Comparison of prevalence, antimicrobial resistance, and occurrence of multidrug-resistant Salmonella in antimicrobial-free and conventional pig production

Conventional swine production evolved to routinely use antimicrobials, and common occurrence of antimicrobial-resistant Salmonella has been reported. There is a paucity of information on the antimicrobial resistance of Salmonella in swine production in the absence of antimicrobial selective pressure. Therefore, we compared the prevalence and antimicrobial resistance of Salmonella isolated from antimicrobial-free and conventional production systems. A total of 889 pigs and 743 carcasses were sampled in the study. Salmonella prevalence was significantly higher among the antimicrobial-free systems (15.2%) than the conventional systems (4.2%) (odds ratio [OR] = 4.23; P < 0.05). Antimicrobial resistance was detected against 10 of the 12 antimicrobials tested. The highest frequency of resistance was found against tetracycline (80%), followed by streptomycin (43.4%) and sulfamethoxazole (36%). Frequency of resistance to most classes of antimicrobials (except tetracycline) was significantly higher among conventional farms than antimicrobial-free farms, with ORs ranging from 2.84 for chloramphenicol to 23.22 for kanamycin at the on-farm level. A total of 28 antimicrobial resistance patterns were detected. A resistance pattern with streptomycin, sulfamethoxazole, and tetracycline (n = 130) was the most common multidrug resistance pattern. There was no significant difference in the proportion of isolates with this pattern between the conventional (19.5%) and the antimicrobial-free systems (18%) (OR = 1.8; P > 0.05). A pentaresistance pattern with ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline was strongly associated with antimicrobial-free groups (OR = 5.4; P = 0.01). While showing the higher likelihood of finding antimicrobial resistance among conventional herds, this study also implies that specific multidrug-resistant strains may occur on antimicrobial-free farms. A longitudinal study with a representative sample size is needed to reach more conclusive results of the associations detected in this study.     

Molecular epidemiology and antimicrobial resistance profiles of Salmonella isolates from dairy heifer calves and adult lactating cows in a Mediterranean pasture-based system of Australia

Dairy cows can be reservoirs of foodborne pathogens such as Salmonella that pose serious public health risks to humans. The study was designed to examine the molecular epidemiology and antimicrobial resistance profiles of Salmonella isolates from dairy heifer calves and adult lactating cows in the pasture-based system of Australia. A total of 838 animals (328 heifer calves and 510 lactating cows) from 22 farms were sampled. Overall, 54 Salmonella isolates were recovered (calves 28/328 and cows 26/510). A herd-level Salmonella prevalence of 50% (95% confidence interval: 31%–69%) was recorded. Within-herd prevalence for Salmonella ranged between 4%–29% and 4%–45% among the heifer calves and adult lactating cows, respectively. Three different serovars were identified with Salmonella Infantis being the most common serovar (n = 33, 61%) followed by Salmonella Kiambu (n = 20, 37.0%) and one isolate of Salmonella Cerro (2%). The highest antimicrobial resistance prevalence of Salmonella isolates was found against streptomycin (n = 31, 57%), followed by cefoxitin (n = 12, 22%), ceftriaxone (n = 2, 4%), and chloramphenicol (n = 1, 2%). Multiple class resistance was observed on 4 isolates against cefoxitin, chloramphenicol, and streptomycin. Multilocus sequence types ST32 (61%), ST309 (37%), and ST367 (2%) were strongly linked to the serovars Salmonella Infantis, Salmonella Kiambu, and Salmonella Cerro, respectively. Whole genome sequencing of Salmonella isolates detected only 2 resistance genes: aac(6′) gene that confers resistance against aminoglycosides among 40.7% of the isolates, and a single isolate positive for the blaDHA-16 gene. Two distinct clusters among the serovars were observed suggesting 2 independent sources of spread. Despite the low prevalence of antimicrobial resistance among Salmonella from the dairy farms, our findings contribute to the regional and national understanding of antimicrobial resistance in dairy herds in Australia. There is need for continued antimicrobial resistance stewardship and surveillance programs to ensure the production of high-quality food products and the long-term protection of both animal and human health.     

