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.     

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.     

Incidence of Salmonellae in Dressed Broiler-Fryer Chickens

Salmonellae were isolated from 72 of 264 broiler-fryer type chickens that had been purchased in retail stores in the Lafayette, Ind., area in 1963. Meat from the tail area and giblet portions were used in sampling. Equal numbers of dressed whole and cut-up birds were positive for salmonellae. Thirteen different serotypes were identified, the more common being Salmonella infantis, S. reading, and S. blockley. Incubation at 43 C of the blended sample in Selenite-F Enrichment broth containing cystine gave a larger number of recoveries than did incubation at 37 C. There was no significant difference between the means for the birds that yielded salmonellae and those that did not in the locally processed group, when compared for numbers of aerobic microorganisms at 37 C, coliforms, or most probable number of enterococci. In a comparison of poultry processed in-state by the five processors included in the study with that processed out-of-state, there was a general trend for a larger number of positive specimens in the locally produced group. The fall season was an exception.     

Salmonella Survival on Pecans as Influenced by Processing and Storage Conditions

Survival of Salmonella senftenberg 775W, S. anatum, and S. typhimurium during exposure to currently practiced, as well as abusive, pecan processing and storage conditions was studied. Thermal treatments normally carried out during the processing of pecans are inadequate to consistently destory salmonellae in highly contaminated inshell nuts. Pecan nut packing tissue was toxic to salmonellae, thus affording some protection against high initial contamination and subsequent survival of the organisms. Examinations of inoculated inshell pecans stored at -18, -7, 5, and 21 C for up to 32 weeks revealed that the extent of survival was inversely correlated to the storage temperature. S. senftenberg 775W and S. anatum were not detectable on inshell nuts after 16 weeks of storage at 21 C. Little decrease in viable population of the three species was noted on inoculated pecan halves stored at -18, -7, and 5 C for 32 weeks. Due to organoleptic quality deterioration in pecan nutmeats at elevated temperatures, sterilization methods other than thermal treatment appear to be required for the elimination of viable salmonellae from pecan nuts.     

The influence of manufacture on the free D-amino acid content of Cheddar cheese

Summary. The changes in the concentration and those of composition of alanine, aspartic acid and glutamic acid enantiomers were investigated during manufacture of Cheddar cheese. The amount of D-alanine increased continuously during ripening following the liberation of L-alanine originated from the proteolysis of milk proteins. There was slightly more D-aspartic and D-glutamic acid in the dry matter of curd after pressing than before pressurization. The D-amino acid content and the ratio of the D-enantiomers related to the total amount of free amino acids differed significantly among cheeses produced with different single-strain starters. The D-amino acid composition changed during manufacture, but the influence of the strain selection was not significant on the D-amino acid pattern.     

Production of lipopeptide antibiotic iturin A using soybean curd residue cultivated with Bacillus subtilis in solid-state fermentation

Bacillus subtilis RB14-CS, which suppresses the growth of various plant pathogens in vitro by producing the lipopeptide antibiotic iturin A, was cultured using soybean curd residue, okara, a by-product of tofu manufacture in solid-state fermentation. After 4 days incubation, iturin A production reached 3,300 mg/kg wet solid material (14 g/kg dry solid material), which is approximately tenfold higher than that in submerged fermentation. When the okara product cultured with RB14-CS was introduced into soil infested with Rhizoctonia solani, which is a causal agent of damping-off of tomato, the disease occurrence was significantly suppressed. After 14 days, the number of RB14-CS cells remained in soil at the initial level, whereas almost no iturin A was detected in soil. As the okara cultured with RB14-CS exhibited functions of both plant disease suppression and nutritional effect on tomato seedlings, this product is expected to contribute to the recycling of the soybean curd residue.     

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.     

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.     

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.     

