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The present study was aimed at evaluating the effect of herbal extracts supplementation, particularly quaternary-benzo(c)phenanthridine alkaloids (QBA), which have been previously demonstrated to have anti-inflammatory, antimicrobial, and immune-modulator effects. We investigated the role of QBA on stress response and Salmonella shedding in finishing pigs transported to the slaughterhouse. A total of 82 pigs were orally challenged with a Salmonella cocktail (day 0) containing Salmonella Meleagridis, Hartford, Bovismorbificans and Newport serovars and randomly assigned to three treatment groups after 2 wks (day [D] 14): T1, in-feed QBA; T2, in-feed and water-soluble QBA; CON, nonsupplemented). Pigs were transported to the slaughterhouse 2 weeks after intervention (D 28) and slaughtered after nearly 19 h (D 29). Saliva, fecal samples, and carcass swabs were collected from all pigs. Salivary cortisol, Salmonella shedding, and carcass contamination were measured. A high positive correlation (Spearman rank correlation coefficient range 0.82-0.93) between salivary cortisol and Salmonella shedding was found after transportation in all groups (p < 0.05). Only the CON group showed an increase in salivary cortisol after transportation (5.48 ng/mL; p < 0.0001) to concentrations that were higher than in T1 (2.73 ng/mL; p = 0.0002) and T2 (1.88 ng/mL; p < 0.0001). Salmonella prevalence and shedding decreased after transportation in pigs receiving the QBA intervention (p < 0.05), whereas the control group showed a significant increase in Salmonella shedding after transportation (p = 0.04). At D 28, pigs in T2 shed lower numbers of Salmonella as compared to T1 (1.3E + 02 CFU/mL versus 8E + 03 CFU/mL; p = 0.002). Additionally, carcass contamination by Salmonella was higher in the CON group than the treated groups (p = 0.01). The findings show QBA intervention was effective in reducing transportation stress of pigs, resulting in reduced Salmonella shedding and positively impacting animal welfare and pork safety.

Nontyphoidal Salmonella infections caused by antimicrobial-resistant (AMR) strains are of great public health concern. We compared the phenotypic and genotypic relationships among temporally and spatially related AMR Salmonella isolates (n=1058) representing several predominant serovars, including Salmonella Typhimurium, Salmonella Typhimurium var. 5-, Salmonella Derby, Salmonella Heidelberg, Salmonella Muenchen, Salmonella Schwarzengrund, and Salmonella Rissen of human clinical cases (n=572), pig (n=212), and farm environment (n=274) origin in North Carolina. Antimicrobial susceptibility testing was performed using the broth microdilution method, and genotypic resistance determinants, including class I and II integrons, were identified. Overall, Salmonella isolates exhibited the highest frequency of resistance to tetracycline (50%), followed by sulfisoxazole (36%) and streptomycin (27%). We identified 16 different antimicrobial resistance genes, including extended spectrum and AmpC β-lactamases-producing genes (bla(TEM), bla(PSE), and bla(CMY-2)), in all the β-lactam- and cephalosporin-resistant Salmonella isolates from humans, pigs, and the environment. Class I integrons of 1-kb and 1.2-kb size were identified from all the three sources (humans, 66%; pigs, 85%; environment, 58%), while Class II integrons of 2-kb size were identified only in pig (10%) and environmental (19%) isolates. We detected genotypic similarity between Salmonella Typhimurium isolated from humans, pigs, and the environment while serovars Derby, Heidelberg, and Muenchen exhibited genotypic diversity. Detection of AMR Salmonella isolates from humans, pigs, and the environment is a concern for clinicians and veterinarians to mitigate the dissemination of AMR Salmonella strains.

