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Aims To understand the genetics involved in surface attachment and biofilm formation of Listeria monocytogenes. Methods and Results An in vitro screen of a Himar1 transposon library of L. monocytogenes strain 15G01 identified three transposants that produced significantly different biofilm levels when compared to the wild-type strain; two mutants exhibited enhanced biofilm formation and one produced less biofilm biomass than the wild-type. The mutant 15G01 mprF::Himar1, which had a transposon insertion in the mprF gene, was selected 29 for further analysis. The mutant produced a more densely populated biofilm on solid surfaces such as 30 stainless steel and polystyrene, as determined using scanning electron and light microscopy. The 15G01 31 mprF::Himar1 mutant remained viable in biofilms, but showed an increase in sensitivity to the cationic 32 antimicrobial gallidermin. The mutant also displayed reduced invasiveness in CaCo-2 intestinal cells, 33 suggesting virulence properties are compromised by the inactivation of mprF. 34 Conclusions 35 Biofilm formation and gallidermin resistance of L. monocytogenes is influenced by mprF, but this trait is 36 associated with a compromise in invasiveness. 37 Significance 38 The presence of pathogenic microorganisms in the food processing environment can cause a significant 39 problem, especially when these microorganisms are established as biofilms. This study shows that the 40 inactivation of the mprF gene results in enhanced biofilm formation and abiotic surface attachment of 41 Listeria monocytogenes.

Listeria monocytogenes is a foodborne human pathogen that causes systemic infection, fetal-placental infection in pregnant women causing abortion and stillbirth and meningoencephalitis in elderly and immunocompromised individuals. This study aimed to analyse L. monocytogenes from different sources from New Zealand (NZ) and to compare them with international strains. We used pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and whole-genome single nucleotide polymorphisms (SNP) to study the population structure of the NZ L. monocytogenes isolates and their relationship with the international strains. The NZ isolates formed unique clusters in PFGE, MLST and whole-genome SNP comparisons compared to the international isolates for which data were available. PFGE identified 31 AscI and 29 ApaI PFGE patterns with indistinguishable pulsotypes being present in seafood, horticultural products and environmental samples. Apart from the Asc0002:Apa0002 pulsotype which was distributed across different sources, other pulsotypes were site or factory associated. Wholegenome analysis of 200 randomly selected L. monocytogenes isolates revealed that lineage II dominated the NZ L. monocytogenes populations. MLST comparison of international and NZ isolates with lineage II accounted for 89% (177 of 200) of the total L. monocytogenes population, while the international representation was 45.3% (1674 of 3473). Rarefaction analysis showed that sequence type richness was greater in NZ isolates compared to international trend, however, it should be noted that NZ isolates predominantly came from seafood, horticulture and their respective processing environments or factories, unlike international isolates where there was a good mixture of clinical, food and environmental isolates.

Listeria monocytogenes is a ubiquitous foodborne pathogen that results in a high rate of mortality in sensitive and immunocompromised people. Contamination of food with L. monocytogenes is thought to occur during food processing, most often as a result of the pathogen producing a biofilm that persists in the environment and acting as the source for subsequent dispersal of cells onto food. A survey of seafoodprocessing plants in New Zealand identified the persistent strain 15G01, which has a high capacity to form biofilms. In this study, a transposon library of L. monocytogenes 15G01 was screened for mutants with altered biofilm formation, assessed by a crystal violet assay, to identify genes involved in biofilm formation. This screen identified 36 transposants that showed a significant change in biofilm formation compared to the wild type. The insertion sites were in 27 genes, 20 of which led to decreased biofilm formation and seven to an increase. Two insertions were in intergenic regions. Annotation of the genes suggested that they are involved in diverse cellular processes, including stress response, autolysis, transporter systems, and cell wall/membrane synthesis. Analysis of the biofilms produced by the transposants using scanning electron microscopy and fluorescence microscopy showed notable differences in the structure of the biofilms compared to the wild type. In particular, inactivation of uvrB and mltD produced coccoid-shaped cells and elongated cells in long chains, respectively, and the mgtB mutant produced a unique biofilm with a sandwich structure which was reversed to the wild-type level upon magnesium addition. The mltD transposant was successfully complemented with the wild-type gene, whereas the phenotypes were not or only partially restored for the remaining mutants. IMPORTANCE The major source of contamination of food with Listeria monocytogenes is thought to be due to biofilm formation and/or persistence in food-processing plants. By establishing as a biofilm, L. monocytogenes cells become harder to eradicate due to their increased resistance to environmental threats. Understanding the genes involved in biofilm formation and their influence on biofilm structure will help identify new ways to eliminate harmful biofilms in food processing environments. To date, multiple genes have been identified as being involved in biofilm formation by L. monocytogenes; however, the exact mechanism remains unclear. This study identified four genes associated with biofilm formation by a persistent strain. Extensive microscopic analysis illustrated the effect of the disruption of mgtB, clsA, uvrB, and mltD and the influence of magnesium on the biofilm structure. The results strongly suggest an involvement in biofilm formation for the four genes and provide a basis for further studies to analyze gene regulation to assess the specific role of these biofilm-associated genes.

