Susceptibility of Escherichia coli isolated from uteri of postpartum dairy cows to antibiotic and environmental bacteriophages. Part II: In vitro antimicrobial activity evaluation of a bacteriophage cocktail and several antibiotics

The use of pathogenic-specific antimicrobials, as proposed by bacteriophage therapy, is expected to reduce the incidence of resistance development. Eighty Escherichia coli isolated from uteri of Holstein dairy cows were phenotypically characterized for antimicrobial resistance to ampicillin, ceftiofur, chloramphenicol, florfenicol, spectinomycin, streptomycin, and tetracycline by broth microdilution method. The lytic activity of a bacteriophage cocktail against all isolates was performed by a similar method. Additionally, the effect of different concentrations of antimicrobials and multiplicities of infections (MOI) of the bacteriophage cocktail on E. coli growth curve was measured. Isolates exhibited resistance to ampicillin (33.7%), ceftiofur (1.2%), chloramphenicol (100%), and florfenicol (100%). All strains were resistant to at least 2 of the antimicrobial agents tested; multidrug resistance (≥3 of 7 antimicrobials tested) was observed in 35% of E. coli isolates. The major multidrug resistance profile was found for ampicillin-chloramphenicol-florfenicol, which was observed in more than 96.4% of the multidrug-resistant isolates. The bacteriophage cocktail preparation showed strong antimicrobial activity against multidrug-resistant E. coli. Multiplicity of infection as low as 10⁻⁴ affected the growth of the E. coli isolates. The ratio of 10 bacteriophage particles per bacterial cell (MOI = 10¹) was efficient in inhibiting at least 50% of all isolates. Higher MOI should be tested in future in vitro studies to establish ratios that completely inhibit bacterial growth during longer periods. All isolates resistant to florfenicol were resistant to chloramphenicol and, because florfenicol was recently introduced into veterinary clinics, this finding suggests that the selection pressure of chloramphenicol, as well as other antimicrobials, may still play a relevant role in the emergence and dissemination of florfenicol resistance in E. coli. The bacteriophage cocktail had a notable capacity to inhibit the in vitro growth of E. coli isolates, and it may be an attractive alternative to conventional treatment of metritis by reducing E. coli in uteri of postpartum dairy cows.     

Prevalence,molecular characterization,and antimicrobial resistance profiles of Listeria monocytogenes,Salmonella enterica,and Escherichia coli O157:H7 on dairy cattle farms in Jordan

This study determined the prevalence, pulsed-field gel electrophoresis profiles, and antimicrobial resistance profile of Listeria monocytogenes, Salmonella enterica, and Escherichia coli O157:H7 isolates from dairy cattle farms in Jordan. Samples from bulk tank milk (n = 305), cattle feces (n = 610), and rectoanal mucosal swabs (n = 610) were collected from 61 dairy cattle farms. We confirmed 32 L. monocytogenes, 28 S. enterica, and 24 E. coli O157:H7 isolates from the samples. The farm-level prevalence (at least 1 positive sample per farm) of L. monocytogenes, S. enterica, and E. coli O157:H7 was 27.9, 19.7, and 23.0%, respectively. The prevalence of L. monocytogenes, S. enterica, and E. coli O157:H7 in bulk tank milk was 7.5, 1.6, and 3.3%, respectively. The prevalence of L. monocytogenes and S. enterica in fecal samples was 1.5 and 3.8%, respectively, and the prevalence of E. coli O157:H7 in rectoanal mucosal swabs was 2.3%. Based on disk diffusion testing, all L. monocytogenes, S. enterica, and E. coli O157:H7 isolates exhibited resistance to at least 1 antimicrobial class. Multidrug resistance (resistance to 3 or more classes of antimicrobials) was exhibited by 96.9% of L. monocytogenes, 91.7% of E. coli O157:H7, and 82.1% of S. enterica isolates. Moreover, 93.8, 79.2, and 57.1% of the L. monocytogenes, E. coli O157:H7, and S. enterica isolates, respectively, were resistant to 5 or more antimicrobial classes. More than 50% of L. monocytogenes isolates were resistant to ampicillin, clindamycin, penicillin, erythromycin, quinupristin–dalfopristin, streptomycin, teicoplanin, linezolid, vancomycin, kanamycin, and tetracycline. More than 50% of S. enterica and E. coli O157:H7 isolates were resistant to ampicillin, cephalothin, nalidixic acid, kanamycin, streptomycin, amoxicillin–clavulanic acid, and tetracycline. The prevalence of the studied pathogens this study was comparable to reports from other countries. The isolated pathogens exhibited a high degree of antimicrobial resistance, suggesting that the bacterial flora of dairy cattle in Jordan are under intense antimicrobial selection pressure. Additional research is required to determine the causes and drivers of resistance, and to develop approaches to mitigating antimicrobial resistance.     

