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Current diagnostic methods for Johne's disease in cattle allow reliable detection of infections with Mycobacterium avium ssp. paratuberculosis (MAP) not before animals are 2 years of age. Applying a flow cytometry-based approach (FCA) to quantify a MAP-specific interferon-gamma (IFN-γ) response in T cell subsets, the present study sought to monitor the kinetics of the cell-mediated immune response in experimentally infected calves. Six MAP-negative calves and six calves, orally inoculated with MAP at 10 days of age, were sampled every 4 weeks for 52 weeks post-inoculation (wpi). Peripheral blood mononuclear cells (PBMC) were stimulated with either purified protein derivatives (PPD) or whole cell sonicates derived from MAP (WCSj), M. avium ssp. avium or M. phlei for 6 days followed by labeling of intracellular IFN-γ in CD4+ and CD8+ T cells. No antigen-specific IFN-γ production was detectable in CD8+ cells throughout and the responses of CD4+ cells of MAP-infected and control calves were similar up to 12 wpi. However, the mean fluorescence intensity (MFI) for the detection of IFN-γ in CD4+ cells after WCSj antigen stimulation allowed for a differentiation of animal groups from 16 wpi onwards. This approach had a superior sensitivity (87.8%) and specificity (86.8%) to detect infected animals from 16 wpi onwards, i.e., in an early infection stage, as compared to the IFN-γ release assay (IGRA). Quantification of specific IFN-γ production at the level of individual CD4+ cells may serve, therefore, as a valuable tool to identify MAP-infected juvenile cattle.
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Linfocitos T CD4-Positivos , Enfermedades de los Bovinos , Citometría de Flujo , Interferón gamma , Mycobacterium avium subsp. paratuberculosis , Paratuberculosis , Animales , Bovinos , Paratuberculosis/inmunología , Paratuberculosis/diagnóstico , Paratuberculosis/microbiología , Mycobacterium avium subsp. paratuberculosis/inmunología , Mycobacterium avium subsp. paratuberculosis/fisiología , Interferón gamma/metabolismo , Citometría de Flujo/veterinaria , Citometría de Flujo/métodos , Enfermedades de los Bovinos/inmunología , Enfermedades de los Bovinos/diagnóstico , Enfermedades de los Bovinos/microbiología , Linfocitos T CD4-Positivos/inmunología , BiomarcadoresRESUMEN
The global spread of plasmid-mediated mobile colistin resistance (mcr) genes threatens the vital role of colistin as a drug of last resort. We investigated whether the recurrent occurrence of specific E. coli pathotypes and plasmids in individual pig farms resulted from the continued presence or repeated reintroduction of distinct E. coli strains. E. coli isolates (n = 154) obtained from three pig farms with at least four consecutive years of mcr detection positive for virulence-associated genes (VAGs) predicting an intestinal pathogenic pathotype via polymerase chain reaction were analyzed. Detailed investigation of VAGs, antimicrobial resistance genes and plasmid Inc types was conducted using whole genome sequencing for 87 selected isolates. Sixty-one E. coli isolates harbored mcr-1, and one isolate carried mcr-4. On Farm 1, mcr-positive isolates were either edema disease E. coli (EDEC; 77.3%) or enterotoxigenic E. coli (ETEC; 22.7%). On Farm 2, all mcr-positive strains were ETEC, while mcr-positive isolates from Farm 3 showed a wider range of pathotypes. The mcr-1.1 gene was located on IncHI2 (Farm 1), IncX4 (Farm 2) or IncX4 and IncI2 plasmids (Farm 3). These findings suggest that various pathogenic E. coli strains play an important role in maintaining plasmid-encoded colistin resistance genes in the pig environment over time.
