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.

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.

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.

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.

Faecal shedding of antimicrobial-resistant Clostridium difficile strains by dogs.

OBJECTIVES: To longitudinally assess the shedding of antimicrobial resistant Clostridium difficile strains by clinically healthy dogs raised at breeding facilities. METHODS: 18 puppies from three different litters (#1, 2 and 3) were sampled weekly from parturition to day 20-55 postpartum. Faecal samples from the mothers of litters #2 and 3 were also available for analysis. Bacterial isolates were ribotyped, tested for in vitro antimicrobial susceptibility and further characterised. RESULTS: C. difficile was recovered from all sampled animals of litters #1 and 2, and a third of puppies from litter #3, but marked differences in C. difficile recovery were detected in different age groups (0-100%). Recovered PCR ribotypes included 056 (22 isolates), 010 (6 isolates), 078 and 213 (2 isolates each), and 009 and 020 (1 isolate each). Different ribotypes were shed by four individual animals. Regardless of their origin and ribotype, all isolates demonstrated full resistance to levofloxacin. Additionally, all but one isolate (belonging to ribotype 078) were resistant to ertapenem, and all ribotype 010 isolates displayed high-level resistance to clindamycin, clarithromycin and erythromycin. A single ribotype 078 isolate showed metronidazole heteroresistance. CLINICAL SIGNIFICANCE: Healthy dogs can shed antimicrobial-resistant C. difficile strains.

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.

Postoperative Clostridium difficile infection with PCR ribotype 078 strain identified at necropsy in five Thoroughbred racehorses.

Clostridium difficile is an important cause of acute enterocolitis in horses. We describe five cases of C difficile infection occurring postoperatively in Thoroughbred racehorses. Following diarrhoea or colic accompanied by a marked increase in packed cell volume (to ≥60 per cent) and leucopenia (≤4000 cells/µl) within two to four days after surgery in all five horses, four of them died or were euthanased because of colitis or severe diarrhoea. In these four horses, necrotising entero-typhlo-colitis was revealed by postmortem examination, and C difficile was recovered from the contents of the small and/or large intestine. The remaining horse was euthanased because of marked decline in general condition and the presence of a lung abscess, from which C difficile was isolated. The horse had had severe postoperative diarrhoea before the onset of respiratory disorder; laboratory tests for C difficile were not performed on the faeces. All C difficile isolates were toxin-A-positive, toxin-B-positive and actin-specific ADP-ribosyltransferase (CDT)-positive. The isolates were indistinguishable by pulsed field gel electrophoresis analysis, PCR ribotyping, and slpA sequence typing, and the slpA sequences and PCR ribotype patterns were identical to those of known PCR type 078. This case sequence might have been healthcare-associated infection, although there was about a four-month interval between each disease onset.

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.

Antimicrobial susceptibility and molecular subtypes of Staphylococcus aureus isolated from pig tonsils and cow's milk in China.

This study investigated and compared the antimicrobial resistance patterns and ribotypes of Staphylococcus aureus isolated from pig tonsils and cow's milk in China. A total of 90 isolates of S. aureus was included: 42 strains were isolated from tonsils of pigs and 48 from half-udder milk. The broth microdilution method and the double-disc diffusion test (D test) were used for antimicrobial susceptibility testing. The mecA gene for methicillin-resistant S. aureus (MRSA) and the ermA, ermB, ermC, and msrA genes for erythromycin-resistant strains were detected by polymerase chain reaction (PCR). The isolates were ribotyped with the Riboprinter system. The highest frequency of resistance was observed with clindamycin (91.1%), followed by penicillin (90.0%), and erythromycin (85.6%). All strains were susceptible to vancomycin and trimethoprim-sulfamethoxazole. The D test showed that 54.5% (42/77) of erythromycin-resistant isolates had the constitutive resistance phenotype and 45.5% (35/77) had the inducible resistance phenotype to clindamycin. A higher proportion of resistance to cephalosporins, macrolides, fluoroquinolones, and pleuromutilins was observed in pig isolates than in milk isolates (P < 0.05). The mecA gene was detected in all MRSA isolates; 89.6% of erythromycin-resistant strains harbored the ermC gene and 16.9% harbored the ermB gene. A total of 35 different ribogroups was found among the isolates investigated; 83.3% of pig strains belonged to 1 cluster with a similarity coefficient of 0.84. In contrast, 3 main clusters were observed among 68.8% of milk strains, which indicates a high degree of host specificity.

