Deep UV excited muscle cell autofluorescence varies with the fibre type.

The rat skeletal muscle consists of four pure types of muscle cells called type I, type IIA, type IIX and type IIB, and their hybrids in different proportions. They differ in their contraction speeds and metabolic pathways. The intracellular composition is adapted to the fibre function and therefore to fibre types. Given that small differences in composition are likely to alter the optical properties of the cells, we studied the impact of the cell type on the fluorescence response following excitation in the deep UV region. Rat soleus and extensor digitorum longus (EDL) muscle fibres, previously identified based on their cell types by immunohistofluorescence analysis, were analyzed by synchrotron fluorescence microspectroscopy on stain-free serial muscle cross-sections. Muscle fibres excited at 275 nm showed differences in the fluorescence emission intensity among fibre types at 302, 325, 346 and 410 nm. The 410/325 ratio decreased significantly with contractile and metabolic features in EDL muscle, in the order of I > IIA > IIX > IIB fibres (p < 0.01). Compared to type I fibres, the 346/302 ratio of IIA fibres decreased significantly in both EDL and soleus muscles (p < 0.01). This study highlights the usefulness of autofluorescence spectral signals to characterize histological cross-sections of muscle fibres with no staining chemicals.

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.

Bacterial adhesion to animal tissues: protein determinants for recognition of extracellular matrix components.

The extracellular matrix (ECM) is present within all animal tissues and organs. Actually, it surrounds the eukaryotic cells composing the four basic tissue types, i.e. epithelial, muscle, nerve and connective. ECM does not solely refer to connective tissue but composes all tissues where its composition, structure and organization vary from one tissue to another. Constituted of the four main fibrous proteins, i.e. collagen, fibronectin, laminin and elastin, ECM components form a highly structured and functional network via specific interactions. From the basement membrane to interstitial matrix, further heterogeneity exists in the organization of the ECM in various tissues and organs also depending on their physiological state. Back to a molecular level, bacterial proteins represent the most significant part of the microbial surface components recognizing adhesive matrix molecules (MSCRAMM). These cell surface proteins are secreted and localized differently in monoderm and diderm-LPS bacteria. While one collagen-binding domain (CBD) and different fibronectin-binding domains (FBD1 to 8) have been registered in databases, much remains to be learned on specific binding to other ECM proteins via single or supramolecular protein structures. Besides theinteraction of bacterial proteins with individual ECM components, this review aims at stressing the importance of fully considering the ECM at supramolecular, cellular, tissue and organ levels. This conceptual view should not be overlooked to rigorously comprehend the physiology of bacterial interaction from commensal to pathogenic species.

Oenococcus sicerae sp. nov., isolated from French cider.

Two Gram-stain-positive, small ellipsoidal cocci, non-motile, oxidase- and catalase-negative, and facultative anaerobic strains (UCMA15228T and UCMA17102) were isolated in France, from fermented apple juices (ciders). The 16S rRNA gene sequence was identical between the two isolates and showed 97 % similarity with respect to the closest related species Oenococcus oeni and O. kitaharae. Therefore, the two isolates were classified within the genus Oenococcus. The phylogeny based on the pheS gene sequences also confirmed the position of the new taxon. DNA-DNA hybridizations based on in silico genome-to-genome comparisons (GGDC) and Average Nucleotide Identity (ANI) values, as well as species-specific PCR, validated the novelty of the taxon. Various phenotypic characteristics such as the optimum temperature and pH for growth, the ability to metabolise sugars, the aptitude to perform the malolactic fermentation, and the resistance to ethanol and NaCl, revealed that the two strains are distinguishable from the other members of the Oenococcus genus. The combined genotypic and phenotypic data support the classification of strains UCMA15228T and UCMA17102 into a novel species of Oenococcus, for which the name O. sicerae sp. nov. is proposed. The type strain is UCMA15228T (=DSM107163T=CIRM-BIA2288T).

Colonisation of Meat by Escherichia coli O157:H7: Investigating Bacterial Tropism with Respect to the Different Types of Skeletal Muscles, Subtypes of Myofibres, and Postmortem Time.

