Influence of temperature on regulation of key virulence and stress response genes in Listeria monocytogenes biofilms.

Temperature is a major determinant of Listeria (L.) monocytogenes adherence and biofilm formation on abiotic surfaces. However, its role on gene regulation of L. monocytogenes mature biofilms has not been investigated. In the present study, we aimed to evaluate the impact of temperature up- and down-shift on L. monocytogenes biofilms gene transcription. L. monocytogenes strain EGD-e biofilms were first developed on stainless steel surfaces in Brain Heart Infusion broth at 20 °C for 48 h. Then, nutrient broth was renewed, and mature biofilms were exposed to 10 °C, 20 °C or 37 °C for 24 h. Biofilm cells were harvested and RNA levels of plcA, prfA, hly, mpl, plcB, sigB, bapL, fbpA, fbpB, lmo2178, lmo0880, lmo0160, lmo1115, lmo 2089, lmo2576, lmo0159 and lmo0627 were evaluated by quantitative RT-PCR. The results revealed an over-expression of all genes tested in biofilm cells compared to planktonic cells. When biofilms were further allowed to proliferate at 20 °C for 24 h, the transcription levels of key virulence, stress response and putative binding proteins genes plcA, sigB, fbpA, fbpB, lmo1115, lmo0880 and lmo2089 decreased. A temperature-dependent transcription for sigB, plcA, hly, and lmo2089 genes was observed after biofilm proliferation at 10 °C or 37 °C. Our findings suggest that temperature differentially affects gene regulation of L. monocytogenes mature biofilms, thus modulating attributes such as virulence, stress response and pathogenesis.

Genomic and Metabolic Characteristics of the Pathogenicity in Pseudomonas aeruginosa.

In recent years, the effectiveness of antimicrobials in the treatment of Pseudomonas aeruginosa infections has gradually decreased. This pathogen can be observed in several clinical cases, such as pneumonia, urinary tract infections, sepsis, in immunocompromised hosts, such as neutropenic cancer, burns, and AIDS patients. Furthermore, Pseudomonas aeruginosa causes diseases in both livestock and pets. The highly flexible and versatile genome of P. aeruginosa allows it to have a high rate of pathogenicity. The numerous secreted virulence factors, resulting from its numerous secretion systems, the multi-resistance to different classes of antibiotics, and the ability to produce biofilms are pathogenicity factors that cause numerous problems in the fight against P. aeruginosa infections and that must be better understood for an effective treatment. Infections by P. aeruginosa represent, therefore, a major health problem and, as resistance genes can be disseminated between the microbiotas associated with humans, animals, and the environment, this issue needs be addressed on the basis of an One Health approach. This review intends to bring together and describe in detail the molecular and metabolic pathways in P. aeruginosa's pathogenesis, to contribute for the development of a more targeted therapy against this pathogen.

Desiccation: An environmental and food industry stress that bacteria commonly face.

Water is essential for all living organisms, for animals as well as for plants and micro-organisms. For these latter, the presence of water or a humid environment with a high air relative humidity (RH) is necessary for their survival and growth. Thus, variations in the availability of water or in the air relative humidity constitute widespread environmental stresses which challenge microorganisms, and especially bacteria. Indeed, in their direct environment, bacteria are often faced with conditions that remove cell-bound water through air-drying of the atmosphere. Bacterial cells are subject to daily or seasonal environmental variations, sometimes going through periods of severe desiccation. This is also the case in the food industry, where air dehumidification treatments are applied after the daily cleaning-disinfection procedures. In plants producing low-water activity products, it is also usual to significantly reduce or eliminate water usage. Periodic desiccation exposure affects bacteria viability and so they require strategies to persist. Negative effects of desiccation are wide ranging and include direct cellular damage but also changes in the biochemical and biophysical properties of cells for which planktonic cells are more exposed than cells in biofilm. Understanding the mechanisms of desiccation adaptation and tolerance has a biological and biotechnological interest. This review gives an overview of the factors influencing desiccation tolerance and the biological mechanisms involved in this stress response.

