Prevalence of shiga toxin-producing Escherichia coli in dairy cattle and their products

The main objective of this review was to assess the role of dairy cattle and their products in human infections with Shiga toxin-producing Escherichia coli (STEC). A large number of STEC strains (e.g., members of the serogroups O26, O91, O103, O111, O118, O145, and O166) have caused major outbreaks and sporadic cases of human illnesses that have ranged from mild diarrhea to the life-threatening hemolytic uremic syndrome. These illnesses were traced to O157 and non-O157 STEC. In most cases, STEC infection was attributed to consumption of ground beef or dairy products that were contaminated with cattle feces. Thus, dairy cattle are considered reservoirs of STEC and can impose a significant health risk to humans. The global nature of food supply suggests that safety concerns with beef and dairy foods will continue and the challenges facing the dairy industry will increase at the production and processing levels. In this review, published reports on STEC in dairy cattle and their products were evaluated to achieve the following specific objectives: 1) to assemble a database on human infections with STEC from dairy cattle, 2) to assess prevalence of STEC in dairy cattle, and 3) to determine the health risks associated with STEC strains from dairy cattle. The latter objective is critically important, as many dairy STEC isolates are known to be of high virulence. Fecal testing of dairy cattle worldwide showed wide ranges of prevalence rates for O157 (0.2 to 48.8%) and non-O157 STEC (0.4 to 74.0%). Of the 193 STEC serotypes of dairy cattle origin, 24 have been isolated from patients with hemolytic uremic syndrome. Such risks emphasize the importance and the need to develop long-term strategies to assure safety of foods from dairy cattle.     

Multidrug residues and antimicrobial resistance patterns in waste milk from dairy farms in Central California

Waste milk (WM) is a common source of feed for preweaned calves in US dairy farms. However, limited information is available about characteristics of this product, including concentration of drug residues and potential hazards from antibiotic-resistant bacteria present in the milk. The aims of this cross-sectional study were to (1) identify and measure the concentration of antimicrobial residues in raw WM samples on dairy farms in the Central Valley of California, (2) survey farm management practices for factors associated with the occurrence of specific antimicrobial residues in raw WM, (3) characterize the antimicrobial resistance patterns of E. coli cultured from raw WM samples, and (4) evaluate the potential association between WM quality parameter and risk of identifying drug residues in milk. A single raw bulk tank WM sample was collected from dairy farms located in California's Central Valley (n = 25). A questionnaire was used to collect information about farm management practices. Waste milk samples were analyzed for a multidrug residue panel using liquid chromatography–tandem mass spectrometry. Bacteria were cultured and antimicrobial resistance was tested using standard techniques; milk quality parameters (fat, protein, lactose, solids-not-fat, somatic cell count, coliform count, and standard plate count) were also measured. Of the 25 samples collected, 15 (60%) contained detectable concentrations of at least 1 antimicrobial. Of the drug residue–positive samples, 44% (11/25) and 16% (4/25) had detectable concentrations of β-lactams and tetracycline, respectively. The most prevalent drug residues were ceftiofur (n = 7, 28%), oxytetracycline (n = 4, 16%), and cephapirin (n = 3, 12%). No significant associations were identified between farm characteristics or management practices and presence of drug residues in WM. In this study, 20% of farms did not pasteurize WM before feeding to calves. Two of the 10 Escherichia coli isolated from WM samples were multidrug resistant. Streptococcus spp. (n = 21, 84%) was the most common genus cultured from WM samples, followed by Staphylococcus spp. (n = 20, 80%) and E.coli (n = 10, 40%). Mycoplasma spp. was cultured from 2 WM samples (n = 2, 8%). The presence of drug residues in WM at concentrations that increase selection of resistant bacteria indicates the need for additional studies targeting on-farm milk treatments to degrade drug residues before feeding to calves. The presence of multidrug-resistant E. coli in WM urges the need for on-farm practices that reduce calf exposure to resistant bacteria, such as pasteurization.     

