Muller-Kauffmann Tetrathionate Broth Research Articles

Letter to the Editor

To Dr. Donnelly, Scientific Editor, Food Microbiology and Safety Section, Consumer demand for healthy, additive-free foods has led to increased consumption of raw salad vegetables in developed countries. Availability of prepared bagged salads has increased, and so have the number of studies on the microbiological quality of minimally processed vegetables ready for consumption. The initial microbial load of vegetables, especially leafy ones, plays a crucial role in the microbiological content of pre-packed salads, yet few studies have been published to this day on microorganisms of fecal origin isolated from raw/untreated vegetables. In 1980, an article, authored by the late Professor J.A. Papadakis of the Athens School of Hygiene and myself (National Salmonella-Shigella Reference Centre of Greece), was published in ‘Hippocrates’ scientific journal of the Medical School of the Univ. of Athens Greece, discussing isolation techniques for the qualitative detection of Salmonella from fresh vegetables (Papadakis and Efstratiou 1980). In Greece the practice of consuming with every meal a side plate of salad made of uncooked fresh vegetables is a widely applied practice. Irrigation with wastewater might result in vegetable contamination with fecal microorganisms. Indeed it had been published (Papavassiliou and others 1967) that vegetables from the central market of Athens were found contaminated with coli-aerogenes bacteria (86.8%) and E. coli (22.3%). In the article by Papadakis and Efstratiou, 538 samples of 7 vegetables (lettuce, tomatoes, green peppers, parsley, spinach, celery, chicory) were examined for the presence of Salmonella spp. The vegetables were locally produced and were bought from green-grocery stores in the Athens area. 300gr of each vegetable was homogenized in 300ml buffered peptone water in a stomacher and incubated at 37 °C for 16 to 20 h. Tomatoes were ‘washed’ in buffered peptone water for 3 to 5 min. Following this pre-enrichment step, enrichment was carried out by inoculation in 3 broths: Muller-Kauffmann tetrathionate broth (1 ml of inoculum in 10 ml broth), Mannitol Selenite F broth (1 ml of inoculum in 10 ml broth), and Rappaport broth modified by Vassiliadis and others (1978) (0.1 ml of inoculum in 10 ml broth). All enrichment media were incubated for 24 h and 48 h. They were streaked out on SS (Salmonella Shigella) agar (Difco) and on brilliant green agar (Oxoid 329 with the addition of 0.25% sodium desoxycholate). The plates were incubated at 37 °C for 24 h, and 4 suspect colonies from each plate, if present, were inoculated into Kligler Iron agar slants. Tubes exhibiting the characteristics of Salmonella were further examined by standard biochemical and serological methods at the National Salmonella-Shigella Reference Centre of Greece. Salmonellae were isolated from six (1.1%) of the examined samples. Only lettuce (2.0%) and parsley (8.6%) were positive. All 3 positive lettuce samples yielded Salmonella Montevideo, although they were purchased 1 wk apart each other, but from the same retailer. The 3 positive parsley samples contained Salmonella Newport, Abony, and Orion. All 6 Salmonella strains were isolated from the modified Rappaport enrichment broth. One strain was isolated from Muller-Kauffmann tetrathionate broth; no Salmonella was isolated from Mannitol Selenite F broth. The conclusion was reached that enrichment in Rappaport medium as modified by Vassiliadis was more appropriate for the isolation of salmonellae from fresh vegetables. Superiority of Rappaport-Vassiliadis enrichment broth in detecting salmonellae in food products and polluted water led to its commercial preparation as dehydrated medium ‘Rappaport-Vassiliadis (RV) enrichment broth’ (Oxoid CM669 and other manufacturers). Should any of your readers be interested in of the original article, which is in English, I would be happy to post a copy if contacted at efstratiou@aegean.gr Sincerely,

Comparison of DNA-extraction methods and Selective Enrichment broths on the detection of Salmonella Typhimurium in swine feces by polymerase chain reaction (PCR)

The aim of this study was to compare different DNA-extraction methods and selective enrichment broths for their effectiveness to detect Salmonella Typhimurium in artificially inoculated swine feces samples (100 CFU/g) by polymerase chain reaction. After enrichment in Rappaport-Vassiliadis, selenite cystine or Muller-Kauffmann tetrathionate, aliquots were used for DNA extraction by three different methods: boiling-centrifugation, phenol-chloroform and salting-out. Aliquots of extracted DNA were then used as template in PCR. The selective enrichment broths had no effect on the efficiency of PCR when boiling-centrifugation and salting-out were used. On the other hand, phenol-chloroform was superior (P<0.05) when combined to Rappaport-Vassiliadis. Considering cost and efficiency parameters, we encourage the use of Muller-Kauffmann tetrathionate broth in combination with boiling-centrifugation DNA-extraction procedure.

