RESUMO
Campylobacter jejuni is one of the major causes of bacterial gastrointestinal disease in humans worldwide. This foodborne pathogen colonizes the intestinal tracts of chickens, and consumption of chicken and poultry products is identified as a common route of transmission. We analyzed two C. jejuni strains after oral challenge with 105 CFU/ml of C. jejuni per chick; one strain was a robust colonizer (A74/C) and the other a poor colonizer (A74/O). We also found extensive phenotypic differences in growth rate, biofilm production, and in vitro adherence, invasion, intracellular survival, and transcytosis. Strains A74/C and A74/O were genotypically similar with respect to their whole genome alignment, core genome, and ribosomal MLST, MLST, flaA, porA, and PFGE typing. The global proteomes of the two congenic strains were quantitatively analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and 618 and 453 proteins were identified from A74/C and A74/O isolates, respectively. Cluster of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that carbon metabolism and motility proteins were distinctively overexpressed in strain A74/C. The robust colonizer also exhibited a unique proteome profile characterized by significantly increased expression of proteins linked to adhesion, invasion, chemotaxis, energy, protein synthesis, heat shock proteins, iron regulation, two-component regulatory systems, and multidrug efflux pump. Our study underlines phenotypic, genotypic, and proteomic variations of the poor and robust colonizing C. jejuni strains, suggesting that several factors may contribute to mediating the different colonization potentials of the isogenic isolates.
Assuntos
Aderência Bacteriana , Proteínas de Bactérias , Biofilmes , Infecções por Campylobacter , Campylobacter jejuni , Galinhas , Genótipo , Fenótipo , Proteoma , Proteômica , Campylobacter jejuni/genética , Campylobacter jejuni/metabolismo , Campylobacter jejuni/crescimento & desenvolvimento , Animais , Galinhas/microbiologia , Infecções por Campylobacter/microbiologia , Infecções por Campylobacter/veterinária , Biofilmes/crescimento & desenvolvimento , Aderência Bacteriana/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Doenças das Aves Domésticas/microbiologia , Tipagem de Sequências Multilocus , Espectrometria de Massas em Tandem , Genoma Bacteriano/genéticaRESUMO
The goal of this study was to test the efficacy of in-package dielectric barrier discharge-cold plasma (DBD-CP) treatment to inactivate poultry-associated spoilage (Pseudomonas fluorescens) and pathogenic (Salmonella enterica Typhimurium, Campylobacter jejuni) bacteria. Liquid cultures of the bacterial isolates were sealed within packages containing ambient air (Trial 1) or modified air (65% O2:30% CO2:5% N2; Trial 2). The packages were subjected to treatment times ranging from 30 to 180 s, and after 24 h incubation at 4 °C, bacterial titers were determined. The DBD-CP system completely inactivated the four isolates tested, although the in-package gas composition and treatment times were isolate-specific. Both C. jejuni isolates were completely inactivated between 30 s (modified air) and 120 s (ambient air), while modified air was required for the complete inactivation of S. typhimurium (90 s) and P. fluorescens (180 s). This DBD-CP system is effective for inactivating major poultry-associated spoilage and pathogenic bacteria in liquid culture, and through this study, system parameters to optimize inactivation were determined. This study demonstrates the potential for DBD-CP treatment to inactivate major bacteria of economic interest to the poultry industry, thus potentially allowing for reduced spoilage (e.g., longer shelf life) and increased safety of poultry products.
