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Plasmid conjugation is a key facilitator of horizontal gene transfer (HGT), and plasmids encoding antibiotic resistance drive the increasing prevalence of antibiotic resistance. In natural, engineered, and clinical environments, bacteria often grow in protective biofilms. Therefore, a better understanding of plasmid transfer in biofilms is needed. Our aim was to investigate plasmid transfer in a biofilm-adapted wrinkly colony mutant of Xanthomonas retroflexus (XRw) with enhanced matrix production and reduced motility. We found that XRw biofilms had an increased uptake of the broad host-range IncP-1ϵ plasmid pKJK5 compared to the wild type (WT). Proteomics revealed fewer flagellar-associated proteins in XRw, suggesting that flagella were responsible for reducing plasmid uptake. This was confirmed by the higher plasmid uptake of non-flagellated fliM mutants of the X. retroflexus wrinkly mutant as well as the wild type. Moreover, testing several flagellar mutants of Pseudomonas putida suggested that the flagellar effect was more general. We identified seven mechanisms with the potential to explain the flagellar effect and simulated them in an individual-based model. Two mechanisms could thus be eliminated (increased distances between cells and increased lag times due to flagella). Another mechanism identified as viable in the modeling was eliminated by further experiments. The possibility of steric hindrance of pilus movement and binding by flagella, reducing the frequency of contact and thus plasmid uptake, proved viable, and the three other viable mechanisms had a reduced probability of plasmid transfer in common. Our findings highlight the important yet complex effects of flagella during bacterial conjugation in biofilms.IMPORTANCEBiofilms are the dominant form of microbial life and bacteria living in biofilms are markedly different from their planktonic counterparts, yet the impact of the biofilm lifestyle on horizontal gene transfer (HGT) is still poorly understood. Horizontal gene transfer by conjugative plasmids is a major driver in bacterial evolution and adaptation, as exemplified by the troubling spread of antibiotic resistance. To either limit or promote plasmid prevalence and dissemination, we need a better understanding of plasmid transfer between bacterial cells, especially in biofilms. Here, we identified a new factor impacting the transfer of plasmids, flagella, which are required for many types of bacterial motility. We show that their absence or altered activity can lead to enhanced plasmid uptake in two bacterial species, Xanthomonas retroflexus and Pseudomonas putida . Moreover, we demonstrate the utility of mathematical modeling to eliminate hypothetical mechanisms., Competing Interests: The authors declare no conflict of interest.

Microorganisms colonizing plant roots co-exist in complex, spatially structured multispecies biofilm communities. However, little is known about microbial interactions and the underlying spatial organization within biofilm communities established on plant roots. Here, a well-established four-species biofilm model (Stenotrophomonas rhizophila, Paenibacillus amylolyticus, Microbacterium oxydans, and Xanthomonas retroflexus, termed as SPMX) was applied to Arabidopsis roots to study the impact of multispecies biofilm on plant growth and the community spatial dynamics on the roots. SPMX co-culture notably promoted root development and plant biomass. Co-cultured SPMX increased root colonization and formed multispecies biofilms, structurally different from those formed by monocultures. By combining 16S rRNA gene amplicon sequencing and fluorescence in situ hybridization with confocal laser scanning microscopy, we found that the composition and spatial organization of the four-species biofilm significantly changed over time. Monoculture P. amylolyticus colonized plant roots poorly, but its population and root colonization were highly enhanced when residing in the four-species biofilm. Exclusion of P. amylolyticus from the community reduced overall biofilm production and root colonization of the three species, resulting in the loss of the plant growth-promoting effects. Combined with spatial analysis, this led to identification of P. amylolyticus as a keystone species. Our findings highlight that weak root colonizers may benefit from mutualistic interactions in complex communities and hereby become important keystone species impacting community spatial organization and function. This work expands the knowledge on spatial organization uncovering interspecific interactions in multispecies biofilm communities on plant roots, beneficial for harnessing microbial mutualism promoting plant growth., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

We report here seven draft genomes of bacterial strains from two Danish wastewater facilities, two of which might be characterized as a new group within the Pseudomonas and Pseudochrobactrum genera, respectively. These genomes will provide useful references for understanding bacterial interactions and horizontal gene transfer within bacterial communities., Competing Interests: The authors declare no conflict of interest.