Prevalence and antimicrobial resistance of Salmonella in retail foods in northern China

A total of 387 retail meat, seafood and milk powder samples were collected from nine cities in northern China in 2005 and screened for the presence of Salmonella. Salmonella strains isolated were subjected to serotyping and antimicrobial susceptibility testing. Salmonella was isolated from 81 (20.9%, 81/387) samples and classified into 23 serotypes. The isolates were frequently resistant to sulfamethoxazole (86.4%), sulfamethoxazole/trimethoprim (48.1%), nalidixic acid (30.9%), tetracycline (19.8%), carboxybenzylpenicillin (17.3%), amoxicillin (17.3%) and ampicillin (16.0%). The multiple resistance (resistance to ≥ 3 antibiotics) was found in 29.6% (n = 24) isolates. Additionally, 4 isolates from chicken displayed the ACSSuTNx profile, resistant to ampicillin, chloramphenicol, streptomycin, sulfonamide, tetracycline and nalidixic acid, in particular, strain HBS084 showing the resistance to as many as 20 antibiotics. Salmonella from chicken showed the higher frequency of antimicrobial resistance. Our findings indicate that in northern China food products of animal origin can be a source of exposure for consumers to multiresistant Salmonella strains.     

Effect of on-farm use of antimicrobial drugs on resistance in fecal Escherichia coli of preweaned dairy calves

Respiratory disease and diarrhea are the 2 most common diseases that result in the use of antimicrobial drugs in preweaned calves. Because the use of drugs in food animals, including dairy calves, has the potential for generating cross-resistance to drugs used in human medicine, it is vital to propose farm practices that foster the judicious use of antimicrobials while assuring animal health and productivity. The objective of this study was to use dairy farm calf treatment records to identify antimicrobial drug treatments in calves and to evaluate their effects on the prevalence of antimicrobial-resistant Escherichia coli from rectal swabs of preweaned dairy calves. Eight farms from central New York participated in the study, 3 farms using individual pen housing management and 5 farms using group pen housing management. Eligible study farms could not add antimicrobial drugs to the milk fed to preweaned calves and were required to have farm records documenting antimicrobial drug treatment of calves from birth to weaning. Three fecal E. coli isolates per calf were tested for susceptibility to 12 antimicrobial drugs using a Kirby-Bauer disk diffusion assay. A total of 473 calves were sampled, from which 1,423 commensal E. coli isolates were tested. Of the 9 antimicrobial drugs used on study farms, only enrofloxacin was significantly associated with reduced antimicrobial susceptibility of E. coli isolates, although treatment with ceftiofur was associated with reduced susceptibility to ceftriaxone. The median numbers of days from treatment with ceftiofur and enrofloxacin to rectal swab sampling of calves were 16 d (range: 1–39) and 12 d (range: 6–44), respectively. At the isolate level, treatment with enrofloxacin resulted in odds ratios of 2 [95% confidence interval (CI): 1–4] and 3 (95% CI: 2–6), respectively, for isolation of nonsusceptible E. coli to nalidixic acid and ciprofloxacin compared with calves not treated with enrofloxacin. Treatment with ceftiofur resulted in an odds ratio of 3 (95% CI: 0.9–12) for isolation of nonsusceptible E. coli to ceftriaxone compared with calves not treated with ceftiofur. Treatment with enrofloxacin resulted in selection of isolates that presented phenotypic resistance to both ciprofloxacin and ceftriaxone. Treatment with ceftiofur resulted in a higher prevalence of isolates resistant to ≥3 antimicrobial drugs (97%) compared with no treatment with ceftiofur (73%). These findings reinforce the necessity for continued implementation of practices at the dairy farm that support the sustainable and judicious use of antimicrobial drugs in dairy calves.     

Introduction of new multidrug-resistant Salmonella enterica strains into commercial dairy herds

A longitudinal observational study of 59 dairy herds was conducted in Washington State to estimate the rate of introduction of new multidrug-resistant (MDR) Salmonella enterica strains onto commercial dairy herds. Samples were collected on these herds over 7 visits separated by intervals of 2 to 4 mo over a period of 15 to 21 mo. Samples were cultured for Salmonella spp. and serogroup, serovar, and antimicrobial susceptibility patterns were identified for MDR Salmonella isolates. Fingerprinting generated by pulsed-field gel electrophoresis (PFGE) using XbaI restriction enzyme digestion generated genotyping profiles for all MDR isolates identified in the study. The rate of new MDR Salmonella strain introduction was 0.9 per herd-year (95% confidence interval: 0.6-1.4). The rates for the most commonly introduced MDR Salmonella serovars were 0.4/herd-year for Typhimurium, 1.2/herd-year for Newport, and 0.1/herd-year for Dublin. Thirty-three of 59 herds (56%) had at least one new MDR Salmonella introduction during the study period. The number of new MDR Salmonella strains acquired by dairy herds ranged from zero to 8. Thirteen of the 59 herds had a history of clinical salmonellosis. Among these 13 herds, 6 herds acquired new MDR Salmonella strains, although these strains were different than historical clinical strains. These data indicate that acquisition of new MDR Salmonella strains by dairy herds was a common event in participating herds, although the number of strains introduced varied greatly among herds.     

