Variation in Biofilm Formation among Strains of Listeria monocytogenes

Contamination of food by Listeria monocytogenes is thought to occur most frequently in food-processing environments where cells persist due to their ability to attach to stainless steel and other surfaces. Once attached these cells may produce multicellular biofilms that are resistant to disinfection and from which cells can become detached and contaminate food products. Because there is a correlation between virulence and serotype (and thus phylogenetic division) of L. monocytogenes, it is important to determine if there is a link between biofilm formation and disease incidence for L. monocytogenes. Eighty L. monocytogenes isolates were screened for biofilm formation to determine if there is a robust relationship between biofilm formation, phylogenic division, and persistence in the environment. Statistically significant differences were detected between phylogenetic divisions. Increased biofilm formation was observed in Division II strains (serotypes 1/2a and 1/2c), which are not normally associated with food-borne outbreaks. Differences in biofilm formation were also detected between persistent and nonpersistent strains isolated from bulk milk samples, with persistent strains showing increased biofilm formation relative to nonpersistent strains. There were no significant differences detected among serotypes. Exopolysaccharide production correlated with cell adherence for high-biofilm-producing strains. Scanning electron microscopy showed that a high-biofilm-forming strain produced a dense, three-dimensional structure, whereas a low-biofilm-forming strain produced a thin, patchy biofilm. These data are consistent with data on persistent strains forming biofilms but do not support a consistent relationship between enhanced biofilm formation and disease incidence.     

Identification of Listeria monocytogenes Determinants Required for Biofilm Formation

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

Microtiter Plate Assay for Assessment of Listeria monocytogenes Biofilm Formation

Listeria monocytogenes has the ability to form biofilms on food-processing surfaces, potentially leading to food product contamination. The objective of this research was to standardize a polyvinyl chloride (PVC) microtiter plate assay to compare the ability of L. monocytogenes strains to form biofilms. A total of 31 coded L. monocytogenes strains were grown in defined medium (modified Welshimer's broth) at 32°C for 20 and 40 h in PVC microtiter plate wells. Biofilm formation was indirectly assessed by staining with 1% crystal violet and measuring crystal violet absorbance, using destaining solution. Cellular growth rates and final cell densities did not correlate with biofilm formation, indicating that differences in biofilm formation under the same environmental conditions were not due to growth rate differences. The mean biofilm production of lineage I strains was significantly greater than that observed for lineage II and lineage III strains. The results from the standardized microtiter plate biofilm assay were also compared to biofilm formation on PVC and stainless steel as assayed by quantitative epifluorescence microscopy. Results showed similar trends for the microscopic and microtiter plate assays, indicating that the PVC microtiter plate assay can be used as a rapid, simple method to screen for differences in biofilm production between strains or growth conditions prior to performing labor-intensive microscopic analyses.     

A Mutation in the luxS Gene Influences Listeria monocytogenes Biofilm Formation

Using a Vibrio harveyi reporter strain, we demonstrated that Listeria monocytogenes secretes a functional autoinducer 2 (AI-2)-like signal. A luxS-deficient mutant produced a denser biofilm and attached to a glass surface 19-fold better than the parent strain. Exogenous AI-2 failed to restore the wild-type phenotype to the mutant. It seems that an intact luxS gene is associated with repression of components required for attachment and biofilm formation.     

Dependence of Continuous-Flow Biofilm Formation by Listeria monocytogenes EGD-e on SOS Response Factor YneA

