Experimental Acquisition, Maintenance, and Transmission of Methicillin-Resistant Staphylococcus aureus by the Common Bed Bug, Cimex lectularius.

Here, we fed bed bugs through a membrane contaminated with methicillin-resistant Staphylococcus aureus (MRSA) at a concentration naturally present on human skin. We then determined the amount of viable MRSA present on their surface and internally over a period of 7 days. We also determined whether bed bugs that fed through the contaminated membrane could transmit MRSA to an uncontaminated membrane when taking a second blood meal 7 days later. Bed bugs acquired MRSA both externally on the cuticle surface as well as internally when feeding. MRSA was found to persist for 7 days both externally and internally in some bed bugs. Furthermore, MRSA replicated internally but not externally. Most importantly, bed bugs were able to transmit MRSA to an uncontaminated membrane feeder in 2 of 3 trials. These findings provide the first experimental support for the hypothesis that bed bugs may contribute to the transmission of MRSA in some settings.

Influenza enhances host susceptibility to non-pulmonary invasive Streptococcus pyogenes infections.

Streptococcus pyogenes (group A streptococcus; GAS) causes a variety of invasive diseases (iGAS) such as bacteremia, toxic shock syndrome, and pneumonia, which are associated with high mortality despite the susceptibility of the bacteria to penicillin ex vivo. Epidemiologic studies indicate that respiratory influenza virus infection is associated with an increase in the frequency of iGAS diseases, including those not directly involving the lung. We modified a murine model of influenza A (IAV)-GAS superinfection to determine if viral pneumonia increased the susceptibility of mice subsequently infected with GAS in the peritoneum. The results showed that respiratory IAV infection increased the morbidity (weight loss) of mice infected intraperitoneally (i.p.) with GAS 3, 5, and 10 d after the initial viral infection. Mortality was also significantly increased when mice were infected with GAS 3 and 5 d after pulmonary IAV infection. Increased mortality among mice infected with virus 5 d prior to bacterial infection correlated with increased dissemination of GAS from the peritoneum to the blood, spleen, and lungs. The interval was also associated with a significant increase in the pro-inflammatory cytokines IFN-γ, IL-12, TNF-α, MCP-1 and IL-27 in sera. We conclude, using a murine model, that respiratory influenza virus infection increases the likelihood and severity of systemic iGAS disease, even when GAS infection does not originate in the respiratory tract.

Codon-Dependent Transcriptional Changes in Response to Tryptophan Limitation in the Tryptophan Auxotrophic Pathogens Chlamydia trachomatis and Streptococcus pyogenes.

Chlamydia trachomatis and Streptococcus pyogenes are among the most prevalent bacterial pathogens of humans. Interestingly, both pathogens are tryptophan (Trp) auxotrophs and must acquire this essential amino acid from their environment. For Chlamydia, an obligate intracellular bacterium, this means scavenging Trp from the host cell in which they reside. For Streptococcus, a primarily extracellular bacterium, this means scavenging Trp from the local environment. In the course of a natural immune response, both pathogens can be exposed to Trp-limiting conditions through the action of the interferon gamma-inducible IDO1 enzyme, which catabolizes Trp to N-formylkynurenine. How these pathogens respond to Trp starvation is incompletely understood. However, we have previously demonstrated that genes enriched in Trp codons were preferentially transcribed in C. pneumoniae during Trp limitation. Chlamydia, but not Streptococcus, lacks a stringent response, which is a global regulon activated by uncharged tRNAs binding in the A site of the ribosome. We hypothesized that the chlamydial response to Trp limitation is a consequence of lacking a stringent response. To test this, we compared global transcription profiles of C. trachomatis to both wild-type and stringent response mutant strains of Streptococcus during Trp starvation. We observed that both Trp auxotrophs respond with codon-dependent changes in their transcriptional profiles that correlate with Trp codon content but not transcript stability. Importantly, the stringent response had no impact on these transcriptional changes, suggesting an evolutionarily conserved adaptation to Trp starvation. Therefore, we have revealed a novel response of Trp auxotrophic pathogens in response to Trp starvation. IMPORTANCE Chlamydia trachomatis and Streptococcus pyogenes are important pathogens of humans. Interestingly, both are auxotrophic for tryptophan and acquire this essential amino acid from the host environment. However, part of the host defense against pathogens includes the degradation of tryptophan pools. Therefore, Chlamydia and Streptococcus are particularly susceptible to tryptophan starvation. Most model bacteria respond to amino acid starvation by using a global regulon called the stringent response. However, Chlamydia lacks a stringent response. Here, we investigated the chlamydial response to tryptophan starvation and compared it to both wild-type and stringent response mutant strains of S. pyogenes to determine what role a functional stringent response plays during tryptophan starvation in these pathogens. We determined that both of these pathogens respond to tryptophan starvation by increasing transcription of tryptophan codon-rich genes. This effect was not dependent on the stringent response and highlights a previously unrecognized and potentially evolutionarily conserved mechanism for surviving tryptophan starvation.