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BACKGROUND: Escherichia coli bacteria capable of producing the toxin Stx2e and possessing F18-fimbriae (edema disease E. coli, EDEC) are considered causative agents of porcine oedema disease. This disease, which usually occurs in piglets shortly after weaning, has a high lethality in affected animals and can lead to high economic losses in piglet rearing. The aim of this cross-sectional field study was to determine the prevalence of EDEC in weaned piglets in Germany at pen and farm levels. RESULTS: Ninety-nine farms with unknown history of infections with shigatoxin-producing E. coli (STEC) and oedema disease were sampled. On each farm, up to five pens were selected for sampling (n = 481). The piglets in these pens were at an age 1-3 weeks after weaning. Single faecal samples (n = 2405) and boot swabs (n = 479) were collected from the floor. On 50 farms, cotton ropes were additionally used to collect oral fluid samples (n = 185) and rope wash out samples (n = 231) from the selected pens. All samples were analyzed by bacterial culture combined with a duplex PCR for the presence of the corresponding genes stx2e and fedA (major subunit protein of F18 fimbriae). In addition, whole DNA specimens extracted from boot swabs, oral fluid samples, and rope wash out samples were directly examined by duplex PCR for DNA of stx2e and fedA. A pen was classified as positive if at least one of the samples, regardless of the technique, yielded a positive result in the PCR, and farms were considered positive if at least one pen was classified as positive. Overall, genes stx2e and fedA were found simultaneously in 24.9% (95% CI 22.1-29.1%) of sampled pens and in 37.4% (95% CI 27.9-47.7%) of sampled farms. Regardless of the presence of F18-fimbriae, Escherichia coli encoding for Stx2e (STEC-2e) were found in 35.1% (95% CI 31.0-39.1%) of the pens and 53.5% (95% CI 44.4-63.6%) of the farms sampled. CONCLUSIONS: Escherichia coli strains considered capable to cause oedema disease in swine (EDEC) are highly prevalent in the surveyed pig producing farms in Germany. Due to intermittent shedding of EDEC and a potentially low within-farm prevalence, we recommend a combination of different sampling techniques for EDEC monitoring at pen and farm levels. Further studies are needed to understand which STEC-2e strains really pose the risk of causing severe porcine disease.
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Strangles, caused by Streptococcus equi ssp. equi (S. equi equi), is a highly infectious and frequent disease of equines worldwide. No data are available regarding the molecular epidemiology of strangles in Indonesia. This study aimed to characterize S. equi equi isolates obtained from suspected strangles cases in Indonesia in 2018. Isolates originated from seven diseased horses on four different farms located in three provinces of Indonesia. Whole genome sequences of these isolates were determined and used for seM typing, multilocus sequence typing (MLST), and core genome MLS typing (cgMLST). Genomes were also screened for known antimicrobial resistance genes and genes encoding for the recombinant antigens used in the commercial Strangvac® subunit vaccine. All seven S. equi equi isolates from Indonesia belonged to ST179 and carried seM allele 166. Isolates differed from each other by only 2 to 14 cgSNPs and built an exclusive sub-cluster within the Bayesian Analysis of Population Structure (BAPS) cluster 2 (BAPS-2) of the S. equi equi cgMLST scheme. All isolates revealed predicted amino acid sequence identity to seven and high similarity to one of the eight antigen fragments contained in Strangvac®. Furthermore, all isolates were susceptible to beta-lactam antibiotics penicillin G, ampicillin, and ceftiofur. Our data suggest that the horses from this study were affected by strains of the same novel sublineage within globally distributed BAPS-2 of S. equi equi. Nevertheless, penicillin G can be used as a first-choice antibiotic against these strains and Strangvac® may also be protective against Indonesian strains.
