Biofilm formation and intra-pulsotype variability of Listeria monocytogenes at temperatures relevant to food processing environments.

Listeria monocytogenes is a foodborne pathogen that can infect both humans and animals and cause noninvasive gastrointestinal listeriosis or invasive listeriosis. The objectives of this study were to determine the genetic diversity of L. monocytogenes; the genes associated with its resistance to antibiotics, benzalkonium chloride (BC), and cadmium chloride (CdCl2); and its biofilm formation. A total of 132 fresh fruits (44 samples) and vegetables (88 samples) were selected for this study. The genetic diversity of the isolates and the genes associated with their antibiotic resistance were determined using PCR amplification; meanwhile, their levels of susceptibility to antibiotics were determined using the agar diffusion method. Their levels of resistance to BC and CdCl2 were determined using the minimum inhibitory concentration method, and their capacity for biofilm formation was evaluated using the crystal violet staining method. A total of 17 L. monocytogenes strains were collected: 12.8% (17/132) from fresh fruits and vegetables in this study. The isolates of L. monocytogenes belonged to phylogenetic groups I.1 (29.4% (5/17); serotype 1/2a) and II.2 (70.5% (12/17); serotype 1/2b); strains containing Listeria pathogenicity islands (LIPIs) were also identified at prevalence rates of 100% for LIPI-1 and LIPI-2 (17/17), 29.4% for LIPI-3 (5/17), and 11.7% for LIPI-4 (2/17). The antibiotic susceptibility tests showed that the L. monocytogenes isolates exhibited six different multiresistant patterns, with multiple antibiotic resistance (MAR) index of ≥0.46 (70.5%; 12/17); additionally, the genes Ide, tetM, and msrA, associated with efflux pump Lde, tetracycline, and ciprofloxacin resistance, were detected at 52.9% (9/17), 29.4% (5/17), and 17.6% (3/17), respectively. The phenotypic tests showed that 58.8% (10/17) of cadmium-resistant L. monocytogenes isolates had a co-resistance of 23.5% (4/17) to BC. Finally, all strains of L. monocytogenes exhibited moderate biofilm production. The results of this study contribute to our understanding of the persistence and genetic diversity of L. monocytogenes strains isolated from fresh fruits and vegetables; in addition, their resistance to CdCl2, which is correlated with co-resistance to BC disinfectant, is helpful for the food industry.

Listeria monocytogenes (L. monocytogenes) is a foodborne pathogen that poses significant risks to public health and food safety. The present study aimed to identify the presence of Listeria spp. in various samples, including pasteurized milk, chicken fillets, and stool samples from pregnant women in Sharkia Governorate, Egypt. Additionally, the study identified the serotypes, virulence-associated genes, antimicrobial resistance patterns, and biofilm formation in L. monocytogenes isolates. Moreover, the antibacterial and anti-biofilm activity of Lactobacillus plantarum ATCC 14917 (L. plantarum) against L. monocytogenes isolates was investigated. A cross-sectional study was conducted from August 2021 to January 2022 to collect 300 samples of pasteurized milk, chicken fillets, and stool from pregnant women admitted to outpatient clinics of hospitals. The results showed that 32.7% of the samples were positive for Listeria spp., including L. innocua (48.9%), L. monocytogenes (26.5%), L. ivanovii (14.3%), L. grayi (5.1%), and L. welshimeri (5.1%). Among all L. monocytogenes isolates, hlyA, actA, inlC, and inlJ virulence-associated genes were detected. However, the virulence genes plcB, iap, and inlA were found in 10 (38.5%), 8 (30.8%), and 25 (96.2%) isolates, respectively. The L. monocytogenes isolates classified into four serotypes (1/2a, 1/2b, 1/2c, and 4b), with 1/2a and 4b each identified in 30.8% of the isolates, while 1/2b and 1/2c were identified in 19.2% of the isolates. All L. monocytogenes isolates showed 100% resistance to streptomycin, kanamycin, and nalidix acid, and 92.3% of isolates showed gentamicin resistance. However, all isolates were susceptible to ampicillin and ampicillin/sulbactam. Multidrug resistance (MDR) was observed in 20 (76.9%) L. monocytogenes isolates. The biofilm formation ability of 26 L. monocytogenes isolates was evaluated at different incubation temperatures. At 4°C, 25°C, and 37°C, 53.8, 69.2, and 80.8% of the isolates, respectively, were biofilm producers. Furthermore, 23.1% were strong biofilm producers at both 4°C and 25°C, while 34.6% were strong biofilm formers at 37°C. Treating L. monocytogenes isolates with L. plantarum cell-free supernatant (CFS) reduced the number of biofilm-producing isolates to 15.4, 42.3, and 53.8% at 4°C, 25°C, and 37°C, respectively. L. plantarum's CFS antibacterial activity was tested against six virulent, MDR, and biofilm-forming L. monocytogenes isolates. At a concentration of 5 μg/mL of L. plantarum CFS, none of the L. monocytogenes isolates exhibited an inhibition zone. However, an inhibition zone was observed against L. monocytogenes strains isolated from pasteurized milk and pregnant women's stools when using a concentration of 10 μg/mL. Transmission electron microscopy (TEM) revealed that L. plantarum CFS induced morphological and intracellular structural changes in L. monocytogenes. In conclusion, this study identified virulent MDR L. monocytogenes isolates with strong biofilm-forming abilities in food products in Egypt, posing significant risks to food safety. Monitoring the prevalence and antimicrobial resistance profile of L. monocytogenes in dairy and meat products is crucial to enhance their safety. Although L. plantarum CFS showed potential antibacterial and anti-biofilm effects against L. monocytogenes isolates, further research is needed to explore its full probiotic potential.

