Spatially localised expression of the glutamate decarboxylase gadB in Escherichia coli O157:H7 microcolonies in hydrogel matrices.

Functional diversity within isogenic spatially organised bacterial populations has been shown to trigger emergent community properties such as stress tolerance. Considering gadB gene encoding a key glutamate decarboxylase involved in E. coli tolerance to acidic conditions, we investigated its expression in hydrogels mimicking the texture of some structured food matrices (such as minced meat or soft cheese). Taking advantage of confocal laser scanning microscopy combined with a genetically-engineered dual fluorescent reporter system, it was possible to visualise the spatial patterns of bacterial gene expression from in-gel microcolonies. In E. coli O157:H7 microcolonies, gadB showed radically different expression patterns between neutral (pH 7) or acidic (pH 5) hydrogels. Differential spatial expression was determined in acidic hydrogels with a strong expression of gadB at the microcolony periphery. Strikingly, very similar spatial patterns of gadB expression were further observed for E. coli O157:H7 grown in the presence of L. lactis. Considering the ingestion of contaminated foodstuff, survival of E. coli O157:H7 to acidic stomachal stress (pH 2) was significantly increased for bacterial cells grown in microcolonies in acidic hydrogels compared to planktonic cells. These findings have significant implications for risk assessment and public health as they highlight inherent differences in bacterial physiology and virulence between liquid and structured food products. The contrasting characteristics observed underscore the need to consider the distinct challenges posed by these food types, thereby emphasising the importance of tailored risk mitigation strategies.

Growth of Listeria monocytogenes is promoted at low temperature when exogenous unsaturated fatty acids are incorporated in its membrane.

Listeria monocytogenes is a psychrotrophic food-borne pathogen mostly associated with consumption of ready-to eat foods. Due to its high prevalence in raw materials, it is fundamental to control its growth at low temperature. In lipid-rich products, fatty acids can be heterogeneously distributed in the food matrix and can be present in the environment immediately surrounding the pathogen. In this study, we sought to understand the impact of exogenous fatty acids on the growth and membrane physiology of L. monocytogenes according to the temperature and strain. We demonstrate that exogenous unsaturated fatty acids promote the growth of L. monocytogenes at 5 °C but not at 37 °C. The level of growth modifications is dependent upon the strain. At 5 °C, there is high incorporation of unsaturated fatty acids, which decreases the weighted-average melting temperature of membrane fatty acids allowing L. monocytogenes to compensate for the decrease in fluidity caused by the temperature, thus leading to increased growth. In contrast, the incorporation of saturated fatty acids decreases membrane fluidity and prevents growth at 5 °C. This study underlines the absolute necessity to understand better the cold adaptation of L. monocytogenes in lipid-rich foods in order to adjust their shelf-life and guarantee their microbiological safety.

Genetic, physiological, and cellular heterogeneities of bacterial pathogens in food matrices: Consequences for food safety.

In complex food systems, bacteria live in heterogeneous microstructures, and the population displays phenotypic heterogeneities at the single-cell level. This review provides an overview of spatiotemporal drivers of phenotypic heterogeneity of bacterial pathogens in food matrices at three levels. The first level is the genotypic heterogeneity due to the possibility for various strains of a given species to contaminate food, each of them having specific genetic features. Then, physiological heterogeneities are induced within the same strain, due to specific microenvironments and heterogeneous adaptative responses to the food microstructure. The third level of phenotypic heterogeneity is related to cellular heterogeneity of the same strain in a specific microenvironment. Finally, we consider how these phenotypic heterogeneities at the single-cell level could be implemented in mathematical models to predict bacterial behavior and help ensure microbiological food safety.

Spatial organisation of Listeria monocytogenes and Escherichia coli O157:H7 cultivated in gel matrices.

