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.

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.

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.

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.

Intraarticular injection of relaxin-2 alleviates shoulder arthrofibrosis.

Arthrofibrosis is a prevalent condition affecting greater than 5% of the general population and leads to a painful decrease in joint range of motion (ROM) and loss of independence due to pathologic accumulation of periarticular scar tissue. Current treatment options are limited in effectiveness and do not address the underlying cause of the condition: accumulation of fibrotic collagenous tissue. Herein, the naturally occurring peptide hormone relaxin-2 is administered for the treatment of adhesive capsulitis (frozen shoulder) and to restore glenohumeral ROM in shoulder arthrofibrosis. Recombinant human relaxin-2 down-regulates type I collagen and α smooth muscle actin production and increases intracellular cAMP concentration in human fibroblast-like synoviocytes, consistent with a mechanism of extracellular matrix degradation and remodeling. Pharmacokinetic profiling of a bolus administration into the glenohumeral joint space reveals the brief systemic and intraarticular (IA) half-lives of relaxin-2: 0.96 h and 0.62 h, respectively. Furthermore, using an established, immobilization murine model of shoulder arthrofibrosis, multiple IA injections of human relaxin-2 significantly improve ROM, returning it to baseline measurements collected before limb immobilization. This is in contrast to single IA (sIA) or multiple i.v. (mIV) injections of relaxin-2 with which the ROM remains constrained. The histological hallmarks of contracture (e.g., fibrotic adhesions and reduced joint space) are absent in the animals treated with multiple IA injections of relaxin-2 compared with the untreated control and the sIA- and mIV-treated animals. As these findings show, local delivery of relaxin-2 is an innovative treatment of shoulder arthrofibrosis.

Rat Model of Adhesive Capsulitis of the Shoulder.

This proposal aims to create an in vivo rat model of adhesive capsulitis for researching potential treatment options for this condition and other etiologies of comparable arthrofibrosis. The model includes extra-articular fixation of the shoulder in rats via scapular to humeral suturing, resulting in a secondary contracture without invading the intra-articular space and resulting in decreased rotational ROM and increased joint stiffness. We used 10 Sprague-Dawley rats for the purpose of this study. Baseline ROM measurements were taken before glenohumeral immobilization. The rats were subjected to 8 weeks of immobilization before the fixation sutures were removed and changes in ROM and joint stiffness were evaluated. To evaluate whether immobilization resulted in a significant reduction in ROM, changes in kinematics were calculated. ROM was measured at each time point in the follow-up period and was compared to the baseline internal and external ROM measurements. In order to evaluate the stiffness, joint kinetics were calculated by determining the differences in torque (text and tint ) needed to reach the initial external rotation of 60° and initial internal rotation of 80°. After the removal of the extra-articular suture fixation on follow-up day 0, we found a 63% decrease in total ROM compared to baseline. We observed continuous improvement until week 5 of follow-up, with the progress slowing down around a 19% restriction. On week 8 of follow-up, there was still an 18% restriction of ROM. Additionally, on follow-up day 0, we found the torque increased by 13.3 Nmm when compared to baseline. On week 8, the total torque was measured to be 1.4 ± 0.2 Nmm higher than initial measurements. This work introduces a rat model of shoulder adhesive capsulitis with lasting reduced ROM and increased stiffness.