Escherichia coli O157:H7 is an enterohaemorrhagic E. coli (EHEC) responsible for serious diseases, especially pediatric, and of great concern for the meat industry. Meat contamination by EHEC occurs at slaughtering, especially at dehiding stage, where bacteria can be transferred from hides to carcasses. The skeletal muscle tissues comprise four major types of myofibres, which differ in their contraction velocity and metabolism. Myofibres are surrounded by the extracellular matrix (ECM). Adhesion of E. coli O157:H7 to meat was investigated considering well-defined types of skeletal muscle and their constituent myofibres as well as postmortem changes in muscle, using fluorescence microscopy and immunohistochemical analyses. By analysing the adhesion of E. coli O157:H7 to model oxidative (soleus) and glycolytic [extensor digitorum longus (EDL)] skeletal muscles, it first appeared that differential adhesion occurred at the surface of these extreme skeletal muscle types. At a cellular level, bacterial adhesion appeared to occur essentially at the ECM. Considering the different constituent myofibres of types I, IIA, IIX and IIB, no significant differences were observed for adhering bacteria. However, bacterial adhesion to the ECM was significantly influenced by postmortem structural modifications of muscle tissues. By providing information on spatial localisation of E. coli O157:H7 on meat, this investigation clearly demonstrated their ability to adhere to skeletal muscle, especially at the ECM, which consequently resulted in their heterogeneous distribution in meat. As discussed, these new findings should help in reassessing and mitigating the risk of contamination of meat, the food chain and ultimately human infection by EHEC.

Hyperspectral deep ultraviolet autofluorescence of muscle fibers is affected by postmortem changes.

After slaughter, muscle cells undergo biochemical and physicochemical changes that may affect their autofluorescence characteristics. The autofluorescent response of different rat extensor digitorum longus (EDL) and soleus muscle fiber types was investigated by deep ultraviolet (UV) synchrotron microspectroscopy immediately after animal sacrifice and after 24 h of storage in a moist chamber at 20 °C. The glycogen content decreased from 23 to 18 µmol/g of fresh muscle in 24 h postmortem. Following a 275 nm excitation wavelength, the spectral muscle fiber autofluorescence response showed discrimination depending upon postmortem time (t0 versus t24 h) on both muscles at 346 and 302 nm and, to a lesser extent, at 408 and 325 nm. Taken individually, all fiber types were discriminated but with variable accuracy, with type IIA showing better separation of t0/t24 h than other fiber types. These results suggest the usefulness of the autofluorescent response of muscle cells for rapid meat-aging characterization.

Colonization of the meat extracellular matrix proteins by O157 and non-O157 enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) are anthropozoonotic agents that range third among food-borne pathogens respective to their incidence and dangerousness in the European Union. EHEC are Shiga-toxin producing E. coli (STEC) responsible for foodborne poisoning mainly incriminated to the consumption of contaminated beef meat. Among the hundreds of STEC serotypes identified, EHEC mainly belong to O157:H7 but non-O157 can represent 20 to 70% of EHEC infections per year. Seven of those serogroups are especially of high-risk for human health, i.e. O26, O45, O103, O111, O121, O145 and O104. While meat can be contaminated all along the food processing chain, EHEC contamination essentially occurs at the dehiding stage of slaughtering. Investigating bacterial colonization to the skeletal-muscle extracellular matrix (ECM) proteins, it appeared that environmental factors influenced specific and non-specific bacterial adhesion of O157 and non-O157 EHEC as well as biofilm formation. Importantly, mechanical treatment (i.e. shaking, centrifugation, pipetting and vortexing) inhibited and biased the results of bacterial adhesion assay. Besides stressing the importance of the protocol to investigate bacterial adhesion to ECM proteins, this study demonstrated that the colonization abilities to ECM proteins vary among EHEC serogroups and should ultimately be taken into consideration to evaluate the risk of contamination for different types of food matrices.

Proteinaceous determinants of surface colonization in bacteria: bacterial adhesion and biofilm formation from a protein secretion perspective.

