Transient and resident pathogens: Intra-facility genetic diversity of Listeria monocytogenes and Salmonella from food production environments.

Food production facilities are often routinely tested over time for the presence of foodborne pathogens (e.g., Listeria monocytogenes or Salmonella enterica subsp. enterica). Strains detected in a single sampling event can be classified as transient; positive findings of the same strain across multiple sampling events can be classified as resident pathogens. We analyzed whole-genome sequence (WGS) data from 4,758 isolates (L. monocytogenes = 3,685; Salmonella = 1,073) from environmental samples taken by FDA from 536 U.S. facilities. Our primary objective was to determine the frequency of transient or resident pathogens within food production facilities. Strains were defined as isolates from the same facility that are less than 50 SNP (single-nucleotide polymorphisms) different from one another. Resident pathogens were defined as strains that had more than one isolate collected >59 days apart and from the same facility. We found 1,076 strains (median = 1 and maximum = 21 strains per facility); 180 were resident pathogens, 659 were transient, and 237 came from facilities that had only been sampled once. As a result, 21% of strains (180/ 839) from facilities with positive findings and that were sampled multiple times were found to be resident pathogens; nearly 1 in 4 (23%) of L. monocytogenes strains were found to be resident pathogens compared to 1 in 6 (16%) of Salmonella strains. Our results emphasize the critical importance of preventing the colonization of food production environments by foodborne pathogens, since when colonization does occur, there is an appreciable chance it will become a resident pathogen that presents an ongoing potential to contaminate product.

Incidence and Growth Potential of Bacillus cereus in Ready-to-Serve Foods.

To simulate temperature abuse, 106 test portions of ready-to-serve moist foods, 12 test portions of rehydrated powdered infant formula, and 18 test portions of nonfat dry milk were incubated for 20 and 24 h at 26°C, and then examined for Bacillus cereus . Of the ready-to-serve moist foods, 88 of 106 were positive for B. cereus at levels ranging from 0.25 to 8.5 × 106/g after 20 h of incubation and from 0.1 to 58 × 106/g after 24 h. All of the powdered milk and 12 of the 15 units of infant formula, representing five brands, were positive, with counts ranging from 0.15 to 5.0 × 106/g in 20 h and 5.0 to 49 × 106 after 24 h. B. cereus counts in the powdered products were low, ranging from 0.09/g for one of two soy-based products to an average of 0.29/g for milk-based products. However, these levels were sufficient to initiate growth of B. cereus in almost every 2-oz serving. Similar results were obtained for rehydrated nonfat milk, with initial B. cereus counts ranging from 0.29 to 1.5/g; at 26°C the counts averaged 3.3 × 107 after 20 h and 5.5 × 107 after 24 h. Counts ranged from 2.0 × 104 to 1.1 × 105 after 9 h in milk and were in excess of 106/g after 10.5 h.

Evaluation of a Reversed Passive Latex Agglutination Test Kit for Clostridium perfringens Enterotoxin.

A reversed passive latex agglutination (RPLA) test kit for Clostridium perfringens enterotoxin (CPE) marketed by the Denka-Seiken Co., Tokyo, Japan, was evaluated by using culture supernatant fluids and extracts from feces of food poisoning patients. Nanograms of CPE were detectable with the assay and the reaction was specific, as shown by parallel activity in a double antibody enzyme-linked immunosorbent assay (ELISA). Although less sensitive, the RPLA method is easier to perform than the ELISA and counterimmunoelectrophoresis, both of which require special test reagents and equipment not generally available.

Improved Media for Sporulation and Enterotoxin Production by Clostridium perfringens.

Sporulation of 24 strains of Clostridium perfringens isolated from stools of food poisoning patients and normal controls was improved by adding sodium carbonate to Duncan-Strong (DS) sporulation medium and replacing starch with raffinose in Taniguti's AEA medium. Viable counts of heat-tolerant spores (75°C for 20 min) were 2 to 186 times greater in modified AEA medium and 2 to 169 times greater in modified DS than in DS medium. Reversed passive latex agglutination assays revealed a corresponding increase in enterotoxin titers in supernatant fluids of the 12 enterotoxigenic strains grown in modified AEA medium and in modified DS medium.

Growth and Toxin Production by Clostridium botulinum in Sauteed Onions.

In October, 1983, sauteed onions in "patty-melt" sandwiches were epidemiologically responsible for a large outbreak of botulism in Peoria, Illinois. Spores of strains of Clostridium botulinum type A, recovered from Spanish onions or from patients who consumed sauteed onions, produced high toxin titers within 48 h from 2 spores/g of onions when experimentally inoculated into sauteed onions. Laboratory strains of C. botulinum type A which normally produce high-titered toxin in culture media yielded very low toxin titers and required 3 to 4 d and an extremely high inoculum of spores/g of onions. Five strains of C. botulinum type A were isolated from 75 raw onions obtained from the Peoria restaurant where the outbreak occurred.

