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Salmonella isolates were recovered from bulk tank milk as part of the National Animal Health Monitoring System (NAHMS) Dairy 2002 and 2007 surveys. In-line milk filters were also tested in the 2007 survey. The objective of this study was to determine the prevalence of antimicrobial resistance among Salmonella enterica isolates from bulk milk and milk filters in the NAHMS Dairy 2002 and 2007 surveys and to further characterize resistant isolates. Susceptibilities to 15 antibiotics were determined for 176 Salmonella isolates of 26 serotypes using an automated antimicrobial susceptibility system. Resistant isolates were screened by PCR for the presence of the extended-spectrum ß-lactamase (blaCMY) gene and class I integrons and further characterized by pulsed-field gel electrophoresis. Thirty isolates (17.0%) representing six S. enterica serotypes exhibited resistance to at least one antimicrobial agent (serotypes Newport [14 of 14 isolates exhibited resistance], Dublin [7 of 7], Typhimurium [3 of 5], Kentucky [4 of 22], Anatum [1 of 13], and Infantis [1 of 2]). Twenty isolates (11.4%), including all 14 Newport, 3 Dublin, 2 Typhimurium, and 1 Infantis isolate, displayed the typical multidrug-resistant, blaCMY-positive (MDR-AmpC) phenotype which included resistance to ampicillin, chloramphenicol, streptomycin, sulfonamide, and tetracycline, plus resistance to amoxicillin-clavulanic acid and extended-spectrum cephalosporins. Five of the MDR-AmpC isolates carried class I integrons (2.8%). Two-enzyme (XbaI and BlnI) pulsed-field gel electrophoresis discerned clades within serotypes and, together with the resistance profiles, identified strains that appeared to have persisted temporally and geographically. These results suggest that there is a low but appreciable risk of infection with MDR Salmonella from consumption of nonpasteurized milk and dairy products. [ABSTRACT FROM AUTHOR]

Although dairy cattle are known reservoirs for salmonellae, cattle that are shedding this organism are often asymptomatic and difficult to identify. A dairy herd that was experiencing a sustained, subclinical outbreak of Salmonella enterica subsp. enterica Cerro was monitored for 2 years. Fecal samples from the lactating cows were collected every 6 to 8 weeks and tested for the presence of Salmonella. Fecal prevalence of Salmonella fluctuated throughout the observation period and ranged from 8 to 88%. Manure composites and water trough samples were collected along with the fecal samples, and bulk milk and milk filters were cultured for the presence of Salmonella on a weekly basis. Over 90% of the manure composites--representing high-animal-traffic areas--were positive at each sampling. Salmonella was detected in 11% of milk samples and in 66% of the milk filters. Results of weekly bulk milk quality testing (i.e., bulk tank somatic cell score, standard plate count, preliminary incubation count) were typically well within acceptable ranges. Milk quality variables had low correlations with herd Salmonella fecal prevalence. When observed over time, sampling period average prevalence of Salmonella in milk filters closely paralleled fecal prevalence of Salmonella in the herd. Based on results of this study, milk filters appear to be an effective method for monitoring shedding prevalence at the herd level. In-line filter testing is also a more sensitive measure of Salmonella, and perhaps other pathogens, in raw milk than testing the milk alone. [ABSTRACT FROM AUTHOR]

Samples of bulk tank milk from dairies across the United States, taken as part of the National Animal Health Monitoring Dairy 2002 survey, were analyzed for the presence of several genes encoding virulence factors associated with enterohemorrhagic forms of Escherichia coli (EHEC) using real-time and conventional PCR assays. Samples from 859 farms in 21 states were collected and enriched in EC medium at 42.5°C to amplify any E. coli present, and DNA was isolated from the resulting biomass. The eaeA gene encoding intimin, a virulence factor associated with enteropathogenic forms of E. coli and EHEC, was detected in 199 (23%) of the samples. Thirty-six samples (4.2%) were positive for eaeA, the gamma allele of the translocated intimin receptor (γ-tir), found in EHEC strains of O157:H7, and one or both shiga-like toxin genes (stx1 and stx2), a combination that may be indicative of the presence of O157:H7 EHEC. Testing these 36 samples with a commercially available real-time PCR kit for detection of O157:H7 indicated that 5 samples could be contaminated with O157:H7. A multiplex PCR to detect the presence of fliC, rfbE, and hlyA genes found in O157:H7 reduced to 2 (0.2% of all samples) the number of samples likely to be contaminated with this organism. A strain of O157:H7 (eaeA+, γ-tir+, stx2+, rfbE+, fliC+, hlyA+) was subsequently isolated from one sample. Thirty-four eaeA-positive samples did not contain detectable γ-tir but did contain one or both of the stx genes suggesting the presence of EHEC strains other than O157:H7. These results indicate a low incidence of O157:H7 in bulk tank milk but suggest that a risk from other enteropathogenic and EHEC forms of E. coli may exist and that PCR targeting virulence factors associated with highly pathogenic forms of E. coli may be an effective means of detecting potential dangers in raw milk. [ABSTRACT FROM AUTHOR]

