Darrell O. Bayles

In-feed antibiotic effects on the swine intestinal microbiome.

Antibiotics have been administered to agricultural animals for disease treatment, disease prevention, and growth promotion for over 50 y. The impact of such antibiotic use on the treatment of human diseases is hotly debated. We raised pigs in a highly controlled environment, with one portion of the littermates receiving a diet containing performance-enhancing antibiotics [chlortetracycline, sulfamethazine, and penicillin (known as ASP250)] and the other portion receiving the same diet but without the antibiotics. We used phylogenetic, metagenomic, and quantitative PCR-based approaches to address the impact of antibiotics on the swine gut microbiota. Bacterial phylotypes shifted after 14 d of antibiotic treatment, with the medicated pigs showing an increase in Proteobacteria (1–11%) compared with nonmedicated pigs at the same time point. This shift was driven by an increase in Escherichia coli populations. Analysis of the metagenomes showed that microbial functional genes relating to energy production and conversion were increased in the antibiotic-fed pigs. The results also indicate that antibiotic resistance genes increased in abundance and diversity in the medicated swine microbiome despite a high background of resistance genes in nonmedicated swine. Some enriched genes, such as aminoglycoside O-phosphotransferases, confer resistance to antibiotics that were not administered in this study, demonstrating the potential for indirect selection of resistance to classes of antibiotics not fed. The collateral effects of feeding subtherapeutic doses of antibiotics to agricultural animals are apparent and must be considered in cost-benefit analyses.

The Contribution of Cytolethal Distending Toxin to Bacterial Pathogenesis

Cytolethal distending toxin (CDT) is a bacterial toxin that initiates a eukaryotic cell cycle block at the G2 stage prior to mitosis. CDT is produced by a number of bacterial pathogens including: Campylobacter species, Escherichia coli, Salmonella enterica serovar Typhi, Shigella dystenteriae, enterohepatic Helicobacter species, Actinobacillus actinomycetemcomitans (the cause of aggressive periodontitis), and Haemophilus ducreyi (the cause of chancroid). The functional toxin is composed of three proteins; CdtB potentiates a cascade leading to cell cycle block, and CdtA and CdtC function as dimeric subunits, which bind CdtB and delivers it to the mammalian cell interior. Once inside the cell, CdtB enters the nucleus and exhibits a DNase I-like activity that results in DNA double-strand breaks. The eukaryotic cell responds to the DNA double-strand breaks by initiating a regulatory cascade that results in cell cycle arrest, cellular distension, and cell death. Mutations in CdtABC that cause any of the three subunits to lose function prevent the bacterial cell from inducing cytotoxicity. The result of CDT activity can differ somewhat depending on the eukaryotic cell types affected. Epithelial cells, endothelial cells, and keratinocytes undergo G2 cell cycle arrest, cellular distension, and death; fibroblasts undergo G1 and G2 arrest, cellular distension, and death; and immune cells undergo G2 arrest followed by apoptosis. CDT contributes to pathogenesis by inhibiting both cellular and humoral immunity via apoptosis of immune response cells, and by generating necrosis of epithelial-type cells and fibroblasts involved in the repair of lesions produced by pathogens resulting in slow healing and production of disease symptoms. Thus, CDT may function as a virulence factor in pathogens that produce the toxin.

Comparison of the Cecal Microbiota of Domestic and Wild Turkeys

The extent to which production methods alter intestinal microbial communities of livestock is currently unknown. As the intestinal microbiota may affect animal health, nutrition, and food safety, a baseline comparison of the cecal communities of domestic and wild turkeys was performed. Oligonucleotide fingerprinting of ribosomal RNA (rRNA) genes (OFRG) of 2,990 16S rRNA clones and dot blot quantification of dominant populations were used to identify the dominant bacterial taxa. Seventy-three percent of all the clones belonged to as yet uncultured genera. However, at a higher phylogenetic level, the OFRG library was composed of 54% Bacteroidetes clones (52% of the domestic library clones, 56% of the wild library clones), 30% Firmicutes clones (33% of the domestic library clones, 32% of the wild library clones), 3% Proteobacteria clones (5% domestic, 2% wild), and 3% Deferribacteres clones (4% domestic, 1% wild). Seven percent of the clones were unidentifiable (6% domestic, 9% wild). Bacteroidetes clones included the genera Alistipes, Prevotella, Megamonas, and Bacteroides. Of the Clostridiales clones, groups IV, IX, and XIV including genera Faecalibacterium, Megasphaera, Phascolarctobacterium, and Papillibacter were predominant. Lactobacillus, Enterococcus, and Streptococcus bacilli were also identified. β- δ- and γ-proteobacterial genera included Acinetobacter, Sutterella, and Escherichia. Deferribacteres clones showed high similarity to Mucispirillum schaedleri. Statistical comparison of the domestic and wild turkey clone libraries indicated similar levels of community richness and evenness despite the fact that the two libraries shared only 30% of the total clone operational taxonomic units. Together these results indicate that although high level taxonomic community structure is similar, high-density turkey production causes considerable divergence of the genera found in the ceca of commercial birds from those of their wild counterparts.

