Recurrence of Clostridium difficile infection in the Western Australian population.

Clostridium difficile, the most common cause of hospital-associated diarrhoea in developed countries, presents major public health challenges. The high clinical and economic burden from C. difficile infection (CDI) relates to the high frequency of recurrent infections caused by either the same or different strains of C. difficile. An interval of 8 weeks after index infection is commonly used to classify recurrent CDI episodes. We assessed strains of C. difficile in a sample of patients with recurrent CDI in Western Australia from October 2011 to July 2017. The performance of different intervals between initial and subsequent episodes of CDI was investigated. Of 4612 patients with CDI, 1471 (32%) were identified with recurrence. PCR ribotyping data were available for initial and recurrent episodes for 551 patients. Relapse (recurrence with same ribotype (RT) as index episode) was found in 350 (64%) patients and reinfection (recurrence with new RT) in 201 (36%) patients. Our analysis indicates that 8- and 20-week intervals failed to adequately distinguish reinfection from relapse. In addition, living in a non-metropolitan area modified the effect of age on the risk of relapse. Where molecular epidemiological data are not available, we suggest that applying an 8-week interval to define recurrent CDI requires more consideration.

Natural products show diverse mechanisms of action against Clostridium difficile.

AIMS: To investigate the mechanisms of action of natural products with bactericidal (cinnamon root powder, peppermint oil, trans-cinnamaldehyde, menthol and zingerone) or bacteriostatic (fresh garlic bulb extract, garlic clove powder, Leptospermum honey and allicin) activity against two Clostridium difficile strains. METHODS AND RESULTS: Bactericidal products significantly reduced intracellular ATP after 1 h (P ≤ 0·01), quantified using the BacTiter-Glo reagent, and damaged the cell membrane, shown by the leakage of both 260-nm-absorbing materials and protein, and the uptake of propidium iodide. Bacteriolysis was not observed, determined by measuring optical density of treated cell suspensions at 620-nm. The effect of three bacteriostatic products on protein synthesis was quantified using an Escherichia coli S30 extract system, with Leptospermum honey (16% w/v) showing significant inhibition (P < 0·01). Lastly, no products showed elevated minimum inhibitory concentrations against antimicrobial-resistant C. difficile, determined by broth microdilution. CONCLUSIONS: Cytoplasmic membrane damage was identified as a mechanism of action that may contribute to the activity of several natural products against C. difficile. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes the possible mechanisms of action of natural products against C. difficile, yet the efficacy in vivo to be determined.

In silico, in vitro and in vivo analysis of putative virulence factors identified in large clostridial toxin-negative, binary toxin- producing C. difficile strains.

The relevance of large clostridial toxin-negative, binary toxin-producing (A-B-CDT+) Clostridium difficile strains in human infection is still controversial. In this study, we investigated putative virulence traits that may contribute to the role of A-B-CDT+C. difficile strains in idiopathic diarrhea. Phenotypic assays were conducted on 148 strains of C. difficile comprising 10 different A-B-CDT+C. difficile ribotypes (RTs): 033, 238, 239, 288, 585, 586, QX143, QX444, QX521 and QX629. A subset of these isolates (n = 53) was whole-genome sequenced to identify genetic loci associated with virulence and survival. Motility studies showed that with the exception of RT 239 all RTs tested were non-motile. C. difficile RTs 033 and 288 had deletions in the F2 and F3 regions of their flagella operon while the F2 region was absent from strains of RTs 238, 585, 586, QX143, QX444, QX521 and QX629. The flagellin and flagella cap genes, fliC and fliD, respectively, involved in adherence and host colonization, were conserved in all strains, including reference strains. All A-B-CDT+C. difficile strains produced at least three extracellular enzymes (deoxyribonuclease, esterase and mucinase) indicating that these are important extracellular proteins. The toxicity of A-B-CDT+C. difficile strains in Vero cells was confirmed, however, pathogenicity was not demonstrated in a mouse model of disease. Despite successful colonization by most strains, there was no evidence of disease in mice. This study provides the first in-depth analysis of A-B-CDT+C. difficile strains and contributes to the current limited knowledge of these strains as a cause of C. difficile infection.