Thermal insulation of the winter cauliflower curd by wrapper-leaves

The role of leaves as insulation for mature curds during exposure to frost has been investigated for winter-harvested cauliflower. The inner, wrapper-leaves and their enclosed air spaces provided significant protection for the curd tissues in mild frost, but under more severe conditions (below – 2.5°C) were ineffective. A relationship between higher wrapper-leaf number and increased insulation was described. The increase in insulation however, was small even with much higher wrapper-leaf numbers. Exposure of the curd surface at maturity was found to prevent the insulating effect of wrapper-leaves, particularly if the diameter of the exposed area was greater than 6 cm. The data also suggested a possible role for the exposed stem of the cauliflower as a site of heat loss that may adversely affect curd temperature. It is postulated that breeding for improved frost resistance by greatly increasing wrapper-leaf number would not be effective, although any reduction in wrapper-leaf number and the presence of tightly wrapped curds should be avoided. Furthermore, exposure of the curd surface at maturity should be kept to a minimum.     

Fate of Escherichia coli O157:H7 during the manufacture of Mozzarella cheese

AIMS: The fate of Escherichia coli O157:H7 was investigated during the manufacture of Mozzarella cheese. METHODS AND RESULTS: The Mozzarella cheese was made from unpasteurized milk which was inoculated to contain ca 10(5) cfu ml(-1)E. coli O157:H7. Two different heating temperatures (70 and 80 degrees C), commonly used during curd stretching, were investigated to determine their effects on the viability of E. coli O157:H7 in Mozzarella cheese. Stretching at 80 degrees C for 5 min resulted in the loss of culturability of E. coli O157:H7 strains, whereas stretching at 70 degrees C reduced the number of culturable E. coli O157:H7 by a factor of 10. CONCLUSIONS: The results show that stretching curd at 80 degrees C for 5 min is effective in controlling E. coli O157:H7 during the production of Mozzarella cheese. Brining and storage at 4 degrees C for 12 h was less effective than the stretching. Significance and Impact of the Study: Mozzarella cheese should be free of E. coli O157:H7 only if temperatures higher than or equal to 80 degrees C are used during milk processing.     

Fate of Salmonella in dry confectionery raw materials

Aims:  To study the behaviour of salmonellae inoculated in the dry, raw materials, related to chocolate confectionery manufacture, as affected by the strain, cell preparation method and storage temperature.
Methods and Results:  Outbreak and non outbreak-associated salmonellae were used for the inoculation of cocoa butter oil, crushed cocoa and hazelnut shells, cocoa beans and almond kernels. Dry matrices were inoculated with lawn-collected and broth-grown cells and were stored at 5 and 21°C for 21 days. Results demonstrated that all strains survived in all dry matrices. Lawn-collected cells survived considerably better than broth-collected cells, at either temperature, and outbreak-associated strains of Salmonella serotype Enteritidis PT30 and Salmonella serotype Oranienburg appeared to be among the best surviving strains.
Conclusions:  The work demonstrated that salmonellae can survive storage for 3–4 weeks in dry raw materials and that survival is dependent on the source of strains, cell preparation and inoculation methodology, and storage temperature.
Significance and Impact of the Study:  The data contributes to understanding the parameters to consider when assessing the risk of dry raw materials as the most likely source of salmonellae in chocolate processing plants. It also demonstrates the importance of implementing effective lethal processes and segregation procedures to prevent cross-contamination to ensure the safety of confectionery products regarding Salmonella .     

Behaviour of Listeria monocytogenes during the traditional manufacture of water-buffalo Mozzarella cheese

F. VILLANI, O. PEPE, G. MAURIELLO, G. MOSCHETTI, L. SANNINO AND S. COPPOLA. 1996. The behaviour of a four-strains mixture of Listeria monocytogenes (strains Scott A, V7, OH and Cal) during the traditional manufacture of water-buffalo Mozzarella cheese was investigated at two levels of inoculation: ca 10⁵and 10³cfu ml^-1of vat milk. No significant change in Listeria counts was observed during the curd ripening (4.0–4.5 h), at the end of which the pH ranged between 4.83 and 4.91. A decrease of about 2 log was observed after stretching of the curd in hot water (95°C), followed by complete elimination of Listeria after 48 and 24 h of storage of the final cheese in the conditioning liquid (skim water resulting from the stretching, pH ca 4.0) with initial high and low contamination of the cheese milk respectively. Results also indicated that a 1.7 log reduction of L. monocytogenes could be achieved during the preparation of the natural whey culture utilized as starter in cheesemaking.     

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.     