Listeria monocytogenes is ubiquitous in nature and a major concern for the food industry, since it is the causal agent of the serious foodborne illness listeriosis. This organism can be introduced through many routes to food-processing environments and may become established on food-processing equipment. Subsequently, food products may become contaminated during processing. In addition, the bacterium can grow at refrigeration temperatures. Biofilms are regarded as important with respect to the survival and growth of microorganisms in the food industry. Microorganisms growing in biofilms are protected against cleaning and disinfection and are difficult to eradicate. L. monocytogenes may grow in biofilms that protect them against environmental stress and can be isolated from surfaces after cleaning and disinfection. In this study, a total of eight L. monocytogenes strains isolated from the meat industry and one reference strain L. monocytogenes ATCC 19111 were studied for their capability to form a biofilm. The biofilm forming behavior of nine L. monocytogenes strains was determined in two different media, Tryptone soya yeast extract broth (TSYEB) or Brain-heart infusion broth (BHI), at temperatures 7 °C, 25 °C, 37 °C, 42 °C for 5 days. The method used to assess biofilm formation was crystal violet staining. All strains were able to form biofilm, but the growth condition affected the levels formed. The lowest biofilm formation was observed at 7 °C. Further, the most effective medium in promoting biofilm production by the L. monocytogenes isolates from meat was BHI medium while for reference strain L. monocytogenes ATCC 19111 it was TSYEB. Incubation temperature was the most significant factor influencing the biofilm production levels, and also the type of used nutritive medium was important factor.

This study focuses on the detection of biofilm formation using Congo Red Agar (CRA) and Crystal Violet Staining (CVS) methods. Biofilms are complex microbial communities formed by bacteria adhering to surfaces, surrounded by extracellular polymeric substances, which provide protection against external stressors. Biofilm formation plays a critical role in infection control and combating antibiotic resistance. In this research, the biofilm production capacities of 24 standard bacterial strains, 11 multidrug-resistant (MDR) strains, 9 foodborne isolates, and 24 clinical isolates were evaluated. The CRA method qualitatively identifies biofilm formation through colony morphology, while the CVS assay quantitatively measures biofilm production. The results demonstrated that 66.18% of the 68 microorganisms tested using CRA were biofilm positive, with Gram-positive bacteria generally exhibiting stronger biofilm production. The CVS assay further provided more precise measurements of biofilm production, identifying strong biofilm producers such as Enterococcus faecalis ATCC 29212, Pseudomonas fluorescens P1, and Staphylococcus aureus ATCC 25923. The study found that while CRA is effective in detecting strong biofilm producers, it has limitations in identifying weaker biofilm producers. In contrast, the CVS assay proved to be more sensitive and reliable. The findings also highlight that variations in biofilm production capacity emphasize the significance of biofilm management in clinical infections and the challenges encountered in treatment.