Salmonella bacteriophage diversity reflects host diversity on dairy farms

Salmonella is an animal and human pathogen of worldwide concern. Surveillance programs indicate that the incidence of Salmonella serovars fluctuates over time. While bacteriophages are likely to play a role in driving microbial diversity, our understanding of the ecology and diversity of Salmonella phages is limited. Here we report the isolation of Salmonella phages from manure samples from 13 dairy farms with a history of Salmonella presence. Salmonella phages were isolated from 10 of the 13 farms; overall 108 phage isolates were obtained on serovar Newport, Typhimurium, Dublin, Kentucky, Anatum, Mbandaka, and Cerro hosts. Host range characterization found that 51% of phage isolates had a narrow host range, while 49% showed a broad host range. The phage isolates represented 65 lysis profiles; genome size profiling of 94 phage isolates allowed for classification of phage isolates into 11 groups with subsequent restriction fragment length polymorphism analysis showing considerable variation within a given group. Our data not only show an abundance of diverse Salmonella phage isolates in dairy farms, but also show that phage isolates that lyse the most common serovars causing salmonellosis in cattle are frequently obtained, suggesting that phages may play an important role in the ecology of Salmonella on dairy farms.     

Association between bulk-tank milk Salmonella antibody level and high calf mortality in Danish dairy herds

Salmonella enterica ssp. enterica Dublin is the most common Salmonella serotype found in the dairy sector in Denmark. Salmonella antibody level in bulk-tank milk (BTM), indicative of Salmonella Dublin infection in the herd, has been recorded regularly in all Danish dairy herds through a surveillance program since 2002. The objective of this study was to investigate whether high BTM Salmonella antibody level was associated with high calf mortality at herd level. Other risk factors for high calf mortality were also investigated: breed, production type (organic vs. conventional), number of animals purchased, herd size, and number of neighbor herds within a 4.9-km radius. Data from the Danish Cattle Database including the Salmonella surveillance program from September 2007 through August 2008 were used. Dairy herds with more than 20 cows were included (n = 4,337). Because of a highly right-skewed distribution of calf mortality with many zero values, calf mortality had to be dichotomized for the analysis. Therefore, in this study, high calf mortality was defined as calf mortality of more than 6.5% for calves aged 1 to 180 d. A logistic analysis was performed to identify risk factors associated with the probability of a herd having high calf mortality. The following factors were significantly associated with high calf mortality: high BTM Salmonella antibody level, odds ratio (OR) = 2.0 (95% confidence interval = 1.6-2.4), organic production OR = 1.4 (95% confidence interval = 1.1-1.7) for organic versus conventional production, and breed. Purchase of 8 or more animals increased the OR of high calf mortality more than purchase of 1 to 7 animals, which again had a higher OR compared with purchase of 0 animals. Because only 14.3% of the population consisted of herds with high BTM Salmonella status, the estimated proportion of herds with high calf mortality could only be reduced from 38.7 to 36.5% by eradicating Salmonella from the Danish cattle population (i.e., a population attributable risk of 2.2%). This showed that although there is a strong association between BTM Salmonella status and calf mortality, the problem with high calf mortality will not be solved by eradicating Salmonella. All other things equal, a population with more Salmonella-infected herds would gain a larger reduction in calf mortality from a Salmonella control campaign. Nevertheless, individual herds with a high within-herd prevalence of Salmonella are likely to benefit, both economically and regarding animal welfare, from controlling pathogenic Salmonella types in cattle.     

Comparison of antimicrobial susceptibility of Staphylococcus aureus isolated from bulk tank milk in organic and conventional dairy herds in the midwestern United States and Denmark

An observational study was conducted to compare the antimicrobial susceptibility patterns of Staphylococcus aureus isolated from bulk tank milk in organic and conventional dairy farms in Wisconsin, United States, and southern Jutland, Denmark. Bulk tank milk samples and data regarding management and production were collected from 30 organic and 30 conventional dairy farms in Wisconsin and 20 organic and 20 conventional dairy farms in Denmark. S. aureus isolates were tested for resistance against 15 antimicrobial agents by semiautomatic microbroth dilution methods in each country. Of the 118 bulk tank milk samples in Wisconsin, 71 samples (60%) yielded at least one S. aureus isolate, and a total of 331 isolates were collected. Of the 40 bulk tank milk samples from Denmark, 27 samples (55%) yielded at least one S. aureus isolate, and a total of 152 isolates were collected. Significant differences between organic and conventional dairies were detected only to ciprofloxacin in Wisconsin and avilamycin in Denmark. Significant differences (P < 0.05) between the two countries were detected in nine antimicrobials. Denmark had a higher probability of having reduced susceptibility to ciprofloxacin and streptomycin (P = 0.015 and 0.003, respectively). Wisconsin isolates had a higher probability of having reduced susceptibility to seven other antimicrobial agents (bacitracin, gentamicin, kanamycin, penicillin, sulphamethoxazole, tetracycline, and trimethoprim). We found small differences between organic and conventional farm types in each country and larger differences between the two national agricultural systems.     

