Antimicrobial properties of three lactic acid bacterial cultures and their cell free supernatants against Listeria monocytogenes.

Control of Listeria monocytogenes in ready-to-eat (RTE) food products is a significant challenge and improved means for control are needed. In this study, the anti-listerial effects of three lactic acid bacteria (LAB) were investigated. Spot-on-lawn assays demonstrated the largest zones of inhibition against L. monocytogenes were produced by the Pediococcus acidilactici strain, with zone diameters ranging from 13 to 18 mm. Minimum inhibitory concentration (MIC) experiments using cell free supernatant (CFS) from the LAB revealed that while two of the strains were effective at inhibiting L. monocytogenes growth only up to a 1:4 dilution, P. acidilactici was able to inhibit growth up to a 1:256 dilution. Survival assays performed at 7°C determined that the P. acidilactici strain was capable of producing a 4.5 log reduction in L. monocytogenes counts and maintaining the reduction for 21 days. The effectiveness of P. acidilactici was reduced under log phase growth, autoclaving for longer than 15 min (121°C and 15 psi), and treatment with proteinase K (25 mg/mL).

A review of the ecology, genomics, and stress response of Listeria innocua and Listeria monocytogenes.

Listeria monocytogenes is a Gram-positive foodborne pathogen responsible for a severe disease occurring in immuno-compromised populations. Foodborne illness caused by L. monocytogenes is a serious public health concern because of the high associated mortality. Study of the closely related, but nonpathogenic Listeria innocua has accounted for a better understanding of the behavior of L. monocytogenes in environments beyond the laboratory. Traditionally, the ecological co-habitation, genomic synteny, and physiological similarity of the two species have supported use of L. innocua for predicting the behavior of L. monocytogenes in farm and food processing environments. However, a careful review of the current literature indicates that in a given situation it may not be prudent to use L. innocua as a surrogate for L. monocytogenes without prior confirmation of their similar phenotypes, as an increasing number of studies have arisen demonstrating differences in L. monocytogenes and L. innocua stress response, and furthermore, there are differences among the L. monocytogenes subgroups. Future research should take into consideration that multiple surrogates might be required to accurately model even a single condition depending on the L. monocytogenes subgroup of interest.

Enhanced inhibition of Listeria monocytogenes by a combination of cold pressed terpeneless Valencia orange oil and antibiotics.

This study was designed to evaluate the ability of cold pressed terpeneless Valencia orange oil (CPTVO) to enhance the effectiveness of antibiotics against 10 strains of Listeria monocytogenes. Disc diffusion assays were performed to determine the effects of CPTVO and two antibiotics with different mechanisms of action (i.e., penicillin and chloramphenicol) individually and in combination with CPTVO. CPTVO alone produced zones ranging from 16.5 to 19.9 mm. Penicillin at 2 or 10 units produced zones ranging from <6 to 13.4 mm, and from 16 to 19.5 mm, respectively. Chloramphenicol at 5 or 30 µg had zones ranging from <6 to 6.9 mm, and from 10.8 to 15.9 mm, respectively. Penicillin (2 and 10 units) plus CPTVO produced zones ranging from 20.2 to 25.3 mm, and from 21.9 to 28 mm, respectively. Chloramphenicol (5 or 30 µg) plus CPTVO produced zones of from 20.1 to 26.6 mm, and from 19.5 to 23.9 mm, respectively. In conclusion, the combination of antibiotics with CPTVO increases their ability to inhibit L. monocytogenes.

Inhibition of Listeria monocytogenes by exposure to a combination of nisin and cold-pressed terpeneless Valencia oil.