Listeria monocytogenes is a ubiquitous foodborne pathogen that results in a high rate of mortality in sensitive and immunocompromised people. Contamination of food with L. monocytogenes is thought to occur during food processing, most often as a result of the pathogen producing a biofilm that persists in the environment and acting as the source for subsequent dispersal of cells onto food. A survey of seafood-processing plants in New Zealand identified the persistent strain 15G01, which has a high capacity to form biofilms. In this study, a transposon library of L. monocytogenes 15G01 was screened for mutants with altered biofilm formation, assessed by a crystal violet assay, to identify genes involved in biofilm formation. This screen identified 36 transposants that showed a significant change in biofilm formation compared to the wild type. The insertion sites were in 27 genes, 20 of which led to decreased biofilm formation and seven to an increase. Two insertions were in intergenic regions. Annotation of the genes suggested that they are involved in diverse cellular processes, including stress response, autolysis, transporter systems, and cell wall/membrane synthesis. Analysis of the biofilms produced by the transposants using scanning electron microscopy and fluorescence microscopy showed notable differences in the structure of the biofilms compared to the wild type. In particular, inactivation of uvrB and mltD produced coccoid-shaped cells and elongated cells in long chains, respectively, and the mgtB mutant produced a unique biofilm with a sandwich structure which was reversed to the wild-type level upon magnesium addition. The mltD transposant was successfully complemented with the wild-type gene, whereas the phenotypes were not or only partially restored for the remaining mutants. IMPORTANCE The major source of contamination of food with Listeria monocytogenes is thought to be due to biofilm formation and/or persistence in food-processing plants. By establishing as a biofilm, L. monocytogenes cells become harder to eradicate due to their increased resistance to environmental threats. Understanding the genes involved in biofilm formation and their influence on biofilm structure will help identify new ways to eliminate harmful biofilms in food processing environments. To date, multiple genes have been identified as being involved in biofilm formation by L. monocytogenes; however, the exact mechanism remains unclear. This study identified four genes associated with biofilm formation by a persistent strain. Extensive microscopic analysis illustrated the effect of the disruption of mgtB, clsA, uvrB, and mltD and the influence of magnesium on the biofilm structure. The results strongly suggest an involvement in biofilm formation for the four genes and provide a basis for further studies to analyze gene regulation to assess the specific role of these biofilm-associated genes.

