Isolation of Salmonellae from Sewage with a New Procedure of Enrichment

Forty samples of sewage on Moore's swabs were examined for the presence of salmonellae. They were first pre-enriched in buffered peptone water. From each pre-enrichment, three enrichments were made: (1) in a new, considerably modified, formula of Rappaport medium (R 10) incubated at 43 °C (R 10/43 °C), (2) in the usual formula (R25) of the same medium at 37 °C (R25/37 °C) and (3) in Muller-Kauffmann's tetrathionate broth at 43 °C (MK/43 °C). Practically the same numbers of swabs were found positive by the first two enrichment procedures, 38 and 39 respectively, while only 17 were found positive by the MK procedure. The R10/43 °C method was superior to the two other procedures; it yielded 103 strains of salmonellae as against 82 with the second Rappaport procedure, and only 25 with the MK/43 °C technique. A similar observation was made concerning the frequency of isolation of different serotypes by the three procedures; the number of the isolated serotypes was 24, 19 and 11, respectively. The new R 10/43 °C method of enrichment had also a much stronger inhibitory effect on the competing bacteria than the two other procedures of enrichment used.     

Evaluation of the Growth of Salmonellae in Rappaport's Broth and in Muller-Kauffmann's Tetrathionate Broth

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°.     

A Comparison of Methods for the Enrichment of Salmonellae from Sheep Faeces

S ummary : A 10-tube MPN technique was used to test the efficiency of nutrient, tetrathionate, mannitol-selenite and mannitol-selenite-cystine broths as enrichment media for detecting salmonellae. Small numbers of broth grown salmonellae could be detected in all 4 media in the presence of 5% of sheep faeces. In naturally infected sheep faeces small numbers of salmonellae were not detected with either nutrient or tetrathionate broths. With mannitol-selenite the sensitivity of salmonella detection increased with both incubation temperature (37–43°) and the addition of faeces. The most sensitive and reliable medium for detecting salmonellae in naturally infected sheep faeces was mannitol-selenite-cystine broth. Neither incubation temperature (37–43°) nor the addition of faeces had a statistically significant effect on its sensitivity.     

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.     

Development and evaluation of two novel oligonucleotide probes based on 16S rRNA sequence for the identification of Salmonella in foods

DNA sequence in the V3 to V6 region of the 16S rRNA gene of Salmonella enteritidis was determined. By comparison of this sequence with those of Escherichia coli and Proteus vulgaris obtained from GenBank/EMBL database, three oligonucleotides termed as 16S I, 16S II and 16S III were synthesized. Hybridization of these oligonucleotides with 325 Salmonella isolates and some non- Salmonella isolates including the Salmonella closely related species of the family of Enterobacteriaceae showed that 16S II could not be used as a Salmonella specific-probe. 16S I and 16S III hybridized with all the Salmonella isolates tested, the former also hybridizing with Citrobacter spp. and the latter hybridizing with Klebsiella pneumoniae as well as Serratia marcescens. Since enrichment of the target cells in food samples was usually required prior to the DNA hybridization assay, the interference from those non- Salmonella isolates could be prevented by enrichment by culturing in lactose-combined tetrathionate (CTET) broth followed by Gram-negative (GN) broth at 37°C and/or 43°C. Such a culture step could inhibit the growth of Klebsiella spp., Ser. marcescens and/or Citrobacter spp. and allowed the specific detection of Salmonella .     

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.     

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.     

Production and stability of pediocin N5p in grape juice medium

The production and stability of pediocin N5p from Pediococcus pentosaceus , isolated from wine, were examined in grape juice medium. Maximum growth and higher titre (4000 U ml^-1) were observed at a initial pH of 7·5 and 30°C. The activity of the inhibitory substance was stable between pH values from 2·0 to 5·0 at 4° and 30°C. At pH 10·0 it was completely inactivated. When submitted to 30 min at 80°, 100° and 115°C, maximal stability was observed at pH 2·0. Ethanol up to 10% did not affect pediocin activity at acid pH, nor did 40–80 mg 1^-1 SO₂, independently or combined with different ethanol concentrations, affect inhibitory activity.     

