Survival of Campylobacter jejuni during in vitro culture with mixed bovine ruminal microorganisms in the presence of methanogen inhibitors.

Foodborne pathogen Campylobacter jejuni has been associated with ruminants. The objectives of this experiment were to determine C. jejuni survivability in mixed in vitro rumen microbial populations and the impact on methane production with or without methane inhibitors 2-bromosulfonate (BES) and/or sodium nitrate. When inoculated into rumen microbial populations without or with 0.5 mM BES, 5.0 mM nitrate or their combination, C. jejuni viability decreased from 4.7 ± 0.1 log10 colony forming units (CFU)/mL after 24 h. Loss of C. jejuni viability was greater (P < 0.05) when incubated under 100% CO2 compared to 50% H2:50% CO2, decreasing 1.46 versus 1.15 log units, respectively. C. jejuni viability was also decreased (P < 0.05) by more than 0.43 log units by the anti-methanogen treatments. Rumen microbial populations produced less methane (P = 0.05) when incubated with than without C. jejuni regardless of whether under 100% CO2 or 50% H2:50% CO2. For either gas phase, nitrate was decreased (13.2 versus 37.9%) by the anti-methanogen treatments versus controls although not always significant. C. jejuni-inoculated populations metabolized 16.4% more (P < 0.05) nitrate under H2:CO2 versus 100% CO2. Apparently, C. jejuni can compete for H2 with methanogens but has limited survivability under rumen conditions.

Methylsulfonylmethane exhibits bacteriostatic inhibition of Escherichia coli, and Salmonella enterica Kinshasa, in vitro.

AIMS: To evaluate antibacterial properties of methylsulfonylmethane (MSM) on Escherichia coli (MDRE21) and Salmonella enterica serovar Kinshasa (SK132). METHODS AND RESULTS: Bacterial proliferation analysis was measured spectrophotometrically during log phase growth with 0, 3, 5, 7, 10, 12 and 16% MSM. To assess the mechanism of inhibition, cultures were grown overnight with 0-16% MSM and enumerated on unmedicated brain-heart infusion agar (BHIA) or BHIA with 0-16% MSM. The long-term viability studies were done to evaluate the impact of 10% MSM. Absorbance data indicated a dose-dependent inhibition from 0 to 16% MSM. There was no growth of MDRE21 or SK132 on BHIA in 10-16% MSM. Both strains enumerated on unmedicated BHIA from overnight cultures with 10-16% MSM were able to partially recover. CONCLUSIONS: Recovery after MSM removal may be indicative of a bacteriostatic mechanism of inhibition. The long-term viability studies illustrated that neither MDRE21 nor SK132 could be rescued from 10% MSM after 5 or 6 days respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: Methylsulfonylmethane antibacterial activity may prove useful during pre or postharvest food safety as a disinfectant. The primary benefit being, its clinical safety to humans. Comparisons to other disinfectants would also need to be done to determine if MSM was superior to those already on the market and would be cost effective.

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.

Inhibition of multidrug-resistant Staphylococci by sodium chlorate and select nitro- and medium chain fatty acid compounds.

AIMS: Determine the antimicrobial effects of 5 µmol ml-1 sodium chlorate, 9 µmol ml-1 nitroethane or 2-nitropropanol as well as lauric acid, myristic acid and the glycerol ester of lauric acid Lauricidin® , each at 5 mg ml-1 , against representative methicillin-resistant staphylococci, important mastitis- and opportunistic dermal-pathogens of humans and livestock. METHODS AND RESULTS: Three methicillin-resistant Staphylococcus aureus and two methicillin-resistant coagulase-negative staphylococci were cultured at 39°C in 5 µmol ml-1 nitrate-supplemented half-strength Brain Heart Infusion broth treated without or with the potential inhibitors. Results revealed that 2-nitropropanol was the most potent and persistent of all compounds tested, achieving 58-99% decreases in mean specific growth rates and maximum optical densities when compared with untreated controls. Growth inhibition did not persist by cultures treated solely with chlorate or nitroethane, with adaptation occurring by different mechanisms after 7 h. Adaptation did not occur in cultures co-treated with nitroethane and chlorate. The medium chain fatty acid compounds had modest effects on all the staphylococci tested except the coagulase-negative Staphylococcus epidermidis strain NKR1. CONCLUSIONS: The antimicrobial activity of nitrocompounds, chlorate and medium chain fatty acid compounds against different methicillin-resistant staphylococci varied in potency. SIGNIFICANCE AND IMPACT OF THE STUDY: Results suggest that differential antimicrobial activities exhibited by mechanistically dissimilar inhibitors against methicillin-resistant staphylococci may yield potential opportunities to combine the treatments to overcome their individual limitations and broaden their activity against other mastitis and dermal pathogens.

