Browsing by Author "Latasa Osta, Cristina"

Now showing 1 - 13 of 13
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    Bap, a biofilm matrix protein of Staphylococcus aureus prevents cellular internalization through binding to GP96 host receptor
    (Public Library of Science, 2012) Valle Turrillas, Jaione; Latasa Osta, Cristina; Gil Puig, Carmen; Toledo Arana, Alejandro; Solano Goñi, Cristina; Penadés, José R.; Lasa Uzcudun, Íñigo; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
    The biofilm matrix, composed of exopolysaccharides, proteins, nucleic acids and lipids, plays a well-known role as a defence structure, protecting bacteria from the host immune system and antimicrobial therapy. However, little is known about its responsibility in the interaction of biofilm cells with host tissues. Staphylococcus aureus, a leading cause of biofilmassociated chronic infections, is able to develop a biofilm built on a proteinaceous Bap-mediated matrix. Here, we used the Bap protein as a model to investigate the role that components of the biofilm matrix play in the interaction of S. aureus with host cells. The results show that Bap promotes the adhesion but prevents the entry of S. aureus into epithelial cells. A broad analysis of potential interaction partners for Bap using ligand overlayer immunoblotting, immunoprecipitation with purified Bap and pull down with intact bacteria, identified a direct binding between Bap and Gp96/GRP94/Hsp90 protein. The interaction of Bap with Gp96 provokes a significant reduction in the capacity of S. aureus to invade epithelial cells by interfering with the fibronectin binding protein invasion pathway. Consistent with these results, Bap deficient bacteria displayed an enhanced capacity to invade mammary gland epithelial cells in a lactating mice mastitis model. Our observations begin to elucidate the mechanisms by which components of the biofilm matrix can facilitate the colonization of host tissues and the establishment of persistent infections.
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    PublicationOpen Access
    Biofilm matrix exoproteins induce a protective immune response against Staphylococcus aureus biofilm infection
    (American Society for Microbiology, 2014) Gil Puig, Carmen; Solano Goñi, Cristina; Burgui Erice, Saioa; Latasa Osta, Cristina; García Martínez, Begoña; Toledo Arana, Alejandro; Lasa Uzcudun, Íñigo; Valle Turrillas, Jaione; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua: IIQ14066.RI1
    The Staphylococcus aureus biofilm mode of growth is associated with several chronic infections that are very difficult to treat due to the recalcitrant nature of biofilms to clearance by antimicrobials. Accordingly, there is an increasing interest in preventing the formation of S. aureus biofilms and developing efficient antibiofilm vaccines. Given the fact that during a biofilm-associated infection, the first primary interface between the host and the bacteria is the self-produced extracellular matrix, in this study we analyzed the potential of extracellular proteins found in the biofilm matrix to induce a protective immune response against S. aureus infections. By using proteomic approaches, we characterized the exoproteomes of exopolysaccharide-based and proteinbased biofilm matrices produced by two clinical S. aureus strains. Remarkably, results showed that independently of the nature of the biofilm matrix, a common core of secreted proteins is contained in both types of exoproteomes. Intradermal administration of an exoproteome extract of an exopolysaccharide-dependent biofilm induced a humoral immune response and elicited the production of interleukin 10 (IL-10) and IL-17 in mice. Antibodies against such an extract promoted opsonophagocytosis and killing of S. aureus. Immunization with the biofilm matrix exoproteome significantly reduced the number of bacterial cells inside a biofilm and on the surrounding tissue, using an in vivo model of mesh-associated biofilm infection. Furthermore, immunized mice also showed limited organ colonization by bacteria released from the matrix at the dispersive stage of the biofilm cycle. Altogether, these data illustrate the potential of biofilm matrix exoproteins as a promising candidate multivalent vaccine against S. aureus biofilm-associated infections.
