Efficacies of Nisin A and Nisin V Semipurified Preparations Alone and in Combination with Plant Essential Oils for Controlling Listeria monocytogenes

The food-borne pathogenic bacterium Listeria is known for relatively low morbidity and high mortality rates, reaching up to 25 to 30%. Listeria is a hardy organism, and its control in foods represents a significant challenge. Many naturally occurring compounds, including the bacteriocin nisin and a number of plant essential oils, have been widely studied and are reported to be effective as antimicrobial agents against spoilage and pathogenic microorganisms. The aim of this study was to investigate the ability of semipurified preparations (SPP) containing either nisin A or an enhanced bioengineered derivative, nisin V, alone and in combination with low concentrations of the essential oils thymol, carvacrol, and trans-cinnamaldehyde, to control Listeria monocytogenes in both laboratory media and model food systems. Combinations of nisin V-containing SPP (25 μg/ml) with thymol (0.02%), carvacrol (0.02%), or cinnamaldehyde (0.02%) produced a significantly longer lag phase than any of the essential oil-nisin A combinations. In addition, the log reduction in cell counts achieved by the nisin V-carvacrol or nisin V-cinnamaldehyde combinations was twice that of the equivalent nisin A-essential oil treatment. Significantly, this enhanced activity was validated in model food systems against L. monocytogenes strains of food origin. We conclude that the fermentate form of nisin V in combination with carvacrol and cinnamaldehyde offers significant advantages as a novel, natural, and effective means to enhance food safety by inhibiting food-borne pathogens such as L. monocytogenes.     

Bioactivity of selected essential oils and some components on Listeria monocytogenes biofilms

Listeria monocytogenes is a Gram-positive bacterium, able to survive and grow in water, soil, agricultural products, various foods and the food-processing environment. The ubiquitous nature of the organism, coupled with its ability to colonise food-processing surfaces by forming biofilms, causes it to be of a major concern to the industry. Increased foodborne pathogen resistance and negative consumer perceptions regarding the use of synthetic bacteriocides, has resulted in natural antimicrobials being sourced from the plant kingdom. The listerial antibiofilm activities of Syzygium aromaticum (clove), Mentha spicata (spearmint), Lippia rehmannii and Cymbopogon citratus (lemongrass) essential oils and their major components were evaluated using the crystal violet assay and confocal scanning laser microscopy. Listerial biofilms treated with S. aromaticum or M. spicata essential oils, or the pure compounds nerol and citral, exhibited a similar biofilm biomass to the positive control. However, the essential oils of lemongrass, L. rehmannii, eugenol and R-carvone caused biofilm enhancement, rather than inhibition. L. rehmannii and lemongrass essential oils did not display any antibiofilm properties. Results obtained were confirmed by microscopic observations indicating either a reduction (inhibition) or an increase (enhancement) in biofilm biomass when exposed to the essential oils or pure compounds. The present study revealed that M. spicata and S. aromaticum essential oils as well as pure citral and nerol are good candidates for further development of ecofriendly disinfectants.     

Novel strategies of essential oils,chitosan, and nano- chitosan for inhibition of multi-drug resistant: E. coli O157:H7 and Listeria monocytogenes

Despite the wide range of available antibiotics, food borne bacteria demonstrate a huge spectrum of resistance. The current study aims to use natural components such as essential oils (EOs), chitosan, and nano-chitosan that have very influential antibacterial properties with novel technologies like chitosan solution/film loaded with EOs against multi-drug resistant bacteria. Two strains of Escherichia coli O157:H7 and three strains of Listeria monocytogenes were used to estimate antibiotics resistance. Ten EOs and their mixture, chitosan, nano-chitosan, chitosan plus EO solutions, and biodegradable chitosan film enriched with EOs were tested as antibacterial agents against pathogenic bacterial strains. Results showed that E. coli O157:H7 51,659 and L. monocytogenes 19,116 relatively exhibited considerable resistance to more than one single antibiotic. Turmeric, cumin, pepper black, and marjoram did not show any inhibition zone against L. monocytogenes; Whereas, clove, thyme, cinnamon, and garlic EOs exhibited high antibacterial activity against L. monocytogenes with minimum inhibitory concentration (MIC) of 250–400 μl 100^?1 ml and against E. coli O157:H7 with an MIC of 350–500 μl 100^?1 ml, respectively. Among combinations, clove, and thyme EOs showed the highest antibacterial activity against E. coli O157:H7 with MIC of 170 μl 100^?1 ml, and the combination of cinnamon and clove EOs showed the strongest antibacterial activity against L. monocytogenes with an MIC of 120 μl 100^?1 ml. Both chitosan and nano-chitosan showed a promising potential as an antibacterial agent against pathogenic bacteria as their MICs were relatively lower against L. monocytogenes than for E. coli O157:H7. Chitosan combined with each of cinnamon, clove, and thyme oil have a more effective antibacterial activity against L. monocytogenes and E. coli O157:H7 than the mixture of oils alone. Furthermore, the use of either chitosan solution or biodegradable chitosan film loaded with a combination of clove and thyme EOs had the strongest antibacterial activity against L. monocytogenes and E. coli O157:H7. However, chitosan film without EOs did not exhibit an inhibition zone against the tested bacterial strains.     