Sixty-one Listeria monocytogenes strains from raw milk were analyzed with an automated repetitive element-based PCR (rep-PCR) system to examine the utility of this system for serotype grouping and to determine whether specific regional relationships could be identified. Results of the similarity analysis revealed two primary clusters of L. monocytogenes isolates. Cluster 2 exclusively contained serogroup 1/2a isolates; however, two 1/2a isolates were also found in cluster 1. Isolates of serogroups 1/2b, 4b, 3b, and 4c were also in cluster 1. Clusters I and 2 were separated at a relative similarity of 86%. Listeria species other than L. monocytogenes (L. ivanovii, L. seeligeri, L. welshimeri, L. grayi, and L. innocua) had similarity scores of less than 80% in pairwise comparisons with the L. monocytogenes isolates. Thus, this method may be useful for species identification once an isolate is characterized as Listeria. When rep-PCR fingerprints of the L. monocytogenes 1/2a isolates were compared, there was no apparent regional grouping. However, discrimination between isolates suggests that the rep-PCR assay might be useful for tracking L. monocytogenes 1/2a and for tracking isolates across regions or within smaller ecological niches. The automated rep-PCR method could not discriminate between serotypes 1/2b and 4b but may be useful for discriminating between 1/2a and other serotypes and for tracking isolates within serotype 1/2a. [ABSTRACT FROM AUTHOR]

Samples of bulk tank milk from dairies across the United States, taken as part of the National Animal Health Monitoring System Dairy 2002 survey, were analyzed for the presence of Salmonella enterica using a commercially available real-time polymerase chain reaction (PCR) kit. Samples from 854 farms in 21 states were collected and enriched in tetrathionate broth to amplify any salmonellae present, and DNA was isolated from the resulting biomass. One hundred one samples (11.8%) were shown to contain Salmonella enterica using the real-time PCR assay, whereas conventional culture techniques detected the pathogen in only 22 (2.6%) of the samples. A conventional PCR assay targeting a different gene from Salmonella enterica confirmed the presence of the organism in 94 of the real-time PCR-positive samples. Thus, assay of milk samples by real-time PCR indicates that the prevalence of Salmonella enterica in US bulk tank milk is substantially higher than previously reported. [ABSTRACT FROM AUTHOR]

The objective of this study was to determine the prevalence of Salmonella, Listeria monocytogenes, and fecal coliforms in bulk tank milk in the United States. As part of the NAHMS Dairy 2002 survey, 861 bulk tank milk samples were collected from farms in 21 states. Milk was directly plated on selective agars for direct bacterial enumeration and was enriched in selective broths to increase detection sensitivity. Somatic cell counts (SCC) and standard plate counts (SPC) were also determined. Coliforms were detected in 95% (818 of 860) of the samples, and the average SCC was 295,000 cells/mL. Twenty-two samples (2.6%) were culture-positive for Salmonella, and 9 serotypes were identified: Montevideo (n = 7), Newport (n = 4), Muenster (n = 2), Meleagridis (n = 2), Cerro (n = 2), 44:Z36 (Z38) (n = 2), Dublin (n = 1), Anatum (n = 1), and 9, 12:nonmotile (n = 1). Listeria monocytogenes was isolated from 56 (6.5%) samples, and serotyping of these isolates yielded 5 serotypes (1/2a, 1/2b, 3b, 4b, and 4c). Of the L. monocytogenes isolates, 93% were serotypes 1/2a, 1/ 2b, and 4b, the most common human clinical isolates. Regional differences in L. monocytogenes and Salmonella prevalence were observed, but more studies are needed to determine the validity of these differences. There were no apparent relationships between SCC or SPC and incidence of Salmonella or L. monocytogenes. Although the prevalence of L. monocytogenes and Salmonella was low, these pathogens represent a potential risk to consumers of raw milk and raw milk products. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance has become a major public health concern, and animal agriculture is often implicated as a source of resistant bacteria. The primary objective of this study was to determine prevalence of antimicrobial resistance in Salmonella and E. coli from healthy animals on dairy farms in Pennsylvania. Confidence intervals (CI) of farm prevalence were calculated for resistance, and the comparisons between different animal groups were adjusted for within-farm clustering. The secondary objective was to identify potential risk factors associated with antimicrobial resistance. In particular, we investigated the correlations between previous antimicrobial use and current resistance in E. coli using multiple logistic regression models. A total of 444 composite manure samples were collected from 80 herds in Pennsylvania. Four age and production groups were included: pre-weaned calves, post-weaned calves, dry cows, and lactating cows. E. coli (n = 2370) and Salmonella (n = 1095), when isolated from the manure composite samples (n = 444), were screened for resistance on antimicrobial supplemented agar. Isolates of interest were further assayed against 14 antimicrobials using a broth microdilution method. Salmonella was isolated from at least one composite manure sample from 51 (67%) farms, with 99% being pan-susceptible, and was detected more frequently in lactating cows and dry cows than pre- and post-weaned calves. The most prevalent Salmonella serogroups were K, C1, and C2. Among the E. coli isolates, resistance was more frequently detected in pre-weaned and post-weaned calves than in dry and lactating cows for all 14 antimicrobials tested (P < 0.05). Multi-drug resistance (resistant to ≥3 antimicrobial classes) in E. coli was detected in 66 farms (83%) and was detected more often in pre-weaned calves (P < 0.05). The blaCTX-M and blaCMY genes were found in cephalosporin-resistant E. coli from 5% and 35% of the farms surveyed, respectively. A number of risk factors, including antimicrobial use, were found to be associated with an increased likelihood of resistance in E. coli, and these associations were not limited to the same antimicrobial classes. The results of this study showed dairy farms, especially pre-weaned calves and their environment, are significant reservoirs for antimicrobial resistance, and antimicrobial use may aggravate the resistance problem. [ABSTRACT FROM AUTHOR]

