Assessing the importance of interspecific interactions in the evolution of microbial communities.

Interspecific interactions play an important role in the establishment of a community phenotype. Furthermore, the evolution of a community can both occur through an independent evolution of the species composing the community and the interactions among them. In this study, we investigated how important the evolution of interspecific interactions was in the evolutionary response of eight two-bacterial species communities regarding productivity. We found evidence for an evolution of the interactions in half of the studied communities, which gave rise to a mean change of 15% in community productivity as compared to what was expected from the individual responses. Even when the interactions did not evolve themselves, they influenced the evolutionary responses of the bacterial strains within the communities, which further affected community response. We found that evolution within a community often promoted the adaptation of the bacterial strains to the abiotic environment, especially for the dominant strain in a community. Overall, this study suggested that the evolution of the interspecific interactions was frequent and that it could increase community response to evolution.

Listeria monocytogenes: Investigation of Fitness in Soil Does Not Support the Relevance of Ecotypes.

Listeria monocytogenes (Lm) is a ubiquitous bacterium that causes the serious foodborne illness listeriosis. Although soil is a primary reservoir and a central habitat for Lm, little information is available on the genetic features underlying the fitness of Lm strains in this complex habitat. The aim of this study was to identify (i) correlations between the strains fitness in soil, their origin and their phylogenetic position (ii) identify genetic markers allowing Lm to survive in the soil. To this end, we assembled a balanced panel of 216 Lm strains isolated from three major ecological compartments (outdoor environment, animal hosts, and food) and from 33 clonal complexes occurring worldwide. The ability of the 216 strains to survive in soil was tested phenotypically. Hierarchical clustering identified three phenotypic groups according to the survival rate (SR): phenotype 1 "poor survivors" (SR < 2%), phenotype 2 "moderate survivors" (2% < SR < 5%) and phenotype 3 "good survivors" (SR > 5%). Survival in soil depended neither on strains' origin nor on their phylogenetic position. Genome-wide-association studies demonstrated that a greater number of genes specifically associated with a good survival in soil was found in lineage II strains (57 genes) than in lineage I strains (28 genes). Soil fitness was mainly associated with variations in genes (i) coding membrane proteins, transcription regulators, and stress resistance genes in both lineages (ii) coding proteins related to motility and (iii) of the category "phage-related genes." The cumulative effect of these small genomic variations resulted in significant increase of soil fitness.

In Vitro Evolution of Listeria monocytogenes Reveals Selective Pressure for Loss of SigB and AgrA Function at Different Incubation Temperatures.

The alternative sigma factor B (σB) contributes to the stress tolerance of the foodborne pathogen Listeria monocytogenes by upregulating the general stress response. We previously showed that σB loss-of-function mutations arise frequently in strains of L. monocytogenes and suggested that mild stresses might favor the selection of such mutations. In this study, we performed in vitro evolution experiments (IVEE) where L. monocytogenes was allowed to evolve over 30 days at elevated (42°C) or lower (30°C) incubation temperatures. Isolates purified throughout the IVEE revealed the emergence of sigB operon mutations at 42°C. However, at 30°C, independent alleles in the agr locus arose, resulting in the inactivation of Agr quorum sensing. Colonies of both sigB mutants and agr mutants exhibited a greyer coloration on 7-days-old agar plates than those of the parental strain. Scanning electron microscopy revealed a more complex colony architecture in the wild type than in the mutant strains. sigB mutant strains outcompeted the parental strain at 42°C but not at 30°C, while agr mutant strains showed a small increase in competitive fitness at 30°C. Analysis of 40,080 L. monocytogenes publicly available genome sequences revealed a high occurrence rate of premature stop codons in both the sigB and agrCA loci. An analysis of a local L. monocytogenes strain collection revealed 5 out of 168 strains carrying agrCA alleles. Our results suggest that the loss of σB or Agr confer an increased competitive fitness in some specific conditions and this likely contributes to the emergence of these alleles in strains of L. monocytogenes. IMPORTANCE To withstand environmental aggressions, L. monocytogenes upregulates a large regulon through the action of the alternative sigma factor B (σB). However, σB becomes detrimental for L. monocytogenes growth under mild stresses, which confer a competitive advantage to σB loss-of-function alleles. Temperatures of 42°C, a mild stress, are often employed in mutagenesis protocols of L. monocytogenes and promote the emergence of σB loss-of-function alleles in the sigB operon. In contrast, lower temperatures of 30°C promote the emergence of Agr loss-of-function alleles, a cell-cell communication mechanism in L. monocytogenes. Our findings demonstrate that loss-of-function alleles emerge spontaneously in laboratory-grown strains. These alleles rise in the population as a consequence of the trade-off between growth and survival imposed by the activation of σB in L. monocytogenes. Additionally, our results demonstrate the importance of identifying unwanted hitchhiker mutations in newly constructed mutant strains.