Listeria monocytogenes was previously shown to form biofilms composed of a network of knitted chains under continuous-flow conditions. Here we show that the SOS response is activated under these conditions and that deletion of its regulon member yneA results in diminished biofilm formation under continuous-flow conditions.The food-borne pathogen Listeria monocytogenes is widely distributed in the environment and is able to grow in soil and on plant materials, thereby facilitating environmental transmission of this pathogen. L. monocytogenes is therefore frequently encountered in food processing facilities, on food contact surfaces, in pipelines, on floors, and in drains, which in turn may result in contamination of food products. It is expected that the formation of biofilms and subsequent dispersal plays an important role in recontamination processes. Biofilms are structured communities of microorganisms adhering to a surface that may be encapsulated within a self-produced protective and adhesive matrix of extracellular polymeric substances (EPS) (9). Most studies of L. monocytogenes biofilm formation focus on biofilm formation under static conditions on polystyrene, glass, or stainless steel surfaces. L. monocytogenes biofilms on polystyrene and glass consist of a homogeneous layer, while on stainless steel L. monocytogenes biofilms consist of single attached cells or microcolonies (2, 6, 11). The small, rod-shaped morphology of these static biofilm cells is very similar to the morphology of planktonic cells. However, L. monocytogenes biofilms formed under continuous-flow conditions, conceivably encountered in industrial pipelines, consist of a dense network of knitted chains composed of elongated cells and surrounding ball-shaped microcolonies (10). Recently, it has been shown that activation of the L. monocytogenes SOS response factor YneA resulted in cell elongation (14). The SOS response is involved in DNA repair, restart of stalled replication forks (3, 8), and mutagenesis (12). It is regulated by RecA (activator) and LexA (repressor) and furthermore contains DNA repair systems and translesion DNA polymerases such as DinB (15). To prevent transaction of the genome during replication fork stalling, septum formation at the midcell is inhibited by YneA, which results in cell elongation (7, 14). Recently, RecA-dependent genetic recombination was described for Pseudomonas aeruginosa biofilm cells harvested from a drip flow reactor, pointing to activation of the SOS response under these conditions (1). In this study, we investigated whether the SOS response is activated during L. monocytogenes EGD-e biofilm formation and whether there is a role for YneA in knitted chain biofilm formation.L. monocytogenes EGD-e (5), its isogenic in-frame ΔrecA and ΔyneA deletion mutants, its yneA complementation mutant, and its recA and yneA promoter reporter mutants (14) were grown in brain heart infusion (BHI; Difco) broth. No significant difference in planktonic growth between wild-type and mutant cultures was observed (results not shown). Continuous-flow biofilm formation experiments were performed as described previously (10) with small modifications. Biofilms were grown in a flow cell (BST FC 281; Biosurface Technologies Corporation) at 20°C, using BHI with a flow rate of 10 ml/h. Static biofilm experiments were performed as described previously (4) with small modifications. Biofilms were grown in BHI in 12-well polystyrene microtiter plates (Greiner) using a 1% inoculum of an overnight-grown culture. For quantification, the biofilm cells were harvested in phosphate-buffered saline (PBS), serially diluted in PBS, and plated on BHI agar. Colonies were enumerated after 2 days of incubation at 30°C. Quantitative real-time PCR analysis was performed as described previously (13) using primers shown in appendix S1 in the supplemental material. Shortly, biofilms were quenched in RNAprotect (Qiagen) following the manufacturer''s protocol and harvested. Expression levels were normalized using the housekeeping genes tpi, rpoB, and 16S rRNA. Biofilm formation experiments and quantitative PCR (Q-PCR) analysis were performed in two independent biological experiments using two replicates each. Statistically significant differences were identified using a two-tailed Student t test (P < 0.05).To investigate activation of L. monocytogenes EGD-e SOS response during continuous-flow biofilm formation, Q-PCR analysis of the SOS response genes recA, lexA, yneA, and dinB and promoter reporter studies using the promoters for recA and yneA were performed (Fig. ​(Fig.1).1). Compared with the reference (planktonic cells from a 48-h liquid