1a. Objectives (from AD-416):
1. Provide data and characterize pathogen prevalence, unique characteristics and trends on antibiotic resistance, subtyping and molecular characterization of foodborne pathogens in food animals. 2. Identify and characterize potential genetic markers within and across serotypes for Salmonella isolated from poultry for rapid identification and diagnosis. 3. Evaluate the role of innovative chemical and/or biological treatments including arsenicals, prebiotics, or ammonium compounds and how they impact the prevalence and type of antimicrobial resistant pathogens or resistance genes.
1b. Approach (from AD-416):
Under current funding, this research is designed to be conducted by a team of two scientists. Because objectives for this project are non-hypothesis driven, specific goals have been established for each objective. Data from this research will be used to assist other Federal agencies in assessing antimicrobial resistance in food animal populations as well as to address a direct need outlined by the National Action Plan for Combatting Antibiotic Resistant Bacteria (CARB) in evaluating potential alternatives to antimicrobials. Data generated on biocide resistance and resistance genes active against chemicals specific to poultry production and processing is a specific concern to USDA-FSIS. Development of technologies for detection of microbial contaminants is a critical need for Federal regulatory agencies.
3. Progress Report:
1. Multidrug resistant Salmonella (n=194) from food animals and food production facilities were sequenced. A manuscript presenting the draft genomes of 185 of those was written and is in internal review. The isolates’ sequences were assembled into 204 plasmids characterizing their replicon types and resistance genes. One hundred additional resistance genes were localized to other mobile genetic elements. Forty historic Salmonella isolates were also sequenced and antimicrobial resistance and virulence genes were analyzed and compared to present day isolates. This data will determine how antimicrobial resistance changes in Salmonella over time, a major goal in characterizing the development of resistance. 2. The role of surface water as a location for the development of resistant bacteria and as a source for its transmission to humans or food animals is an important gap in food safety and public health knowledge. The third year of seasonal monitoring collected water samples with the Upper Oconee Watershed Network at 100 different locations along the Oconee River. Salmonella, E. coli, and Enterococcus isolated from the samples were subtyped, characterized for antimicrobial resistance, DNA fingerprinted, and tested for virulence genes. An extended-spectrum beta-lactamase (ESBL)-producing E. coli was isolated. ß-lactam antimicrobials are the first choice of drugs for infections caused by Enterobacteriaceae such as E. coli and ESBL-producing bacteria are an emerging threat to human medicine. Occurrence of this antimicrobial resistant bacteria in surface water suggests that water can serve as a reservoir for resistance and may play a role in its transmission. 3. International collaborations with researchers from Nigeria, Egypt, and Pakistan are on-going. Genomes of nine ESBL-producing E. coli from a study of isolates from poultry, cattle, swine, and clinical samples were sequenced and submitted as a bioproject to the National Center for Biotechnology Information. Analysis found several ESBL-producing genes. The study found that although some isolates from the human and chicken sources in Egypt shared characteristics, more isolates from humans were resistant to the ß-lactams than those from poultry. Information from underfunded countries is important for evaluating ESBL gene dissemination and for monitoring emergence of new genes. Staphylococcus (S.) aureus were isolated from table eggs in Pakistan and methicillin-resistant S. aureus (MRSA) were characterized. MRSA were recovered from 11% of the eggs, and all were multidrug resistant. The MRSA isolates were the genotype of MRSA that cause human infections. Results from this study showed that MRSA are present in table eggs which may be transmitted to humans. 4. Genomic analysis is being used to identify and characterize potential genetic markers within and across serotypes for Salmonella isolated from poultry and other animals for rapid identification and diagnosis. Comparative genomic analysis of 14 Salmonella serotypes from swine revealed the presence of 27 unique resistance genes which may be used to differentiate Salmonella from different commodities. Study of genetic differences among Enterococcus (E.) cecorum associated with outbreaks of enterococcal spondylitis in broilers which causes hind limb paralysis continued. This directly effects the poultry industry as outbreaks can result in 10-15% mortality. Comparative genomics identified virulence genes and antimicrobial resistance genes in ten clinical E. cecorum. This sub-project is expected to identify virulence genes specific for clinical E. cecorum which will allow development of diagnostic tests to detect them on poultry farms. Long-term products may include a vaccine candidate to protect broilers and roosters from disease caused by E. cecorum. 5. Multidrug resistant Salmonella were evaluated for resistance to non-antimicrobial chemicals/solutions (biocides) used in poultry processing. This study detected resistance to biocides including arsenic compounds; arsB, a gene encoding arsenic resistance was present in the resistant strains, but was missing or inactivated by a mutation in the sensitive strains. Eight Salmonella strains responsible for major outbreaks in the U.S. were also assayed and arsenic resistance was detected in two isolates. Overall, no correlation was detected between biocide resistance and antimicrobial resistance; however, only two hundred multidrug-resistant isolates were assayed, so this result is preliminary. 6. Development of a high-throughput assay to determine susceptibility of Salmonella to 17 biocides is on-going. This method will be made publicly available for use by animal production facilities and for scientific inquires. The biocide plate is currently being tested with resistant E. coli isolates and will be tested with resistant enterococci to detect resistance to any of these compounds in those bacterial genera. Biocide testing coupled with investigation of the genetics leading to the resistance will give a better understanding of the role of biocides in bacterial contamination of meat and development of antimicrobial resistance.
