Bacteriology of Dehydrated Space Foods

The initial bacteriological requirement established in 1964 for space foods by the U.S. Army Natick Laboratories are: a total aerobic plate count ( 300,000), chocolate ice cream cubes (20,000), and each of four samples of chocolate candy (12,000 to 61,000); (ii) coliforms: two out of three vanilla milk drinks (16 and 127) and one beef hash bar (14); (iii) fecal coliforms: one sample of chicken soup and gravy base positive; (iv) fecal streptococci: two samples of peanut cubes (40 and 108), coconut cubes (75), chicken soup and gravy base (2,650), beef soup and gravy base (33), and five out of six flavored milk drinks (23 to 300); (v) salmonellae: one each of chicken and beef soup and gravy base were positive.     

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.     

Enzyme-linked immunosorbent assay for detection of Salmonella lipopolysaccharide in poultry specimens

An enzyme-linked immunosorbent assay (ELISA) for detection of salmonellae was developed and evaluated by using artificially contaminated specimens of poultry feed, feces, litter, or carcass rinsings, and naturally contaminated water samples. Specimens containing salmonellae of serogroups B or C2 inhibited the binding of polyvalent anti-O serum to microtiter plate wells coated with lipopolysaccharide of Salmonella typhimurium (serogroup B) or Salmonella albany (serogroup C2), respectively. Treatment of specimens with Rhozyme 41 (a protease) inhibited nonspecific reactions. The ELISA detected 106 of 111 culture-positive specimens contaminated with salmonellae of serogroups B or C2. Nineteen of 20 specimens containing salmonellae of serogroup C1 and all of 36 culture-negative specimens were ELISA negative. All seven water samples that contained salmonellae of serogroups B or C2, including three that were culture positive only after delayed secondary enrichment, were ELISA positive. Seven of the nine water samples that contained salmonellae of other serogroups, and all 38 culture-negative samples, were ELISA negative. The ELISA was simple to perform, produced results in 48 h, and was more economical than culture methods.     

Enzyme-linked immunosorbent assay for detection of Salmonella lipopolysaccharide in poultry specimens.

An enzyme-linked immunosorbent assay (ELISA) for detection of salmonellae was developed and evaluated by using artificially contaminated specimens of poultry feed, feces, litter, or carcass rinsings, and naturally contaminated water samples. Specimens containing salmonellae of serogroups B or C2 inhibited the binding of polyvalent anti-O serum to microtiter plate wells coated with lipopolysaccharide of Salmonella typhimurium (serogroup B) or Salmonella albany (serogroup C2), respectively. Treatment of specimens with Rhozyme 41 (a protease) inhibited nonspecific reactions. The ELISA detected 106 of 111 culture-positive specimens contaminated with salmonellae of serogroups B or C2. Nineteen of 20 specimens containing salmonellae of serogroup C1 and all of 36 culture-negative specimens were ELISA negative. All seven water samples that contained salmonellae of serogroups B or C2, including three that were culture positive only after delayed secondary enrichment, were ELISA positive. Seven of the nine water samples that contained salmonellae of other serogroups, and all 38 culture-negative samples, were ELISA negative. The ELISA was simple to perform, produced results in 48 h, and was more economical than culture methods.     

Growth of Salmonella on sprouting alfalfa seeds as affected by the inoculum size, native microbial load and Pseudomonas fluorescens 2-79

Aims: To investigate the growth of salmonellae on sprouting alfalfa seeds as affected by the inoculum size, microbial load and Pseudomonas fluorescens 2–79. Methods and Results: Alfalfa seeds pre‐inoculated with ≤10¹–10³ CFU g^?1 of salmonellae and with or without Ps. fluorescens 2–79 were sprouted in glass jars and the population of salmonellae were determined daily for up to 6 days. The population of salmonellae on germinating seeds reached the maximum 2–3 days after sprouting when total bacterial count reached the maximum (10⁹ CFU g^?1). The population of salmonellae on sprouting seeds not treated with Ps. fluorescens 2–79 showed a net increase of 3–4 log units. However, the population of salmonellae on alfalfa seeds treated with Ps. fluorescens 2–79 showed a net increase of only 1–2 log units. Disinfection of seeds with calcium hypochlorite enhanced the growth of salmonellae. Conclusions: Treatment of seeds with Ps. fluorescens 2–79 reduced the growth of salmonellae by 2–3 log units. Significance and Impact of the Study: The potential of Ps. fluorescens 2–79 as a biological agent for use in control of salmonellae on sprouting seeds was demonstrated and warrants further investigation.     

