Hypovirulent Listeria monocytogenes strains are less frequently recovered than virulent strains on PALCAM and Rapid' L. mono media

Several selective media have been developed to detect Listeria monocytogenes contaminated foodstuffs. Polymyxin-acriflavine-LiCl-ceftazidime-aesculin-mannitol (PALCAM) and Oxford media, required for the EN ISO method 11 290-1, are used for the detection of Listeria spp. in 2 days based on the expression of esculinase activity. Selective agar media such as Rapid' L. mono and Agar Listeria according to Ottaviani and Agosti (ALOA), based on the activity of phosphatidylinositol phospholipase C (PI-PLC) that allows the specific detection of L. monocytogenes in 2 days, are also used. However, no medium can assess the level of virulence of L. monocytogenes strains. Using a plaque-forming assay followed by subcutaneous footpad inoculation in mice, 15 virulent, 8 hypovirulent and 17 avirulent strains were discriminated among L. monocytogenes strains mainly originating from food (36/40). Their growth was tested on the four selective media. After 2 days, the number of colony forming units (cfu) of all the virulent strains was significantly superior to the number obtained with avirulent strains on all the four media tested, and superior to the number obtained with hypovirulent strains on PALCAM and Oxford media. These results showed a relationship between the level of virulence of L. monocytogenes strains and their growth on the selective agar media tested. Moreover, 1 out of 8 hypovirulent and 5 out of 17 avirulent strains did not grow on Rapid' L. mono medium, and 1 hypovirulent and 8 avirulent strains grew but did not express PI-PLC activity during the 7 days of incubation. The lack of detection of PI-PLC activity on Rapid' L. mono was not related to a gene mutation since these strains expressed enzymatic activity on ALOA medium, which detected up to 92% of the hypo- and avirulent strains. In contrast, some of these strains without growth or enzymatic activity expression would not be detected with PALCAM and Rapid' L. mono in foodstuffs on the second day.     

SigB plays a major role in Listeria monocytogenes tolerance to bile stress

The ability of Listeria monocytogenes to tolerate high levels of bile stress is critical to its successful infection and colonization in the human gastrointestinal tract. L. monocytogenes encodes bile salt hydrolase by a bsh gene which plays a significant role in hydrolyzing high concentrations of bile salt when L. monocytogenes grows under hypoxemic condition. As the bsh promoter contains consensus SigB and PrfA binding sites, we investigated the role of SigB (σ^B) and PrfA in L. monocytogenes tolerance against bile stress by comparing the survival of isogenic deletion mutants of L. monocytogenes EGD_ΔsigB, EGD_ΔprfA and EGD_ΔprfAΔsigB with their parent strain EGD at high levels of bile salt. Our results show that the sigB deletion significantly reduced the MICs of bile salt for EGD_ΔsigB and EGD_ΔprfAΔsigB (2.6% and 2.2% vs 3.5% in wild type strain EGD), while the growth rates of these two sigB deletion mutants (EGD_ΔsigB and EGD_ΔprfAΔsigB) were affected the most in the presence of 3% bile salt. Pre-exposure to alkali (pH 9.0) and osmotic (0.3 M NaCl) stresses for a short period of time (30 min) resulted in improved growth of L. monocytogenes as well as its prfA-sigB isogenic mutants even under sublethal concentrations of bile salt, while pre-exposure to acid pH (pH 4.5) failed to provide cross-protection against subsequent bile stress. Furthermore, the sigB gene had more remarkable influence than that of prfA on bsh expression, as much lower levels of bsh transciption were observed in EGD_ΔsigB and EGD_ΔprfAΔsigB. Meanwhile, bsh expression in the deletion mutants did not respond to elevated levels of bile salt. These data indicate that σ^B might play a crucial role in Listeria survival under bile salt environment in the gastrointestinal tract before its successful colonization, invasion and intracellular propagation.     

