Clostridioides difficile as a Potential Pathogen of Importance to One Health: A Review.

Clostridioides difficile (basonym Clostridium) is a bacterial enteropathogen associated with cases of C. difficile infection that can result in pseudomembranous colitis, rapid fluid loss, and death. For decades following its isolation, C. difficile was thought to be a solely nosocomial pathogen, being isolated from individuals undergoing antimicrobial therapy and largely affecting elderly populations. More recently, C. difficile spores have been identified in the broader environment, including in food-producing animals, soil, and food matrices, in both ready-to-eat foods and meat products. Furthermore, evidence has emerged of hypervirulent ribotypes (RTs), such as RT078, similar to those cultured in asymptomatic carriers, also being identified in these environments. This finding may reflect on adaptations arising in these bacteria following selection pressures encountered in these niches, and which occurs due to an increase in antimicrobial usage in both clinical and veterinary settings. As C. difficile continues to adapt to new ecological niches, the taxonomy of this genus has also been evolving. To help understand the transmission and virulence potential of these bacteria of importance to veterinary public health, strategies applying multi-omics-based technologies may prove useful. These approaches may extend our current understanding of this recognized nosocomial pathogen, perhaps redefining it as a zoonotic bacterium. In this review, a brief background on the epidemiological presentation of C. difficile will be highlighted, followed by a review of C. difficile in food-producing animals and food products. The current state of C. difficile taxonomy will provide evidence of Clade 5 (ST11/RT078) delineation, as well as background on the genomic elements linked to C. difficile virulence and ongoing speciation. Recent studies applying second- and third-generation sequencing technologies will be highlighted, and which will further strengthen the argument made by many throughout the world regarding this pathogen and its consideration within a One Health dimension.

PCR-ribotype distribution of Clostridium difficile in Irish pigs.

Clostridium difficile is an important enteric pathogen in humans causing infections in the healthcare environment and the community. Carriage of C. difficile and C. difficile-related enterocolitis has been reported in piglets worldwide. The aim of this study was to investigate the rates of C. difficile isolation from pigs in Ireland. Faecal samples from piglet litters and sows were collected from six farms in 2015. The sows were non-diarrhoeal at the time of sampling. The diarrhoeal status of the piglets was unknown. C. difficile was isolated from 34/44 (77%) of piglet litter samples and from 33/156 (21%) of sow samples. The isolation rate in sows varied from 3 to 39% and in piglet litters from 72 to 86% depending on farm location. Toxin A and toxin B were present in 99% (66/67) of isolates; and binary toxin in 85% (57/67). Only PCR-ribotypes 078 (88%) and 193 (12%) were identified in piglets. Seven PCR-ribotypes were detected in sow C. difficile isolates: PCR-ribotypes 078 (67%), 050 (12%), 014/020 (6%), 015 (6%), 029 (3%), 035 (3%) and 193 (3%). This study shows that toxigenic C. difficile strains such as PCR-ribotype 078 can be commonly isolated from pigs at different geographical locations in Ireland. Since PCR-ribotype 078 is frequently found in humans in Ireland, this highlights the potential for interspecies transmission.

Immunoreactive cell wall proteins of Clostridium difficile identified by human sera.

Clostridium difficile is a leading cause of nosocomial infection in the developed world, causing antibiotic-associated disease in susceptible populations. The identity of immunogenic proteins is important in understanding the pathogenesis of disease and in the design of vaccines. This study analysed the sera of six patients infected during a hospital outbreak of a C. difficile ribotype 017 strain. Using a proteomics-based approach, cell wall proteins were separated by two-dimensional PAGE, and immunoreactive proteins were revealed by reaction with patient sera. The identity of immunoreactive proteins was established by MS. Forty-two different proteins were identified in total. All patient sera reacted with at least one component of the surface-layer protein (SLP), four reacted with both components (high- and low-molecular-mass SLPs), and five reacted with one other cell wall protein, suggesting that these are immunodominant antigens. The role of these proteins as potential vaccine candidates and their roles in pathogenesis deserve further study.

Emergence and control of fluoroquinolone-resistant, toxin A-negative, toxin B-positive Clostridium difficile.