Subproteomic signature comparison of in vitro selected fluoroquinolone resistance and ciprofloxacin stress in Salmonella Typhimurium DT104B.

BACKGROUND: Fluoroquinolone resistance in nontyphoidal Salmonella is a situation of serious and international concern, particularly in S. Typhimurium DT104B multiresistant strains. Although known to be multifactorial, fluoroquinolone resistance is still far from a complete understanding. METHODS: Subproteome changes between an experimentally selected fluoroquinolone-resistant strain (Se6-M) and its parent strain (Se6), and also in Se6-M under ciprofloxacin (CIP) stress, were evaluated in order to give new insights into the mechanisms involved. Proteomes were compared at the intracellular and membrane levels by a 2-DE~LC-MS/MS and a shotgun LC-MS/MS approach, respectively. RESULTS: In total, 35 differentially abundant proteins were identified when comparing Se6 with Se6-M (25 more abundant in Se6 and 10 more abundant in Se6-M) and 82 were identified between Se6-M and Se6-M+CIP (51 more abundant in Se6-M and 31 more abundant under ciprofloxacin stress). CONCLUSION: Several proteins with known and possible roles in quinolone resistance were identified which provide important information about mechanism-related differential protein expression, supporting the current knowledge and also leading to new testable hypotheses on the mechanism of action of fluoroquinolone drugs.

Critical review on biofilm methods.

Biofilms are widespread in nature and constitute an important strategy implemented by microorganisms to survive in sometimes harsh environmental conditions. They can be beneficial or have a negative impact particularly when formed in industrial settings or on medical devices. As such, research into the formation and elimination of biofilms is important for many disciplines. Several new methodologies have been recently developed for, or adapted to, biofilm studies that have contributed to deeper knowledge on biofilm physiology, structure and composition. In this review, traditional and cutting-edge methods to study biofilm biomass, viability, structure, composition and physiology are addressed. Moreover, as there is a lack of consensus among the diversity of techniques used to grow and study biofilms. This review intends to remedy this, by giving a critical perspective, highlighting the advantages and limitations of several methods. Accordingly, this review aims at helping scientists in finding the most appropriate and up-to-date methods to study their biofilms.

Inactivation of the SecA2 protein export pathway in Listeria monocytogenes promotes cell aggregation, impacts biofilm architecture and induces biofilm formation in environmental condition.

Listeria monocytogenes has a dichotomous lifestyle, existing as an ubiquitous saprophytic species and as an opportunistic intracellular pathogen. Besides its capacity to grow in a wide range of environmental and stressful conditions, L. monocytogenes has the ability to adhere to and colonize surfaces. Morphotype variation to elongated cells forming rough colonies has been reported for different clinical and environmental isolates, including biofilms. This cell differentiation is mainly attributed to the reduced secretion of two SecA2-dependent cell-wall hydrolases, CwhA and MurA. SecA2 is a non-essential SecA paralogue forming an alternative translocase with the primary Sec translocon. Following investigation at temperatures relevant to its ecological niches, i.e. infection (37°C) and environmental (20°C) conditions, inactivation of this SecA2-only protein export pathway led, despite reduced adhesion, to the formation of filamentous biofilm with aerial structures. Compared to the wild type strain, inactivation of the SecA2 pathway promoted extensive cell aggregation and sedimentation. At ambient temperature, this effect was combined with the abrogation of cell motility resulting in elongated sedimented cells, which got knotted and entangled together in the course of filamentous-biofilm development. Such a cell differentiation provides a decisive advantage for listerial surface colonization under environmental condition. As further discussed, this morphotypic conversion has strong implication on listerial physiology and is also of potential significance for asymptomatic human/animal carriage.

Identification of novel GAPDH-derived antimicrobial peptides secreted by Saccharomyces cerevisiae and involved in wine microbial interactions.

