Nutrient-rich Broth Research Articles

Validation of the MicroSnap Coliform and E. coli Test System for Enumeration and Detection of Coliforms and E. coli in a Variety of Foods.

The MicroSnap Coliform and E. coli system was devised to give rapid enumeration and detection of coliforms and/or Escherichia coli strains in a sample of food within an 8 h working shift. The method measures β-galactosidase and β-glucuronidase enzymes using novel bioluminogenic substrates which develop an output light signal proportional to the concentration of enzyme discovered. The assay uses two different phases to determine the enzyme concentration. The first phase is an enrichment of the sample in a nutrient-rich broth device at 37 ± 0.5°C. After 6 or 8 h, an aliquot is taken from the enrichment device and injected into the Coliform Detection Device, which is assayed in a luminometer after 10 min of incubation at 37 ± 0.5°C. Samples testing positive in the Coliform Detection Device can be subsequently assayed specifically for E. coli using the E. coli Detection Device. The relative light unit output from the detection device is proportional to the bacterial concentration when the incubation was initiated, which is proportional to the contamination level in the matrix being assessed. The MicroSnap Coliform and E. coli system was evaluated for both quantitative and qualitative analysis of coliforms and E. coli in a variety of foods. Three different luminometers were used in the analysis, each of which has different functionalities and different sensitivities. The MicroSnap method showed good correlation with the appropriate corresponding reference method for enumeration of coliforms and E. coli. A statistically significant difference was seen in detection of E. coli in milk, as reported by the independent laboratory. The reference method reported higher mean Log10 CFU counts than the MicroSnap method; however, no significant differences were seen between the MicroSnap system and reference methods for any of the other matrixes. Inclusivity testing was conducted on 25 different non-E. coli coliforms and 25 different E. coli strains, and exclusivity testing was conducted on 30 different species of nontarget organisms. Two E. coli strains were not detected in the Coliform Detection Device after 8 h on one of the instruments. All other inclusivity strains tested were detected after 8 h of incubation. None of the exclusivity strains were detected. The lot-to-lot and kit stability studies showed no statistical differences between lots or over the term of the shelf-life. Robustness studies indicate that the timing of incubation for the detection phase is critical for correct system functioning.

Transcriptome Sequence and Plasmid Copy Number Analysis of the Brewery Isolate Pediococcus claussenii ATCC BAA-344T during Growth in Beer

Growth of specific lactic acid bacteria in beer leads to spoiled product and economic loss for the brewing industry. Microbial growth is typically inhibited by the combined stresses found in beer (e.g., ethanol, hops, low pH, minimal nutrients); however, certain bacteria have adapted to grow in this harsh environment. Considering little is known about the mechanisms used by bacteria to grow in and spoil beer, transcriptome sequencing was performed on a variant of the beer-spoilage organism Pediococcus claussenii ATCC BAA-344T (Pc344-358). Illumina sequencing was used to compare the transcript levels in Pc344-358 growing mid-exponentially in beer to those in nutrient-rich MRS broth. Various operons demonstrated high gene expression in beer, several of which are involved in nutrient acquisition and overcoming the inhibitory effects of hop compounds. As well, genes functioning in cell membrane modification and biosynthesis demonstrated significantly higher transcript levels in Pc344-358 growing in beer. Three plasmids had the majority of their genes showing increased transcript levels in beer, whereas the two cryptic plasmids showed slightly decreased gene expression. Follow-up analysis of plasmid copy number in both growth environments revealed similar trends, where more copies of the three non-cryptic plasmids were found in Pc344-358 growing in beer. Transcriptome sequencing also enabled the addition of several genes to the P . claussenii ATCC BAA-344T genome annotation, some of which are putatively transcribed as non-coding RNAs. The sequencing results not only provide the first transcriptome description of a beer-spoilage organism while growing in beer, but they also highlight several targets for future exploration, including genes that may have a role in the general stress response of lactic acid bacteria.