Antimicrobial resistance patterns of bovine Salmonella enterica isolates submitted to the Wisconsin Veterinary Diagnostic Laboratory: 2006–2015

Salmonellosis on the dairy continues to have a significant effect on animal health and productivity and in the United States. Additionally, Salmonella enterica ssp. enterica causes an estimated 1.2 million cases of human illness annually. Contributing to the morbidity and mortality in both human and domestic animal species is emergence of antimicrobial resistance by Salmonella species and increased incidence of multidrug-resistant isolates. This study describes serotype distribution and the antimicrobial resistance patterns for various Salmonella serotypes isolated from bovine samples submitted to the Wisconsin Veterinary Diagnostic Laboratory (WVDL) over the past 10 yr. Salmonella serotyping and antimicrobial susceptibility testing data were obtained from the laboratory information management system at WVDL. Data from accessions were limited to bovine samples submitted to the WVDL between January 2006 and June 2015 and those that had both a definitive serotype and complete results for antimicrobial susceptibility testing. A total of 4,976 isolates were identified. Salmonella enterica ser. Dublin was the most prevalent serotype identified among bovine samples submitted to the WVDL, accounting for a total of 1,153 isolates (23% of total isolates) over the study period. Along with Dublin, Salmonella enterica ser. Cerro (795, 16%), Newport (720, 14%), Montevideo (421, 8%), Kentucky (419, 8%), and Typhimurium (202, 4%) comprised the top 6 most commonly isolated serotypes during that time. Overall, resistance of bovine Salmonella isolates in the study population remained stable, although decreases in resistance were noted for gentamicin, neomycin, and trimethoprim sulfamethoxazole during the study period. All isolates remained susceptible to enrofloxacin. These data show that antimicrobial susceptibility for bovine Salmonella has changed in the population served by WVDL in the past 10 yr. This information is important for understanding Salmonella disease ecology in Wisconsin. Our findings are also relevant for animal and public health by improving informed antimicrobial use, new drug development, and regulation of their use in food animals.     

Incidence and antibiotic resistance of Salmonella spp. on raw chicken carcasses

The current study was carried out to detect Salmonella spp. contamination on chicken carcasses and to determine the antibiotic susceptibility profiles and serotype distribution of the isolates. A total of 200 packaged fresh raw chicken samples sold at retail in different markets in central Anatolia were analysed between April 2005 and March 2006. Salmonella spp. was detected in 34% (68/200) of samples using cultural technique and were confirmed by PCR. Ten Salmonella serovars were identified; predominant ones included Typhimurium, Infantis and Heidelberg. All of the Salmonella spp. isolates tested, exhibited resistance to one or more antimicrobial agents used. Resistance to penicillin, oxacillin, clindamycin, vancomycin, erythromycin and ampicillin were evident 100%, 97%, 97%, 92.6%, 89.7% and 85.2%, respectively. Also resistance to tetracycline (67.6%), streptomycin (61.7%), neomycin (55.8%) and cephalothin (52.9%) was observed but a small percentage of the isolates demonstrated resistance to gentamicin (14.7%), chloramphenicol (10.2%), cefotaxime (2.9%) and amikacin (2.9%). As a result, high prevalence of Salmonella spp. and the relatively high resistance among the bacteria tested could pose public health and therapeutic problems in consumers as potential vehicle of resistant Salmonella foodborne infections. To avoid Salmonella contamination, hygienic rules of slaughter and poultry meat processing must be rigorously observed and antibiotic use must be controlled by governmental agencies to prevent increased resistance of antibiotics.     

Antimicrobial resistance in Campylobacter coli isolated from pigs in two provinces of China

