Genetic Influences on Salmonella Formation

heritable Influences on Salm sensationlla FormationIHF Gene Influences Salmonella Enteritidis Biofilm FormationIntegration Host chemical element out(p) (IHF) is pregnant for biofilm system by Salmonella enterica EnteritidisBruna Leite, Catierine Hirsch Werle, Camila Pinheiro do Carmo, Diego Borin Nbrega, Guilherme Paier Milanez, Cristina E. Alv atomic number 18z-Martinez, Marcelo BrocchiAbstractSalmonella enterica Enteritidis forms biofilms and survives in agricultural environments where it infects bird and testis. Once established, biofilms are difficult to eradicate, due to their naughty oppositeness compa violent to planktonic stalls, ca utilize serious problems in industry and human beings health. In this subject field, we evaluated biofilm ecesis in wild-type separate outs of S. enterica Enteritidis and in ihf pas seuls employing different microbiology techniques. Our data indicate that ihf magnetic variations intro impaired biofilm governing body, with a r educed of matrix formation and a decline in CFU and metabolous activity. Phe nonypic analysis indicated a deficiency in curli fimbriae flavour and in cellulose lap and pellicle formation. These results show that IHF has a restrictive single-valued function in biofilm formation in S. enterica Enteritidis.Keywords Biofilm, Salmonella enterica Enteritidis, Polysaccharide matrix, Curli fimbriae, kioskulose, Integration Host Factor. first appearanceA biofilm is defined as a bacteriuml colony aider to a solid surface, which secretes a protective exopolysaccharide matrix. Every natural unwavering surface is a potential substrate for microbial biofilms. These sessile cellular microbial consortia are embedded within self- crapd extracellular polymeric substances (EPS). In food handling facilities, biofilms brook be oddly badThe baron to form biofilms is in any(prenominal) case an important factor in the severity of S. Enterica. S. enterica subspecies I serovar Enteritidis i s a leading convey of salmonellosis worldwide, and has emerged as one of the virtually important foodborne pathogens for humans. It is mainly associated with consumption of contaminated meat and eggs of poultry. A number of studies flip instituted that S. enterica is capable of forming biofilms on a wide variety of contact surfaces, and the formation of biofilms may improve the ability of these organisms to resist stresses much(prenominal) as desiccation, extreme temperatures, antibiotics, and antiseptics. Biofilm formation in allows S. enterica to survive for wide periods in a poultry farm environment and to contaminate poultry meat and eggs, which remain the leading vehicles of salmonellosis outbreaksMany factors are involved in biofilm development. Curli fimbriae and cellulose are the major components of biofilm organize by S. enterica, whereas capsular polysaccharide, other polysaccharide-rich compounds such as lipopolyssaccharide (LPS), and a large secreted protein, Bap A, likewise contrisolelye to biofilm formation. Several regulatory components involved in biofilm formation incur been identifiedThe expression of curli fimbriae and cellulose grass be assayed phe nonypicalally by increase enteric bacteria on congo red indicator platesBacteria may live in planktonic form in liquid media or as biofilms on biotic or abiotic surfaces. They need to adjust their constituenttic programs in order to discombobulate from one lifestyle to another. The production of bacterial products and behaviours associated with environmental adaptation mustiness be tightly coordinated to optimize the energy consumption. In bacteria, gene expression regulation is exerted primarily at the level of transcription fountain using a large array of transcription factors whose concentrations and activities change depending on specific environmental or metabolic signals. Topological changes in desoxyribonucleic acid also influence promoter recognition, open complex format ion, and gene expressionNucleoid-associated proteins ( snoozes) are global regulators of gene expression in bacteria. They alter the topology of deoxyribonucleic acid by bending, bridging, or wrapping it, leading to deoxyribonucleic acid transactions and three-fold cellular effects that culminate in the modulation of gene expression. Integration-host factor (IHF) is a dimeric NAP that binds deoxyribonucleic acid in a sequence-specific manner and introduces curvatures of up to 180, which influence many aspects of bacterial physiology, including global gene expression, DNA topology, site-specific recombination, and DNA replication. In E. coli and S. enterica Typhimurium, the two IHF subunits-IHF and IHF- deal assemble as hetero- or