Characterization of Paeniclostridium sordellii Metalloproteinase-1 in vitro and in an experimental model of infection.

OBJECTIVE: Paeniclostridium sordellii is a pathogen that causes rapidly fatal infections characterized by severe edema, extreme leukemoid reaction and lack of an innate immune response. We recently identified a metalloproteinase of P. sordellii-1 (Mcs1) that cleaves human vascular cell adhesion molecule 1, an adhesion molecule important to hematopoietic precursor retention and leukocyte diapedesis. In the current study, we further characterize Mcs1 activity and investigate its role in pathogenesis. METHODS: Mcs1 peptide cleavage sequence and activity conditions were identified using a semi-quantitative fluorescence-based reporter assay. Additional host targets for Mcs1 protease activity were tested and confirmed by gel electrophoreses and western blots. Finally, Mcs1 knock out (ΔMcs1) and complemented (cMcs1) strains were developed for assessment in our animal model of myonecrosis. RESULTS: Data show that Mcs1 prefers aliphatic amino acid residues, I or L, especially when adjacent to negatively charged or noncharged-polar residues. In vitro, Mcs1 cleaved or partially cleaved human cell adhesion molecules, E-selectin and intracellular adhesion molecule-1 (ICAM-1), and mediators of innate immune infection defense, complement protein-3 and antimicrobial peptide LL-37. In vivo, infection with the ΔMcs1 P. sordellii strain had little effect on animal survival, tissue destruction or circulating white blood cell counts compared to wild type and cMcs1 strains. CONCLUSIONS: Similar to proteolytic virulence factors from other pathogens, Mcs1 is a promiscuous protease that cleaves multiple human-host factors. Despite minimal impact of Mcs1 on the murine model of P. sordellii infection, it is worth considering its role in humans and other animal models.

Emerging erythromycin and clindamycin resistance in group A streptococci: Efficacy of linezolid and tedizolid in experimental necrotizing infection.

OBJECTIVES: Clindamycin (CLI) and erythromycin (ERY) resistance is increasing among group A streptococci (GAS) causing invasive disease and alternative treatments are urgently required. In this study, the efficacy of the newer oxazolidinone tedizolid (TZD) was compared with the first drug in this class, linezolid (LNZ), in experimental murine myonecrosis caused by ERY-susceptible/CLI-susceptible (ERYS/CLIS) or ERY- resistant/CLI-resistant (ERYR/CLIR) GAS. METHODS: Normal adult outbred Swiss Webster female mice (10 per group) were infected intramuscularly with ERYS/CLIS (ATCC 12384) or ERYR/CLIR (15-003) GAS. Treatments began 4 h post-infection and continued for 72 h. TZD and LNZ (10, 20 and 40 mg/kg) were given intraperitoneally every 12 h. Saline, penicillin (PEN), CLI and ERY were given every 6 h. Survival and infection severity signs and symptoms were followed for 12 days. RESULTS: Both GAS strains were susceptible to LNZ, TZD and PEN; strain 15-003 was confirmed as constitutively resistant to ERY and CLI. Blood levels following a 40 mg/kg dose of LZD and TZD were 30.9 ± 4.0 µg/mL and 21.9 ± 5.3 µg/mL, respectively. Both TZD and LNZ were highly efficacious for the treatment of severe experimental myonecrosis caused by ERYS/CLIS and, importantly, ERYR/CLIR GAS. CONCLUSION: In the current era of emerging macrolide/lincosamide resistance among GAS, these data support the use of TZD and LNZ as first-line antibiotics for the treatment of life-threatening GAS infections in humans.

Fidaxomicin reduces early toxin A and B production and sporulation in Clostridium difficilein vitro.