The spatial organisation of bacterial pathogens in food matrices remains poorly understood, but is important in improving risk assessment and preventing infection of consumers by contaminated foodstuff. By combining confocal laser scanning microscopy with genetic fluorescent labelling of Listeria monocytogenes and Escherichia coli O157:H7, it was possible to investigate the spatial patterns of colonisation of both foodborne pathogens in gel matrices, alone or in combination, in various environmental conditions. Increasing low melting point agarose (LMPA) concentrations triggers the transition between a motile single-cell lifestyle to a sessile population spatially organised as microcolonies. The size, number and morphology of microcolonies were highly affected by supplementations in NaCl or lactic acid, two compounds frequently used in food products. Strikingly, single-cell motility was partially restored at higher LMPA concentration in the presence of lactic acid for Escherichia coli O157:H7 and in the presence of NaCl for Listeria monocytogenes. Co-culture of both species in the hydrogel affected pathogen colonisation features; Listeria monocytogenes was better able to colonise gel matrices containing lactic acid in the presence of Escherichia coli O157:H7. Altogether, this investigation provides insights into the spatial distribution and structural dynamics of bacterial pathogens in gel matrices. Potential impacts on food safety are discussed.

Rapid assessment and prediction of the efficiency of two preservatives against S. aureus in cosmetic products using High Content Screening-Confocal Laser Scanning Microscopy.

Most cosmetic products are susceptible to microbiological spoilage due to contaminations that could happen during fabrication or by consumer's repetitive manipulation. The composition of cosmetic products must guarantee efficient bacterial inactivation all along with the product shelf life, which is usually assessed by challenge-tests. A challenge-test consists in inoculating specific bacteria, i.e. Staphylococcus aureus, in the formula and then investigating the bacterial log reduction over time. The main limitation of this method is relative to the time-consuming protocol, where 30 days are needed to obtain results. In this study, we have proposed a rapid alternative method coupling High Content Screening-Confocal Laser Scanning Microscopy (HCS-CLSM), image analysis and modeling. It consists in acquiring real-time S. aureus inactivation kinetics on short-time periods (typically 4h) and in predicting the efficiency of preservatives on longer scale periods (up to 7 days). The action of two preservatives, chlorphenesin and benzyl alcohol, was evaluated against S. aureus at several concentrations in a cosmetic matrix. From these datasets, we compared two secondary models to determine the logarithm reduction time (Dc) for each preservative concentration. Afterwards, we used two primary inactivation models to predict log reductions for up to 7 days and we compared them to observed log reductions. The IQ model better fits datasets and the Q value gives information about the matrix level of interference.

The Role of Biofilms in the Development and Dissemination of Microbial Resistance within the Food Industry.

Biofilms are multicellular sessile microbial communities embedded in hydrated extracellular polymeric matrices. Their formation is common in microbial life in most environments, while those formed on food-processing surfaces are of considerable interest in the context of food hygiene. Biofilm cells express properties that are distinct from planktonic ones, in particular, notorious resistance to antimicrobial agents. Thus, a special feature of biofilms is that, once they have been developed, they are hard to eradicate, even when careful sanitization procedures are regularly applied. A great deal of ongoing research has investigated how and why surface-attached microbial communities develop such resistance, and several mechanisms are to be acknowledged (e.g., heterogeneous metabolic activity, cell adaptive responses, diffusion limitations, genetic and functional diversification, and microbial interactions). The articles contained in this Special Issue deal with biofilms of some important food-related bacteria (including common pathogens such as Salmonella enterica, Listeria monocytogenes, and Staphylococcus aureus, as well as spoilage-causing spore-forming bacilli), providing novel insights on their resistance mechanisms and implications, together with novel methods (e.g., use of protective biofilms formed by beneficial bacteria, enzymes) that could be used to overcome such resistance and thus improve the safety of our food supply and protect public health.

Ferulic Acid and Eugenol Have Different Abilities to Maintain Their Inhibitory Activity Against Listeria monocytogenes in Emulsified Systems.