Swine are the primary reservoir for foodborne illness associated with Yersinia enterocolitica. The use of antimicrobials in animal agriculture has been hypothesized as having a potential role in the increase in prevalence of zoonotic pathogens. The objective of this study was to compare the frequency of Y. enterocolitica fecal shedding in swine reared on farms with conventional antimicrobial use policies to farms that were antimicrobial free (ABF). Swine farms were selected from three regions in the United States. In each region, farms were categorized based on antimicrobial use policy. Fecal samples were collected from pigs on-farm within 48 h of harvest. The overall proportion of Y. enterocolitica and ail-harboring Y. enterocolitica-positive pigs was 10.9% and 4.0%, respectively. There were increased odds (odds ratio [OR] 6.8, 95% confidence interval [CI] 3.46-13.28) for a pig to be Y. enterocolitica positive if it was reared on an ABF farm as compared to a conventional farm. There was no significant association between farm antimicrobial use policy and isolation of an ail-harboring Y. enterocolitica from an individual pig (OR 1.8, 95% CI 0.90-3.61). The association of antimicrobial use policy with Y. enterocolitica shedding in feces should be interpreted cautiously, as antimicrobial use cannot be separated from other management factors (e.g., confinement or outdoor housing), which may be associated with risk of Y. enterocolitica in swine.

Although Staphylococcus aureus has been implicated as the main Staphylococcus species causing human food poisoning, recent studies have shown that coagulase-negative Staphylococcus could also harbor enterotoxin-encoding genes. Such organisms are often present in goat milk and are the most important mastitis-causing agents. Therefore, this study aimed to investigate the occurrence of enterotoxin-encoding genes among coagulase-positive (CoPS) and coagulase-negative (CoNS) staphylococci isolated from raw goat milk produced in the semi-arid region of Paraiba, the most important region for goat milk production in Brazil. Enterotoxin-encoding genes were screened in 74 staphylococci isolates (30 CoPS and 44 CoNS) by polymerase chain reaction targeting the genes sea, seb, sec, sed, see, seg, seh, and sei. Enterotoxin-encoding genes were found in nine (12.2%) isolates, and four different genes (sea, sec, seg, and sei) were identified amongst the isolates. The most frequent genes were seg and sei, which were often found simultaneously in 44.5% of the isolates. The gene sec was the most frequent among the classical genes, and sea was found only in one isolate. All CoPS isolates (n=7) harboring enterotoxigenic genes were identified as S. aureus. The two coagulase-negative isolates were S. haemolyticus and S. hominis subsp. hominis and they harbored sei and sec genes, respectively. A higher frequency of enterotoxin-encoding genes was observed amongst CoPS (23.3%) than CoNS (4.5%) isolates (p<0.05), reinforcing the importance of S. aureus as a potential foodborne agent. However, the potential risk posed by CoNS in goat milk should not be ignored because it has a higher occurrence in goat milk and enterotoxin-encoding genes were detected in some isolates.

Yersinia enterocolitica is an important foodborne pathogen, and pigs are recognized as a major reservoir and potential source of pathogenic strains to humans. A total of 172 Y. enterocolitica recovered from conventional and antimicrobial-free pig production systems from different geographic regions (North Carolina, Ohio, Michigan, Wisconsin, and Iowa) were investigated to determine their pathogenic significance to humans. Phenotypic and genotypic diversity of the isolates was assessed using antibiogram, serogrouping, and amplified fragment length polymorphism (AFLP). Carriage of chromosomal and plasmid-borne virulence genes were investigated using polymerase chain reaction. A total of 12 antimicrobial resistance patterns were identified. More than two-thirds (67.4%) of Y. enterocolitica were pan-susceptible, and 27.9% were resistant against β-lactams. The most predominant serogroup was O:3 (43%), followed by O:5 (25.6%) and O:9 (4.1%). Twenty-two of 172 (12.8%) isolates were found to carry Yersinia adhesion A (yadA), a virulence gene encoded on the Yersinia virulence plasmid. Sixty-nine (40.1%) isolates were found to carry ail gene. The ystA and ystB genes were detected in 77% and 26.2% of the strains, respectively. AFLP genotyping of isolates showed wide genotypic diversity and were grouped into nine clades with an overall genotypic similarity of 66.8-99.3%. AFLP analysis revealed that isolates from the same production system showed clonal relatedness, while more than one genotype of Y. enterocolitica circulates within a farm.