Quantifying Listeria monocytogenes prevalence and concentration in minced pork meat and estimating performance of three culture media from presence/absence microbiological testing using a deterministic and stochastic approach

Listeria monocytogenes poses a serious threat to public health, and the majority of cases of human listeriosis are associated with contaminated food. Reliable microbiological testing is needed for effective pathogen control by food industry and competent authorities. The aims of this work were to estimate the prevalence and concentration of L. monocytogenes in minced pork meat by the application of a Bayesian modeling approach, and also to determine the performance of three culture media commonly used for detecting L. monocytogenes in foods from a deterministic and stochastic perspective. Samples (n = 100) collected from local markets were tested for L. monocytogenes using in parallel the PALCAM, ALOA and RAPID'L.mono selective media according to ISO 11290-1:1996 and 11290-2:1998 methods. Presence of the pathogen was confirmed by conducting biochemical and molecular tests. Independent experiments (n = 10) for model validation purposes were performed. Performance attributes were calculated from the presence–absence microbiological test results by combining the results obtained from the culture media and confirmative tests. Dirichlet distribution, the multivariate expression of a Beta distribution, was used to analyze the performance data from a stochastic perspective. No L. monocytogenes was enumerated by direct-plating (<10 CFU/g), though the pathogen was detected in 22% of the samples. L. monocytogenes concentration was estimated at 14–17 CFU/kg. Validation showed good agreement between observed and predicted prevalence (error = −2.17%). The results showed that all media were best at ruling in L. monocytogenes presence than ruling it out. Sensitivity and specificity varied depending on the culture-dependent method. None of the culture media was perfect in detecting L. monocytogenes in minced pork meat alone. The use of at least two culture media in parallel enhanced the efficiency of L. monocytogenes detection. Bayesian modeling may reduce the time needed to draw conclusions regarding L. monocytogenes presence and the uncertainty of the results obtained. Furthermore, the problem of observing zero counts may be overcome by applying Bayesian analysis, making the determination of a test performance feasible.     

Liquid and solid selective differential media for the detection and enumeration of L. monocytogenes and other Listeria spp

A selective and differential medium (PALCAM agar) was elaborated for the isolation and enumeration of Listeria monocytogenes. PALCAM is based on Columbia agar with 0.05% glucose made selective by the addition of 0.001% polymyxin B, 0.0005% acriflavin, 1.5% lithium chloride and 0.002% ceftazidime. The diagnostic traits were attained by the incorporation of (i) 0.08% aesculin and 0.05% ferric salt; and (ii) 1% mannitol plus 0.008% phenol red. PALCAM recovered test strains of L. monocytogenes and other Listeria spp quantitatively and suppressed most other bacteria of common occurrence in fresh food. L. monocytogenes colonies were approximately 2 mm grey-green with a black sunken centre and a black halo on a cherry-red background. The occasional Enterococcus or Staphylococcus strains developing on the medium gave rise to grey colonies with a brown-green halo or yellow colonies with a yellow halo. PALCAM was the preferred medium out of 13 tested Listeria selective agars in current use. A similar differential enrichment broth, L-PALCAMY was developed based on peptone yeast extract broth with 2.5% egg yolk emulsion. The diagnostic traits and inhibitors used in this medium were the same as in PALCAM agar, through in different concentrations. Growth rate and cellcrop of L. monocytogenes in L-PALCAMY were of the same order as in Columbia broth. The growth of the majority of other bacteria of common occurrence in fresh foods was inhibited. The medium recovered L. monocytogenes more effectively from severely contaminated food than other current enrichment media.     