Characterization of antimicrobial resistance in Salmonella enterica serovar Typhimurium isolates from food animals in the U.S.

Salmonella enterica subspecies enteric serovar Typhimurium is the second most common serovar implicated in human diseases in the United States. In this study, 120 S. Typhimurium isolates from animal sources were characterized by antimicrobial susceptibility testing, antimicrobial resistance gene detection and plasmid analysis. Overall, 94 (78%) of the isolates were resistant to one or more antimicrobials and 63 (53%) were resistant to at least five antimicrobials. Resistance was most commonly observed to streptomycin (62%), sulfisoxazole (62%), or tetracycline (61%). When resistance was detected, a corresponding resistance gene was detected in 89% of cases. Class 1 integrons were detected in 51 isolates, all which contained the aadA2 gene. A plasmid Inc group was detected in 68 (57%) isolates. Thirty nine (57%) of these isolates were resistant to 5 or more antimicrobials.     

Escherichia coli of poultry food origin as reservoir of sulphonamide resistance genes and integrons

The antimicrobial resistance phenotype and genotype, the flanking regions of sulphonamide resistance genes and the integrons were analyzed in 166 Escherichia coli isolates recovered from poultry meat in Tunisia. High percentages of resistance were detected to ampicillin, streptomycin, nalidixic acid, sulphonamide and tetracycline (66-95%), and lower percentages to gentamicin, amoxicillin-clavulanic acid and cefoxitin (1-4%). The bla_TEM, tet(A)/tet(B), aph(3′)-Ia, aac(6′)-Ib-cr, aac(3)-II and cmlA genes were identified in 92, 82, 29, 2, 2 and 7 isolates, respectively. Class 1 and/or class 2 integrons were detected in 52% of E. coli isolates and five different gene cassette arrangements were identified in the variable regions of class 1 integrons, which included antimicrobial resistance determinants. Sixty-eight isolates contained the sul1 gene and 37 of them presented this gene into a class 1 integron structure. The sul3 gene was detected associated with non-classic class 1 integrons in 4 out of 46 sul3-positive isolates. The sul2 gene was detected in 66 isolates, 51 of them were linked to strA/B genes in seven different genetic structures. Seventy-three-per-cent of integron-positive isolates presented resistance to at least five different antimicrobial families versus 38.7% of integron-negative isolates. Our study highlights the role of commensal E. coli isolates from poultry meat as an important reservoir for sulphonamide resistance genes and integrons carrying antimicrobial resistance genes.     

Antimicrobial resistance and prevalence of virulence factor genes in fecal Escherichia coli of Holstein calves fed milk with and without antimicrobials

Diarrhea in calves has a significant effect on the dairy industry. A common management practice for preventing or decreasing the effects of such disease in preweaned calves is by the use of antimicrobials in milk or milk replacer. In this study, Escherichia coli antimicrobial resistance in fecal samples collected from calves 2 to 8 d of age that had or had not received antimicrobials in the milk and that had or had not signs of diarrhea by inspection of fecal consistency were investigated. Specifically, resistance of E. coli isolates to individual antimicrobials, multiresistance patterns, and presence of virulence factors were analyzed. Escherichia coli isolates were tested for susceptibility to 12 antimicrobials by use of a Kirby-Bauer disk diffusion assay. The study was conducted at 3 farms, 1 administering growth-promoting antimicrobials (GPA) in the milk and 2 not using GPA in the milk (NGPA). All isolates were susceptible to ciprofloxacin and cefepime. From the total isolates tested, 84% (n = 251) were resistant to at least 2 antimicrobials and 81% (n = 251) were resistant to 3 or more antimicrobials. When antimicrobial resistance was compared between GPA and NGPA, it was observed that the GPA group had higher odds of antimicrobial resistance for most of the individual antimicrobials tested. No significant correlation of virulence factors in GPA or NGPA and diarrheic or non-diarrheic (control) fecal samples was found. Of the 32 virulence factors evaluated, 21 were detected in the study population; the incidence of only 1 virulence factor was statistically significant in each of the diarrheic status (diarrheic or non-diarrheic) and treatment status (NGPA or GPA) groups. Phylogenetic analysis based on the nucleotide sequence of the DNA gyrase gene (gyrB) from 31 fecal E. coli isolates revealed 3 main clades.     

