Amy E. Bryant

Impact of antibiotics on expression of virulence-associated exotoxin genes in methicillin-sensitive and methicillin-resistant Staphylococcus aureus.

Extracellular protein toxins contribute to the pathogenesis of a wide variety of Staphylococcus aureus infections. The present study investigated the effects that cell-wall active antibiotics and protein-synthesis inhibitors have on transcription and translation of genes for Panton-Valentine leukocidin, alpha-hemolysin, and toxic-shock syndrome toxin 1, in both methicillin-sensitive and methicillin-resistant S. aureus. Subinhibitory concentrations of nafcillin induced and prolonged mRNA for Panton-Valentine leukocidin, alpha-toxin, and toxic-shock syndrome toxin 1 and increased toxin production. In contrast, clindamycin and linezolid markedly suppressed translation, but not transcription, of toxin genes. These results suggest (1) that protein-synthesis inhibition is an important consideration in the selection of antimicrobial agents to treat serious infections caused by toxin-producing gram-positive pathogens and (2) that, by inducing and enhancing toxin production, inadvertent use of beta-lactam antibiotics to treat methicillin-resistant S. aureus infections may contribute to worse outcomes.

Virulence studies on chromosomal alpha-toxin and theta-toxin mutants constructed by allelic exchange provide genetic evidence for the essential role of alpha-toxin in Clostridium perfringens-mediated gas gangrene.

The pathogenesis of clostridial myonecrosis, or gas gangrene, involves the growth of the anaerobic bacterium Clostridium perfringens in the infected tissues and the elaboration of numerous extracellular toxins and enzymes. The precise role of each of these toxins in tissue invasion and necrosis has not been determined. To enable genetic approaches to be used to study C. perfringens pathogenesis we developed an allelic exchange method which involved the transformation of C. perfringens cells with a suicide plasmid carrying a gene insertionally inactivated with an erythromycin‐resistance determinant. The frequency with which double reciprocal crossover events were observed was increased to a workable level by increasing the amount of homologous DNA located on either side of the inactivated gene. Allelic exchange was used to isolate mutations in the‘chromosomal pfoA gene, which encodes an oxygen‐labile haemolysin known as Θ‐toxin or perfringolysin O. and in the chromosomal pic gene, which encodes the α‐toxin or phospholipase C. The resultant mutants failed to produce detectable Θ‐toxin or α‐toxin activity, respectively, and could be complemented by recombinant plasmids that carried the respective wild‐type genes. The resultant strains were virulence tested in a mouse myonecrosis model. The results showed that the pic mutants had demonstrably reduced virulence and therefore provided definitive genetic evidence for the essential role of α‐toxin in gas gangrene or clostridial myonecrosis.

Identification and molecular analysis of a locus that regulates extracellular toxin production in Clostridium perfringens

The anaerobic bacterium Clostridium perfringens mediates clostridial myonecrosis, or gas gangrene, by producing a number of extracellular toxins and enzymes. Transposon mutagenesis with Tn916 was used to isolate a pleiotropic mutant of C. perfringens that produced reduced levels of phospholipase C, protease and sialidase, and did not produce any detectable perfringolysin O activity. Southern hybridization revealed that a single copy of Tn916 had inserted into a 2.7 kb Hindlll fragment in the C. perfringens chromosome. A 4.3 kb Pstl fragment, which spanned the Tn916 insertion site, was cloned from the wild‐type strain. When subcloned into a shuttle vector and introduced into C. perfringens this fragment was able to complement the Tn916‐derived mutation. Transformation of the mutant with plasmids containing the 2.7 kb Hindlll fragment, or the 4.3 kb Pstl fragment resulted in toxin and enzyme levels greater than or equal to those of the wild‐type strain. The Pstl fragment was sequenced and found to potentially encode seven open reading frames, two of which appeared to be arranged in an operon and shared sequence similarity with members of two‐component signal transduction systems. The putative virR gene encoded a protein with a deduced molecular weight of 30140, and with sequence similarity to activators in the response regulator family of proteins. The next gene, virS, into which Tn916 had inserted, was predicted to encode a membrane‐spanning protein with a deduced molecular weight of 51 274. The putative VirS protein had sequence similarity to sensor proteins and also contained a histidine residue highly conserved in the histidine protein kinase family of sensor proteins. Virulence studies carried out using a mouse model implicated the virS gene in the pathogenesis of histotoxic C. perfringens infections. It was concluded that a two‐component sensor regulator system that activated the expression of a number of extracellular toxins and enzymes involved In virulence had been cloned and sequenced. A model that described the regulation of extracellular toxin production in C. perfringens was constructed.

