Listeria Monocytogenes Strains Research Articles

Evaluation of the Formation of Single- and Double-Species Biofilms on Intraventricular Catheters by Strains of Staphylococcus aureus, Listeria monocytogenes and Escherichia coli with K1 Antigen

Background: Intraventricular catheters (IVCs) are an important diagnostic and therapeutic tool. They are used in patients after traumatic brain injury, intracranial haemorrhage, ventricular, or hydrocephalus. However, the use of IVCs may lead to the development of bacterial brain and cerebrovascular infections associated with the presence of microorganisms in cerebrospinal fluid (ventriculitis). What is especially dangerous for the patients health is the formation of biofilms by microorganisms on the catheters. Objectives: The aim of the study was to evaluate the intensity of single- and double-species biofilms formation on IVCs by Staphylococcus aureus (SAU), Escherichia coli (ECO) with K1 antigen, and Listeria monocytogenes (LMO). Methods: The research material was 15 strains of S. aureus, L. monocytogenes, and E. coli with K1 antigen, each. Evaluation of biofilm formation was performed on sterile fragments of IVCs with 1 cm length. The intensity of biofilm formation by the tested strains was determined with the quantitative method using sonication for removal of bacteria from IVCs surface. Biofilm formation ability was verified individually for each of the tested bacteria strain and for SAU + LMO, SAU + ECO, LMO + ECO combinations. Randomly selected fragments of biofilm-coated catheters were subjected to scanning electron microscopy (SEM). Results: The number of bacteria isolated from single-species biofilms on the IVCs surface ranged from 3.79 × 107 do 8.95 × 107 CFU × cm-2, depending on the species of microorganism. The strongest biofilm formed ECO with K1 antigen strains and the weakest - LMO strains. The scomposition of multi-species biofilms significantly influences the intensity of biofilm formation by strains from the examined species. In the SAU + LMO combination, strains from both species formed biofilm more intensively than in single-species variant. In turn, ECO has shown a significant inhibitory effect on biofilm formation by other tested species. Conclusions: It was stated that bacteria form biofilm on IVCs and it is a significant medical problem. The species composition of multi-species biofilms significantly influences the number of individual bacteria recovered from this biofilm.

Modelling the fate and serogroup variability of persistent Listeria monocytogenes strains on grated cheese at different storage temperatures

Processed cheese from cow's milk is one of the most consumed dairy products worldwide. Since this product is defined as ready-to-eat, foodborne pathogens such as Listeria monocytogenes can represent a health concern for susceptible populations. In this study, the individual and combined kinetic behaviour of four L. monocytogenes serogroups (namely, 1/2a, 1/2b, 1/2c and 4b) persistently isolated from a Spanish cheesemaking factory was modelled on grated cheese at different isothermal conditions (4 and 12 °C) during a 120-days period. The serogroup variability was characterized over the storage time by the isolation and identification of the different serogroups in the cocktail containing the four strains. This processed cheese did not support the growth of L. monocytogenes during storage. Survival patterns described by the log-linear type model indicated a high variability of L. monocytogenes serotypes at the tested temperatures: L. monocytogenes serogroup 4b showed a more rapid decrease rate at 4 °C than at 12 °C, while the opposite trend was found for the rest of serogroups and the L. monocytogenes cocktail containing all the strains. Survival rate of L. monocytogenes serogroup 1/2c at 4 °C was 0.007 log CFU/d being the most resistant serotype while at 12 °C, serogroup 1/2a showed the lowest survival rate (0.011 log CFU/d), thus showing a prolonged survival at this temperature. This study highlights the potential implications of L. monocytogenes contamination in processed cheese and shows that serogroup variability should be considered when evaluating survival patterns in contaminated products. Finally, the predictive models developed here could be useful to assist food operators to set specific storage conditions and formulations to avoid L. monocytogenes growth and survival in grated cheeses.