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    PublicationOpen Access
    Cloning, nucleotide sequencing, and analysis of the AcrAB-TolC efflux pump of enterobacter cloacae and determination of its involvement in antibiotic resistance in a clinical isolate
    (American Society for Microbiology, 2007) Pérez, Astrid; Canle, Delia; Latasa Osta, Cristina; Poza, Margarita; Beceiro, Alejandro; Tomás, María del Mar; Fernández, Ana; Mallo, Susana; Pérez, Sonia; Molina, Francisca; Villanueva González, Rosa; Lasa Uzcudun, Íñigo; Bou, Germán; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
    Enterobacter cloacae is an emerging clinical pathogen that may be responsible for nosocomial infections. Management of these infections is often difficult, owing to the high frequency of strains that are resistant to disinfectants and antimicrobial agents in the clinical setting. Multidrug efflux pumps, especially those belonging to the resistance-nodulation-division family, play a major role as a mechanism of antimicrobial resistance in gram-negative pathogens. In the present study, we cloned and sequenced the genes encoding an AcrAcB-TolC-like efflux pump from an E. cloacae clinical isolate (isolate EcDC64) showing a broad antibiotic resistance profile. Sequence analysis showed that the acrR, acrA, acrB, and tolC genes encode proteins that display 79.8%, 84%, 88%, and 82% amino acid identities with the respective homologues of Enterobacter aerogenes and are arranged in a similar pattern. Deletion of the acrA gene to yield an AcrA-deficient EcDC64 mutant (EcΔacrA) showed the involvement of AcrAB-TolC in multidrug resistance in E. cloacae. However, experiments with an efflux pump inhibitor suggested that additional efflux systems also play a role in antibiotic resistance. Investigation of several unrelated isolates of E. cloacae by PCR analysis revealed that the AcrAB system is apparently ubiquitous in this species.
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    PublicationOpen Access
    Coordinated cyclic-di-GMP repression of salmonella motility through YcgR and cellulose
    (American Society for Microbiology, 2013) Zorraquino Salvo, Violeta; García Martínez, Begoña; Latasa Osta, Cristina; Echeverz Sarasúa, Maite; Toledo Arana, Alejandro; Valle Turrillas, Jaione; Lasa Uzcudun, Íñigo; Solano Goñi, Cristina; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua: 1312/2010
    Cyclic di-GMP (c-di-GMP) is a secondary messenger that controls a variety of cellular processes, including the switch between a biofilm and a planktonic bacterial lifestyle. This nucleotide binds to cellular effectors in order to exert its regulatory functions. In Salmonella, two proteins, BcsA and YcgR, both of them containing a c-di-GMP binding PilZ domain, are the only known c-di-GMP receptors. BcsA, upon c-di-GMP binding, synthesizes cellulose, the main exopolysaccharide of the biofilm matrix. YcgR is dedicated to c-di-GMP-dependent inhibition of motility through its interaction with flagellar motor proteins. However, previous evidences indicate that in the absence of YcgR, there is still an additional element that mediates motility impairment under high c-di-GMP levels. Here we have uncovered that cellulose per se is the factor that further promotes inhibition of bacterial motility once high c-di-GMP contents drive the activation of a sessile lifestyle. Inactivation of different genes of the bcsABZC operon, mutation of the conserved residues in the RxxxR motif of the BcsA PilZ domain, or degradation of the cellulose produced by BcsA rescued the motility defect of ΔycgR strains in which high c-di-GMP levels were reached through the overexpression of diguanylate cyclases. High c-di-GMP levels provoked cellulose accumulation around cells that impeded flagellar rotation, probably by means of steric hindrance, without affecting flagellum gene expression, exportation, or assembly. Our results highlight the relevance of cellulose in Salmonella lifestyle switching as an architectural element that is both essential for biofilm development and required, in collaboration with YcgR, for complete motility inhibition.
