Effects of Isotricha, Dasytricha, Entodinium, and total fauna on ruminal fermentation and duodenal flow in wethers fed different diets

The objective was to measure rumen fermentation and duodenal flow of amino acids and nonammonia N components in five groups of five ruminally and duodenally cannulated wethers that were fauna-free, or inoculated with the ciliate protozoa genera of Isotricha, Dasytricha, Entodinium, or a normal population (total) of fauna. They were used in two 25-d periods and fed a haycrop-based diet in the first period and a corn silage-based diet in the second period. Feces, duodenal digesta, and rumen contents were sampled in each period and analyzed. The number of Entodinium in wethers containing the Entodinium monofauna was higher than the total protozoa numbers, including Entodinium, in wethers containing total fauna population. The type of diet or fauna did not affect total volatile fatty acid concentrations in rumen fluid. The ammonia N concentration in rumen fluid was higher in wethers containing total fauna (25 mg/100 ml) than in fauna-free wethers fed the two diets (18 and 12 mg/ 100 ml). In comparison with the respective fauna-free wethers, the concentration of ammonia in wethers containing Entodinium was higher when fed the corn silage diet, but not different when fed the haycrop diet. Ruminal presence of total fauna or Entodinium decreased the nonammonia N by 16 and 17%, and total amino acid flows from the stomach by 20 and 19%, respectively. Flow of bacteria N was decreased in wethers fed the two diets when Entodinium or total fauna were present in the rumen. The presence of Isotricha resulted in higher flow of bacteria N in wethers fed the haycrop diet, but the presence of Dasytricha resulted in higher bacteria N flow in wethers fed the corn silage diet. Entodinium was the most detrimental of ciliate protozoa species concerning protein nutrition of the host ruminant.     

Escherichia coli O157:H7 populations in ruminants can be reduced by orange peel product feeding

Foodborne pathogenic bacteria such as Escherichia coli O157:H7 are threats to the safety of beef. Citrus peel and dried orange pulp are by-products from citrus juice production that have natural antimicrobial effects and are often incorporated into least-cost ration formulations for beef and dairy cattle. This study was designed to determine if orange peel and pulp affected E. coli O157:H7 populations in vivo. Sheep (n = 24) were fed a cracked corn grain-based diet that was supplemented with a 50-50 mixture of dried orange pellet and fresh orange peel to achieve a final concentration (dry matter basis, wt/wt) of 0, 5, or 10% pelleted orange peel (OP) for 10 days. Sheep were artificially inoculated with 10(10) CFU of E. coli O157:H7 by oral dosing. Fecal shedding of E. coli O157:H7 was measured daily for 5 days after inoculation, after which all animals were humanely euthanized. At 96 h postinoculation, E. coli O157:H7 shedding was reduced (P < 0.05) in sheep fed 10% OP. Populations of inoculated E. coli O157:H7 were reduced by OP treatment throughout the gastrointestinal tract; however, this reduction reached significant levels in the rumen (P < 0.05) of sheep fed 10% OP diets. Cecal and rectal populations of E. coli O157:H7 were reduced (P < 0.05) by inclusion of both 5 and 10% OP diets. Our results demonstrate that orange peel products can be used as a preharvest intervention strategy as part of an integrated pathogen reduction scheme.     

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.     

Evaluation of increased incubation temperature and cefixime-tellurite treatment for the isolation of Escherichia coli O157:H7 from minced beef

To improve enrichment and isolation of Escherichia coli O157:H7, this study evaluated increased incubation temperature and cefixime-tellurite (CT) on five strains of each of the following bacteria, E. coli, Hafnia alvei, Enterobacter spp., Citrobacter freundii and E. coli O157:H7, and two strains of E. coli O157:nH7. These were grown in pure culture in LST broth with varying cefixime-tellurite concentrations. A range of incubation temperatures from 37 to 46 degrees C was investigated for the inhibition of cohabitant microorganisms. Minced beef, spiked with E. coli O157:H7 and cohabitant microorganisms was investigated. Increased incubation temperature (42 degrees C) and treatment with half of the prescribed amount of cefixime-tellurite by BAM for SMAC agar in enrichment step were the most effective in selectively growing E. coli O157:H7. The results show that E. coli O157:H7 is more resistant to these two conditions than the other cohabitant bacteria.     