High pressure processing (HPP) and thermal treatments are food preservation technologies used by the food industry to inactivate Listeria monocytogenes. However, the safe shelf-life of treated products could be compromised by the presence and/or recovery of surviving bacteria. The aim of the present study was to evaluate the effect of these technologies on the invasion capacity of L. monocytogenes. Eleven L. monocytogenes strains (serovars 1/2a,1/2b,1/2c and 4b) isolated from food processing plants and food products were grown in culture media, associated with Greenshell™ mussel surfaces and subjected (or not) to HPP (350 MPa, 2 min) or mild heating (MH, 55 °C, 10 min). The invasiveness of the strains was compared immediately after processing and after 14 days of storage at 8 °C using human intestinal epithelial Caco-2 cells. All strains on mussels had decreased invasion capacity compared with those coming directly from culture media. Invasiveness of L. monocytogenes also decreased immediately after pressurization but returned to the rate in untreated samples after 14 days of refrigerated storage. In contrast, MH did not affect invasion capacity, and treated samples showed the same rates as non-MH treated ones. Lineage I strains were more invasive than lineage II strains after both mild processing treatments. Based on these results, the risk of L. monocytogenes infection by the consumption of treated mussels will not be increased, as the invasiveness of possible survivors is not increased.

Aim To isolate and characterize listeriaphages from seafood environments. Methods and Results Listeriaphages (phages) isolated from seafood environments were distinguished by physical and biological techniques including restriction digestion of phage DNA. Three phages belonged to order Caudovirales and showed psychrotrophic characteristics. The phages had broad host ranges against 23 Listeria strains by productive infection or at least by adsorption. At 15 ± 1°C, adsorption rate constants of the three phages ranged from 8·93 × 10−9 to 3·24 × 10−11 ml min−1 across different Listeria monocytogenes strains. In indicator hosts, the mean burst sizes of phages LiMN4L, LiMN4p and LiMN17 were c. 17, 17 and 11 plaque-forming units (PFU) per cell, respectively, at 15 ± 1°C. The respective latent periods were c. 270 min for phages LiMN4p and LiMN17, whereas for phage LiMN4L, it was c. 240 min. Conclusions The three virulent psychrotrophic phages isolated from seafood-processing environments had broad host ranges and low productive replication. These characteristics suggest that the phages may be suitable as passive biocontrol agents against seafood-borne L. monocytogenes. Significance and Impact of the Study This is the first report on the isolation of autochthonous virulent listeriaphages from seafood-processing environments and information on single-step replication and adsorption characteristics of such listeriaphages.

Contamination of mussels with the human pathogen Listeria monocytogenes occurs during processing in the factory, possibly from bacteria persisting in the factory's indoor and outdoor areas. In this study, a selection of persistent (n = 8) and sporadic (n = 8) L. monocytogenes isolates associated with mussel-processing premises in New Zealand were investigated for their phenotypic and genomic characteristics. To identify traits that favour or contribute to bacterial persistence, biofilm formation, heat resistance, motility and recovery from dry surfaces were compared between persistent and sporadic isolates. All isolates exhibited low biofilm formation at 20 °C, however, at 30 °C persistent isolates showed significantly higher biofilm formation after 48 h using cell enumeration and near significant difference using the crystal violet assay. All 16 isolates were motile at 20 °C and 30 °C and motility was fractionally higher for sporadic isolates, but no significant difference was observed. We found persistent isolates tend to exhibit greater recovery after incubation on dry surfaces compared to sporadic isolates. Two of the three most heat-resistant isolates were persistent, while four of five isolates lacking heat resistance were sporadic isolates. Comparison of genome sequences of persistent and sporadic isolates showed that there was no overall clustering of persistent or sporadic isolates, and that differences in prophages and plasmids were not associated with persistence. Our results suggest a link between persistence and biofilm formation, which is most likely multifactorial, combining subtle phenotypic and genotypic differences between isolates.

