Defining the phylogenetics and resistome of the major Clostridioides difficile ribotypes circulating in Australia.

Clostridioides difficile infection (CDI) remains a significant public health threat globally. New interventions to treat CDI rely on an understanding of the evolution and epidemiology of circulating strains. Here we provide longitudinal genomic data on strain diversity, transmission dynamics and antimicrobial resistance (AMR) of C. difficile ribotypes (RTs) 014/020 (n=169), 002 (n=77) and 056 (n=36), the three most prominent C. difficile strains causing CDI in Australia. Genome scrutiny showed that AMR was uncommon in these lineages, with resistance-conferring alleles present in only 15/169 RT014/020 strains (8.9 %), 1/36 RT056 strains (2.78 %) and none of 77 RT002 strains. Notably, ~90 % of strains were resistant to MLSB agents in vitro, but only ~5.9 % harboured known resistance alleles, highlighting an incongruence between AMR genotype and phenotype. Core genome analyses revealed all three RTs contained genetically heterogeneous strain populations with limited evidence of clonal transmission between CDI cases. The average number of pairwise core genome SNP (cgSNP) differences within each RT group ranged from 23.3 (RT056, ST34, n=36) to 115.6 (RT002, ST8, n=77) and 315.9 (RT014/020, STs 2, 13, 14, 49, n=169). Just 19 clonal groups (encompassing 40 isolates), defined as isolates differing by ≤2 cgSNPs, were identified across all three RTs (RT014/020, n=14; RT002, n=3; RT056, n=2). Of these clonal groups, 63 % (12/19) comprised isolates from the same Australian State and 37 % (7/19) comprised isolates from different States. The low number of plausible transmission events found for these major RTs (and previously documented populations in animal and environmental sources/reservoirs) points to widespread and persistent community sources of diverse C. difficile strains as opposed to ongoing nationwide healthcare outbreaks dominated by a single clone. Together, these data provide new insights into the evolution of major lineages causing CDI in Australia and highlight the urgent need for enhanced surveillance, and for public health interventions to move beyond the healthcare setting and into a One Health paradigm to effectively combat this complex pathogen.

Genomic epidemiology and transmission dynamics of recurrent Clostridioides difficile infection in Western Australia.

Recurrent cases of Clostridioides difficile infection (rCDI) remain one of the most common and serious challenges faced in the management of CDI. The accurate distinction between a relapse (caused by infection with the same strain) and reinfection (caused by a new strain) has implications for infection control and prevention, and patient therapy. Here, we used whole-genome sequencing to investigate the epidemiology of 94 C. difficile isolates from 38 patients with rCDI in Western Australia. The C. difficile strain population comprised 13 sequence types (STs) led by ST2 (PCR ribotype (RT) 014, 36.2%), ST8 (RT002, 19.1%) and ST34 (RT056, 11.7%). Among 38 patients, core genome SNP (cgSNP) typing found 27 strains (71%) from initial and recurring cases differed by ≤ 2 cgSNPs, suggesting a likely relapse of infection with the initial strain, while eight strains differed by ≥ 3 cgSNPs, suggesting reinfection. Almost half of patients with CDI relapse confirmed by WGS suffered episodes that occurred outside the widely used 8-week cut-off for defining rCDI. Several putative strain transmission events between epidemiologically unrelated patients were identified. Isolates of STs 2 and 34 from rCDI cases and environmental sources shared a recent evolutionary history, suggesting a possible common community reservoir. For some rCDI episodes caused by STs 2 and 231, within-host strain diversity was observed, characterised by loss/gain of moxifloxacin resistance. Genomics improves discrimination of relapse from reinfection and identifies putative strain transmission events among patients with rCDI. Current definitions of relapse and reinfection based on the timing of recurrence need to be reconsidered.

Spore-Forming Clostridium (Clostridioides) difficile in Wastewater Treatment Plants in Western Australia.