Transcriptome Sequencing, and Rapid Development and Application of SNP Markers for the Legume Pod Borer Maruca vitrata (Lepidoptera: Crambidae)

The legume pod borer, Maruca vitrata (Lepidoptera: Crambidae), is an insect pest species of crops grown by subsistence farmers in tropical regions of Africa. We present the de novo assembly of 3729 contigs from 454- and Sanger-derived sequencing reads for midgut, salivary, and whole adult tissues of this non-model species. Functional annotation predicted that 1320 M. vitrata protein coding genes are present, of which 631 have orthologs within the Bombyx mori gene model. A homology-based analysis assigned M. vitrata genes into a group of paralogs, but these were subsequently partitioned into putative orthologs following phylogenetic analyses. Following sequence quality filtering, a total of 1542 putative single nucleotide polymorphisms (SNPs) were predicted within M. vitrata contig assemblies. Seventy one of 1078 designed molecular genetic markers were used to screen M. vitrata samples from five collection sites in West Africa. Population substructure may be present with significant implications in the insect resistance management recommendations pertaining to the release of biological control agents or transgenic cowpea that express Bacillus thuringiensis crystal toxins. Mutation data derived from transcriptome sequencing is an expeditious and economical source for genetic markers that allow evaluation of ecological differentiation.

Postinfectious irritable bowel syndrome: a long-term consequence of bacterial gastroenteritis.

Irritable bowel syndrome (IBS) is a commonly diagnosed disease characterized by gastrointestinal symptoms that may be associated with psychological illness and emotional problems. The prevalence rate worldwide for IBS ranges from 10 to 20% and is higher for women than for men. IBS imposes a substantial financial burden on both patients and employers because of increased medical costs and decreased work productivity. Recent studies indicate that inflammatory processes involving the gastrointestinal tract are strongly correlated with IBS. Acute bacterial gastroenteritis has been linked with the onset of symptoms in approximately 15% of patients diagnosed with IBS; these cases have been called postinfectious IBS. Organisms commonly associated with postinfectious IBS include the foodborne pathogens Campylobacter, Escherichia coli, Salmonella, and Shigella. The pathologic changes associated with postinfectious IBS are likely due to inflammatory reactions induced by the infecting organisms. Postinfectious IBS should be recognized as a potential long-term consequence of foodborne gastroenteritis.

The Application and Performance of Single Nucleotide Polymorphism Markers for Population Genetic Analyses of Lepidoptera

Microsatellite markers are difficult to apply within lepidopteran studies due to the lack of locus-specific PCR amplification and the high proportion of “null” alleles, such that erroneous estimations of population genetic parameters often result. Herein single nucleotide polymorphism (SNP) markers are developed from Ostrinia nubilalis (Lepidoptera: Crambidae) using next generation expressed sequence tag (EST) data. A total of 2742 SNPs were predicted within a reference assembly of 7414 EST contigs, and a subset of 763 were incorporated into 24 multiplex PCR reactions. To validate this pipeline, 5 European and North American sample sites were genotyped at 178 SNP loci, which indicated 84 (47.2%) were in Hardy–Weinberg equilibrium. Locus-by-locus FST, analysis of molecular variance, and STRUCTURE analyses indicate significant genetic differentiation may exist between European and North American O. nubilalis. The observed genetic diversity was significantly lower among European sites, which may result from genetic drift, natural selection, a genetic bottleneck, or ascertainment bias due to North American origin of EST sequence data. SNPs are an abundant source of mutation data for molecular genetic marker development in non-model species, with shared ancestral SNPs showing application within closely related species. These markers offer advantages over microsatellite markers for genetic and genomic analyses of Lepidoptera, but the source of mutation data may affect the estimation of population parameters and likely need to be considered in the interpretation of empirical data.