Outgrowth and Toxin Production by Clostridium botulinum in Bottled Chopped Garlic.

The ability of Clostridium botulinum types A and B spores to grow and produce toxin in commercially bottled chopped garlic in soybean oil was investigated. Eight type A and seven type B strains of C. botulinum , mostly of vegetable origin, were used as inocula. Various numbers of spores were inoculated directly into the jars containing garlic, incubated at 35°C and sampled for organoleptic acceptance and presence of toxin every 5th d. In parallel studies conducted at room temperature, jars were sampled at 15-d intervals. At 35°C, when 1 spore/g of garlic was used as inoculum, toxin was produced in 15 d by type A and in 20 d by type B strains. At room temperature, five spores of type A or B per g of garlic produced toxin throughout 75 d. Even when highly toxic, garlic looked and smelled acceptable. Five strains of C. botulinum type A were isolated from 115 bulbs of fresh garlic.

Growth of Salmonella spp. in Cantaloupe, Watermelon, and Honeydew Melons.

The ability of Salmonella spp. to grow on the interior tissues of cantaloupe, watermelon, and honeydew melons was investigated. Pieces of rind-free melons (pH 5.90-6.67) and tryptic soy broth (TSB, pH 5.90) were inoculated with a mixed culture (approximately 100 CFU/g or ml) containing equal proportions of five species of Salmonella ( S. anatum , S. Chester , S. havana , S. poona , and S. senftenberg ). Inoculated melon pieces and TSB were incubated for 24 h at 5 or 23°C. Viable populations of salmonellae were determined by surface plating test portions on Hektoen enteric agar. Results indicated that Salmonella growth was rapid and prolific on the melons and in TSB at 23°C incubation. Final populations on watermelons were approximately 1.0 log10 greater than populations on cantaloupe and honeydew and in TSB. Although viable Salmonella populations on melons and in TSB did not increase during the 24-h incubation at 5°C, little or no decrease in viable populations was observed.

Bacillus cereus Contamination of Seeds and Vegetable Sprouts Grown in a Home Sprouting Kit.

Sprouting seeds (alfalfa, mung bean and wheat) were purchased at local health food stores and examined for Bacillus cereus by the official AOAC method. Of 98 units collected, 56 (57%) were positive for B. cereus at levels ranging from 3 to >500 per g. Population levels of B. cereus on sprouts grown from naturally contaminated seeds in a home sprouting kit ranged from a mean of log10 3.72 for alfalfa to 5.39 for wheat; the log10 mean for mung bean sprouts was 4.52. Washing contaminated sprouts for 10 min with warm tap water as recommended by the manufacturer of the sprouting kits reduced the B. cereus count for mung bean sprouts by approximately one log unit but was less effective for wheat sprouts. B. cereus populations large enough to cause food poisoning (>105/g) frequently remained on wheat sprouts even after three wash cycles, and significant numbers of viable B. cereus remained on wheat sprouts even after cooking for 20 min.

Enumeration and Characterization of Clostridium perfringens Spores in the Feces of Food Poisoning Patients and Normal Controls.

The fecal spore enumeration method for confirming Clostridium perfringens as the cause of food poisoning was evaluated using strains implicated in nine outbreaks in the United States. Confirmed spore counts from 66 stool specimens were made on tryptose-sulfite-cycloserine (TSC) without egg yolk and trypticase soy-sheep blood (TSB) agars. Counts from outbreak stools on TSC agar ranged from 2.0 × 104 to 3.5 × 108 (mean ≥ 1.4 × 106/g) as compared with <103 to 5.0 × 105/g (overall mean 9.5 × 103/g) from normal stools. Similar results were obtained with TSB agar. Isolates from seven of the nine outbreaks were nonhemolytic and produced ≥100 ng enterotoxin/ml in spore broth, as measured by an enzyme-linked immunosorbent assay. Spores in stools from six of the outbreaks were heat-resistant and survived heating for 30 to 60 min at 100°C in cooked meat medium. Strains from the three remaining outbreaks were heat-sensitive and survived heating for only 15 min at 100°C. Enterotoxigenic isolates from all but one of the outbreaks were serotyped. In all instances, the predominant strain in specimens from an outbreak was of the same serotype, indicating that it was the causative strain. Reexamination of five specimens from each of three outbreaks after storage at -20°C for 6 months showed only a minimal reduction in the spore counts.

