High Production of LukMF' in Staphylococcus aureus Field Strains Is Associated with Clinical Bovine Mastitis

Staphylococcus aureus, a major cause of bovine mastitis, produces a wide range of immune-evasion molecules. The bi-component leukocidin LukMF' is a potent killer of bovine neutrophils in vitro. Since the role of LukMF' in development of bovine mastitis has not been studied in natural infections, we aimed to clarify whether presence of the lukM-lukF' genes and production levels of LukMF' are associated with clinical severity of the disease. Staphylococcus aureus was isolated from mastitis milk samples (38 clinical and 17 subclinical cases) from 33 different farms. The lukM-lukF' genes were present in 96% of the isolates. Remarkably, 22% of the lukM-lukF'-positive S. aureus isolates displayed a 10-fold higher in vitro LukMF' production than the average of the lower-producing ones. These high producing isolates were cultured significantly more frequently from clinical than subclinical mastitis cases. Also, the detection of LukM protein in milk samples was significantly associated with clinical mastitis and high production in vitro. The high producing LukMF' strains all belonged to the same genetic lineage, spa-type t543. Analysis of their global toxin gene regulators revealed a point mutation in the Repressor of toxins (rot) gene which results in a non-functional start codon, preventing translation of rot. This mutation was only identified in high LukMF' producing isolates and not in low LukMF' producing isolates. Since rot suppresses the expression of various toxins including leukocidins, this mutation is a possible explanation for increased LukMF' production. Identification of high LukMF' producing strains is of clinical relevance and can potentially be used as a prognostic marker for severity of mastitis

Colonization of Extramammary Sites with Mastitis-Associated S. aureus Strains in Dairy Goats.

Staphylococcus aureus ( S. aureus), a major mastitis pathogen in dairy goats, is classified as a contagious pathogen. Although previous research has shown that extramammary body sites can be colonized with S. aureus, it is unknown whether these sites are reservoirs for intramammary infections. The aim of this research was to determine whether extramammary sites can be colonized with mastitis-associated S. aureus strains in dairy goats. Milk samples were collected from 207 primiparous goats and from 120 of these goats, extramammary site samples (hock, groin, nares, vulva and udder) were collected from a large commercial dairy goat herd in the Netherlands during four sampling visits. Extramammary site swabs and milk samples were (selectively) cultured and S. aureus isolates were spa genotyped. The prevalence of colonization of the extramammary sites at goat level was 51.7% and the prevalence of S. aureus intramammary infections was 7.2%. The nares were colonized most frequently (45%), while the groin area was colonized the least (2.5%). Six spa genotypes were identified in this herd and there was no significant difference in the distribution of spa genotypes between the milk or the extramammary sites ( p = 0.141). Both in the extramammary sites and in the milk, spa genotypes t544 (82.3% and 53.3%) and t1236 (22.6% and 33.3%) were the dominant genotypes. These results show that in goats, extramammary sites, particularly the nares, are frequently colonized with mastitis-associated S. aureus strains. Extramammary sites may, thus, be a source of S. aureus intramammary infections that are not targeted by the intervention measures aimed at preventing transmission from infected udder glands

Association of Staphylococcus aureus genotypes with milk or colonization of extramammary sites in Dutch dairy cattle indicates strain variation in reservoirs for intramammary infections

