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Klebsiella pneumoniae infections have become a growing threat for human health. The lack of understanding of the relationship between antibiotic resistance, mucoviscosity, and biofilm formation in clinical isolates impedes our abilities to effectively predict K. pneumoniae infection outcomes. These traits are also associated with fitness in natural populations and more specifically within a host. The Multidrug-Resistant Organism Repository and Surveillance Network offers a unique opportunity into the genetic and phenotypic variabilities in the K. pneumoniae isolates encountered in the clinics today. To this end, we compared the genetic profiles of these isolates with the phenotypic biofilm formation abilities, percent mucoviscosity, and growth rates. We found most isolates formed limited biofilm, although a select group of isolates could form extremely robust biofilms. Variation in biofilm formation could not be explained by difference in growth rate, suggesting specific genetic and physical determinants. Interestingly, the most mucoid strains in the populations were lacking the genetic element regulating the mucoid phenotype and three of these isolates were able to form robust biofilms. There was a significant phenotype-genotype correlation with decreased biofilm formation and an insertion sequence in the transcriptional activator of the type III fimbrial system. Finally, confocal microscopy highlighted the structural and spatial heterogeneity of biofilm among the most robust biofilm formers not detected by traditional methods. The combination of phenotypic, genomic and image analyses allowed us to reveal an unexpected phenotypic diversity and an intricate relation between growth, mucoviscosity and specific virulence-associated genetic determinants.

Bacterial evolution is affected by mobile genetic elements like phages and conjugative plasmids, offering new adaptive traits while incurring fitness costs. Their infection is affected by the bacterial capsule. Yet, its importance has been difficult to quantify because of the high diversity of confounding mechanisms in bacterial genomes such as anti-viral systems and surface receptor modifications. Swapping capsule loci between Klebsiella pneumoniae strains allowed us to quantify their impact on plasmid and phage infection independently of genetic background. Capsule swaps systematically invert phage susceptibility, revealing serotypes as key determinants of phage infection. Capsule types also influence conjugation efficiency in both donor and recipient cells, a mechanism shaped by capsule volume and conjugative pilus structure. Comparative genomics confirmed that more permissive serotypes in the lab correspond to the strains acquiring more conjugative plasmids in nature. The least capsule-sensitive pili (F-like) are the most frequent in the species' plasmids, and are the only ones associated with both antibiotic resistance and virulence factors, driving the convergence between virulence and antibiotics resistance in the population. These results show how traits of cellular envelopes define slow and fast lanes of infection by mobile genetic elements, with implications for population dynamics and horizontal gene transfer., (© 2024. The Author(s).)

Klebsiella variicola is an emergent human pathogen causing diverse infections, some of which in the urinary tract. However, little is known about the evolution and maintenance of genetic diversity in this species, the molecular mechanisms and their population dynamics. Here, we characterized the emergence of a novel rdar-like ( r ough and d ry) morphotype which is contingent both on the genetic background and the environment. We show that mutations in either the nitrogen assimilation control gene (nac) or the type III fimbriae regulator, mrkH , suffice to generate rdar-like colonies. These morphotypes are primarily selected for the reduced inter-cellular aggregation as a result of MrkH loss-of-function which reduces type 3 fimbriae expression. Additionally, these clones also display increased growth rate and reduced biofilm formation. Direct competitions between rdar and wild type clones show that mutations in mrkH provide large fitness advantages. In artificial urine, the morphotype is under strong negative frequency-dependent selection and can socially exploit wild type strains. An exhaustive search for mrkH mutants in public databases revealed that ca 8% of natural isolates analysed had a truncated mrkH gene many of which were due to insertions of IS elements, including a reported clinical isolate with rdar morphology. These strains were rarely hypermucoid and often isolated from human, mostly from urine and blood. The decreased aggregation of these mutants could have important clinical implications as we hypothesize that such clones could better disperse within the host allowing colonisation of other body sites and potentially leading to systemic infections., Competing Interests: Authors declare that we do not have any competing financial interests in relation to the work described., (© The Author(s) 2023. Published by Oxford University Press on behalf of FEMS.)

Adaptation to one environment can often generate phenotypic and genotypic changes which impact the future ability of an organism to thrive in other environmental conditions. In the context of host-microbe interactions, biofilm formation can increase survival rates in vivo upon exposure to stresses, like the host's immune system or antibiotic therapy. However, how the generic process of adaptation impacts the ability to form biofilm and how it may change through time has seldomly been studied. To do so, we used a previous evolution experiment with three strains of the Klebsiella pneumoniae species complex, in which we specifically did not select for biofilm formation. We observed that changes in the ability to form biofilm happened very fast at first and afterwards reverted to ancestral levels in many populations. Biofilm changes were associated to changes in population yield and surface polysaccharide production. Genotypically, mutations in the tip adhesin of type III fimbriae (mrkD) or the fim switch of type I fimbriae were shaped by nutrient availability during evolution, and their impact on biofilm formation was dependent on capsule production. Analyses of natural isolates revealed similar mutations in mrkD, suggesting that such mutations also play an important role in adaptation outside the laboratory. Our work reveals that the latent evolution of biofilm formation, and its temporal dynamics, depend on nutrient availability, the genetic background and other intertwined phenotypic and genotypic changes. Ultimately, it suggests that small differences in the environment can alter an organism's fate in more complex niches like the host., (© 2023. Springer Nature Limited.)

