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.     

An improved ELISA method for the detection of Salmonella typhimurium

The applicability of enzyme-linked immunosorbent assay (ELISA) for the detection of salmonellas in foodstuffs was investigated. Several factors affecting the sensitivity of the ELISA, such as the type of protein used for plate post-coating, the method of antibody labelling, and accelerators for antigen-antibody and enzyme-substrate reactions, were studied. Labelling of the antibody with horseradish peroxidase and the use of o-phenylenediamine as substrate in the detection system were demonstrated to be most suitable for the enzyme assay. Based on these findings, an improved ELISA method was developed for the detection of Salmonella typhimurium. The improved technique was able to detect as few as 5 x 10(4)-10(5) cell/ml of salmonellas, and about 24 h were required to enrich the bacteria in food samples and to perform the test. With some modifications, the ELISA assay could reach a very high level of sensitivity and provide excellent reproducibility.     

Evaluation of the Vitek Immunodiagnostic Assay System (VIDAS) for the detection of Salmonella in foods

A Salmonella Assay using the Vitek Immunodiagnostic Assay System (VIDAS) was compared with a conventional cultural method (CCM) for the detection of salmonellas in 141 samples of artificially and naturally contaminated foods. There was an overall agreement of 92.9% between the methods. The productivity of the VIDAS Salmonella Assay (VSA) was not improved using an alternative enrichment protocol for the detection of Salmonella in 12 raw meat samples.
The sensitivity and specificity of the VSA was assessed using pure cultures of salmonellas and non-salmonellas. The detection limit was 1.8 times 10⁶ salmonellas ml^-1 in M-broth and some Citrobacter freundii strains gave false-positive results.
Using an immunomagnetic separation (IMS) technique and an abbreviated cultural enrichment, the VSA results could be obtained a day earlier than the standard VSA method.     

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.     

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.     

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.     

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.     

Limitations of the enzyme-linked immunosorbent assay for routine determination of legume inoculant quality

Peat inoculants containing strains of either Rhizobium or Bradyrhizobium spp. were used to determine correlations between cell numbers and A₄₀₅ values obtained with double antibody sandwich enzyme-linked immunosorbent assay (DAS ELISA) and indirect ELISA. ELISA values of inoculants containing strains of Rhizobium were weak and non-specific; with Bradyrhizobium spp. strains, readings were higher and cross-reactions negligible when heated inoculant suspensions were allowed to stand for 3 h before ELISA determinations were made. With soybean inoculant, correlation coefficients of r = 0.93 and 0.83 were obtained with DAS and indirect ELISA, respectively. A linear curve relating log cell numbers to A₄₀₅ values was used to determine the reliability of DAS ELISA values obtained over 2 years with tests on commercially produced soybean inoculants. In the range 5 times 10⁸-ca 3 times 10⁹ cells/g inoculant, DAS ELISA estimates closely followed plate counts but no significant correlation was found when inoculants contained >ca 3 times 10⁹ cells/g. With a minimum requirement of 1 times 10⁹ cells/g inoculant, discrepancies between DAS ELISA estimates and plate counts obtained with inoculants produced with gamma-irradiated peat would have resulted in the erroneous rejection or acceptance of 14.5% of all inoculants tested, based on DAS ELISA estimates. With inoculants produced with steam-sterilized peat, which was unfavourable for survival of strain WB1, 70.0% of the inoculants rejected because of low plate counts would have been acceptable on the basis of DAS ELISA estimates.     

Polymerase chain reaction detection and speciation of Campylobacter upsaliensis and C. helveticus in human faeces and comparison with culture techniques

A polymerase chain reaction (PCR) assay based on the 16S rRNA gene and an improved DNA extraction procedure were developed for the direct detection and differentiation of Campylobacter upsaliensis and C. helveticus in seeded human faeces. The PCR assay was compared with culture detection by a membrane filter (MF) technique and on selective agar (SA) containing 8 mg l⁻¹ cefoperazone. Both MF culture and the PCR assay detected 10⁵ colony-forming units (cfu) g⁻¹ faeces. Selective agar culture of some strains could detect as few as 10³ cfu g⁻¹ faeces. However, some strains were susceptible to cefoperazone and either failed to grow or were detected only with reduced sensitivity in the presence of the antibiotic. Detection by MF and SA both required 48–96 h incubation in a microaerobic atmosphere and did not specifically identify the isolate. By contrast, the PCR assay could be completed within 8 h and accurately identified the two phenotypically similar species, C. upsaliensis and C. helveticus.     