Zoonotic Transfer of Clostridium difficile Harboring Antimicrobial Resistance between Farm Animals and Humans.

The emergence of Clostridium difficile as a significant human diarrheal pathogen is associated with the production of highly transmissible spores and the acquisition of antimicrobial resistance genes (ARGs) and virulence factors. Unlike the hospital-associated C. difficile RT027 lineage, the community-associated C. difficile RT078 lineage is isolated from both humans and farm animals; however, the geographical population structure and transmission networks remain unknown. Here, we applied whole-genome phylogenetic analysis of 248 C. difficile RT078 strains from 22 countries. Our results demonstrate limited geographical clustering for C. difficile RT078 and extensive coclustering of human and animal strains, thereby revealing a highly linked intercontinental transmission network between humans and animals. Comparative whole-genome analysis reveals indistinguishable accessory genomes between human and animal strains and a variety of antimicrobial resistance genes in the pangenome of C. difficile RT078. Thus, bidirectional spread of C. difficile RT078 between farm animals and humans may represent an unappreciated route disseminating antimicrobial resistance genes between humans and animals. These results highlight the importance of the "One Health" concept to monitor infectious disease emergence and the dissemination of antimicrobial resistance genes.

High prevalence of Clostridium difficile on retail root vegetables, Western Australia.

AIMS: The incidence of community-associated Clostridium difficile infection (CA-CDI) in Australia has increased since mid-2011. With reports of clinically important C. difficile strains being isolated from retail foods in Europe and North America, a foodborne source of C. difficile in cases of CA-CDI is a possibility. This study represents the first to investigate the prevalence and genotypes of C. difficile in Australian retail vegetables. METHODS AND RESULTS: A total of 300 root vegetables grown in Western Australia (WA) were collected from retail stores and farmers' markets. Three vegetables of the same kind bought from the same store/market were treated as one sample. Selective enrichment culture, toxin profiling and PCR ribotyping were performed. Clostridium difficile was isolated from 30% (30/100) of pooled vegetable samples, 55·6% of organic potatoes, 50% of nonorganic potatoes, 22·2% of organic beetroots, 5·6% of organic onions and 5·3% of organic carrots. Over half (51·2%, 22/43) the isolates were toxigenic. Many of the ribotypes of C. difficile isolated were common among human and Australian animals. CONCLUSIONS: Clostridium difficile could be found commonly on retail root vegetables of WA. This may be potential sources for CA-CDI. SIGNIFICANCE AND IMPACT OF THE STUDY: This study enhances knowledge of possible sources of C. difficile in the Australian community, outside the hospital setting.

Effects of natural products on several stages of the spore cycle of Clostridium difficile in vitro.

AIMS: To investigate the effect of natural products on the spore cycle of Clostridium difficile in vitro. METHODS AND RESULTS: Twenty-two natural products were investigated using four C. difficile strains. Effects on sporulation, determined using microscopy and a conventional spore recovery assay, showed that fresh onion bulb extract (6·3% v v-1 ) and coconut oil (8% v v-1 ) inhibited sporulation in all four isolates by 66-86% and 51-88%, respectively, compared to untreated controls. Fresh ginger rhizome extract (25% v v-1 ) was also inhibitory, although to a lesser extent. Using a standard spore germination and outgrowth assay, germination was unaffected by the 22 products, whereas outgrowth was significantly reduced by artichoke extract (18·8 mg ml-1 ), fresh onion bulb extract (25% v v-1 ), Leptospermum honeys (8% w v-1 ) and allicin (75 mg ml-1 ; P < 0·01). Sporicidal activity, investigated using a standard plate recovery assay, was minimal. CONCLUSIONS: Three of the 22 natural products (13%) showed inhibitory effects on sporulation of C. difficile and six products (27%) reduced vegetative outgrowth of C. difficile. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows the potential of natural products to inhibit different stages of C. difficile sporulation and encourages further investigation in this field.

Comparative Genome Analysis and Global Phylogeny of the Toxin Variant Clostridium difficile PCR Ribotype 017 Reveals the Evolution of Two Independent Sublineages.