Characterization of antibiotic and disinfectant susceptibility profiles among Pseudomonas aeruginosa veterinary isolates recovered during 1994-2003.

AIMS: To evaluate susceptibility of Pseudomonas aeruginosa veterinary isolates to antibiotics and disinfectants. METHODS AND RESULTS: Pseudomonas aeruginosa isolates collected from dogs (n = 155) and other animals (n = 20) from sixteen states during 1994-2003 were tested for susceptibility. Most isolates were resistant to twenty-one antimicrobials tested, and the highest prevalence of resistance was to ß-lactams (93.8%) and sulphonamides (93.5%). Fluoroquinolone resistance did not increase from 1994 to 2003. Ciprofloxacin and enrofloxacin had a 5 and 16% prevalence of resistance, respectively, while sarafloxacin and nalidixic acid had a prevalence of resistance of 97 and 98%, respectively. Strains were pan-resistant to triclosan and chlorhexidine, were highly resistant to benzalkonium chloride and demonstrated high susceptibility to other disinfectants. Didecyldimethylammonium chloride was the most active ammonium chloride. Inducible resistance was observed to cetyl ammonium halides, chlorhexidine and benzyl ammonium chlorides, which formulate disinfectants used in veterinary clinics and dairies. Organic acid inhibition was associated with the dissociated acid species. CONCLUSIONS: Dissociated organic acids appear able to inhibit Ps. aeruginosa, and rates of fluoroquinolone resistance merit sustained companion animal isolate surveillance. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of Ps. aeruginosa susceptibility to 24 disinfectants and illustrates the high resistance of Ps. aeruginosa to both antibiotics and disinfectants.

Competitive effect of commensal faecal bacteria from growing swine fed chlortetracycline-supplemented feed on ß-haemolytic Escherichia coli strains with multiple antimicrobial resistance plasmids.

AIMS: To determine the differences in competitive fitness among Escherichia coli strains with different plasmid profiles when grown in suspension with commensal faecal bacteria from growing swine fed chlortetracycline-supplemented or unsupplemented diets. METHODS AND RESULTS: Five multiple drug-resistant (MDR) E. coli strains that possessed 0, 2, 6 or 8 plasmids were inoculated into anoxic faecal cultures from swine fed an unsupplemented (control) or chlortetracycline (50 g ton(-1))-supplemented (experimental) diet. On days 21 of chlortetracycline supplementation, faecal growth competition studies were performed. MDR E. coli were enumerated at 0, 6 and 24 h. The plasmid-free strain was below culturable limits in both the control and experimental cultures by 24 h. For each plasmid-bearing strain, there was no statistically significant difference in population CFU ml(-1) (P < 0.05) between the control and experimental cultures. CONCLUSIONS: There was no significant effect on the faecal microflora, owing to the inclusion of chlortetracycline, in the swine diets, that affected the growth of E. coli in the competition studies employed. Furthermore, these results suggest that the cost of maintaining plasmids in these E. coli strains had little influence on survivability. SIGNIFICANCE AND IMPACT OF STUDY: Mutations that led to antimicrobial resistance may have a greater impact on survivability than multiple plasmid carriage.

Influence of sprinklers, used to alleviate heat stress, on faecal shedding of E. coli O157:H7 and Salmonella and antimicrobial susceptibility of Salmonella and Enterococcus in lactating dairy cattle.