There is growing evidence that shows Clostridium (Clostridioides) difficile is a pathogen of One Health importance with a complex dissemination pathway involving animals, humans, and the environment. Thus, environmental discharge and agricultural recycling of human and animal waste have been suspected as factors behind the dissemination of Clostridium difficile in the community. Here, the presence of C. difficile in 12 wastewater treatment plants (WWTPs) in Western Australia was investigated. Overall, C. difficile was found in 90.5% (114/126) of raw sewage influent, 48.1% (50/104) of treated effluent, 40% (2/5) of reclaimed irrigation water, 100% (38/38) of untreated biosolids, 95.2% (20/21) of anaerobically digested biosolids, and 72.7% (8/11) of lime-amended biosolids. Over half of the isolates (55.3% [157/284]) were toxigenic, and 97 C. difficile ribotypes (RTs) were identified, with RT014/020 the most common (14.8% [42/284]). Thirteen C. difficile isolates with the toxin gene profile A+ B+ CDT+ (positive for genes coding for toxins A and B and the binary C. difficile transferase toxin [CDT]) were found, including the hypervirulent RT078 strain. Resistance to the antimicrobials fidaxomicin, vancomycin, metronidazole, rifaximin, amoxicillin-clavulanate, meropenem, and moxifloxacin was uncommon; however, resistance to clindamycin, erythromycin, and tetracycline was relatively frequent at 56.7% (161/284), 14.4% (41/284), and 13.7% (39/284), respectively. This study revealed that toxigenic C. difficile was commonly encountered in WWTPs and being released into the environment. This raises concern about the possible spillover of C. difficile into animal and/or human populations via land receiving the treated waste. In Western Australia, stringent measures are in place to mitigate the health and environmental risk of recycling human waste; however, further studies are needed to elucidate the public health significance of C. difficile surviving the treatment processes at WWTPs. IMPORTANCE Clostridium difficile infection (CDI) is a leading cause of antimicrobial-associated diarrhea in health care facilities. Extended hospital stays and recurrences increase the cost of treatment and morbidity and mortality. Community-associated CDI (CA-CDI) cases, with no history of antimicrobial use or exposure to health care settings, are increasing. The isolation of clinically important C. difficile strains from animals, rivers, soil, meat, vegetables, compost, treated wastewater, and biosolids has been reported. The objective of this study was to characterize C. difficile in wastewater treatment plants (WWTPs) in Australia. We found that C. difficile can survive the treatment processes of WWTPs, and toxigenic C. difficile was being released into the environment, becoming a potential source/reservoir for CA-CDI.

Activity of newest generation ß-lactam/ß-lactamase inhibitor combination therapies against multidrug resistant Pseudomonas aeruginosa.

Multidrug resistant (MDR) P. aeruginosa accounts for 35% of all P. aeruginosa isolated from respiratory samples of patients with cystic fibrosis (CF). The usefulness of ß-lactam antibiotics for treating CF, such as carbapenems and later generation cephalosporins, is limited by the development of antibacterial resistance. A proven treatment approach is the combination of a ß-lactam antibiotic with a ß-lactamase inhibitor. New ß-lactam/ß-lactamase inhibitor combinations are available, but data are lacking regarding the susceptibility of MDR CF-associated P. aeruginosa (CFPA) to these new combination therapies. In this study we determined MIC values for three new combinations; imipenem-relebactam (I-R), ceftazidime-avibactam (CZA), and ceftolozane-tazobactam (C/T) against MDR CFPA (n = 20). The MIC90 of I-R, CZA, and C/T was 64/4, 32/4, and 16/8 (all µg/mL), respectively. The susceptibility of isolates to imipenem was not significantly improved with the addition of relebactam (p = 0.68). However, susceptibility to ceftazidime was significantly improved with the addition of avibactam (p < 0.01), and the susceptibility to C/T was improved compared to piperacillin/tazobactam (p < 0.05) These data provide in vitro evidence that I-R may not be any more effective than imipenem monotherapy against MDR CFPA. The pattern of susceptibility observed for CZA and C/T in the current study was similar to data previously reported for non-CF-associated MDR P. aeruginosa.

Development of 1,2,4-Oxadiazole Antimicrobial Agents to Treat Enteric Pathogens within the Gastrointestinal Tract.