The effect of sublethal concentrations of benzalkonium chloride on invasiveness and intracellular proliferation of Listeria monocytogenes

Listeria monocytogenes, which survives improper cleaning and disinfection processes are a major source of recontamination of food products, where they can survive or even multiply until consumption. In this study we investigated the effect of sublethal concentration of benzalkonium chloride (BAC) on the virulence of L. monocytogenes using human intestinal, hepatocytic and macrophage-like cells, three cell types involved in the human infection pathway of L. monocytogenes. Therefore we used four different strains, the type strain EGDe and three isolates from food or food processing environment, which revealed different susceptibility to BAC. Incubation of L. monocytogenes with sublethal concentrations of BAC reduced the invasiveness of all four strains in all three types of cells, respectively. However, a significant decrease could only be observed in the strains more sensitive to BAC. Furthermore sublethal concentration of BAC increased significantly the intracellular proliferation for all four strains in all three different human cell types. Interestingly, this effect of disinfectants on the virulence of L. monocytogenes was maintained even after 24 h incubation in a nutrient-rich broth, which mimics the food matrix. In conclusion we showed that exposure of L. monocytogenes to sublethal concentrations of disinfectants, although reducing invasion, supports the survival and growth of intracellular bacteria.This study underlines the importance of proper disinfection in food processing environment to limit the number of L. monocytogenes foodborne illnesses.

Optimization of Human Granulocyte Macrophage-Colony Stimulating Factor (hGM-CSF) Expression Using Asparaginase and Xylanase Gene’s Signal Sequences in Escherichia coli

The toxicity of the recombinant protein towards the expression host remains a significant deterrent for bioprocess development. In this study, the expression of human granulocyte macrophage-colony stimulating factor (hGM-CSF), which is known to be toxic to its host, was enhanced many folds using a combination of genetic and bioprocess strategies in Escherichia coli. The N terminus attachment of endoxylanase and asparaginase signal sequences from Bacillus subtilis and E. coli, respectively, in combination with and without His-tag, considerably improved expression levels. Induction and media optimization studies in shake flask cultures resulted in a maximal hGM-CSF concentration of 365 mg/L in the form of inclusion bodies (IBs) with a specific product yield (Y (P/X)) of 120 mg/g dry cell weight in case of the asparaginase signal. Culturing the cells in nutrient rich Terrific broth maintained the specific product yields (Y (P/X)) while a 6.6-fold higher volumetric concentration of both product and biomass was obtained. The purification and refolding steps were optimized resulting in a 95% pure protein with a fairly high refolding yield of 45%. The biological activity of the refolded protein was confirmed by a cell proliferation assay on hGM-CSF dependent human erythroleukemia TF-1 cells. This study demonstrated that this indeed is a viable route for the efficient production of hGM-CSF.

Attenuation ofMycobacterium tuberculosisFunctionally Disrupted in a Fatty Acyl–Coenzyme A Synthetase GenefadD5

One key adaptation that Mycobacterium tuberculosis established to survive long term in vivo is a reliance on lipids as an energy source. M. tuberculosis H37Rv has 36 fadD genes annotated as putative fatty acyl-coenzyme A (CoA) synthetase genes, which encode enzymes that activate fatty acids for metabolism. One such gene, fadD5 (Rv0166), is located within the mce1 operon, a cluster of genes associated with M. tuberculosis persistence. We disrupted the putative fatty acid-binding site of fadD5 in H37Rv M. tuberculosis. No significant differences were found in the growth of the mutant and wild-type strains in vitro in nutrient-rich broth or in activated RAW264.7 cells. However, the fadD5 mutant was diminished in growth in minimal medium containing mycolic acid but not other long-chain fatty acids. C57BL/6 mice infected with the fadD5 mutant survived significantly longer than those infected with the wild type, and the mutant never attained the plateau phase of infection in mouse lungs. Infection in the steady-state phase was maintained for up to 168 days at a level that was 1-2 logs less than that noted in the wild type. These observations raise the rather intriguing possibility that FadD5 may serve to recycle mycolic acids for the long-term survival of the tubercle bacilli.

Heterologous expression of a tannic acid-inducible laccase3 of Cryphonectria parasitica in Saccharomyces cerevisiae

