Development of a 24 h screen to detect viable salmonellas in faeces

A 24 h screen to detect viable salmonellas in faeces was developed by studying growth dynamics of salmonellas and competing flora in combinations of enrichment media and artificially-inoculated pig faeces. Muller-Kauffmann tetrathionate (MK) broth, incubated overnight at 42°C, maintained the lowest ratio of salmonella: competing flora and identified all inoculated samples. A 4 h postenrichment in M broth plus novobiocin reduced the number of false-positive results in subsequent ELISAs. Adjusting the negative cut-off values and incubation time of the chromogenic substrate from that recommended in the ELISA instructions reduced the rate of false-positive results further and allowed the detection of 10³ salmonellas per ml in the presence of up to 10⁷ ml⁻¹ aerobic-competing cells. Suspension of faeces diluted 1 in 2 and 1 in 5, rather than 1 in 10 in MK broth did not necessitate further adjustments to the ELISA baseline values. The proposed screen protocol is an overnight incubation of faeces suspended 1 in 10 in MK broth, a 1 in 100 subculture into M broth plus 10 μg ml⁻¹ novobiocin (MbN) for 4 h, steam inactivation of MbN cultures and testing by ELISA, and can detect three salmonella cells per g faeces.     

Detection of salmonellas in confectionery products by conductance

A modified lysine decarboxylase broth has been developed which could be used with a Bactometer M123 to differentiate salmonellas from other bacteria by the characteristics of the conductance detection curve. The medium was used in combination with a selenite cystine trimethylamine oxide dulcitol medium to screen 50 strains of salmonellas and 42 strains of other organisms to establish detection curve magnitude and rate values which could be used to identify curves specific to salmonellas. The combination of media detected all salmonellas tested except Salmonella pullorum . The two media were used to screen 100 inoculated product samples with the Bactometer instrument, in parallel with traditional plating procedures, and using various combinations of pre-enrichment and selective enrichment incubation periods. After 24 h pre-enrichment, the Bactometer system detected more positive samples than the conventional plating procedures after pre-enrichment and selective enrichment. It is considered that these media used in parallel in the Bactometer after conventional pre-enrichment could provide a 48 h screening procedure for salmonellas with a sensitivity comparable to present plating procedures.     

Detection of salmonellas in confectionery products by conductance

A modified lysine decarboxylase broth has been developed which could be used with a Bactometer M123 to differentiate salmonellas from other bacteria by the characteristics of the conductance detection curve. The medium was used in combination with a selenite cystine trimethylamine oxide dulcitol medium to screen 50 strains of salmonellas and 42 strains of other organisms to establish detection curve magnitude and rate values which could be used to identify curves specific to salmonellas. The combination of media detected all salmonellas tested except Salmonella pullorum. The two media were used to screen 100 inoculated product samples with the Bactometer instrument, in parallel with traditional plating procedures, and using various combinations of pre-enrichment and selective enrichment incubation periods. After 24 h pre-enrichment, the Bactometer system detected more positive samples than the conventional plating procedures after pre-enrichment and selective enrichment. It is considered that these media used in parallel in the Bactometer after conventional pre-enrichment could provide a 48 h screening procedure for salmonellas with a sensitivity comparable to present plating procedures.     

Evaluation of enhanced haemolysis agar for detection of Listeria spp. and L. monocytogenes from production lines of fresh to cold-smoked fish.

Enhanced haemolysis agar (EHA) was compared to the two conventional Listeria isolation agars Oxford and PALCAM for its ability to detect Listeria spp. from production lines of fresh to cold-smoked fish. The ability of EHA for distinguishing L. monocytogenes colonies from other Listeria spp. was also evaluated.A total of 243 fish and environmental samples were analysed. Overall, 42 samples were found to contain Listeria spp. Only 34 samples were positive simultaneously by the three plating media. Two samples considered to be negative by the two conventional agars were found to be positive after isolation on EHA. All three selective agars were shown to be less effective in recovering Listeria spp. after primary enrichment in half-Fraser broth, compared to secondary enrichment in Fraser broth after 24 and 48 h.From 79 Listeria but presumptive negative L. monocytogenes colonies, EHA identified correctly 76 Listeria spp. and presented three false-negative results_three colonies further identified as L. monocytogenes but showing no noticeable haemolysis on EHA. Twenty-three of the thirty-three L. monocytogenes presumptive positive colonies, were confirmed positive and ten were identified as L. seeligeri.Despite its ability of distinguishing L. monocytogenes from the other Listeria spp., unless it is produced as a commercial medium, EHA cannot be an alternative to time-consuming classical identification because the preparation of this medium is both time and labour intensive.     