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.     

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.     

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.     

Influence of farming methods on microbiological contamination and prevalence of resistance to antimicrobial drugs in isolates from beef

The presence of Escherichia coli, Staphylococcus aureus, Listeria monocytogenes and Salmonella spp. was determined in 75 samples of conventional beef and in 75 samples of organic beef. All samples came from cattle slaughtered and processed in the same slaughterhouse and quartering room. A total of 180 E. coli, 180 S. aureus and 98 L. monocytogenes strains were analyzed by an agar disk diffusion assay for their resistance to 11 antimicrobials, for the case of E. coli and S. aureus, or 9 antimicrobials, for the case of L. monocytogenes. Salmonella spp. were not isolated from any of the beef samples. No significant differences in prevalence were obtained for any of the bacterial species tested between organic and conventional beef. E. coli isolated from organic beef exhibited significant differences in antimicrobial resistance against 5 of the 11 antimicrobials tested as compared to isolates recovered from conventional beef. In the case of S. aureus, these differences were only found for 3 of the 11 antimicrobials tested and for L. monocytogenes, no differences were obtained between isolates obtained from organic or conventional beef. Although no significant differences were obtained in microbiological contamination, E. coli and S. aureus isolates from organically farmed beef samples showed significantly lower rates of antimicrobial resistance in E. coli and S. aureus isolates.     

Comparison of antimicrobial resistance patterns and phage types of Salmonella Typhimurium isolated from pigs,pork and humans in Belgium between 2001 and 2006

Infections with non-typhoid Salmonella represent a major problem in industrialized countries.The emergence and spread of antimicrobial-resistant pathogens, among them Salmonella, has become a serious health hazard worldwide. One of the most commonly isolated non-typhoid Salmonella serovars in pigs, pork and humans is Salmonella Typhimurium. In this study the comparison of the incidences of resistance to nine antimicrobials, resistance patterns and phage types between S. Typhimurium isolated from pigs (n = 581), pork (n = 255) and humans (n = 1870) in Belgium in the period 2001 to 2006 was performed.Resistance to the antimicrobials ampicillin, chloramphenicol, streptomycin, sulfonamides and tetracycline was frequently observed and varied between 23.5% and 83.1%. Resistance ranged from 15.6% to 20.7% for the combination trimethoprim–sulfonamides and from 3.4% to 5.8% for nalidixic acid. Resistance to the critical important antimicrobials cephalosporins and fluoroquinolones was found sporadically (≤ 1.2%). Resistance to the different antimicrobials was observed to be similar in S. Typhimurium isolates from the various origins. Twenty-seven antimicrobial resistance patterns representing in total 75.2%, 89.0% and 89.6% of the isolates from pigs, pork and humans respectively were found to be common among the three groups and 73 combinations antimicrobial resistance pattern/phage type were found to be common among pork and human isolates, representing 70.1% of the pork isolates and 51.0% of the human isolates. The high percentage of isolates that have a common resistance pattern, and in a less pronounced way a common combination phage type/resistance pattern, are in agreement with the hypothesis of transfer of antimicrobial resistant Salmonella from pigs via the consumption of pork to humans as one of the possible pathways. The most prevalent combination in Belgium within both the pork isolates (7.4%) and the human isolates (13.2%) was S. Typhimurium DT104 resistant to ampicillin, chloramphenicol, streptomycine, sulfonamides and tetracycline.     

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.     

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.     

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.     

Feeding of waste milk to Holstein calves affects antimicrobial resistance of Escherichia coli and Pasteurella multocida isolated from fecal and nasal swabs

