Jessica L. Danzeisen

Modulations of the Chicken Cecal Microbiome and Metagenome in Response to Anticoccidial and Growth Promoter Treatment

With increasing pressures to reduce or eliminate the use of antimicrobials for growth promotion purposes in production animals, there is a growing need to better understand the effects elicited by these agents in order to identify alternative approaches that might be used to maintain animal health. Antibiotic usage at subtherapeutic levels is postulated to confer a number of modulations in the microbes within the gut that ultimately result in growth promotion and reduced occurrence of disease. This study examined the effects of the coccidiostat monensin and the growth promoters virginiamycin and tylosin on the broiler chicken cecal microbiome and metagenome. Using a longitudinal design, cecal contents of commercial chickens were extracted and examined using 16S rRNA and total DNA shotgun metagenomic pyrosequencing. A number of genus-level enrichments and depletions were observed in response to monensin alone, or monensin in combination with virginiamycin or tylosin. Of note, monensin effects included depletions of Roseburia, Lactobacillus and Enterococcus, and enrichments in Coprococcus and Anaerofilum. The most notable effect observed in the monensin/virginiamycin and monensin/tylosin treatments, but not in the monensin-alone treatments, was enrichments in Escherichia coli. Analysis of the metagenomic dataset identified enrichments in transport system genes, type I fimbrial genes, and type IV conjugative secretion system genes. No significant differences were observed with regard to antimicrobial resistance gene counts. Overall, this study provides a more comprehensive glimpse of the chicken cecum microbial community, the modulations of this community in response to growth promoters, and targets for future efforts to mimic these effects using alternative approaches.

Salmonella Pathogenicity and Host Adaptation in Chicken-Associated Serovars

SUMMARY Enteric pathogens such as Salmonella enterica cause significant morbidity and mortality. S. enterica serovars are a diverse group of pathogens that have evolved to survive in a wide range of environments and across multiple hosts. S. enterica serovars such as S. Typhi, S. Dublin, and S. Gallinarum have a restricted host range, in which they are typically associated with one or a few host species, while S. Enteritidis and S. Typhimurium have broad host ranges. This review examines how S. enterica has evolved through adaptation to different host environments, especially as related to the chicken host, and continues to be an important human pathogen. Several factors impact host range, and these include the acquisition of genes via horizontal gene transfer with plasmids, transposons, and phages, which can potentially expand host range, and the loss of genes or their function, which would reduce the range of hosts that the organism can infect. S. Gallinarum, with a limited host range, has a large number of pseudogenes in its genome compared to broader-host-range serovars. S. enterica serovars such as S. Kentucky and S. Heidelberg also often have plasmids that may help them colonize poultry more efficiently. The ability to colonize different hosts also involves interactions with the hosts immune system and commensal organisms that are present. Thus, the factors that impact the ability of Salmonella to colonize a particular host species, such as chickens, are complex and multifactorial, involving the host, the pathogen, and extrinsic pressures. It is the interplay of these factors which leads to the differences in host ranges that we observe today.

Comparative genomics and phylogeny of the IncI1 plasmids: A common plasmid type among porcine enterotoxigenic Escherichia coli

Increasing reports of multidrug resistance conferred by conjugative plasmids of Enterobacteriaceae necessitate a better understanding of their evolution. One such group is the narrow-host-range IncI1 plasmid type, known for their ability to carry genes encoding resistance to extended-spectrum beta lactamases. The focus of this study was to perform comparative sequencing of IncI1 plasmids from porcine enterotoxigenic Escherichia coli (ETEC), isolated irrespective of antimicrobial susceptibility phenotype. Five IncI1 plasmids of porcine ETEC origin and one IncI1 plasmid from a Salmonella enterica serovar Kentucky isolate from a healthy broiler chicken were sequenced and compared to existing IncI1 plasmid sequences in an effort to better understand the overall genetic composition of the IncI1 plasmid lineages. Overall, the sequenced porcine ETEC IncI1 plasmids were divergent from other sequenced IncI1 plasmids based upon multiple means of inferred phylogeny. High occurrences of IncI1 and IncA/C plasmid-associated genes and the blaTEM and blaCMY-2 beta lactamase genes were observed among porcine ETEC. However, the presence of blaTEM and blaCMY-2 did not strongly correlate with IncI1 plasmid possession, suggesting that these plasmids in porcine ETEC are not primarily associated with the carriage of such resistance genes. Overall, this work suggests a conservation of the IncI1 plasmid backbone among sequenced plasmids with a single locus for the acquisition of accessory genes, such as those associated with antimicrobial resistance. Furthermore, the high occurrence of IncI1 and IncA/C plasmids among clinical E. coli from commercial swine facilities is indicative of extensive horizontal gene transfer among porcine ETEC.