Comparison of Four Different Methods for Salmonella Detection in Fecal Samples of Porcine Origin

Performances of four detection methods were evaluated for recovery of Salmonella spp. in naturally contaminated fecal specimens of porcine origin. The NMKL 71 method consisted of enrichment in Rappaport-Vassiliadis broth and plating on xylose-lysine-desoxycholate medium, whereas the SP-VG-M002 method relied on a Diasalm enrichment followed by streaking on xylose-lysine-tergitol 4 agar (XLT-4). The VIDAS SLM method was composed of double enrichment in Muller-Kauffmann tetrathionate broth and in M broths before processing in a VIDAS device. If the results were positive, the VIDAS ICS immunoenrichment was performed and the result transferred onto three different selective media. The VIDAS ICS protocol is an immunoconcentration step followed by plating on XLT-4. Seventy-eight samples were tested with all four methods simultaneously, leading to 34 positive samples with at least one method. For this assay, VIDAS SLM revealed 31 positive samples (91.2%), whereas the average positive percentage of the three other methods was 37.3% (P < 0.001). Two-paired comparisons with the VIDAS SLM method were also performed. McNemar values were systematically highly significant (P < 0.001). The proportion of agreement was significantly inferior (P < 0.05) for the comparison of VIDAS ICS and VIDAS SLM (68.7%) compared with the two other paired comparisons (average percentage, 81.5%). The conclusion reached by this trial is that VIDAS SLM significantly improves the recovery of Salmonella in naturally contaminated fecal specimens. For the paired-comparisons, NMKL 71 and SP-VG-M002 were comparable in terms of efficiency, whereas the VIDAS ICS protocol, as established by the manufacturer for food samples only, seemed less efficient than the other two.

Evaluation of a 24-Hour Bioluminescent Enzyme Immunoassay for the Rapid Detection of Salmonella in Chicken Carcass Rinses

A bioluminescent enzyme immunoassay (BEIA), using Salmonella-specific monoclonal antibody M183 for capture and biotinylated monoclonal antibody M183 for detection, was developed with InteLite AB streptavidin-biotinylated firefly luciferase complex as a reporter. Salmonella cultures were preenriched in buffered peptone water with shaking for 6 h at 37°C and then selectively enriched in Muller-Kauffmann tetrathionate (MKTT) broth and modified semisolid Rappaport-Vassiliadis (MSRV) medium for 16 h at 42°C. After enrichment, the total test time for the BEIA was 1.5 h. The analytical sensitivity of the BEIA ranged from 6.0 × 102 CFU/ml to 1.2 × 105 CFU/ml in MKTT and from 1.4 × 105 to 2.3 × 106 CFU/ml in MSRV using six Salmonella serovars prevalent in Canada. With enrichment cultures, the BEIA detected 1 CFU of Salmonella Typhimurium and Salmonella Enteritidis in 25 ml of chicken rinses. Representative strains of 10 Salmonella serovars were detected, and cross-reactivity was not observed with 25 non-Salmonella foodborne bacteria. The BEIA performance was assessed by testing 420 poultry samples, which were analyzed in parallel with the standard MSRV culture method. The BEIA detected 117 (27.88%) Salmonella-positive samples, whereas the standard MSRV culture method detected 124 (29.5%). The BEIA had a sensitivity of 64.5% and a specificity of 87.5% compared to the standard MSRV culture method. However, similar specificities and sensitivities were obtained when the standard MSRV culture method was compared to the BEIA (sensitivity = 68.4% and specificity = 85.5%). Neither method detected 100% of the Salmonella found in the samples tested, and statistical analyses indicated no significant difference between the two methods. In summary, the BEIA offers another alternative for the detection of Salmonella, with the additional advantage of providing a 24-h test for detecting Salmonella in chicken carcass rinses. The results obtained in this research indicate that tests are still needed for the isolation and detection of Salmonella that will establish the true prevalence of Salmonella in chicken samples.

Comparison of classical isolation protocols with a 24 h screen to detect viable salmonellas in faeces

A 24 h screen which detects three viable salmonella cells per g of faeces was compared with classical isolation procedures for their ability to identify salmonella-positive samples from a pig rearing unit. The screen involved an overnight enrichment in Muller-Kauffmann tetrathionate (MK) broth, subculture for 4 h in M broth containing 10 micrograms ml-1 novobiocin, followed by detection of the presence of salmonellas by BacTrace and Salmonella-tek ELISAs. The classical protocols were: (1) an overnight and 48 h incubation in MK or selenite cysteine broth; or (2) overnight incubation in buffered peptone water and 24 h subculture in Rappaport-Vassiliadis broth (BPW-RV). Salmonellas were isolated from the broth cultures on xylose lysine deoxycholate and brilliant green agars. Thirty four of 100 samples were positive for salmonellas but no single isolation protocol identified all of them. The best of the classical isolation protocols, 48 h incubation in MK broth, identified 27 (79%) of the 34 positive samples whilst the screen identified 26 (76%) of the 34 positive samples. False-positive results were obtained from all isolation protocols except BPW-RV.

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 degrees 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 10(3) salmonellas per ml in the presence of up to 10(7) 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 micrograms 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.