DNA sequence and analysis of a 90.1-kb plasmid in Shiga toxin-producing Escherichia coli (STEC) O145:NM 83-75☆

Shiga toxin-producing Escherichia coli (STEC) strains belonging to serogroup O145 are important emerging food-borne pathogens responsible for sporadic cases and outbreaks of hemorrhagic colitis and hemolytic uremic syndrome. A large plasmid carried by STEC O145:NM strain 83-75 and named pO145-NM was sequenced, and the genes were annotated. pO145-NM is 90,103bp in size and carries 89 open reading frames. Four genes/regions in pO145-NM encode for STEC virulence factors, including toxB (protein involved in adherence), espP (a serine protease), katP (catalase peroxidase), and the hly (hemolysin) gene cluster. These genes have also been identified in large virulence plasmids found in other STEC serogroups, including O26, O157, O111, and O103. pO145-NM carries the espPα subtype that is associated with STEC strains that cause more severe disease. Phylogenetic analyses of HlyB, EspP, and ToxB in various STEC strains showed a high degree of similarity of these proteins in E. coli serotypes O145:NM, O26:H11/H-, O111:NM/H-, and O157:H7 potentially placing these STEC into a related group.

Genomic and proteomic characterization of SuMu, a Mu-like bacteriophage infecting Haemophilus parasuis

BackgroundHaemophilus parasuis, the causative agent of Glässer’s disease, is prevalent in swine herds and clinical signs associated with this disease are meningitis, polyserositis, polyarthritis, and bacterial pneumonia. Six to eight week old pigs in segregated early weaning herds are particularly susceptible to the disease. Insufficient colostral antibody at weaning or the mixing of pigs with heterologous virulent H. parasuis strains from other farm sources in the nursery or grower-finisher stage are considered to be factors for the outbreak of Glässer’s disease. Previously, a Mu-like bacteriophage portal gene was detected in a virulent swine isolate of H. parasuis by nested polymerase chain reaction. Mu-like bacteriophages are related phyologenetically to enterobacteriophage Mu and are thought to carry virulence genes or to induce host expression of virulence genes. This study characterizes the Mu-like bacteriophage, named SuMu, isolated from a virulent H. parasuis isolate.ResultsCharacterization was done by genomic comparison to enterobacteriophage Mu and proteomic identification of various homologs by mass spectrometry. This is the first report of isolation and characterization of this bacteriophage from the Myoviridae family, a double-stranded DNA bacteriophage with a contractile tail, from a virulent field isolate of H. parasuis. The genome size of bacteriophage SuMu was 37,151 bp. DNA sequencing revealed fifty five open reading frames, including twenty five homologs to Mu-like bacteriophage proteins: Nlp, phage transposase-C-terminal, COG2842, Gam-like protein, gp16, Mor, peptidoglycan recognition protein, gp29, gp30, gpG, gp32, gp34, gp36, gp37, gpL, phage tail tube protein, DNA circulation protein, gpP, gp45, gp46, gp47, COG3778, tail fiber protein gp37-C terminal, tail fiber assembly protein, and Com. The last open reading frame was homologous to IS1414. The G + C content of bacteriophage SuMu was 41.87% while its H. parasuis host genome’s G + C content was 39.93%. Twenty protein homologs to bacteriophage proteins, including 15 structural proteins, one lysogeny-related and one lysis-related protein, and three DNA replication proteins were identified by mass spectrometry. One of the tail proteins, gp36, may be a virulence-related protein.ConclusionsBacteriophage SuMu was characterized by genomic and proteomic methods and compared to enterobacteriophage Mu.

The In-Feed Antibiotic Carbadox Induces Phage Gene Transcription in the Swine Gut Microbiome