Listeria monocytogenes is the causative agent of listeriosis in humans. Its wide distribution in the environment is one of the main reasons it is a source of food poisoning. L. monocytogenes also forms biofilms, making cleaning and sanitation difficult. Microtiter plate assays have previously been used to assess biofilm formation of L. monocytogenes and to evaluate the antimicrobial activity of disinfectants in suspension. In this study, we have used a microplate assay to evaluate the minimum sanitizer concentrations required for a 5 log10 CFU/ml decrease of L. monocytogenes cells in suspension and in biofilm. Twenty-one sanitizers were tested against 20 strains of L. monocytogenes. The results showed that all tested sanitizers achieved a 5-log10 reduction of viable cells in suspension at concentrations below the manufacturers' recommended concentrations, of a biguanide-based product. When tested against L. monocytogenes in biofilm, only the peroxyacetic acid, chlorine dioxide and acidified sodium chorite-based products gave a 5-log10 decrease, within or close to the manufacturers' recommended concentrations. No relationship was observed between susceptibility to chemical sanitizers and other characteristics of the isolates: pulsotypes, sources, biofilm forming ability or persistence. This work suggests that caution must be taken in selecting an appropriate sanitizer for use in food processing plants and an effective cleansing programme is required to ensure thorough inactivation and removal of L. monocytogenes in biofilms.

Considering the lack of information available in Brazil concerning listeriosis, the present study aimed to analyze phenotypic and genotypically Listeria monocytogenes clinical isolates from the Southwestern region of the State of São Paulo, Brazil. From January 1995 to May 2005, thirteen isolates of L. monocytogenes from twelve patients with listeriosis were sent to the Adolfo Lutz Institute (Campinas Regional Laboratory, São Paulo, Brazil). The antimicrobial susceptibility of the isolates was evaluated by the microdilution broth method for ampicillin, gentamicin, trimethoprim, sulfamethoxazole and vancomycin. They were also serotyped (Denka Seiken, Japan), and sub-typed by PFGE employing ApaI and AscI and the American CDC protocol. None of the isolates showed resistance to the antibiotics tested; however, seven of them presented increased values for sulfamethoxazole MICs. Ten isolates belonged to serotype 4b, two to serotype 1/2a and only one to serotype 1/2b. After digestion with ApaI and AscI, the strains were distributed in 3 different groups according to their profile. It was observed that the spatial or temporally unrelated strains exhibited similar PFGE profiles, indicating a possible clonal relationship among them. No Brasil, dados sobre a ocorrência de surtos ou casos esporádicos de listeriose são raros, assim como estudos que tratem da caracterização de cepas de L. monocytogenes isoladas destes episódios. Desta forma, o presente estudo teve por objetivo avaliar feno e genotipicamente 13 cepas de Listeria monocytogenes isoladas de 12 casos clínicos de listeriose ocorridos em 6 municípios da região sudoeste do Estado de São Paulo, Brasil, no período de janeiro de 1995 a maio de 2005. As cepas foram submetidas ao teste de sensibilidade a cinco antimicrobianos (ampicilina, gentamicina, trimetoprim, sulfametoxazol e vancomicina) pelo método de microdiluição em caldo; à sorotipagem (Denka Seiken, Japão) e à determinação do perfil de macro-restrição por eletroforese em campo pulsado (PFGE, Pulsenet, CDC, USA, 2001). Nenhuma das cepas foi resistente aos antimicrobianos testados; entretanto, sete tiveram valores da CIM aumentados para o sulfametoxazol. Dez cepas pertenciam ao sorotipo 4b, duas ao sorotipo 1/2a e apenas uma ao sorotipo 1/2b. Com o emprego da técnica de PFGE e as enzimas Apa I e Asc I as cepas foram separadas em três grupos diferentes, de acordo com seus perfis moleculares. É interessante destacar que cepas não relacionadas (temporal ou espacialmente) apresentaram perfis moleculares semelhantes indicando uma possível relação clonal entre elas.

Listeria monocytogenes 15G01, a strain belonging to the persistent pulsotype 5132, was isolated from a seafood processing plant in New Zealand. Simple monoculture assays using crystal violet staining showed good biofilm formation for this strain and it was therefore chosen to be further investigated in regard to its biofilm forming ability. To evaluate its behaviour in different conditions commonly encountered in food processing environments, biofilm assays and growth studies were performed using common laboratory media under a range of temperatures (20 °C, 30 °C and 37 °C). Furthermore, the effects of incubation time and different environmental conditions including static, dynamic and anaerobic incubation on biofilm formation were investigated. Changes in the environmental conditions resulted in different biofilm phenotypes of L. monocytogenes 15G01. We demonstrated that increasing temperature and incubation time led to a higher biofilm mass and that dynamic incubation has little effect on biofilm formation at 37 °C but encourages biofilm formation at 30 °C. Biofilm production at 20 °C was minimal regardless of the medium used. We furthermore observed that anaerobic environment led to reduced biofilm mass at 30 °C for all tested media but not at 37 °C. Biofilm formation could not be narrowed down to one factor but was rather dependent on multiple factors with temperature and medium having the biggest effects.