PURPOSE: Fidaxomicin, a macrocyclic antibiotic, has been approved for the treatment of Clostridium difficile infection (CDI). Previous work by our group has demonstrated that some antibiotics at sub-inhibitory concentrations stimulate early toxin production and sporulation by C. difficile. Prior studies revealed that fidaxomicin, when added to late stationary-phase organisms, reduced exotoxin production and spore formation by C. difficile. However, the ability of fidaxomicin to trigger early virulence factor production and spore formation has never been investigated. METHODOLOGY: Sub-inhibitory concentrations of the RNA synthesis inhibitor fidaxomicin (1/4×, 1/8×, 1/16× MIC) were added immediately to lag-phase cultures of historical (strain 9689) and epidemic BI/NAP1/027 (strain 5325) strains of C. difficile, and their effects on sporulation and toxin A (TcdA) and toxin B (TcdB) production were compared.Results/Key findings. Even at sub-inhibitory concentrations, all doses of fidaxomicin reduced both TcdA and TcdB gene expression and protein production in the historical and epidemic C. difficile strains. Fidaxomicin also dose-dependently reduced viable spore production by the 9689 and 5325 strains. Reductions in spore formation were also observed in both strains treated with tigecycline and vancomycin. However, all concentrations of metronidazole stimulated a ~2 log increase in spore production by the 5325 isolate. CONCLUSION: The ability of fidaxomicin to suppress early exotoxin production and endospore formation by historical and epidemic strains of C. difficile may explain its clinical success in treating severe and recurrent cases of CDI disease.

Spontaneous C. septicum gas gangrene: A literature review.

As the infectious disease paradigm undergoes a subtle shift, unusual infections associated with malignancy and immunosuppression are being increasingly reported. Spontaneous or non-traumatic Clostridium septicum infection is one such unusual infection which has gained prominence. This article aims to understand the pathophysiology, clinical manifestations and current trends in diagnosing and treating this rare but deadly infection. To understand the multifactorial causation of this infection a review of published cases of spontaneous C. septicum gas gangrene was performed and a total of 94 such cases were identified. Several factors were analyzed for each case: age, infection location and underlying illness, presenting signs and symptoms, neutropenia, gross pathology of the colon, antibiotic use, surgical intervention, and survival. A known or occult malignancy was present in 71% patients and an overall mortality of 67% was observed.

Experimental identification and computational characterization of a novel extracellular metalloproteinase produced by Clostridium sordellii.

Clostridium sordellii is a lethal pathogen for both animals and humans. Severe capillary leakage, toxic shock syndrome, and an extreme leukemoid reaction (LR), are hallmark features of C. sordellii infections and contribute to its high mortality rate. Here we report the discovery of a previously unknown and uncharacterized metalloproteinase of C. sordellii (referred as Mcs1) that cleaves human vascular cell adhesion molecule (VCAM)-1 in vitro, an adhesion molecule critical to hematopoietic precursor retention and leukocyte diapedesis. We successfully identified the open reading frame encoding Mcs1 within the ATCC 9714 genome and developed an Δmcs1 mutant strain using the ClosTron mutagenesis technology. No VCAM-1 proteolysis was observed from exotoxins collected from mutant strain cultures. Using advanced protein structural modeling and molecular dynamics simulation techniques, the 3D molecular structure and conformational features of Mcs1 were also characterized. Our data demonstrates that Mcs1 proteolytic activity is controlled by the electrostatic interactions between Glu113 and Arg227 residues and the gating motions within its cleft region. This pilot interdisciplinary investigation provided crucial experimental evidence of the existence of Mcs1 in C. sordellii and molecular insights into its 3D structure and proteolytic activity. These findings have the potential to help advance new therapeutics and diagnostics against deadly C. sordellii infections. Follow-up in vitro and in vivo work is under way to further characterize Mcs1 enzymatic kinetics and its role in C. sordellii pathogenesis.

Effects of delayed NSAID administration after experimental eccentric contraction injury - A cellular and proteomics study.

BACKGROUND: Acute muscle injuries are exceedingly common and non-steroidal anti-inflammatory drugs (NSAIDs) are widely consumed to reduce the associated inflammation, swelling and pain that peak 1-2 days post-injury. While prophylactic use or early administration of NSAIDs has been shown to delay muscle regeneration and contribute to loss of muscle strength after healing, little is known about the effects of delayed NSAID use. Further, NSAID use following non-penetrating injury has been associated with increased risk and severity of infection, including that due to group A streptococcus, though the mechanisms remain to be elucidated. The present study investigated the effects of delayed NSAID administration on muscle repair and sought mechanisms supporting an injury/NSAID/infection axis. METHODS: A murine model of eccentric contraction (EC)-induced injury of the tibialis anterior muscle was used to profile the cellular and molecular changes induced by ketorolac tromethamine administered 47 hr post injury. RESULTS: NSAID administration inhibited several important muscle regeneration processes and down-regulated multiple cytoprotective proteins known to inhibit the intrinsic pathway of programmed cell death. These activities were associated with increased caspase activity in injured muscles but were independent of any NSAID effect on macrophage influx or phenotype switching. CONCLUSIONS: These findings provide new molecular evidence supporting the notion that NSAIDs have a direct negative influence on muscle repair after acute strain injury in mice and thus add to renewed concern about the safety and benefits of NSAIDS in both children and adults, in those with progressive loss of muscle mass such as the elderly or patients with cancer or AIDS, and those at risk of secondary infection after trauma or surgery.

