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In vitro inhibition of avian pathogenic Enterococcus cecorum isolates by probiotic Bacillus strains.

ABSTRACT: Enterococcus cecorum is a commensal bacteria and opportunistic pathogen that can cause outbreaks of Enterococcal spondylitis ("kinky back") in poultry, with a growing concern worldwide. Numerous Bacillus-based probiotic strains are commercially available with proven effects in supporting gut health and growth performance, but efficacy against pathogenic E. cecorum is unknown. This study compared the in vitro inhibitory potential of cell-free supernatants (CFSs) of 18 Bacillus strains (14 commercial probiotic strains, 1 internal negative control and 3 type strains) on the growth of 9 clinical E. cecorum isolates. Standardized biomass cultures of live Bacillus were harvested and filtered to obtain CFSs. Inhibitory potential against E. cecorum isolates was assessed via a microdilution assay in which the final pathogen concentration was ? 104 CFU/mL. Absorbance (OD) was measured every 15 min for 15 h and used to calculate percentage growth inhibition at an OD equivalent to 0.4 in the positive control (PC) (pathogen but no CFS), and growth delay vs. PC. Growth kinetic responses of pathogen isolate-Bacillus strain combinations ranged from total pathogen inhibition to partial inhibition, lag in growth, no effect, or increased growth vs. PC. Percentage inhibition of individual isolates varied markedly among Bacillus strains, from 100% to -100% (growth promotion as recorded for the type strain) (B. amyloliquefaciens DSM7T). Five B. amyloliquefaciens CFSs produced higher average inhibition rates (>75%) than 2 out of 3 Bacillus licheniformis CFSs (-2.5, and -8.39% vs. PC, respectively) and 1 out of 2 Bacillus subtilis CFSs (7.3% vs. PC) (P < 0.05). Commercial strain 3AP4 exhibited the highest average percentage inhibition vs. PC (85.0% ± 7.9) and the most consistent inhibitory effect across pathogen isolates. The findings indicate that some commercially available poultry probiotic Bacillus strains are effective at inhibiting pathogenic E. cecorum in vitro, but effects are highly strain and pathogen isolate-dependent. Further work is required to confirm effects in vivo and isolate the inhibitory substances.

SUBMITTER: Medina Fernandez S

PROVIDER: S-EPMC6527510 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Project description:BackgroundEnterococcus cecorum (EC) infection currently is one of the most important bacterial diseases of modern broiler chickens but can also affect ducks or other avian species. However, little is known concerning pathogenesis of EC and most studies concentrate on examinations of EC strains from broilers only. The objective of this study was to compare pathogenic and commensal EC strains from different animal species concerning different phenotypic and genotypic traits.ResultsPathogenic and commensal EC strains were not clearly separated from each other in a phylogenetic tree based on partial sequences of the 16S-rRNA-gene and also based on the fatty acid profile determined with gas chromatography. C12:0, C14:0, C15:0, C16:0, C17:0, C18:0, C18:1 w7c, C18:1 w9c and C20:4 w6,9,12,15c were detected as the major fatty acids. None of the 21 pathogenic EC strains was able to utilize mannitol, while 9 of 29 commensal strains were mannitol positive. In a dendrogram based on MALDI-TOF MS data, pathogenic strains were not clearly separated from commensal isolates. However, significant differences concerning the prevalence of several mass peaks were confirmed between the two groups. Two different antisera were produced but none of the serotypes was predominantly found in the pathogenic or commensal EC isolates. Enterococcal virulence factors gelE, esp, asa1, ccf, hyl and efaAfs were only detected in single isolates via PCR. No virulence factor was found significantly more often in the pathogenic isolates. The chicken embryo lethality of the examined EC isolates varied from 0 up to 100%. The mean embryo lethality in the pathogenic EC isolates was 39.7%, which was significantly higher than the lethality of the commensal strains, which was 18.9%. Additionally, five of the commensal isolates showed small colony variant growth, which was never reported for EC before.ConclusionsPathogenic and commensal EC isolates from different animal species varied in chicken embryo lethality, in their ability to metabolize mannitol and probably showed divergent mass peak patterns with MALDI-TOF MS. These differences may be explained by a separate evolution of pathogenic EC isolates. Furthermore, different serotypes of EC were demonstrated for the first time.