culture), all four tested SOS response genes were upregulated in wild-type strain cells isolated from a 48-h continuous-flow biofilm (P < 0.05, t test), but not in cells isolated from a 48-h static biofilm (Fig. ​(Fig.1A).1A). Furthermore, the ΔrecA mutant strain did not show upregulation of yneA and the other SOS response genes during continuous-flow biofilm formation, indicating that RecA is required for activation of the SOS response during continuous-flow biofilm formation. Furthermore, continuous-flow biofilm formation also resulted in visible expression of enhanced green fluorescent protein (EGFP) for both yneA and recA promoter reporters (Fig. 1B and C). Expression of EGFP was not observed for these promoter reporters in planktonic cells grown in liquid culture or during static biofilm formation (results not shown). These results indicate that the SOS response is specifically activated during continuous-flow biofilm formation.Open in a separate windowFIG. 1.Activation of the SOS response during biofilm formation. (A) The graph shows differential expression of four SOS response genes in the wild-type and ΔrecA mutant strain between 48-h stationary-phase cultures (black), 48-h static biofilms (light gray), and 48-h continuous-flow biofilms (dark gray). Expression for each SOS response gene in the wild-type 48-h stationary-phase cultures is set at 1. (B and C) Micrographs show fluorescence (1) and phase-contrast (2) pictures of cells expressing EGFP from the recA (B) and yneA (C) promoters after 48 h of biofilm formation in BHI at 20°C under continuous-flow conditions.The impact of RecA and YneA on biofilm formation was assessed using the wild-type strain and in-frame ΔyneA and ΔrecA strains (Fig. ​(Fig.2).2). Both ΔrecA and ΔyneA mutants showed a significant deficiency in total biofilm produced under continuous-flow conditions (P < 0.05, t test), which was approximately 100-fold lower than that of the wild-type strain. No significant difference in static biofilm formation between wild-type and mutant strains was observed. Apparently, YneA and RecA are not required for static biofilm formation, which is in line with the lack of activation of the SOS response under these conditions. The wild-type, ΔyneA, and ΔrecA strains were microscopically examined during continuous-flow biofilm formation (Fig. ​(Fig.3).3). Analysis of the number of adherent cells 1 h after the start of the experiment did not reveal differences between the wild-type strain and the two mutants, which indicates that initial attachment is similar. After 24 h, the wild-type strain biofilm appeared to be composed of a complex structure of elongated cells forming a network of knitted chains, which after 48 h had developed into a denser network containing ball-shaped microcolonies. These results are in concordance with the study by Rieu et al. (10). However, both ΔyneA and ΔrecA mutant strains showed only some patches of adherent cells after 24 h, which developed into very small microcolonies after 48 h. Thus, formation of elongated cells in a network of knitted chains was not observed for these mutants. These results indicate that RecA and YneA are required to form this type of biofilm. To verify the specific role of YneA in continuous-flow biofilm formation, a yneA complementation mutant was constructed, which indeed showed biofilm formation capacity similar to that of the wild type, under both continuous-flow and static conditions (results not shown).Open in a separate windowFIG. 2.Comparative analysis of biofilm formation between wild-type strain and ΔrecA and ΔyneA mutants under continuous-flow and static conditions. The graph shows the amount of biofilm produced by wild-type and mutant strains after 48 h of biofilm formation at 20°C under continuous-flow (dark gray) and static (light gray) conditions. *, significantly different from wild-type strain (P < 0.05, t test).Open in a separate windowFIG. 3.Knitted chain biofilm formation under continuous-flow conditions is dependent on RecA and YneA. The micrographs show biofilms formed after 1, 24, and 48 h in BHI at 20°C for the wild-type strain (A), the ΔrecA mutant strain (B), and the ΔyneA mutant strain (C).This study established a clear link between the SOS response and knitted chain biofilm formation under continuous-flow conditions. RecA-dependent activation of the SOS response and in particular of yneA under continuous-flow conditions resulted in cell elongation and the formation of knitted chain biofilms. The signals that activate the L. monocytogenes SOS response are currently being studied and may provide tools for control of biofilm formation under continuous-flow conditions.      