Ce travail visait à étudier et comparer les patrons de résistance antimicrobienne et les ribotypes d'isolats de Staphylococcus aureus provenant des amygdales de porc et du lait de vache en Chine. Un total de 90 isolats de S. aureus était inclus : 42 souches provenaient des amygdales de porcs et 48 provenant de lait de 2 des 4 quartiers. La méthode de microdilution en bouillon et l'épreuve de double diffusion en disque (test D) ont été utilisées pour déterminer la sensibilité aux antibiotiques. Le gène mecA des S. aureus résistants à la méthicilline (MRSA) et les gènes ermA, ermB, ermC, et msrA des souches résistantes à l'érythromycine ont été détectés par réaction d'amplification en chaîne par la polymérase (PCR). Les isolats ont été ribotypés avec le système Riboprinter. La fréquence de résistance la plus élevée a été observée avec la clindamycine (91,1 %), suivie de la pénicilline (90,0 %) et l'érytrhomycine (85,6 %). Toutes les souches étaient sensibles à la vancomycine et au trimethoprime-sulfaméthoxazole. Le test D a montré que 54,5 % (42/77) des isolats résistants à l'érythromycine avaient le phénotype constitutif de résistance et 45,5 % (35/77) avaient le phénotype de résistance inductible à la clindamycine. Une fréquence plus élevée de résistance aux céphalosporines, aux macrolides, aux fluoroquinolones, et aux pleuromutilines était observée chez les isolats porcins comparativement aux isolats laitiers (P < 0,05). Le gène mecA a été détecté à partir de tous les isolats MRSA; 89,6 % des souches résistantes à l'érythromycine portaient le gène ermC et 16,9 portaient le gène ermB. Un total de 35 ribogroupes différents a été trouvé parmi les isolats étudiés; 83,3 % des souches porcines appartenaient à 1 regroupement avec un coefficient de similarité de 0,84. En contrepartie, 3 regroupements principaux ont été observés parmi les 68,8 % d'isolats provenant de lait, ce qui indique un degré élevé de spécificité d'hôte.(Traduit par Docteur Serge Messier).

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.

Prevalence of Clostridium difficile in horses.

Fecal samples were collected to establish the apparent prevalence of Clostridium difficile shedding in Standardbred and Thoroughbred racehorses housed at 4 racetracks and 2 breeding facilities, and in horses admitted to a referral large animal clinic. Forty-one (7.59%) of 540 racetrack horses, seven (5.83%) of 120 breeding farm horses, and four (4.88%) out of 82 horses admitted to the referral clinic were culture-positive for C. difficile. An overall fecal culture prevalence of 7.01% for C. difficile was identified in 742 fecal samples. PCR-ribotyping and toxin gene identification was performed and seventeen 17 PCR-ribotypes were identified among the 52 C. difficile isolates.

Listeriosis outbreak in dairy cattle caused by an unusual Listeria monocytogenes serotype 4b strain.

