Polyhexamethylene biguanide promotes adaptive cross-resistance to gentamicin in Escherichia coli biofilms.

Antimicrobial resistance is a critical public health issue that requires a thorough understanding of the factors that influence the selection and spread of antibiotic-resistant bacteria. Biocides, which are widely used in cleaning and disinfection procedures in a variety of settings, may contribute to this resistance by inducing similar defense mechanisms in bacteria against both biocides and antibiotics. However, the strategies used by bacteria to adapt and develop cross-resistance remain poorly understood, particularly within biofilms -a widespread bacterial habitat that significantly influences bacterial tolerance and adaptive strategies. Using a combination of adaptive laboratory evolution experiments, genomic and RT-qPCR analyses, and biofilm structural characterization using confocal microscopy, we investigated in this study how Escherichia coli biofilms adapted after 28 days of exposure to three biocidal active substances and the effects on cross-resistance to antibiotics. Interestingly, polyhexamethylene biguanide (PHMB) exposure led to an increase of gentamicin resistance (GenR) phenotypes in biofilms formed by most of the seven E. coli strains tested. Nevertheless, most variants that emerged under biocidal conditions did not retain the GenR phenotype after removal of antimicrobial stress, suggesting a transient adaptation (adaptive resistance). The whole genome sequencing of variants with stable GenR phenotypes revealed recurrent mutations in genes associated with cellular respiration, including cytochrome oxidase (cydA, cyoC) and ATP synthase (atpG). RT-qPCR analysis revealed an induction of gene expression associated with biofilm matrix production (especially curli synthesis), stress responses, active and passive transport and cell respiration during PHMB exposure, providing insight into potential physiological responses associated with adaptive crossresistance. In addition, confocal laser scanning microscopy (CLSM) observations demonstrated a global effect of PHMB on biofilm architectures and compositions formed by most E. coli strains, with the appearance of dense cellular clusters after a 24h-exposure. In conclusion, our results showed that the PHMB exposure stimulated the emergence of an adaptive cross-resistance to gentamicin in biofilms, likely induced through the activation of physiological responses and biofilm structural modulations altering gradients and microenvironmental conditions in the biological edifice.

Bacillus subtilis NDmed, a model strain for biofilm genetic studies.

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.

Biofilms as protective cocoons against biocides: from bacterial adaptation to One Health issues.

Bacteria in the food chain mostly live in communities associated with surfaces known as biofilms, which confer specific survival and adaptive abilities. In such communities, the bacteria mostly exhibit higher tolerance to external stress, and their recurrent exposure along the food chain to biocides used during cleaning and disinfection procedures raises concern about the adaptation routes they develop, both at single-cell and communal levels. In recent years, an increasing number of research subjects have focused on understanding the specific features of biofilms that enable bacterial populations to adapt to biocide exposure within a 'protective cocoon'. The first part of this review concentrates on the diversity of adaptive strategies, including structural modulation of these biofilms, physiological response or the acquisition of genetic resistance. The second part discusses the possible side effects of biofilm adaptation to biocides on antimicrobial cross-resistance, virulence and colonization features from a One Health perspective.

Migration of surface-associated microbial communities in spaceflight habitats.

Astronauts are spending longer periods locked up in ships or stations for scientific and exploration spatial missions. The International Space Station (ISS) has been inhabited continuously for more than 20 years and the duration of space stays by crews could lengthen with the objectives of human presence on the moon and Mars. If the environment of these space habitats is designed for the comfort of astronauts, it is also conducive to other forms of life such as embarked microorganisms. The latter, most often associated with surfaces in the form of biofilm, have been implicated in significant degradation of the functionality of pieces of equipment in space habitats. The most recent research suggests that microgravity could increase the persistence, resistance and virulence of pathogenic microorganisms detected in these communities, endangering the health of astronauts and potentially jeopardizing long-duration manned missions. In this review, we describe the mechanisms and dynamics of installation and propagation of these microbial communities associated with surfaces (spatial migration), as well as long-term processes of adaptation and evolution in these extreme environments (phenotypic and genetic migration), with special reference to human health. We also discuss the means of control envisaged to allow a lasting cohabitation between these vibrant microscopic passengers and the astronauts.

Presence and Depletion of Sulfadiazine, Trimethoprim, and Oxytetracycline into Feathers of Treated Broiler Chickens and Impact on Antibiotic-Resistant Bacteria.