Saccharomyces cerevisiae plays a primordial role in alcoholic fermentation and has a vast worldwide application in the production of fuel-ethanol, food and beverages. The dominance of S. cerevisiae over other microbial species during alcoholic fermentations has been traditionally ascribed to its higher ethanol tolerance. However, recent studies suggested that other phenomena, such as microbial interactions mediated by killer-like toxins, might play an important role. Here we show that S. cerevisiae secretes antimicrobial peptides (AMPs) during alcoholic fermentation that are active against a wide variety of wine-related yeasts (e.g. Dekkera bruxellensis) and bacteria (e.g. Oenococcus oeni). Mass spectrometry analyses revealed that these AMPs correspond to fragments of the S. cerevisiae glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein. The involvement of GAPDH-derived peptides in wine microbial interactions was further sustained by results obtained in mixed cultures performed with S. cerevisiae single mutants deleted in each of the GAPDH codifying genes (TDH1-3) and also with a S. cerevisiae mutant deleted in the YCA1 gene, which codifies the apoptosis-involved enzyme metacaspase. These findings are discussed in the context of wine microbial interactions, biopreservation potential and the role of GAPDH in the defence system of S. cerevisiae.

Activity of Epsilon-poly-L-lysine against Multidrug-Resistant Pseudomonas aeruginosa and Klebsiella pneumoniae Isolates of Urinary Tract Infections.

Pseudomonas aeruginosa and Klebsiella pneumoniae are notorious for their resistance to antibiotics and propensity for biofilm formation, posing significant threats to human health. Epsilon-poly-L-lysine (ε-PL) emerges as a naturally occurring antimicrobial poly(amino acid), which positions it as a prospective agent for addressing challenges linked to multidrug resistance. ε-PL symbolizes a promising avenue in the pursuit of efficacious therapeutic strategies and warrants earnest consideration within the realm of clinical treatment. Thus, our objective was to determine the antibiotic susceptibility profiles of 38 selected P. aeruginosa and ESBL-producing K. pneumoniae clinical isolates and determine the ability of ε-PL to inhibit biofilm formation. After PCR analysis, detection of genes related to ß-lactamases was observed among the selected isolates of P. aeruginosa [blaSPM (35.7%), blaKPC (35.7%), blaSHV (14.3%), blaCTX-M (14.3%), blaOXA (14.3%), blaTEM (7.1%), blaPER (7.1%), blaVIM (7.1%), and blaVIM-2 (7.1%)] and K. pneumoniae [blaCTX-M (91.7%), blaTEM (83.3%), blaKPC (16.7%), blaNDM (12.5%), and blaOXA (4.2%)]. The results of testing the activity of ε-PL against the clinical isolates showed relatively high minimum inhibitory concentrations (MICs) for the P. aeruginosa (range: 8-64 µg/mL) and K. pneumoniae isolates (range: 16-32 µg/mL). These results suggest the need for prior optimization of ε-PL concerning its viability as an alternative to antibiotics for treating infections caused by P. aeruginosa and K. pneumoniae of clinical origin. It is noteworthy that, in the context of a low antibiotic discovery rate, ε-PL could play a significant role in this quest, considering its low toxicity and the unlikely development of resistance. Upon exposure to ε-PL, P. aeruginosa and K. pneumoniae isolates exhibited a reduction in biofilm production, with ε-PL concentration showing an inverse relationship, particularly in isolates initially characterized as strong or moderate producers, indicating its potential as a natural antimicrobial agent with further research needed to elucidate optimal concentrations and application methods across different bacterial species. Further research is needed to optimize its use and explore its potential in various applications.

Determination of Antimicrobial Resistance and the Impact of Imipenem + Cilastatin Synergy with Tetracycline in Pseudomonas aeruginosa Isolates from Sepsis.