Development of a loop-mediated isothermal amplification assay for detecting Listeria monocytogenes prfA in milk

A rapid and sensitive loop-mediated isothermal amplification (LAMP) assay was developed for detecting Listeria monocytogenes prfA in milk. The inclusivity of 23 L. monocytogenes and the exclusivity of 16 non-L. monocytogenes strains were both 100% in the assay. The limit of detection (LoD) of the LAMP assay in Listeria enrichment broth (LEB) was 2.22 CFU/mL after 12 h and 24 h of incubation. The LoDs of the LAMP assay in LEB with artificially contaminated milk (LEB-M) incubated for 12 h (2.22×10¹ CFU/mL) and 24 h (2.22 CFU/mL) were lower than those of the PCR and real-time PCR assays. Comparison of the LoDs in LEB with those in LEB-M showed that the LAMP assay was less influenced by the milk compounds than the real-time PCR assay. Our results indicate that the LAMP assay can be utilized as a potential screening tool for L. monocytogenes in milk.     

Habituation to organic acid anions induces resistance to acid and bile in Listeria monocytogenes

We evaluated the intrinsic and inducible resistance of four human pathogenic strains of Listeria monocytogenes to acid and bile, factors associated with virulence. Cells were grown in media at pH 7.4, or in media at pH 6.0 containing 0 (HCl control) or 4.75 mM of different organic acids, harvested at stationary or mid log phase, and challenged for 1 h in acid or bile. Stationary phase cells were intrinsically more resistant to either challenge than log phase cells, and large differences between strains were evident among the latter. Compared to the HCl control, habituation to log phase with organic acids induced significant (p < 0.05) and meaningful (≥ 1 log) increases in acid resistance of three of four strains tested, and in bile resistance of two strains suggesting that exposure to organic acid anions may enhance virulence in L. monocytogenes.     

Simultaneous detection of Listeria monocytogenes and Salmonella by multiplex PCR in cooked ham

A multiplex PCR (m-PCR) assay with an internal amplification control (IAC) was developed for the simultaneous detection of Salmonella spp. and Listeria monocytogenes through invA and prfA genes, respectively. To ensure the detection of the pathogens in cooked ham, samples were enriched in both buffered peptone-water and Half Fraser broth. Subsequently, equal volumes of enrichment broths were mixed and DNA purification was performed prior to m-PCR reaction, saving considerable time and effort. The m-PCR also proved to be very useful as a simple and ready-to-go method for simultaneous confirmation of presumptive L. monocytogenes and Salmonella spp. colonies directly from agar plates without any DNA extraction steps.     

Reduced growth of Listeria monocytogenes in two model cheese microcosms is not associated with individual microbial strains

Two model antilisterial microbial communities consisting of two yeasts, two Gram positive and two Gram negative bacteria, and originating from Livarot cheese smear were previously designed. They were used in the present study to analyse the impact of microbial population dynamics on growth of Listeria monocytogenes in cheese microcosm. Specific culture media and PCR primers were developed for simultaneous culture-dependent and real-time PCR quantification of strains belonging to Marinomonas sp., Paenibacillus sp., Staphylococcus equorum, Arthrobacter arilaitensis, Pseudomonas putida, Serratia liquefaciens, Candida natalensis, and Geotrichum candidum, in cheese microcosms. All strains were enumerated after 3, 5, 8 and 14 days at 15 °C. They established well at high counts in all cheese microcosms. Growth dynamics for all strains in presence of L. monocytogenes WSLC 1685 were compared to those of microbial communities obtained by omitting in turn one of the six members of the initial community. The growth of the microbial strains was neither markedly disturbed by Listeria presence nor by the removal of each strain in turn. Furthermore, these communities had a significant reducing effect on growth of L. monocytogenes independently of pH, as confirmed by mathematical modelling. A barrier effect was observed, that could be explained by specific competition for nutrients.     