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    PublicationOpen Access
    A DIVA vaccine strain lacking RpoS and the secondary messenger c-di-GMP for protection against salmonellosis in pigs
    (BioMed Central, 2020) Gil Puig, Carmen; Latasa Osta, Cristina; García Ona, Enrique; Lázaro, Isidro; Labairu, Javier; Echeverz Sarasúa, Maite; Burgui Erice, Saioa; García Martínez, Begoña; Lasa Uzcudun, Íñigo; Solano Goñi, Cristina; Ciencias de la Salud; Osasun Zientziak; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa; Gobierno de Navarra / Nafarroako Gobernua, IIM 13329.RI1
    Salmonellosis is the second most common food-borne zoonosis in the European Union, with pigs being a major reservoir of this pathogen. Salmonella control in pig production requires multiple measures amongst which vaccination may be used to reduce subclinical carriage and shedding of prevalent serovars, such as Salmonella enterica serovar Typhimurium. Live attenuated vaccine strains offer advantages in terms of enhancing cell mediated immunity and allowing inoculation by the oral route. However, main failures of these vaccines are the limited cross-protection achieved against heterologous serovars and interference with serological monitoring for infection. We have recently shown that an attenuated S. Enteritidis strain (ΔXIII) is protective against S. Typhimurium in a murine infection model. ΔXIII strain harbours 13 chromosomal deletions that make it unable to produce the sigma factor RpoS and synthesize cyclic-di-GMP (c-di-GMP). In this study, our objectives were to test the protective effects of ΔXIII strain in swine and to investigate if the use of ΔXIII permits the discrimination of vaccinated from infected pigs. Results show that oral vaccination of pre-weaned piglets with ΔXIII cross-protected against a challenge with S. Typhimurium by reducing faecal shedding and ileocaecal lymph nodes colonization, both at the time of weaning and slaughter. Vaccinated pigs showed neither faecal shedding nor tissue persistence of the vaccine strain at weaning, ensuring the absence of ΔXIII strain by the time of slaughter. Moreover, lack of the SEN4316 protein in ΔXIII strain allowed the development of a serological test that enabled the differentiation of infected from vaccinated animals (DIVA).
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    PublicationOpen Access
    Effect of transcriptional activators SoxS, RobA, and RamA on expression of multidrug efflux pump AcrAB-TolC in enterobacter cloacae
    (American Society for Microbiology, 2012) Pérez, Astrid; Poza, Margarita; Aranda, Jesús; Latasa Osta, Cristina; Medrano, Francisco Javier; Tomás, María del Mar; Romero, Antonio; Lasa Uzcudun, Íñigo; Bou, Germán; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
    Control of membrane permeability is a key step in regulating the intracellular concentration of antibiotics. Efflux pumps confer innate resistance to a wide range of toxic compounds such as antibiotics, dyes, detergents, and disinfectants in members of the Enterobacteriaceae. The AcrAB-TolC efflux pump is involved in multidrug resistance in Enterobacter cloacae. However, the underlying mechanism that regulates the system in this microorganism remains unknown. In Escherichia coli, the transcription of acrAB is upregulated under global stress conditions by proteins such as MarA, SoxS, and Rob. In the present study, two clinical isolates of E. cloacae, EcDC64 (a multidrug-resistant strain overexpressing the AcrAB-TolC efflux pump) and Jc194 (a strain with a basal AcrAB-TolC expression level), were used to determine whether similar global stress responses operate in E. cloacae and also to establish the molecular mechanisms underlying this response. A decrease in susceptibility to erythromycin, tetracycline, telithromycin, ciprofloxacin, and chloramphenicol was observed in clinical isolate Jc194 and, to a lesser extent in EcDC64, in the presence of salicylate, decanoate, tetracycline, and paraquat. Increased expression of the acrAB promoter in the presence of the above-described conditions was observed by flow cytometry and reverse transcription-PCR, by using a reporter fusion protein (green fluorescent protein). The expression level of the AcrAB promoter decreased in E. cloacae EcDC64 derivates deficient in SoxS, RobA, and RamA. Accordingly, the expression level of the AcrAB promoter was higher in E. cloacae Jc194 strains overproducing SoxS, RobA, and RamA. Overall, the data showed that SoxS, RobA, and RamA regulators were associated with the upregulation of acrAB, thus conferring antimicrobial resistance as well as a stress response in E. cloacae. In summary, the regulatory proteins SoxS, RobA, and RamA were cloned and sequenced for the first time in this species. The involvement of these proteins in conferring antimicrobial resistance through upregulation of acrAB was demonstrated in E. cloacae.