Multispecies biofilms are important models for studying the evolution of microbial interactions. Co-cultivation of Xanthomonas retroflexus (XR) and Paenibacillus amylolyticus (PA) systemically leads to the appearance of an XR wrinkled mutant (XRW), increasing biofilm production. The nature of this new interaction and the role of each partner remain unclear. We tested the involvement of secreted molecular cues in this interaction by exposing XR and XRW to PA or its supernatant and analysing the response using RNA-seq, colony-forming unit (CFU) estimates, biofilm quantification, and microscopy. Compared to wild type, the mutations in XRW altered its gene expression and increased its CFU number. These changes matched the reported effects for one of the mutated genes: a response regulator part of a two-component system involved in environmental sensing. When XRW was co-cultured with PA or its supernatant, the mutations effects on XRW gene expression were masked, except for genes involved in sedentary lifestyle, being consistent with the higher biofilm production. It appears that the higher biofilm production was the result of the interaction between the genetic context (mutations) and the biotic environment (PA signals). Regulatory genes involved in environmental sensing need to be considered to shed further light on microbial interactions., (© 2023 The Authors. Environmental Microbiology published by Applied Microbiology International and John Wiley & Sons Ltd.)

Mixed species biofilms exhibit increased tolerance to numerous stresses compared to single species biofilms. The aim of this study was to examine the effect of grazing by the heterotrophic protist, Tetrahymena pyriformis, on a mixed species biofilm consisting of Pseudomonas aeruginosa, Pseudomonas protegens, and Klebsiella pneumoniae. Protozoan grazing significantly reduced the single species K. pneumoniae biofilm, and the single species P. protegens biofilm was also sensitive to grazing. In contrast, P. aeruginosa biofilms were resistant to predation. This resistance protected the otherwise sensitive members of the mixed species biofilm consortium. Rhamnolipids produced by P. aeruginosa were shown to be the primary toxic factor for T. pyriformis. However, a rhamnolipid-deficient mutant of P. aeruginosa (P. aeruginosa Δ rhlAB ) maintained grazing resistance in the biofilm, suggesting the presence of at least one additional protective mechanism. P. aeruginosa with a deleted gene encoding the type III secretion system also resisted grazing. A transposon library was generated in the Δ rhlAB mutant to identify the additional factor involved in community biofilm protection. Results indicated that the Pseudomonas Quinolone Signal (PQS), a quorum sensing signaling molecule, was likely responsible for this effect. We confirmed this observation by showing that double mutants of Δ rhlAB and genes in the PQS biosynthetic operon lost grazing protection. We also showed that PQS was directly toxic to T. pyriformis . This study demonstrates that residing in a mixed species biofilm can be an advantageous strategy for grazing sensitive bacterial species, as P. aeruginosa confers community protection from protozoan grazing through multiple mechanisms. IMPORTANCE Biofilms have been shown to protect bacterial cells from predation by protists. Biofilm studies have traditionally used single species systems, which have provided information on the mechanisms and regulation of biofilm formation and dispersal, and the effects of predation on these biofilms. However, biofilms in nature are comprised of multiple species. To better understand how multispecies biofilms are impacted by predation, a model mixed-species biofilm was here exposed to protozoan predation. We show that the grazing sensitive strains K. pneumonia and P. protogens gained associational resistance from the grazing resistant P. aeruginosa. Resistance was due to the secretion of rhamnolipids and quorum sensing molecule PQS. This work highlights the importance of using mixed species systems.

