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 μg 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.     

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.     

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.     

Microbiological composition of raw milk from selected farms in the Camembert region of Normandy

Raw milk from 27 farms was sampled over 6 months for listerias, salmonellas, Yersinia enterocolitica and campylobacters. Total bacterial counts and somatic cell counts were measured. Lactococci, lactobacilli, dextran-producing leuconostocs, Brevibacterium linens , yeasts and moulds, Staphylococcus aureus and other Micrococcaceae, Pseudomonas , coliforms, Escherichia coli , enterococci, Clostridium perfringens and spores of anaerobic lactate-fermenting bacteria were also counted. Pseudomonas (2000 cfu ml⁻¹), lactococci (760 cfu ml⁻¹) and Micrococcaceae (720 cfu ml⁻¹) were the most numerous groups. Lactic acid bacteria were detected in all samples. Coliforms were present in most samples, but 84% of samples had counts <100 cfu ml⁻¹. Staphylococcus aureus was detected in 62% of milks, the average count was 410 cfu ml⁻¹. About 80% of supplies had ≤10 E. coli cfu ml⁻¹ and all samples had 1 Cl. perfringens cfu ml⁻¹. Two of the tested milks were positive for salmonellas (2·9%), four were positive for Listeria monocytogenes (5·8%), 25 for Yersinia enterocolitica (36%) and one for campylobacters (1·4%).     

Viable but non-culturable salmonellas in soil

P.E. TURPIN, K.A. MAYCROFT, C.L. ROWLANDS AND E.M.H. WELLINGTON. 1993. An enzyme-linked immunosorbent assay (ELISA) and a microwell fluorescent antibody (FA) direct count method have been developed for the monitoring of salmonellas in soil. Both methods have a minimum detection level of ca 10⁶ cells per gram of soil. The FA direct count method gave a linear recovery for the inoculum range 10⁶–10⁹ cells per gram of soil. When monitored by plate counts the survival of salmonellas was greater in a sterile than in a non-sterile soil. Evidence was found for the production of viable but non-culturable salmonellas in non-sterile soil; plate counts dropped rapidly with time, but FA direct counts and ELISA remained level. The salmonella cells became progressively smaller and rounder with time. Dead salmonella cells introduced into soil rapidly disappeared.     

Detection of Clostridium botulinum types A, B, E and F in foods by PCR and DNA probe

A PCR procedure was developed for the detection of Clostridium botulinum in foods. PCR products were detected in agarose gels and by Southern hybridization. The sensitivity of PCR was tested in broth cultures and in canned asparagus, dry cured ham and honey. The sensitivity of the method in broth was high (2·1–8·1 cfu ml⁻¹) for types A and B, but rather low (10⁴ cfu ml⁻¹) for types E and F. However, after enrichment at 37°C for 18 h, it was possible to detect Cl. botulinum types A, B, E and F in food samples at initial levels of about 1 cfu 10 g⁻¹ of food. This PCR detection protocol provides a sensitive and relatively rapid technique for the routine detection of Cl. botulinum in foods.     

Antibacterial activity of bovine lactoferrin and its peptides against enterohaemorrhagic Escherichia coli O157:H7

The antimicrobial activities of bovine lactoferrin (bLF), its pepsin hydrolysate (bLFH) and the active peptide lactoferricin^® B (LFcinB) against four clinical isolates of enterohaemorrhagic Escherichia coli O157:H7 were studied. The MICs against these isolates were 3 mg ml⁻¹ for bLF, 0·1–0·2 mg ml⁻¹ for bLFH and 8–10 μg ml⁻¹ for LFcinB in 1% Bactopeptone broth. LFcinB killed these bacteria within 3 h at concentrations above 10 μg ml⁻¹. Transmission electron microscopy findings suggested that LFcinB acts on the bacterial surface and affects cytoplasmic contents. LFcinB was shown to influence the levels of verotoxins in the culture supernatant fluid of an E. coli 0157:H7 strain. These results demonstrate that E. coli O157:H7 strains are susceptible to the antimicrobial effects of bLF and its peptides.     

Bacteriological stability and growth kinetics of Pseudomonas aeruginosa in bottled water

Bacteriological stability of water bottled in plastic containers and the growth kinetics of Pseudomonas aeruginosa were determined. Samples of water from the source, water to be bottled, finished product and sterile water bottled in non-returnable and returnable containers were analysed for aerobic colony count, coliforms, Escherichia coli and Ps. aeruginosa. The samples were examined for up to 30 d storage. Aerobic colony count increased 6 d after bottling to between 10³ and 10⁵ cfu ml⁻¹. Coliforms and E. coli were not found in any sample. Pseudomonas aeruginosa was isolated from commercial products bottled in returnable plastic containers due to the contamination from the containers and the subsequent multiplication utilizing trace nutrients. The predominant Ps. aeruginosa strains showed high doubling time (26 h) due to competition from the accompanying flora. In the absence of competing flora Ps. aeruginosa reached higher density than the maximum reached by aerobic flora, with a doubling time of only 3·6 h. After 30 d storage, this micro-organism was predominant.     

