Minimum inhibitory concentrations of frequently used antibiotics against Escherichia coli and Trueperella pyogenes isolated from uteri of postpartum dairy cows

The objective of this study was to determine minimum inhibitory concentrations (MIC) of frequently used antimicrobials for Escherichia coli and Trueperella pyogenes isolated from postpartum bovine uteri of cows with acute puerperal metritis (APM, n = 67), cows suspected to have APM (n = 37), and healthy cows (n = 37) and to evaluate possible differences in MIC according to clinical signs. Cows with APM had reddish-brown, fetid vaginal discharge and rectal temperature (RT) ≥39.5°C within 21 d in milk; cows suspected to have APM had either reddish-brown, fetid vaginal discharge or RT ≥39.5°C within 21 d in milk; and healthy cows had neither fetid discharge nor RT ≥39.5°C. Samples were collected from cows on commercial dairy herds (n = 7) using the cytobrush technique. A total of 37 T. pyogenes isolates and 85 E. coli isolates were tested. Ceftiofur, a third-generation cephalosporin that is often used to treat APM, was the focus of analysis. Trueperella pyogenes and E. coli were isolated more often from samples of cows with APM (46 and 90%, respectively) compared with samples from healthy cows (19 and 54%, respectively). Regarding cows suspected to have APM, T. pyogenes and E. coli were numerically more often isolated (30 and 70%, respectively) than in healthy cows (19 and 54%, respectively). Minimum inhibitory concentrations of ceftiofur were low. For T. pyogenes and E. coli, MIC₅₀ (concentration that inhibited growth of 50% of isolates) were 0.25 and 0.5 µg/mL and MIC₉₀ (concentration that inhibited growth of 90% of isolates) were 0.5 and 1 µg/mL, respectively. Although ceftiofur inhibited all T. pyogenes at the highest concentration tested (64 µg/mL), the growth of 5.9% of E. coli was not impaired. Recently, ampicillin has been suggested as an alternative treatment for APM. Although the T. pyogenes isolates exhibited low MIC in general (MIC₅₀ ≤0.015 µg/mL and MIC₉₀ = 0.06 µg/mL) and 81.1% of all T. pyogenes could be inhibited at the lowest ampicillin concentration tested, 11.8% of the E. coli isolates were not impaired at the highest concentration (64 µg/mL) tested in this study. The MIC₅₀ and MIC₉₀ of E. coli were 4 and ≥128 µg/mL, respectively. We detected no difference in the MIC distributions of ceftiofur or ampicillin among isolates from the 3 APM groups. In summary, E. coli with high MIC against ceftiofur as well as against ampicillin were found in this study.     

Short communication: Fluoroquinolone susceptibility of Staphylococcus aureus strains isolated from caprine clinical mastitis in southeast Spain

The antibiotic susceptibility of 32 strains of Staphylococcus aureus isolated from the mastitic milk of dairy goats was evaluated. The antibiotics tested were 3 fluoroquinolones that have been developed especially for use in veterinary medicine: danofloxacin, marbofloxacin, and orbifloxacin. Minimum inhibitory concentration (MIC) tests were performed according to the microdilution broth method. The MIC₉₀ (concentration at which 90% inhibition is achieved) values obtained were 0.5, 1, and 1 mg/L for danofloxacin, marbofloxacin, and orbifloxacin, respectively. Danofloxacin was the most active fluoroquinolone tested against Staph. aureus strains isolated from milk; however, specific testing is required before using these drugs as therapy for the control of clinical mammary infections in goats.     

