Interactions of organic acids with vancomycin-resistant Enterococcus faecium isolated from community wastewater in Texas.

AIMS: Investigate the interactions of organic acids (OAs), acetic, butyric, citric, formic, lactic and propionic acid against 50 Gram-positive vancomycin-resistant Enterococcus faecium (VRE) strains to determine whether pH, undissociated or dissociated acid forms correlate with bacterial inhibition. METHODS AND RESULTS: Concentrations of undissociated and dissociated OAs at the molar minimum inhibitory concentrations (MICM s) of the VRE were calculated using the Henderson-Hasselbalch equation. The pH at the MICM s of all VRE strains against acetic, butyric, formic and propionic acids was similar, 4·66 ± 0·07, but there was a 1·1 pH unit difference for all six OAs. Inhibition of VRE by all six OAs did not appear to be solely dependent on pH or on the undissociated OA species. The inhibition of VRE by all six dissociated acids was within Δ = 3·1 mmol l-1 . CONCLUSIONS: Vancomycin-resistant Enterococcus faecium inhibition correlated with the dissociated OA species. A small decrease in the concentration of the dissociated OAs from optimum may result in allowing VRE strains to escape disinfection. SIGNIFICANCE AND IMPACT OF THE STUDY: When an OA is used to disinfect VRE strains, the concentration of the dissociated OA should be carefully controlled. A concentration of at least 20 mmol l-1 dissociated OA should be maintained when disinfecting VRE.

Prevalence of salmonella in neck skin and bone of chickens.

Bone-in and boneless parts, such as drumsticks, are used in ground chicken production. In addition, neck skin is used as a source of fat in ground products. Contaminated chicken neck skin and bones containing internalized Salmonella are potential sources of this pathogen in ground chicken. This study determined the prevalence of Salmonella and serotype distribution in drumstick bones and neck skin of postchill chicken carcasses. One week prior to slaughter, chicken houses (n = 26) at nine farms were tested for the presence of Salmonella, using the boot sock method. Chicken flocks from these houses originated from Salmonella-positive breeders. Eight Salmonella-positive chicken flocks and one flock with undetermined Salmonella status were monitored through processing. Three hundred postchill drumsticks and 299 neck skin samples were analyzed for Salmonella prevalence. Skin samples were rinsed and stomached prior to analysis. Bones were extracted from the drumsticks, external surfaces were sterilized, and bones were crushed for analysis. One Salmonella isolate from each positive sample was serogrouped. Half of the isolates representing different sample types were serotyped. Overall, Salmonella was found in 0.8, 21.4, and 80.1% of bone marrow, neck skin, and farms, respectively. Prevalence of Salmonella on rinsed skin samples (2.3%) and stomached skin samples (20.7%) differed significantly (P ≤ 0.05). Serogroups B, C2, D, and E were found at 23.4, 31.9, 11.7, and 29.8%, respectively. Six Salmonella serotypes were identified: Liverpool (37.9%), Kentucky (27.6%), and Typhimurium (27.6%) were isolated most frequently from neck skin; the two bone isolates were Kentucky; and more than 50% of the farm isolates were Kentucky and Ouakam. Salmonella-contaminated neck skin might be a more significant source of this contamination in ground chicken than Salmonella internalized in bones.

Effect of essential oil compound on shedding and colonization of Salmonella enterica serovar Heidelberg in broilers.

The objectives of this study were to determine the effect of an essential oil blend (EO; carvacrol, thymol, eucalyptol, lemon) administered in drinking water on the performance, mortality, water consumption, pH of crop and ceca, and Salmonella enterica serovar Heidelberg fecal shedding and colonization in broiler birds following Salmonella Heidelberg challenge and feed withdrawal. Chicks were randomly assigned to water treatments containing 0.05, 0.025, or 0.0125% EO or untreated controls. Treatments were administered in drinking water on 0 to 7 and 35 to 42 d. One-half of the chicks were challenged with Salmonella Heidelberg and placed in pens with unchallenged chicks on d 1. Performance, mortality, water consumption, and pH were determined during the 42-d study. Prevalence of Salmonella Heidelberg was determined on drag swabs (0, 14, and 42 d) and in the ceca and crops (42 d). The 0.05% EO administered in drinking water significantly (P < 0.05) reduced Salmonella Heidelberg colonization in crops of challenged birds, significantly lowered the feed conversion ratio, and increased weight gain compared with controls. The 0.025% and 0.015% EO in drinking water significantly lowered the feed conversion ratio and increased weight gain compared with controls, but did not significantly reduce Salmonella Heidelberg colonization in the crops. The EO in drinking water did not significantly reduce Salmonella Heidelberg colonization in ceca or fecal shedding in broilers. The EO used in the study may control Salmonella Heidelberg contamination in crops of broilers when administered in drinking water and therefore may reduce the potential for cross-contamination of the carcass when the birds are processed.