The extent to which carriage of antibiotic resistant bacteria in food animals is responsible for the burden of antibiotic resistance in human infections is currently not well known. Thus, there is a need to further evaluate the genomic diversity of multidrug resistant (MDR) bacteria and the microbial communities in which they are found within these animals. To further evaluate the prevalence of antibiotic resistance in the feces of dairy animals, we sequenced the genomes of 160 antibiotic resistant Escherichia coli and 28 fecal metagenomes isolated from adult dairy cows, veal calves, and pre-weaned calves. E. coli isolates were collected from composite fecal samples and fecal grab samples taken directly from the animal. Isolates were screened for susceptibility to a panel of 14 antibiotics, and 150 showing resistance to at least one antibiotic and 10 antibiotic-susceptible isolates were selected for whole genome shotgun sequencing using a paired-end sequencing approach on an Illumina NextSeq 500. Twenty-eight fecal grab samples collected from animals shedding variable levels of antibiotic resistant E. coli were selected for shotgun metagenomics sequencing. Phylogenetic relationships among the E. coli genomes were inferred using a maximum likelihood approach in RAxML, and sequence types were determined in silico. At least four E. coli isolates from each of the major phylogroups were detected. The most frequently detected sequence types were ST88 (10% of all isolates), ST10 (9%), and ST117 (6%). Within these sequence types, isolates from different animals on the same farm and isolates from different farms were closely related, indicating that some strains of antibiotic resistant E. coli may be circulating among dairy herds within the same region. Interestingly, several MDR ST117 isolates were closely related to human ExPEC isolates and isolates recovered from chickens with colibacillosis. Metagenomic analyses indicated that for all samples tetracycline resistance genes were the most frequently identified resistance genes (61% of all resistance genes), followed by aminoglycoside (20%), macrolide (8%), beta-lactam (3%), sulfonamide (0.8%), and trimethoprim (0.6%) resistance genes. In veal calf fecal samples trimethoprim and fluoroquinolone resistance genes were more frequently detected than in pre-weaned dairy calf samples. Future work will identify genomic features that may confer persistence of high frequency antibiotic resistant E. coli strains and sequence types in the dairy cow hindgut and factors that influence the presence of antibiotic resistance in pre-weaned calves. [ABSTRACT FROM AUTHOR]

In this article the authors respond to a comment on their article titled "Effects of Temperature on Bacterial Transport and Destruction in Bioretention Media: Field and Laboratory Evaluations," published in the 2012 issue. They state that they thank the author for pointing out other studies of urban runoff that have similarly observed input concentrations of fecal coliform bacteria.