Genome Sequences and Phylogenetic Analysis of K88- and F18-Positive Porcine Enterotoxigenic Escherichia coli

Porcine enterotoxigenic Escherichia coli (ETEC) continues to result in major morbidity and mortality in the swine industry via postweaning diarrhea. The key virulence factors of ETEC strains, their serotypes, and their fimbrial components have been well studied. However, most studies to date have focused on plasmid-encoded traits related to colonization and toxin production, and the chromosomal backgrounds of these strains have been largely understudied. Here, we generated the genomic sequences of K88-positive and F18-positive porcine ETEC strains and examined the phylogenetic distribution of clinical porcine ETEC strains and their plasmid-associated genetic content. The genomes of porcine ETEC strains UMNK88 and UMNF18 were both found to contain remarkable plasmid complements containing known virulence factors, potential novel virulence factors, and antimicrobial resistance-associated elements. The chromosomes of these strains also possessed several unique genomic islands containing hypothetical genes with similarity to classical virulence factors, although phage-associated genomic islands dominated the accessory genomes of these strains. Phylogenetic analysis of 78 clinical isolates associated with neonatal and porcine diarrhea revealed that a limited subset of porcine ETEC lineages exist that generally contain common toxin and fimbrial profiles, with many of the isolates belonging to the ST10, ST23, and ST169 multilocus sequencing types. These lineages were generally distinct from existing human ETEC database isolates. Overall, most porcine ETEC strains appear to have emerged from a limited subset of E. coli lineages that either have an increased propensity to carry plasmid-encoded virulence factors or have the appropriate ETEC core genome required for virulence.

Genome Analysis and Phylogenetic Relatedness of Gallibacterium anatis Strains from Poultry

Peritonitis is the major disease problem of laying hens in commercial table egg and parent stock operations. Despite its importance, the etiology and pathogenesis of this disease have not been completely clarified. Although avian pathogenic Escherichia coli (APEC) isolates have been incriminated as the causative agent of laying hen peritonitis, Gallibacterium anatis are frequently isolated from peritonitis lesions. Despite recent studies suggesting a role for G. anatis in the pathogenesis of peritonitis, little is known about the organism’s virulence mechanisms, genomic composition and population dynamics. Here, we compared the genome sequences of three G. anatis isolates in an effort to understand its virulence mechanisms and identify novel antigenic traits. A multilocus sequence typing method was also established for G. anatis and used to characterize the genotypic relatedness of 71 isolates from commercial laying hens in Iowa and 18 international reference isolates. Genomic comparisons suggest that G. anatis is a highly diverse bacterial species, with some strains possessing previously described and potential virulence factors, but with a core genome containing several antigenic candidates. Multilocus sequence typing effectively distinguished 82 sequence types and several clonal complexes of G. anatis, and some clones seemed to predominate among G. anatis populations from commercial layers in Iowa. Biofilm formation and resistance to antimicrobial agents was also observed in several clades. Overall, the genomic diversity of G. anatis suggests that multiple lineages exist with differing pathogenic potential towards birds.

Succession of the turkey gastrointestinal bacterial microbiome related to weight gain

Because of concerns related to the use of antibiotics in animal agriculture, antibiotic-free alternatives are greatly needed to prevent disease and promote animal growth. One of the current challenges facing commercial turkey production in Minnesota is difficulty obtaining flock average weights typical of the industry standard, and this condition has been coined “Light Turkey Syndrome” or LTS. This condition has been identified in Minnesota turkey flocks for at least five years, and it has been observed that average flock body weights never approach their genetic potential. However, a single causative agent responsible for these weight reductions has not been identified despite numerous efforts to do so. The purpose of this study was to identify the bacterial community composition within the small intestines of heavy and light turkey flocks using 16S rRNA sequencing, and to identify possible correlations between microbiome and average flock weight. This study also sought to define the temporal succession of bacteria occurring in the turkey ileum. Based upon 2.7 million sequences across nine different turkey flocks, dominant operational taxonomic units (OTUs) were identified and compared between the flocks studied. OTUs that were associated with heavier weight flocks included those with similarity to Candidatus division Arthromitus and Clostridium bartlettii, while these flocks had decreased counts of several Lactobacillus species compared to lighter weight flocks. The core bacterial microbiome succession in commercial turkeys was also defined. Several defining markers of microbiome succession were identified, including the presence or abundance of Candidatus division Arthromitus, Lactobacillus aviarius, Lactobacillus ingluviei, Lactobacillus salivarius, and Clostridium bartlettii. Overall, the succession of the ileum bacterial microbiome in commercial turkeys proceeds in a predictable manner. Efforts to prevent disease and promote growth in the absence of antibiotics could involve target dominant bacteria identified in the turkey ileum that are associated with increased weight gain.