Microbial analysis of commercially available US Queso Fresco

Queso Fresco (QF), a fresh Hispanic-style cheese, is often associated with Listeria monocytogenes outbreaks and recalls. Queso Fresco's susceptibility to bacterial contamination is partially due to its high pH and moisture content as well as Listeria's tolerance for the salt content typical for QF. Nine different brands of US QF, 2 packages from 4 different lots (to account for temporal variability), were sampled. The pH, salt content, and moisture content were analyzed in addition to microbial testing including yeasts and molds, coliforms, lactic acid bacteria enumeration, and L. monocytogenes counts. The cheeses were also inoculated with a cocktail of 5 food and human isolates of food-borne outbreak-associated Listeria monocytogenes strains to evaluate how the differences between brands influenced Listeria growth. Three of the cheeses underwent additional genus-level microbial analysis using extracted 16S rDNA, allowing for phylogenetic analysis between bacterial taxa including diversity and relative abundance. We found little variation between the sampled QF pH (range = 6.62-6.86), salt content (1.53-2.01%), and moisture content (43.90-54.50%). Yeasts and molds were below the detection limit of enumeration in all of the cheeses and coliforms were below the detection limit across the first 3 lots, but were detected at varying levels in the fourth lot (>3.0 most probable number/g) for 3 of the brands. Listeria monocytogenes was not isolated after enrichment in any of the samples. All cheeses tested positive for the presence of lactic acid bacteria, with only 1 of the cheeses being labeled as produced with added cultures having substantial counts. Fourteen days after inoculation with L. monocytogenes, at least 2.5 log10 cfu/g of growth was found for all QF brands stored at 4°C. Microbial genus analysis showed that, among the 3 brands, the microbial community was more similar within brand than when compared with the other 2 brands. Thermus, Anoxybacillus, and Streptococcus accounted for the dominant genera of brands A, B, and C, respectively. These variations within the microbial community may account for sensory differences and help manufacturers determine quality control consistency more readily than culture-based methods.

An essential role for hfq involved in biofilm formation and virulence in serotype 4b Listeria monocytogenes

Regulator factor Hfq has been widely detected among both Gram-positive and Gram-negative bacteria; however, its role in Gram-positive bacteria is less well established and varies among species. In Listeria monocytogenes (Lm), an organism able to adapt to a range of environments and live both saprobiotic and parasitic lifestyles, the role of Hfq is not fully understood. Serotype 4b Listeria monocytogenes strains associated with the majority of listeriosis outbreak, while the function of hfq in serotype 4b strains still not referenced. Here, we constructed hfq deletion and reversion mutants of serotype 4b Lm NTSN and analysed the biological characteristics both in vitro and in vivo. The deletion of hfq resulted in a growth deficiency in medium containing 4.5% ethanol or 1% Triton X-100, and the growth of the mutant was significantly reduced at 4 °C. Furthermore, the hfq deletion dramatically decreased biofilm formation in BHI medium and gastric fluid medium, and reduced the invasion and replication rate into the Caco-2BBe cells and RAW264.7 cells. However, complementation restored the wild-type phenotype. Importantly, mouse infection experiments demonstrated that hfq played a more important role in the colonisation and virulence in serotype 4b strain Lm NTSN than in the serotype 1/2a strain Lm EGDe. Taken together, these results demonstrated that hfq is a novel factor associated with biofilm formation, and plays an essential role in the stress response and pathogenisis in serotype 4b strain Lm NTSN. Our data provide the basis for further research into the function of Hfq in serotype 4b Listeria monocytogenes.

Whole-Genome Shotgun Sequencing of Three Listeria monocytogenes Strains Isolated from a Ready-to-Eat Salad-Producing Facility in Switzerland.

Ready-to-eat (RTE) raw foods harbor the risk of transmitting Listeria monocytogenes from the environment to the consumer. We isolated three strains from a facility producing RTE salad. These strains were used to perform challenge tests on different RTE salad products. Here, we present the shotgun genome sequences of all three of these strains.