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Introduction: The global emergence of plasmid-mediated colistin resistance is threatening the efficacy of colistin as one of the last treatment options against multi-drug resistant Gram-negative bacteria. To date, ten mcr-genes (mcr-1 to mcr-10) were reported. While mcr-1 has disseminated globally, the occurrence of mcr-2 was reported scarcely. Methods and results: We determined the occurrence of mcr-1 and mcr-2 genes among Escherichia coli isolates from swine and performed detailed genomic characterization of mcr-2-positive strains. In the years 2010-2017, 7,614 porcine E. coli isolates were obtained from fecal swine samples in Europe and isolates carrying at least one of the virulence associated genes predicting Shiga toxin producing E. coli (STEC), enterotoxigenic E. coli (ETEC) or enteropathogenic E. coli (EPEC) were stored. 793 (10.4%) of these isolates carried the mcr-1 gene. Of 1,477 additional E. coli isolates obtained from sheep blood agar containing 4 mg/L colistin between 2018 and 2020, 36 (2.4%) isolates were mcr-1-positive. In contrast to mcr-1, the mcr-2 gene occurred at a very low frequency (0.13%) among the overall 9,091 isolates. Most mcr-2-positive isolates originated from Belgium (n = 9), one from Spain and two from Germany. They were obtained from six different farms and revealed multilocus sequence types ST10, ST29, ST93, ST100, ST3057 and ST5786. While the originally described mcr-2.1 was predominant, we also detected a new mcr-2 variant in two isolates from Belgium, which was termed mcr-2.8. MCR-2 isolates were mostly classified as ETEC or ETEC-like, while one isolate from Spain represented an atypical enteropathogenic E. coli (aEPEC; eae+). The ST29-aEPEC isolate carried mcr-2 on the chromosome. Another eight isolates carried their mcr-2 gene on IncX4 plasmids that resembled the pKP37-BE MCR-2 plasmid originally described in Belgium in 2015. Three ST100 E. coli isolates from a single farm in Belgium carried the mcr-2.1 gene on a 47-kb self-transmissible IncP type plasmid of a new IncP-1 clade. Discussion: This is the first report of mcr-2 genes in E. coli isolates from Germany. The detection of a new mcr-2 allele and a novel plasmid backbone suggests the presence of so far undetected mcr-2 variants and mobilizable vehicles.
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Pathogenic bacteria, such as enteropathogenic Escherichia coli (EPEC) and enterotoxigenic E. coli (ETEC), cause diarrhea in mammals. In particular, E. coli colonizes and infects the gastrointestinal tract via type 1 fimbriae (T1F). Here, the major zymogen granule membrane glycoprotein 2 (GP2) acts as a host cell receptor. GP2 is also secreted by the pancreas and various mucous glands, interacting with luminal type 1 fimbriae-positive E. coli. It is unknown whether GP2 isoforms demonstrate specific E. coli pathotype binding. In this study, we investigated interactions of human, porcine, and bovine EPEC and ETEC, as well as commensal E. coli isolates with human, porcine, and bovine GP2. We first defined pathotype- and host-associated FimH variants. Second, we could prove that GP2 isoforms bound to FimH variants to various degrees. However, the GP2-FimH interactions did not seem to be influenced by the host specificity of E. coli. In contrast, soluble GP2 affected ETEC infection and phagocytosis rates of macrophages. Preincubation of the ETEC pathotype with GP2 reduced the infection of cell lines. Furthermore, preincubation of E. coli with GP2 improved the phagocytosis rate of macrophages. Our findings suggest that GP2 plays a role in the defense against E. coli infection and in the corresponding host immune response. IMPORTANCE Infection by pathogenic bacteria, such as certain Escherichia coli pathotypes, results in diarrhea in mammals. Pathogens, including zoonotic agents, can infect different hosts or show host specificity. There are Escherichia coli strains which are frequently transmitted between humans and animals, whereas other Escherichia coli strains tend to colonize only one host. This host specificity is still not fully understood. We show that glycoprotein 2 is a selective receptor for particular Escherichia coli strains or variants of the adhesin FimH but not a selector for a species-specific Escherichia coli group. We demonstrate that GP2 is involved in the regulation of colonization and infection and thus represents a molecule of interest for the prevention or treatment of disease.