The objective of this study was to determine persistence of clonal strains from farm to retail by assessing the clonal relatedness of Campylobacter coli isolated on farm, peri-harvest, and at processing from 11 individually identified pigs. Phenotypic (antimicrobial susceptibility) and genotypic (pulsed field gel electrophoresis [PFGE] and multi-locus sequence typing [MLST]) characterization of isolates was conducted. There was high genetic diversity of Campylobacter isolates from on-farm fecal samples. Campylobacter isolates from farm, post-evisceration, hide, and carcass samples showed similar phenotypes and belonged to the same genotypic clusters based on PFGE and sequence types (STs) based on MLST. Five STs that have not been previously reported were identified (ST-4083, ST-4084, ST-4085, ST-4086, ST-4087). Despite high genotypic diversity of C. coli on farm, retail meat products were consistently contaminated with isolates of the same STs, particularly ST854 and ST1056, as isolates collected from previous stages confirming persistence of strains from pre- to post-harvest.

The objective of this study was to investigate the association between ceftiofur use policy in finishing swine barns and recovery of fecal Escherichia coli or Salmonella spp. resistant to ceftriaxone. The study population included 54 finishing swine barns from three companies located in North Carolina. The barns were each classified according to their reported therapeutic ceftiofur use rates of "Rare," "Moderate," and "Common." Fecal samples from the barns were cultured for the presence of E. coli and Salmonella spp. resistant to ceftriaxone using selective media designed to recover rare organisms expressing the AmpC β-lactamase phenotype. A total of 1899 swine fecal samples yielded 1193 E. coli (63%) resistant to ceftriaxone. Recovery rates by ceftiofur use classification were 45% for Rare, 73% for Moderate, and 68% Common ceftiofur use groups. Barns reporting Rare ceftiofur use had a lower odds of recovery of E. coli (OR=0.32; p<0.001) resistant to ceftriaxone compared to Common use barns. The overall Salmonella spp. prevalence was 63.8% (n=714). Of these, 65 Salmonella were resistant to ceftriaxone with the highest rate (6%) found in the Common ceftiofur use group, followed by Rare (4.1%) and Moderate (0.15%). The odds of recovery of Salmonella resistant to ceftriaxone were similar for barns with ceftiofur use classified as Rare and Common. Samples from barns with ceftiofur use classified as Moderate had a lower odds (OR=0.02; p<0.01) of recovery of Salmonella resistant to ceftriaxone than barns classified as Common. Our result is consistent with the hypothesis that the use of ceftiofur in finishing swine barns, beyond its rare application, may influence the recovery of enteric E. coli with resistance to cephalosporin drugs, although other unmeasured factors appear to be important in the recovery of cephalosporin-resistant Salmonella. The dissemination of enteric bacteria with resistance to cephalosporins has the potential to impact both veterinary and human therapeutic treatment options.

We conducted a study to compare the prevalence and antimicrobial resistance profile of Campylobacter isolated from 34 farm-slaughter pair cohorts of pigs raised in conventional and antimicrobial-free (ABF) production systems. Isolates originated from four different states of two geographic regions (region 1--Ohio and Michigan; region 2--Wisconsin and Iowa). A total of 838 fecal and 1173 carcass samples were examined. Campylobacter isolates were speciated using multiplex polymerase chain reaction targeting ceuE and hipO genes. The minimum inhibitory concentration was determined using agar dilution to a panel of six antimicrobials: chloramphenicol, erythromycin, gentamicin, ciprofloxacin, nalidixic acid, and tetracycline. Campylobacter spp. was isolated from 472 of 838 pigs (56.3%). Campylobacter prevalence did not vary significantly based on production system (conventional [58.9%] and ABF [53.7%], odds ratio [OR] 1.4, 95% confidence interval [CI] 0.8-2.6, p = 0.24) or geographic region (region 1 [54.1%] and region 2 [58.2%], OR 1.02, 95% CI 0.6-1.9, p = 0.92). At slaughter plant, Campylobacter prevalence varied based on processing stages (19.4% at pre-evisceration, 25.3% at postevisceration, and 3.2% at postchill). Resistance was common to tetracycline (64.5%), erythromycin (47.9%), and nalidixic acid (23.5%). Campylobacter isolates from conventional production systems were more likely to be erythromycin resistant than from ABF (OR 3.2, 95% CI 1.4-7.2, p = 0.01). The proportion of ciprofloxacin-resistant Campylobacter coli isolates were 3.7% and 1.2% from ABF and conventional production systems, respectively. Thirty-seven out of 1257 C. coli (2.9%) were resistant to both erythromycin and ciprofloxacin, drugs of choice for treatment of invasive human campylobacteriosis. The finding of ciprofloxacin resistance, particularly from ABF herds, has significant implications on the potential role of risk factors other than mere antimicrobial use for production purposes.

