2010 Annual Report
1a.Objectives (from AD-416)
1) Use antibiotic resistance data obtained from the Collaboration on Animal Health and Food Safety Epidemiology (CAHFSE) and the National Antimicrobial Resistance Monitoring System - Enteric Bacteria (NARMS) programs and poultry studies to identify sources, reservoirs and amplifiers of resistant food borne and commensal bacteria, as well as the path of dissemination of these resistant bacteria in food producing animals and poultry. Results may be used for risk assessment and in developing mitigation strategies. .
2) Map the spread of antimicrobial resistance throughout the US using molecular epidemiology and population genetic studies of antimicrobial resistant bacterial isolates, including participation in USDA VetNet. .
3) Analyze and differentiate antimicrobial resistance mechanisms, both phenotypically and genotypically, and rapidly identify resistant strains.
1b.Approach (from AD-416)
Under current funding, this research is designed to be conducted by a team of five scientists, each focusing on one particular organism or area. Each SY will design a specific research plan maximizing collaborations within the Unit structure. Although independent research will be conducted, a majority of experiments will be interactive, minimizing the need to repeat experimental samplings, particularly in the field. This research format will also maximize acquisition of data which will provide insight of the interaction between bacterial populations within the host and/or environment, particularly those interactions involving food borne zoonotic and commensal bacteria. Three SYs will focus on the molecular aspects of AR, particularly in Campylobacter, Salmonella and commensal bacteria (E. coli and enterococci). Critical to the molecular research will be epidemiologic studies provided by the CAHFSE program and ecologic (field and environment) studies which will not only provide a source of isolates for the molecular studies, but will also determine prevalence and dissemination of AR attributes within production settings, the environment, and among bacterial populations. Another significant source of isolates will be available from the NARMS program. These isolates will be well characterized to the serotype level and antimicrobial resistance phenotype. Additionally, all isolates will have been subjected to PFGE analysis to determine relatedness among isolates. Specific genotypic characterization will be conducted. Pathogenic studies involving bacterial strains collected from the CAHFSE and the NARMS programs, as well as those which have been genetically modified in the laboratory, will provide information regarding virulence (or lack thereof) associated with the acquisition of AR. Additionally, transfer of resistance genes may be studied under these environments.
Significant advances for detecting the source of antimicrobial resistance was determined for Salmonella and E. coli. Using an antimicrobial resistance microarray developed in the Unit, common resistance genes were found in Salmonella and E. coli isolated from swine indicating that these bacteria share a common bacterial source for resistance genes. The genetic source of multi-drug resistance (MDR) was investigated using replicon multiplex PCR and a plasmid microarray, both developed in the Unit. These techniques detected MDR plasmids from both Salmonella and E. coli. E. coli isolates were genetically diverse by plasmid type, antimicrobial resistance, and genetic typing using Pulsed-Field Gel Electrophoresis (PFGE). MDR plasmids from both bacteria are being sequenced using high-throughput sequencing to determine if they are the primary source of MDR in Salmonella and E. coli. Other studies on Salmonella found that culture methods influenced Salmonella serotypes recovered from various samples and that European Union and U.S. poultry sampling protocols both produced false negatives, but no significant difference in Salmonella prevalence.
Studies on detection and reduction of Campylobacter were conducted. Campylobacter colonized young chicks through various routes, but chlorine dioxide reduced Campylobacter numbers during feather removal. Real-time PCR was excellent for detecting Campylobacter from carcass rinsates.
Two separate studies on dairy cattle conducted by the National Animal Health Monitoring System (NAHMS) and Cornell University found that resistant enterococci were prevalent in dairy cattle fecal samples and that enterococci may be a reservoir of resistance for other bacteria. Other common sources of antimicrobial resistant bacteria were found in a study on prevalence of resistant bacteria from companion animals. MDR E. coli from both dogs and cats were detected as well as Methicillin-Resistant Staphylococcus aureus (MRSA). MRSA isolates from companion animals were genetically different than MRSA isolated from humans from the same geographic area. Preliminary results suggest that companion animals may serve as a reservoir of resistant bacteria that may also be transferred to humans.
