Listeria Monocytogenes Genes Research Articles

Some virulence genes and biofilm formation capabilities of Listeria monocytogenes isolates from different sources

In this study, it was aimed to determine the biofilm-forming abilities of both clinical and food-borne isolates of Listeria monocytogenes, to investigate the presence of nine different virulence genes, and to consider the current threat status of this agent. A total of 28 isolates, 21 from food and seven from clinical origin, were used in the study. To determine the biofilm formation abilities of isolates, two different methods namely “tube adherence” and “microplate” were used respectively. For the determination of nine different virulence genes of Listeria monocytogenes (inlA, inlC, inlJ, hylA, luxS, flaA, prfA, inlB, actA), the method of polymerase chain reaction (PCR) was used. As a result, all isolates were found to be able to form a biofilm to varying degrees by both tube and microplate methods. These two methods were similar in terms of their results. All nine different virulence gene regions were detected at various rates in the isolates. Whereas the genes directly related to biofilm formation for the isolates weren't detected, to form biofilm was observed. The virulence genes detected in clinical origin isolates were proportionally higher than in food-borne isolates (except for flaA and prfA gene regions). It was concluded that bacteria of Listeria monocytogenes continue to form biofilm and carry virulence genes regardless they are from food or clinical origin. Also, food-borne contaminations continue to be a severe threat to human health. So, to prevent listeriosis cases of both humans and animals should be taken required precautions and all cases should be considered carefully.

Investigating the Effect of Melittin Peptide in Preventing Biofilm Formation, Adhesion and Expression of Virulence Genes in Listeria monocytogenes.

Listeria monocytogenes is a notable food-borne pathogen that has the ability to create biofilms on different food processing surfaces, making it more resilient to disinfectants and posing a greater risk to human health. This study assessed melittin peptide's anti-biofilm and anti-pathogenicity effects on L. monocytogenes ATCC 19115. Melittin showed minimum inhibitory concenteration (MIC) of 100μg/mL against this strain and scanning electron microscopy images confirmed its antimicrobial efficacy. The OD measurement demonstrated that melittin exhibited a strong proficiency in inhibiting biofilms and disrupting pre-formed biofilms at concentrations ranging from 1/8MIC to 2MIC and this amount was 92.59 ± 1.01% to 7.17 ± 0.31% and 100% to 11.50 ± 0.53%, respectively. Peptide also reduced hydrophobicity and self-aggregation of L. monocytogenes by 35.25% and 14.38% at MIC. Melittin also significantly reduced adhesion to HT-29 and Caco-2 cells by 61.33% and 59%, and inhibited invasion of HT-29 and Caco-2 cells by 49.33% and 40.66% for L. monocytogenes at the MIC value. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) revealed melittin's impact on gene expression, notably decreasing inlB (44%) and agrA (45%) gene expression in L. monocytogenes. flaA and hly genes also exhibited reduced expression. Also, significant changes were observed in sigB and prfA gene expression. These results underscore melittin's potential in combating bacterial infections and biofilm-related challenges in the food industry.

Detection of Some Virulence Factors of Listeria Species Isolated from Chicken and their Products

