Fate of salmonellas and competing flora in meat sample enrichments in buffered peptone water and in Muller-Kauffmann's tetrathionate medium

Studies have been carried out in which growth patterns of a Salmonella sp. and competing micro-organisms, especially other Enterobacteriaceae, were followed during pre-enrichment in buffered peptone water (BPw) and subsequent selective enrichment in tetrathionate broth (TBB). Pre-enrichment cultures were inoculated with minced meat and three reference samples containing nalidixic acid-resistant salmonellas. Irrespective of their initial numbers in BPw, Enterobacteriaceae increased to 10⁸/ml or more. During incubation in TBB at 43C, numbers of lactose-positive Enterobacteriaceae decreased in most enrichments which resulted in a positive salmonella isolation, but remained constant in the majority of those that did not. Levels of lactose-negative Enterobacteriaceae did not decrease in most salmonella-positive tests, but did so in half of the salmonella-negative ones. In the salmonella-positive tests the numbers of salmonellas had increased to 10³–10⁷/ml in BPw and after transfer to TBB slowly reached 10⁴/ml or more. In all cases the numbers of salmonellas exceeded those of the competing flora on brilliant green agar (BGA). In the salmonella-negative tests the numbers of salmonellas had increased less in BPw and decreased in most of the TBB enrichments. In none of these negative tests did the numbers of salmonellas exceed those of the competing flora on BGA. Escherichia coli dominated in most of the salmonella-negative tests. The results suggest more influence of lactose-positive than lactose-negative Enterobacteriaceae on the detection of salmonellas. The effect of competing microorganisms seems to depend not only upon their initial numbers, but also upon the types that can interact with salmonellas during selective enrichment.     

Increased recovery of salmonellae from environmental samples enriched with buffered peptone water.

The incidence and persistence of salmonellae in weather pools on the top of Stone Mountain were investigated with lactose and buffered peptone water used as pre-enrichment broths. A total of 162 samples were collected from 16 weather pools over a 3-month period. The use of buffered peptone water increased the recovery of salmonellae by approximately 25%. The combined use of direct enrichment in tetrathionate broth containing brilliant green dye and pre-enrichment in buffered peptone water followed by enrichment in tetrathionate broth made it possible to detect all 37 of the contaminated samples. All of the isolates were Salmonella bareilly, the only serotype recovered in a previous study. All but one of the isolations were made from moist or wet samples. S. bareilly was isolated from rabbit dung and litter collected near the weather pools, but attempts to trap rabbits for study were unsuccessful. Random samples taken along a side of the mountain yielded S. bareilly in weather pools within the upper third of the mountain; below this level, S. weslaco and S. memphis were recovered, but not S. bareilly.     

Increased recovery of salmonellae from environmental samples enriched with buffered peptone water.

The incidence and persistence of salmonellae in weather pools on the top of Stone Mountain were investigated with lactose and buffered peptone water used as pre-enrichment broths. A total of 162 samples were collected from 16 weather pools over a 3-month period. The use of buffered peptone water increased the recovery of salmonellae by approximately 25%. The combined use of direct enrichment in tetrathionate broth containing brilliant green dye and pre-enrichment in buffered peptone water followed by enrichment in tetrathionate broth made it possible to detect all 37 of the contaminated samples. All of the isolates were Salmonella bareilly, the only serotype recovered in a previous study. All but one of the isolations were made from moist or wet samples. S. bareilly was isolated from rabbit dung and litter collected near the weather pools, but attempts to trap rabbits for study were unsuccessful. Random samples taken along a side of the mountain yielded S. bareilly in weather pools within the upper third of the mountain; below this level, S. weslaco and S. memphis were recovered, but not S. bareilly.     

Comparison of Muller-Kauffmann tetrathionate broth with Rappaport-Vassiliadis (RV) medium for the isolation of salmonellas from sewage sludge

The Rappaport-Vassiliadis (RV, modification of Rappaport's) medium was superior to Muller-Kauffmann tetrathionate broth (MKT) for the enrichment and isolation of salmonellas from sewage sludge samples, either naturally contaminated or artificially inoculated with salmonella. Isolation rates for RV (42°C) were higher even when MKT (43°C) performance was improved by the use of brilliant green sulphamandelate agar. Kinetic results show that the suppression of the competing flora was poorer in MKT than in RV. The use of high inoculum to medium volume ratios (e.g. 1:5000) increased the isolation rates from RV and kinetic data explain why this was so.     