The animal arm of the National Antimicrobial Resistance Monitoring System (NARMS) database now contains antimicrobial susceptibility patterns for over 58,000 Salmonella, 6,500 Campylobacter, 22,500 generic E. coli and 11,000 Enterococcus. Of those, 2100 Salmonella and 500 Campylobacter were submitted to VetNet in FY10. The VetNet database was also used to identify new PFGE patterns from Salmonella isolated from broiler carcass rinses.
Antimicrobial susceptibility testing and species determination of Campylobacter isolates from the Food Safety Inspection Service Young Chicken Baseline Study. Antimicrobial susceptibility testing and speciation of Campylobacter isolates from the Food Safety Inspection Service (FSIS) Chicken Parts Shakedown and Baseline Studies was conducted on raw chicken parts. FSIS is currently collecting chicken part samples as part of a pilot/shakedown prior to starting the actual baseline study. Currently, approximately 100 Campylobacter isolates have been identified as either C. jejuni (75.5 %) or C. coli (24.5 %), a species ratio typically observed from chicken carcass rinsates post-chill. Tetracycline resistance appeared most often among the isolates (47 %). About a fifth of the isolates (19 %) were resistant to the fluoroquinolone, ciprofloxacin, which was banned in 2005 from use in poultry. Resistance levels were < 5 % for azithromycin, erythromycin, clindamycin, and gentamicin. This data can be used in the development of risk-based sampling programs and regulatory decisions.
Routes for Campylobacter coli colonization of the intestinal tract of chicks. Newly hatched chicks may be exposed to significant levels of Campylobacter from various sources in the hatchery and grow-out environments. Once Campylobacter reach the ceca of the young chick, they are able to multiply to high levels in a relatively short time period. The young chick then contaminates the environment by excreting large numbers of Campylobacter through both cecal and fecal droppings which in turn can result in the contamination of other birds in the grow-out facility. In this study, Campylobacter coli, naturally gentamicin resistant, was introduced into day-of hatch chicks through various body openings (mouth, nasal passage, eye and cloaca) at three different inoculum levels (approximately 20, 200 and 2000 CFU/bird) to determine which of the routes and numbers of Campylobacter would result in the highest level of colonization in the chicks. Inoculum levels at least approximately 200 CFU/bird or greater readily colonized birds from all four routes of entry. Nasal passage was the least likely route for colonization. These data suggest that Campylobacter can readily colonize day of hatch birds via various routes and contaminate the environment through shedding in feces. Therefore, multiple intervention strategies may be required to interrupt exposure and/or colonization with Campylobacter.
Application of chlorine dioxide to lessen bacterial contamination during broiler defeathering. Due to escape of contaminated gut contents during feather removal at slaughter, the number of Campylobacter spp. recovered from broiler carcasses increases during feather removal. Chlorine dioxide (ClO2) is approved for use as an antimicrobial treatment during poultry processing. A study was designed to test if application of ClO2 during feather removal could prevent the expected increase in Campylobacter numbers on carcasses. Chlorine dioxide de-feathering significantly decreased the number of Campylobacter by almost log 3 CFU per mL and had an additional effect on lowering of Escherichia coli numbers. Application of ClO2 during feather removal may have potential to mitigate the increase in bacterial contamination associated with broiler de-feathering.
Characterization of multidrug-resistant Escherichia coli by plasmid replicon typing and Pulsed-Field Gel Electrophoresis. Plasmid replicon typing is a multiplex PCR-based method that can be used to classify 18 of the 26 known plasmid types in Enterobacteriaceae. The objective of this study was to analyze plasmid replicon types, antimicrobial phenotypes, and genetic relationships of 35 multidrug-resistant (MDR) Escherichia coli. A total of 35 E. coli isolates from seven different animal sources were selected based on antimicrobial phenotypes of clinically important drugs. Pulsed-Field Gel Electrophoresis (PFGE) was used to determine genetic relationships among the E. coli isolates. Multiplex PCR was used to examine the replicon (i.e. Inc) types. Linkage analysis was conducted for isolate antimicrobial resistance, replicon types and animal sources. A high degree of genotypic diversity was observed, with 34 different PFGE types found among the 35 isolates examined. A total of 15 different plasmid replicon types were detected and all isolates carried multiple replicon types. Overall Inc B/O plasmids were the most common type identified followed by Inc FIB. Significant linkage of Inc B/O was seen with trimethoprim-sulfamethoxazole while Inc FIB was significantly linked with chloramphenicol. Significant linkage was also found with amoxicillin-clavulanic acid, ampicillin, cefoxitin, tetracycline, and ceftiofur, the drugs used as the main criteria in selecting the E. coli isolates. The isolate animal sources did not cluster according to PFGE type. The importance of commensal E. coli as reservoirs of plasmids carrying antimicrobial resistance is emphasized by these results. Additional research will determine the extent of diversity and linkage associations among plasmids carrying antimicrobial resistance genes in E. coli.