Colonization of the gastrointestinal (GI) tract with pathogenic bacteria is an important risk factor for the development of certain potentially severe and life-threatening healthcare-associated infections, yet efforts to develop effective decolonization agents have been largely unsuccessful thus far. Herein, we report modification of the 1,2,4-oxadiazole class of antimicrobial compounds with poorly permeable functional groups in order to target bacterial pathogens within the GI tract. We have identified that the quaternary ammonium functionality of analogue 26a results in complete impermeability in Caco-2 cell monolayers while retaining activity against GI pathogens Clostridioides difficile and multidrug-resistant (MDR) Enterococcus faecium. Low compound recovery levels after oral administration in rats were observed, which suggests that the analogues may be susceptible to degradation or metabolism within the gut, highlighting a key area for optimization in future efforts. This study demonstrates that modified analogues of the 1,2,4-oxadiazole class may be potential leads for further development of colon-targeted antimicrobial agents.

Genetically related Clostridium difficile from water sources and human CDI cases revealed by whole-genome sequencing.

Clostridium difficile isolates from the environment are closely related to those from humans, indicating a possible environmental transmission route for C. difficile infection (CDI). In this study, C. difficile was isolated from 47.3% (53/112) of lake/pond, 23.0% (14/61) of river, 20.0% (3/15) of estuary and 0.0% (0/89) of seawater samples. The most common toxigenic strain isolated was C. difficile PCR ribotype (RT) 014/020 (10.5%, 8/76). All water isolates were susceptible to fidaxomicin, metronidazole, rifaximin, amoxicillin/clavulanic acid, moxifloxacin and tetracycline. Resistance to vancomycin, clindamycin, erythromycin and meropenem was detected in 5.3% (4/76), 26.3% (20/76), 1.3% (1/76) and 6.6% (5/76) of isolates, respectively. High-resolution core-genome analysis was performed on RT 014/020 isolates of water origin and 26 clinical RT 014/020 isolates from the same year and geographical location. Notably, both human and water strains were intermixed across three sequence types (STs), 2, 13 and 49. Six closely related groups with ≤10 core-genome single nucleotide polymorphisms were identified, five of which comprised human and water strains. Overall, 19.2% (5/26) of human strains shared a recent genomic relationship with one or more water strains. This study supports the growing hypothesis that environmental contamination by C. difficile plays a role in CDI transmission.

Whole-genome sequencing links Clostridium (Clostridioides) difficile in a single hospital to diverse environmental sources in the community.

AIMS: To investigate if Clostridium (Clostridioides) difficile infection (CDI), traditionally thought of as hospital-acquired, can be genomically linked to hospital or community environmental sources, and to define possible importation routes from the community to the hospital. METHODS AND RESULTS: In 2019, C. difficile was isolated from 89/300 (29.7%) floor and 96/300 (32.0%) shoe sole samples at a tertiary hospital in Western Australia. Non-toxigenic C. difficile ribotype (RT) 010 predominated among floor (96.6%) and shoe sole (73.2%) isolates, while toxigenic RT 014/020 was most prevalent among contemporaneous clinical cases (33.0%) at the hospital. Whole-genome sequencing and high-resolution core genome single nucleotide polymorphism (cgSNP) analysis on C. difficile strains from hospital and community sources showed no clinical C. difficile RT 014/020 strains were genetically related, and evidence of frequent long-distance, multi-directional spread between humans, animals and the environment. In addition, cgSNP analysis of environmental RT 010 strains suggested transportation of C. difficile via shoe soles. CONCLUSIONS: While C. difficile RT 014/020 appears to spread via routes outside the healthcare system, RT 010 displayed a pattern of possible importation from the community into the hospital. SIGNIFICANCE AND IMPACT OF STUDY: These findings suggest developing community-based infection prevention and control strategies could significantly lower rates of CDI in the hospital setting.

Cationic Peptidomimetic Amphiphiles Having a N-Aryl- or N-Naphthyl-1,2,3-Triazole Core Structure Targeting Clostridioides (Clostridium) difficile: Synthesis, Antibacterial Evaluation, and an In Vivo C. difficile Infection Model.