The current study was conducted to determine the occurrence of Listeria species in live chicken and some chicken products (fillets, burger and luncheon). In addition, determine the antibiogram profile of Listeria strains and detection of some virulence genes of Listeria monocytogenes which recovered from the examined samples. The study included 100 samples including cloacal swabs from broiler chicken (n =25) from 5 different broiler chicken farms beside as well as chicken products (25 samples each of chicken fillets, burger and luncheon) that were randomly purchased from supermarkets. Isolation and identification of Listeria was performed according to the standard protocol provided by ISO (2017) using Listeria selective agar (Oxford formulation) with Listeria selective supplement then the suspected colonies were identified using a panel of biochemical tests. It was recorded that the recovery rate of Listeria species from the examined samples was 24, 20, 8 and 16%, in cloacal swabs, chicken fillets, burger and luncheon, respectively with an overall incidence of 17%. In addition, biochemical identification of the recovered Listeria isolates revealed the presence of L. monocytogenes, L. innocua, L. ivanovii, L. seeligeri and L. grayi with various rates where L. monocytogenes scored the highest rate 29.4% (5 isolates). Antimicrobial susceptibility of Listeria strains (n=17) clarified that the highest antibiotic resistance was recorded when using streptomycin (82.4%) followed by Neomycin (76.5%) then Doxycycline and Cefotaxime (52.9%) while the lowest resistance was noticed when using Amikacin (11.8%) then Ciprofloxacin (35.3%). Finally, molecular detection of virulence genes in the identified L. monocytogenes strains (n= 5) clarified the detection of iap and actA genes in the five isolates while hylA gene was detected in three isolates only. At the end, results obtained by this study emphasize a significant occurrence of resistant Listeria in chicken and chicken products including processed animal-originated foods commonly purchased by the public in including; chicken fillets, burger and luncheon. Seriously, the presence of such resistant bacteria endangers human health by making routine antibiotic courses useless in the treatment of such infections. Finally, data proved that retail foods of animal origin pose a risk to human health.

The Occurrence of Serotypes and Virulence Genes of Listeria monocytogenes in Various Food Products

Background: Given that controlling Listeria contamination is very important in food chain system, the knowledge of their prevalence in food is very important. Therefore, this study aims to examine the prevalence of important Listeria species in various food types and evaluate serotype distribution, as well as the study of the virulence factors of L. monocytogenes.
 Methods: During July 2018 and January 2020, 900 food samples were collected in the North of Iran, including beef, chicken, fish, shrimp, milk and yogurt, green vegetables, mixed vegetable salad, Olivier salad, and cottage cheese. After isolation and identification steps, each bacterial DNA was extracted. Then, using specific primers, species, serotypes, and virulence genes of Listeria isolates were evaluated in the samples by Polymerase Chain Reaction (PCR) method.
 Results: The test results of 136 samples (15.1%) were positive for Listeria spp. and the most contaminated food was beef (35%) followed by chicken (29%) and green vegetables (23%). The most isolated Listeria spp. was L. monocytogenes and L. ivanovii. Among L. monocytogenes isolates, the dominant serogroups were 1/2a and 4b; furthermore, all of the isolates of this species harbored four virulence genes, including hlyA, plc, iap, and actA.
 Conclusion: These reports highlighted the importance of food safety in various food products, particularly raw meats and vegetables. Moreover, contamination of healthy foods such as fish and vegetables with Listeria is an indicator of public health.

Study the Antimicrobial Resistance Genes of Listeria Monocytogenes Isolated From Industrial and Clinical Samples in Iraq

Multi-drug resistance in Listeria monocytogenes is considered a major public health problem associated with foodborne outbreaks and causes high hospitalization and mortality rates. This study aimed to investigate the antimicrobial resistant genes among Listeria monocytogenes isolated from meat and clinical samples. Phenotypically, the isolates were tested for their susceptibility against the 12 most commonly used antimicrobials in veterinary and human therapy via the disc diffusion method, while conventional PCR was performed to study the presence or absence of 14 resistance genes predicted in L. monocytogenes isolates. The study established that 30(66.66%) of L. monocytogenes isolates showed phenotypic multi-drug resistance against at least three antimicrobial classes. Furthermore, high resistance frequencies were reported among commonly used antibiotics for listeriosis therapy. The present study revealed that the investigated isolates show resistance against tetracycline 33(73.3%), ampicillin 29(64.4%), penicillin 28(62.2%), erythromycin 26(57.8%), and gentamycin, clindamycin and vancomycin 24(53.3% each). Of the 45 L. monocytogenes isolates studied, 37(82.2%) were phenotypically susceptible to meropenem, followed by ciprofloxacin 36(80.0%) and SXT 30(66.7%). PCR amplification of antimicrobial resistance genes established the occurrence of antibiotic resistance genes in all studied L. monocytogenes isolates. Notably, 41(91.11%) of these isolates exhibited more than five resistance genes. Surprisingly, penA and ampC were detected in all L. monocytogenes strains 45(100%), followed by ermB 44(97.8%), tetA 38(84.4%), tetG 32(71.1%), and vanB 30(66.7%). Moreover, vanA 22(48.9%) and tetB 18(40.0%) were detected less frequently. The lowest incidences of resistance genes were observed in L. monocytogenes carrying tetD 4(8.9%) and cmlA 6(13.3%). In conclusion, the study demonstrates that the majority of L. monocytogenes from human and meat samples displayed a high index of resistance to a variety of agents used for clinical listeriosis treatment adding further burden to the existing global antibiotic resistance problem.

