Prevalence of Antimicrobial Resistant and Extended-Spectrum Beta-Lactamase-producing Escherichia coli in Dairy Cattle Farms in East Tennessee.

Antimicrobials have been widely used in dairy farms to prevent and control dairy cattle diseases since 1960s. This led to the emergence of antimicrobial resistant bacteria (ARB) that, along with their antimicrobial resistance genes (ARGs), can spread from dairy farms to humans. Therefore, regular antimicrobial resistance (AMR) monitoring is important to implement proper mitigation measures. The objective of this study was to determine the prevalence of AMR and extended-spectrum beta-lactamases (ESBLs)-producing Escherichia coli in dairy cattle. A cross-sectional study was conducted in four dairy cattle farms (A-D) in East Tennessee. A total of 80 samples consisting of 20 samples each of bulk tank milk, feces, dairy cattle manure-amended soil, and prairie soil adjacent to the farms were collected and cultured for the isolation of E. coli. Tetracycline (TETr)-, third-generation cephalosporin (TGCr)- and nalidixic acid (NALr)-resistant E. coli (n = 88) were isolated and identified on agar media supplemented with TET, cefotaxime, and NAL, respectively. TGCr E. coli were tested for ESBLs and other coselected ARGs. TETr (74%, n = 88) was the most common, followed by TGCr (20%) and NALr (8%). Farms had significant (p < 0.001) differences: the highest prevalence of TGCr (55%) and TETr (100%) were observed in farm D, while all NALr isolates were from farm C. Over 83% of TGCr isolates (n = 18) harbored ESBL gene blaCTX-M. Majority (78%) of the E. coli isolates were multidrug-resistant (MDR), being positive for beta-lactams (blaCTX-M), TETs tet(A), tet(B), tet(M)), sulfonamides (sul2), aminoglycosides (strA), and phenicols (floR). This study indicated the widespread occurrence of MDR ESBLs-E. coli in dairy cattle farms. AMR surveillance of more dairy farms and identification of farm-level risk factors are important to mitigate the occurrence and spread of ARB of significant public health importance, such as ESBLs-E. coli.

Efficacy of novel staphylococcal surface associated protein vaccines against Staphylococcus aureus and non-aureus staphylococcal mastitis in dairy cows.

Mastitis is an inflammation of the mammary gland commonly caused by bacteria or fungi. Staphylococcus aureus is a major bacterium that causes mastitis in dairy cows. Non-aureus staphylococci are also increasingly reported, with Staphylococcus chromogenes being the most common species. Current staphylococcal mastitis control programs are not fully effective, and treatment with antibiotics is not sustainable. Non-antibiotic sustainable control tools, such as effective vaccines, are critically needed. We previously developed S. aureus surface-associated proteins (SASP) and S. chromogenes surface-associated proteins (SCSP) vaccines that conferred partial protective effects. We hypothesized that vaccination with SASP or SCSP would reduce the incidence of S. aureus mastitis throughout the lactation period. The objective of this study was to evaluate the efficacy of SASP and SCSP vaccines against S. aureus and non-aureus staphylococcal mastitis under natural exposure over 300 days of lactation. Pregnant Holstein dairy cows (n = 45) were enrolled and assigned to receive SASP (n = 15) or SCSP (n = 16) vaccines or unvaccinated control (n = 14). Cows were vaccinated with 1.2 mg of SASP or SCSP with Emulsigen-D adjuvant. Control cows were injected with phosphate-buffered saline with Emulsigen-D adjuvant. Three vaccine injections were given subcutaneously at 60, 40, and 20 days before the expected calving. Booster vaccinations were given at 120 and 240 days in milk. Cows were monitored for mastitis at quarter and cow levels, staphylococcal mastitis incidence, changes in serum and milk anti-SASP and anti-SCSP antibody titers, bacterial counts in milk, adverse reactions, milk yield and milk somatic cells count over 300 days of lactation. The SCSP vaccine conferred a significant reduction in the incidence of staphylococcal mastitis. Milk and serum anti-SASP and anti-SCSP antibody titers were increased in the vaccinated cows compared to unvaccinated control cows. Anti-SASP and anti-SCSP antibody titers decreased at about 120 days in milk, indicating the duration of immunity of about four months. In conclusion, the SASP and SCSP vaccines conferred partial protection from natural infection.