Analysis of antimicrobial resistance genes in bacteria co-isolated from swine fecal samples. To determine if identical antimicrobial resistance genes can be detected in different bacterial species isolated from the same fecal sample. Salmonella, E. coli, Campylobacter and Enterococcus species were isolated from feces collected on farm from healthy swine from 2003 to 2006 by the Collaboration on Animal Health and Food Safety Epidemiology (CAHFSE) swine pilot project. Forty-nine fecal samples yielded isolates of all four bacterial species. Each isolate was analyzed for the presence of 775 antimicrobial resistance genes by microarray. Multiple resistance genes were found in most of the isolates. Statistical analysis determined that Salmonella and E. coli isolated from the same fecal sample had identical genes detected at a significant level. This indicates that Salmonella and E. coli may have a common source for acquiring antimicrobial resistance genes or may exchange resistance genes in the swine environment. This data suggests that these common genetic elements in different bacteria may serve as a reservoir for resistance in important pathogens such as Salmonella. These genetic elements require further studies to understand their amplification and spread in food animals.
Development of microarrays for detection of multi-drug resistant plasmids in Salmonella and E. coli. To develop a DNA microarray for analysis of multi-drug resistant (MDR) plasmids responsible for high levels of antimicrobial resistance in Salmonella and E. coli. A DNA microarray was constructed containing probes for ~500 genes commonly found in different plasmid types. This technique allows the rapid analysis of the genetic element that is responsible for wide spread MDR Salmonella in the U.S. This tool is being used for analysis in multiple Unit projects and with collaborators in other agencies to define the evolution and spread of these MDR plasmids. These studies include: MDR Salmonella clinical isolates from animals collected in 2005 by National Veterinary Services Laboratory (NVSL); MDR Salmonella isolates collected from 1997 to 2009 by the National Antimicrobial Resistance Monitoring System (NARMS); MDR E. coli isolates collected from 1999 to 2009 by NARMS; MDR Salmonella collected in 2007 by NARMS partners in FDA, CDC, and Canada; MDR Acinetobacter isolates from wounded soldiers returning from Iraq; and MDR Salmonella Javiana from FDA. These studies will allow us to monitor the spread of MDR plasmids in Salmonella and determine their threat to human and animal health.
Sequence analysis of multi-drug resistant plasmids from Salmonella and E. coli. To determine if the majority of multi-drug resistance (MDR) found in National Antimicrobial Resistance Monitoring System (NARMS) Salmonella isolates is due to the MDR encoding plasmids. Multiple studies conducted by the Unit and other researchers have associated the MDR phenotype with the Inc A/C plasmid genotype in the U.S. To better define the impact of this MDR plasmid on human and animal health, high-throughput sequencing (HTS) is underway to determine the evolution of these MDR plasmids and how they spread in Salmonella and E. coli. Thirty representative MDR Salmonella and 12 MDR E. coli plasmid samples are being sequenced using Roche 454 technology with collaborators at ARS, Clay Center, NB. Data from these experiments will allow us to understand the evolutionary factors that may drive the spread of these MDR plasmids in bacteria found in animals. Preliminary information indicates that these plasmid encode metal and sanitizer resistance genes that may give bacteria that possess them a survival advantage in the animal environment or in the meat processing plant environment. These could be targets for either future interventions or points where intervention strategies may need to be modified to address bacteria that carry these MDR plasmids. In addition, collaboration with ARS scientists at the Eastern Regional Research Center is also addressing small plasmids which also encode antimicrobial resistance genes. This study complements the large MDR plasmid study and ensures that all aspects of antimicrobial resistance spread due to plasmids are addressed.