Transcriptome mapping of pAR060302, a blaCMY-2-positive broad-host-range IncA/C plasmid

ABSTRACT The multidrug resistance-encoding plasmids belonging to the IncA/C incompatibility group have recently emerged among Escherichia coli and Salmonella enterica strains in the United States. These plasmids have a unique genetic structure compared to other enterobacterial plasmid types, a broad host range, and a propensity to acquire large numbers of antimicrobial resistance genes via their accessory regions. Using E. coli strain DH5α harboring the prototype IncA/C plasmid pAR060302, we sought to define the baseline transcriptome of IncA/C plasmids under laboratory growth and in the face of selective pressure. The effects of ampicillin, florfenicol, or streptomycin exposure were compared to those on cells left untreated at logarithmic phase using Illumina platform-based RNA sequencing (RNA-Seq). Under growth in Luria-Bertani broth lacking antibiotics, much of the backbone of pAR060302 was transcriptionally inactive, including its putative transfer regions. A few plasmid backbone genes of interest were highly transcribed, including genes of a putative toxin-antitoxin system and an H-NS-like transcriptional regulator. In contrast, numerous genes within the accessory regions of pAR060302 were highly transcribed, including the resistance genes floR, bla CMY-2, aadA, and aacA. Treatment with ampicillin or streptomycin resulted in no genes being differentially expressed compared to controls lacking antibiotics, suggesting that many of the resistance-associated genes are not differentially expressed due to exposure to these antibiotics. In contrast, florfenicol treatment resulted in the upregulation of floR and numerous chromosomal genes. Overall, the transcriptome mapping of pAR060302 suggests that it mitigates the fitness costs of carrying resistance-associated genes through global regulation with its transcriptional regulators.

Clonal dissemination of Enterobacter cloacae harboring blaKPC-3 in the upper midwestern United States.

ABSTRACT Carbapenemase-producing, carbapenem-resistant Enterobacteriaceae, or CP-CRE, are an emerging threat to human and animal health, because they are resistant to many of the last-line antimicrobials available for disease treatment. Carbapenemase-producing Enterobacter cloacae harboring blaKPC-3 recently was reported in the upper midwestern United States and implicated in a hospital outbreak in Fargo, North Dakota (L. M. Kiedrowski, D. M. Guerrero, F. Perez, R. A. Viau, L. J. Rojas, M. F. Mojica, S. D. Rudin, A. M. Hujer, S. H. Marshall, and R. A. Bonomo, Emerg Infect Dis 20:1583–1585, 2014, http://dx.doi.org/10.3201/eid2009.140344). In early 2009, the Minnesota Department of Health began collecting and screening CP-CRE from patients throughout Minnesota. Here, we analyzed a retrospective group of CP-E. cloacae isolates (n = 34) collected between 2009 and 2013. Whole-genome sequencing and analysis revealed that 32 of the strains were clonal, belonging to the ST171 clonal complex and differing collectively by 211 single-nucleotide polymorphisms, and it revealed a dynamic clone under positive selection. The phylogeography of these strains suggests that this clone existed in eastern North Dakota and western Minnesota prior to 2009 and subsequently was identified in the Minneapolis and St. Paul metropolitan area. All strains harbored identical IncFIA-like plasmids conferring a CP-CRE phenotype and an additional IncX3 plasmid. In a single patient with multiple isolates submitted over several months, we found evidence that these plasmids had transferred from the E. cloacae clone to an Escherichia coli ST131 bacterium, rendering it as a CP-CRE. The spread of this clone throughout the upper midwestern United States is unprecedented for E. cloacae and highlights the importance of continued surveillance to identify such threats to human health.

Separate F-Type Plasmids Have Shaped the Evolution of the H30 Subclone of Escherichia coli Sequence Type 131.