Catabolic activity and biofilm formation of foodborne Listeria monocytogenes strains

Listeria monocytogenes is a major foodborne pathogen causing increased morbidity worldwide. It forms resistant biofilm structures in food processing facilities after sanitization, consequently creating a public health concern. Many studies on the metabolism and transmission of L. monocytogenes has provided insights into its intracellular infection process, however there is limited understanding on the substrate utilization of the bacteria. Therefore, the main objective of this study was to investigate the carbon and nitrogen substrate catabolism and the biofilm forming potential of 3 Malaysian L. monocytogenes strains (LM41, LM92 and LM115) previously isolated from ready-to-eat foods. Biolog Phenotype Microarray (PM) system was used to study the catabolic activity of the foodborne strains in 190 carbon and 380 nitrogen sources. PM analysis showed that the carbon and nitrogen catabolic activity of L. monocytogenes strains were considerably limited and these strains utilised Tween 40 and Tween 80, which are commonly used for the sanitation in food and meat processing industries. Furthermore, all 3 strains showed strong biofilm forming potential in nutrient-rich and nutrient-limited media, irrespective of the serogroups. The data generated could be utilised to develop alternative measure to inhibit biofilm formation in L. monocytogenes in the food processing environment.

Effect of electron beam and gamma radiation on drug-susceptible and drug-resistant Listeria monocytogenes strains in salmon under different temperature.

To investigate the effect of gamma radiation and high energy electron beam doses on the inactivation of antibiotic-susceptible and antibiotic-resistant Listeria monocytogenes strains inoculated on the surface of raw salmon fillets stored at different temperature (-20, 4 and 25°C). The population of bacteria strains resistance to penicillin, ampicillin, meropenem, erythromycin and trimethoprim-sulfamethoxazole was generated. When using gamma irradiation, the theoretical lethal dose ranged from 1·44 to 5·68kGy and for electron beam the values ranged from 2·99 to 6·83kGy. The theoretical lethal dose for both radiation methods was higher for antibiotic-resistant strains. Gamma radiation proved to be a more effective method for extending salmon fillet shelf-life. The evaluation of pulsed-field gel electrophoresis electrophoregram revealed that the repair of radiation-caused DNA damage occurred faster in antibiotic-resistant L. monocytogenes strains. The number of live L. monocytogenes cells, 40h after irradiation, also was higher in antibiotic-resistant strain suspension. The present study showed that gamma radiation was more effective in the elimination of the tested micro-organisms and food preservation, than a high energy electron beam. The antibiotic-resistant L. monocytogenes strains were more resistant to both radiation methods. There are a lot of research on the effect of radiation on the number of bacteria in food products. However, there is almost no information about the effect of strain properties, such as drug susceptibility, virulence, etc., on their resistance to ionizing radiation. An increasing number of drug resistant bacterial strains isolated from food, encourages to take up this research subject.

Sequence-Based Characterization of Listeria monocytogenes Strains Isolated from Domestic Retail Meat in the Tokyo Metropolitan Area of Japan.

The level of Listeria monocytogenes contamination of domestic retail meat in Tokyo, Japan, was assessed by comparison of isolates from 2004 to 2007 with those isolated before 2003. The overall prevalence of L. monocytogenes among these samples significantly diminished over time (1998-2003, 28.0%; 2004-2007, 17.6%) reflecting a significant decrease in the frequency of contamination of beef. Serotype 1/2a was isolated most frequently, reflecting a change in the predominant serotype in pork from 1/2c to 1/2a. We performed a simple genetic subtyping method based on 3 genes, iap, sigB, and actA, as well as traditional multilocus sequence typing to classify the allele types (ATs). No extensive variation among sequence types was detected. However, increased genetic diversity among the ATs of the 3 genes in the 2004-2007 isolates was evident. We identified AT 26 of the iap gene, which was not previously reported in Japanese isolates, and 6 ATs of the sigB gene, including 4 with nonsense mutations not currently registered in L. monocytogenes DNA databases. sigB is an evolutionally conserved gene that plays a role in the stress response. Our results indicate that the sigB gene may be relatively unstable among L. monocytogenes strains circulating in Japan.

Lactobacillus plantarum 5BG Survives During Refrigerated Storage Bio-Preserving Packaged Spanish-Style Table Olives (cv. Bella di Cerignola).