The extracellular capsule is a virulence factor present in many facultative pathogens, but its role in antimicrobial resistance remains controversial. To shed light on this debate, we tested six antibiotics on four Klebsiella pneumoniae species complex strains. Noncapsulated strains exhibited increased tolerance to polymyxins, but not to other antibiotics, as measured using the MIC. Our results urge caution on the use of therapeutic agents that target the capsule and may result in selection for its inactivation.

Many bacterial genomes carry prophages whose induction can eliminate competitors. In response, bacteria may become resistant by modifying surface receptors, by lysogenization, or by other poorly known processes. All these mechanisms affect bacterial fitness and population dynamics. To understand the evolution of phage resistance, we co-cultivated a phage-sensitive strain (BJ1) and a polylysogenic Klebsiella pneumoniae strain (ST14) under different phage pressures. The population yield remained stable after 30 days. Surprisingly, the initially sensitive strain remained in all populations and its frequency was highest when phage pressure was strongest. Resistance to phages in these populations emerged initially through mutations preventing capsule biosynthesis. Protection through lysogeny was rarely observed because the lysogens have increased death rates due to prophage induction. Unexpectedly, the adaptation process changed at longer time scales: the frequency of capsulated cells in BJ1 populations increased again because the production of the capsule was fine-tuned, reducing the ability of phage to absorb. Contrary to the lysogens, these capsulated-resistant clones are pan-resistant to a large panel of phages. Intriguingly, some clones exhibited transient non-genetic resistance to phages, suggesting an important role of phenotypic resistance in coevolving populations. Our results show that interactions between lysogens and sensitive strains are shaped by antagonistic co-evolution between phages and bacteria. These processes may involve key physiological traits, such as the capsule, and depend on the time frame of the evolutionary process. At short time scales, simple and costly inactivating mutations are adaptive, but in the long term, changes drawing more favorable trade-offs between resistance to phages and cell fitness become prevalent., Competing Interests: OR, Jd, ER No competing interests declared, (© 2023, Rendueles et al.)

Type VI secretion systems (T6SS), recently described in hypervirulent K. pneumoniae (hvKp) strains, are involved in bacterial warfare but their role in classical clinical strains (cKp) has been little investigated. In silico analysis indicated the presence of T6SS clusters (from zero to four), irrespective of the strains origin or virulence, with a high prevalence in the K. pneumoniae species (98%). In the strain CH1157, two T6SS-apparented pathogenicity islands were detected, T6SS-1 and -2, harboring a phospholipase-encoding gene (tle1) and a potential new effector-encoding gene named tke (Type VI Klebsiella effector). Tle1 expression in Escherichia coli periplasm affected cell membrane permeability. T6SS-1 isogenic mutants colonized the highest gastrointestinal tract of mice less efficiently than their parental strain, at long term. Comparative analysis of faecal 16S sequences indicated that T6SS-1 impaired the microbiota richness and its resilience capacity. Oscillospiraceae family members could be specific competitors for the long-term gut establishment of K. pneumoniae., (© 2022. The Author(s).)

Ecological causes of developmental evolution, for example from predation, remain much investigated, but the potential importance of latent phenotypes in eco-evo-devo has received little attention. Using the predatory bacterium Myxococcus xanthus, which undergoes aggregative fruiting body development upon starvation, we tested whether adaptation to distinct growth environments that do not induce development latently alters developmental phenotypes under starvation conditions that do induce development. In an evolution experiment named MyxoEE-3, growing M. xanthus populations swarmed across agar surfaces while adapting to conditions varying at factors such as surface stiffness or prey identity. Such ecological variation during growth was found to greatly impact the latent evolution of development, including fruiting body morphology, the degree of morphological trait correlation, reaction norms, degrees of developmental plasticity and stochastic diversification. For example, some prey environments promoted retention of developmental proficiency whereas others led to its systematic loss. Our results have implications for understanding evolutionary interactions among predation, development and motility in myxobacterial life cycles, and, more broadly, how ecology can profoundly shape the evolution of developmental systems latently rather than by direct selection on developmental features., (© 2022. The Author(s).)