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.     

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.     

Sandwich capture ELISA by a murine monoclonal antibody against a genus-specific LPS epitope for the detection of different common serotypes of salmonellas.

A sandwich capture ELISA based on a murine monoclonal antibody against a genus-specific epitope in the outer core region of the Salmonella lipopolysaccharide is described for the detection of different common serotypes of salmonellas. Four h broth cultures of seven standard and 24 wild strains of salmonellas were all detected by the capture ELISA while overnight broth cultures of 21 non-salmonella standard strains were all negative. The capture ELISA detected 1 ng/ml of Ra lipopolysaccharide, 10(6)/ml of a smooth wild strain of Salm. typhimurium, and 1120 cells of Salm. heidelberg after enrichment culture for 4 h.     

Dynamics of salmonella isolation with modified Rappaport's medium (R10)

Enhanced growth of salmonellas in Rappaport's medium as modified by Vassiliadis et al. (1976) after pre-enrichment in buffered peptone water during the first 6 h was obtained by replacement of tryptone by soya peptone. The competing bacteria, i.e. those which grow on brilliant green agar and which may interfere with the isolation of salmonellas when Rappaport's medium (R10) incubated at 43°C is used for enrichment were inhibited or reduced in numbers when the normal amount of 5 g soya peptone/litre was used. When the amount was increased to 10 g/l, growth occurred, mainly of Enterobacter and Klebsiella species. The isolation of salmonellas was found to be largely dependent on the number and the ratio of their competitors. Every measure taken to reduce the number of competitive bacteria increases the possibility of isolating salmonellas. This explains the effect of improved selectivity of Rappaport's medium when small inocula are used. Rapid onset of growth of salmonellas by employing soya peptone introduces the possibility of using shorter incubation times than 48 h.     

Laboratory studies on salmonella-contaminated cheese involved in a major outbreak of gastroenteritis

A major outbreak of gastroenteritis was traced to Cheddar cheese contaminated with Salmonella typhimurium. There were no significant differences in pH values of the contaminated (mean pH 5.31) and non-contaminated (mean pH 5.39) cheese. The isolation rates of Salm. typhimurium were about the same when cheese samples were homogenized in lactose broth, lactose broth containing 1% Tween 80, or in aqueous 2% sodium citrate. Salmonella typhimurium was isolated regardless of preenrichment in lactose broth, but required selective enrichment in selenite cystine or tetrathionate brilliant green broth. There were no marked differences in the isolation rates obtained with different selective enrichment media, or after incubation at 36 degrees and 43 degrees C for 24 or 48 h. Contaminated samples of cheese failed to yield Salm. typhimurium consistently despite large and multiple samplings; samples from the interior of cheese blocks yielded positive results more frequently than the samples from the exterior. The number of Salm. typhimurium in factory sealed blocks as well as in samples obtained from the homes of known cases of salmonellosis was found to range from less than 3/100 g to 9/100 g of cheese. The infective dose of Salm. typhimurium in contaminated cheese was probably no greater than 10(4) organisms, and a rapid decline in numbers of Salm. typhimurium must have occurred subsequent to the outbreak.     

Enrichment in Muller-Kauffmann's Broth and Rappaport's Broth from Buffered Peptone Water in the Isolation of Salmonellae from Minced Meat

Four hundred and ninetysix samples of minced meat were examined for the presence of salmonellae. The methods of isolation used in the study were: (a) the standardized direct technique of enrichment in Muller-Kauffmann's broth incubated at 43· for 24 and 48 h, described by Edel & Kampelmacher (1969), (b) secondary enrichment in Rappaport's broth from the former medium after 24 h incubation, and (c) pre-enrichments in buffered peptone water incubated at 37·, followed by enrichments in Muller-Kauffmann's and in Rappaport's broth. These last procedures resulted in the higher rates of isolation of salmonellae with a slight, but not statistically significant, superiority of the enrichment in Rappaport's medium.     

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 10(8)/ml or more. During incubation in TBB at 43 degrees C, 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 10(3)-10(7)/ml in BPw and after transfer to TBB slowly reached 10(4)/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.     