MULTIPLEX DETECTION OF ESCHERICHIA COLI AND SALMONELLA ENTERITIDIS BY USING QUANTUM DOT-LABELED ANTIBODIES

In this study, we demonstrated the simultaneous detection of Escherichia coli and Salmonella enteritidis, by coupling immunomagnetic separation (IMS) with quantum dots (QDs) labeling. QDs having different emission wavelengths were conjugated with anti- E. coli and anti- Salmonella antibodies. QD–antibody conjugates were used to label immunomagnetically separated bacteria and the fluorescence intensities were measured for enumerations of both species. The concentrations of primary antibodies used in IMS, the ratio of QDs to antibodies during the conjugation and the concentration of QD–antibody conjugates used in labeling were optimized to enhance the sensitivity of the assay. After labeling bacteria with QDs, the quenching observed between bead–bacteria complex and QDs was eliminated by separating QDs from the complex using sodium dodecyl sulfate solution. The fluorescence intensities due to the capturing of different concentrations of bacteria were measured and the working ranges were found to be 5 × 10² to 5 × 10⁵ cfu/mL for E. coli and 4  ×  10² to 4  ×  10⁵ cfu/mL for S. enteritidis .

PRACTICAL APPLICATIONS

In this study, antibody-conjugated multicolor quantum dots (QDs) were used for simultaneous detection of Escherichia coli and Salmonella enteritidis . The results of this study indicate that QD labels can be used in multiplex, rapid and selective detection of bacteria with detection limits comparable with those of many novel methods in cases where the assay conditions are optimized. Furthermore, the assay can be modified for the simultaneous detection of more than two species through using QD labels having different emission wavelengths.     

Detection of low levels of specific Salmonella species by fluorescent antibodies and flow cytometry

The use of fluorescently-labelled monoclonal antibodies, with detection by multi-parameter flow cytometry, was investigated for the rapid detection of salmonellas in pure cultures. Accurate detection of specific Salmonella serotypes was demonstrated down to levels of below 10⁴ cells ml^-1 (within 30 min) and 1 cell ml^-1 (after 6 h non-selective pre-enrichment). This level of sensitivity was attainedeven in the presence of high levels of other bacterial species that would otherwise have interfered with the results. With combinations of different antibodies, each with a unique fluorescent label, simultaneous analysis for two species was possible.     

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.     

Improved PCR detection sensitivity of Erwinia carotovora subsp. atroseptica in potato tuber peel extract by prior enrichment on a selective medium

A simple and sensitive method was developed to replace the need for complex and laborious DNA extraction to remove inhibitory substances in potato tuber peel extract before detection of Erwinia carotovora subsp. atroseptica (Eca) by PCR. Eca was enriched by a factor of 10⁵ when peel extract was inoculated onto a selective medium, CVP, and incubated at 27°C for 24 h. Bacterial micro-colonies which developed were suspended in 500 μl of water and the bacteria diluted in water 100-fold, or 10-fold followed by washing by centrifugation, before PCR testing. The sensitivity of detection obtained with the former was ca 10¹–10² cells ml⁻¹ and with the latter ca 10¹ cells ml⁻¹, when different numbers of streptomycin-resistant Eca strain were added to peel extract from three Eca-free potato cultivars. The method was validated and the sensitivity confirmed relative to two different commonly used Eca detection methods using naturally contaminated tubers.     

Time-resolved fluoroimmunoassay as a method for detection of Erwinia chrysanthemi in potato peel extracts

J.M. VAN DER WOLF. 1993. Time-resolved fluoroimmunoassay (TR-FIA) was compared with double antibody sandwich (DAS)-ELISA for detection of Erwinia chrysanthemi antigens in potato peel extracts. Pure cultures were used to optimize TR-FIA with respect to the microplate washing procedure and dilution buffer compositions.
The detection threshold level for spiked potato peel extracts with the optimized TR-FIA format was 10⁵ cells ml^-1 as for the detection level of DAS-ELISA. The signal to background ratios at concentrations above 10⁵ cells ml^-1 were higher with TR-FIA than with DAS-ELISA. The dynamic range of TR-FIA was also superior to that of DAS-ELISA.
It can be concluded that TR-FIA is an attractive alternative to DAS-ELISA as a detection method for Erw. chrysanthemi, especially when quantification is required.     

Quantitative detection of meat spoilage bacteria by using the polymerase chain reaction (PCR) and an enzyme linked immunosorbent assay (ELISA)

A quantitative PCR-ELISA for the rapid enumeration of bacteria in refrigerated raw meat has been developed using primers designed from conserved regions in the 16S ribosomal RNA gene (rRNA). Amplified PCR products generated using a digoxigenin-labelled primer were automatically hybridized to a biotinylated probe included in the PCR reaction. The hybridization was performed as part of the PCR programme. The biotin-digoxigenin hybrids were quantified by an enzyme-linked immunosorbent assay (ELISA). Streptavidin bound to the wells of a microtitre plate was used to capture the biotin-digoxigenin-labelled fragments that were detected with a peroxidase anti-digoxigenin conjugate. Subsequent enzymic conversion of substrate gave distinct absorbance differences when assaying meat samples containing bacteria in the range 10²–10⁷ cfu cm⁻². The detection threshold for the PCR-ELISA assay developed in this work is 10² cfu cm⁻².     