Listeria monocytogenes is an important foodborne pathogen and its control in foods is a significant challenge. This study evaluated the effectiveness of nisin and cold-pressed terpeneless Valencia oil (CPTVO) on limiting L. monocytogenes growth. Disk diffusion assays were performed to determine the effects of CPTVO and nisin individually and in combination. Together, these antimicrobials produced a zone of inhibition that was significantly larger (P < 0.05) than zones correlating to CPTVO or nisin individually. Furthermore, L. monocytogenesΔsigB had an increased sensitivity to the combination treatment. Growth experiments performed in brain heart infusion (BHI) broth revealed the effects of nisin and CPTVO, individually and in combination on L. monocytogenes growth rate. When L. monocytogenes was grown in BHI containing 0.025% CPTVO and 26 IU/mL nisin, no growth inhibition was observed relative to the control. However, exposure to CPTVO at 0 h followed by the introduction of nisin at 15 h resulted in a statistically significant (P < 0.05) reduction in growth. This approach to inhibiting L. monocytogenes has potential as an all-natural, generally-recognized-as-safe multiple hurdle intervention that may be applicable for ready-to-eat products in which L. monocytogenes is likely to cause foodborne illness.

Efficacy of cold-pressed terpeneless Valencia oil and its primary components on inhibition of Listeria species by direct contact and exposure to vapors.

This study used disk diffusion assays to evaluate the effectiveness of cold-pressed terpeneless Valencia oil (CPTVO) and its primary components (linalool, citral, and decanal) at inhibiting Listeria via direct contact or exposure to vapors. In general, all Listeria strains tested responded similarly to CPTVO and its components. Direct contact with linalool produced zones of inhibition that were significantly smaller (P < 0.0001) than those associated with all other antimicrobials tested. Zones of inhibition for sealed plates were significantly larger (P < 0.0001) than those observed for unsealed plates, although the method for sealing the plates was insignificant. Exposure to CPTVO vapors resulted in zones of inhibition that were significantly smaller than those resulting from decanal vapors (P < 0.0001). The difference observed between the zones of inhibition produced by antimicrobial exposure via vapors or direct contact was only slightly significant (P = 0.02). Antimicrobial essential oil (EO) vapors may be an effective alternative to direct contact EOs to safely and effectively inhibit microorganisms while minimizing undesired organoleptic changes sometimes associated with EO contact. CPTVO and its primary components, decanal and citral, may have potential in the food industry as all natural, generally recognized as safe antimicrobials used in modified atmosphere packaging designed to inhibit Listeria without requiring direct contact with food products.

Listeria monocytogenes: antibiotic resistance in food production.

Listeria monocytogenes is an opportunistic human pathogen that causes listeriosis, a disease that mainly affects the immunocompromised, the elderly, infants, and pregnant women. Listeriosis has become increasingly common in the last 25 years since the first foodborne outbreak was noted. Treatment for listeriosis currently consists primarily of supportive therapy in conjunction with the use of intravenous antibiotics. Antibiotics have been commercially available for over 60 years for treatment of a myriad of clinical diseases. Bacteria resistant to antibiotics have been developing over this same period. This review seeks to elucidate the extent of antibiotic resistance in L. monocytogenes, the possible transmission mechanisms, and contributing factors to distribution of antibiotic resistance among Listeria species, and possible control strategies.

Listeria marthii sp. nov., isolated from the natural environment, Finger Lakes National Forest.

Four isolates (FSL S4-120(T), FSL S4-696, FSL S4-710, and FSL S4-965) of Gram-positive, motile, facultatively anaerobic, non-spore-forming bacilli that were phenotypically similar to species of the genus Listeria were isolated from soil, standing water and flowing water samples obtained from the natural environment in the Finger Lakes National Forest, New York, USA. The four isolates were closely related to one another and were determined to be the same species by whole genome DNA-DNA hybridization studies (>82 % relatedness at 55 degrees C and >76 % relatedness at 70 degrees C with 0.0-0.5 % divergence). 16S rRNA gene sequence analysis confirmed their close phylogenetic relatedness to Listeria monocytogenes and Listeria innocua and more distant relatedness to Listeria welshimeri, L. seeligeri, L. ivanovii and L. grayi. Phylogenetic analysis of partial sequences for sigB, gap, and prs showed that these isolates form a well-supported sistergroup to L. monocytogenes. The four isolates were sufficiently different from L. monocytogenes and L. innocua by DNA-DNA hybridization to warrant their designation as a new species of the genus Listeria. The four isolates yielded positive reactions in the AccuProbe test that is purported to be specific for L. monocytogenes, did not ferment L-rhamnose, were non-haemolytic on blood agar media, and did not contain a homologue of the L. monocytogenes virulence gene island. On the basis of their phenotypic characteristics and their genotypic distinctiveness from L. monocytogenes and L. innocua, the four isolates should be classified as a new species within the genus Listeria, for which the name Listeria marthii sp. nov. is proposed. The type strain of L. marthii is FSL S4-120(T) (=ATCC BAA-1595(T) =BEIR NR 9579(T) =CCUG 56148(T)). L. marthii has not been associated with human or animal disease at this time.

