RESUMO
Gut bacterial communities have a profound influence on the health of humans and animals. Early-life gut microbial community structure influences the development of immunological competence and susceptibility to disease. For the Thoroughbred racehorse, the significance of early-life microbial colonisation events on subsequent health and athletic performance is unknown. Here we present data from a three-year cohort study of horses bred for racing designed to explore interactions between early-life gut bacterial community structure, health events in later life and athletic performance on the racetrack. Our data show that gut bacterial community structure in the first months of life predicts the risk of specific diseases and athletic performance up to three years old. Foals with lower faecal bacterial diversity at one month old had a significantly increased risk of respiratory disease in later life which was also associated with higher relative abundance of faecal Pseudomonadaceae. Surprisingly, athletic performance up to three years old, measured by three different metrics, was positively associated with higher faecal bacterial diversity at one month old and with the relative abundance of specific bacterial families. We also present data on the impact of antibiotic exposure of foals during the first month of life. This resulted in significantly lower faecal bacterial diversity at 28 days old, a significantly increased risk of respiratory disease in later life and a significant reduction in average prize money earnings, a proxy for athletic performance. Our study reveals associations between early-life bacterial community profiles and health events in later life and it provides evidence of the detrimental impact of antimicrobial treatment in the first month of life on health and performance outcomes in later life. For the first time, this study demonstrates a relationship between early-life gut bacterial communities and subsequent athletic performance that has implications for athletes of all species including humans.
Assuntos
Fezes , Microbioma Gastrointestinal , Cavalos , Animais , Fezes/microbiologia , Doenças dos Cavalos/microbiologia , Desempenho Atlético , Bactérias/classificação , Bactérias/genética , Masculino , FemininoRESUMO
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 90â¯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 ß-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.
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Anti-Infecciosos , Doenças do Cão , Pioderma , Infecções Estafilocócicas , Cães , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Staphylococcus , Pioderma/tratamento farmacológico , Pioderma/veterinária , Pioderma/microbiologia , Anti-Infecciosos/uso terapêutico , Testes de Sensibilidade Microbiana/veterinária , Doenças do Cão/tratamento farmacológico , Doenças do Cão/microbiologia , Infecções Estafilocócicas/veterináriaRESUMO
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.
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Antibacterianos , Infecções por Escherichia coli , Antibacterianos/farmacologia , Biologia Computacional/métodos , Farmacorresistência Bacteriana/genética , Escherichia coli , Infecções por Escherichia coli/diagnóstico , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Testes de Sensibilidade MicrobianaRESUMO
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, in vitro 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 ex vivo 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.IMPORTANCE 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 in vivo research models.
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Fermentação/fisiologia , Microbioma Gastrointestinal/fisiologia , Intestino Grosso/microbiologia , Modelos Biológicos , Animais , Ácidos Graxos/metabolismo , Ácidos Graxos Voláteis/metabolismo , Fezes/microbiologia , Cavalos , Técnicas In Vitro/métodos , Intestino Grosso/química , Intestino Grosso/fisiologiaRESUMO
OBJECTIVES: To analyse the biological effects of a 1920 nm endovenous laser (EVL) on extra-fascial great saphenous vein (GSV) in vitro. METHODS: A 10 cm length of a large tributary bypassing a hypoplastic segment of the GSV (sometimes called an "extra-fascial GSV") was obtained during routine varicose vein surgery. The length was treated in five sections with different LEEDs (0 (control), 20, 40, 60, and 80 J/cm) with a 1920 nm EVL at 4W power, in a novel in vitro treatment model. The biological effects were assessed by histological staining of the samples for haematoxylin and eosin (HE) and Martius Scarlet Blue (MSB), and by immunofluorescent detection of p-p53 and VCAM-1. RESULTS: Histological analysis showed significant structural damage at LEEDs above 60 J/cm, especially in the intima and media, with the treatment at 80 J/cm causing perforation of the vein wall. In addition, there was a significant increase in p-p53 expression in treated tissue at 60 and 80 J/cm. CONCLUSIONS: Using this ex vivo model, the results indicate that in vitro treatment with a 1920 nm EVL, at or above an LEED of 60 J/cm and 4 W power, causes significant vein wall cell death reaching deep into the media by a combination of direct thermal damage and apoptosis. A wavelength of 1920 nm appears to be effective for the endovenous ablation of truncal veins.