Meta-analysis of the effects of sulfate versus hydroxy trace mineral source on nutrient digestibility in dairy and beef cattle

Trace mineral (TM) source can potentially alter nutrient digestibility through effects on microbial populations. A meta-analysis was conducted to determine whether sulfate versus hydroxy (IntelliBond) sources of supplemental Cu, Zn, and Mn had any effect on dry matter intake (DMI), dry matter digestibility, and neutral detergent fiber (NDF) digestibility. All available cattle studies (8 studies, 12 comparisons) were used to estimate the effect size (hydroxy mean − sulfate mean). Factors included in the analysis were method of digestibility analysis (total collection, marker-based, or 24 h in situ), study design (randomized design or Latin square), beef (n = 5) versus dairy (n = 7) cattle, and days on treatment; these factors were retained when P < 0.05. Dry matter digestibility was increased by hydroxy TM in beef (1.64 ± 0.35 units) but not in dairy models (0.16 ± 0.13 units) relative to sulfate TM. The NDF digestibility increased significantly with hydroxy versus sulfate TM, but digestibility assessment method influenced this response. Studies using total collection or undigested NDF as a flow marker showed a significant increase (2.68 ± 0.40 units and 1.08 ± 0.31 units, respectively) in NDF digestibility for hydroxy versus sulfate TM; but studies utilizing 24-h in situ incubation did not detect any change (−0.03 ± 0.23 units). These observations may reveal differences in precision of measurement or may indicate mineral effects beyond the rumen; total collection is considered the gold standard method. Hydroxy TM did not affect DMI per animal or per unit of body weight relative to sulfate TM. In conclusion, feeding hydroxy versus sulfate TM does not appear to affect DMI but, depending on type of cattle and method of measurement, can increase dry matter digestibility and NDF digestibility, which may be explained by differences in solubility of the TM sources in rumen, differentially affecting fermentation.     

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.     

Escherichia coli O26 in minced beef: prevalence, characterization and antimicrobial resistance pattern

Verocytotoxin-producing Escherichia coli (VTEC) non-O157 serogroups are among the most important emerging food-borne pathogen groups. In particular, the O26 serogroup is able to cause a large spectrum of illnesses in humans which have a significant public health impact as they may range from haemorrhagic colitis (HC) to haemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). It is known that VTEC organisms are associated with animal reservoirs, i.e. ruminants, and foods of animal origin, especially undercooked meat and raw milk, are often involved in outbreaks. In this study, 250 minced beef samples collected at retail outlets in southern Italy were tested for the presence of E. coli O26 and the isolates were characterized and studied for their antimicrobial resistance properties. Three minced beef samples (1.2%) tested positive for E. coli O26; one isolate per positive sample was characterized. One isolate harboured the genes encoding for virulence factors intimin (eaeA) and enterohaemolysin (hlyA), while none presented verocytotoxin-encoding genes (stx1 and stx2) and all were negative at the verotoxicity assay. All the isolates showed resistance properties to at least four antimicrobial agents tested and two were multi-drug resistant (MDR). Although no verocytotoxin-encoding genes were found in the isolates, the presence of potentially pathogenic E. coli O26 strains in minced beef points to the need for proper hygiene during meat production to reduce the risk of food-borne illnesses and transmission of MDR organisms via foods to humans. This paper is the first report on the presence and characterization of E. coli O26 in minced beef marketed in Italy.     

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 drug resistance of Salmonella and Escherichia coli isolates from cattle feces, hides, and carcasses