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 DeltaRD30 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.

Contributions of six lineage-specific internalin-like genes to invasion efficiency of Listeria monocytogenes.

Listeria monocytogenes strains are divided into at least three lineages, which seem to differ in virulence. Internalins are surface-attached or secreted proteins that encode leucine-rich repeats, and L. monocytogenes encodes species-specific as well as lineage-specific internalin and internalin-like genes. Internalins A and B have previously been shown to be critical for L. monocytogenes host cell invasion. Transcription of selected internalins is regulated by the virulence gene regulator PrfA and/or the stress-responsive alternative sigma factor sigma(B). We hypothesized that lineage-specific internalin-like genes may contribute to differential virulence and niche adaptation of the L. monocytogenes lineages. Initial quantitative real time, reverse transcriptase PCR (RT-PCR) showed that the six selected lineage-specific internalin-like genes were transcribed in cells grown at 16 degrees and 37 degrees C. Lineage-specific internalin-like gene, lineage II (lsiIIX) showed significantly higher transcript levels in log-phase cells grown at 37 degrees C as compared to 16 degrees C. The gene lsiIA was preceded by a putative sigma(B)-dependent promoter and showed sigma(B)-dependent transcription. None of the null mutants in lineage-specific internalin-like genes differed from their respective parent strain in ability to invade either human intestinal epithelial or hepatocyte-like cell lines. All three mutants in lineage I-specific internalin-like genes exhibited the same growth condition-dependent invasion phenotype as their parent strain ( approximately 1.5 log higher invasion efficiency when grown at 30 degrees C without aeration versus 37 degrees C with aeration). Despite structural similarities to internalins with known roles in host cell attachment and invasion, none of the six lineage-specific internalin-like genes characterized here appear to contribute to invasion. Combined with the observation that some nonpathogenic Listeria species also carry internalin genes, our findings suggest a broad role of Listeria internalins, not limited to attachment and invasion of human cells. Due to the wide host range of L. monocytogenes and the fact that transcription of internalin-like genes can differ considerably depending on growth condition, elucidating the function of different internalins and internalin-like genes will remain a challenge.

Growth and persistence of Listeria monocytogenes isolates on the plant model Arabidopsis thaliana.

While the majority of human listeriosis cases appear to be linked to consumption of processed ready-to-eat foods (e.g., deli meats), a few listeriosis outbreaks have been linked to consumption of contaminated vegetables. In this study, we assessed four isolates representing the major Listeria monocytogenes lineages for their abilities to attach to and grow on Arabidopsis thaliana, a well-characterized plant model. When plants were dipped for 5min into 3ml of water containing 8.8logCFU of L. monocytogenes and rinsed repeatedly, L. monocytogenes was recovered from the leaves at densities from 1.52 to 2.17logCFU/cm(2). Ten days after exposure, bacterial numbers had increased over initial numbers by 2.60-2.95logCFU/cm(2). Using L. monocytogenes expressing GFP, bacteria were visualized in the intercellular spaces of A. thaliana leaves, suggesting internalization through stomata. These data indicate that L. monocytogenes can rapidly attach to and multiply on plant surfaces and colonize intercellular spaces in A. thaliana leaves where it may be protected from sanitation treatments. When A. thaliana seeds were exposed to L. monocytogenes, between 4.23 and 4.57logCFU/cm(2) were recovered from leaves 7 days post-germination, suggesting that contaminated seeds can produce contaminated plants. Overall, our study demonstrates that prevention of L. monocytogenes contamination of plants throughout growing stages is critical, consistent with recommendations for other produce-transmitted foodborne pathogens.