The toolbox available for microbiologists to study interspecies interactions is rapidly growing, and with continuously more advanced instruments, we are able to expand our knowledge on establishment and function of microbial communities. However, unravelling molecular interspecies interactions in complex biological systems remains a challenge, and interactions are therefore often studied in simplified communities. Here we perform an in-depth characterization of an observed interspecies interaction between two co-isolated bacteria, Xanthomonas retroflexus and Paenibacillus amylolyticus . Using microsensor measurements for mapping the chemical environment, we show how X. retroflexus promoted an alkalization of its local environment through degradation of amino acids and release of ammonia. When the two species were grown in proximity, the modified local environment induced a morphological change and growth of P. amylolyticus followed by sporulation. 2D spatial metabolomics enabled visualization and mapping of the degradation of oligopeptide structures by X. retroflexus and morphological changes of P. amylolyticus through e.g. the release of membrane-associated metabolites. Proteome analysis and microscopy were used to validate the shift from vegetative growth towards sporulation. In summary, we demonstrate how environmental profiling by combined application of microsensor, microscopy, metabolomics and proteomics approaches can reveal growth and sporulation promoting effects resulting from interspecies interactions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Herschend, Ernst, Koren, Melnik, da Silva, Røder, Damholt, Hägglund, Svensson, Sørensen, Kühl, Dorrestein and Burmølle.)

Drought severely restricts plant production and global warming is further increasing drought stress for crops. Much information reveals the ability of individual microbes affecting plant stress tolerance. However, the effects of emergent bacterial community properties on plant drought tolerance remain largely unexplored. Here, we inoculated Arabidopsis plants in vivo with a four-species bacterial consortium (Stenotrophomonas rhizophila, Xanthomonas retroflexus, Microbacterium oxydans, and Paenibacillus amylolyticus, termed as SPMX), which is able to synergistically produce more biofilm biomass together than the sum of the four single-strain cultures, to investigate its effects on plant performance and rhizo-microbiota during drought. We found that SPMX remarkably improved Arabidopsis survival post 21-day drought whereas no drought-tolerant effect was observed when subjected to the individual strains, revealing emergent properties of the SPMX consortium as the underlying cause of the induced drought tolerance. The enhanced drought tolerance was associated with sustained chlorophyll content and endogenous abscisic acid (ABA) signaling. Furthermore, our data showed that the addition of SPMX helped to stabilize the diversity and structure of root-associated microbiomes, which potentially benefits plant health under drought. These SPMX-induced changes jointly confer an increased drought tolerance to plants. Our work may inform future efforts to engineer the emergent bacterial community properties to improve plant tolerance to drought., (© 2021. The Author(s).)

The crystal violet assay is widely used for biofilm quantitation despite its toxicity and variability. Here, we instead combine fluorescence labelling with the Cytation 5 multi-mode plate reader, to enable simultaneous acquisition of both quantitative and imaging biofilm data. This high-throughput method produces more robust data and provides information about morphology and spatial species organization within the biofilm., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Plasmids facilitate rapid bacterial adaptation by shuttling a wide variety of beneficial traits across microbial communities. However, under non-selective conditions, maintaining a plasmid can be costly to the host cell. Nonetheless, plasmids are ubiquitous in nature where bacteria adopt their dominant mode of life - biofilms. Here, we demonstrate that biofilms can act as spatiotemporal reserves for plasmids, allowing them to persist even under non-selective conditions. However, under these conditions, spatial stratification of plasmid-carrying cells may promote the dispersal of cells without plasmids, and biofilms may thus act as plasmid sinks., (© 2021. The Author(s).)

Temperate ϕH20-like phages are repeatedly identified at geographically distinct areas as free phage particles or as prophages of the fish pathogen Vibrio anguillarum. We studied mutants of a lysogenic isolate of V. anguillarum locked in the quorum-sensing regulatory modes of low (ΔvanT) and high (ΔvanO) cell densities by in-frame deletion of key regulators of the quorum-sensing pathway. Remarkably, we find that induction of the H20-like prophage is controlled by the quorum-sensing state of the host, with an eightfold increase in phage particles per cell in high-cell-density cultures of the quorum-sensing-deficient ΔvanT mutant. Comparative studies with prophage-free strains show that biofilm formation is promoted at low cell density and that the H20-like prophage stimulates this behavior. In contrast, the high-cell-density state is associated with reduced prophage induction, increased proteolytic activity, and repression of biofilm. The proteolytic activity may dually function to disperse the biofilm and as a quorum-sensing-mediated antiphage strategy. We demonstrate an intertwined regulation of phage-host interactions and biofilm formation, which is orchestrated by host quorum-sensing signaling, suggesting that increased lysogeny at high cell density is not solely a strategy for phages to piggy-back the successful bacterial hosts but is also a host strategy evolved to take control of the lysis-lysogeny switch to promote host fitness.

