Dual-species biofilm of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> on stainless steel surface

Listeria monocytogenes is a Gram-positive bacterium commonly associated with foodborne diseases. Due its ability to survive under adverse environmental conditions and to form biofilm, this bacterium is a major concern for the food industry, since it can compromise sanitation procedures and increase the risk of post-processing contamination. Little is known about the interaction between L. monocytogenes and Gram-negative bacteria on biofilm formation. Thus, in order to evaluate this interaction, Escherichia coli and L. monocytogenes were tested for their ability to form biofilms together or in monoculture. We also aimed to evaluate the ability of L. monocytogenes 1/2a and its isogenic mutant strain (ΔprfA ΔsigB) to form biofilm in the presence of E. coli. We assessed the importance of the virulence regulators, PrfA and σ^B, in this process since they are involved in many aspects of L. monocytogenes pathogenicity. Biofilm formation was assessed using stainless steel AISI 304 #4 slides immersed into brain heart infusion broth, reconstituted powder milk and E. coli preconditioned medium at 25 °C. Our results indicated that a higher amount of biofilm was formed by the wild type strain of L. monocytogenes than by its isogenic mutant, indicating that prfA and sigB are important for biofilm development, especially maturation under our experimental conditions. The presence of E. coli or its metabolites in preconditioned medium did not influence biofilm formation by L. monocytogenes. Our results confirm the possibility of concomitant biofilm formation by L. monocytogenes and E. coli, two bacteria of major significance in the food industry.     

Biofilm formation by different serological variants of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> in association with <Emphasis Type="Italic">Bacillus pumilus</Emphasis>

Ability of Listeria monocytogenes serological variants belonging to phylogenetic lines I and II to form biofilms on glass, both in monoculture and in a consortium with Bacillus pumilus, was shown. After 72 h, both L. monocytogenes serological variants formed mature biofilms both in monoculture and in association with bacilli. The presence of B. pumilus in Listeria biofilms resulted in alteration of L. monocytogenes colony morphology, decrease in their enzymatic activity and aghesive capacity, enhanced virulence and hemolytic activity, and cell motility observed at 37°С. Importantly, all of these modifications of the biological characteristics were of a phenotypic nature and were restored when joint incubation of bacteria was terminated.     

Factors affecting survival of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> and <Emphasis Type="Italic">Listeria innocua</Emphasis> in soil samples

We investigated the ability of several strains of L. monocytogenes and Listeria innocua strains to survive in local soil samples in vitro. Survival of three L. monocytogenes strains, EGDe, CD83, and CD1038, and three L. innocua strains, CLIP, FH2117, FH2152, was monitored in soil samples by direct enumeration of colony-forming units on selective agar. The study did not demonstrate any species-specific difference in soil survival, and all Listeria strains exhibited a marked decline in numbers over time. Bioluminescence imaging approaches to detect lux-tagged strains in soil proved largely ineffective, most likely due to the reduced metabolic activity of strains in this environment. We investigated the influence of specific factors including the presence of a background microbiota, growth temperature, moisture and strain motility upon persistence in this environment. A sequenced L. monocytogenes strain, EGDe, was capable of active growth in sterile soil yet exhibited a decline in the presence of the normal soil microbiota. Furthermore, greater survival was seen at lower incubation temperatures in normal soil. Finally, we demonstrated a direct correlation between motility and survival of L. monocytogenes in soil with highly motile L. monocytogenes strains exhibiting greater soil survival than non-motile mutants.     

Linearized kinetic model of<Emphasis Type="Italic"> Listeria monocytogenes</Emphasis> biofilm growth

In this study the dynamics of biofilm formation on aluminum has been investigated. The process of cell growth has been observed using fluorescence microscopy. It has been confirmed that the process of biofilm formation can be represented as a sum of two separate processes: cell adhesion and colony proliferation. The derived set of equations describes kinetics of surface population growth and characteristic times for adsorption and combined growth processes, including characteristic time for the nutrient supply depletion. All equations contain variables based on the fundamental characteristics of bacterial population and can be easily determined from the experimental data or estimated theoretically. The developed theoretical model allows obtaining realistic values for population growth time and characteristic time for nutrient limitation occurrence during the biofilm development. Resulting equations qualitatively describe the biofilm formation process, and allow predicting microbial kinetics in the batch reactor system and determining critical values of the process parameters.     