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Escherichia coli Enteropatógena , Escherichia coli Enterotoxigénica , Infecciones por Escherichia coli , Animales , Bovinos , Diarrea/microbiología , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/veterinaria , Fimbrias Bacterianas/metabolismo , Mamíferos , Glicoproteínas de Membrana/metabolismo , Vesículas Secretoras/metabolismo , PorcinosRESUMEN
Cattle and other ruminants are primary reservoirs for Shiga toxin-producing Escherichia coli (STEC) strains which have a highly variable, but unpredictable, pathogenic potential for humans. Domestic swine can carry and shed STEC, but only STEC strains producing the Shiga toxin (Stx) 2e variant and causing edema disease in piglets are considered pathogens of veterinary medical interest. In this chapter, we present general diagnostic workflows for sampling livestock animals to assess STEC prevalence, magnitude, and duration of host colonization. This is followed by detailed method protocols for STEC detection and typing at genetic and phenotypic levels to assess the relative virulence exerted by the strains.
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Enfermedades de los Bovinos , Infecciones por Escherichia coli , Toxina Shiga II/metabolismo , Escherichia coli Shiga-Toxigénica , Enfermedades de los Porcinos , Animales , Bovinos , Enfermedades de los Bovinos/diagnóstico , Enfermedades de los Bovinos/metabolismo , Enfermedades de los Bovinos/microbiología , Infecciones por Escherichia coli/diagnóstico , Infecciones por Escherichia coli/metabolismo , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/veterinaria , Escherichia coli Shiga-Toxigénica/clasificación , Escherichia coli Shiga-Toxigénica/aislamiento & purificación , Escherichia coli Shiga-Toxigénica/metabolismo , Escherichia coli Shiga-Toxigénica/patogenicidad , Porcinos , Enfermedades de los Porcinos/diagnóstico , Enfermedades de los Porcinos/metabolismo , Enfermedades de los Porcinos/microbiologíaRESUMEN
[This corrects the article DOI: 10.1016/j.vas.2017.04.001.].
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Plasmid-mediated resistance to carbapenems and colistin in Enterobacteriaceae represents an emerging public health threat. Although animals have been identified as a relevant source of multidrug-resistant (MDR) bacteria, there are only a few reports on the presence of carbapenemases in animal isolates. In this study, 7850 faecal Escherichia coli isolates obtained from 2160 pigs were screened for carbapenem non-susceptibility using Mueller-Hinton agar supplemented with meropenem. Eleven isolates showed growth on meropenem-containing agar but only two proved positive by PCR for a carbapenemase gene, namely blaOXA-48-like. The two isolates were obtained from different pigs housed at the same farm in Italy and were not genetically related by multilocus sequence typing (MLST), comprising ST359 and ST641. Whole-genome sequencing revealed the presence of blaOXA-181 in both isolates; in addition, the colistin resistance gene mcr-1 and aminoglycoside resistance gene armA were found in one isolate. The blaOXA-181 resistance gene was located on a 51.5-kb non-conjugative plasmid of replicon type IncX3 and the mcr-1 gene on a 33.3-kb transferable IncX4 plasmid. The high nucleotide similarity (>99%) of plasmids pEcIHIT31346-OXA-181 and pEcIHIT31346-MCR-1 to published plasmids from various human and animal sources suggests that specific antibiotic resistance plasmids are circulating among E. coli strains worldwide and across vertebrate species barriers. Although carbapenems are not licensed for use in livestock and the overall prevalence of carbapenemases in porcine E. coli appears to be low, the current findings indicate that even pigs can host MDR strains with accumulated plasmid-mediated resistance against several last-line antibiotics.