The aim of this study was to determine the prevalence, antimicrobial resistance and molecular epidemiology of Campylobacter coli isolated from swine in China. A total of 190 C. coli isolates obtained from two slaughter houses and ten conventional pig farms in Shandong (SD, n = 95) and Ningxia (NX, n = 95) provinces were tested for their susceptibility to 14 antimicrobials. A high prevalence (> 95%) of ciprofloxacin and tetracycline-resistant strains was observed in both SD and NX. The erythromycin and clindamycin resistance rates of C. coli from NX (ERY: 54.7% CLI: 43.2%) were higher than those from SD (ERY: 37.9%, CLI: 35.8%). A significant difference (P < 0.05) was observed in erythromycin resistance rate, but not (P > 0.05) in clindamycin resistance rate. while the resistance rates of ampicillin and kanamycin in NX (AMP: 34.7%, KAN: 43.2%) were significantly lower (P < 0.05) than those in SD (AMP: 51.6%, KAN: 71.6%). None of the tested isolates were resistant to phenicols. The majority of the isolates from both provinces (SD: 80% and NX: 73.7%) showed multi-drug resistance profiles. The point mutations of A2075G in the 23S rRNA and C257T in the gyrA gene were detected in 98% (87/89) of macrolide resistant isolates and all ciprofloxacin resistant isolates, respectively. In addition, all tetracycline-resistant isolates harbored the tet(O) gene. The high prevalence of antimicrobial resistance in C. coli strains derived from pigs in China was observed and was likely due to the extensive use of various antimicrobials. Prudent use of antimicrobial agents on farms should be further emphasized to control the dissemination of antimicrobial resistant C. coli.     

The role of animal movement, including off-farm rearing of heifers, in the interherd transmission of multidrug-resistant Salmonella

Fifty-nine commercial dairy farms were sampled 7 times over 15 to 21 mo to determine the role of animal movement, including off-farm rearing of heifers, in the interherd transmission of multidrug-resistant (MDR) Salmonella spp. Farm management data were collected by on-site inspections and questionnaires on herd management practices before and after the study. Forty-four percent (26/59) of herds did not acquire any new MDR Salmonella strains. The number of newly introduced MDR Salmonella strains acquired by the remaining 56% (33/59) of herds ranged from 1 to 8. Logistic regression models indicated that off-farm heifer raising, including contract heifer raising where heifers commingle with cattle from other farms [commingled heifers, odds ratio (OR) = 8.9, 95% confidence interval (CI): 2.4, 32.80], and herd size per 100-animal increment (herd size, OR = 1.04, 95% CI, 1.01, 1.05) were significantly associated with the introduction of new MDR Salmonella strains. The negative binomial regression similarly revealed that commingled heifers [relative risk (RR) = 2.3, 95% CI: 1.1, 4.7], herd size per 100 animals (RR = 1.02, 95% CI, 1.01, 1.03), and a history of clinical salmonellosis diagnosed before the study (RR = 2.5, 95% CI, 1.3, 5.0) were significantly associated with the number of new MDR Salmonella strains that were introduced. Factors not associated with the introduction of new MDR Salmonella strains were housing of heifers and cows in the same close-up pen, a common hospital-maternity pen, and the number of purchased cattle. This study highlights the role of animal movement in the interherd transmission of MDR Salmonella spp.     

Herd-level relationship between antimicrobial use and presence or absence of antimicrobial resistance in gram-negative bovine mastitis pathogens on Canadian dairy farms

Concurrent data on antimicrobial use (AMU) and resistance are needed to contain antimicrobial resistance (AMR) in bacteria. The present study examined a herd-level association between AMU and AMR in Escherichia coli (n = 394) and Klebsiella species (n = 139) isolated from bovine intramammary infections and mastitis cases on 89 dairy farms in 4 regions of Canada [Alberta, Ontario, Québec, and Maritime Provinces (Prince Edward Island, Nova Scotia, and New Brunswick)]. Antimicrobial use data were collected using inventory of empty antimicrobial containers and antimicrobial drug use rate was calculated to quantify herd-level AMU. Minimum inhibitory concentrations (MIC) were determined using Sensititre National Antimicrobial Resistance Monitoring System (NARMS) gram-negative MIC plate (Trek Diagnostic Systems Inc., Cleveland, OH). Isolates were classified as susceptible, intermediate, or resistant. Intermediate and resistant category isolates were combined to form an AMR category, and multivariable logistic regression models were built to determine herd-level odds of AMR to tetracycline, ampicillin, cefoxitin, chloramphenicol, trimethoprim-sulfamethoxazole combination, sulfisoxazole, streptomycin and kanamycin in E. coli isolates. In the case of Klebsiella species isolates, logistic regression models were built for tetracycline and sulfisoxazole; however, no associations between AMU and AMR in Klebsiella species were observed. Ampicillin-intermediate or -resistant E. coli isolates were associated with herds that used intramammarily administered cloxacillin, penicillin-novobiocin combination, and cephapirin used for dry cow therapy [odds ratios (OR) = 26, 32, and 189, respectively], and intramammary ceftiofur administered for lactating cow therapy and systemically administered penicillin (OR = 162 and 2.7, respectively). Use of systemically administered penicillin on a dairy farm was associated with tetracycline and streptomycin-intermediate or -resistant E. coli isolates (OR = 5.6 and 2.8, respectively). Use of cephapirin and cloxacillin administered intramammarily for dry cow therapy was associated with increasing odds of having at least 1 kanamycin-intermediate or -resistant E. coli isolate at a farm (OR = 8.7 and 9.3, respectively). Use of systemically administered tetracycline and ceftiofur was associated with cefoxitin-intermediate or -resistant E. coli (OR = 0.13 and 0.16, respectively); however, the odds of a dairy herd having at least 1 cefoxitin-intermediate or -resistant E. coli isolate due to systemically administered ceftiofur increased with increasing average herd parity (OR = 3.1). Association between herd-level AMU and AMR in bovine mastitis coliforms was observed for certain antimicrobials. Differences in AMR between different barn types and geographical regions were not observed.     

