Prevalence of mcr-1 in the Cecal Contents of Food Animals in the United States.

A survey of 2,003 cecal content samples from chickens, turkeys, cattle, and swine at slaughter facilities in the United States was conducted to estimate the prevalence of the mcr-1 gene conferring resistance to colistin in Enterobacteriaceae Two cecal samples from swine had Escherichia coli with IncI2 plasmids bearing the mcr-1 gene.

Antimicrobial Susceptibility and Plasmid Replicon Typing of Salmonella enterica Serovar Kentucky Isolates Recovered from Broilers.

Salmonella Kentucky has become the predominant serovar recovered from broilers slaughtered in the United States, and the prevalence of antimicrobial resistance (AMR) has increased dramatically in this serovar. Relationships between AMR, genotype, and plasmid replicon types were characterized for 600 Salmonella Kentucky isolates recovered from chicken carcasses from 2004 to 2013. Pulsed-field gel electrophoresis cluster analysis revealed 112 unique types sharing 79% similarity. Over half of the isolates studies were assigned to two large clusters (unique restriction patterns) consisting of 190 (A) and 151 (B) isolates. The remaining (n = 259) more diverse isolates (110 unique patterns) shall be designated cluster C for discussion. Clusters A had significantly more (p < 0.05) isolates resistant to streptomycin (68.4%) and tetracycline (91.6%) compared to cluster C (50.6% and 40.9% to streptomycin and tetracycline, respectively) or cluster B, which had the least (p < 0.05) resistance (11.9% and 13.2% to streptomycin and tetracycline, respectively). In addition, there was segregation of plasmid replicon types among clusters. Cluster A had significantly more (p < 0.05) replicon type FIB (90.5%) compared to cluster C (37.1%), which had significantly more compared to cluster B (10.6%). Cluster B had significantly more (p < 0.05) replicon type I1 (87.4%) compared to cluster C (68.7%), which had significantly more (p < 0.05) compared to cluster A (32.6%). Cluster C harbored significantly more (p < 0.05) HI2 replicon type (18.1%) compared to clonal clusters A (1.6%) or B (1.3%). The prevalence of plasmid replicon type A/C did not differ among clusters (A, 0.5%; B, 2.0%; C, 0.4%). Both streptomycin and tetracycline resistance were significantly linked (p < 0.05) to plasmid replicon type FIB. In addition, replicon type HI2 was also significantly linked (p < 0.05) to streptomycin resistance. We conclude that the dramatic increase in streptomycin and tetracycline resistance among Salmonella Kentucky isolated from poultry is due to the expansion of strains harboring plasmid replicon types FIB and HI2.

Colonization of a newly constructed commercial chicken further processing plant with Listeria monocytogenes.

This study was undertaken to determine potential sources of Listeria monocytogenes in a newly constructed chicken further processing plant and document the eventual colonization of the facility by this pathogen. To ascertain the colonization status of the plant, floor drains were sampled after a production shift and again after a cleanup shift on roughly a monthly basis for 21 months. Potential sources of L. monocytogenes to the plant included incoming raw meat, incoming fresh air, and personnel. Nearby environment and community samples were also examined. All L. monocytogenes detected were subjected to DNA sequence-based subtyping. L. monocytogenes was not detected in the plant before the commencement of processing operations. Within 4 months, several subtypes of L. monocytogenes were detected in floor drains, both before and after cleaning and sanitizing operations. No L. monocytogenes was detected on filters for incoming air, samples associated with plant employees, or a nearby discount shopping center. One subtype of L. monocytogenes was detected in a natural stream near the plant; however, this subtype was never detected inside the plant. Eight subtypes of L. monocytogenes were detected in raw meat staged for further processing; one of the raw meat subtypes was indistinguishable from a persistent drain subtype recovered after cleaning on eight occasions in four different drains. Poultry further processing plants are likely to become colonized with L. monocytogenes; raw product is an important source of the organism to the plant.

Analysis of risk factors associated with Salmonella spp. isolated from U.S. feedlot cattle.