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Objectives: To assess stability and contribution of a large ESBL-encoding IncI1 plasmid to intestinal colonization by Escherichia coli O104:H4 in two different mammalian hosts. Methods: Specific-pathogen-free 3-4-day-old New Zealand White rabbits and conventionally reared 6-week-old weaned lambs were orally infected with WT E. coli O104:H4 or the ESBL-plasmid-cured derivative, and the recovery of bacteria in intestinal homogenates and faeces monitored over time. Results: Carriage of the ESBL plasmid had differing impacts on E. coli O104:H4 colonization of the two experimental hosts. The plasmid-cured strain was recovered at significantly higher levels than WT during late-stage colonization of rabbits, but at lower levels than WT in sheep. Regardless of the animal host, the ESBL plasmid was stably maintained in virtually all in vivo passaged bacteria that were examined. Conclusions: These findings suggest that carriage of ESBL plasmids has distinct effects on the host bacterium depending upon the animal species it encounters and demonstrates that, as for E. coli O157:H7, ruminants could represent a potential transmission reservoir.
Assuntos
Escherichia coli O104/genética , Escherichia coli O104/patogenicidade , Interações entre Hospedeiro e Microrganismos , Coelhos/microbiologia , Ovinos/microbiologia , Animais , Fezes/microbiologia , Intestinos , Plasmídeos , Especificidade da Espécie , beta-LactamasesRESUMO
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.
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Controle de Doenças Transmissíveis/métodos , Escherichia coli Êntero-Hemorrágica/isolamento & purificação , Infecções por Escherichia coli/diagnóstico , Infecções por Escherichia coli/prevenção & controle , Escherichia coli Shiga Toxigênica/isolamento & purificação , Animais , Escherichia coli Êntero-Hemorrágica/patogenicidade , Humanos , Escherichia coli Shiga Toxigênica/patogenicidade , Zoonoses/diagnóstico , Zoonoses/prevenção & controleRESUMO
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(-1) were 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.
Assuntos
Antibacterianos/metabolismo , Cefalosporinas/metabolismo , Indústria de Laticínios , Leite/química , Resíduos , Animais , Biodegradação Ambiental , Bovinos , Escherichia coli/crescimento & desenvolvimento , Fermentação , Temperatura Alta , Modelos Biológicos , beta-Lactamases/metabolismoRESUMO
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.
Assuntos
Diarreia/tratamento farmacológico , Infecções por Escherichia coli/tratamento farmacológico , Lactobacillus plantarum/fisiologia , Lactulose/administração & dosagem , Probióticos/administração & dosagem , Doenças dos Suínos/tratamento farmacológico , Simbióticos/análise , Animais , Colo/microbiologia , Diarreia/sangue , Diarreia/metabolismo , Diarreia/microbiologia , Escherichia coli Enterotoxigênica/fisiologia , Infecções por Escherichia coli/sangue , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Feminino , Lactulose/metabolismo , Masculino , Probióticos/análise , Suínos , Doenças dos Suínos/sangue , Doenças dos Suínos/metabolismo , Doenças dos Suínos/microbiologia , Fator de Necrose Tumoral alfa/sangue , DesmameRESUMO
The presence of 10 virulence genes was examined using polymerase chain reaction (PCR) in 365 European O157 and non-O157 Escherichia coli isolates associated with verotoxin production. Strain-specific PCR data were analysed using hierarchical clustering. The resulting dendrogram clearly separated O157 from non-O157 strains. The former clustered typical high-risk seropathotype (SPT) A strains from all regions, including Sweden and Spain, which were homogenous by Cramer's V statistic, and strains with less typical O157 features mostly from Hungary. The non-O157 strains divided into a high-risk SPTB harbouring O26, O111 and O103 strains, a group pathogenic to pigs, and a group with few virulence genes other than for verotoxin. The data demonstrate SPT designation and selected PCR separated verotoxigenic E. coli of high and low risk to humans; although more virulence genes or pulsed-field gel electrophoresis will need to be included to separate high-risk strains further for epidemiological tracing.
