Bifidobacteria comprise a significant proportion of the human gut microbiota. Several bifidobacterial strains are currently used as therapeutic interventions, claiming various health benefits by ...acting as probiotics. However, the precise mechanisms by which they maintain habitation within their host and consequently provide these benefits are not fully understood. Here we show that Bifidobacterium breve UCC2003 produces a cell surface-associated exopolysaccharide (EPS), the biosynthesis of which is directed by either half of a bidirectional gene cluster, thus leading to production of one of two possible EPSs. Alternate transcription of the two opposing halves of this cluster appears to be the result of promoter reorientation. Surface EPS provided stress tolerance and promoted in vivo persistence, but not initial colonization. Marked differences were observed in host immune response: strains producing surface EPS (EPS+) failed to elicit a strong immune response compared with EPS-deficient variants. Specifically, EPS production was shown to be linked to the evasion of adaptive B-cell responses. Furthermore, presence of EPS+ B. breve reduced colonization levels of the gut pathogen Citrobacter rodentium. Our data thus assigns a pivotal and beneficial role for EPS in modulating various aspects of bifidobacterial–host interaction, including the ability of commensal bacteria to remain immunologically silent and in turn provide pathogen protection. This finding enforces the probiotic concept and provides mechanistic insights into health-promoting benefits for both animal and human hosts.
Salmonella enterica serovar Paratyphi B variant Java sequence type 28 is prevalent in poultry and poultry meat. We investigated the evolutionary relatedness between sequence type 28 strains from ...Europe and Latin America using time-resolved phylogeny and principal component analysis. We sequenced isolates from Colombia, Guatemala, Costa Rica, and the Netherlands and complemented them with publicly available genomes from Europe, Africa, and the Middle East. Phylogenetic time trees and effective population sizes (N
) showed separate clustering of strains from Latin America and Europe. The separation is estimated to have occurred during the 1980s. N
of strains increased sharply in Europe around 1995 and in Latin America around 2005. Principal component analysis on noncore genes showed a clear distinction between strains from Europe and Latin America, whereas the plasmid gene content was similar. Regardless of the evolutionary separation, similar features of resistance to β-lactams and quinolones/fluoroquinolones indicated parallel evolution of antimicrobial resistance in both regions.
Host Defense Peptides (HDPs) such as cathelicidins are multifunctional effectors of the innate immune system with both antimicrobial and pleiotropic immunomodulatory functions. Chicken cathelicidin-2 ...(CATH-2) has multiple immunomodulatory effects in vitro and the D-amino acid analog of this peptide has been shown to partially protect young chicks from a bacterial infection. However, the mechanisms responsible for CATH-2 mediated in vivo protection have not been investigated so far. In this study, D-CATH-2 was administered in ovo and the immune status and microbiota of the chicks were investigated at 7 days posthatch to elucidate the in vivo mechanisms of the peptide. In three consecutive studies, no effects on numbers and functions of immune cells were found and only small changes were seen in gene expression of Peripheral Blood Mononuclear Cells (PBMCs). In two studies, intestinal microbiota composition was determined which was highly variable, suggesting that it was strongly influenced by environmental factors. In both studies, in ovo D-CATH-2 treatment caused significant reduction of Ruminococcaceae and Butyricicoccus in the cecum and Escherichia/Shigella in both ileum and cecum. In conclusion, this study shows that, in the absence of an infectious stimulus, in ovo administration of a CATH-2 analog alters the microbiota composition but does not affect the chicks' immune system posthatch.
Intramammary infections (IMI) with Staphylococcus aureus are a common cause of bovine mastitis and can result in both clinical (CM) or subclinical mastitis (SCM). Although bacterial isolates of S. ...aureus differ in their virulence potential it is largely unclear which bacterial virulence factors are responsible for increased clinical severity. We performed a genome wide association study and used a generalized linear mixed model to investigate the correlation between gene carriage, lineage and clinical outcome of IMI in a collection of S. aureus isolates from cattle with CM (n = 125) and SCM (n = 151) from 11 European countries. An additional aim was to describe the genetic variation of bovine S. aureus in Europa. The dominant lineages in our collection were clonal complex (CC) 151 (81/276, 29.3%), CC97 (54/276, 19.6%), CC479 (32/276, 11.6%) and CC398 (19/276, 6.9%). Virulence and antimicrobial resistance (AMR) gene carriage was highly associated with CC. Among a selection of nine virulence and AMR genes, CC151, CC479 and CC133 carried more virulence genes than other CCs, and CC398 was associated with AMR gene carriage. Whereas CC151, CC97 were widespread in Europe, CC479, CC398 and CC8 were only found in specific countries. Compared to CC151, CC479 was associated with CM rather than SCM (OR 3.62; 95% CI 1.38-9.50) and the other CCs were not. Multiple genes were associated with CM, but due to the clustering within CC of carriage of these genes, it was not possible to differentiate between the effect of gene carriage and CC on clinical outcome of IMI. Nevertheless, this study demonstrates that characterization of S. aureus CC and virulence genes helps to predict the likelihood of the occurrence of CM following S. aureus IMI and highlights the potential benefit of diagnostics tools to identify S. aureus CC during bovine mastitis.