Horizontal Gene Transfer and Loss of Serotype-Specific Genes in Listeria monocytogenes Can Lead to Incorrect Serotype Designations with a Commonly-Employed Molecular Serotyping Scheme.

Listeria monocytogenes is a Gram-positive, facultative intracellular foodborne pathogen capable of causing severe, invasive illness (listeriosis). Three serotypes, 1/2a, 1/2b, and 4b, are leading contributors to human listeriosis, with 4b including the major hypervirulent clones. The multiplex PCR scheme developed by Doumith and collaborators employs primers targeting specific lineages (e.g., lineage II-specific lmo0737, lineage I-specific LMOf2365_2059) or serotypes (e.g., serotype 4b-specific LMOf2365_1900). The Doumith scheme (DS) is extensively employed for molecular serotyping of L. monocytogenes due to its high accuracy, relative ease, and affordability. However, for certain strains, the DS serotype designations are in conflict with those relying on antibody-based schemes or whole-genome sequence (WGS) analysis. In the current study, all 27 tested serotype 4b strains with sequence type 782 (ST782) within the hypervirulent clonal complex 2 (CC2) were designated 1/2b/3b using the DS. These strains lacked the serotype 4b-specific gene LMOf2365_1900, while retaining LMOf2365_2059, which, together with prs, yields the DS 1/2b/3b profile. Furthermore, 15 serotype 1/2a strains of four STs, mostly from water, were designated 1/2b/3b using the DS. These strains lacked the lmo0737 cassette but harbored genomic islands with LMOf2365_2059, thus yielding the DS 1/2b/3b profile. Lastly, we investigated a novel, dual 1/2a-1/2b profile obtained using the DS with 21 serotype 1/2a strains of four STs harboring both the lmo0737 cassette and genomic islands with LMOf2365_2059. The findings suggest that for certain strains and clones of L. monocytogenes the DS designations should be viewed with caution and complemented with alternative tools, e.g., traditional serotyping or WGS analysis. IMPORTANCE Listeria monocytogenes is a foodborne pathogen responsible for severe illness (listeriosis), especially in pregnant women and their fetuses, immunocompromised individuals, and the elderly. Three serotypes, 1/2a, 1/2b, and 4b, account for most human listeriosis, with certain serotype 4b clonal complexes (CCs) overrepresented in human disease. Serotyping remains extensively employed in Listeria epidemiologic investigations, and a multiplex PCR-based serotyping scheme is widely used. However, the PCR gene targets can be lost or gained via horizontal gene transfer, leading to novel PCR profiles without known serotype designations or to incorrect serotype assignments. Thus, an entire serotype 4b clone of the hypervirulent CC2 would be misidentified as serotype 1/2b, and several strains of serotype 1/2a would be identified as serotype 1/2b. Such challenges are especially common in novel clones from underexplored habitats, e.g., wildlife and surface water. The findings suggest caution in application of molecular serotyping, while highlighting Listeria's diversity and potential for horizontal gene transfer.

