Microbially mediated growth suppression and death of salmonella in composted sewage sludge

The role of compost microflora in the suppression of salmonella regrowth in composted sewage sludge was investigated. Microbial inhibition studies of salmonella growth were conducted on nutrient agar, in composts that had been subjected to different temperatures in compost piles, and in radiation sterilized composts inoculated with selected fractions of the compost microflora. Agar assays of inhibition indicated that bacteria and actinomycetes were not suppressive to salmonellae, but a few fungi were. However, compost inoculation assays showed consistently that fungi were not suppressive, but bacteria and actinomycetes were. In compost inoculation assays, microbial antagonists, when present, either killed salmonellae or reduced their growth rate. No suppression of salmonellae occurred in compost taken from 70°C compost-pile zones despite the presence and growth of many types of microbes. With greater numbers and kinds of microbes in 55°C compost, salmonella growth was suppressed 100–10,000-fold. Salmonellae died when inoculated into compost from unheated zones (25–40°C) of piles. Prior colonization of compost with only noncoliform gram-negative bacteria suppressed salmonellae growth 3,000-fold. Coliforms when inoculated prior to salmonellae accounted for 75% of salmonella die-off. Mesophilic curing to allow colonization of curing piles in their entirety by gram-negative bacteria, especially coliforms, should be an effective way to prevent repopulation by salmonellae.     

Identification of sources of microbial pathogens on cantaloupe rinds from pre-harvest to post-harvest operations

Foodborne illness outbreaks involving cantaloupes have increased dramatically in the past 15 years in the United States and other countries. The need for the identification of the microbial sources that contaminate cantaloupe rinds has been raised by various investigators. This study was undertaken to identify the agricultural, industrial and human sources of microbial contamination from the pre- to post-harvest operations of cantaloupes grown at ten different farms in southern Texas. Results indicate that irrigation water contained a wide range of microorganisms that could cause human illness and were able to survive on the rind of cantaloupes before, at, and after harvesting. Fungi, total aerobic bacteria and total coliform bacteria were not completely eliminated by chlorinated water in the disinfection tanks of the six packinghouses under investigation. There were significant (P < 0.05) reductions on rind populations of fungi and total aerobic bacteria as well as drastic reductions in total coliform bacteria on the rinds after the disinfection and rinsing steps in all packing facilities. There was no evidence of the presence of Escherichia coli O157:H7 on packed cantaloupes across packinghouses. Less than a geometric mean of 1 c.f.u. cm⁻² of salmonellae were detected on the surface of packed cantaloupes in two of the packinghouses, and approximately ten times more salmonellae were found on the packed fruit processed in the remaining packinghouses. A similar trend was observed with listeriae. Results suggested that microbial loads originating from river water may survive on the rind or may re-infest cantaloupes after the disinfection and rinsing process at the packinghouses. Disinfection techniques and aseptic handling of cantaloupes at the packing facilities need a closer evaluation to ensure a safe product.     

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.     

Evaluation of an X-ray microprobe technique as a possible aid to detect salmonellae

The X-ray microprobe was examined as a possible aid in the detection of salmonellae. Results obtained indicate that specific bacterial antigen (Salmonella) increased in phosphorus and sulfur after reaction with the fluorescein isothiocyanate tagged specific antibody. Nonspecific antigen (Escherichia coli) did not increase in phosphorus and sulfur after reaction with fluorescein isothiocyanate tagged anti-Salmonella antibody. The technique developed supports the hypothesis that X-ray microprobe analysis may be useful in detecting salmonellae, or other bacteria, by determining increases in the elemental constituents of bacterial cells when reacted with elemental-tagged antibodies.     

Isolation of salmonellae and other potential pathogens from the freshwater aquarium snail Ampullaria.

The freshwater aquarium snail (Ampullaria spp.) was demonstrated to carry as many as 10(8) viable mesophilic bacteria per g of meat plus shell. Some 16 genera of bacteria were identified, with gram negatives predominating. Enrichment culture techniques enabled the isolation of salmonellae from 24 to 42 lots of 200 g each. The salmonellae comprised eight different serotypes, including Salmonella newport, Salmonella saint-paul, and Salmonella infantis. This association of salmonellae with snails may contribute to cases of human salmonellosis, since other aquarium species have already been shown to contribute to many such cases. The snails were also found to commonly harbor Pseudomonas aeruginosa and, occasionally, Edwardsiella tarda.     

