Prevalence and Genetic Basis of Antimicrobial Resistance in Non-aureus Staphylococci Isolated from Canadian Dairy Herds.

Emergence and spread of antimicrobial resistance is a major concern for the dairy industry worldwide. Objectives were to determine: (1) phenotypic and genotypic prevalence of drug-specific resistance for 25 species of non-aureus staphylococci, and (2) associations between presence of resistance determinants and antimicrobial resistance. Broth micro-dilution was used to determine resistance profiles for 1,702 isolates from 89 dairy herds. Additionally, 405 isolates were sequenced to screen for resistance determinants. Antimicrobial resistance was clearly species-dependent. Resistance to quinupristin/dalfopristin was common in Staphylococcus gallinarum (prevalence of 98%), whereas S. cohnii and S. arlettae were frequently resistant to erythromycin (prevalence of 63 and 100%, respectively). Prevalence of resistance was 10% against ß-lactams and tetracyclines. In contrast, resistance to antimicrobials critically important for human medicine, namely vancomycin, fluoroquinolones, linezolid and daptomycin, was uncommon (< 1%). Genes encoding multidrug-resistance efflux pumps and resistance-associated residues in deducted amino acid sequences of the folP gene were the most frequent mechanisms of resistance, regardless of species. The estimated prevalence of the mecA gene was 17% for S. epidermidis. Several genes, including blaZ, mecA, fexA, erm, mphC, msrA, and tet were associated with drug-specific resistance, whereas other elements were not. There were specific residues in gyrB for all isolates of species intrinsically resistant to novobiocin. This study provided consensus protein sequences of key elements previously associated with resistance for 25 species of non-aureus staphylococci from dairy cattle. These results will be important for evaluating effects of interventions in antimicrobial use in Canadian dairy herds.

Prevalence of non-aureus staphylococci species causing intramammary infections in Canadian dairy herds.

Non-aureus staphylococci (NAS), the microorganisms most frequently isolated from bovine milk worldwide, are a heterogeneous group of numerous species. To establish their importance as a group, the distribution of individual species needs to be determined. In the present study, NAS intramammary infection (IMI) was defined as a milk sample containing ≥1,000 cfu/mL in pure or mixed culture that was obtained from a cohort of cows assembled by the Canadian Bovine Mastitis Research Network. Overall, 6,213 (6.3%) of 98,233 quarter-milk samples from 5,149 cows and 20,305 udder quarters were associated with an NAS IMI. Of the 6,213 phenotypically identified NAS isolates, 5,509 (89%) were stored by the Canadian Bovine Mastitis Research Network Mastitis Pathogen Collection and characterized using partial sequencing of the rpoB housekeeping gene, confirming 5,434 isolates as NAS. Prevalence of each NAS species IMI was estimated using Bayesian models, with presence of a specific NAS species as the outcome. Overall quarter-level NAS IMI prevalence was 26%. The most prevalent species causing IMI were Staphylococcus chromogenes (13%), Staphylococcus simulans (4%), Staphylococcus haemolyticus (3%), Staphylococcus xylosus (2%), and Staphylococcus epidermidis (1%). The prevalence of NAS IMI as a group was highest in first-parity heifers and was evenly distributed throughout cows in parities ≥2. The IMI prevalence of some species such as S. chromogenes, S. simulans, and S. epidermidis differed among parities. Overall prevalence of NAS IMI was 35% at calving, decreased over the next 10 d, and then gradually increased until the end of lactation. The prevalence of S. chromogenes, Staphylococcus gallinarum, Staphylococcus cohnii, and Staphylococcus capitis was highest at calving, whereas the prevalence of S. chromogenes, S. haemolyticus, S. xylosus, and S. cohnii increased during lactation. Although the overall prevalence of NAS IMI was similar across barn types, the prevalence of S. simulans, S. xylosus, S. cohnii, Staphylococcus saprophyticus, S. capitis, and Staphylococcus arlettae IMI was higher in tiestall barns; the prevalence of S. epidermidis IMI was lowest; and the prevalence of S. chromogenes and Staphylococcus sciuri IMI was highest in bedded-pack barns. Staphylococcus simulans, S. epidermidis, S. xylosus, and S. cohnii IMI were more prevalent in herds with intermediate to high bulk milk somatic cell count (BMSCC) and S. haemolyticus IMI was more prevalent in herds with high BMSCC, whereas other common NAS species IMI were equally prevalent in all 3 BMSCC categories. Distribution of NAS species IMI differed among the 4 regions of Canada. In conclusion, distribution differed considerably among NAS species IMI; therefore, accurate identification (species level) is essential for studying NAS epidemiology.