Staphylococcus aureus (S. aureus) is a major mastitis pathogen with a detrimental impact on udder health in dairy cattle. Although S. aureus is generally classified as a contagious mastitis pathogen, control measures aimed at preventing contagious transmission are not always effective. Previous studies showed that various extramammary sites can be colonized with S. aureus and could be a reservoir for S. aureus intramammary infections (IMI). The aim of this study was to determine the prevalence of S. aureus extramammary site colonization in Dutch dairy cattle and to compare the spa genotypes of S. aureus isolates from IMI to spa genotypes of isolates from extramammary sites. Six herds were visited and from cows with a composite milk somatic cell count ≥200,000 cells/mL quarter milk samples and swabs from various extramammary sites (hock, groin, udder cleft, nares, and feces) were taken. Extramammary site samples were processed by a two-step high salt selective culture and presence of S. aureus was confirmed by femA PCR. S. aureus isolates from milk and extramammary sites were compared by spa typing. The cow level colonization varied from 0% to 73%, and the prevalence of IMI in the sampled cows varied from 0% to 61% between herds. The extramammary site with the highest prevalence of colonization was the hock (23%) and the lowest prevalence of colonization was found for the nares (5%) and feces (5%). Spa typing of S. aureus isolates from either extramammary sites or milk showed that in most herds there were one or two predominant S. aureus spa genotypes present. Different S. aureus spa genotypes could be categorized into three groups based on the distribution between milk or extramammary sites: i) predominantly milk associated, ii) associated with both milk and extramammary sites, and iii) associated with extramammary sites. In conclusion, we showed that the prevalence of extramammary site colonization differed significantly between herds and extramammary sites and that specific S. aureus spa genotypes were associated with milk (IMI) or extramammary site colonization. Extramammary S. aureus reservoirs could be a source for IMI that cannot be eradicated by intervention measures aimed at contagious mastitis pathogens

A survey of foot disinfection practices for control of bovine digital dermatitis; evaluating solution depth, footbath hygiene, and the potential of footbaths as infection reservoirs for Treponema species

Bovine digital dermatitis remains a widespread endemic disease of dairy cattle worldwide. Footbathing is commonly used as a control measure and has significant economic and environmental impact. There are few studies documenting footbathing practices on dairy farms, or evaluating their suitability for achieving foot disinfection. This study describes footbathing practices on 32 farms observed in the United Kingdom, Ireland, and the Netherlands. We measured solution depth throughout footbathing and observed levels below 7cm on 9/32 farms, which leads to inadequate foot coverage. Solution depth was associated with the number of cow passages, decreasing by 1.2cm for every 100 cow passages. We also describe levels of organic matter content (g/L) throughout footbathing as a proxy for footbath hygiene. Our data indicates that almost half of footbaths (15/32) became contaminated above the 20g/L threshold to which veterinary biocides are tested for efficacy, and that organic matter content is associated with the number of cow passages per liter of footbathing solution provided. A multivariable mixed model predicted that one liter of footbathing solution per cow should be sufficient to prevent excess contamination. As a further measure of hygiene, we tested a subset of footbath samples to quantify the amount of DNA present from the Treponema species which are considered instrumental in the etiology of digital dermatitis. We did not detect Treponema DNA in footbath samples, suggesting they are unlikely to act as infection reservoirs for this disease. Multivariable mixed models including farm identity as a random effect demonstrated that for both change in solution depth and organic matter content the effect of farm-level factors was large. Because of the magnitude of this farm effect, applying model predictions will not translate to adequate solution depth and hygiene on all farms. Our data highlights the importance of footbath auditing on individual farms

Within-Household Transmission and Bacterial Diversity of Staphylococcus pseudintermedius.

Staphylococcus pseudintermedius can be transmitted between dogs and their owners and can cause opportunistic infections in humans. Whole genome sequencing was applied to identify the relatedness between isolates from human infections and isolates from dogs in the same households. Genome SNP diversity and distribution of plasmids and antimicrobial resistance genes identified related and unrelated isolates in both households. Our study shows that within-host bacterial diversity is present in S. pseudintermedius, demonstrating that multiple isolates from each host should preferably be sequenced to study transmission dynamics

Genomic analysis of European bovine Staphylococcus aureus from clinical versus subclinical mastitis