The diarrheal pathogen Clostridium difficile consists of at least six distinct evolutionary lineages. The RT017 lineage is anomalous, as strains only express toxin B, compared to strains from other lineages that produce toxins A and B and, occasionally, binary toxin. Historically, RT017 initially was reported in Asia but now has been reported worldwide. We used whole-genome sequencing and phylogenetic analysis to investigate the patterns of global spread and population structure of 277 RT017 isolates from animal and human origins from six continents, isolated between 1990 and 2013. We reveal two distinct evenly split sublineages (SL1 and SL2) of C. difficile RT017 that contain multiple independent clonal expansions. All 24 animal isolates were contained within SL1 along with human isolates, suggesting potential transmission between animals and humans. Genetic analyses revealed an overrepresentation of antibiotic resistance genes. Phylogeographic analyses show a North American origin for RT017, as has been found for the recently emerged epidemic RT027 lineage. Despite having only one toxin, RT017 strains have evolved in parallel from at least two independent sources and can readily transmit between continents.

A population-based longitudinal study of Clostridium difficile infection-related hospitalization in mid-age and older Australians.

Clostridium difficile is the principal cause of infectious diarrhoea in hospitalized patients. We investigated the incidence and risk factors for hospitalization due to C. difficile infection (CDI) in older Australians. We linked data from a population-based prospective cohort study (the 45 and Up Study) of 266 922 adults aged ⩾45 years recruited in New South Wales, Australia to hospitalization and death records for 2006-2012. We estimated the incidence of CDI hospitalization and calculated days in hospital and costs per hospitalization. We also estimated hazard ratios (HR) for CDI hospitalization using Cox regression with age as the underlying time variable. Over a total follow-up of 1 126 708 person-years, 187 adults had an incident CDI hospitalization. The crude incidence of CDI hospitalization was 16·6/100 000 person-years, with a median hospital stay of 6 days, and a median cost of AUD 6102 per admission. Incidence increased with age and year of follow-up, with a threefold increase for 2009-2012. After adjustment, CDI hospitalization rates were significantly lower in males than females (adjusted HR 0·6, 95% confidence interval 0·4-0·7). CDI hospitalization rates increased significantly over 2009-2012. There is a need to better understand the increasing risk of CDI hospitalization in women.

Prevalence of Clostridium difficile infection and colonization in a tertiary hospital and elderly community of North-Eastern Peninsular Malaysia.

Little is known about Clostridium difficile infection (CDI) in Asia. The aims of our study were to explore (i) the prevalence, risk factors and molecular epidemiology of CDI and colonization in a tertiary academic hospital in North-Eastern Peninsular Malaysia; (ii) the rate of carriage of C. difficile among the elderly in the region; (iii) the awareness level of this infection among the hospital staffs and students. For stool samples collected from hospital inpatients with diarrhea (n = 76) and healthy community members (n = 138), C. difficile antigen and toxins were tested by enzyme immunoassay. Stool samples were subsequently analyzed by culture and molecular detection of toxin genes, and PCR ribotyping of isolates. To examine awareness among hospital staff and students, participants were asked to complete a self-administered questionnaire. For the hospital and community studies, the prevalence of non-toxigenic C. difficile colonization was 16% and 2%, respectively. The prevalence of CDI among hospital inpatients with diarrhea was 13%. Out of 22 C. difficile strains from hospital inpatients, the toxigenic ribotypes 043 and 017 were most common (both 14%). In univariate analysis, C. difficile colonization in hospital inpatients was significantly associated with greater duration of hospitalization and use of penicillin (both P < 0·05). Absence of these factors was a possible reason for low colonization in the community. Only 3% of 154 respondents answered all questions correctly in the awareness survey. C. difficile colonization is prevalent in a Malaysian hospital setting but not in the elderly community with little or no contact with hospitals. Awareness of CDI is alarmingly poor.

Antimicrobial activity of natural products against Clostridium difficile in vitro.