Listeria monocytogenes is a foodborne pathogen of special concern for ready-to-eat food producers. The control of its presence is a critical step in which food-grade sanitizers play an essential role. L. monocytogenes is believed to persist in food processing environments in biofilms, exhibiting less susceptibility to sanitizers than planktonic cells. This study aimed to test the susceptibility of L. monocytogenes in planktonic culture and biofilm to three commercial food-grade sanitizers and to benzalkonium chloride; together with the genetic subtyping of the isolates. L. monocytogenes isolates were collected from raw materials, final products and food-contact surfaces during a 6-year period from a ready-to-eat meat-producing food industry and genetically characterized. Serogrouping and pulsed-field gel electrophoresis (PFGE) revealed genetic variability and differentiated L. monocytogenes isolates in three clusters. The biofilm-forming ability assay revealed that the isolates were weak biofilm producers. L. monocytogenes strains were susceptible both in the planktonic and biofilm form to oxidizing and ethanol-based compounds and to benzalkonium chloride, but not to quaternary ammonium compound. A positive association of biofilm-forming ability and LD90 values for quaternary ammonium compound and benzalkonium chloride was found. This study highlights the need for preventive measures improvement and for a conscious selection and use of sanitizers in food-related environments to control Listeria monocytogenes.

Listeria monocytogenes is the causative agent of the foodborne disease listeriosis, which is often fatal in susceptible individuals. The persistence of L. monocytogenes in the food production environment (FPE), defined as the repeated isolation of the same strain over several months, leads to the reoccurrence of food product recalls and listeriosis outbreaks. Recently, it was revealed that the plasmids of L. monocytogenes are highly conserved and distributed among persistent strains in the FPE and that some plasmids enhanced survival when cells were exposed to FPE-associated stress conditions. Much is known of the chromosomally-encoded molecular mechanisms of L. monocytogenes that increase tolerance to FPE-associated stress conditions. However, our understanding of the stress response roles of L. monocytogenes plasmids is limited. A more comprehensive understanding of the L. monocytogenes survival mechanisms that enable its persistence in food and FPEs might ultimately improve regulations to mitigate L. monocytogenes contamination of food products. Transcriptome sequencing is a standard method used to identify genes that are important for the response to stresses relevant to food production. We conducted differential gene expression analysis of plasmid-harboring L. monocytogenes strains in response to diverse FPE-relevant stress conditions. We show significant shifts in plasmid gene expression and that many plasmid genes, mainly those with no predicted putative function, are differentially expressed in diverse conditions. We also reveal that plasmid-harbored non-coding RNAs are highly prevalent in Listeria plasmids and are significantly induced during stress response. Our transcriptome data will serve as a foundation for future studies by highlighting which plasmid genes may be involved in stress response and could be used for intervention targets to alleviate the problematic persistence of L. monocytogenes. Ultraviolet (UV) light is used in food production to mitigate microbial contamination. Plasmid-encoded UV tolerance mechanisms have been described in bacteria other than L. monocytogenes. We compared CFUs of wildtype L. monocytogenes strains against plasmid-cured strains post UV exposure and revealed that L. monocytogenes plasmids significantly increased survival. From sequence analysis, we identified two candidate genes that may be involved in UV stress response and can be used in future functional characterization studies. Finally, we cloned and expressed two L. monocytogenes plasmid genes that were significantly induced during lactic acid stress, the clpL of pLM6179 (previously shown to have a role in heat stress), and the mco gene of pLMR479a. Previously, our group found that the plasmids pLM6179 and pLMR479a significantly increased survival during lactic acid and hydrogen peroxide stress exposure. Thus, we investigated whether the clpL and mco genes are important in response to lactic acid and hydrogen peroxide stress on a single gene level. Our data show that the clpL gene of L. monocytogenes plasmids enhanced growth in media containing hydrogen peroxide, demonstrating that the clpL gene has an important role in other FPE-associated stress conditions in addition to heat stress. We were not able to elucidate a potential role for the mco gene in the lactic acid and hydrogen peroxide stress response. However, we provide several suggestions for improving the experimental design and encourage future research to reveal if clpL and mco genes are suitable candidates for targets to combat L. monocytogenes contamination in the FPE. The mco and clpL genes are conserved among persistent L. monocytogenes strains’ plasmids. Thus, work pertaining to their function is relevant for multiple L. monocytogenes strains that colonize the FPE.