Prevalence and characterization of Escherichia coli O157:H7 isolates from meat and meat products sold in Amathole District,Eastern Cape Province of South Africa

Meat and meat products have been implicated in outbreaks of Escherichia coli O157:H7 in most parts of the world. In the Amathole District Municipality of the Eastern Cape Province of South Africa, a large number of households consume meat and meat products daily, although the microbiological quality of these types of food is questionable. The present study investigated the prevalence of E. coli O157:H7 isolated from selected meat and meat products (45 samples each of biltong, cold meat, mincemeat, and polony) sold in this area. Strains of E. coli O157:H7 were isolated by enrichment culture and confirmed by polymerase chain reaction (PCR). Also investigated were the antibiogram profiles of the E. coli O157:H7 isolates. Five (2.8%) out of 180 meat and meat products examined were positive for E. coli O157:H7 that carried the fliC_H7, rfbE_O157, and eaeA genes. Two of the E. coli O157:H7 isolates were resistant against all the eight antibiotics tested. To prevent E. coli O157:H7 infections, meat and meat products such as biltong, cold meat, mincemeat and polony should be properly handled, and packed in sterile polyvinyl wrappers.     

Investigation of some factors affecting the antibacterial activity of rosemary extracts in food models by a food microdilution method

The activity of rosemary phenolic extracts against Listeria monocytogenes and Escherichia coli was evaluated in 50% food models of meat, vegetable and dairy products in relation to some factors that can affect MIC (minimal inhibitory concentration) values (inoculum level, proportion of food, temperature) using a new food microdilution method. It was shown that the interactions of meat and milk components with plant extracts reduced the antibacterial effectiveness of rosemary extracts. MIC values for L. monocytogenes were lower than for E. coli in all tested conditions. A lower inoculation level caused a decrease in MIC values for E. coli but an increase in MIC values for L. monocytogenes in control media. In food models, MIC values were higher or equal to MIC values in control media regardless of bacterial type. We showed that the food microdilution method represents a simple, rapid, reproducible and inexpensive method for testing the antimicrobial efficiency of plant extracts in food systems.     

Antimicrobial activity of crude extracts from actinomycetes against mastitis pathogens

The emergence of antimicrobial resistance to commonly used antibiotics has necessitated the development of new antimicrobial products. Crude extracts produced by actinomycetes contain antimicrobial metabolites that can inhibit bacterial growth. The objective of our study was to evaluate the antimicrobial activity of crude extracts (Caat1–54 and CaatP5–8) produced by actinomycetes against isolates of Staphylococcus aureus, Staphylococcus chromogenes, Streptococcus dysgalactiae, and Streptococcus uberis, which were obtained from the milk of cows affected by mastitis in 23 dairy herds. Twenty isolates of each bacterial species were used to define minimum inhibitory concentrations (MIC) of both crude extracts and ceftiofur (positive control). The MIC₅₀ and MIC₉₀ were defined at the concentration required to inhibit the growth of 50 and 90% of bacterial isolates tested, respectively. The MIC results were evaluated by survival analysis. Staphylococcus aureus isolates presented MIC₉₀ of Caat 1–54 ≥6.25 µg/mL, ceftiofur ≥12.5 µg/mL, and Caat P5–8 ≥100 µg/mL. Streptococcus uberis presented MIC₉₀ of ceftiofur ≥0.39 µg/mL, Caat 1–54 ≥50 µg/mL, and Caat P5–8 ≥100 µg/mL. Staphylococcus chromogenes isolated from subclinical mastitis presented MIC₉₀ of Caat 1–54 ≥0.78 µg/mL and ceftiofur and Caat P5–8 of ≥6.25 and ≥100 µg/mL, respectively. Streptococcus dysgalactiae isolated from clinical mastitis presented similar MIC₉₀ values between antimicrobials tested (ceftiofur, Caat 1–54, and Caat P-58), but these values (≥100 µg/mL) were higher than the values obtained from other pathogens evaluated in the present study. Our results indicate that Caat 1–54 and Caat P5–8 crude extracts present in vitro antimicrobial activity against isolates of Staph. aureus, Staph. chromogenes, Strep. dysgalactiae, and Strep. uberis isolated from clinical and subclinical mastitis.     