Natural phenolic compounds are found in large quantities in plants and plant extracts and byproducts from agro-industries. They could be used to ensure food quality and safety due to their antimicrobial properties demonstrated in systems such as culture media. The aim of this study was to evaluate the ability of two natural phenolic compounds, ferulic acid and eugenol, to maintain their inhibitory activity against the growth of Listeria monocytogenes in an oil-in-water emulsion, simulating a complex food system. The minimum inhibitory concentration (MIC) of each phenolic compound was first determined in culture medium, consisting of TS broth and an added emulsifier. Whey proteins and Tween 80 increased the MIC of the antimicrobial activity of eugenol. The MIC of ferulic acid was less affected by the addition of Tween 80. The inhibitory activities of both phenolic compounds were then compared at the same concentration in emulsions and their corresponding aqueous phases by following the growth of L. monocytogenes by plate counting. In emulsified systems, eugenol lost the high inhibitory activity observed in the aqueous phase, whereas ferulic acid retained it. The partition coefficient (logPoct/wat) appears to be a key factor. Eugenol (logPoct/wat = 2.61) dispersed in the aqueous phase intercalates into the bacterial membrane and has high antimicrobial activity. In contrast, it likely preferentially partitions into the lipid droplets when dispersed in an emulsion, consequently losing its antimicrobial activity. As ferulic acid is more hydrophilic, a higher proportion probably remains in the aqueous phase of the emulsion, retaining its antimicrobial activity.

Inhibitory activity of phenolic acids against Listeria monocytogenes: Deciphering the mechanisms of action using three different models.

Phenolic compounds are well known for their antimicrobial activity. They may provide an interesting solution to ensure food safety by preventing the growth of foodborne pathogens while addressing the wishes of consumers for the use of natural preservatives in food and favoring the reuse of agro-industry byproducts. However, their mechanism of action is still not very well understood. Here, we aimed to decipher the complex mechanism of action of eight phenolic acids by decomposing their effects, such as the general effect of the decrease of extracellular pH (γ(pH)) and specific inhibitory effects of the undissociated (γ(Au)) and dissociated (γ(Ad)) forms. We thus developed three different models and applied them to a dataset of Listeria monocytogenes growth rates experimentally obtained in the presence of various concentrations of phenolic acids at several pHs. The model that best fits the dataset was selected for each phenolic acid to explore the potential mechanisms. The results show that the antimicrobial activity is mainly due to the effect of the undissociated forms, except for chlorogenic and gallic acids, for which the antimicrobial activity is mainly due to a decrease in extracellular pH. In addition, the dissociated forms of p-coumaric and ferulic acids show significant inhibitory activity.

Characterization of Bacterial Membrane Fatty Acid Profiles for Biofilm Cells.

When exposed to environmental stresses, bacteria can modulate its fatty acid composition of membrane phospholipids in order to optimize membrane fluidity. Characterization of bacterial membrane fatty acid profiles is thus an interesting indicator of cellular physiological state. The methodology described here aims to improve the recovering of biofilm cells for the characterization of their fatty acid profiles. The saponification reagent is directly applied on the whole biofilm before the removal of cells from the inert surface. In this way, maximum of the cells and their fatty acids can be recovered from the deepest layers of the biofilm.

Impact of Bacterial Membrane Fatty Acid Composition on the Failure of Daptomycin To Kill Staphylococcus aureus.

Daptomycin is a last-resort membrane-targeting lipopeptide approved for the treatment of drug-resistant staphylococcal infections, such as bacteremia and implant-related infections. Although cases of resistance to this antibiotic are rare, increasing numbers of clinical, in vitro, and animal studies report treatment failure, notably against Staphylococcus aureus The aim of this study was to identify the features of daptomycin and its target bacteria that lead to daptomycin treatment failure. We show that daptomycin bactericidal activity against S. aureus varies significantly with the growth state and strain, according to the membrane fatty acid composition. Daptomycin efficacy as an antibiotic relies on its ability to oligomerize within membranes and form pores that subsequently lead to cell death. Our findings ascertain that daptomycin interacts with tolerant bacteria and reaches its membrane target, regardless of its bactericidal activity. However, the final step of pore formation does not occur in cells that are daptomycin tolerant, strongly suggesting that it is incapable of oligomerization. Importantly, membrane fatty acid contents correlated with poor daptomycin bactericidal activity, which could be manipulated by fatty acid addition. In conclusion, daptomycin failure to treat S. aureus is not due to a lack of antibiotic-target interaction, but is driven by its capacity to form pores, which depends on membrane composition. Manipulation of membrane fluidity to restore S. aureus daptomycin bactericidal activity in vivo could open the way to novel antibiotic treatment strategies.