Multiplex PCR for simultaneous detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 in meat samples

A multiplex PCR method was developed for simultaneous detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 in meat samples. DNA detection sensitivity for this method was 10(3) CFU/ml for each pathogen. When this protocol was used for the detection of each of the above pathogenic bacteria in spiked pork samples, 1 cell per 25 g of inoculated sample could be detected within 30 h. In the samples of naturally contaminated meat, Salmonella spp., L. monocytogenes, and E. coli O157:H7 were detected over the same time period. Excellent agreement was obtained for the results of multiplex PCR and the conventional culture method, which suggests that the multiplex PCR is a reliable and useful method for rapid screening of meat products for Salmonella spp., L. monocytogenes, and E. coli O157:H7 contamination.     

Acquisition of antibiotic resistance plasmids by enterohemorrhagic Escherichia coli O157:H7 within rumen fluid

The emergence of antibiotic resistance among important foodborne pathogens like Escherichia coli O157:H7 has become an important issue with regard to food safety. In contrast to the case for Salmonella, antibiotic resistance has been slow in its development in E. coli O157:H7 despite the presence of mobile antibiotic resistance genes in other E. coli organisms that inhabit the same animal host. We set out to determine if rumen fluid influences the transfer of plasmid-mediated, antibiotic resistance to E. coli O157:H7. A commensal E. coli strain from a dairy cow was transformed with conjugative R plasmids and served as the donor in matings with naladixic acid-resistant E. coli O157:H7. R plasmids were transferred from the donor E. coli strain to E. coli O157:H7 in both Luria-Bertani (LB) broth and rumen fluid. R plasmids were transferred at a higher frequency to E. coli O157:H7 during 6 h of incubation in rumen fluid at rates comparable to those in LB broth, indicating that conditions in rumen fluid favor the transfer of the plasmids to E. coli O157. This finding suggests that the cow's rumen is a favorable environment for the genetic exchange of plasmids between microflora and resident E. coli O157:H7 in the bovine host.     

Bactericidal effect of sodium chlorate on Escherichia coli O157:H7 and Salmonella typhimurium DT104 in rumen contents in vitro

Escherichia coli O157:H7 and Salmonella Typhimurium DT104 are important foodborne pathogens affecting the beef and dairy industries and strategies are sought to rid these organisms from cattle at slaughter. Both pathogens possess respiratory nitrate reductase that also reduces chlorate to the lethal chlorite ion. Because most anaerobes lack respiratory nitrate reductase, we hypothesized that chlorate may selectively kill E. coli O157:H7 and Salmonella Typhimurium DT104 but not potentially beneficial anaerobes. In support of this hypothesis, we found that concentrations of E. coli O157:H7 and Salmonella Typhimurium DT104 were reduced from approximately 1,000,000 colony forming units (CFU) to below our level of detection (< or = 10 CFU) following in vitro incubation (24 h) in buffered ruminal contents (pH 6.8) containing 5 mM added chlorate. In contrast, chlorate had little effect on the most probable number (mean +/- SD) of total culturable anaerobes (ranging from 9.9 +/- 0.72 to 10.7 +/- 0.01 log10 cells/ml). Thus, chlorate was bactericidal to E. coli O157:H7 and Salmonella Typhimurium DT104 but not to potentially beneficial bacteria. The bactericidal effect of chlorate was concentration dependent (less at 1.25 mM) and markedly affected by pH (more bactericidal at pH 6.8 than pH 5.6).     

Fecal shedding and rumen growth of Escherichia coli O157:H7 in fasted calves.

Nine weaned calves aged from 8 to 12 weeks were fitted with rumen cannulas and were inoculated by cannula with 10(10) CFU of a five-strain mixture of nalidixic acid-resistant Escherichia coli O157:H7. Six calves were fasted for 48 h on days 15 and 16 and days 22 and 23 after inoculation. Samples of rumen contents and feces were obtained daily to enumerate E. coli O157:H7 populations and to determine rumen volatile fatty acid (VFA) concentrations and rumen pH. Fasting resulted in a marked decrease in rumen VFA concentrations from a mean of 135 mmol/liter before the fast to a mean of 35 mmol/liter during the second day of the fast. However, there was no correlation between daily VFA concentration and daily rumen or fecal numbers of E. coli O157:H7 in any of the calves. Fasting generally had no significant effect on the rumen or fecal numbers of E. coli O157:H7. The exception was a single fasted calf that experienced a 3-log(10) CFU/g increase in fecal shedding during and after the first fast. Despite the consistent changes in VFA concentrations in fasted calves, the fluctuations in rumen numbers of E. coli O157:H7 in the rumen of fasted calves were minimal. At the end of the experiment, E. coli O157:H7 was detected in either the rumen or omasum in two of three control calves at necropsy and in either the rumen or reticulum in five of six fasted calves. E. coli O157:H7 was detected in the colon in two of three control calves and in six of six fasted calves at necropsy. These results suggest that in cattle already shedding E. coli O157:H7, feed withdrawal and the associated changes in rumen pH and VFA concentrations have little effect on fecal shedding and rumen proliferation of E. coli O157:H7.     