Kinetics of resuscitation and growth of L. monocytogenes as a tool to select appropriate enrichment conditions as a prior step to rapid detection methods

Rapid methods still rely on a prior (shortened) enrichment step before application. Quantitative information is a prerequisite for understanding the resuscitation kinetics of the growth during the enrichment step. In this study various basal and newly introduced selective enrichment broths were evaluated. First, growth parameters (λ, μ_max) of both healthy and sub-lethally injured cells were determined. Next, a selection of enrichment broths was compared for their capacity to support detection within 24 h of low numbers of Listeria monocytogenes in artificially and naturally contaminated food samples. Detection was performed either by phage protein-based capture (Listeria Capture kit, Profos, Regensburg, Germany) combined with plating on chromogenic medium or by fluorescence in situ hybridization (FISH) using the VIT-Listeria kit (Vermicon, Munich, Germany). Kinetics of resuscitation and growth of L. monocytogenes in various enrichment broths showed that for detection of low numbers of sub-lethally injured L. monocytogenes cells at least an overnight enrichment was needed. A selective enrichment broth was needed to enable proliferation of L. monocytogenes within the indigenous bacterial flora present in foods. However, combination of an appropriate enrichment condition with advanced detection techniques may enable a 24 h detection of L. monocytogenes.     

Incidence of Listeria monocytogenes in different food products commercialized in Portugal

Several types of food products on sale in Portugal, were examined for the presence of Listeria monocytogenes. Secondary enrichments, in Fraser broth, were analysed by the mini-Vidas LMO, enzyme-linked fluorescent immunoassay technique. Positive samples were confirmed by isolation on Oxford and PALCAM selective agars followed by biochemical characterization. Of 1035 samples, 72 (7.0%) were positive for L. monocytogenes, the majority being from raw products (milk, meat, fish, flour) although some heat-processed or fermented foods (ready-to-eat) were also positive. In Portugal, a predilection for fresh cheese was indicated as a potential risk for consumers.     

Development of a nucleic acid lateral flow immunoassay for simultaneous detection of <Emphasis Type="Italic">Listeria</Emphasis> spp. and <Emphasis Type="Italic">Listeria</Emphasis><Emphasis Type="Italic">monocytogenes</Emphasis> in food

We present a new nucleic acid lateral flow immunoassay (NALFIA) for the assessment of listeria contamination. The detection procedure starts with enrichment of sample in Half Fraser broth (24 h). Following isolation of DNA, a duplex PCR is performed with two labelled primer sets, one generic and directed to a specific sequence of the gene encoding 16S rRNA from Listeria spp. and the other specific and directed to a part of the prfA gene encoding the central virulence gene regulator from the food pathogen Listeria monocytogenes (3.5 h). The PCR solution is directly added to the one-step assay device and the appearance of a grey/black line is indicative of the presence of specific amplicons (max 15 min). In all tests performed, the method correctly identified L. monocytogenes and strains of Listeria spp. PCR material of over 20 food samples was tested by NALFIA. The method proved to be useful for the detection of L. monocytogenes in different kinds of food samples.     

Performance of Three Culture Media Commonly Used for Detecting Listeria monocytogenes

Listeria monocytogenes poses a serious threat to public health, and the majority of cases of human listeriosis are associated with contaminated food. Reliable microbiological testing is needed for effective control of this pathogen by the food industry and competent authorities. The aim of this study was to determine the performance of three culture media commonly used for detecting L. monocytogenes in foods. Minced pork meat samples (n = 100) were subjected to microbiological testing for L. monocytogenes according to International Organization for Standardization methods 11290-1:1996 and 11290-2:1998 using PALCAM, ALOA, and RAPID'L. mono culture media in parallel. Presence of the pathogen was confirmed by conducting biochemical and molecular tests on the presumptive L. monocytogenes colonies. Performance attributes of sensitivity, specificity, positive and negative predictive values, positive and negative likelihood ratios, diagnostic odds ratios, error odds ratios, receiving operating characteristic (ROC) curve, and area under this curve were calculated from the presence-absence microbiological test results by combining the results obtained from the culture media and confirmative tests. PALCAM had the best performance in terms of positive predictive value (i.e., a positive result indicates high probability of L. monocytogenes presence) but not in terms of sensitivity (i.e., the ability of the medium to detect the pathogen when present). RAPID'L. mono was the most sensitive medium. None of the culture media were perfect for detecting L. monocytogenes in minced pork meat alone. The pathogen was detected in 16, 19, and 26% (apparent prevalence) of the samples by PALCAM, ALOA, and RAPID'L. mono, respectively, although the true prevalence of the pathogen was 22%. These findings indicate that the use of a single culture medium may lead to erroneous determination of the prevalence of L. monocytogenes.     