A comparison of standard cultural methods for the detection of foodborne Salmonella

The sensitivity of the standard cultural method of the International Organization for Standardization (ISO 6579 and ISO 3565 combined) was compared to that of the Health Protection Branch (HPB) procedure for the detection of foodborne Salmonella. Of 195 foods tested, 84 (43.1%) were found to contain salmonellae by one or more cultural conditions. Of these, 75 (89.3%) and 68 (81.0%) were identified by the ISO and HPB methods, respectively. The apparent lack of agreement between both methods likely stemmed from the low indigenous numbers of salmonellae in several food homogenates, and unequal transfer of the target microorganism into homologous ISO and HPB pre-enrichment broths. The sensitivities of the commercially available Muller-Kauffmann tetrathionate broth (MKTBG 43, Oxoid CM343), and a closely-related medium prepared with Oxoid CM29 tetrathionate base varied from 86.9 to 89.3%, and were deemed equivalent to that obtained with the ISO formulation of MKTBG 43 (89.3%). Comparatively fewer contaminated samples were identified from selenite cystine (SC 35) and selenite brilliant green (SBG 35) enrichment cultures (82.1–83.3%). The high selectivity and saccharide-independent response of the bismuth sulfite agar medium warrants its consideration as a mandatory plating medium in ISO methodologies for the effective detection of typical and atypical biotypes of foodborne Salmonella spp.

Sensitivity and Specificity of Different Methods for the Isolation of Salmonella from Pigs

Three different selective enrichment media, Rappaport-Vassiliadis broth (RV), selenite broth (SB) and Müller-Kauffmann tetrathionate broth (MKTB), in combination with plating on modified brilliant green agar (BGA), were compared for the isolation of Salmonella from samples of pig feces. These conventional methods were also compared with a new ELISA kit in conjunction with RV and SB enrichment. Of the conventional methods, enrichment in RV had a higher sensitivity and selectivity than SB and MKTB. Recovery of S. typhimurium from MKTB was significantly poorer than recovery of other serotypes. The combination of RV enrichment and ELISA was as good as the conventional method involving RV enrichment, with a similar high sensitivity and specificity.

Performance of Several Enrichment Media in the Isolation of Salmonellae from Liquid Egg Products

Ninety-seven samples of raw liquid whole egg and egg yolk were analyzed for the presence of Salmonella; 51 samples (52%) were found positive. A comparative study was conducted on the performance of seven selective enrichment procedures in the isolation of Salmonella from liquid egg products: selenite-cystine broth incubated at 37°C and 43°C, Muller-Kauffmann tetrathionate broth at 43°C, modified Rappaport medium RIO/100 and RIO/10 also incubated at 43°C, the experimental broth of Greenwood et al. incubated at 37° and 43°C. The best results were obtained with tetrathionate broth which detected 96% of all positive samples. Differences in the rate of isolation by the tetrathionate broth, selenite-cystine broth, modified Rappaport medium RIO/100 and the experimental broth of Greenwood et al., all incubated at 43°C, were not significant as determined by paired χ2 test. Minor results were obtained with selenite-cystine broth and the experimental broth of Greenwood et al., both incubated at 37°C. Modified Rappaport medium RIO/100 proved to be more efficient than RIO/10.

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.

SALMONELLA CONTAMINATION OF MEAT AND BONE MEAL

The production of meat and bone meal from 8 rendering plants was examined for the presence of salmonellas. Of 164 samples of final product 114 (69.5%) were contaminated with salmonellas. Of 65 samples, collected at various points from the production line 35 (53.8%), and of 95 samples collected from the processing environment 79 (83.1%) were found to be contaminated with salmonellas. A total of 41 serotypes were found with S. havana, S. eimsbuettel, S. ohio and S. singapore being the most frequently isolated. Pre-enrichment of 25g samples in buffered peptone water, followed by enrichment in mannitol selenite cystine broth at 42 degrees C and Muller-Kauffmann Tetrathionate Broth at 37 degrees C and plating on Bismuth Sulphite Agar was found to yield 98.5% of the salmonella-positive samples.

Salmonella surveillance with reference to pigs – Cardiff abattoir, 1968–1975

Between 1968 and 1975, well over 3000 samples of pig faeces and 407 samples of pig mesenteric glands were examined for salmonellas. A very wide range of serotypes was isolated. The material from this survey was used to evaluate certain aspects of salmonella isolations. The factors studied were: selective motility enrichment, size of inoculum of faeces, comparison of drain swab with animal sample and comparison of selenite F broth with Muller-Kauffmann tetrathionate broth. The conclusions drawn from these investigations allowed an efficient routine to be developed during the survey.

FURTHER STUDIES ON THE INFLUENCE OF VARIATIONS IN THE ENRICHMENT METHOD FOR THE DETECTION OF SALMONELLAE.

Faeces and mesenteric lymph nodes of pigs were examined for salmonellae. Two jars of Muller-Kauffmann's tetrathionate broth and two jars of Osborn-Stoke's selenite — brilliant green medium were inoculated. After both 24 hr and 72 hr incubation periods subinoculations on Kauffmann's brilliant green — phenol red medium were made in duplicate from each jar.