Salmonellae and pollution indicator bacteria in municipal and food processing effluents and the Cornwallis River

A study was conducted to determine the incidence of fecal coliforms, fecal streptococci, Aeromonas hydrophilia, and Salmonella spp. in the waste discharges of seven sewage treatment plants, four fruit and vegetable canneries, a meat packing plant, a poultry processing plant, and a potato processing plant located along the Cornwallis River in Nova Scotia, Canada. Surface water samples were also collected from 13 locations in the river to assess the impact of these waste discharges on the receiving water quality. The results showed that the final effluents from most of the sewage treatment and processing plants were of very poor bacteriological quality, with the number of indicator bacteria comparable with those found in raw sewage. Salmonellae were isolated from the effluents of the meat and poultry plants and five of the seven sewage treatment plants surveyed. No salmonellae were detected in the effluents of the fruit and vegetable canneries. The impact of the discharge of untreated municipal and food processing wastes on the Cornwallis River water quality was evidenced by the recovery from river water of five Salmonella serotypes, and the high fecal coliform counts which exceeded recommended limit for bathing and shellfish harvesting.     

Incidence of salmonellae in fecal samples of production swine and swine at slaughter plants in the United States in 1978.

Nine swine slaughter plants and 19 swine production units were randomly selected for sampling from the six highest swine-producing states representing a total of 64% of the United States swine production. Three composites of 10 fresh swine fecal samples were obtained from each slaughter plant, representing three different farm sources of swine. Two composite fecal samples were collected from two different production pens from each production unit. Samples were analyzed for salmonellae. Isolated salmonellae were biochemically and serologically identified and tested for antibiotic susceptibility and resistance transfer ability. Salmonellae were recovered from swine at seven of the nine slaughter plants and 16 of the 27 composites of slaughter swine. Of the 19 production units, 3 had swine shedding salmonellae. Resistances found included streptomycin, tetracycline, and sulfadiazine. Of the 52 total isolates tested, 71% had some level of antibiotic resistance. Only 3 of 37 resistant isolated could transfer resistance under the conditions used.     

Multiyear study of sludge application to farmland: prevalence of bacterial enteric pathogens and antibody status of farm families

We describe our experience with the isolation of salmonellae from sewage sludge from four treatment plants in different geographic areas of Ohio. Over 3 years, we isolated salmonellae 50 times from 311 sludge samples. Most isolations were made after enrichment in Selenite broth (BBL Microbiology Systems, Cockeysville, Md.). The largest proportion of isolations came from the plant serving the population of Columbus, a large metropolitan area. A significantly greater number of isolations from this plant were made during the first quarter of the year. Twenty-one different serotypes were isolated, along with five untypable strains. The most frequently isolated serotype was Salmonella infantis. Five of the strains were multiply resistant to antibiotics. We also describe the prevalence of antibodies to salmonellae in members of the families residing on the farms in the study. It was found that antibodies to group C salmonellae predominated.     

Multiyear study of sludge application to farmland: prevalence of bacterial enteric pathogens and antibody status of farm families.

We describe our experience with the isolation of salmonellae from sewage sludge from four treatment plants in different geographic areas of Ohio. Over 3 years, we isolated salmonellae 50 times from 311 sludge samples. Most isolations were made after enrichment in Selenite broth (BBL Microbiology Systems, Cockeysville, Md.). The largest proportion of isolations came from the plant serving the population of Columbus, a large metropolitan area. A significantly greater number of isolations from this plant were made during the first quarter of the year. Twenty-one different serotypes were isolated, along with five untypable strains. The most frequently isolated serotype was Salmonella infantis. Five of the strains were multiply resistant to antibiotics. We also describe the prevalence of antibodies to salmonellae in members of the families residing on the farms in the study. It was found that antibodies to group C salmonellae predominated.     