BACKGROUND: Clostridium difficile is a major cause of infectious diarrhea in hospitalized patients. Between August 2003 and January 2004, we experienced an increase in the incidence of C. difficile-associated disease. We describe the investigation into and management of the outbreak in this article. METHODS: A total of 73 consecutive patients with nosocomial C. difficile-associated diarrhea were identified. C. difficile isolates were characterized using toxin-specific enzyme immunoassays, a tissue-culture fibroblast cytotoxicity assay, polymerase chain reaction (PCR), and antimicrobial susceptibility tests. Rates of recurrence and of C. difficile colitis were recorded. Changes in antibiotic use and infection control policies were documented. RESULTS: The incidence of C. difficile-associated diarrhea peaked at 21 cases per 1,000 patient admissions. Of the C. difficile isolates recovered, 85 (95%) were identical toxin A-negative and toxin B-positive strains, corresponding to toxinotype VIII and PCR ribotype 017. All clonal isolates were resistant to multiple antibiotics, including ofloxacin, ciprofloxacin, levofloxacin, moxifloxacin, and gatifloxacin (minimum inhibitory concentrations [MICs] of greater than 32 micro g/mL) and erythromycin, clarithromycin, and clindamycin (MICs of greater than 256 micro g/mL). Recurrent C. difficile-associated disease occurred in 26 (36%) of the patients. At least 10 (14%) of the patients developed C. difficile colitis. Additional infection control measures introduced included the use of ward memos, a hand-hygiene awareness campaign, increased environmental cleaning, attention to prescribing practices for antibiotics, increased awareness of diarrheal illness, and early isolation of affected patients. Total use of fluoroquinolones did not change throughout the study period. Despite persistence of this toxin-variant strain, the incidence of C. difficile-associated disease in our institution decreased to fewer than 5 cases per 1,000 admissions. CONCLUSIONS: We report on the emergence of a fluoroquinolone- and clindamycin-resistant, toxin A-negative, and toxin B-positive strain of C. difficile associated with an outbreak of C. difficile-associated disease in our institution during a 6-month period. We found that careful attention to improvement of infection control interventions was the most important means of controlling this nosocomial pathogen.

Toxin A-negative, toxin B-positive Clostridium difficile.

Clostridium difficile is a major cause of infectious diarrhea in hospitalized patients. Many pathogenic strains of Clostridium difficile produce two toxins TcdA and TcdB, both of which are pro-inflammatory and enterotoxic in human intestine. Clinically relevant toxin A-negative, toxin B-positive (A(-)B(+)) strains of Clostridium difficile that cause diarrhea and colitis in humans have been isolated with increasing frequency worldwide. This perspective describes these important toxin variant strains and highlights the need to use Clostridium difficile diagnostic methods that can detect both TcdA and TcdB.

High-level resistance to moxifloxacin and gatifloxacin associated with a novel mutation in gyrB in toxin-A-negative, toxin-B-positive Clostridium difficile.

OBJECTIVES: To determine the mechanism of high-level resistance to fluoroquinolone antimicrobials in toxin-A-negative, toxin-B-positive (A- B+) Clostridium difficile isolates. METHODS: Following culture 16-23S PCR ribotyping was used to determine genomic relationships between A- B+ C. difficile isolates. Antimicrobial susceptibilities were determined using Etests in the presence and absence of the efflux pump inhibitors reserpine (20 microg/mL), L-phenylalanine-L-arginine-beta-naphthylamide (PAbetaN; 20 microg/mL) and verapamil (100 microg/mL). Genomic regions including the quinolone-resistance-determining-region (QRDR) of gyrA and gyrB were amplified and characterized. RESULTS: PCR ribotyping profiles identified one major cluster of A- B+ C. difficile, universally resistant to the fluoroquinolones tested (ofloxacin, ciprofloxacin, levofloxacin, moxifloxacin and gatifloxacin; MICs > 32 mg/L). All isolates with high-level resistance had a transversion mutation (A-->T) resulting in the amino acid substitution Asp-426-->Val in gyrB. Non-clonal isolates were susceptible to moxifloxacin and gatifloxacin (MICs 0.3 and 0.4 mg/L, respectively) with reduced susceptibility to levofloxacin (MIC 3 mg/L) consistent with the wild-type genotype. The MICs for resistant isolates were not significantly affected by the addition of any of the efflux pump inhibitors. No amino acid substitutions were identified in the QRDR of gyrA. CONCLUSIONS: High-level resistance to fluoroquinolones in A- B+ C. difficile is associated with a novel transversion mutation in gyrB. The emergence of universal resistance in different C. difficile strain types may be a factor promoting outbreaks in hospitals.

Multi-drug resistance in Salmonella enterica: efflux mechanisms and their relationships with the development of chromosomal resistance gene clusters.