Pseudomonas aeruginosa is among the most ubiquitous bacteria in the natural world, exhibiting metabolic and physiological versatility, which makes it highly adaptable. Imipenem + cilastatin and tetracycline are antibiotic combinations commonly used to treat infections caused by P. aeruginosa, including serious infections such as sepsis. In the context of bacterial infections, biofilm, formed by bacterial cells surrounded by extracellular substances forming a matrix, plays a pivotal role in the resistance of P. aeruginosa to antibiotics. This study aimed to characterize a representative panel of P. aeruginosa isolates from septicemias, assessing their susceptibility to various antibiotics, specifically, imipenem + cilastatin and tetracycline, and the impact of these treatments on biofilm formation. Results from antibiotic susceptibility tests revealed sensitivity in most isolates to six antibiotics, with four showing near or equal to 100% sensitivity. However, resistance was observed in some antibiotics, albeit at minimal levels. Notably, tetracycline showed a 100% resistance phenotype, while imipenem + cilastatin predominantly displayed an intermediate phenotype (85.72%), with some resistance (38.1%). Microdilution susceptibility testing identified effective combinations against different isolates. Regarding biofilm formation, P. aeruginosa demonstrated the ability to produce biofilms. The staining of microtiter plates confirmed that specific concentrations of imipenem + cilastatin and tetracycline could inhibit biofilm production. A significant proportion of isolates exhibited resistance to aminoglycoside antibiotics because of the presence of modifying genes (aac(3)-II and aac(3)-III), reducing their effectiveness. This study also explored various resistance genes, unveiling diverse resistance mechanisms among P. aeruginosa isolates. Several virulence genes were detected, including the las quorum-sensing system genes (lasI and lasR) in a significant proportion of isolates, contributing to virulence factor activation. However, genes related to the type IV pili (T4P) system (pilB and pilA) were found in limited isolates. In conclusion, this comprehensive study sheds light on the intricate dynamics of P. aeruginosa, a remarkably adaptable bacterium with a widespread presence in the natural world. Our findings provide valuable insights into the ongoing battle against P. aeruginosa infections, highlighting the need for tailored antibiotic therapies and innovative approaches to combat biofilm-related resistance.

The Impact of the Virulence of Pseudomonas aeruginosa Isolated from Dogs.

Pseudomonas aeruginosa is a pathogenic bacterium that can cause serious infections in both humans and animals, including dogs. Treatment of this bacterium is challenging because some strains have developed multi-drug resistance. This study aimed to evaluate the antimicrobial resistance patterns and biofilm production of clinical isolates of P. aeruginosa obtained from dogs. The study found that resistance to various ß-lactam antimicrobials was widespread, with cefovecin and ceftiofur showing resistance in 74% and 59% of the isolates tested, respectively. Among the aminoglycosides, all strains showed susceptibility to amikacin and tobramycin, while gentamicin resistance was observed in 7% of the tested isolates. Furthermore, all isolates carried the oprD gene, which is essential in governing the entry of antibiotics into bacterial cells. The study also investigated the presence of virulence genes and found that all isolates carried exoS, exoA, exoT, exoY, aprA, algD, and plcH genes. This study compared P. aeruginosa resistance patterns worldwide, emphasizing regional understanding and responsible antibiotic use to prevent multi-drug resistance from emerging. In general, the results of this study emphasize the importance of the continued monitoring of antimicrobial resistance in veterinary medicine.

Study of Antimicrobial Resistance, Biofilm Formation, and Motility of Pseudomonas aeruginosa Derived from Urine Samples.