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    PublicationOpen Access
    Evaluation of a Salmonella strain lacking the secondary messenger c-di-GMP and RpoS as a live oral vaccine
    (Public Library of Science, 2016) Latasa Osta, Cristina; Echeverz Sarasúa, Maite; García Ona, Enrique; Burgui Erice, Saioa; Casares, Noelia; Hervás Stubbs, Sandra; Lasarte, Juan José; Lasa Uzcudun, Íñigo; Solano Goñi, Cristina; García Martínez, Begoña; Gil Puig, Carmen; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua: IIM 13329.RI1
    Salmonellosis is one of the most important bacterial zoonotic diseases transmitted through the consumption of contaminated food, with chicken and pig related products being key reservoirs of infection. Although numerous studies on animal vaccination have been performed in order to reduce Salmonella prevalence, there is still a need for an ideal vaccine. Here, with the aim of constructing a novel live attenuated Salmonella vaccine candidate, we firstly analyzed the impact of the absence of cyclic-di-GMP (c-di-GMP) in Salmonella virulence. Cdi-GMP is an intracellular second messenger that controls a wide range of bacterial processes, including biofilm formation and synthesis of virulence factors, and also modulates the host innate immune response. Our results showed that a Salmonella multiple mutant in the twelve genes encoding diguanylate cyclase proteins that, as a consequence, cannot synthesize c-di-GMP, presents a moderate attenuation in a systemic murine infection model. An additional mutation of the rpoS gene resulted in a synergic attenuating effect that led to a highly attenuated strain, referred to as ΔXIII, immunogenic enough to protect mice against a lethal oral challenge of a S. Typhimurium virulent strain. ΔXIII immunogenicity relied on activation of both antibody and cell mediated immune responses characterized by the production of opsonizing antibodies and the induction of significant levels of IFN-γ, TNF- α, IL-2, IL-17 and IL-10. ΔXIII was unable to form a biofilm and did not survive under desiccation conditions, indicating that it could be easily eliminated from the environment. Moreover, ΔXIII shows DIVA features that allow differentiation of infected and vaccinated animals. Altogether, these results show ΔXIII as a safe and effective live DIVA vaccine
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    PublicationOpen Access
    Genetic reductionist approach for dissecting individual roles of GGDEF proteins within the c-di-GMP signaling network in Salmonella
    (National Academy of Sciences, 2009) Solano Goñi, Cristina; García Martínez, Begoña; Latasa Osta, Cristina; Toledo Arana, Alejandro; Zorraquino Salvo, Violeta; Valle Turrillas, Jaione; Casals, Joan; Pedroso, Enrique; Lasa Uzcudun, Íñigo; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
    Bacteria have developed an exclusive signal transduction system involving multiple diguanylate cyclase and phosphodiesterase domain-containing proteins (GGDEF and EAL/HD-GYP, respectively) that modulate the levels of the same diffusible molecule, 3 -5 -cyclic diguanylic acid (c-di-GMP), to transmit signals and obtain specific cellular responses. Current knowledge about c-di- GMP signaling has been inferred mainly from the analysis of recombinant bacteria that either lack or overproduce individual members of the pathway, without addressing potential compensatory effects or interferences between them. Here, we dissected c-di-GMP signaling by constructing a Salmonella strain lacking all GGDEF-domain proteins and then producing derivatives, each restoring 1 protein. Our analysis showed that most GGDEF proteins are constitutively expressed and that their expression levels are not interdependent. Complete deletion of genes encoding GGDEFdomain proteins abrogated virulence, motility, long-term survival, and cellulose and fimbriae synthesis. Separate restoration revealed that 4 proteins from Salmonella and 1 from Yersinia pestis exclusively restored cellulose synthesis in a c-di-GMP–dependent manner, indicating that c-di-GMP produced by different GGDEF proteins can activate the same target. However, the restored strain containing the STM4551-encoding gene recovered all other phenotypes by means of gene expression modulation independently of c-di-GMP. Specifically, fimbriae synthesis and virulence were recovered through regulation of csgD and the plasmid-encoded spvAB mRNA levels, respectively. This study provides evidence that the regulation of the GGDEF-domain proteins network occurs at 2 levels: a level that strictly requires c-di-GMP to control enzymatic activities directly, restricted to cellulose synthesis in our experimental conditions, and another that involves gene regulation for which c-di-GMP synthesis can be dispensable.