This paper proposes bio-preservation as a tool to assure quality and safety of Spanish-style table olives cv. Bella di Cerignola. Lactobacillus plantarum 5BG was inoculated in ready to sell olives packaged in an industrial plant by using a half-volume brine (4% NaCl; 2% sucrose). The samples were stored at 4°C. The survival of the inoculated strain, the microbiological quality, the sensory scores and the survival of a strain of Listeria monocytogenes inoculated in brines were evaluated. The persistence of the Lb. plantarum bio-preserving culture was confirmed on olives (≥6.5 Log CFU/g) and in brine (≥7 Log CFU/ml). Bio-preserved olives (SET1) showed a better sensory profile than chemically acidified control olives (SET2) and the texture was the real discriminative parameter among samples. Bio-preserved olives recorded better scores during storage because of their ability to retain good hardness, crunchiness, and fibrousness without cracks. The inoculation of Lb. plantarum positively acted on the safety of olives, as the D-value of L. monocytogenes was reduced from 40 (SET2) to 5 days (SET1). In conclusion, Lb. plantarum 5BG and the physico-chemical conditions achieved in the settled procedure are suitable for the industrial packaging of Bella di Cerignola table olives, improving the process by halving brining volumes and avoiding chemical stabilizers, and significantly reducing the salt concentration. The final product is also safely preserved for almost 5 months as suggested by the reduction of the survival rate of L. monocytogenes.

The effect of blood on the ability of biofilm formation by Listeria monocytogenes strains

Introduction: Listeria monocytogenes strains are widespread in the natural environment and are the etiological factor of listeriosis. Food is the main source of L. monocytogenes. Secondary contamination of food products which results from the formation of biofilm by L. monocytogenes on the surfaces of the processing devices makes an essential problem. Material and methods: We evaluated the ability of biofilm formation in media supplemented with sheep blood (in a volume of 5.0, 10.0, 20.0 and 50.0%) for the reference strain L. monocytogenes ATCC 7644 and strains isolated from the human blood and carrots on the polypropylene surface. Results: The strain isolated from blood most efficiently formed a biofilm with increasing blood sheep’s addition (nutrient broth — 4.87 × 105 CFU × cm-2, blood supplement 50.0% (v/v) — 3.4 × 107 CFU × cm-2).The number of L. monocytogenes recovered from the biofilm for the reference strain and the strain from carrot increased to 20.0% by volume of the blood addition (3.07 × 105 CFU × cm-2 and 4.03 × 105 CFU × cm-2 respectively — nutrient broth; 1.1 × 107 CFU × cm-2 and 9.23 × 106 CFU × cm-2 — blood supplement 20.0% (v/v)). Decrease in the number of cells recovered from the biofilm for the reference strain and the one isolated from carrot has been demonstrated at 50.0% addition of blood into nutrient medium (8.8 × 106 CFU × cm-2 and 7.87 × 106 CFU × cm-2 respectively). Conclusion: The addition of sheep blood to the medium at concentrations up to 20.0% increases the number of cells recovered from the biofilm for all studied L. monocytogenes strains whereas medium with 50.0% blood stimulates the biofilm formation only by the strain isolated from blood.

Individual and combined efficacies of mild heat and ultraviolet-c radiation against Escherichia coli O157:H7, Salmonella enterica, and Listeria monocytogenes in coconut liquid endosperm