Assuntos
Desinfecção/métodos , Microbiologia de Alimentos/métodos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/fisiologia , Gases em Plasma , Animais , Viabilidade Microbiana/efeitos dos fármacos , Aves DomésticasRESUMO
The use of antibiotics in agroecosystems has been implicated in the rise in antibiotic resistance (AR), which can affect environmental, animal, and human health. To determine the environmental impact of antibiotic use in agroecosystems, appropriate background levels of AR in agricultural environments in the absence of antibiotic application must be determined. Therefore, to determine background levels of AR in broiler production, four target microbes (, , , and ) were isolated from 15 all-natural, antibiotic-free, pasture-raised broiler flocks from six farms within the southeastern United States. The AR profiles of these isolates were characterized using the CDC National Antimicrobial Resistance Monitoring System for Enteric Bacteria (NARMS), and these resistance patterns were compared across target microbes and farms and throughout the life cycle of the flocks along the farm-to-fork continuum. Antibiotic resistances were most prevalent in and and least prevalent in . Although and were isolated from the same farms and characterized using the same NARMS plates, they exhibited distinct AR profiles, with demonstrating clear farm-specific resistance patterns. Multidrug resistance rates (three or more antibiotics), in order of prevalence, were (63.9%), (36.0%), (12.7%), and (1.4%). The results of this study demonstrate the variability in background AR among major food safety-related microbes, even when isolated from similar production and processing samples from the same farms, and indicate the need for the proper design of future broiler production studies to account for this highly dynamic background AR.
Assuntos
Antibacterianos/farmacologia , Galinhas , Resistência Microbiana a Medicamentos , Animais , Bactérias/genética , Bactérias/isolamento & purificação , Humanos , Prevalência , Sudeste dos Estados UnidosRESUMO
Campylobacter jejuni is a Gram-negative spiral rod bacterium and is the leading but underreported bacterial food-borne pathogen that causes human campylobacteriosis worldwide. Raw or undercooked poultry products are regarded as a major source for human infection. C. jejuni flagella have been implicated in colonization and adhesion to the mucosal surface of chicken gastrointestinal tracts. Therefore, flagellar proteins would be the excellent targets for further investigation. In this report, we used the recombinant technology to generate a battery of C. jejuni flagellar proteins, which were purified by His tag affinity chromatography and determined antigenic profiles of these recombinant flagellar proteins using sera from chickens older than 6 weeks of age. The immunoblot results demonstrate that each chicken serum reacted to various numbers of recombinant flagellar proteins. Among these recombinant proteins, chicken sera reacted predominantly to the FlgE1, FlgK, FlhF, FliG and FliY proteins. These antibody screening results provide a rationale for further evaluation of these recombinant flagellar proteins as potential vaccines for chickens to improve food safety as well as investigation of host immune response to C. jejuni.
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Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/imunologia , Campylobacter jejuni/imunologia , Proteínas Recombinantes/imunologia , Animais , Antígenos de Bactérias/genética , Campylobacter jejuni/genética , Galinhas , Escherichia coli/genética , Flagelos/química , Immunoblotting , Proteínas Recombinantes/genéticaRESUMO
The recent multistate outbreak of a multidrug-resistant (MDR) Salmonella Heidelberg strain from commercial poultry production highlights the need to better understand the reservoirs of these zoonotic pathogens within the commercial poultry production and processing environment. As part of a larger study looking at temporal changes in microbial communities within the major water tanks within a commercial processing facility, this paper identifies and characterizes Salmonella enterica isolated from the water in a final scalder tank at 3 times during a typical processing day: prior to the birds entering the tank (start), halfway through the processing day (mid), and after the final birds were scalded (end). Over 3 consecutive processing days, no Salmonella were recovered from start-of-day water samples, while a total of 56 Salmonella isolates were recovered from the mid-day and end-of-day scalder water samples. Traditional and newer PCR-based serotyping methods eventually identified these isolates as either group C3 S. Kentucky (n=45) and group B S. Heidelberg (n=11). While none of the S. Kentucky isolates possessed any resistances to the antimicrobials tested, all S. Heidelberg isolates were found to be multidrug resistant to 5 specific antimicrobials representing 3 antimicrobial classes. Due to the potential public health impact of S. Heidelberg and the recent nationwide poultry-associated outbreak of multidrug-resistant S. Heidelberg, future studies should focus on understanding the transmission and environmental growth dynamics of this serotype within the commercial poultry processing plant environment.