Phenolic compounds can delay the oxidation of polyunsaturated fatty acids and the growth of Listeria monocytogenes: structure-activity relationships.

BACKGROUND: Phenolic compounds present a potential solution to ensure food quality and safety. Indeed, they can limit oxidation reactions and bacterial growth in food products. Although their antioxidant mechanisms of action are well known, their antibacterial ones are less well understood, especially in light of their chemical structures. The aim of this study was first to quantify both aspects of a series of natural phenolic compounds and then link these activities to their chemical structure. RESULTS: We evaluated antioxidant activity by measuring the capacity of phenolic compounds to delay free linoleic acid oxidation caused by the action of a hydrophilic azo-radical initiator (AAPH). We evaluated antibacterial activity by measuring the growth inhibition of Listeria monocytogenes and determining the non-inhibitory and minimum inhibitory concentrations for each compound. Compounds with ortho-diphenolic structures were the best antioxidants, whereas those belonging to the simple phenol category were the best antibacterial compounds. CONCLUSION: The physico-chemical properties of the compounds influenced both activities but not in the same way. The chemical environment of the phenolic group and the presence of delocalization structures are the most important parameters for antioxidant activity, whereas the partition coefficient, logP, is one of the most important factors involved in antibacterial activity. © 2018 Society of Chemical Industry.

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.

The Biofilm Lifestyle Involves an Increase in Bacterial Membrane Saturated Fatty Acids.

Biofilm formation on contact surfaces contributes to persistence of foodborne pathogens all along the food and feed chain. The specific physiological features of bacterial cells embedded in biofilms contribute to their high tolerance to environmental stresses, including the action of antimicrobial compounds. As membrane lipid adaptation is a vital facet of bacterial response when cells are submitted to harsh or unstable conditions, we focused here on membrane fatty acid composition of biofilm cells as compared to their free-growing counterparts. Pathogenic bacteria (Staphylococcus aureus, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella Typhimurium) were cultivated in planktonic or biofilm states and membrane fatty acid analyses were performed on whole cells in both conditions. The percentage of saturated fatty acids increases in biofilm cells in all cases, with a concomitant decrease of branched-chain fatty acids for Gram-positive bacteria, or with a decrease in the sum of other fatty acids for Gram-negative bacteria. We propose that increased membrane saturation in biofilm cells is an adaptive stress response that allows bacteria to limit exchanges, save energy, and survive. Reprogramming of membrane fluidity in biofilm cells might explain specific biofilm behavior including bacterial recalcitrance to biocide action.

Hydrosol of Thymbra capitata Is a Highly Efficient Biocide against Salmonella enterica Serovar Typhimurium Biofilms.