In vivo Tn<Emphasis Type="Italic">5</Emphasis>-based transposon mutagenesis of Streptomycetes

This paper reports the in vivo expression of the synthetic transposase gene tnp(a) from a hyperactive Tn5 tnp gene mutant in Streptomyces coelicolor. Using the synthetic tnp(a) gene adapted for Streptomyces codon usage, we showed random insertion of the transposon into the Streptomycetes genome. The insertion frequency for the hyperactive Tn5 derivative is 98% of transformed S. coelicolor cells. The random transposition has been confirmed by the recovery of ~1.1% of auxotrophs. The Tn5 insertions are stably inherited in the absence of apramycin selection. The transposon contains an apramycin resistance selection marker and an R6Kγ origin of replication for transposon rescue. We identified the transposon insertion loci by random sequencing of 14 rescue plasmids. The majority of insertions (12 of 14) were mapped to putative open-reading frames on the S. coelicolor chromosome. These included two new regulatory genes affecting S. coelicolor growth and actinorhodin biosynthesis.     

<Emphasis Type="Italic">Listeria </Emphasis><Emphasis Type="Italic">monocytogenes</Emphasis> internalins bind to the human intestinal mucin MUC2

Listeria monocytogenes cross the intestinal barrier causing systemic infections with high mortality rates. Intestinal infection triggers release of intestinal mucus. We show that three L. monocytogenes internalins, InlB, InlC and InlJ all bound to MUC2 (the major component of intestinal mucus), but not to the cell surface mucin MUC1. Binding was strongest to InlB>InlC>InlJ (P < 0.001). Listerial internalins are characterized by their internalin domain, composed by leucine rich repeats (LRR) followed by an immunogloblin-like region. We report here that the internalin domain of the InlJ protein also bound MUC2, suggesting that an internalin domain is sufficient to bind to MUC2.     

Adaptation and cross-adaptation of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> and <Emphasis Type="Italic">Salmonella enterica</Emphasis> to poultry decontaminants

Information on the potential for acquired reduced susceptibility of bacteria to poultry decontaminants occurring is lacking. Minimal Inhibitory Concentrations (MICs) were established for assessing the initial susceptibility and the adaptative and cross-adaptative responses of four bacterial strains (Listeria monocytogenes serovar l/2a, L. monocytogenes serovar 4b, Salmonella enterica serotype Typhimurium, and S. enterica serotype Enteritidis) to four poultry decontaminants (trisodium phosphate, acidified sodium chlorite -ASC-, citric acid, and peroxyacetic acid). The initial susceptibility was observed to differ among species (all decontaminants) and between Salmonella strains (ASC). These inter- and intra-specific variations highlight (1) the need for strict monitoring of decontaminant concentrations to inactivate all target pathogens of concern, and (2) the importance of selecting adequate test strains in decontamination studies. MICs of ASC (0.17±0.02 to 0.21±0.02 mg/ml) were higher than the U.S. authorized concentration when applied as a pre-chiller or chiller solution (0.05 to 0.15 mg/ml). Progressively increasing decontaminant concentrations resulted in reduced susceptibility of strains. The highest increase in MIC was 1.88 to 2.71-fold (ASC). All decontaminants were shown to cause cross-adaptation of strains between both related and unrelated compounds, the highest increase in MIC being 1.82-fold (ASC). Our results suggest that the in-use concentrations of ASC could, in certain conditions, be ineffective against Listeria and Salmonella strains. The adaptative and cross-adaptative responses of strains tested to poultry decontaminants are of minor concern. However, the observations being presented here are based on in vitro studies, and further research into practical applications are needed in order to confirm these findings.     

Manipulating the <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> genome using <Emphasis Type="Italic">mariner</Emphasis> transposons

Tc1, one of the founding members of the Tc1/mariner transposon superfamily, was identified in the nematode Caenorhabditis elegans more than 25 years ago. Over the years, Tc1 and other endogenous mariner transposons became valuable tools for mutagenesis and targeted gene inactivation in C. elegans. However, transposition is naturally repressed in the C. elegans germline by an RNAi-like mechanism, necessitating the use of mutant strains in which transposition was globally derepressed, which causes drawbacks such as uncontrolled proliferation of the transposons in the genome and accumulation of background mutations. The more recent mobilization of the Drosophila mariner transposon Mos1 in the C. elegans germline circumvented the problems inherent to endogenous transposons. Mos1 transposition strictly depends on the expression of the Mos transposase, which can be controlled in the germline using inducible promoters. First, Mos1 can be used for insertional mutagenesis. The mobilization of Mos1 copies present on an extrachromosomal array results in the generation of a small number of Mos1 genomic insertions that can be rapidly cloned by inverse PCR. Second, Mos1 insertions can be used for genome engineering. Triggering the excision of a genomic Mos1 insertion causes a chromosomal break, which can be repaired by transgene-instructed gene conversion. This process is used to introduce specific changes in a given gene, such as point mutations, deletions or insertions of a tag, and to create single-copy transgenes.     