Contamination can occur at a number of stages during farm-to-fork processing. Preharvest intervention is an ongoing area of interest in reduction of risk of foodborne illness. This study examined risk factors associated with detection of Salmonella from cattle in U.S. feedlots. During two visits to 73 feedlots, 25 fresh fecal samples were collected from each of three pen floors. Associations between management and demographic factors and culture status were evaluated using logistic regression. Factors positively associated with culture-positive status included brewers' grains (odds ratio [OR] = 26.35; confidence interval [CI], 10.33-67.20), corn gluten (OR = 10.35; CI, 5.98-17.91), or cottonseed hulls (OR = 8.34; CI, 3.58-19.42) in the ration, and sourcing animals in a pen from multiple herds of origin (OR = 5.17; CI, 2.32-11.51). Factors negatively associated with positive culture status included urea (OR = 0.27; CI, 0.16-0.44), alfalfa, clover, or sorghum silage (OR = 0.31; CI, 0.12-0.79), and antimicrobials of the tetracycline class in the ration (within 2 weeks before sampling, OR = 0.04 and CI, 0.02-0.09; more than 2 weeks before sampling, OR = 0.23 and CI, 0.06-0.80). Since 18.3% of positive samples were on a single operation, a second model was constructed after excluding data from this operation. Three additional variables were retained in the second model, including grain-processing method (OR for dry roll, cracked, or unprocessed grain = 2.99; CI, 1.55-5.75), soybean meal (OR = 2.74; CI, 1.58-4.75), and use of a coccidiostat in the ration (OR for no coccidiostat = 4.50; CI, 2.03-10.01). Considering the increasing use of by-products of the biofuel industry as feeds, further investigation of the association between feeding brewers' grains and corn gluten and Salmonella recovery is warranted.

23S rRNA gene mutations contributing to macrolide resistance in Campylobacter jejuni and Campylobacter coli.

The genetic basis of macrolide resistance in Campylobacter coli (n = 17) and C. jejuni (n = 35) isolates previously subjected to in vivo selective pressure was investigated to determine if the number of copies of 23S rRNA genes with macrolide-associated mutations affects the minimum inhibitory concentration (MIC) of macrolides. Sequence data for domain V of the 23S rRNA gene revealed that two macrolide-resistant C. coli isolates had adenine-->guanine transitions at position 2059 (A2059G, Escherichia coli numbering). One of the two isolates had the A2059G transition in only two of the three gene copies. Among the macrolide-resistant C. jejuni isolates (n = 9), two different point mutations within domain V were observed. Three macrolide-resistant C. jejuni isolates had A2059G transitions. One of these three C. jejuni isolates had the A2059G transition in only two of the three gene copies. Six macrolide-resistant C. jejuni isolates had an adenine-->cytosine transversion at position 2058 (A2058C, E. coli numbering) in all three copies of the 23S rRNA gene. Campylobacter jejuni isolates with the A2058C transversion had higher erythromycin MICs (>256 microg/mL) compared to C. jejuni isolates with A2059G transitions (64-128 microg/mL). In addition, the C. jejuni and C. coli isolates with only two copies of the 23S rRNA gene having A2059G substitutions had lower macrolide MICs compared to isolates with all three copies of the gene mutated. No isolates were observed having only one copy of the 23S rRNA gene with a mutation. Sequence analysis of ribosomal proteins L4 (rplD) and L22 (rplV) indicated that ribosomal protein modifications did not contribute to macrolide resistance among the collection of Campylobacter examined.

Antimicrobial drug resistance of fecal Escherichia coli and Salmonella spp. isolates from United States dairy cows.

Antimicrobial resistance is a growing concern for public and animal health. Threats to public health could come from the transfer of pathogens from animals to people via indirect contact such as through food or by direct contact with animals. In addition, concern has been raised for the potential transfer of resistance determinants from animals to humans through commensal bacterial flora such as Escherichia coli. Isolates of E. coli and Salmonella spp. from dairy cows on farms in 21 states were evaluated for resistance to a panel of 16 antimicrobial drugs. Resistance patterns for E. coli were compared to those of Salmonella spp. when they were isolated concurrently on the same farm or from the same fecal sample. Overall, most of the E. coli isolates (85.3%) and Salmonella spp. isolates (87.2%) were susceptible to all antimicrobials in the panel. The resistance profiles for E. coli with and without concurrent isolation of Salmonella were comparable with the exception of tetracycline resistance, which was more common among the E. coli isolated with Salmonella spp. The resistance patterns for E. coli and Salmonella spp. isolated concurrently were not significantly different for any of the antimicrobials evaluated. The data from this study demonstrate that the majority of commensal E. coli and Salmonella spp. recovered from feces of dairy cows harbored no resistance to a broad range of antimicrobial drugs. Further studies are indicated to better understand the factors that influence the frequency of resistance in commensal E. coli and Salmonella spp. on dairy operations.

Development of macrolide-resistant Campylobacter in broilers administered subtherapeutic or therapeutic concentrations of tylosin.