Plant Growth Promoting Bacterial Consortium Induces Shifts in Indigenous Soil Bacterial Communities and Controls Listeria monocytogenes in Rhizospheres of Cajanus cajan and Festuca arundinacea.

The rhizosphere is a dynamic and complex interface between plant roots and microorganisms. Owing to exudates, a web of interactions establishes among the microbial members of this micro-environment. The present study explored the impact of a bacterial consortium (Azotobacter chroococcum, Bacillus megaterium and Pseudomonas fluorescens, ABP), on the fate of a human pathogen, Listeria monocytogenes EGD-e, in soil and in the rhizospheres of Cajanus cajan and Festuca arundinacea, in addition to its plant growth promoting effect. The study further assessed the impact these bioinoculants exert on the autochthonous soil bacterial communities. Experiments in sterilised soil inoculated with bioinoculants and L. monocytogenes revealed the inhibition of L. monocytogenes by approximately 80-fold compared to that without the consortium. Subsequently, experiments were conducted in non-sterile soil microcosms planted with C. cajan and F. arundinacea, and in bulk soil. The consortium led to a significant increase in plant growth in both plants and prevented growth of L. monocytogenes. However, the presence of resident soil bacterial communities overshadowed this inhibitory effect, and a sharp decline in L. monocytogenes populations (5-6 log reduction) was recorded under non-sterile soil conditions. A shift in the soil resident bacterial communities was observed upon amendment with the bioinoculants. A significant increase of potential Plant Growth Promoting Rhizobacteria (PGPR) and biocontrol agents was observed, while the abundance of potential phytopathogens dropped. The present study opens up new avenues for the application of such a consortium given their dual benefits of plant growth promotion and restricting phytopathogens as well as human pathogen.

Plants as a realized niche for Listeria monocytogenes.

Listeria monocytogenes is a human pathogen. It is the causative agent of listeriosis, the leading cause of bacterial-linked foodborne mortality in Europe and elsewhere. Outbreaks of listeriosis have been associated with the consumption of fresh produce including vegetables and fruits. In this review we summarize current data providing direct or indirect evidence that plants can serve as habitat for L. monocytogenes, enabling this human pathogen to survive and grow. The current knowledge of the mechanisms involved in the interaction of this bacterium with plants is addressed, and whether this foodborne pathogen elicits an immune response in plants is discussed.

LisRK is required for optimal fitness of Listeria monocytogenes in soil.

Listeria monocytogenes is a food-borne pathogen responsible for the disease listeriosis. It is ubiquitously found in the environment and soil is one of its natural habitats. Listeria monocytogenes is highly capable of coping with various stressful conditions. We hypothesized that stress-responsive two-component systems such as LisRK might contribute to the adaptation of L. monocytogenes to the soil environment. Indeed, investigations of the population dynamics of wild-type and mutant strains suggest an important role of LisRK for optimal fitness of L. monocytogenes in sterile soil. Results from non-sterile soil showed that the parental strain was capable of surviving longer than mutant strains lacking lisRK or genes encoding the LisRK-regulated LhrC small RNAs (sRNAs), suggesting that LisRK as well as the LhrC sRNAs were important for survival. Transcription of five LisRK-regulated genes was assessed after 1 h incubation in sterile soil. We observed that LisRK and the LhrC sRNAs contribute to the upregulation of lmo2522 in the soil environment. Notably, lmo2522 encodes an equivalent of the resuscitation promoting factors, Rpfs, in actinobacteria. Collectively, our study demonstrates that LisRK is important for growth and survival in sterile and non-sterile soil and suggests a role for LisRK-regulation of Lmo2522 in resuscitation from dormancy in the soil environment.