Clostridial Gas Gangrene: Evidence That α and θ Toxins Differentially Modulate the Immune Response and Induce Acute Tissue Necrosis

: The rapid extension of necrosis and an absence of polymorphonuclear leukocytes (PMNL) at the site of infection are two hallmarks of Clostridium perfringens gas gangrene. While both alpha and theta toxins profoundly affect PMNL function and viability in vitro, their roles in muscle destruction and impairment of the inflammatory response in vivo have not been investigated. Comparative histopathologic examinations were performed on animals infected with either wild-type C. perfringens, or isogenic, toxin-deficient mutants of C. perfringens. Tissue destruction was modest in animals infected with the alpha toxin-deficient mutant; destruction was more pronounced in tissues infected with the theta toxin-deficient mutant or the wild-type strain. alpha and theta toxins also displayed differing abilities to modulate the inflammatory response. Histopathologic studies in which recombinant toxins were injected together with killed, washed C. perfringens further substantiated these tissue-destructive and differential antiinflammatory effects.

Life-threatening clostridial infections.

Life-threatening soft tissue infections caused by Clostridium species have been described in the medical literature for hundreds of years largely because of their fulminant nature, distinctive clinical presentations and complex management issues. The Clostridium species perfringens, septicum and histolyticum are the principal causes of trauma-associated gas gangrene and their incidence increases dramatically in times of war, hurricanes, earthquakes and other mass casualty conditions. Recently, there has also been an increased incidence of spontaneous gas gangrene caused by Clostridium septicum in association with gastrointestinal abnormalities and neutropenia. Similarly, over the last 15 years there has been increased recognition of a toxic shock-like syndrome associated with Clostridium sordellii in individuals skin-popping black tar heroin, in women undergoing childbirth or other gynecologic procedures including medically-induced abortion. Like their cousins Clostridium tetanus and Clostridium botulinum, the pathogenesis of these clostridial infections is largely the consequence of potent exotoxin production. Strategies to inhibit toxin production, neutralize circulating toxins and prevent their interaction with cells of the innate immune response are sorely needed. Recent studies have elucidated novel targets that may hold promise for newer therapeutic modalities.

Group A Streptococcal Myonecrosis: Increased Vimentin Expression after Skeletal-Muscle Injury Mediates the Binding of Streptococcus pyogenes

Necrotizing fasciitis and myonecrosis caused by invasive infection with group A streptococci (GAS) are life-threatening conditions that have reemerged worldwide. Half of all GAS myonecrosis cases have no known portal of entry; yet, for unknown reasons, infection becomes established precisely at the site of a prior, nonpenetrating minor injury, such as a muscle strain. We hypothesized that GAS establishes infection by binding to surface molecules that are up-regulated on injured skeletal-muscle cells. Here, we isolated and identified vimentin as the major skeletal-muscle GAS-binding protein. Furthermore, we found that vimentin expression was up-regulated on injured skeletal-muscle cells in vitro and was expressed in muscle tissues from a patient with GAS myonecrosis who died of streptococcal toxic shock syndrome. These findings provide a molecular mechanism to explain the development of severe GAS soft-tissue infections at the sites of prior minor muscle trauma. This understanding may provide a basis for novel preventive strategies or therapies for patients with this devastating infection.

Immunization with the C-domain of α-toxin prevents lethal infection, localizes tissue injury, and promotes host response to challenge with Clostridium perfringens

Clostridium perfringens gas gangrene is characterized by rapid tissue destruction, impaired host response, and, often, death. Phospholipase C (alpha -toxin) is the virulence factor most responsible for these pathologies. The present study investigated the efficacy of active immunization with the C-terminal domain of alpha -toxin (Cpa247-370) in a murine model of gas gangrene. Primary end points of the study were survival, progression of infection, and tissue perfusion. Secondary end points, which were based on findings of histologic evaluation of tissues, included the extent of tissue destruction and microvascular thrombosis, as well as the magnitude of the tissue inflammatory response. Survival among C-domain-immunized animals was significantly greater than that among sham-immunized control animals. Furthermore, immunization with the C-domain localized the infection and prevented ischemia of the feet. Histopathologic findings demonstrated limited muscle necrosis, reduced microvascular thrombosis, and enhanced granulocytic influx in C-domain-immunized mice. We conclude that immunization with the C-domain of phospholipase C is a viable strategy for the prevention of morbidity and mortality associated with C. perfringens gas gangrene.