Prevalence of ColE1-Like plasmids and kanamycin resistance genes in Salmonella enterica serovars. To expand previous studies in assessing the prevalence of the isolates harboring ColE1-like plasmids carrying the aph gene responsible for kanamycin resistance (KANR) in Salmonella isolates. One hundred and two KANR Salmonella isolates collected through the National Antimicrobial Resistance Monitoring System (NARMS) in 2005 were screened by PCR using ColE1 primer sets. ColE1 positive isolates were further studied by restriction mapping. ColE1 typing primers designed in this study successfully identified three groups of ColE1-like plasmids and will aid in selection of isolates for sequencing. All 7 (100%) of the isolates that were serovar Typhimurium and serovar Typhimurium variant 5-, penta+Kan-resistant, and positive for ColE1 by PCR, were also associated with the DT104 phage type. The mobility and high copy number of the ColE1 plasmid family warrants close monitoring of its prevalence and transmission among food animals.
Development of assays for the genetic characterization of Escherichia albertii. Polymerase chain reaction (PCR) based tests coupled with sequencing was studied for positive genetic identification of Escherichia albertii from other closely related organisms. We have started analysis of isolates from Food Safety Inspection Service (FSIS) carcass rinses to positively identify Escherichia albertii. Escherichia albertii is a possible zoonotic and animal pathogen and identification and tracking of this organism may be important. Escherichia albertii has been linked to avian deaths and it is closely related to Shigella and Escherichia coli; but other than sequencing the whole organism, no definitive identification methods are available. These methods currently under study could be used for quick and easy identification of Escherichia albertii.
Prevalence of antimicrobial resistant Escherichia coli from companion animals. The contribution of dogs and cats as reservoirs of antimicrobial resistant E. coli remains largely undefined. This is increasingly important considering the possible transfer of bacteria from companion animals to the human host. In this study, dogs and cats from veterinary clinics were screened for the presence of E. coli. A total of 317 E. coli were isolated from nasal, teeth, rectal, belly and hindquarter sites of 155 dogs and 121 cats. Approximately 19.2% and 24.4% of E. coli from dogs and cats, respectively, were resistant to antimicrobials. The highest level of resistance was to ampicillin for both groups of animals. Multi-drug resistance (MDR) (resistance >2 antimicrobials) was observed in 36 isolates which were resistant to as few as two and as many as ten antimicrobials. Results from this study indicate that healthy dogs and cats are a source of antimicrobial resistant E. coli and may act as a reservoir of antimicrobial resistance that can be transferred to the human host.
Sensitivity and selectivity of cultivation methods for recovery of Salmonella serotype Enteriditis. Salmonella serotype Enteritidis (SE) can be difficult to isolate from broiler breeder flock environmental samples even when hatchery progeny chicks are positive. This is due, in part, to the fact that Salmonella serotypes exhibit different growth characteristics during sample enrichment. Different cultivation methods were evaluated using pure culture, mixed Salmonella serotypes and in the presence of non-Salmonella microflora. Although no difference in SE growth was apparent in pure cultures, the addition of other Salmonella, feces, or fluff significantly reduced SE growth. However, other, non Enteritidis serogroups could still be recovered from the different culture methods. It is apparent that cultivation methods select for particular serogroups and may allow particular serotypes to outgrow others. This would negatively influence the probability of recovering certain serotypes present in lower numbers within the sample and may explain why SE was not detected from any of the non-spiked cultivation methods.
Culture methods can influence the Salmonella serotype diversity seen from broiler carcass rinsates. Salmonella diversity on broiler carcasses has received significant attention in recent years due in large part to emphasis on the predominance of certain serotypes associated in human illness. The objective of this study was to evaluate Salmonella diversity from broiler carcass rinsates by two cultivation methods. External carcass rinses were evaluated using a pre-enrichment followed by transfer into either Gram negative (GN) or tetrathionate (TET) broth. After incubation, aliquots from each broth were plated onto BGS and XLT4 plates. When available, three typical colonies were picked per plate for identification and serotyping. A total of 236 Salmonella isolates comprising eight different serotypes were recovered. S. Kentucky and S. Berta were the two prominent serotypes recovered from both plant visits while S. Kiambu and S. Mbandaka were second most prevalent from visits 1 and 2, respectively. Significantly more S. Kentucky was recovered from GN broth, while more S. Heidelberg and S. Senftenberg were isolated from TET. S. Kentucky was also recovered significantly more often from XLT4 than BGS when originating from TET broth. S. Kiamba was only detected on XLT4 plates. Salmonella diversity on broiler carcasses was evident and differed depending on culture method used. The enrichment and plating media combinations, along with number of colonies selected, influenced the serotypes recovered and demonstrated the bias that may occur during cultivation of Salmonella.