A listeriosis outbreak, in dairy cattle, with a high case mortality and acute death after onset of symptoms was investigated using gross pathology and bacteriologic approaches, including molecular characterization of a clinical Listeria monocytogenes isolate. In a herd of 315 animals, 9 animals showed clinical symptoms consistent with listeriosis, including 3 animals that died within 2-4 days after acute onset of clinical signs, 4 animals that were euthanized, and 2 that survived. Initial EcoRI ribotyping and serotyping indicated that this outbreak was caused by an unusual L. monocytogenes serotype 4b strain, which was classified into lineage III. Further characterization of this isolate by DNA sequencing-based subtyping methods indicated that the strain responsible for this outbreak represented a unique genotype as supported by its classification into a new sigB allelic type, which has not been identified previously among >290 isolates, and by compelling phylogenetic evidence. While lineage III isolates are generally rare, they seem to be more common among L. monocytogenes isolates from animals with clinical signs of listeriosis. This is the first report of a particularly severe clinical course of disease associated with infection by a lineage III strain. The high prevalence of Listeria spp., including L. monocytogenes, in the farm environments may favor emergence and evolution of novel, and possibly more virulent, L. monocytogenes strains. Continued monitoring of animal listeriosis cases and outbreaks may not only improve animal health but also aid in the early discovery of newly emerging L. monocytogenes strains.

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.

Administration of Bacillus coagulans in calves: recovery from faecal samples and evaluation of functional aspects of spores.

An investigation was carried out into the recovery from calf faeces of Bacillus coagulans spores added to the feed as probiotic. For this purpose, Bacillus coagulans spores (9 log10 CFU g⁻¹) were given daily to 10 calves during the whole farming periods; another 10 calves acted as controls. Throughout the trial the faecal spore counts were significantly (P < 0.01) higher in the treated group than in the controls (averaging 2.1 x 105 vs 3.7 x 104 CFU g⁻¹). Bacterial cells were recovered from faecal samples and ribotyping matched the strain isolated from faecal sample to the clone administered to the animals. In addition, the recovered cells were found to maintain their functionality aspects of acid production, survival in artificial gastric juice and in the presence of bile, and attachment to human intestinal epithelial cells. The results further elucidate the fate of spore formers administered to calves, and this will help in the development of new species-specific nutritional strategies.

Genetic diversity of porcine Pasteurella multocida strains from the respiratory tract of healthy and diseased swine.

A total of 382 porcine Pasteurella multocida strains, isolated from cases of pneumonia and progressive atrophic rhinitis (PAR) as well as from clinically healthy pigs of more than 150 German husbandries were characterized by detection of virulence-associated genes (VAGs) and ribotyping to understand the relationships between "commensal" and "pathogenic" strains, enabling a rational choice of vaccine strains. The diversity of the strains according to VAGs was low and mainly limited to capsular type genes (capA: 53.4%; capD: 45.8%; capF: 0.3%; cap-negative: 0.5%; hssB: 95.3%), dermonecrotoxin gene toxA (3.4%), as well as adhesion-related genes pfhaB (20.9%) and hgbB (84.3%). Ribotyping identified 13 patterns, but the vast majority of strains (95.8%) clustered in only three of these, namely IA-1 (45.5%), IA-7 (30.1%), and IIA-1 (20.2%). Pattern IA-1 was associated with capD(+) strains (93.6%) and harboured the majority of toxA(+) strains (84.6%). Pattern IA-7 mostly contained pfhaB(-), toxA(-)capA(+) strains (93.9%), while pattern IIA-1 was predominantly composed of pfhaB(+), toxA(-)capA(+) strains (87.0%). Clinical strains associated with pneumonia or PAR shared the above mentioned major ribotypes in comparable proportions with strains derived from healthy pigs, suggesting P. multocida to act more as an opportunistic than as an obligate pathogen in pigs. The limited number of subpopulations may either reflect a recent evolution of P. multocida in pigs or a selection by means of horizontal transfer of capsular genes, toxA or pfhaB. These data enforce further phylogenetic and epidemiological studies, examining the properties of different subpopulations of porcine P. multocida strains as well as factors of the porcine hosts themselves, which might be involved in disease susceptibility.