The Fluorescent Antibody Technique for the Detection of Salmonella in Routine Use

S ummary : The direct and indirect fluorescent antibody technique (FAT) were compared with cultural methods for detecting salmonellae in meat products, animal feedingstuffs, poultry carcase swabs, giblets and poultry plant and equipment swabs. Salmonellae were not isolated from meat products and fluorescent cells were not seen on slides prepared by either FAT. The indirect and direct FAT recorded 13% and 9% respectively, false positive results, with samples of animal feedingstuffs, but the direct FAT recorded a single false negative result. Salmonellae were not isolated from poultry carcase swabs but 3% and 4·5% respectively, of false positive results were obtained with the indirect and direct FAT. Salmonellae were isolated from both giblet samples and poultry plant swabs and both gave rise to false negative FAT results. Preliminary studies of the efficacy of the FAT for screening animal faecal material for salmonellae indicated that no single combination of enrichment broth and FAT gives unequivocal results, but the staining of smears from tetrathionate broth by either FAT gives rise to a high percentage of false negative results.     

Salmonellae Associated with Further-processed Turkey Products

"Further-processed" turkey products, prepared from chilled, eviscerated, and thawed carcasses at two commercial turkey-processing plants, were evaluated, for the presence of salmonellae. These organisms were isolated from swab samples from 12% of chilled, eviscerated turkey carcasses, 27% of finished products, and 24% of processing equipment. The same serotypes as those found throughout a plant on any one visit were recovered from 31% of rinse-samples taken from hands and gloves of processing personnel. Salmonellae were found in samples taken on 37 of 48 visits; a greater number of recoveries were made on days when freshly killed turkeys were processed (87%) than when frozen-defrosted carcasses were processed (59%). The predominant serotype isolated from meat and environment usually changed from visit to visit. Salmonella sandiego and Salmonella anatum were the most frequent among 23 serotypes recovered. Most of the isolated serotypes are commonly associated with turkeys and have been incriminated as causative agents of human salmonellosis. The implication is that further-processed turkey products, if inadequately cooked by the consumer and if improperly refrigerated between the time of manufacture and consumption, could directly transmit salmonellae. These same products might also contaminate other foods by introducing salmonellae into food-preparation areas.     

Sources of Listeria monocytogenes Contamination in a Cold-Smoked Rainbow Trout Processing Plant Detected by Pulsed-Field Gel Electrophoresis Typing

Sites of Listeria monocytogenes contamination in a cold-smoked rainbow trout (Oncorhynchus mykiss) processing plant were detected by sampling the production line, environment, and fish at different production stages. Two lots were monitored. The frequency of raw fish samples containing L. monocytogenes was low. During processing, the frequency of fish contaminated with L. monocytogenes clearly rose after brining, and the most contaminated sites of the processing plant were the brining and postbrining areas. A total of 303 isolates from the raw fish, product, and the environment were characterized by pulsed-field gel electrophoresis (PFGE). PFGE yielded nine pulsotypes, which formed four clusters. The predominating L. monocytogenes pulsotypes of the final product were associated with brining and slicing, whereas contaminants of raw fish were not detected in the final product. Air-mediated contamination in the plant could not be proved. In accordance with these results, an L. monocytogenes eradication program was planned. The use of hot steam, hot air, and hot water seemed to be useful in eliminating L. monocytogenes. None of the control samples taken in the 5 months after the eradication program was implemented contained L. monocytogenes.     

One group of genetically similar Listeria monocytogenes strains frequently dominates and persists in several fish slaughter- and smokehouses

Contamination of foods with the human pathogen Listeria monocytogenes may occur during processing, and the purpose of this study was to determine whether genetically similar strains colonize different processing plants or whether specific persistent strains are unique to each processing plant. We hypothesized that specific L. monocytogenes strains may be better adapted to specific environmental niches in the processing environment. L. monocytogenes contamination patterns were identified by the collection of 686 and 267 samples from the processing environments: raw fish and products of four fish smokehouses and four fish slaughterhouses, respectively. Samples were collected both during production and after cleaning and disinfection. Typically, these samplings were separated by 1 to 3 months. Sampling sites were targeted toward areas likely to harbor the bacterium. L. monocytogenes was isolated from 213 samples, and one strain from each positive sample was typed by RAPD (random amplified polymorphic DNA) analysis with four different primers. The 213 strains were divided into 37 RAPD types. One RAPD type was predominant; 86 of 213 strains belonged to this type. This type was found in three smokehouses and two slaughterhouses and was predominant in three of these plants. A subset of 35 strains was also analyzed by amplified fragment length polymorphism typing, which confirmed the genetic similarity of the groups. Moreover, strains of the dominant RAPD type were indistinguishable from strains isolated frequently from smoked fish products 10 years ago. One smokehouse was surveyed for a year and a half, and the dominant RAPD type persisted throughout the survey period and accounted for 94 of 118 isolates. Our study indicates that strains of L. monocytogenes that are genetically very closely related may be especially adapted to colonizing the processing equipment or especially resistant to cleaning and disinfection.     