Signaling Peptide SpoV Is Essential for Streptococcus pyogenes Virulence, and Prophylaxis with Anti-SpoV Decreases Disease Severity.

Streptococcal peptide of virulence (SpoV) is a Streptococcus pyogenes (group A streptococcus (GAS))-specific peptide that is important for GAS survival in murine blood, and the expression of the virulence factors streptolysin O (slo) and streptolysin S (sagA). We used a spoV mutant in isolate MGAS315 to assess the contribution of the SpoV peptide to virulence by using a murine model of invasive disease and an ex vivo human model (Lancefield assay). We then used antibodies to SpoV in both models to evaluate their ability to decrease morbidity and mortality. Results showed that SpoV is essential for GAS virulence, and targeting the peptide has therapeutic potential.

The Streptococcus pyogenes signaling peptide SpoV regulates streptolysin O and enhances survival in murine blood.

Streptococcus pyogenes (Group A Streptococcus, GAS) is a human pathogen that causes a wide range of diseases. For successful colonization within a variety of host niches, GAS must sense and respond to environmental changes. Intercellular communication mediated by peptides is one way GAS coordinates gene expression in response to diverse environmental stressors, which enhances bacterial survival and contributes to virulence. Using peptidomics we identified SpoV (Streptococcal peptide controlling virulence) in culture supernatant fluids. SpoV is a secreted peptide encoded near the gene encoding the extracellular cholesterol-dependent cytolysin streptolysin O (slo) The addition of synthetic SpoV peptide derivatives, but not control peptides, increased slo transcript abundance in an M49 isolate but not in an M3 isolate. Deletion of spoV decreased slo transcript abundance, extracellular SLO protein levels, and SLO-specific hemolytic activity. Complementation of the spoV mutant increased slo transcript abundance. Lastly, a spoV mutant was deficient in the ability to survive in murine blood compared to the parental strain. Moreover, pre-incubation of the spoV mutant with synthetic SpoV peptide derivatives increased GAS survival. Our findings show that slo expression is regulated, in part, by the GAS-specific signaling peptide SpoV.IMPORTANCEGAS secretes signaling peptides that can alter gene expression and impact virulence. We used peptidomics to identify a signaling peptide designated SpoV. Further, we showed that SpoV altered the expression of the cholesterol-dependent cytolysin SLO. Peptide signaling plays an important regulatory role during disease progression among several bacterial pathogens, including GAS. The therapeutic potential of manipulating peptide-controlled regulatory networks is an attractive option for the development of novel therapeutic strategies that disrupt virulence gene expression.

Immunotherapy targeting the Streptococcus pyogenes M protein or streptolysin O to treat or prevent influenza A superinfection.