Coselection of Cadmium and Benzalkonium Chloride Resistance in Conjugative Transfers from Nonpathogenic Listeria spp. to Other Listeriae

Resistance to the quaternary ammonium disinfectant benzalkonium chloride (BC) may be an important contributor to the ability of Listeria spp. to persist in the processing plant environment. Although a plasmid-borne disinfectant resistance cassette (bcrABC) has been identified in Listeria monocytogenes, horizontal transfer of these genes has not been characterized. Nonpathogenic Listeria spp. such as L. innocua and L. welshimeri are more common than L. monocytogenes in food processing environments and may contribute to the dissemination of disinfectant resistance genes in listeriae, including L. monocytogenes. In this study, we investigated conjugative transfer of resistance to BC and to cadmium from nonpathogenic Listeria spp. to other nonpathogenic listeriae, as well as to L. monocytogenes. BC-resistant L. welshimeri and L. innocua harboring bcrABC, along with the cadmium resistance determinant cadA2, were able to transfer resistance to other nonpathogenic listeriae as well as to L. monocytogenes of diverse serotypes, including strains from the 2011 cantaloupe outbreak. Transfer among nonpathogenic Listeria spp. was noticeably higher at 25°C than at 37°C, whereas acquisition of resistance by L. monocytogenes was equally efficient at 25 and 37°C. When the nonpathogenic donors were resistant to both BC and cadmium, acquisition of cadmium resistance was an effective surrogate for transfer of resistance to BC, suggesting coselection between these resistance attributes. The results suggest that nonpathogenic Listeria spp. may behave as reservoirs for disinfectant and heavy metal resistance genes for other listeriae, including the pathogenic species L. monocytogenes.     

Effects of Essential Oils from Eucalyptus globulus Leaves on Soil Organisms Involved in Leaf Degradation

The replacement of native Portuguese forests by Eucalyptus globulus is often associated with deleterious effects on terrestrial and aquatic communities. Several studies have suggested that such a phenomenon is linked with the leaf essential oils released into the environment during the Eucalyptus leaf degradation process. However, to date, the way these compounds affect leaf degradation in terrestrial systems i.e. by direct toxic effects to soil invertebrates or indirectly by affecting food of soil fauna, is still unknown. In order to explore this question, the effect of essential oils extracted from E. globulus leaves on terrestrial systems was investigated. Fungal growth tests with species known as leaf colonizers (Mucor hiemalis, Alternaria alternata, Penicillium sp., Penicillium glabrum and Fusarium roseum) were performed to evaluate the antifungal effect of essential oils. In addition, a reproduction test with the collembolans Folsomia candida was done using a gradient of eucalyptus essential oils in artificial soil. The influence of essential oils on feeding behaviour of F. candida and the isopods Porcellio dilatatus was also investigated through food avoidance and consumption tests. Eucalyptus essential oils were lethal at concentrations between 2.5–20 µL/mL and inhibited growth of all fungal species between 1.25–5 µL/mL. The collembolan reproduction EC₅₀ value was 35.0 (28.6–41.2) mg/kg and both collembola and isopods preferred leaves without oils. Results suggested that the effect of essential oils in leaf processing is related to direct toxic effects on fungi and soil fauna and to indirect effects on the quality and availability of food to soil invertebrates.     