Assuntos
Escherichia coli O157/classificação , Escherichia coli O157/patogenicidade , Escherichia coli Shiga Toxigênica/classificação , Escherichia coli Shiga Toxigênica/patogenicidade , Virulência/genética , Animais , Análise por Conglomerados , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/microbiologia , Escherichia coli O157/genética , Proteínas de Escherichia coli/genética , Europa (Continente)/epidemiologia , Humanos , Prevalência , Ovinos , Escherichia coli Shiga Toxigênica/genética , SuínosRESUMO
Enteric bacteria with resistance to third and fourth generation cephalosporin antibiotics, especially Escherichia coli bearing the blaCTX-M gene, have been detected in a wide range of food producing animals. However, commercial vaccines for these organisms are not currently available. An autogenous vaccine was prepared from E. coli bearing the blaCTX-M-14 gene and evaluated as a potential control measure to reduce shedding and dissemination of these organisms in cattle. Calves (n=30) received either an autogenous vaccine prepared from E. coli serotype O33 bearing the blaCTX-M-14 gene or a placebo by intramuscular injection on three separate occasions. Two weeks after the final vaccination, all calves were challenged by oral gavage with the O33 CTX-M-14 strain of E. coli (1×10(10) CFU). Faeces, intestinal mucosa and blood samples were taken for enumeration of total and CTX-M-14 E. coli and for assessment of the humoral immune response. The cumulative number of total E. coli excreted at 7 days post-challenge was significantly (p=0.006) lower in the vaccinated group than the placebo group. However, there was no significant difference in the shedding of either CTX-M-14 E. coli or total E. coli between vaccinated and placebo calves throughout the study period. The systemic immune response to E. coli O33 antigen was tested by ELISA and was significantly higher (p<0.001) in vaccinated than placebo calves. However, there was no significant difference in the mucosal immune response. These findings do not support the use of autogenous vaccination for the control of CTX-M-14 E. coli in calves.
Assuntos
Autovacinas/uso terapêutico , Doenças dos Bovinos/prevenção & controle , Infecções por Escherichia coli/veterinária , Vacinas contra Escherichia coli/uso terapêutico , Animais , Derrame de Bactérias , Bovinos , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/microbiologia , Resistência às Cefalosporinas/genética , Escherichia coli/genética , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/prevenção & controle , Fezes/microbiologia , Plasmídeos/genéticaRESUMO
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.
Assuntos
Vírus da Hepatite E/fisiologia , Replicação Viral , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Vírus da Hepatite E/genética , Vírus da Hepatite E/ultraestrutura , Hepatócitos/virologia , Humanos , Microscopia Eletrônica , RNA Viral/análise , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , Cultura de Vírus/métodosRESUMO
A candidate live vaccine for avian pathogenic Escherichia coli (APEC) was constructed from a virulent field APEC 078 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 078 aroA mutant were protected against a challenge at 6 wk of age by virulent APEC strains.
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Galinhas , Infecções por Escherichia coli/veterinária , Vacinas contra Escherichia coli/efeitos adversos , Escherichia coli/imunologia , Fímbrias Bacterianas/fisiologia , Doenças das Aves Domésticas/prevenção & controle , Perus , Animais , Escherichia coli/genética , Infecções por Escherichia coli/prevenção & controle , Vacinas contra Escherichia coli/administração & dosagem , Flagelos/fisiologia , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/efeitos adversosRESUMO
Escherichia coli O115 has been isolated from healthy sheep and was shown to be associated with attaching-effacing (AE) lesions in the large intestine. Following previous observations of interactions between E. coli O157 and O26, the aim of the present study was to assess what influence an O115 AE E. coli (AEEC) would have on E. coli O157 colonisation in vitro and in vivo. We report that E. coli O115- and O157-associated AE lesions were observed on HEp-2 cells and on the mucosa of ligated ovine spiral colon. In single strain inoculum, E. coli O115 associated intimately with HEp-2 cells and the spiral colon in greater numbers than E. coli O157:H7. However, in mixed inoculum studies, the number of E. coli O115 AE lesions was significantly reduced suggesting negative interference by E. coli O157. Use of the ligated colon model in the present work has allowed in vitro observations to be extended and confirmed whilst using a minimum of experimental animals. The findings support a hypothesis that some AEEC can inhibit adhesion of other AEEC in vivo. The mechanisms involved may prove to be of utility in the control of AE pathovars.
