Bacterial virulence plays a crucial role in MRSA sepsis

Bacterial sepsis is a major global cause of death. However, the pathophysiology of sepsis has remained poorly understood. In industrialized nations, Staphylococcus aureus represents the pathogen most commonly associated with mortality due to sepsis. Because of the alarming spread of antibiotic resistance, anti-virulence strategies are often proposed to treat staphylococcal sepsis. However, we do not yet completely understand if and how bacterial virulence contributes to sepsis, which is vital for a thorough assessment of such strategies. We here examined the role of virulence and quorum-sensing regulation in mouse and rabbit models of sepsis caused by methicillin-resistant S. aureus (MRSA). We determined that leukopenia was a predictor of disease outcome during an early critical stage of sepsis. Furthermore, in device-associated infection as the most frequent type of staphylococcal blood infection, quorum-sensing deficiency resulted in significantly higher mortality. Our findings give important guidance regarding anti-virulence drug development strategies for the treatment of staphylococcal sepsis. Moreover, they considerably add to our understanding of how bacterial sepsis develops by revealing a critical early stage of infection during which the battle between bacteria and leukocytes determines sepsis outcome. While sepsis has traditionally been attributed mainly to host factors, our study highlights a key role of the invading pathogen and its virulence mechanisms.     

The role of virulence determinants in community-associated MRSA pathogenesis

The recent emergence of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) marked a quantum change in the biology and epidemiology of a major human pathogen. Various virulence determinants unique to CA-MRSA have been uncovered recently, which shed light on how these strains spread easily and sustainably among humans and frequently cause severe disease. The role of the Panton Valentine leukocidin (PVL) in CA-MRSA pathogenesis is a matter of much debate. Although epidemiological data have indicated a role for PVL in the CA-MRSA disease process, recent data from relevant animal models indicate that PVL does not impact virulence of prevalent CA-MRSA strains. Identifying specialized pathogenic traits of CA-MRSA remains a challenge that will yield new diagnostic tools and therapeutic targets for drug and vaccine development. Here, we discuss the roles of PVL, the arginine catabolic mobile element and phenol-soluble modulins in the pathogenesis of prevalent CA-MRSA strains.     

MRSA epidemic linked to a quickly spreading colonization and virulence determinant

The molecular processes underlying epidemic waves of methicillin-resistant Staphylococcus aureus (MRSA) infection are poorly understood(1). Although a major role has been attributed to the acquisition of virulence determinants by horizontal gene transfer(2), there are insufficient epidemiological and functional data supporting that concept. We here report the spread of clones containing a previously extremely rare(3,4) mobile genetic element–encoded gene, sasX. We demonstrate that sasX has a key role in MRSA colonization and pathogenesis, substantially enhancing nasal colonization, lung disease and abscess formation and promoting mechanisms of immune evasion. Moreover, we observed the recent spread of sasX from sequence type 239 (ST239) to invasive clones belonging to other sequence types. Our study identifies sasX as a quickly spreading crucial determinant of MRSA pathogenic success and a promising target for therapeutic interference. Our results provide proof of principle that horizontal gene transfer of key virulence determinants drives MRSA epidemic waves.     

Transcriptional analysis of antibiotic resistance and virulence genes in multiresistant hospital-acquired MRSA

The staphylococcal chromosome cassette mec cannot solely explain the multiresistance phenotype or the relatively mild virulence profile of hospital-acquired methicillin-resistant Staphylococcus aureus (HA-MRSA). This study reports that several multiresistant HA-MRSA strains differently expressed genes that may support antibiotic resistance, modify the bacterial surface and influence the pathogenic process. Genes encoding efflux pumps (norA, arsB, emrB) and the macrolide resistance gene ermA were found to be commonly expressed by HA-MRSA strains, but not in the archetypal MRSA strain COL. At equivalent cell density, the agr system was considerably less activated in all MRSA strains (including COL) in comparison with a prototypic antibiotic-susceptible strain. These results are in contrast to those observed in recent community-acquired MRSA isolates and may partly explain how multiresistant HA-MRSA persist in the hospital setting.     