To determine patterns of cross-contamination and antibiotic susceptibility of microorganisms commonly associated with cattle, 60 cattle shipped to a commercial abattoir (20 in each of three separate trial periods) were followed through processing. Samples for bacterial isolation were collected from the feces and hides immediately before shipping, from the hides at the abattoir after exsanguination, and from the carcasses before evisceration and in the cooler. Samples were cultured for Salmonella and non-type-specific Escherichia coli. Salmonella was identified in 33.9% (n = 20) of the fecal samples and on 37.3% (n = 22) of the hides before shipment. At the abattoir, the proportion of hides from which Salmonella was isolated increased (P < 0.001) to 84.2% (48 hides). Nonspecific E. coli and Salmonella were recovered from 40.4 and 8.3% of preevisceration carcass samples, respectively. No Salmonella or nonspecific E. coli were recovered from hotbox carcass samples. Isolates were tested for antimicrobial drug susceptibility. For nonspecific E. coli, 80.3% (n = 270) of the isolates were resistant to at least one antimicrobial drug. For Salmonella, 97% (n = 101) of the isolates were resistant to at least one antimicrobial drug; however, only 4.0% were resistant to two or more. The most common resistance was to sulfamethoxazole. These results indicate that the presence of microorganisms resistant to antimicrobial drugs is common in cattle and beef. Further studies are needed to identify the sources and causes of this drug resistance.     

Changes in the antimicrobial resistance profiles of Salmonella isolated from the same Michigan dairy farms in 2000 and 2009

This study compared the antimicrobial resistance profiles of Salmonella collected from the same Michigan, USA dairy farms between the years 2000 and 2009. The specific objective was to understand the type and distribution of changes in antimicrobial resistance that occurred within farms over the past 10 years. Multinomial, multilevel models were constructed to estimate the differences in minimum inhibitory concentrations (MICs) between years. The MICs of most antimicrobials were significantly lower in 2009 than in 2000, but were higher for amikacin and gentamicin. Decreases in MICs were in part due to changes in the prevalence of multidrug resistant strains, but were also distributed across the susceptible population of isolates. The type and direction of within-farm changes in MICs were similar for the majority of farms. These results suggest a decrease in antimicrobial resistance and/or a change in the population structure of Salmonella that colonize dairy farms in Michigan.     

Estimates of daily oxygen consumption,carbon dioxide and methane emissions,and heat production for beef and dairy cattle using spot gas sampling

A simulation study was conducted to examine accuracy of estimating daily O₂ consumption, CO₂ and CH₄ emissions, and heat production (HP) using a spot sampling technique and to determine optimal spot sampling frequency (FQ). Data were obtained from 3 experiments where daily O₂ consumption, emissions of CO₂ and CH₄, and HP were measured using indirect calorimetry (respiration chamber or headbox system). Experiment 1 used 8 beef heifers (ad libitum feeding; gaseous exchanges measured every 30 min over 3 d in respiration chambers); Experiment 2 used 56 lactating Holstein-Friesian cows (restricted feeding; gaseous exchanges measured every 12 min over 3 d in respiration chambers); Experiment 3 used 12 lactating Jersey cows (ad libitum feeding; gaseous exchanges measured every hour for 1 d using headbox style chambers). Within experiment, averages of all measurements (FQALL) and averages of measurements selected at time points with 12, 8, 6, or 4 spot sampling FQ (i.e., sampling every 2, 3, 4, and 6 h in a 24-h cycle, respectively; FQ12, FQ8, FQ6, and FQ4, respectively) were compared. Within study a mixed model was used to compare gaseous exchanges and HP among FQALL, FQ12, FQ8, FQ6, and FQ4, and an interaction of dietary treatment by FQ was examined. A regression model was used to evaluate accuracy of spot sampling within study [i.e., FQALL (observed) vs. FQ12, FQ8, FQ6, or FQ4 (estimated)]. No interaction of diet by FQ was observed for any variables except for CH₄ production in experiment 1. No FQ effect was observed for gaseous exchanges and HP except in experiment 2 where CO₂ production was less (5,411 vs. 5,563 L/d) for FQ4 compared with FQALL, FQ12, and FQ8. A regression analysis between FQALL and each FQ within study showed that slopes and intercepts became farther from 1 and 0, respectively, for almost all variables as FQ decreased. Most variables for FQ12 and FQ8 had root mean square prediction error (RMSPE) less than 10% of the mean and concordance correlation coefficient (CCC) greater than 0.80, and RMSPE increased and CCC decreased as FQ decreased. When a regression analysis was conducted with combined data from the 3 experiments (mixed model with study as a random effect), results agreed with those from the analysis for the individual studies. Prediction errors increased and CCC decreased as FQ decreased. Generally, all the estimates from FQ12, FQ8, FQ6, and FQ4 had RMSPE less than 10% of the means and CCC greater than 0.90 except for FQ6 and FQ4 for O₂ consumption and CH₄ production. In conclusion, the spot sampling simulation with 3 indirect calorimetry experiments indicated that FQ of at least 8 samples (every 3 h in a 24-h cycle) was required to estimate daily O₂ consumption, CO₂ and CH₄ production, and HP and to detect changes in those in response to dietary treatments. This sampling FQ may be considered when using techniques that measure spot gas exchanges such as the GreenFeed and face mask systems.     