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Infecciones por Escherichia coli/veterinaria , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Escherichia coli/aislamiento & purificación , beta-Lactamasas/genética , Animales , Antibacterianos/farmacología , Carbapenémicos/farmacología , Colistina/farmacología , Conjugación Genética , Farmacorresistencia Bacteriana , Escherichia coli/clasificación , Infecciones por Escherichia coli/microbiología , Granjas , Transferencia de Gen Horizontal , Italia , Tipificación de Secuencias Multilocus , Plásmidos/análisis , PorcinosAsunto(s)
Antibacterianos/farmacología , Colistina/farmacología , Farmacorresistencia Bacteriana/genética , Enterobacteriaceae/genética , Proteínas de Escherichia coli/genética , Técnicas de Amplificación de Ácido Nucleico/métodos , Plásmidos/genética , Enterobacteriaceae/efectos de los fármacos , Enterobacteriaceae/aislamiento & purificación , HumanosRESUMEN
The ability of enteropathogenic Escherichia coli (EPEC) to express virulence factor genes and develop attaching and effacing (AE) lesions is inhibited in acidic environmental conditions. This inhibition is due to the activation of transcription factor GadX, which upregulates expression of glutamic acid decarboxylase (Gad). Gad, in turn, produces γ-aminobutyric acid (GABA), which was recently shown to have a beneficial effect on the jejunal epithelium in vitro due to increased mucin-1 levels. In the present study, we sought to test whether forced GadX activation/overexpression abolishes virulence associated features of EPEC and provokes increased GABA production. EPEC strains were isolated from diarrheic pigs and submitted to activation of GadX by acidification as well as gadX overexpression via an inducible expression vector plasmid. GABA concentrations in the growth medium, ability for adhesion to porcine intestinal epithelial cells (IPEC-J2) and virulence gene expression were determined. Growth in acidified media led to increased GABA levels, upregulated gadA/B expression and downregulated mRNA synthesis of the bacterial adhesin intimin. EPEC strains transformed with the gadX gene produced 2.1-3.4-fold higher GABA levels than empty-vector controls and completely lost their ability to adhere to IPEC-J2 cells and to induce actin accumulation. We conclude that intensified gadX activation can abolish the ability of EPEC to adhere to the intestinal epithelium by reducing the expression of major virulence genes.
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Sequence type 131 (ST131) is one of the predominant Escherichia coli lineages among extraintestinal pathogenic E. coli (ExPEC) that causes a variety of diseases in humans and animals and frequently shows multidrug resistance. Here, we report the first genome sequence of an ST131-ExPEC strain from poultry carrying the plasmid-encoded colistin resistance gene mcr-1.
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Multidrug-resistant Escherichia coli encoding CTX-M-type extended-spectrum ß-lactamases (ESBLs) are isolated in increasing numbers from humans, companion animals and livestock, raising concern regarding the exchange and spread of isolates in these populations. In this study, whole-genome sequencing of CTX-M-15-producing E. coli isolates recently sampled from humans, companion animals, livestock and farm environments was performed. In total, 26 different sequence types (STs) were detected, of which ST410 was the most frequent and was the only ST present in all populations studied. Five clades (designated A-E) were detected within the ST410 isolates. In particular, isolates of clade B were present in all four populations and had core genomes that differed by less than 70 single nucleotide polymorphisms (SNPs). Isolates of clades B and C were also clonally marked, exhibiting identical chromosomal insertions of blaCTX-M-15 at distinct loci. These data provide strong evidence for the clonal dissemination of specific clades of CTX-M-15-producing E. coli ST410 in human and animal populations.