This study determined the inactivation kinetic parameters of selected pathogens in heat, ultraviolet-C and combined heat-UV-C treated coconut liquid endosperm. Separate cocktails of Escherichia coli O157:H7, Salmonella enterica serovars, and Listeria monocytogenes strains were inoculated into coconut liquid endosperm (pH 5.15, TSS 4.4oBx, TA 0.062% malic acid, extinction coefficient (ε) at 254 nm of 0.0154 cm−1) for inactivation studies. Result showed that all organisms generally exhibited a log-linear heat inactivation behavior (R2 0.81–0.99). The E. coli O157:H7 cocktail (D55 = 19.75 min, D57 = 10.79 min, D60 = 3.38 min, and D63 = 0.46 min) was found to be significantly more resistant (P > 0.05) than the tested cocktail of L. monocytogenes (D55 = 11.68 min, D57 = 4.53 min, D60 = 1.82 min and D63 = 0.26 min) and S. enterica cocktail (D55 = 3.08 min, D57 = 2.60 min, D60 = 0.89 min and D63 = 0.25 min). Despite the differences in DT values, computed z values for L. monocytogenes cocktail (5.12 ± 0.43 °C) and E. coli O157:H7 cocktail (4.95 ± 0.12 °C) were not significantly different (P > 0.05), but were both significantly (P < 0.05) lower than that of S. enterica cocktail (7.10 ± 0.15 °C). All test organisms also exhibited a generally log-linear UV-C inactivation behavior (R2 0.90–0.99) with E. coli O157:H7 cocktail (DUV-C = 25.26 mJ/cm2) demonstrating greatest resistance to UV-C than S. enterica (DUV-C = 24.65 mJ/cm2) and L. monocytogenes (DUV-C = 17.30 mJ/cm2) cocktails. The D55 values of each organism cocktail were used to calculate for the 3-log reduction heating process schedules, during which UV-C treatments were simultaneously applied. Lethal rates (F values) calculations in the combined processes revealed that within the 3-log reduction heating processes, co-exposure of UV-C resulted in 5.62 to 6.20 log reductions in the test organism populations. Heating caused 69.3, 97.2, and 67.4% of the reduction in E. coli O157:H7, S. enterica and L. monocytogenes cocktails, respectively. These results can be used as baseline data in the establishment of mild heat treatment in combination with UV-C process schedules for coconut liquid endosperm and other similar products.

Benzalkonium chloride tolerance of Listeria monocytogenes strains isolated from a meat processing facility is related to presence of plasmid-borne bcrABC cassette.

Listeria monocytogenes is a serious foodborne pathogen capable of persisting in food processing environments. Tolerance to disinfectants used in industrial settings constitutes an important factor of Listeria survival. In the present study, the mechanism of tolerance to benzalkonium chloride (BAC) was investigated in 77 L.monocytogenes isolates from a meat facility. By PCR approach, the mdrL and lde chromosomal efflux pump genes were detected in all isolates. No isolate was positive for qacH and emrE genes. However, the bcrABC cassette was present in 17 isolates of serogroup IIa possessing the same AscI/ApaI pulsotype, the operon being localized on a plasmid. The significant relation of BAC tolerance with bcrABC presence was confirmed as all bcrABC positive isolates showed the highest minimal inhibitory concentration (MIC) values for BAC and increased sensitivity to BAC was observed after plasmid curing. No effect of the efflux pump inhibitor reserpine on BAC tolerance in bcrABC positive strains was observed in contrast to all bcrABC negative strains. Lower ethidium bromide efflux in bcrABC positive isolates compared to bcrABC negative and plasmid-cured L.monocytogenes isolates was observed. The expression of bcrABC genes was BAC-induced. The confirmed effect of bcrABC to increased BAC tolerance, coupled with its plasmid location, may be an important factor in potential dissemination of the biocide resistance among Listeria species. The understanding of molecular mechanisms of biocide tolerance should help to improve control measures to prevent further spread of L.monocytogenes in food production environments with frequent use of BAC.

Population Genetic Structure of Listeria monocytogenes Strains Isolated From the Pig and Pork Production Chain in France.