homo-dimers. at that place is also evidence indicating that the different dimeric forms of IHF regulate different but lapping sets of genesBased on the global regulatory role of IHF, we hypothesized that this NAP can influence or directly regulate gen es involved in biofilm formation in S. enterica Enteritidis. This hypothesis is supported by foregoing observations demonstrating that IHF activates curli production in S. enterica Typhimurium. Therefore, in this study, we evaluated the role of IHF genes in the initial stages of biofilm formation in S. Enteritidis. To this end, we performed phenotypic studies using isogenic deletion mutants of individual ihf genes (ihfA or ihfB) and a paradigm mutant telephone line with deletions in both IHF subunits (ihfAB double mutant).Materials and mannersbacterial examinesIn this study, the S. enterica Enteritidis wild-type physical body PT4SEn (IOC4647) provided a by the Fundao Oswaldo Cruz (FIOCRUZ, Rio de Janeiro, Brazil) was used. The draft genome of this strain was recently produce (Milanez et al. 2016). It was found to be pathogenic in a mouse good example assay (Carmo et al., unpublished results). The mutants of S. Enteritidis PT4SEn were previously constructed (Carmo et al., un published results) by deletion of ihf genes using the lambda Red system by transduction with P22HT phages. Mutant strains were designated as S. enterica Enteritidis PT4SEn ihfA, PT4SEn ihfB, and PT4SEn ihfAB.Bacterial step-up conditions and storageBacteria were cultivated in Luria-Bertani broth (LB) and on Luria-Bertani agar (LBA) plates prepared according to the method of Sambrook and Russell. All strains were stored at -80C in 30% glycerolAll strains were inoculated from fresh LBA plates into 15 mL LB and grown for 18 2 h at 37C in an orbital shaker at 140 rpm. Cells were harvested by centrifugation (for 5 min at 9,500 g and 4C) and resuspended in NaCl (0.9%) adjusted to 0.5 McFarland outdo equivalent to 1.5 108 cells/mL prior to use in subsequent assays. complementation of S. enterica Enteritidis ihfA and ihfB mutantsSequences corresponding to the ihfA and ihfB genes and their regulatory regions were obtained by PCR from the PT4SEn genome using the primers listed in Table 1 . The DNA fragments were cloned in the pACYC184 sender (New England Biolabs, USA) betwixt the NcoI and EcoRI restriction sites (restriction enzyme sites in the DNA fragments were introduced via the primers) and the vector was subsequently electroporated into the measureive S. enterica Enteritidis mutant strains. Cloning, PCR amplification, electroporation, plasmid extraction, and agarose gel electrophoresis were performed as suggested by Sambrook and Russell ( two hundred1). After DNA purification using the Wizard Genomic DNA Purification Kit (Promega Corporation, Madison, USA), Sanger sequencing was performed using 3730XL Applied Biosystems (Foster City, California, USA) by the High surgical operation Technologies Central Laboratory in Life Sciences (LACTAD, University of Campinas UNICAMP, Campinas, Brazil).Biofilm formation on polystyrene platesBiofilms were formed in 96-well plates (Cell Culture Plate, Nest, Biotechnology Co, mainland China) containing 200 L of cell abateme nt (1 106 cells/mL) of S. enterica Enteritidis PT4SEn wild-type or mutant strains in LB supplemented with 0.25% of glucose. Plates were incubated at 37C with orbital shaking at 140 rpm for 48, 72, and 120 h. At the end of the brooding period, planktonic cells were carefully removed, and biofilms were washed twice with 200 L of saline solution (0.9% NaCl).The crystal purplish spotting method was used to assess total biofilm biomass. Each well of the biofilm plates was incubated with 200 L of methanol for 15 minutes. Subsequently, methanol was removed and 1% (v/v) crystal violet solution was added, followed by a 5-min incubation period. Wells were washed with distilled irrigate and finally 33% (v/v) acetic acid was added. The absorbance was mensural at 570 nm.The colorimetric method based on the reduction of XTT (2,3- bis(2-methoxy-4-nitro-5-sulfophenyl)-5-(phenylamino)carbonyl-2H tetrazolium hydroxide Sigma-Aldrich, USA) was used to nail down cell activity (XTT is converted to a glossed formazan salt in the presence of metabolic activity). To each well of the biofilm plate, 200 L of a solution containing 200 mg/L of XTT and 20 mg/L of phenazinemethosulphate (PMS Sigma-Aldrich, Ukraine) was added. Microtiter plates were incubated for 3 h at 37C in the dark. The absorbance was measured at 490 nm.To assess the number of viable cells in biofilms, 200 L of saline solution was added to each well before removal of the biofilm by scraping. For each sample, an aliquot of 1 mL (5 wells) was sonicated (20 s with 22% of bountifulness Ultrasonic Processor, Cole-Parmer, Illinois, USA) to promote biofilm disruption. The number of colony forming units (CFU) in biofilms was contumacious by performing 10-fold serial dilutions in saline solution, plating on LBA plates in triplicate, and incubating for 24 h.Scanning electron micros re-create (SEM) of biofilm cellsBiofilms of S. enterica Enteritidis PT4SEn wild-type and mutant strains formed in 24-well plates (Well Cell Cul ture Cluster, Costar) were dehydrated by a 15-min immersion in change magnitude ethanol concentrations (70, 95, and 100% ethanol v/v) and placed in sealed desiccators. The samples were attach on aluminium stubs with carbon tape, sputter-coated with gold, and analysed with a JEOL JSM-5800LV scanning microscope. All experiments were carried out in duplicate.Biofilm formation at the air-liquid port wineBiofilm formation at the air-liquid interface was assessed in S. enterica Enteritidis PT4SEn strains by inoculation of LB cultures without NaCl, followed by incubation at 28C without shaking. Every day for 10 geezerhood, each keep apart was visually examined for pellicle formation. Experiments were performed in triplicate.Expression of curli fimbriaeBacterial colony morphology of S. enterica Enteritidis PT4SEn wild-type and mutant strains was analysed on LB agar without NaCl, supplemented with Congo red (1.01340.0025, Sigma-Aldrich, Germany 40 g/mL) and Coomassie brilliant blue G (B 0770-5G, Sigma-Aldrich, China 20 g/mL). Bacterial cultures were spread on agar plates and the colour and degree of colony rugosity were determined afterwards 96 h of yield at 28C. Images were captured with a camera (Nikon P500) and under an HBO 100 Carl Zeiss Illuminating microscope system.Cellulose productionThe fluorescence exhibited by bacteria after growth of S. enterica Enteritidis PT4SEn wild-type and mutant strains in LB plates with Calcofluor (Fluorescent Brightener 28 F3543-1G, Sigma-Aldrich, China 200 g/mL) served as an indicator of cellulose production. Fluorescence was analysed visually using an UV light (366 nm) after 48 h of growth at 37C.Statistical analysisData were analysed using STATA software, version 13.0 (Stata Corp, College Station, TX, USA). Data from all assays were compared using one-way analysis of variance (ANOVA). Sidaks adjustment for multiple equations was performed after a real fitting. The significance level was set at 5%.Resultsihf mutants displa y reduced viability, biomass, and metabolic activityA decrease of rough 1-2 log10 in number of viable cells was ascertained for the ihf mutants in comparison with the wild-type S. enterica Enteritidis PT4SEn strain by CFU counting (Figure 1-A). The differences observed were statistically significant (P 0.05) for all periods of fourth dimension evaluated. The introduction of the pACYC184 plasmid carrying ihfA or ihfB was primarily associated with an increase in CFUs, but complementation did not completely restore the determine to those obtained with the wild-type strain. No statistical differences were observed at 48 and 72 h of incubation between ihfAc and the wild-type strain. The same observation is valid for ihfB after 120 h of incubation (Figure 1-A). These results show that the restoration of ihfA or ihfB gene copies in mutant strains is generally associated with an increase in CFUs in biofilms.The total biofilm biomass, assessed by CV staining of S. enterica Enteritidis P T4SEn and mutant strains is presented in Figure 1-B. An increase in biomass is observed for the wild-type strain over time. However, this effect was not observed for the correspondent PT4SEn ihfAB double mutant. no(prenominal) of the mutants presented an increase in biofilm matrix stringency at 48 and 72 h of incubation (P 0.05). The complemented PT4SEn ihfA and ihfB mutants (ihfAc and ihfBc) showed an increase in total biofilm biomass in comparison to the non-complemented mutants (Figure 1-B).All mutant strains exhibited a significant reduction in metabolic activity measured by the XTT assay for cells in biofilm (P 0.05). In addition, the double mutant (ihfAB) showed the greatest reduction in metabolic activity at 72 and 120 h (Figure 1-C).ihf genes are essential for biofilm structureTo gain characterize biofilm formation and structure in strains lacking ihf genes, we performed scanning electron microscopy (SEM) analysis of cells in biofilms. As shown in Figure 2, the absence of ihfA or ihfB drastically affects biofilm formation, as null mutants of S. enterica Enteritidis PT4SEn (Figure 2-D, E and F) exhibited a low meat of matrix and small number of cells compared to the wild-type (Figure 2-A). Complementation of ihf gene