The parasitoid factor in the virulence and spread of lepidopteran baculoviruses

Insect parasitoids and baculoviruses play important roles in the natural and strategic biological control of insects. The two parasites are frequent competitors within common hosts and much research has focused on the negative impact that baculoviral host infections have on parasitoids. This review summarizes the impacts that parasitoids may have on the virulence and spread of lepidopteran baculoviruses. By changing host behavior and development, parasitoids have been shown to decrease baculovirus virulence and productivity within parasitized baculovirus-susceptible hosts; however, studies of the tools used by hymenopteran parasitoids to overcome their hosts' immune systems, suggest that parasitoids may, in some cases, facilitate baculoviral infections in less susceptible hosts. Laboratory and field research have demonstrated that parasitoids can mechanically transmit bacuioviruses between insects, and in this way, increase the efficacy of the viruses. Instances of new, more virulent isolates of baculoviruses have been recorded from specifically parasitoid-targeted hosts suggesting other possible benefits from the transmission or activation of baculoviruses by parasitoids.     

MRSA lineage USA300 isolated from bloodstream infections exhibit altered virulence regulation

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Identification of novel cytolytic peptides as key virulence determinants for community-associated MRSA

Methicillin-resistant Staphylococcus aureus (MRSA) remains a major human pathogen. Traditionally, MRSA infections occurred exclusively in hospitals and were limited to immunocompromised patients or individuals with predisposing risk factors. However, recently there has been an alarming epidemic caused by community-associated (CA)-MRSA strains, which can cause severe infections that can result in necrotizing fasciitis or even death in otherwise healthy adults outside of healthcare settings. In the US, CA-MRSA is now the cause of the majority of infections that result in trips to the emergency room. It is unclear what makes CA-MRSA strains more successful in causing human disease compared with their hospital-associated counterparts. Here we describe a class of secreted staphylococcal peptides that have a remarkable ability to recruit, activate and subsequently lyse human neutrophils, thus eliminating the main cellular defense against S. aureus infection. These peptides are produced at high concentrations by standard CA-MRSA strains and contribute significantly to the strains' ability to cause disease in animal models of infection. Our study reveals a previously uncharacterized set of S. aureus virulence factors that account at least in part for the enhanced virulence of CA-MRSA.     

A bacterial regulatory RNA attenuates virulence,spread and human host cell phagocytosis

Staphylococcus aureus pathogenesis is directed by regulatory proteins and RNAs. We report the case of an RNA attenuating virulence and host uptake, possibly to sustain commensalism. A S. aureus sRNA, SprC (srn_3610), reduced virulence and bacterial loads in a mouse infection model. S. aureus deleted for sprC became more virulent and increased bacterial dissemination in colonized animals. Conversely, inducing SprC expression lowered virulence and the bacterial load. Without sprC, S. aureus phagocytosis by monocytes and macrophages was higher, whereas bacteria were internalized at lower yields when SprC expression was stimulated. Without sprC, higher internalization led to a greater number of extracellular bacteria, facilitating colonization. SprC expression decreased after phagocytosis, concurring with the facilitated growth of bacteria lacking the sRNA in the presence of an oxidant. The major staphylococcal autolysin facilitates S. aureus uptake by human phagocytes. ATL proved to be negatively regulated by SprC. The SprC domains involved in pairing with atl mRNA were analyzed. The addition of ATL reduced phagocytosis of bacteria lacking sprC with no effects on wild-type bacterial uptake, implying that SprC influences phagocytosis, at least in part, by controlling ATL. Since the control of SprC on ATL was modest, other factors must contribute to atl regulation.     

Molecular mechanisms of virus spread and virion components as tools of virulence. A review

Despite of differences in replication strategy among virus families, some basic principles have remained similar. Analogous mechanisms govern virus entry into cells and the use of enzymes which direct the replication of the virus genome. The function of many cell surface receptors (such as glycosoaminoglycans, glycoproteins, proteins) which interact with viral capsid proteins or envelope glycoproteins has recently been elucidated. The list of cellular receptors (Table I) is still far from being final. The capsid components, similarly as the envelope glycoproteins, may form specific pocket like sites, which interact with the cell surface receptors. Neutralizing antibodies usually react with antigenic domains adjacent to the receptor binding site(s) and hamper the close contact inevitable for virion attachment. In the case of more complex viruses, such as herpes simplex virus, different viral glycoproteins interact with several cellular receptors. At progressed phase of adsorption the virions are engulfed into endocytic vesicles and the virion fusion domain(s) become(s) activated. The outer capsid components of reoviruses which participate in adsorption and fusion may get activated already in the lumen of digestive tract, i.e. before their engulfment by resorptive epithelium cells. Activation of the hydrophobic fusion domain(s) is a further important step allowing to pass through the lipid bilayer when penetrating the cell membrane in order to reach the cytosol. Activation of the virion fusion domain is accomplished by a conformation change, which occurs at acid pH (influenza virus hemagglutinin, sigma 1 protein of the reovirus particle) and/or after protease treatment. The herpes simplex virus fusion factors (gD and gH) undergo conformation changes by a pH-independent mechanism triggered due to interaction with the cell surface receptor(s) and mediated by mutual interactions with the viral envelope glycoproteins. The virion capsid or envelope components participating in the entry and membrane fusion are not the only tools of virulence. The correct function of virus coded proteins, which participate in replication of the viral genome, and/or in the supply of necessary nucleotides, may be very essential. In the case of enteroviruses, which RNA interacts with ribosomes directly, the correct configuration of the non-coding viral RNA sequence is crucial for initiation of translation occurring in the absence of the classical "cap" structure.     