BackgroundA tannic acid-inducible and mycoviral-regulated laccase3 (lac3) from the chestnut blight fungus Cryphonectria parasitica has recently been identified, but further characterization was hampered because of the precipitation of protein products by tannic acid supplementation. The present study investigated the heterologous expression of the functional laccase3 using a yeast Saccharomyces cerevisiae.ResultsLaccase activity in the culture broth of transformants measured using a laccase-specific substrate suggested that the lac3 gene was successfully expressed and the corresponding protein product secreted into the culture media. In addition, activity staining and Western blot analysis of a native gel revealed that the enzyme activity co-existed with the protein product specific to anti-laccase3 antibody, confirming that the cloned lac3 gene is responsible for the laccase activity. When transformants were grown on plates containing tannic acid-supplemented media, brown coloration was observed around transformed cells, indicating the oxidation of tannic acid. However, the enzymatic activity was measurable only in the selective ura- media and was negligible in nonselective nutrient-rich culture conditions. This was in part because of the increased plasmid instability in the nonselective media. Moreover, the protein product of lac3 appears to be sensitive to the cultured nonselective nutrient-rich broth, because a rapid decline in enzymatic activity was observed when the cultured broth of ura- media was mixed with that of nonselective nutrient-rich broth. In addition, constitutive expression of the lac3 gene resulted in a reduced cell number of the lac3 transformants compared to that of vector-only transformed control. However, the presence of recombinant vector without lac3 induction did not affect the growth of transformants.ConclusionsThe results suggest that expression of the lac3 gene has an inhibitory effect on the growth of transformed S. cerevisiae and that the controlled expression of lac3 is appropriate for the possible application of recombinant yeast to the treatment of phenolic compounds.

HtaA Is an Iron-Regulated Hemin Binding Protein Involved in the Utilization of Heme Iron in Corynebacterium diphtheriae

Many human pathogens, including Corynebacterium diphtheriae, the causative agent of diphtheria, use host compounds such as heme and hemoglobin as essential iron sources. In this study, we examined the Corynebacterium hmu hemin transport region, a genetic cluster that contains the hmuTUV genes encoding a previously described ABC-type hemin transporter and three additional genes, which we have designated htaA, htaB, and htaC. The hmu gene cluster is composed of three distinct transcriptional units. The htaA gene appears to be part of an iron- and DtxR-regulated operon that includes hmuTUV, while htaB and htaC are transcribed from unique DtxR-regulated promoters. Nonpolar deletion of either htaA or the hmuTUV genes resulted in a reduced ability to use hemin as an iron source, while deletion of htaB had no effect on hemin iron utilization in C. diphtheriae. A comparison of the predicted amino acid sequences of HtaA and HtaB showed that they share some sequence similarity, and both proteins contain leader sequences and putative C-terminal transmembrane regions. Protein localization studies with C. diphtheriae showed that HtaA is associated predominantly with the cell envelope when the organism is grown in minimal medium but is secreted during growth in nutrient-rich broth. HtaB and HmuT were detected primarily in the cytoplasmic membrane fraction regardless of the growth medium. Hemin binding studies demonstrated that HtaA and HtaB are able to bind hemin, suggesting that these proteins may function as cell surface hemin receptors in C. diphtheriae.

Resuscitation of Seventeen-year Stressed Salmonella typhimurium

Abstract Salmonella enterica serovar typhimurium was stressed by incubation in seawater microcosms for seventeen years. The microcosms were prepared in such a way as to allow progressive evaporation of the water. Despite being introduced into the sterile seawater at very high concentrations, the Salmonella rapidly declined to levels undetectable by plate counts on nutrient agar. After two years of starvation, about half of the seawater volume had evaporated from each microcosm, and salt crystals appeared. Inoculation of the salty suspension did not result in any culturable strains in selective and non-selective media. However, incubation of samples in nutrient-rich broth, without supplemental growth factors, allowed resuscitation of stressed cells, yielding colonies that remained viable for extended periods of time. After three years the total volume of water had evaporated from each microcosm, and only salt crystals remained. These microcosms were then incubated at room temperature for seventeen years. Resuscitation of VBNC Salmonella from the salt crystals was conducted in vitro and in vivo; recovery occurring after incubation in both nutrient broth and in mice. Recovery occurred in the Salmonella administered orally into the mice, but not in those administered by intra-peritoneal injection. The identity of initial, stressed and revived strains was confirmed by DGGE analysis and sequencing of 16S RNA gene fragments.

Resuscitation of eleven-year VBNC Citrobacter

Citrobacter freundii strain WA1 was stressed by incubation in seawater microcosms for eleven years. After two years of starvation, no culturable strain was observed. Incubation of samples in nutrient-rich broth medium not supplemented with growth factors, however, allowed resuscitation of VBNC cells so that subsequent plating yielded observable colonies for significantly extended periods of time. Recovery of VBNC Citrobacter freundii was obtained by incubation in nutrient broth even after eleven years of starvation. To see whether the samples contained the same strain of Citrobacter freundii inoculated 11 years ago. The complete 16S rRNA gene was PCR amplified and sequenced from initial, stressed and revived strains of Citrobacter freundii strain WA1.The 16S rRNA gene sequences from eleven-year stressed strains were homologous with a high degree of similarity to the GenBank reference strain and were identical to each other.