Pseudomonas aeruginosa causes urinary tract infections associated with catheters by forming biofilms on the surface of indwelling catheters. Therefore, controlling the spread of the bacteria is crucial to preventing its transmission in hospitals and the environment. Thus, our objective was to determine the antibiotic susceptibility profiles of twenty-five P. aeruginosa isolates from UTIs at the Medical Center of Trás-os-Montes and Alto Douro (CHTMAD). Biofilm formation and motility are also virulence factors studied in this work. Out of the twenty-five P. aeruginosa isolates, 16% exhibited multidrug resistance, being resistant to at least three classes of antibiotics. However, the isolates showed a high prevalence of susceptibility to amikacin and tobramycin. Resistance to carbapenem antibiotics, essential for treating infections when other antibiotics fail, was low in this study, Notably, 92% of the isolates demonstrated intermediate sensitivity to ciprofloxacin, raising concerns about its efficacy in controlling the disease. Genotypic analysis revealed the presence of various ß-lactamase genes, with class B metallo-ß-lactamases (MBLs) being the most common. The blaNDM, blaSPM, and blaVIM-VIM2 genes were detected in 16%, 60%, and 12% of the strains, respectively. The presence of these genes highlights the emerging threat of MBL-mediated resistance. Additionally, virulence gene analysis showed varying prevalence rates among the strains. The exoU gene, associated with cytotoxicity, was found in only one isolate, while other genes such as exoS, exoA, exoY, and exoT had a high prevalence. The toxA and lasB genes were present in all isolates, whereas the lasA gene was absent. The presence of various virulence genes suggests the potential of these strains to cause severe infections. This pathogen demonstrated proficiency in producing biofilms, as 92% of the isolates were found to be capable of doing so. Currently, antibiotic resistance is one of the most serious public health problems, as options become inadequate with the continued emergence and spread of multidrug-resistant strains, combined with the high rate of biofilm production and the ease of dissemination. In conclusion, this study provides insights into the antibiotic resistance and virulence profiles of P. aeruginosa strains isolated from human urine infections, highlighting the need for continued surveillance and appropriate therapeutic approaches.

Specific Proteomic Identification of Collagen-Binding Proteins in Escherichia coli O157:H7: Characterisation of OmpA as a Potent Vaccine Antigen.

Escherichia coli is a versatile commensal species of the animal gut that can also be a pathogen able to cause intestinal and extraintestinal infections. The plasticity of its genome has led to the evolution of pathogenic strains, which represent a threat to global health. Additionally, E. coli strains are major drivers of antibiotic resistance, highlighting the urgent need for new treatment and prevention measures. The antigenic and structural heterogeneity of enterohaemorrhagic E. coli colonisation factors has limited their use for the development of effective and cross-protective vaccines. However, the emergence of new strains that express virulence factors deriving from different E. coli diarrhoeagenic pathotypes suggests that a vaccine targeting conserved proteins could be a more effective approach. In this study, we conducted proteomics analysis and functional protein characterisation to identify a group of proteins potentially involved in the adhesion of E. coli O157:H7 to the extracellular matrix and intestinal epithelial cells. Among them, OmpA has been identified as a highly conserved and immunogenic antigen, playing a significant role in the adhesion phenotype of E. coli O157:H7 and in bacterial aggregation. Furthermore, antibodies raised against recombinant OmpA effectively reduced the adhesion of E. coli O157:H7 to intestinal epithelial cells. The present work highlights the role of OmpA as a potent antigen for the development of a vaccine against intestinal pathogenic E. coli.

Effect of subtherapeutic and therapeutic sulfamethazine concentrations on transcribed genes and translated proteins involved in Microbacterium sp. C448 resistance and degradation.

Microbacterium sp. C448, isolated from a soil regularly exposed to sulfamethazine (SMZ), can use various sulphonamide antibiotics as the sole carbon source for growth. The basis for the regulation of genes encoding the sulphonamide metabolism pathway, the dihydropteroate synthase sulphonamide target (folP), and the sulphonamide resistance (sul1) genes is unknown in this organism. In the present study, the response of the transcriptome and proteome of Microbacterium sp. C448 following exposure to subtherapeutic (33 µM) or therapeutic (832 µM) SMZ concentrations was evaluated. Therapeutic concentration induced the highest sad expression and Sad production, consistent with the activity of SMZ degradation observed in cellulo. Following complete SMZ degradation, Sad production tended to return to the basal level observed prior to SMZ exposure. Transcriptomic and proteomic kinetics were concomitant for the resistance genes and proteins. The abundance of Sul1 protein, 100-fold more abundant than FolP protein, did not change in response to SMZ exposure. Moreover, non-targeted analyses highlighted the increase of a deaminase RidA and a putative sulphate exporter expression and production. These two novel factors involved in the 4-aminophenol metabolite degradation and the export of sulphate residues formed during SMZ degradation, respectively, provided new insights into the Microbacterium sp. C448 SMZ detoxification process.