Inefficient in vitro killing of virulent or nonvirulent Salmonella typhimurium by murine polymorphonuclear neutrophils

The bactericidal capability of murine peritoneal polymorphonuclear neutrophils against virulent and nonvirulent Salmonella typhimurium was examined in an in vitro system. Although preincubation of the bacteria in specific murine antiserum elicited greater chemiluminescence from phagocytizing neutrophils than did incubation in normal murine serum, antiserum did not enhance ingestion, as less than 5% of the challenge was taken up by neutrophils under any of the conditions studied. Nonvirulent salmonellae showed a transient decrease in viable numbers early during in vitro incubation with or without intact neutrophils. Virulent salmonellae, however, were able to multiply without a lag period except when these bacteria were pretreated with antiserum and incubated in association with intact murine neutrophils. Results of these in vitro studies suggest that the murine polymorphonuclear neutrophil and antisalmonella antibody must act together to effect neutrophil-associated bactericidal activity against virulent salmonellae, and thus, that the neutrophil alone does not play a major role in the protection of unvaccinated, sensitive mice from disease caused by S. typhimurium.     

Elevated-Temperature Technique for the Isolation of Salmonella from Streams

With a modified Moore swab for sampling bacteria, Salmonella organisms were recovered consistently from surface waters when the enrichment broths of SBG-sulfa and tetrathionate and Brilliant Green Agar plates were incubated at 41.5 C. When an equal portion of the same swab sample was incubated at 37.0 C, no salmonellae were detected. By use of the elevated-temperature technique, salmonellae were recovered from stream sites having relatively low total coliform densities of 2,200 per 100 ml and fecal coliform densities of 220 per 100 ml. These pathogens were isolated in water sampled as far as 73 river miles and 4 days time of travel downstream from the source of pollution under an ice cover that averaged 2.5 ft (76.2 cm) in thickness.     

Direct detection of Salmonella spp. in estuaries by using a DNA probe.

A method for direct detection of Salmonella spp. in water was developed by using a commercially available DNA probe. Particulate DNA was extracted from 500- to 1,500-ml water samples collected from New York Harbor and Chesapeake Bay and used as a substrate for a salmonella-specific DNA probe in dot blot assays. The method detected salmonellae in water samples from 12 of 16 sites, including 6 sites where salmonellae could not be cultured. The specificity of the probe was evaluated, and cross-hybridization, although negligible, was used to set detection limits for the assay. Salmonella DNA bound the probe quantitatively, and from these results Salmonella DNA in the total particulate DNA in environmental samples could be estimated. The data obtained in this study indicate that Salmonella spp. often are not detected in water samples by culture methods, even when they are present in significant numbers.     

Direct detection of Salmonella spp. in estuaries by using a DNA probe

A method for direct detection of Salmonella spp. in water was developed by using a commercially available DNA probe. Particulate DNA was extracted from 500- to 1,500-ml water samples collected from New York Harbor and Chesapeake Bay and used as a substrate for a salmonella-specific DNA probe in dot blot assays. The method detected salmonellae in water samples from 12 of 16 sites, including 6 sites where salmonellae could not be cultured. The specificity of the probe was evaluated, and cross-hybridization, although negligible, was used to set detection limits for the assay. Salmonella DNA bound the probe quantitatively, and from these results Salmonella DNA in the total particulate DNA in environmental samples could be estimated. The data obtained in this study indicate that Salmonella spp. often are not detected in water samples by culture methods, even when they are present in significant numbers.     

Salmonellae as an Index of Pollution of Surface Waters

Screening enrichments of surface water specimens by means of a polyvalent fluorescent antibody reagent for the salmonellae yielded approximately 60% more positive specimens than was obtained by cultural procedures. It is not known what fraction of the excess of fluorescent antibody-positive over culturally positive specimens represents staining of non-salmonellae or non-arizonae as opposed to the staining of non-cultivatable organisms of these two genera. Cotton gauze and rayon-polypropylene fiber swabs were equally sensitive for collecting salmonellae from the streams examined. Tetrathionate enrichment incubated at 41.5 C appeared to be superior to selenite-cystine for isolation of salmonellae from surface waters. Twenty-eight serotypes of Salmonella and two serotypes of Arizona were identified in the 121 positive specimens. In water rated moderately polluted, 65% of all specimens tested were positive; in minimally polluted waters, 38% were positive; and in unpolluted streams, 44% were positive.     

Proflavine-mediated inactivation of Salmonella dublin exposed to visible sunlight in natural fresh water

The survival of Salmonella dublin exposed to visible sunlight, and heterotrophic bacteria in freshwater microcosms in the presence and absence of the photosensitizer proflavine, was studied. Enumeration of S. dublin and the heterotrophic bacteria showed that in both illuminated and nonilluminated systems (without proflavine) the bacteria remained viable and culturable for at least 6 days. The optimal proflavine concentration (no effect in the dark and a maximal photoinactivation of salmonellae after irradiation) was 2 mg l(-1). In contrast to S. dublin, the heterotrophic bacteria overcame the initial inhibitory effect of proflavine. The possible use of photosterilization against contamination with pathogenic bacteria in water model ecosystems, is discussed.     