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Aderência Bacteriana/fisiologia , Colo/microbiologia , Infecções por Escherichia coli/veterinária , Escherichia coli/fisiologia , Doenças dos Ovinos/microbiologia , Animais , Colo/patologia , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/patologia , Técnicas Imunoenzimáticas/veterinária , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Microscopia Eletrônica de Transmissão/veterinária , Ovinos/microbiologia , Doenças dos Ovinos/patologiaRESUMO
The number and proportion of CTX-M positive Escherichia coli organisms were determined in feces from cattle, chickens, and pigs in the United Kingdom to provide a better understanding of the risk of the dissemination of extended-spectrum ß-lactamase (ESBL) bacteria to humans from food animal sources. Samples of bovine (n = 35) and swine (n = 20) feces were collected from farms, and chicken cecal contents (n = 32) were collected from abattoirs. There was wide variation in the number of CTX-M-positive E. coli organisms detected; the median (range) CFU/g were 100 (100 × 10(6) to 1 × 10(6)), 5,350 (100 × 10(6) to 3.1 × 10(6)), and 2,800 (100 × 10(5) to 4.7 × 10(5)) for cattle, chickens, and pigs, respectively. The percentages of E. coli isolates that were CTX-M positive also varied widely; median (range) values were 0.013% (0.001 to 1%) for cattle, 0.0197% (0.00001 to 28.18%) for chickens, and 0.121% (0.0002 to 5.88%) for pigs. The proportion of animals designated high-density shedders (≥1 × 10(4) CFU/g) of CTX-M E. coli was 3/35, 15/32, and 8/20 for cattle, chickens, and pigs, respectively. We postulate that high levels of CTX-M E. coli in feces facilitate the dissemination of bla(CTX-M) genes during the rearing of animals for food, and that the absolute numbers of CTX-M bacteria should be given greater consideration in epidemiological studies when assessing the risks of food-borne transmission.
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Derrame de Bactérias , Escherichia coli/enzimologia , Escherichia coli/isolamento & purificação , Fezes/microbiologia , beta-Lactamases/biossíntese , Animais , Carga Bacteriana , Bovinos , Galinhas , Microbiologia Ambiental , Microbiologia de Alimentos , Humanos , Suínos , Reino UnidoRESUMO
Verotoxigenic Escherichia coli (VTEC) serotype O157:H7 strains from a Swedish cattle prevalence study (n=32), and livestock-derived strains linked to human disease (n=13), were characterized by microarray and PCR detection of virulence genes. The overall aim of the study was to investigate the distribution of known virulence determinants and determine which genes are linked to increased pathogenicity in humans. A core set of 18 genes or gene variants were found in all strains, while seven genes were variably present. This suggests that the majority of VTEC O157:H7 found in Swedish cattle carry a broad repertoire of virulence genes and should be considered potentially harmful to humans. A single virulence gene type was significantly associated with strains linked to human disease cases (P=0.012), but no genetic trait to explain the increased virulence of this genotype could be found.