Anti-TNF Agents Restrict Adherent-invasive Escherichia coli Replication Within Macrophages Through Modulation of Chitinase 3-like 1 in Patients with Crohn's Disease.

BACKGROUND AND AIMS: The mechanism of action of anti-tumour necrosis factor [anti-TNF] agents could implicate macrophage modulation in Crohn's disease [CD]. As CD macrophages are defective in controlling CD-associated adherent-invasive Escherichia coli [AIEC], anti-TNF agents could limit AIEC replication within macrophages. We assessed the effect of anti-TNF agents on AIEC survival within monocyte-derived macrophages [MDMs] from CD patients and attempted to identify the proteins involved. METHODS: Peripheral blood MDMs were obtained from 44 CD patients [22 with and 22 without anti-TNF agents]. MDMs were infected with reference strain AIEC-LF82. Proteomic analysis was performed before and 6 h after AIEC-LF82 infection. RESULTS: AIEC-LF82 survival was lower in MDMs from CD patients receiving anti-TNF agents compared to those who did not [-73%, p = 0.006]. After AIEC-LF82 infection, the levels of CD82 [p = 0.007], ILF3 [Interleukin enhancer-binding factor 3; p = 0.001], FLOT-1 [Flotillin-1; p = 0.007] and CHI3L1 [Chitinase 3-like 1; p = 0.035] proteins were different within CD-MDMs depending on anti-TNF exposure. FLOT-1 [ϱ = -0.44; p = 0.038] and CHI3L1 [ϱ = 0.57, p = 0.006] levels were inversely and positively correlated with AIEC survival within MDMs from CD patients with or without anti-TNF, respectively. We observed a dose-dependent decrease of AIEC-LF82 survival after adjunction of anti-TNF within MDMs, inducing an increase of FLOT-1 and decrease of CHI3L1 mRNA levels. Neutralization of intra-macrophagic CHI3L1 protein using anti-CHI3L1 antibodies reduced AIEC survival within macrophages 6 h after infection [p < 0.05]. CONCLUSION: Anti-TNF agents are able to restrict replication of pathobionts, such as AIEC, within macrophages by modulating FLOT-1 and CHI3L1 expression in CD patients.

Deep impact of the inactivation of the SecA2-only protein export pathway on the proteosurfaceome of Listeria monocytogenes.