Comparison of Muller-Kauffmann tetrathionate broth with Rappaport-Vassiliadis (RV) medium for the isolation of salmonellas from sewage sludge

The Rappaport-Vassiliadis (RV, modification of Rappaport's) medium was superior to Muller-Kauffmann tetrathionate broth (MKT) for the enrichment and isolation of salmonellas from sewage sludge samples, either naturally contaminated or artificially inoculated with salmonella. Isolation rates for RV (42 degrees C) were higher even when MKT (43 degrees C) performance was improved by the use of brilliant green sul-phamandelate agar. Kinetic results show that the suppression of the competing flora was poorer in MKT than in RV. The use of high inoculum to medium volume ratios (e.g. 1:5000) increased the isolation rates from RV and kinetic data explain why this was so.     

Comparison of two commercial preparations of buffered peptone water for the recovery and growth of Salmonella bacteria from foods

This study compared the performance of two commercial preparations of buffered peptone water. Performance was assessed in terms of ability to resuscitate and recover low numbers of stressed cells, buffering capacity, growth of Salmonella bacteria in pure culture and growth of Salmonella in food pre-enrichments. Although both the preparations of BPW had similar chemical compositions, differences in their recovery performance were found. Brand A recovered significantly higher numbers of heat-injured Salmonella (mean = 0.57 log10 cfu ml(-1) difference) in pure culture compared with brand B when dealing with very low inoculum levels. Although brand B had higher buffering capacity, the pH at the end of the pre-enrichment was found to be similar in both media, even in foods such as milk powder which showed the greatest decline in pH. Both brands were comparable in their ability to grow unstressed Salmonella from different food types. In unstressed cell studies, similar cell numbers were recovered at the end of a 24 h incubation period from both media, although brand B yielded a higher biomass. In the food study with unstressed cells, performance was related more to the food type and the likely association between this and the level and type of competitor organisms present, rather than to the brand of medium used.     

Efficacy of supplemented buffered peptone water for the isolation of Campylobacter jejuni and C. coli from broiler retail products

Broiler retail samples (n=113) were analyzed to determine (i) the effectiveness of buffered peptone water (BPW) supplemented with blood and antibiotics for the isolation of Campylobacter jejuni and C. coli, (ii) if a 1:4 enrichment ratio performs similarly as a 1:9 ratio, and (iii) if BPW is similar to Bolton broth for enumeration of Campylobacter spp. in retail broiler meat using the most probably number (MPN) procedure. Chi-square comparison showed that BPW performed similarly as Bolton broth (P< or =0.05) for Campylobacter isolation in breast tenders, boneless breasts, split breasts and skin samples. However, BPW showed a lower detection rate (P> or =0.05) for thighs and boneless thighs. When the results were combined, BPW performed similarly as Bolton broth for the isolation of Campylobacter spp. (P< or =0.05). BPW at an enrichment ratio of 1:4 was statistically similar to Bolton broth or BPW at a ratio of 1:9. No differences were observed between the MPN data from Bolton broth and the MPN data from BPW (P< or =0.50). A multiplex PCR assay revealed that ca. 48% of the isolates obtained from Bolton broth and 59% of the isolates obtained with BPW were C. coli. Both Bolton broth and BPW allowed for the growth of C. jejuni and C. coli from the same sample. Remarkably, a large genomic variability was observed by PFGE analysis of the isolates collected from the same sample with Bolton broth or BPW, which confirms that more than one genotype can successfully multiply during enrichment and be recoverable on agar plates. These findings suggest that BPW could be used as an enrichment medium for isolation of Campylobacter from retail broiler samples. The implications of the high number of C. coli isolates found in this study is discussed.     

A comparison of isolation procedures for salmonellas from polluted water using two forms of Rappaport's medium

The efficiency of Rappaport's broth ( RB10 ) and Rappaport's broth containing novobiocin ( NRB10 ) were compared for the isolation of salmonellas from polluted water, both as direct enrichment media and after pre-enrichment in buffered peptone water. Ninety samples were examined and 41 were found to contain salmonellas by at least one of the procedures used. Direct inoculation of the sample into RB10 resulted in the recovery of salmonellas from only 29.3% of the samples found to be positive. The use of NRB10 as a direct enrichment medium increased the percentage recovery to 78.0% of the total positive samples. Pre-enrichment in buffered peptone water allowed the recovery of salmonellas from a total of 41 samples whereas direct enrichment recovered them from only 32. No significant difference was demonstrated in the efficiencies of RB10 and NRB10 in recovering salmonellas after pre-enrichment in buffered peptone water. Three selective agars were used: no significant difference in their ability to recover salmonellae was demonstrated.     