Caracterização fenotípica e molecular de amostras de Burkholderia mallei isoladas na Região Nordeste do Brasil / Phenotypic and molecular characterization of Burkholderia mallei isolated in northeastern Brazil

Objetivou-se com este trabalho realizar o estudo bioquímico e molecular de amostras de Burkholderia mallei isoladas de eqüídeos com diagnóstico clínico e sorológico para o mormo e provenientes da Região Metropolitana do Recife-PE e Zona da Mata dos Estados de Alagoas e Pernambuco. Foram realizadas as técnicas microbiológicas para o isolamento e identificação fenotípica de B. mallei e as técnicas moleculares de ribotipagem-PCR e RAPD-PCR. Das oito amostras estudadas, quatro apresentaram pequenas variações fenotípicas. Nas técnicas moleculares, as amostras formaram quatro grupos de diferentes perfis ribotípicos, demonstrando também quatro perfis genotípicos. Houve associação nos resultados da Ribotipagem-PCR e RAPD-PCR. As variações nos perfis ribotípicos e genotípicos foram associadas às diferentes regiões estudadas. De acordo com os resultados obtidos, conclui-se que as pequenas variações bioquímicas não estão associadas aos diferentes perfis moleculares e que essas diferenças demonstram uma heterogeneidade que está associada à procedência das amostras, indicando que a infecção nos animais ocorre por clones diferentes das amostras analisadas.

The objective of this paper was to study the molecular performance and phenotypic characterization of Burkholderia mallei isolated from horses with clinical and serological diagnosis of glanders, originating from the Metropolitan District of Recife and Zona da Mata of Pernambuco and Alagoas. The isolation and biochemical identification of B. mallei was carried out by microbiological and molecular techniques of PCR-fingerprinting and RAPD-PCR. From the eight samples studied, four showed little phenotype variations. In the molecular tests, the samples formed 4 groups of different ribotype profiles and 4 genotype profiles. There was some association of PCR-fingerprinting with RAPD-PCR results. It was concluded that the slight biochemical variations were not associated with different molecular profiles. They also indicated that these differences show heterogeneity associated with the origin of the sample, indicating that the infection was caused by clones of different strains and that the polymorphism of DNA observed could make it difficult to choose one standard strain for an immune prophylactic treatment of glanders.

Coagulase-negative staphylococci isolated from bovine extramammary sites and intramammary infections in a single dairy herd.

Isolates of various species of coagulase-negative staphylococci (CNS) from extramammary swab samples were compared with isolates of bovine mastitis CNS species. Swab samples were taken from perineum skin and udder skin, teat apices and teat canals of lactating dairy cows of the research dairy herd of the University of Helsinki in 1999 and 2002. In addition, hands of herd staff and liners of teat cups were sampled for CNS. CNS isolates from milk samples of subclinical or clinical mastitis in the same herd were collected during 1998-2002. Species identification was performed using phenotyping (API Staph ID 32 test) and by constructing a 16 and 23S rRNA RFLP library (ribotyping). Based on phenotype, 84% of mastitis isolates and 57% of extramammary isolates were identified at species level with >90% probability. Ribotype patterns formed 24 clusters, and 15 of them included a CNS type strain. If the ribotype clusters contained isolates of both extramammary and mastitis origin, they were further typed using pulsed-field gel electrophoresis (PFGE). The predominant CNS species in mastitis, based both on phenotyping and genotyping, were Staph. chromogenes and Staph. simulans. Phenotyping failed to identify half of the extramammary isolates. Based on phenotyping, Staph. equorum and Staph. sciuri, and based on ribotyping, Staph. succinus and Staph. xylosus, were the predominant CNS species in extramammary samples. The most common species in milk samples, Staph. chromogenes, was also isolated from several extramammary samples, and five out of ten pulsotypes were shared between mastitis and extramammary isolates, indicating that strains from udder skin are highly similar. The second commonest mastitis species, Staph. simulans, was isolated only from three extramammary samples, indicating that Staph. simulans may be more specifically associated with mastitis. Consequently, the origin of CNS mastitis may vary depending on the causing CNS species.