The use of milk containing antimicrobial residues in calf feeding programs has been shown to select for resistant fecal Escherichia coli in dairy calves. However, information is scarce about the effects of feeding calves waste milk (WM) on the prevalence of multidrug-resistant bacteria. The objective of this study was to determine the antimicrobial resistance patterns of fecal E. coli and nasal Pasteurella multocida isolates from calves fed either milk replacer (MR) or WM in 8 commercial dairy farms (4 farms per feeding program). Fecal and nasal swabs were collected from 20 ± 5 dairy calves at 42 ± 3.2 d of age, and from 10 of these at approximately 1 yr of age in each study farm to isolate the targeted bacteria. Furthermore, resistance of E. coli isolates from calf-environment and from 5 calves at birth and their dams was also evaluated in each study farm. Resistances were tested against the following antimicrobial agents: amoxicillin-clavulanic acid, ceftiofur, colistin, doxycycline (DO), enrofloxacin (ENR), erythromycin, florfenicol, imipenem, and streptomycin. A greater number of fecal E. coli resistant to ENR, florfenicol, and streptomycin and more multidrug-resistant E. coli phenotypes were isolated in feces of calves fed WM than in those fed MR. However, the prevalence of fecal-resistant E. coli was also influenced by calf age, as it increased from birth to 6 wk of age for ENR and DO and decreased from 6 wk to 1 yr of age for DO regardless of the feeding program. From nasal samples, an increase in the prevalence of colistin-resistant P. multocida was observed in calves fed WM compared with those fed MR. The resistance patterns of E. coli isolates from calves and their dams tended to differ, whereas similar resistance profiles among E. coli isolates from farm environment and calves were observed. The findings of this study suggest that feeding calves WM fosters the presence of resistant bacteria in the lower gut and respiratory tracts of dairy calves.     

Sheep and goats are reservoirs of colistin resistant Escherichia coli that co-resist critically important antimicrobials: First study from Jordan

There is a knowledge gap on colistin-resistant and mobilized colistin-resistant (mcr) Escherichia coli in sheep and goats worldwide. This study determined the prevalence and antimicrobial resistance of mobilized colistin-resistant (mcr) E. coli in dairy sheep and goat flocks in Jordan. A total of 948 milk samples were collected from 155 flocks across Jordan. The milk samples were pre-enriched in MacConkey broth and then plated on MacConkey agar supplemented with 8 mg/l colistin and the presence of mcr-1 gene in the isolates was tested by polymerase chain reaction. In total, 1,158 E. coli isolates were colistin resistant, with 74.8% herd-level prevalence and 39.5% individual animal prevalence. Sixty-one (5.3%) of 1,158 phenotypically colistin-resistant E. coli harbored mcr-1, with 23.9% herd-level prevalence and 6.6% individual animal prevalence. About 77.1% of the mcr-1 E. coli resisted one or more antimicrobial classes and 37.7% were multidrug resistant. More than 30% of the mcr-1 positive E. coli isolates were resistant to nalidixic acid, tetracycline, ceftazidime, cefpodoxime and ampicillin, whereas 8–20% of the isolates were resistant to cefotaxime, ceftriaxone, gentamicin, tobramycin, sulfamethoxazole-trimethoprim, and amoxicillin-clavulanic acid. This study indicates that small ruminants are reservoirs of colistin-resistant and mcr-1 positive E. coli that exhibit co-resistance to critically important antimicrobials which would pose public health issue and calls for the necessary banning of the use of colistin in small ruminants.     

Prevalence and antimicrobial susceptibility of gram-negative bacteria isolated from bovine mastitis between 2003 and 2008 in Korea

The objective of this study was to assess trends in the prevalence and distribution of gram-negative bacteria isolated from bovine mastitis and their antimicrobial susceptibilities during a 6-yr period between 2003 and 2008 in Korea. Escherichia coli, Pseudomonas fluorescens, Klebsiella pneumoniae, Enterobacter cloacae, Acinetobacter lwoffi/junii, Pseudomonas aeruginosa, and Serratia marcescens were the most commonly observed pathogens during this period. Generally, gram-negative bacteria showed low susceptibilities to most of the antimicrobials tested in this study, except amikacin and gentamicin. Although these 2 aminoglycosides were broadly active against gram-negative bacteria, less than half of those bacteria showed susceptibilities to streptomycin. The β-lactams, except piperacillin, had the lowest activity among antimicrobials tested in this study. Susceptibilities to chloramphenicol and trimethoprim were fairy high in all genera of gram-negative bacteria, except Acinetobacter spp. and Pseudomonas spp., whereas relatively high resistance to tetracycline was observed uniformly among gram-negative bacteria. There was no significant change in the prevalence of bacterial and the proportion of antimicrobial resistance among gram-negative bacteria isolates during a 6-yr period.     

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.     