AIMS: To investigate the antimicrobial activity of various natural products against Clostridium difficile in vitro. METHODS AND RESULTS: The antibacterial activity of 20 natural products was determined by the agar well diffusion and broth microdilution assays against four C. difficile strains, three comparator organisms and four gastrointestinal commensal organisms. Of the raw natural products, garlic juice had the highest activity. The most active processed products were peppermint oil and the four pure compounds trans-cinnamaldehyde, allicin, menthol and zingerone. Furthermore, Bacteroides species had similar susceptibility to C. difficile to most natural products; however, Lactobacillus casei was less susceptible. The combined effect of natural products with vancomycin or metronidazole was determined using the conventional checkerboard titration method and the fractional inhibitory concentration index was calculated. The results showed a possible synergism between trans-cinnamaldehyde and vancomycin and partial synergy between trans-cinnamaldehyde and metronidazole. CONCLUSIONS: The study indicates a range of antimicrobial activity of natural products against C. difficile and suggests that they may be useful as alternative or complementary treatments for C. difficile infection (CDI), particularly as most are able to be given orally. SIGNIFICANCE AND IMPACT OF THE STUDY: This study encourages further investigation of natural products for treatment of CDI.

Australasian Society of Infectious Diseases updated guidelines for the management of Clostridium difficile infection in adults and children in Australia and New Zealand.

The incidence of Clostridium difficile infection (CDI) continues to rise, whilst treatment remains problematic due to recurrent, refractory and potentially severe nature of disease. The treatment of C. difficile is a challenge for community and hospital-based clinicians. With the advent of an expanding therapeutic arsenal against C. difficile since the last published Australasian guidelines, an update on CDI treatment recommendations for Australasian clinicians was required. On behalf of the Australasian Society of Infectious Diseases, we present the updated guidelines for the management of CDI in adults and children.

Emergence and spread of predominantly community-onset Clostridium difficile PCR ribotype 244 infection in Australia, 2010 to 2012.

We describe an Australia-wide Clostridium difficile outbreak in 2011 and 2012 involving the previously uncommon ribotype 244. In Western Australia, 14 of 25 cases were community-associated, 11 were detected in patients younger than 65 years, 14 presented to emergency/outpatient departments, and 14 to non-tertiary/community hospitals. Using whole genome sequencing, we confirm ribotype 244 is from the same C. difficile clade as the epidemic ribotype 027. Like ribotype 027, it produces toxins A, B, and binary toxin, however it is fluoroquinolone-susceptible and thousands of single nucleotide variants distinct from ribotype 027. Fifteen outbreak isolates from across Australia were sequenced. Despite their geographic separation, all were genetically highly related without evidence of geographic clustering, consistent with a point source, for example affecting the national food chain. Comparison with reference laboratory strains revealed the outbreak clone shared a common ancestor with isolates from the United States and United Kingdom (UK). A strain obtained in the UK was phylogenetically related to our outbreak. Follow-up of that case revealed the patient had recently returned from Australia. Our data demonstrate new C. difficile strains are an on-going threat, with potential for rapid spread. Active surveillance is needed to identify and control emerging lineages.

Diversity of nontypeable Haemophilus influenzae strains colonizing Australian Aboriginal and non-Aboriginal children.

Nontypeable Haemophilus influenzae (NTHI) strains are responsible for respiratory-related infections which cause a significant burden of disease in Australian children. We previously identified a disparity in NTHI culture-defined carriage rates between Aboriginal and non-Aboriginal children (42% versus 11%). The aim of this study was to use molecular techniques to accurately determine the true NTHI carriage rates (excluding other culture-identical Haemophilus spp.) and assess whether the NTHI strain diversity correlates with the disparity in NTHI carriage rates. NTHI isolates were cultured from 595 nasopharyngeal aspirates collected longitudinally from asymptomatic Aboriginal (n=81) and non-Aboriginal (n=76) children aged 0 to 2 years living in the Kalgoorlie-Boulder region, Western Australia. NTHI-specific 16S rRNA gene PCR and PCR ribotyping were conducted on these isolates. Confirmation of NTHI by 16S rRNA gene PCR corrected the NTHI carriage rates from 42% to 36% in Aboriginal children and from 11% to 9% in non-Aboriginal children. A total of 75 different NTHI ribotypes were identified, with 51% unique to Aboriginal children and 13% unique to non-Aboriginal children (P<0.0001). The strain richness (proportion of different NTHI ribotypes) was similar for Aboriginal (19%, 65/346) and non-Aboriginal children (19%, 37/192) (P=0.909). Persistent carriage of the same ribotype was rare in the two groups, but colonization with multiple NTHI strains was more common in Aboriginal children than in non-Aboriginal children. True NTHI carriage was less than that estimated by culture. The Aboriginal children were more likely to carry unique and multiple NTHI strains, which may contribute to the chronicity of NTHI colonization and subsequent disease.