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Doenças dos Bovinos/epidemiologia , Infecções por Escherichia coli/veterinária , Escherichia coli O157/genética , Análise de Sequência com Séries de Oligonucleotídeos/veterinária , Animais , Bovinos , Doenças dos Bovinos/microbiologia , Eletroforese em Gel de Campo Pulsado , Infecções por Escherichia coli/microbiologia , Escherichia coli O157/patogenicidade , Genes Bacterianos/genética , Humanos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reação em Cadeia da Polimerase , Prevalência , Suécia/epidemiologia , Virulência/genéticaRESUMO
The development of novel intervention strategies for the control of zoonoses caused by bacteria such as Salmonella spp. in livestock requires appropriate experimental models to assess their suitability. Here, a novel porcine intestinal in vitro organ culture (IVOC) model utilizing cell crown (CC) technology (CCIVOC) (Scaffdex) was developed. The CCIVOC model was employed to investigate the characteristics of association of S. enterica serovar Typhimurium strain SL1344 with porcine intestinal tissue following exposure to a Lactobacillus plantarum strain. The association of bacteria to host cells was examined by light microscopy and electron microscopy (EM) after appropriate treatments and staining, while changes in the proteome of porcine jejunal tissues were investigated using quantitative label-free proteomics. Exposure of porcine intestinal mucosal tissues to L. plantarum JC1 did not reduce the numbers of S. Typhimurium bacteria associating to the tissues but was associated with significant (P < 0.005) reductions in the percentages of areas of intestinal IVOC tissues giving positive staining results for acidic mucins. Conversely, the quantity of neutrally charged mucins present within the goblet cells of the IVOC tissues increased significantly (P < 0.05). In addition, tubulin-α was expressed at high levels following inoculation of jejunal IVOC tissues with L. plantarum. Although L. plantarum JC1 did not reduce the association of S. Typhimurium strain SL1344 to the jejunal IVOC tissues, detection of increased acidic mucin secretion, host cytoskeletal rearrangements, and proteins involved in the porcine immune response demonstrated that this strain of L. plantarum may contribute to protecting the pig from infections by S. Typhimurium or other pathogens.
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Interações Hospedeiro-Patógeno , Intestinos/microbiologia , Intestinos/patologia , Lactobacillus plantarum/crescimento & desenvolvimento , Lactobacillus plantarum/imunologia , Salmonella typhimurium/crescimento & desenvolvimento , Salmonella typhimurium/patogenicidade , Animais , Aderência Bacteriana , Carga Bacteriana , Intestinos/química , Microscopia , Mucinas/análise , Técnicas de Cultura de Órgãos , Proteoma/análise , Suínos , Tubulina (Proteína)/análiseRESUMO
AIMS: To determine the effect of various enrofloxacin dose regimes on the colonization and selection of resistance in Campylobacter jejuni strain 81116P in experimentally colonized chickens. METHODS AND RESULTS: Two experiments were undertaken, in which 14-day-old chickens were colonized with 1 × 10(7) -1 × 10(9 ) CFU g(-1) Camp. jejuni strain 81116P and then treated with enrofloxacin at 12-500 ppm in drinking water for various times. Caecal colonization levels were determined at various time-points after start-of-treatment, and the susceptibility of recovered isolates to ciprofloxacin was monitored. Resistance was indicated by growth on agar containing 4 µg ml(-1) ciprofloxacin, MICs of 16 µg ml(-1) and the Thr86Ile mutation in gyrA. Enrofloxacin at doses of 12-250 ppm reduced Camp. jejuni colonization over the first 48-72 h after start-of-treatment. The degree of reduction in colonization was dose, but not treatment time, dependent. In all cases, maximal colonization was re-established within 4-6 days. Fluoroquinolone-resistant organisms were recoverable within 48 h of start-of-treatment; after a further 24 h all recovered isolates were resistant. In contrast, a dose of 500 ppm enrofloxacin reduced colonization to undetectable levels within 48 h, and the treated birds remained Campylobacter negative throughout the remaining experimental period. By high pressure liquid chromatography, for all doses, the maximum concentrations of enrofloxacin and ciprofloxacin in the caecal contents were detected at the point of treatment completion. Thereafter, levels declined to undetectable by 7 days post-treatment withdrawal. CONCLUSIONS: In a model using chickens maximally colonized with Camp. jejuni 81116P, treatment with enrofloxacin, at doses of 12-250 ppm in drinking water, enables the selection, and clonal expansion, of fluoroquinolone-resistant organisms. However, this is preventable by treatment with 500 ppm of enrofloxacin. SIGNIFICANCE AND IMPACT OF THE STUDY: Treatment of chickens with enrofloxacin selects for resistance in Camp. jejuni in highly pre-colonized birds. However, a dose of 500 ppm enrofloxacin prevented the selection of resistant campylobacters.