Antimicrobial Resistance of Major Bacterial Pathogens from Dairy Cows with High Somatic Cell Count and Clinical Mastitis.

Mastitis is the most prevalent and economically important disease caused by different etiological agents, which leads to increased somatic cell count (SCC) and low milk quality. Treating mastitis cases with antimicrobials is essential to reduce SCC and improve milk quality. Non-prudent use of antimicrobials in dairy farms increased the development of antimicrobial resistant bacteria. This study's objectives were (1) to isolate and identify etiological agents of mastitis and (2) to determine antimicrobial resistance profiles of bacterial isolates. A total of 174 quarter milk samples from 151 cows with high SCC and clinical mastitis from 34 dairy farms in Tennessee, Kentucky, and Mississippi were collected. Bacterial causative agents were determined by bacteriological and biochemical tests. The antimicrobial resistance of bacterial isolates against 10 commonly used antimicrobials was tested. A total of 193 bacteria consisting of six bacterial species, which include Staphylococcus aureus, Streptococcus uberis, Streptococcus dysgalactiae, Escherichia coli, Klebsiella oxytoca and Klebsiella pneumoniae were isolated. Staphylococcus aureus was the predominant isolate followed by Strep. spp., E. coli, and Klebsiella spp. Results of this study showed that Gram-negatives (E. coli and Klebsiella spp.) were more resistant than Gram-positives (Staph. aureus and Streptococcus spp.). Continuous antimicrobial resistance testing and identification of reservoirs of resistance traits in dairy farms are essential to implement proper mitigation measures.

Experimental Staphylococcus aureus Mastitis Infection Model by Teat Dipping in Bacterial Culture Suspension in Dairy Cows.

Mastitis is inflammation of mammary glands usually caused by bacteria such as Staphylococcus aureus. Dairy cows are susceptible to mastitis during early dry and transition periods. Effective vaccine is needed during these periods. One of the limitations to develop an effective vaccine against S. aureus is the absence of good infection model. Intramammary infusion (IMIF) with S. aureus has been used as an infection model to test vaccine efficacy. IMIF is reliable in causing mastitis, but it bypasses physical barriers, non-specific natural defenses, and immunity in the teat canal. IMIF also transfers a large number of bacteria into the intramammary area at once. The objective of this study was to develop S. aureus IMIF model that mimics natural infection. Eight Holstein dairy cows were randomly divided into two groups of experimental (n = 5) and control (n = 3) cows. All teats of experimental cows were dipped in S. aureus culture suspension, whereas that of control cows were dipped in phosphate-buffered saline. Results showed that four of five cows were infected with challenge strain by day 3 of the challenge. The remaining cow was infected with Staphylococcus chromogenes. In conclusion, an experimental S. aureus intramammary infection can be induced by teat dipping into bacterial suspension.

Comparison of clonal relatedness and antimicrobial susceptibility of fecal Escherichia coli from healthy dogs and their owners.

OBJECTIVE: To determine prevalence of within-household sharing of fecal Escherichia coli between dogs and their owners on the basis of pulsed-field gel electrophoresis (PFGE), compare antimicrobial susceptibility between isolates from dogs and their owners, and evaluate epidemiologic features of cross-species sharing by use of a questionnaire. SAMPLE POPULATION: 61 healthy dog-owner pairs and 30 healthy control humans. PROCEDURES: 3 fecal E coli colonies were isolated from each participant; PFGE profiles were used to establish relatedness among bacterial isolates. Susceptibility to 17 antimicrobials was determined via disk diffusion. A questionnaire was used to evaluate signalment, previous antimicrobial therapy, hygiene, and relationship with dog. RESULTS: A wide array of PFGE profiles was observed in E coli isolates from all participants. Within-household sharing occurred with 9.8% prevalence, and across-household sharing occurred with 0.3% prevalence. No behaviors were associated with increased clonal sharing between dog and owner. No differences were found in susceptibility results between dog-owner pairs. Control isolates were more likely than canine isolates to be resistant to ampicillin and trimethoprim-sulfamethoxazole. Owners and control humans carried more multdrug-resistant E coli than did dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Within-household sharing of E coli was detected more commonly than across-household sharing, but both direct contact and environmental reservoirs may be routes of cross-species sharing of bacteria and genes for resistance. Cross-species bacterial sharing is a potential public health concern, and good hygiene is recommended.