Characterization of Methicillin-Resistant Staphylococcus aureus (MRSA) from companion animals and humans. The contribution of companion animals as reservoirs of MRSA remains largely undefined. This is increasingly important considering the possibility of transfer of bacteria from companion animals to the human host. In this study, MRSA from companion dogs and cats were isolated and characterized and compared to those from humans in the Athens, GA area. MRSA were isolated from various sites on ill and healthy animals; MRSA isolates from humans were obtained from a local hospital. All isolates from companion animals were of the same staphylococcal chromosome cassette (SCC) mec (methicillin) type which was different from that of the human MRSA isolates. Susceptibility of MRSA isolates was performed to determine the range of resistance; all isolates from companion animals exhibited the same resistance pattern among the 18 antimicrobials tested. Also, a new multi-locus sequence type (MLST) was detected among the MRSA isolates. Resistance patterns for human MRSA varied, but did contain some of the same antimicrobial resistance genes as MRSA from companion animals. MRSA causes severe to fatal infections among high risk populations and healthy individuals. Sources of community-acquired MRSA infections vary and the current study shows that companion animals may harbor the organism. (National Program Component 1.1, Problem Statement 1.1.5: Antibiotic Resistance)
Antimicrobial susceptibility testing of food-borne pathogens by the animal arm of the National Antimicrobial Resistance Monitoring System (NARMS). Antimicrobial susceptibility testing of food-borne pathogens by the animal arm of NARMS was conducted on Salmonella, Campylobacter, E. coli and Enterococcus from animal and environmental sources. Since 1996, the animal arm of NARMS has been based in the USDA-ARS at the RRC in Athens, GA. Salmonella was selected as the sentinel organism and antimicrobial resistance in over 58,000 Salmonella isolates has been determined. Campylobacter, generic E. coli and Enterococcus were added in subsequent years resulting in over 6,500, 22,500 and 11,000, respectively, antimicrobial susceptibility patterns for each of those bacteria. Veterinary Diagnostic Laboratories served as diagnostic sentinel sites through 2006. This is the only national program for surveillance of resistant bacteria in animals in the U.S. and provides critical information regarding the prevalence and distribution of antimicrobial resistant bacteria in on-farm animals, from diagnostic submissions, and from raw product collected from federally inspected slaughter and processing plants.
Prevalence, species distribution, and antimicrobial resistance of enterococci isolated from US dairy cattle. The contribution of food animals as reservoirs of antimicrobial resistant enterococci remains largely undefined. This is increasingly important considering the possibility of transfer of bacteria from food animals to humans via the food chain. In this study, prevalence and antimicrobial resistance of enterococci in fecal samples from U.S. dairy operations participating in the 2007 National Animal Health Monitoring System (NAHMS) survey of dairy cattle health and management practices was examined. The NAHMS Dairy 2007 study represented 79.5% of U.S. dairy operations and 82.5% of U.S. dairy cows and was conducted in 17 states. Approximately 30-35 healthy cows were sampled on each of 122 dairy operations from the end of February through August 2007. Of the 122 operations sampled, 118 (96.7%) had at least one dairy cow positive for enterococci and 88.7% (637/718) of dairy cattle fecal samples were positive for enterococci. Enterococcus hirae, E. faecalis, and E. faecium were the most prevalent enterococcal species isolated. The highest levels of resistance were to lincomycin, flavomycin, and tetracycline; none of the isolates were resistant to chloramphenicol, gentamicin, linezolid, tigecycline, or vancomycin. Results from this study indicate that fecal samples from U.S. dairy cattle contain high numbers of antimicrobial resistant enterococci and may act as a reservoir of antimicrobial resistant enterococci that can be transferred to the human host via the food chain.