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    Inhibiting the two‑component system GraXRS with verteporfin to combat Staphylococcus aureus infections
    (Nature Research, 2020) Prieto Mariscal, Juana María; Rapún Araiz, Beatriz; Gil Puig, Carmen; Penadés, José R.; Lasa Uzcudun, Íñigo; Latasa Osta, Cristina; Ciencias de la Salud; Osasun Zientziak
    Infections caused by Staphylococcus aureus pose a serious and sometimes fatal health issue. With the aim of exploring a novel therapeutic approach, we chose GraXRS, a Two-Component System (TCS) that determines bacterial resilience against host innate immune barriers, as an alternative target to disarm S. aureus. Following a drug repurposing methodology, and taking advantage of a singular staphylococcal strain that lacks the whole TCS machinery but the target one, we screened 1.280 offpatent FDA-approved drug for GraXRS inhibition. Reinforcing the connection between this signaling pathway and redox sensing, we found that antioxidant and redox-active molecules were capable of reducing the expression of the GraXRS regulon. Among all the compounds, verteporfin (VER) was really efficient in enhancing PMN-mediated bacterial killing, while topical administration of such drug in a murine model of surgical wound infection significantly reduced the bacterial load. Experiments relying on the chemical mimicry existing between VER and heme group suggest that redox active residue C227 of GraS participates in the inhibition exerted by this FDA-approved drug. Based on these results, we propose VER as a promising candidate for sensitizing S. aureus that could be helpful to combat persistent or antibiotic-resistant infections.
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    PublicationOpen Access
    Novel strategies for treating biofilm and MRSA associated infections
    (2021) Prieto Mariscal, Juana María; Latasa Osta, Cristina; Ciencias de la Salud; Osasun Zientziak
    En esta tesis hemos trabajado en dos estrategias, un cribado fenotípico y otro frente a una diana específica, que podrían ayudar a ampliar las alternativas terapéuticas frente a S. aureus. En el primer capítulo, nos hemos centrado en la capacidad de esta bacteria para formar biofilms, y en el problema que esto implica. En el segundo capítulo, nos hemos fijado un objetivo alternativo para 'desarmar' a S. aureus, centrándonos en la participación del sistema de dos componentes (TCS) GraXRS en la capacidad de S. aureus para contrarrestar las barreras del sistema inmune innato del huésped.
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    PublicationOpen Access
    Overexpression of the recA gene decreases oral but not intraperitoneal fitness of Salmonella enterica
    (American Society for Microbiology, 2010) Medina-Ruiz, Laura; Campoy Sánchez, Susana; Latasa Osta, Cristina; Cardenas, Paula; Alonso, Juan Carlos; Barbé, Jordi; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
    Transcription of the Salmonella enterica recA gene is negatively controlled by the LexA protein, the repressor of the SOS response. The introduction of a mutation (recAo6869) in the LexA binding site, in the promoter region of the S. enterica ATCC 14028 recA gene, allowed the analysis of the effect that RecA protein overproduction has on the fitness of this virulent strain. The fitness of orally but not intraperitoneally inoculated recAo6869 cells decreased dramatically. However, the SOS response of this mutant was induced normally, and there was no increase in the sensitivity of the strain toward DNA-damaging agents, bile salts, or alterations in pH. Nevertheless, S. enterica recAo6869 cells were unable to swarm and their capacity to cross the intestinal epithelium was significantly reduced. The swarming deficiency in recAo6869 cells is independent of the flagellar phase. Moreover, swimming activity of the recAo6869 strain was not diminished with respect to the wild type, indicating that the flagellar synthesis is not affected by RecA protein overproduction. In contrast, swarming was recovered in a recAo6869 derivative that overproduced CheW, a protein known to be essential for this function. These data demonstrate that an equilibrium between the intracellular concentrations of RecA and CheW is necessary for swarming in S. enterica. Our results are the first to point out that the SOS response plays a critical role in the prevention of DNA damage by abolishing bacterial swarming in the presence of a genotoxic compound.