Clostridioides (also known as Clostridium) difficile is a Gram-positive anaerobic, spore producing bacterial pathogen that causes severe gastrointestinal infection in humans. The current chemotherapeutic options are inadequate, expensive, and limited, and thus inexpensive drug treatments for C. difficile infection (CDI) with improved efficacy and specificity are urgently needed. To improve the solubility of our cationic amphiphilic 1,1'-binaphthylpeptidomimetics developed earlier that showed promise in an in vivo murine CDI model we have synthesized related compounds with an N-arytriazole or N-naphthyltriazole moiety instead of the 1,1'-biphenyl or 1,1'-binaphthyl moiety. This modification was made to increase the polarity and thus water solubility of the overall peptidomimetics, while maintaining the aromatic character. The dicationic N-naphthyltriazole derivative 40 was identified as a C. difficile-selective antibacterial with MIC values of 8 µg/mL against C. difficile strains ATCC 700057 and 132 (both ribotype 027). This compound displayed increased water solubility and reduced hemolytic activity (32 µg/mL) in an in vitro hemolysis assay and reduced cytotoxicity (CC50 32 µg/mL against HEK293 cells) relative to lead compound 2. Compound 40 exhibited mild efficacy (with 80% survival observed after 24 h compared to the DMSO control of 40%) in an in vivo murine model of C. difficile infection by reducing the severity and slowing the onset of disease.

Molecular Characterization of, and Antimicrobial Resistance in, Clostridioides difficile from Thailand, 2017-2018.

Antimicrobial resistance (AMR) plays an important role in the pathogenesis and spread of Clostridioides difficile infection (CDI). Many antimicrobials, such as fluoroquinolones, have been associated with outbreaks of CDI globally. This study characterized AMR among clinical C. difficile strains in Thailand, where antimicrobial use remains inadequately regulated. Stool samples were screened for tcdB and positives were cultured. C. difficile isolates were characterized by toxin profiling and PCR ribotyping. Antimicrobial susceptibility testing was performed by agar incorporation, and whole-genome sequencing and AMR genotyping were performed on a subset of strains. There were 321 C. difficile strains isolated from 326 stool samples. The most common toxigenic ribotype (RT) was RT 017 (18%), followed by RTs 014 (12%) and 020 (7%). Resistance to clindamycin, erythromycin, moxifloxacin, and rifaximin was common, especially among RT 017 strains. AMR genotyping revealed a strong correlation between resistance genotype and phenotype for moxifloxacin and rifaximin. The presence of erm-class genes was associated with high-level clindamycin and erythromycin resistance. Point substitutions in the penicillin-binding proteins were not sufficient to confer meropenem resistance, but a Y721S substitution in PBP3 was associated with a 4.37-fold increase in meropenem minimal inhibitory concentration. No resistance to metronidazole, vancomycin, or fidaxomicin was observed.

Antimicrobial resistance surveillance of Clostridioides difficile in Australia, 2015-18.

BACKGROUND: Clostridioides difficile was listed as an urgent antimicrobial resistance (AMR) threat in a report by the CDC in 2019. AMR drives the evolution of C. difficile and facilitates its emergence and spread. The C. difficile Antimicrobial Resistance Surveillance (CDARS) study is nationwide longitudinal surveillance of C. difficile infection (CDI) in Australia. OBJECTIVES: To determine the antimicrobial susceptibility of C. difficile isolated in Australia between 2015 and 2018. METHODS: A total of 1091 strains of C. difficile were collected over a 3 year period by a network of 10 diagnostic microbiology laboratories in five Australian states. These strains were tested for their susceptibility to nine antimicrobials using the CLSI agar incorporation method. RESULTS: All strains were susceptible to metronidazole, fidaxomicin, rifaximin and amoxicillin/clavulanate and low numbers of resistant strains were observed for meropenem (0.1%; 1/1091), moxifloxacin (3.5%; 38/1091) and vancomycin (5.7%; 62/1091). Resistance to clindamycin was common (85.2%; 929/1091), followed by resistance to ceftriaxone (18.8%; 205/1091). The in vitro activity of fidaxomicin [geometric mean MIC (GM) = 0.101 mg/L] was superior to that of vancomycin (1.700 mg/L) and metronidazole (0.229 mg/L). The prevalence of MDR C. difficile, as defined by resistance to ≥3 antimicrobial classes, was low (1.7%; 19/1091). CONCLUSIONS: The majority of C. difficile isolated in Australia did not show reduced susceptibility to antimicrobials recommended for treatment of CDI (vancomycin, metronidazole and fidaxomicin). Resistance to carbapenems and fluoroquinolones was low and MDR was uncommon; however, clindamycin resistance was frequent. One fluoroquinolone-resistant ribotype 027 strain was detected.