Short communication: turbidity as an indicator of Escherichia coli presence in water troughs on cattle farms

Studies have shown that water-drinking troughs are an important source of Escherichia coli infection on cattle farms, and a study was designed to provide farmers with an easy-to-use tool to monitor trough contamination and help determine when to empty and clean water troughs. A total of 164 water troughs were sampled on 33 cattle farms, and the on-farm turbidity tester results were found to be significantly correlated with laboratory turbidity results. Turbidity was associated with E. coli concentration, although the association was not linear. Emptying the troughs within a week of sampling was shown to reduce the turbidity score, but no linear association between time of emptying and E. coli concentration was discovered. A turbidity score of 4 was set as a cutoff point for when to clean a trough, yielding a sensitivity of 0.94 and a specificity of 0.03 for identifying a level of E. coli concentration that was more likely to contain E. coli O157 (>5,800 cfu/100 mL).     

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.     

Effects of feeding pasteurized waste milk to dairy calves on phenotypes and genotypes of antimicrobial resistance in fecal Escherichia coli isolates before and after weaning

The aim of this study was to evaluate the effects of feeding pasteurized waste milk (pWM) to calves on antimicrobial resistance of fecal Escherichia coli at both phenotypic and genotypic levels. Fifty-two Holstein female calves (3 ± 1.3 d of age) were fed 1 of the 2 different types of milk: milk replacer (MR) without antimicrobials or pWM with β-lactam residues until weaning at 49 d of age. Fecal swabs of all calves were obtained on d 0, 35, and 56 of the study and 3 E. coli isolates per sample were studied. Phenotypic resistance was tested by the disk diffusion method against a panel of 12 antimicrobials. A total of 13 resistance genes consisting of β-lactam, sulfonamide, tetracycline, and aminoglycoside families were examined by PCR. Feeding pWM to calves increased the presence of phenotypic resistance to ampicillin, cephalotin, ceftiofur, and florfenicol in fecal E. coli compared with MR-fed calves. However, the presence of resistance to sulfonamides, tetracyclines, and aminoglycosides was common in dairy calves independent of their milk-feeding source, suggesting other factors apart from the feeding source are involved in the emergence of antimicrobial resistance.     

Understanding farmers' and veterinarians' behavior in relation to antimicrobial use and resistance in dairy cattle: A systematic review