New Insights into the Colonization and Release Processes of Xenorhabdus nematophila and the Morphology and Ultrastructure of the Bacterial Receptacle of Its Nematode Host, Steinernema carpocapsae

We present results from epifluorescence, differential interference contrast, and transmission electron microscopy showing that Xenorhabdus nematophila colonizes a receptacle in the anterior intestine of the infective juvenile (IJ) stage of Steinernema carpocapsae. This region is connected to the esophagus at the esophagointestinal junction. The process by which X. nematophila leaves this bacterial receptacle had not been analyzed previously. In this study we monitored the movement of green fluorescent protein-labeled bacteria during the release process. Our observations revealed that Xenorhabdus colonizes the distal region of the receptacle and that exposure to insect hemolymph stimulated forward movement of the bacteria to the esophagointestinal junction. Continued exposure to hemolymph caused a narrow passage in the distal receptacle to widen, allowing movement of Xenorhabdus down the intestine and out the anus. Efficient release of both the wild type and a nonmotile strain was evident in most of the IJs incubated in hemolymph, whereas only a few IJs incubated in nutrient-rich broth released bacterial cells. Incubation of IJs in hemolymph treated with agents that induce nematode paralysis dramatically inhibited the release process. These results suggest that bacterial motility is not required for movement out of the distal region of the receptacle and that hemolymph-induced esophageal pumping provides a force for the release of X. nematophila out of the receptacle and into the intestinal lumen.

The role of nutrient presence on the adhesion kinetics of Burkholderia cepacia G4g and ENV435g

The adhesion kinetics of Burkholderia cepacia G4g and ENV435g have been investigated in a radial stagnation point flow (RSPF) system under well-controlled hydrodynamics and solution chemistry. The sensitivity of adhesion behavior to nutrient condition was also examined. Supplementary cell characterization techniques were conducted to evaluate the viability, hydrophobicity, electrophoretic mobility, size, and charge density of cells grown in both nutrient rich Luria broth (LB) and nutrient poor basal salts medium (BSM). Comparable adhesion kinetics were observed for the wild-type (G4g) and mutant (ENV435g) grown in the same medium; however, the attachment efficiency increased with the level of nutrient presence for both cell types by approximately 60%. Nutrient condition altered deposition due to its impact on the surface charge characteristics and size of the cells. Adhesion behavior was consistent with expectations based on classical Derjaguin–Landau–Verwey–Overbeek (DLVO) theory for colloidal interactions, as the adhesion efficiency increased with ionic strength. However, the results also suggest the involvement of non-DLVO type interactions that influence cell adhesion. Systematic experimentation with B. cepacia in the RSPF system demonstrated that the ENV435g mutant is not “adhesion deficient”; rather, adhesion for both the G4g and ENV435g was a function of the nutrient condition and resulting cell surface chemistry.

Quantitative Effects of Carbohydrates and Aromatic Amino Acids on Clostridium botulinum Toxin Gene Expression Using a Rapid Competitive RT/PCR Assay

A rapid competitive RT/PCR assay was developed to determine the effects of nutrients on Clostridium botulinum type E toxin gene expression. The type E strain (EVH) was grown in a nutrient-rich broth containing 1% glucose (base medium). Toxin gene expression was quantified at both mid and late exponential phases of growth. It was found that toxin encoding mRNA levels were highly growth phase dependent with elevated levels found in late exponential phase compared to mid exponential phase. Changing the carbohydrate source had a smaller effect on toxin encoding mRNA levels but as earlier results have suggested, toxin encoding mRNA levels show a strong correlation with type E growth rate. The results have important implications for the food industry whereby risk of type E botulism could be correlated to the nutrient composition of the contaminated food or assessed from C. botulinum growth rates in challenged foodstuffs.