A clonal lineage of Escherichia coli known as ST131 has emerged as a dominating strain type causing extraintestinal infections in humans. The evolutionary history of ST131 E. coli is now well understood. However, the role of plasmids in ST131’s evolutionary history is poorly defined. This study utilized real-time, single-molecule sequencing to compare plasmids from various current and historical lineages of ST131. From this work, it was determined that a series of plasmid gains, losses, and recombinational events has led to the currently circulating plasmids of ST131 strains. These plasmids appear to have evolved to acquire similar gene clusters on multiple occasions, suggesting possible plasmid-mediated convergent evolution leading to evolutionary success. These plasmids also appear to be better suited to exist in specific strains of ST131 due to coadaptive mutations. Overall, a series of events has enabled the evolution of ST131 plasmids, possibly contributing to the lineage’s success. ABSTRACT The extraintestinal pathogenic Escherichia coli (ExPEC) H30 subclone of sequence type 131 (ST131-H30) has emerged abruptly as a dominant lineage of ExPEC responsible for human disease. The ST131-H30 lineage has been well described phylogenetically, yet its plasmid complement is not fully understood. Here, single-molecule, real-time sequencing was used to generate the complete plasmid sequences of ST131-H30 isolates and those belonging to other ST131 clades. Comparative analyses revealed separate F-type plasmids that have shaped the evolution of the main fluoroquinolone-resistant ST131-H30 clades. Specifically, an F1:A2:B20 plasmid is strongly associated with the H30R/C1 clade, whereas an F2:A1:B− plasmid is associated with the H30Rx/C2 clade. A series of plasmid gene losses, gains, and rearrangements involving IS26 likely led to the current plasmid complements within each ST131-H30 sublineage, which contain several overlapping gene clusters with putative functions in virulence and fitness, suggesting plasmid-mediated convergent evolution. Evidence suggests that the H30Rx/C2-associated F2:A1:B− plasmid type was present in strains ancestral to the acquisition of fluoroquinolone resistance and prior to the introduction of a multidrug resistance-encoding gene cassette harboring blaCTX-M-15. In vitro experiments indicated a host strain-independent low frequency of plasmid transfer, differential levels of plasmid stability even between closely related ST131-H30 strains, and possible epistasis for carriage of these plasmids within the H30R/Rx lineages. IMPORTANCE A clonal lineage of Escherichia coli known as ST131 has emerged as a dominating strain type causing extraintestinal infections in humans. The evolutionary history of ST131 E. coli is now well understood. However, the role of plasmids in ST131’s evolutionary history is poorly defined. This study utilized real-time, single-molecule sequencing to compare plasmids from various current and historical lineages of ST131. From this work, it was determined that a series of plasmid gains, losses, and recombinational events has led to the currently circulating plasmids of ST131 strains. These plasmids appear to have evolved to acquire similar gene clusters on multiple occasions, suggesting possible plasmid-mediated convergent evolution leading to evolutionary success. These plasmids also appear to be better suited to exist in specific strains of ST131 due to coadaptive mutations. Overall, a series of events has enabled the evolution of ST131 plasmids, possibly contributing to the lineage’s success.

Temporal Relationships Exist Between Cecum, Ileum, and Litter Bacterial Microbiomes in a Commercial Turkey Flock, and Subtherapeutic Penicillin Treatment Impacts Ileum Bacterial Community Establishment

Gut health is paramount for commercial poultry production, and improved methods to assess gut health are critically needed to better understand how the avian gastrointestinal tract matures over time. One important aspect of gut health is the totality of bacterial populations inhabiting different sites of the avian gastrointestinal tract, and associations of these populations with the poultry farm environment, since these bacteria are thought to drive metabolism and prime the developing host immune system. In this study, a single flock of commercial turkeys was followed over the course of 12 weeks to examine bacterial microbiome inhabiting the ceca, ileum, and corresponding poultry litter. Furthermore, the effects of low-dose, growth-promoting penicillin treatment (50 g/ton) in feed on the ileum bacterial microbiome were also examined during the early brood period. The cecum and ileum bacterial communities of turkeys were distinct, yet shifted in parallel to one another over time during bird maturation. Corresponding poultry litter was also distinct yet more closely represented the ileal bacterial populations than cecal bacterial populations, and also changed parallel to ileum bacterial populations over time. Penicillin applied at low dose in feed significantly enhanced early weight gain in commercial poults, and this correlated with predictable shifts in the ileum bacterial populations in control versus treatment groups. Overall, this study identified the dynamics of the turkey gastrointestinal microbiome during development, correlations between bacterial populations in the gastrointestinal tract and the litter environment, and the impact of low-dose penicillin on modulation of bacterial communities in the ileum. Such modulations provide a target for alternatives to low-dose antibiotics.