Effects of diet on rumen proliferation and fecal shedding of Escherichia coil O157:H7 in calves

Calves inoculated with Escherichia coli O157:H7 and fed either a high-roughage or high-concentrate diet were evaluated for rumen proliferation and fecal shedding of E. coli O157:H7. Calves fed the high-roughage diet had lower mean rumen volatile fatty acid concentrations and higher rumen pH values than did calves fed the high-concentrate diet. Despite these differences in rumen conditions, the calves fed the high-roughage diet did not have greater rumen populations of E. coli O157: H7 and did not exhibit increased or longer fecal shedding compared with the calves fed the high-concentrate diet. Two calves shedding the highest mean concentrations of E. coli O157:H7 were both fed the high-concentrate diet. There was a significant (P < 0.05) positive correlation between fecal shedding and rumen volatile fatty acid concentration in calves fed a high-concentrate diet. The effects of diet on E. coli O157:H7 proliferation and acid resistance were investigated using an in vitro rumen fermentation system. Rumen fluid collected from steers fed a high-roughage diet, but not from steers fed a high-concentrate diet, supported the proliferation of E. coli O157:H7. Rumen fluid from steers fed a high-concentrate diet rapidly induced acid resistance in E. coli O157:H7. The impact of diet on fecal shedding of E. coli O157:H7 is still unclear and may depend on dietary effects on fermentation in the colon and on diet-induced changes in the resident microflora. However, rapid development of acid tolerance by E. coli O157:H7 in the rumens of calves fed high-concentrate diets, allowing larger populations to survive passage through the acidic abomasum to proliferate in the colon, may be one factor that influences fecal shedding in cattle on feed.     

Escherichia coli O157:H7 in faeces from cattle, sheep and pigs in the southwest part of Norway during 1998 and 1999

During a 2-year period from January 1998 to December 1999, intestinal content from 1541 cattle, 665 sheep and 1976 pigs were analysed for Escherichia coli O157:H7 using the immunomagnetic separation procedure. The animals originated from 848, 605 and 832 herds from the southwest part of Norway, respectively. E. coli O157:H7 was present in three samples from cattle from different herds, giving a herd prevalence of 0.35% and an animal prevalence of 0.19%. From pigs, E. coli O157:H7 was isolated from two pigs from different herds, giving a herd prevalence of 0.24% and an animal prevalence of 0.1%. A follow-up study revealed another positive testing pig from one of these herds. E. coli O157:H7 was not found from any of the 665 investigated sheep. By PCR analysis, all six E. coli O157:H7 isolates were shown to contain the genes encoding Shiga toxin 2 (stx2), the intimin protein (eae) and the H7 flagellum (fliC-H7). One of the cattle isolates also harboured the Shiga toxin 1 encoding (stx1) gene. The six isolates were differentiated into three pulse-field gel electrophoresis profiles. The results indicate that the occurrence of E. coli O157:H7 in cattle, sheep and pigs in the southwest part of Norway is low compared to other European countries.     

Inhibition of pathogens on fresh produce by ultraviolet energy

Ultraviolet energy at a wavelength of 253.7 nm (UVC) was investigated for its bactericidal effects on the surface of Red Delicious apples, leaf lettuce and tomatoes inoculated with cultures of Salmonella spp. or Escherichia coli O157:H7. Inoculated samples were subjected to different doses ranging from 1.5 to 24 mW/cm(2) of UVC and enumerated on tryptic soy agar plus 0.05 g/l nalidixic acid to determine effective log reductions of microbial populations. UVC applied to apples inoculated with E. coli O157:H7 resulted in the highest log reduction of approximately 3.3 logs at 24 mW/cm(2). Lower log reductions were seen on tomatoes inoculated with Salmonella spp. (2.19 logs) and green leaf lettuce inoculated with both Salmonella spp. and E. coli O157:H7 (2.65 and 2.79, respectively). No significant statistical difference (p>0.05) was seen in the ability of UVC to inactivate a higher population of either Salmonella spp. or E. coli O157:H7 on the surface of green leaf lettuce. No significant difference was seen among the use of different doses applied to the surface of fresh produce for reduction of E. coli O157:H7 or Salmonella spp. (p>0.05). The use of UVC may prove to be beneficial in protecting the safety of fruits and vegetables in conjunction with Good Agricultural Practices and Good Manufacturing Practices.     