Comparison of four agar plating media with and without added novobiocin for isolation of salmonellae from beef and deboned poultry meat

Four plating media, Hektoen enteric (HE), xylose-lysine deoxycholate (XLD), tryptic soy-xylose-lysine (TSXL), and tryptic soy-brillant green (TSBG) agars with and without 10 mg of added novobiocin per ml, were evaluated for recovery of Salmonella from roast beef and deboned turkey. Colonies producing a reaction typical of H(2)S-positive salmonellae (alkaline with black centers) were picked. On the media without novobiocin, from 109 determinations on 75 samples, number of salmonellae found and false-positives were, respectively: HE-13, 58; XLD-17, 18; TSXL-23, 0; TSBG-22, 7. When novobiocin was present the corresponding results were: HE-17, 24; XLD-21, 2; TSXL-23, 3; TSBG-20, 7. A total of 25 determinations were positive on one or more agars. False-positives on HE and XLD without novobiocin were predominantly Proteus, which were almost totally eliminated by addition of 10 mg of novobiocin per liter. If alkaline H(2)S-negative colonies had been considered, many more false-positives would have been found on HE and XLD but not on TSBG or TSXL. Addition of novobiocin markedly improved isolations of salmonellae from XLD and HE and reduced the number of false-positives. Addition of novobiocin did not improve performance of TSXL and slightly impaired differentiation of salmonellae from Citrobacter on TSBG. XLD with novobiocin and TSXL are highly specific for H(2)S-positive salmonellae, and the appearance of Salmonella-like colonies on these media can be considered a presumptive test for H(2)S-positive salmonellae.     

Occurrence, growth, and suppression of salmonellae in composted sewage sludge

Composted sewage sludge may be used to improve soil quality, but there remains some doubt concerning the microbiological safety of the product. Sewage sludge composts from 30 municipalities were sampled, and four samples (12%) contained salmonellae (two contained fewer than 0.3/g, and the other two had 21/g and 1.7 X 10(4)/g). All 30 composts were inoculated with salmonellae; the populations decreased at a specific death rate of about 0.15 h-1 over 24 h at 36 degrees C. In irradiation-sterilized composts inoculated with salmonellae, the salmonellae grew at a rate of 0.65 doublings per h for over 24 h. Growth and death rates were found to be moisture and flora associated. The growth or death rates for antibiotic-resistant salmonellae were not different from those of nonresistant strains. It was concluded that the active indigenous flora of compost establishes a homeostatic barrier to colonization by salmonellae, and in the absence of competing flora, reinoculated salmonellae may grow to potentially hazardous densities. The active microflora of moist composts eliminated contaminating salmonellae (10(5)/g) after 6 weeks.     

Effects of in vitro growth phase on the pathogenesis of Salmonella typhimurium in mice

The growth phase of a bacterial (Salmonella typhimurium) culture was shown to have pronounced effects on the pathogenic properties of the harvested bacteria. Salmonellae obtained from a culture in primary (exponential) growth phase (PP) were more readily cleared from the blood and more readily killed by phagocytes than were salmonellae obtained from a more slowly growing secondary growth phase (SP) culture. PP salmonellae were observed to cause death of mice sooner than SP salmonellae. This appeared to be because the more rapid growth of PP, as compared to SP, salmonellae continued in the liver and spleen for several hours following intravenous injection, and more than compensated for their high in vivo death rate. As a result, within 4 h there were approximately 10-fold more live salmonellae in the spleens and livers of mice that had received PP, as compared to SP, salmonellae. This 10-fold difference was maintained until the death of the mice, indicating that after the first 4 h post-inoculation, the net in vivo growth of the salmonellae was the same regardless of their growth phase in the inoculating culture. This transition between PP and SP salmonellae occurred long before a dense stationary phase culture was obtained. Salmonellae grown in minimal media exhibited the biological properties of SP salmonellae and never entered as rapid a growth phase as did salmonellae in complete media.     

Occurrence, growth, and suppression of salmonellae in composted sewage sludge.