The enumeration of thermotrophic types amongst the Enterobacteriaceae colonizing perishable foods

A total of 41 pure cultures of Enterobacteriaceae, comprising 32 thermotrophic and nine psychrotrophic strains, pathogens or marker organisms, were examined for numbers of colony forming units obtained at 37° and 42°5°C (thermotrophs) and 30°C (psychrotrophs), when surface-plated on a rich infusion agar and violet red bile agar. In addition 42 food and water samples, collected in a rural area of the Philippines, were examined by surface inoculating violet red bile AIPC (agar immersion plating and contact; 'dip') slides and incubating at 37° and 42°5°C. At 42°5°C there was almost total recovery of the thermotrophic Enterobacteriaceae, whereas the psychrotrophic strains were completely suppressed. At 37°C the psychrotrophs were only slightly inhibited. The Philippine foods, predominantly cooked meals, milk and drinking water, appeared to be significantly colonized by thermotrophic Enterobacteriaceae. It is concluded that incubation at 42°5°C satisfactorily selects enteropathogenic and other enteric Enterobacteriaceae while suppressing the psychrotrophic types which are mainly of vegetable origin. It is emphasized that, regardless of the temperature used, a resuscitation procedure for Enterobacteriaceae populations that have incurred sublethal injury in food has to precede counts on or in the usual selective media.     

Rapid and sensitive method for detection of Salmonella strains using a combination of polymerase chain reaction and reverse dot-blot hybridization

Abstract Cell-free extracts of Thiobacillus acidophilus catalysed the stoichiometric conversion of tetrathionate to thiosulphate, sulphur and two protons. The pH optimum of the enzyme activity was 3.0 and its temperature optimum 40°C. The enzyme was unstable at 30 and 40°C, at which its activity decreased to zero within 100 and 20 h, respectively. Enzyme activity was not affected by incubation for 1 week on ice or by freezing and thawing of the extract. The K _m for tetrathionate was 0.3 mM. Enzyme activity was stimulated by ammonium sulphate up to a concentration of 1M. The results indicate that trithionate hydrolase cannot account for the observed conversion of tetrathionate.     

Comparison of the Effects of Liquid Medium Repair and the Incorporation of Catalase in MacConkey Type Media on the Recovery of Enterobacteriaceae Sublethally Stressed by Freezing

Nine pure cultures of species of Enterobacteriaceae were stressed by rapid freezing in tryptone soya broth (TSB) to — 22°C and subsequent storage at that temperature for 7 d. About one to two log cycles kill and at least one additional log cycle sublethal impairment was achieved. Numbers of colonies of these cultures in poured plates of violet red bile glucose (VRBG) agar, with 67 u/ml of catalase added at 47°C, were only slightly higher than those in plain VRBG, both incubated overnight at 30°C. Two hours incubation of TSB suspensions at 17–25° C resulted in almost complete restoration of the ability of cells to develop colonies in VRBG, without, however, leading to any significant multiplication.
Similar experiments with 32 samples of frozen minced meat, 27 samples of frozen surface water, 18 of frozen chicken liver and 14 of fresh sausage substantiated the results obtained in the studies on pure cultures.
In the experiments with the nine pure cultures the influence of the nutrient composition of the solid enumeration media: 'minimal' agar, TSB agar (TSBA) and Mueller-Hinton agar with Polyvitex nutrient supplement (MHA), on the recovery of Enterobacteriaceae stressed by freezing was also studied. Colony numbers in TSBA and MHA were virtually identical. The glucose mineral salts medium led to lower recovery, indicating that so-called 'minimal medium recovery' of stressed bacterial populations is not a common phenomenon.     

Efficiency of different enrichment and isolation procedures for the detection of Salmonella serotypes in edible offal

Rapid detection systems for Salmonella in foodstuffs are currently being developed. However, existing standards still call for application of traditional methods employing pre-enrichment followed by selective enrichment and isolation. The efficacy of various methods was tested using 264 chicken and lamb organ meats. Pre-enrichment was carried out in Tryptone Soy Broth (TSB) and enrichment in Tetrathionate Brilliant Green Broth (TTB) at 37°C, Selenite Broth with Brilliant Green and Sulphapyridine at 37°C and 43°C, and Rappaport-Vassiliadis Broth (RV 10) at 42°C. The isolation media were Brilliant Green Agar (BGA), Deoxycholate Citrate Agar, Hektoen Enteric Agar (HEA) and Salmonella-Shigella Agar.
Enrichment in RV/42°C followed by isolation on BGA as recommended by ISO standard no. 6579 and enrichment in TTB/37°C followed by isolation in HEA, no longer recommended by that standard, produced the best results. Low percentages of positive samples and difficulties in detecting Salmonella are the result of interference by competing organisms (Enterobacteriaceae) and the number of salmonellas present after enrichment.
A total of 528 samples (TSB, eggs, lamb liver and chicken liver) were inoculated with Salm. enteritidis, Salm. kapemba and Salm. virchow , and the preceding experiment was repeated. All the TSB and egg samples tested positive, but the percentage of positive samples from the lamb and chicken liver was only 81–92%. Recovery of the salmonellas did not depend upon the method employed or the serotype inoculated but instead on interference by competing flora and the numbers of Salmonella present in the samples.     

