Comparison of methods for isolating Salmonella bacteria from faeces of naturally infected pigs
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A series of experiments was conducted using faecal samples collected from commercial swine farms to evaluate the effects of variation in methods used for the detection of Salmonella bacteria. The primary objective of the studies was to compare the protocols routinely used in two laboratories in the USA. The studies included five experiments comparing the enrichment protocols used routinely in the respective laboratories (Method 1: 10 g faeces—buffered peptone water (BPW) pre‐enrichment—selective enrichment in Rappaport/Vassiliadis (RV) broth; Method 2: ∼1g faeces—primary enrichments in tetrathionate and Hajna GN broths—secondary enrichment in RV broth). The effects of enrichment temperatures (37 vs 42 °C) using RV broth (two experiments) and delayed secondary enrichment (four experiments) were also evaluated. Direct comparison of Method 1 and Method 2 indicated comparable results. However, when compared using faecal samples of equal weight, the Method 2 enrichment protocol was more sensitive for detecting Salmonella bacteria than the Method 1 protocol. Enrichment in RV at 42 °C was superior to 37 °C, particularly for samples that were pre‐enriched in BPW. Delayed secondary enrichment increased detection of Salmonella bacteria in swine faeces. These results highlight the imperfect sensitivity of culture methods, and the need for researchers to consider the sensitivity of bacteriological methods in the design and interpretation of the results of epidemiologic studies based on faecal cultureKeywords:
Tetrathionate
Enrichment culture
Rappaport
Detection of Salmonella is a key point in veterinary salmonella research and surveillance programs. In the conventional isolation method, the selective enrichment is a critical step because suppresses competitive rnicroflora and permits Salmonella to proliferate. Several selective enrichment broths have been compared for the recovery of Salmonella. In many studies Rappaport-Vassiliadis medium (RV) has been found more productive than MollerKauffmann tetrathionate broth (MKT) and selenite cystine broth (SC) for analyzing meat products, feces and animal feed (1,2). Moreover, significant difference was found in the recovery of Salmonella from food, when MKT medium was incubated at 42°C.
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Brilliant green
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The relative efficiency of three selective enrichment broths (Muller-Kauffmann tetrathionate. Rappaport's and selenite F) was investigated for the isolation of salmonellae from seagull cloacal swabs. Pre-enrichment in buffered peptone water was employed throughout the study, which involved the examination of 560 gulls, sixty (10.7%) of which were found to be carrying salmonellae. Rappaport's broth as modified by Vassiliadis for incubation at 43 degrees C (Vassiliadis et al. 1976) yielded the highest number of positive swabs (57) and the widest range of serotypes. It was significantly more efficient that either selenite F or tetrathionate broth, although the results obtained with Rappaport's broth incubated at 37 and 43 degrees C did not differ significantly (P greater than 0.5). Eleven serotypes were isolated during the study, the most prevalent being Salmonella virchow.
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S ummary . The behaviour of 70 strains of salmonellae belonging to 44 serotypes in Rappaport's broth and in Muller‐Kauffmann's tetrathionate broth was examined. With an inoculum of 5–25 cells, 5 strains did not grow in Rappaport's medium, 2 multiplied slowly and 63 grew strongly in 24 h. With an inoculum of 100–500 organisms all but one strain grew readily in 24 h. In Muller–Kauffmann's tetrathionate broth inoculated with pure cultures of salmonellae, growth of many strains was markedly inhibited, in the absence of added faeces, at 37° and 43°. This inhibition was more severe with light inocula at 43°. The addition of 0.05% (w/v) of salmonella‐free human faeces to Muller–Kauffmann's tetrathionate broth, did not stimulate growth of salmonellae. In contrast, the addition of 5% (w/v) of human stools to this medium resulted in a heavy growth of the added salmonellae, especially at 43°.