Monoclonal antibody detection of Pseudomonas spp. in refrigerated meat by an indirect ELISA

Monoclonal antibodies generated against live cells of Pseudomonas fluorescens have been used in an indirect ELISA format for the detection of Pseudomonas spp. in refrigerated meat. The detection threshold for the ELISA assay developed in this work was 10⁴ cfu cm⁻².     

Detection of Yersinia enterocolitica in food by PCR amplification

A polymerase chain reaction (PCR) assay was developed for detection of pathogenic, virulent strains of Yersinia enterocolitica . By using both virulence loci virF and ail as markers for pathogenicity, detection of species with a virulence factor present was possible. DNA preparation in the presence of hexadecyl trimethy ammonium bromide (CTAB) was followed by two 44 cycle amplification reactions, one for each of the markers. As few as 10² Y. enterocolitica cells were detected in ground pork in the presence of 10⁵–10⁶ bacteria of other species. The described PCR assay provides a sensitive robust assay for the detection of virulent Y. enterocolitica in food.     

Experimental enzyme-linked amperometric immunosensors for the detection of salmonellas in foods

Two enzyme-linked amperometric immunosensors specific for salmonellas were developed as rapid methods for quantifying and detecting these organisms in pure cultures and foods. Both used alkaline phosphatase as the enzyme reporter molecule but one system used phenyl phosphate as the substrate followed by the electrochemical detection of phenol at a polarized platinum electrode. The other system incorporated an enzyme amplification step and relied on the electrochemical detection of a reduced mediator, ferrocyanide. Both assays were rapid (4 h) and specific and generated salmonella-dependent signals above 10⁴ cfu/ml (phenyl phosphate system) or 10⁵ cfu/ml (enzyme amplified system) in pure cultures and samples of several foods, although the results with beef samples showed considerable variation. Both systems were able to detect low (1–5 cfu/g or /ml) numbers of salmonellas in foods after non-selective (18 h) and selective (22 h) enrichment steps but four samples, out of 147, gave false positive results. False positive results were eliminated by reducing the enrichment steps to 6 h and 18 h respectively (90 samples).     

Detection of bacterial antigens using immuno-PCR

E. KAKIZAKI, T. YOSHIDA, H. KAWAKAMI, M. OSETO, T. SAKAI AND M. SAKAI. 1996. A new and very sensitive antigen detection technique, immuno-polymerase chain reaction (immuno-PCR), was developed. This method is basically similar to the enzyme-linked immunosorbent assay which detects an antigen-antibody reaction, but instead of an enzyme being conjugated to an antibody, a DNA fragment is used and this DNA can be amplified by PCR. We applied this method to the detection of the fish pathogen, Pasteurella piscicida , in naturally infected yellowtail. Using immuno-PCR, 3.4 cfu ml⁻¹ of bacteria could be detected. In comparison, ELISA detected only 3.4 × 10⁴ cfu ml⁻¹. Immuno-PCR is a powerful method for detection of pathogens in host tissues.     

Rapid detection of pathogenic Vibrio parahaemolyticus by a sensitive and specific duplex PCR-hybridization probes assay using LightCycler

Aims:  To develop a real-time polymerase chain reaction (PCR) hybridization probe assay for rapid and specific detection of thermostable direct haemolysin-producing Vibrio parahaemolyticus.
Methods and Results:  Primers and hybridization probes were designed to target the toxR and tdh2 genes. Mismatches were introduced in the tdh2 primers for specific amplification of the target. The 3' ends of donor probes for both genes were labelled with fluorescein. The 5' ends of recipient probes for tdh2 and toxR were labelled with LC Red 640 and LC Red 705, respectively. The real-time assay was evaluated against conventional biochemical tests and the KAP-RPLA kit (Kanagawa phenomenon detection kit by reverse passive latex agglutination). toxR and tdh2 were detected in 100% and 91% of clinical V. parahaemolyticus isolates ( n  = 118), respectively. Specificity and sensitivity of the real-time assay for toxR and tdh2 were 100%, respectively. Dynamic range of detection for toxR was 10⁷–10¹ CFU ml⁻¹ and that for tdh2 was 10⁷–10⁴ CFU ml⁻¹.
Conclusions:  The LightCycler assay described is sensitive and highly specific for detection of pathogenic V. parahaemolyticus in a single reaction tube within 80 min.
Significance and Impact of the Study:  The assay developed allows accurate detection of pathogenic V. parahaemolyticus , which is valuable for rapid tracing of infection source during outbreaks.