Listeria monocytogenes Biofilm-Associated Protein (BapL) May Contribute to Surface Attachment of L. monocytogenes but Is Absent from Many Field Isolates

Listeria monocytogenes is a food-borne pathogen capable of adhering to a range of surfaces utilized within the food industry, including stainless steel. The factors required for the attachment of this ubiquitous organism to abiotic surfaces are still relatively unknown. In silico analysis of the L. monocytogenes EGD genome identified a putative cell wall-anchored protein (Lmo0435 [BapL]), which had similarity to proteins involved in biofilm formation by staphylococci. An insertion mutation was constructed in L. monocytogenes to determine the influence of this protein on attachment to abiotic surfaces. The results show that the protein may contribute to the surface adherence of strains that possess BapL, but it is not an essential requirement for all L. monocytogenes strains. Several BapL-negative field isolates demonstrated an ability to adhere to abiotic surfaces equivalent to that of BapL-positive strains. BapL is not required for the virulence of L. monocytogenes in mice.     

Inhibition of Listeria monocytogenes by Food-Borne Yeasts

Many bacteria are known to inhibit food pathogens, such as Listeria monocytogenes, by secreting a variety of bactericidal and bacteriostatic substances. In sharp contrast, it is unknown whether yeast has an inhibitory potential for the growth of pathogenic bacteria in food. A total of 404 yeasts were screened for inhibitory activity against five Listeria monocytogenes strains. Three hundred and four of these yeasts were isolated from smear-ripened cheeses. Most of the yeasts were identified by Fourier transform infrared spectroscopy. Using an agar-membrane screening assay, a fraction of approximately 4% of the 304 red smear cheese isolates clearly inhibited growth of L. monocytogenes. Furthermore, 14 out of these 304 cheese yeasts were cocultivated with L. monocytogenes WSLC 1364 on solid medium to test the antilisterial activity of yeast in direct cell contact with Listeria. All yeasts inhibited L. monocytogenes to a low degree, which is most probably due to competition for nutrients. However, one Candida intermedia strain was able to reduce the listerial cell count by 4 log units. Another four yeasts, assigned to C. intermedia (three strains) and Kluyveromyces marxianus (one strain), repressed growth of L. monocytogenes by 3 log units. Inhibition of L. monocytogenes was clearly pronounced in the cocultivation assay, which simulates the conditions and contamination rates present on smear cheese surfaces. We found no evidence that the unknown inhibitory molecule is able to diffuse through soft agar.     

Changes in aroma profiles of 11 Indian Ocimum taxa during plant ontogeny

The genus Ocimum is an important source of many essential oils and aromatic chemicals used principally in the food and cosmetic industries. The aromas (compositions) of essential oils of genus Ocimum are characterized by a great chemical variability affecting the commercial value of this genus. This study was conducted to evaluate the ontogenic variations in aroma profiles of 11 Indian Ocimum taxa belonging to 5 Ocimum species, viz. O. basilicum L., O. americanum L., O. gratissimum L., O. tenuiflorum L., and O. kilimandscharicum Guerke, grown in foot hills of Uttarakhand, India during rain-autumn cropping season. Essential oil yield was found to vary significantly during different phenophases: from 0.34 to 0.87 % in four landraces of O. basilicum, 0.16 to 0.38 % in ‘Green and Purple’ of O. tenuiflorum, 0.23 to 0.43 % in O. americanum, 0.34 to 0.78 % in O. kilimandscharicum and 0.48 to 0.68 % in O. gratissimum. The variation in composition of the essential oils was analyzed by gas chromatography with flame ionization detection and gas chromatography with mass spectrometry. Analyses of the essential oils led to the identification of 95 constituents forming 93.8–99.7 % of the total essential oil compositions, with phenyl propanoids (≤0.1–90.0 %) and monoterpenoids (2.8–95.5 %) as the prevalent components. Substantial variations in essential oil yield, and qualitative and quantitative compositions due to different stages of plants growth were noticed. These results indicated that the knowledge of appropriate growth stage is very important to obtain higher essential oil yield and desired active constituents of Ocimum, which were used extensively in food, cosmetics and allied industries.     