Prevalence of Salmonella on raw poultry at retail markets in China.

Data regarding Salmonella on raw poultry are very limited in China. The objective of this study was to determine the prevalence of Salmonella on raw poultry at the retail level in six provinces and two national cities in China. Whole chicken carcasses (n = 1,152) were collected from three types of retail markets (large, small, and wet). All samples were analyzed for the presence of Salmonella by using the U.S. Department of Agriculture, Food Safety Inspection Service method. Of 1,152 chicken samples, overall Salmonella prevalence was 52.2%. The highest prevalence was observed in Guangxi Province (65.3%), next in Guangdong Province (64.6%), and then in Beijing (63.9%), Shaanxi Province (50.7%), Henan Province (47.9%), Shanghai (44.4%), and Fujian Province (42.4%), and lowest prevalence was observed in Sichuan Province (38.9%). Salmonella prevalence was significantly different among the six provinces and two national cities. Salmonella prevalence was highest in the wet markets (54.4%) compared with the large markets (50.3%) and the small markets (52.1%), but differences were not significant (P > 0.05). Good manufacturing practices, good agricultural practices, and hazard analysis critical control point systems for Salmonella control in poultry production at the farm, processing, and retail level should be implemented.

Assessing the similarity of antimicrobial resistance phenotypes among fecal Escherichia coli isolates from two aggregated occupational cohorts of humans versus swine using cluster analysis and multivariate statistics.

Statistical methods employed to analyze antimicrobial resistance (AR) phenotypic data have largely focused on multiple individual antimicrobial resistance outcomes without considering the pharmacologic and biological dependence among these data. In our 3-year longitudinal study, the relationship between AR phenotype of E. coli isolates from integrated multi-site group cohorts of humans and swine and the following risk factors: host-species (human versus swine) and human vocation (swine-workers versus non-workers) was assessed; first, by using cluster analysis techniques and then multivariate generalized estimating equation (GEE) models. Human sewage wastewater draining from occupation-specific housing and swine fecal E. coli isolates (n=3,113 and 3,428, respectively) were tested for antimicrobial susceptibility to 15 agents on the 2003 NARMS panel using the Sensititre system (Trek Diagnostics, Cleveland, OH). The MIC values for each isolate were interpreted according to standardized breakpoints into resistant or susceptible. The phenotypic data (n=6,541) were cluster-analyzed using Ward's minimum variance with Jaccard's distance measure. The multivariate relationships of E. coli cluster membership with the risk factors in the study were assessed using a multivariate GEE model in a SAS((R)) macro to adjust for the multiple cluster dependencies as well as adjusting for response dependencies within each unit location. The cluster solution that best described our entire dataset and where the multivariate GEE model converged was 14. In general, the adjusted odds-ratios of the multiple clusters (i.e., 14 clusters) for human isolates were significantly (P<0.05) at a higher odds of being in the pansusceptible cluster (OR=12.8), and also in clusters that contained high levels of resistance to amoxicillin/clavulanic acid, ampicillin, cefoxitin, nalidixic acid, sulfisoxazole, and/or trimethoprim/sulfamethoxazole, when compared to swine isolates. The adjusted odds-ratios of the multiple clusters for non-swine worker isolates were at significantly (P<0.05) higher risk of being in the pansusceptible cluster (OR=13.6) compared to swine-worker isolates (OR=12.1) (swine isolates were the referent group). In general, the adjusted odds-ratios of the multiple clusters for swine-worker E. coli isolates were significantly (P<0.05) at higher odds of being in multi-resistant clusters (defined as resistant to >or=3 antimicrobial agents) as compared to non-swine worker isolates. Considering vocation, swine-worker E. coli isolates exhibited increased odds of falling in multi-drug resistance clusters compared to those isolates arising from non-swine-workers.

Quantification of the bla(CMY-2) in feces from beef feedlot cattle administered three different doses of ceftiofur in a longitudinal controlled field trial.

