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Background Early weaning and intensive farming practices predispose piglets to the development of infectious and often lethal diseases, against which antibiotics are used. Besides contributing to the build-up of antimicrobial resistance, antibiotics are known to modulate the gut microbial composition. As an alternative to antibiotic treatment, studies have previously investigated the potential of probiotics for the prevention of postweaning diarrhea. In order to describe the post-weaning gut microbiota, and to study the effects of two probiotics formulations and of intramuscular antibiotic treatment on the gut microbiota, we sampled and processed over 800 faecal time-series samples from 126 piglets and 42 sows. Results Here we report on the largest shotgun metagenomic dataset of the pig gut lumen microbiome to date, consisting of >8 Tbp of shotgun metagenomic sequencing data. The animal trial, the workflow from sample collection to sample processing, and the preparation of libraries for sequencing, are described in detail. We provide a preliminary analysis of the dataset, centered on a taxonomic profiling of the samples, and a 16S-based beta diversity analysis of the mothers and the piglets in the first 5 weeks after weaning. Conclusions This study was conducted to generate a publicly available databank of the faecal metagenome of weaner piglets aged between 3 and 9 weeks old, treated with different probiotic formulations and intramuscular antibiotic treatment. Besides investigating the effects of the probiotic and intramuscular antibiotic treatment, the dataset can be explored to assess a wide range of ecological questions with regards to antimicrobial resistance, host-associated microbial and phage communities, and their dynamics during the aging of the host.

BackgroundEarly weaning and intensive farming practices predispose piglets to the development of infectious and often lethal diseases, against which antibiotics are used. Besides contributing to the build-up of antimicrobial resistance, antibiotics are known to modulate the gut microbial composition. Studies have previously investigated the effects of probiotics as alternatives to antibiotic treatment for the prevention of post-weaning diarrhea. In order to describe the post-weaning gut microbiota, and the effects of two probiotics formulations and of intramuscular antibiotic treatment on the gut microbiota, we processed over 800 faecal time-series samples from 126 piglets and 42 sows, generating over 8Tbp of metagenomic shotgun sequence data. Here we describe the animal trial procedures, the generation of our metagenomic dataset and the analysis of the microbial community composition using a phylogenetic framework.ResultsFactors such as age, litter effects, and breed, by significantly correlating with gut microbial community shifts, can be major confounding factors in the assessment of treatment effects. Intramuscular antibiotic treatment and probiotic treatments were found to correlate with alpha and beta diversity, as well as with a transient establishment of Mollicutes and Lactobacillales, respectively. We found the abundance of certain taxa to correlate with weight gain.ConclusionsOur findings demonstrate that breed, litter, and age, are important contributors to variation in the community composition, and that treatment effects of the antibiotic and probiotic treatments were subtle, while host age was the dominant factor in shaping the gut microbiota of piglets after weaning. The current study shows, by means of a phylogenetic diversity framework, that the post-weaning pig gut microbiome appears to follow a highly structured developmental program with characteristic post-weaning changes that can distinguish hosts that were born as little as two days apart in the second month of life.

BackgroundIntensive farming practices can increase exposure of animals to infectious agents against which antibiotics are used. Besides leading to antimicrobial resistance (AMR), orally administered antibiotics are well known to cause dysbiosis. To counteract dysbiotic effects, numerous studies in the past two decades sought to understand whether probiotics are a valid tool to help re-establish a healthy gut microbial community after antibiotic treatment. However, although dysbiotic effects of antibiotics are well investigated, little is known about the effects of intramuscular antibiotic treatment on the gut microbiome and a few studies attempted to study treatment effects using phylogenetic diversity analysis techniques. In this study we sought to determine the effects of two probiotic- and one intramuscularly administered antibiotic treatment on the developing gut microbiome of post-weaning piglets between their 3rd and 9th week of life.MethodsShotgun metagenomic sequences from over 800 faecal time-series samples derived from 126 piglets and 42 sows were analysed in a phylogenetic framework to characterise the developing gut microbial community composition of post-weaning piglets. We assessed the effects of intramuscular antibiotic treatment and probiotic oral treatment on the diversity of these gut microbial communities using alpha and beta diversity measures.ResultsDifferences between individual hosts such as breed, litter, and age, were found to be important contributors to variation in the community composition. Host age was the dominant factor in shaping the gut microbiota of piglets after weaning. The post-weaning pig gut microbiome appeared to follow a highly structured developmental program with characteristic post-weaning changes that can distinguish hosts that were born as little as two days apart in the second month of life. Treatment effects of the antibiotic and probiotic treatments were found but were subtle and included a higher representation of Mollicutes associated with intramuscular antibiotic treatment, and an increase of Lactobacillus associated with probiotic treatment.DiscussionThe discovery of correlations between experimental factors and microbial community composition is more commonly addressed with OTU-based methods and rarely analysed via phylogenetic diversity measures. The latter method, although less intuitive than the former, suffers less from library size normalization biases, and it proved to be instrumental in this study for the discovery of correlations between microbiome composition and host-, and treatment factors.