Antimicrobial resistance profiles of 5 common bovine mastitis pathogens in large Chinese dairy herds

The prevalence of antimicrobial resistance (AMR) is increasing in human and animal pathogens, becoming a concern worldwide. However, prevalence and characteristics of AMR of bovine mastitis pathogens in large Chinese dairy herds are still unclear. Therefore, our objective was to determine the AMR profile of bacteria isolated from clinical mastitis in large (>500 cows) Chinese dairy herds. A total of 541 isolates of the 5 most common species, Staphylococcus aureus (n = 103), non-aureus staphylococci (NAS; n = 107), Streptococcus species (n = 101), Klebsiella species (n = 130), and Escherichia coli (n = 100), isolated from bovine clinical mastitis on 45 dairy farms located in 10 provinces of China were included. Presence of AMR was determined by minimum inhibitory concentrations using the microdilution method. Prevalence of multidrug resistance (resistance to >2 antimicrobials) was 27% (148/541). A very wide distribution of minimum inhibitory concentrations was screened in all isolates, including Staph. aureus isolates, which were resistant to penicillin (66%). In addition, NAS (30%) were more resistant than Staph. aureus to oxacillin (84%), penicillin (62%), tetracycline (34%), and clindamycin (33%). Prevalence of resistance to tetracycline was high (59%) in Streptococcus spp. Additionally, prevalence of resistance of both E. coli and Klebsiella spp. was high to amoxicillin/clavulanate potassium (81 and 38%, respectively), followed by tetracycline (only Klebsiella spp. 32%). A high proportion (27%) of isolates were multidrug resistant; the most frequent combinations were clindamycin-cefalexin-tetracycline or enrofloxacin-cefalexin-penicillin patterns for Staph. aureus; enrofloxacin-oxacillin-penicillin-tetracycline patterns for NAS; clindamycin-enrofloxacin-tetracycline patterns for Streptococcus spp.; amoxicillin/clavulanate potassium-ceftiofur-polymyxin B patterns for Klebsiella spp.; and amoxicillin/clavulanate potassium-ceftiofur-polymyxin B patterns for E. coli. Resistance for 4 kinds of antimicrobials highly critical for human medicine, including daptomycin, vancomycin, imipenem, and polymyxin B, ranged from 0 to 24%. In conclusion, prevalence of AMR in mastitis pathogens was high on large Chinese dairy farms, potentially jeopardizing both antimicrobial efficacy and public health. Results of this study highlighted the need for improvements in antimicrobial stewardship and infection control programs in large Chinese dairy farms to reduce emergence of AMR.     

Antimicrobial susceptibility and characterization of extended-spectrum β-lactamases in Escherichia coli isolated from bovine mastitic milk in South Korea from 2012 to 2015

In this study, we aimed to assess trends in antimicrobial resistance and to investigate the characteristics of extended-spectrum β-lactamase (ESBL)-producing isolates from bovine mastitic milk from 2012 to 2015. A total of 374 Escherichia coli isolates were analyzed (154 in 2012, 113 in 2013, 76 in 2014, and 31 in 2015). No consistent trends in antimicrobial resistance of E. coli isolates occurred during the 4-yr period. The most frequently observed resistance was tetracycline (23.3%), followed by streptomycin (17.1%), ampicillin (16.6%), neomycin (11.8%), and trimethoprim/sulfamethoxazole (11.2%). Multidrug resistance was observed in 15.5% of isolates. Among these isolates, 15 (4.0%) carried one or more bla_CTX-M and AmpC ESBL genes from 11 different farms, including bla_CTX-M-15 at 4 farms, bla_CTX-M-3 at 2 farms, bla_CTX-M-1 at 3 farms, and bla_CMY-2 at 3 farms. This study is the first report of bla_CTX-M-3-producing E. coli in dairy milk. Transfer of ESBL was observed in 3 bla_CTX-M-3-producing isolates, 1 bla_CTX-M-1-producing isolate, and all 3 bla_CMY-2-producing isolates. Almost all bla_CTX-M-15 and bla_CTX-M-1 genes possessed an insertion sequence, ISECP1, upstream of the bla_CTX-M gene. Identical pulsed-field gel electrophoresis profiles were also observed in bla_CTX-M-producing E. coli from the same farm. These results suggested that ESBL might spread by both clonal and horizontal spread in dairy farms in South Korea. Although no significant changes occurred in the antimicrobial resistance of E. coli during the 4-yr study period, the resistance rates and presence of ESBL were high compared with those in other countries. Thus, these findings suggest the importance of control measures for E. coli, particularly ESBL-producing bacteria, on dairy farms to reduce treatment failure and transmission to humans.