Investigation of Antimicrobial Resistance Genes in Listeria monocytogenes from 2010 through to 2021

Antimicrobial resistance (AMR) is a serious public health issue. Due to resistance to current antibiotics and a low rate of development of new classes of antimicrobials, AMR is a leading cause of death worldwide. Listeria monocytogenes is a deadly foodborne pathogen that causes listeriosis for the immunocompromised, the elderly, and pregnant women. Unfortunately, antimicrobial resistance has been reported in L. monocytogenes. This study conducted the first comprehensive statistical analysis of L. monocytogenes isolate data from the National Pathogen Detection Isolate Browser (NPDIB) to identify the trends for AMR genes in L. monocytogenes. Principal component analysis was firstly used to project the multi-dimensional data into two dimensions. Hierarchical clustering was then used to identify the significant AMR genes found in L. monocytogenes samples and to assess changes during the period from 2010 through to 2021. Statistical analysis of the data identified fosX, lin, abc-f, and tet(M) as the four most common AMR genes found in L. monocytogenes. It was determined that there was no increase in AMR genes during the studied time period. It was also observed that the number of isolates decreased from 2016 to 2020. This study establishes a baseline for the ongoing monitoring of L. monocytogenes for AMR genes.

Multiple Anti-microbial Resistance Profile and Molecular Detection of Some Virulence ‎Genes of Listeria Monocytogenes Isolated from Fresh Raw Meat Retailed in Zaria, ‎Northwestern Nigeria ‎

Multiple Anti-microbial Resistance Profile and Molecular Detection of Some Virulence ‎Genes of Listeria Monocytogenes Isolated from Fresh Raw Meat Retailed in Zaria, ‎Northwestern Nigeria ‎

Growth and Expression of Virulence Genes of Listeria monocytogenes during the Processing of Dry-Cured Fermented "Salchichón" Manufactured with a Selected Lactilactobacillus sakei.

Simple SummaryDuring the ripening process of the dry-cured fermented sausage “salchichón”, Listeria monocytogenes could fail to be eliminated. In addition, the food safety criterion for L. monocytogenes in the European Union sets up a maximum level of 100 units of this microorganism per gram in ready-to-eat products throughout their shelf-life. Thus, since L. monocytogenes could be present in this product, it is necessary to evaluate the impact of the dry-cured fermented processing in the potential virulence of this pathogen, even considering the possible effect of the usual microbiota (lactic-acid bacteria) of “salchichón”. In this work, the effect of the processing of “salchichón”, inoculated with a selected strain of Lactilactobacillus sakei, on the growth of L. monocytogenes and on the expression of its virulence genes, was evaluated. The processing of “salchichón” provoked a relevant reduction in L. monocytogenes, but this pathogen was not completely eliminated. However, a downregulation in the expression of the tested virulence genes was found, which could suppose a reduction in the pathogenic effect of this microorganism. These findings could be of great interest to consider the dry-cured ripening of “salchichón” as a safe process to control the pathogen L. monocytogenes.The effect of the dry-cured fermented processing of “salchichón” inoculated with a selected strain of Lactilactobacillus sakei (205) on the growth and transcriptional response of three virulence genes (plcA, hly, and iap) of Listeria monocytogenes was evaluated. For this, three different batches of “salchichón” were analyzed: batch B (inoculated only with L. sakei), batch L (inoculated only with L. monocytogenes), and batch L + B (inoculated with both microorganisms). Sausages were ripened for 90 days according to a traditional industrial process. The processing of “salchichón” provoked a reduction in L. monocytogenes counts of around 2 log CFU/g. The downregulation of the expression of the three genes was found at the end of ripening when the water activity (aw) of “salchichón” was <0.85 aw. The combined effect on the reduction in L. monocytogenes counts together with the downregulation in the expression of the virulence genes throughout the “salchichón” processing could be of great interest to control the hazard caused by the presence of this pathogenic bacterium.