deletions by a wild-type copy of the corresponding gene promoted a significant restoration of biofilm formation (Figure 2-B and C).Pellicle formation at the air-liquid interfaceTo advertise characterize the mutant strains with respect to their ability to form biofilms we analysed the biofilm formation at the air-liquid interface of cultures of the different strains. Cultures of the wild-type strain formed a thick and rigid pellicle after 10 days of growth (Figure 3-A). On the other hand, PT4SEn ihfA or PT4SEn ihfB mutant strains formed a less compact and fragile pellicle (not shown). Interestingly, the double mutant strain PT4SEn ihfAB did not form a visible pellicle at all at the air-liquid interface. Instead, cell deposition was ob served at the bottom of the tube (Figure 3-B). Complementation with the wild-type copy of ihfA and ihfB restored the phenotype of the single mutants (PT4SEn ihfAc and PT4SEn ihfBc strains), which now formed a thick and rigid pellicle (not shown).Curli and celluloseSince curli and cellulose are important components in biofilm formation, we evaluated the role of IHF on their production. To this end, colony morphology was analysed on LBA plates supplemented with Congo red and Coomassie brilliant blue, as previously described.. enterica Enteritidis PT4SEn wild-type and PT4SEn ihfA and ihfB complemented strains exhibited a phenotype legitimate with curli fimbriae and cellulose production, with red, dry, and rough (rdar) colony morphology (Figure 4-A to D). However, the PT4SEn ihfA, PT4SEn ihfB, and PT4SEn ihfAB mutants of S. enterica Enteritidis did not display the same colour and roughness, but instead exhibited a similar, but not identical, smooth and color (saw) morphotype, indicati ng a deficiency in the expression of curli fimbriae and probably also of cellulose (Figure 4-E to H). The expression of cellulose was also tested by screening the colonies for Calcofluor binding Cellulose production was observed for all strains evaluated by this method, except for the double mutant ihfAB that was not light under an UV light source and was considered a poor manufacturer of cellulose (Figure 5).DiscussionThe presence of microorganisms on food contact surfaces is one of the most common courtships of food spoilage and transmission of foodborne diseases. Inadequate cleaning and disinfection of food-processing environments is the cause of major economic losses and represents a serious danger to public health. The ability of microorganisms to adhere and form biofilms makes disinfection even more difficult and ambitious Infections with Salmonella enterica Enteritidis represent a major health problem and a significant burden on the food industry. About 80% of the infecti ons are caused by biofilm formation In the matrix of a biofilm, bacteria grow on either biotic or abiotic surfaces, attaching to the surface and to each other, conferring resistance to liberty responses as well as healthful agents As a consequence, antimicrobial treatments typically fail to eradicate biofilms. The need to create effective therapies to prevent biofilm infections is a pressing challenge in the food industryThe growing interest in understanding the regulatory network of gene activities during the enactment from a planktonic to a sessile cellular lifestyle, prompted us to check up on the role of IHF in S. enterica Enteritidis biofilm formation. IHF has an important role in the regulation of gene expression and environment adaptability of S. Enterica Therefore, S. Enteritidis deletion mutants for ihfA, ihfB, or both genes (ihfAB) were employed in different assays to analyse biofilm formation. The logic behind this approach is based on the fact that IHF can act as a homodimer (IHF or IHF) or as a heterodimer (IHF) The results presented here indicate an important role of this NAP in the formation of biofilms in S. enterica Enteritidis.All typical biofilm characteristics analysed in this study (CFU, biomass, and cellular metabolic activity) were significantly decreased in S. enterica Enteritidis mutant strains for ihfA, ihfB, or ihfAihfB. The biofilms formed by mutant strains exhibited a decreased matrix density compared with the wild-type strain. Therefore, these results indicate that IHF can influence the initial stage of biofilm formation by S. enterica Enteritidis, as the matrix is necessary in this phase. This is also supported by CV staining and SEM.The colony morphotypes observed in Congo red among wild-type and complemented strains exhibited the rdar morphotype, an indication of curli and cellulose production, patch the mutant strains exhibited a similar but not identical saw morphotype, suggesting an altered expression of curli and