Antibacterial effect of plant-derived antimicrobials on major bacterial mastitis pathogens in vitro

The objective of this study was to investigate the antimicrobial effect of plant-derived antimicrobials including trans-cinnamaldehyde (TC), eugenol, carvacrol, and thymol on major bacterial mastitis pathogens in milk. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the aforementioned compounds on Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Staphylococcus aureus, and Escherichia coli were determined. In addition, the bactericidal kinetics of TC on the aforementioned pathogens and the persistence of the antimicrobial activity of TC in milk over a period of 2 wk were investigated. All 4 plant-derived molecules exhibited antimicrobial activity against the 5 mastitis pathogens tested, but TC was most effective in killing the bacteria. The MIC and MBC of TC on Staph. aureus, E. coli, and Strep. uberis were 0.1 and 0.45%, respectively, whereas that on Strep. agalactiae and Strep. dysgalactiae were 0.05 and 0.4%, respectively. The MIC and MBC of the other 3 molecules ranged from 0.4 to 0.8% and 0.8 to 1.5%, respectively. In time-kill assays, TC at the MBC reduced the bacterial pathogens in milk by 4.0 to 5.0 log₁₀ cfu/mL and to undetectable levels within 12 and 24 h, respectively. The antimicrobial effect of TC persisted for the duration of the experiment (14 d) without any loss of activity. Results of this study suggest that TC has the potential to be evaluated as an alternative or adjunct to antibiotics as intramammary infusion to treat bovine mastitis.     

Baicalin inhibits Escherichia coli isolates in bovine mastitic milk and reduces antimicrobial resistance

In this study, we aimed to evaluate the inhibitory effect of baicalin on Escherichia coli in vitro and the effects of baicalin treatment on antimicrobial resistance of the E. coli isolates. Through isolation, purification, and identification, a total of 56 E. coli strains were isolated from 341 mastitic milk samples. The study of inhibition effect of baicalin on the E. coli strains in vitro was focused on permeability and morphology of the isolates using an alkaline phosphatase kit and scanning electron microscopy. Furthermore, the resistance spectrum of the isolates to the common antimicrobial agents was tested at sub-minimum inhibitory concentrations of baicalin by the agar dilution method. Extended-spectrum β-lactamase and plasmid-mediated quinolone resistance genes were amplified by PCR before and after incubation with baicalin. The results revealed that baicalin has certain inhibitory effects on the isolates in vitro. The alkaline phosphatase enzyme activity was significantly increased from 1.246 to 2.377 U/100 mL, and the surface of E. coli was concave and shriveled. Analysis of the resistance spectrum and PCR amplification showed that, after administration with baicalin, the sensitivity of most strains to the selected antimicrobial agents was enhanced. Strikingly, the drug-resistant genes from 71.43% (40/56) of these isolates were found to have drug-resistant genes to different extents. Altogether, the current study confirmed both the inhibitory effect on Escherichia coli in vitro and the reduction of antimicrobial resistance by baicalin. This is the first comprehensive study to report on baicalin, a traditional Chinese medicine that acts on E. coli isolated from the mastitic milk samples.     

Antimicrobial activities of polyhexamethylene biguanide against biofilm-producing Prototheca bovis causing bovine mastitis

Prototheca spp. is a frequent cause of bovine mastitis and is highly resistant to commonly used disinfectants. This study aimed to: (1) evaluate the antimicrobial activity of polyhexamethylene biguanide (PHMB) against mastitis-causing Prototheca spp., and (2) evaluate the biofilm production ability of Prototheca spp. A total of 85 Prototheca bovis and 2 Prototheca blaschkeae isolates from bovine mastitis cases were submitted to biofilm production assays and antimicrobial susceptibility tests against PHMB and disinfectants commonly used in dairy herds (chlorhexidine digluconate, povidone-iodine, sodium dichloroisocyanurate, and sodium hypochlorite). The minimal inhibitory concentration (MIC) and minimal algicidal concentration (MAC) were determined by microdilution assays. We observed that PHMB (MIC₉₀: ≥2 µg/mL and MAC₉₀: ≥4 µg/mL) and chlorhexidine gluconate (MIC₉₀ and MAC₉₀: ≥2 µg/mL) presented the highest antimicrobial activity against P. bovis isolates, followed by sodium dichloroisocyanurate (MIC₉₀ and MAC₉₀: ≥1,400 µg/mL), sodium hypochlorite (MIC₉₀ and MAC₉₀: ≥2,800 µg/mL), and povidone-iodine (MIC₉₀ and MAC₉₀: ≥3,200 µg/mL). Concerning P. blaschkeae isolates, PHMB (MIC and MAC ≥1 µg/mL) and chlorhexidine gluconate (MIC and MAC ≥1 µg/mL) were the disinfectants that presented the lowest concentration values required to inhibit the isolates. Regarding biofilms formation, 63.5% (n = 54/85) of the P. bovis isolates were classified as strong, 28.3% (n = 24/85) moderate, and 8.2% (n = 7/85) weak biofilm producers. In contrast, the P. blaschkeae isolates were classified as weak and moderate biofilm producers. These findings suggest that PHMB has the potential to be used for teat and milking-equipment disinfection for the prevention of mastitis-causing Prototheca spp. in dairy herds.     