The valorization of poultry byproducts, like feathers (processed to feather meal), in animal feed could contribute to the presence of veterinary drugs, including antibiotics. An animal study was carried out to study the fate of sulfadiazine, trimethoprim, and oxytetracycline in feathers, plasma, and droppings of broiler chickens. Cage and floor housing, different from current farm practices, were studied. Samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A longer presence of antibiotics was observed in feathers compared to plasma, with sulfadiazine being present the most. The internal presence (via blood) and the external presence (via droppings) of antibiotics in/on feathers were shown. Analysis of Escherichia coli populations, from droppings and feathers, highlighted that resistant bacteria could be transferred from droppings to feathers in floor-housed animals. The overall results suggest that feathers are a potential reservoir of antimicrobial residues and could contribute to the selection of antibiotic-resistant bacteria in the environment, animals, and humans.

FepR as a Central Genetic Target in the Adaptation to Quaternary Ammonium Compounds and Cross-Resistance to Ciprofloxacin in Listeria monocytogenes.

The foodborne pathogen, Listeria monocytogenes, (Lm), frequently undergoes selection pressure associated with the extensive use of disinfectants, such as quaternary ammonium compounds, which are widely used in food processing plants. The repeated exposure to sub-inhibitory biocide concentrations can induce increased tolerance to these compounds, but can also trigger the development of antibiotic resistance, and both increase the risk of food contamination and persistence in food production environments. Although the acquisition of genes can explain biocide tolerance, the genetic mechanisms underlying the adaptive cross-resistance to antibiotics remain unclear. We previously showed that repeated exposure to benzalkonium chloride (BC) and didecyldimethyl ammonium chloride (DDAC) led to reduced susceptibility to ciprofloxacin in Lm strains from diverse sources. Here, we compared the genomes of 16 biocide-adapted and 10 parental strains to identify the molecular mechanisms of fluoroquinolone cross-resistance. A core genome SNP analysis identified various mutations in the transcriptional regulator fepR (lmo2088) for 94% of the adapted strains and mutations in other effectors at a lower frequency. FepR is a local repressor of the MATE fluoroquinolone efflux pump FepA. The impact of the mutations on the structure and function of the protein was assessed by performing in silico prediction and protein homology modeling. Our results show that 75% of the missense mutations observed in fepR are located in the HTH domain of the protein, within the DNA interaction site. These mutations are predicted to reduce the activity of the regulator, leading to the overexpression of the efflux pump responsible for the ciprofloxacin-enhanced resistance.

Genomic elements located in the accessory repertoire drive the adaptation to biocides in Listeria monocytogenes strains from different ecological niches.

In response to the massive use of biocides for controlling Listeria monocytogenes (hereafter Lm) contaminations along the food chain, strains showing biocide tolerance emerged. Here, accessory genomic elements were associated with biocide tolerance through pangenome-wide associations performed on 197 Lm strains from different lineages, ecological, geographical and temporal origins. Mobile elements, including prophage-related loci, the Tn6188_qacH transposon and pLMST6_emrC plasmid, were widespread across lineage I and II food strains and associated with tolerance to benzalkonium-chloride (BC), a quaternary ammonium compound (QAC) widely used in food processing. The pLMST6_emrC was also associated with tolerance to another QAC, the didecyldimethylammonium-chloride, displaying a pleiotropic effect. While no associations were detected for chemically reactive biocides (alcohols and chlorines), genes encoding for cell-surface proteins were associated with BC or polymeric biguanide tolerance. The latter was restricted to lineage I strains from animal and the environment. In conclusion, different genetic markers, with polygenic nature or not, appear to have driven the Lm adaptation to biocide, especially in food strains but also from animal and the environment. These markers could aid to monitor and predict the spread of biocide tolerant Lm genotypes across different ecological niches, finally reducing the risk of such strains in food industrial settings.

A European-wide dataset to uncover adaptive traits of Listeria monocytogenes to diverse ecological niches.