Listeria monocytogenes presents a dimorphism associated to the SecA2 activity with cells having a normal rod shape or a dysmorphic elongated filamentous form. Besides variation of the cell and colony morphotype, this cell differentiation has profound ecophysiological and physiopathological implications with collateral effects on virulence and pathogenicity, biotope colonisation, bacterial adhesion and biofilm formation. This suggests the SecA2-only protein export could influence the listerial cell surface, which was investigated first by characterising its properties in L. monocytogenes wt and ΔsecA2. The degree of hydrophilicity and Lewis acid-base properties appeared significantly affected upon SecA2 inactivation. As modification of electrostatic properties would owe to modification in the composition of cell-surface proteins, the proteosurfaceome was further investigated by shotgun label-free proteomic analysis with a comparative relative quantitative approach. Following secretomic analysis, the protein secretion routes of the identified proteins were mapped considering the cognate transport and post-translocational maturation systems, as well as protein categories and subcellular localisation. Differential protein abundance profiles coupled to network analysis revealed the SecA2 dependence of 48 proteins, including some related to cell envelope biogenesis, translation and protein export, which could account for modifications of adhesion and surface properties of L. monocytogenes upon SecA2 inactivation. This investigation unravelled the profound influence of SecA2 activity on the cell surface properties and proteosurfaceome of L. monocytogenes, which provides advanced insights about its ecophysiopathology. SIGNIFICANCE: L. monocytogenes is a foodborne zoonotic pathogen and etiological agent of human listeriosis. This species presents a cellular dimorphism associated to the SecA2 activity that has profound physiopathological and ecophysiological implications with collateral effects on bacterial virulence and colonisation. To explore the influence of the SecA2-only protein export on the listerial cell, the surface properties of L. monocytogenes expressing or depleted of SecA2 was characterised by microelectrophoresis, microbial affinity to solvents and contact angles analyses. As modifications of hydrophilicity and Lewis acid-base electrostatic properties would owe to modification in the composition of cell-surface proteins, the proteinaceous subset of the surfaceome, i.e. the proteosurfaceome, was investigated further by shotgun label-free proteomic analysis. This subproteome appeared quite impacted upon SecA2 inactivation with the identification of proteins accounting for modifications in the cell surface properties. The profound influence of SecA2 activity on the cell surface of L. monocytogenes was unravelled, which provides advanced insights about its ecophysiopathology.

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.

Molecular biology of surface colonization by Listeria monocytogenes: an additional facet of an opportunistic Gram-positive foodborne pathogen.

The opportunistic and facultative intracellular pathogenic bacterium Listeria monocytogenes causes a rare but severe foodborne disease called listeriosis, the outcome of which can be fatal. The infection cycle and key virulence factors are now well characterized in this species. Nonetheless, this knowledge has not prevented the re-emergence of listeriosis, as recently reported in several European countries. Listeria monocytogenes is particularly problematic in the food industry since it can survive and multiply under conditions frequently used for food preservation. Moreover, this foodborne pathogen also forms biofilms, which increase its persistence and resistance in industrial production lines, leading to contamination of food products. Significant differences have been reported regarding the ability of different isolates to form biofilms, but no clear correlation can be established with serovars or lineages. The architecture of listerial biofilms varies greatly from one strain to another as it ranges from bacterial monolayers to the most recently described network of knitted chains. While the role of polysaccharides as part of the extracellular matrix contributing to listerial biofilm formation remains elusive, the importance of eDNA has been demonstrated. The involvement of flagella in biofilm formation has also been pointed out, but their exact role in the process remains to be clarified because of conflicting results. Two cell-cell communication systems LuxS and Agr have been shown to take part in the regulation of biofilm formation. Several additional molecular determinants have been identified by functional genetic analyses, such as the (p)ppGpp synthetase RelA and more recently BapL. Future directions and questions about the molecular mechanisms of biofilm formation in L. monocytogenes are further discussed, such as correlation between clonal complexes as revealed by MLST and biofilm formation, the swarming over swimming regulation hypothesis regarding the role of the flagella, and the involvement of microbial surface components recognizing adhesive matrix molecules in the colonization of abiotic and biotic surfaces.

Extraction and Preparation of Listeria monocytogenes Subproteomes for Mass Spectrometry Analysis.

Proteomics has become an essential tool to answer biologists' questions. For bacteriologists, the proteome of bacteria is much less complex than that of eukaryotic organisms. However, not all the different cell "compartments" are easily accessible, and the analysis of cell envelope proteins is particularly challenging. For the Gram-positive bacterium Listeria monocytogenes, one of the main foodborne pathogen microorganisms, the study of surface proteins is crucial to better understand the mechanisms of pathogenicity, as well as adaptation/resistance to and persistence in hostile environments. The evolution of proteomic techniques, and particularly the possibility of separating and analyzing complex protein samples by off-gel (LC-MS/MS) versus in-gel (two-dimensional electrophoresis) approach, has opened the doors to new extraction and preparation methods to target the different subproteomes. Here, we describe three procedures to prepare and analyze intracellular, exocellular, and cell surface proteins: (1) the cell fractionation, based on cell broken and separation of protein subfractions by differential centrifugation; (2) the biotinylation, based on the labeling of cell surface proteins and their selective extraction; and (3) the enzymatic shaving by the action of trypsin on intact cells. These complementary methods allow to encompass all L. monocytogenes subproteomes for general profiling or target studies and could be applicable to other Gram-positive bacteria.