Habitat Disturbances Modulate the Barrier Effect of Resident Soil Microbiota on Listeria monocytogenes Invasion Success.

Microbial communities are continuously exposed to the arrival of alien species. In complex environments such as soil, the success of invasion depends on the characteristics of the habitat, especially the diversity and structure of the residing bacterial communities. While most data available on microbial invasion relies on experiments run under constant conditions, the fate of invading species when the habitat faces disturbances has not yet been addressed. Here, we designed experiments to assess the consequences of habitat disturbance on the success of ongoing microbial invasion. We investigated (i) if disturbance-induced alterations in resident microbial communities could mitigate or facilitate invasion of Listeria monocytogenes, (ii) if disturbance itself could either improve or reduce the invader's fitness and (iii) if the invading species alters the structure of indigenous microbial communities. Our data show that environmental disturbances affect invasion patterns of L. monocytogenes in soils. Intriguingly, successful invasion was recorded in a regimen of disturbances that triggered small changes in microbial community structure while maintaining high bacterial diversity. On the opposite, dramatic decline of the invader was recorded when disturbance resulted in emergence of specific communities albeit concomitant with a diversity loss. This suggests that community composition is more important than its diversity when it comes to prevent the establishment of an invading species. Finally, shifts in bacterial communities during the disturbance event were strengthened by the presence of the invader indicating a major impact of invasion on microbial diversity when the habitat faces disturbance.

Evidence of Biocontrol Activity of Bioinoculants Against a Human Pathogen, Listeria monocytogenes.

Due to rhizodeposits and various microbial interactions, the rhizosphere is an extremely dynamic system, which provides a conductive niche not only for bacteria beneficial to plants but also for those that might pose a potential threat to humans. The importance of bioinoculants as biocontrol agents to combat phytopathogens has been widely recognized. However, little information exists with respect to their role in inhibiting human pathogens in the rhizosphere. The present study is an attempt to understand the impact of an established bacterial consortium, Azotobacter chroococcum, Bacillus megaterium, and Pseudomonas fluorescens, on the survivability of Listeria monocytogenes in the rhizosphere of Cajanus cajan and Festuca arundinacea. An experiment conducted in Hoagland's medium in the presence of C. cajan demonstrated that the presence of bioinoculants impaired growth of L. monocytogenes compared to that observed in their absence. On the other hand, in the presence of F. arundinacea, no significant differences were observed in the population dynamics of L. monocytogenes in the presence or absence of the bioinoculants. Agar plate assay through cross streak method revealed the inhibition of L. monocytogenes by bioinoculants. Potential bioactive compounds were identified by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). These results suggest that agricultural amendments can act as protective agents against human pathogens while enforcing plant growth promotion.

Investigation of the roles of AgrA and σB regulators in Listeria monocytogenes adaptation to roots and soil.

Little is known about the regulatory mechanisms that ensure the survival of the food-borne bacterial pathogen Listeria monocytogenes in the telluric environment and on roots. Earlier studies have suggested a regulatory overlap between the Agr cell-cell communication system and the general stress response regulator σB. Here, we investigated the contribution of these two systems to root colonisation and survival in sterilised and biotic soil. The ability to colonise the roots of the grass Festuca arundinacea was significantly compromised in the double mutant (∆agrA∆sigB). In sterile soil at 25°C, a significant defect was observed in the double mutant, suggesting some synergy between these systems. However, growth was observed and similar population dynamics were shown in the parental strain, ΔagrA and ΔsigB mutants. In biotic soil at 25°C, viability of the parental strain declined steadily over a two-week period highlighting the challenging nature of live soil environments. Inactivation of the two systems further decreased survival. The synergistic effect of Agr and σB was stronger in biotic soil. Transcriptional analysis confirmed the expected effects of the mutations on known Agr- and σB-dependent genes. Data highlight the important role that these global regulatory systems play in the natural ecology of this pathogen.

Exploring Listeria monocytogenes Transcriptomes in Correlation with Divergence of Lineages and Virulence as Measured in Galleria mellonella.