Listeria monocytogenes (Lm) is a ubiquitous bacterium that causes listeriosis, a serious foodborne illness. In the nature-to-human transmission route, Lm can prosper in various ecological niches. Soil and decaying organic matter are its primary reservoirs. Certain clonal complexes (CCs) are over-represented in food production and represent a challenge to food safety. To gain new understanding of Lm adaptation mechanisms in food, the genetic background of strains found in animals and environment should be investigated in comparison to that of food strains. Twenty-one partners, including food, environment, veterinary and public health laboratories, constructed a dataset of 1484 genomes originating from Lm strains collected in 19 European countries. This dataset encompasses a large number of CCs occurring worldwide, covers many diverse habitats and is balanced between ecological compartments and geographic regions. The dataset presented here will contribute to improve our understanding of Lm ecology and should aid in the surveillance of Lm. This dataset provides a basis for the discovery of the genetic traits underlying Lm adaptation to different ecological niches.

The coordinated population redistribution between Bacillus subtilis submerged biofilm and liquid-air pellicle.

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 Biofilms: Structural Plasticity and Emerging Properties.

Microbial biofilms are found everywhere and can be either beneficial or detrimental, as they are involved in crucial ecological processes and in severe chronic infections. The functional properties of biofilms are closely related to their three-dimensional (3D) structure, and the ability of microorganisms to collectively and dynamically shape the community spatial organization in response to stresses in such biological edifices. A large number of works have shown a relationship between the modulation of the spatial organization and ecological interactions in biofilms in response to environmental fluctuations, as well as their emerging properties essential for nutrient cycling and bioremediation processes in natural environments. On the contrary, numerous studies have emphasized the role of structural rearrangements and matrix production in the increased tolerance of bacteria in biofilms toward antimicrobials. In these last few years, the development of innovative approaches, relying on recent technological advances in imaging, computing capacity, and other analytical tools, has led to the production of original data that have improved our understanding of this close relationship. However, it has also highlighted the need to delve deeper into the study of cell behavior in such complex communities during 3D structure development and maturation- from a single-cell to a multicellular scale- to better control or harness positive and negative impacts of biofilms. For this Special Issue, the interplay between biofilm emerging properties and their 3D spatial organization considering different models, from single bacteria to complex environmental communities, and various environments, from natural ecosystems to industrial and medical settings are addressed.

Selection of a Gentamicin-Resistant Variant Following Polyhexamethylene Biguanide (PHMB) Exposure in Escherichia coli Biofilms.

Antibiotic resistance is one of the most important issues facing modern medicine. Some biocides have demonstrated the potential of selecting resistance to antibiotics in bacteria, but data are still very scarce and it is important to better identify the molecules concerned and the underlying mechanisms. This study aimed to assess the potential of polyhexamethylene biguanide (PHMB), a widely used biocide in a variety of sectors, to select antibiotic resistance in Escherichia coli grown in biofilms. Biofilms were grown on inox coupons and then exposed daily to sublethal concentrations of PHMB over 10 days. Antibiotic-resistant variants were then isolated and characterized phenotypically and genotypically to identify the mechanisms of resistance. Repeated exposure to PHMB led to the selection of an E. coli variant (Ec04m1) with stable resistance to gentamycin (8-fold increase in minimum inhibitory concentration (MIC) compared to the parental strain. This was also associated with a significant decrease in the growth rate in the variant. Sequencing and comparison of the parental strain and Ec04m1 whole genomes revealed a nonsense mutation in the aceE gene in the variant. This gene encodes the pyruvate dehydrogenase E1 component of the pyruvate dehydrogenase (PDH) complex, which catalyzes the conversion of pyruvate to acetyl-CoA and CO2. A growth experiment in the presence of acetate confirmed the role of this mutation in a decreased susceptibility to both PHMB and gentamicin (GEN) in the variant. This work highlights the potential of PHMB to select resistance to antibiotics in bacteria, and that enzymes of central metabolic pathways should be considered as a potential target in adaptation strategies, leading to cross-resistance toward biocides and antibiotics in bacteria.

Comparison of the Genetic Features Involved in Bacillus subtilis Biofilm Formation Using Multi-Culturing Approaches.

Surface-associated multicellular assemblage is an important bacterial trait to withstand harsh environmental conditions. Bacillus subtilis 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. B. subtilis 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 B. subtilis submerged biofilm formation and their link with other multicellular models at the air interface. NDmed, an undomesticated B. subtilis strain isolated from a hospital, has demonstrated the ability to produce highly structured immersed biofilms when compared to strains classically used for studying B. subtilis biofilms. In this contribution, we have conducted a multi-culturing comparison (between macrocolony, swarming, pellicle, and submerged biofilm) of B. subtilis 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., tasA, epsA-O, cap, ypqP) caused a negative impact on all biofilm phenotypes but favored swarming motility on semi-solid surfaces. Mutation of bslA, 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 lytF, 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 lytABC mutant. In addition, B. subtilis NDmed with sinR 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.