The importance of microbial biofilms has been well-recognized for several decades, and focus is now shifting towards investigating multispecies biofilm communities rather than mono- or dual-species biofilms. Therefore, the demand for techniques that provide a sufficient amount of information at adequate resolution is increasing. One major challenge for multispecies studies is that diversity and spatial organization often lead to a high degree of spatial and chemical heterogeneity. Many current approaches do not account for such heterogeneity and therefore only provide average information (-omics techniques in particular), which could obscure important information about the community. Here, we bring attention to the issues of heterogeneity when analysing synthetic multi-species biofilms, in vitro, and the importance of multi-scale approaches. We provide an overview of current and newer approaches that can be applied to biofilm communities, in order to elucidate interactions at the appropriate scale., (© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Attachment of microorganisms to food contact surfaces and the subsequent formation of biofilms may cause equipment damage, food spoilage and even diseases. Mixed-species biofilms are ubiquitous in the food industry and they generally exhibit higher resistance to disinfectants and antimicrobials compared to single-species biofilms. The physiology and metabolic activity of microorganisms in mixed-species biofilms are however rather complicated to study, and despite targeted research efforts, the potential role of mixed-species biofilms in food industry is still rather unexplored. In this review, we summarize recent studies in the context of bacterial social interactions in mixed-species biofilms, resistance to disinfectants, detection methods, and potential novel strategies to control the formation of mixed-species biofilms for enhanced food safety and food quality.

Multispecies biofilms are structured and spatially defined communities, where interspecies interactions impact assembly and functionality. Here, we compared the spatial organization and growth of bacterial cells in differently composed biofilm communities over time to determine links between interspecies interactions and selection for biofilm phenotypes of individual species. An established model community consisting of Stenotrophomonas rhizophila, Xanthomonas retroflexus, Microbacterium oxydans and Paenibacillus amylolyticus was used. It was found that interspecies interactions led to varying levels of selection for a new colony phenotype of X. retroflexus, depending on the presence/absence of other species. When M. oxydans was absent, X. retroflexus was not able to establish in the top layers of the biofilm, which led to selection for a hyper-matrix forming phenotype of X. retroflexus that successfully established in the biofilm top layers. No such phenotypic X. retroflexus variants were identified in the presence of M. oxydans. These findings indicate that interspecies interactions may lead to favourable localization of individual species in a multispecies biofilm and thereby reduce selection for competitive phenotypes., (© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Bacteriophages (phages) have been shaping bacterial ecology and evolution for millions of years, for example, by selecting for defence strategies. Evidence supports that bacterial biofilm formation is one such strategy and that biofilm-mediated protection against phage infection depends on maturation and composition of the extracellular matrix. Interestingly, studies have revealed that phages can induce and strengthen biofilms. Here we review interactions between bacteria and phages in biofilms, discuss the underlying mechanisms, the potential of phage therapy for biofilm control, and emphasize the importance of considering biofilms in future phage research. This is especially relevant as biofilms are associated with increased tolerance towards antibiotics and are implicated in the majority of chronic infections., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The arrival order of different species to a habitat can strongly impact community assembly and succession dynamics, thus influencing functionality. In this study, we asked how prior colonization of one community member would influence the assembly of a synergistic multispecies biofilm community grown in vitro . We expected that the prior arrival would confer an advantage, in particular for good biofilm formers. Yet, we did not know if the cohabitants would be impaired or benefit from the pre-colonization of one member, depending on its ability to form biofilm. We used a consortium consisting of four soil bacteria; Stenotrophomonas rhizophila, Xanthomonas retroflexus, Microbacterium oxydans and Paenibacillus amylolyticus . This consortium has been shown to act synergistically when grown together, thus increasing biofilm production. The results showed that the two good biofilm formers gained a fitness advantage (increase in abundance) when allowed prior colonization on an abiotic surface before the arrival of their cohabitants. Interestingly, the significantly higher number of the pre-colonized biofilm formers did not affect the resulting composition in the subsequent biofilm after 24 h.