Composted sewage sludge may be used to improve soil quality, but there remains some doubt concerning the microbiological safety of the product. Sewage sludge composts from 30 municipalities were sampled, and four samples (12%) contained salmonellae (two contained fewer than 0.3/g, and the other two had 21/g and 1.7 X 10(4)/g). All 30 composts were inoculated with salmonellae; the populations decreased at a specific death rate of about 0.15 h-1 over 24 h at 36 degrees C. In irradiation-sterilized composts inoculated with salmonellae, the salmonellae grew at a rate of 0.65 doublings per h for over 24 h. Growth and death rates were found to be moisture and flora associated. The growth or death rates for antibiotic-resistant salmonellae were not different from those of nonresistant strains. It was concluded that the active indigenous flora of compost establishes a homeostatic barrier to colonization by salmonellae, and in the absence of competing flora, reinoculated salmonellae may grow to potentially hazardous densities. The active microflora of moist composts eliminated contaminating salmonellae (10(5)/g) after 6 weeks.     

Bacteriological Survey of Frozen Meat and Gravy Produced at Establishments Under Federal Inspection

During visits to 34 federally inspected establishments producing frozen meat and gravy, 541 production line samples and 535 finished product units were collected for bacteriological analyses. It was found that more than 70% of the sets of finished product (10 units/set) produced under good manufacturing practices had: (i) four or fewer coliform-positive units, (ii) two or fewer Escherichia coli-positive units, (iii) three or fewer Staphylococcus aureus-positive units, and (iv) an aerobic plate count of fewer than 50,000/g (geometric mean of 10 units). All finished product units were negative for salmonellae.     

Delayed secondary enrichment for the isolation of salmonellae from broiler chickens and their environment.

Specimens collected from six broiler flocks were cultured for salmonellae by three methods. (i) For direct enrichment, the specimen was homogenized, and 1 ml of the homogenate was inoculated into tetrathionate-brillant green broth; (ii) for preenrichment, liquid specimens and homogenates were incubated at 37 degrees C, and on the next day 1 ml was inoculated into tetrathionate-brillant green broth; and (iii) for delayed secondary enrichment, incubated preenrichment cultures were held at room temperature for 7 to 10 days and then subcultured to fresh tetrathionate-brilliant green broth. All tetrathionate-brilliant green broth cultures were incubated at 42 degrees C for 24 to 48 h before plating. Significantly more isolations of salmonellae were obtained by delayed secondary enrichment than by direct enrichment or preenrichment. Salmonellae were isolated from 417 of 2,283 (18.3%) samples of litter, intestinal contents, and feces cultured by all three methods. Of these positive specimens, direct enrichment detected 208 (49.9%), preenrichment detected 282 (67.6%), and delayed secondary enrichment detected 373 (89.4%). Of 896 specimens of swabs and rinse fluids that were cultured by preenrichment and delayed secondary enrichment, 259 (28.9%) yielded salmonellae. Delayed secondary enrichment detected 254 (98.1%) of these, and preenrichment detected 147 (56.8%). A total of 23 serotypes of salmonellae were identified. The greater effectiveness of delayed secondary enrichment for the isolation of salmonellae was not likely due to the selection of certain serotypes or to an increased inhibition of competing flora.     

Detection of Salmonellae in the Environment

The incidence of salmonellae in contrasting environments was compared in this study. Samples collected from or near surface waters in a lush hardwood forest yielded four salmonellae serotypes from six culturally positive samples. A total of 76 samples collected from the top of a granite outcropping over a 3-month period yielded 10 positive samples. Only two salmonellae serotypes were isolated, and one of these was isolated only once. The nature of the sample material had no significant effect on the detection of salmonellae from the two sampling sites. However, the presence or absence of visible moisture in the sample significantly affected the recovery of salmonellae. The results showed that even a harsh environment such as that found on top of Stone Mountain may serve as an ecological niche for the survival and transmission of salmonellae.     

