Close contact between pets and owners provides the opportunity for transmission of antimicrobial resistant organisms like extended-spectrum beta-lactamase (ESBL)/AmpC beta-lactamase (AmpC)-producing ...Enterobacteriaceae, posing a risk to public health.
To investigate whether raw feed is a risk factor for household cats to shed ESBL-producing Enterobacteriaceae, a cohort study was designed. Additionally, raw and non-raw commercial pet food products were screened for the presence of ESBL-producing Enterobacteriaceae.
Weekly fecal samples of 17 cats in the control group and 19 cats in the exposed group were collected for three weeks and analyzed for the presence of ESBL-producing Enterobacteriaceae. Questionnaires were obtained to determine additional risk factors. Fecal samples were cultured on MacConkey agar supplemented with 1 mg/L cefotaxime. PCR and sequence analysis was used for screening for ESBL genes in suspected isolates. Pet food samples were cultured in LB broth supplemented with 1 mg/L cefotaxime and processed as described above.
In the cohort study, ESBL-producing bacteria were isolated from 3 of 51 (5.9%) samples in the control group compared to 37 of 57 (89.5%) samples in the exposed group. A significant association was found between ESBL shedding and feeding raw pet food products (OR = 31.5). No other risk factors were identified in this study. ESBL-producing Enterobacteriaceae were isolated from 14 of 18 (77.8%) raw pet food products and 0 of 35 non-raw pet food products.
This study shows a strong association between shedding of ESBL-producing bacteria in household cats and feeding raw pet food. Raw pet food was often contaminated with ESBL-producing Enterobacteriaceae.
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 (10
4
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.
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.
To explore the dynamics of faecal ESBL/AmpC shedding in dairy cattle and farmers, a study was conducted to examine changes in shedding by individual animals, as well as environmental exposure, and to ...study the association between antimicrobial use (AMU) and ESBL/AmpC shedding.
The study comprised a cross-sectional survey of 20 farms and a 1 year follow-up of 10 farms. Faecal samples were cultured by both direct inoculation on MacConkey agar + 1 mg/L cefotaxime (MC+) and enrichment in LB-broth + 1 mg/L cefotaxime with subsequent inoculation on MC+. Dust samples were collected using electrostatic dustfall collectors (EDCs). Human faecal samples were collected by the farmers. Presence of ESBL/AmpC genes was screened for by PCR and sequencing. Using mixed effects logistic regression, ORs were determined and population-attributable fractions (PAFs) calculated subsequently.
In Phase 1, 8/20 farms were positive for ESBL/AmpC and, with 2 negative farms, were selected for Phase 2. Transient shedding of dominant allele variants was observed in the animals. EDCs and human faecal samples did not reflect what was observed in the animals. AMU was related to shedding of ESBLs in the next sampling moment OR 14.6 (95% CI 3.0-80.0) and the PAF of AMU was 0.36 (95% CI 0.08-0.77). Calves fed with colostrum from cows on dry-off therapy was not a risk factor OR 1.7 (95% CI 0.7-4.9, P = 0.28).
The presence of ESBL/AmpC could only be partly explained by AMU. No link was shown between shedding in cattle and humans or the environment. Interventions should focus on prevention of introduction.
, 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
genes and production levels of LukMF' are associated with clinical severity of the disease.
was isolated from mastitis milk samples (38 clinical and 17 subclinical cases) from 33 different farms. The
-
genes were present in 96% of the isolates. Remarkably, 22% of the
-positive
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,
-type t543. Analysis of their global toxin gene regulators revealed a point mutation in the Repressor of toxins (
) gene which results in a non-functional start codon, preventing translation of
. This mutation was only identified in high LukMF' producing isolates and not in low LukMF' producing isolates. Since
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.
(
), 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
, 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
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
isolates were
genotyped. The prevalence of colonization of the extramammary sites at goat level was 51.7% and the prevalence of
intramammary infections was 7.2%. The nares were colonized most frequently (45%), while the groin area was colonized the least (2.5%). Six
genotypes were identified in this herd and there was no significant difference in the distribution of
genotypes between the milk or the extramammary sites (
= 0.141). Both in the extramammary sites and in the milk,
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
strains. Extramammary sites may, thus, be a source of
intramammary infections that are not targeted by the intervention measures aimed at preventing transmission from infected udder glands.
Staphylococcus pseudintermedius is an opportunistic pathogen in dogs and cats and occasionally causes infections in humans. S. pseudintermedius is often resistant to multiple classes of ...antimicrobials. It requires a reliable detection so that it is not misidentified as S. aureus. Phenotypic and currently-used molecular-based diagnostic assays lack specificity or are labour-intensive using multiplex PCR or nucleic acid sequencing. The aim of this study was to identify a specific target for real-time PCR by comparing whole genome sequences of S. pseudintermedius and non-pseudintermedius.Genome sequences were downloaded from public repositories and supplemented by isolates that were sequenced in this study. A Perl-script was written that analysed 300-nt fragments from a reference genome sequence of S. pseudintermedius and checked if this sequence was present in other S. pseudintermedius genomes (n = 74) and non-pseudintermedius genomes (n = 138). Six sequences specific for S. pseudintermedius were identified (sequence length between 300-500 nt). One sequence, which was located in the spsJ gene, was used to develop primers and a probe. The real-time PCR showed 100% specificity when testing for S. pseudintermedius isolates (n = 54), and eight other staphylococcal species (n = 43). In conclusion, a novel approach by comparing whole genome sequences identified a sequence that is specific for S. pseudintermedius and provided a real-time PCR target for rapid and reliable detection of S. 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.
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