The extracellular capsule is a major virulence factor, but its ubiquity in free-living bacteria with large environmental breadths suggests that it shapes adaptation to novel niches. Yet, how it does so, remains unexplored. Here, we evolve three Klebsiella strains and their capsule mutants in parallel. Their comparison reveals different phenotypic and genotypic evolutionary changes that alter virulence-associated traits. Non-capsulated populations accumulate mutations that reduce exopolysaccharide production and increase biofilm formation and yield, whereas most capsulated populations become hypermucoviscous, a signature of hypervirulence. Hence, adaptation to novel environments primarily occurs by fine-tuning expression of the capsular locus. The same evolutionary conditions selecting for mutations in the capsular gene wzc leading to hypermucoviscosity also result in increased susceptibility to antibiotics by mutations in the ramA regulon. This implies that general adaptive processes outside the host can affect capsule evolution and its role in virulence and infection outcomes may be a by-product of such adaptation., (© 2022. The Author(s).)

While the major virulence factors for Vibrio cholerae, the cause of the devastating diarrheal disease cholera, have been extensively studied, the initial intestinal colonization of the bacterium is not well understood because non-human adult animals are refractory to its colonization. Recent studies suggest the involvement of an interbacterial killing device known as the type VI secretion system (T6SS). Here, we tested the T6SS-dependent interaction of V. cholerae with a selection of human gut commensal isolates. We show that the pathogen efficiently depleted representative genera of the Proteobacteria in vitro, while members of the Enterobacter cloacae complex and several Klebsiella species remained unaffected. We demonstrate that this resistance against T6SS assaults was mediated by the production of superior T6SS machinery or a barrier exerted by group I capsules. Collectively, our data provide new insights into immunity protein-independent T6SS resistance employed by the human microbiota and colonization resistance in general., (© 2021. The Author(s).)

Mobile genetic elements (MGEs) drive genetic transfers between bacteria using mechanisms that require a physical interaction with the cellular envelope. In the high-priority multidrug-resistant nosocomial pathogens (ESKAPE), the first point of contact between the cell and virions or conjugative pili is the capsule. While the capsule can be a barrier to MGEs, it also evolves rapidly by horizontal gene transfer (HGT). Here, we aim at understanding this apparent contradiction by studying the covariation between the repertoire of capsule genes and MGEs in approximately 4,000 genomes of Klebsiella pneumoniae (Kpn). We show that capsules drive phage-mediated gene flow between closely related serotypes. Such serotype-specific phage predation also explains the frequent inactivation of capsule genes, observed in more than 3% of the genomes. Inactivation is strongly epistatic, recapitulating the capsule biosynthetic pathway. We show that conjugative plasmids are acquired at higher rates in natural isolates lacking a functional capsular locus and confirmed experimentally this result in capsule mutants. This suggests that capsule inactivation by phage pressure facilitates its subsequent reacquisition by conjugation. Accordingly, capsule reacquisition leaves long recombination tracts around the capsular locus. The loss and regain process rewires gene flow toward other lineages whenever it leads to serotype swaps. Such changes happen preferentially between chemically related serotypes, hinting that the fitness of serotype-swapped strains depends on the host genetic background. These results enlighten the bases of trade-offs between the evolution of virulence and multidrug resistance and caution that some alternatives to antibiotics by selecting for capsule inactivation may facilitate the acquisition of antibiotic resistance genes (ARGs)., Competing Interests: The authors have declared that no competing interests exist.

The fitness cost associated with the production of bacterial capsules is considered to be offset by the protection provided by these extracellular structures against biotic aggressions or abiotic stress. However, it is unknown if the capsule contributes to fitness in the absence of these. Here, we explored conditions favouring the maintenance of the capsule in Klebsiella pneumoniae, where the capsule is known to be a major virulence factor. Using short-term experimental evolution on different Klebsiella strains, we showed that small environmental variations have a strong impact on the maintenance of the capsule. Capsule inactivation is frequent in nutrient-rich, but scarce in nutrient-poor media. Competitions between wild-type and capsule mutants in nine different strains confirmed that the capsule is costly in nutrient-rich media. Surprisingly, these results also showed that the presence of a capsule provides a clear fitness advantage in nutrient-poor conditions by increasing both growth rates and population yields. The comparative analyses of the wild-type and capsule mutants reveal complex interactions between the environment, genetic background and serotype even in relation to traits known to be relevant during pathogenesis. In conclusion, our data suggest there are novel roles for bacterial capsules yet to be discovered and further supports the notion that the capsule's role in virulence may be a by-product of its contribution to bacterial adaptation outside the host.