Prevalence of urovirulence genes cnf, hlyD, sfa/foc, and papGIII in fecal Escherichia coli from healthy dogs and their owners.

OBJECTIVE: To determine the prevalence of 4 urovirulence genes in fecal Escherichia coli isolates from healthy dogs and their owners and to determine whether detection of E coli strains with these genes was associated with a history of urinary tract infection (UTI). SAMPLE POPULATION: 61 healthy dog-owner pairs and 30 healthy non-dog owners. PROCEDURES: A fecal specimen was obtained from each participant, and 3 colonies of E coli were isolated from each specimen. A multiplex PCR assay was used to detect 4 genes encoding virulence factors: cytotoxic necrotizing factor (cnf), hemolysin (hlyD), s-fimbrial and F1C fimbriae adhesin (sfa/foc), and pilus associated with pyelonephritis G allele III (papGIII). Human participants completed a questionnaire to provide general information and any history of UTI for themselves and, when applicable, their dog. RESULTS: 26% (16/61) of dogs, 18% (11/61) of owners, and 20% (6/30) of non-dog owners had positive test results for >or= 1 E coli virulence gene. One or more genes were identified in fecal E coli isolates of both dog and owner in 2% (1/61) of households. There was no difference in the detection of any virulence factor between dog-owner pairs. Female owner history of UTI was associated with detection of each virulence factor in E coli strains isolated from their dogs' feces. CONCLUSIONS AND CLINICAL RELEVANCE: Dogs and humans harbored fecal E coli strains possessing the genes cnf, hlyD, sfa/foc, and papGIII that encode urovirulence factors. It was rare for both dog and owner to have fecal E coli strains with these virulence genes.

Comparison of Staphylococcus aureus surface protein extraction methods and immunogenicity.

Staphylococcus aureus is the major contagious bovine mastitis pathogen and has no effective vaccine. Strain variation and limited knowledge of common immunogenic antigen/s are among major constraints for developing effective vaccines. S. aureus cell surface proteins that are exposed to the host immune system constitute good vaccine candidates. The objective of this study was to compare two novel S. aureus surface protein extraction methods with biotinylation method and evaluate immune-reactivity of extracted proteins. Surface proteins were extracted from nine genetically distinct S. aureus strains from cases of bovine mastitis. After extraction, bacterial cell integrity was examined by Gram staining and electron microscopy to determine if extraction methods caused damage to cells that may release non-surface proteins. The extracted proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and evaluated for immune-reactivity using western blot. Results showed that all three extraction methods provided multiple protein bands on SDS-PAGE. Western blot result showed several immunoreactive surface proteins, in which some proteins strongly (well-resolved, thick, dark, and intense band) reacted across the nine strains tested. The three methods are valid for the extraction of surface proteins and hexadecane, and cholic acid methods are more feasible than biotinylation since both are easier, cheaper, and have minor effects on the bacterial cell. Strongly immune-reactive surface proteins may serve as potential candidates for a vaccine to control S. aureus mastitis in dairy cows.

Effect of omega-3 polyunsaturated fatty acid (n-3 PUFA) supplementation to lactating sows on growth and indicators of stress in the postweaned pig1,2.