The Listeria monocytogenes Homolog of the Escherichia coli era Gene Is Involved in Adhesion to Inert Surfaces

Two transposon-insertional mutants of Listeria monocytogenes showing smaller viable surface-attached cell populations after disinfection with N,N-didecyl-N,N-dimethylammonium chloride were identified. In both mutants, transposon Tn917-lac was found to be inserted into the same gene, lmo1462, which is homologous to the essential Escherichia coli era gene. Both L. monocytogenes lmo1462-disrupted mutants displayed lower growth rates, as was also shown for several E. coli era mutants, and the lmo1462 gene was able to complement the growth defect of an E. coli era mutant. We showed that the disruption of lmo1462 decreased the ability of L. monocytogenes cells to adhere to stainless steel. Our results suggest that this era-like gene is involved in adhesion and contributes to the presence of L. monocytogenes on surfaces.     

Development of a Novel Selective and Differential Medium for the Isolation of Listeria monocytogenes

A new medium (lecithin and levofloxacin [LL] medium) is described for the isolation of Listeria monocytogenes from food samples. LL medium includes lecithin from soybeans for the detection of phosphatidylinositol-specific phospholipase C (PI-PLC) and phosphatidylcholine-specific phospholipase C (PC-PLC) produced by L. monocytogenes. Levofloxacin is incorporated to inhibit the growth of microorganisms other than L. monocytogenes, especially Bacillus cereus, shown to possess PI-PLC and PC-PLC activities. L. monocyogenes produced white colonies with a halo on LL medium, whereas Listeria innocua appeared as white colonies without a halo. Levofloxacin at 0.20 mg/liter completely inhibited the growth of B. cereus, while the growth of L. monocytogenes was unaffected. In the second phase of the study, the sensitivity and the specificity of LL medium were compared to those of modified Oxford agar (MOX) and two chromogenic media (Brilliance Listeria agar and CHROMagar Listeria), using a total of 250 food samples. From 200 unspiked food samples, the specificity of LL medium (96.0%) was superior to that of MOX (72.0%) and similar to the specificities of Brilliance Listeria agar (96.5%) and CHROMagar Listeria (94.5%). From 50 spiked food samples, LL medium and CHROMagar Listeria represented the highest sensitivities (96.0%), followed by Brilliance Listeria agar (92.0%) and MOX (54.0%). Also, LL medium showed the highest confirmation rate (98.8%), followed by Brilliance Listeria agar (98.7%), CHROMagar Listeria (98.3%), and MOX (52.0%). On the basis of its good specificity and cost effectiveness, LL medium is useful for the isolation of L. monocytogenes from food samples.     

Effect of Octenidine Hydrochloride on Planktonic Cells and Biofilms of Listeria monocytogenes