Distribution of non-aureus staphylococci species in udder quarters with low and high somatic cell count, and clinical mastitis.

The effect of non-aureus staphylococci (NAS) in bovine mammary health is controversial. Overall, NAS intramammary infections (IMI) increase somatic cell count (SCC), with an effect categorized as mild, mostly causing subclinical or mild to moderate clinical mastitis. However, based on recent studies, specific NAS may affect the udder more severely. Some of these apparent discrepancies could be attributed to the large number of species that compose the NAS group. The objectives of this study were to determine (1) the SCC of quarters infected by individual NAS species compared with NAS as a group, culture-negative, and major pathogen-infected quarters; (2) the distribution of NAS species isolated from quarters with low SCC (<200,000 cells/mL) and high SCC (≥200,000 cells/mL), and clinical mastitis; and (3) the prevalence of NAS species across quarters with low and high SCC. A total of 5,507 NAS isolates, 3,561 from low SCC quarters, 1,873 from high SCC quarters, and 73 from clinical mastitis cases, were obtained from the National Cohort of Dairy Farms of the Canadian Bovine Mastitis Research Network. Of quarters with low SCC, high SCC, or clinical mastitis, 7.6, 18.5, and 4.3% were NAS positive, respectively. The effect of NAS IMI on SCC was estimated using mixed-effect linear regression; prevalence of NAS IMI was estimated using Bayesian analyses. Mean SCC of NAS-positive quarters was 70,000 cells/mL, which was higher than culture-negative quarters (32,000 cells/mL) and lower than major pathogen-positive quarters (129,000 to 183,000 cells/mL). Compared with other NAS species, SCC was highest in quarters positive for Staphylococcus capitis, Staphylococcus gallinarum, Staphylococcus hyicus, Staphylococcus agnetis, or Staphylococcus simulans. In NAS-positive quarters, Staphylococcus xylosus (12.6%), Staphylococcus cohnii (3.1%), and Staphylococcus equorum (0.6%) were more frequently isolated from quarters with low SCC than other NAS species, whereas Staphylococcus sciuri (14%) was most frequently isolated from clinical mastitis cases. Finally, in NAS-positive quarters, Staphylococcus chromogenes, S. simulans, Staphylococcus epidermidis, and Staphylococcus haemolyticus were isolated with similar frequency from among low SCC and high SCC quarters and clinical mastitis cases. Staphylococcus chromogenes, S. simulans, S. xylosus, S. haemolyticus, S. epidermidis, S. agnetis, Staphylococcus arlettae, S. capitis, S. gallinarum, S. sciuri, and Staphylococcus warneri were more prevalent in high than in low SCC quarters. Because the NAS are a large, heterogeneous group, considering them as a single group rather than at the species, or even subspecies level, has undoubtedly contributed to apparent discrepancies among studies as to their distribution and importance in IMI and mastitis.

Comprehensive Phylogenetic Analysis of Bovine Non-aureus Staphylococci Species Based on Whole-Genome Sequencing.

Non-aureus staphylococci (NAS), a heterogeneous group of a large number of species and subspecies, are the most frequently isolated pathogens from intramammary infections in dairy cattle. Phylogenetic relationships among bovine NAS species are controversial and have mostly been determined based on single-gene trees. Herein, we analyzed phylogeny of bovine NAS species using whole-genome sequencing (WGS) of 441 distinct isolates. In addition, evolutionary relationships among bovine NAS were estimated from multilocus data of 16S rRNA, hsp60, rpoB, sodA, and tuf genes and sequences from these and numerous other single genes/proteins. All phylogenies were created with FastTree, Maximum-Likelihood, Maximum-Parsimony, and Neighbor-Joining methods. Regardless of methodology, WGS-trees clearly separated bovine NAS species into five monophyletic coherent clades. Furthermore, there were consistent interspecies relationships within clades in all WGS phylogenetic reconstructions. Except for the Maximum-Parsimony tree, multilocus data analysis similarly produced five clades. There were large variations in determining clades and interspecies relationships in single gene/protein trees, under different methods of tree constructions, highlighting limitations of using single genes for determining bovine NAS phylogeny. However, based on WGS data, we established a robust phylogeny of bovine NAS species, unaffected by method or model of evolutionary reconstructions. Therefore, it is now possible to determine associations between phylogeny and many biological traits, such as virulence, antimicrobial resistance, environmental niche, geographical distribution, and host specificity.