Preliminary characterization of microflora of Comté cheese

The evolution of the microflora of three Comté cheeses made in duplicate with raw milk from three different sources was followed during ripening. The same starter was used with each type of milk. The comparison of the cheeses did not reveal any significant difference in the development of the microflora. Starter lactic acid bacteria ( Streptococcus thermophilus and Lactobacillus helveticus) , which are added at the beginning of manufacture, decreased quickly in the first stages of ripening supporting the hypothesis of cell autolysis. Other micro-organisms, i.e. homofermentative and heterofermentative lactobacilli ( Lact. delbrueckii ssp. lactis , Lact. paracasei ssp. paracasei , Lact. rhamnosus and Lact. fermentum ), pediococci, enterococci and propionibacteria grew in cheese from small numbers in fresh curd. The characterization of Strep. thermophilus by pulsed-field gel electrophoresis showed that wild strains were also able to grow in the curd. The values for the genome size of 11 Strep. thermophilus strains determined in this investigation were in the range of 1·8–2·3 Mbp. The potential role of starter and raw milk microflora in cheese flavour development was considered.     

Temporal analyses of the distribution and diversity of Salmonella in natural biofilms

The diversity and distribution of salmonellae in freshwater biofilms were analyzed at a fine scale (i.e. in 20 locations from a 324 cm² area) for two sites in San Marcos, TX. A concrete storm water overflow channel (City Park) was sampled 4 times and a concrete surface in the spring-fed headwaters of the San Marcos River (Spring Lake) 5 times between April and September 2009, and each biofilm sample analyzed by a combination of traditional enrichment methods and molecular techniques. PCR detection of the invA gene, that encodes a protein of a type III secretion system present in salmonellae, after semi-selective enrichment of salmonellae was achieved in biofilms from all 20 locations at the City Park site, with locations generally being positive 2-3 times out of 4 sampling times for a total of 59% positive samples. InvA gene fragment detection in biofilms was less frequent for the 5 sampling times and 20 locations from the Spring Lake site (18% of all samples), with 1 sampling time being entirely negative and 8 locations remaining negative throughout the study. Rep-PCR fingerprinting of 491 Salmonella isolates obtained from both sites resulted in 30 distinct profiles, with 26 and 7 profiles retrieved from City Park and Spring Lake samples, respectively, and thus with 3 profiles present at both sites, and multiple strains frequently obtained from single locations at both sites. The composition of Salmonella strains in the area analyzed changed in time with large differences between early (April, June) and late sampling times (September) within and among sites, except for one strain (S12) that was present at almost all sampling times at both sites, though often at different locations within the area analyzed. These results demonstrate the presence of salmonellae in natural biofilms and a significant micro-heterogeneity with differences in diversity and persistence of salmonellae.     

Causal Relationship between Microbial Ecology Dynamics and Proteolysis during Manufacture and Ripening of Protected Designation of Origin (PDO) Cheese Canestrato Pugliese

Pyrosequencing of the 16S rRNA gene, community-level physiological profiles determined by the use of Biolog EcoPlates, and proteolysis analyses were used to characterize Canestrato Pugliese Protected Designation of Origin (PDO) cheese. The number of presumptive mesophilic lactococci in raw ewes'' milk was higher than that of presumptive mesophilic lactobacilli. The numbers of these microbial groups increased during ripening, showing temporal and numerical differences. Urea-PAGE showed limited primary proteolysis, whereas the analysis of the pH 4.6-soluble fraction of the cheese revealed that secondary proteolysis increased mainly from 45 to 75 days of ripening. This agreed with the concentration of free amino acids. Raw ewes'' milk was contaminated by several bacterial phyla: Proteobacteria (68%; mainly Pseudomonas), Firmicutes (30%; mainly Carnobacterium and Lactococcus), Bacteroidetes (0.05%), and Actinobacteria (0.02%). Almost the same microbial composition persisted in the curd after molding. From day 1 of ripening onwards, the phylum Firmicutes dominated. Lactococcus dominated throughout ripening, and most of the Lactobacillus species appeared only at 7 or 15 days. At 90 days, Lactococcus (87.2%), Lactobacillus (4.8%; mainly Lactobacillus plantarum and Lactobacillus sakei), and Leuconostoc (3.9%) dominated. The relative utilization of carbon sources by the bacterial community reflected the succession. This study identified strategic phases that characterized the manufacture and ripening of Canestrato Pugliese cheese and established a causal relationship between mesophilic lactobacilli and proteolysis.     