Molecular typing of Salmonella and Campylobacter using Pulsed-Field Gel Electrophoresis (PFGE) by USDA VetNet. USDA VetNet was modeled after PulseNet USA, the national molecular subtyping network for food-borne disease surveillance to subtype zoonotic pathogens submitted to the animal arm of the National Antimicrobial Resistance Monitoring System (NARMS). Additionally, USDA VetNet PFGE patterns are compared to PulseNet PFGE patterns for surveillance and investigation of food-borne illness outbreaks. Currently the program includes PFGE patterns for nontyphoidal Salmonella and Campylobacter isolated from diagnostic animal specimens, healthy farm animals, and carcasses of food-producing animals at slaughter. VetNet maintains two PFGE databases, one for Salmonella and one for Campylobacter. PFGE Tagged Image File Format (TIFF) images, demographic information, and antimicrobial resistance profiles for approximately 2,100 Salmonella isolates and 500 Campylobacter isolates were added to the databases this year. The establishment of USDA VetNet enhances the mission of the agriculture and public health communities in surveillance and investigation of food borne illness outbreaks.
Pulsed-Field Gel Electrophoresis (PFGE) and antimicrobial resistance patterns of Salmonella serotypes isolated from broiler external carcass rinses. Pulsed-field Gel Electrophoresis (PFGE) and antibiogram patterns have been used to evaluate the diversity within and between individual isolates within Salmonella serotypes. The objectives of the study were to evaluate the PFGE and antimicrobial resistance patterns of Salmonella broiler carcass rinse isolates and compare pattern similarity to those isolates found in the USDA-VetNet database. Salmonella isolates (n=176) originated from post-pick broiler carcass rinse samples. Antimicrobial resistance diversity as well as the PFGE diversity was studied for eight different S. enterica serotypes: Agona; Berta; Heidelberg; 4,5,12:i:-; Kentucky; Kiambu; Mbandaka; and Senftenberg Overall, eight different antibiogram patterns were observed across all isolates. S. Kentucky isolates exhibited the greatest heterogeneity with six different antimicrobial resistance patterns within 13 different PFGE patterns. Isolates among both serotypes were pan-susceptible. Compared to patterns within the USDA-VetNet database, a total of ten new Salmonella PFGE XbaI patterns were identified with six new patterns originating from S. Kentucky isolates. The S. Kiambu isolates all had a unique XbaI pattern that has a slight band shift difference from a S. Kiambu pattern only seen twice before in diagnostic cattle samples. The identification of new patterns suggests that further work needs to be conducted on how culture methods can influence recovery of certain Salmonella serotypes as well as diversity within those serotypes. The isolates from this study originated from cultivation method combinations that are not commonly used by regulatory agencies to recover Salmonella from poultry carcasses.
Comparison of sampling methods for microbiological evaluation of broiler carcasses. Comparing prevalence data from country-to-country is difficult when the sampling protocols for detecting Salmonella on poultry differ. The European Union (E.U.) uses a three carcass composite of neck skin totaling 25g while in the United States (U.S.), the whole carcass is rinsed with 400 ml of buffered peptone water but only 30 ml is analyzed. The purpose of this study was to evaluate a whole carcass rinse (WCR) and a neck skin excision (NS) procedure for Salmonella and Escherichia coli isolation from the same broiler carcass. No significant difference in Salmonella prevalence was found between the samples processed by the two methods, but a significant difference in Salmonella prevalence was observed for carcasses sampled pre- and post-chill. Both procedures produced many false-negative Salmonella results. A significant difference between the two sampling methods was seen, however, the WCR samples had higher E. coli and coliform counts than did the NS samples. Given the lack of sensitivity of both methods, it is not surprising that both methods produced false negatives. If a validated, sensitive, and accurate sampling and testing procedure were used worldwide, researchers would be better able to directly compare Salmonella prevalence data among countries.
Prevalence of antimicrobial resistant bacteria from dairy cattle in the northeast United States. In collaboration with Quality Milk Production Services at Cornell University, environmental and fecal samples were taken from three different dairy cattle farms in the Northeast US on a quarterly basis. These samples were analyzed for the presence of Enterococcus, E. coli, Salmonella, and Campylobacter. Recovered isolates were speciated and tested for antimicrobial susceptibility. As expected, E. coli and Enterococcus were recovered most often (90% and 84%, respectively), followed by Campylobacter (32%), and Salmonella (13 %). Overall, environmental samples were only slightly less positive than individual fecal samples. The only resistance observed among Campylobacter isolates was to tetracycline (80 %). Antimicrobial susceptibility testing of the other organisms is currently being conducted. These data are useful for determining persistence and transmission of bacteria as well as for developing mitigation strategies.