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Antibacterianos/farmacología , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/veterinaria , Escherichia coli/clasificación , Escherichia coli/enzimología , Fluoroquinolonas/farmacología , beta-Lactamasas/metabolismo , Animales , Perros , Farmacorresistencia Bacteriana Múltiple , Microbiología Ambiental , Escherichia coli/aislamiento & purificación , Infecciones por Escherichia coli/epidemiología , Variación Genética , Genoma Bacteriano , Genotipo , Alemania/epidemiología , Humanos , Ganado , Epidemiología Molecular , Tipificación de Secuencias Multilocus , Análisis de Secuencia de ADNRESUMEN
Neonatal calf diarrhea is still one of the most important diseases in calf rearing, and severe diarrhea has a marked effect on animal welfare. Furthermore, significant economic losses can result from this disease due to high mortality rates, high medical costs, and low weight gain. To avoid a fatal outcome of the disease, it is crucial that vulnerable calves are identified as early as possible. Interleukin-6 is described as an early and reliable prognostic marker in several diseases. In this study, 20 scouring calves were tested by ELISA for their IL-6 serum concentrations. Samples were collected twice, at the beginning of diarrhea and 7 to 10d later. Regarding the clinical outcome after 7 to 10d, calves were classified as recovered or nonrecovered. A receiver operating characteristic analysis was conducted to determine the prognostic value of IL-6 for the progress of clinical symptoms. At the beginning of diarrhea, the IL-6 concentration was significantly higher in nonrecovering calves compared with those that recover 7 to 10d after the onset of diarrhea. Interleukin-6 proved to be a useful additional parameter in the clinical examination. High initial IL-6 values can support the decision for closer monitoring and an adapted therapeutic strategy for the respective calves. This may help to prevent unnecessary animal suffering and reduce economic losses.
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Enfermedades de los Bovinos/diagnóstico , Interleucina-6/sangre , Animales , Animales Recién Nacidos , Bovinos , Diarrea/diagnóstico , Diarrea/veterinaria , Heces , PronósticoRESUMEN
BACKGROUND: Post-weaning diarrhoea (PWD), due to Escherichia coli, is an important cause of economic losses to the pig industry primarily as a result of mortality and worsened productive performance. In spite of its relevance, recent data about the prevalence of virulence genes and pathotypes among E. coli isolates recovered from cases of PWD in Europe are scarce. RESULTS: This study investigates the prevalence of fimbrial and toxin genes of E. coli by PCR among 280 farms with PWD across Europe. A total of 873 samples collected within the first 48 h after the onset of PWD (occurring 7-21 days post weaning) were submitted to the laboratory for diagnostic purposes. Isolation and identification of E. coli were performed following standard bacteriological methods and PCR assays for the detection of genes encoding for fimbriae (F4, F5, F6, F18 and F41) and toxins (LT, STa, STb and Stx2e). The prevalence of fimbriae and toxins among E. coli isolates from cases of PWD was: F4 (45.1 %), F18 (33.9 %), F5 (0.6 %), F6 (0.6 %), F41 (0.3 %), STb (59.1 %), STa (38.1 %), LT (31.9 %) and Stx2e (9.7 %). E. coli isolates carrying both fimbrial and toxin genes were detected in 52.5 % of the cases (178 out of 339 isolates), with 94.9 % of them being classified as enterotoxigenic E. coli (ETEC). The most common virotype detected was F4, STb, LT. CONCLUSIONS: This study confirms that ETEC is frequently isolated in pig farms with PWD across Europe, with F4- and F18-ETEC variants involved in 36.1 % and 18.2 % of the outbreaks, respectively.
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Cattle are the most important reservoir for enterohemorrhagic Escherichia coli (EHEC), a subset of shigatoxigenic E. coli (STEC) capable of causing life-threatening infectious diseases in humans. In cattle, Shiga toxins (Stx) suppress the immune system thereby promoting long-term STEC shedding. First infections of animals at calves' age coincide with the lack of Stx-specific antibodies. We hypothesize that vaccination of calves against Shiga toxins prior to STEC infection may help to prevent the establishment of a persistent type of infection. The objectives of this study were to generate recombinant Shiga toxoids (rStx1mut & rStx2mut) by site-directed mutagenesis and to assess their immunomodulatory, antigenic, and immunogenic properties. Cultures of bovine primary immune cells were used as test systems. In ileal intraepithelial lymphocytes both, recombinant wild type Stx1 (rStx1WT) and rStx2WT significantly induced transcription of IL-4 mRNA. rStx1WT and rStx2WT reduced the expression of Stx-receptor CD77 (syn. Globotriaosylceramide, Gb3) on B and T cells from peripheral blood and of CD14 on monocyte-derived macrophages. At the same concentrations, rStx1mut and rStx2mut exhibited neither of these effects. Antibodies in sera of cattle naturally infected with STEC recognized the rStxmut toxoids equally well as the recombinant wild type toxins. Immunization of calves with rStx1mut plus rStx2mut led to induction of antibodies neutralizing Stx1 and Stx2. While keeping their antigenicity and immunogenicity recombinant Shiga toxoids are devoid of the immunosuppressive properties of the corresponding wild type toxins in cattle and candidate vaccines to mitigate long-term STEC shedding by the reservoir host.