Production of a nisin Z/pediocin mixture by pH-controlled mixed-strain batch cultures in supplemented whey permeate

The conditions for high production of nisin Z and pediocin during pH-controlled, mixed-strain batch cultures in a supplemented whey permeate medium with Lactococcus lactis subsp. lactis biovar. diacetylactis UL719, a nisin Z producer strain, and variant T5 of Pediococcus acidilactici UL5, a pediocin-producing strain resistant to high concentrations of nisin, were studied. Mixed cultures were performed at 37 °C and pH 5·5 by first inoculating with variant T5 and then with L. diacetylactis UL719 after 8 h incubation, and were compared with single-strain batch cultures. High productions of both nisin Z and pediocin were obtained after 18 or 16 h incubation during mixed cultures, with titres of 3000 and 730 AU ml⁻¹, or 1060 and 1360 AU ml⁻¹, respectively, corresponding to approximately 75 and 55, or 25 and 100 mg l⁻¹ of pure nisin Z and pediocin, respectively. In pure cultures, nisin Z and pediocin productions were higher than in mixed cultures, and maximum activities were obtained after 10 h incubation, with approximately 10 000 AU ml⁻¹ (250 mg l⁻¹ pure nisin Z) and 2500 AU ml⁻¹ (190 mg l⁻¹ pure pediocin). During mixed cultures, significant pediocin degradation was observed in the culture supernatant fluid after 16 h incubation, together with a sharp drop in Ped. acidilactici UL5 cell viability. In the test conditions, the feasibility of producing a nisin/pediocin mixture by mixed-strain fermentation was demonstrated. The bacteriocin mixture produced in a supplemented whey permeate medium could be used as a natural food-grade biopreservative with a broad activity spectrum.     

Increased ELISA sensitivity using a modified extraction buffer for detection of Xanthomonas campestris pv. vesicatoria in leaf tissue

In vitro and in planta sensitivity of an indirect enzyme-linked immunoassaytechnique, using a monoclonal antibody specific for the lipopolysaccharide (LPS) of Xanthomonas campestris pv. vesicatoria , was increased 10-foldby using a newextraction buffer (gl of : KH₂PO₄, 2; NaHPO₄, 11·5; EDTAdisodium, 0·14; thimerosal, 0·02; and lysozyme, 0·2). The procedure improvedsensitivity without increasing background levels. In vitro , the limit of detection wasbetween 1×10⁷ and 1×10⁸ cells ml⁻¹ with the conventionalextraction buffer phosphate-buffered saline (PBS) and less than 1×10⁶ cells ml⁻¹ when lysozyme extraction buffer was substituted for PBS. In comparing 22 X. c.vesicatoria strains, absorbance readings were increased close to three-fold with the lysozymeextraction buffer as opposed to PBS. When leaf tissue extract was spiked with the bacterium, thelimit of detection was 1×10⁷ cfu ml⁻¹ and 1×10⁸ cfu ml⁻¹ with the lysozyme solution and PBS, respectively, as the extraction buffers. Whenusing the lysozyme extraction buffer in combination with a commercial amplification system, thelimit of detection was decreased to less than 1×10⁵ cfu ml⁻¹ in leaftissue. The addition of the lysozyme and EDTA to the phosphate buffer resulted in release of asignificant quantity of LPS and concomitant dramatic increase in sensitivity. The new procedure,termed lysozyme ELISA (L-ELISA), should increase sensitivity of ELISA reactions where LPS isthe reacting epitope.     

Use of flow cytometry for enumeration of Mycoplasma mycoides subsp. mycoides large-colony type in broth medium

Aims:  The potential of using flow cytometry (FC) in combination with a fluorescent dye (SYBR green-I) for rapidly estimating Mycoplasma mycoides subSPS. mycoides large-colony type (MmmLC) in broth culture was examined.
Methods and Results:  The FC analysis was performed by staining the MmmLC cells with a fluorescent dye, SYBR green-I (SYBR), and the results were compared with plate count method (colony forming units, – CFUs). There was a good correlation (linear regression, r ² = 0·93) between mycoplasma counts determined by FC (cells ml⁻¹) and by traditional plate count method (CFU ml⁻¹). The lowest bacterial concentration detected by FC and traditional plate count was of the order of 10⁴ cells ml⁻¹ and 10³ CFU ml⁻¹, respectively. FC method allowed results in 20–30 min, whereas at least 24 h were necessary to obtain results with the traditional plate count method (CFU).
Conclusion:  Growth rates of MmmLC in broth medium determined by FC were highly reproducible and correlated well with mycoplasma counts assessed by the plate count method.
Significance and Impact of the Study:  These findings suggest that FC could be a good alternative to replace other time-consuming techniques that are currently used to enumerate mycoplasma in broth medium, such as plate count method (CFU).     