Validation of apple cider pasteurization treatments against Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes.

Time and temperature pasteurization conditions common in the Wisconsin cider industry were validated using a six-strain cocktail of Escherichia coli O157:H7 and acid-adapted E. coli O157:H7 in pH- and degrees Brix-adjusted apple cider. Strains employed were linked to outbreaks (ATCC 43894 and 43895, C7927, and USDA-FSIS-380-94) or strains engineered to contain the gene for green fluorescent protein (pGFP ATCC 43894 and pGFP ATCC 43889) for differential enumeration. Survival of Salmonella spp. (CDC 0778. CDC F2833, and CDC H0662) and Listeria monocytogenes (H0222, F8027, and F8369) was also evaluated. Inoculated cider of pH 3.3 or 4.1 and 11 or 14 degrees Brix was heated under conditions ranging from 60 degrees C for 14 s to 71.1 degrees C for 14 s. A 5-log reduction of nonadapted and acid-adapted E. coli O157:H7 was obtained at 68.1 degrees C for 14 s. Lower temperatures, or less time at 68.1 degrees C, did not ensure a 5-log reduction in E. coli O157:H7. A 5-log reduction was obtained at 65.6 degrees C for 14 s for Salmonella spp. L. monocytogenes survived 68.1 degrees C for 14 s, but survivors died in cider within 24 h at 4 degrees C. Laboratory results were validated with a surrogate E coli using a bench-top plate heat-exchange pasteurizer. Results were further validated using fresh unpasteurized commercial ciders. Consumer acceptance of cider pasteurized at 68.1 degrees C for 14 s (Wisconsin recommendations) and at 71.1 degrees C for 6 s (New York recommendations) was not significantly different. Hence, we conclude that 68.1 degrees C for 14 s is a validated treatment for ensuring adequate destruction of E. coli O157:H7, Salmonella spp., and L. monocytogenes in apple cider.     

Validation of Dry Cured Ham Process for Control of Pathogens

ABSTRACT: The dry curing process for hams to control Salmonella spp., Escherichia coli O157:H7, Listeria monocytogenes , and Staphylococcus aureus was evaluated. Fresh hams, surface inoculated with each microorganism, were processed by a commercial style process. There was no significant (p < 0.05) difference in reduction of microbial populations between ham sampling locations (cushion, butt, hock). Interaction of salt concentration (8%), pH (5.5), ham storage temperature (20 °C), and ham aw (0.92) limited staphylococcal proliferation. Mean log reduction of Salmonella spp., E. coli O157:H7 and L. monocytogenes populations on inoculated hams after 69 d of curing were 5.5, 5.5, and 4.0 CFU/cm², respectively and after 120 d were 5.7, 5.5, and 4.8 CFU/cm², respectively. Keywords: dry cured ham, Salmonella, Esherichia coli O157:H7, Listeria monocytogenes, Staphylococcus aureus     

Prevalence and molecular characterization of Escherichia coli O157:H7 by multiple-locus variable-number tandem repeat analysis and pulsed-field gel electrophoresis in three sheep farming operations in California

A year-long study was conducted to determine the fecal prevalence of Escherichia coli O157:H7 in three sheep ranches. Strain diversity and persistence were compared with multiple-locus variable-number tandem repeat analysis and pulsed-field gel electrophoresis. Ranch C, a feedlot, consisted of young sheep raised predominantly on a high-grain diet. The other two sites consisted of sheep raised on native pasture and a combination of native and irrigated pasture. Forty fecal samples were collected every month from each ranch. Samples were examined for E. coli O157:H7 by immunomagnetic separation and culture of the magnetic beads onto selective media. Detection of virulence markers in positive isolates was determined by PCR. E. coli O157:H7 was isolated from 100 (22.7%) of 440 fecal samples collected from ranch C. On ranch B, 9 (1.9%) of the 480 fecal samples were positive for the pathogen, while none of the samples from ranch A were positive. On ranch C, the odds of detecting E. coli O157:H7 was 3.2 times greater during the warmer months compared with the cooler months of the year. There was no association between days spent in the feedlot and fecal prevalence of the pathogen (P = 0.62). Most multiple-locus variable-number tandem repeat analysis types were isolated only once from ranch C (14 of 23), but several strains were isolated over 4 to 6 months, often in many intervening negative months. This study revealed that the prevalence of E. coli O157:H7 can be high in some sheep ranches in California, especially in feedlots where young sheep are fed predominantly high-grain rations.     