Bacterial drug resistance represents one of the most crucial problems in present day antibacterial chemotherapy. Of particular concern to public health is the continuing worldwide epidemic spread of Salmonella enterica serovar Typhimurium phage type DT104 harbouring a genomic island called Salmonella genomic island I (SGI-1). This island contains an antibiotic gene cluster conferring resistance to ampicillin, chloramphenicol, florfenicol, streptomycin, sulfonamides and tetracyclines. These resistance genes are assembled in a mosaic pattern, indicative of several independent recombinational events. The mobility of SGI-1 coupled with the ability of various antibiotic resistance genes to be integrated and lost from the chromosomal resistance locus allows for the transfer of stable antibiotic resistance to most of the commonly used antibiotics and adaptation to new antibiotic challenges. This, coupled with the incidence of increasing fluoroquinolone resistance in these strains increases the risk of therapeutic failure in cases of life-threatening salmonellosis. Fluoroquinolone resistance has largely been attributed to mutations occurring in the genes coding for intracellular targets of these drugs. However, efflux by the AcrAB-TolC multi-drug efflux pump has recently been shown to directly contribute to fluoroquinolone resistance. Furthermore, the resistance to chloramphenicol-florfenicol and tetracyclines in DT104 isolates, is due to interaction between specific transporters for these antibiotics encoded by genes mapping to the SGI-1 and the AcrAB-TolC tripartite efflux pump. The potential for the use of efflux pump inhibitors to restore therapeutic efficacy to fluoroquinolones and other antibiotics offers an exciting developmental area for drug discovery.

Characterization of a collection of Enterobacter sakazakii isolates from environmental and food sources.

Enterobacter sakazakii has emerged as a rare cause of neonatal meningitis, septicemia and enterocolitis. Contaminated infant milk formula (IMF) has been identified as one infection route. A small number of clinical outbreaks have been epidemiologically linked to IMF contaminated post-pasteurization during manufacture and/or mishandled when reconstituted. Currently no agreed standardized typing protocol has been developed to trace E. sakazakii. The objectives of this study were to apply biochemical and genetic methods to characterize 51 environmental and food E. sakazakii isolates and 6 E. sakazakii type strains. Isolates were presumptively identified using biochemical profiles based on API 20E and ID32E methods and by culture on differential selective Druggan Forsythe Iversen (DFI) agar. Identification was subsequently confirmed by real time polymerase chain reaction (PCR). All but one of the isolates was identified as E. sakazakii by biochemical profiling. One isolate was identified as Escherichia vulneris by ID 32E and as Pantoea agglomerans by API 20E. All isolates produced green/blue colonies on DFI medium characteristic of this organism. Real time PCR could differentiate between E. sakazakii, Enterobacter spp. and other Enterobacteriacae. Analysis of RAPD banding patterns revealed 3 major clusters of E. sakazakii. There was a large degree of diversity noted amongst the remaining isolates. Our findings indicate that RAPD may be applied as a useful and reliable tool for direct comparison of E. sakazakii isolates providing traceability through the infant formula food chain.

Toxigenic C. difficile induced inflammatory marker expression by human intestinal epithelial cells is asymmetrical.

Clostridium difficile infection of the intestinal epithelium and consequent pseudomembranous colitis is an important cause of morbidity and mortality. Pathogenesis has been ascribed exclusively to toxin production. Using in vitro models of human intestinal epithelial layers, we show that exposure to toxigenic C. difficile upregulates epithelial expression of IL-8 and ICAM-1, two molecules important in neutrophil chemoattraction and adhesion and subsequent inflammation. IL-8 production was also stimulated by toxin-containing supernatants. C difficile induced IL-8 release was inhibited by specific antiserum. Increased ICAM-1 expression only occurred after basolateral exposure to C. difficile while apical exposure had no effect on ICAM-1 expression. However, transepithelial electrical resistance was impaired by apical exposure to bacterial suspensions. We suggest that apical exposure to C. difficile induces changes in epithelial layer integrity which allows the bacteria and/or the toxin access to the basolateral compartment where pathogenic inflammatory mechanisms are activated.

Antimicrobial resistance in nontyphoidal Salmonella from food sources in Colombia: evidence for an unusual plasmid-localized class 1 integron in serotypes Typhimurium and Anatum.