Listeria monocytogenes wall teichoic acid decoration in virulence and cell‐to‐cell spread

Wall teichoic acid (WTA) comprises a class of glycopolymers covalently attached to the peptidoglycan of gram positive bacteria. In Listeria monocytogenes, mutations that prevent addition of certain WTA decorating sugars are attenuating. However, the steps required for decoration and the pathogenic process interrupted are not well described. We systematically examined the requirement for WTA galactosylation in a mouse oral‐virulent strain by first creating mutations in four genes whose products conferred resistance to a WTA‐binding bacteriophage. WTA biochemical and structural studies indicated that galactosylated WTA was directly required for bacteriophage adsorption and that mutant WTA lacked appreciable galactose in all except one mutant – which retained a level ca. 7% of the parent. All mutants were profoundly attenuated in orally infected mice and were impaired in cell‐to‐cell spread in vitro. Confocal microscopy of cytosolic mutants revealed that all expressed ActA on their cell surface and formed actin tails with a frequency similar to the parent. However, the mutant tails were significantly shorter – suggesting a defect in actin based motility. Roles for the gene products in WTA galactosylation are proposed. Identification and interruption of WTA decoration pathways may provide a general strategy to discover non‐antibiotic therapeutics for gram positive infections. © 2016 John Wiley & Sons Ltd     

Community-acquired MRSA and pig-farming

Background

Female genital tuberculosis is an uncommon disease that is rarely diagnosed in developed countries.

Case presentation

A 61-year-old postmenopausal woman who had undergone surgery and treated with adjuvant chemotherapy for infiltrating ductal carcinoma of the breast five years ago, presented with bloody vaginal discharge, fatigue, weight loss, and low grade fevers at night for two months. Histological examination of the endometrium, done based on the suspicion of a second primary cancer due to the tamoxifen therapy, revealed a granulomatous reaction. Liquid and solid mycobacterial cultures of the tissues were performed. Although the acid fast staining was negative, the liquid culture was positive for Mycobacterium tuberculosis. Involvement of other systems was not detected. The patient was treated with a three-drug antituberculosis regimen for 9 months and recovered fully.

Conclusion

Female genital tuberculosis is a rare but curable disease that should be included in the differential diagnosis of women with menstrual problems. Early diagnosis is important and may prevent unnecessary invasive procedures for the patient.     

N1L is an ectromelia virus virulence factor and essential for in vivo spread upon respiratory infection

The emergence of zoonotic orthopoxvirus infections and the threat of possible intentional release of pathogenic orthopoxviruses have stimulated renewed interest in understanding orthopoxvirus infections and the resulting diseases. Ectromelia virus (ECTV), the causative agent of mousepox, offers an excellent model system to study an orthopoxvirus infection in its natural host. Here, we investigated the role of the vaccinia virus ortholog N1L in ECTV infection. Respiratory infection of mice with an N1L deletion mutant virus (ECTVΔN1L) demonstrated profound attenuation of the mutant virus, confirming N1 as an orthopoxvirus virulence factor. Upon analysis of virus dissemination in vivo, we observed a striking deficiency of ECTVΔN1L spreading from the lungs to the livers or spleens of infected mice. Investigating the immunological mechanism controlling ECTVΔN1L infection, we found the attenuated phenotype to be unaltered in mice deficient in Toll-like receptor (TLR) or RIG-I-like RNA helicase (RLH) signaling as well as in those missing the type I interferon receptor or lacking B cells. However, in RAG-1(-/-) mice lacking mature B and T cells, ECTVΔN1L regained virulence, as shown by increasing morbidity and virus spread to the liver and spleen. Moreover, T cell depletion experiments revealed that ECTVΔN1L attenuation was reversed only by removing both CD4(+) and CD8(+) T cells, so the presence of either cell subset was still sufficient to control the infection. Thus, the orthopoxvirus virulence factor N1 may allow efficient ECTV infection in mice by interfering with host T cell function.     