Development of a microtitre ELISA to quantify development of Cryptosporidium parvum in vitro

Abstract An in situ enzyme-linked immunosorbent assay (ELISA) was developed to evaluate growth of Cryptosporidium parvum in vitro. Ninety-six-well tissue culture microtitre plates were each seeded with 4.0 X 104 human ileocecal adenocarcinoma (HCT-8) cells, then infected with CsCl-purified oocysts 24 h later. The growth medium consisted of RPMI 1640 supplemented with 10% fetal bovine serum, 15 mM HEPES (JV-2-hydroxyethylpiperazine N −2-ethanesulfonic acid), 50 mM glucose, 1 μg ml⁻¹ folic acid, 4 μg ml⁻¹ 4-aminobenzoic acid, 2 μg ml⁻¹ pantothenic acid and 35 μg ml⁻¹ ascorbic acid. Incubation conditions were at 37 ° C in a 5% CO₂/95% humidified air incubator. Oocysts were allowed to excyst in situ so that sporozoites could infect cells directly. Monolayers were then washed, new medium added, and infected cells re-incubated. Levels of infection were assessed 48 h later using a rat anti-C. parvum polyvalent antiserum directed against purified parasite membranes, followed by a goat anti-rat IgG conjugated to horseradish peroxidase and 3,3',5,5'-tetramethyl-benzidine as substrate. Using various parasite inoculating doses and incubation times, optimal results were obtained using a 90-min exposure of host cells to 2.5−3.0 × 10⁴ oocysts/well. Evaluation of various concentrations of four anti-microbials (monensin, lasalocid, paromomycin and sulfadimethoxine) in the system resulted in the acquisition of precise dose-response curves for each compound.     

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.     

A note on the effect of the lactoperoxidase systems on salmonellas in vitro and in vivo

The lactoperoxidase system (LPS), a natural bactericidal system in milk, was investigated for its activity against salmonellas in vivo and in vitro. In acidified raw milk, in which the LPS was supplemented with an exogenous supply of H₂O₂, the numbers of salmonellas decreased rapidly. Different salmonella serotypes were affected to the same extent; rough strains, however, were more susceptible than smooth strains. When calves were fed on fresh milk, containing the LPS, and challenged with Salmonella typhimurium in doses of either 10⁹ or 10¹⁰, the clinical findings and salmonella excretion patterns were similar to those of control calves fed on heated milk. It was concluded that further studies, perhaps in the field, are necessary to evaluate LPS as a possible non-antibiotic system to control salmonellosis.     

A study of isolation procedures for multiple infections of Salmonella and Arizona in a wild marsupial, the quokka (Setonix brachyurus)

Rectal swabs and faeces were used in the regular sampling for salmonellas and Arizonas from a heavily-infected population of a marsupial, the quokka ( Setonix brachyurus ). The media used were strontium selenite A and strontium chloride B enrichment broths, with subculture onto modified bismuth sulphite agar and deoxycholate citrate agar. A study of sampling, enrichment, sub-culture and colony selection procedures produced an optimal scheme giving high yields but consistent with reasonable economy of time and materials. A three-swab sample was taken and inoculated into the two enrichment media, and with each enrichment subjected to three subcultures. The absolute efficiency of this procedure was greater than 80% (and confirmed by a serological method), compared with only 67% for a single swab in a single enrichment. Recovery of some serotypes depended on the media used; e.g. Arizonas could not be recovered satisfactorily from strontium chloride B enrichment. Faeces samples were found to be greatly superior to rectal swabs for detecting salmonellas and arizonas but they were less convenient in field studies. In a comparison of rectal swabs and faeces samples where the actual concentration of salmonellas was known, it was found that the efficiency of rectal swabs approached 100% if there were more than 10³ salmonellas/g faeces, but this declined to approximately 50% if there were 10²-10³ salmonellas/g faeces and only 25% if there were less than 10² salmonellas/g faeces. A new statistical procedure was introduced for comparing the number of isolations from two methods, and this should be of use in similar methodological studies.     