Distribution of 13 virulence genes among clinical and environmental Aeromonas spp. in Western Australia.

We evaluated the pathogenic potential of 98 clinical and 31 environmental Aeromonas isolates by detecting the presence of 13 virulence genes using a polymerase chain reaction (PCR)-based method. The majority (96 %) of the strains contained at least one of the virulence genes. The overall distribution was aerA/haem (77 %), alt (53 %), lafA (51 %), ast (39 %), flaA (32 %), aspA (29 %), vasH (26 %), ascV (16 %) and aexT (13 %). No amplification products were detected for the genes encoding a bundle-forming pilus (BfpA and BfpG) or a Shiga-like toxin (stx-1 and stx-2). Five or more virulence genes were detected in 42 % of environmental and 24 % of clinical isolates. Among the major species, 48 % of A. hydrophila and 42 % of A. dhakensis isolates harboured five or more virulence genes compared with 19 % in A. veronii bv. sobria and none in A. caviae isolates. Our results suggest that, in Western Australia, strains of A. dhakensis and A. hydrophila are potentially more virulent than those of A. veronii bv. sobria and A. caviae, although the pathogenic potential of Aeromonas spp. is probably strain- rather than species-dependent.

Clostridium difficile infection diagnosis in a paediatric population: comparison of methodologies.

The increasing incidence of Clostridium difficile infection (CDI) in paediatric hospitalised populations, combined with the emergence of hypervirulent strains, community-acquired CDI and the need for prompt treatment and infection control, makes the rapid, accurate diagnosis of CDI crucial. We validated commonly used C. difficile diagnostic tests in a paediatric hospital population. From October 2011 to January 2012, 150 consecutive stools were collected from 75 patients at a tertiary paediatric hospital in Perth, Western Australia. Stools were tested using: C. Diff Quik Chek Complete, Illumigene C. difficile, GeneOhm Cdiff, cycloserine cefoxitin fructose agar (CCFA) culture, and cell culture cytotoxin neutralisation assay (CCNA). The reference standard was growth on CCFA or Cdiff Chromagar and PCR on isolates to detect tcdA, tcdB, cdtA, and cdtB. Isolates were PCR ribotyped. The prevalence of CDI was high (43 % of patients). Quik Chek Complete glutamate dehydrogenase (GDH) demonstrated a low negative predictive value (NPV) (93 %). Both CCNA and Quik Chek Complete toxin A/B had poor sensitivity (33 % and 29 % respectively). Molecular methods both had 89 % sensitivity. Algorithms using GDH + Illumigene or GeneOhm reduced the sensitivity to 85 % and 83 % respectively. Ribotype UK014/20 predominated. GDH NPV and GeneOhm and Illumigene sensitivities were reduced compared with adult studies. Quik Chek Complete and CCNA cannot reliably detect toxigenic CDI. A GDH first algorithm showed reduced sensitivity. In a high prevalence paediatric population, molecular methods alone are recommended over the use of GDH algorithm or culture and CCNA, as they demonstrate the best test performance characteristics.

Clostridioides difficile colonization and infection in a cohort of Australian adults with cystic fibrosis.