To tackle antimicrobial resistance, it is vital that farmers' and veterinarians' antimicrobial use behaviors and attitudes toward resistance are understood so that we can identify how beliefs and motives influence practices. Current literature details qualitative and quantitative research that explores the knowledge, attitudes and perceptions of dairy farmers and veterinarians with respect to antimicrobial resistance and antimicrobial practices, and the reported findings are varied and conflicting. Our objective was to conduct a systematic review to assess the evidence and knowledge gaps in the published literature. We identified articles via database searches of Embase, Medline, PubMed, Scopus, and Web of Science; we limited findings to published articles available in English with no publication year restrictions. Article screening was conducted at 3 levels: title, abstract, and full text. Of the 349 articles identified, 35 were retained for systematic review. Transparency of reporting was assessed for each study using the Consolidated Criteria for Reporting Qualitative Research (COREQ) framework. Quality was assessed using the Critical Appraisal Skills Programme qualitative checklist. Findings relating to dairy farmers' and veterinarians' knowledge, attitudes, and perceptions on antimicrobial resistance and practices were thematically analyzed. The comprehensiveness of reporting was variable: studies reported 5 to 26 of the 32 COREQ checklist items. Five key themes emerged from the data: knowledge and awareness of antimicrobial resistance; factors influencing farmer and veterinarian decision-making; perceived barriers and facilitators to reduced antimicrobial use; perceived responsibility for antimicrobial resistance; and the role of the farmer and veterinarian relationship in reducing antimicrobial use. Awareness of prudent antimicrobial use was not uniform between studies. Many factors influence farmers' and veterinarians' decisions to use antimicrobials, including animal welfare and available resources. The farmer–veterinarian relationship is a potential barrier or facilitator of reduced antimicrobial use, depending on the perceived relationship dynamic. Encouraging collaboration between farmers and veterinarians could lead to shared responsibility for reducing antimicrobial use. This review provided a coherent picture of what is currently known and identified gaps in the current knowledge to inform future behavioral intervention research. Increased knowledge, skill development, resources, engagement, and further research to address the gaps we identified are the main recommendations to effectively overcome barriers, elicit appropriate behavior change, and achieve reduced antimicrobial use in dairy cattle.     

Population structure of rumen Escherichia coli associated with subacute ruminal acidosis (SARA) in dairy cattle

Previous studies indicated that only subacute ruminal acidosis (SARA), induced by feeding a high-grain diet, is associated with an inflammatory response and increased abundance of Escherichia coli in the rumen. We hypothesized that ruminal E. coli in grain pellet-induced SARA carried virulence factors that potentially contribute to the immune activation during SARA. One hundred twenty-nine E. coli isolates were cultured from the rumens of 8 cows (4 animals per treatment) in which SARA had been nutritionally induced by feeding a high-grain diet (GPI-SARA) or a diet containing alfalfa pellets (API-SARA). The population structure of the E. coli was evaluated with the ABD genotyping system and repetitive sequence-based (rep)-PCR fingerprinting. Twenty-five virulence factors were evaluated with PCR. Escherichia coli numbers were higher in the GPI-SARA treatment than in the API-SARA treatment. The genetic structure of the E. coli was significantly different between SARA challenge models. Isolates from GPI-control (46%), API-control (70%), and API-SARA (53%) were closely related and fell into one cluster, whereas isolates from GPI-SARA (54%) grouped separately. The ABD typing indicated a shift from an A-type E. coli population to a B1-type population only due to GPI-SARA. Of the 25 virulence factors tested, curli fiber genes were highly associated with GPI. Curli fibers were first identified in E. coli mastitis isolates and are potent virulence factors that induce a range of immune responses. Results suggest that under low rumen pH conditions induced by a grain diet, there is a burst in the number of E. coli with virulence genes that can take advantage of these rumen conditions to trigger an inflammatory response.     

Occurrence and antibiotic resistance of Escherichia coli,Staphylococcus aureus and Bacillus cereus in raw milk and dairy products in Turkey

In this survey, 150 samples of raw milk, white cheese and ice cream from three different dairy‐processing plants in Ankara were analysed to find out if they were contaminated with Escherichia coli, Staphylococcus aureus or Bacillus cereus. The highest contamination percentages were found in raw milk samples as follows: B. cereus (90%), E. coli (74%) and S. aureus (56%) followed by cheese (70% B. cereus, 60% E. coli, and 48% S. aureus) and ice cream (56% E. coli, 36% S. aureus and 20% B. cereus). The survey showed that 2% of cheese samples were contaminated with E. coli O157. It was also found that the numbers of S. aureus and E. coli in raw milk, cheese and ice cream samples exceeded the numbers permitted under the Turkish Food Codex (TFC). The number of B. cereus in raw milk, cheese and ice cream samples was lower than the limit given in the TFC standards. The study also showed that E. coli and S. aureus exhibit resistance to ampicillin, penicillin, tetracycline, erythromycin, gentamicin and trimethoprim/sulfamethoxazole. Escherichia coli isolates also showed resistance to chloramphenicol and ciprofloxacin but none of them exhibited resistance to cefotaxime. All S. aureus isolates were found to be susceptible to cefotaxime, chloramphenicol, and ciprofloxacin. Bacillus cereus isolates were found to be resistant to ampicillin, penicillin and trimethoprim/sulfamethoxazole and sensitive to cefotaxime, chloramphenicol, ciprofloxacin erythromycin, gentamicin and tetracycline.     

