Summary
Escherichia coli comprises a highly diverse group of Gram‐negative bacteria and is a common member of the intestinal microflora of humans and animals. Generally, such colonization is ...asymptomatic; however, some E. coli strains have evolved to become pathogenic and thus cause clinical disease in susceptible hosts. One pathotype, the Shiga toxigenic E. coli (STEC) comprising strains expressing a Shiga‐like toxin is an important foodborne pathogen. A subset of STEC are the enterohaemorrhagic E. coli (EHEC), which can cause serious human disease, including haemolytic uraemic syndrome (HUS). The diagnosis of EHEC infections and the surveillance of STEC in the food chain and the environment require accurate, cost‐effective and timely tests. In this review, we describe and evaluate tests now in routine use, as well as upcoming test technologies for pathogen detection, including loop‐mediated isothermal amplification (LAMP) and whole‐genome sequencing (WGS). We have considered the need for improved diagnostic tools in current strategies for the control and prevention of these pathogens in humans, the food chain and the environment. We conclude that although significant progress has been made, STEC still remains an important zoonotic issue worldwide. Substantial reductions in the public health burden due to this infection will require a multipronged approach, including ongoing surveillance with high‐resolution diagnostic techniques currently being developed and integrated into the routine investigations of public health laboratories. However, additional research requirements may be needed before such high‐resolution diagnostic tools can be used to enable the development of appropriate interventions, such as vaccines and decontamination strategies.
The intestinal microbiota of the horse, an animal of huge economic and social importance worldwide, is essential to the health of the animal. Understanding the intestinal ecosystem and its dynamic ...interaction with diet and dietary supplements currently requires the use of experimental animals, with consequent welfare and financial constraints. Here, we describe the development and assessment, using multiple analytical platforms, of a three-vessel, continuous-flow,
model of the equine hindgut. After inoculation of the model with fresh horse feces, the bacterial communities established in each vessel had a taxonomic distribution similar to that of the source animal. Short-chain fatty acid (SCFA) and branched-chain fatty acid (BCFA) production within the model at steady state was consistent with the expected bacterial function, although higher concentrations of some SCFA/BCFA relative to those in the
gut content were apparent. We demonstrate the intermodel repeatability and the ability of the model to capture some aspects of individual variation in bacterial community profiles. The findings of this proof-of-concept study, including recognition of the limitions of the model, support its future development as a tool for investigating the impact of disease, nutrition, dietary supplementation, and medication on the equine intestinal microbiota.
The equine gut model that we have developed and describe has the potential to facilitate the exploration of how the equine gut microbiota is affected by diet, disease, and medication. It is a convenient, cost-effective, and welfare-friendly alternative to
research models.
► We setup a 3D cells culture able to support HEV viral replication. ► HEV RNA was detected by real time-RT-PCR during all the course of the experiments. ► HEV RNA progeny was detectable by real time ...RT-PCR demonstrating the viral replication. ► HEV like particle were detected by EM.
Hepatitis E is an acute, viral hepatitis epidemic in developing regions, but which is detected with increasing frequency in sporadic form in developed regions. Pigs and possibly some other mammals are considered reservoirs of zoonotic infection with hepatitis E virus (HEV). However, whilst the relative significance of potential transmission routes from pigs to people is still unclear, the consumption of raw or undercooked pig meat has been implicated as a source of HEV infection. The lack of information about HEV zoonotic transmission is due in part to the difficulties of in vitro propagation of HEV. The Rotating Wall Vessel (RVW) has been described as a useful tool for the culture of cell lines in a 3-dimensional (3D) configuration. The aim of this work was to develop a 3D cell culture system for HEV to facilitate studies into the viability of virions contaminating pig tissues. This study, demonstrated that HEV can replicate efficiently in the RWV in human hepatoblastoma PLC/PRF/5 cells for up to 5 months not only by real time RT-PCR but also by detection of complete virions via electron microscopy. Furthermore, the replication of HEV progeny was observed by detecting HEV RNA by RT-PCR. The progeny were able to infect fresh 3D cultures, showing that this method is able to produce infectious hepatitis E virions.