UNLABELLED: Salmonella is recognized as one of the most significant enteric foodborne bacterial pathogens. In recent years, the resistance of pathogens to biocides and other environmental stresses, especially when they are embedded in biofilm structures, has led to the search for and development of novel antimicrobial strategies capable of displaying both high efficiency and safety. In this direction, the aims of the present work were to evaluate the antimicrobial activity of hydrosol of the Mediterranean spice Thymbra capitata against both planktonic and biofilm cells of Salmonella enterica serovar Typhimurium and to compare its action with that of benzalkonium chloride (BC), a commonly used industrial biocide. In order to achieve this, the disinfectant activity following 6-min treatments was comparatively evaluated for both disinfectants by calculating the concentrations needed to achieve the same log reductions against both types of cells. Their bactericidal effect against biofilm cells was also comparatively determined by in situ and real-time visualization of cell inactivation through the use of time-lapse confocal laser scanning microscopy (CLSM). Interestingly, results revealed that hydrosol was almost equally effective against biofilms and planktonic cells, whereas a 200-times-higher concentration of BC was needed to achieve the same effect against biofilm compared to planktonic cells. Similarly, time-lapse CLSM revealed the significant advantage of the hydrosol to easily penetrate within the biofilm structure and quickly kill the cells, despite the three-dimensional (3D) structure of Salmonella biofilm. IMPORTANCE: The results of this paper highlight the significant antimicrobial action of a natural compound, hydrosol of Thymbra capitata, against both planktonic and biofilm cells of a common foodborne pathogen. Hydrosol has numerous advantages as a disinfectant of food-contact surfaces. It is an aqueous solution which can easily be rinsed out from surfaces, it does not have the strong smell of the essential oil (EO) and it is a byproduct of the EO distillation procedure without any industrial application until now. Consequently, hydrosol obviously could be of great value to combat biofilms and thus to improve product safety not only for the food industries but probably also for many other industries which experience biofilm-related problems.

Adaptation of the wine bacterium Oenococcus oeni to ethanol stress: role of the small heat shock protein Lo18 in membrane integrity.

Malolactic fermentation in wine is often carried out by Oenococcus oeni. Wine is a stressful environment for bacteria because ethanol is a toxic compound that impairs the integrity of bacterial membranes. The small heat shock protein (sHsp) Lo18 is an essential actor of the stress response in O. oeni. Lo18 prevents the thermal aggregation of proteins and plays a crucial role in membrane quality control. Here, we investigated the interaction between Lo18 and four types of liposomes: one was prepared from O. oeni grown under optimal growth conditions (here, control liposomes), one was prepared from O. oeni grown in the presence of 8% ethanol (here, ethanol liposomes), one was prepared from synthetic phospholipids, and one was prepared from phospholipids from Bacillus subtilis or Lactococcus lactis. We observed the strongest interaction between Lo18 and control liposomes. The lipid binding activity of Lo18 required the dissociation of oligomeric structures into dimers. Protein protection experiments carried out in the presence of the liposomes from O. oeni suggested that Lo18 had a higher affinity for control liposomes than for a model protein. In anisotropy experiments, we mimicked ethanol action by temperature-dependent fluidization of the liposomes. Results suggest that the principal determinant of Lo18-membrane interaction is lipid bilayer phase behavior rather than phospholipid composition. We suggest a model to describe the ethanol adaptation of O. oeni. This model highlights the dual role of Lo18 in the protection of proteins from aggregation and membrane stabilization and suggests how modifications of phospholipid content may be a key factor determining the balance between these two functions.

Plant-derived compounds as natural antimicrobials to control paper mill biofilms.

Biofilms can cause severe problems in industrial paper mills, particularly of economic and technological types (clogging of filters, sheet breaks or holes in the paper, machine breakdowns, etc.). We present here some promising results on the use of essential oil compounds to control these biofilms. Biofilms were grown on stainless-steel coupons with a microbial white water consortium sampled from an industrial paper mill. Five essential oil compounds were screened initially in the laboratory in terms of their antimicrobial activity against planktonic cells and biofilms. The three most active compounds were selected and then tested in different combinations. The combination finally selected was tested at the pilot scale to confirm its efficiency under realistic conditions. All the compounds tested were as active against biofilms as they were against planktonic cells. The most active compounds were thymol, carvacrol, and eugenol, and the most efficient combination was thymol-carvacrol. At a pilot scale, with six injections a day, 10 mM carvacrol alone prevented biocontamination for at least 10 days, and a 1 mM thymol-carvacrol combination enabled a 67 % reduction in biofilm dry matter after 11 days. The use of green antimicrobials could constitute a very promising alternative or supplement to the treatments currently applied to limit biofilm formation in the environment of paper mill machines.