Bacteriophages as Antimicrobial Agents? Proteomic Insights on Three Novel Lytic Bacteriophages Infecting ESBL-Producing Escherichia coli.

With the emergence of multiresistant bacteria, the use of bacteriophages is gaining renewed interest as potential antimicrobial agents. The aim of this study was to analyze the structure of three lytic bacteriophages infecting Escherichia coli (SD1, SD2, and SD3) using a gel-based proteomics approach and the cellular response of this bacterium to phage SD1 infection at the proteome level. The combination of the results of 1-DE and 2-DE followed by mass spectrometry led to the identification of 3, 14, and 9 structure proteins for SD1, SD2, and SD3 phages, respectively. Different protein profiles with common proteins were noticed. We also analyzed phage-induced effects by comparing samples from infected cells to those of noninfected cells. We verified important changes in E. coli proteins expression during phage SD1 infection, where there was an overexpression of proteins involved in stress response. Our results indicated that viral infection caused bacterial oxidative stress and bacterial cells response to stress was orchestrated by antioxidant defense mechanisms. This article makes an empirical scientific contribution toward the concept of bacteriophages as potential antimicrobial agents. With converging ecological threats in the 21st century, novel approaches to address the innovation gaps in antimicrobial development are more essential than ever. Further research on bacteriophages is called for in this broader context of planetary health and integrative biology.

The Secretome landscape of Escherichia coli O157:H7: Deciphering the cell-surface, outer membrane vesicle and extracellular subproteomes.

Among diarrheagenic E. coli (DEC), enterohaemorrhagic E. coli (EHEC) are the most virulent anthropozoonotic agents. The ability of bacterial cells to functionally interact with their surrounding essentially relies on the secretion of different protein effectors. To experimentally determine the repertoire of extracytoproteins in E. coli O157:H7, a subproteomic analysis was performed not only considering the extracellular milieu but the cell surface and outer membrane vesicles. Following a secretome-based approach, the proteins trafficking from the interior to the exterior of the cell were depicted considering cognate protein transport systems and subcellular localisation. Label-free quantitative analysis of the proteosurfaceome, proteovesiculome and exoproteome from E. coli O157:H7 grown in three different nutrient media revealed differential protein expression profiles and allowed defining the core and variant subproteomes. Network analysis further revealed the higher abundance of some protein clusters in chemically defined medium over rich complex medium, especially related to some outer membrane proteins, ABC transport and Type III secretion systems. This first comprehensive study of the EHEC secretome unravels the profound influence of environmental conditions on the extracytoplasmic proteome, provides new insight in the physiology of E. coli O157:H7 and identifies potentially important molecular targets for the development of preventive strategies against EHEC/STEC. SIGNIFICANCE: Escherichia coli O157:H7 is responsible for severe diarrhoea especially in young children. Despite years of investigations, the global view of the extracytoplasmic proteins expressed in this microorganism was eluded. To provide the first comprehensive view of the secretome landscape of E. coli O157:H7, the exoproteome, proteosurfaceome and proteovesiculome were profiled using growth conditions most likely to induce changes in bacterial protein secretion. The profound influence of growth conditions on the extracytoplasmic proteome was unravelled and allowed identifying the core and variant subproteomes. Besides new insight in the physiology of enterohaemorrhagic E. coli, these proteins potentially constitute important molecular targets for the development of preventive strategies.