AIMS: To determine the effect of sprinklers on faecal shedding of Escherichia coli (E. coli) O157:H7 and Salmonella in lactating dairy cattle. METHODS AND RESULTS: Sprinklers were applied to lactating dairy cattle on two farms at either the feedbunk or in the holding pen prior to milking. Faecal samples were collected approx. 1 and 4 weeks following initiation of sprinkler treatments for culture of E. coli O157:H7, Salmonella and Enterococcus. No treatment differences were observed for E. coli O157:H7. Salmonella was higher (P = 0.11) in the control treatment on day-7 whereas on day-28, the bunk sprinklers increased the number of Salmonella positive cows. Salmonella prevalence decreased (P = 0.0001) on day-5 and when examined across days in cows exposed to sprinklers prior to milking. Antimicrobial susceptibility screening found very few isolates that were multi-drug resistant. All Enterococcus isolates were susceptible to vancomycin. CONCLUSIONS: This study demonstrated a significant decrease in faecal prevalence of Salmonella in lactating cattle following exposure to sprinklers administered prior to milking. SIGNIFICANCE AND IMPACT OF THE STUDY: Identification of current dairy management techniques that are also effective in reducing on-farm prevalence of pathogenic bacteria could have significant food safety and environmental implications.

Dietary lactose and its effect on the disease condition of necrotic enteritis.

Clostridium perfringens is the etiologic agent of necrotic enteritis (NE) and is ubiquitous in nature. The incidence of NE has increased in countries and commercial companies that have stopped using antibiotic growth promoters. The mechanisms of colonization of C. perfringens and the factors involved in onset of NE are not fully understood. Previously, our laboratory has demonstrated that lactose could potentially reduce Salmonella and C. perfringens in ceca of poultry. In the present investigation, we hypothesized that dietary lactose would reduce the clinical signs of NE and could be used as an alternative to antibiotics. In experiment 1, day-of-hatch broilers were fed either a nonlactose control diet, a diet with 2.5% lactose, or a diet with 4.5% lactose throughout the experiment. Birds were administered C. perfringens (10(7) cfu/mL) daily via oral gavage for 3 consecutive days starting on d 17. When evaluating the intestinal lesions associated with NE, birds fed 2.5% lactose had significantly lower (P < 0.05) lesion scores (0.70 +/- 0.52) compared with the control (1.55 +/- 0.52) or the 4.5% lactose (1.60 +/- 0.52). The data from the microbial analysis showed that the addition of lactose did not affect any bacterial populations when compared with the control birds that did not receive dietary lactose over the 21-d evaluation. The overall lesion scores in experiment 2 were significantly (P < 0.05) reduced in birds fed 2.5% lactose compared with the birds fed the control diet with mean lesion scores of 1.10 +/- 0.73 and 1.80 +/- 0.73, respectively. These experiments suggest that lactose could be used as a potential alternative to growth-promoting antibiotics to help control this costly disease.

Transferability of antimicrobial resistance from multidrug-resistant Escherichia coli isolated from cattle in the USA to E. coli and Salmonella Newport recipients.

OBJECTIVES: This study aimed to evaluate conjugative transfer of cephalosporin resistance among 100 strains of multidrug-resistant Escherichia coli (MDRE) to Salmonella enterica serotype Newport and E. coli DH5α recipients. METHODS: Phenotypic and genotypic profiles were determined for MDRE as well as for Salmonella Newport (trSN) and E. coli DH5α (trDH) transconjugants. RESULTS: Of 95 MDRE donor isolates, 26 (27%) and 27 (28%) transferred resistance to trSN and trDH recipients, respectively. A total of 27 MDRE (27%) were confirmed as extended-spectrum ß-lactamase (ESBL)-producers based on the double-disk synergy assay and whole-genome sequencing (WGS). WGS was performed on 25 of the ESBL-producing isolates, showing that 2 isolates carried blaCTX-M-6, 22 possessed blaCTX-M-32 and 1 was negative for blaCTX-M genes. Fourteen of the ESBLs sequenced were qnrB19. Differential transfer of IncA/C and IncN from MDRE32 was observed between trSN32 and trDH32. IncN-positive trDH32 displayed an ESBL phenotype, whereas IncA/C-positive trSN32 displayed an AmpC phenotype. The rate of ESBL transfer to trSN and trDH recipients was 11% and 96%, respectively. CONCLUSIONS: Twenty-seven MDRE were phenotypically identified as ESBL-producers. WGS of 25 MDRE revealed that 2 and 22 isolates carried blaCTX-M-6 and blaCTX-M-32, respectively. One multidrug-resistant isolate exhibited conversion from an AmpC phenotype to an ESBL phenotype with the transfer of only the IncN plasmid. The rate of resistance transfer to Salmonella or E. coli recipients was nearly identical. However, the ESBL phenotype was transferred with significantly greater prevalence to E. coli compared with Salmonella Newport (96% and 11%, respectively).