Campylobacter jejuni is a gastrointestinal pathogen that is able to modify membrane and periplasmic proteins by the N-linked addition of a 7-residue glycan at the strict attachment motif (D/E)XNX(S/T). Strategies for a comprehensive analysis of the targets of glycosylation, however, are hampered by the resistance of the glycan-peptide bond to enzymatic digestion or β-elimination and have previously concentrated on soluble glycoproteins compatible with lectin affinity and gel-based approaches. We developed strategies for enriching C. jejuni HB93-13 glycopeptides using zwitterionic hydrophilic interaction chromatography and examined novel fragmentation, including collision-induced dissociation (CID) and higher energy collisional (C-trap) dissociation (HCD) as well as CID/electron transfer dissociation (ETD) mass spectrometry. CID/HCD enabled the identification of glycan structure and peptide backbone, allowing glycopeptide identification, whereas CID/ETD enabled the elucidation of glycosylation sites by maintaining the glycan-peptide linkage. A total of 130 glycopeptides, representing 75 glycosylation sites, were identified from LC-MS/MS using zwitterionic hydrophilic interaction chromatography coupled to CID/HCD and CID/ETD. CID/HCD provided the majority of the identifications (73 sites) compared with ETD (26 sites). We also examined soluble glycoproteins by soybean agglutinin affinity and two-dimensional electrophoresis and identified a further six glycosylation sites. This study more than doubles the number of confirmed N-linked glycosylation sites in C. jejuni and is the first to utilize HCD fragmentation for glycopeptide identification with intact glycan. We also show that hydrophobic integral membrane proteins are significant targets of glycosylation in this organism. Our data demonstrate that peptide-centric approaches coupled to novel mass spectrometric fragmentation techniques may be suitable for application to eukaryotic glycoproteins for simultaneous elucidation of glycan structures and peptide sequence. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

Mycoplasma hyopneumoniae is the causative pathogen of porcine enzootic pneumonia, an economically significant disease that disrupts the mucociliary escalator in the swine respiratory tract. Expression of Mhp107, a P97 paralog encoded by the gene mhp107, was confirmed using ESI-MS/MS. To investigate the function of Mhp107, three recombinant proteins, F1Mhp107, F2 Mhp107, and F3Mhp107, spanning the N-terminal, central, and C-terminal regions of Mhp107 were constructed. Colonization of swine by M. hyopneumoniae requires adherence of the bacterium to ciliated cells of the respiratory tract. Recent studies have identified a number of M. hyopneumoniae adhesins that bind heparin, fibronectin, and plasminogen. F1Mhp107 was found to bind porcine heparin (KD ∼90 nM) in a dose-dependent and saturable manner, whereas F3Mhp107 bound fibronectin (K D ∼180 nM) at physiologically relevant concentrations. F1 Mhp107 also bound porcine plasminogen (KD = 24 nM) in a dose-dependent and physiologically relevant manner. Microspheres coated with F3Mhp107 mediate adherence to porcine kidney epithelial-like (PK15) cells, and all three recombinant proteins (F1Mhp107-F3 Mhp107) bound swine respiratory cilia. Together, these findings indicate that Mhp107 is a member of the multifunctional M. hyopneumoniae adhesin family of surface proteins and contributes to both adherence to the host and pathogenesis. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

