Esculin hydrolysis negative and TcdA-only producing strains of Clostridium (Clostridioides) difficile from the environment in Western Australia

Background and Aims: Clostridium (Clostridiodes) difficile clade 3 ribotype (RT) 023 strains that fail to produce black colonies on bioMérieux ChromID agar have been reported, as well as variant strains of C. difficile that produce only toxin A. We have recently isolated strains of C. difficile from the environment in Western Australia (WA) with similar characteristics. The objective of this study was to characterize these strains. It was hypothesized that a putative β-glucosidase gene was lacking in these strains of C. difficile, including RT 023, leading to white colonies. Methods and Results: A total of 17 environmental isolates of C. difficile from garden soil and compost, and gardening shoe soles in Perth, WA, failed to produce black colonies on ChromID agar. MALDI-TOF MS analysis confirmed these strains as C. difficile. Four strains contained only a tcdA gene (A+B−CDT−) by PCR and were a novel RT (QX 597). All isolates were susceptible to all antimicrobials tested except one with low-level resistance to clindamycin (MIC = 8 mg/L). The four tcdA-positive strains were motile. All isolates contained neither bgl locus but only bgl K or a putative β-glucosidase gene by PCR. Whole-genome sequencing showed the 17 strains belonged to novel multi-locus sequence types 632, 848, 849, 850, 851, 852 and 853, part of the evolutionarily divergent clade C-III. Four isolates carried a full-length tcdA but not tcdB nor binary toxin genes. Conclusions: ChromID C. difficile agar is used for the specific detection of C. difficile in the samples. To date, all strains except RT 023 strains from clinical samples hydrolyse esculin. This is the first report to provide insights into the identification of esculin hydrolysis negative and TcdA-only producing (A+B−CDT−) strains of C. difficile from environmental samples. Significance and Impact of the Study: White colonies of C. difficile from environmental samples could be overlooked when using ChromID C. difficile agar, leading to false-negative results, however, whether these strains are truly pathogenic remains to be proven

Evolutionary and genomic insights into Clostridioides difficile sequence type 11: A diverse zoonotic and antimicrobial-resistant lineage of global One Health importance

Clostridioides difficile (Clostridium difficile) sequence type 11 (ST11) is well established in production animal populations worldwide and contributes considerably to the global burden of C. difficile infection (CDI) in humans. Increasing evidence of shared ancestry and genetic overlap of PCR ribotype 078 (RT078), the most common ST11 sublineage, between human and animal populations suggests that CDI may be a zoonosis. We performed whole-genome sequencing (WGS) on a collection of 207 ST11 and closely related ST258 isolates of human and veterinary/environmental origin, comprising 16 RTs collected from Australia, Asia, Europe, and North America. Core genome single nucleotide variant (SNV) analysis identified multiple intraspecies and interspecies clonal groups (isolates separated by ≤2 core genome SNVs) in all the major RT sublineages: 078, 126, 127, 033, and 288. Clonal groups comprised isolates spread across different states, countries, and continents, indicative of reciprocal long-range dissemination and possible zoonotic/anthroponotic transmission. Antimicrobial resistance genotypes and phenotypes varied across host species, geographic regions, and RTs and included macrolide/lincosamide resistance (Tn6194 [ermB]), tetracycline resistance (Tn6190 [tetM] and Tn6164 [tet44]), and fluoroquinolone resistance (gyrA/B mutations), as well as numerous aminoglycoside resistance cassettes. The population was defined by a large “open” pan-genome (10,378 genes), a remarkably small core genome of 2,058 genes (only 19.8% of the gene pool), and an accessory genome containing a large and diverse collection of important prophages of the Siphoviridae and Myoviridae. This study provides novel insights into strain relatedness and genetic variability of C. difficile ST11, a lineage of global One Health importance. IMPORTANCE: Historically, Clostridioides difficile (Clostridium difficile) has been associated with life-threatening diarrhea in hospitalized patients. Increasing rates of C. difficile infection (CDI) in the community suggest exposure to C. difficile reservoirs outside the hospital, including animals, the environment, or food. C. difficile sequence type 11 (ST11) is known to infect/colonize livestock worldwide and comprises multiple ribotypes, many of which cause disease in humans, suggesting CDI may be a zoonosis. Using high-resolution genomics, we investigated the evolution and zoonotic potential of ST11 and a new closely related ST258 lineage sourced from diverse origins. We found multiple intra- and interspecies clonal transmission events in all ribotype sublineages. Clones were spread across multiple continents, often without any health care association, indicative of zoonotic/anthroponotic long-range dissemination in the community. ST11 possesses a massive pan-genome and numerous clinically important antimicrobial resistance elements and prophages, which likely contribute to the success of this globally disseminated lineage of One Health importance

