Adriana Pedroso

Antimicrobial Resistance-Conferring Plasmids with Similarity to Virulence Plasmids from Avian Pathogenic Escherichia coli Strains in Salmonella enterica Serovar Kentucky Isolates from Poultry

ABSTRACT Salmonella enterica, a leading cause of food-borne gastroenteritis worldwide, may be found in any raw food of animal, vegetable, or fruit origin. Salmonella serovars differ in distribution, virulence, and host specificity. Salmonella enterica serovar Kentucky, though often found in the food supply, is less commonly isolated from ill humans. The multidrug-resistant isolate S. Kentucky CVM29188, isolated from a chicken breast sample in 2003, contains three plasmids (146,811 bp, 101,461 bp, and 46,121 bp), two of which carry resistance determinants (pCVM29188_146 [strAB and tetRA] and pCVM29188_101 [blaCMY-2 and sugE]). Both resistance plasmids were transferable by conjugation, alone or in combination, to S. Kentucky, Salmonella enterica serovar Newport, and Escherichia coli recipients. pCVM29188_146 shares a highly conserved plasmid backbone of 106 kb (>90% nucleotide identity) with two virulence plasmids from avian pathogenic Escherichia coli strains (pAPEC-O1-ColBM and pAPEC-O2-ColV). Shared avian pathogenic E. coli (APEC) virulence factors include iutA iucABCD, sitABCD, etsABC, iss, and iroBCDEN. PCR analyses of recent (1997 to 2005) S. Kentucky isolates from food animal, retail meat, and human sources revealed that 172 (60%) contained similar APEC-like plasmid backbones. Notably, though rare in human- and cattle-derived isolates, this plasmid backbone was found at a high frequency (50 to 100%) among S. Kentucky isolates from chickens within the same time span. Ninety-four percent of the APEC-positive isolates showed resistance to tetracycline and streptomycin. Together, our findings of a resistance-conferring APEC virulence plasmid in a poultry-derived S. Kentucky isolate and of similar resistance/virulence plasmids in most recent S. Kentucky isolates from chickens and, to lesser degree, from humans and cattle highlight the need for additional research in order to examine the prevalence and spread of combined virulence and resistance plasmids in bacteria in agricultural, environmental, and clinical settings.

The chicken gastrointestinal microbiome

The domestic chicken is a common model organism for human biological research and of course also forms the basis of a global protein industry. Recent methodological advances have spurred the recognition of microbiomes as complex communities with important influences on the health and disease status of the host. In this minireview, we provide an overview of the current state of knowledge of the chicken gastrointestinal microbiome focusing on spatial and temporal variability, the presence and importance of human pathogens, the influence of the microbiota on the immune system, and the importance of the microbiome for poultry nutrition. Review and meta-analysis of public data showed cecal communities dominated by Firmicutes and Bacteroides at the phylum level, while at finer levels of taxonomic resolution, a phylogenetically diverse assemblage of microorganisms appears to have similar metabolic functions that provide important benefits to the host as inferred from metagenomic data. This observation of functional redundancy may have important implications for management of the microbiome. We foresee advances in strategies to improve gut health in commercial operations through management of the intestinal microbiota as an alternative to in-feed subtherapeutic antibiotics, improvements in pre- and probiotics, improved management of polymicrobial poultry diseases, and better control of human pathogens via colonization reduction or competitive exclusion strategies.

Can Probiotics Improve the Environmental Microbiome and Resistome of Commercial Poultry Production