OCCURRENCE OF ESCHERICHIA COLIO157:H7/O157, LISTERIA MONOCYTOGENES AND SALMONELLA SPP. IN BEEF SLAUGHTERHOUSE ENVIRONMENTS, EQUIPMENT AND WORKERS

Escherichia coli O157/O157:H7, Listeria monocytogenes and Salmonella spp. are major foodborne pathogens and the emergence of these pathogens has been reported in many countries. The aim of this study was to investigate contamination of the beef slaughterhouse environment, equipment and workers with E. coli O157/O157:H7, L. monocytogenes and Salmonella spp. For this study, 500 swab samples were taken from 19 different points in five privately owned slaughterhouses, their periphery, slaughterhouse equipment and slaughterhouse employees. The presence of E. coli O157:H7/O157, L. monocytogenes and Salmonella spp. was determined with the application of the immunomagnetic separation method. Our study showed that the swabs taken from the five slaughterhouses contained E. coli O157:H7 in the environment, equipment, abattoir workers and water with a frequency 0.31, 1, 1.42 and 0%, respectively; while E. coli O157 was evident in the environment, equipment, abattoir workers and water with a ratio of 15, 10, 10 and 0%, respectively; L. monocytogenes was detected in the environment, equipment, abattoir workers and water at a ratio of 4.37, 15, 5.71 and 0%, respectively; and Salmonella spp. occurrence in the environment, equipment, abattoir workers and water at a ratio of 3.43, 16, 11.42, and 0%, respectively. Implementing hazard analysis critical control point principles in work procedures would definitely reduce the gross contamination occurring in abattoirs.

PRACTICAL APPLICATIONS

This study has revealed the effect of personnel and equipment on the contamination routes of E. coli O157:H7, Listeria monocytogenes and Salmonella spp. in meat in slaughterhouses and showed that especially L. monocytogenes and Salmonella spp. may pose a higher risk than E. coli O157:H7 in slaughterhouses.     

用寡核苷酸微阵列芯片方法检测常见的食源性致病微生物

研制了1种高通量检测食品中常见致病微生物的寡核苷酸微阵列芯片。该芯片在种的水平可鉴别金黄色葡萄球菌、阪崎肠杆菌、肠出血性大肠埃希氏菌O157:H7、霍乱弧茵、空肠弯曲杆菌、大肠弯曲杆菌、单核细胞增生李斯特菌、乙型洛血型链球菌和副溶血性弧菌;在属的水平可鉴别弯曲肠杆菌属、沙门氏菌属和李斯特氏菌属。研究中利用16S rDNA和沙门氏菌、O157:H7和副溶血性弧菌的特异基因序列设计引物和探针。用参比菌株和实验室自分离的菌株检测该芯片的特异性,结果表明该芯片的特异性良好,在所检测的菌株之间无交叉反应,与同属的其他菌株之间也不存在交叉反应。在鲜活海产品的24 h增菌液中添加参比菌株,当样品增菌液中的目的菌的量达到10~6～10~7 CFU/mL时,芯片可以准确检测出目的菌。     

Reduction of Listeria monocytogenes, Salmonella spp., and Escherichia coli O157:H7 During Processing of Country-cured Hams

ABSTRACT: The country-cured ham process, including curing, equalization, cold-smoked or nonsmoked, and aging up to 6 mo, was validated and showed its effectiveness in achieving a 6-log reduction of Listeria monocytogenes, Salmonella spp., and Escherichia coli O157:H7. The viable counts of L. monocytogenes populations decreased to below detection levels after 206 d, Salmonella populations required 122 d, and E. coli O157:H7 required 66 d. However, L. monocytogenes -inoculated hams were positive and Salmonella spp-inoculated and E. coli O157:H7-inoculated hams were negative following enrichment procedures at the end of the aging process. Therefore, the survival of L. monocytogenes on country-cured ham represents a risk.     

