Distribution and characterization of Clostridium difficile isolated from dogs in Japan.

We collected 204 nondiarrhoeic canine fecal samples and isolated 68 Clostridium difficile strains from 62 of these samples. Strains were grouped into 29 PCR ribotypes. Only 47% of the strains were toxigenic.

Serotype and antimicrobial resistance patterns of Salmonella isolates from commercial birds and poultry environment in Mississippi.

To obtain information about Salmonella from commercial birds and poultry environments within Mississippi, 50 Salmonella enterica isolates were collected and characterized by intergenic sequence ribotyping (ISR) serotyping and by determining antimicrobial resistance. ISR assigned serotype to all 50 Salmonella enterica isolates whereas the Kauffman-White-LeMinor antibody-based scheme assigned serotype to 48. Agreement between both methods was K = 89.58. Within the set, 12 serotypes were detected. The antimicrobial resistance patterns (ARP) of 12 serotypes, namely Enteritidis, Typhimurium, Kentucky, Bredeney, Mbandaka, Saintpaul, Montevideo, Cubana, Lille, Senftenberg, Johannesburg, and one serotype UN0094, were determined using minimum inhibitory concentration values. The antibiograms demonstrated differences between Salmonella serotypes and among isolates of the same serotype. All isolates were 100% susceptible to enrofloxacin and trimethoprim/sulfamethoxazole. The number of antimicrobials to which the isolates were resistant ranged from two to nine. Twenty-two different ARPs were identified and ARP1, with resistance to spectinomycin and sulfadimethoxine, was most frequently observed. Forty isolates (80%) were resistant to three or more antimicrobials and were thus designated multidrug resistant. Detection of a unique serotype, and variation in antibiograms within the set, demonstrates that it is important to survey isolates periodically from a region to follow epidemiologic trends.

Novel molecular type of Clostridium difficile in neonatal pigs, Western Australia.

Clostridium difficile causes neonatal enteritis in piglets; strains of PCR ribotype 078 are most commonly identified. We investigated C. difficile prevalence in piglets in Australia and isolated a novel strain with a unique pathogenicity locus. In a mouse infection model, this strain produced more weight loss than did a ribotype 078 strain.

Infrequent intrahousehold transmission of Clostridioides difficile between pet owners and their pets.

Companion animals have been shown to carry Clostridioides difficile strains that are similar or identical to strains found in people, and a small number of studies have shown that pets carry genetically identical C. difficile isolates as their owners, suggesting inter-species transmission. However, the directionality of transmission is ultimately unknown, and the frequency with which animals acquire C. difficile following their owners' infection is unclear. The goal of this study was to assess how often pets belonging to people with C. difficile infection carry genetically related C. difficile isolates. We enrolled pet owners from two medical institutions (University of Pennsylvania Health System (UPHS) and The Ohio State University Wexner Medical Center (OSUWMC)) who had diarrhoea with or without positive C. difficile assays and tested their faeces and their pets' faeces for C. difficile using both anaerobic culture and PCR assays. When microorganisms were obtained from both the owner and pet and had the same toxin profile or ribotype, isolates underwent genomic sequencing. Faecal samples were obtained from a total of 59 humans, 72 dogs and 9 cats, representing 47 complete households (i.e. where a sample was available from the owner and at least one pet). Of these, C. difficile was detected in 30 humans, 10 dogs and 0 cats. There were only two households where C. difficile was detected in both the owner and pet. In one of these households, the C. difficile isolates were of different toxin profiles/ribotypes (A+/B+ / RT 499 from the owner, A-/B- / RT PR22386 from the dog). In the other household, the isolates were genetically identical (one SNP difference). Interestingly, the dog from this household had recently received a course of antibiotics (cefpodoxime and metronidazole). Our findings suggest that inter-species transmission of C. difficile occurs infrequently in households with human C. difficile infections.

Clonality and virulence traits of Escherichia coli associated with haemorrhagic septicaemia in turkeys.