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Proteínas Bacterianas/genética , Enfermedades de los Bovinos/inmunología , Infecciones por Escherichia coli/veterinaria , Vacunas contra Escherichia coli/inmunología , Escherichia coli Shiga-Toxigénica/inmunología , Toxoides/farmacología , Animales , Proteínas Bacterianas/metabolismo , Bovinos , Enfermedades de los Bovinos/microbiología , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/microbiología , Vacunas contra Escherichia coli/efectos adversos , Masculino , Mutagénesis Sitio-Dirigida/veterinaria , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vacunas Sintéticas/efectos adversos , Vacunas Sintéticas/inmunologíaRESUMEN
Escherichia (E.) coli producing extended-spectrum beta-lactamases (ESBLs) are an increasing problem for public health. The success of ESBLs may be due to spread of ESBL-producing bacterial clones, transfer of ESBL gene-carrying plasmids or exchange of ESBL encoding genes on mobile elements. This makes it difficult to identify transmission routes and sources for ESBL-producing bacteria. The objectives of this study were to compare the distribution of genotypic and phenotypic properties of E. coli isolates from different animal and human sources collected in studies in the scope of the national research project RESET. ESBL-producing E. coli from two longitudinal and four cross-sectional studies in broiler, swine and cattle farms, a cross-sectional and a case-control study in humans and diagnostic isolates from humans and animals were used. In the RESET consortium, all laboratories followed harmonized methodologies for antimicrobial susceptibility testing, confirmation of the ESBL phenotype, specific PCR assays for the detection of bla(TEM), bla(CTX), and bla(SHV) genes and sequence analysis of the complete ESBL gene as well as a multiplex PCR for the detection of the four major phylogenetic groups of E. coli. Most ESBL genes were found in both, human and non-human populations but quantitative differences for distinct ESBL-types were detectable. The enzymes CTX-M-1 (63.3% of all animal isolates, 29.3% of all human isolates), CTX-M-15 (17.7% vs. 48.0%) and CTX-M-14 (5.3% vs. 8.7%) were the most common ones. More than 70% of the animal isolates and more than 50% of the human isolates contained the broadly distributed ESBL genes bla(CTX-M-1), bla(CTX-M-15), or the combinations bla(SHV-12)+bla(TEM) or bla(CTX-M-1)+bla(TEM). While the majority of animal isolates carried bla(CTX-M-1) (37.5%) or the combination bla(CTX-M-1)+bla(TEM) (25.8%), this was the case for only 16.7% and 12.6%, respectively, of the human isolates. In contrast, 28.2% of the human isolates carried bla(CTX-M-15) compared to 10.8% of the animal isolates. When grouping data by ESBL types and phylogroups bla(CTX-M-1) genes, mostly combined with phylogroup A or B1, were detected frequently in all settings. In contrast, bla(CTX-M-15) genes common in human and animal populations were mainly combined with phylogroup A, but not with the more virulent phylogroup B2 with the exception of companion animals, where a few isolates were detectable. When E. coli subtype definition included ESBL types, phylogenetic grouping and antimicrobial susceptibility data, the proportion of isolates allocated to common clusters was markedly reduced. Nevertheless, relevant proportions of same subtypes were detected in isolates from the human and livestock and companion animal populations included in this study, suggesting exchange of bacteria or bacterial genes between these populations or a common reservoir. In addition, these results clearly showed that there is some similarity between ESBL genes, and bacterial properties in isolates from the different populations. Finally, our current approach provides good insight into common and population-specific clusters, which can be used as a basis for the selection of ESBL-producing isolates from interesting clusters for further detailed characterizations, e.g. by whole genome sequencing.