Listeriosis is caused by the food-borne pathogen Listeria monocytogenes , which can be found in seafood and processing plants. To evaluate the risk to human health associated with seafood production in New Zealand, multi-virulence-locus sequence typing (MVLST) was used to define the sequence types (STs) of 31 L. monocytogenes isolates collected from seafood-processing plants, 15 from processed foods, and 6 from human listeriosis cases. The STs of these isolates were then compared with those from a collection of seafood isolates and epidemic strains from overseas. A total of 17 STs from New Zealand clustered into two lineages: seafood-related isolates in lineages I and II and all human isolates in lineage II. None of the New Zealand STs matched previously described STs from other countries. Isolates (belonging to ST01-N and ST03-N) from mussels and their processing environments, however, were identical to those of sporadic listeriosis cases in New Zealand. ST03-N isolates (16 from mussel-processing environments, 2 from humans, and 1 from a mussel) contained an inlA premature stop codon (PMSC) mutation. Therefore, the levels of invasiveness of 22 isolates from ST03-N and the three other common STs were compared using human intestinal epithelial Caco-2 cell lines. STs carrying inlA PMSCs, including ST03-N isolates associated with clinical cases, had a low invasion phenotype. The close relatedness of some clinical and environmental strains, as revealed by identical MVLST profiles, suggests that local and persistent environmental strains in seafood-processing environments pose a potential health risk. Furthermore, a PMSC in inlA does not appear to give L. monocytogenes a noninvasive profile.

Listeria-infecting bacteriophages (listeriaphages) can be used to control Listeria monocytogenes in the food industry. However, the sensitivity of many of seafood-borne Listeria strains to phages has not been reported. This research investigated the host ranges of three listeriaphages (FWLLm1, FWLLm3 and FWLLm5) by the formation of lytic zones and plaques on host lawns and in vitro lysis kinetics of listeriaphage FWLLm3. The study also predicted the phage titres required to lyse host cells. The host ranges of the phages were determined using 50 L. monocytogenes strains, of which 48 were isolated from the seafood industry and two from clinical cases. Of the 50 strains, 36 were tested at 25 and 30 ℃ and the remainder (14) at 15 and 25 ℃. Based on the formation of either discrete plaques or lytic zones (host kill zones), the host ranges of FWLLm1, FWLLm3 and FWLLm5 were about 87%, 81% and 87%, respectively, at 25 ℃. Six L. monocytogenes strains from the seafood environment were insensitive to all three phages, while the other seafood strains (42) were phage-sensitive. The adsorption rate constant ( k value) of listeriaphage FWLLm3 was between 1.2 × 10−9 and 1.6 × 10−9 ml/min across four host strains in tryptic soy broth at 25 ℃. The cultures (at 3–4 log colony-forming unit (CFU/ml) were completely lysed ( 8.7 log phage-forming units (PFU/ml) for 30 min. Re-growth of phage-infected cultures was not detected after 24 h. The effective empirical phage titre was similar to the calculated titre using a kinetic model. Results indicate the potential use of the three phages for controlling L. monocytogenes strains in seafood processing environments.