Food animal production systems have become more consolidated and integrated, producing large, concentrated animal populations and significant amounts of fecal waste. Increasing use of manure and litter as a more “natural” and affordable source of fertilizer may be contributing to contamination of fruits and vegetables with foodborne pathogens. In addition, human and animal manure have been identified as a significant source of antibiotic resistance genes thereby serving as a disseminator of resistance to soil and waterways. Therefore, identifying methods to remediate human and animal waste is critical in developing strategies to improve food safety and minimize the dissemination of antibiotic resistant bacteria. In this study, we sought to determine whether withdrawing antibiotic growth promoters or using alternatives to antibiotics would reduce the abundance of antibiotic resistance genes or prevalence of pathogens in poultry litter. Terminal restriction fragment length polymorphism (T-RFLP) paired with high throughput sequencing was used to evaluate the bacterial community composition of litter from broiler chickens that were treated with streptogramin growth-promoting antibiotics, probiotics, or prebiotics. The prevalence of resistance genes and pathogens was determined from sequencing results or PCR screens of litter community DNA. Streptogramin antibiotic usage did not elicit statistically significant differences in Shannon diversity indices or correlation coefficients among the flocks. However, T-RFLP revealed that there were inter-farm differences in the litter composition that was independent of antibiotic usage. The litter from all farms, regardless of antibiotic usage, contained streptogramin resistance genes (vatA, vatB, and vatE), macrolide-lincosamide-streptogramin B resistance genes (ermA and ermB), the tetracycline resistance gene tetM and class 1 integrons. There was inter-farm variability in the distribution of vatA and vatE with no statistically significant differences with regards to usage. Bacterial diversity was higher in litter when probiotics or prebiotics were administered to flocks but as the litter aged, diversity decreased. No statistically signficant differences were detected in the abundance of class 1 integrons where 3%–5% of the community was estimated to harbor a copy. Abundance of pathogenic Clostridium species increased in aging litter despite the treatment while the abundance of tetracycline-resistant coliforms was unaffected by treatment. However some treatments decreased the prevalence of Salmonella. These findings suggest that withdrawing antibiotics or administering alternatives to antibiotics can change the litter bacterial community and reduce the prevalence of some pathogenic bacteria, but may not immediately impact the prevalence of antibiotic resistance.

rpoS-Regulated Core Genes Involved in the Competitive Fitness of Salmonella enterica Serovar Kentucky in the Intestines of Chickens

ABSTRACT Salmonella enterica serovar Kentucky has become the most frequently isolated serovar from poultry in the United States over the past decade. Despite its prevalence in poultry, it causes few human illnesses in the United States. The dominance of S. Kentucky in poultry does not appear to be due to single introduction of a clonal strain, and its reduced virulence appears to correlate with the absence of virulence genes grvA, sseI, sopE, and sodC1. S. Kentuckys prevalence in poultry is possibly attributable to its metabolic adaptation to the chicken cecum. While there were no difference in the growth rate of S. Kentucky and S. Typhimurium grown microaerophilically in cecal contents, S. Kentucky persisted longer when chickens were coinfected with S. Typhimurium. The in vivo advantage that S. Kentucky has over S. Typhimurium appears to be due to differential regulation of core Salmonella genes via the stationary-phase sigma factor rpoS. Microarray analysis of Salmonella grown in cecal contents in vitro identified several metabolic genes and motility and adherence genes that are differentially activated in S. Kentucky. The contributions of four of these operons (mgl, prp, nar, and csg) to Salmonella colonization in chickens were assessed. Deletion of mgl and csg reduced S. Kentucky persistence in competition studies in chickens infected with wild-type or mutant strains. Subtle mutations affecting differential regulation of core Salmonella genes appear to be important in Salmonellas adaptation to its animal host and especially for S. Kentuckys emergence as the dominant serovar in poultry.

Effect of in ovo administration of an adult-derived microbiota on establishment of the intestinal microbiome in chickens