Positional Isomers of Biphenyl Antimicrobial Peptidomimetic Amphiphiles.

Small-molecule antimicrobial peptidomimetic amphiphiles represent a promising class of novel antimicrobials with the potential for widespread therapeutic application. To investigate the role of spatial positioning for key hydrophobic and hydrophilic groups on the antimicrobial efficacy and selectivity, positional isomers of the lead biphenyl antimicrobial peptidomimetic compound 1 were synthesized and subjected to microbial growth inhibition and mammalian toxicity assays. Positional isomer 4 exhibited 4-8× increased efficacy against the pathogenic Gram-negative bacteria Pseudomonas aeruginosa and Escherichia coli (MIC = 2 µg/mL), while isomers 2, 3, and 7 exhibited a 4× increase in activity against Acinetobacter baumannii (MIC = 4 µg/mL). Changes in molecular shape had a significant impact on Gram-negative antibacterial efficacy and the resultant spectrum of activity, whereas all structural isomers exhibited significant efficacy (MIC = 0.25-8 µg/mL) against Gram-positive bacterial pathogens (e.g., methicillin-resistant Staphylococcus aureus, Streptococcus pneumoniae, and Enterococcus faecalis).

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.

Mild or Malign: Clinical Characteristics and Outcomes of Clostridium difficile Infection in Thailand.

Little is known about the clinical characteristics of Clostridium difficile infection (CDI) in Asia in general, and Thailand specifically, with a few studies suggesting that the disease may be milder than elsewhere. This study aimed to describe CDI in Thailand, evaluate treatment options and their outcomes, and explore possible protective factors responsible for any unique disease characteristics. From 2015 to 2018, 469 patients were included in the study. All patients had their stools tested for the tcdB gene by direct PCR and detection of toxigenic C. difficile by culture. C. difficile isolates were subjected to toxin gene profiling and ribotyping, and patient medical records were reviewed retrospectively. There were 248 and 221 patients included in CDI and control groups, respectively. The CDI group had a higher overall 30-day mortality rate than the control group (21% versus 14%, P = 0.046), but only 2 deaths (1%) were directly attributable to CDI. Metronidazole treatment was not inferior to vancomycin in this population, and vancomycin was associated with a higher 30-day mortality rate (P = 0.047). The prevalence of severe CDI and disease outcomes were not different between patients infected with A-B+ C. difficile and A+B+ C. difficile strains or between patients with and without colonization by nontoxigenic C. difficile Besides C. difficile-specific tests, neither a single laboratory result nor a combination of results was predictive of CDI. In conclusion, CDI in Thailand was relatively mild, and metronidazole remained an effective treatment option for these mild infections.

Antimicrobial Susceptibilities of Clostridium difficile Isolates from 12 Asia-Pacific Countries in 2014 and 2015.