Survival of probiotic adjunct cultures in cheese and challenges in their enumeration using selective media

Various selective media for enumerating probiotic and cheese cultures were screened, with 6 media then used to study survival of probiotic bacteria in full-fat and low-fat Cheddar cheese. Commercial strains of Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus paracasei, or Bifidobacterium lactis were added as probiotic adjuncts. The selective media, designed to promote growth of certain lactic acid bacteria (LAB) over others or to differentiate between LAB, were used to detect individual LAB types during cheese storage. Commercial strains of Lactococcus, Lactobacillus, and Bifidobacterium spp. were initially screened on the 6 selective media along with nonstarter LAB (NSLAB) isolates. The microbial flora of the cheeses was analyzed during 9 mo of storage at 6°C. Many NSLAB were able to grow on media presumed selective for Lactococcus, Bifidobacterium spp., or Lb. acidophilus, which became apparent after 90 d of cheese storage, Between 90 and 120 d of storage, bacterial counts changed on media selective for Bifidobacterium spp., suggesting growth of NSLAB. Appearance of NSLAB on Lb. casei selective media [de man, Rogosa, and Sharpe (MRS) + vancomycin] occurred sooner (30 d) in low-fat cheese than in full-fat control cheeses. Differentiation between NSLAB and Lactococcus was achieved by counting after 18 to 24 h when the NSLAB colonies were only pinpoint in size. Growth of NSLAB on the various selective media during aging means that probiotic adjunct cultures added during cheesemaking can only be enumerated with confidence on selective media for up to 3 or 4 mo. After this time, growth of NSLAB obfuscates enumeration of probiotic adjuncts. When adjunct Lb. casei or Lb. paracasei cultures are added during cheesemaking, they appear to remain at high numbers for a long time (9 mo) when counted on MRS + vancomycin medium, but a reasonable probability exists that they have been overtaken by NSLAB, which also grow readily on this medium. Enumeration using multiple selective media can provide insight into whether it is the actual adjunct culture or a NSLAB strain that is being enumerated.     

Bayesian inference for quantifying Listeria monocytogenes prevalence and concentration in minced pork meat from presence/absence microbiological testing

The purpose of this work was to estimate the prevalence and concentration of Listeria monocytogenes in minced pork meat by the application of a Bayesian modeling approach. Samples (n?=?100) collected from local markets were tested for L.?monocytogenes using in parallel the PALCAM, ALOA and RAPID'L.mono selective media. Presence of the pathogen was confirmed through biochemical and molecular tests. Independent experiments (n?=?10) for validation purposes were performed. No L.?monocytogenes was enumerated by direct-plating (<10?CFU/g), though the pathogen was detected in 22% of the samples. Sensitivity and specificity varied depending on the culture method. L.?monocytogenes concentration was estimated at 14-17?CFU/kg. Validation showed good agreement between observed and predicted prevalence (error?=?-2.17%). The use of at least two culture media in parallel enhanced the efficiency of L.?monocytogenes detection. Bayesian modeling may reduce the time needed to draw conclusions regarding L.?monocytogenes presence and the uncertainty of the results obtained.     