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.     

Proteome of Salmonella typhimurium SL1344: identification of novel abundant cell envelope proteins and assignment to a two-dimensional reference map.

Forty-nine cell envelope proteins of Salmonella typhimurium SL1344 have been identified by microsequencing and assigned to a two-dimensional reference map. Ten of the sequenced proteins appear to be novel. Several others closely match currently hypothetical proteins or proteins found in other bacteria but not previously reported in salmonellae.     

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.     

Role of Microbial Iron Transport Compounds in the Bacterial Spoilage of Eggs

Microbial iron transport compounds, belonging either to the hydroxamate family excreted by pseudomonads, or to the phenolate family excreted by salmonellae, reverse the bacteriostatic effect of conalbumin on the growth of these bacteria in egg white. The presence of microgram quantities of these compounds permits both salmonellae and pseudomonads to reach dense populations in egg white. The role of these iron transport compounds in bacterial egg spoilage is discussed.     

Survival of Salmonella enterica in freshwater and sediments and transmission by the aquatic midge Chironomus tentans (Chironomidae: Diptera)

Survival of a nalidixic acid-resistant strain of Salmonella enterica serovar Typhimurium mr-DT-104 in water and sediments was tested using artificially contaminated aquaria. Water samples remained culture positive for salmonella for up to 54 days. Sediment samples were culture positive up to 119 days. In addition, potential mechanisms for spreading salmonella in the environments by chironomid larvae and adults were tested. We evaluated the acquisition of mr-DT-104 by chironomids from contaminated aquatic sediments and subsequent spread to uncontaminated sediments. Larval chironomids raised in contaminated sediments became culture positive, and the bacteria were carried over to adults after emergence. Contamination of clean sediments by chironomid larvae was not demonstrated. These findings clearly suggest that mr-DT-104 serovar organisms can survive in aquatic sediments for at least several months. Uptake of salmonellae by chironomid larvae and adults suggests that they are possible vectors of mr-DT-104 in both aquatic and terrestrial environments, although the role of larval defecation in movement of bacteria to new sediments was not demonstrated.     

Salmonellosis in mice infected with Mycobacterium tuberculosis BCG. I. Role of endotoxin in infection

In agreement with previous observations, mice dying of salmonellosis were found to have a relatively constant number of Salmonella cells in their carcasses (ca. 5 x 10(9)). This number was not the result of bacterial overgrowth in moribund animals and therefore appears to be related to lethality. Similar numbers of salmonellae were recovered from the carcasses of infected mice which had previously been rendered hyperreactive to endotoxin either by infection with M. tuberculosis BCG or by adrenalectomy. In BCG mice, desensitization to endotoxin did not occur during the infection and, therefore, at death these mice contained a number of bacteria which would be equivalent to 1,000 ld(50) of endotoxin. Although the number of bacteria recovered from normal mice is roughly equivalent to a lethal quantity of endotoxin, this is obviously not the case in hyperreactive mice. Therefore, the relationship between lethality and 5 x 10(9) salmonellae cannot be explained by their endotoxin content. Nevertheless, when hyperreactive BCG mice are challenged parenterally with Salmonella, the endotoxin content of the inoculum may markedly influence the course of the infection.     

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.     

Enumeration of Salmonellae in Table Eggs,Pasteurized Egg Products,and Egg-Containing Dishes by Using Quantitative Real-Time PCR

Salmonellae are a major cause of food-borne outbreaks in Europe, with eggs and egg products being identified as major sources. Due to the low levels of salmonellae in eggs and egg products, direct quantification is difficult. In the present study, enrichment quantitative real-time PCR (qPCR) was employed for enumeration of salmonellae in different matrices: table eggs, pasteurized egg products, and egg-containing dishes. Salmonella enterica serovar Enteritidis and S. enterica serovar Tennessee were used to artificially contaminate these matrices. The results showed a linear regression between the numbers of salmonellae and the quantification cycle (C_q) values for all matrices used, with the exception of pasteurized egg white. Standard curves were constructed by using both stationary-phase cells and heat-stressed cells, with similar results. Finally, this method was used to evaluate the fate of salmonellae in two egg-containing dishes, long egg and tiramisu, at abused refrigeration temperatures, and results indicated the growth of bacteria over a 1-week period. In conclusion, enrichment qPCR was shown to be reliable for enumeration of salmonellae in different egg products.     

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.