1. Cryptic antimicrobial resistance in rare Salmonella serotypes. Salmonella enterica is an important foodborne pathogen that causes gastroenteritis and enteric fever in humans. The more than two thousand serovars of Salmonella differ greatly in their ability to cause disease in humans and in their resistance to antimicrobials. To characterize antimicrobial resistance in rare Salmonella serotypes, ARS researchers in Athens, Georgia, sequenced the genomes of Salmonella serovar Bardo, Blockley, Orion, Putten and subspecies diarizonae serovar IIIb. As expected, draft genomes of multidrug resistant serovars contained resistance genes that corresponded with the resistance phenotype. However, a cryptic aminoglycoside resistance gene was also detected. The cryptic aminoglycoside resistance gene was also found in serovars that did not exhibit any phenotypic resistance to any antimicrobial including aminoglycosides. The information generated from the analysis of the genomes will be useful for predicting phenotypic resistance and future comparative analyses will improve understanding of genome evolution and multidrug resistance in Salmonella.
2. Expansion of the Comprehensive Antibiotic Resistance Database. The development of antimicrobial resistant (AMR) bacteria in food animals presents a threat to food safety and human and animal health. DNA sequencing of genes or whole bacterial genomes has been used to identify and characterize AMR genes; unknown DNA sequences are identified using a database of known AMR genes. The ability to detect and analyze AMR genes is highly dependent on using a complete and carefully annotated database of known AMR genes. Researchers from Ontario, Canada, and ARS, Athens, Georgia, have developed the Comprehensive Antibiotic Resistance Database (CARD; http://arpcard.mcmaster.ca). CARD is a manually curated database containing high quality reference AMR genetic data including genes and proteins. Its design allows the development of novel genome analysis tools, such as the Resistance Gene Identifier (RGI) for prediction of resistance genes from whole genome sequences. CARD’s RGI has been used to analyze thousands of bacterial genomes from hundreds of research and clinical studies, resulting in AMR data used in a wide variety of research. CARD is a crucial tool in research used to combat AMR; its development and continual update is key to understanding AMR and discovering ways to reduce AMR impact in humans, animals, and the environment.
3. Multidrug resistant Escherichia coli in Nigeria. An aim of the National Action Plan for Combatting Antimicrobial Resistant bacteria is to gather country-specific and regional information on drivers of antimicrobial resistance. ARS researchers in Athens, Georgia, in collaboration with Olabisi Onabanjo University, Lagos, Nigeria screened clinical isolates of Escherichia coli from Lagos, Nigeria, for resistance to antimicrobials. A majority of the E. coli that were resistant to antimicrobials were also multidrug resistant exhibiting resistance to as many as 16 antimicrobials in a single strain. Identical genetic types were detected among isolates from different samples suggesting that isolates were shared among the population. Results of this study demonstrate the presence of circulating multidrug resistant E. coli in the Nigerian community. Monitoring antimicrobial resistance in developing countries is necessary to develop worldwide prevention and control strategies for combatting antimicrobial resistance.
4. Survival of Salmonella in diverse environments. Salmonella senses its surroundings and changes the genes it expresses to adapt and survive in diverse environments where it can contaminate food and cause foodborne illness in humans. How Salmonella controls this gene expression is only partially understood. Bacteria can control expression of specific genes through sigma factors which bind to DNA in a specific region of a gene and initiates expression. Sigma54 is unique because after binding to the region of DNA, it will not work until it senses the correct environmental conditions. ARS researchers in Athens, Georgia, in collaboration with the University of Georgia, Athens, Georgia, conducted experiments to activate all Sigma54 dependent genes in Salmonella without any environmental signals. This allowed the identification of all of the genes controlled by Sigma54 in the Sigma54 group, suggesting new roles for Sigma54 in this pathogen. This data is critical to understand how pathogens respond rapidly to new environments enabling them to persist on farms, live in animals, survive in processing plants, contaminate food and infect humans. This knowledge provides approaches to identify ways to disrupt this adaptation and prevent Salmonella from being a food safety threat.
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