Cost effective control of avian diseases and food borne pathogens remains a high priority for all sectors of the poultry industry with cleansing and disinfection, vaccination and competitive ...exclusion approaches being used widely. Previous studies showed that
Bacillus subtilis PY79
hr was an effective competitive exclusion agent for use in poultry to control avian pathogenic
Escherichia coli serotype O78:K80. Here we report experiments that were undertaken to test the efficacy of
B. subtilis PY79
hr in the control of
Salmonella enterica serotype Enteritidis and
Clostridium perfringens in young chickens. To do this, 1-day-old and 20-day-old specific pathogen free (SPF) chicks were dosed with a suspension of
B. subtilis spores prior to challenge with
S. Enteritidis (S1400) and
C. perfringens, respectively. For both challenge models, a single oral inoculum of 1×10
9 spores given 24
h prior to challenge was sufficient to suppress colonisation and persistence of both
S. Enteritidis and
C. perfringens. In particular, the faecal shedding of
S. Enteritidis, as measured by a semi-quantitative cloacal swabbing technique, was reduced significantly for the 36 days duration of the experiment.
B. subtilis persisted in the intestine although with decreasing numbers over the same period. These data add further evidence that
B. subtilis spores may be effective agents in the control of avian diseases and food borne pathogens.
Abstract
Improvements in cost and speed of next generation sequencing (NGS) have provided a new pathway for delivering disease diagnosis, molecular typing, and detection of antimicrobial resistance ...(AMR). Numerous published methods and protocols exist, but a lack of harmonisation has hampered meaningful comparisons between results produced by different methods/protocols vital for global genomic diagnostics and surveillance. As an exemplar, this study evaluated the sensitivity and specificity of five well-established in-silico AMR detection software where the genotype results produced from running a panel of 436
Escherichia coli
were compared to their AMR phenotypes, with the latter used as gold-standard. The pipelines exploited previously known genotype–phenotype associations. No significant differences in software performance were observed. As a consequence, efforts to harmonise AMR predictions from sequence data should focus on: (1) establishing universal minimum to assess performance thresholds (e.g. a control isolate panel, minimum sensitivity/specificity thresholds); (2) standardising AMR gene identifiers in reference databases and gene nomenclature; (3) producing consistent genotype/phenotype correlations. The study also revealed limitations of in-silico technology on detecting resistance to certain antimicrobials due to lack of specific fine-tuning options in bioinformatics tool or a lack of representation of resistance mechanisms in reference databases. Lastly, we noted user friendliness of tools was also an important consideration. Therefore, our recommendations are timely for widespread standardisation of bioinformatics for genomic diagnostics and surveillance globally.
A candidate live vaccine for avian pathogenic Escherichia coli (APEC) was constructed from a virulent field APEC O78 strain by mutation of the aroA gene. The mutant was highly similar to the parent ...wild-type strain in respect of colony morphology, motility, growth in suspension, hemagglutination, Congo Red binding, HEp-2 cell adhesion, and the elaboration of surface antigens type 1 fimbriae and flagella, although production of curli fimbriae was reduced marginally. The mutant proved avirulent when inoculated into 1-day-old chicks by spray application and when presented again in the drinking water at 7 days of age. Chickens and turkeys vaccinated with an O78 aroA mutant were protected against a challenge at 6 wk of age by virulent APEC strains. Eficacia de una vacuna viva atenuada de Escherichia coli O78∶K80 en pollos y pavos. Se elaboró una vacuna viva para Escherichia coli patógena aviar (APEC) a partir de una cepa de campo patógena para las aves y virulenta O78 mediante mutación del gene aroA. La mutante fue muy similar a la cepa silvestre original con respecto a la morfología de la colonia, motilidad, crecimiento en suspensión, hemaglutinación, afinidad al rojo Congo, adhesión a células HEp-2 y la elaboración de antígenos de superficie tipo 1 fimbria y flagelo, aunque la producción de fimbrias rizadas se redujo marginalmente. La mutante no mostró virulencia cuando se inoculó en pollitos de un día vía aerosol, ni cuando se administró nuevamente a los 7 días de edad, vía agua de bebida. Los pollos y pavos vacunados con la mutante O78 aroA fueron protegidos contra el desafío a las 6 semanas de edad con una cepa de E. coli patógena aviar virulenta.