Technical note: Antimicrobial susceptibility of Portuguese isolates of Staphylococcus aureus and Staphylococcus epidermidis in subclinical bovine mastitis

To evaluate the antimicrobial resistance traits of staphylococci responsible for subclinical bovine mastitis in Portugal, the minimum inhibitory concentrations (MIC) of 7 antimicrobial agents, frequently administered for mastitis treatment, were determined for 30 Staphylococcus aureus and 31 Staphylococcus epidermidis field isolates. β-Lactamase production was detected through the use of nitrocefin-impregnated discs. The MIC that inhibited 90% of the isolates tested (MIC₉₀) of penicillin, oxacillin, cefazolin, gentamicin, sulfamethoxazole/trimethoprim, oxytetracycline, and enrofloxacin were, respectively, 4, 0.5, 1, 1, 0.25, 0.25, and 0.06 μg/mL for Staph. aureus and ≥64, 8, 1, 32, ≥64, ≥64, and 0.06 μg/mL for Staph. epidermidis. All Staph. aureus isolates showed susceptibility to oxacillin, cefazolin, gentamicin, sulphamethoxazole/trimethoprim, and enrofloxacin. β-Lactamase production was detected in 20 of these isolates (66.7%), all of which were resistant to penicillin. Of the 31 Staph. epidermidis tested, 24 (77.4%) were β-lactamase positive. All isolates were susceptible to both cefazolin and enrofloxacin. Nine Staph. epidermidis isolates were resistant to oxacillin, with MIC values ranging from 4 to 8 μg/mL. The MIC values of 5 antimicrobial agents tested were higher than those reported in other countries. Enrofloxacin was the only exception, showing lower MIC values compared with other reports. Overall, the antimicrobial agents tested in our study, with the exception of penicillin, were active against the 61 isolates studied.     

Negatively controlled,randomized clinical trial to evaluate intramammary treatment of nonsevere,gram-negative clinical mastitis