Animal rehabilitation centres provide a unique opportunity to study the microbiome of wild animals because subjects will be handled for their treatment and can therefore be sampled longitudinally. ...However, rehabilitation may have unintended consequences on the animals' microbiome because of a less varied and suboptimal diet, possible medical treatment and exposure to a different environment and human handlers. Our study describes the gut microbiome of two large seal cohorts, 50 pups (0-30 days old at arrival) and 23 weaners (more than 60 days old at arrival) of stranded harbour seals admitted for rehabilitation at the Sealcentre Pieterburen in the Netherlands, and the effect of rehabilitation on it. Faecal samples were collected from all seals at arrival, two times during rehabilitation and before release. Only seals that did not receive antimicrobial treatment were included in the study. The average time in rehabilitation was 95 days for the pups and 63 days for the weaners. We observed that during rehabilitation, there was an increase in the relative abundance of some of the Campylobacterota spp and Actinobacteriota spp. The alpha diversity of the pups' microbiome increased significantly during their rehabilitation (p-value <0.05), while there were no significant changes in alpha diversity over time for weaners. We hypothesize that aging is the main reason for the observed changes in the pups' microbiome. At release, the sex of a seal pup was significantly associated with the microbiome's alpha (i.e., Shannon diversity was higher for male pups, p-value <0.001) and beta diversity (p-value 0.001). For weaners, variation in the microbiome composition (beta diversity) at release was partly explained by sex and age of the seal (p-values 0.002 and 0.003 respectively). We mainly observed variables known to change the gut microbiome composition (e.g., age and sex) and conclude that rehabilitation in itself had only minor effects on the gut microbiome of seal pups and seal weaners.
Antimicrobial-resistance (AMR) genes in bacteria are often carried on plasmids and these plasmids can transfer AMR genes between bacteria. For molecular epidemiology purposes and risk assessment, it ...is important to know whether the genes are located on highly transferable plasmids or in the more stable chromosomes. However, draft whole-genome sequences are fragmented, making it difficult to discriminate plasmid and chromosomal contigs. Current methods that predict plasmid sequences from draft genome sequences rely on single features, like
-mer composition, circularity of the DNA molecule, copy number or sequence identity to plasmid replication genes, all of which have their drawbacks, especially when faced with large single-copy plasmids, which often carry resistance genes. With our newly developed prediction tool RFPlasmid, we use a combination of multiple features, including
-mer composition and databases with plasmid and chromosomal marker proteins, to predict whether the likely source of a contig is plasmid or chromosomal. The tool RFPlasmid supports models for 17 different bacterial taxa, including
,
and
, and has a taxon agnostic model for metagenomic assemblies or unsupported organisms. RFPlasmid is available both as a standalone tool and via a web interface.
Abstract Objectives Antimicrobials can select for antimicrobial-resistant bacteria. After treatment the active compound is excreted through urine and faeces. As some antimicrobials are chemically ...stable, recirculation of subinhibitory concentrations of antimicrobials may occur due to coprophagic behaviour of animals such as chickens. Methods The persistence of three antimicrobials over time and their potential effects on antimicrobial resistance were determined in four groups of broilers. Groups were left untreated (control) or were treated with amoxicillin (unstable), doxycycline or enrofloxacin (stable). Antimicrobials were extracted from the faecal samples and were measured by LC-MS/MS. We determined the resistome genotypically using shotgun metagenomics and phenotypically by using Escherichia coli as indicator microorganism. Results Up to 37 days after treatment, doxycycline and enrofloxacin had concentrations in faeces equal to or higher than the minimal selective concentration (MSC), in contrast to the amoxicillin treatment. The amoxicillin treatment showed a significant difference (P ≤ 0.01 and P ≤ 0.0001) in the genotypic resistance only directly after treatment. On the other hand, the doxycycline treatment showed approximately 52% increase in phenotypic resistance and a significant difference (P ≤ 0.05 and P ≤ 0.0001) in genotypic resistance throughout the trial. Furthermore, enrofloxacin treatment resulted in a complete non-WT E. coli population but the quantity of resistance genes was similar to the control group, likely because resistance is mediated by point mutations. Conclusions Based on our findings, we suggest that persistence of antimicrobials should be taken into consideration in the assessment of priority classification of antimicrobials in livestock.