Salmonella is a prominent foodborne pathogen in the US and dairy cattle have frequently been identified as reservoirs for this organism. Here we describe a long-term outbreak of Salmonella enterica subsp. enterica Cerro that was detected in a Pennsylvania dairy herd during routine monitoring as part of a longitudinal study of several dairy herds. An initial analysis of individual fecal grab samples from the milking herd indicated a very low prevalence of Salmonella (1/102) and Salmonella was not detected in 10 environmental samples. In a subsequent sampling 6 mo later, Salmonella was isolated from 43.5% (47/108) of the cows and 40% of collected environmental samples (10/25). The Salmonella isolated in the first sampling was serovar Typhimurium var. Copenhagen, however isolates from the second herd sampling were serovars Cerro (91.5%) and Kentucky (8.5%). Sporadic environmental sampling between these initial complete milking herd samplings yielded serotypes Kentucky, Typhimurium var. Copenhagen, and Cerro and clinical salmonellosis was observed in several cows shedding Typhimurium var. Copenhagen. The herd was sampled nine additional times over the next 14 mo and fecal prevalence of Salmonella ranged from 8.4% to 89%. Prevalence remained high (58 to 69%) over the following 8 mo, showed evidence of decline over the next 3 mo, and then increased again to 89%. Cerro was the only serotype isolated during this period. Bulk milk was cultured weekly during this 22-mo period and Salmonella was isolated from 5.3% (5/94) of milk samples. For the last 14 mo of the study, milk filters were also cultured for Salmonella on a weekly basis and 65% (37/57) of the filters tested positive. There were no significant health or production concerns in the herd during this outbreak. Based on these observations we conclude that sub-clinical Salmonella enterica subsp. enterica Cerro infection can be persistent in a milking herd. [ABSTRACT FROM AUTHOR]

A phosphotriesterase (PTE) capable of hydrolyzing organophosphate esters was purified from Escherichia coli strain DH-5Error! Bookmark not defined.alpha carrying a cloned opd gene from Flavobacterium. The effects of temperature and metal ion concentrations on enzyme stability and activity were investigated. Optimum conditions for PTE's catalytic activity were determined to be 35 deg. C and pH 8.5. Protein-metal equilibrium binding experiments showed that PTE could accommodate two equivalents of Co2+ or Zn2+ ions. PTE protein was found to have higher affinity for Co2+. In addition,Co2+ was found to possess the most positive effects in maintaining and restoring PTE's stability and catalytic activity when compared to other divalent metal ions. Assessment of the feasibility of PTE operation in a practical environment was performed in a system designed to mimic a continuously stirred tank reactor (CSTR) with different solution compositions in the flow reservoir. PTE was deactivated in 24 hours when the inflow solution contained 5% ethanol or 1 mM EDTA, while it retained one third of its initial activity in a deionized water stream. When the inflow solution contained 1 mM Co2+, PTE was found to retain activity throughout the 24-hour experiment. [ABSTRACT FROM AUTHOR]

Streptomyces sp. PS1/5 was able to degrade atrazine in soil while growing on a mixture of chitin and ground cornstalks as carbon and nitrogen source. The addition of triple superphosphate greatly stimulatedthe degradation of atrazine by Streptomyces sp. PS1/5, while the addition of lime stimulated the degradation to a lesser degree. Ferrous sulfate had a small positive effect on atrazine degradation. Atrazinein soil was reduced from 20 g g-1 dry soil (20 ppm) to 6 ppm within 30 days in soil amended with 8% chitin, 2% cornstalks and 0.1% triple superphosphate and incubated at 25 deg. C. The results indicate that it may be possible to use bacteria of the genus Streptomyces to degrade pesticide residues in contaminated soils. [ABSTRACT FROM AUTHOR]

The objective of this study was to assess the presence of a Listeria monocytogenes-containing biofilm in milking equipment as a potential source of bulk tank milk contamination on a dairy farm where milk contamination had been previously documented. Samples were collected from milking equipment and milking parlor premises on 4 occasions and analyzed for the presence of L. monocytogenes. Pulsed-field gel electrophoresis (PFGE) typing was conducted on L. monocytogenes isolates from the milking equipment, parlor and storage room floors, bulk tank milk, and in-line milk filters. Pieces from milk meters and rubber liners were obtained to visually assess the presence of a biofilm using scanning electron microscopy. A total of 6 (15%), 4 (25%), and 1 (6%) samples were culture-positive for L. monocytogenes in the first, second, and third sample collection, respectively. Two samples were L. monocytogenes hly PCR-positive but were culture-negative in the fourth sample collection. Combined AscI and ApaI restriction analysis yielded 6 PFGE types for 15 L. monocytogenes isolates obtained from milking equipment, parlor, bulk tank milk, and milk filters. A predominant and persistent PFGE type (PFGE type T) was observed among these L. monocytogenes isolates (9/15 isolates). Scanning electron microscopy of samples from the bottom cover of 2 milk meters showed the presence of individual and clusters of bacteria, mainly associated with surface scratches. The presence of a bacterial biofilm was observed on the bottom covers of the 2 milk meters. Prevention of the establishment of biofilms in milking equipment is a crucial step in fulfilling the requirement of safe, high-quality milk. [ABSTRACT FROM AUTHOR]