Exposure to Quaternary Ammonium Compounds Selects Resistance to Ciprofloxacin in Listeria monocytogenes.

In this contribution, the antimicrobial susceptibility toward 11 antibiotics and four biocides of a panel of 205 Listeria monocytogenes (Lm) strains isolated from different ecological niches (i.e., food, animals and natural environment) was evaluated. The impact of exposure to biocides on the antibiotic susceptibilities of Lm was also investigated. Lm strains isolated from food exhibited overall a lower susceptibility (higher minimal inhibitory concentrations, MIC) for ammonium quaternary compounds (QACs) and peracetic acid (PAC) than strains isolated from animals and natural environments. Conversely, the ecological origins of Lm strains did not significantly affect their susceptibilities towards antibiotics. Interestingly, repeated exposure to QACs recurrently led to a decrease in susceptibility toward ciprofloxacin (CIP), a fluoroquinolone antibiotic, largely used in human medicine. Moreover, these lower levels of susceptibility to CIP remained stable in most Lm strains even after subcultures without biocide selection pressure, suggesting an adaptation involving modifications at the genetic level. Results underlined the ability of Lm to adapt to biocides, especially QACs, and the potential link between this adaptation and the selection of resistance toward critical antibiotics such as ciprofloxacin. These data support a potential role of the extensive use of QACs from "farm to fork" in the selection of biocide and antibiotic resistance in pathogenic bacteria such as Lm.

Emergence of a Synergistic Diversity as a Response to Competition in Pseudomonas putida Biofilms.

Genetic diversification through the emergence of variants is one of the known mechanisms enabling the adaptation of bacterial communities. We focused in this work on the adaptation of the model strain Pseudomonas putida KT2440 in association with another P. putida strain (PCL1480) recently isolated from soil to investigate the potential role of bacterial interactions in the diversification process. On the basis of colony morphology, three variants of P. putida KT2440 were obtained from co-culture after 168 h of growth whereas no variant was identified from the axenic KT2440 biofilm. The variants exhibited distinct phenotypes and produced biofilms with specific architecture in comparison with the ancestor. The variants better competed with the P. putida PCL1480 strain in the dual-strain biofilms after 24 h of co-culture in comparison with the ancestor. Moreover, the synergistic interaction of KT2440 ancestor and the variants led to an improved biofilm production and to higher competitive ability versus the PCL1480 strain, highlighting the key role of diversification in the adaptation of P. putida KT2440 in the mixed community. Whole genome sequencing revealed mutations in polysaccharides biosynthesis protein, membrane transporter, or lipoprotein signal peptidase genes in variants.

Impact of cleaning and disinfection procedures on microbial ecology and Salmonella antimicrobial resistance in a pig slaughterhouse.

To guarantee food safety, a better deciphering of ecology and adaptation strategies of bacterial pathogens such as Salmonella in food environments is crucial. The role of food processing conditions such as cleaning and disinfection procedures on antimicrobial resistance emergence should especially be investigated. In this work, the prevalence and antimicrobial resistance of Salmonella and the microbial ecology of associated surfaces communities were investigated in a pig slaughterhouse before and after cleaning and disinfection procedures. Salmonella were detected in 67% of samples and isolates characterization revealed the presence of 15 PFGE-patterns belonging to five serotypes: S.4,5,12:i:-, Rissen, Typhimurium, Infantis and Derby. Resistance to ampicillin, sulfamethoxazole, tetracycline and/or chloramphenicol was detected depending on serotypes. 16S rRNA-based bacterial diversity analyses showed that Salmonella surface associated communities were highly dominated by the Moraxellaceae family with a clear site-specific composition suggesting a persistent colonization of the pig slaughterhouse. Cleaning and disinfection procedures did not lead to a modification of Salmonella susceptibility to antimicrobials in this short-term study but they tended to significantly reduce bacterial diversity and favored some genera such as Rothia and Psychrobacter. Such data participate to the construction of a comprehensive view of Salmonella ecology and antimicrobial resistance emergence in food environments in relation with cleaning and disinfection procedures.