Wet brewers' grains or bean curd pomance as partial replacement of soybean meal for lactating cows

The purpose of this study was to evaluate the effects of supplementation of the by-product, brewers' grains and the bean curd pomance, on the performance of lactating cows and their ruminal characteristics. Through this, we wanted to increase resource utilization and to eliminate pollution from these by-products. Thirty-two Holstein lactating cows were allocated randomly into four dietary treatment groups. The experimental diets were formulated to be isoenergetic and isonitrogenous according to National Research Council (1989), and containing 35% corn silage, 20% alfalfa hay and 45% concentrates on DM basis. The dietary treatments consisted of the inclusion of different by-products sources. These included soybean bean (as the control), brewers' grains, bean curd pomance, and the mixed by-products (containing the same amount of brewers' grains and bean curd pomance). The experimental feeding lasted for eight weeks after one week of adaptation. In addition, four rumen cannulated Holstein cows were used in a 4×4 Latin square with 10-day period for collecting rumen samples. Results showed that cows that were fed the bean curd pomance diet produced significantly more milk than those that were fed the brewers' grains diet or the mixed by-product diet. The cows on the control diet produced significantly less milk than the other treatment groups (P<0.05). Both the control and brewers' grains groups consumed more feed than those in the bean curd pomance and mixed by-products diet groups (P<0.05). The cows that were fed the mixed by-products diet produced a significantly lower percentage of milk fat, total solids, milk protein and milk lactose than the others (P<0.05). The milk lactose percentage was significantly higher for cows that were fed the bean curd pomance than those fed the brewers' grains diet (P<0.05). Cows that were fed the bean curd pomance produced significantly more milk fat, total solids, milk protein and milk lactose than the others (P<0.05). Cows that were fed the mixed by-products diet produced significantly lower amount of milk fat, total solids and milk protein than the control and brewers' grains group (P<0.05). These diets did not significantly influence the body weight of the cows. It, however, significantly influenced the ruminal characteristics (P<0.05).     

Microbial Ecology Dynamics Reveal a Succession in the Core Microbiota Involved in the Ripening of Pasta Filata Caciocavallo Pugliese Cheese

Pyrosequencing of the 16S rRNA targeting RNA, community-level physiological profiles made with Biolog EcoPlates, proteolysis, and volatile component (VOC) analyses were mainly used to characterize the manufacture and ripening of the pasta filata cheese Caciocavallo Pugliese. Plate counts revealed that cheese manufacture affected the microbial ecology. The results agreed with those from culture-independent approaches. As shown by urea-PAGE, reverse-phase high pressure liquid chromatography (RP-HPLC), and free-amino-acid (FAA) analyses, the extent of secondary proteolysis mainly increased after 30 to 45 days of ripening. VOCs and volatile free fatty acids (VFFA) were identified by a purge-and-trap method (PT) and solid-phase microextraction (SPME) coupled with gas chromatography-mass spectrometry (GC-MS), respectively. Except for aldehydes, the levels of most of VOCs and VFFA mainly increased from 30 to 45 days onwards. As shown through pyrosequencing analysis, raw cows'' milk was contaminated by Firmicutes (53%), Proteobacteria (39%), Bacteroidetes (7.8%), Actinobacteria (0.06%), and Fusobacteria (0.03%), with heterogeneity at the genus level. The primary starter Streptococcus thermophilus dominated the curd population. Other genera occurred at low incidence or sporadically. The microbial dynamics reflected on the overall physiological diversity. At 30 days, a microbial succession was clearly highlighted. The relative abundance of Streptococcus sp. and especially St. thermophilus decreased, while that of Lactobacillus casei, Lactobacillus sp., and especially Lactobacillus paracasei increased consistently. Despite the lower relative abundance compared to St. thermophilus, mesophilic lactobacilli were the only organisms positively correlated with the concentration of FAAs, area of hydrophilic peptide peaks, and several VOCs (e.g., alcohols, ketones, esters and all furans). This study showed that a core microbiota was naturally selected during middle ripening, which seemed to be the main factor responsible for cheese ripening.