Fifty-five clinical isolates of avian pathogenic Escherichia coli (APEC) from seven outbreaks of acute haemorrhagic septicaemia in turkeys were characterized by serotyping, plasmid profiling including restriction analysis with HindIII, ribotyping with EcoRI and HindIII, multilocus sequence typing (MLST) and virulence profiling. A clonal relationship was demonstrated for each outbreak according to serotype, plasmid profiling, ribotyping, and MLST. In addition, isolates demonstrated highly similar virulence profiles, as all isolates were positive for F11 pili and possessed genes encoding aerobactin (iucD), increased serum survival (iss), temperature-sensitive haemagglutinin (tsh) and colicin V plasmid operon genes (cva/cvi). However, only 20% of the isolates produced colicin V and 42% exhibited serum resistance. All strains with O group O111 and a single O18ac strain (demonstrating non-clonal DNA profiles) were positive for enteroaggregative heat-stabile toxin (EAST1), while isolates of a single outbreak all possessed the enteroaggregative toxin gene (astA). All isolates were negative for genes encoding verocytotoxins (vtx/stx), iron-repressible protein (irp2), P-fimbria (papC), invasion plasmid antigen (ipaH), attaching and effacing gene (eae), enterohaemolysin (ehxA), and enterotoxins LT, STIa (ST(p)) and STIb (ST(h)). In conclusion, highly similar virulence profiles were demonstrated for isolates of E. coli associated with a single well-defined lesion type of colibacillosis in turkeys; acute haemorrhagic septicaemia. The isolates obtained, however, demonstrated a different phylogenetic background, underlining the importance of using well-defined strain collections for characterization of APEC pathotypes.

Clostridium difficile and methicillin-resistant Staphylococcus aureus shedding by slaughter-age pigs.

BACKGROUND: Clostridium difficile and methicillin-resistant Staphylococcus aureus are critical human pathogens and of increasing concern in food animals. Because of the apparent impact of age on prevalence of these organisms, studies of slaughter age pigs are important when considering the potential for contamination of food. This study evaluated C. difficile and MRSA shedding by slaughter age pigs from farms across Canada. RESULTS: Clostridium difficile was isolated from 30/436 (6.9%) samples from 15/45 (33%) farms. After adjusting for clustering at the herd level, the prevalence was 3.4%. Ribotype 078 (toxinotype V, North American Pulsotype 7) was the most common strain, accounting for 67% of isolates. MRSA was isolated from 21/460 (4.6%) pigs from 5/46 (11%) farms. The prevalence in pigs after adjusting for clustering at the herd level was 0.2%. Seven different spa types were identified, with 3 related spa types (t011, t034, new) accounting for 16 (76%) consistent with ST398 predominating. Both MRSA and C. difficile samples were collected from 45 farms. Both MRSA and C. difficile were detected on 2 (4.4%), with C. difficile only on 13 (29%), MRSA only on 3 (6.7%) and neither on 27 (60%). CONCLUSIONS: The prevalence of C. difficile and MRSA in slaughter age pigs was relatively low, particularly in comparison with studies involving younger pigs. The predominance of C. difficile ribotype 078 and MRSA ST398 was not surprising, but there was diversity in strain types and the majority of isolates of both organisms were strains that can be found in humans. While the prevalence of C. difficile and MRSA in slaughter age pigs was relatively low, there is clearly potential for contamination of meat from healthy pigs carrying this pathogen into slaughterhouses.

Comparison of Clostridioides difficile strains from animals and humans: First results after introduction of C. difficile molecular typing and characterization at the Istituto Zooprofilattico Sperimentale of Piemonte, Liguria e Valle d'Aosta, Italy.

PCR ribotypes (RTs027 and 078) are known causes of Clostridioides difficile infection (CDI) in humans. Molecular typing and characterization of 39 C. difficile strains isolated from samples from humas and animals in 2016-2018 indicated an overlap of RTs between community-acquired patients (CA-CDI) and domestic animals from the same geographical area; 14 RTs were identified: 12 RTs were positive for toxins A/B; RT078, RT080 and RT126 were also positive for binary toxin (CDT). Most of the RTs from the animals (RTs020, 078, 106, 126) were also detected in the samples from humans. Strains grouped into three clusters: cluster I included prevalently human strains, mainly RT 018; clusters II and III included strains from humans and animals, mainly RT078 and RT020. The CA-CDI strains suggested animals as a reservoir of C. difficile isolated together with other microorganisms from animals, highlighting the association of enteric pathogens as a cause of infection and death.

Prevalence of Clostridium difficile in retail pork.

Clostridium difficile was isolated from 1.8% (7/393) of retail pork samples obtained from 4 Canadian provinces. Five ribotypes and 3 toxinotypes were identified. Three isolates were indistinguishable from the international outbreak strain ribotype 027 and were toxinotype III. Although the implications for food safety practices remain elusive, the frequency of toxigenic isolates and isolates indistinguishable from known human pathogenic strains suggests contaminated pork may be a source of C. difficile in humans.

Molecular Diagnosis of Mycoplasma Species Infection in Camels Using Semi-nested PCR.