Pulsed field,ribotype and integron analysis of multidrug resistant isolates of Salmonella enterica serovar Newport

Twenty-one isolates of Salmonella enterica serovar Newport were evaluated for their antimicrobial resistance, pulsed field gel electrophoresis (PFGE) profiles, ribotype profiles, and their integron profiles. Antimicrobial resistance profiles indicated that 20 of the 21 isolates were resistant to the following antibiotics: amoxicillin–clavulanic acid (AMOX/CA), ampicillin (AMPC), cefoxitin (CFOX), ceftiofur (TIO), cephalothin (CRIN), chloramphenicol (CHL), streptomycin (STR), tetracycline (TET), and sulfamethoxazole (SMX). Five isolates showed resistance to gentamycin (GEN) and kanamycin (KAN). Trimethoprim–sulfamethoxazole (SMX/TMP) resistance was observed in six isolates. Eight of the twenty one isolates showed intermediate resistance to ceftriaxone (CTRX), with one isolate exhibiting complete resistance. PFGE clearly resolved the Salmonella Newport isolates into nine distinct clusters, and a good congruence was observed between PFGE and antibiotic resistance patterns. Automated riboprinting clearly distinguished between antibiotic resistant and sensitive strains of Salmonella Newport, and resolved the isolates into two ribogroups. One group consisted of the multidrug resistant isolates, and the other grouping contained the sensitive isolate. Three different integrons (1.0, 1.2, and 1.8 kb) were observed in many of the isolates, and several isolates contained more than one integron. Restriction fragment length polymorphisms (RFLP) indicated that integrons of the same size were indistinguishable.When integron analysis and ribotype analysis were used in conjunction, four subtypes of multidrug resistant Salmonella Newport isolates were clearly defined. These results demonstrate the possibility of utilizing automated ribotyping and integron analysis to rapidly subtype multidrug resistant Salmonella Newport isolates.     

Effect of preweaned dairy calf housing system on antimicrobial resistance in commensal Escherichia coli

Group housing of preweaned dairy calves is a growing practice in the United States. The objective of this practice is to increase the average daily gain of calves in a healthy and humane environment while reducing labor requirements. However, feeding protocols, commingling of calves, and occurrence of disease in different calf-housing systems may affect the prevalence of antimicrobial drug-resistant bacteria. This study evaluated the effect of a group pen-housing system and individual pen-housing system on antimicrobial resistance trends in fecal Escherichia coli of preweaned dairy calves and on the prevalence of environmental Salmonella. Twelve farms from central New York participated in the study: 6 farms using an individual pen-housing system (IP), and 6 farms using a group pen-housing system (GP). A maximum of 3 fecal E. coli isolates per calf was tested for susceptibility to 12 antimicrobial drugs using a Kirby-Bauer disk diffusion assay. Calves in GP had a significantly higher proportion of E. coli resistant to ciprofloxacin and nalidixic acid, whereas calves in IP had a significantly higher proportion of E. coli resistant to ampicillin, ceftiofur, gentamycin, streptomycin, and tetracycline. Calf-housing system had an effect on resistance to individual antimicrobial drugs in E. coli, but no clear-cut advantage to either system was noted with regard to overall resistance frequency. No outstanding difference in the richness and diversity of resistant phenotypes was observed between the 2 calf-housing systems.     

Relationship between antimicrobial drug usage and antimicrobial susceptibility of gram-positive mastitis pathogens