Occurrence, antimicrobial resistance, serotyping and virulence genes of Listeria monocytogenes isolated from foods

Listeria monocytogenes is a pathogen contaminated food, it is the cause of listeriosis worldwide. The aims of this study were to investigate the occurrence, antimicrobial resistance, serotyping and virulence genes of L. monocytogenes isolated from foods in Meknes city of Morocco. From June 2017 to May 2018, 520 food samples were randomly collected from a traditional market and two overcrowded popular neighborhoods (Lahdim and Hamria) and subjected to the detection of L. monocytogenes. Then, the antimicrobial susceptibility of the isolated strains were evaluated using the standard disk diffusion method and the determination of serotypes and virulence genes was performed by PCR. The results showed the detection of L. monocytogenes in fifteen (2.9%) of 520 samples, including three (5.7%) isolates in traditional whey, raw minced meat and raw sausage, two (3.8%) in raw milk and one (1.9%) in smen (traditional butter), raw bovine meat, raw poultry meat and raw fish, while salads and rayeb (traditional coagulated milk) were not contaminated. Among the fifteen isolated L. monocytogenes, nine (60%) belonged to the serogroup (1/2a, 1/2c, 3a and 3c), two (13.3%) belonged to the serogroup (1/2b, 3b, 4b and 4d) and four (26.6%) do not belong to any studied serogroup. Furthermore, fifteen (100%) isolates showed the presence of actA gene, fourteen (93.3%) harbored hlyA, prfA and plcB genes, thirteen (86.7%) carried inlA and inlC genes and twelve (80%) showed inlJ gene. The antimicrobial susceptibility analysis showed that the isolated strains were more resistant to amoxicillin/clavulanic acid (67.0%), erythromycin (60.0%), sulphamethoxazole (40.0%), ampicillin and sulphamethoxazole/trimethoprim (33.0%) and tetracycline (20.0%). Furthermore, 66.7% (10/15) were multidrug-resistant. From this study, we can conclude that foods marketed in Meknes city were contaminated by multidrug-resistant strains of L. monocytogenes harboring virulence genes, which may cause a serious risk to public health.

Identification of Listeria monocytogenes Genes Contributing to Oxidative Stress Resistance under Conditions Relevant to Host Infection.

The Gram-positive bacterium Listeria monocytogenes survives in environments ranging from the soil to the cytosol of infected host cells. Key to L. monocytogenes intracellular survival is the activation of PrfA, a transcriptional regulator that is required for the expression of multiple bacterial virulence factors. Mutations that constitutively activate prfA (prfA* mutations) result in high-level expression of multiple bacterial virulence factors as well as the physiological adaptation of L. monocytogenes for optimal replication within host cells. Here, we demonstrate that L. monocytogenesprfA* mutants exhibit significantly enhanced resistance to oxidative stress in comparison to that of wild-type strains. Transposon mutagenesis of L. monocytogenesprfA* strains resulted in the identification of three novel gene targets required for full oxidative stress resistance only in the context of PrfA activation. One gene, lmo0779, predicted to encode an uncharacterized protein, and two additional genes known as cbpA and ygbB, encoding a cyclic di-AMP binding protein and a 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase, respectively, contribute to the enhanced oxidative stress resistance of prfA* strains while exhibiting no significant contribution in wild-type L. monocytogenes Transposon inactivation of cbpA and lmo0779 in a prfA* background led to reduced virulence in the liver of infected mice. These results indicate that L. monocytogenes calls upon specific bacterial factors for stress resistance in the context of PrfA activation and thus under conditions favorable for bacterial replication within infected mammalian cells.

Effect of E-beam treatment on expression of virulence and stress-response genes of Listeria monocytogenes in dry-cured ham