The objective of this negatively controlled, randomized clinical trial was to examine clinical outcomes of 2-d or 8-d treatment using an approved intramammary (IMM) product containing ceftiofur hydrochloride compared with no antimicrobial treatment of nonsevere, gram-negative cases of clinical mastitis (CM). Additionally, we contrasted clinical outcomes of cases caused by Escherichia coli (n = 56) or Klebsiella pneumoniae (n = 54). Cases (n = 168) of nonsevere (abnormal milk or abnormal milk and udder) CM were randomly assigned to receive 2 d (n = 56) or 8 d (n = 56) of IMM ceftiofur or assigned to a negative control group (n = 56). At enrollment, quarter milk samples were collected and used for on-farm culture, somatic cell count (SCC), and confirmatory microbiological analysis. Quarter milk samples were collected weekly from 7 to 28 d after enrollment for microbiological and SCC analysis. Clinical outcomes were followed for 90 d or until the end of lactation (follow-up period, FUP). Overall, no significant differences in quarter-level recurrence of CM (32% for negative control, 34% for the 2-d treatment, and 32% for the 8-d treatment), culling (18% for negative control, 12% for 2-d treatment, and 11% for 8-d treatment), voluntary dry-off of affected quarters (20% for negative control, 30% for 2-d treatment, and 27% for 8-d treatment), days until return to normal milk (4.2 days for negative control, 4.8 days for 2-d treatment, 4.5 days for 8-d treatment), weekly quarter-SCC during the FUP (6.1, 6.3, and 6.0 log₁₀SCC for the negative control, 2-d, and 8-d treatments, respectively), or daily milk yield during the FUP (37.1, 36.3, and 37.6 kg/cow per day for the negative control, 2-d, and 8-d treatments, respectively) were observed among experimental groups. Days of discarded milk were greater for cows assigned to 8-d IMM ceftiofur (11.1 d) than for cows assigned to 2-d (6.9 d) or cows assigned to negative control (5.6 d). Bacteriological cure (BC) at 14 and 21 d after enrollment was greater in cows assigned to 8-d (89%) and 2-d (84%) treatment than in cows assigned to negative control (67%), but this outcome was confounded by pathogen. For CM caused by Kleb. pneumoniae, BC was greater for quarters assigned to receive treatment (combined 2-d and 8-d groups; 74% BC) than for quarters assigned to negative control (18%). In contrast, no differences in BC were observed for CM caused by E. coli (97–98%). Culling and voluntary dry-off of affected quarters were significantly greater for cows with quarters affected by Kleb. pneumoniae (22% culled, 39% voluntary dry-off of quarters) than for cows with quarters affected with E. coli (7% culled, 11% voluntary dry-off of quarters). Overall, use of IMM ceftiofur did not result in improvement of most clinical outcomes, but differences between E. coli and Kleb. pneumoniae were evident. In contrast to E. coli, Kleb. pneumoniae caused chronic intramammary infection and induced worse clinical outcomes. Intramammary antibiotic treatment of most mild and moderate cases of CM caused by E. coli is not necessary, but more research is needed to identify which quarters affected by Kleb. pneumoniae may benefit from antimicrobial therapy.     

Antibiotic susceptibility of members of the Lactobacillus acidophilus group using broth microdilution and molecular identification of their resistance determinants

The range of antibiotic susceptibility to 13 antibiotics in 101 strains of the Lactobacillus acidophilus group was examined using the lactic acid bacteria susceptibility test medium (LSM) and broth microdilution. Additionally, microarray analysis and PCR were applied to identify resistance genes responsible for the displayed resistant phenotypes in a selection of strains. In general, narrow as well as broad unimodal and bimodal MIC distributions were observed for the Lactobacillus acidophilus group and the tested antimicrobial agents. Atypically resistant strains could be determined by visual inspection of the obtained MIC ranges for ampicillin, chloramphenicol, clindamycin, erythromycin, quinupristin/dalfopristin, streptomycin and tetracycline. For most of these atypically resistant strains underlying resistance determinants were found. To our knowledge erm(A) was detected in lactobacilli for the first time within this study. Data derived from this study can be used as a basis for reviewing present microbiological breakpoints for categorization of susceptible and resistant strains within the Lactobacillus acidophilus group to assess the safety of microorganisms intended for use in food and feed applications.     

The hygienic condition of manufacturing beef destined for the manufacture of hamburger patties

The hygienic condition of the manufacturing beef collected at six carcass breaking plants for dispatch to hamburger patty manufacturing plants was examined. At each plant, 24×1kg samples of meat were selected at random from the product being collected into bulk containers. Total aerobic, coliform andEscherichia colicounts per gram were enumerated for each sample. The log mean (X) and standard deviation (s.d.) were calculated for the log₁₀values of each set of 24 counts, on the assumption that the distribution of counts approximated the log normal. A value for the log₁₀of the arithmetic mean (log A) was calculated for each set from the values for X and s.d. Log A values for total, coliform andE. colicounts ranged from 3.5 to 4.9, 0.7 to 3.0 and 0.2 to 2.6 log₁₀cfu g⁻¹, respectively. For each set of samples, there was a weak or no correlation between log₁₀values for total counts and those for coliforms orE. coli, but correlations between log₁₀values for coliform andE. colicounts ranged from non (R²=0.19) to close (R²=0.97). The results show that there are large differences between plants in the numbers ofE. coliin the manufacturing beef which they produce. The differences inE. colinumbers are not reliably reflected by differences between total or coliform counts in product from different plants. In addition, the findings indicate that manufacturing beef obtained from culled cow carcasses may generally be less heavily contaminated withE. colithan the trimmings obtained from the carcasses of feedlotted steers. Clearly, some current processes for the production of manufacturing beef destined for hamburger patty manufacture are poorly controlled with respect to minimizing the contamination of product withE. coliand, presumably, other faecal bacteria. The methods used in this study offer a means of objectively identifying such hygienically inadequate processes.     