The influence of subminimal inhibitory concentrations of benzalkonium chloride on biofilm formation by Listeria monocytogenes

The present study aimed to assess the activity of cell-free supernatant (CFS) containing bacteriocins on the formation and maintenance of biofilms developed by Listeria monocytogenes, and the associated effect of bacteriocins and ethylene-diamine-tetra-acetic acid (EDTA) on the formed biofilm. CFS from 9 lactic acid bacteria (LAB) strains was tested for inhibitory activity against 85 L. monocytogenes isolates and 21 LAB strains. Then, 12 L. monocytogenes strains were selected based on genetic profiles and sensitivity to CFS and were subjected to an in vitro assay to assess biofilm formation in microtiter plates, considering different culture media and incubation conditions. Based on these results, 6 L. monocytogenes strains were subjected to the same in vitro procedure to assess biofilm formation, being co-inoculated with CFS. In addition, these strains were subjected to the same in vitro procedure, modified by adding the CFS after biofilm formation. Relevant decrease in biofilm formation was observed in the first experiment, but CFS added after biofilm formation did not eliminate them. CFS from Lactobacillus curvatus ET31 were selected due to its anti-biofilm activity, being associated to EDTA at different concentrations and tested for biofilm control of three strains of L. monocytogenes, using the same in vitro procedure described previously. Concentrated bacteriocin presented poor performance in eliminating formed biofilms, and EDTA concentration presented no evident interference on biofilm elimination. Twelve selected L. monocytogenes strains were positive for investigated virulence makers and negative for luxS gene, recognized as being involved in biofilm formation. Selected L. monocytogenes strains were able to produce biofilms under different conditions. CFSs have the potential to prevent biofilm formation, but they were not able to destroy already formed biofilms. Nevertheless, low concentrations of CFS combined with EDTA caused a relevant reduction in already formed biofilms, but this association was not able to eliminate them. The activity of selected CFS was demonstrated against L. monocytogenes-formed biofilms, being more effective when associated to EDTA at different concentrations.

Tracking of Listeria monocytogenes in meat establishment using Whole Genome Sequencing as a food safety management tool: A proof of concept

Diversity assessment of Listeria monocytogenes biofilm formation: Impact of growth condition, serotype and strain origin

Klebsiella pneumoniae is a prominent pathogen causing life-threatening bloodstream infections. Although biofilm formation and resistance to human serum are well-recognized virulence traits, their interrelatedness during K. pneumoniae bloodstream infections remains unclear. Here, we hypothesize that biofilm production is related to K. pneumoniae's ability to thrive in human serum and, therefore, may predict the strains' ability for serum survival. We analyzed 57 clinical, genetically diverse classical K. pneumoniae strains and characterized their survival and biofilm-producing ability in human serum. Serum survival patterns revealed three serum resistance categories-Low, Mid, and High. In addition, the biofilm biomass produced by the strains correlated with their serum resistance level (P < 0.001), and 3D biofilm visualization using confocal microscopy further confirmed that biofilm extracellular polysaccharide substances and biomass patterns were consistent with the serum resistance categories. Moreover, we revealed a direct correlation between the level of biofilm formation and the strain's serum survival level (R2 = 0.696), a prerequisite for systemic K. pneumoniae dissemination. As biofilm formation in serum reflects both survival and biofilm-forming ability, we assessed biofilm formation in defined modified basal medium (BM2), to rule out serum-mediated killing, and discovered a strong and significant association between the serum resistance category and BM2 biofilm biomass (P < 0.0001). By applying regression models, we discovered that biofilm formation serves as a significant predictor for bacterial survival in serum. Overall, our findings establish biofilm production in K. pneumoniae as a biomarker of serum survival and may open a new avenue for predicting bloodstream infection risk in clinical settings.IMPORTANCEBloodstream infections caused by Klebsiella pneumoniae are devastating life-threatening infections worldwide. Understanding the survival strategies of K. pneumoniae in the bloodstream is critical for elucidating key aspects of bacterial pathogenicity and developing new diagnostic and therapeutic modalities. Although serum survival is a recognized virulence trait necessary to thrive in the bloodstream, the relationship between serum resistance and biofilm formation, a multicellular organization that may protect bacteria from bloodstream stressors, remains poorly understood. In this article, we demonstrate biofilm production in human serum by clinical classical K. pneumoniae strains for the first time and discovered a direct correlation between the level of biofilm biomass formation and the degree of serum survival in human serum and in defined modified basal medium. These findings offer insights into the importance of biofilm production in K. pneumoniae serum resistance and may be used to develop future therapeutic strategies targeting bloodstream infections.