A comparison of isolation procedures for salmonellas from polluted water using two forms of Rappaport's medium

F ricker , C.R. 1984. A comparison of isolation procedures for salmonellas from polluted water using two forms of Rappaport's medium. Journal of Applied Bacteriology 56 , 305–309.
The efficiency of Rappaport's broth (RB10) and Rappaport's broth containing novobiocin (NRB10) were compared for the isolation of salmonellas from polluted water, both as direct enrichment media and after pre-enrichment in buffered peptone water. Ninety samples were examined and 41 were found to contain salmonellas by at least one of the procedures used. Direct inoculation of the sample into RB10 resulted in the recovery of salmonellas from only 29.3% of the samples found to be positive. The use of NRB10 as a direct enrichment medium increased the percentage recovery to 78.0% of the total positive samples. Pre-enrichment in buffered peptone water allowed the recovery of salmonellas from a total of 41 samples whereas direct enrichment recovered them from only 32. No significant difference was demonstrated in the efficiencies of RB10 and NRB10 in recovering salmonellas after pre-enrichment in buffered peptone water. Three selective agars were used; no significant difference in their ability to recover salmonellae was demonstrated.     

Evaluation of an enzyme immunoassay technique for the detection of salmonellas in minced meat

A comparison was made between a commercially available enzyme immunoassay (ELISA) and various cultural procedures for detecting salmonellas in minced meat contaminated with a standard inoculum. To detect salmonellas by the ELISA technique, it was necessary to modify the recommended baseline for spectrophotometric measurements to avoid false-positive results. The incidence of false-negatives was no greater than that obtained with a standard isolation procedure. Both methods were affected by the competing microflora.     

A note on the performance of Rappaport's medium, compared with Rappaport-Vassiliadis broth, in the isolation of salmonellas from meat products, after pre-enrichment

Salmonellas were isolated from meat products using a slightly modified Rappaport's enrichment medium (R25), Rappaport-Vassiliadis procedure (Rappaport's broth containing 10 ml instead of 30 ml of Malachite Green solution and incubated at 43^oC instead of 37^oC), and Muller-Kauffmann's tetrathionate broth. From 255 samples, 89 were found positive with the Rappaport-Vassiliadis procedure, 83 with the R25 broth, whereas only 43 were positive with Muller-Kauffmann broth. It is concluded that the R25 medium may be used as an alternative to the more effective Rappaport-Vassiliadis broth when the only available incubation temperature is 37^oC.     

Acid-base and respiratory properties of a buffered bovine erythrocyte perfusion medium

Current research in organ physiology often utilizes in situ or isolated perfused tissues. We have characterized a perfusion medium associated with excellent performance characteristics in perfused mammalian skeletal muscle. The perfusion medium consisting of Krebs-Henseleit buffer, bovine serum albumin, and fresh bovine erythrocytes was studied with respect to its gas-carrying relationships and its response to manipulation of acid-base state. Equilibration of the perfusion medium at base excess of -10, -5, 0, 5, and 10 mmol X L-1 to humidified gas mixtures varying in their CO2 and O2 content was followed by measurements of perfusate hematocrit, hemoglobin concentration, pH, Pco2, Cco2, Po2, and percent oxygen saturation. The oxygen dissociation curve was similar to that of mammalian bloods, having a P50 of 32 Torr (1 Torr = 133.3 Pa), Hill's constant n of 2.87 +/- 0.15, and a Bohr factor of -0.47, showing the typical Bohr shifts with respect to CO2 and pH. The oxygen capacity was calculated to be 190 mL X L-1 blood. The carbon dioxide dissociation curve was also similar to that of mammalian blood. The in vitro nonbicarbonate buffer capacity (delta [HCO3-] X delta pH-1) at zero base excess was -24.6 and -29.9 mmol X L-1 X pH-1 for the perfusate and buffer, respectively. The effects of reduced oxygen saturation on base excess and pH of the medium were quantified. The data were used to construct an acid-base alignment diagram for the medium, which may be used to quantify the flux of nonvolatile acid or base added to the venous effluent during tissue perfusions.     