Listeria monocytogenes is an ubiquitous pathogenic bacterium, transmissible to humans through the consumption of contaminated food. The pork production sector has been hit hard by a series of L. monocytogenes-related food poisoning outbreaks in France. An overview of the diversity of strains circulating at all levels of the pork production chain, from pig farming (PF) to finished food products (FFP), is needed to identify the contamination routes and improve food safety. Until now, no typing data has been available on strains isolated across the entire pig and pork production chain. Here, we analyzed the population genetic structure of 687 L. monocytogenes strains isolated over the last 20 years in virtually all the French départements from three compartments of this production sector: PF, the food processing environment (FPE), and FFP. The genetic structure was described based on Multilocus sequence typing (MLST) clonal complexes (CCs). The CCs were obtained by mapping the PFGE profiles of the strains. The distribution of CCs was compared firstly between the three compartments and then with CCs obtained from 1106 strains isolated from other food production sectors in France. The predominant CCs of pig and pork strains were not equally distributed among the three compartments: the CC37, CC59, and CC77 strains, rarely found in FPE and FFP, were prevalent in PF. The two most prevalent CCs in the FPE and FFP compartments, CC9 and CC121, were rarely or never detected in PF. No CC was exclusively associated with the pork sector. Three CCs (CC5, CC6, and CC2) were considered ubiquitous, because they were observed in comparable proportions in all food production sectors. The two most prevalent CCs in all sectors were CC9 and CC121, but their distribution was disparate. CC9 was associated with meat products and food products combining several food categories, whereas CC121 was not associated with any given sector. Based on these results, CC121 is likely able to colonize a larger diversity of food products than CC9. Both CCs being associated with the food production suggests, that certain processing steps, such as slaughtering or stabilization treatments, favor their settlement and the recontamination of the food produced.

Listeria monocytogenes strains show large variations in competitive growth in mixed culture biofilms and suspensions with bacteria from food processing environments

Interactions and competition between resident bacteria in food processing environments could affect their ability to survive, grow and persist in microhabitats and niches in the food industry. In this study, the competitive ability of L. monocytogenes strains grown together in separate culture mixes with other L. monocytogenes (L. mono mix), L. innocua (Listeria mix), Gram-negative bacteria (Gram− mix) and with a multigenera mix (Listeria + Gram− mix) was investigated in biofilms on stainless steel and in suspensions at 12 °C. The mixed cultures included resident bacteria from processing surfaces in meat and salmon industry represented by L. monocytogenes (n = 6), L. innocua (n = 5) and Gram-negative bacteria (n = 6; Acinetobacter sp., Pseudomonas fragi, Pseudomonas fluorescens, Serratia liquefaciens, Stenotrophomonas maltophilia). Despite hampered in growth in mixed cultures, L. monocytogenes established in biofilms with counts at day nine between 7.3 and 9.0 log per coupon with the lowest counts in the Listeria + G− mix that was dominated by Pseudomonas. Specific L. innocua inhibited growth of L. monocytogenes strains differently; inhibition that was further enhanced by the background Gram-negative microbiota. In these multispecies and multibacteria cultures, the growth competitive effects lead to the dominance of a strong competitor L. monocytogenes strain that was only slightly inhibited by L. innocua and showed strong competitive abilities in mixed cultures with resident Gram-negative bacteria. The results indicates complex patterns of bacterial interactions and L. monocytogenes inhibition in the multibacteria cultures that only partially depend on cell contact and likely involve various antagonistic and bacterial tolerance mechanisms. The study indicates large variations among L. monocytogenes in their competitiveness under multibacterial culture conditions that should be considered in further studies towards understanding of L. monocytogenes persistence in food processing facilities.

The effectiveness of radiant catalytic ionization in inactivation ofListeria monocytogenesplanktonic and biofilm cells from food and food contact surfaces as a method of food preservation

The aim of the study was to evaluate the microbicidal effectiveness of radiant catalytic ionization (RCI) against Listeria monocytogenes strains in the form of planktonic cells and biofilm on food products and food contact surfaces as a method of food preservation. The study material comprised six strains of L.monocytogenes, isolated from food. Samples of different types of food available by retail (raw carrot, frozen salmon filets, soft cheese) and the fragments of surfaces (stainless steel AISI 304, rubber, milled rock tiles, polypropylene) were used in the experiment. The obtained results showed the effectiveness of RCI in the inactivation of both forms of the tested L. monocytogenes strains on all the surfaces. The effectiveness of RCI for biofilm forms was lower as compared with planktonic forms. The PRR value ranged from 18·19 to 99·97% for planktonic form and from 3·92 to 70·10% for biofilm. The RCI phenomenon induces the inactivation of L.monocytogenes on surfaces of food and materials used in the processing industry to a varying degree, depending on the manner of surface contamination, the properties of the contaminated materials as well as on the origin of the strain and the properties of surrounding dispersive environment in which the micro-organisms were suspended. Searching of new actions aimed at the reduction of the microbial contamination of food and food contact surfaces are extremely important. RCI method has been already described as an effective technique of microbial and abiotic pollution removal from air. However, our studies provide new, additional data related to evaluation the RCI efficacy against microbes on different surfaces, both in planktonic and biofilm form.