Clostridium (Clostridioides) difficile causes toxin-mediated diarrhea and pseudomembranous colitis, primarily among hospital inpatients. Outbreaks of C. difficile infection (CDI) have been caused by strains with acquired antimicrobial resistance, particularly fluoroquinolone resistance, including C. difficile ribotype (RT) 027 in North America and Europe and RT 017, the most common strain in Asia. Despite being the most common cause of hospital-acquired infection in high-income countries, and frequent misuse of antimicrobials in Asia, little is known about CDI in the Asia-Pacific region. We aimed to determine the antimicrobial susceptibility profiles of a collection of C. difficile isolates from the region. C. difficile isolates (n = 414) from a 2014 study of 13 Asia-Pacific countries were tested for susceptibility to moxifloxacin, amoxicillin-clavulanate, erythromycin, clindamycin, rifaximin, metronidazole, vancomycin, and fidaxomicin according to the Clinical and Laboratory Standards Institute's agar dilution method. All isolates were susceptible to metronidazole, vancomycin, amoxicillin-clavulanate, and fidaxomicin. Moxifloxacin resistance was detected in all countries except Australia, all RT 369 and QX 239 strains, and 92.7% of RT 018 and 70.6% of RT 017 strains. All C. difficile RT 012, 369, and QX 239 strains were also resistant to erythromycin and clindamycin. Rifaximin resistance was common in RT 017 strains only (63.2%) and was not detected in Australian, Japanese, or Singaporean isolates. In conclusion, antimicrobial susceptibility of C. difficile varied by strain type and by country. Multiresistance was common in emerging RTs 369 and QX 239 and the most common strain in Asia, RT 017. Ongoing surveillance is clearly warranted.

Antimicrobial resistance in Clostridium difficile ribotype 017.

Introduction: Antimicrobial resistance (AMR) played an important role in the initial outbreaks of Clostridium difficile infection (CDI) in the 1970s. C. difficile ribotype (RT) 017 has emerged as the major strain of C. difficile in Asia, where antimicrobial use is poorly regulated. This strain has also caused CDI outbreaks around the world for almost 30 years. Many of these outbreaks were associated with clindamycin and fluoroquinolone resistance. AMR and selective pressure is likely to be responsible for the success of this RT and may drive future outbreaks.Areas covered: This narrative review summarizes the prevalence and mechanisms of AMR in C. difficile RT 017 and transmission of these AMR mechanisms. To address these topics, reports of outbreaks due to C. difficile RT 017, epidemiologic studies with antimicrobial susceptibility results, studies on resistance mechanisms found in C. difficile and related publications available through Pubmed until September 2019 were collated and the findings discussed.Expert opinion: Primary prevention is the key to control CDI. This should be achieved by developing antimicrobial stewardship in medical, veterinary and agricultural practices. AMR is the key factor that drives CDI outbreaks, and methods for the early detection of AMR can facilitate the control of outbreaks.

Molecular epidemiology of Clostridium difficile isolated from piglets.

Information on the epidemiology of C. difficile infection (CDI) in South-East Asian countries is limited, as is data on possible animal reservoirs of C. difficile in the region. We investigated the prevalence and molecular epidemiology of C. difficile in piglets and the piggery environment in Thailand and Malaysia. Piglet rectal swabs (n = 224) and piggery environmental specimens (n = 23) were collected between 2015 and 2016 from 11 farms located in Thailand and Malaysia. All specimens were tested for the presence of C. difficile with toxigenic culture. PCR assays were performed on isolates to determine the ribotype (RT), and the presence of toxin genes. Whole genome sequencing was used on a subset of isolates to determine the evolutionary relatedness of RT038 (the most prevalent RT identified) common to pigs and humans from Thailand and Indonesia. C. difficile was recovered from 35% (58/165) and 92% (54/59) of the piglets, and 89% (8/9) and 93% (13/14) of the environmental specimens from Thailand and Malaysia, respectively. All strains from Thailand, and 30 strains from Malaysia (23 piglet and 7 environmental isolates) were non-toxigenic. To our knowledge, this is the first and only report with a complete lack of toxigenic C. difficile among piglets, a feature which could have a protective effect on the host. The most common strain belonged to RT038 (ST48), accounting for 88% (51/58) of piglet and 78% (7/9) of environmental isolates from Thailand, and all 30 isolates tested from Malaysia. Piglet RT038 isolates from Thailand and Malaysia differed by only 18 core-genome single nucleotide variants (cgSNVs) and both were, on average, 30 cgSNVs different from the human strains from Thailand and Indonesia, indicating a common ancestor in the last two decades.

High prevalence of Clostridium difficile in soil, mulch and lawn samples from the grounds of Western Australian hospitals.