The objective of this longitudinal controlled trial was to quantitatively compare carriage of a gene encoding for ceftiofur-resistance (bla(CMY-2)), standardized to a reference gene (16SrRNA), among total community DNA extracted from fecal samples collected from cattle treated with three different dose regimens of ceftiofur crystalline-free acid (CCFA) versus those untreated (controls). Sixty-one steers were assigned to three treatment regimens and housed in six pens. In each pen, five steers were treated and five were controls (one of the pens had six controls). CCFA administration was as follows: two-thirds dose treatment (4.4 mg/kg, on day 0), single-dose treatment (6.6 mg/kg, on day 0), and three-dose treatment (6.6 mg/kg, on days 0, 6, and 13). Fecal samples were collected on days 0, 3, 7, 10, 14, 18, 21, and 28. The gene copy numbers/gram of feces for bla(CMY-2) and 16SrRNA were determined in total community DNA samples using quantitative real-time PCR. The relationships between the quantities of standardized bla(CMY-2), nonstandardized bla(CMY-2), and nonstandardized 16SrRNA, and the explanatory variables (treatment, time, and treatment x time) were assessed using repeated measures mixed models. There were significant differences in each of the three models with respect to each explanatory variable. Overall, while steers administered three doses and two-thirds dose of CCFA had significantly higher quantities of nonstandardized bla(CMY-2) than controls, the standardized values were lower. The administration of CCFA in feedlot cattle may provide selection pressure favoring higher levels of bla(CMY-2) carriage, but this may also lead to concurrent reductions in the total bacterial population (as reflected by lowered 16SrRNA) during the treatment period.

Relationship between level of antibiotic use and resistance among Escherichia coli isolates from integrated multi-site cohorts of humans and swine.

The objective of this longitudinal ecological study was to examine the relationship between the prevalence of antibiotic-resistant (AR) commensal Escherichia coli isolates from both monthly human wastewater and composite swine fecal samples and the concurrent aggregated monthly antibiotic use recorded within each host species in multi-site vertically integrated swine and human populations. In addition, human vocation (swine worker versus non-swine worker), swine production group, and season were examined as potential confounding variables. Human and swine E. coli isolates (n=2469 human and 2310 swine, respectively) were tested for antimicrobial susceptibility using a commercial broth microdilution system. In the human population, among swine workers the relative odds of tetracycline resistance were increased significantly for tetracycline (class) drug use at the third quartile and above of mean monthly dosage (MMD) (OR=1.8) as compared to the referent category (non-use). The relative odds of ciprofloxacin resistance were significantly increased for ciprofloxacin use in non-swine workers (OR=5.5) as compared to the referent (non-use). The relative odds of tetracycline resistance were increased significantly for chlortetracycline use in medicated feed for the upper tertile of MMD category (OR=2.9) as compared to the referent category (no use) across all swine production groups. While high variability among seasonal samples over the 3-year period was observed, no common seasonal trends relating to antibiotic use and prevalence of resistance over the 3-year period were apparent. The overall effects of concurrent human and swine antibiotic use on AR E. coli levels were inconsistent and modest in this study.

Longitudinal study of antimicrobial resistance among Escherichia coli isolates from integrated multisite cohorts of humans and swine.

In a 3-year longitudinal study, we examined the relationship between the seasonal prevalence of antimicrobial-resistant (AR) Escherichia coli isolates from human wastewater and swine fecal samples and the following risk factors: the host species, the production type (swine), the vocation (human swine workers, non-swine workers, and slaughter plant workers), and the season, in a multisite, vertically integrated swine and human population representative of a closed agri-food system. Human and swine E. coli (n = 4,048 and 3,429, respectively) isolates from wastewater and fecal samples were tested for antimicrobial susceptibility, using the Sensititre broth microdilution system. There were significant (P < 0.05) differences among AR E. coli prevalence levels of (i) the host species, in which swine isolates were at higher risk for resistance to tetracycline, kanamycin, ceftiofur, gentamicin, streptomycin, chloramphenicol, sulfisoxazole, and ampicillin; (ii) the swine production group, in which purchased boars, nursery piglets, and breeding boars isolates had a higher risk of resistance to streptomycin and tetracycline; and iii) the vocation cohorts, in which swine worker cohort isolates exhibited lower sulfisoxazole and cefoxitin prevalence than the non-swine worker cohorts, while the slaughter plant worker cohort isolates exhibited elevated cefoxitin prevalence compared to that of non-swine workers. While a high variability was observed among seasonal samples over the 3-year period, no significant temporal trends were apparent. There were significant differences in the prevalence levels of multidrug-resistant isolates between host species, with swine at a higher risk of carrying multidrug-resistant strains than humans. Considering vocation, slaughter plant workers were at higher risk of exhibiting multidrug-resistant E. coli than non-swine workers.