Fecal prevalence and diversity of Salmonella species in lactating dairy cattle in four states.

Salmonella is one of the most serious foodborne pathogenic bacteria in the United States, causing an estimated 1.3 million human illnesses each year. Dairy cows can be reservoirs of foodborne pathogenic bacteria, including Salmonella spp.; it is estimated that from 27 to 31% of dairy herds across the United States are colonized by Salmonella. The present study was designed to examine the occurrence of Salmonella spp. on dairies and to examine the serotypic diversity of Salmonella isolates on sampled dairies from across the United States. Fecal samples (n = 60 per dairy) were collected from 4 dairies in each of 4 states for a total of 960 fecal samples representing a total population of 13,200 dairy cattle. In the present study, 93 of 960 samples (9.96%) collected were culture-positive for Salmonella enterica. At least one Salmonella fecal-shedding cow was found in 9 of the 16 herds (56%) and the within-herd prevalence varied in our study from 0% in 7 herds to a maximum of 37% in 2 herds, with a mean prevalence among Salmonella-positive herds of 17%. Seventeen different serotypes were isolated, representing 7 different Salmonella serogroups. There were 2 or more different serogroups and serotypes present on 7 of the 9 Salmonella-positive farms. Serotypes Montevideo and Muenster were the most frequent and widespread. From our data, it appears that subclinical colonization with Salmonella enterica is relatively common on dairy farms and is represented by diverse serotypes on US dairy farms.

Persistence of a vancomycin-resistant Enterococcus faecium in an anaerobic continuous-flow culture of porcine microflora in the presence of subtherapeutic concentrations of vancomycin.

Recombined porcine continuous-flow culture (RPCF) maintained in a continuous-flow fermentation system is effective in protecting neonatal and weaned pigs against infection by enteropathogens. In the current study, we demonstrate the effect of RPCF on vancomycin-resistant enterococci (VRE) in the presence and absence of subtherapeutic levels of vancomycin. Also examined was the ability of VRE to transfer vancomycin resistance to endogenous Enterococcus faecalis 137.1. When RPCF was challenged with VRE, the rate of VRE clearance was dependent on the method of challenge. In the control experiment, RPCF was challenged with 7.0 log10/CFU/ml VRE. Clearance of VRE from the culture was observed within 7 days at a rate of 1.44 log10/day. RPCF containing 0.001 microg/ml vancomycin cleared VRE at a slightly lower rate of 0.94 log10/day. RPCF containing 0.01 microg/ml or 0.1 microg/ml vancomycin reduced the level of VRE from 7.0 log10/CFU/ml to 2.0 log10/CFU/ml within 9 days, but failed to clear the VRE after 24 days. During the period of decline, the VRE clearance rate for the 0.01 microg/ml and 0.1 microg/ml vancomycin-treated cultures was 0.52 log10/day, and 0.53 log10/day, respectively. E. faecalis 137.1 endogenous to RPCF did not acquire the vancomycin resistance genes throughout the experiment as evidenced by direct selection, ribotyping, and pulsed-field gel electrophoresis.

Recent pre-harvest supplementation strategies to reduce carriage and shedding of zoonotic enteric bacterial pathogens in food animals.

Food-borne bacterial illnesses strike more than 76 million North Americans each year. Many of these illnesses are caused by animal-derived foodstuffs. Slaughter and processing plants do an outstanding job in reducing bacterial contamination after slaughter and during further processing, yet food-borne illnesses still occur at an unacceptable frequency. Thus, it is imperative to widen the window of action against pathogenic bacteria. Attacking pathogens on the farm or in the feedlot will improve food safety all the way to the consumer's fork. Because of the potential improvement in overall food safety that pre-harvest intervention strategies can provide, a broad range of preslaughter intervention strategies are currently under investigation. Potential interventions include direct anti-pathogen strategies, competitive enhancement strategies and animal management strategies. Included in these strategies are competitive exclusion, probiotics, prebiotics, antibiotics, antibacterial proteins, vaccination, bacteriophage, diet, and water trough interventions. The parallel and simultaneous application of one or more preslaughter strategies has the potential to synergistically reduce the incidence of human food-borne illnesses by erecting multiple hurdles, thus preventing entry of pathogens into the food chain. This review emphasizes work with Escherichia coli O157:H7 to illustrate the various strategies.