Bacteria often live in communities of mixed species embedded in a self-produced extracellular matrix of polysaccharides, proteins and DNA, termed biofilms. The BioFlux microfluidic flow system is useful for studying biofilm formation in different media under flow. However, analyzing the architecture and maturation of biofilms under flow requires a proper seeding, which can prove difficult when working with bacteria of different sizes, motile bacteria or aiming for a high number of replicates. Here we developed an efficient protocol that exploits viscosity tuning and seeding indicator dyes to improve seeding and allow for high-throughput examination and visualization of consistent mono- and mixed-species biofilm developments under flow.

Microbial communities primarily consist of multiple species that affect one another's fitness both directly and indirectly. This study showed that the cocultivation of Paenibacillus amylolyticus and Xanthomonas retroflexus exhibited facultative mutualistic interactions in a static environment, during the course of which a new adapted phenotypic variant of X. retroflexus appeared. Although the emergence of this variant was not directly linked to the presence of P. amylolyticus, its establishment in the coculture enhanced the productivity of both species due to mutations that stimulated biofilm formation. The mutations were detected in genes encoding a diguanylate cyclase predicted to synthesise cyclic-di-GMP. Examinations of the biofilm formed in cocultures of P. amylolyticus and the new variant of X. retroflexus revealed a distinct spatial organisation: P. amylolyticus only resided in biofilms in association with X. retroflexus and occupied the outer layers. The X. retroflexus variant therefore facilitated increased P. amylolyticus growth as it produced more biofilm biomass. The increase in X. retroflexus biomass was thus not at the expense of P. amylolyticus, demonstrating that interspecies interactions can shape diversification in a mutualistic coculture and reinforce these interactions, ultimately resulting in enhanced communal performance.

The composition and development of naturally occurring microbial communities are defined by a complex interplay between the community and the surrounding environment and by interactions between community members. Intriguingly, these interactions can in some cases cause synergies, where the community is able to outperform its single-species constituents. However, the underlying mechanisms driving community interactions are often unknown and difficult to identify due to high community complexity. Here, we show how opposite pH drift induced by specific community members leads to pH stabilization of the microenvironment, acting as a positive interspecies interaction, driving in vitro community synergy in a model consortium of four coisolated soil bacteria, Microbacterium oxydans , Xanthomonas retroflexus , Stenotrophomonas rhizophila , and Paenibacillus amylolyticus We use microsensor pH measurements to show how individual species change the local pH microenvironment and how cocultivation leads to a stabilized pH regime over time. Specifically, in vitro acid production from P. amylolyticus and alkali production primarily from X. retroflexus led to an overall pH stabilization of the local environment over time, which in turn resulted in enhanced community growth. This specific type of interspecies interaction was found to be highly dependent on medium type and concentration; however, similar pH drift from the individual species could be observed across medium variants. IMPORTANCE Understanding interspecies interactions in bacterial communities is important for unraveling species dynamics in naturally occurring communities. These dynamics are fundamental for identifying evolutionary drivers and for the development of efficient biotechnological industry applications. Recently, pH interplay among community members has been identified as a factor affecting community development, and pH stabilization has been demonstrated to result in enhanced community growth. The use of model communities in which the effect of changing pH level can be attributed to specific species contributes to the investigation of community developmental drivers. This contributes to assessment of the extent of emergent behavior and members' contributions to community development. Here, we show that pH stabilization of the microenvironment in vitro in a synthetic coisolated model community results in synergistic growth. This observation adds to the growing diversity of community interactions leading to enhanced community growth and hints toward pH as a strong driver for community development in diverse environments., (Copyright © 2018 American Society for Microbiology.)