As for many opportunistic pathogens, the virulence potential of Listeria monocytogenes is highly heterogeneous between isolates and correlated, to some extent, with phylogeny and gene repertoires. In sharp contrast with copious data on intraspecies genome diversity, little is known about transcriptome diversity despite the role of complex genetic regulation in pathogenicity. The current study implemented RNA sequencing to characterize the transcriptome profiles of 33 isolates under optimal in vitro growth conditions. Transcript levels of conserved single-copy genes were comprehensively explored from several perspectives, including phylogeny, in silico-predicted virulence category based on epidemiological multilocus sequence typing (MLST) data, and in vivo virulence phenotype assessed in Galleria mellonella Comparing baseline transcriptomes between isolates was intrinsically more complex than standard genome comparison because of the inherent plasticity of gene expression in response to environmental conditions. We show that the relevance of correlation analyses and their statistical power can be enhanced by using principal-component analysis to remove the first level of irrelevant, highly coordinated changes linked to growth phase. Our results highlight the major contribution of transcription factors with key roles in virulence to the diversity of transcriptomes. Divergence in the basal transcript levels of a substantial fraction of the transcriptome was observed between lineages I and II, echoing previously reported epidemiological differences. Correlation analysis with in vivo virulence identified numerous sugar metabolism-related genes, suggesting that specific pathways might play roles in the onset of infection in G. mellonellaIMPORTANCEListeria monocytogenes is a multifaceted bacterium able to proliferate in a wide range of environments from soil to mammalian host cells. The accumulated genomic data underscore the contribution of intraspecies variations in gene repertoire to differential adaptation strategies between strains, including infection and stress resistance. It seems very likely that the fine-tuning of the transcriptional regulatory network is also a key component of the phenotypic diversity, albeit more difficult to investigate than genome content. Some studies reported incongruity in the basal transcriptome between isolates, suggesting a putative relationship with phenotypes, but small isolate numbers hampered proper correlation analyses with respect to their characteristics. The present study is the embodiment of the promising approach that consists of analyzing correlations between transcriptomes and various isolate characteristics. Statistically significant correlations were found with phylogenetic groups, epidemiological evidence of virulence potential, and virulence in Galleria mellonella larvae used as an in vivo model.

Survival of Listeria monocytogenes in Soil Requires AgrA-Mediated Regulation.

In a recent paper, we demonstrated that inactivation of the Agr system affects the patterns of survival of Listeria monocytogenes (A.-L. Vivant, D. Garmyn, L. Gal, and P. Piveteau, Front Cell Infect Microbiol 4:160, http://dx.doi.org/10.3389/fcimb.2014.00160). In this study, we investigated whether the Agr-mediated response is triggered during adaptation in soil, and we compared survival patterns in a set of 10 soils. The fate of the parental strain L. monocytogenes L9 (a rifampin-resistant mutant of L. monocytogenes EGD-e) and that of a ΔagrA deletion mutant were compared in a collection of 10 soil microcosms. The ΔagrA mutant displayed significantly reduced survival in these biotic soil microcosms, and differential transcriptome analyses showed large alterations of the transcriptome when AgrA was not functional, while the variations in the transcriptomes between the wild type and the ΔagrA deletion mutant were modest under abiotic conditions. Indeed, in biotic soil environments, 578 protein-coding genes and an extensive repertoire of noncoding RNAs (ncRNAs) were differentially transcribed. The transcription of genes coding for proteins involved in cell envelope and cellular processes, including the phosphotransferase system and ABC transporters, and proteins involved in resistance to antimicrobial peptides was affected. Under sterilized soil conditions, the differences were limited to 86 genes and 29 ncRNAs. These results suggest that the response regulator AgrA of the Agr communication system plays important roles during the saprophytic life of L. monocytogenes in soil.

The Agr communication system provides a benefit to the populations of Listeria monocytogenes in soil.