Competitive exclusion of Salmonella from the gut of neonatal and weaned pigs.

Our laboratory has developed a bacterial competitive-exclusion (CE) culture against enteropathogens (which are considered human foodborne pathogens) for use in swine. In this article, we document the effects of this CE culture, PCF1, on cecal colonization by and fecal shedding of Salmonella Choleraesuis in neonatal and weaned pigs and its effects on the horizontal transmission of this pathogen between weaned penmates. Piglets treated with the PCF1 culture twice within their first day of life and challenged with Salmonella 48 h after birth shed Salmonella at a significantly (P < 0.05) lower rate than did control pigs in experiment 1. Significant reductions of the pathogen were also observed in the cecum, the cecal contents, the ileocolic junction, and the colon contents (P < 0.05). In experiment 2, culture of the cecal contents and lymph nodes revealed a significant reduction in Salmonella isolated from PCF1-treated pigs (P < 0.05). Pigs in experiment 3 were treated as pigs in experiments 1 and 2 were: however, they were followed through day 10 postweaning. Significant reductions in shedding were noted for treated groups both pre- and postweaning (P < 0.05). Experiments 4 and 5 assessed the effects of PCF1 treatment on the horizontal transmission of Salmonella between littermates that were followed through day 14 postweaning. In these experiments, litters were divided into untreated contacts (UC), untreated seeders (US), treated contacts (TC), and treated seeders (TS). Overall, TC in experiment 4 shed Salmonella at a significantly lower rate than UC and US did (P < 0.05). In experiment 5, the transmission of Salmonella was significantly reduced for litters in which TS or TC were present, as evidenced by reduced shedding of Salmonella by both treated and untreated animals within these litters (P < 0.05). TS shed less often than US did, resulting in reduced levels of Salmonella shedding by both treated and untreated contacts (P < 0.05). Litters containing both TC and UC or both TC and US also shed Salmonella at lower rates than did litters in which only UC and US were present (P < 0.05).

Genotypic variation among arcobacter isolates from a farrow-to-finish swine facility.

Arcobacter spp. were isolated from nursing sows and developing pigs on three farms of a farrow-to-finish swine operation and market-age pigs at slaughter. Isolates were identified by polymerase chain reaction and genotypic fragment patterns were examined by pulsed-field gel electrophoresis (PFGE). Incidences of Arcobacter-positive samples increased progressively as the pigs aged, resulting in all of the pens at the end of the growth cycle in the finishing barn containing Arcobacter-positive feces. However, only 10 of 350 cecal samples from slaughtered pigs were positive. There was little similarity between genotypic patterns for Arcobacter collected from the three farms. The level of genotypic variation revealed by PFGE suggested that pigs in this farrow-to-finish operation were colonized by multiple Arcobacter parent genotypes that may have undergone genomic rearrangement, common to members of Campylobacteraceae, during successive passages through the animals. Additionally, the level of genotypic diversity seen among Arcobacter isolates from farms of a single farrow-to-finish swine operation suggests an important role for genotypic phenotyping as a source identification and monitoring tool during outbreaks.

Colicin concentrations inhibit growth of Escherichia coli O157:H7 in vitro.

Escherichia coli O157:H7 is a virulent foodborne pathogen that causes severe human illness and inhabits the intestinal tract of food animals. Colicins are antimicrobial proteins produced by E. coli strains that inhibit or kill other E. coli. In the present Study, the efficacy of three pore-forming colicins (El, N, and A) were quantified in vitro against E. coli O157:H7 strains 86-24 and 933. Colicins E1 and N reduced the growth of E. coli O157:H7 strains, but the efficacy of each colicin varied among strains. Colicin E1 was more effective against both strains of E. coli O157:H7 than colicins A and N and reduced (P < 0.05) populations of E. coli O157:H7 at concentrations <0.1 microg/ml. These potent antimicrobial proteins may potentially provide an effective and environmentally sound preharvest strategy to reduce E. coli O157:H7 in food animals.