Viral infections complicated by a bacterial infection are typically referred to as coinfections or superinfections. Streptococcus pyogenes, the group A streptococcus (GAS), is not the most common bacteria associated with influenza A virus (IAV) superinfections but did cause significant mortality during the 2009 influenza pandemic even though all isolates are susceptible to penicillin. One approach to improve the outcome of these infections is to use passive immunization targeting GAS. To test this idea, we assessed the efficacy of passive immunotherapy using antisera against either the streptococcal M protein or streptolysin O (SLO) in a murine model of IAV-GAS superinfection. Prophylactic treatment of mice with antiserum to either SLO or the M protein decreased morbidity compared to mice treated with non-immune sera; however, neither significantly decreased mortality. Therapeutic use of antisera to SLO decreased morbidity compared to mice treated with non-immune sera but neither antisera significantly reduced mortality. Overall, the results suggest that further development of antibodies targeting the M protein or SLO may be a useful adjunct in the treatment of invasive GAS diseases, including IAV-GAS superinfections, which may be particularly important during influenza pandemics.

The Streptococcus pyogenes fibronectin/tenascin-binding protein PrtF.2 contributes to virulence in an influenza superinfection.

Influenza A virus (IAV) and Streptococcus pyogenes (the group A Streptococcus; GAS) are important contributors to viral-bacterial superinfections, which result from incompletely defined mechanisms. We identified changes in gene expression following IAV infection of A549 cells. Changes included an increase in transcripts encoding proteins with fibronectin-type III (FnIII) domains, such as fibronectin (Fn), tenascin N (TNN), and tenascin C (TNC). We tested the idea that increased expression of TNC may affect the outcome of an IAV-GAS superinfection. To do so, we created a GAS strain that lacked the Fn-binding protein PrtF.2. We found that the wild-type GAS strain, but not the mutant, co-localized with TNC and bound to purified TNC. In addition, adherence of the wild-type strain to IAV-infected A549 cells was greater compared to the prtF.2 mutant. The wild-type strain was also more abundant in the lungs of mice 24 hours after superinfection compared to the mutant strain. Finally, all mice infected with IAV and the prtF.2 mutant strain survived superinfection compared to only 42% infected with IAV and the parental GAS strain, indicating that PrtF.2 contributes to virulence in a murine model of IAV-GAS superinfection.

Binding host proteins to the M protein contributes to the mortality associated with influenza-Streptococcus pyogenes superinfections.

The mortality associated with influenza A virus (IAV) is often due to the development of secondary bacterial infections known as superinfections. The group A streptococcus (GAS) is a relatively uncommon cause of IAV superinfections, but the mortality of these infections is high. We used a murine model to determine whether the surface-localized GAS M protein contributes to the outcome of IAV-GAS superinfections. A comparison between wild-type GAS and an M protein mutant strain (emm3) showed that the M3 protein was essential to virulence. To determine whether the binding, or recruitment, of host proteins to the bacterial surface contributed to virulence, GAS was suspended with BALF collected from mice that had recovered from a sub-lethal infection with IAV. Following intranasal inoculation of naïve mice, the mortality associated with the wild-type strain, but not the emm3 mutant strain, was greater compared to mice inoculated with GAS suspended with either BALF from uninfected mice or PBS. Further analyses showed that both albumin and fibrinogen (Fg) were more abundant in the respiratory tract 8 days after IAV infection, that M3 bound both proteins to the bacterial surface, and that suspension of GAS with either protein increased GAS virulence in the absence of antecedent IAV infection. Overall, the results showed that M3 is essential to the virulence of GAS in an IAV superinfection and suggested that increased abundance of albumin and Fg in the respiratory tract following IAV infection enhanced host susceptibility to secondary GAS infection.

The Association between Invasive Group A Streptococcal Diseases and Viral Respiratory Tract Infections.

Viral infections of the upper respiratory tract are associated with a variety of invasive diseases caused by Streptococcus pyogenes, the group A streptococcus, including pneumonia, necrotizing fasciitis, toxic shock syndrome, and bacteremia. While these polymicrobial infections, or superinfections, are complex, progress has been made in understanding the molecular basis of disease. Areas of investigation have included the characterization of virus-induced changes in innate immunity, differences in bacterial adherence and internalization following viral infection, and the efficacy of vaccines in mitigating the morbidity and mortality of superinfections. Here, we briefly summarize viral-S. pyogenes superinfections with an emphasis on those affiliated with influenza viruses.