In this study, we investigated whether the Agr communication system of the pathogenic bacterium Listeria monocytogenes was involved in adaptation and competitiveness in soil. Alteration of the ability to communicate, either by deletion of the gene coding the response regulator AgrA (response-negative mutant) or the signal pro-peptide AgrD (signal-negative mutant), did not affect population dynamics in soil that had been sterilized but survival was altered in biotic soil suggesting that the Agr system of L. monocytogenes was involved to face the complex soil biotic environment. This was confirmed by a set of co-incubation experiments. The fitness of the response-negative mutant was lower either in the presence or absence of the parental strain but the fitness of the signal-negative mutant depended on the strain with which it was co-incubated. The survival of the signal-negative mutant was higher when co-cultured with the parental strain than when co-cultured with the response-negative mutant. These results showed that the ability to respond to Agr communication provided a benefit to listerial cells to compete. These results might also indicate that in soil, the Agr system controls private goods rather than public goods.

Listeria monocytogenes, a down-to-earth pathogen.

Listeria monocytogenes is the causative agent of the food-borne life threatening disease listeriosis. This pathogenic bacterium received much attention in the endeavor of deciphering the cellular mechanisms that underlie the onset of infection and its ability to adapt to the food processing environment. Although information is available on the presence of L. monocytogenes in many environmental niches including soil, water, plants, foodstuff and animals, understanding the ecology of L. monocytogenes in outdoor environments has received less attention. Soil is an environmental niche of pivotal importance in the transmission of this bacterium to plants and animals. Soil composition, microbial communities and macrofauna are extrinsic edaphic factors that direct the fate of L. monocytogenes in the soil environment. Moreover, farming practices may further affect its incidence. The genome of L. monocytogenes presents an extensive repertoire of genes encoding transport proteins and regulators, a characteristic of the genome of ubiquitous bacteria. Postgenomic analyses bring new insights in the process of soil adaptation. In the present paper focussing on soil, we review these extrinsic and intrinsic factors that drive environmental adaptation of L. monocytogenes.

Microbial diversity and structure are drivers of the biological barrier effect against Listeria monocytogenes in soil.

Understanding the ecology of pathogenic organisms is important in order to monitor their transmission in the environment and the related health hazards. We investigated the relationship between soil microbial diversity and the barrier effect against Listeria monocytogenes invasion. By using a dilution-to-extinction approach, we analysed the consequence of eroding microbial diversity on L. monocytogenes population dynamics under standardised conditions of abiotic parameters and microbial abundance in soil microcosms. We demonstrated that highly diverse soil microbial communities act as a biological barrier against L. monocytogenes invasion and that phylogenetic composition of the community also has to be considered. This suggests that erosion of diversity may have damaging effects regarding circulation of pathogenic microorganisms in the environment.

Listeria monocytogenes differential transcriptome analysis reveals temperature-dependent Agr regulation and suggests overlaps with other regulons.

Listeria monocytogenes is a ubiquitous, opportunistic pathogenic organism. Environmental adaptation requires constant regulation of gene expression. Among transcriptional regulators, AgrA is part of an auto-induction system. Temperature is an environmental cue critical for in vivo adaptation. In order to investigate how temperature may affect AgrA-dependent transcription, we compared the transcriptomes of the parental strain L. monocytogenes EGD-e and its ΔagrA mutant at the saprophytic temperature of 25°C and in vivo temperature of 37°C. Variations of transcriptome were higher at 37°C than at 25°C. Results suggested that AgrA may be involved in the regulation of nitrogen transport, amino acids, purine and pyrimidine biosynthetic pathways and phage-related functions. Deregulations resulted in a growth advantage at 37°C, but affected salt tolerance. Finally, our results suggest overlaps with PrfA, σB, σH and CodY regulons. These overlaps may suggest that through AgrA, Listeria monocytogenes integrates information on its biotic environment.

Changes in gene expression during adaptation of Listeria monocytogenes to the soil environment.

Listeria monocytogenes is a ubiquitous opportunistic pathogen responsible for listeriosis. In order to study the processes underlying its ability to adapt to the soil environment, whole-genome arrays were used to analyse transcriptome modifications 15 minutes, 30 minutes and 18 h after inoculation of L. monocytogenes EGD-e in soil extracts. Growth was observed within the first day of incubation and large numbers were still detected in soil extract and soil microcosms one year after the start of the experiment. Major transcriptional reprofiling was observed. Nutrient acquisition mechanisms (phosphoenolpyruvate-dependent phosphotransferase systems and ABC transporters) and enzymes involved in catabolism of specific carbohydrates (ß-glucosidases; chitinases) were prevalent. This is consistent with the overrepresentation of the CodY regulon that suggests that in a nutrient depleted environment, L. monocytogenes recruits its extensive repertoire of transporters to acquire a range of substrates for energy production.