An evaluation of the use of 4-methylumbelliferyl-β-D-glucuronide (MUG) in different solid media for the detection and enumeration of Escherichia coli in foods

The use of 4-methylumbelliferyl-β- D -glucuronide (MUG) in different solid media for the detection and enumeration of Escherichia coli in foods was evaluated by testing the effects of different substrate concentrations (50 or 100 μg ml⁻¹), incubation temperatures (37 or 41·5°C) and incubation times (8, 12, 24 and 48 h). Different kinds of foods, both naturally and artificially contaminated, were analysed. The use of selective media without differential substances and an incubation time of 24 h seem to be worthy of recommendation. In this case an incubation temperature of 37°C would be preferred and the MUG concentration could be reduced to 50 μg ml⁻¹. Incubation times shorter than 24 h, which may cause a loss of sensitivity, require higher incubation temperatures (41·5°C) and MUG concentration (100 μg ml⁻¹).     

Conjugal transfer at natural population densities in a microcosm simulating an estuarine environment

Abstract Estuarine microcosms were used to follow conjugal transfer of a broad host range IncP1 plasmid from a Pseudomonas putida donor to indigenous bacteria. Donor cells were added at a concentration similar to the natural abundance of bacteria in the water column (10⁶ cells ml⁻¹). Transfer was not detected in any of the test microcosms (calculated limit of detection of 10⁻⁷ and 10⁻⁴ transconjugants donor⁻¹ in water column and sediment, respectively), with the exception of transfer to an isogenic recipient (added at 10⁵ cells ml⁻¹) in sediments of controls that had been inoculated with both donors and recipients. The same plasmid was transferred with high efficiencies (10⁻¹ to 10⁻³) to a variety of recipients in filter and broth matings. These results suggest that if conjugal gene transfer occurred, it was at efficiencies that were not detectable in estuarine microcosms simulating natural population densities.     

Use of impedance measurements to estimate numbers of antibiotic resistant Salmonella strains

Impedance detection times were compared with traditional plating methods for enumerating antibiotic-resistant strains of Salmonella stanley, Salm. thompson and Salm. infantis grown in laboratory medium and pork slurry. The correleation of log₁₀ counts of salmonellas with detection times was highly significant ( r = -0·96 for broth and r = -0·94 for slurries. The confidence limits (± og₁₀ 1·0 for broth and ± log₁₀ 1·65 for slurry) indicated that detection times could reliably be used as a rapid means of enumerating salmonellas when large numbers of counts of known strains are required for growth studies. Use of antibiotic-resistant strains also permitted their selective detection by impedance from the natural spoilage flora of pork slurry when the same antibiotics were incorporated in the detection medium.     

Effect of high-temperature, short-time (HTST) pasteurization on milk containing low numbers of Mycobacterium paratuberculosis

The efficacy of high-temperature, short-time (HTST) pasteurization (72 °C/15 s) when low numbers (≤ 10³ cfu ml ⁻¹ ) of Mycobacterium paratuberculosis are present in milk was investigated. Raw cows' milk spiked with Myco. paratuberculosis (10³ cfu ml⁻¹, 10² cfu ml⁻¹, 10 cfu ml⁻¹, and 10 cfu 50 ml⁻¹) was subjected to HTST pasteurization using laboratory pasteurizing units. Ten bovine strains of Myco. paratuberculosis were tested in triplicate. Culture in BACTEC Middlebrook 12B radiometric medium detected acid-fast survivors in 14·8% and 10% of HTST-pasteurized milk samples at the 10³ and 10² cfu ml⁻¹ inoculum levels, respectively, whereas conventional culture on Herrold's egg yolk medium containing mycobactin J detected acid-fast survivors in only 3·7% and 6·7% of the same milk samples. IS900-based PCR confirmed that these acid-fast survivors were Myco. paratuberculosis . No viable Myco. paratuberculosis were isolated from HTST-pasteurized milk initially containing either 10 cfu ml⁻¹ or 10 cfu 50 ml⁻¹.     

Antimicrobial action of essential oils : the effect of dimethylsulphoxide on the activity of cinnamon oil

Fifty-one essential oils extracted from plants of known origin were tested for their antimicrobial activity against three bacteria, Pseudomonas aeruginosa , Staphylococcus aureus , Escherichia coli and four yeasts, Torulopsis utilis , Schizosaccharomyces pombe , Candida albicans and Saccharomyces cerevisiae using the drop diffusion method. All showed antimicrobial activity against at least one of the micro-organisms. Following this preliminary screening, 13 essential oils showing antimicrobial activity against at least five of the micro-organisms were tested in the range 50 μg ml⁻¹ to 500 μg ml⁻¹ using broth micro dilution techniques with dimethylsulphoxide (DMSO) as a dispersing solvent. The concentration of most of the oils required for total inhibition of growth was >500 μg ml⁻¹. Further studies on the antimicrobial action of cinnamon oil in the range 10–150 μg ml⁻¹ showed that 50-fold higher activity was found when no dispersing solvent was used.