A series of three cases of severe Clostridium difficile infection in Australia associated with a binary toxin producing clade 2 ribotype 251 strain

Three patients with severe Clostridium difficile infection (CDI) caused by an unusual strain of C. difficile, PCR ribotype (RT) 251, were identified in New South Wales, Australia. All cases presented with severe diarrhoea, two had multiple recurrences and one died following a colectomy. C. difficile RT251 strains were isolated by toxigenic culture. Genetic characterisation was performed using techniques including toxin gene profiling, PCR ribotyping, whole genome sequencing (WGS), in-silico multi-locus-sequence-typing (MLST) and core-genome single nucleotide variant (SNV) analyses. Antimicrobial susceptibility was determined using an agar incorporation method. In vitro toxin production was confirmed by Vero cell cytotoxicity assay and pathogenicity was assessed in a murine model of CDI. All RT251 isolates contained toxin A (tcdA), toxin B (tcdB) and binary toxin (cdtA and cdtB) genes. Core-genome analyses revealed the RT251 strains were clonal, with 0–5 SNVs between isolates. WGS and MLST clustered RT251 in the same evolutionary clade (clade 2) as RT027. Despite comparatively lower levels of in vitro toxin production, in the murine model RT251 infection resembled RT027 infection. Mice showed marked weight loss, severe disease within 48 h post-infection and death. All isolates were susceptible to metronidazole and vancomycin. Our observations suggest C. difficile RT251 causes severe disease and emphasise the importance of ongoing surveillance for new and emerging strains of C. difficile with enhanced virulence

Clinical and laboratory features of invasive community-onset methicillin-resistant Staphylococcus aureus infection: a prospective case–control study

Differences between the features of invasive community-onset methicillin-resistant Staphylococcus aureus (cMRSA) and methicillin-susceptible S. aureus (cMSSA) infections are incompletely understood. Fifty-seven patients with invasive cMRSA infection were prospectively identified at two teaching hospitals; for each cMRSA case, two cases of invasive cMSSA infection acted as controls. The primary outcome was 30-day all-cause mortality. Patients with invasive cMRSA infection were more likely to be Aboriginal (25% vs. 14%, age-adjusted odds ratio [OR] 2.5, p=0.037), reside in a long-term care facility and/or have been hospitalised in the previous year (51% vs. 34%, p=0.04) and less likely to have endocarditis (2% vs. 12%, p=0.02) or require admission to an intensive care unit or high-dependency area (7% vs. 21%, p=0.02). All-cause mortality at 30 days was similar in the cMRSA and cMSSA groups (9% vs. 7%, p=0.68). Panton-Valentine leukocidin (PVL) genes were detected in a similar proportion of cMRSA and cMSSA isolates (32% vs. 27%, p=0.49) and the presence of PVL genes was associated with younger age (35 years vs. 55 years, p<0.001), Aboriginal ethnicity (38% vs. 10%, p<0.001), skin and soft-tissue infection (54% vs. 19%, p<0.001), lower illness severity at presentation (SAPS II score 9 vs. 21, p=0.001) and shorter hospitalisation (9 days vs. 24 days, p<0.001). Patients with "PVL-positive" and "PVL-negative" S. aureus infection had similar 30-day all-cause mortality (4% vs. 9%, p=0.28). Few clinical features differentiated patients with invasive cMRSA infection from those with infection caused by cMSSA. Invasive "PVL-positive" S. aureus infection was associated with less morbidity but similar mortality to "PVL-negative" infection

Surveillance for antimicrobial resistance in Australian isolates of Clostridium difficile, 2013–14

Objectives: The objective of this study was to determine the activity of fidaxomicin and comparator antimicrobials against Clostridium difficile isolated from patients with C. difficile infection (CDI) in Australian hospitals and in the community. Methods: One private and one public laboratory from five states in Australia submitted a total of 474 isolates/PCR-positive stool samples during three collection periods in August-September 2013 (n=175), February-March 2014 (n=134) and August-September 2014 (n=165). Isolate identification was confirmed by selective culture for C. difficile and a proportion of isolates from each state were characterized by PCR for toxin genes and PCR ribotyping. MICs of fidaxomicin and eight comparator antimicrobials were determined for all isolates using agar methodology. Results: Site collection yielded 440 isolates of C. difficile and PCR revealed a heterogeneous strain population comprising 37 different PCR ribotypes (RTs), 95% of whichwere positive for tcdA and tcdB (A+B+). The most common RTs were 014 (29.8%) and 002 (15.9%). Epidemic RT 027 was not identified; however, small numbers of virulent RTs 078 and 244 were found. Resistance to vancomycin, metronidazole and fidaxomicin was not detected and resistance to moxifloxacin was very low (3.4%). Fidaxomicin showed potent in vitro activity against all 440 isolates (MIC50/MIC90 0.03/0.12 mg/L) and was superior to metronidazole (MIC50/MIC90 0.25/0.5 mg/L) and vancomycin (MIC50/MIC90 1/2 mg/L). Conclusions: These data confirm the potent in vitro activity of fidaxomicin against C. difficile. Moreover, this study provides an important baseline for ongoing long-term surveillance of antimicrobial resistance and prospective tracking of prominent and emerging strain types