Escherichia coli O157:H7 populations in sheep can be reduced by chlorate supplementation.

Ruminant animals are a natural reservoir of the foodborne pathogen Escherichia coli O157:H7. Some foodborne pathogens (e.g., E. coli) are equipped with a nitrate reductase that cometabolically reduces chlorate. The intracellular reduction of chlorate to chlorite kills nitrate reductase-positive bacteria; however, species that do not reduce nitrate are not affected by chlorate. Therefore, it has been suggested that ruminants be supplemented with chlorate prior to shipment for slaughter in order to reduce foodborne illnesses in human consumers. Sheep (n = 14) were fed a high-grain ration and were experimentally infected with E. coli O157:H7. These sheep were given an experimental product (XCP) containing the equivalent of either 2.5 mM NaNO3 and 100 mM NaCl (control sheep; n = 7) or 2.5 mM NaNO3 and 100 mM NaClO3 (chlorate [XCP]-treated sheep; n = 7). Control and XCP-treated sheep were treated for 24 h; XCP treatment reduced the population of inoculated E. coli O157:H7 (P < 0.05) from 10(2), 10(5), and 10(5) CFU/g in the rumen, cecum, and rectum, respectively, to < 10(1) CFU/g in all three sections of the gastrointestinal tract. The number of sheep testing positive for E. coli O157:H7 was significantly reduced by XCP treatment. In a similar fashion, total E. coli and coliforms were also reduced (P < 0.05) in all three compartments of the intestinal tract. Intestinal pH, total volatile fatty acid production, and the acetate/propionate ratio were unaffected by XCP treatment. On the basis of these results, it appears that chlorate treatment can be an effective method for the reduction of E. coli O157:H7 populations in ruminant animals immediately prior to slaughter.

Sodium chlorate supplementation reduces E. coli O157:H7 populations in cattle.

Cattle are a natural reservoir of the food-borne pathogen Escherichia coli O157:H7. Therefore, strategies that reduce E. coli O157:H7 prior to slaughter will reduce human exposures to this virulent pathogen. When bacteria that can anaerobically respire on nitrate (e.g., E. coli) are exposed to chlorate, they die because the intracellular enzyme nitrate reductase converts nitrate to nitrite, but also co-metabolically reduces chlorate to cytotoxic chlorite. Because chlorate is bactericidal only against nitrate reductase-positive bacteria, it has been suggested that chlorate supplementation be used as a strategy to reduce E. coli O157:H7 populations in cattle prior to harvest. Cattle (n = 8) were fed a feedlot-style high-grain diet experimentally infected with three strains of E. coli O157:H7. Cattle were given access to drinking water supplemented with 2.5 mM KNO3 and 100 mM NaCl (controls; n = 4) or 2.5 mM KNO3 and 100 mM NaClO3 (chlorate-treated; n = 4). Sodium chlorate treatment for 24 h reduced the population of all E. coli O157:H7 strains approximately two logs (10(4) to 10(2)) in the rumen and three logs (10(6) to 10(3)) in the feces. Chlorate treatment reduced total coliforms and generic E. coli from 106 to 10(4) in the rumen and by two logs throughout the rest of the gastrointestinal tract (ileum, cecum, colon, and rectum). Chlorate treatment reduced E. coli O157:H7 counts throughout the intestinal tract but did not alter total culturable anaerobic bacterial counts or the ruminal fermentation pattern. Therefore, it appears that chlorate supplementation is a viable potential strategy to reduce E. coli O157:H7 populations in cattle prior to harvest.

Utilization of the nitrate reductase enzymatic pathway to reduce enteric pathogens in chickens.