Porcine enzootic pneumonia is a chronic respiratory disease that affects swine. The etiological agent of the disease, Mycoplasma hyopneumoniae, is a bacterium that adheres to cilia of the swine respiratory tract, resulting in loss of cilia and epithelial cell damage. A M. hyopneumoniae protein P116, encoded by mhp108, was investigated as a potential adhesin. Examination of P116 expression using proteomic analyses observed P116 as a full-length protein and also as fragments, ranging from 17 to 70 kDa in size. A variety of pathogenic bacterial species have been shown to bind the extracellular matrix component fibronectin as an adherence mechanism. M. hyopneumoniae cells were found to bind fibronectin in a dose-dependent and saturable manner. Surface plasmon resonance was used to show that a recombinant C-terminal domain of P116 bound fibronectin at physiologically relevant concentrations (KD 24 ± 6 nM). Plasmin(ogen)-binding proteins are also expressed by many bacterial pathogens, facilitating extracellular matrix degradation. M. hyopneumoniae cells were found to also bind plasminogen in a dose-dependent and saturable manner; the C-terminal domain of P116 binds to plasminogen (KD 44 ± 5 nM). Plasminogen binding was abolished when the C-terminal lysine of P116 was deleted, implicating this residue as part of the plasminogen binding site. P116 fragments adhere to the PK15 porcine kidney epithelial-like cell line and swine respiratory cilia. Collectively these data suggest that P116 is an important adhesin and virulence factor of M. hyopneumoniae. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

Campylobacter jejuni is a major gastrointestinal pathogen that colonizes host mucosa via interactions with extracellular matrix proteins, such as fibronectin (Fn). Fn-binding is mediated by a 37 kDa outer membrane protein termed Campylobacter adherence Factor (CadF). The outer membrane protein profile of a recent gastrointestinal C. jejuni clinical isolate (JHH1) was analysed using 2-DE and MS. Several spots were identified as products of the cadF gene. These included mass and pI variants of 34 and 30 kDa, as well as 24 kDa (CadF(24)) and 22 kDa (CadF(22)) mass variants. CadF variants were fully characterized by MALDI-TOF MS and MALDI-MS/MS. These data confirmed that CadF forms re-folding variants resulting in spots with lower mass and varying pI that are identical at the amino acid sequence level and are not modified post-translationally. CadF(22) and CadF(24), however, were characterized as N-terminal, membrane-associated polypeptides resulting from cleavage between serine(195) and leucine(196), and glycine(201) and phenylalanine(202), respectively. These variants were more abundant in the virulent (O) isolate of C. jejuni NCTC11168 when compared with the avirulent (genome sequenced) isolate. Hexahistidine fusion constructs of full-length CadF (34 kDa), CadF(24), and the deleted C-terminal OmpA domain (14 kDa; CadF(14)) were created in Escherichia coli. Recombinant CadF variants were probed against patient sera and revealed that only full-length CadF retained reactivity. Binding assays showed that CadF(24) retained Fn-binding capability, while CadF(14) did not bind Fn. These data suggest that the immunogenic epitope of CadF is cleaved to generate smaller Fn-binding polypeptides, which are not recognized by the host humoral response. CadF cleavage therefore may be associated with virulence in C. jejuni.

The first Australian isolate of Salmonella enterica serovar Paratyphi B d -tartrate-utilizing (dT + ) that is resistant to ampicillin, chloramphenicol, florfenicol, streptomycin, spectinomycin, sulfonamides, and tetracycline (ApCmFlSmSpSuTc) and contains SGI1 was isolated from a patient with gastroenteritis in early 1995. This is the earliest reported isolation globally. The incidence of infections caused by these SGI1-containing multiply antibiotic-resistant S. enterica serovar Paratyphi B dT + strains increased during the next few years and occurred sporadically in all states of Australia. Several molecular criteria were used to show that the early isolates are very closely related to one another and to strains isolated during the following few years and in 2000 and 2003 from home aquariums and their owners. Early isolates from travelers returning from Indonesia shared the same features. Thus, they appear to represent a true clone arising from a single cell that acquired SGI1. Some minor differences in the resistance profiles and molecular profiles also were observed, indicating the ongoing evolution of the clone, and phage type differences were common, indicating that this is not a useful epidemiological marker over time. Three isolates from 1995, 1998, and 1999 contained a complete sul1 gene but were susceptible to sulfamethoxazole due to a point mutation that creates a premature termination codon. This SGI1 type was designated SGI1-R. The loss of resistance genes also was examined. When strains were grown for many generations in the absence of antibiotic selection, the loss of SGI1 was not detected. However, variants SGI1-C (resistance profile SmSpSu) and SGI1-B (resistant to ApSu), which had lost part of the integron, arose spontaneously, presumably via homologous recombination between duplications in the In104 complex integron.