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Anti-Infecciosos/farmacologia , Farmacorresistência Bacteriana Múltipla , Doenças das Aves Domésticas/epidemiologia , Salmonelose Animal/epidemiologia , Salmonella enterica/efeitos dos fármacos , Salmonella enterica/genética , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Contagem de Colônia Microbiana/veterinária , DNA Intergênico/genética , DNA Intergênico/metabolismo , Eletroforese em Gel de Campo Pulsado/veterinária , Temperatura Alta , Testes de Sensibilidade Microbiana/veterinária , Dados de Sequência Molecular , Doenças das Aves Domésticas/microbiologia , RNA Ribossômico 5S/genética , RNA Ribossômico 5S/metabolismo , Salmonelose Animal/microbiologia , Salmonella enterica/isolamento & purificação , Salmonella enterica/metabolismo , Análise de Sequência de DNA/veterinária , Estados Unidos/epidemiologia , ÁguaRESUMO
Campylobacter jejuni, a Gram-negative rod bacterium, is the leading causative agent of human acute bacterial gastroenteritis worldwide. Consumption and handling of raw or undercooked poultry are regarded as a major source for human infection. Because bacterial chemotaxis guides microorganisms to colonization and invasion in the host cells, proteins involved in chemotactic processes can be novel targets for vaccine development. In this communication, we report amplification, cloning and expression of the C. jejuni chemotactic proteins in an Escherichia coli expression system. A total of 15 chemotactic protein genes were successfully expressed. These recombinant proteins were confirmed by nucleotide sequencing, SDS-PAGE analysis and immunoblot analysis of six-His and hemagglutinin tags. Twelve recombinant chemotactic proteins were further tested whether they were antigenic using sera from broiler chickens older than 4 weeks. The immunoblot results show that each chicken serum reacted to a variety of the recombinant proteins, but all sera reacted to the Cjj0473 gene product (annotated as a methyl-accepting chemotaxis protein), suggesting that anti-Campylobacter antibodies may be prevalent in the poultry population. These antibody screening results provide a rationale for further evaluation of the Cjj0473 protein as a potential vaccine for broilers to improve human food safety.
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Proteínas de Bactérias/imunologia , Campylobacter jejuni/imunologia , Galinhas/imunologia , Proteínas de Membrana/imunologia , Animais , Anticorpos Antibacterianos/sangue , Proteínas de Bactérias/genética , Quimiotaxia , Escherichia coli/metabolismo , Proteínas de Membrana/genética , Proteínas Quimiotáticas Aceptoras de Metil , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologiaRESUMO
The complete nucleotide sequence was determined for a cryptic plasmid, pTIW94, recovered from several Campylobacter jejuni isolates from wild birds in the southeastern United States. pTIW94 is a circular molecule of 3860 nucleotides, with a G+C content (31.0%) similar to that of many Campylobacter spp. genomes. A typical origin of replication, with iteron sequences, was identified upstream of DNA sequences that demonstrated similarity to replication initiation proteins. A total of five open reading frames (ORFs) were identified; two of the five ORFs demonstrated significant similarity to plasmid pCC2228-2 found within Campylobacter coli. These two ORFs were similar to essential replication proteins RepA (100%; 26/26 aa identity) and RepB (95%; 327/346 aa identity). A third identified ORF demonstrated significant similarity (99%; 421/424 aa identity) to the MOB protein from C. coli 67-8, originally recovered from swine. The other two identified ORFs were either similar to hypothetical proteins from other Campylobacter spp., or exhibited no significant similarity to any DNA or protein sequence in the GenBank database. Promoter regions (-35 and -10 signal sites), ribosomal binding sites upstream of ORFs, and stem-loop structures were also identified within the plasmid. These results demonstrate that pTIW94 represents a previously un-reported small cryptic plasmid with unique sequences as well as highly similar sequences to other small plasmids found within Campylobacter spp., and that this cryptic plasmid is present among Campylobacter spp. recovered from different genera of wild birds.