The objective of this study was to analyze relationships between usage of antimicrobial drugs on dairy farms and results of antimicrobial susceptibility testing of mastitis pathogens. Exposure to selected antimicrobial drugs (n = 10) was standardized by calculation of the number of defined daily doses used per cow. Farms (n = 40) were categorized based on amount of antimicrobial exposure: organic (no usage); conventional-low usage (conventional farms not using or using less than or equal to the first quartile of use of each compound); and conventional-high usage (conventional farms using more than the first quartile of a particular compound). The minimum inhibitory concentration (MIC) of selected antimicrobial drugs was determined using a commercial microbroth dilution system for isolates of Staphylococcus aureus (n = 137), coagulase-negative staphylococci (CNS, n = 294), and Streptococcus spp. (n = 95) obtained from subclinical mastitis infections. Most isolates were inhibited at the lowest dilution tested of most antimicrobial drugs. Survival curves for Staph. aureus and CNS demonstrated heterogeneity in MIC based on the amount of exposure to penicillin and pirlimycin. For CNS, farm type was associated with the MIC of ampicillin and tetracycline. For Streptococcus spp., farm type was associated with MIC of pirlimycin and tetracycline. For all mastitis pathogens studied, the MIC of pirlimycin increased with increasing exposure to defined daily doses of pirlimycin. The level of exposure to most other antimicrobial drugs was not associated with MIC of mastitis pathogens. A dose-response effect between antimicrobial exposure and susceptibility was observed for some pathogen-antimicrobial combinations, but exposure to other antimicrobial drugs commonly used for prevention and treatment of mastitis was not associated with resistance.     

Exposure to antimicrobials through the milk diet or systemic therapy is associated with a transient increase in antimicrobial resistance in fecal Escherichia coli of dairy calves

The objective of this prospective cohort study was to describe the relationship between exposure to antimicrobials, through both the milk diet and systemic therapy, and to describe antimicrobial resistance of fecal Escherichia coli in dairy calves pre- and postweaning. A convenience sample of 15 Minnesota dairy farms was chosen, representing 3 equal cohorts of milk diet fed to preweaned calves: medicated milk replacer (MMR), nonmedicated milk replacer (NMR), or pasteurized nonsaleable milk (PNM). Five newborn calves were enrolled on each farm, with fecal samples collected from each calf at 1, 3, 5, and 16 wk of age. After isolation, 3 colonies of E. coli were randomly selected from each sample to determine antimicrobial susceptibility by minimum inhibitory concentration (Sensititer, Thermo Scientific, Waltham, MA) to 8 antimicrobials in 8 classes. The isolate was given an antimicrobial resistance score (ARS) according to the number of antimicrobial classes to which it was resistant. Any isolate resistant to 3 or more antimicrobials was defined as being multidrug resistant (MDR). Relationships between ARS and MDR (dependent variables) and possible explanatory variables were analyzed using mixed multivariable linear and logistic regression models, respectively, with critical P-values adjusted for multiple contrasts. Seventy percent of isolates were resistant to sulfadimethoxine. For wk 1 and 3, the mean ARS values were greatest for fecal E. coli from calves fed MMR or PNM compared with NMR, with no difference in ARS values between the MMR and PNM groups at either time point. At wk 5, the mean ARS value was greatest for fecal E. coli from calves fed MMR (3.56 ± 0.45; mean ± SE), intermediate for calves fed PNM (2.64 ± 0.45), and lowest for calves fed NMR (1.54 ± 0.45). However, by wk 16, the mean ARS values were ≤1.0 and did not differ among milk diets. Evaluation of the proportion of isolates with MDR mirrored the results of the ARS analysis (MDR more prevalent in MMR and PNM groups preweaning; no difference among milk diets at 16 wk). There was a tendency for an increase in ARS at wk 5 (1.28 ± 0.70), and the odds for MDR in fecal E. coli were estimated to be 5.2 (95% confidence interval = 0.67, 35.7) and 101.1 (95% confidence interval = 1.15, >999.9) higher at wk 3 and 5 if the calf was treated with a systemic antimicrobial within the 14-d period before sampling. These findings suggest that exposure to antimicrobials through the milk diet or systemic therapy may result in a transient increase in resistance in fecal E. coli, but once the antimicrobial pressure is removed, susceptible E. coli are able to flourish again, resulting in an overall decrease in resistance.     

A survey of mastitis pathogens including antimicrobial susceptibility in southeastern Australian dairy herds