Selection of recently isolated colicinogenic Escherichia coli strains inhibitory to Escherichia coli O157:H7

Escherichia coli strains were screened for their ability to inhibit E. coli O157:H7. An initial evaluation of 18 strains carrying previously characterized colicins determined that only colicin E7 inhibited all of the E. coli O157:H7 strains tested. A total of 540 strains that had recently been isolated from humans and nine different animal species (cats, cattle, chickens, deer, dogs, ducks, horses, pigs, and sheep) were tested by a flip-plating technique. Approximately 38% of these strains were found to inhibit noncolicinogenic E. coli K12 strains. The percentage of potentially colicinogenic E. coli per animal species ranged from 14% for horse isolates to 64% for sheep strains. Those isolates that inhibited E. coli K12 were screened against E. coli O157:H7, and 42 strains were found to be capable of inhibiting all 22 pathogenic strains tested. None of these 42 strains produced bacteriophages, and only 24 isolates inhibited serotype O157:H7 in liquid culture. The inhibitory activity of these strains was completely eliminated by treatment with proteinase K. When mixtures of these 24 colicinogenic strains were grown in anaerobic continuous culture, the four-strain E. coli O157:H7 population was reduced at a rate of 0.25 log10 cells per ml per h, which was fivefold faster than the washout rate. Two strains originally isolated from cat feces (F16) and human feces (H30) were identified by repetitive sequences polymerase chain reaction as the predominant isolates in continuous cultures. The results of this work indicate that animal species other than cattle can be sources of anti-O157 colicinogenic strains, and these results also lead to the identification of at least two isolates that could potentially be used in preharvest control strategies.     

Detection of Bacteroidales fecal indicators and the zoonotic pathogens E. coli 0157:H7, salmonella, and campylobacter in river water

Bacteroidales host-specific PCR offers a rapid method of diagnosing fecal pollution in water and identifying sources of input. To assess human health risks from exposure to fecal pathogens, however, Bacteroidales markers should be detectable when pathogens are present. To determine if Bacteroidales general, human-, ruminant-, and swine-specific markers correlate with certain fecal pathogens, we conducted a retrospective study on water samples for which the presence of E. coli O157:H7, Salmonella spp., and Campylobacter spp. had been determined. We found a positive relationship between detection of the Bacteroidales general fecal marker and presence of the pathogens. Detection of ruminant-specific markers predicted E. coli O157: H7 occurrence. There was a significant increase in the likelihood of detecting Salmonella when a ruminant marker was present, and Campylobacter spp. when human markers were present. For pathogens such as E. coli O157: H7 that are strongly associated with particular hosts, Bacteroidales host-specific markers can estimate the likelihood of pathogen occurrence, enabling more accurate health risk assessments.     

Survey of retail alfalfa sprouts and mushrooms for the presence of Escherichia coil O157:H7, Salmonella,and Listeria with BAX,and evaluation of this polymerase chain reaction-based system with experimentally contaminated samples

BAX, a polymerase chain reaction (PCR)-based pathogen detection system, was used to survey retail sprouts and mushrooms for contamination with Escherichia coli O157:H7, Salmonella, Listeria spp., and Listeria monocytogenes. No Salmonella or E. coli O157:H7 was detected in the 202 mushroom and 206 alfalfa sprout samples screened. L. monocytogenes was detected in one sprout sample, and seven additional sprout samples tested positive for the genus Listeria. BAX also detected Listeria species in 17 of the mushroom samples. Only 6 of 850 PCR assays (0.7%) failed to amplify control DNA, and therefore reagent failures and the inhibition of PCR by plant compounds were rare. The sensitivity of the detection system was evaluated by assaying samples inoculated with 10 CFU of each of the pathogens. One hundred seventy-two alfalfa sprout samples were inoculated with E. coli O157:H7, and two sets of 130 samples were experimentally contaminated with Salmonella Enteritidis and L. monocytogenes. The frequency of detection depended on the protocols used for inoculation and culturing. Inoculation of samples with approximately 10 CFU from frozen stocks yielded detection rates of 87.5 and 94.5% for L. monocylogenes and Salmonella Enteritidis, respectively, in mushrooms. The corresponding rates for alfalfa sprouts were 94.5 and 76.3%. The E. coli O157:H7 detection rate was 100% for mushrooms but only 48.6% for sprouts when standard BAX culture protocols were used. The substitution of an overnight incubation in modified E. coli medium for the 3-h brain heart infusion incubation increased the rate of E. coli O157:H7 detection to 75% for experimentally contaminated sprouts. The detection rate was 100% when E. coli O157:H7 cells from a fresh overnight culture were used for the inoculation. Test sensitivity is therefore influenced by the type of produce involved and is probably related to the growth of pathogens in the resuscitation and enrichment media.