Complete Genome Sequence of Listeria monocytogenes Strain MR310, Isolated from a Pastured-Flock Poultry Farm System.

ABSTRACTInvestigation of Listeria monocytogenes transmission from environmental sources associated with pasture-raised chickens to poultry products is needed to determine ways to prevent potential foodborne illness. Here, we report the complete genome sequence of Listeria monocytogenes MR310, one of the isolates from a pastured-flock poultry management system.

Listeria innocua and Listeria monocytogenes strains from dairy plants behave similarly in biofilm sanitizer testing

Five L. innocua and five L. monocytogenes, including persistent and non-persistent isolates collected from Gorgonzola processing plants, were compared regarding their biofilm-forming ability and biofilm susceptibility to two hydrogen peroxide (HP) based disinfectants in use at the plants. No significant difference in biofilm-forming ability by both species was observed (P > 0.05) in crystal violet and viable count assays. The susceptibility to disinfectants was determined in simulated clean and soiled biofilm forming conditions, by growing biofilms, respectively, in 1/10 diluted TSB-YE and in TSB-YE. The results showed no significant differences between species or conditions (P > 0.05) regardless of whether the isolates were classified as persistent or non-persistent. A hierarchical clustering based on Principal Component Analysis performed on the tested variables, indicated the presence of two major clusters. Isolates from both species were allocated in both clusters, suggesting that they behaved in a similar way in response to the tested conditions. This study showed that biofilms of L. innocua could monitor the effectiveness of HP-based disinfectants. Moreover, biofilms of L. innocua from the same environment could be used as surrogates of L. monocytogenes in sanitizer-based biofilm eradication trials simulating dairy processing environments, whenever the use of the pathogen is not an option.

A phase 2 trial of CRS-207 and pembrolizumab in adults with recurrence of metastatic gastric, gastroesophageal junction (GEJ), or esophageal adenocarcinomas.

TPS200 Background: CRS-207 is a live, attenuated, double-deleted Listeria monocytogenes (LADD) strain with a mesothelin expression cassette inserted. CRS-207 has a well-established, manageable toxicity profile and can elicit mesothelin-specific cell-mediated immunity. Clinical activity of CRS-207 has been shown in a separate Phase 1 trial of mesothelioma. In gastroesophageal (GE) cancers, mesothelin expression is estimated between 30% – 50% and has been correlated with poor prognosis. Preclinical models suggest the combination of LADD-based immunotherapeutics and a PD1 inhibitor may induce more sustained anti-tumor responses than either LADD or PD1 inhibitor monotherapy. GE cancers are responsive to PD1 inhibitors in a subset of patients; this trial proposes to evaluate the efficacy of CRS-207 combined with pembrolizumab in patients with relapsed GE cancer, and to correlate clinical activity with mesothelin expression level. Methods: This Phase 2, open-label, single-arm, multicenter clinical study (NCT 03122548) will enroll approximately 79 subjects at 20 sites. Adults with histologically-confirmed, advanced gastric, GEJ, or esophageal adenocarcinomas are eligible. Subjects must have received 1 or 2 prior treatment regimens, which must have included a platinum and a fluoropyrimidine. Subjects must have disease progression with measurable tumors. A pre-treatment biopsy of either the primary tumor or metastatic site is required prior to dosing. Subjects with prior exposure to checkpoint inhibitors or other immunotherapies are excluded. From cycle 1 to 4 during the treatment and evaluation period, pembrolizumab (IV 200 mg) and CRS-207 (IV 1 x 109 CFU) are both administered in 3-week cycles. Afterward, pembrolizumab will be administered once every 3 weeks; CRS-207 will be given once every 6 weeks. CT scans will be performed every 6 weeks to monitor disease status. The primary endpoint is objective response rate defined by RECIST 1.1. Clinical trial information: NCT03122548.