Sandwich capture ELISA by a murine monoclonal antibody against a genus-specific LPS epitope for the detection of different common serotypes of salmonellas

D.CHOI, R.S.W. TSANG AND M.H. NG. 1992. A sandwich capture ELISA based on a murine monoclonal antibody against a genus-specific epitope in the outer core region of the Salmonella lipopolysaccharide is described for the detection of different common serotypes of salmonellas. Four h broth cultures of seven standard and 24 wild strains of salmonellas were all detected by the capture ELISA while overnight broth cultures of 21 non-salmonella standard strains were all negative. The capture ELISA detected 1 ng/ml of Ra lipopolysaccharide, 10⁶/ml of a smooth wild strain of Salm. typhimurium , and 1120 cells of Salm. heidelberg after enrichment culture for 4 h.     

Evaluation of Culture Media for the Isolation of Salmonellas from Sewage Sludge

Ten samples of sewage sludge were examined by various methods for the isolation of salmonellas using three types of enrichment broth: Muller-Kauffmann Tetrathionate Broth (MKTB), Selenite F Broth (SFB), and Brilliant Green Broth (BGB), two temperatures (37°C and 43°C) and three selective media: Deoxycholate Citrate Agar incubated aerobically (DCA), and anaerobically (DCA(N)), Brilliant Green Agar (BGA), and Bismuth Sulphite Agar (BSA). The results suggest that a combination of pre-enriched MKTB incubated at 37°C and plated on to BGA at 24 and 48 h was the best method, but when examining contaminated material such as sewage sludge, it appears unwise to rely on one single method.     

A real-time PCR assay for the detection of Campylobacter jejuni in foods after enrichment culture

A real-time PCR assay was developed for the quantitative detection of Campylobacter jejuni in foods after enrichment culture. The specificity of the assay for C. jejuni was demonstrated with a diverse range of Campylobacter species, related organisms, and unrelated genera. The assay had a linear range of quantification over six orders of magnitude, and the limit of detection was approximately 12 genome equivalents. The assay was used to detect C. jejuni in both naturally and artificially contaminated food samples. Ninety-seven foods, including raw poultry meat, offal, raw shellfish, and milk samples, were enriched in blood-free Campylobacter enrichment broth at 37 degrees C for 24 h, followed by 42 degrees C for 24 h. Enrichment cultures were subcultured to Campylobacter charcoal-cefoperazone-deoxycholate blood-free selective agar, and presumptive Campylobacter isolates were identified with phenotypic methods. DNA was extracted from enrichment cultures with a rapid lysis method and used as the template in the real-time PCR assay. A total of 66 samples were positive for C. jejuni by either method, with 57 samples positive for C. jejuni by subculture to selective agar medium and 63 samples positive in the real-time PCR assay. The results of both methods were concordant for 84 of the samples. The total time taken for detection from enrichment broth samples was approximately 3 h for the real-time PCR assay, with the results being available immediately at the end of PCR cycling, compared to 48 h for subculture to selective agar. This assay significantly reduces the total time taken for the detection of C. jejuni in foods and is an important model for other food-borne pathogens.     

APPLICATION OF POLYMERASE CHAIN REACTION-OLIGONUCLEOTIDE LIGATION ASSAY FOR THE DETECTION OF SALMONELLAE IN PROCESSED MEAT, POULTRY, FISH, AND PET FOODS

We have tested a rapid and sensitive DNA-based assay for the detection of Salmonella serovars in a number of different processed meat, fish, poultry, and pet food samples. This technique uses an enrichment broth cultivation followed by a Salmonella-specific polymerase chain reaction (PCR) and oligonucleotide ligation assay (OLA) to specifically detect amplified PCR products in an ELISA-based microtiter plate format. The combined cultivation and PCR-OLA techniques were compared with a conventional culture method and with DNA hybridizations of PCR products for the detection of Salmonella bacteria. Eighty-one different processed meat, poultry, and pet food samples were screened for the presence of Salmonella serovars after 24 h and 48 h of enrichment broth cultivation. After 24 h of incubation, one ground turkey sample was positive by both culture and PCR-OLA (100% sensitivity and 100% specificity). After 48 h of incubation, two additional samples (ground beef and a dog food sample) were positive by both culture and PCR-OLA (100% sensitivity and 100% specificity), and three other samples (two ground beef samples and one ground turkey) were positive only by PCR-OLA (96.1% specificity). All positive PCR-OLA results were confirmed in DNA hybridizations with an oligonucleotide specific for the amplified PCR product. When compared to conventional culture, the combined 48 h enrichment and PCR-OLA had a positive predictive value of 50% and a negative predictive value of 100%. We concluded that a combined cultivation and PCR-OLA could be used as a sensitive and specific presumptive screening method for detecting Salmonella serovars in processed meat, fish, poultry, and pet foods.     