OBJECTIVE To determine effects of in ovo administration of a probiotic on development of the intestinal microbiota of 2 genetic lineages (modern and heritage) of chickens. SAMPLE 10 newly hatched chicks and 40 fertile eggs to determine intestinal microbiota at hatch, 900 fertile eggs to determine effects of probiotic on hatchability, and 1,560 chicks from treated or control eggs. PROCEDURES A probiotic competitive-exclusion product derived from adult microbiota was administered in ovo to fertile eggs of both genetic lineages. Cecal contents and tissues were collected from embryos, newly hatched chicks, and chicks. A PCR assay was used to detect bacteria present within the cecum of newly hatched chicks. Fluorescence in situ hybridization and vitality staining were used to detect viable bacteria within intestines of embryos. The intestinal microbiota was assessed by use of 16S pyrosequencing. RESULTS Microscopic evaluation of embryonic cecal contents and tissues subjected to differential staining techniques revealed viable bacteria in low numbers. Development of the intestinal microbiota of broiler chicks of both genetic lineages was enhanced by in ovo administration of adult microbiota. Although the treatment increased diversity and affected composition of the microbiota of chicks, most bacterial species present in the probiotic were transient colonizers. However, the treatment decreased the abundance of undesirable bacterial species within heritage lineage chicks. CONCLUSIONS AND CLINICAL RELEVANCE In ovo inoculation of a probiotic competitive-exclusion product derived from adult microbiota may be a viable method of managing development of the microbiota and reducing the prevalence of pathogenic bacteria in chickens.

Sodium butyrate improved performance while modulating the cecal microbiota and regulating the expression of intestinal immune-related genes of broiler chickens

ABSTRACT This study evaluated the effect of sodium butyrate (SB) on performance, expression of immune‐related genes in the cecal tonsils, and cecal microbiota of broiler chickens when dietary energy and amino acids concentrations were reduced. Day‐old male Ross 708 broiler chicks were fed dietary treatments in a 3 × 2 factorial design (8 pens per treatment) with 3 dietary formulations (control diet; reduction of 2.3% of amino acids and 60 kcal/kg; and reduction of 4.6% of amino acids and 120 kcal/kg) with or without the inclusion of 0.1% of SB. Feed intake (FI), body weight gain (BW gain), and feed conversion ratio (FCR) were recorded until 28 d of age. From 14 to 28 d, there was an interaction of nutrient density by SB (P = 0.003) wherein BW gain of birds fed SB was impaired less by the energy/amino acids reduction than unsupplemented birds. A similar result was obtained from 1 to 28 d (P = 0.004). No interaction (P < 0.05) between nutrient density by SB was observed for FCR. Nutritional density of the diets and SB modified the structure, composition, and predicted function of the cecal microbiota. The nutritionally reduced diet altered the imputed function performed by the microbiota and the SB supplementation reduced these variations, keeping the microbial function similar to that observed in chickens fed a control diet. The frequency of bacterial species presenting the butyryl‐CoA: acetate CoA‐transferase gene increased in the microbiota of chickens fed a nutritionally reduced diet without SB supplementation, and was not changed by nutrient density of the diet when supplemented with SB (interaction; P = 0.01). SB modulated the expression of immune related genes in the cecal tonsils; wherein SB upregulated the expression of A20 in broilers fed control diets (P < 0.05) and increased IL‐6 expression (P < 0.05). These results show that SB had positive effects on the productive performance of broilers fed nutritionally reduced diets, partially by modulating the cecal microbiota and exerting immune‐modulatory effects.

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

Current commercial PCRs tests for identifying Salmonella target genes unique to this genus. However, there are two species, six subspecies, and over 2,500 different Salmonella serovars, and not all are equal in their significance to public health. For example, finding S. enterica subspecies IIIa Arizona on a table egg layer farm is insignificant compared to the isolation of S. enterica subspecies I serovar Enteritidis, the leading cause of salmonellosis linked to the consumption of table eggs. Serovars are identified based on antigenic differences in lipopolysaccharide (LPS)(O antigen) and flagellin (H1 and H2 antigens). These antigenic differences are the outward appearance of the diversity of genes and gene alleles associated with this phenotype. We have developed an allelotyping, multiplex PCR that keys on genetic differences between four major S. enterica subspecies I serovars found in poultry and associated with significant human disease in the US. The PCR primer pairs were targeted to key genes or sequences unique to a specific Salmonella serovar and designed to produce an amplicon with size specific for that gene or allele. Salmonella serovar is assigned to an isolate based on the combination of PCR test results for specific LPS and flagellin gene alleles. The multiplex PCRs described in this article are specific for the detection of S. enterica subspecies I serovars Enteritidis, Hadar, Heidelberg, and Typhimurium. Here we demonstrate how to use the multiplex PCRs to identify serovar for a Salmonella isolate.