Fate of food-borne pathogens on modified-atmosphere packaged meat and fish

The fate of several food-borne pathogens on modified-atmosphere-packaged (MAP) meat and fish has been examined. In no instance was growth of the pathogen in MAP greater than that in the appropriate control.     

Fate of Listeria monocytogenes in processed meat products during refrigerated storage

The fate of Listeria monocytogenes during refrigerated storage was determined on several processed meat products, including ham, bologna, wieners, sliced chicken, sliced turkey, fermented semidried sausage, bratwurst, and cooked roast beef. The meats were surface inoculated with a five-strain mixture of less than or equal to 200 or ca. 10(5) L. monocytogenes cells per package, vacuum packaged, and stored at 4.4 degrees C. Survival or growth of listeriae was determined for up to 12 weeks of storage or until the product was spoiled. The organism survived but did not grow on summer sausage, grew only slightly on cooked roast beef, grew well on some wiener products but not on others, grew well (10(3) to 10(5) CFU/g increase within 4 weeks) on ham, bologna, and bratwurst, and grew exceptionally well (10(3) to 10(5) CFU/g increase within 4 weeks) on sliced chicken and turkey. The rate of growth depended largely upon the type of product and the pH of the product. Growth was most prolific on processed poultry products. The organism generally grew well on meats near or above pH 6 and poorly or not at all on products near or below pH 5. These results indicate the importance of preventing postprocessing contamination of L. monocytogenes in a variety of ready-to-eat meat products.     

Fate of salmonellas in the alimentary tract of chicks pre-treated with a mature caecal microflora to increase colonization resistance

Oral challenge with a strain of Salmonella kedougou , resistant to nalidixic acid, gave a time-course for salmonella survival in the the alimentary tract of chicks pre-treated with a caecal culture from an eight-week-old bird to increase colonization resistance. In untreated, control chicks, salmonella colonization of the caeca resulted in counts of > 10⁶/g within 48 h and a mean generation time of 1·6 h. With treated birds, however, the salmonellas failed to multiply in the caeca and decreased to a low level over a 48-h period, thus suggesting a mainly bacteriostatic effect. Pretreatment of chicks with the caecal culture also reduced the proportion of salmonella-positive crop samples. Growth of the salmonella in a feed-slurry system resembling moistened crop contents occurred rapidly at 37°C but was prevented by addition of the treatment culture, a decline in numbers coinciding with Lactobacillus populations of ca 10⁹/g and a pH value of 5·5. Incorporation of a feed decontaminant, 1% formic acid (pH 4·0), rapidly eliminated both salmonellas and organisms added from the treatment culture.     

Fate of salmonellas in the alimentary tract of chicks pre-treated with a mature caecal microflora to increase colonization resistance

Oral challenge with a strain of Salmonella kedougou, resistant to nalidixic acid, gave a time-course for salmonella survival in the alimentary tract of chicks pre-treated with a caecal culture from an eight-week-old bird to increase colonization resistance. In untreated, control chicks, salmonella colonization of the caeca resulted in counts of greater than 10(6)/g within 48 h and a mean generation time of 1.6 h. With treated birds, however, the salmonellas failed to multiply in the caeca and decreased to a low level over a 48-h period, thus suggesting a mainly bacteriostatic effect. Pre-treatment of chicks with the caecal culture also reduced the proportion of salmonella-positive crop samples. Growth of the salmonella in a feed-slurry system resembling moistened crop contents occurred rapidly at 37 degrees C but was prevented by addition of the treatment culture, a decline in numbers coinciding with Lactobacillus populations of ca 10(9)/g and a pH value of 5.5. Incorporation of a feed decontaminant, 1% formic acid (pH 4.0), rapidly eliminated both salmonellas and organisms added from the treatment culture.     

Use of a commercially available ELISA kit for detection of salmonellas in water

The accuracy and specificity of a commercially available ELISA kit (Locate, Rhone-Poulenc Diagnostics) were assessed when applied to enrichment cultures of naturally contaminated water and sewage. The kit showed 66/180 samples positive by both culture and ELISA. The ELISA showed six positives which could not be confirmed by culture.