Inhibition of Clostridium perfringens using Bacteriophages and Bacteriocin Producing Strains.

In this study, we isolated and characterized a bacteriocin-producing strain and two bacteriophages (P4, A3), showing antimicrobial effects against Clostridium perfringens, from chicken and swine feces by the spot-on-the lawn antagonism method. The selected strain was identified as Streptococcus hyointestinalis by 16S rRNA gene sequencing. The bacteriocin from the isolated strain exhibited strong inhibitory activity against four strains of C. perfringens and all the tested strains of Listeria monocytogenes, and the bacteriocin were highly heat- and pH-stable even at pH 2, pH 10 and 121℃ for 15 min. We also evaluated the combined effects of the isolated bacteriocin and phages. Combining the phage treatments and bacteriocin resulted in a synergetic effect compared with the phage or the bacteriocin alone. In addition, during the probiotic test, the bacteriocin-producing S. hyointestinalis B19 strain reduced the population of C. perfringens significantly. Treatment with S. hyointestinalis B19 and a cocktail of lytic bacteriophages eradicated the C. perfringens KCTC 3269T, completely. Consequently, the isolated bacteriocin and bacteriophages represent candidates for effective biocontrol of C. perfringens, and bacteriocin-producing S. hyointestinalis B19 is a potential probiotic candidate for use in domestic animals.

Survival of Listeria monocytogenes during storage of soft cheeses Galotyri and Touloumotyri at 5°C and 12°C

Soft spreadable cheeses Galotyri and Touloumotyri were inoculated with Listeria monocytogenes strains Scott A or California (CA), to contain 3.6-7.5 x 105 CFU/g of cheese and stored at 5°C and 12°C f or 30d and 40d respectively. The initial pH of the cheeses Galotyri and Touloumotyri were 4.35 (S.D.±0.08) and 4.53 (S.D. ±0.06) respectively, and remained relatively constant during the entire experiment. Fat in dry mater was 50.06% (S.D. ±1.16) in Calotyri and 37.43% (S.D. ±0.62) in Touloumotyri cheese. Values of moisture content of cheeses were 68.04% (S.D. ±0.36) and 73.01% (S.D.±0.14) respectively. The NaCl content for Galotyri and Touloumotyri cheeses was 1.54% (S.D.±0.12) and 1.03% (S.D. ±0.10) respectively. Duplicate samples of cheeses were used for enumeration of L. monocytogenes and pH. Listeria counts were obtained by surface plating on lithium chloride phenylethanol moxalactam agar (LPM). Selected Listeria colonies were confirmed biochemically. Viability loss of L. monocytogenes in Galotyri cheese was rapid, but the pathogen survived for more than 40 d in Touloumotyri cheese. During storage of Galotyri cheese at 5°C and 12°C, population of both strains of L. monocytogenes decreased significantly (P&lt;0.01) to less than 10 viable cells/g of cheese, within 7 to 16 d for 7 trials. At that time population of L. monocytogenes decreased by 2.9-4.55 Log10 CFU/g. Estimated D-value was 2.17 d (S.D. ±1.02) for the 7 trials. Only for one trial L. monocytogenes was counted up to 24 d, but was not survived more than 26 d. The reduction of population was statistically significant (P&lt;0.01) for this trial also. Cold enrichment up to 8 weeks of samples in which L. monocytogenes was not counted, indicated that the pathogen survived for 16 d up to 27 d in a l l trials. Both strains of L. monocytogenes survived for more than 40 d during storage of Touloumotyri cheese at 5°C and 12°C. Population of L. monocytogenes decreased significantly (P&lt;0.05) by 0.98 to 1.96 Log10 CFU/g during storage of Touloumotyri cheese at 5 °C and 12 °C. After 40d of storage of cheese, populations of the pathogen ranged from 5.3 to 51 χΙΟ3 CFU/g. Calculation indicated that survivors of the pathogen represent the 1.1% up to 10.6% of the initial population of L. monocytogenes inoculated to the cheese.