The development of a biofilm constitutes a survival strategy by providing bacteria a protective environment safe from stresses such as microbicide action and can thus lead to important health-care ...problems. In this study, biofilm resistance of a Bacillus subtilis strain (called hereafter ND(medical)) recently isolated from endoscope washer-disinfectors to peracetic acid was investigated and its ability to protect the pathogen Staphylococcus aureus in mixed biofilms was evaluated. Biocide action within Bacillus subtilis biofilms was visualised in real time using a non-invasive 4D confocal imaging method. The resistance of single species and mixed biofilms to peracetic acid was quantified using standard plate counting methods and their architecture was explored using confocal imaging and electronic microscopy. The results showed that the ND(medical) strain demonstrates the ability to make very large amount of biofilm together with hyper-resistance to the concentration of PAA used in many formulations (3500 ppm). Evidences strongly suggest that the enhanced resistance of the ND(medical) strain was related to the specific three-dimensional structure of the biofilm and the large amount of the extracellular matrix produced which can hinder the penetration of peracetic acid. When grown in mixed biofilm with Staphylococcus aureus, the ND(medical) strain demonstrated the ability to protect the pathogen from PAA action, thus enabling its persistence in the environment. This work points out the ability of bacteria to adapt to an extremely hostile environment, and the necessity of considering multi-organism ecosystems instead of single species model to decipher the mechanisms of biofilm resistance to antimicrobials agents.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Biofilms constitute the prevalent way of life for microorganisms in both natural and man-made environments. Biofilm-dwelling cells display greater tolerance to antimicrobial agents than those that ...are free-living, and the mechanisms by which this occurs have been investigated extensively using single-strain axenic models. However, there is growing evidence that interspecies interactions may profoundly alter the response of the community to such toxic exposure. In this paper, we propose an overview of the studies dealing with multispecies biofilms resistance to biocides, with particular reference to the protection of pathogenic species by resident surface flora when subjected to disinfectants treatments. The mechanisms involved in such protection include interspecies signaling, interference between biocides molecules and public goods in the matrix, or the physiology and genetic plasticity associated with a structural spatial arrangement. After describing these different mechanisms, we will discuss the experimental methods available for their analysis in the context of complex multispecies biofilms.
Abstract
Bacillus subtilis
can form various types of spatially organised communities on surfaces, such as colonies, pellicles and submerged biofilms. These communities share similarities and ...differences, and phenotypic heterogeneity has been reported for each type of community. Here, we studied spatial transcriptional heterogeneity across the three types of surface-associated communities. Using RNA-seq analysis of different regions or populations for each community type, we identified genes that are specifically expressed within each selected population. We constructed fluorescent transcriptional fusions for 17 of these genes, and observed their expression in submerged biofilms using time-lapse confocal laser scanning microscopy (CLSM). We found mosaic expression patterns for some genes; in particular, we observed spatially segregated cells displaying opposite regulation of carbon metabolism genes (
gapA
and
gapB
), indicative of distinct glycolytic or gluconeogenic regimes coexisting in the same biofilm region. Overall, our study provides a direct comparison of spatial transcriptional heterogeneity, at different scales, for the three main models of
B. subtilis
surface-associated communities.
A better understanding of the impact of antibiotics on bacteria is required to increase the efficiency of antibiotic treatments and to slow the emergence of resistance. Using Escherichia coli, we ...examined how bacteria exposed to sublethal concentrations of ampicillin adjust gene expression patterns and metabolism to simultaneously deal with the antibiotic-induced damage and maintain rapid growth. We found that the treated cells increased energy production, as well as translation and macromolecular repair and protection. These responses are adaptive, because they confer increased survival not only to lethal ampicillin treatment but also to non-antibiotic lethal stresses. This robustness is modulated by nutrient availability. Because different antibiotics and other stressors induce the same set of responses, we propose that it constitutes a general core hormetic stress response. It is plausible that this response plays an important role in the robustness of bacteria exposed to antibiotic treatments and constant environmental fluctuations in natural environments.