Preliminary study of certain serotypes, genetic and antimicrobial resistance profiles of verotoxigenic Escherichia coli (VTEC) isolated in Bosnia and Germany from cattle or pigs and their products

The aim of this study was to gather more information on the spread of VTEC serotypes, genetic profiles and resistance patterns from pigs or pork and from cattle or beef in different areas, and to improve detection of the source of outbreaks with a wider data pool. Of 130 Escherichia coli samples isolated from a cattle slaughter house and beef retail products in Sarajevo, Bosnia and Herzegovina (BiH), seven were identified as verotoxigenic (VTEC). In comparison, 22 VTEC of 264 E. coli isolates were isolated from bovine faeces (14) and beef products (8) from Germany. Furthermore 23 VTEC of 76 isolates were identified from pig carcasses (10), faeces (9) and pork products (4) from Germany. Gene detection and serotyping were carried out in our laboratory and in the National Reference Laboratory. Antimicrobial resistance was tested with the dilution method in microtitre plates. All porcine isolates belonged to serotypes thus far not associated with human disease. Bovine VTEC were either serotypes commonly associated with human diseases (O157:H7, O103:H2, O157:H-) or rare serotypes. One serotype (O96:H19) was found only in isolates from Sarajevo. Most German VTEC, especially those of porcine origin, had only vtx2 genes, whereas all Bosnian isolates had vtx1 and vtx2 genes. The eae gene was found only in "classical" VTEC serotypes. All 52 VTEC (100%) investigated were resistant to the three sulfonamides tested; porcine isolates were mainly resistant to oxytetracycline (43%) and chlortetracycline (37%), bovine isolates mainly to trimethoprim/sulfamethoxazole and ampicillin (10% each). If sulfonamide resistances are disregarded, more than half (53.8%) of porcine VTEC were multiresistant and one-fourth (25%) of German bovine isolates, but none of the Bosnian bovine isolates. The results show the considerable spread of resistances in VTEC. These results also point out the necessity of gathering data from different geographical areas in order to be able to identify typical local variations in serotypes or gene expression and thus to trace human infections more quickly to their source.     

Characterization of antimicrobial resistance of Escherichia coli recovered from foods of animal and fish origin in Korea

Antimicrobial-resistant Escherichia coli is transferred from food-producing animals to humans through the food chain. We investigated the prevalence of antimicrobial resistance and resistance determinants and characterized the integrons of foodborne E. coli in Korea. In total, 162 E. coli isolates from commercial foods (raw meat, fish, and processed foods) were collected by the National Antimicrobial Resistance Management Program from 2004 to 2006. Susceptibility to 20 antibiotics was tested by disk diffusion, and resistance determinants were detected using PCR and genomic sequence analysis. The isolates were highly resistant to antibiotics commonly used in livestock farming. Resistance to tetracycline (74.7%) was the most frequently observed, followed by streptomycin (71%) and ampicillin (51.2%). Class 1 integrons were detected in 13 isolates (8%), and nine of these integrons were located on conjugative plasmids. None of the isolates produced extended-spectrum β -lactamase. One isolate (0.6%) harbored bla(CMY-2), which was located on a conjugative plasmid. Although the qnr gene was not detected, aac(6'9)-Ib-cr was present in two isolates (1.2%). This is the first report of aac(6'9)-Ib-cr in food isolates. Three or four amino acid substitutions at positions 83 and 87 in gyrA and at positions 80 and/or 84 in parC were found in six isolates, representing high resistance to ciprofloxacin (MIC ≥ 16 mg/liter). These results suggest that E. coli isolates carrying resistance genes and integrons are present in the Korean food chain.     