Various adverse conditions can trigger defensive mechanisms in Listeria monocytogenes that can increase the virulence of surviving cells. The objective of this study was to evaluate the expression of one stress-response (sigB) and three virulence (plcA, hly, and iap) genes in L. monocytogenes exposed to a sub lethal dose of E-beam irradiation in dry-cured ham. To accomplish this, dry-cured ham slices (10 g) were immersed in a 109 CFU/mL suspension of L. monocytogenes strain S4-2 and subsequently irradiated with 1, 2, or 3 kGy. After irradiation, samples were stored at 7 °C or 15 °C for 30 days. Absolute gene expression levels were determined by RT-qPCR, and numbers of surviving Listeria cells were assessed by microbial counts after different storage times (0, 7, 15, and 30 days). At 7 °C, after E-beam treatment at doses of 2 or 3 kGy, Listeria gene expression significantly increased (p ≤ 0.05) up to day 15. Listeria counts decreased with increasing dosage. The relationship between absolute gene expression and the number of surviving Listeria cells could indicate that sublethal doses of E-beam irradiation can increase expression of the genes studied. We observed no significant influence of storage time or temperature on gene expression (p > 0.05). Listeria that survives E-beam treatment may display increased virulence, constituting a significant potential public health risk.

Antimicrobial Susceptibility, Serotyping, and Molecular Characterization of Antibiotic Resistance Genes in Listeria monocytogenes Isolated from Pregnant Women with a History of Abortion.

Background:Listeria monocytogenes show high mortality among pregnant women and newborns. This study aimed to detect L. monocytogenes in pregnant women with a history of abortion and assess the serotypes, antibiotic susceptibility patterns, and its resistance genes.Methods:Overall, 400 vaginal swabs were taken from pregnant women with a history of abortion in the past few years in a tertiary care hospital in Tehran, Iran, during 2015–2018. Antibiotics susceptibility to a panel of 10 antibiotics was determined using the standard disk diffusion method and the isolates serotyped by the agglutination method. The antimicrobial-resistant isolates were also screened for the presence of tetM, ermB and dfrD genes by PCR.Results:Overall, 22 L. monocytogenes isolates were identified. High rates of resistance were observed for trimethoprim (50%; n=11), sulphamethoxazole (50%; n=11), tetracycline (45.45%; n=10) and gentamicin (36.36%; n=8). From 22 L. monocytogenes isolates, 13 (59.10 %), 5 (22.73%), 3 (13.63%) and 1 (4.54%) belonged to serotypes 4b, 1/2a, 1/2b, and 3c, respectively. The genetic determinant tetM was detected in 70% of the tetracycline-resistant isolates. Out of 11 trimethoprim-resistant isolates, 27.27% isolates contained dfrD. Moreover, the ermB gene was found in 83.33% of the erythromycin-resistant isolates.Conclusion:Ampicillin and partly penicillin consider to be suitable antimicrobial agents to treat human listeriosis. Moreover, due to resistance against many antibiotics, it is necessary to continue monitoring and managing antimicrobial resistance.

Phylogenetic Analysis and Antibiotics Resistance of Listeria Monocytogenes Contaminating Chicken Meat in Surabaya, Indonesia.

The objective of this study was to identify the phylogenetic analysis and antibiotic resistance of Listeria monocytogenes contaminating chicken meat in Surabaya. 60 chicken meat samples were collected from supermarkets, mobile vendors, and traditional markets in Surabaya. A selective medium is used for isolation and identification of Listeria monocytogenes by chopping 25 grams of the chicken meat and to put it into the sterilized Erlenmeyer flasks. Some methods were used for the identification procedures, such as biochemical and morphological tests, antibiotic resistance test, PCR, and sequencing; also a phylogenetic analysis was conducted by a neighbor-joining analysis using Genetix Mac ver 8.0 with hlyA genes of Listeria monocytogenes recorded in GenBank, such as Lineage I (KC808543), Lineage II (AY229462, AY229346, AY229499, and AY229404), Lineage III (KJ504139, HQ686043, KJ504116, and DQ988349), and Lineage IV (EU840690, EF030606). The result shows that the prevalence of L. monocytogenes in Surabaya contaminating the chicken meat samples from the supermarkets was 10% (2/20), from the mobile vendors was 0/20 (0%), and from the traditional markets was 5% (1/20). It was seen from the band at 456 bp fragment. Furthermore, three isolates found in Surabaya were included in the new lineages which were resistant to old-generation antibiotics such as sulfamethonazole-trimetophrim (SXT) and amoxyllin sulbactam (MAS), but they were still sensitive to new-generation antibiotics such as cefotaxime (CTX) and meropenem (MEM).