BACKGROUND: Little is known about Clostridioides difficile infection (CDI) in patients with cystic fibrosis (CF). The aim of this study was to investigate the prevalence, molecular epidemiology and risk factors for CDI in asymptomatic and symptomatic adults with CF in Western Australia. METHODS: Faecal samples from symptomatic and asymptomatic patients were prospectively collected and tested for the presence of C. difficile by toxigenic culture. Ribotyping was performed by established protocols. Logistic regression analysis was performed to analyse the risk factors for C. difficile colonization and infection. Extensive environmental sampling was performed within the CF clinic in Perth. RESULTS: The prevalence rates of asymptomatic toxigenic and non-toxigenic C. difficile colonization were 30% (14/46 patients) and 24% (11/46 patients), respectively. Fifteen ribotypes (RTs) of C. difficile were identified, of which non-toxigenic RT 039 was the most common. Among the symptomatic patients, the prevalence of toxigenic CDI was 33% (11/33 patients). Impaired glucose tolerance/diabetes mellitus and duration of intravenous antibiotic use in the past 12 months were significantly associated with increased risk of asymptomatic toxigenic C. difficile carriage and CDI. A trend towards higher CF transmembrane conductance regulator modulator treatment was observed in the CDI group. Extensive environmental sampling showed no evidence of toxigenic C. difficile contamination within the CF clinic. CONCLUSIONS: A high prevalence of asymptomatic carriage of toxigenic C. difficile was observed in adults with CF, comparable with that observed in the symptomatic CF population. There was no evidence of direct person-to-person transmission.

Clostridium difficile and One Health.

BACKGROUND: For over four decades, Clostridium difficile has been a significant enteric pathogen of humans. It is associated with the use of antimicrobials that generally disrupt the microbiota of the gastrointestinal tract. Previously, it was thought that C. difficile was primarily a hospital-acquired infection; however, with the emergence of community-associated cases, and whole-genome sequencing suggesting the majority of the hospital C. difficile infection (CDI) cases are genetically distinct from one another, there is compelling evidence that sources/reservoirs of C. difficile outside hospitals play a significant role in the transmission of CDI. OBJECTIVES: To review the 'One Health' aspects of CDI, focusing on how community sources/reservoirs might be acting as a conduit in the transfer of C. difficile between animals and humans. The importance of a One Health approach in managing CDI is discussed. SOURCES: A literature search was performed on PubMed and Web of Science for relevant papers published from 1 January 2000 to 10 July 2019. CONTENT: We present evidence that demonstrates transmission of C. difficile in hospitals from asymptomatic carriers to symptomatic CDI patients. The source of colonization is most probably community reservoirs, such as foods and the environment, where toxigenic C. difficile strains have frequently been isolated. With high-resolution genomic sequencing, the transmission of C. difficile between animals and humans can be demonstrated, despite a clear epidemiological link often being absent. The ways in which C. difficile from animals and humans can disseminate through foods and the environment are discussed, and an interconnected transmission pathway for C. difficile involving food animals, humans and the environment is presented. IMPLICATIONS: Clostridium difficile is a well-established pathogen of both humans and animals that contaminates foods and the environment. To manage CDI, a One Health approach with the collaboration of clinicians, veterinarians, environmentalists and policy-makers is paramount.

Status of vaccine research and development for Clostridium difficile.

Clostridium difficile associated disease is fundamentally associated with dysbiosis of the gut microbiome as a consequence of antibiotic use. This is because this sporulating, obligate anaerobe germinates and proliferates rapidly in the dysbiotic gut, which is an indirect consequence of their use. During its growth, C. difficile produces two toxins, toxin A (TcdA) and toxin B (TcdB), which are responsible for the majority of clinical symptoms associated with the disease. Three parenterally delivered vaccines, based on detoxified or recombinant forms of these toxins, have undergone or are undergoing clinical trials. Each offers the opportunity to generate high titres of toxin neutralising antibodies. Whilst these data suggest these vaccines may reduce primary symptomatic disease, they do not in their current form reduce the capacity of the organism to persist and shed from the vaccinated host. The current progress of vaccine development is considered with advantages and limitations of each highlighted. In addition, several alternative approaches are described that seek to limit C. difficile germination, colonisation and persistence. It may yet prove that the most effective treatments to limit infection, disease and spread of the organism will require a combination of therapeutic approaches. The potential use and efficacy of these vaccines in low and middle income countries will be depend on the development of a cost effective vaccine and greater understanding of the distribution and extent of disease in these countries.