The use of antimicrobials in food animal production, particularly those commonly used to treat infections in humans, has become a source of debate in recent years. However, limited data are available regarding the development of resistance following the subtherapeutic or therapeutic administration of antimicrobials in animal production. The objective of this study was to evaluate the effect of the administration of therapeutic and subtherapeutic concentrations of tylosin on the erythromycin susceptibility of Campylobacter jejuni and Campylobacter coli isolated from the ceca of treated broilers. In three replicated studies, day-of-hatch chicks were exposed to macrolide-susceptible C. jejuni or C. coli. At 2 weeks of age, tylosin was administered at subtherapeutic (22 ppm, continuously in the diet) or therapeutic concentrations (529 ppm, in the drinking water for 5 days). Broilers were sacrificed weekly. Total and erythromycin-resistant Campylobacter spp. were enumerated from individual ceca plus cecal contents. Overall erythromycin resistance was observed at a higher frequency (P < 0.01) among C. coli isolates (70.8%) than among C. jejuni isolates (36.8%) following tylosin administration. Across Campylobacter species, erythromycin resistance was observed at a higher frequency (P < 0.001) when tylosin was administered at subtherapeutic (62.7%) than at therapeutic (11.4%) concentrations. Subtherapeutic administration resulted in the recovery of 83.3 and 56.1% erythromycin-resistant isolates compared with only 33.3 and 7.9% of the isolates expressing erythromycin resistance following the administration of therapeutic concentrations for C. coli and C. jejuni, respectively. Further studies are needed to determine the factors involved in the apparent difference in the acquisition of macrolide resistance in C. coli compared with C. jejuni.

Complete Genome Sequence of a Colistin Resistance Gene (mcr-1)-Bearing Isolate of Escherichia coli from the United States.

Transmissible colistin resistance conferred by the mcr-1 gene-bearing IncI2 plasmid has been recently reported in Escherichia coli in the United States. We report here the completed genome sequence of a second E. coli strain isolated from swine in the United States that carried the mcr-1 gene on an IncI2-type plasmid.

Colistin Resistance mcr-1-Gene-Bearing Escherichia coli Strain from the United States.

Transmissible colistin resistance in the form of an mcr-1-gene-bearing plasmid has been recently reported in Enterobacteriaceae in several parts of the world. We report here the completed genome sequence of an Escherichia coli strain isolated from swine in the United States that carried the mcr-1 gene on an IncI2-type plasmid.

Genetic relatedness of high-level aminoglycoside-resistant enterococci isolated from poultry carcasses.

Approximately 46% (75/162) or poultry enterococci collected between 1999 and 2000 exhibited high-level resistance to gentamicin (minimum inhibitory concentration [MIC] > or = 500 microg/ml), kanamycin (MIC > or = 500 microg/ml), or streptomycin (MIC > or = 1000 microg/ml). Forty-one percent of the isolates were resistant to kanamycin (n = 67), whereas 23% and 19% were resistant to genramicin (n = 37) and streptomycin (n = 31), respectively. The predominant species identified was Enterococcus faecium (n = 105), followed by Enterococcus faecalis (n = 40) and Enterococcus durans (n = 8). Using polymerase chain reaction, the isolates were examined for the presence of 10 aminoglycoside resistance genes [ant(6)-Ia, ant(9)-Ia, ant(4')-Ia, aph(3')-IIIa, aph(2")-Ib, aph(2")-Ic, aph(2")-Id, aac(6')-Ie-aph(2")-Ia, and aac(6')-Ii]. Five aminoglycoside resistance genes were detected, most frequently aac(6')-Ii and ant(6)-Ia from E. faecium. Seven E. faecalis isolates resistant to gentamicin, kanamycin, or streptomycin were negative for all genes tested, indicating that additional resistance genes may exist. Phylogenetic analysis revealed that the isolates were genetically different with little clonality. These data indicate that enterococci from poultry are diverse and contain potentially unidentified aminoglycoside resistance genes.

Antimicrobial susceptibility patterns of Salmonella isolates from cattle in feedlots.

OBJECTIVE: To evaluate the antimicrobial susceptibility patterns of Salmonella isolates from feedlot cattle. DESIGN: Cross-sectional study. SAMPLE POPULATION: 263 Salmonella isolates. PROCEDURES: Fecal samples were collected from the floor of 2 pens in each of 100 feedlots. Two hundred eighty Salmonella isolates were recovered after bacteriologic culture from 38 pens. Of these, 263 isolates were available for antimicrobial susceptibility testing to 16 antimicrobials, using microbroth dilution breakpoint plates. RESULTS: Less than 5% of isolates were resistant to any of the antimicrobials tested, with the exception of sulfamethoxazole (15; 5.7%) and tetracycline (61; 23.2%). Most isolates (197; 74.9%) were susceptible to all antimicrobials tested, whereas 18 (6.8%) were resistant to 2 or more antimicrobials. The percentage of isolates with resistance to any antimicrobial varied by serotype. The percentage of isolates resistant to various antimicrobials was not related to concurrent use of antimicrobials in the feed. CONCLUSIONS AND CLINICAL RELEVANCE: With the exception of tetracycline and sulfamethoxazole, resistance of Salmonella isolates to any of the antimicrobials was uncommon. Most isolates were susceptible to all antimicrobials tested. Antimicrobial resistance was not related to the presence of antimicrobials in the ration being fed at the time of sample collection.