Laboratory-based surveillance of Clostridium difficile Infection in Australian health care and community settings, 2013 to 2018

In the early 2000s, a binary toxin (CDT)-producing strain of Clostridium difficile, ribotype 027 (RT027), caused extensive outbreaks of diarrheal disease in North America and Europe. This strain has not become established in Australia, and there is a markedly different repertoire of circulating strains there compared to other regions of the world. The C. difficile Antimicrobial Resistance Surveillance (CDARS) study is a nationwide longitudinal surveillance study of C. difficile infection (CDI) in Australia. Here, we describe the molecular epidemiology of CDI in Australian health care and community settings over the first 5 years of the study, 2013 to 2018. Between 2013 and 2018, 10 diagnostic microbiology laboratories from five states in Australia participated in the CDARS study. From each of five states, one private (representing community) and one public (representing hospitals) laboratory submitted isolates of C. difficile or PCR-positive stool samples during two collection periods per year, February-March (summer/autumn) and August-September (winter/spring). C. difficile was characterized by toxin gene profiling and ribotyping. A total of 1,523 isolates of C. difficile were studied. PCR ribotyping yielded 203 different RTs, the most prevalent being RT014/020 (n = 449; 29.5%). The epidemic CDT+ RT027 (n = 2) and RT078 (n = 6), and the recently described RT251 (n = 10) and RT244 (n = 6) were not common, while RT126 (n = 17) was the most prevalent CDT+ type. A heterogeneous C. difficile population was identified. C. difficile RT014/020 was the most prevalent type found in humans with CDI. Continued surveillance of CDI in Australia remains critical for the detection of emerging strain lineages

Expression Suppression and Activity Inhibition of TRPM7 Regulate Cytokine Production and Multiple Organ Dysfunction Syndrome During Endotoxemia: A New Target for Sepsis

Background: Main pathological features detected during sepsis and endotoxemia include over-secretion of pro-inflammatory cytokines and multiorgan dysfunction syndrome (MODS). Unfortunately, current clinical efforts to treat sepsis are unsatisfactory, and mortality remains high. Interestingly, transient receptor potential (TRP) melastatin 7 (TRPM7) ion channel controlling Ca2+ and Mg2+ permeability is involved in cytokine production and inflammatory response. Furthermore, TRPM7 downregulation has been shown to alleviate local symptoms in some models of sepsis, but its effects at a systemic level remain to be explored. Objective: To test whether TRPM7 mediates cytokine production and MODS during endotoxemia. Methods: Endotoxemic and sham-endotoxemic rats were subjected to pharmacological inhibition of TRPM7 using carvacrol, or to expression suppression by adenovirus delivery of shRNA (AdV(shTRPM7)). Then, cytokine and MODS levels in the blood were measured. Results: Inhibition of TRPM7 with carvacrol and suppression with AdV(shTRPM7 )were both efficient in inhibiting the over-secretion of pro-inflammatory cytokines TNF-alpha, IL-1 beta, IL-6, and IL-12 in endotoxemic rats, without inducing downregulation in blood levels of anti-inflammatory cytokines IL-10 and IL-4. Additionally, the use of carvacrol and AdV(shTRPM7) significantly prevented liver and pancreas dysfunction, altered metabolic function, and hypoglycemia, induced by endotoxemia. Furthermore, muscle mass wasting and cardiac muscle damage were also significantly reduced by the use of carvacrol and AdV(shTRPM7) in endotoxemic rats. Conclusion: Our results indicate TRPM7 ion channel as a key protein regulating inflammatory responses and MODS during sepsis. Moreover, TRPM7 appears as a novel molecular target for the management of sepsis