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•Sublethal stresses induce a general core hormetic stress response•Energy metabolism and translation are increased by sublethal antibiotic treatments•Antibiotic-treated cells deplete amino acids faster than untreated cells•Antibiotic-treated cells induce the stringent response earlier than untreated cells
Mathieu et al. report that sublethal antibiotic treatments induce a general core hormetic stress response, which endows bacteria with capacity to survive different subsequent lethal stresses. It is plausible that this response plays an important role in the robustness of bacteria exposed to antibiotic treatments and constant environmental fluctuations in nature.
Surface-associated multicellular assemblage is an important bacterial trait to withstand harsh environmental conditions.
is one of the most studied Gram-positive bacteria, serving as a model for the ...study of genetic pathways involved in the different steps of 3D biofilm formation.
biofilm studies have mainly focused on pellicle formation at the air-liquid interface or complex macrocolonies formed on nutritive agar. However, only few studies focus on the genetic features of
submerged biofilm formation and their link with other multicellular models at the air interface. NDmed, an undomesticated
strain isolated from a hospital, has demonstrated the ability to produce highly structured immersed biofilms when compared to strains classically used for studying
biofilms. In this contribution, we have conducted a multi-culturing comparison (between macrocolony, swarming, pellicle, and submerged biofilm) of
multicellular communities using the NDmed strain and mutated derivatives for genes shown to be required for motility and biofilm formation in pellicle and macrocolony models. For the 15 mutated NDmed strains studied, all showed an altered phenotype for at least one of the different culture laboratory assays. Mutation of genes involved in matrix production (i.e.,
,
,
,
) caused a negative impact on all biofilm phenotypes but favored swarming motility on semi-solid surfaces. Mutation of
, a gene coding for an amphiphilic protein, affected the stability of the pellicle at the air-liquid interface with no impact on the submerged biofilm model. Moreover, mutation of
an autolysin gene required for cell separation, had a greater effect on the submerged biofilm model than that formed at aerial level, opposite to the observation for
mutant. In addition,
NDmed with
mutation formed wrinkled macrocolony, less than that formed by the wild type, but was unable to form neither thick pellicle nor structured submerged biofilm. The results are discussed in terms of the relevancy to determine whether genes involved in colony and pellicle formation also govern submerged biofilm formation, by regarding the specificities in each model.
Bacillus subtilis is a widely used bacterial model to decipher biofilm formation, genetic determinants and their regulation. For several years, studies were conducted on colonies or pellicles formed ...at the interface with air, but more recent works showed that non-domesticated strains were able to form thick and structured biofilms on submerged surfaces. Taking advantage of time-lapse confocal laser scanning microscopy, we monitored bacterial colonization on the surface and observed an unexpected biphasic submerged biofilm development. Cells adhering to the surface firstly form elongated chains before being suddenly fragmented and released as free motile cells in the medium. This switching coincided with an oxygen depletion in the well which preceded the formation of the pellicle at the liquid-air interface. Residual bacteria still associated with the solid surface at the bottom of the well started to express matrix genes under anaerobic metabolism to build the typical biofilm protruding structures.
Microbial life abounds on surfaces in both natural and industrial environments, one of which is the food industry. A solid substrate, water and some nutrients are sufficient to allow the construction ...of a microbial fortress, a so-called biofilm. Survival strategies developed by these surface-associated ecosystems are beginning to be deciphered in the context of rudimentary laboratory biofilms. Gelatinous organic matrices consisting of complex mixtures of self-produced biopolymers ensure the cohesion of these biological structures and contribute to their resistance and persistence. Moreover, far from being just simple three-dimensional assemblies of identical cells, biofilms are composed of heterogeneous sub-populations with distinctive behaviours that contribute to their global ecological success. In the clinical field, biofilm-associated infections (BAI) are known to trigger chronic infections that require dedicated therapies. A similar belief emerging in the food industry, where biofilm tolerance to environmental stresses, including cleaning and disinfection/sanitation, can result in the persistence of bacterial pathogens and the recurrent cross-contamination of food products. The present review focuses on the principal mechanisms involved in the formation of biofilms of food-borne pathogens, where biofilm behaviour is driven by its three-dimensional heterogeneity and by species interactions within these biostructures, and we look at some emergent control strategies.