Characterization of natural isolate Lactobacillus acidophilus BGRA43 useful for acidophilus milk production

Lactobacillus acidophilus BGRA43 was selected from a set of human origin isolates of Lact. acidophilus strains for the highest growth rates and antagonistic effect against both Gram-positive and Gram-negative bacteria. The strain BGRA43 also exhibited an inhibitory effect on the growth of Clostridium sporogenes. Inhibition of this strain seems to be due to lactic acid production rather than hydrogen peroxide or bacteriocin. Growth of Lact. acidophilus BGRA43 in non-fat skim milk for 6 h at 37 °C resulted in a lowering of the pH value to 4·53. Besides the fast acidification, this strain generated a high viscosity of skim milk. These characteristics make the strain BGRA43 attractive for acidophilus milk production. Lactobacillus acidophilu s BGRA43 produces an extracellular proteinase. Whole cells efficiently degraded casein for 3 h at 37 °C especially α- and β-casein fractions. Total DNA isolated from the strain BGRA43 did not show any hybridization with lactococcal proteinase probes indicating that this strain produces a distinctive proteinase.     

A medium for the isolation, enumeration and rapid presumptive identification of injured Clostridium perfringens and Bacillus cereus

A blood-free egg yolk medium (BCP) containing pyruvate, inositol, mannitol and a bromocresol purple indicator in a nutrient agar base has been developed to initiate the growth of Clostridium perfringens . It is comparable to blood agar for the growth of normal, chilled stored vegetative cells and heat-injured spores of Cl. perfringens and Bacillus cereus . It has the advantage over blood agar in exhibiting presumptive evidence of Cl. perfringens (production of lecithinase and inositol fermentation) after an overnight incubation at 43°C-45°C. Pyruvate, catalase and other hydrogen peroxide degraders were found to remove toxins rapidly formed in media exposed to air and light. Free radical scavengers of superoxide, hydroxyl ions and singlet oxygen were ineffective. Without scavengers the formation of 10–20 μg/ml hydrogen peroxide in the exposed medium was indicated and found lethal to injured Cl. perfringens .
The BCP medium has been used successfully for the rapid identification and enumeration of Cl. perfringens in foods and faeces from food poisoning outbreaks and cases of suspected infectious diarrhoea. Greater recovery of severely injured vegetative Cl. perfringens could be obtained by pre-incubation at 37°C of inoculated media for 2–4 h followed by overnight incubation at 43°C-45°C. Tryptose-sulphite-cyclo-serine and Shahidi-Ferguson-perfringens agar base were found to inhibit the growth of several strains of injured vegetative Cl. perfringens . This was not completely overcome by the addition of pyruvate. The inclusion of mannitol also allows the medium to be used for the presumptive identification of B. cereus . Growth and lecithinase activity are profuse on BCP. Heat-injured spores are recovered equally well on BCP and blood agar. A scheme for the identification of some other clos-tridia on BCP is presented.     

Growth of Bacteria on Meat at Room Temperatures

The development of spoilage flora and the growth of individual psychrotrophs and pathogens on meat held at 20 or 30°C were studied. Under aerobic conditions psychrotrophic pseudomonads accounted for 60% of the spoilage flora at 20°C, but <20% at 30°C where they were displaced by species of Acinetobacter and Enterobacteriaceae which included both mesophilic and psychrotrophic strains. Mesophiles dominated the anaerobic spoilage flora at 30°C when clostridia were the major species, but at 20° the flora contained mesophiles and psychrotrophs in similar proportions and was dominated by Enterobacteriaceae. These results were largely predictable from the growth rate data for individual organisms.
Interactions between species occurred more frequently at 20°C than at 30°C. When pathogenic species were grown at 20 or 30°Cin competition with equal numbers of psychrotrophic spoilage organisms, no interactions were observed. When pathogens were grown in competition with high numbers of psychrotrophs, only Lactobacillus growing anaerobically inhibited Salmonella typhimurium and Escherichia coli , but other pathogens were inhibited to varying degrees depending on the competing species and the incubation conditions. In general, the degree of inhibition was greater at 20 than at 30°C and facultative organisms were more susceptible under anaerobic than aerobic conditions. It appears that the cumulative stresses of low pH, suboptimal temperatures and competition with large numbers of saprophytic organisms can inhibit many of the pathogens likely to be present on meat. The organisms least affected by the conditions on meat surfaces, Salmonella and Esch. coli , are likely to be the main hazards on meat of normal pH held at room temperatures.     