Extracellular iron reductase activity produced by Listeria monocytogenes

Little is known about how pathogenic microorganisms that do not produce low-molecular-weight iron-chelating agents, termed siderophores, acquire iron from their environment. We have identified an extracellular enzyme produced by Listeria monocytogenes that can mobilize iron from a variety of iron-chelate complexes via reduction of the metal. The iron reductase requires Mg²⁺, flavin mononucleotide (FMN), and reduced nicotinamide adenine dinucleotide (NADH) for activity. Saturation kinetics were found when initial velocity studies of iron reduction were carried out as a function of variable FMN concentrations in the presence of 100 μM NADH and 10 mM Mg²⁺. Hyperbolic kinetics were also found when these studies were repeated as a function of variable NADH concentrations along with 20 μM FMN and 10 mM Mg²⁺. This process of extracellular reduction, in all likelihood, could be involved in the mobilization of iron from soils and aqueous environments and from host tissues in pathogenic processes. This is the first report of the extracellular enzymic reduction of iron by microorganisms. Received: 12 March 1996 / Accepted: 16 April 1996     

Control of Listeria monocytogenes in a Biofilm by Competitive-Exclusion Microorganisms

Biofilms from drains in food processing facilities with a recent history of no detectable Listeria monocytogenes in floor drains were cultured for microorganisms producing antilisterial metabolites. A total of 413 microbial isolates were obtained from 12 drain biofilm samples and were assayed at 15 and 37°C for activities that were bactericidal or inhibitory to L. monocytogenes, by two agar plate assays. Twenty-one of 257 bacterial isolates and 3 of 156 yeast isolates had antilisterial activity. All 24 isolates which produced metabolites inhibitory to L. monocytogenes were assayed for antilisterial activity in coinoculated broth cultures containing tryptic soy broth with yeast extract (TSB-YE). A five-strain mixture of 10³ CFU of L. monocytogenes/ml and 10⁵ CFU of the candidate competitive-exclusion microorganism/ml was combined in TSB-YE and incubated at 37°C for 24 h, 15°C for 14 days, 8°C for 21 days, and 4°C for 28 days. Substantial inhibition of L. monocytogenes growth (4 to 5 log CFU/ml) was observed for nine bacterial isolates at 37°C, two at 15 and 8°C, and three at 4°C. The inhibitory isolates were identified as Enterococcus durans (six isolates), Lactococcus lactis subsp. lactis (two isolates), and Lactobacillus plantarum (one isolate). The anti-L. monocytogenes activity of these isolates was evaluated in biofilms of L. monocytogenes on stainless steel coupons at 37, 15, 8, and 4°C. Results revealed that two isolates (E. durans strain 152 and L. lactis subsp. lactis strain C-1-92) were highly inhibitory to L. monocytogenes (growth inhibition of >5 log₁₀ CFU of L. monocytogenes/cm²). These two bacterial isolates appear to be excellent competitive-exclusion candidates to control L. monocytogenes in biofilms at environmental temperatures of 4 to 37°C.     

Morphotypic Conversion in Listeria monocytogenes Biofilm Formation: Biological Significance of Rough Colony Isolates

Adherence to a stainless steel surface selected isolates of Listeria monocytogenes with enhanced surface colonization abilities and a change in phenotype from the common smooth colony morphology to a succession of rough colony morphotypes. Growth in broth culture of the best-adapted, surface-colonizing rough colony morphotype gave a smooth colony revertant. Comparative analysis revealed that the smooth and rough variants had similar phenotypic and biochemical characteristics (e.g., identical growth rates and tolerances to antibiotics and environmental stressors). Rough colony isolates, however, failed to coordinate motility or induce autolysis. The defect in autolysis of rough colony isolates, which involved impaired cellular localization of several peptidoglycan-degrading enzymes, including cell wall hydrolase A (CwhA), suggested a link to a secretory pathway defect. The genetic basis for the impairment was studied at the level of the accessory secretory pathway component SecA2. DNA sequencing of the secA2 gene in smooth and rough colony isolates found no mutations in the coding or promoter regions. Analysis of SecA2 expression with an integrated secA2-FLAG tag construct found the protein to be upregulated in the rough and revertant backgrounds compared to the parental smooth colony isolate. A compensatory mechanism involving the SecA2 secretion pathway components is postulated to control smooth to rough interconversion of L. monocytogenes. Such phenotypic variation may enhance the ability of this opportunistic pathogen to colonize environments as diverse as processing surfaces, food products, and animal hosts.     