Assuntos
Aves/microbiologia , Campylobacter jejuni/genética , Plasmídeos/genética , Animais , Sequência de Bases , Mapeamento Cromossômico , Primers do DNA/genética , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Regiões Promotoras Genéticas/genética , Análise de Sequência de DNA , Homologia de Sequência , Sudeste dos Estados Unidos , Especificidade da EspécieRESUMO
Regulatory methods for detection of the foodborne protozoan parasite Cyclospora cayetanensis must be specific and sensitive. To that end, we designed and evaluated (in a single laboratory validation) a novel and improved primer/probe combination (Mit1C) for real-time PCR detection of C. cayetanensis in produce. The newly developed primer/probe combination targets a conserved region of the mitochondrial genome of C. cayetanensis that varies in other closely related organisms. The primer/probe combination was evaluated both in silico and using several real-time PCR kits and polymerases against an inclusivity/exclusivity panel comprised of a variety of C. cayetanensis oocysts, as well as DNA from other related Cyclospora spp. and closely related parasites. The new primer/probe combination amplified only C. cayetanensis, thus demonstrating specificity. Sensitivity was evaluated by artificially contaminating cilantro, raspberries, and romaine lettuce with variable numbers (200 and 5) of C. cayetanensis oocysts. As few as 5 oocysts were detected in 75%, 67.7%, and 50% of the spiked produce samples (cilantro, raspberries, and romaine lettuce), respectively, all uninoculated samples and no-template real-time PCR controls were negative. The improved primer/probe combination should prove an effective analytical tool for the specific detection of C. cayetanensis in produce.
Assuntos
Coriandrum , Cyclospora , Ciclosporíase , Rubus , Animais , Cyclospora/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Oocistos , Ciclosporíase/diagnóstico , Ciclosporíase/parasitologiaRESUMO
Introduction: With more public interest in consuming locally grown produce, small specialty crop farms (SSCF) are a viable and growing segment of the food production chain in the United States. Methods: The goal of this study was to investigate the genomic diversity of Campylobacter isolated from dairy manure (n = 69) collected from 10 SSCF in Northeast Ohio between 2018 and 2020. Results: A total of 56 C. jejuni and 13 C. coli isolates were sequenced. Multi-locus sequence typing (MLST) identified 22 sequence types (STs), with ST-922 (18%) and ST-61 (13%) predominant in C. jejuni and ST-829 (62%) and ST-1068 (38%) predominant in C. coli. Interestingly, isolates with similar genomic and gene contents were detected within and between SSCF over time, suggesting that Campylobacter could be transmitted between farms and may persist in a given SSCF over time. Virulence-associated genes (n = 35) involved in the uptake and utilization of potassium and organic compounds (succinate, gluconate, oxoglutarate, and malate) were detected only in the C. jejuni isolates, while 45 genes associated with increased resistance to environmental stresses (capsule production, cell envelope integrity, and iron uptake) were detected only in the C. coli isolates. Campylobacter coli isolates were also sub-divided into two distinct clusters based on the presence of unique prophages (n = 21) or IncQ conjugative plasmid/type-IV secretion system genes (n = 15). Campylobacter coli isolates harbored genes associated with resistance to streptomycin (aadE-Cc; 54%) and quinolone (gyrA-T86I; 77%), while C. jejuni had resistance genes for kanamycin (aph3'-IIIa; 20%). Both species harbored resistance genes associated with ß-lactam (especially, blaOXA-193; up to 100%) and tetracycline (tetO; up to 59%). Discussion/Conclusion: Our study demonstrated that Campylobacter genome plasticity associated with conjugative transfer might provide resistance to certain antimicrobials and viral infections via the acquisition of protein-encoding genes involved in mechanisms such as ribosomal protection and capsule modification.