Dietary omega-3 polyunsaturated fatty acids (n-3 PUFA) are precursors for lipid metabolites that reduce inflammation. Two experiments were conducted to test the hypothesis that enriching the sow diet in n-3 PUFA during late gestation and throughout lactation reduces stress and inflammation and promotes growth in weaned pigs. A protected fish oil product (PFO; Gromega) was used to enrich the diet in n-3 PUFA. In the initial experiment, time-bred gilts were fed a gestation and lactation diet supplemented with 0% (control; n = 5), 0.25% (n = 4), 0.5% (n = 4), or 1% (n = 5) PFO from 101 ± 2 d of gestation to day 16 of lactation. Adding 1% PFO to the diet increased the n-3:n-6 PUFA ratio in colostrum and milk compared with controls (P = 0.05). A subsequent experiment was performed to determine whether supplementing the sow diet with 1% PFO improved growth and reduced circulating markers of acute inflammation and stress in the offspring. Plasma was harvested from piglets (16 per treatment group) on day 0 (d of weaning) and days 1 and 3 postweaning. Pigs from the 1% PFO treatment group weighed more (P = 0.03) on day 3 postweaning and had a greater (P ˂ 0.05) n-3:n-6 PUFA ratio in plasma on each day sampled compared with 0% PFO controls. There was an overall treatment effect on plasma total cortisol (P = 0.03) and haptoglobin (P = 0.04), with lesser concentrations in pigs on the 1% PFO diet. Plasma corticosteroid-binding globulin (CBG) concentrations were not different between treatment groups but were less (P ˂ 0.001) on days 1 and 3 when compared with day 0. The resultant free cortisol index [FCI (cortisol/CBG)] was less (P = 0.02) on days 1 and 3 for pigs from the 1% treatment group compared with the controls. An ex vivo lipopolysaccharide (LPS) challenge of whole blood collected on days 0 and 1 was used to determine whether 1% PFO attenuated release of inflammatory cytokines (IL-1ß, IL-6, and TNF-α). Blood from pigs within the 1% PFO treatment group tended (P = 0.098) to have a lesser mean concentration of TNF-α in response to LPS compared with blood from controls. These results suggest that providing a PFO supplement as 1% of the diet to sows beginning in late gestation and during lactation can increase the n-3:n-6 PUFA ratio in their offspring, which may improve growth and reduce the acute physiological stress response in the pigs postweaning.

Characterization of antimicrobial resistance patterns and class 1 integrons in Escherichia coli O26 isolated from humans and animals.

Antimicrobial resistance patterns and the prevalence of antimicrobial resistance genes and class 1 integrons in 35 Escherichia coli O26 isolated from humans and food-producing animals were evaluated. All isolates were resistant to cefaclor, cefalothin and sulfonamide and were susceptible to amikacin, gentamicin, cefmetazole, cefotaxime, ceftriaxone, ciprofloxacin, norfloxacin and trimethoprim. Most isolates were resistant to aztreonam, ampicillin, tetracycline, streptomycin and kanamycin. All ampicillin- and streptomycin-resistant E. coli O26 carried ampC and strA-strB gene sequences, respectively. Florfenicol- and chloramphenicol-resistant isolates carried floR but not cmlA. Class1 integrons were identified in 14% of E. coli O26 isolates. To our knowledge, this is the first report describing the presence of multiple antimicrobial resistance genes in E. coli O26 isolated from human and animal origins.

Phenotypic and genotypic antimicrobial resistance patterns of Escherichia coli isolated from dairy cows with mastitis.

Pulsed field gel electrophoresis (PFGE) patterns, susceptibility to 26 antimicrobial agents used in veterinary and human medicine, and prevalence of antimicrobial resistance genes of Escherichia coli isolated from cows with mastitis were evaluated. Among 135 E. coli isolates, PFGE analysis revealed 85 different genetic patterns. All E. coli were resistant to two or more antimicrobials in different combinations. Most E. coli were resistant to antimicrobials used in veterinary medicine including ampicillin (98.4%, >or=32 microg/ml) and many E. coli were resistant to streptomycin (40.3%, >or=64 microg/ml), sulfisoxazole (34.1%, >or=512 microg/ml), and tetracycline (24.8%, >or=16 microg/ml). Most E. coli were resistant to antimicrobials used in human medicine including aztreonam (97.7%, >or=32 microg/ml) and cefaclor (89.9%, >or=32 microg/ml). Some E. coli were resistant to nitrofurantoin (38%, >or=128 microg/ml), cefuroxime (22.5%, >or=32 microg/ml), fosfomycin (17.8%, >or=256 microg/ml). All E. coli were susceptible to ciprofloxacin and cinoxacin. Almost 97% (123 of 127) of ampicillin-resistant isolates carried ampC. Eleven of 52 (21.2%) streptomycin-resistant isolates carried strA, strB and aadA together and 29 streptomycin-resistant isolates (55.8%) carried aadA alone. Among 44 sulfisoxazole-resistant E. coli, 1 isolate (2.3%) carried both sulI and sulII, 12 (27.3%) carried sulI and 10 (22.7%) isolates carried sulII. Among 32 tetracycline-resistant isolates, 14 (43.8%) carried both tetA and tetC and 14 (43.8%) carried tetC. Results of this study demonstrated that E. coli from cows with mastitis were genotypically different, multidrug resistant and carried multiple resistance genes. These bacteria can be a reservoir for antimicrobial resistance genes and can play a role in the dissemination of antimicrobial resistance genes to other pathogenic and commensal bacteria in the dairy farm environment.