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Culture based methods are commonly employed to detect pathogens in food and environmental samples. These methods are time consuming and complex, requiring multiple nonselective and selective enrichment broths, and usually take at least one week to recover and identify pathogens. Improving pathogen detection in foods is a primary goal for regulatory agencies and industry. Salmonella detection in food relies on a series of culture steps in broth formulations optimized to resuscitate Salmonella and reduce the abundance of competitive bacteria. Examples of nonselective pre-enrichment broths used to isolate Salmonella from food include Lactose, Universal Pre-enrichment, Buffered Peptone Water and Trypticase Soy broths. Tetrathionate and Rappaport-Vassiliadis broths are employed after a 24-hour nonselective enrichment to select for Salmonella and hamper the growth of competitive bacteria. In this study, we tested a new formulation of Tetrathionate broth that lacks brilliant green dye and has lower levels of Tetrathionate. We employed this Tetrathionate broth formulation in conjunction with a 6-hour nonselective pre-enrichment period and determined that Salmonella recovery was possible one day earlier than standard food culture methods. We tested the shortened culture method in different nonselective enrichment broths, enumerated Salmonella in the non-selective enrichments, and used 16S rRNA gene sequencing to determine the proportional abundances of Salmonella in the Tetrathionate and Rappaport-Vassiliadis selective enrichments. Together these data revealed that a 6-hour nonselective pre-enrichment reduces the levels of competitive bacteria inoculated into the selective Tetrathionate and Rappaport-Vassiliadis selective broths, enabling the recovery of Salmonella one day earlier than standard culture enrichment methods.
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Tetrathionate broth (Müller-Kauffmann) and Rappaport's medium were compared in isolating Salmonella from meat- and bone-meal, receiving waters, sewage, and sludge. The results (Table I) show no difference in isolations from meat- and bone-meal, receiving waters, or sewage, while in studying sludge isolations were significantly less frequent from Rappaport's medium than from tetrathionate broth. The Salmonella positive plates, however, were easier to read when inoculated from Rappaport's medium than from tetrathionate broth.
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To determine the appropriate method for isolating Salmonella enterica, we compared the growth of S. enterica serovars using three selective enrichment media. S. enterica was more successfully isolated from artificially contaminated fecal samples after enrichment in Hajna tetrathionate broth or modified semisolid Rappaport agar than in Rappaport broth. Since most bacteria (other than motile S. enterica) do not migrate on modified semisolid Rappaport agar, the growth characteristics of S. enterica can be interpreted easily and quickly. Two S. enterica isolates did not migrate on modified semisolid Rappaport agar, but did grow in Hajna tetrathionate broth, which suggests that the combined use of these selective enrichment media is appropriate for isolating S. enterica.
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Food microbiology
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ABSTRACT Results obtained with tetrathionate broth (TBB) using the standard method and Rappaport‐Vassiliadis medium (RV) did not differ markedly with respect to the proportion of positive samples or the sero‐ and phage types of salmonellae isolated from minced beef and pork. TBB, based on the commercial dehydrated medium of Oxoid (CM 343), gave fewer positive samples while that of Merck (Art. No. 5172) was equivalent to RV. In individual experiments results were sometimes markedly different. Results obtained for reference samples of minced meat, with salmonellae added, demonstrated that the chances of detecting salmonellae increase with increasing numbers present initially. At the same time it was also demonstrated that the presence of minced meat and its competing microflora have an unpredictable effect on the detection of salmonellae.
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National standard
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R hodes , P., Q uesnel , L.B. & C ollard , P. 1985. Growth kinetics of mixed culture in salmonella enrichment media. Journal of Applied Bacteriology 59 , 231–237. A technique for investigating the kinetics of salmonella enrichment is reported. Its use with four enrichment media (Rappaport' medium, Muller‐Kauffmann tetra‐thionate broth (MKT) tetrathionate broth and selenite F) is described and the effect of elevated temperature on the growth kinetics shown. Rappaport' medium at 37d̀C and MKT at either 37d̀C or 42d̀C were far superior to selenite F and tetrathionate broth in their selective properties and, with the exception of Rappaport' medium, the use of elevated temperature increased the selectivity of the media.
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Bacterial growth
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