RESUMO
BACKGROUND: During the Rift Valley fever (RVF) epidemic of 2006-2007 in eastern Africa, spatial mapping of the outbreaks across Kenya, Somalia, and Tanzania was performed and the RVF viruses were isolated and genetically characterized. METHODS: Following confirmation of the RVF epidemic in Kenya on 19 December 2006 and in Tanzania on 2 February 2007, teams were sent to the field for case finding. Human, livestock, and mosquito specimens were collected and viruses isolated. The World Health Organization response team in Kenya worked with the WHO's polio surveillance team inside Somalia to collect information and specimens from Somalia. RESULTS: Seven geographical foci that reported hundreds of livestock and >25 cases in humans between December 2006 and June 2007 were identified. The onset of RVF cases in each epidemic focus was preceded by heavy rainfall and flooding for at least 10 days. Full-length genome analysis of 16 RVF virus isolates recovered from humans, livestock, and mosquitoes in 5 of the 7 outbreak foci revealed 3 distinct lineages of the viruses within and across outbreak foci. CONCLUSION: The findings indicate that the sequential RVF epidemics in the region were caused by multiple lineages of the RVF virus, sometimes independently activated or introduced in distinct outbreak foci.
Assuntos
Surtos de Doenças , Febre do Vale de Rift/epidemiologia , Febre do Vale de Rift/virologia , Vírus da Febre do Vale do Rift/genética , África Oriental/epidemiologia , Animais , Culicidae/virologia , Bases de Dados de Ácidos Nucleicos , Geografia , Humanos , Chuva , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Febre do Vale de Rift/transmissão , Vírus da Febre do Vale do Rift/isolamento & purificação , Fatores de Risco , Análise de Sequência , Organização Mundial da SaúdeRESUMO
Campylobacter spp. are the leading cause of bacterial foodborne infections in both developed and developing countries. The food commodities primarily attributed to campylobacteriosis include raw milk, poultry, seafood, and fresh produce. Furthermore, insects, animal/bird fecal material, and agricultural water have been shown to be the sources of Campylobacter contamination in these commodities. Both established and emerging species of Campylobacter have been recovered from food and environmental sources. Therefore, optimal detection and isolation of Campylobacter spp., including the emerging species, is critical for improved surveillance, prevention, and traceback of Campylobacter outbreaks. This review focuses on the existing variability in Campylobacter enrichment and isolation procedures used by researchers and regulatory agencies worldwide, for various matrices. Additionally, the challenges associated with developing and validating new culture, molecular, and immunological methods for rapid and sensitive Campylobacter detection are discussed.
Assuntos
Infecções por Campylobacter , Campylobacter , Animais , Infecções por Campylobacter/diagnóstico , Infecções por Campylobacter/microbiologia , Galinhas , Fezes/microbiologia , Microbiologia de Alimentos , Aves Domésticas/microbiologia , ÁguaRESUMO
BACKGROUND: Because biotechnological uses of bacteriophage gene products as alternatives to conventional antibiotics will require a thorough understanding of their genomic context, we sequenced and analyzed the genomes of four closely related phages isolated from Clostridium perfringens, an important agricultural and human pathogen. RESULTS: Phage whole-genome tetra-nucleotide signatures and proteomic tree topologies correlated closely with host phylogeny. Comparisons of our phage genomes to 26 others revealed three shared COGs; of particular interest within this core genome was an endolysin (PF01520, an N-acetylmuramoyl-L-alanine amidase) and a holin (PF04531). Comparative analyses of the evolutionary history and genomic context of these common phage proteins revealed two important results: 1) strongly significant host-specific sequence variation within the endolysin, and 2) a protein domain architecture apparently unique to our phage genomes in which the endolysin is located upstream of its associated holin. Endolysin sequences from our phages were one of two very distinct genotypes distinguished by variability within the putative enzymatically-active domain. The shared or core genome was comprised of genes with multiple sequence types belonging to five pfam families, and genes belonging to 12 pfam families, including the holin genes, which were nearly identical. CONCLUSIONS: Significant genomic diversity exists even among closely-related bacteriophages. Holins and endolysins represent conserved functions across divergent phage genomes and, as we demonstrate here, endolysins can have significant variability and host-specificity even among closely-related genomes. Endolysins in our phage genomes may be subject to different selective pressures than the rest of the genome. These findings may have important implications for potential biotechnological applications of phage gene products.