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    PublicationOpen Access
    Relevant role of fibronectin-binding proteins in Staphylococcus aureus biofilm-associated foreign-body infections
    (American Society for Microbiology, 2009) Vergara Irigaray, Marta; Valle Turrillas, Jaione; Merino Barberá, Nekane; Latasa Osta, Cristina; García Martínez, Begoña; Ruiz de los Mozos Aliaga, Igor; Solano Goñi, Cristina; Toledo Arana, Alejandro; Penadés, José R.; Lasa Uzcudun, Íñigo; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua
    Staphylococcus aureus can establish chronic infections on implanted medical devices due to its capacity to form biofilms. Analysis of the factors that assemble cells into a biofilm has revealed the occurrence of strains that produce either a polysaccharide intercellular adhesin/poly-N-acetylglucosamine (PIA/PNAG) exopolysaccharide- or a protein-dependent biofilm. Examination of the influence of matrix nature on the biofilm capacities of embedded bacteria has remained elusive, because a natural strain that readily converts between a polysaccharide- and a protein-based biofilm has not been studied. Here, we have investigated the clinical methicillin (meticillin)-resistant Staphylococcus aureus strain 132, which is able to alternate between a proteinaceous and an exopolysaccharidic biofilm matrix, depending on environmental conditions. Systematic disruption of each member of the LPXTG surface protein family identified fibronectin-binding proteins (FnBPs) as components of a proteinaceous biofilm formed in Trypticase soy broth-glucose, whereas a PIA/PNAG-dependent biofilm was produced under osmotic stress conditions. The induction of FnBP levels due to a spontaneous agr deficiency present in strain 132 and the activation of a LexA-dependent SOS response or FnBP overexpression from a multicopy plasmid enhanced biofilm development, suggesting a direct relationship between the FnBP levels and the strength of the multicellular phenotype. Scanning electron microscopy revealed that cells growing in the FnBP-mediated biofilm formed highly dense aggregates without any detectable extracellular matrix, whereas cells in a PIA/PNAG-dependent biofilm were embedded in an abundant extracellular material. Finally, studies of the contribution of each type of biofilm matrix to subcutaneous catheter colonization revealed that an FnBP mutant displayed a significantly lower capacity to develop biofilm on implanted catheters than the isogenic PIA/PNAG-deficient mutant.
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    PublicationOpen Access
    Salmonella biofilm development depends on the phosphorylation status of RcsB
    (American Society for Microbiology, 2012) Latasa Osta, Cristina; García Martínez, Begoña; Echeverz Sarasúa, Maite; Toledo Arana, Alejandro; Valle Turrillas, Jaione; Campoy Sánchez, Susana; García del Portillo, Francisco; Solano Goñi, Cristina; Lasa Uzcudun, Íñigo; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua: IIM13329.RI1
    The Rcs phosphorelay pathway is a complex signaling pathway involved in the regulation of many cell surface structures in enteric bacteria. In response to environmental stimuli, the sensor histidine kinase (RcsC) autophosphorylates and then transfers the phosphate through intermediary steps to the response regulator (RcsB), which, once phosphorylated, regulates gene expression. Here, we show that Salmonella biofilm development depends on the phosphorylation status of RcsB. Thus, unphosphorylated RcsB, hitherto assumed to be inactive, is essential to activate the expression of the biofilm matrix compounds. The prevention of RcsB phosphorylation either by the disruption of the phosphorelay at the RcsC or RcsD level or by the production of a nonphosphorylatable RcsB allele induces biofilm development. On the contrary, the phosphorylation of RcsB by the constitutive activation of the Rcs pathway inhibits biofilm development, an effect that can be counteracted by the introduction of a nonphosphorylatable RcsB allele. The inhibition of biofilm development by phosphorylated RcsB is due to the repression of CsgD expression, through a mechanism dependent on the accumulation of the small noncoding RNA RprA. Our results indicate that unphosphorylated RcsB plays an active role for integrating environmental signals and, more broadly, that RcsB phosphorylation acts as a key switch between planktonic and sessile life-styles in Salmonella enterica serovar Typhimurium.