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Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/veterinaria , Escherichia coli/clasificación , Escherichia coli/enzimología , beta-Lactamasas/análisis , beta-Lactamasas/clasificación , Animales , Bovinos , Pollos , ADN Bacteriano/química , ADN Bacteriano/genética , Escherichia coli/genética , Escherichia coli/aislamiento & purificación , Genotipo , Humanos , Pruebas de Sensibilidad Microbiana , Fenotipo , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Porcinos , beta-Lactamasas/genéticaRESUMEN
BACKGROUND: Multiresistant Gram-negative bacteria producing extended-spectrum ß-lactamases (ESBLs) are an emerging problem in human and veterinary medicine. This study focused on comparative molecular characterization of ß-lactamase and ESBL-producing Enterobacteriaceae isolates from central Hesse in Germany. Isolates originated from humans, companion animals (dogs and cats) and horses. RESULTS: In this study 153 (83.6%) of the human isolates (n = 183) and 163 (91.6%) of the animal isolates (n = 178) were confirmed as ESBL producers by PCR and subsequent sequencing of the PCR amplicons. Predominant ESBL subtypes in human and animal samples were CTX-M-15 (49.3%) and CTX-M-1 (25.8%) respectively. Subtype blaCTX-M-2 was found almost exclusively in equine and was absent from human isolates. The carbapenemase OXA-48 was detected in 19 ertapenem-resistant companion animal isolates in this study. The Plasmid-encoded quinolone resistance (PMQR) gene aac('6)-Ib-cr was the most frequently detected antibiotic- resistance gene present in 27.9% of the human and 36.9% of the animal ciprofloxacin-resistant isolates. Combinations of two or up to six different resistance genes (penicillinases, ESBLs and PMQR) were detected in 70% of all isolates investigated. The most frequent species in this study was Escherichia coli (74%), followed by Klebsiella pneumoniae (17.5%), and Enterobacter cloacae (4.2%). Investigation of Escherichia coli phylogenetic groups revealed underrepresentation of group B2 within the animal isolates. CONCLUSIONS: Isolates from human, companion animals and horses shared several characteristics regarding presence of ESBL, PMQR and combination of different resistance genes. The results indicate active transmission and dissemination of multi-resistant Enterobacteriaceae among human and animal populations.
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Farmacorresistencia Bacteriana Múltiple , Enterobacteriaceae/efectos de los fármacos , Enterobacteriaceae/enzimología , beta-Lactamasas/genética , Animales , ADN Bacteriano/química , ADN Bacteriano/genética , Enterobacteriaceae/aislamiento & purificación , Genotipo , Alemania , Caballos , Humanos , Mascotas , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADNAsunto(s)
Enfermedades de los Bovinos/microbiología , Diarrea/veterinaria , Infecciones por Escherichia coli/veterinaria , Escherichia coli/enzimología , Escherichia coli/aislamiento & purificación , Toxina Shiga/genética , beta-Lactamasas/genética , Animales , Bovinos , Diarrea/microbiología , Escherichia coli/clasificación , Escherichia coli/genética , Infecciones por Escherichia coli/microbiología , Genotipo , Análisis por Micromatrices , Pruebas de Sensibilidad Microbiana , Análisis de Secuencia por Matrices de Oligonucleótidos , Plásmidos/análisis , Serogrupo , Toxina Shiga/metabolismo , beta-Lactamasas/metabolismoRESUMEN
We characterized ESBL-producing Escherichia coli isolates from diseased dog, cat and human sources for their plasmid content. Plasmids with different Inc groups and combinations of resistance genes were detected in these isolates. The pan-genome of the plasmid-associated genes was found to be large, indicating diversity of the gene pool among the plasmids. No commonly occurring plasmids with similar gene content in isolates from dog, cats and humans were detected.