Molecular identification and antimicrobial susceptibility of Nocardia spp. isolated from bovine mastitis in Brazil.

Nocardia spp. infections can cause severe damage to the mammary gland due to suppurative pyogranulomatous lesions and lack of clinical cure in response to conventional antimicrobial therapy. Although Nocardia infections are considered relatively uncommon in cows, there has been an apparent worldwide increase in the incidence of bovine mastitis caused by Nocardia spp, perhaps due to environmental transmission of this ubiquitous pathogen. The objectives of present study were to determine: (i) species distribution of 80 Nocardia isolates involved in bovine mastitis (based on molecular methods); and (ii) antimicrobial susceptibility pattern of all isolates from three geographical areas in Brazil. In this study, Nocardia nova (80%) was the most frequently isolated species, followed by Nocardia farcinica (9%). Additionally, Nocardia puris, Nocardia cyriacigeorgica, Nocardia veterana, Nocardia africana, and Nocardia arthritidis were detected using 16S rRNA sequencing. This is apparently the first report of N. puris, N. veterana, N. cyriacigeorgica, N. arthritidis and N. africana in association with bovine mastitis. Based on the disk diffusion test, isolates were most frequently resistant to cloxacillin (75%), ampicillin (55%) and cefoperazone (47%), whereas few Nocardia spp. were resistant to amikacin, cefuroxime or gentamicin.

Screening of antigenemia and isolation of Cryptococcus neoformans and C. gattii from cloaca and crop of birds in the state of Paraná, Brazil / Antigenemia e tentativa de isolamento de Cryptococcus neoformans e C. gattii a partir da cloaca e inglúvio de aves no Estado do Paraná

Cryptococcus neoformans and C. gattii are associated with dry bird excreta but rarely recovered from birds' digestive tract. The objective of the present study was (1) to verify the existence of C. neoformans and C. gattii in crop and cloaca of wildlife and captivity birds hypothesizing about a possible primary source of this yeast in the excreta, and (2) to determine the fungi's invasive capability in avian species through latex agglutination. For that purpose, 172 cloacal and 77 crop samples of domestic pigeon, Passerine, and Psittacine birds were collected. None of these samples was positive, suggesting that the yeast is not saprobiotic in the digestive tract of these birds. Only one out of 82 serum samples collected from pigeons and Psittacine birds was positive (title 1:2) showing that Cryptococcus sp. probably has a low invasive capability in birds, and is thus considered only a dry excreta colonizer.

Cryptococcus neoformans e C. gattii são frequentemente isolados de excretas de aves, entretanto ocorre pouca recuperação desse fungo a partir do trato gastrintestinal. Os objetivos desse estudo foram verificar a existência de C. neoformans e C. gattii no inglúvio e na cloaca de aves de vida livre e cativeiro, avaliando uma possível fonte primária desta levedura nas excretas e determinar a capacidade invasiva do fungo em aves por meio da aglutinação em látex. Para tanto, foram coletadas 172 amostras de cloaca e 77 de inglúvio de pombos- domésticos, Passeriformes e Psittaciformes. Nenhuma amostra se mostrou positiva, sugerindo-se que o fungo não é saprobiótico do aparelho digestório destas aves. Das 82 amostras de soro colhidas a partir de pombos-domésticos e Psittaciformes, somente uma obtida a partir de pombo-doméstico se mostrou positiva (titulação 1:2), demonstrando que Cryptococcus sp. apresenta baixa capacidade invasiva em aves, sendo, portanto, considerado somente um colonizador de excretas ressequidas.