Food products carry bacteria unless specifically sterilised. These bacteria can be pathogenic, commensal or associated with food spoilage, and may also be resistant to antimicrobials. Current methods ...for detecting bacteria on food rely on culturing for specific bacteria, a time-consuming process, or 16S rRNA metabarcoding that can identify different taxa but not their genetic content. Directly sequencing metagenomes of food is inefficient as its own DNA vastly outnumbers the bacterial DNA present. We optimised host DNA depletion enabling efficient sequencing of food microbiota, thereby increasing the proportion of non-host DNA sequenced 13-fold (mean; range: 1.3–40-fold) compared to untreated samples. The method performed best on chicken, pork and leafy green samples which had high mean prokaryotic read proportions post-depletion (0.64, 0.74 and 0.74, respectively), with lower mean prokaryotic read proportions in salmon (0.50) and prawn samples (0.19). We show that bacterial compositions and concentrations of antimicrobial resistance (AMR) genes differed by food type, and that salmon metagenomes were influenced by the production/harvesting method. The approach described in this study is an efficient and effective method of identifying and quantifying the predominant bacteria and AMR genes on food.
•Method optimised for depleting host DNA from foods of animal and plant origin to maximise metagenome sequencing of microbes.•Chicken, leafy greens, pork, prawns and salmon contained AMR gene concentrations from 104-1010 AMR genes/gram of food.•Aquacultured salmon had distinct microbial taxa from those caught in the wild.
•Wild birds and poultry are the main sources of Campylobacter in surface water.•Livestock density, water type and season influence the animal source contributions.•Wild bird-borne Campylobacter ...strains predominate in recreational waters.•Poultry-borne Campylobacter strains are more common in high poultry density areas.
Campylobacter jejuni and C. coli, the primary agents of human bacterial gastroenteritis worldwide, are widespread in surface water. Several animal sources contribute to surface water contamination with Campylobacter, but their relative contributions thus far remained unclear. Here, the prevalence, genotype diversity, and potential animal sources of C. jejuni and C. coli strains in surface water in the Netherlands were investigated. It was also assessed whether the contribution of the different animal sources varied according to surface water type (i.e. agricultural water, surface water at discharge points of wastewater treatment plants WWTPs, and official recreational water), season, and local livestock (poultry, pig, ruminant) density. For each surface water type, 30 locations spread over six areas with either high or low density of poultry, ruminants, or pigs, were sampled once every season in 2018-2019. Campylobacter prevalence was highest in agricultural waters (77%), and in autumn and winter (74%), and lowest in recreational waters (46%) and in summer (54%). In total, 76 C. jejuni and 177 C. coli water isolates were whole-genome sequenced. Most C. coli water isolates (78.5%) belonged to hitherto unidentified clones when using the seven-locus sequence type (ST) scheme, while only 11.8% of the C. jejuni isolates had unidentified STs. The origin of these isolates, as defined by core-genome multi-locus sequence typing (cgMLST), was inferred by comparison with Campylobacter strain collections from meat-producing poultry, laying hens, adult cattle, veal calves, small ruminants, pigs, and wild birds. Water isolates were mainly attributed to wild birds (C. jejuni: 60.0%; C. coli: 93.7%) and meat-producing poultry (C. jejuni: 18.9%; C. coli: 5.6%). Wild bird contribution was high among isolates from recreational waters and WWTP discharge points, and in areas with low poultry (C. coli) or high ruminant (C. jejuni) densities. The contribution of meat-producing poultry was high in areas with high density of poultry, springtime, agricultural waters and WWTP discharge points. While wild birds and poultry were the main contributors to Campylobacter contamination in surface water, their contribution differed significantly by water type, season, and local poultry and ruminant densities.
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The features contributing to differences in pathogenicity of the Campylobacter fetus subspecies are unknown. Putative factors involved in pathogenesis are located in genomic islands that encode a ...type IV secretion system (T4SS) and fic domain (filamentation induced by cyclic AMP) proteins, which may disrupt host cell processes. In the genomes of 27 C. fetus strains, three phylogenetically-different T4SS-encoding regions (T4SSs) were identified: one was located in both the chromosome and in extra-chromosomal plasmids; one was located exclusively in the chromosome; and one exclusively in extra-chromosomal plasmids. We observed that C. fetus strains can contain multiple T4SSs and that homologous T4SSs can be present both in chromosomal genomic islands (GI) and on plasmids in the C. fetus strains. The GIs of the chromosomally located T4SS differed mainly by the presence of fic genes, insertion sequence elements and phage-related or hypothetical proteins. Comparative analysis showed that T4SS sequences, inserted in the same locations, were conserved in the studied C. fetus genomes. Using phylogenetic analysis of the T4SSs, it was shown that C. fetus may have acquired the T4SS regions from other Campylobacter species by horizontal gene transfer. The identified T4SSs and fic genes were found in Cff and Cfv strains, although the presence of T4SSs and fic genes were significantly associated with Cfv strains. The T4SSs and fic genes could not be associated with S-layer serotypes or geographical origin of the strains.
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