Growth and Toxin Production by Clostridium botulinum in Sliced Raw Potatoes Under Vacuum With and Without Sulfite.

The ability of Clostridium botulinum type A or B spores to grow and produce toxin in fresh raw potatoes under vacuum with or without sulfite at 22°C was investigated. Fresh, peeled, sliced potatoes, untreated or dipped for 2 min in sulfite (NaHSO3) and drained, were surface-inoculated at several levels with a mixture of C. botulinum spores, either type A or B, and placed in oxygen-impermeable bags (200 g/bag) that were then vacuum-sealed and incubated at room temperature (22°C). Toxicity was tested on days 0, 3, 4, 5 and 6. After incubation, the potatoes were blended and centrifuged, and the millipore-filtered supernatant fluid was injected intraperitoneally into mice. Sensory evaluation, except taste, was also performed. Potatoes inoculated with C. botulinum type A spores, but untreated with NaHSO3 became toxic in 3 days, which coincided with the sensory evaluation, "Unfit for human consumption." However, despite inoculum size or residual SO2 levels, potatoes treated with NaHSO3 appeared acceptable for human consumption through day 6, even though they were toxic after 4 days of incubation. Toxicity from type B spores occurred later and in fewer test samples than type A. Again, the potatoes appeared acceptable but were toxic. Thus, although NaHSO3 markedly extended the consumer acceptability of peeled, sliced, raw potatoes at the abuse temperature, it did not inhibit outgrowth and toxin production by C. botulinum under these same conditions.

Differences and Similarities Among Proteolytic and Nonproteolytic Strains of Clostridium botulinum Types A, B, E and F: A Review.

Cultures of Clostridium botulinum types A, B, E and F, which are responsible for human botulism, fall into two groups with different characteristics unrelated to toxin type. These groups differ primarily with respect to proteolysis, but also have different somatic and spore antigens and DNA; the heat resistance of their spores, their growth at low temperatures and their salt tolerance also differ. All known type A strains are proteolytic and all type E strains are non proteolytic, but types B and F have some proteolytic and some nonproteolytic strains. Although proteolytic strains can activate their own toxins, nonproteolytic strains cannot do so and therefore require trypsinization for maximum toxicity. Proteolytic strains are unable to grow at temperatures below 10 C, but have relatively high salt tolerance and spores of high heat resistance. Nonproteolytic strains can grow at 3.3 C and have a lower salt tolerance; their spores have a much lower heat resistance than those of proteolytic strains.

Heat Resistance of Clostridium botulinum Type B Spores Grown from Isolates from Commercially Canned Mushrooms 1.

The heat resistance of ten Clostridium botulinum type B spore crops was determined in mushroom puree and 0.067M Sorenson phosphate buffer (pH 7). The spore crops were grown from Clostridium botulinum isolates obtained from commercially canned mushrooms. The D-values for all of the C. botulinum spore crops were overall slightly higher in the buffer than in mushroom puree. The mean D(110.0 C)-value for the ten spore crops in buffer was 1.17 min and for the spores in mushroom puree the mean D(110.0 C)-value was 0.78 min. The mean D(115.6 C)-value in buffer for the ten spore crops was 0.24 min compared to a mean D(115.6 C)-value of 0.19 min for spores in mushroom puree. The C. botulinum type B spores tested in this study had a heat resistance that was less than the classical heat resistance for C. botulinum spores.

Heat Resistance of Clostridium botulinum Type G in Phosphate Buffer.

The heat resistance of two strains of Clostridium botulinum type G in phosphate buffer was studied by the thermal death time (TDT) tube method and the thermal destruction rate (TDR) method. The strains were estimated to have one highly heat-resistant spore among approximately 100 spores or 10,000 relatively heat-labile spores. The heat-labile spores were studied by the TDR method and the heat-resistant spores by the TDT tube method. Decimal reduction times (D) for the heat-labile spores were determined by the slopes of the survivor curves. D values for strain 89 ranged from 0.6 min at 190°F to 6.9 min at 170°F and for strain 2739 from 0.9 min at 200°F to 5.9 min at 180°F. Thermal destruction curves for the heat-labile spores gave z values of 24.0 and 17.5 for two spore stocks of strain 89 and 26.0 for strain 2739. D values for the heat-resistant spores, calculated from the combined data of replicate experiments by the Schmidt probability method, ranged from 0.29 min at 240°F to 1.51 min at 210°F for strain 89 and from 0.25 min at 240°F to 1.48 min at 210°F for strain 2739. Extrapolated to 250°F, the thermal destruction curves of the heat-resistant spores gave D250 values of 0.14 to 0.19 min. The thermal destruction curves of the heat-resistant spores were very flat, however, with z values of 37.9 and 49.1 for the two spore stocks of strain 89 and 37.7 for strain 2739. Low-acid canned food processes will provide the same margin of safety for type G as for other proteolytic strains of C. botulinum but ultra high processing temperatures probably will not.