Biofilms of a Bacillus subtilis hospital isolate protect Staphylococcus aureus from biocide action.

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.

Deciphering biofilm structure and reactivity by multiscale time-resolved fluorescence analysis.

In natural, industrial and medical environments, microorganisms mainly live as structured and organised matrix-encased communities known as biofilms. In these communities, microorganisms demonstrate coordinated behaviour and are able to perform specific functions such as dramatic resistance to antimicrobials, which potentially lead to major public health and industrial problems. It is now recognised that the appearance of such specific biofilm functions is intimately related to the three-dimensional organisation of the biological edifice, and results from multifactorial processes. During the last decade, the emergence of innovative optical microscopy techniques such as confocal laser scanning microscopy in combination with fluorescent labelling has radically transformed imaging in biofilm research, giving the possibility to investigate non-invasively the dynamic mechanisms of formation and reactivity of these biostructures. In this chapter, we discuss the contribution of fluorescence analysis and imaging to the study at different timescales of various processes: biofilm development (hours to days), antimicrobial reactivity within the three-dimensional structure (minutes to hours) or molecular diffusion/reaction phenomena (pico- to milliseconds).

The spatial architecture of Bacillus subtilis biofilms deciphered using a surface-associated model and in situ imaging.

The formation of multicellular communities known as biofilms is the part of bacterial life cycle in which bacteria display cooperative behaviour and differentiated phenotypes leading to specific functions. Bacillus subtilis is a Gram-positive bacterium that has served for a decade as a model to study the molecular pathways that control biofilm formation. Most of the data on B. subtilis biofilms have come from studies on the formation of pellicles at the air-liquid interface, or on the complex macrocolonies that develop on semi-solid nutritive agar. Here, using confocal laser scanning microcopy, we show that B. subtilis strains of different origins are capable of forming biofilms on immersed surfaces with dramatically protruding "beanstalk-like" structures with certain strains. Indeed, these structures can reach a height of more than 300 µm with one undomesticated strain from a medical environment. Using 14 GFP-labeled mutants previously described as affecting pellicle or complex colony formation, we have identified four genes whose inactivation significantly impeded immersed biofilm development, and one mutation triggering hyperbiofilm formation. We also identified mutations causing the three-dimensional architecture of the biofilm to be altered. Taken together, our results reveal that B. subtilis is able to form specific biofilm features on immersed surfaces, and that the development of these multicellular surface-associated communities involves regulation pathways that are common to those governing the formation of pellicle and/or complex colonies, and also some specific mechanisms. Finally, we propose the submerged surface-associated biofilm as another relevant model for the study of B. subtilis multicellular communities.

Induction of fatty acid composition modifications and tolerance to biocides in Salmonella enterica serovar Typhimurium by plant-derived terpenes.

To enhance food safety and stability, the food industry tends to use natural antimicrobials such as plant-derived compounds as an attractive alternative to chemical preservatives. Nonetheless, caution must be exercised in light of the potential for bacterial adaptation to these molecules, a phenomenon previously observed with other antimicrobials. The aim of this study was to characterize the adaptation of Salmonella enterica serovar Typhimurium to sublethal concentrations of four terpenes extracted from aromatic plants: thymol, carvacrol, citral, and eugenol, or combinations thereof. Bacterial adaptation in these conditions was demonstrated by changes in membrane fatty acid composition showing (i) limitation of the cyclization of unsaturated fatty acids to cyclopropane fatty acids when cells entered the stationary phase and (ii) bacterial membrane saturation. Furthermore, we demonstrated an increased cell resistance to the bactericidal activity of two biocides (peracetic acid and didecyl dimethyl ammonium bromide). The implications of membrane modifications in terms of hindering the penetration of antimicrobials through the bacterial membrane are discussed.