Application of SYBR green real-time PCR assay for specific detection of Salmonella spp. in dairy farm environmental samples.

The objective of this study was to develop and evaluate a SYBR Green 1 real-time PCR method for the specific detection of Salmonella spp. in dairy farm environmental samples. Previously reported 119-bp invA gene was selected for specificity, and 124 Salmonella spp. including type strains and 116 non-Salmonella strains were evaluated. All Salmonella strains tested were invA-positive and all non-salmonella strains yielded no amplification products. The melting temperature (Tm=79 degrees C) was consistently specific for the amplicon. Correlation coefficients of standard curves constructed using the threshold cycle (C(T)) versus copy numbers of Salmonella Enteritidis showed good linearity in broth (R2=0.994; slope=3.256) and sterilized milk (R2=0.988; slope=3.247), and the minimum levels of detection were >10(2) and >10(3) colony forming units (CFU)/ml, respectively. To validate the real-time PCR assay, an experiment was conducted with both spiked and naturally contaminated samples. Lagoon water, feed/silage, bedding soil, and bulk tank milk samples obtained from dairy farms were spiked with 10(0) to 10(5) CFU/ml of Salmonella Enteritidis. Sensitivities for detecting Salmonella in these sources were 10(3) to 10(4) CFU/ml of inoculums in broth without enrichment. Detection limits were reduced to <10 CFU/ml of inoculum in broth after 18 h enrichment. Ninety-three environmental samples including fecal slurry, feed/silage, lagoon water, drinking water, bulk tank milk, farm soil, and bedding soil were analyzed for the presence of Salmonella by real-time PCR, results were compared with those obtained by conventional culture methods. All samples analyzed were negative for Salmonella by both real-time PCR and standard culture method. No false positive or false negative results were detected.

Efficacy of extended pirlimycin therapy for treatment of experimentally induced Streptococcus uberis intramammary infections in lactating dairy cattle.

Streptococcus uberis is an important cause of mastitis in dairy cows throughout the world, particularly during the dry period, around the time of calving, and during early lactation. Strategies for controlling S. uberis mastitis have not received adequate research attention and are therefore poorly defined and inadequate. Objectives of the present study were to evaluate the efficacy of extended therapy regimens with pirlimycin for treatment of experimentally induced S. uberis intramammary infections in lactating dairy cows during early lactation and to evaluate the usefulness of the S. uberis experimental infection model for evaluating antimicrobial efficacy in dairy cows. The efficacy of extended pirlimycin intramammary therapy regimens was investigated in 103 mammary glands of 68 dairy cows that became infected following experimental challenge with S. uberis during early lactation. Cows infected with S. uberis in one or both experimentally challenged mammary glands were randomly allocated to three groups, representing three different treatment regimens with pirlimycin, including 2-day (n = 21 cows, 31 mammary quarters), 5-day (n = 21 cows, 32 quarters), and 8-day (n = 26 cows, 40 quarters). For all groups, pirlimycin was administered at a rate of 50 mg of pirlimycin hydrochloride via intramammary infusion. A cure was defined as an experimentally infected mammary gland that was treated with pirlimycin and was bacteriologically negative for the presence of S. uberis at 7, 14, 21, and 28 days after treatment. Experimental S. uberis intramammary infections were eliminated in 58.1% of the infected quarters treated with the pirlimycin 2-day regimen, 68.8% for the 5-day regimen, and 80.0% for the 8-day regimen. Significant differences (P <.05) in efficacy were observed between the 2-day and 8-day treatment regimens. The number of somatic cells in milk decreased significantly following therapy in quarters for which treatment was successful in eliminating S. uberis. However, there was no evidence to suggest that extended therapy with pirlimycin resulted in a greater reduction in somatic cell counts in milk than the 2-day treatment. The S. uberis experimental infection model was a rapid and effective means of evaluating antimicrobial efficacy during early lactation at a time when mammary glands are highly susceptible to S. uberis intramammary infection.