The long-known resistance to pathogens provided by host-associated microbiota fostered the notion that adding protective bacteria could prevent or attenuate infection. However, the identification of endogenous or exogenous bacteria conferring such protection is often hindered by the complexity of host microbial communities. Here, we used zebrafish and the fish pathogen Flavobacterium columnare as a model system to study the determinants of microbiota-associated colonization resistance. We compared infection susceptibility in germ-free, conventional and reconventionalized larvae and showed that a consortium of 10 culturable bacterial species are sufficient to protect zebrafish. Whereas survival to F. columnare infection does not rely on host innate immunity, we used antibiotic dysbiosis to alter zebrafish microbiota composition, leading to the identification of two different protection strategies. We first identified that the bacterium Chryseobacterium massiliae individually protects both larvae and adult zebrafish. We also showed that an assembly of 9 endogenous zebrafish species that do not otherwise protect individually confer a community-level resistance to infection. Our study therefore provides a rational approach to identify key endogenous protecting bacteria and promising candidates to engineer resilient microbial communities. It also shows how direct experimental analysis of colonization resistance in low-complexity in vivo models can reveal unsuspected ecological strategies at play in microbiota-based protection against pathogens.

Klebsiella species are able to colonize a wide range of environments and include worrisome nosocomial pathogens. Here, we sought to determine the abundance and infectivity of prophages of Klebsiella to understand how the interactions between induced prophages and bacteria affect population dynamics and evolution. We identified many prophages in the species, placing these taxa among the top 5% of the most polylysogenic bacteria. We selected 35 representative strains of the Klebsiella pneumoniae species complex to establish a network of induced phage-bacteria interactions. This revealed that many prophages are able to enter the lytic cycle, and subsequently kill or lysogenize closely related Klebsiella strains. Although 60% of the tested strains could produce phages that infect at least one other strain, the interaction network of all pairwise cross-infections is very sparse and mostly organized in modules corresponding to the strains' capsule serotypes. Accordingly, capsule mutants remain uninfected showing that the capsule is a key factor for successful infections. Surprisingly, experiments in which bacteria are predated by their own prophages result in accelerated loss of the capsule. Our results show that phage infectiousness defines interaction modules between small subsets of phages and bacteria in function of capsule serotype. This limits the role of prophages as competitive weapons because they can infect very few strains of the species complex. This should also restrict phage-driven gene flow across the species. Finally, the accelerated loss of the capsule in bacteria being predated by their own phages, suggests that phages drive serotype switch in nature.

Background: Evolution in one selective environment often latently generates phenotypic change that is manifested only later in different environments, but the complexity of behavior important to fitness in the original environment might influence the character of such latent-phenotype evolution. Using Myxococcus xanthus, a bacterium possessing two motility systems differing in effectiveness on hard vs. soft surfaces, we test (i) whether and how evolution while swarming on one surface-the selective surface-latently alters motility on the alternative surface type and (ii) whether patterns of such latent-phenotype evolution depend on the complexity of ancestral motility, specific ancestral motility genotypes and/or the selective surface of evolution. We analysze an experiment in which populations established from three ancestral genotypes-one with both motility systems intact and two others with one system debilitated-evolved while swarming across either hard or soft agar in six evolutionary treatments. We then compare motility-phenotype patterns across selective vs. alternative surface types., Results: Latent motility evolution was pervasive but varied in character as a function of the presence of one or two functional motility systems and, for some individual-treatment comparisons, the specific ancestral genotype and/or selective surface. Swarming rates on alternative vs. selective surfaces were positively correlated generally among populations with one functional motility system but not among those with two. This suggests that opportunities for pleiotropy and epistasis generated by increased genetic complexity underlying behavior can alter the character of latent-phenotype evolution. No tradeoff between motility performance across surface types was detected in the dual-system treatments, even after adaptation on a surface on which one motility system dominates strongly over the other in driving movement, but latent-phenotype evolution was instead idiosyncratic in these treatments. We further find that the magnitude of stochastic diversification at alternative-surface swarming among replicate populations greatly exceeded diversification of selective-surface swarming within some treatments and varied across treatments., Conclusion: Collectively, our results suggest that increases in the genetic and mechanistic complexity of behavior can increase the complexity of latent-phenotype evolution outcomes and illustrate that diversification manifested during evolution in one environment can be augmented greatly by diversification of latent phenotypes manifested later.