Seventy-two isolates representing 18 serotypes recovered from various food samples collected in Colombia were tested for antimicrobial susceptibilities. The collection was further characterized for extended-spectrum cephalosporin, aminoglycoside, and tetracycline resistance markers. Multidrug resistant (MDR) isolates were further investigated for class 1 integrons and were evaluated for the presence of conjugative plasmids along with a determination of the incompatibility group by polymerase chain reaction (PCR). Antibiogram analysis showed that the incidence rate of ceftiofur resistance was moderately high (15%). A similar level of resistance to neomycin and oxytetracycline (11% and 10%, respectively) was also observed. There was a high prevalence of gene cassettes as part of one or more class 1 integrons (61%), many of which contained determinants that contributed to the resistance profile. Class 1 integrons identified in MDR Salmonella enterica serotypes Typhimurium and Anatum isolates were characterized. Sequencing identified several incomplete open reading frames (ORFs) as part of a gene cassette (bla-( imp-13 ), dfr7, blr1088, and aac8) along with a complete gene cassette (bla-(oxa2)) in each case. A mosaic of gene cassettes was identical in the two Salmonella serotypes. These integrons were located to a conjugative replicon. Plasmid profiling and incompatibility typing identified three plasmids belonging to Inc groups A/C, P, and W. Our study highlights the role of integrons, contributing to a MDR phenotype that is capable of dissemination to other bacteria.

Antimicrobial resistance in isolates of Salmonella spp. from pigs and the characterization of an S. Infantis gene cassette.

One hundred and seven Salmonella isolates of various serotypes were investigated for resistance to a panel of nine antimicrobial agents by standardized methods. Thirty four isolates were susceptible to the selected antimicrobial agents. Thirty-six (of 107) were resistant to three or more antimicrobial agents and defined as multidrug resistant (MDR). Salmonella Typhimurium was the most resistant serotype. All resistant isolates were examined for the presence of class 1 integrons. Thirty-two integron-associated gene cassettes (of varying sizes) were identified. A 1,000-bp amplicon similar to that flanking the distal region of the Salmonella Genomic Island (SGI)-1 in Salmonella Typhimurium was detected in a majority of the S. Typhimurium isolates in this study. In contrast, a 1,800-bp amplicon was identified in all Salmonella Infantis isolates. This amplicon was completely characterized in one isolate. The presence of class 1 integrons in Salmonella spp. in pigs may be important if these zoonotic pathogens were to enter the food chain.

Clostridium difficile toxoid vaccine in recurrent C. difficile-associated diarrhea.

BACKGROUND & AIMS: Recurrent C difficile -associated diarrhea (CDAD) is associated with a lack of protective immunity to C difficile toxins. A parenteral C difficile vaccine containing toxoid A and toxoid B was reported previously to be safe and immunogenic in healthy volunteers. Our aim was to examine whether the vaccine is also well tolerated and immunogenic in patients with recurrent CDAD. METHODS: Subjects received 4, 50-microg intramuscular inoculations of the C difficile vaccine over an 8-week period. Serum antitoxin antibodies were measured by ELISA, and toxin neutralizing activity was evaluated using the tissue culture cytotoxin assay. RESULTS: Three patients with multiple episodes of recurrent CDAD were vaccinated. Two of the 3 showed an increase in serum IgG antitoxin A antibodies (3-fold and 4-fold increases, respectively) and in serum IgG antitoxin B antibodies (52-fold and 20-fold, respectively). Both also developed cytotoxin neutralizing activity against toxin A and toxin B. Prior to vaccination, the subjects had required nearly continuous treatment with oral vancomycin for 7, 9, and 22 months, respectively, to treat recurrent episodes of CDAD. After vaccination, all 3 subjects discontinued treatment with oral vancomycin without any further recurrence. CONCLUSIONS: A C difficile toxoid vaccine induced immune responses to toxins A and B in patients with CDAD and was associated with resolution of recurrent diarrhea. The results of this study support the feasibility of active vaccination against C difficile and its toxins in high-risk individuals but must be validated in larger, randomized, controlled trials.

Human antibody response to surface layer proteins in Clostridium difficile infection.

Clostridium difficile is a major cause of infectious diarrhoea in hospitalised patients. Surface layer proteins (SLPs) are the most abundant surface localised proteins expressed by C. difficile. The aim of this study was to examine the humoral immune response to C. difficile SLPs and its potential role in protection from C. difficile associated diarrhoea (CDAD). Serum antibodies to SLPs from C. difficile were measured by ELISA in a cohort of 146 patients (55 patients with CDAD, 34 asymptomatic carriers, and 57 controls). No significant difference was detected in serum IgM, IgA or IgG antibody levels between cases, carriers or control groups at any of the time points tested. However, patients with recurrent episodes of C. difficile diarrhoea had significantly lower IgM-anti-SLP levels than patients with a single episode on days 1, 3, 6 and 9 (p = 0.05, p = 0.009, p = 0.02, p = 0.049). The adjusted odds ratio for recurrent diarrhoea associated with a low day 3 serum IgM anti-SLP antibody level was 24.5 (95% confidence interval; 1.6-376.3). Further studies which examine the specific anti-SLP antibody responses to the colonising strain are warranted to determine if immune responses to C. difficile SLPs play a role in protection from CDAD.