Assuntos
Clostridium perfringens/virologia , Evolução Molecular , Genômica , Siphoviridae/genética , Amidoidrolases/química , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Biologia Computacional , Simulação por Computador , Endopeptidases/química , Genótipo , Cadeias de Markov , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Estrutura Terciária de Proteína , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de SequênciaRESUMO
Campylobacter spp. are small, asaccharolytic bacteria exhibiting unique nutritional and environmental requirements. Campylobacter spp. exist as commensal organisms in some animal species, yet are estimated to be the most common causative agents of foodborne illness in humans. C. jejuni is most often associated with poultry, while C. coli are more frequently associated with swine. Temperature has been suggested to trigger potential colonization or virulence factors in C. jejuni, and recent studies have demonstrated temperature-dependent genes are important to colonization. It is possible that temperature-dependent colonization factors are in part responsible for the species-specific colonization characteristics of C. coli also. We determined utilization of 190 different sole carbon substrates by C. coli ATCC 49941 at 37 and 42°C using phenotype microarray (PM) technology. Temperature did affect amino acid utilization. L-asparagine and L-serine allowed significantly (P = 0.004) more respiration by C. coli ATCC 49941 at the lower temperature of 37°C as compared to 42°C. Conversely, L-glutamine was utilized to a significantly greater extent (P = 0.015) at the higher temperature of 42°C. Other organic substrates exhibited temperature-dependent utilization including succinate, D,L-malate, and propionate which all supported active respiration by C. coli to a significantly greater extent at 42°C. Further investigation is needed to determine the basis for the temperature-dependent utilization of substrates by Campylobacter spp. and their possible role in species-specific colonization.
Assuntos
Campylobacter coli/metabolismo , Campylobacter coli/efeitos da radiação , Carbono/metabolismo , Temperatura , Aminoácidos/metabolismo , Técnicas de Tipagem Bacteriana , Metabolismo dos Carboidratos , Ácidos Carboxílicos/metabolismo , Análise em MicrossériesRESUMO
Here, we report the draft genome sequences of robust (A74/C_24-3) and poor (A74/O_2-2) chicken-colonizing Campylobacter jejuni isolates. Whole-genome sequence analyses of these isolates will be helpful in facilitating further studies to identify genetic factors used in chicken colonization.
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Most rapid identification methods for Campylobacter are designed to detect thermotolerant Campylobacter jejuni (C. jejuni) and Campylobacter coli (C. coli). A growing number of thermosensitive Campylobacter species are now gaining recognition as emerging human pathogens. Methods are lacking for the rapid screening of these emerging species. Loop-mediated Isothermal Amplification (LAMP) is a nucleic acid amplification method that allows for the rapid and cost-effective detection of bacteria. Degenerate primers against the 16S rRNA sequences for C. jejuni, C. coli, C. lari, C. upsaliensis, C. ureolyticus, C. fetus, C. gracilis, C. rectus, and C. concisus were designed. Isothermal amplification was conducted using ATCC reference strains at 68 °C for 30 min using WarmStart® Colorimetric LAMP reagents. Positive reactions were indicated by a color change from pink to yellow; specificity to Campylobacter was confirmed using a restriction enzyme digest (RsaI). The developed LAMP reaction was specific for the reference strains, which was confirmed against an exclusivity panel that consisted of other enteric pathogens, including E. coli, Salmonella, Shigella, Helicobacter, and Arcobacter. This method was also evaluated for the detection of C. jejuni, C. coli, and C. lari in primary enrichment media from artificially contaminated fresh spinach samples. The LAMP method provides an option to rapidly screen for the presence of pathogenic Campylobacter spp. in field surveillance and trace-back analysis.