English

Staphylococcus sp. isolated from oil-contaminated soil was grown in 1% drilling fluid base oil, HDF- 2000, as a sole source of carbon and energy. The organism has strong affinity for the substrate, growing at the rate of 0.16 h-1. It uses adherence and emulsification as mechanisms for oil uptake. In a nutrient-rich marine broth, base oil (up to 2.0% v/v) and glucose (up to 1.6% w/v) have no significant effect on the growth rates. This showed that the Staphylococcus sp. is a strong primary utilizer of the base oil and has potential for application in bioremediation processes involving oil-based drilling fluids. Key words : Drilling fluid base oil, Staphylococcus sp., biodegradation African Journal of Biotechnology Vol.2(9) 2003: 293-295

Controls on Fe reduction and mineral formation by a subsurface bacterium

The reductive dissolution of FeIII (hydr)oxides by dissimilatory iron-reducing bacteria (DIRB) could have a large impact on sediment genesis and Fe transport. If DIRB are able to reduce FeIII in minerals of high structural order to carry out anaerobic respiration, their range could encompass virtually every O 2-free environment containing FeIII and adequate conditions for cell growth. Previous studies have established that Shewanella putrefaciens CN32, a known DIRB, will reduce crystalline Fe oxides when initially grown at high densities in a nutrient-rich broth, conditions that poorly model the environments where CN32 is found. By contrast, we grew CN32 by batch culture solely in a minimal growth medium. The stringent conditions imposed by the growth method better represent the conditions that cells are likely to encounter in their natural habitat. Furthermore, the expression of reductases necessary to carry out dissimilatory Fe reduction depends on the method of growth. It was found that under anaerobic conditions CN32 reduced hydrous ferric oxide (HFO), a poorly crystalline FeIII mineral, and did not reduce suspensions containing 4 mM FeIII in the form of poorly ordered nanometer-sized goethite (α-FeOOH), well-ordered micron-sized goethite, or nanometer-sized hematite (α-Fe 2O 3) crystallites. Transmission electron microscopy (TEM) showed that all minerals but the micron-sized goethite attached extensively to the bacteria and appeared to penetrate the outer cellular membrane. In the treatment with HFO, new FeII and FeIII minerals formed during reduction of HFO-Fe in culture medium containing 4.0 mmol/L P i (soluble inorganic P), as observed by TEM with energy-dispersive X-ray spectroscopy, selected area electron diffraction, and X-ray diffraction. The minerals included magnetite (Fe 3O 4), goethite, green rust, and vivianite [Fe 3(PO 4) 2 · 8H 2O]. Vivianite appeared to be the stable end product and the mean coherence length was influenced by the rate of FeIII reduction. When P i was 0.4 mol/L under otherwise identical conditions, goethite was the only mineral observed to form, and less Fe 2+ was produced overall. Hence, the ability of DIRB to reduce Fe (hydr)oxides may be limited when the bacteria are grown under nutrient-limited conditions, and the minerals that result depend on the vigor of FeIII reduction.

Resuscitation of Salmonella enterica serovar typhimurium and enterohemorrhagic Escherichia coli from the viable but nonculturable state by heat-stable enterobacterial autoinducer.

Salmonella enterica serovar Typhimurium and enterohemorrhagic Escherichia coli were stressed by prolonged incubation in water microcosms until it was no longer possible to observe colony formation when samples were plated on nonselective medium. Overnight incubation of samples in nutrient-rich broth medium supplemented with growth factors, however, allowed resuscitation of stressed and viable but nonculturable cells so that subsequent plating yielded observable colonies for significantly extended periods of time. The growth factors were (i) the trihydroxamate siderophore ferrioxamine E (for Salmonella only), (ii) the commercially available antioxidant Oxyrase, and (iii) the heat-stable autoinducer of growth secreted by enterobacterial species in response to norepinephrine. Analysis of water microcosms with the Bioscreen C apparatus confirmed that these supplements enhanced recovery of cells in stressed populations; enterobacterial autoinducer was the most effective, promoting resuscitation in populations that were so heavily stressed that ferrioxamine E or Oxyrase had no effect. Similar results were observed in Bioscreen analysis of bacterial populations stressed by heating. Patterns of resuscitation of S. enterica serovar Typhimurium rpoS mutants from water microcosms and heat stress were qualitatively similar, suggesting that the general stress response controlled by the sigma(s) subunit of RNA polymerase plays no role in autoinducer-dependent resuscitation. Enterobacterial autoinducer also resuscitated stressed populations of Citrobacter freundii and Enterobacter agglomerans.

Identification of novel hrp-regulated genes through functional genomic analysis of the Pseudomonas syringae pv. tomato DC3000 genome.