Isolation of Salmonellae From Food Samples: VI. Comparison of Methods for the Isolation of Salmonella From Egg Products

The recovery of salmonellae from egg products was studied, by use of three different enrichment procedures: (i) selenite broth, (ii) selenite broth containing 10% sterile feces, and (iii) the lactose pre-enrichment procedure. Brilliant Green Agar was used throughout as the recovery medium. Although the lactose pre-enrichment methodology promoted Salmonella recovery from samples containing small numbers of dormant organisms, the efficiency of this enrichment method is adversely affected by unfavorable coliform-Salmonella ratios. Under such conditions, early subculture of lactose broth into selenite broth is indicated. Selenite broth containing 10% sterile feces was more efficient than the lactose pre-enrichment methodology in promoting the growth of “dormant” salmonellae. Albumen adversely affected recovery of salmonellae from selenite broth, whereas whole egg and egg yolk enhanced Salmonella recovery from this medium. The selenite-feces medium presents a solution to the major problems encountered in the detection of salmonellae in egg products and offers an approach to a single medium in which food-borne salmonellae will manifest themselves with a minimum of laboratory manipulation.     

Evidence of association of salmonellae with tomato plants grown hydroponically in inoculated nutrient solution

The possibility of uptake of salmonellae by roots of hydroponically grown tomato plants was investigated. Within 1 day of exposure of plant roots to Hoagland nutrient solution containing 4.46 to 4.65 log(10) CFU of salmonellae/ml, the sizes of the pathogen populations were 3.01 CFU/g of hypocotyls and cotyledons and 3.40 log(10) CFU/g of stems for plants with intact root systems (control) and 2.55 log(10) CFU/g of hypocotyls and cotyledons for plants from which portions of the roots had been removed. A population of > or =3.38 log(10) CFU/g of hypocotyls-cotyledons, stems, and leaves of plants grown for 9 days was detected regardless of the root condition. Additional studies need to be done to unequivocally demonstrate that salmonellae can exist as endophytes in tomato plants grown under conditions that simulate commonly used agronomic practices.     

Comparison of Four Agar Plating Media with and Without Added Novobiocin for Isolation of Salmonellae from Beef and Deboned Poultry Meat

Four plating media, Hektoen enteric (HE), xylose-lysine deoxycholate (XLD), tryptic soy-xylose-lysine (TSXL), and tryptic soy-brillant green (TSBG) agars with and without 10 mg of added novobiocin per ml, were evaluated for recovery of Salmonella from roast beef and deboned turkey. Colonies producing a reaction typical of H₂S-positive salmonellae (alkaline with black centers) were picked. On the media without novobiocin, from 109 determinations on 75 samples, number of salmonellae found and false-positives were, respectively: HE—13, 58; XLD—17, 18; TSXL—23, 0; TSBG—22, 7. When novobiocin was present the corresponding results were: HE—17, 24; XLD—21, 2; TSXL—23, 3; TSBG—20, 7. A total of 25 determinations were positive on one or more agars. False-positives on HE and XLD without novobiocin were predominantly Proteus, which were almost totally eliminated by addition of 10 mg of novobiocin per liter. If alkaline H₂S-negative colonies had been considered, many more false-positives would have been found on HE and XLD but not on TSBG or TSXL. Addition of novobiocin markedly improved isolations of salmonellae from XLD and HE and reduced the number of false-positives. Addition of novobiocin did not improve performance of TSXL and slightly impaired differentiation of salmonellae from Citrobacter on TSBG. XLD with novobiocin and TSXL are highly specific for H₂S-positive salmonellae, and the appearance of Salmonella-like colonies on these media can be considered a presumptive test for H₂S-positive salmonellae.     

Growth of Salmonella typhimurium in Skim Milk Concentrates

The influence of various levels of skim milk solids and temperature on the duration of lag phase, growth rate, and extent of growth of Salmonella typhimurium was investigated. The effect on growth of salmonellae (and a strain of Escherichia coli) of reduced pressure at a constant solids level and under conditions simulating vacuum condensation of skim milk was also studied. S. typhimurium grew when inoculated into skim milk solutions ranging from 10 to 60% solids and over a temperature range of 23 to 44 C. At 10 to 12 C, growth was evident only in the 10% skim milk. As the total solids level was increased or incubation temperature was deviated from the optimum, or both, there was an increase in the lag phase and generation time of salmonellae. A lower cell population also resulted. The generation time at 37 C of S. typhimurium incubated at atmospheric pressure was approximately one-half that in skim milk concentrates held under reduced pressure. In addition, a slightly longer lag phase and lower cell yield characterized the growth under reduced pressure. Concentration of skim milk had little or no effect on viability of salmonellae or E. coli when the vapor temperature in the vacuum pan was below the maximum growth temperature for salmonellae. Increasing the vapor temperature to 48 C caused a two-log reduction in viable organisms during the concentrating period (65 min).     