Bacterial colonization of biotic or abiotic surfaces results from two quite distinct physiological processes, namely bacterial adhesion and biofilm formation. Broadly speaking, a biofilm is defined as the sessile development of microbial cells. Biofilm formation arises following bacterial adhesion but not all single bacterial cells adhering reversibly or irreversibly engage inexorably into a sessile mode of growth. Among molecular determinants promoting bacterial colonization, surface proteins are the most functionally diverse active components. To be present on the bacterial cell surface, though, a protein must be secreted in the first place. Considering the close association of secreted proteins with their cognate secretion systems, the secretome (which refers both to the secretion systems and their protein substrates) is a key concept to apprehend the protein secretion and related physiological functions. The protein secretion systems are here considered in light of the differences in the cell-envelope architecture between diderm-LPS (archetypal Gram-negative), monoderm (archetypal Gram-positive) and diderm-mycolate (archetypal acid-fast) bacteria. Besides, their cognate secreted proteins engaged in the bacterial colonization process are regarded from single protein to supramolecular protein structure as well as the non-classical protein secretion. This state-of-the-art on the complement of the secretome (the secretion systems and their cognate effectors) involved in the surface colonization process in diderm-LPS and monoderm bacteria paves the way for future research directions in the field.

In vitro colonization of the muscle extracellular matrix components by Escherichia coli O157:H7: the influence of growth medium, temperature and pH on initial adhesion and induction of biofilm formation by collagens I and III.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 are responsible for repeated food-poisoning cases often caused by contaminated burgers. EHEC infection is predominantly a pediatric illness, which can lead to life-threatening diseases. Ruminants are the main natural reservoir for EHEC and food contamination almost always originates from faecal contamination. In beef meat products, primary bacterial contamination occurs at the dehiding stage of slaughtering. The extracellular matrix (ECM) is the most exposed part of the skeletal muscles in beef carcasses. Investigating the adhesion to the main muscle fibrous ECM proteins, insoluble fibronectin, collagen I, III and IV, laminin-α2 and elastin, results demonstrated that the preceding growth conditions had a great influence on subsequent bacterial attachment. In the tested experimental conditions, maximal adhesion to fibril-forming collagens I or III occurred at 25°C and pH 7. Once initially adhered, exposure to lower temperatures, as applied to meat during cutting and storage, or acidification, as in the course of post-mortem physiological modifications of muscle, had no effect on detachment, except at pHu. In addition, dense biofilm formation occurred on immobilized collagen I or III and was induced in growth medium supplemented with collagen I in solution. From this first comprehensive investigation of EHEC adhesion to ECM proteins with respect to muscle biology and meat processing, new research directions for the development of innovative practices to minimize the risk of meat contamination are further discussed.

Exoproteomic analysis of the SecA2-dependent secretion in Listeria monocytogenes EGD-e.

As part of the Sec translocase, the accessory ATPase SecA2 is present in some pathogenic Gram-positive bacteria. In Listeria monocytogenes, deletion of secA2 results in filamentous cells that form rough colonies and have lower virulence. However, only a few proteins have been identified that are secreted by this pathway. This investigation aims to provide the first exoproteomic analysis of the SecA2-dependent secretion in L. monocytogenes EGD-e. By using media and temperatures relevant to bacterial physiology, we demonstrated that the rough colony and elongated bacterial cell morphotypes are highly dependent on growth conditions. Subsequently, comparative exoproteomic analyses of the ΔsecA2 versus wt strains were performed in chemically defined medium at 20°C and 37°C. Analyzing the proteomic data following the secretomics-based method, part of the proteins appeared routed towards the Sec pathway and exhibited an N-terminal signal peptide. For another significant part, they were primarily cytoplasmic proteins, thus lacking signal peptide and with no predictable secretion pathway. In total, 13 proteins were newly identified as secreted via SecA2, which were essentially associated with cell-wall metabolism, adhesion and/or biofilm formation. From this comparative exoproteomic analysis, new insights into the L. monocytogenes physiology are discussed in relation to its saprophytic and pathogenic lifestyle.