Plasmid DNA delivery by D-alanine-deficient Listeria monocytogenes

Optimal DNA vaccine efficacy requires circumventing several obstacles, including low immunogenicity, a need for adjuvant, and the costs of purifying injection grade plasmid DNA. Bacterial delivery of plasmid DNA may provide an efficient and low-cost alternative to plasmid purification and injection. Also, the bacterial vector may exhibit potential as an immune adjuvant in vivo. Thus, we elected to examine the use of cell-wall-deficient Listeria monocytogenes as a DNA delivery vehicle in vitro. First, the D-alanine-deficient (Deltadal-dat) L. monocytogenes strain DP-L3506, which undergoes autolysis inside eukaryotic host cells in the absence of D-alanine, was transformed with a plasmid encoding green fluorescent protein (GFP) under control of the CMV promoter (pAM-EGFP). Then COS-7 and MC57G cell lines were infected with the transformed DP-L3506 at various multiplicities of infection (MOI) in the presence or absence of D-alanine. Subsequent GFP expression was observed in both cell lines by 24 h post-infection with DP-L3506(pAM-EGFP). Notably, no GFP positive cells were observed when D-alanine was omitted. Although transfection efficiency initially increased as a result of D-alanine supplementation, high concentration or long-term supplementation led to sustained bacterial growth that killed the infected host cells, resulting in fewer GFP-expressing cells. Thus, efficient DNA delivery by transformed bacteria must balance bacterial invasion and survival with target cell health and survival.     

Carbon Catabolite Control is Important for Listeria monocytogenes Biofilm Formation in Response to Nutrient Availability

The foodborne pathogen Listeria monocytogenes has the ability to develop biofilm in food-processing environment, which becomes a major concern for the food safety. The biofilm formation is strongly influenced by the availability of nutrients and environmental conditions, and particularly enhanced in poor minimal essential medium (MEM) containing glucose rather than in rich brain heart infusion (BHI) broth. To gain better insight into the conserved protein expression profile in these biofilms, the proteomes from biofilm- and planktonic-grown cells from MEM with 50?mM glucose or BHI were compared using two-dimensional polyacrylamide gel electrophoresis followed by MALDI-TOF/TOF analysis. 47 proteins were successfully identified to be either up (19 proteins) or down (28 proteins) regulated in the biofilm states. Most (30 proteins) of them were assigned to the metabolism functional category in cluster of orthologous groups of proteins. Among them, up-regulated proteins were mainly associated with the pentose phosphate pathway and glycolysis, whereas a key enzyme CitC involved in tricarboxylic acid cycle was down-regulated in biofilms compared to the planktonic states. These data implicate the importance of carbon catabolite control for L. monocytogenes biofilm formation in response to nutrient availability.     

Species-specific detection of Listeria monocytogenes by DNA amplification

The polymerase chain reaction was used to detect and specifically identify Listeria monocytogenes. A 174-bp region of the listeriolysin O gene was shown to be specifically amplified in L. monocytogenes but not in other species of Listeria or in a number of other gram-positive and gram-negative organisms. Less than 50 organisms could routinely be detected by a procedure involving two rounds of 35 amplification cycles each and without the need for subsequent hybridization with labeled probes.     

Assessment of the Roles of LuxS, S-Ribosyl Homocysteine, and Autoinducer 2 in Cell Attachment during Biofilm Formation by Listeria monocytogenes EGD-e