Assuntos
Antibacterianos/farmacologia , Campylobacter jejuni/efeitos dos fármacos , Galinhas/microbiologia , Fluoroquinolonas/farmacologia , Animais , Campylobacter jejuni/crescimento & desenvolvimento , Campylobacter jejuni/isolamento & purificação , Ceco/microbiologia , Ciprofloxacina/farmacologia , Farmacorresistência Bacteriana , EnrofloxacinaRESUMO
Recently, it has been reported that Salmonella secrete flagellin in response to host produced lysophospholipids. However, this monomer of the bacterial flagella activates Toll-like receptor 5 (TLR5) in the innate immune system. The objective of this study was to examine the role of flagellin expression during infection of species-specific macrophages (MPhi) which either expressed or lacked TLR5. Initially, TLR5-activity was confirmed in bovine MPhi using Salmonella typhimurium derived-flagellin. Within these cells, recombinant FliC induced a potent CXCL8 response when compared to the heterogeneous (FliC/FljB) form of purified flagellin. Furthermore, neither form of flagellin induced nitrite secretion which was subsequently detected after exposing bovine MPhi to LPS in the presence of IFN-gamma. Flagellin enhanced the accumulation of Salmonella enteritidis in TLR5-positive bovine and human MPhi which was independent of adhesion in bovine MPhi. In contrast, murine MPhis which lacked TLR5 were equally susceptible to hosting S. enteritidis, with or without flagellin. However, lack of flagellin in S. typhimurium marginally inhibited bacterial accumulation in bovine MPhi, where FljB and FliC compensated for the lack of each other. This study suggests that flagellin may be inducing TLR5-dependent internalisation mechanisms in Mcapital EF, Cyrillic which vary qualitatively between different species and Salmonella serotypes.
Assuntos
Flagelina/metabolismo , Macrófagos/metabolismo , Infecções por Salmonella/imunologia , Salmonella enteritidis/imunologia , Salmonella typhimurium/imunologia , Animais , Bovinos , Contagem de Colônia Microbiana , Flagelina/genética , Flagelina/imunologia , Humanos , Imunidade Inata , Imunização , Interleucina-8/genética , Interleucina-8/metabolismo , Lipopolissacarídeos/metabolismo , Macrófagos/imunologia , Macrófagos/microbiologia , Macrófagos/patologia , Infecções por Salmonella/genética , Infecções por Salmonella/metabolismo , Salmonella enteritidis/patogenicidade , Salmonella typhimurium/patogenicidade , Especificidade da Espécie , Receptor 5 Toll-Like/biossíntese , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Células U937RESUMO
Escherichia fergusonii has been associated with a wide variety of intestinal and extra-intestinal infections in both humans and animals but, despite strong circumstantial evidence, the degree to which the organism is responsible for the pathologies identified remains uncertain. Thirty isolates of E. fergusonii collected between 2003 and 2004 were screened using an Escherichia coli virulence gene array to test for the presence of homologous virulence genes in E. fergusonii. The iss (increased serum survival) gene was present in 13/30 (43%) of the test strains and the prfB (P-related fimbriae regulatory) and ireA (siderophore receptor IreA) genes were also detected jointly in 3/30 (10%) strains. No known virulence genes were detected in 14/30 (47%) of strains. Following confirmatory PCR and sequence analysis, the E. fergusoniiprfB, iss and ireA genes shared a high degree of sequence similarity to their counterparts in E. coli, and a particular resemblance was noted with the E. coli strain APEC O1 pathogenicity island. In tissue culture adherence assays, nine E. fergusonii isolates associated with HEp-2 cells with a 'localised adherence' or 'diffuse adherence' phenotype, and they proved to be moderately invasive. The E. fergusonii isolates in this study possess both some phenotypic and genotypic features linked to known pathotypes of E. coli, and support existing evidence that strains of E. fergusonii may act as an opportunistic pathogens, although their specific virulence factors may need to be explored.