Project description:In recent years, pathogenic strains of Enterococcus cecorum (EC) have emerged as a causing agent of septicemia and skeletal infection in broiler chickens with a high economic impact worldwide. Although research has been conducted, many aspects of the pathogenesis of the EC-associated disease are still unknown. In the present study, an experimental infection model was established in broiler chickens. Two different EC strains (EC14 and EC15) were compared in two different concentrations of each strain (2 × 106 and 2 × 108 colony-forming units per milliliter (CFU/mL)) after oral infection of one-day-old chicks. Clinical signs and gross lesions of the EC-associated disease were monitored in the following seven weeks. Although both EC strains were originally isolated from clinical disease outbreaks and had a high embryonic lethality, only EC14 successfully induced the typical course of the EC-associated disease with characteristic clinical signs and gross lesions. In total, 23% of the birds in the two EC14-groups were EC-positive in extraintestinal organs on culture, and no differences were found between the two infectious doses. EC14 was frequently detected via real-time PCR in the free thoracic vertebra (FTV) and femoral heads without any detectable gross lesions. The number of EC positive spleens from infected broilers was comparable using bacterial isolation and a specific real-time PCR. Interestingly, EC15 was not detected in extraintestinal organs, although birds in the EC15 groups were colonized by EC in the ceca after experimental infection. The present study represents first proof that virulence differs among EC strains in experimentally infected chickens, and emphasizes the need to further characterize virulence factors and pathogenic mechanisms of EC. The strain EC14 at a dose of 106 CFU is suitable for reproduction of the EC-associated disease. The experimental infection model reported here provides the basis for further research on the EC pathogenesis and possible prevention and intervention strategies.

Project description:Enterococcus cecorum (EC) is known as a commensal in the intestines of mammals and birds. However, it has been described as an emerging pathogen in poultry industry worldwide. The aim of this study was to analyze and compare EC isolated from clinical material collected from poultry groups with different production purposes. The genetic diversity among pathogenic EC in relation to each specific poultry type was examined. In total, 148 isolates from independent infection outbreaks (2011-2016) were used: 76 broiler chickens (CB), 37 broiler breeders (BB), 23 layers (CL), 7 waterfowl (W) and 5 turkey (T) flocks (1 isolate/1 flock). We provided age ranges at diagnosis of EC-infection for 5 poultry groups. Isolates obtained from CB were significantly more frequently retrieved from bone marrow, joints, spine, and contrary to BB, CL less frequently retrieved from respiratory system. The study showed differences between EC of various poultry types in relation to 10/32 (31.3%) biochemical parameters. EC isolates from CB were significantly more often positive for βGAL, βNAG, MLZ, and less often positive for PAL and βMAN than isolates from other poultry types. However, BB and W isolates showed higher ability to metabolise mannitol than CB, CL, and T. CB isolates showed lower ability to survive at 60°C. Only chicken EC-isolates harbored virulence genes: CB (8.1%) > BB (3.4%) > CL (2%). No specific pulsotype of EC was associated with a specific poultry. One or several various (up to 6) genetic types of EC may be involved in outbreaks in CB flocks within one year in one region. Outbreaks reported in following years in the same region were usually caused by a distinct set of EC-genetic types. PFGE results indicated at the genetic heterogeneity among pathogenic isolates involved in outbreaks in relation to each poultry type. To our best knowledge, this is the first study which provides a comparison between clinical EC from 5 poultry groups. The study provides a new insight into EC as pathogen of different bird species. The obtained data may be useful in further studies on EC-infections more focused on a specific type of poultry.

Project description:Enterococcus cecorum is an emerging pathogen responsible for osteomyelitis, spondylitis, and femoral head necrosis causing animal suffering and mortality and requiring antimicrobial use in poultry. Paradoxically, E. cecorum is a common inhabitant of the intestinal microbiota of adult chickens. Despite evidence suggesting the existence of clones with pathogenic potential, the genetic and phenotypic relatedness of disease-associated isolates remains little investigated. Here, we sequenced and analyzed the genomes and characterized the phenotypes of more than 100 isolates, the majority of which were collected over the last 10 years from 16 French broiler farms. Comparative genomics, genome-wide association studies, and the measured susceptibility to serum, biofilm-forming capacity, and adhesion to chicken type II collagen were used to identify features associated with clinical isolates. We found that none of the tested phenotypes could discriminate the origin of the isolates or the phylogenetic group. Instead, we found that most clinical isolates are grouped phylogenetically, and our analyses selected six genes that discriminate 94% of isolates associated with disease from those that are not. Analysis of the resistome and the mobilome revealed that multidrug-resistant clones of E. cecorum cluster into a few clades and that integrative conjugative elements and genomic islands are the main carriers of antimicrobial resistance. This comprehensive genomic analysis shows that disease-associated clones of E. cecorum belong mainly to one phylogenetic clade. IMPORTANCE Enterococcus cecorum is an important pathogen of poultry worldwide. It causes a number of locomotor disorders and septicemia, particularly in fast-growing broilers. Animal suffering, antimicrobial use, and associated economic losses require a better understanding of disease-associated E. cecorum isolates. To address this need, we performed whole-genome sequencing and analysis of a large collection of isolates responsible for outbreaks in France. By providing the first data set on the genetic diversity and resistome of E. cecorum strains circulating in France, we pinpoint an epidemic lineage that is probably also circulating elsewhere that should be targeted preferentially by preventive strategies in order to reduce the burden of E. cecorum-related diseases.

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In vitro inhibition of avian pathogenic Enterococcus cecorum isolates by probiotic Bacillus strains.

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