Alpha toxin from Clostridium perfringens induces proinflammatory changes in endothelial cells.

Alpha toxin from Clostridium perfringens type A, a phospholipase C, has been implicated in many of the localized and systemic features of gas gangrene. We demonstrated that human endothelial cells synthesize two vasoactive lipids, platelet-activating factor (PAF) and prostacyclin, in response to alpha toxin treatment. The stimulated synthesis of PAF required the enzymatic activity of the toxin and subsequent protein kinase C activation. Alpha toxin-treated endothelial cells accumulated the products of the phospholipase C reaction, diacylglycerol and ceramide, and exhibited a decrease in the enzymatic precursors phosphatidylcholine and sphingomyelin. Furthermore, the temporal accumulation of PAF depended on the concentration of the toxin in the overlying medium and was blocked in the presence of a neutralizing antibody. The cultured endothelial cells also exhibited enhanced neutrophil adhesion in response to alpha toxin which was mediated through the PAF receptor and P-selectin. P-selectin expression by endothelial cells and extravascular neutrophil accumulation were also observed in tissue sections from alpha toxin-injected Sprague-Dawley rats. These endothelial cell-mediated processes are important in maintaining vascular homeostasis and, when activated in a dysregulated manner by C. perfringens alpha toxin, may contribute to localized and systemic manifestations of gas gangrene including enhanced vascular permeability, localized neutrophil accumulation, and myocardial dysfunction.

Clostridial Gas Gangrene. II. Phospholipase C—Induced Activation of Platelet gpIIbIIIa Mediates Vascular Occlusion and Myonecrosis in Clostvidium perfringens Gas Gangrene

Clostridium perfringens gas gangrene is a fulminant infection, and radical amputation remains the single best treatment. It has been hypothesized that rapid tissue destruction is related to tissue hypoxia secondary to toxin-induced vascular obstruction, and previous studies demonstrated that phospholipase C (PLC) caused a rapid and irreversible decrease in skeletal muscle blood flow that paralleled the formation of intravascular aggregates of activated platelets, fibrin, and leukocytes. In this study, flow cytometry demonstrated that PLC stimulated platelet/neutrophil aggregation in a gpIIbIIIa-dependent fashion. Pretreatment of animals with heparin or depletion of leukocytes reduced blood-flow deficits, and aggregate formation caused by PLC. It is concluded that fulminant tissue destruction in gas gangrene results from profound attenuation of blood flow caused by PLC-induced, gpIIbIIIa-mediated formation of heterotypic platelet/polymorphonuclear leukocyte aggregates. Therapeutic strategies that target gpIIbIIIa may prevent vascular occlusion, maintain tissue viability, and provide an alternative to radical amputation for patients with this infection.

In Vitro Production of Panton-Valentine Leukocidin among Strains of Methicillin-Resistant Staphylococcus aureus Causing Diverse Infections

BACKGROUND Community-acquired methicillin-resistant Staphylococcus aureus strains have recently been associated with severe necrotizing infections. Greater than 75% of these strains carry the genes for Panton-Valentine leukocidin (PVL), suggesting that this toxin may mediate these severe infections. However, to date, studies have not provided evidence of toxin production. METHODS Twenty-nine community-acquired methicillin-resistant Staphylococcus aureus and 2 community-acquired methicillin-susceptible S. aureus strains were collected from patients with infections of varying severity. Strains were analyzed for the presence of lukF-PV and SCCmecA type. PVL production in lukF-PV gene-positive strains was measured by ELISA, and the amount produced was analyzed relative to severity of infection. RESULTS Only 2 of the 31 strains tested, 1 methicillin-resistant Staphylococcus aureus abscess isolate and 1 nasal carriage methicillin-susceptible S. aureus isolate, were lukF-PV negative. All methicillin-resistant Staphylococcus aureus strains were SCCmec type IV. PVL was produced by all strains harboring lukF-PV, although a marked strain-to-strain variation was observed. Twenty-six (90%) of 29 strains produced 50-350 ng/mL of PVL; the remaining strains produced PVL in excess of 500 ng/mL. The quantity of PVL produced in vitro did not correlate with severity of infection. CONCLUSIONS Although PVL likely plays an important role in the pathogenesis of these infections, its mere presence is not solely responsible for the increased severity. Factors that up-regulate toxin synthesis in vivo could contribute to more-severe disease and worse outcomes in patients with community-acquired methicillin-resistant Staphylococcus aureus infection.