prob ably also of cellulose. In fact, bacterial growth in calcofluor-containing medium indicated that the single ihf-mutants were able to produce cellulose, but the ihf-double mutant exhibited some deficiency in the production of this polysaccharide.Previously, Gerstel, Park, and Rmling demonstrated that the ihfAB double mutant of two S. enterica Typhimurium strains caused a reduction in CsgD expression and an altered rdar morphotype suggesting a role for IHF in curli expression in S. enterica Typhimurium. Curli is expressed by two divergent operons, csgBAC and csgDEFG. CsgD is a major regulator of curli expression and biofilm formation. This gene activates transcription of csgA and csgB that encodes the major (CsgA) and the minor (CsgB) curli subunits In addition, csgD also regulates cellulose production Therefore, IHF plays an important role in biofilm formation in S. enterica Typhimurium. Our results demonstrate a similar role for IHF in the biofilm formation of S. enterica Enteritidi s. Despite high genetic similarity, the Enteritidis and Typhimurium serovars differ in various ecological and host-relationship parameters However, the regulation of biofilm formation by IHF in both serovars suggests that IHF plays a central role in S. enterica biofilm biogenesis. However, additional studies of IHF function on biofilm biogenesis in other S. enterica serovars are needed to just clarify this question. In addition, the single ihf mutants also exhibited a phenotypic alteration in biofilm formation, indicating that both subunits are necessary for captivate biofilm production. In our results, all the ihf mutants showed a deficiency for curli fimbriae production by phenotypic tests. To some extent, a deficiency in cellulose production was also observed, particularly in the double ihf-mutant.The complementation of the ihfA and ihfB mutants by the introduction of a pACYC184 plasmid carrying the wild-type genes reverted the deficiency in biofilm biomass, cell metabolism, an d CFUs, but in the majority of the tests the values did not reach those observed for the wild-type strain. This is probably due to a dot effect of IHF or IHF, despite the low copy number (about 15 copies per cell) of the plasmid used. In fact, the expression of ihf genes is finely regulated and depends on the growth phaseThe two operons bcsABZC and bcsEFG are responsible for cellulose biosynthesis in both S. enterica Enteritidis and S. enterica Typhimurium. This was demonstrated by the construction of non-polar mutants of bcsC and bcsE genes that formed a fragile pellicle at the air-liquid interface of LB medium The same authors also showed that cellulose-deficient mutants were more raw to chlorine treatments, indicating that the deficiency in the production of extracellular matrix can leave the cells more susceptible to the action of some chemical agents. In our study, IHF mutant strains formed a less compact pellicle in LB compared to wild-type strains. In addition, the ihf doub le mutant did not form an air pellicle at all, suggesting a role for IHF in the expression of cellulose. These findings corroborate a previous study in which S. enterica Typhimurium ihfAB mutants exhibited reduced bcsC transcription when evaluated by microarray analysis, but further studies are needed to better characterize the underlying molecular mechanisms.Karaca, N Akcelik, and M Akcelik (2013) also evaluated pellicle formation at the air-liquid interface of 31 S. enterica isolates. They showed that the growth rate of isolates with a rigid pellicle was greater than that of the ones forming a fragile pellicle. Biofilm production at the air-liquid interface can facilitate and contribute to gas exchange, while enabling the acquisition of nutrients and water from the liquid phase. Biofilms at air-liquid and solid-air interfaces can cause serious problems in industrial water systems.In conclusion, our results indicate that IHF has an important regulatory role in biofilm formation of S. enterica serovar Enteritidis. Moreover, both IHF subunits appear to have a role in this process. Our data pave the way for further studies investigating the mechanisms involved in the regulation of biofilm formation by IHF.AcknowledgementsThis work was supported by grants from Fundao de Amparo Pesquisa do Estado de So Paulo (FAPESP 2014/13412-8) and Conselho Nacional de Desenvolvimento Cientfico e Tecnolgico (CNPq), Brazil. BL, DBN, and GPM were supported by a FAPESP fellowship (FAPESP 2012/25426-8, 2012/10608-3, and 2012/05382-6, respectively). CHW and CPC were supported by fellowships from CNPq (141629/2012-6 and 140786/2012-0, respectively). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest or conflict.