Listeria monocytogenes is a food-borne pathogen capable of forming biofilms and persisting in food processing environments for extended periods of time, thereby potentially contaminating foods. The efficacy of octenidine hydrochloride (OH) for inactivating planktonic cells and preformed biofilms of L. monocytogenes was investigated at 37, 21, 8, and 4°C in the presence and absence of organic matter (rehydrated nonfat dry milk). OH rapidly killed planktonic cells and biofilms of L. monocytogenes at all four temperatures. Moreover, OH was equally effective in killing L. monocytogenes biofilms on polystyrene and stainless steel matrices in the presence and absence of organic matter. The results underscore OH''s ability to prevent establishment of L. monocytogenes biofilms by rapidly killing planktonic cells and to eliminate preformed biofilms, thus suggesting that it could be used as a disinfectant to prevent L. monocytogenes from persisting in food processing environments.Listeria monocytogenes is a major bacterial pathogen (2), accounting for approximately 28% of the deaths resulting from food-borne illnesses in the United States (22). It is widespread in nature and occurs in soil, vegetation, fecal matter, sewage, water, and animal feed (14). Because it is ubiquitous, L. monocytogenes is frequently isolated from foods and food processing environments (13, 23), thereby presenting a significant challenge to the food industry. Several studies have shown that L. monocytogenes is capable of adhering to food contact surfaces, such as glass, stainless steel, rubber, and polystyrene (6, 11, 28). Upon attachment to such surfaces, L. monocytogenes establishes biofilms and persists for long periods of time in the food processing environment (18, 30). This potentially poses a food safety hazard since biofilms are an important source of contamination of food products that come into contact with them. In addition, biofilms also protect the underlying bacteria from desiccation, antimicrobials, and sanitizing agents (7, 16). Thus, eradication of L. monocytogenes biofilms in processing plants is critical for improving food safety.When problems with persistent L. monocytogenes are encountered in food processing facilities, plant hygiene and sanitation are emphasized (31). This involves preventing the establishment of L. monocytogenes biofilms in the food processing environment and reducing contamination of product contact surfaces. A variety of cleaners and disinfectants, including quaternary ammonium compounds and hypochlorite, have been evaluated for this purpose (20). Although these compounds are approved by the Food and Drug Administration for use as disinfectants in processing plants, they are not effective in killing L. monocytogenes (24, 25), especially in the presence of soil or organic matter and at low temperatures. Therefore, there is a need for an effective disinfectant that can eliminate listerial biofilms in the presence of organic matter at a wide range of temperatures. Octenidine hydrochloride (OH) is a positively charged bispyridinamine that exhibits antimicrobial activity against plaque-producing organisms, such as Streptococcus mutans and Streptococcus sanguis (5). Toxicity studies with a variety of species have shown that OH is not absorbed through mucous membranes and the gastrointestinal tract, and there have been no reports of carcinogenicity, genotoxicity, or mutagenicity of this compound (17, 19, 29).The objective of this study was to investigate the efficacy of OH for inactivating planktonic cells and preformed biofilms of L. monocytogenes at 37, 21, 8, and 4°C in the presence and absence of organic matter on two matrices, polystyrene and stainless steel.     

Isolation and characterization of Listeria monocytogenes from tropical seafood of Kerala, India

Listeria monocytogenes, which is an intracellular pathogen, causes various illnesses in human as well as in animals. The pathogenicity of this organism depends upon the presence of different virulence genes. A total of 324 tropical seafood and fishery environmental samples were screened for L. monocytogenes. The incidence of the human pathogenic species L. monocytogenes was 1.2 % of the samples. Listeria spp. was detected in 32.3, 27.1, and 5 % of fresh, frozen, and dry fish samples, respectively. Listeria innocua was found to be the most prevalent species of Listeria in the tropical seafood and environmental samples of Kerala. Listeria monocytogenes and L. innocua isolates were confirmed by multiplex PCR. L. monocytogenes isolates from the four positive samples showed phosphatidylinositol-specific phospholipase C reaction on Chromocult® Listeria selective agar. Molecular characterization of L. monocytogenes isolates for virulence genes revealed the presence of β-hemolysin (hly), plcA, actA, metalloprotease (mpl), iap and prfA genes in all the isolates recovered from the positive samples.     

Reservoirs of Listeria Species in Three Environmental Ecosystems

Soil and water are suggested to represent pivotal niches for the transmission of Listeria monocytogenes to plant material, animals, and the food chain. In the present study, 467 soil and 68 water samples were collected in 12 distinct geological and ecological sites in Austria from 2007 to 2009. Listeria was present in 30% and 26% of the investigated soil and water samples, respectively. Generally, the most dominant species in soil and water samples were Listeria seeligeri, L. innocua, and L. ivanovii. The human- and animal-pathogenic L. monocytogenes was isolated exclusively from 6% soil samples in regions A (mountainous region) and B (meadow). Distinct ecological preferences were observed for L. seeligeri and L. ivanovii, which were more often isolated from wildlife reserve region C (Lake Neusiedl) and from sites in proximity to wild and domestic ruminants (region A). The higher L. monocytogenes detection and antibiotic resistance rates in regions A and B could be explained by the proximity to agricultural land and urban environment. L. monocytogenes multilocus sequence typing corroborated this evidence since sequence type 37 (ST37), ST91, ST101, and ST517 were repeatedly isolated from regions A and B over several months. A higher L. monocytogenes detection and strain variability was observed during flooding of the river Schwarza (region A) and Danube (region B) in September 2007, indicating dispersion via watercourses.     