Listeria monocytogenes is a food-borne pathogen which causes listeriosis and is difficult to eradicate from seafood processing environments; therefore, more effective control methods need to be developed. This study investigated the effectiveness of three bacteriophages (LiMN4L, LiMN4p and LiMN17), individually or as a three-phage cocktail at ≈9 log10 PFU/ml, in the lysis of three seafood-borne L. monocytogenes strains (19CO9, 19DO3 and 19EO3) adhered to a fish broth layer on stainless steel coupon (FBSSC) and clean stainless steel coupon (SSC), in 7-day biofilm, and dislodged biofilm cells at 15 ± 1 °C. Single phage treatments (LiMN4L, LiMN4p or LiMN17) decreased bacterial cells adhered to FBSSC and SSC by ≈3–4.5 log units. Phage cocktail reduced the cells on both surfaces (≈3.8–4.5 and 4.6–5.4 log10 CFU/cm2, respectively), to less than detectable levels after ≈75 min (detection limit = 0.9 log10 CFU/cm2). The phage cocktail at ≈5.8, 6.5 and 7.5 log10 PFU/cm2 eliminated Listeria contamination (≈1.5–1.7 log10 CFU/cm2) on SSC in ≈15 min. One-hour phage treatments (LiMN4p, LiMN4L and cocktail) in three consecutive applications resulted in a decrease of 7-day L. monocytogenes biofilms (≈4 log10 CFU/cm2) by ≈2–3 log units. Single phage treatments reduced dislodged biofilm cells of each L. monocytogenes strain by ≈5 log10 CFU/ml in 1 h. The three phages were effective in controlling L. monocytogenes on stainless steel either clean or soiled with fish proteins which is likely to occur in seafood processing environments. Phages were more effective on biofilm cells dislodged from the surface compared with undisturbed biofilm cells. Therefore, for short-term phage treatments of biofilm it should be considered that some disruption of the biofilm cells from the surface prior to phage application will be required.

Greenshell™ mussels are New Zealand's largest seafood export species. Some export markets require compliance with 'zero' tolerance legislation for Listeria monocytogenes in 25 g of product. Even though individually quick frozen (IQF) mussel products are labeled 'to be cooked', and are not classified as ready-to-eat, some markets still require them to comply with the strict policy. Three mussel processing plants were assessed for the pattern of L. monocytogenes contamination on raw material, environment, food contact surfaces, and in the final product. Cultures (n = 101) obtained from an industrial Listeria monitoring program from August 2007 to June 2009 were characterized by serotyping and pulsed field gel electrophoresis. Using the crystal violet method, isolates were assessed for their ability to form biofilms. This work confirmed the presence of L. monocytogenes in raw and processed product, and the importance of cross-contamination from external and internal environments. Processing plants had L. monocytogenes pulsotypes that were detected more than once over 6 months. No correlation was found between biofilm-forming ability and persistent isolates. Two pulsotypes (including a persistent one), were previously isolated in human cases of listeriosis in New Zealand, but none of the pulsotypes matched those involved in international outbreaks.

Listeria monocytogenes is a cause of concern to food industries, mainly for those producing ready-to-eat (RTE) products. This microorganism can survive processing steps such as curing and cold smoking and is capable of growing under refrigeration temperatures. Its presence in RTE fish products with extended shelf life may be a risk to the susceptible population. One example of such a product is gravlax salmon; a refrigerated fish product not exposed to listericidal processes and was the subject of this study. In order to evaluate the incidence and dissemination of L. monocytogenes 415 samples were collected at different steps of a gravlax salmon processing line in São Paulo state, Brazil. L. monocytogenes was confirmed in salmon samples (41%), food contact surfaces (32%), non-food contact surfaces (43%) and of food handlers' samples (34%), but could not be detected in any ingredient. 179 L. monocytogenes isolates randomly selected were serogrouped and typed by PFGE. Most of L. monocytogenes strains belonged to serogroup 1 (73%). 61 combined pulsotypes were found and a dendrogram identified six clusters: most of the strains (120) belonged to cluster A. It was suggested that strains arriving into the plant via raw material could establish themselves in the processing environment contaminating the final product. The wide dissemination of L. monocytogenes in this plant indicates that a great effort has to be taken to eliminate the microorganism from these premises, even though it was not observed multiplication of the microorganism in the final product stored at 4ºC up to 90 days.