Pseudomonas syringae pv. tomato (Pst) strain DC3000 infects the model plants Arabidopsis thaliana and tomato, causing disease symptoms characterized by necrotic lesions surrounded by chlorosis. One mechanism used by Pst DC3000 to infect host plants is the type III protein secretion system, which is thought to deliver multiple effector proteins to the plant cell. The exact number of type III effectors in Pst DC3000 or any other plant pathogenic bacterium is not known. All known type III effector genes of P. syringae are regulated by HrpS, an NtrC family protein, and the HrpL alternative sigma factor, which presumably binds to a conserved cis element (called the "hrp box") in the promoters of type III secretion-associated genes. In this study, we designed a search motif based on the promoter sequences conserved in 12 published hrp operons and putative effector genes in Pst DC3000. Seventy-three predicted genes were retrieved from the January 2001 release of the Pst DC3000 genome sequence, which had 95% genome coverage. The expression of the 73 genes was analysed by microarray and Northern blotting, revealing 24 genes/operons (including eight novel genes), the expression of which was consistently higher in hrp-inducing minimal medium than in nutrient-rich Luria-Bertani broth. Expression of all eight genes was dependent on the hrpS gene. Most were also dependent on the hrpL gene, but at least one was dependent on the hrpS gene, but not on the hrpL gene. An AvrRpt2-based type III translocation assay provides evidence that some of the hrpS-regulated novel genes encode putative effector proteins.

Characterization of spontaneous gacS and gacA regulatory mutants of Pseudomonas fluorescens biocontrol strain CHAO.

In Pseudomonasfluorescens strain CHAO, the response regulator gene gacA controls expression of extracellular enzymes and antifungal secondary metabolites, which are important for this strain's biocontrol activity in the plant rhizosphere. Two Tn5 insertion mutants of strain CHA0 that had the same pleiotropic phenotype as gacA mutants were complemented by the gacS sensor kinase gene of P. syringae pv. syringae as well as that of P. fluorescens strain Pf-5, indicating that both transposon insertions had occurred in the gacS gene of strain CHA0. This conclusion was supported by Southern hybridisation using a gacS probe from strain Pf-5. Overexpression of the wild-type gacA gene partially compensated for the gacS mutation, however, the overexpressed gacA gene was not stably maintained, suggesting that this is deleterious to the bacterium. Strain CHA0 grown to stationary phase in nutrient-rich liquid media for several days accumulated spontaneous pleiotropic mutants to levels representing 1.25% of the population; all mutants lacked key antifungal metabolites and extracellular protease. Half of 44 spontaneous mutants tested were complemented by gacS, the other half were restored by gacA. Independent point and deletion mutations arose at different sites in the gacA gene. In competition experiments with mixtures of the wild type and a gacA mutant incubated in nutrient-rich broth, the mutant population temporarily increased as the wild type decreased. In conclusion, loss of gacA function can confer a selective advantage on strain CHA0 under laboratory conditions.

Rapid and economical detection of Salmonella enteritidis in eggs by the polymyxin-cloth enzyme immunoassay

A rapid, simple and economical procedure for the detection of Salmonella enteritidis in eggs was developed. The contents of whole eggs inoculated with low numbers of S. enteritidis were mixed with a minimal volume of a nutrient-rich broth (1:2 ratio of egg to broth) and incubated overnight. The lipopolysaccharide (LPS) antigens of S. enteritidis were extracted by heating in the presence of cholate. The antigens were captured on polymyxincoated polyester cloth, and the captured antigens were detected by sequential reactions with anti-serogroup D1 rabbit antiserum, anti-rabbit antibody-peroxidase conjugate and tetramethylbenzidine substrate solution. This polymyxin-cloth enzyme immunoassay (polymyxin-CEIA) was highly specific for salmonellae bearing the factor 0:9 antigen, reacting in the assay of 19 S. enteritidis strains tested, including two rough isolates, but not with salmonellae lacking the factor 0:9 antigen or non- Salmonella bacteria. The threshold sensitivity of the polymyxin-CEIA for S. enteritidis suspensions was ca. 10 6 cfu ml . This combined enrichment culture and polymyxin-CEIA required less than 24 h to complete and detected as few as 1–2 S. enteritidis cfu inoculated into a whole egg. This procedure should facilitate the routine monitoring of S. enteritidis in large numbers of egg samples.