Efficacy of extended pirlimycin hydrochloride therapy for treatment of environmental Streptococcus spp and Staphylococcus aureus intramammary infections in lactating dairy cows.

Fifty-one chronically infected lactating dairy cows were used to evaluate the efficacy of extended pirlimycin therapy regimens for treatment of intramammary infections by environmental Streptococcus spp and Staphylococcus aureus. Cows (n = 47) with one or more infected mammary quarters were blocked by parity and randomly allocated to one of three groups for treatment with pirlimycin (50 mg/mammary quarter) as follows: one treatment per day for 2 days (n = 36 infected mammary quarters); one treatment per day for 5 days (n = 36 infected mammary quarters); and one treatment per day for 8 days (n = 20 infected mammary quarters). Four cows with nine infected mammary quarters were included as untreated controls. Milk samples from each mammary quarter were collected 7 days before treatment, immediately before treatment, and weekly for 4 weeks after the final treatment for microbiological evaluation. A bacteriologic cure was defined as a treated, infected quarter that was bacteriologically negative for the presence of previously identified bacteria at weekly intervals after treatment. Efficacy of pirlimycin therapy against intramammary infections caused by environmental Streptococcus spp and S. aureus was 44.4%, 61.1%, and 95.0% for the 2-, 5-, and 8-day treatment regimens, respectively. None of the infections in the untreated control quarters was cured. Significant differences in efficacy were detected between all pirlimycin groups and the untreated control group, between the 8- and 2-day treatment regimens, and between the 8-day and 5-day treatment regimens (P < or = .05). Results of this study indicate that extended pirlimycin therapy was effective in eliminating intramammary infections caused by environmental streptococci and S. aureus in lactating dairy cows.

Intramammary infections in heifers during early lactation following intramammary infusion of pirlimycin hydrochloride or penicillin-novobiocin at the first milking after parturition.

A study was conducted to determine whether intramammary antibiotic treatment of heifer mammary glands following the first milking after calving was effective for reducing the percentage of mammary quarters infected during early lactation. Jersey and Holstein heifers from two research herds were assigned to one of three treatment groups: (1) no intramammary infusion following the first milking after parturition, (2) intramammary infusion of all quarters with pirlimycin hydrochloride following the first milking after parturition and (3) intramammary infusion of all quarters with novobiocin sodium plus penicillin G procaine following the first milking after parturition. Almost 93% of Jersey heifers (40/43) and 73.1% of quarters (125/171) were infected at the first milking. Almost 77% of quarters (33/43) were cured following treatment with pirlimycin, 61.8% (21/34) were cured following treatment with penicillin-novobiocin and 39.6% (19/48) of infections were eliminated spontaneously in the untreated control group. Significantly fewer infections were observed in pirlimycin or penicillin-novobiocin treated mammary glands of Jersey heifers during early lactation than in untreated control mammary glands. Almost 89% of Holstein heifers (32/36) and 52.8% of quarters (76/144) were infected at the first milking. About 57% (12/21) of quarters were cured following treatment with pirlimycin, 41.4% (12/29) were cured following treatment with penicillin-novobiocin and 23.1% (6/26) of infections were eliminated spontaneously in the untreated negative control group. Significantly fewer infections were observed in pirlimycin treated mammary glands of Holstein heifers during early lactation than in untreated control mammary glands. However, no significant differences were observed following penicillin-novobiocin treatment of Holstein heifers after the first milking of lactation compared with untreated control quarters. Coagulase-negative staphylococci, Streptococcus uberis and Streptococcus dysgalactiae subsp dysgalactiae were isolated most frequently in heifers from both herds.

Prevalence of enterotoxin and toxic shock syndrome toxin genes in Staphylococcus aureus isolated from milk of cows with mastitis.