Evidence of autoinduction heterogeneity via expression of the Agr system of Listeria monocytogenes at the single-cell level.

To investigate if the primary function of the Agr system of Listeria monocytogenes is to monitor cell density, we followed Agr expression in batch cultures, in which the autoinducer concentration was uniform, and in biofilms. Expression was heterogeneous, suggesting that the primary function of Agr is not to monitor population density.

Communication and autoinduction in the species Listeria monocytogenes: A central role for the agr system.

In order to withstand changes in their environment, bacteria have evolved mechanisms to sense the surrounding environment, integrate these signals and adapt their physiology to thrive under fluctuating conditions. Among these mechanisms, the ability of bacteria to exchange information between cells has become a dynamic field of interest for microbiologists over the past four decades. First described by Nelson et al.,1 this phenomenon often referred as either cell-cell communication, Quorum Sensing and/or AutoInduction involves the synthesis of small signal molecules called autoinducers. These signal molecules may be sensed by the bacterial population in the vicinity and induce regulation of gene expression. To date, three major communication systems have been described in bacteria. In this mini-review, we discuss the involvement of known communication systems in the transmission of information in the species Listeria monocytogenes. We will also discuss the latest findings on the role of communication in the regulation by Listeria monocytogenes of major adaptive strategies.

The impact of growth history and flagellation on the adhesion of various Listeria monocytogenes strains to polystyrene.

The contribution of growth history and flagella to adhesion of Listeria monocytogenes was analysed. An in-frame deletion on the flagellin encoding gene (flaA) was performed in L. monocytogenes EGD-e to compare its adhesion ability with the parental strain, after cultivation at various pH values and temperatures. The pH, as well as the temperature, affected the adhesion of L. monocytogenes EGD-e. In addition, the adhesion of L. monocytogenes EGD-e was reduced in energy-depressed cells. Conversely, the physicochemical bacterial surface characteristics affected by growth history did not influence the adhesion. Adhesion variations observed among environmental and clinical strains was attributed to the flagella. The naturally aflagellated strains resulted in an adhesion capacity similar to that observed for mutants and parental strains cultivated under flagellum expression repressing conditions. However, L. monocytogenes is able to adhere to inert surfaces through a residual adhesion process without flagella. All these observations emphasize the importance to consider the food environmental factors in the risk assessment of L. monocytogenes in food industry.

Listeria monocytogenes EGD-e biofilms: no mushrooms but a network of knitted chains.

Listeria monocytogenes is a food pathogen that can attach on most of the surfaces encountered in the food industry. Biofilms are three-dimensional microbial structures that facilitate the persistence of pathogens on surfaces, their resistance toward antimicrobials, and the final contamination of processed goods. So far, little is known about the structural dynamics of L. monocytogenes biofilm formation and its regulation. The aims of this study were, by combining genetics and time-lapse laser-scanning confocal microscopy (LSCM), (i) to characterize the structural dynamics of L. monocytogenes EGD-e sessile growth in two nutritional environments (with or without a nutrient flow), and (ii) to evaluate the possible role of the L. monocytogenes agr system during biofilm formation by tracking the spatiotemporal fluorescence expression of a green fluorescent protein (GFP) reporter system. In the absence of nutrient flow (static conditions), unstructured biofilms composed of a few layers of cells that covered the substratum were observed. In contrast, when grown under dynamic conditions, L. monocytogenes EGD-e biofilms were highly organized. Indeed, ball-shaped microcolonies were surrounded by a network of knitted chains. The spatiotemporal tracking of fluorescence emitted by the GFP reporter system revealed that agr expression was barely detectable under static conditions, but it progressively increased during 40 h under dynamic conditions. Moreover, spatial analysis revealed that agr was expressed preferentially in cells located outside the microcolonies. Finally, the in-frame deletion of agrA, which encodes a transcriptional regulator, resulted in a decrease in initial adherence without affecting the subsequent biofilm development.