Capsules allow bacteria to colonize novel environments, to withstand numerous stresses, and to resist antibiotics. Yet, even though genetic exchanges with other cells should be adaptive under such circumstances, it has been suggested that capsules lower the rates of homologous recombination and horizontal gene transfer. We analysed over one hundred pan-genomes and thousands of bacterial genomes for the evidence of an association between genetic exchanges (or lack thereof) and the presence of a capsule system. We found that bacteria encoding capsules have larger pan-genomes, higher rates of horizontal gene transfer, and higher rates of homologous recombination in their core genomes. Accordingly, genomes encoding capsules have more plasmids, conjugative elements, transposases, prophages, and integrons. Furthermore, capsular loci are frequent in plasmids, and can be found in prophages. These results are valid for Bacteria, independently of their ability to be naturally transformable. Since we have shown previously that capsules are commonly present in nosocomial pathogens, we analysed their co-occurrence with antibiotic resistance genes. Genomes encoding capsules have more antibiotic resistance genes, especially those encoding efflux pumps, and they constitute the majority of the most worrisome nosocomial bacteria. We conclude that bacteria with capsule systems are more genetically diverse and have fast-evolving gene repertoires, which may further contribute to their success in colonizing novel niches such as humans under antibiotic therapy., Competing Interests: The authors have declared that no competing interests exist.

Genetically similar cells of the soil bacterium Myxococcus xanthus cooperate at multiple social behaviours, including motility and multicellular development. Another social interaction in this species is outer membrane exchange (OME), a behaviour of unknown primary benefit in which cells displaying closely related variants of the outer membrane protein TraA transiently fuse and exchange membrane contents. Functionally incompatible TraA variants do not mediate OME, which led to the proposal that TraA incompatibilities determine patterns of intercellular cooperation in nature, but how this might occur remains unclear. Using natural isolates from a centimetre-scale patch of soil, we analyse patterns of TraA diversity and ask whether relatedness at TraA is causally related to patterns of kin discrimination in the form of both colony-merger incompatibilities (CMIs) and interstrain antagonisms. A large proportion of TraA functional diversity documented among global isolates is predicted to be contained within this cm-scale population. We find evidence of balancing selection on the highly variable PA14-portion of TraA and extensive transfer of traA alleles across genomic backgrounds. CMIs are shown to be common among strains identical at TraA, suggesting that CMIs are not generally caused by TraA dissimilarity. Finally, it has been proposed that interstrain antagonisms might be caused by OME-mediated toxin transfer. However, we predict that most strain pairs previously shown to exhibit strong antagonisms are incapable of OME due to TraA dissimilarity. Overall, our results suggest that most documented patterns of kin discrimination in a natural population of M. xanthus are not causally related to the TraA sequences of interactants., (© 2018 John Wiley & Sons Ltd.)

Extracellular capsules constitute the outermost layer of many bacteria, are major virulence factors, and affect antimicrobial therapies. They have been used as epidemiological markers and recently became vaccination targets. Despite the efforts to biochemically serotype capsules in a few model pathogens, little is known of their taxonomic and environmental distribution. We developed, validated, and made available a computational tool, CapsuleFinder, to identify capsules in genomes. The analysis of over 2500 prokaryotic genomes, accessible in a database, revealed that ca. 50% of them-including Archaea-encode a capsule. The Wzx/Wzy-dependent capsular group was by far the most abundant. Surprisingly, a fifth of the genomes encode more than one capsule system-often from different groups-and their non-random co-occurrence suggests the existence of negative and positive epistatic interactions. To understand the role of multiple capsules, we queried more than 6700 metagenomes for the presence of species encoding capsules and showed that their distribution varied between environmental categories and, within the human microbiome, between body locations. Species encoding capsules, and especially those encoding multiple capsules, had larger environmental breadths than the other species. Accordingly, capsules were more frequent in environmental bacteria than in pathogens and, within the latter, they were more frequent among facultative pathogens. Nevertheless, capsules were frequent in clinical samples, and were usually associated with fast-growing bacteria with high infectious doses. Our results suggest that capsules increase the environmental range of bacteria and make them more resilient to environmental perturbations. Capsules might allow opportunistic pathogens to profit from empty ecological niches or environmental perturbations, such as those resulting from antibiotic therapy, to colonize the host. Capsule-associated virulence might thus be a by-product of environmental adaptation. Understanding the role of capsules in natural environments might enlighten their function in pathogenesis.

An atypically large outbreak of Elizabethkingia anophelis infections occurred in Wisconsin. Here we show that it was caused by a single strain with thirteen characteristic genomic regions. Strikingly, the outbreak isolates show an accelerated evolutionary rate and an atypical mutational spectrum. Six phylogenetic sub-clusters with distinctive temporal and geographic dynamics are revealed, and their last common ancestor existed approximately one year before the first recognized human infection. Unlike other E. anophelis, the outbreak strain had a disrupted DNA repair mutY gene caused by insertion of an integrative and conjugative element. This genomic change probably contributed to the high evolutionary rate of the outbreak strain and may have increased its adaptability, as many mutations in protein-coding genes occurred during the outbreak. This unique discovery of an outbreak caused by a naturally occurring mutator bacterial pathogen provides a dramatic example of the potential impact of pathogen evolutionary dynamics on infectious disease epidemiology.