Dynamics of salmonella isolation with modified Rappaport's medium (R10)

Enhanced growth of salmonellas in Rappaport's medium as modified by Vassiliadis et al. (1976) after pre-enrichment in buffered peptone water during the first 6 h was obtained by replacement of tryptone by soya peptone. The competing bacteria, i.e. those which grow on brilliant green agar and which may interfere with the isolation of salmonellas when Rappaport's medium (R10) incubated at 43 degrees C is used for enrichment were inhibited or reduced in numbers when the normal amount of 5 g soya peptone/litre was used. When the amount was increased to 10 g/l, growth occurred, mainly of Enterobacter and Klebsiella species. The isolation of salmonellas was found to be largely dependent on the number and the ratio of their competitors. Every measure taken to reduce the number of competitive bacteria increases the possibility of isolating salmonellas. This explains the effect of improved selectivity of Rappaport's medium when small inocula are used. Rapid onset of growth of salmonellas by employing soya peptone introduces the possibility of using shorter incubation times 48 h.     

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.     

AN ELECTRICAL TESTING METHOD FOR THE DETECTION OF LISTERIA SPECIES IN CONFECTIONERY PRODUCTS

An electrical testing method for the detection of Listeria spp. in confectionery products and associated raw materials was developed in which samples can be negatively screened within 48h. The method involves a 24h resuscitation in Listeria enrichment broth followed by a 24h test in a Bactometer M128 using a broth (Claremont broth) developed from Oxford agar. The comparative study involved analysis of 511 samples (chocolate, dairy, cereal, nut and fruit products) tested by the Interim Australian Standard method (AS) and the Bactometer method (BM). The sensitivity and specificity of the BM for samples §1 Listeria/g was 100% and 99.8%, respectively, when compared to the AS method. When samples containing < 1 Listeria/g sample were added to the analysis, the sensitivity and specificity marginally dropped (98.3% and 99.6%). The electrical capacitance method is rapid and easy to perform, with a negative result being available within 48h making it a viable tool in a positive release QA program in food manufacturing factories.     

A Comparison of the Fluorescent Antibody Method and a Standardized Cultural Method for the Detection of Salmonellas

The results of routine use of the indirect fluorescent antibody (FA) technique using the Spicer-Edwards H antisera set are reported for a range of agricultural and food samples. The FA technique was used on samples after the pre-enrichment incubation period in the proposed ISO method for isolation of salmonellas. The numbers of FA false positive samples ( ca. 5% overall) and FA false negative samples ( ca. 1·3%) were low, but some originally FA false positive results were later shown to be false negative cultural results.     

Automated detection of Salmonella spp. in foods.

An automated method to detect salmonellae in foods was developed and tested in food samples intentionally contaminated with the test organisms. Liquid eggs, shell eggs, dry eggs, skim milk and chicken were spiked with Salmonella enteritidis, S. typhimurium or S. newport to yield 2 to 25 CFU per 25 g or ml of sample. Following pre-enrichment in universal pre-enrichment broth at 42 degrees C for 6 h (eggs and milk) or 16 h (chicken), Salmonella cells were captured by immunomagnetic beads coated with Salmonella antibody (Vicam, Watertown, MA). The beads were transferred to selective liquid media containing carbohydrate (dulcitol or xylose), amino acid (lysine or ornithine), and H2S indicator, and incubated at 42 degrees C in the BioSys instrument (MicroSys, Ann Arbor, MI). Salmonella positive samples were identified by black discoloration of the media during incubation, while negative samples remained colorless. These color changes were recorded by the instrument. All the artificially contaminated samples tested positive within 15-18 h, while control samples remained negative during 24 h incubation. The results agreed with standard identification procedures. A total of 24 h was required to detect 2 to 25 CFU of the pathogen in 25 g or ml of eggs and milk, and up to 36 h in chicken, compared to 72 h in the standard methods.