Prevalence of binary toxin positive Clostridium difficile in diarrhoeal humans in the absence of epidemic ribotype 027

Virulence of Clostridium difficile is primarily attributed to the large clostridial toxins A and B while the role of binary toxin (CDT) remains unclear. The prevalence of human strains of C. difficile possessing only CDT genes (A¯B¯CDT +) is generally low (\u3c 5%), however, this genotype is commonly found in neonatal livestock both in Australia and elsewhere. Zoonotic transmission of C. difficile has been suggested previously. Most human diagnostic tests will not detect A¯B¯CDT + strains of C. difficile because they focus on detection of toxin A and/or B. We performed a prospective investigation into the prevalence and genetic characteristics of A¯B¯CDT + C. difficile in symptomatic humans. All glutamate dehydrogenase or toxin B gene positive faecal specimens from symptomatic inpatients over 30 days (n = 43) were cultured by enrichment, and C. difficile PCR ribotypes (RTs) and toxin gene profiles determined. From 39 culture-positive specimens, 43 C. difficile isolates were recovered, including two A¯B¯CDT + isolates. This corresponded to an A¯B¯CDT + prevalence of 2/35 (5.7%) isolates possessing at least one toxin, 2/10 (20%) A¯B¯+ isolates, 2/3 CDT + isolates and 1/28 (3.6%) presumed true CDI cases. No link to Australian livestock-associated C. difficile was found. Neither A¯B¯CDT + isolate was the predominant A¯B¯CDT + strain found in Australia, RT 033, nor did they belong to toxinotype XI. Previous reports infrequently describe A¯B¯CDT + C. difficile in patients and strain collections but the prevalence of human A¯B¯CDT + C. difficile is rarely investigated. This study highlights the occurrence of A−B−CDT+ strains of C. difficile in symptomatic patients, warranting further investigations of its role in human infection

Antimicrobial susceptibility of Clostridium difficile isolated from food and environmental sources in Western Australia

We recently reported a high prevalence of Clostridium difficile in retail vegetables, compost and lawn in Western Australia. The objective of this study was to investigate the antimicrobial susceptibility of previously isolated food and environmental C. difficile isolates from Western Australia. A total of 274 C. difficile isolates from vegetables, compost and lawn were tested for susceptibility to a panel of 10 antimicrobial agents (fidaxomicin, vancomycin, metronidazole, rifaximin, clindamycin, erythromycin, amoxicillin/clavulanic acid, moxifloxacin, meropenem and tetracycline) using the agar incorporation method. Fidaxomicin was the most potent agent (MI

Evaluation of the Cepheid® Xpert® C. difficile binary toxin (BT) diagnostic assay

Strains of Clostridium difficile producing only binary toxin (CDT) are found commonly in animals but not humans. However, human diagnostic tests rarely look for CDT. The Cepheid Xpert C. difficile BT assay detects CDT with equal sensitivity (≥92%) in human and animal faecal samples

A Phenotypically Silent vanB2 Operon Carried on a Tn1549-Like Element in Clostridium difficile

In the last decade, Clostridium difficile infection (CDI) has reached an epidemic state with increasing incidence and severity in both health care and community settings. Vancomycin is an important first-line therapy for CDI, and the emergence of resistance would have significant clinical consequences. In this study, we describe for the first time a vanB2 vancomycin resistance operon in C. difficile, isolated from an Australian veal calf at slaughter. The operon was carried on an ~42-kb element showing significant homology and synteny to Tn1549, a conjugative transposon linked with the emergence and global dissemination of vancomycin-resistant enterococci (VRE). Notably, the C. difficile strain did not show any reduced susceptibility to vancomycin in vitro (MIC, 1 mg/liter), possibly as a result of an aberrant vanRB gene. As observed for other anaerobic species of the animal gut microbiota, C. difficile may be a reservoir of clinically important vancomycin resistance genes

Evolutionary and Genomic Insights into Clostridioides difficile Sequence Type 11: a Diverse Zoonotic and Antimicrobial-Resistant Lineage of Global One Health Importance

