinately regulate stress response programs with virulence factor expression. The mechanism by which bacteria recognize these stresses and subsequently induce transcriptional responses remains not well understood. In this study, we showed that LiaFSR senses cell envelope stress through colocalization of LiaF and LiaS with the group A Streptococcus (GAS) ExPortal and is activated in direct response to ExPortal disruption by antimicrobials or human antimicrobial peptides. Our studies shed new light on the sensing of cell envelope stress in Gram-positive bacteria and may contribute to the development of therapies targeting these processes.

Unexpected relationships between frequency of antimicrobial resistance, disease phenotype and emm type in group A Streptococcus.

Despite universal susceptibility to ß-lactams, resistance to second-line antimicrobials (e.g. erythromycin) is increasingly common among group A Streptococcus (GAS). To better understand the frequency of regional GAS antimicrobial resistance, we screened a previously described GAS strain collection from Houston, TX, USA, for resistance to commonly used antimicrobials. A total of 100/929 (10.8 %) showed resistance to at least one antimicrobial. Tetracycline resistance was identified in 52 (5.6 %) GAS strains. The cumulative frequency of erythromycin and clindamycin resistance [macrolide (M) and macrolide-lincosamide-streptogramin (MLS) phenotypes] was greatest among invasive GAS strains (9.9 %) compared to that of strains derived from any other infection type (5.9 %, P=0.045). We identified emm types 11, 75, 77 and 92 as the only emm types with high (e.g. >50 %) within-emm type resistance and contributing to the majority (24/26; 92 %) of erythromycin/clindamycin resistance in invasive GAS. High-frequency resistance emm types were also significantly overrepresented in invasive GAS strains as indicated by invasive index. We performed whole-genome sequencing to define genetic elements associated with resistance among emm types 11, 75, 77 and 92. Diverse mobile elements contributed to GAS resistance including transposons, integrative conjugative elements, prophage and a plasmid. Phylogenetic analysis suggests recent clonal emergence of emm92 GAS strains. Our findings indicate that less frequently encountered GAS emm types disproportionately contribute to resistance phenotypes, are defined by diverse mobile genetic elements and may favour invasive disease.