We estimated the proportion of retail beef and pork products containing bla(CMY)-mediated third-generation cephalosporin resistance in commensal Escherichia coli and Salmonella enterica. Samples were obtained from 50 grocery stores located in two U.S. states. From each store, 20 fresh meat products were purchased, including 7 packages of ground beef, 3 packages of beef steak, 6 packages of pork chops, and 4 packages of pork ribs. The resulting 1000 packages of fresh meat product were individually screened for the presence of E. coli or Salmonella harboring bla(CMY). Over 8% of all retail meat packages contained E. coli with bla(CMY), whereas 4% contained Salmonella and only 0.5% contained Salmonella with bla(CMY). Retail pork products more frequently yielded E. coli with bla(CMY) than did beef products (12.2% vs. 4.0%; p = 0.06). Salmonella were also recovered more frequently from pork than beef (5.8% vs. 2.4%; p < 0.01). In addition, all five Salmonella isolates with bla(CMY) were recovered from pork products. Our data suggest that enteric bacteria carrying bla(CMY) are frequently present in fresh retail meat products. However, we did not quantify the number of resistant bacteria present in these products, which makes the public health implications of this result unclear.

CTX-M extended-spectrum β-lactamases are enzymes produced by bacteria that are capable of inhibiting the antimicrobial effects of cephalosporin drugs. Recently, the first domestically acquired Salmonella in the United States expressing bla(CTX-M) was reported. This is a concern because expanded-spectrum cephalosporins are the treatment of choice for invasive Gram-negative infections, including salmonellosis in children. Because Salmonella transmission is primarily foodborne, there is also concern that resistant enteric bacteria from livestock can be transferred through the food supply chain to consumers. bla(CTX-M) has not been previously identified in bacterial isolates from food animal populations in the United States. We report the recovery of CTX-M-type extended-spectrum β-lactamases from fecal Escherichia coli of sick and healthy dairy cattle in Ohio. Four individual fecal samples yielded E. coli isolates representing three clonal strains that carried bla(CTX-M) on transferable plasmids. Two distinguishable plasmids were identified, each encoding bla(CTX-M-1) or bla(CTX-M-79). Transferrable bla(CTX-M) genes in bovine E. coli have the potential to serve as a reservoir of resistance for pathogens and may represent a public health concern.

The objective of this cross-sectional study was to compare the prevalence of Salmonella and antimicrobial-resistant Salmonella, as well as investigate the distribution of this pathogen in organic and conventional broiler poultry farms. Fecal (n = 420), feed (n = 140), and drinking water (n = 140) samples were collected from birds at 3 and 8 weeks of age for 2-flock cycles. One house was sampled per farm at three organic and four conventional broiler farms from the same company in North Carolina. All samples were analyzed for the presence of Salmonella using selective enrichment techniques. Further phenotypic (antimicrobial susceptibility) and genotypic (pulsed-field gel electrophoresis [PFGE]) testing were performed. Salmonella prevalences in fecal samples were 5.6% (10/180) and 38.8% (93/240) from organic and conventional farms, respectively. From feed, 5.0% (3/60) and 27.5% (22/80) of the samples were positive for Salmonella from organic and conventional farms, respectively. None of the water samples were positive for Salmonella. Seventy isolates were characterized by antimicrobial susceptibility and PFGE types. The two most common resistance phenotypes were single resistance to streptomycin (36.2% [25/58]: conventional; 25% [3/12] organic), and multidrug resistance to six antimicrobial agents: ampicillin-streptomycin-amoxicillin/clavulanic acid-cephalothin-ceftiofur-cefoxitin (AmStAxChCfFx; 39.7%: conventional only). Genotypic analysis using PFGE showed clonality among isolates within and between the two types of farms. The results of our study suggest that within this poultry company, the prevalence of fecal Salmonella was lower in certified-organic birds than in conventionally raised birds, and the prevalence of antimicrobial-resistant Salmonella was also higher in conventionally raised birds than in certified-organic birds.