Previous reports have shown that some bacteria, including Salmonella, use a dissimilatory nitrate reductase enzyme pathway (NREP) in anaerobic environments. This enzyme reduces nitrate to nitrite and has been shown to cometabolize chlorate to cytotoxic chlorite. The present investigations were performed to evaluate the susceptibility of a competitive exclusion culture (CE) to the experimental chlorate product (ECP). A commercially available CE product was evaluated for its nitrate reductase activity and therefore its chlorate sensitivity. Individual isolates (in triplicate) were cultured in 10 mL of Viande Levure broth containing 5 mM sodium nitrate or 10 mM sodium chlorate. Bacterial growth (optical density at 625 nm) was measured and 1-mL aliquots were removed concurrently for colorimetric determination of nitrate content at 0, 3, 6, and 24 h. Of the 15 different facultative strains, 11 had slight NREP utilization, 3 had moderate NREP utilization, and the remainder were NREP negative (with slight and moderate NREP utilization: >0.1 to <1.0 mM and >1.0 mM nitrate used within 6 h, respectively). Of the obligate anaerobes evaluated, 3 had slight NREP utilization and the remainder were NREP negative. In vivo studies utilizing both products (CE and ECP) in a horizontal transmission challenge model (seeders + contacts) showed significant reductions in Salmonella from 5.37 to 1.76 log10 cfu/g and 3.94 to 0.07 log10 cfu/g, respectively. The combined effect of the CE culture and an ECP are effective in killing these food-borne pathogens.

Competitive exclusion of a glycopeptide-resistant Enterococcus faecium in the presence of vancomycin but not equivalent concentrations of tylosin or gentamicin.

The effect of subtherapeutic concentrations of antibiotics (10.0 and 40.0 microg/mL of vancomycin, gentamicin, and tylosin) on the efficacy of a mixed anaerobe culture of chicken microflora (CCF) was studied in a continuous-flow fermentation system. Efficacy of CCF posttreatment was assessed by challenge with glycopeptide-resistant Enterococcus faecium (GRE) at 6.0 log10 cfu/mL. Bacterial enumeration of endogenous CCF isolates, volatile fatty acid (VFA) analysis, and challenge with GRE indicated that CCF efficacy was affected by all antibiotic treatments. Although CCF treated with 10.0 microg/mL of vancomycin eliminated GRE13 at a rate of 0.61 log10 cfu/ mL per day, it was unable to eliminate E. coli, a gram-negative challenge organism. All other antibiotic treatments allowed GRE persistence at approximately 2.0 to 6.5 log10 cfu/mL. All antibiotic-treated cultures had decreased concentrations of acetic and propionic acids. Our data suggest that low concentrations of antimicrobials may adversely affect the microbial ecology of gut microflora with respect to its ability to exclude exogenous bacteria. Moreover, gentamicin had an adverse effect on the inhibitory stringency of CCF even though it showed little anti-anaerobic activity against CCF strict anaerobes in pure culture. Verification of the results in live animals will be necessary to determine if antimicrobial treatment could compromise the effectiveness of normal microflora to serve as a natural host defense against infection.

Evaluation of immunosuppressants and dietary mechanisms in an experimental disease model for necrotic enteritis.

Clostridium perfringens (CP) is the etiologic agent of necrotic enteritis (NE). Clinical signs of this disease include depression, decreased appetite, diarrhea, and severe necrosis of the intestinal tract. Understanding the disease progression of NE has been difficult due to its complexity and the involvement of multiple factors (dietary components, immunosuppression, and mechanical irritation of the gut) that appear to contribute to this syndrome. In the present investigation, day-of-hatch broilers were fed a 55% wheat diet and randomly assigned to 1 of 8 groups. Treatments included positive control (CP challenge only), commercial coccidia vaccine (CCV), commercial bursal disease vaccine (CBDV), or the combination of CCV and CBDV, and an appropriate negative control for each (vaccinated and not challenged). Challenged treatment groups received 10(7) cfu of CP twice daily. When compared with controls, broilers in each treatment group had increased (P < or = 0.05) lesion scores, with mean scores of 1.05 and 2.05 in the CP and CBDV + CP treatments, respectively. When compared with controls, the incidence of CP increased (P < or = 0.05) in all treatment groups (73 and 100% in the CCV + CP and CBDV + CP treatment groups, respectively). Compared with controls, percentage mortality increased (P < or = 0.05) from 2% to 26 and 34% in the CP and CBDV + CP treatment groups, respectively. Results of this study indicate that the methodology used provides a good model for studying NE.