Investigating the prevalence of resistance genes in Listeria monocytogenes isolated from food and clinical samples

Investigating the prevalence of resistance genes in Listeria monocytogenes isolated from food and clinical samples

Potential Ad Hoc Markers of Persistence and Virulence in Canadian Listeria monocytogenes Food and Clinical Isolates

The Listeria monocytogenes gene inlA, encoding a surface virulence protein, was examined for the presence of premature stop codon (PMSC) mutations in 82 isolates obtained by the Canadian Food Inspection Agency (CFIA) from foods and food contact surfaces. These mutations were coanalyzed for the presence of stress survival islet 1 (SSI-1) and for the abilities of the isolates to invade Caco-2 intestinal epithelial cells and form biofilms on polystyrene. PMSC mutations were present in one-third of the isolates (predominantly those of serogroup 1/2a), and their presence was correlated with a noninvasive phenotype. The presence of SSI-1 and the ability to form biofilms were also linked to the 1/2a serogroup. Serogroup 4b isolates lacked inlA PMSC mutations and were invasive, but neither formed biofilms nor carried SSI-1. To expand upon these experimental findings, an in silico analysis was performed on L. monocytogenes genomes from Canadian databases of 278 food isolates and 607 clinical isolates. The prevalence of inlA PMSC mutations in genomes of food isolates was significantly higher (P < 0.0001) than that in clinical isolates. Also, a three-codon deletion in inlA associated with a hyperinvasive phenotype was more prevalent in genomes from clinical isolates (primarily of clonal complex 6, serogroup 4b) than in those from food isolates (P < 0.001). In contrast, SSI-1 was significantly overrepresented (P < 0.001) in genomes from food isolates. We propose the hypothesis that SSI-1 and inlA play a role in the evolution of Canadian L. monocytogenes strains into either a virulent (represented by serogroup 4b clinical isolates) or an environmentally persistent (represented by serogroup 1/2a food isolates) phenotype. The combined presence of SSI-1 and inlA PMSC mutations have potential for use as genetic markers for risk assessment when L. monocytogenes is recovered from foods, indicating low potential for pathogenesis.

Antilisterial Effect and Influence on Listeria monocytogenes Gene Expression of Enterocin or Enterococcus faecalis in Sliced Dry-Cured Ham Stored at 7°C

In this study, we focused on the effect of an enterocin or an Enterococcus faecalis strain added onto sliced dry-cured ham that was artificially inoculated with Listeria monocytogenes and stored at 7°C. The population of L. monocytogenes and the expression of five genes were monitored throughout the storage period. A persistent and a nonpersistent strain were tested, and both were influenced by the presence of the enterocin; both populations were reduced by more than 2 Log CFU/g after 14 days compared with the control, noninoculated ham. The presence of E. faecalis, a bacteriocin-producing lactic acid bacterium, had a less pronounced effect on the viable counts for both strains. Concerning gene expression, a common trend observed for both strains in the presence of enterocin was the down-regulation of genes tested after 30 min of storage at 7°C. For the remainder of the storage period, the expression fluctuated but was mostly reduced. Similarly, the presence of E. faecalis led to an overall down-regulation of genes. The effect on gene expression of both enterocin and E. faecalis was more pronounced on the nonpersistent L. monocytogenes strain. Although the potential of a bacteriocin and a bacteriocin-producing microorganism to control L. monocytogenes was confirmed, this study highlights that gene expression may be influenced and needs to be evaluated when considering such biopreservation interventions.