Staphylococcus aureus isolated from milk of cows with mastitis were evaluated for the prevalence of 16 enterotoxin genes (sea-see and seg-seq) and toxic shock syndrome toxin gene (tsst-1). Of 78 S. aureus examined, 73 (93.6%) were positive for one or more enterotoxin genes and these were divided into 36 groups by the presence of different enterotoxin genes. Enterotoxin genes including sen (84.6%), sem (71.8%), sei (60.3%) and sed (52.6%) were found frequently, while seg (24.4%), seq (16.7%), seo (12.8%), and seb (1.3%) were found at lower frequencies. Toxic shock syndrome toxin (tsst-1) gene was detected in 20 (25.6%) isolates and was always found in combination with other enterotoxin genes. The majority (88.5%) harbored more than one enterotoxin gene in different combinations. Eight S. aureus isolates (10.3%) were positive for sed, sei, sem, and sen; six (7.7%) possessed sed, seg, sei, sem, sen, and tsst-1; five (6.4%) had sei, sem, and sen; and four (5.1%) had sei, and sen. One isolate was positive for seb along with other SE genes including sed, seh, sem, sen, seq, and tsst-1. None of the isolates carried other enterotoxin genes (sea, sec, see, sej, sek, sel, and sep). PFGE profiles revealed 15 distinct pulsotypes among the 78 S. aureus isolates evaluated. PFGE and enterotoxin gene profiles did not match with each other because a single pulsotype carried different combinations of enterotoxin genes. The majority of S. aureus isolated from milk of mastitic cows carried newly described SE genes sem, sen and sei along with classical SE genes, sed and tsst-1. This is the first report describing the high prevalence of newly described enterotoxin genes, sem and sen in S. aureus from bovine mastitis. The high prevalence of enterotoxin genes and tsst-1 in S. aureus may be important as it is relevant to udder pathogenicity and food hygiene.

Phenotypic and genetic markers for serotype-specific detection of Shiga toxin-producing Escherichia coli O26 strains from North America.

Phenotypic and genetic markers of Shiga toxin-producing Escherichia coli (STEC) O26 from North America were used to develop serotype-specific protocols for detection of this pathogen. Carbohydrate fermentation profiles and prevalence of gene sequences associated with STEC O26 (n = 20) were examined. Non-STEC O26 (n = 17), E. coli O157 (n = 20), E. coli O111 (n = 22), and generic E. coli (n = 21) were used as comparison strains. Effects of supplements: cefixime-tellurite, 4-methylumbelliferyl-beta-D-glucuronide (MUG) and chromogenic additives (5-bromo4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal), 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide (X-GlcA) and o-nitrophenyl-beta-D-galactopyranoside (ONPG), added to isolation agar media were examined. Tests for presence of gene sequences encoding beta intimin (eae beta), Shiga toxin 1 and 2 (stx1 and stx2), H7 flagella (flicCh7), enterohemolysin (ehlyA), O26 somatic antigen (wzx), and high pathogenicity island genes (irp2 and fyuA) were conducted using multiplex polymerase chain reaction. Pulsed-field gel electrophoresis (PFGE) of XbaI restriction endonuclease genomic DNA digests was used to establish clonality among E. coli O26 strains. Of the 26 carbohydrates tested, only rhamnose had diagnostic value. Rhamnose non-fermenters included STEC O26 (100%), non-STEC O26 (40%), generic E. coli (29%), E. coli O111 (23%), and E. coli O157 (0%). Rhamnose non-fermenting colonies growing on Rhamnose-McConkey agar supplemented with X-GlcA, X-Gal, or ONPG, respectively, were blue, white, or faint yellow, whereas rhamnose-fermenters were red. Blue colonies from X-GlcA-containing media were the most well-defined and easiest to pick for further tests. All STEC O26 were MUG-fluorescent, while STEC O157 (n = 18) were non-fluorescent. E. coli O111 and generic E. coli strains were either MUG-positive or-negative. Serotype-specific detection of STEC O26 was achieved by selecting cefixime-tellurite-resistant, MUG-fluorescent, rhamnose-nonfermenting colonies, which carried stx1, eae beta, irp2, and wzx gene sequences. STEC O26 prevalence in dairy farm environmental samples determined using the developed isolation and genetic detection protocols was 4%. PFGE indicated the presence of one major cluster of E. coli O26 with 72-100% DNA fragment-length digest similarity among test strains. The serotype-specific detection methods described herein have potential for routine application in STEC O26 diagnosis.