The objectives for this study were to (1) describe the pathogen profile in quarters from cows with clinical mastitis and in cows with subclinical mastitis in southeastern Australia; and (2) describe antimicrobial susceptibility among isolated pathogens. As a secondary objective, we aimed to compare antimicrobial resistance prevalence in pathogens isolated from clinical and subclinical mastitis samples. A convenience sample of dairy herds (n = 65) from 4 regions in southeastern Australia (Gippsland, Northern Victoria, Tasmania, Western Victoria) were invited to submit milk samples from cows with clinical and subclinical mastitis over a 14-mo period (January 2011 to March 2012). Farmers were instructed to collect aseptic quarter milk samples from the first 10 cases of clinical mastitis for each month of the study. In addition, farmers submitted composite milk samples from cows with subclinical mastitis at 1 or 2 sampling occasions during the study period. Aerobic culture and biochemical tests were used to identify isolates. Isolates were classified as susceptible, intermediate, or resistant to a panel of antimicrobial agents based on the zone of growth inhibition around antimicrobial-impregnated disks, with antimicrobial resistance (AMR) classified as nonsusceptibility by combining intermediate and resistant groups into a single category. Generalized linear mixed models were used to compare the prevalence of AMR between clinical and subclinical mastitis isolates. For clinical mastitis samples (n = 3,044), 472 samples (15.5%) were excluded for contamination. Of the remaining samples (n = 2,572), the most common results were Streptococcus uberis (39.2%), no growth (27.5%), Staphylococcus aureus (10.6%), Escherichia coli (8.4%), and Streptococcus dysgalactiae (6.4%). For subclinical mastitis samples (n = 1,072), 425 (39.6%) were excluded due to contamination. Of the remaining samples (n = 647), the most common results were no growth (29.1%), Staph. aureus (29.1%), and Strep. uberis (21.6%). The prevalence of AMR among common isolates was low for the majority of antimicrobial agents. Exploratory analysis found that the probability of Staph. aureus demonstrating resistance to penicillin was 5.16 times higher (95% confidence interval: 1.68, 15.88) in subclinical isolates relative to clinical Staph. aureus isolates. A similar association was observed for amoxicillin with subclinical Staph. aureus isolates being 4.70 times (95% confidence interval: 1.49, 14.75) more likely to be resistant than clinical Staph. aureus isolates. We concluded that the most common bacteria causing clinical mastitis in dairy herds in Australia is likely to be Strep. uberis, whereas Staph. aureus is likely to be the most common cause of subclinical mastitis. Despite decades of antimicrobial use to control these organisms, AMR appears to be uncommon.     

Genetic characterization of antimicrobial resistance in Salmonella enterica serovars isolated from dairy cattle in Wisconsin

Salmonella enterica is a pathogen of humans and animals, and is one of the most frequent causes of bacterial foodborne illness worldwide. People consuming contaminated foods or working with infected livestock have the potential to become infected with Salmonella and may require antimicrobial therapy. Antimicrobial resistance in Salmonella has become a problem worldwide due in part to the inappropriate use of antimicrobial agents in human and veterinary medicine. In this study, forty-five Salmonella isolates from diagnostic fecal samples of Wisconsin dairy cattle were serotyped and characterized by antimicrobial susceptibility testing using agar disk diffusion, antimicrobial resistance gene detection by PCR, plasmid analysis and conjugation studies. The predominant serovars detected were Kentucky, Newport, Typhimurium, Cerro, Dublin and Montevideo. Over half (51%) of all isolates were resistant to at least one antimicrobial agent, and 29% were resistant to 8–10 drugs. The most commonly observed resistance phenotypes were to streptomycin (44%), tetracycline (42%), sulfisoxazole (40%), chloramphenicol (35%), ampicillin (33%), and cefoxitin (33%). When resistance was detected phenotypically, a corresponding resistance gene was detected 86.2% of the time. Plasmids ranging in size from < 8 to 165 kb were detected in 45% of the isolates. A greater understanding of the factors associated with antimicrobial resistance in Salmonella should provide insights into the factors that contribute to the development of resistant pathogens during dairy production, which in turn can lead to strategies to minimize the spread of antimicrobial resistant Salmonella in the food supply.     

Characterization of bacterial susceptibility isolates in sixteen dairy farms in Taiwan

Bacteria were isolated from dairy cows, dairy farm environments, and dairy workers in 2 geographically different areas of eastern and northern Taiwan. Isolates were evaluated for antimicrobial susceptibility and the phylogenetics of isolated Escherichia coli O157:H7 were characterized. A total of 1,346 bacteria were identified, including 226 E. coli, 30 Pseudomonas spp. (7 Pseudomonas aeruginosa), 259 other gram-negative bacteria, 271 Enterococcus spp., 314 Staphylococcus spp., 195 Streptococcus spp., and 51 other gram-positive bacteria. Among them, 88% (1,184/1,346) of the isolates were resistant to sulfadimethoxine. The percentages of gram-negative bacteria resistant to oxy-tetracycline and streptomycin were 48% (249/515) and 78% (404/515), respectively. Gram-positive bacteria isolated from eastern Taiwan, the least polluted region of Taiwan, were found to have greater antimicrobial resistance than those isolated from northern Taiwan. Two E. coli O157:H7 from 2 different geographical areas were isolated. Both were vt2-positive but vt1-negative and had phylogenetic similarities of 82 and 67%, respectively, compared with previous isolates. Information on antimicrobial susceptibility revealed from this dairy farm survey may serve as a baseline for future studies and may also highlight the need to formulate better regulation strategies for the safe use of antimicrobials on food-producing farms.