Evolutionary adaptation can be achieved by mechanisms accessible to all organisms, including faster growth and interference competition, but self-generated motility offers additional possibilities. We tested whether 55 populations of the bacterium Myxococcus xanthus that underwent selection for increased fitness at the leading edge of swarming colonies adapted by swarming faster toward unused resources or by other means. Populations adapted greatly but diversified markedly in both swarming phenotypes and apparent mechanisms of adaptation. Intriguingly, although many adapted populations swarm intrinsically faster than their ancestors, numerous others do not. Some populations evolved interference competition toward their ancestors, whereas others gained the ability to facultatively increase swarming rate specifically upon direct interaction with ancestral competitors. Our results both highlight the diverse range of mechanisms by which actively motile organisms can adapt evolutionarily and help to explain the high levels of swarming-phenotype diversity found in local soil populations of M. xanthus.

Elizabethkingia anophelis is an emerging pathogen involved in human infections and outbreaks in distinct world regions. We investigated the phylogenetic relationships and pathogenesis-associated genomic features of two neonatal meningitis isolates isolated 5 years apart from one hospital in Central African Republic and compared them with Elizabethkingia from other regions and sources. Average nucleotide identity firmly confirmed that E. anophelis, E. meningoseptica and E. miricola represent demarcated genomic species. A core genome multilocus sequence typing scheme, broadly applicable to Elizabethkingia species, was developed and made publicly available (http://bigsdb.pasteur.fr/elizabethkingia). Phylogenetic analysis revealed distinct E. anophelis sublineages and demonstrated high genetic relatedness between the African isolates, compatible with persistence of the strain in the hospital environment. CRISPR spacer variation between the African isolates was mirrored by the presence of a large mobile genetic element. The pan-genome of E. anophelis comprised 6,880 gene families, underlining genomic heterogeneity of this species. African isolates carried unique resistance genes acquired by horizontal transfer. We demonstrated the presence of extensive variation of the capsular polysaccharide synthesis gene cluster in E. anophelis. Our results demonstrate the dynamic evolution of this emerging pathogen and the power of genomic approaches for Elizabethkingia identification, population biology and epidemiology.

Diverse forms of kin discrimination, broadly defined as alteration of social behavior as a function of genetic relatedness among interactants, are common among social organisms from microbes to humans. However, the evolutionary origins and causes of kin-discriminatory behavior remain largely obscure. One form of kin discrimination observed in microbes is the failure of genetically distinct colonies to merge freely upon encounter. Here, we first use natural isolates of the highly social bacterium Myxococcus xanthus to show that colony-merger incompatibilities can be strong barriers to social interaction, particularly by reducing chimerism in multicellular fruiting bodies that develop near colony-territory borders. We then use experimental laboratory populations to test hypotheses regarding the evolutionary origins of kin discrimination. We show that the generic process of adaptation, irrespective of selective environment, is sufficient to repeatedly generate kin-discriminatory behaviors between evolved populations and their common ancestor. Further, we find that kin discrimination pervasively evolves indirectly between allopatric replicate populations that adapt to the same ecological habitat and that this occurs generically in many distinct habitats. Patterns of interpopulation discrimination imply that kin discrimination phenotypes evolved via many diverse genetic mechanisms and mutation-accumulation patterns support this inference. Strong incompatibility phenotypes emerged abruptly in some populations but strengthened gradually in others. The indirect evolution of kin discrimination in an asexual microbe is analogous to the indirect evolution of reproductive incompatibility in sexual eukaryotes and linguistic incompatibility among human cultures, the commonality being indirect, noncoordinated divergence of complex systems evolving in isolation.

Positively frequency-dependent selection is predicted from theory to promote diversity in patchily structured populations and communities, but empirical support for this prediction has been lacking. Here, we investigate frequency-dependent selection among isolates from a local natural population of the highly social bacterium Myxococcus xanthus. Upon starvation, closely related cells of M. xanthus cooperate to construct multicellular fruiting bodies, yet recently diverged genotypes co-residing in a local soil population often antagonize one another during fruiting-body development in mixed groups. In the experiments reported here, both fitness per se and strong forms of interference competition exhibit pervasive and strong positive frequency dependence (PFD) among many isolates from a centimeter-scale soil population of M. xanthus. All strains that compete poorly at intermediate frequency are shown to be competitively dominant at high frequency in most genotype pairings during both growth and development, and strongly so. Interference competition is often lethal and appears to be contact dependent rather than mediated by diffusible compounds. Finally, we experimentally demonstrate that positively frequency-dependent selection maintains diversity when genotype frequencies vary patchily in structured populations. These results suggest that PFD contributes to the high levels of local diversity found among M. xanthus social groups in natural soil populations by reinforcing social barriers to cross-territory invasion and thereby also promotes high within-group relatedness. More broadly, our results suggest that potential roles of PFD in maintaining patchily distributed diversity should be investigated more extensively in other species., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Bacterial biofilms are dense and often mixed-species surface-attached communities in which bacteria coexist and compete for limited space and nutrients. Here we present the different antagonistic interactions described in biofilm environments and their underlying molecular mechanisms, along with ecological and evolutionary insights as to how competitive interactions arise and are maintained within biofilms.