Identification of Listeria monocytogenes Determinants Required for Biofilm Formation

Listeria monocytogenes is a Gram-positive, food-borne pathogen of humans and animals. L. monocytogenes is considered to be a potential public health risk by the U.S. Food and Drug Administration (FDA), as this bacterium can easily contaminate ready-to-eat (RTE) foods and cause an invasive, life-threatening disease (listeriosis). Bacteria can adhere and grow on multiple surfaces and persist within biofilms in food processing plants, providing resistance to sanitizers and other antimicrobial agents. While whole genome sequencing has led to the identification of biofilm synthesis gene clusters in many bacterial species, bioinformatics has not identified the biofilm synthesis genes within the L. monocytogenes genome. To identify genes necessary for L. monocytogenes biofilm formation, we performed a transposon mutagenesis library screen using a recently constructed Himar1 mariner transposon. Approximately 10,000 transposon mutants within L. monocytogenes strain 10403S were screened for biofilm formation in 96-well polyvinyl chloride (PVC) microtiter plates with 70 Himar1 insertion mutants identified that produced significantly less biofilms. DNA sequencing of the transposon insertion sites within the isolated mutants revealed transposon insertions within 38 distinct genetic loci. The identification of mutants bearing insertions within several flagellar motility genes previously known to be required for the initial stages of biofilm formation validated the ability of the mutagenesis screen to identify L. monocytogenes biofilm-defective mutants. Two newly identified genetic loci, dltABCD and phoPR, were selected for deletion analysis and both ΔdltABCD and ΔphoPR bacterial strains displayed biofilm formation defects in the PVC microtiter plate assay, confirming these loci contribute to biofilm formation by L. monocytogenes.     

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the gastrointestinal phase of listeriosis due to the lack of a small animal model that closely mimics human disease. This paper describes a novel mouse model for oral transmission of L. monocytogenes. Using this model, mice fed L. monocytogenes-contaminated bread have a discrete phase of gastrointestinal infection, followed by varying degrees of systemic spread in susceptible (BALB/c/By/J) or resistant (C57BL/6) mouse strains. During the later stages of the infection, dissemination to the gall bladder and brain is observed. The food borne model of listeriosis is highly reproducible, does not require specialized skills, and can be used with a wide variety of bacterial isolates and laboratory mouse strains. As such, it is the ideal model to study both virulence strategies used by L. monocytogenes to promote intestinal colonization, as well as the host response to invasive food borne bacterial infection.     

Chemical composition and antifungal activity of the essential oils of Lippia rehmannii from South Africa

Lippia rehmannii H.Pearson (Verbenaceae) is an aromatic bush, indigenous to the northern parts of South Africa. As far as could be ascertained, the essential oil composition has not been previously reported and forms the subject of this investigation. Aerial parts of the shrub were collected from two localities in Gauteng, South Africa, and the isolated essential oils were analysed by gas chromatography. Citral, a mixture of the E- and Z-isomers, was found to be the main constituent of the oils, while borneol, camphor, neryl acetate, isocaryophyllene, p-cymene, β-caryophyllene and β-caryophyllene oxide were other major compounds present. Oil compositions, within and between the two localities, did not differ significantly. The in vitro antifungal activity of L. rehmannii essential oil was compared to that of Cympopogon citratus (lemongrass) and pure citral, against a number of pre- and postharvest fungal food pathogens. At a concentration of 3000 µL/L, lemongrass oil and pure citral caused complete growth inhibition of all the pathogens tested. Lippia rehmannii, containing less citral than lemongrass oil, was effective at this concentration against the majority of pathogens, but only partially restricted the growth of Lasiodiplodia theobromae and Botrytis cinerea. This finding suggests that citral may be largely responsible for the observed antifungal activities. Essential oil from L. rehmannii appears to be a good candidate for the in vitro control of Fusarium oxysporum and Rhizoctonia solani and application of these oils in the field should be investigated.     