Abstract: Intramammary infections (IMI) with Staphylococcus aureus are a common cause of bovine mastitis and can result in both clinical (CM) or subclinical mastitis (SCM). Although bacterial isolates of S. aureus differ in their virulence potential it is largely unclear which bacterial virulence factors are responsible for increased clinical severity. We performed a genome wide association study and used a generalized linear mixed model to investigate the correlation between gene carriage, lineage and clinical outcome of IMI in a collection of S. aureus isolates from cattle with CM (n = 125) and SCM (n = 151) from 11 European countries. An additional aim was to describe the genetic variation of bovine S. aureus in Europa. The dominant lineages in our collection were clonal complex (CC) 151 (81/276, 29.3%), CC97 (54/276, 19.6%), CC479 (32/276, 11.6%) and CC398 (19/276, 6.9%). Virulence and antimicrobial resistance (AMR) gene carriage was highly associated with CC. Among a selection of nine virulence and AMR genes, CC151, CC479 and CC133 carried more virulence genes than other CCs, and CC398 was associated with AMR gene carriage. Whereas CC151, CC97 were widespread in Europe, CC479, CC398 and CC8 were only found in specific countries. Compared to CC151, CC479 was associated with CM rather than SCM (OR 3.62; 95% CI 1.38–9.50) and the other CCs were not. Multiple genes were associated with CM, but due to the clustering within CC of carriage of these genes, it was not possible to differentiate between the effect of gene carriage and CC on clinical outcome of IMI. Nevertheless, this study demonstrates that characterization of S. aureus CC and virulence genes helps to predict the likelihood of the occurrence of CM following S. aureus IMI and highlights the potential benefit of diagnostics tools to identify S. aureus CC during bovine mastitis

Colonization of Extramammary Sites with Mastitis-Associated <i>S. aureus</i> Strains in Dairy Goats

Staphylococcus aureus (S. aureus), a major mastitis pathogen in dairy goats, is classified as a contagious pathogen. Although previous research has shown that extramammary body sites can be colonized with S. aureus, it is unknown whether these sites are reservoirs for intramammary infections. The aim of this research was to determine whether extramammary sites can be colonized with mastitis-associated S. aureus strains in dairy goats. Milk samples were collected from 207 primiparous goats and from 120 of these goats, extramammary site samples (hock, groin, nares, vulva and udder) were collected from a large commercial dairy goat herd in the Netherlands during four sampling visits. Extramammary site swabs and milk samples were (selectively) cultured and S. aureus isolates were spa genotyped. The prevalence of colonization of the extramammary sites at goat level was 51.7% and the prevalence of S. aureus intramammary infections was 7.2%. The nares were colonized most frequently (45%), while the groin area was colonized the least (2.5%). Six spa genotypes were identified in this herd and there was no significant difference in the distribution of spa genotypes between the milk or the extramammary sites (p = 0.141). Both in the extramammary sites and in the milk, spa genotypes t544 (82.3% and 53.3%) and t1236 (22.6% and 33.3%) were the dominant genotypes. These results show that in goats, extramammary sites, particularly the nares, are frequently colonized with mastitis-associated S. aureus strains. Extramammary sites may, thus, be a source of S. aureus intramammary infections that are not targeted by the intervention measures aimed at preventing transmission from infected udder glands

<i>Enterococcus cecorum</i> lesion strains are less sensitive to the hostile environment of albumen and more resistant to lysozyme compared to cloaca strains

Enterococcus cecorum lesion strains cause more embryonic mortality after inoculation into the albumen of embryonated eggs compared to cloaca strains. We hypothesized that these strain differences are a result of differences in sensitivity to the antimicrobial effects of the albumen. In this study, the sensitivity of 14 lesion strains and 14 cloaca strains to albumen from 12-day incubated and non-incubated eggs was assessed. A major antimicrobial protein of the albumen is lysozyme and, therefore, the lysozyme resistance of all strains was determined. Albumen from 12-day incubated and non-incubated eggs was inoculated with four cloaca strains and four lesion strains (104 CFU/tube). Based on the results, in a subsequent experiment, all 28 strains were inoculated only into albumen from non-incubated eggs. For all strains, the minimal inhibitory concentrations (MIC) of lysozyme were determined using an agar dilution method and the growth rates in broth with (500 and 2000 µg/ml) or without lysozyme were assessed. Compared to cloaca strains, lesion strains had 320 times higher odds of being reisolated from albumen (95% confidence interval: 55–3962) and had 8.5 times higher reisolation ratios (reisolation dose/inoculation dose) (95% confidence interval: 4.8–15.3). Thirteen cloaca strains had a MIC ranging from 1000 to 8000 µg/ml, while one cloaca strain and all lesion strains were resistant to the lysozyme concentrations tested. Growth rates of cloaca strains were decreased more by lysozyme compared to lesion strains. In conclusion, lesion strains had higher survival rates in egg albumen and were more resistant to lysozyme compared to cloaca strains. Egg albumen inhibits Enterococcus cecorum cloaca strains more than lesion strains.Enterococcus cecorum lesion strains are resistant to high concentrations of lysozyme.Lysozyme resistance could enhance survival in albumen and body fluids. Egg albumen inhibits Enterococcus cecorum cloaca strains more than lesion strains. Enterococcus cecorum lesion strains are resistant to high concentrations of lysozyme. Lysozyme resistance could enhance survival in albumen and body fluids.</p