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.     

Short communication: In vitro antimicrobial susceptibility of Mycoplasma agalactiae strains isolated from dairy goats

This study examined the susceptibility to several antimicrobials of 28 isolates of Mycoplasma agalactiae obtained from goats in a region (southeastern Spain) where contagious agalactia is endemic. For each isolate, the minimum inhibitory concentration (MIC) against 12 antimicrobials of the quinolone, macrolide, aminoglycoside, and tetracycline families was determined. The antimicrobials with the lowest MIC were enrofloxacin, ciprofloxacin, tylosin, and doxycycline, all with MIC₉₀ (concentration at which growth of 90% of the isolates is inhibited) <1 µg/mL. Norfloxacin (a quinolone) showed a wide MIC range (0.1–12.8 µg/mL), suggesting a resistance mechanism toward this antimicrobial that was not elicited by enrofloxacin or ciprofloxacin (the other quinolones tested). Erythromycin showed the highest MIC₉₀ such that its use against Mycoplasma agalactiae is not recommended. Finally, Mycoplasma agalactiae isolates obtained from goat herds with clinical symptoms of contagious agalactia featured higher MIC₉₀ and MIC₅₀ (concentration at which growth of 50% of the isolates is inhibited) values for many of the antimicrobials compared with isolates from asymptomatic animals. The relationship between the extensive use of antimicrobials in herds with clinical contagious agalactia and variations in MIC requires further study.     

Antimicrobial resistance in <Emphasis Type="Italic">Escherichia coli</Emphasis> strains isolated from organic and conventional pork meat: a comparative survey

Mean counts of Escherichia coli were determined for 54 samples of organic pork meat, and in 67 samples of conventional pork meat. Up to three E. coli strains from each sample were analysed by an agar disk diffusion assay for their resistance to ampicillin, chloramphenicol, cephalotin, doxycycline, enrofloxacin, gentamicin, nitrofurantoin, sulfisoxazole and streptomycin by the agar disk diffusion method. Results indicated that the presence of E. coli was significantly (P < 0.05) higher in organic pork meat as compared to conventional pork meat. Isolates from organic pork meat exhibited lower levels of antimicrobial resistance against ampicillin (P < 0.0001), doxycycline (P < 0.0001) and sulfisoxazole (P < 0.0001), as compared to isolates from conventional meat. Moreover, presence of multi-resistant E. coli strains was significantly (P < 0.0001) higher in conventional pork meat as compared to organic pork meat, the largest differences being observed for isolates resistant to combinations of ampicillin, sulfisoxazol and/or doxycycline. Organically-farmed pork samples showed significantly lower development of antimicrobial resistance in E. coli, thus contributing to reduce the development and spread of antimicrobial resistance among these food-borne bacteria.     

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.     

Aerobic bacterial, coliform, Escherichia coli and Staphylococcus aureus counts of raw and processed milk from selected smallholder dairy farms of Zimbabwe