BACKGROUND AND OBJECTIVE: Bacteriological isolation and identification of Mycoplasma species is difficult and time-consuming, therefore, molecular identification of Mycoplasma using PCR targeting specific genes is considered a specific and sensitive method for identification. The aim of current study was to isolate, characterize Mycoplasma infection in dromedary camels in Saudi Arabia. MATERIALS AND METHODS: Nasal swabs were randomly collected from 93 camels and tested for Mycoplasma by sequencing of their 16S rRNA genes using universal primers. RESULTS: The 93 samples, 24 were positive for Mycoplasma. However, no positive results were obtained using species-specific primers for Mycoplasma arginine, M. bovis or M. mycoides subsp. mycoides, thus, 16S rDNA sequencing methods and semi-nested PCR were employed. Sequences were matched to those in GenBank and phylogenetic analysis was performed. Mycoplasma edwardii (77-84% similarity with Mycoplasma edwardii ATCC 23462) and one isolate of Mycoplasma yeastsii (100% similarity with M. yeastsii GM274B) were identified. Further, some Mycoplasma species were identified as previously uncultured. The incidence of Mycoplasma infection in camels in Taif city, Saudi Arabia, was approximately 26%. CONCLUSION: This study provides insights into the accuracy and efficiency of PCR and universal primers for the detection and identification of Mycoplasma, thereby circumventing conventional culturing methods that require several days to complete and exhibit low accuracy.

Characterization of four Escherichia albertii isolates collected from animals living in Antarctica and Patagonia.

Escherichia albertii is a recently discovered species with a limited number of well characterized strains. The aim of this study was to characterize four of the E. albertii strains, which were among 41 identified Escherichia strains isolated from the feces of living animals on James Ross Island, Antarctica, and Isla Magdalena, Patagonia. Sequencing of 16S rDNA, automated ribotyping, and rep-PCR were used to identify the four E. albertii isolates. Phylogenetic analyses based on multi-locus sequence typing showed these isolates to be genetically most similar to the members of E. albertii phylogroup G3. These isolates encoded several virulence factors including those, which are characteristic of E. albertii (cytolethal distending toxin and intimin) as well as bacteriocin determinants that typically have a very low prevalence in E. coli strains (D, E7). Moreover, E. albertii protein extracts caused cell cycle arrest in human cell line A375, probably because of cytolethal distending toxin activity.

Molecular analysis of Clostridium difficile isolates recovered from horses with diarrhea.

Clostridium difficile is an important cause of diarrhea in horses, causing sporadic and epidemic disease of varying severity. This study evaluated the molecular characteristics of 48 C. difficile isolates recovered from diarrheic horses admitted to a veterinary hospital by using PCR-ribotyping and toxin gene profile. Additionally, feces were tested for the presence of C. difficile toxin A/B via enzyme immunosorbant assay (EIA) in 38 horses. The toxin genes tcdA, tcdB and cdtB were present in 27 (56.25%), 35 (72.91%) and 2 (4.1%) strains, respectively. Eight isolates (16.6%) were A(-)B(+) variants. Thirteen of forty-eight isolates (27.0%) did not posses any toxin genes (A(-)B(-)CDT(-)). A positive EIA result was reported in 17 (44%) of the cases. There was no association between the presence of different ribotypes or strains and toxin gene(s) profiles and the clinical outcome.

Natural and experimental infection of neonatal calves with Clostridium difficile.

Clostridium difficile toxins were associated with calf diarrhea in a recent retrospective study; however, no causal relationship has been prospectively investigated. This infection study tested whether the oral inoculation of neonatal calves with a toxigenic strain of C. difficile (PCR-ribotype 077) results in enteric disease. Fourteen 6-24 h old male colostrums-fed Holstein calves, received either three doses of C. difficile (1.4 x 10(8) +/- 3.5 x 10(8) cfu) (n = 8) or sterile culture broth (n = 6). Calves were euthanized on day 6 or after the onset of diarrhea, whichever came first. Fecal and intestinal samples were blindly cultured for C. difficile, and tested for its toxin A/B (C. difficile TOX A/B II ELISA, Techlab). PCR-ribotyping was used to compare inoculated and recovered isolates. Diarrhea was observed in all control calves and 3/8 of inoculated calves (p = 0.03), but it did not occur in calves that tested positive for C. difficile toxins. Fecal toxins were identified only from two controls. PCR-ribotyping confirmed the presence of C. difficile PCR-ribotype 077 in samples of all inoculated calves, but not from controls. The identification of five other PCR-ribotypes in 3/8 (37.5%) and 2/6 (33.3%) of inoculated and control calves, respectively, indicated early natural infection (< or = 24h of age). Five of 14 cecal samples had C. difficile (p = 0.01). In conclusion, the oral administration of C. difficile PCR-ribotype 077 to neonatal calves resulted in fecal/intestinal colonization but not in detection of toxins, or signs of enteric disease. Further studies are required to investigate the clinical relevance of C. difficile in calves.