Determination of optimized isolation and prevalence estimates for Clostridium difficile from healthy food animals. Two isolation methods were compared for isolation of Clostridium difficile from food animal feces. The single alcohol shock method (SS) used selective enrichment followed by alcohol shock and isolation on two different agar plates. The double alcohol shock method (DS) used alcohol shock prior to and after selective enrichment followed by isolation on the same agars as above. A total of 55 swine fecal samples, 32 dairy cattle fecal samples, and 188 beef cattle fecal samples were positive for C. difficile by either method. However, the DS method was significantly better than SS for the recovery of C. difficile in swine feces while the SS was significantly better than the DS for the recovery of C. difficile in beef cattle feces. There was no significant difference between methods for the recovery of C. difficile from dairy cattle feces. This study demonstrates that food animals may harbor C. difficile and provides critical information that isolation methods may not have universal application across animal species.
Presence of Clostridium difficile toxin genes in isolates recovered from healthy bovine and swine. Clostridium difficile infections represent an important human health problem in healthcare settings; however, community-acquired cases are emerging. Evidence suggests that food animals and contaminated meat could be possible transmission vectors for C. difficile-associated disease (CDAD). We used PCR to examine 478 C. difficile isolates from healthy, on-farm food animal feces (beef cattle, swine, and dairy cattle) and from the dairy farm environment for the presence of toxin genes. About 63% of the isolates were capable of producing both toxins A and B. Significantly more potentially toxigenic (A+B+) strains were recovered from dairy fecal, dairy environmental, and swine fecal isolates compared to beef fecal isolates. Overall, C. difficile from beef isolates were most likely to be non-toxigenic (A-B-) or of the variant types compared to the other samples. This research suggests that healthy swine, dairy cattle, and the dairy environment, may serve as sources to transmit virulent C. difficile strains to humans via the food supply chain.
Evaluation of molecular typing methods for Clostridium difficile. Semi-Automated Repetitive Extragenic Palindromic-Polymerase Chain Reaction (REP-PCR) as a molecular typing method for Clostridium difficile was evaluated for its reliability for typing Clostridium difficile by comparing results to those obtained by Pulsed-Field Gel Electrophoresis (PFGE). The relationship between C. difficile isolated from food animals and human was also determined. A total of 221 isolates of C. difficile from swine, dairy cattle, environmental samples, and humans were typed by REP-PCR and PFGE. One hundred nineteen and 82 distinct patterns were identified by REP-PCR and PFGE, respectively. REP-PCR and PFGE results were concordant for 32 isolates. Clustering of the remaining 188 isolates differed. Overall, 20 isolates (9%) from animals were indistinguishable from one human isolate by REP-PCR whereas 40 isolates (18%) from animals were indistinguishable from one or more human isolates by PFGE. REP-PCR was more rapid and less labor-intensive than PFGE, and may be an alternate to PFGE for determining strain relatedness of C. difficile isolates. A high percentage of indistinguishable isolates of human and animal was also observed. This suggests that interspecies or other forms of animal-to-human transmission may occur.
Evaluation of two commercial Real-Time PCR assays for detecting Campylobacter in broiler carcass rinses. Traditional plating methods are reliable means for Campylobacter identification from poultry samples, but automated gene-based detection systems now available can reduce assay time, data collection and analysis. Bio-Rad and DuPont Qualicon recently introduced Campylobacter assays for their real-time PCR instruments. We evaluated the utility of these assays compared to standard plating and enumeration methods routinely used in our laboratory. Two replicates of 40 broiler carcass rinses collected before and after defeathering at a commercial processing plant were tested. All samples were positive for Campylobacter by direct plating of rinses: log10 cfu values ranged from 0.24-4.61. In contrast, the Bio-Rad iQ-Check assay returned 60-72.5% positives on direct rinses; the Qualicon BAX® Q7 test produced 60-85% positives with direct rinse samples. Using aliquots of 24 hr enrichment broth from rinses in the real-time assays significantly improved detection: the Bio-Rad test had 95-100% positive while the Qualicon assay found 90-95% positive. Traditional detection methods for Campylobacter can be very time consuming, often requiring days to complete. Commercial real-time PCR instruments for the identification of Campylobacter offer more rapid, simplified alternatives to standard culture-based techniques. With minor modifications to reduce PCR inhibition, these assays would have significant applications for studies involving Campylobacter detection from complex sample material.
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