In the Origin of Species , Charles R. Darwin [Darwin C (1859) On the Origin of Species ] proposed that the struggle for existence must be most intense among closely related species by means of their functional similarity. It has been hypothesized that this similarity, which results in resource competition, is the driver of the evolution of antagonism among bacteria. Consequently, antagonism should mostly be prevalent among phylogenetically and metabolically similar species. We tested the hypothesis by screening for antagonism among all possible pairwise interactions between 67 bacterial species from 8 different environments: 2,211 pairs of species and 4,422 interactions. We found a clear association between antagonism and phylogenetic distance, antagonism being most likely among closely related species. We determined two metabolic distances between our strains: one by scoring their growth on various natural carbon sources and the other by creating metabolic networks of predicted genomes. For both metabolic distances, we found that the probability of antagonism increased the more metabolically similar the strains were. Moreover, our results were not compounded by whether the antagonism was between sympatric or allopatric strains. Intriguingly, for each interaction the antagonizing strain was more likely to have a wider metabolic niche than the antagonized strain: that is, larger metabolic networks and growth on more carbon sources. This indicates an association between an antagonistic and a generalist strategy., Competing Interests: The authors declare no conflict of interest.

Microorganisms frequently co-exist in matrix-embedded multispecies biofilms. Within biofilms, interspecies interactions influence the spatial organization of member species, which likely play an important role in shaping the development, structure and function of these communities. Here, a reproducible four-species biofilm, composed of Stenotrophomonas rhizophila, Xanthomonas retroflexus, Microbacterium oxydans and Paenibacillus amylolyticus, was established to study the importance of individual species spatial organization during multispecies biofilm development. We found that the growth of species that are poor biofilm formers, M. oxydans and P. amylolyticus, were highly enhanced when residing in the four-species biofilm. Interestingly, the presence of the low-abundant M. oxydans (0.5% of biomass volume) was observed to trigger changes in the composition of the four-species community. The other three species were crucially needed for the successful inclusion of M. oxydans in the four-species biofilm, where X. retroflexus was consistently positioned in the top layer of the mature four-species biofilm. These findings suggest that low abundance key species can significantly impact the spatial organization and hereby stabilize the function and composition of complex microbiomes., (© 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Social interactions in which bacteria respond to one another by modifying their phenotype are central determinants of microbial communities. It is known that interspecific interactions influence the biofilm phenotype of bacteria; a phenotype that is central to the fitness of bacteria. However, the underlying role of fundamental ecological factors, specifically coexistence and phylogenetic history, in biofilm formation remains unclear. This study examines how social interactions affect biofilm formation in multi-species co-cultures from five diverse environments. We found prevalence of increased biofilm formation among co-cultured bacteria that have coexisted in their original environment. Conversely, when randomly co-culturing bacteria across these five consortia, we found less biofilm induction and a prevalence of biofilm reduction. Reduction in biofilm formation was even more predominant when co-culturing bacteria from environments where long-term coexistence was unlikely to have occurred. Phylogenetic diversity was not found to be a strong underlying factor but a relation between biofilm induction and phylogenetic history was found. The data indicates that biofilm reduction is typically correlated with an increase in planktonic cell numbers, thus implying a behavioral response rather than mere growth competition. Our findings suggest that an increase in biofilm formation is a common adaptive response to long-term coexistence., (© 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Interspecies interactions are essential for the persistence and development of any kind of complex community, and microbial biofilms are no exception. Multispecies biofilms are structured and spatially defined communities that have received much attention due to their omnipresence in natural environments. Species residing in these complex bacterial communities usually interact both intra- and interspecifically. Such interactions are considered to not only be fundamental in shaping overall biomass and the spatial distribution of cells residing in multispecies biofilms, but also to result in coordinated regulation of gene expression in the different species present. These communal interactions often lead to emergent properties in biofilms, such as enhanced tolerance against antibiotics, host immune responses, and other stresses, which have been shown to provide benefits to all biofilm members not only the enabling sub-populations. However, the specific molecular mechanisms of cellular processes affecting spatial organization, and vice versa, are poorly understood and very complex to unravel. Therefore, detailed description of the spatial organization of individual bacterial cells in multispecies communities can be an alternative strategy to reveal the nature of interspecies interactions of constituent species. Closing the gap between visual observation and biological processes may become crucial for resolving biofilm related problems, which is of utmost importance to environmental, industrial, and clinical implications. This review briefly presents the state of the art of studying interspecies interactions and spatial organization of multispecies communities, aiming to support theoretical and practical arguments for further advancement of this field.