Comparative genomics of phenotypic antimicrobial resistances in methicillin-resistant Staphylococcus pseudintermedius of canine origin

Staphylococcus pseudintermedius is an important pathogen in dogs. Since 2004, methicillin- resistant S. pseudintermedius (MRSP) isolates, often multidrug resistant, have been observed in dogs in the Netherlands. This study aims to link the observed resistance phenotypes in canine MRSP to genotypic antimicrobial resistance markers, and to study the phylogeny of MRSP by genomic comparisons. The genomes of fifty clinical isolates of MRSP from dogs from the Netherlands were sequenced. The resistance genes were identified, and for twenty one different antimicrobials their presence and sequence were associated with the resistance phenotypes. In case of observed discrepancies, the genes were aligned with reference genes. Of the phenotypic resistances, 98.3% could be explained by the presence of an associated resistance gene or point mutation. Discrepancies were mainly resistance genes present in susceptible isolates; 43.8% (7/16) were explained by an insertion, deletion or mutation in the gene. In relation with the resistance gene presence or absence, a single-nucleotide polymorphism (SNP) based phylogeny was constructed to define the population dynamics. The resistance gene content differed according to clonal complex, from very conserved (CC45), to partly conserved (CC71) to highly diverse (CC258) resistance gene patterns. In conclusion, this study shows that the antimicrobial genotype from whole genome sequencing is highly predictive of the resistance phenotype in MRSP. Interestingly, the observed clonal complexes of MRSP isolates were linked with resistance gene patterns</p

Comparative genomics of phenotypic antimicrobial resistances in methicillin-resistant Staphylococcus pseudintermedius of canine origin

Staphylococcus pseudintermedius is an important pathogen in dogs. Since 2004, methicillin- resistant S. pseudintermedius (MRSP) isolates, often multidrug resistant, have been observed in dogs in the Netherlands. This study aims to link the observed resistance phenotypes in canine MRSP to genotypic antimicrobial resistance markers, and to study the phylogeny of MRSP by genomic comparisons. The genomes of fifty clinical isolates of MRSP from dogs from the Netherlands were sequenced. The resistance genes were identified, and for twenty one different antimicrobials their presence and sequence were associated with the resistance phenotypes. In case of observed discrepancies, the genes were aligned with reference genes. Of the phenotypic resistances, 98.3% could be explained by the presence of an associated resistance gene or point mutation. Discrepancies were mainly resistance genes present in susceptible isolates; 43.8% (7/16) were explained by an insertion, deletion or mutation in the gene. In relation with the resistance gene presence or absence, a single-nucleotide polymorphism (SNP) based phylogeny was constructed to define the population dynamics. The resistance gene content differed according to clonal complex, from very conserved (CC45), to partly conserved (CC71) to highly diverse (CC258) resistance gene patterns. In conclusion, this study shows that the antimicrobial genotype from whole genome sequencing is highly predictive of the resistance phenotype in MRSP. Interestingly, the observed clonal complexes of MRSP isolates were linked with resistance gene patterns