Formation of bacterial biofilm communities leads to profound physiological modifications and increased physical and metabolic exchanges between bacteria. It was previously shown that bioactive molecules produced within the biofilm environment contribute to bacterial interactions. Here we describe new pore-forming colicin R, specifically produced in biofilms formed by the natural isolate Escherichia coli ROAR029 but that cannot be detected under planktonic culture conditions. We demonstrate that an increased SOS stress response within mature biofilms induces SOS-dependent colicin R expression. We provide evidence that colicin R displays increased activity against E. coli strains that have a reduced lipopolysaccharide length, such as the pathogenic enteroaggregative E. coli LF82 clinical isolate, therefore pointing to lipopolysaccharide size as an important determinant for resistance to colicins. We show that colicin R toxicity toward E. coli LF82 is increased under biofilm conditions compared with planktonic susceptibility and that release of colicin R confers a strong competitive advantage in mixed biofilms by rapidly outcompeting sensitive neighboring bacteria. This work identifies the first biofilm-associated colicin that preferentially targets biofilm bacteria. Furthermore, it indicates that the study of antagonistic molecules produced in biofilm and multispecies contexts could reveal unsuspected, ecologically relevant bacterial interactions influencing population dynamics in natural environments.

Antivirulence strategies targeting bacterial behavior, such as adhesion and biofilm formation, are expected to exert low selective pressure and have been proposed as alternatives to biocidal antibiotic treatments to avoid the rapid occurrence of bacterial resistance. Here, we tested this hypothesis using group 2 capsule polysaccharide (G2cps), a polysaccharidic molecule previously shown to impair bacterium-surface interactions, and we investigated the nature of bacterial resistance to a nonbiocidal antibiofilm strategy. We screened an Escherichia coli mutant library for an increased ability to form biofilm in the presence of G2cps, and we identified several mutants displaying partial but not total resistance to this antibiofilm polysaccharide. Our genetic analysis showed that partial resistance to G2cps results from multiple unrelated mutations leading to modifications in surface physicochemical properties that counteract the changes in ionic charge and Lewis base properties induced by G2cps. Moreover, some of the identified mutants harboring improved biofilm formation in the presence of G2cps were also partially resistant to other antibiofilm molecules. This study therefore shows that alterations of bacterial surface properties mediate only partial resistance to G2cps. It also experimentally validates the potential value of nonbiocidal antibiofilm strategies, since full resistance to antibiofilm compounds is rare and potentially unlikely to arise in clinical settings.

The influence of microorganisms growing as sessile communities in a large number of human infections has been extensively studied and recognized for 30-40 years, therefore warranting intense scientific and medical research. Nonetheless, mimicking the biofilm-life style of bacteria and biofilm-related infections has been an arduous task. Models used to study biofilms range from simple in vitro to complex in vivo models of tissues or device-related infections. These different models have progressively contributed to the current knowledge of biofilm physiology within the host context. While far from a complete understanding of the multiple elements controlling the dynamic interactions between the host and biofilms, we are nowadays witnessing the emergence of promising preventive or curative strategies to fight biofilm-related infections. This review undertakes a comprehensive analysis of the literature from a historic perspective commenting on the contribution of the different models and discussing future venues and new approaches that can be merged with more traditional techniques in order to model biofilm-infections and efficiently fight them.

Bacterial extracellular polysaccharides have been shown to mediate many of the cell-to-cell and cell-to-surface interactions that are required for the formation, cohesion and stabilization of bacterial biofilms. However, recent studies have identified several bacterial polysaccharides that inhibit biofilm formation by a wide spectrum of bacteria and fungi both in vitro and in vivo. This review discusses the composition, modes of action and potential biological roles of antibiofilm polysaccharides recently identified in bacteria and eukarya. Some of these molecules may have technological applications as antibiofilm agents in industry and medicine., (© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Multi-species biofilm communities are environments in which complex but ill understood exchanges between bacteria occur. Although monospecies cultures are still widely used in the laboratory, new approaches have been undertaken to study interspecies interactions within mixed communities. This review describes our current understanding of competitive relationships involving nonbiocidal biosurfactants, enzymes, and metabolites produced by bacteria and other microorganisms. These molecules target all steps of biofilm formation, ranging from inhibition of initial adhesion to matrix degradation, jamming of cell-cell communications, and induction of biofilm dispersion. This review presents available data on nonbiocidal molecules and provides a new perspective on competitive interactions within biofilms that could lead to antibiofilm strategies of potential biomedical interest., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