Evaluation of the Salmonellae Fluoro-Kit for Fluorescent-Antibody Staining

An evaluation of the newly developed Clinical Sciences, Inc. Salmonellae Fluoro-Kit, which attempts to standardize the various aspects of the fluorescent-antibody (FA) procedure, was performed with 120 naturally contaminated human food, animal feed, and raw material samples. The Association of Official Analytical Chemists (AOAC) method for the detection of salmonellae was used as the control method. The Fluoro-Kit was found to be simple and conveniento to use. The results of this preliminary study show an industrially acceptable rate of recovery of salmonellae by using the Fluoro-Kit in comparison with the A.O.A.C. method. The Fluoro-Kit shows promise as a rapid, salmonellae FA screening method. Problems originally encountered in the application of the Fluoro-Kit are discussed. According to the manufacturer, strict adherence to the now revised procedures included in the Fluoro-Kit will control these problems.     

Incidence of Salmonellae in Animal Feed and the Effect of Pelleting on Content of Enterobacteriaceae

During a survey of meat and bone meals, fish meals and poultry feeds salmonellae were found only in samples of meat and bone meal, for which 19% were Salmonella -positive. MPN estimations on 2 meat and bone meal samples gave figures of 6 and 39 salmonellae/ 100 g, respectively. Sampling of poultry feed before and after pelleting showed that this process gave up to 1000-fold reduction in numbers of Enterobacteriaceae depending on the type of processing involved.     

Longevity of Salmonella typhimurium in Tilapia aurea and water from pools fertilized with swine waste.

Salmonella typhimurium declined rapidly when inoculated into Tilapia aurea culture pools fertilized with fresh swine waste. Within the water column, a 95% decline of viable cells occurred during the first 6 h. Isolation of viable salmonellae was possible at 16 days post-inoculation, but not at 32 days. Similarly, salmonellae could be detected in the viscera and epithelium of T. aurea at 16 days, although not at 32 days. Salmonellae were not isolated from the fish flesh, nor was there evidence of septicemic infection.     

Elucidation of Listeria monocytogenes contamination routes in cold-smoked salmon processing plants detected by DNA-based typing methods

The contamination routes of Listeria monocytogenes in cold-smoked salmon processing plants were investigated by analyzing 3,585 samples from products (produced in 1995, 1996, 1998, and 1999) and processing environments (samples obtained in 1998 and 1999) of two Danish smokehouses. The level of product contamination in plant I varied from 31 to 85%, and no L. monocytogenes was found on raw fish (30 fish were sampled). In plant II, the levels of both raw fish and product contamination varied from 0 to 25% (16 of 185 raw fish samples and 59 of 1,000 product samples were positive for L. monocytogenes). A total of 429 strains of L. monocytogenes were subsequently compared by random amplified polymorphic DNA (RAPD) profiling, and 55 different RAPD types were found. The RAPD types detected on the products were identical to types found on the processing equipment and in the processing environment, suggesting that contamination of the final product (cold-smoked salmon) in both plants (but primarily in plant I) was due to contamination during processing rather than to contamination from raw fish. However, the possibility that raw fish was an important source of contamination of the processing equipment and environment could not be excluded. Contamination of the product occurred in specific areas (the brining and slicing areas). In plant I, the same RAPD type (RAPD type 12) was found over a 4-year period, indicating that an established in-house flora persisted and was not eliminated by routine hygienic procedures. In plant II, where the prevalence of L. monocytogenes was much lower, no RAPD type persisted over long periods of time, and several different L. monocytogenes RAPD types were isolated. This indicates that persistent strains may be avoided by rigorous cleaning and sanitation; however, due to the ubiquitous nature of the organism, sporadic contamination occurred. A subset of strains was also typed by using pulsed-field gel electrophoresis and amplified fragment length polymorphism profiling, and these methods confirmed the type division obtained by RAPD profiling.     