Historically, Clostridioides difficile (Clostridium difficile) has been associated with life-threatening diarrhea in hospitalized patients. Increasing rates of C. difficile infection (CDI) in the community suggest exposure to C. difficile reservoirs outside the hospital, including animals, the environment, or food. C. difficile sequence type 11 (ST11) is known to infect/colonize livestock worldwide and comprises multiple ribotypes, many of which cause disease in humans, suggesting CDI may be a zoonosis. Using high-resolution genomics, we investigated the evolution and zoonotic potential of ST11 and a new closely related ST258 lineage sourced from diverse origins. We found multiple intra- and interspecies clonal transmission events in all ribotype sublineages. Clones were spread across multiple continents, often without any health care association, indicative of zoonotic/anthroponotic long-range dissemination in the community. ST11 possesses a massive pan-genome and numerous clinically important antimicrobial resistance elements and prophages, which likely contribute to the success of this globally disseminated lineage of One Health importance.Clostridioides difficile (Clostridium difficile) sequence type 11 (ST11) is well established in production animal populations worldwide and contributes considerably to the global burden of C. difficile infection (CDI) in humans. Increasing evidence of shared ancestry and genetic overlap of PCR ribotype 078 (RT078), the most common ST11 sublineage, between human and animal populations suggests that CDI may be a zoonosis. We performed whole-genome sequencing (WGS) on a collection of 207 ST11 and closely related ST258 isolates of human and veterinary/environmental origin, comprising 16 RTs collected from Australia, Asia, Europe, and North America. Core genome single nucleotide variant (SNV) analysis identified multiple intraspecies and interspecies clonal groups (isolates separated by ≤2 core genome SNVs) in all the major RT sublineages: 078, 126, 127, 033, and 288. Clonal groups comprised isolates spread across different states, countries, and continents, indicative of reciprocal long-range dissemination and possible zoonotic/anthroponotic transmission. Antimicrobial resistance genotypes and phenotypes varied across host species, geographic regions, and RTs and included macrolide/lincosamide resistance (Tn6194 [ermB]), tetracycline resistance (Tn6190 [tetM] and Tn6164 [tet44]), and fluoroquinolone resistance (gyrA/B mutations), as well as numerous aminoglycoside resistance cassettes. The population was defined by a large “open” pan-genome (10,378 genes), a remarkably small core genome of 2,058 genes (only 19.8% of the gene pool), and an accessory genome containing a large and diverse collection of important prophages of the Siphoviridae and Myoviridae. This study provides novel insights into strain relatedness and genetic variability of C. difficile ST11, a lineage of global One Health importance

Prevalence of binary toxin positive <i>Clostridium difficile</i> in diarrhoeal humans in the absence of epidemic ribotype 027

<div><p>Virulence of <i>Clostridium difficile</i> is primarily attributed to the large clostridial toxins A and B while the role of binary toxin (CDT) remains unclear. The prevalence of human strains of <i>C</i>. <i>difficile</i> possessing only CDT genes (A⁻B⁻CDT⁺) is generally low (< 5%), however, this genotype is commonly found in neonatal livestock both in Australia and elsewhere. Zoonotic transmission of <i>C</i>. <i>difficile</i> has been suggested previously. Most human diagnostic tests will not detect A⁻B⁻CDT⁺ strains of <i>C</i>. <i>difficile</i> because they focus on detection of toxin A and/or B. We performed a prospective investigation into the prevalence and genetic characteristics of A⁻B⁻CDT⁺ <i>C</i>. <i>difficile</i> in symptomatic humans. All glutamate dehydrogenase or toxin B gene positive faecal specimens from symptomatic inpatients over 30 days (<i>n</i> = 43) were cultured by enrichment, and <i>C</i>. <i>difficile</i> PCR ribotypes (RTs) and toxin gene profiles determined. From 39 culture-positive specimens, 43 <i>C</i>. <i>difficile</i> isolates were recovered, including two A⁻B⁻CDT⁺ isolates. This corresponded to an A⁻B⁻CDT⁺ prevalence of 2/35 (5.7%) isolates possessing at least one toxin, 2/10 (20%) A⁻B⁻ isolates, 2/3 CDT⁺ isolates and 1/28 (3.6%) presumed true CDI cases. No link to Australian livestock-associated <i>C</i>. <i>difficile</i> was found. Neither A⁻B⁻CDT⁺ isolate was the predominant A⁻B⁻CDT⁺ strain found in Australia, RT 033, nor did they belong to toxinotype XI. Previous reports infrequently describe A⁻B⁻CDT⁺ <i>C</i>. <i>difficile</i> in patients and strain collections but the prevalence of human A⁻B⁻CDT⁺ <i>C</i>. <i>difficile</i> is rarely investigated. This study highlights the occurrence of A⁻B⁻CDT⁺ strains of <i>C</i>. <i>difficile</i> in symptomatic patients, warranting further investigations of its role in human infection.</p></div