Detection by hyperspectral imaging of shiga toxin-producing Escherichia coli serogroups O26, O45, O103, O111, O121, and O145 on rainbow agar.

The U.S. Department of Agriculture, Food Safety Inspection Service has determined that six non-O157 Shiga toxin-producing Escherichia coli (STEC) serogroups (O26, O45, O103, O111, O121, and O145) are adulterants in raw beef. Isolate and phenotypic discrimination of non-O157 STEC is problematic due to the lack of suitable agar media. The lack of distinct phenotypic color variation among non-O157serogroups cultured on chromogenic agar poses a challenge in selecting colonies for confirmation. In this study, visible and near-infrared hyperspectral imaging and chemometrics were used to detect and classify non-O157 STEC serogroups grown on Rainbow agar O157. The method was first developed by building spectral libraries for each serogroup obtained from ground-truth regions of interest representing the true identity of each pixel and thus each pure culture colony in the hyperspectral agar-plate image. The spectral library for the pure-culture non-O157 STEC consisted of 2,171 colonies, with spectra derived from 124,347 of pixels. The classification models for each serogroup were developed with a k nearest-neighbor classifier. The overall classification training accuracy at the colony level was 99%. The classifier was validated with ground beef enrichments artificially inoculated with 10, 50, and 100 CFU/ml STEC. The validation ground-truth regions of interest of the STEC target colonies consisted of 606 colonies, with 3,030 pixels of spectra. The overall classification accuracy was 98%. The average specificity of the method was 98% due to the low false-positive rate of 1.2%. The sensitivity ranged from 78 to 100% due to the false-negative rates of 22, 7, and 8% for O145, O45, and O26, respectively. This study showed the potential of visible and near-infrared hyperspectral imaging for detecting and classifying colonies of the six non-O157 STEC serogroups. The technique needs to be validated with bacterial cultures directly extracted from meat products and positive identification of colonies by using confirmatory tests such as latex agglutination tests or PCR.

Impact of heat stress on the fecal shedding patterns of Salmonella enterica Typhimurium DT104 and Salmonella enterica infantis by 5-week-old male broilers.

The objective of this study was to determine if there is an impact of heat stress of broiler chickens on number and survival of two types of Salmonella shed in the chicken's feces after an oral challenge. The data from this study indicate that heat stress did not result in higher levels or longer survival of Salmonella spp. shed in feces. It is possible that the duration or intensity of the heat stress employed was not sufficient or that heat stress does not alter the number or survivability for these particular strains of Salmonella spp. Feces stored at room temperature after collection, resulted in the numbers of both strains of Salmonella increasing by one to three logs in the first week. This finding indicates that there could be an increase in environmental contamination under certain conditions.

Effects of tylosin use on erythromycin resistance in enterococci isolated from swine.

The effect of tylosin on erythromycin-resistant enterococci was examined on three farms; farm A used tylosin for growth promotion, farm B used tylosin for treatment of disease, and farm C did not use tylosin for either growth promotion or disease treatment. A total of 1,187 enterococci were isolated from gestation, farrowing, suckling, nursery, and finishing swine from the farms. From a subset of those isolates (n = 662), 59% (124 out of 208), 28% (80 out of 281), and 2% (4 out of 170) were resistant to erythromycin (MIC >/= 8 microg/ml) from farms A, B, and C, respectively. PCR analysis and Southern blotting revealed that 95% (65 out of 68) of isolates chosen from all three farms for further study were positive for ermB, but all were negative for ermA and ermC. By using Southern blotting, ermB was localized to the chromosome in 56 of the isolates while 9 isolates from farms A and B contained ermB on two similar-sized plasmid bands (12 to 16 kb). Pulsed-field gel electrophoresis revealed that the isolates were genetically diverse and represented a heterogeneous population of enterococci. This study suggests that although there was resistance to a greater number of enterococcal isolates on a farm where tylosin was used as a growth promotant, resistant enterococci also existed on a farm where no antimicrobial agents were used.