Variability of Listeria monocytogenes strains in biofilm formation on stainless steel and polystyrene materials and resistance to peracetic acid and quaternary ammonium compounds

Listeria monocytogenes is a ubiquitous bacterium capable of colonising and persisting within food production environments (FPEs) for many years, even decades. This ability to colonise, survive and persist within the FPEs can result in food product cross-contamination, including vulnerable products such as ready to eat food items. Various environmental and genetic elements are purported to be involved, with the ability to form biofilms being an important factor. In this study we examined various mechanisms which can influence colonisation in FPEs. The ability of isolates (n = 52) to attach and grow in biofilm was assessed, distinguishing slower biofilm formers from isolates forming biofilm more rapidly. These isolates were further assessed to determine if growth rate, exopolymeric substance production and/or the agr signalling propeptide influenced these dynamics and could promote persistence in conditions reflective of FPE. Despite no strong association with the above factors to a rapid colonisation phenotype, the global transcriptome suggested transport, energy production and metabolism genes were widely upregulated during the initial colonisation stages under nutrient limited conditions. However, the upregulation of the metabolism systems varied between isolates supporting the idea that L. monocytogenes ability to colonise the FPEs is strain-specific.

To assess the ability of Listeria monocytogenes to form biofilm on different food-contact surfaces with regard to different temperatures, cellular hydrophobicity and motility. Forty-four L. monocytogenes strains from food and food environment were tested for biofilm formation by crystal violet staining. Biofilm levels were significantly higher on glass at 4, 12 and 22 degrees C, as compared with polystyrene and stainless steel. At 37 degrees C, L. monocytogenes produced biofilm at significantly higher levels on glass and stainless steel, as compared with polystyrene. Hydrophobicity was significantly (P < 0.05) higher at 37 degrees C than at 4, 12 and 22 degrees C. Thirty (68.2%) of 44 strains tested showed swimming at 22 degrees C and 4 (9.1%) of those were also motile at 12 degrees C. No correlation was observed between swimming and biofilm production. L. monocytogenes can adhere to and form biofilms on food-processing surfaces. Biofilm formation is significantly influenced by temperature, probably modifying cell surface hydrophobicity. Biofilm formation creates major problems in the food industry because it may represent an important source of food contamination. Our results are therefore important in finding ways to prevent contamination because they contribute to a better understanding on how L. monocytogenes can establish biofilms in food industry and therefore survive in the processing environment.

Introduction. Staphylococcus aureus (SA) and Staphylococcus epidermidis (SE) are the most common pathogens from the genus Staphylococcus causing biofilm-associated infections. Generally, biofilm-associated infections represent a clinical challenge. Bacteria in biofilms are difficult to eradicate due to their resistance and serve as a reservoir for recurring persistent infections.Gap Statement. A variety of protocols for in vitro drug activity testing against staphylococcal biofilms have been introduced. However, there are often fundamental differences. All these differences in methodical approaches can then be reflected in the form of discrepancies between results.Aim. In this study, we aimed to develop optimal conditions for staphylococcal biofilm formation on pegs. The impact of peg surface modification was also studied.Methodology. The impact of tryptic soy broth alone or supplemented with foetal bovine serum (FBS) or human plasma (HP), together with the impact of the inoculum density of bacterial suspensions and the shaking versus the static mode of cultivation, on total biofilm biomass production in SA and SE reference strains was studied. The surface of pegs was modified with FBS, HP, or poly-l-lysine (PLL). The impact on total biofilm biomass was evaluated using the crystal violet staining method and statistical data analysis.Results. Tryptic soy broth supplemented with HP together with the shaking mode led to crucial potentiation of biofilm formation on pegs in SA strains. The SE strain did not produce biofilm biomass under the same conditions on pegs. Preconditioning of peg surfaces with FBS and HP led to a statistically significant increase in biofilm biomass formation in the SE strain.Conclusion. Optimal cultivation conditions for robust staphylococcal biofilm formation in vitro might differ among different bacterial strains and methodical approaches. The shaking mode and supplementation of cultivation medium with HP was beneficial for biofilm formation on pegs for SA (ATCC 29213) and methicillin-resistant SA (ATCC 43300). Peg conditioning with HP and PLL had no impact on biofilm formation in either of these strains. Peg coating with FBS showed an adverse effect on the biofilm formation of these strains. By contrast, there was a statistically significant increase in biofilm biomass production on pegs coated with FBS and HP for SE (ATCC 35983).