Contributions to selected phenotypic characteristics of large species- and lineage-specific genomic regions in Listeria monocytogenes

We hypothesized that genomic regions specific to Listeria monocytogenes or selected L. monocytogenes strains may contribute to virulence and phenotypic differences among the strains. A whole genome alignment of two completed L. monocytogenes genomes and the one completed Listeria innocua genome initially identified 28 genomic regions of difference (RD) > 4 kb that were found in one or both L. monocytogenes genomes, but absent from the non-pathogenic L. innocua. In silico analyses using an additional 18 draft L. monocytogenes genomes showed that (i) 15 RDs were found in all or most L. monocytogenes genomes; (ii) three RDs were found in all or most lineage I genomes, but absent from lineage II genomes; and (iii) four RDs were found in all lineage II genomes, but no lineage I genomes. Null mutants in two L. monocytogenes-specific RDs (RD16 and RD30; found in most L. monocytogenes) and the lineage II-specific RD25 showed no evidence for impaired invasion or intracellular growth in selected tissue culture cells. Although, in pH 5.5 minimal media, the ΔRD30 null mutant showed reduced ability to compete with its parent strain, indicating that RD30 may have a role in L. monocytogenes growth under limited nutrient conditions at acidic pH.     

Comparison of two chromogenic media for the detection of Listeria monocytogenes with the plating media recommended by EN/DIN 11290-1

The efficacy of two selective chromogenic culture media, Agar Listeria Ottaviani and Agosti (ALOA) and RAPID' L. mono for the detection of Listeria monocytogenes in food, was compared with that of an official culture method according to the EN/DIN 11290-01 and -02 protocols [corresponding to the section 35 LMBG (German Food Act) method]. A total of 310 pre-packed ready-to-eat food samples (100 of graved and cold smoked salmon, 130 of different raw and cooked sausages and 80 of delicatessen and mixed salads) were examined. L. monocytogenes was identified in 52 investigated salmon samples. Using two chromogenic media, 50 samples were found positive for L. monocytogenes. Compared to the reference method there were no false-positive results. By the EN/DIN 11290-01 culture procedure after the selective enrichment in Fraser broth 12 out of 130 samples of sausages were positive for L. monocytogenes. These 12 samples were also positive for L. monocytogenes with the chromogenic medium RAPID' L. mono. One sample was false negative with ALOA. Three additional samples were found positive with ALOA and four with RAPID' L. mono. The standard method was inadequate to confirm these samples as positive. Listeria spp. were isolated from 7 samples of mixed salads with both methods. One, 3 and 3 samples were found to contain L. monocytogenes, L. innocua and L. seeligeri, respectively. Both chromogenic media enabled a rapid and specific detection of L. monocytogenes within 24h after enrichment. Visual detection of pathogenic L. monocytogenes and other Listeria spp. was easier on chromogenic media.     

Comparison of plating media for recovery of total and virulent genotypes of Vibrio vulnificus in U.S. market oysters

Vibrio vulnificus is the leading cause of seafood associated mortality in the United States and is generally associated with consumption of raw oysters. Two genetic markers have emerged as indicators of strain virulence, 16S rDNA type B (rrnB) and virulence correlated gene type C (vcgC). While much is known about the distribution of V. vulnificus in oysters, a limited number of studies have addressed the more virulent subtypes. Therefore, the goals of this study were to (1) determine the suitability of media for recovery of total and virulent genotypes of V. vulnificus and (2) evaluate the geographical and seasonal distribution of these genotypes. Market oysters from across the United States and the strains isolated from them during a year-long study in 2007 were used. For media evaluation, VVA and CPC + were compared using direct plating of oyster tissues while mCPC and CPC + were compared for isolation following MPN enrichment. Representative isolates from each media/method were tested for rrn and vcg types to determine their seasonal and geographical distribution. No statistically significant difference was observed between VVA/CPC + or mCPC/CPC + for isolation of total or virulent (rrnB/vcgC) genotypes of V. vulnificus. Overall, 32% of recovered isolates possessed the virulent genotype. The prevalence of these genotypes was highest in oysters from the Gulf Coast during Oct–Dec, and demonstrated a statistically significant geographical and seasonal pattern. This is the first report on the distribution of virulent V. vulnificus genotypes across the United States, which provides novel insight into the occurrence of this pathogen.