Assuntos
Infecções por Campylobacter , Campylobacter , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Campylobacter/genética , Campylobacter/isolamento & purificação , Infecções por Campylobacter/diagnóstico , Infecções por Campylobacter/microbiologia , Primers do DNA , DNA Bacteriano , Humanos , RNA Ribossômico 16S , Sensibilidade e EspecificidadeRESUMO
The use of defined primers for polymerase chain reaction (PCR) amplification of interspersed repetitive DNA elements present at distinct locations in prokaryotic genomes is referred to as repetitive element sequence based-polymerase chain reaction (rep-PCR). The initial discovery of repetitive extragenic palindromic (REP) elements occurred in the genomes of Escherichia coli and Salmonella. The family of REP elements is generally between 33 and 40 bp in length, has 500 to 1,000 copies per genome, and comprises about 1% of the bacterial genomes of E. coli or Salmonella. The amplified DNA fragments, when separated by electrophoresis, constitute a genomic fingerprint that can be employed for subspecies discrimination and strain delineation of bacteria and fungi. The application of rep-PCR to microbes has proven a discriminatory and reproducible tool for microbial subtype analyses and for microbial ecology investigations.
Assuntos
DNA Bacteriano/genética , Microbiologia de Alimentos , Sequências Repetidas Invertidas , Epidemiologia Molecular/métodos , Reação em Cadeia da Polimerase/métodos , Campylobacter/genética , Campylobacter/isolamento & purificação , Campylobacter/patogenicidade , Clostridium/genética , Clostridium/isolamento & purificação , Clostridium/patogenicidade , DNA Bacteriano/isolamento & purificação , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Escherichia coli/patogenicidade , Genoma Bacteriano , Humanos , Salmonella/genética , Salmonella/isolamento & purificação , Salmonella/patogenicidadeRESUMO
While conventionally grown poultry continues to dominate the U. S. poultry industry, there is an increasing demand for locally-grown, "all natural" alternatives. The use of next generation sequencing allows for not only the gross (e.g., community structure) but also fine-scale (e.g., taxa abundances) examination of these complex microbial communities. This data provides a better understanding of how a pasture flock's microbiome changes throughout the production life cycle and how that change in microbial ecology changes foodborne pathogens in alternative poultry production systems. In order to understand this ecology better, pooled broiler samples were taken during the entire flock life cycle, from pre-hatch gastrointestinal samples (N = 12) to fecal samples from the brood (N = 5), and pasture (N = 10) periods. Additional samples were taken during processing, including skin and feather rinsates (N = 12), ceca (N = 12), and whole carcass rinses (N = 12), and finally whole carcasss rinsates of final products (N = 3). Genomic DNA was extracted, 16S rDNA microbiome sequencing was conducted (Illumina MiSeq), and microbiomes were analyzed and compared using QIIME 1.9.1 to determine how microbiomes shifted throughout production continuum, as well as what environmental factors may be influencing these shifts. Significant microbiome shifts occurred during the life cycle of the pasture broiler flock, with the brood and pasture fecal samples and cecal samples being very distinct from the other pre-hatch, processing, and final product samples. Throughout these varied microbiomes, there was a stable core microbiome containing 13 taxa. Within this core microbiome, five taxa represented known foodborne pathogens (Salmonella, Campylobacter) or potential/emerging pathogens (Pseudomonas, Enterococcus, Acinetobacter) whose relative abundances varied throughout the farm-to-fork continuum, although all were more prevalent in the fecal samples. Additionally, of the 25 physiochemical and nutrient variables measured from the fecal samples, the carbon to nitrogen ratio was one of the most significant variables to warrant further investigations because it impacted both general fecal microbial ecology and Campylobacter and Enterococcus taxa within the core fecal microbiomes. These findings demonstrate the need for further longitudinal, farm-to-fork studies to understand the ecology of the microbial ecology of pasture production flocks to improve animal, environmental, and public health.