A cross sectional study was conducted to enumerate total viable bacteria (TBC), coliforms, Escherichia coli and Staphylococcus aureus in raw (n = 120) and processed (n = 20) milk from individual farms from three smallholder dairy schemes of Zimbabwe between October, 2009 and February, 2010. Data on management factors were collected using a structured questionnaire. A standard pour plate technique was used to enumerate total viable bacteria, while for coliforms, E. coli and S. aureus, counts were assessed by the spread plate technique. The association of total viable bacterial counts and management factors was assessed using univariable and a linear regression model. The log₁₀ TBC for raw milk differed significantly (P < 0.05) amongst the schemes with the lowest (5.6 ± 4.7 log₁₀ cfu/ml) and highest (6.7 ± 5.8 log₁₀ cfu/ml) recorded from Marirangwe and Nharira respectively. The mean log₁₀ of TBC of processed milk (6.6 ± 6.0 log₁₀ cfu/ml) were marginally higher than those of raw milk (6.4 ± 5.6 log₁₀ cfu/ml) but not significant (P > 0.05). The coliform, E. coli and S. aureus counts for raw milk significantly differed (P < 0.05) amongst the study areas. The variation in TBC, coliforms, E. coli and S. aureus counts amongst the schemes could be attributed to differences in milking hygiene where farms with more access to training and monitoring of microbiological quality of milk had lower counts. Linear regression analysis revealed dairy scheme, delivery time and season of milking as independently associated with increased TBC of raw milk. The high TBC of raw and processed milk generally indicated low levels of milking hygienic practices, and high level of post-processing contamination, respectively. The high TBC, coliform, E. coli and S. aureus counts of both raw and processed milk may present a public health hazard. Thus, educating the farmers on general hygienic practices, quickening the delivery of milk to collection centres, or availing cooling facilities on-farm will improve the microbiological quality and safety of milk.     

Short communication: Quantitative PCR coupled with sodium dodecyl sulfate and propidium monoazide for detection of culturable Escherichia coli in milk

Escherichia coli has been frequently reported as a major foodborne bacterium contaminating raw milk or pasteurized milk. Therefore, the aim of this study was to explore a quantitative real-time PCR (qPCR) technique combined with sodium dodecyl sulfate (SDS) and propidium monoazide (PMA) to detect culturable E. coli in milk. An internal amplification control was also added into this reaction system as an indicator of false-negative results. The inclusivity and exclusivity of the primers were tested using DNA from 7 E. coli and 14 other bacterial strains. The concentrations of SDS and PMA were determined according to plate counts and quantitative cycle values of qPCR, respectively. A standard curve was established using series diluted E. coli DNA. The reliability and specificity of this method were further determined by the detection of E. coli in spiked milk. The results showed that the optimal concentrations of SDS and PMA were 100 µg/mL and 40 μM, respectively. A standard curve with a good linear relationship (coefficient of determination = 0.997; amplification efficiency = 100.5%) was obtained. Compared with conventional PCR and PMA-qPCR, the SDS-PMA-qPCR assay was more specific and sensitive in culturable E. coli detection. Therefore, we evaluated and improved the SDS-PMA-qPCR method for detecting culturable E. coli in milk.     

Enhancement Antimicrobial Activity of Hyphothiocyanite Using Carrot Against Staphylococcus Aureus and Escherichia Coli

Hypothiocyanite has been known as antimicrobial agent that was generated from lactoperoxidase system (LPOS) but its antimicrobial activity was low against pathogenic bacteria in milk. This research has been done to enhance the antimicrobial activity of hypothiocyanite against pathogenic bacteria commonly found in milk: Staphylococcus aureus and Escherichia coli by addition of carrot extract. The result showed that carrot extract was able to enhance the antimicrobial activity of hypothiocyanite strongly against E. coli, however less enhancement has been found in the antibacterial activity of hypothiocyanite against S. aureus.     

Antibacterial effects of non-thermal dielectric barrier discharge plasma against Escherichia coli and Vibrio parahaemolyticus on the surface of wooden chopping board

In this study, the effects of atmospheric dielectric barrier discharge (DBD) plasma treatment (1.1 kV, 43 kHz, 5–30 min, N₂: 1.5 lpm) were investigated for the inactivation of Escherichia coli and Vibrio parahaemolyticus on wooden chopping board (WCB) surfaces. A reduction of 0.4–1.6 and 0.4–1.3 log CFU/coupon was observed for E. coli and V. parahaemolyticus, respectively, on the WCB surface by DBD plasma treatment. The first-order kinetics model was used to generate linear survival curves and calculate D-values and R² for the bacteria; D-values were 41.0 min and 59.8 min, and R² values were 0.94 and 0.97 for E. coli and V. parahaemolyticus, respectively. Based on these findings, DBD plasma could potentially serve as a new antimicrobial method for wooden cutting boards.