Phenotypic and genotypic characterization of Paenibacillus larvae isolates.

Paenibacillus larvae is the causative agent of American Foulbrood (AFB), a severe disease of honeybees (Apis melifera). The aim of this work was to develop a strategy for the subtyping and the epidemiological analysis of P. larvae. Phenotypic characterisation, susceptibility to several antibiotics, electrophoresis of whole bacterial proteins, rep-PCR, ribotyping and DGGE were assessed using a collection of P. larvae isolates from different Uruguayan and Argentinean locations. Results indicated that there are two P. larvae genotypes circulating in Uruguay ERIC I-BOX A (worldwide distributed) and ERIC I-BOX C (exclusively detected in Argentina until this study). These results suggest that P. larvae isolates had moved between Argentina and Uruguay, probably through the Uruguay River. Patterns of whole bacterial proteins, DGGE and ribotyping did not improve the P. larvae intraspecific discrimination. Antibiotic susceptibility assays showed that 100% isolates were OTC-sensitive and 22% (belonging to ERIC I-BOX A group) were sulfisoxazole-resistant. This work may contribute to the elucidation of basic aspects related to the epidemiology of AFB in Uruguay and in the region.

Phenotypic and molecular epidemiologic evaluation of a Nocardia farcinica mastitis epizootic.

Nineteen putative Nocardia farcinica isolates epidemiologically associated with intramammary products containing neomycin were obtained from clinical cases in a Canada-wide mastitis epizootic. Epidemiologic investigations were unable to identify the mechanism for transmission. To evaluate the hypotheses generated (intrinsic versus extrinsic contamination) and to confirm the identity of N. farcinica, we compared these isolates phenotypically (biochemicals and antimicrobial susceptibility studies) and genotypically (16S rRNA gene sequencing analysis, chromosomal DNA and ribotyping profiles) with the type and reference strains of N. farcinica. Results of biochemical studies and 16S rRNA gene sequencing identified the isolates as N. farcinica. Results of chromosomal DNA and ribotyping profiles and antimicrobial resistance to amikacin indicated all were a unique clone of N. farcinica that differed from the control isolates. Our study suggests the epizootic was caused by transmission of a unique clone of N. farcinica through intrinsically contaminated dry cow intramammary products rather than an extrinsic source.

Pheno- and genotyping of Staphylococcus epidermidis isolated from bovine milk and human skin.

The purpose of this study was to improve our knowledge concerning the epidemiology and strain diversity of Staphylococcus epidermidis isolated from bovine milk in commercial dairy herds. A total of 341 S. epidermidis isolates obtained from cows' milk (317), farmers (17) and patients (7) were characterized. Of these 105 isolates were from cows' milk in two farms, where also 17 isolates were sampled from farmers. The remaining 212 isolates from cows' milk were from 170 farms. All isolates were examined by antimicrobial susceptibility, whereas 202 were examined by pulsed-field gel electrophoresis (PFGE) and 122 by ribotyping. PFGE showed single patterns in the human strains with one exception; one strain was categorised as the same clone as four of the milk strains. PFGE divided 73 of the milk strains into 62 different patterns. The PFGE method had high discriminatory power and shows that many different S. epidermidis types exist in milk samples. Antibiotic resistance patterns matched the SmaI profiles closely in the two herds, but poorly in the routinely collected milk samples. Isolates from herd 1 showed one to five patterns, depending on the typing method used. Isolates from the milker's skin showed one pattern, which was identical to the most common pattern found in the milk isolates. Isolates from herd 2 showed three to four patterns, two of these being identical to skin isolates from the milker. As dairy cows are not a natural host for S. epidermidis the results suggest a human source of these udder infections.

Fatal enterocolitis in Asian elephants (Elephas maximus) caused by Clostridium difficile.

Two cases of fatal enteritis caused by Clostridium difficile in captive Asian elephants are reported from an outbreak affecting five females in the same zoo. Post mortem examination including histopathology demonstrated fibrinonecrotic enterocolitis. C. difficile was isolated by selective cultivation from two dead and a third severely affected elephant. Four isolates were obtained and found positive for toxin A and B by PCR. All isolates were positive in a toxigenic culture assay and toxin was demonstrated in the intestinal content from one of the fatal cases and in a surviving but severely affected elephant. PCR ribotyping demonstrated that the C. difficile isolates shared an identical profile, which was different from an epidemiologically unrelated strain, indicating that the outbreak was caused by the same C. difficile clone. It is speculated that the feeding of large quantities of broccoli, a rich source of sulforaphane, which has been shown to inhibit the growth of many intestinal microorganisms may have triggered a subsequent overgrowth by C. difficile. This is the first report of C. difficile as the main cause of fatal enterocolitis in elephants. The findings emphasize the need to regard this organism as potentially dangerous for elephants and caution is recommended concerning antibiotic treatment and feeding with diets containing antimicrobials, which may trigger an expansion of a C. difficile population in the gut.