Characterization of internalin genes in Listeria monocytogenes from food and humans, and their association with the invasion of Caco-2 cells

BackgroundInternalins are surface proteins that are utilized by Listeria monocytogenes to facilitate its invasion into human intestinal epithelial cells. The expression of a full-length InlA is one of essential virulence factors for L. monocytogenes to cross the intestinal barrier in order to invade epithelial cells.ResultsIn this study, the gene sequences of inlA in 120 L. monocytogenes isolates from food (n = 107) and humans (n = 13) were analyzed. Premature stop codon (PMSC) mutations in inlA were identified in 51 isolates (50 from food and 1 from human). Six mutation types of PMSCs were identified. Among the 51 isolates with PMSCs in inlA, there were 44 serogroup 1/2c, 3c isolates from food, of which seven belonged to serogroups 1/2a, 3a. A total of 153,382 SNPs in 2247 core genes from 42 genomes were identified and used to construct a phylogenetic tree. Serotype 1/2c isolates with inlA PMSC mutations were grouped together. Cell culture studies on 21 isolates showed that the invasion to Caco-2 cells was significantly reduced among isolates with inlA PMSC mutations compared to those without PMSC mutations (P < 0.01). The PMSC mutations in inlA correlated with the inability of the L. monocytogenes isolates to invade Caco-2 cells (Pearson’s coefficient 0.927, P < 0.01).ConclusionOverall, the study has revealed the reduced ability of L. monocytogenes to invade human intestinal epithelial cells in vitro was linked to the presence of PMSC mutations in inlA. Isolates with PMSC mutations shared the same genomic characteristics indicating the genetic basis on the potential virulence of L. monocytogenes invasion.

Combined effect of temperature, water activity and salt content on the growth and gene expression of Listeria monocytogenes in a dry-cured ham model system

The combined effect of temperature, water activity (aw) and NaCl content, usually found in dry-cured ham, on the growth and expression of the virulence and stress-related genes of Listeria monocytogenes was evaluated in a dry-cured ham model system. The highest growth of this pathogen was observed at 15 °C and, at 0.98 and 0.96 aw values. At 0.94 and 0.92 aw values, moderate NaCl levels stimulated the L. monocytogenes growth and repressed the expression of the four tested genes. At 7 °C, the expression of the plcA gene was favored while at 15 °C the hly and iap genes were activated. Preventive actions based on temperature, aw and salt content should be taken to minimise risks associated with growth and gene expression of L. monocytogenes in dry-cured ham.

Characterization of a recombinant aminopeptidase Lmo1711 from Listeria monocytogenes

We aimed to obtain the recombinant aminopeptidase encoded by Listeria monocytogenes (L. monocytogenes) gene lmo1711, and characterized the enzyme. First, the amino acid sequences of Lmo1711 from L. monocytogenes EGD-e and its homologues in other microbial species were aligned and the putative active sites were analyzed. The putative model of Lmo1711 was constructed through the SWISS-MODEL Workspace. Then, the plasmid pET30a-Lmo1711 was constructed and transformed into E. coli for expression of the recombinant Lmo1711. The his-tagged soluble protein was purified using the nickel-chelated affinity column chromatography. With the amino acid-p-nitroaniline as the substrate, Lmo1711 hydrolyzed the substrate to free p-nitroaniline monomers, whose absorbance measured at 405 nm reflected the aminopeptidase activity. The specificity of Lmo1711 to substrates was then examined by changing various substrates, and the effect of metal ions on the catalytic efficiency of this enzyme was further determined. Based on the bioinformatics data, Lmo1711 is a member of the M29 family aminopeptidases, containing a highly conserved catalytic motif (Glu-Glu-His-Tyr-His-Asp) with typical structure arrangements of the peptidase family. The recombinant Lmo1711 with a size of about 49.3 kDa exhibited aminopeptidase activity and had a selectivity to the substrates, with the highest degree of affinity for leucine-p-nitroaniline. Interestingly, the enzymatic activity of Lmo1711 can be activated by Cd²⁺, Zn²⁺, and is strongly stimulated by Co²⁺. We here, for the first time demonstrate that L. monocytogenes lmo1711 encodes a cobalt-activated aminopeptidase of M29 family.