Despite being considered a major hospital-associated pathogen for many years, Clostridium difficile has been isolated increasingly from people without hospital contact. In this study, we investigated the prevalence of C. difficile in the immediate outdoor environment of several hospitals in Perth, Western Australia, to provide further insight into potential sources of community-acquired C. difficile infection. Over 6 months, a total of 159 samples consisting of soil, mulch, lawn and sand were collected from outdoor surroundings of four different old (age>50 years) and new (age<10 years) hospitals. Samples were cultured in a C. difficile selective enrichment broth. Toxin gene profiling using PCR, and PCR ribotyping, was performed on all C. difficile recovered. C. difficile was isolated from 96 of the 159 samples (60.4%). Of the 112 isolates, 33 (29.5%) were toxigenic and 49 (43.8%) were identified as novel strains. Ribotypes (RTs) 014/020 (14.3%) and 010 (13.4%) constituted the highest proportion of isolates. Interestingly, RT 017, a strain endemic to the Asia-Pacific region (but not Australia), was found in a newly laid lawn. This study adds to existing knowledge of potential sources of C. difficile in Western Australia. More research is required to determine the route of transmission of C. difficile from community sources into the hospital.

Clostridium difficile ribotype 017 - characterization, evolution and epidemiology of the dominant strain in Asia.

Clostridium difficile ribotype (RT) 017 is an important toxigenic C. difficile RT which, due to a deletion in the repetitive region of the tcdA gene, only produces functional toxin B. Strains belonging to this RT were initially dismissed as nonpathogenic and circulated largely undetected for almost two decades until they rose to prominence following a series of outbreaks in the early 2000s. Despite lacking a functional toxin A, C. difficile RT 017 strains have been shown subsequently to be capable of causing disease as severe as that caused by strains producing both toxins A and B. While C. difficile RT 017 strains can be found in almost every continent today, epidemiological studies suggest that the RT is endemic in Asia and that the global spread of this MLST clade 4 lineage member is a relatively recent event. C. difficile RT 017 transmission appears to be mostly from human to human with only a handful of reports of isolations from animals. An important feature of C. difficile RT 017 strains is their resistance to several antimicrobials and this has been documented as a possible factor driving multiple outbreaks in different parts of the world. This review summarizes what is currently known regarding the emergence and evolution of strains belonging to C. difficile RT 017 as well as features that have allowed it to become an RT of global importance.

Cationic biaryl 1,2,3-triazolyl peptidomimetic amphiphiles targeting Clostridioides (Clostridium) difficile: Synthesis, antibacterial evaluation and an in vivo C. difficile infection model.

Clostridioides (formerly Clostridium) difficile is a Gram-positive anaerobic bacterial pathogen that causes severe gastrointestinal infection in humans. The current chemotherapeutic options are vastly inadequate, expensive and limited; this results in an exorbitant medical and financial burden. New, inexpensive chemotherapeutic treatments for C. difficile infection with improved efficacy are urgently needed. A streamlined synthetic pathway was developed to allow access to 38 novel mono- and di-cationic biaryl 1,2,3-triazolyl peptidomimetics with increased synthetic efficiency, aqueous solubility and enhanced antibacterial efficacy. The monocationic arginine derivative 28 was identified as a potent, Gram-positive selective antibacterial with MIC values of 4 µg/mL against methicillin-resistant Staphylococcus aureus and 8 µg/mL against C. difficile. Furthermore, the dicationic bis-triazole analogue 50 was found to exhibit broad-spectrum activity with substantial Gram-negative efficacy against Acinetobacter baumannii (8 µg/mL), Pseudomonas aeruginosa (8 µg/mL) and Klebsiella pneumoniae (16 µg/mL); additionally, compound 50 displayed reduced haemolytic activity (<13%) in an in vitro haemolysis assay. Membrane-disruption assays were conducted on selected derivatives to confirm the membrane-active mechanism of action inherent to the synthesized amphiphilic compounds. A comparative solubility assay was developed and utilized to optimize the aqueous solubility of the compounds for in vivo studies. The biaryl peptidomimetics 28 and 67 were found to exhibit significant efficacy in an in vivo murine model of C. difficile infection by reducing the severity and slowing the onset of disease.