icsB: a Shigella flexneri virulence gene necessary for the lysis of protrusions during intercellular spread

Shigella flexneri causes bacillary dysentery by invading epithelial cells of the colonic mucosa. We have characterized the icsB gene which is located on the virulence plasmid pWR100. After inactivation of icsB, the mutant strain remained invasive, but formed abnormally small plaques on HeLa cell monolayers, colonized only the peripheral cells of Caco-2 islets, and was unable to provoke a keratoconjunctivitis in guinea-pigs. Examination of infected HeLa cells showed that the icsB mutant was able to lyse the phagocytic vacuole and to form protrusions at the surface of infected cells, but, unlike the wild type, remained trapped in protrusions surrounded by two membranes. These results indicate that IcsB is involved in the lysis of the protrusions, a step necessary for intercellular spread.     

Seasonality of MRSA infections

Using MRSA isolates submitted to our hospital microbiology laboratory January 2001–March 2010 and the number of our emergency department (ED) visits, quarterly community-associated (CA) and hospital-associated (HA) MRSA infections were modeled using Poisson regressions. For pediatric patients, approximately 1.85x (95% CI 1.45x–2.36x, adj. p<0.0001) as many CA-MRSA infections per ED visit occurred in the second two quarters as occurred in the first two quarters. For adult patients, 1.14x (95% CI 1.01x–1.29x, adj.p = 0.03) as many infections per ED visit occurred in the second two quarters as in the first two quarters. Approximately 2.94x (95% CI 1.39x–6.21x, adj.p = 0.015) as many HA-MRSA infections per hospital admission occurred in the second two quarters as occurred in the first two quarters for pediatric patients. No seasonal variation was observed among adult HA-MRSA infections per hospital admission. We demonstrated seasonality of MRSA infections and provide a summary table of similar observations in other studies.     

Pathogenicity and virulence

Invertebrate pathologists have multiple definitions for the terms pathogenicity and virulence, and these definitions vary across disciplines that focus on host-pathogen interactions. We surveyed various literatures, including plant pathology, invertebrate pathology, evolutionary biology, and medicine, and found most define pathogenicity as the broader term, which incorporates virulence. Virulence is seen as the severity of disease manifestation that can only be measured in infected individuals. These definitions readily apply to both lethal and non-lethal diseases. Invertebrate pathologists commonly use dose-response bioassays to estimate LD(50) or LC(50) (dose or concentration needed to kill 50% of hosts exposed). These bioassays measure pathogenicity if the bioassay includes a transmission component, and measure virulence if the bioassay is measured in infected individuals only. Another common bioassay estimate is LT(50) (median time to death of infected hosts), which is a measure of virulence as long as survivors are not included in its calculation.     

Molecular identification and genotyping of MRSA isolates

The aim of this study was to identify and characterize 97 methicillin-resistant Staphylococcus aureus (MRSA) isolates. Two conventional multiplex PCR assays, a real-time PCR assay and two PCR-based genotyping techniques including the spa - and hypervariable region (HVR)-typing methods were used to identify and characterize 97 MRSA strains isolated between April 2006 to September 2007 from the Steve Biko Academic Hospital. All MRSA isolates were positive for 16S rRNA gene, 99% were positive for the mec A gene and 4% positive for the Panton–Valentine leukocidin (PVL) gene. Staphylococcal cassette chromosome mec (SCC mec ) typing showed 67% of isolates were SCC mec II [health-care-associated MRSA (HA-MRSA)], 14% were SCC mec III (HA-MRSA) and 4% were SCC mec IVd [community-associated MRSA (CA-MRSA)]. These CA-MRSA isolates showed a prevalence of 100% for the PVL gene. Using spa typing, three distinct clusters could be identified while HVR typing revealed six different clusters. CA-MRSA isolates were clustered together using spa and HVR typing. This study showed the prevalence of the CA-MRSA strains, PVL genes, the SCC mec types and the clonality of the MRSA strains. The high prevalence of the PVL gene in CA-MRSA isolates already residing in intensive care units was alarming and indicated the emergence of new MRSA lineages with a particular fitness for community and hospital transmission.