The high prevalence and significance of multispecies biofilms have now been demonstrated in various bacterial habitats with medical, industrial, and ecological relevance. It is highly evident that several species of bacteria coexist and interact in biofilms, which highlights the need for evaluating the approaches used to study these complex communities. This review focuses on the establishment of multispecies biofilms in vitro, interspecies interactions in microhabitats, and how to select communities for evaluation. Studies have used different experimental approaches; here we evaluate the benefits and drawbacks of varying the degree of complexity. This review aims to facilitate multispecies biofilm research in order to expand the current limited knowledge on interspecies interactions., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Here, we report the draft genome sequence of Psychrobacter cibarius strain W1, which was isolated at a slaughterhouse in Denmark. The 3.63-Mb genome sequence was assembled into 241 contigs., (Copyright © 2016 Raghupathi et al.)

We report here the first draft genome sequence ofKocuria variansG6, which was isolated from a meat chopper at a small slaughterhouse in Denmark. The 2.90-Mb genome sequence consists of 95 contigs and contains 2,518 predicted protein-coding genes., (Copyright © 2016 Raghupathi et al.)

We report the 3.09 Mb draft genome sequence ofKocuria palustrisW4, isolated from a slaughterhouse in Denmark., (Copyright © 2016 Herschend et al.)

We report the draft genome sequences of twoPseudoclavibacter helvolusstrains. Strain G8 was isolated from a meat chopper and strain W3 isolated from the wall of a small slaughterhouse in Denmark. The two annotated genomes are 3.91 Mb and 4.00 Mb in size, respectively., (Copyright © 2016 Raghupathi et al.)

We report here the draft genome sequences ofKocuria salsiciaG1 andKocuria rhizophilaG2, which were isolated from a meat chopper at a small slaughterhouse in Denmark. The two annotated genomes are 2.99 Mb and 2.88 Mb in size, respectively., (Copyright © 2016 Herschend et al.)

We report the draft genome sequence ofArthrobacter antarcticusstrain W2, which was isolated from a wall of a small slaughterhouse in Denmark. The 4.43-Mb genome sequence was assembled into 170 contigs., (Copyright © 2016 Herschend et al.)

Bacterial attachment and biofilm formation can lead to poor hygienic conditions in food processing environments. Furthermore, interactions between different bacteria may induce or promote biofilm formation. In this study, we isolated and identified a total of 687 bacterial strains from seven different locations in a meat processing environment and evaluated their biofilm formation capability. A diverse group of bacteria was isolated and most were classified as poor biofilm producers in a Calgary biofilm device assay. Isolates from two sampling sites, the wall and the meat chopper, were further examined for multispecies biofilm formation. Eight strains from each sampling site were chosen and all possible combinations of four member co-cultures were tested for enhanced biofilm formation at 15 °C and 24 °C. In approximately 20% of the multispecies consortia grown at 15 °C, the biofilm formation was enhanced when comparing to monospecies biofilms. Two specific isolates (one from each location) were found to be present in synergistic combinations with higher frequencies than the remaining isolates tested. This data provides insights into the ability of co-localized isolates to influence co-culture biofilm production with high relevance for food safety and food production facilities., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Horizontal gene transfer by conjugation has been reported to increase overall biofilm formation. Biofilm is considered a hot spot for plasmid transfer, and it has been found that social interactions during biofilm formation can increase the biomass. In this study, we demonstrate a contrast to previous studies by showing that the conjugative IncP-1 plasmid pKJK5 influences biofilm formation negatively. The results showed that a co-culture (Pseudomonas putida, Kluyvera sp., and Escherichia coli) formed significantly more biofilm than the strains did individually. When pKJK5 was inserted into P. putida, biofilm formation was significantly reduced compared with the co-culture without plasmid. A nonconjugative version of pKJK5 was also used, and the biofilm formation was restored. Visualization with the BioFlux 1000 facility showed that the presence of pKJK5-containing P. putida in the co-culture led to a changed biofilm structure, where the cells showed a higher tendency to attach to other cells rather than surfaces. This study thus indicates that the presence of conjugative plasmids in some species may decrease the surface-associated biofilm formation of a mixed co-culture by facilitating cell-cell attachment with reduced surface attachment as the consequence., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)