Salmonella enterica is an important foodborne pathogen, and contamination of surface and ground water that may result from various human activities, such as animal production and urbanization, may contribute to the public health burden. The aims of this study was to determine the sources of Salmonella contamination in four different types of watersheds and to assess the relative contribution of multidrug-resistant strains. Eighty-six water samples collected from four different watershed systems, including those impacted by swine production (n = 12), residential/industrial (n = 34), crop agriculture (n = 12), and forestry (n = 28), were cultured for Salmonella and further characterized by serotyping, antimicrobial susceptibility testing, and pulsed-field gel electrophoresis genotyping. Salmonella prevalence was high in all four watersheds: residential/industrial area (58.8%), forestry (57.1%), crop agriculture (50%), and swine production (41.7%). Majority of the Salmonella isolates (87.1%) were pansusceptible. Multidrug resistance up to eight antimicrobials (R-type: AmStTeAxChCeKmGm) was detected in water samples that originated from swine production systems only. Serovars identified included Anatum, Gaminara, and Inverness (18.3% each) and Muenchen and Newport (8.7% each), Bredeny (7.6%), and Montevideo (6.8%). Pulsed-field gel electrophoresis analysis indicated genotypic relatedness among Salmonella recovered from residential/industrial and forestry-associated watersheds (genotypic cluster types A, C, D, E, F, G, H, and J), sites with relatively close geographic proximity. Swine-production-associated isolates were distinctly different from the others (genotypic cluster types B and I), corroborating the phenotypic findings. Overall, the findings suggest that all the various watersheds, including natural forest, remain important contributors of Salmonella contamination. While swine-production-associated water samples were not found to have a disproportionately high prevalence, it was the most important reservoir of multidrug-resistant strains.

A total of 360 spatially and temporally related Campylobacter isolates, including 168 from clinical human cases (Campylobacter jejuni n = 148; Campylobacter coli n = 20) and 192 from retail meats (C. jejuni n = 114; C. coli n = 78), were analyzed for antimicrobial susceptibilities, virulence, and genotypic profiles. Ciprofloxacin-resistant C. jejuni was observed in 13.5% and 19% of the isolates from humans and retail chicken breasts, respectively. Antimicrobial resistance to ciprofloxacin and erythromycin was detected in C. coli isolates recovered from 29% and 16.6% of retail meats and 15% and 5% humans, respectively. Overall, virulence determinants were more prevalent in Campylobacter isolates recovered from retail meats than from humans. C. jejuni isolates from humans were significantly associated with the rakR, dnaJ, and pld genes, whereas C. coli isolates from retail meats were associated with the dnaJ, pld, and virB11 virulence genes. Genotyping of 262 C. jejuni isolates using pulsed-field gel electrophoresis revealed a total of 186 unique SmaI patterns, with 14% of patterns composed of isolates recovered from retail meats and ill humans. All unique groups with indistinguishable SmaI patterns were further analyzed by a second restriction enzyme (KpnI), which revealed limited overlap between isolates from different sources. Significant association between doxycycline-resistant C. jejuni strains recovered from humans and different virulence genes (e.g., cdtB) was identified at the statistical level but not at the genotypic level. In conclusion, significant differences observed in the distribution of antimicrobial resistance profiles, virulence determinants, and genotypic diversity among C. jejuni and C. coli isolates indicate that there are sources other than retail meats that may also contribute to human Campylobacter infections.