Influence of Incubation Temperature and Sodium Heptadecyl Sulfate (Tergitol No. 7) on the Isolation of Salmonellae from Pork Sausage

Cultures of 68 samples of fresh pork sausage purchased locally were incubated at 37 and 43 C, with and without Tergitol No. 7 (sodium heptadecyl sulfate) added to the tetrathionate-Brilliant Green enrichment broth. The results indicated an advantage in incubating the tetrathionate broth at 43 C rather than 37 C in attempting to isolate salmonellae from pork sausage. Without Tergitol, more samples were positive at 43 C than at 37 C, but with Tergitol there was no difference. The higher temperature suppressed the competing gram-negative bacteria and permitted Salmonella to grow in relatively pure culture, thus providing an advantage for isolating and identifying the organisms. Tergitol dispersed and emulsified the fat which improved the isolation of Salmonella when the cultures were incubated at 37 C but not at 43 C. Brilliant Green-sulfadiazine agar was superior to bismuth sulfite agar for isolating salmonellae from tetrathionate broth cultures of fresh pork sausage.     

Antimicrobial activity of food-related Penicillium sp. against pathogenic bacteria in laboratory media and a cheese model system

Food-related Penicillium species ( n ep fy1 = rs 34) and Geotrichum candidum ( n = 11) grown on Czapek Dox and brie agar were tested for their ability to suppress growth of pathogenic bacteria. Ten out of 13 P. camemberti showed antagonistic activity while the other species did not interact significantly with the bacterial growth. The order of inhibition was: Gram-negative bacteria and Bacillus cereus > Listeria monocytogenes , Lactococcus sp. > Micrococcus sp. whereas Lactobacillus sp., Staphylococcus aureus and some Micrococcus sp. were unaffected. When Salmonella typhimurium was inoculated together with P. camemberti P25 in brie agar, bacterial growth was inhibited during the first 6 d of incubation whereafter growth started. The inhibition of L. monocytogenes was similar but less pronounced. The antimicrobial activity produced by P. camemberti P25 and L84 was enhanced with increasing amount of sucrose in the medium. The activity was increased at low pH and destroyed at pH above 8. It was detectable at 15°C but not at 37°C indicating that volatile metabolites might be involved. No significant accumulation of organic acids and no secondary metabolites such as mycotoxins were detected. HSGC-MS analysis indicated that acetaldehyde, benzaldehyde, 3-methylbutanal and 1-octen-3-ol were produced by P. camemberti during the period when inhibitory activity was observed. Pure acetaldehyde and benzaldehyde were shown to be inhibitory to L. monocytogenes and Salm. typhimurium when grown at 15°C and pH 5·5 and 7·0.     

Experimental study on the growth of Salmonella and coli-aerogenes bacteria in pure and mixed cultures,in a fermentor,in two different enrichment media: Sodium selenite and tetrathionate broth

Summary Different serotypes of Salmonella and coli-aerogenes bacteria were grown in a fermentor at +43°C. The Culture media used were composed of two different nutrient broths, one supplemented with sodium selenite, the other with potassium tetrathionate. The growth of both bacteria and the following types of mixed bacteria was studied: Escherichia coli-Salmonella brancaster and Klebsiella pneumoniae-Salmonella brancaster. During the first 10 h of incubation, sodium tetrathionate broth inhibited the multiplication of coli-aerogenes bacteria. Similar results were obtained when these bacteria were grown with S. brancaster and incubated for 18 h, regardless of the original concentration of each of the bacteria. In the presence of sodium selenite, after 18 h of incubation growth was inhibited in E. coli, but not in the other coli-aerogenes bacteria.     

The development of a conductimetric assay for automated detection of metabolically active soft rot Erwinia spp. in potato tuber peel extracts

Four media were tested for their ability to detect the soft rot potato pathogens Erwinia chrysanthemi (Ech) and Erwinia carotovora ssp. atroseptica (Eca) in potato tubers by means of automated conductance measurements. The specificity of the conductimetric assays was determined by testing a set of different Erwinia spp. and potato-associated saprophytes, including the genera Pseudomonas, Bacillus, Enterobacter and Flavobacterium. All bacteria tested produced conductance responses in Special Peptone Yeast Extract, whereas in minimal medium with L-asparagine only Erwinia spp. and Pseudomonas spp. were able to generate large conductance responses. In minimal medium supplemented with glucose and trimethylamine- N -oxide only Enterobacteriaceae, Erwinia spp. included, generated conductance responses, while with pectate as sole carbon source only Erwinia spp. produced distinct conductance responses. The pectate medium proved to be particularly useful for specific automated conductimetric detection of Erwinia spp. in potato peel extracts. Within 48 h, the detection threshold of the conductimetric assay for Eca varied between 10² and 10³ cfu per ml peel extract at both incubation temperatures of 20° and 26°C. Ech was detected at concentrations of 10⁴–10⁵ or 10³–10⁴ cfu ml^-1 at 20° and 26°C, respectively. To eliminate 'false'-positive reactions in conductimetry caused by Erwinia carotovora ssp. carotovora , results of the conductance measurements have to be confirmed by other techniques, like serology or DNA assays.