Extracellular protease in <Emphasis Type="Italic">Actinomycetes</Emphasis> culture supernatants inhibits and detaches <Emphasis Type="Italic">Staphylococcus</Emphasis><Emphasis Type="Italic">aureus</Emphasis> biofilm formation

Bacterial biofilms are associated with chronic infections due to their resistance to antimicrobial agents. Staphylococcus aureus is a versatile human pathogen and can form biofilms on human tissues and diverse medical devices. To identify novel biofilm inhibitors of S. aureus, the supernatants from a library of 458 Actinomycetes strains were screened. The culture supernatants (1% v/v) of more than 10 Actinomycetes strains inhibited S. aureus biofilm formation by more than 80% without affecting the growth. The culture supernatants of these biofilm-reducing Actinomycetes strains contained a protease (equivalent to 0.1 μg proteinase K ml⁻¹), which both inhibited S. aureus biofilm formation and detached pre-existing S. aureus biofilms. This study suggests that protease treatment could be a feasible tool to reduce and eradicate S. aureus biofilms.     

Characterization of the <Emphasis Type="Italic">codY</Emphasis> gene and its influence on biofilm formation in <Emphasis Type="Italic">Bacillus </Emphasis><Emphasis Type="Italic">cereus</Emphasis>

The foodborne pathogen Bacillus cereus can form biofilms on various food contact surfaces, leading to contamination of food products. To study the mechanisms of biofilm formation by B. cereus, a Tn5401 library was generated from strain UW101C. Eight thousand mutants were screened in EPS, a low nutrient medium. One mutant (M124), with a disruption in codY, developed fourfold less biofilm than the wild-type, and its defective biofilm phenotype was rescued by complementation. Addition of 0.1% casamino acids to EPS prolonged the duration of biofilms in the wild-type but not codY mutant. When decoyinine, a GTP synthesis inhibitor, was added to EPS, biofilm formation was decreased in the wild-type but not the mutant. The codY mutant produced three times higher protease activity than the wild-type. Zymogram and SDS-PAGE data showed that production of the protease (∼130 kDa) was repressed by CodY. Addition of proteinase K to EPS decreased biofilm formation by the wild-type. Using a dpp-lacZ fusion reporter system, it was shown that that the B. cereus CodY can sense amino acids and GTP levels. These data suggest that by responding to amino acids and intracellular GTP levels CodY represses production of an unknown protease and is involved in biofilm formation.     

Subtyping of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> isolates by <Emphasis Type="Italic">actA</Emphasis> gene sequencing,PCR-fingerprinting,and cell-invasion assay

Analysis of actA gene sequence polymorphism has been shown to be an effective and relatively inexpensive method for subtyping Listeria monocytogenes isolates, allowing the division of the population of this species into two deeply separate lineages. This sequence-based method as well as PCR-mediated fingerprinting were applied here for the differentiation of 49 isolates of food and clinical origin. Correlation between these two typing approaches was high. Both methods divided the isolates into two lineages, designated I (33 isolates) and II (16 isolates). All the 33 lineage I isolates were assigned to the same, or closely related, six clusters by both typing methods. For the lineage II isolates, PCR fingerprinting was found to be more discriminatory. The isolates were characterized by cell invasion assay. All highly invasive isolates were assigned to lineage I, which constituted a heterogeneous group also containing low-invasive isolates. High-invasive isolates were not found in the genetically determined lineage II. A particular actA cluster, designated Ha, contained all the isolates showing the lowest invasiveness. A common trait of the isolates belonging to this cluster was the presence of a threonine-441 of the deduced ActA sequence instead of the alanine-441 present in the remaining isolates. Thirteen human isolates were classified to lineage I and five to lineage II. A PCR-based method can therefore differentiate L. monocytogenes isolates in accordance with the current phylogenetic model of the evolution of this species.     