Common Characteristics of the Swiss and Argentine Strains of Clostridium botulinum Type G 1.

Five strains of Clostridium botulinum type G of human origin, from Switzerland, were compared with two strains isolated from soil in Argentina. The Swiss and Argentine strains are the only type G strains isolated to date. Characteristics compared were toxigenicity, sporulation, proteolysis and carbohydrate fermentation. High toxin titers were produced in trypticase-peptone-glucose-yeast extract broth incubated anaerobically at 30°C for 10 d. Sporulation occurred in three strains incubated anaerobically on soil extract agar at 35°C for 15 d. Different concentrations of soil extract in the medium promoted sporulation of different strains. Toxins of the Swiss and Argentine strains showed identical patterns for trypsin activation, reaction to A-F antitoxin and neutralization by antitoxin prepared from strain 89G. All seven strains showed delayed proteolytic activity but failed to ferment any of the sugars tested.

Clostridium botulinum Spores in Corn Syrup and Other Syrups.

A total of 738 bottles of corn syrup and other products containing corn syrup (e.g., pancake, maple, waffle, and table syrups) was examined by a membrane filtration procedure for the presence of Clostridium botulinum spores. One each of 354 light and 271 dark corn syrups was presumptively positive for C. botulinum type A spores, but subsequent testing of the entire contents of both bottles proved negative. All other 113 syrups were negative. Results showed that corn syrup and other syrups currently on the market are not food sources of C. botulinum spores for infants.

Evaluation of Unacidified Products Bottled in Oil for Outgrowth and Toxin Production by Clostridium botulinum.

A variety of unacidified products bottled in oil or water were investigated for their ability to support growth and toxin production by Clostridium botulinum . The products were inoculated with a mixture of five strains of C. botulinum type A spores (about 50 spores/g or ml) and incubated at room temperature (23°C). At monthly intervals the organoleptic acceptability of the products, as determined by appearance, odor, and texture, was evaluated and a portion of each sample was removed, diluted 1:2 in gel-phosphate buffer, and injected intraperitoneally into mice. At the end of 4 months the drained solids of each sample were macerated with a minimal amount of buffer and centrifuged; the clear extracts were then injected into mice. None of the products tested supported growth and toxin production by C. botulinum .

Outgrowth of Clostridium botulinum in Shredded Cabbage at Room Temperature Under a Modified Atmosphere.

The ability of Clostridium botulinum types A and B spores to grow and produce toxin in shredded cabbage at room temperature under a modified atmosphere was investigated. Seven type A and seven type B strains of C. botulinum , mostly of vegetable origin, were used as inocula. Shredded cabbage in high barrier bags, 250 g/bag, was inoculated with various numbers of spores, sealed under a modified atmosphere of 70% CO2 and 30% N2 and incubated at room temperature. Duplicate bags were examined for organoleptic acceptability and the presence of toxin from day 3 by blending the entire contents of each bag and injecting mice with dilutions of the extracts. Toxic extracts were typed with appropriate antitoxins. Only type A spores grew and produced toxin in the cabbage. An inoculum of approximately 100-200 type A spores/g of cabbage, whether in single strains or in various combinations, produced toxin on days 4, 5, and 6, while the cabbage was still organoleptically acceptable, as determined by appearance, odor, and texture.

Immunodiffusion Detection of Clostridium botulinum Colonies.

Toxic colonies of Clostridium botulinum types A, B, E and F were detected by precipitating toxin around the colonies on agar containing antitoxin. Incorporating antitoxin in a gel diffusion agar overlay after colonies had developed was unsatisfactory for detection of type E, but worked well for all types when added directly to the plating medium. Addition of 0.6 IU of antitoxin per ml of agar gave satisfactory results with all types except type E. Zones of precipitation were produced by proteolytic strains of types A, B and F incubated at 35°C for 3 d and by nonproteolytic strains of types B and F incubated at 28°C for 4 d; type E required 1.2 IU of antitoxin per ml of agar and 5 d of incubation at 35°C. Nontoxigenic putrefactive anaerobes produced no zones of precipitation with any of the antitoxins, and toxic colonies of C. botulinum mixed among them were easily distinguished. This method was used successfully for selecting type B colonies from plates containing toxic enrichment cultures of tomato juice.