Evaluation of immunomagnetic separation in combination with ALOA Listeria chromogenic agar for the isolation and identification of Listeria monocytogenes in ready-to-eat foods

A study was conducted to evaluate the performance of the ALOA (chromogenic media) in combination with immunomagnetic separation (IMS) for the detection of Listeria monocytogenes in ready-to-eat food products. IMS-ALOA method was found to be equivalent to Health Canada's reference culture method as well as comparable to BAX-PCR method in terms of the sensitivity of the methods for the detection of L. monocytogenes in ready-to-eat foods such as turkey roast, beef roast, mixed vegetable salads, potato and egg salad, soft cheese and smoked salmon. The IMS-ALOA method gave 100% sensitivity in the inclusivity tests with 42 pure L. monocytogenes strains. Exclusivity testing with five other species of Listeria genus and 29 pure non-L. monocytogenes strains from 21 different genera showed 97% specificity. The method was able to detect L. monocytogenes at levels near or below 1 cfu/25 g regulatory limit in ready-to-eat food matrices after 24 h enrichment, with a turnaround time of 3 days compared to 7-8 days for culture method. IMS-ALOA method is a valuable alternate test method for the screening of L. monocytogenes in a variety of foods especially ready-to-eat foods.     

Antibacterial Activity of Subtilosin Alone and Combined with Curcumin, Poly-Lysine and Zinc Lactate Against Listeria monocytogenes Strains

In this paper, the antibacterial effects of the Bacillus amyloliquefaciens-produced bacteriocin subtilosin, both alone and in combination with curcumin, ε-poly-l-lysine (poly-lysine), or zinc lactate, were examined against Listeria monocytogenes. Results indicated that subtilosin inhibits both of the studied bacterial strains, Scott A (wild-type, nisin sensitive) and NR30 (nisin resistant). However, L. monocytogenes Scott A was more sensitive to subtilosin and pure curcumin. In addition, subtilosin was more active at an acidic pH. Subtilosin in combination with encapsulated curcumin displayed partial synergy against L. monocytogenes ScottA. It also had synergistic activity against both L. monocytogenes Scott A and L. monocytogenes NR30 when combined with zinc lactate. Only an additive effect was observed for subtilosin when combined with non-encapsulated curcumin or poly-lysine against the mentioned strains. Thus, using the combination of subtilosin with curcumin, poly-lysine, or zinc lactate, a lower effective dose can be used to control L. monocytogenes infection. Our findings suggest that subtilosin could be used as alternative bacteriocin to nisin, providing an opportunity to use a novel natural and efficacious biopreservative against L. monocytogenes in food preservation. This is the first report on the effects of the combination of subtilosin with natural antimicrobials on L. monocytogenes.     

Bias in the Listeria monocytogenes Enrichment Procedure: Lineage 2 Strains Outcompete Lineage 1 Strains in University of Vermont Selective Enrichments

Listeria monocytogenes can be isolated from a range of food products and may cause food-borne outbreaks or sporadic cases of listeriosis. L. monocytogenes is divided into three genetic lineages and 13 serotypes. Strains of three serotypes (1/2a, 1/2b, and 4b) are associated with most human cases of listeriosis. Of these, strains of serotypes 1/2b and 4b belong to lineage 1, whereas strains of serotype 1/2a and many other strains isolated from foods belong to lineage 2. L. monocytogenes is isolated from foods by selective enrichment procedures and from patients by nonselective methods. The aim of the present study was to investigate if the selective enrichment procedure results in a true representation of the subtypes of L. monocytogenes present in a sample. Eight L. monocytogenes strains (four lineage 1 strains and four lineage 2 strains) and one Listeria innocua strain grew with identical growth rates in the nonselective medium brain heart infusion (BHI), but differed in their growth rate in the selective medium University of Vermont medium I (UVM I). When coinoculated in UVM I, some strains completely outgrew other strains. This outcome was dependent on the lineage of L. monocytogenes rather than the individual growth rate of the strains. When inoculated at identical cell densities in UVM I, L. innocua outcompeted L. monocytogenes lineage 1 strains but not lineage 2 strains. In addition, lineage 2 L. monocytogenes strains outcompeted lineage 1 L. monocytogenes strains in all combinations tested, indicating a bias in strains selected by the enrichment procedures. Bias also occurred when coinoculating two lineage 2 or lineage 1 strains; however, it did not appear to correlate with origin (clinical versus food). Identical coinoculation experiments in BHI suggested that the selective compounds in UVM I and II influenced this bias. The results of the present study demonstrate that the selective procedures used for isolation of L. monocytogenes may not allow a true representation of the types present in foods. Our results could have a significant impact on epidemiological studies, as lineage 1 strains, which are often isolated from clinical cases of listeriosis, may be suppressed during enrichment by other L. monocytogenes lineages present in a food sample.