ABSTRACT Carbadox is a quinoxaline-di-N-oxide antibiotic fed to over 40% of young pigs in the United States that has been shown to induce phage DNA transduction in vitro; however, the effects of carbadox on swine microbiome functions are poorly understood. We investigated the in vivo longitudinal effects of carbadox on swine gut microbial gene expression (fecal metatranscriptome) and phage population dynamics (fecal dsDNA viromes). Microbial metagenome, transcriptome, and virome sequences were annotated for taxonomic inference and gene function by using FIGfam (isofunctional homolog sequences) and SEED subsystems databases. When the beta diversities of microbial FIGfam annotations were compared, the control and carbadox communities were distinct 2 days after carbadox introduction. This effect was driven by carbadox-associated lower expression of FIGfams (n = 66) related to microbial respiration, carbohydrate utilization, and RNA metabolism (q < 0.1), suggesting bacteriostatic or bactericidal effects within certain populations. Interestingly, carbadox treatment caused greater expression of FIGfams related to all stages of the phage lytic cycle 2 days following the introduction of carbadox (q ≤0.07), suggesting the carbadox-mediated induction of prophages and phage DNA recombination. These effects were diminished by 7 days of continuous carbadox in the feed, suggesting an acute impact. Additionally, the viromes included a few genes that encoded resistance to tetracycline, aminoglycoside, and beta-lactam antibiotics but these did not change in frequency over time or with treatment. The results show decreased bacterial growth and metabolism, prophage induction, and potential transduction of bacterial fitness genes in swine gut bacterial communities as a result of carbadox administration. IMPORTANCE FDA regulations on agricultural antibiotic use have focused on antibiotics that are important for human medicine. Carbadox is an antibiotic not used in humans but frequently used on U.S. pig farms. It is important to study possible side effects of carbadox use because it has been shown to promote bacterial evolution, which could indirectly impact antibiotic resistance in bacteria of clinical importance. Interestingly, the present study shows greater prophage gene expression in feces from carbadox-fed animals than in feces from nonmedicated animals 2 days after the initiation of in-feed carbadox treatment. Importantly, the phage genetic material isolated in this study contained genes that could provide resistance to antibiotics that are important in human medicine, indicating that human-relevant antibiotic resistance genes are mobile between bacteria via phages. This study highlights the collateral effects of antibiotics and demonstrates the need to consider diverse antibiotic effects whenever antibiotics are being used or new regulations are considered. FDA regulations on agricultural antibiotic use have focused on antibiotics that are important for human medicine. Carbadox is an antibiotic not used in humans but frequently used on U.S. pig farms. It is important to study possible side effects of carbadox use because it has been shown to promote bacterial evolution, which could indirectly impact antibiotic resistance in bacteria of clinical importance. Interestingly, the present study shows greater prophage gene expression in feces from carbadox-fed animals than in feces from nonmedicated animals 2 days after the initiation of in-feed carbadox treatment. Importantly, the phage genetic material isolated in this study contained genes that could provide resistance to antibiotics that are important in human medicine, indicating that human-relevant antibiotic resistance genes are mobile between bacteria via phages. This study highlights the collateral effects of antibiotics and demonstrates the need to consider diverse antibiotic effects whenever antibiotics are being used or new regulations are considered.

Emerging Infectious Disease Implications of Invasive Mammalian Species: The Greater White-Toothed Shrew (Crocidura russula) Is Associated With a Novel Serovar of Pathogenic Leptospira in Ireland.

The greater white-toothed shrew (Crocidura russula) is an invasive mammalian species that was first recorded in Ireland in 2007. It currently occupies an area of approximately 7,600 km2 on the island. C. russula is normally distributed in Northern Africa and Western Europe, and was previously absent from the British Isles. Whilst invasive species can have dramatic and rapid impacts on faunal and floral communities, they may also be carriers of pathogens facilitating disease transmission in potentially naive populations. Pathogenic leptospires are endemic in Ireland and a significant cause of human and animal disease. From 18 trapped C. russula, 3 isolates of Leptospira were cultured. However, typing of these isolates by standard serological reference methods was negative, and suggested an, as yet, unidentified serovar. Sequence analysis of 16S ribosomal RNA and secY indicated that these novel isolates belong to Leptospira alstonii, a unique pathogenic species of which only 7 isolates have been described to date. Earlier isolations were limited geographically to China, Japan and Malaysia, and this leptospiral species had not previously been cultured from mammals. Restriction enzyme analysis (REA) further confirms the novelty of these strains since no similar patterns were observed with a reference database of leptospires. As with other pathogenic Leptospira species, these isolates contain lipL32 and do not grow in the presence of 8-azagunaine; however no evidence of disease was apparent after experimental infection of hamsters. These isolates are genetically related to L. alstonii but have a novel REA pattern; they represent a new serovar which we designate as serovar Room22. This study demonstrates that invasive mammalian species act as bridge vectors of novel zoonotic pathogens such as Leptospira.