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To examine whether there is a relationship between the degree of Campylobacter contamination observed in product lots of retail Icelandic broiler chicken carcasses and the incidence of human disease, 1,617 isolates from 327 individual product lots were genetically matched (using the flaA short variable region [SVR[) to 289 isolates from cases of human campylobacteriosis whose onset was within approximately 2 weeks from the date of processing. When there was genetic identity between broiler isolates and human isolates within the appropriate time frame, a retail product lot was classified as implicated in human disease. According to the results of this analysis, there were multiple clusters of human disease linked to the same process lot or lots. Implicated and nonimplicated retail product lots were compared for four lot descriptors: lot size, prevalence, mean contamination, and maximum contamination (as characterized by direct rinse plating). For retail product distributed fresh, Mann-Whitney U tests showed that implicated product lots had significantly (P = 0.0055) higher mean contamination than nonimplicated lots. The corresponding median values were 3.56 log CFU/carcass for implicated lots and 2.72 log CFU/carcass for nonimplicated lots. For frozen retail product, implicated lots were significantly (P = 0.0281) larger than nonimplicated lots. When the time frame was removed, retail product lots containing Campylobacter flaA SVR genotypes also seen in human disease had significantly higher mean and maximum contamination numbers than lots containing no genotypes seen in human disease for both fresh and frozen product. Our results suggest that cases of broiler-borne campylobacteriosis may occur in clusters and that the differences in mean contamination levels may provide a basis for regulatory action that is more specific than a presence-absence standard.
Assuntos
Infecções por Campylobacter/epidemiologia , Campylobacter/classificação , Campylobacter/isolamento & purificação , Galinhas/microbiologia , Contaminação de Alimentos , Carne/microbiologia , Animais , Técnicas de Tipagem Bacteriana , Campylobacter/genética , Infecções por Campylobacter/microbiologia , Análise por Conglomerados , Contagem de Colônia Microbiana , DNA Bacteriano/química , DNA Bacteriano/genética , Flagelina/genética , Genótipo , Humanos , Islândia/epidemiologia , Incidência , Epidemiologia Molecular , Dados de Sequência Molecular , Análise de Sequência de DNARESUMO
Rapid molecular techniques that evaluate eggs for the presence of foodborne pathogens is an essential component to poultry food safety monitoring. Interestingly, it is not just table eggs that contribute to outbreaks of foodborne disease. Broiler layer production actively contributes to sustaining of foodborne pathogens within a flock. The surface contamination of production eggs with invasive pathogens such as Salmonella enterica, Campylobacter jejuni, and Listeria monocytogenes during embryogenesis results in gastrointestinal tract (GIT) colonization. Pathogens that secure a niche within the GIT during embryonic development are nearly impossible to eradicate from the food chain. Therefore, current monitoring paradigms are not comprehensive because they fail to capture the presence of invasive pathogens within the embryonic GIT rapidly. By developing tools to recognize the pathogens' presence in the GIT during embryogenesis, producers are then able to spot evaluate broiler eggs for their potential risk as carriers of foodborne pathogens. In this study a novel qPCR assay was developed to semi-quantify pathogen load relative to total bacterial burden. Eggs sampled from three independent production broiler flocks of different ages were assayed for S. enterica (invA), C. jejuni (HipO), and L. monocytogenes (HlyA) against total microbial load (16s). The eggs were sampled at 1-day post-set within each flock, 2 weeks post-set, after vaccination (at 2.5 weeks) and 1-day post-hatch. The eggs were washed, and the yolk and embryonic chick GIT were collected. The DNA was extracted and subjected to a qPCR assay. The results confirm a novel technique for pathogen monitoring relative to total bacterial load and a unique method for monitoring the dynamics of foodborne pathogen invasion throughout broiler egg production.
RESUMO
Currently, there is no universally accepted standard media or method for the recovery of Campylobacter species. This is likely due to the ubiquity of the organism in nature, the complex sample matrices from which the organism is often recovered, as well as the fragile/viable-but nonculturable state the organism assumes in response to stress. The use of a sterile filter placed upon a nonselective Brucella Agar Blood Plate (BAB), followed by incubation at 37 °C in a hydrogen-containing atmosphere (Campycheck), is one method to recover stressed and emerging Campylobacter spp. from complex environmental matrices; however, this technique does not currently allow for the enumeration of the recovered organisms. Enumeration is performed using serial dilutions of sample homogenate plated onto modified Campy-Cefex media followed by incubation at either 37 °C or 42 °C in a microaerobic atmosphere.