Comparison of selected disinfectants efficiency against <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> biofilm formed on various surfaces

Listeria monocytogenes is a main etiological factor of listeriosis, spread mainly by food products. In recent years, an increasing number of patients with listeriosis and an augmentation in L. monocytogenes antibiotic resistance, e.g. to penicillin and ampicillin, has been reported. The aim of the study was to characterise the L. monocytogenes strains isolated from fish-processed food products. Species identification, based on the multiplex-PCR reaction, was performed, and the genetic similarity of the isolates was analysed with the RAPD technique. The strains, in the form of planktonic cells and a biofilm, were subjected to drug-susceptibility analysis, and the effect of disinfectants on the bacillus cells was evaluated. All of the analysed strains were of the Listeria monocytogenes species. Three genetically distant strains were detected, i.e. Lm I, Lm II and Lm III. Approximately 66.6% penicillin-resistant and 66.6% cotrimoxazole-resistant strains were found. No erythromycin-resistant strain was detected. The Lm II strain was simultaneously resistant to four antibiotics, i.e. penicillin, ampicillin, meropenem and cotrimoxazole. The strongest biofilm was formed on aluminium foil and the weakest on rubber. The tested disinfectant antibiofilm effectiveness was related to the type of surface. The most effective agent was paracetic acid and hydrogen peroxide (elimination rate 5.10–6.62 log CFU?×?cm^?2 and 5.70–7.39 log CFU?×?cm^?2 after 1- and 5-min exposure, respectively) and the least—sodium hydroxide (elimination rate 0.52–1.20 log CFU?×?cm^?2 and 0.98–1.81 log CFU?×?cm^?2 after 1- and 5-min exposure, respectively). Further studies on a greater number of L. monocytogenes strains are recommended.     

Comparative Genomic Analysis of the <Emphasis Type="Italic">sigB</Emphasis> Operon in <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> and in Other Gram-Positive Bacteria

The stress-responsive, alternative sigma factor ^B has been described in members of three Gram-positive genera, Bacillus, Listeria, and Staphylococcus. In these bacteria, ^B appears to play an important role in facilitating rapid adaptation to and survival in stressful environments. ^B activity is regulated through a complex system of phosphatases and kinases encoded by rsb (regulator of sigma B) genes. We describe the sigB operon structure for the facultative intracellular pathogen Listeria monocytogenes and apply this sequence as well as other previously described sigB operon sequences to probe the evolution and functional conservation of the ^B stress response system among different Gram-positive bacteria. While ^B as well as two Rsbs (RsbS and RsbT) are highly conserved (73%, 84%, and 79% average amino acid [aa] identities, respectively), the predicted aa sequences of the other Rsb proteins showed less conservation (62–71% aa identities). Furthermore, the sigB operon structure varies among bacterial species. Bacterial species differ in the numbers and identities of rsb genes encoded in their genomes. We thus conclude that the ^B stress-response system as represented by the sigB operon has diverged in both its overall components as well as in the sequences of its individual proteins, even among closely related bacterial species. Differential evolution of this stress response system among various genera may represent a strategy that enables bacteria to adapt cellular response and survival systems to a variety of stress conditions.     

Inactivation of barotolerant strains of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 by ultra high pressure and <Emphasis Type="Italic">tert</Emphasis>-butylhydroquinone combination

Antimicrobial efficacy of ultra-high-pressure (UHP) can be enhanced by application of additional hurdles. The objective of this study was to systematically assess the enhancement in pressure lethality by TBHQ treatment, against barotolerant strains of Escherichia coli O157:H7 and Listeria monocytogenes. Two L. monocytogenes Scott A and the barotolerant OSY-328 strain, and two E. coli O157:H7 strains, EDL-933 and its barotolerant mutant, OSY-ASM, were tested. Cell suspensions containing TBHQ (50 ppm, dissolved in dimethyl sulfoxide) were pressurized at 200 to 500 MPa (23+/-2 degrees C) for 1 min, plated on tryptose agar and enumerated the survivors. The TBHQ-UHP combination resulted in synergistic inactivation of both pathogens, with different degrees of lethality among strains. The pressure lethality threshold, for the combination treatment, was lower for E. coli O157:H7 (> or = 200 MPa) than for L. monocytogenes (> 300 MPa). E. coli O157:H7 strains were extremely sensitive to the TBHQ-UHP treatment, compared to Listeria strains. Interestingly, a control treatment involving DMSO-UHP combination consistently resulted in higher inactivation than that achieved by UHP alone, against all strains tested. However, sensitization of the pathogens to UHP by the additives (TBHQ in DMSO) was prominently greater for UHP than DMSO. Differences in sensitivities to the treatment between these two pathogens may be attributed to discrepancies in cellular structure or physiological functions.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.