The bla(CMY-2) family of the ampC beta-lactamase genes confer broad-spectrum resistance to beta-lactam antimicrobials, including ceftriaxone and ceftiofur, as well as to beta-lactamase inhibitors, such as clavulanic acid. Organisms with the bla(CMY-2) phenotype have been recovered from the environment and from retail meat products, posing a potential public health risk. The objectives of this study were to sequence the bla(CMY-2) gene from Escherichia coli and Salmonella enterica from multiple sources that had a reduced susceptibility to ceftriaxone and to determine the effect of observed mutations in the bla(CMY-2) gene on the antimicrobial resistance phenotype (spectrum and minimum inhibitory concentration/susceptibility patterns) of the isolates. The bla(CMY-2) genes from 52 bacterial isolates were sequenced for this study. Sixty-two percent (32/52) were E. coli and 38% (20/52) were S. enterica. Of the 32 E. coli isolates, 30 were found to carry a beta-lactamase gene that was 100% homologous to bla(CMY-2). One of the E. coli isolates was found to contain a gene that was 90% homologous to bla(CMY-2). This isolate also had lower minimum inhibitory concentrations to tetracyclines, streptomycin, and the sulfonamide antimicrobials than are commonly expected for isolates containing the bla(CMY-2). Of the 20 genes obtained from Salmonella isolates, 8 (40%) were found to be homologous to bla(CMY-2), with no altered susceptibility phenotypes observed.

Salmonella enterica serovar Heidelberg has been recognized as one of the most common serovar associated with foodborne infections in the United States. It is also frequently isolated from nonhuman sources and has increasingly shown resistance to various antimicrobial agents. The present study was undertaken to identify the predominant antimicrobial resistance phenotypes and genotypes of Salmonella Heidelberg (n = 95) isolates of human, swine, and turkey origin. Antimicrobial susceptibility was done using Kirby-Bauer disk diffusion method with a panel of 12 antimicrobials. Pulsed-field gel electrophoresis genotyping was used to determine the diversity of the isolates. The antimicrobial resistance genes and carriage of Class 1 and 2 integrons were determined by polymerase chain reaction. All Salmonella Heidelberg isolates from swine were resistant to one or more of the antimicrobials tested and the majority (73.3%) showed multidrug resistance to streptomycin, tetracycline, and kanamycin (R-type: StTeKm). About 80% of the Salmonella Heidelberg isolates of human origin were pan-susceptible, however, one isolate showed multidrug resistance to 10 of 12 antimicrobials tested. Among the multidrug-resistant (MDR) Salmonella Heidelberg isolates, Class 1 integrons with variable sizes of 1.2 to 1.5 kb were detected in six isolates (three each) from humans and swine. DNA sequencing revealed that Class 1 integrons of both human and swine origin carried a gene encoding aminoglycoside adenyltransferase (aadA). Resistance genes identified in other loci include aphA1-Iab, strA, bla(TEM), and tetA (B). Both human and swine MDR strains of Salmonella Heidelberg carried the resistance phenotypes on self-transferable plasmids. Dendrogram analysis of pulsotypes indicated possible clonality of Salmonella Heidelberg between isolates of human and swine origin. The findings in this study indicate the increasing significance of swine as reservoirs of emerging MDR serovars, such as MDR Salmonella Heidelberg, is of public health significance.

There has been a growing niche-market, outdoor, antimicrobial-free (ABF) swine production system in the last few years prompted by consumers' demand for a more "natural" pork product. The impact of such production systems on reemergence of current and historically significant swine-associated pathogens has not been determined. The objectives of the current study were to determine and compare Salmonella, Toxoplasma, and Trichinella seropositivity in two swine production systems: outdoor ABF and intensive indoor production systems. These three foodborne pathogens represent those with the highest importance for pork consumption. A total of 675 serum samples from three participating states, Wisconsin, North Carolina, and Ohio, were investigated. We found significantly higher seroprevalence of Salmonella and Toxoplasma from ABF herds (54% and 7%, respectively) than conventional (39% and 1%, respectively) (p = 0.001). Two pigs, both from ABF herds, were found to be seropositive for Trichinella. The results from this preliminary study suggest that all three pathogens were more commonly present in pigs that were reared in an ABF, outdoor, niche-market type of environment than the conventional, indoor-reared herds though there were some geographical variation in Salmonella. This warrants a robust epidemiologic study to determine the role of various risk factors in the two production systems that may lead to persistence of bacterial (Salmonella) pathogens and reemergence of parasites (such as Trichinella) of historical significance.