LuxS is responsible for the production of autoinducer 2 (AI-2), which is involved in the quorum-sensing response of Vibrio harveyi. AI-2 is found in several other gram-negative and gram-positive bacteria and is therefore considered a good candidate for an interspecies communication signal molecule. In order to determine if this system is functional in the gastrointestinal pathogen Listeria monocytogenes EGD-e, an AI-2 bioassay was performed with culture supernatants. The results indicated that this bacterium produces AI-2 like molecules. A potential ortholog of V. harveyi luxS, lmo1288, was found by performing sequence similarity searches and complementation experiments with Escherichia coli DH5α, a luxS null strain. lmo1288 was found to be a functional luxS ortholog involved in AI-2 synthesis. Indeed, interruption of lmo1288 resulted in loss of the AI-2 signal. Although no significant differences were observed between Lux1 and EGD-e with regard to planktonic growth (at 10°C, 15°C, 25°C, and 42°C), swimming motility, and phospholipase and hemolytic activity, biofilm culture experiments showed that under batch conditions between 25% and 58% more Lux1 cells than EGD-e cells were attached to the surface depending on the incubation time. During biofilm growth in continuous conditions after 48 h of culture, Lux1 biofilms were 17 times denser than EGD-e biofilms. Finally, our results showed that Lux1 accumulates more S-adenosyl homocysteine (SAH) and S-ribosyl homocysteine (SRH) in culture supernatant than the parental strain accumulates and that SRH, but not SAH or AI-2, is able to modify the number of attached cells.     

Biofilm formation by Salmonella spp. and Listeria monocytogenes on plastic surface

AIMS: To investigate the biofilm formation by 122 Salmonella spp. and 48 Listeria monocytogenes strains on a plastic surface. METHODS: Quantification of biofilm formation was performed in brain heart infusion (BHI), trypcase soya broth (TSB), meat broth (MB) and 1/20 diluted trypcase soya broth (1/20-TSB) in plastic microtitre plates. RESULTS: All tested Salmonella spp. and L. monocytogenes strains produced biofilm in a suitable medium. However, the quantities of biofilm produced by Salmonella spp. were greater than those produced by tested L. monocytogenes strains. The nutrient content of the medium significantly influenced the quantity of produced biofilm. Diluted TSB was the most effective in promoting biofilm production by Salmonella spp., followed by TSB, while the least quantity of biofilm was formed in BHI and MB. L. monocytogenes produced the highest quantities of biofilm in BHI, followed by TSA, then MB, and the least quantities of biofilm were produced in 1/20-TSB. CONCLUSIONS: Salmonella spp. produces more biofilm in nutrient-poor medium, while L. monocytogenes produce more biofilm in nutrient-rich medium.     

Role of host GTPases in infection by Listeria monocytogenes

The bacterial pathogen Listeria monocytogenes induces internalization into mammalian cells and uses actin‐based motility to spread within tissues. Listeria accomplishes this intracellular life cycle by exploiting or antagonizing several host GTPases. Internalization into human cells is mediated by the bacterial surface proteins InlA or InlB. These two modes of uptake each require a host actin polymerization pathway comprised of the GTPase Rac1, nucleation promotion factors, and the Arp2/3 complex. In addition to Rac1, InlB‐mediated internalization involves inhibition of the GTPase Arf6 and participation of Dynamin and septin family GTPases. After uptake, Listeria is encased in host phagosomes. The bacterial protein GAPDH inactivates the human GTPase Rab5, thereby delaying phagosomal acquisition of antimicrobial properties. After bacterial‐induced destruction of the phagosome, cytosolic Listeria uses the surface protein ActA to stimulate actin‐based motility. The GTPase Dynamin 2 reduces the density of microtubules that would otherwise limit bacterial movement. Cell‐to‐cell spread results when motile Listeria remodel the host plasma membrane into protrusions that are engulfed by neighbouring cells. The human GTPase Cdc42, its activator Tuba, and its effector N‐WASP form a complex with the potential to restrict Listeria protrusions. Bacteria overcome this restriction through two microbial factors that inhibit Cdc42‐GTP or Tuba/N‐WASP interaction.     

Species-specific detection of Listeria monocytogenes by DNA amplification.

The polymerase chain reaction was used to detect and specifically identify Listeria monocytogenes. A 174-bp region of the listeriolysin O gene was shown to be specifically amplified in L. monocytogenes but not in other species of Listeria or in a number of other gram-positive and gram-negative organisms. Less than 50 organisms could routinely be detected by a procedure involving two rounds of 35 amplification cycles each and without the need for subsequent hybridization with labeled probes.