The beneficial contribution of commensal bacteria to host health and homeostasis led to the concept that exogenous non-pathogenic bacteria called probiotics could be used to limit disease caused by pathogens. However, despite recent progress using gnotobiotic mammal and invertebrate models, mechanisms underlying protection afforded by commensal and probiotic bacteria against pathogens remain poorly understood. Here we developed a zebrafish model of controlled co-infection in which germ-free zebrafish raised on axenic living protozoa enabled the study of interactions between host and commensal and pathogenic bacteria. We screened enteric fish pathogens and identified Edwardsiella ictaluri as a virulent strain inducing a strong inflammatory response and rapid mortality in zebrafish larvae infected by the natural oro-intestinal route. Using mortality induced by infection as a phenotypic read-out, we pre-colonized zebrafish larvae with 37 potential probiotic bacterial strains and screened for survival upon E. ictaluri infection. We identified 3 robustly protective strains, including Vibrio parahaemolyticus and 2 Escherichia coli strains. We showed that the observed protective effect of E. coli was not correlated with a reduced host inflammatory response, nor with the release of biocidal molecules by protective bacteria, but rather with the presence of specific adhesion factors such as F pili that promote the emergence of probiotic bacteria in zebrafish larvae. Our study therefore provides new insights into the molecular events underlying the probiotic effect and constitutes a potentially high-throughput in vivo approach to the study of the molecular basis of pathogen exclusion in a relevant model of vertebrate oro-intestinal infection.

Unlabelled: Bacterial biofilms often form multispecies communities in which complex but ill-understood competition and cooperation interactions occur. In light of the profound physiological modifications associated with this lifestyle, we hypothesized that the biofilm environment might represent an untapped source of natural bioactive molecules interfering with bacterial adhesion or biofilm formation. We produced cell-free solutions extracted from in vitro mature biofilms formed by 122 natural Escherichia coli isolates, and we screened these biofilm extracts for antiadhesion molecules active on a panel of Gram-positive and Gram-negative bacteria. Using this approach, we showed that 20% of the tested biofilm extracts contained molecules that antagonize bacterial growth or adhesion. We characterized a compound, produced by a commensal animal E. coli strain, for which activity is detected only in biofilm extract. Biochemical and genetic analyses showed that this compound corresponds to a new type of released high-molecular-weight polysaccharide whose biofilm-associated production is regulated by the RfaH protein. We demonstrated that the antiadhesion activity of this polysaccharide was restricted to Gram-positive bacteria and that its production reduced susceptibility to invasion and provided rapid exclusion of Staphylococcus aureus from mixed E. coli and S. aureus biofilms. Our results therefore demonstrate that biofilms contain molecules that contribute to the dynamics of mixed bacterial communities and that are not or only poorly detected in unconcentrated planktonic supernatants. Systematic identification of these compounds could lead to strategies that limit pathogen surface colonization and reduce the burden associated with the development of bacterial biofilms on medical devices., Importance: We sought to demonstrate that bacterial biofilms are reservoirs for unknown molecules that antagonize bacterial adhesion. The use of natural strains representative of Escherichia coli species biodiversity showed that nonbiocidal antiadhesion polysaccharides are frequently found in mature biofilm extracts (bacterium-free suspensions which contain soluble molecules produced within the biofilm). Release of an antiadhesion polysaccharide confers a competitive advantage upon the producing strain against clinically relevant pathogens such as Staphylococcus aureus. Hence, exploring the biofilm environment provides a better understanding of bacterial interactions within complex communities and could lead to improved control of pathogen colonization.

The composition of the eukaryotic community and the three-dimensional structure of diverse phototrophic microbial mats from two hot springs in Iceland (Seltun and Hveradalir geothermal areas) were explored by comparing eukaryotic assemblages from microbial mats. Samples were collected in July 2007 from 15 sampling stations along thermal and pH gradients following both hot springs. Physicochemical data revealed high variability in terms of pH (ranging from 2.8 to 7), with high concentrations of heavy metals, including up to 20 g Fe/l, 80 mg Zn/l, 117 mg Cu/l, and 39 mg Ni/l at the most acidic sampling points. Phylogenetic analysis of 18S rDNA genes revealed a diversity of sequences related to several taxa, including members of the Bacillariophyta, Chlorophyta, Rhodophyta, and Euglenophyta phyla as well as ciliates, amoebae, and stramenopiles. The closest relatives to some of the sequences detected came from acidophilic organisms, even when the samples were collected at circumneutral water locations. Electron microscopy showed that most of the microecosystems analyzed were organized as phototrophic microbial mats in which filamentous cyanobacteria usually appeared as a major component. Deposits of amorphous minerals rich in silica, iron, and aluminium around the filaments were frequently detected.