Identification of Salmonella with the O-1 Bacteriophage

The O-1 bacteriophage test of Cherry et al. (1954) for the presumptive identification of salmonellae in the diagnostic laboratory was investigated. A phage lysate with a titer of 10(12) plaque-forming units per ml was found to be optimal. This preparation lysed 98.2% of Salmonella strains tested, while maintaining its high specificity for salmonellae. Gram-negative organisms other than salmonellae were resistant to the O-1 phage; however, 5.9% of Escherichia coli strains tested were susceptible. The O-1 phage test is a simple, rapid, inexpensive, sensitive, and specific procedure for the identification of salmonellae in the diagnostic laboratory. A presumptive identification is obtained 1 day earlier than with conventional biochemical tests.     

Isolation of Shigellae: VI. Performance of Media with Stool Specimens

The efficiencies of three enrichment broths and four plating media for isolation of enteric pathogens were compared from 1,117 stool specimens. Direct streaking proved to be inferior to enrichment, detecting only 50% of the salmonellae and 61% of the shigellae. By contrast, Selenite Broth (SF) found 90% of the total salmonellae isolates and 82% of the shigellae isolates. Gram-Negative Broth (GN) found 82% and 85%, respectively, but Tetrathionate found only 60% and 39%. Thus, SF and GN were comparable for both salmonellae and shigellae and significantly better than Tetrathionate Broth for both. The plating media compared were MacConkey (MAC), deoxycholate citrate (DC), xylose lysine deoxycholate (XLD), and xylose lysine Brilliant Green (XLBG) Agars. Of the total salmonellae isolated, XLD produced 94%; XLBG, 71%; MAC, 55%; and DC, only 35%. Of shigellae, XLD found 89%; MAC, 75%; XLBG, 63%; and DC, but 27%. The efficacy of XLD is observed to be almost threefold that of DC. The most successful combination of media for the detection of fecal pathogens was GN or SF enrichment broths streaked to XLD plates. These analyses resulted in the isolation of 118 strains of salmonellae and 33 of shigellae.     

Enteropathogenic bacteria in frozen chicken.

Eighty-two samples of frozen chicken from retail stores were examined for the presence of Campylobacter, Yersinia enterocolitica, and salmonellae. Aerobic plate counts and numbers of coliform bacteria at 37 degrees C were determined. Campylobacter fetus subsp. jejuni was found in 22% of the samples, Y. enterocolitica was found in 24.5% and Salmonella typhimurium was found in one sample (1.2%). The isolated strains of Y. enterocolitica belonged to serotypes 4, 5b, 6, and 8. Aerobic plate counts and numbers of coliform bacteria at 37 degrees C were not found to be noticeably higher in samples containing pathogens than in pathogen-free samples. This investigation showed that chicken does contain other pathogenic bacteria than salmonellae. Campylobacter and Y. enterocolitica were isolated in much higher frequencies than Salmonella.     

Survival of Salmonellae During Pepperoni Manufacture

Survival of salmonellae in artificially contaminated beef-pork mixtures (approximately 10(4) salmonellae/g) was studied in pepperoni prepared by either a natural flora or lactic starter culture fermentation or in nonfermented sausages. The pepperoni did not become salmonellae free during the usual commercial 15 to 30-day drying period. Salmonella dublin was present in all products, fermented or unfermented, after 42 to 43 days of drying. At a lower level of contamination, 10(3)/g, S. dublin could not be recovered from starter culture-fermented pepperoni after 14 days of drying but persisted in the natural flora-fermented sausage. S. typhimurium (initial count, 10(4)/g) was absent after 42 days of drying when starter culture was used to ferment the pepperoni, but was still present in the natural flora-fermented and unfermented products. S. dublin, host adapted to cattle, or S. choleraesuis, host adapted to swine, had similar survival patterns in beef pork, or beef-pork pepperoni. Heating salmonellae contaminated beef-pork pepperoni (after fermantation but before drying) to an internal temperature of 60 C (trichinae inactivating) eliminated the food-borne pathogen from the sausage product.