Vancomycin-resistant Enterococcus faecium strains isolated from community wastewater from a semiclosed agri-food system in Texas.

Vancomycin-resistant Enterococcus faecium strains (VRE) were isolated from human wastewater but not swine fecal waste from a semiclosed agri-food system in Texas. Forty-nine VRE isolates possessed vanA, and one possessed vanB. Twenty-one pulsed-field gel electrophoresis types were identified and segregated into three groups. There was evidence of clonal dissemination among geographically separated sites.

Patterns of antimicrobial resistance among commensal Escherichia coli isolated from integrated multi-site housing and worker cohorts of humans and swine.

We examined antimicrobial resistance (AR) phenotypes among commensal Escherichia coli isolated from fecal matter of humans and swine housed in a semi-closed and uniquely integrated multi-site farrow-to-plate operation. Aggregate cohorts of humans consisted of (1) "control" groups of consumers, (2) groups of swine workers, and (3) groups of slaughter-plant workers. Analyses of cross-sectional AR data from 472 human and 376 swine isolates are presented. AR phenotypes among these isolates were compared by (1) host species, (2) facility location, (3) facility type, (4) housing (human) or production (swine) cohort, and (5) sample collection period and time of day. There were significant (p < 0.05) differences in isolates among host-species with swine uniformly at greater risk for (1) AR to four specific antimicrobials (kanamycin, streptomycin, sulfamethoxazole, tetracycline), and (2) multiple resistance phenotypes (p < 0.0001). Facility type and unit location were more often associated with AR differences among swine isolates than among human isolates. Swine production group was significantly associated with AR prevalence (p < 0.05) for nine antimicrobials; in general, purchased boars, suckling piglets, weaned piglets, and lactating sows were at higher risk of AR. There was no significant (p > 0.05) association of human occupational/consumer cohort with AR prevalence. Several unique AR phenotypes were detected in each of the human- and swine-intake groups. These data establish baseline characteristics for an on-going 3-year longitudinal study designed to further characterize AR phenotype and genotype in this population. Host-, facility-, and cohort-specific data demonstrate that sufficient prevalence differences exist to permit the future quantification of AR transmission, should it occur. Based on these cross-sectional data, occupational exposure to either swine-rearing or swine-slaughter facilities does not appear to be associated with the prevalence of phenotypic resistance among the commensal fecal E. coli isolated from this integrated system.

Effect of antibiotics in milk replacer on fecal shedding of Escherichia coli O157:H7 in calves.

The objective of this study was to compare the concentration and duration of fecal shedding of Escherichia coli O157:H7 between calves fed milk replacer with or without antibiotic (oxytetracycline and neomycin) supplementation. Eighteen 1-wk-old Holstein calves were orally inoculated with a strain of E. coli O157:H7 (3.6 x 10(8) cfu/calf) made resistant to nalidixic acid (NA). Rectal samples were obtained three times weekly for 8 wk following oral inoculation. Fecal shedding of NA-resistant E. coli O157:H7 was quantified by direct plating or detected by selective enrichment procedure. Eight weeks after inoculation, calves were killed, necropsied, and tissues (tonsils, retropharyngeal and mesenteric lymph nodes, and Peyer's patches) and gut contents (rumen, omasum, abomasum, ileum, cecum, colon, and rectum) were sampled to quantify or detect NA-resistant E. coli O157:H7. The percentage of calves shedding NA-resistant E. coli O157:H7 in the feces in the antibiotic-fed group was higher (P < 0.001) early in the study period (d 6 and 10) compared with the control group fed no antibiotics. There was no difference between treatment and control groups in the concentration of E. coli O157 in feces that were positive at quantifiable concentrations. A comparison of the duration of fecal shedding between treated and untreated calves showed no significant difference between groups. At necropsy, E. coli O157:H7 was recovered from the rumen and omasum of one calf in the control group and from retropharyngeal lymph node and Peyer's patch of two calves in the antibiotic group. Supplementation of milk replacer with antibiotics may increase the probability of E. coli O157:H7 shedding in dairy calves, but the effect seems to be of low magnitude and short duration.