Staphylococcus pseudintermedius is the most common cause of pyoderma in dogs. We validated a point-of-care (PoC) test based on colorimetric loop-mediated isothermal amplification (LAMP) for rapid S. ...pseudintermedius identification and susceptibility testing for first line antimicrobials for systemic treatment of canine pyoderma, i.e., lincosamides, first generation cephalosporins and amoxicillin clavulanate. Newly designed LAMP primers targeting clinically relevant resistance genes were combined with a previously validated set of primers targeting spsL for species identification. After laboratory validation on 110 clinical isolates, we assessed the performance of the test on 101 clinical specimens using routine culture and susceptibility testing as a reference standard. The average hands-on and turnaround times for the PoC test were 30 and 90 min, respectively. The assay showed sensitivity and specificity near 100% for both species identification and susceptibility testing when performed on bacterial cultures or clinical specimens in the laboratory. However, the PoC test yielded less accurate results when performed on-site by clinical staff (92% sensitivity and 64% specificity for species identification, 67% sensitivity and 96% specificity for β-lactam susceptibility, and 83% sensitivity and 71% specificity for lincosamide susceptibility). These results indicate that the PoC test should be adapted to a user-friendly technology to facilitate performance and interpretation of results by clinical staff. If properly developed, the test would allow veterinarians to gain rapid information on antimicrobial choice, limiting the risk of treatment failure and facilitating adherence to antimicrobial use guidelines in small animal veterinary dermatology.
•A test was developed for direct antibiotic susceptibility testing on skin samples.•The average turnaround time for the assay was 90 min.•The test showed nearly 100% sensitivity and specificity in the laboratory.•Diagnostic accuracy was lower under field conditions.
AIMS: The aims of this work were to develop a model of dairy farm waste milk and to investigate methods for the bioremediation of milk containing cefquinome residues. METHODS AND RESULTS: ...Unpasteurized milk and UHT milk that had both been spiked with cefquinome at a concentration of 2 μg ml⁻¹were used as a model for waste milk containing cephalosporin residues. Adjustment of the spiked UHT milk to pH 10 or treatment with conditioned medium from bacterial growth producing cefotaximase, were the most effective methods for decreasing the cefquinome concentrations within 24 h. A large‐scale experiment (10 l of cefquinome‐spiked unpasteurized milk) suggested that fermentation for 22 h at 37°C followed by heating at 60°C for 2 h was sufficient to decrease cefquinome concentrations to below the limit of quantification (<125 μg kg⁻¹) and to kill the majority of the enriched bacterial population. CONCLUSIONS: One or a combination of the bioremediation methods described may have potential as a practical treatment for dairy farm waste milk. SIGNIFICANCE AND IMPACT OF THE STUDY: Treatment of waste milk to decrease cephalosporin residue concentrations and also to kill bacteria prior to feeding to dairy calves could decrease the risk of selection for ESBL bacteria on dairy farms.
The potential of a prebiotic oligosaccharide lactulose, a probiotic strain of Lactobacillus plantarum, or their synbiotic combination to control postweaning colibacillosis in pigs was evaluated using ...an enterotoxigenic Escherichia coli (ETEC) K88 oral challenge. Seventy-two weanlings were fed four diets: a control diet (CTR), that diet supplemented with L. plantarum (2 × 10(10) CFU · day(-1)) (LPN), that diet supplemented with 10 g · kg(-1) lactulose (LAC), or a combination of the two treatments (SYN). After 7 days, the pigs were orally challenged. Six pigs per treatment were euthanized on days 6 and 10 postchallenge (PC). Inclusion of lactulose improved the average daily gain (ADG) (P < 0.05) and increased lactobacilli (P < 0.05) and the percentage of butyric acid (P < 0.02) in the colon. An increase in the ileum villous height (P < 0.05) and a reduction of the pig major acute-phase protein (Pig-MAP) in serum (P < 0.01) were observed also. The inclusion of the probiotic increased numbers of L. plantarum bacteria in the ileum and colon (P < 0.05) and in the total lactobacilli in the colon and showed a trend to reduce diarrhea (P = 0.09). The concentrations of ammonia in ileal and colonic digesta were decreased (P < 0.05), and the villous height (P < 0.01) and number of ileal goblet cells (P < 0.05) increased, at day 10 PC. A decrease in plasmatic tumor necrosis factor alpha (TNF-α) (P < 0.01) was also seen. The positive effects of the two additives were combined in the SYN treatment, resulting in a complementary synbiotic with potential to be used to control postweaning colibacillosis.
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