Campylobacter jejuni is the leading cause of food- and water-borne illness world-wide. The membrane-associated proteome of a recent C. jejuni gastrointestinal isolate (JHH1) was generated by sodium carbonate precipitation and ultracentrifugation followed by 2-DE and MALDI-TOF MS as well as 2-DLC (strong cation exchange followed by RP chromatography) of trypsin digests coupled to MS/MS (2-DLC/MS/MS). 2-DE/MS identified 77 proteins, 44 of which were predicted membrane proteins, while 2-DLC/MS/MS identified 432 proteins, of which 206 were predicted to be membrane associated. Atotal of 453 unique proteins (27.4% ofthe C. jejuni theoretical proteome), including 187 bona fide membrane proteins were identified in this study. Membrane proteins were also compared between C. jejuni JHH1 and ATCC 700297 to identify factors potentially associated with increased gastrointestinal virulence. We identified 28 proteins that were significantly (>two-fold) more abundant in, or unique to, JHH1, including eight proteins involved in chemotaxis signal transduction and flagellar motility, the amino acid-binding surface antigens CjaA and CjaC, and four outer membrane proteins (OMPs) of unknown function (Cj0129c, Cj1031, Cj1279c, and Cj1721c). Immunoblotting using convalescent patient sera generated postgastrointestinal infection revealed 13 (JHH1) and 12 (ATCC 700297) immunoreactive proteins. These included flagellin (FlaA) and CadF as well as Omp18, Omp50, Cj1721c, PEB1A, PEB2, and PEB4A. This study provides a comprehensive analysis of membrane-associated proteins from C. jejuni. © 2008 Wiley-VCH Verlag GmbH & Co. KGaA.

Mycoplasma hyopneumoniaeis the causative agent of porcine enzootic pneumonia, a chronic and economically significant respiratory disease that affects swine production worldwide.M. hyopneumoniaeadheres to and adversely affects the function of ciliated epithelial cells of the respiratory tract, and the cilium adhesin (Mhp183, P97) is intricately but not exclusively involved in this process. Although binding of pathogenic bacteria to glycosaminoglycans is a recognized step in pathogenesis, knowledge of glycosaminoglycan-binding proteins inM. hyopneumoniaeis lacking. However, heparin and other sulfated polysaccharides are known to block the binding ofM. hyopneumoniaeto purified swine respiratory cilia. In this study, four regions within the cilium adhesin were examined for the ability to bind heparin. Cilium adhesin fragments comprising 653 amino acids of the N terminus and 301 amino acids of the C terminus (containing two repeat regions, R1 and R2) were cloned and expressed. These fragments bound heparin in a dose-dependent and saturable manner with physiologically significant binding affinities of 0.27 ± 0.02 μM and 1.89 ± 0.33 μM, respectively. Heparin binding of both fragments was strongly inhibited by the sulfated polysaccharides fucoidan and mucin but not by chondroitin sulfate B. When the C-terminal repeat regions R1 and R2 were cloned separately and expressed, heparin-binding activity was lost, suggesting that both regions are required for heparin binding. The ability of the cilium adhesin to bind heparin indicates that this molecule plays a multifunctional role in the adherence ofM. hyopneumoniaeto host respiratory surfaces and therefore has important implications with respect to the pathogenesis of this organism.

Two typical coliforms from an intestinal biopsy from an adult case of bloody diarrhoea carried genes encoding intimin-beta, stx(1) and ehxA, and produced verocytotoxin 1 and enterohaemolysin in culture. Both were biochemically typical Escherichia coli O5 :H(-), apart from producing urease. Such O5 isolates represent a human pathogenic E. coli lineage.