Survey of Salmonellae in mesenteric lymph nodes, gallbladder wall and jejunum from healthy pigs slaughtered in Taiwan

A study made in Taiwan showed that 23.3 per cent of healthy slaughter pigs were infected with salmonellae. Samples of gallbladder wall/liver, mesenteric lymph nodes and jejunum wall were found to yield salmonellae in 16.4, 5.2 and 11.6 per cent of cases respectively. Salmonellae isolated belonged to nine different serotypes, S. london being the serotype most frequently isolated (26.8%) followed by S. anatum, S. panama (16.1% each) and S. typhimurium (12.5%). Of the total number of salmonellae isolated 68.5 per cent were detected simultaneously on brilliant-green phenol-red lactose sucrose agar (BGA) and desoxycholate agar (DCA), whilst 18.5 per cent were detected only on DCA and 13 per cent only on BGA plates.     

One Group of Genetically Similar Listeria monocytogenes Strains Frequently Dominates and Persists in Several Fish Slaughter- and Smokehouses

Contamination of foods with the human pathogen Listeria monocytogenes may occur during processing, and the purpose of this study was to determine whether genetically similar strains colonize different processing plants or whether specific persistent strains are unique to each processing plant. We hypothesized that specific L. monocytogenes strains may be better adapted to specific environmental niches in the processing environment. L. monocytogenes contamination patterns were identified by the collection of 686 and 267 samples from the processing environments: raw fish and products of four fish smokehouses and four fish slaughterhouses, respectively. Samples were collected both during production and after cleaning and disinfection. Typically, these samplings were separated by 1 to 3 months. Sampling sites were targeted toward areas likely to harbor the bacterium. L. monocytogenes was isolated from 213 samples, and one strain from each positive sample was typed by RAPD (random amplified polymorphic DNA) analysis with four different primers. The 213 strains were divided into 37 RAPD types. One RAPD type was predominant; 86 of 213 strains belonged to this type. This type was found in three smokehouses and two slaughterhouses and was predominant in three of these plants. A subset of 35 strains was also analyzed by amplified fragment length polymorphism typing, which confirmed the genetic similarity of the groups. Moreover, strains of the dominant RAPD type were indistinguishable from strains isolated frequently from smoked fish products 10 years ago. One smokehouse was surveyed for a year and a half, and the dominant RAPD type persisted throughout the survey period and accounted for 94 of 118 isolates. Our study indicates that strains of L. monocytogenes that are genetically very closely related may be especially adapted to colonizing the processing equipment or especially resistant to cleaning and disinfection.     

Elucidation of Listeria monocytogenes Contamination Routes in Cold-Smoked Salmon Processing Plants Detected by DNA-Based Typing Methods

The contamination routes of Listeria monocytogenes in cold-smoked salmon processing plants were investigated by analyzing 3,585 samples from products (produced in 1995, 1996, 1998, and 1999) and processing environments (samples obtained in 1998 and 1999) of two Danish smokehouses. The level of product contamination in plant I varied from 31 to 85%, and no L. monocytogenes was found on raw fish (30 fish were sampled). In plant II, the levels of both raw fish and product contamination varied from 0 to 25% (16 of 185 raw fish samples and 59 of 1,000 product samples were positive for L. monocytogenes). A total of 429 strains of L. monocytogenes were subsequently compared by random amplified polymorphic DNA (RAPD) profiling, and 55 different RAPD types were found. The RAPD types detected on the products were identical to types found on the processing equipment and in the processing environment, suggesting that contamination of the final product (cold-smoked salmon) in both plants (but primarily in plant I) was due to contamination during processing rather than to contamination from raw fish. However, the possibility that raw fish was an important source of contamination of the processing equipment and environment could not be excluded. Contamination of the product occurred in specific areas (the brining and slicing areas). In plant I, the same RAPD type (RAPD type 12) was found over a 4-year period, indicating that an established in-house flora persisted and was not eliminated by routine hygienic procedures. In plant II, where the prevalence of L. monocytogenes was much lower, no RAPD type persisted over long periods of time, and several different L. monocytogenes RAPD types were isolated. This indicates that persistent strains may be avoided by rigorous cleaning and sanitation; however, due to the ubiquitous nature of the organism, sporadic contamination occurred. A subset of strains was also typed by using pulsed-field gel electrophoresis and amplified fragment length polymorphism profiling, and these methods confirmed the type division obtained by RAPD profiling.     