Interleukin-8 expression by mammary gland endothelial and epithelial cells following experimental mastitis infection with E. coli.

Epithelial and endothelial cells play a pivotal role in initiating and controlling the movement of leukocytes into tissues during inflammation through the production of cytokines and chemokines such as interleukin-8 (IL-8). In situ hybridization with an IL-8 riboprobe was used to determine IL-8 mRNA expression by mammary gland epithelial and endothelial cells in cows with experimental Escherichia coli mastitis. Epithelial cells of the gland, especially surrounding the alveoli, had increased IL-8 mRNA levels at all time points at which tissue samples were collected (8, 12, and 24h) after E. coli challenge. Levels of IL-8 expression in the epithelial cells decreased at 24h post-infection. IL-8 expression by mammary gland endothelial cells was low, but did increase slightly at 24h post-infection. Both epithelial and endothelial cells of the mammary gland can contribute to the production of IL-8 that is typically seen in coliform mastitis.

Ribotyping of Streptococcus uberis from a dairy's environment, bovine feces and milk.

Streptococcus uberis is a major cause of bovine mastitis and infections commonly result from environmental exposure to the pathogen. To identify specific sources of mastitis-causing S. uberis strains, samples were collected monthly from the environment and feces of dry cows in a grazing herd. Environmental and fecal strains of S. uberis were compared to those found in milk. S. uberis was detected in 63% of 94 environmental samples, including water, soil, plant matter, bedding material, flies, and hay, in 23% of 107 fecal samples, and in 4% of 787 milk samples. Automated PvuII ribotyping revealed 48 ribotypes among 266 isolates. Per sample, up to five ribotypes were detected. The distribution of ribotypes did not differ significantly among environmental, fecal and milk samples. Specific environmental sources or strains of udder-pathogenic S. uberis were not identified. Fecal shedding was not persistent and did not differ between dry-off and calving. The proportion of fecal samples containing S. uberis was highest during the summer grazing season. S. uberis was common in farm soil (31 of 35 samples) but not in non-farm soil (0 of 11 samples). We hypothesize that fecal shedding of S. uberis may play a role in maintenance of S. uberis populations in the dairy ecosystem.

Antimicrobial susceptibility and molecular characterization of avian pathogenic Escherichia coli isolates.

Ninety-five avian pathogenic Escherichia coli (APEC) isolates recovered from diagnosed cases of avian colibacillosis from North Georgia between 1996 and 2000 were serotyped and examined for typical virulence-factors, susceptibility to antimicrobials of human and veterinary significance, and genetic relatedness. Twenty different serotypes were identified, with O78 being the most common (12%). The majority of the avian E. coli isolates (60%), however, were non-typeable with standard O antisera. Eighty-four percent of isolates were PCR positive for the temperature-sensitive hemagglutinin (tsh) gene and 86% positive for the increased serum survival (iss) gene. Multiple antimicrobial-resistant phenotypes (> or =3 antimicrobials) were observed in 92% of E. coli isolates, with the majority of isolates displaying resistance to sulfamethoxazole (93%), tetracycline (87%), streptomycin (86%), gentamicin (69%), and nalidixic acid (59%). Fifty-six E. coli isolates displaying resistance to nalidixic acid were co-resistant to difloxacin (57%), enrofloxacin (16%), gatifloxacin (2%), and levofloxacin (2%). DNA sequencing revealed point mutations in gyrA (Ser83-Leu, Asp87-Tyr, Asp87-Gly, Asp87-Ala), gyrB (Glu466-Asp, Asp426-Thr), and parC (Ser80-Ile, Ser80-Arg). No mutations were observed in parE. Twelve of the quinolone-resistant E. coli isolates were tolerant to cyclohexane, a marker for upregulation of the acrAB multi-drug resistance efflux pump. Quinolone-resistant isolates were further genetically characterized via ribotyping. Twenty-two distinct ribogroups were identified, with 61% of isolates clustering into four major ribogroups, indicating that quinolone resistance has emerged among multiple avian pathogenic E. coli serogroups and chromosomal backgrounds.