•Ability to form/join microbial biofilms impacts foodborne pathogen tolerance.•Biofilm tolerance to biocides and stress requires the development of new hygienic strategies.•Interspecific interactions influence pathogen fitness/survival in biofilms.•Spatial modelling as a tool to decipher biofilm structure/function relationships.
The Bacillus subtilis strain NDmed was isolated from an endoscope washer-disinfector in a medical environment. NDmed can form complex macrocolonies with highly wrinkled architectural structures on ...solid medium. In static liquid culture, it produces thick pellicles at the interface with air as well as remarkable highly protruding ‘‘beanstalk-like’’ submerged biofilm structures at the solid surface. Since these mucoid submerged structures are hyper-resistant to biocides, NDmed has the ability to protect pathogens embedded in mixed-species biofilms by sheltering them from the action of these agents. Additionally, this non-domesticated and highly biofilm forming strain has the propensity of being genetically manipulated. Due to all these properties, the NDmed strain becomes a valuable model for the study of B. subtilis biofilms. This review focuses on several studies performed with NDmed that have highlighted the sophisticated genetic dynamics at play during B. subtilis biofilm formation. Further studies in project using modern molecular tools of advanced technologies with this strain, will allow to deepen our knowledge on the emerging properties of multicellular bacterial life.
Bacillus subtilis has been extensively used to study the molecular mechanisms behind the development and dispersal of surface bacterial multicellular communities. Well-structured spatially organised ...communities (colony, pellicle, and submerged biofilm) share some similarities, but also display considerable differences at the structural, chemical and biological levels. To unveil the spatial transcriptional heterogeneity between the different communities, we analysed by RNA-seq nine spatio-physiological populations selected from planktonic and spatially organised communities. This led to a global landscape characterisation of gene expression profiles uncovering genes specifically expressed in each compartmental population. From this mesoscale analysis and using fluorescent transcriptional reporter fusions, 17 genes were selected and their patterns of expression reported at single cell scale with time-lapse confocal laser scanning microscopy (CLSM). Derived kymographs allowed to emphasise spectacular mosaic gene expression patterns within a biofilm. A special emphasis on oppositely regulated carbon metabolism genes (gapA and gapB) permitted to pinpoint the coexistence of spatially segregated bacteria under either glycolytic or gluconeogenic regime in a same biofilm population. Altogether, this study gives novel insights on the development and dispersal of B. subtilis surface-associated communities.
This work shows that the combination of two-dimensional (2D) and three-dimensional (3D) analyses of images acquired by confocal laser scanning microscopy facilitates the quantitative spatiotemporal ...characterization of architectures formed by Listeria monocytogenes biofilms. In particular, the analysis of structural features such as maximum thickness, biovolume, areal porosity and maximum diffusion distance allowed elucidating differences in biofilm formation of three L. monocytogenes strains (L1A1, CECT5873 and CECT4032).
The analysis showed a common sequence for all strains. In the first phase, independent clusters evolve to interconnected clusters and honeycomb-like structures. Flat biofilms characterized the second phase. The structures disappear in the third phase.
Nevertheless, the duration of the phases differed from strain to strain. L1A1 strain exhibited the slowest dynamics and the thickest biofilms while the strain CECT4032 presented the faster dynamics and the thinnest biofilms. Also, the number of dead cells varies significantly from strain to strain.
From the results of the analysis, it can be concluded that 2D parameters are critical to differentiating morphological features while 3D parameters ease the interpretation and comparative study of the different phases during the life cycle of biofilms.
•2D and 3D quantitative image analyses allow characterizing the dynamics of biofilms.•2D parameters identify structures and appearance and location of dead cells.•3D parameters evaluate thickness and biovolume facilitating life cycle studies.•Three Listeria monocytogenes strains follow common patterns during life cycle.•Magnitude and duration of patterns are different: the role of time is critical.
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