A novel murine model of Clostridium sordellii myonecrosis: Insights into the pathogenesis of disease.

Clostridium sordellii infections have been reported in women following natural childbirth and spontaneous or medically-induced abortion, injection drug users and patients with trauma. Death is rapid and mortality ranges from 70 to 100%. Clinical features include an extreme leukemoid reaction, the absence of fever, and only minimal pain or erythema at the infected site. In the current study, we developed a murine model of C. sordellii soft tissue infection to elucidate the pathogenic mechanisms. Mice received 0.5, 1.0 or 2.0 × 10(6) CFU C. sordellii (ATCC 9714 type strain) in the right thigh muscle. All doses caused fatal infection characterized by intense swelling of the infected limb but no erythema or visible perfusion deficits. Survival rates and time to death were inoculum dose-dependent. Mice developed a granulocytic leukocytosis with left shift, the onset of which directly correlated with disease severity. Histopathology of infected tissue showed widespread edema, moderate muscle damage and minimal neutrophil infiltration. Circulating levels of granulocyte colony-stimulating factor (G-CSF), soluble tumor necrosis factor receptor I (sTNF-RI) and interlukin-6 (IL-6) were significantly increased in infected animals, while TNF-α, and IL-1ß levels were only mildly elevated, suggesting these host factors likely mediate the leukocytosis and innate immune dysfunction characteristic of this infection. Thus, this model mimics many of the salient features of this infection in humans and has allowed us to identify novel targets for intervention.

The roles of injury and nonsteroidal anti-inflammatory drugs in the development and outcomes of severe group A streptococcal soft tissue infections.

PURPOSE OF REVIEW: This review summarizes clinical and basic science evidence linking trauma and nonsteroidal anti-inflammatory drug (NSAID) use to initiation and progression of severe group A streptococcal (GAS) soft tissue infection. RECENT FINDINGS: New evidence includes recent clinical series and controlled studies that lend support to an NSAID/GAS association, basic science studies that demonstrate unique roles for nonpenetrating injury and NSAID administration in initiation of cryptogenic GAS infection and experimental studies showing that nonselective NSAIDs accelerate disease progression and limit antibiotic efficacy in established GAS soft tissue infections. Potential mechanisms for these processes are discussed. SUMMARY: NSAIDs are important anti-inflammatory and analgesic drugs; however, new experimental data suggest that nonselective NSAIDs do more than simply mask the signs and symptoms of developing GAS infection. A more thorough understanding of the triadic interplay of injury-triggered immune signaling, GAS soft tissue infection and NSAIDs is of significant clinical importance and could shift the current paradigm of pain management to avert the consequences of such devastating infections.

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.

Identification and characterization of bicistronic speB and prsA gene expression in the group A Streptococcus.

Severe, invasive group A streptococcal infections have reemerged worldwide, and extracellular toxins, including streptococcal pyrogenic exotoxin B (SpeB), have been implicated in pathogenesis. The genetic regulation of SpeB is not fully understood, and the mechanisms involved in the processing of the protoxin to its enzymatically active form have not been definitively established. The present work demonstrated that the genes encoding SpeB (speB) and a peptidyl-prolyl isomerase (prsA) constitute an operon with transcription initiated from two promoters upstream of speB. Further, the speB-prsA operon was transcribed as a bicistronic mRNA. This finding is in contrast to the generally accepted notion that speB is transcribed only as a monocistronic gene. In addition, prsA has its own promoter, and transcription from this promoter starts in early log phase, prior to the transcription of speB. Genomic disruption of prsA decreased the production of enzymatically active SpeB but not the level of the pro-SpeB zymogen. Taken together, these results demonstrate that prsA is required for production of fully mature, enzymatically active SpeB.