Biorefinery for heterogeneous organic waste using microbial electrochemical technology.

Environmental biorefineries aim to produce biofuels and platform biomolecules from organic waste. To this end, microbial electrochemical technologies theoretically allow controlled microbial electrosynthesis (MES) of organic molecules to be coupled to oxidation of waste. Here, we provide a first proof of concept and a robust operation strategy for MES in a microbial electrolysis cell (MEC) fed with biowaste hydrolysates. This strategy allowed stable operation at 5 A/m2 for more than three months in a labscale reactor. We report a two to four-fold reduction in power consumption compared to microbial electrosynthesis with water oxidation at the anode. The bioelectrochemical characterizations of the cells were used to compute energy and matter balances for biorefinery scenarios in which anaerobic digestion (AD) provides the electricity and CO2 required for the MEC. Calculations shows that up to 22% of electrons (or COD) from waste may be converted to organic products in the AD-MEC process.

Exploring Foodborne Pathogen Ecology and Antimicrobial Resistance in the Light of Shotgun Metagenomics.

In this chapter, applications of shotgun metagenomics for taxonomic profiling and functional investigation of food microbial communities with a focus on antimicrobial resistance (AMR) were overviewed in the light of last data in the field. Potentialities of metagenomic approach, along with the challenges encountered for a wider and routinely use in food safety was discussed.

Spatial Organization Plasticity as an Adaptive Driver of Surface Microbial Communities.

Biofilms are dynamic habitats which constantly evolve in response to environmental fluctuations and thereby constitute remarkable survival strategies for microorganisms. The modulation of biofilm functional properties is largely governed by the active remodeling of their three-dimensional structure and involves an arsenal of microbial self-produced components and interconnected mechanisms. The production of matrix components, the spatial reorganization of ecological interactions, the generation of physiological heterogeneity, the regulation of motility, the production of actives enzymes are for instance some of the processes enabling such spatial organization plasticity. In this contribution, we discussed the foundations of architectural plasticity as an adaptive driver of biofilms through the review of the different microbial strategies involved. Moreover, the possibility to harness such characteristics to sculpt biofilm structure as an attractive approach to control their functional properties, whether beneficial or deleterious, is also discussed.

Whole Proteome Analyses on Ruminiclostridium cellulolyticum Show a Modulation of the Cellulolysis Machinery in Response to Cellulosic Materials with Subtle Differences in Chemical and Structural Properties.

Lignocellulosic materials from municipal solid waste emerge as attractive resources for anaerobic digestion biorefinery. To increase the knowledge required for establishing efficient bioprocesses, dynamics of batch fermentation by the cellulolytic bacterium Ruminiclostridium cellulolyticum were compared using three cellulosic materials, paper handkerchief, cotton discs and Whatman filter paper. Fermentation of paper handkerchief occurred the fastest and resulted in a specific metabolic profile: it resulted in the lowest acetate-to-lactate and acetate-to-ethanol ratios. By shotgun proteomic analyses of paper handkerchief and Whatman paper incubations, 151 proteins with significantly different levels were detected, including 20 of the 65 cellulosomal components, 8 non-cellulosomal CAZymes and 44 distinct extracytoplasmic proteins. Consistent with the specific metabolic profile observed, many enzymes from the central carbon catabolic pathways had higher levels in paper handkerchief incubations. Among the quantified CAZymes and cellulosomal components, 10 endoglucanases mainly from the GH9 families and 7 other cellulosomal subunits had lower levels in paper handkerchief incubations. An in-depth characterization of the materials used showed that the lower levels of endoglucanases in paper handkerchief incubations could hypothetically result from its lower crystallinity index (50%) and degree of polymerization (970). By contrast, the higher hemicellulose rate in paper handkerchief (13.87%) did not result in the enhanced expression of enzyme with xylanase as primary activity, including enzymes from the "xyl-doc" cluster. It suggests the absence, in this material, of molecular structures that specifically lead to xylanase induction. The integrated approach developed in this work shows that subtle differences among cellulosic materials regarding chemical and structural characteristics have significant effects on expressed bacterial functions, in particular the cellulolysis machinery, resulting in different metabolic patterns and degradation dynamics.