Occurrence of Campylobacter spp., Salmonella spp. and shiga toxin-producing Escherichia coli in inline milk filters from Swedish dairy farms

This study investigated the occurrence of shiga toxin-producing Escherichia coli (STEC), thermotolerant Campylobacter spp. and Salmonella spp. in Swedish dairy milk. A total of 302 inline milk filters were analyzed. Salmonella was not isolated from any filters. Polymerase chain reaction screening detected thermotolerant Campylobacter in 30.5% of the milk filters analyzed and it was isolated from 12.6% of filters. The stx genes (stx ₁, stx ₂, or both) were screened from 71% of the filters and STEC was isolated from 14% of these. Of the STEC isolates, 21 contained the stx ₁ gene, 19 the stx ₂ gene, and five a combination of both stx ₁ and stx ₂ genes. Whole genome sequence typing on 34 of the 45 STEC showed that they belonged to 21 different serotypes, of which STEC O145:H28 was the most common (2%). STEC O157:H7 was only found from one (0.3%) of the filters. A combination of stx ₂ and eae genes was found from 0.7% of the total number of inline milk filters analyzed, while stx _2a was found in 24% of the whole genome-sequenced isolates. There was a significant positive correlations between number of animals per farm and presence of pathogens on milk filters.     

Occurrence of methicillin-resistant Staphylococcus aureus in dairy cattle herds,related swine farms,and humans in contact with herds

In this study we investigated the circulation of methicillin-resistant Staphylococcus aureus (MRSA) in 2 dairy cattle farms (farm A and B), previously identified as MRSA-positive in bulk tank milk samples, and epidemiologically related to swine farms. Collected specimens included quarter milk samples and nasal swabs from dairy cows, pig nasal swabs collected at both the farm and slaughterhouse level, environmental dust samples, and human nasal swabs from the farms’ owners and workers. The prevalence of MRSA was estimated at the herd level by testing quarter milk samples. The prevalence of MRSA was 4.8% (3/63; 95% confidence interval = 0–10.2%) and 60% (33/55; 95% confidence interval = 47.05–72.95) in farm A and B, respectively. In farm A, MRSA was also isolated from humans, pigs sampled at both farm and slaughterhouse level, and from environmental samples collected at the pig facilities. The dairy cattle facilities of farm A tested negative for MRSA. In farm B, MRSA was isolated from environmental dust samples in both the cattle and pig facilities, whereas nasal swabs collected from cows and from humans tested negative. Sixty-three selected MRSA isolates obtained from different sources in farm A and B were genetically characterized by multilocus sequence typing, spa-typing, ribosomal spacer-PCR, and also tested for the presence of specific virulence genes and for their phenotypical antimicrobial susceptibility by broth microdilution method. Different clonal complex (CC) and spa-types were identified, including CC398, CC97, and CC1, CC already reported in livestock animals in Italy. The MRSA isolates from quarter milk of farm A and B mostly belonged to CC97 and CC398, respectively. Both lineages were also identified in humans in farm A. The CC97 and CC398 quarter milk isolates were also identified as genotype GTBE and GTAF by ribosomal spacer-PCR respectively, belonging to distinct clusters with specific virulence and resistance patterns. The GTBE and GTAF clusters also included swine, environmental, and human isolates from both farms. A high heterogeneity in the genetic and phenotypic profiles was observed in environmental isolates, in particular from farm B. These results demonstrate the possibility of a dynamic sharing and exchange of MRSA lineages or genotypes between different species and farm compartments in mixed-species farms. The risk of transmission between swine and related dairy cattle herds should be considered. Our findings also confirm the zoonotic potential of livestock-associated MRSA and underline the importance of applying biosecurity measures and good hygiene practices to prevent MRSA spread at the farm level and throughout the food production chain.