Isolation of Salmonellae from Pork Carcasses

Four hundred and twenty pork carcasses from four abattoirs were examined for the presence of salmonellae by use of swabbing-enrichment techniques and contact plate methods. Carcasses from only one abattoir were found to be contaminated by swabbing-enrichment (23.3%) and contact plate (17.9%) methods. The area of the skin side of the ham, near the anal opening, was determined to be the area to examine for isolating salmonellae from pork carcasses with the greatest frequency. The most frequently isolated species of salmonellae in this study were Salmonella derby, S. anatum, S. typhimurium, and S. indiana.     

Bacteriological Survey of Fresh Pork Sausage Produced at Establishments Under Federal Inspection

At the time of manufacture, 75% of 67 sets of finished fresh pork sausage collected in 44 plants had aerobic plate counts in the range of 500,000 or fewer/g; 88% contained 100 or fewer E. coli/g; and 75% contained 100 or fewer S. aureus/g (geometric means of 10 samples). Salmonellae were isolated from 28% of 529 samples of pork trimmings used for sausage, and from 28% of 560 finished sausage samples. Semiquantitative analysis revealed that salmonellae were at low levels; more than 80% of the salmonellae-positive samples were positive only in 25-g portions (negative in 1.0- and 0.1-g portions).     

Salmonella in Natural Animal Casings

Destruction of salmonellae in inoculated and naturally contaminated natural animal casings was studied. Salmonellae were effectively destroyed (99.999%) in inoculated hog casings after exposure for 24 h to saturated brine at pH 4.0 and 10.0 adjusted with acetic acid and sodium hydroxide, respectively. Treatment of inoculated hog and sheep casings in saturated brine or saturated brine with citric acid was not nearly as effective as brine containing acetic acid or sodium hydroxide. Salmonellae in naturally contaminated hog casings were virtually eliminated after 21 days of storage in crystalline sodium chloride. Salmonella in sheep and hog casings were eliminated after 7 days of storage in crystalline salt. Treatment of naturally contaminated hog casings with glycerin-salt or sorbitol-salt solutions was not as effective in destroying salmonellae as treatment with crystalline salt.     

Small pet aquarium frogs as a source of Salmonella.

Salmonellae were isolated from 21% of the samples of freshwater aquarium frogs tested and from 25% of the samples of aquarium water containing these frogs. The salmonellae were Salmonella arizonae, S. bovis-morbificans, S. hadar, S. saint-paul, S. typhimurium, and S. worthington. These isolations were made over a period of 9 months and from three different cities. This association of salmonellae with frogs may contribute to cases of human salmonellosis since other aquarium species have already been shown to contribute to such cases.     

Fate of Salmonella enterica serovar Typhimurium on carrots and radishes grown in fields treated with contaminated manure composts or irrigation water

Three different types of compost, PM-5 (poultry manure compost), 338 (dairy cattle manure compost), and NVIRO-4 (alkaline-pH-stabilized dairy cattle manure compost), and irrigation water were inoculated with an avirulent strain of Salmonella enterica serovar Typhimurium at 10(7) CFU g(-1) and 10(5) CFU ml(-1), respectively, to determine the persistence of salmonellae in soils containing these composts, in irrigation water, and also on carrots and radishes grown in these contaminated soils. A split-plot block design plan was used for each crop, with five treatments (one without compost, three with each of the three composts, and one without compost but with contaminated water applied) and five replicates for a total of 25 plots for each crop, with each plot measuring 1.8 x 4.6 m. Salmonellae persisted for an extended period of time, with the bacteria surviving in soil samples for 203 to 231 days, and were detected after seeds were sown for 84 and 203 days on radishes and carrots, respectively. Salmonella survival was greatest in soil amended with poultry compost and least in soil containing alkaline-pH-stabilized dairy cattle manure compost. Survival profiles of Salmonella on vegetables and soil samples contaminated by irrigation water were similar to those observed when contamination occurred through compost. Hence, both contaminated manure compost and irrigation water can play an important role in contaminating soil and root vegetables with salmonellae for several months.