Immunoassay method to check the flagellin mediated binding of <Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis> to polystyrene

Plate count and spectrophotometric methods have been used to asses the ability of an organism to attach to different surfaces and form biofilms. In the present study we report a highly sensitive, specific and quick method to check the role of flagellin in bacterial adhesion to polystyrene. Flagellin from Stenotrophomonas maltophilia showed high affinity for polystyrene (P < 0.05), which decreased on pretreatment of flagellin with anti-flagellin in a dilution dependent manner. In an enzyme immunoassay format a positive correlation was detected between the anti-flagellin dilutions and flagellin attachment to polystyrene (correlation coefficient +0.860155). These evidences conclusively prove the involvement of flagella in the adhesion of S. maltophilia to polystyrene surface and enzyme immunoassay, a quick and reliable method to check this phenomenon.     

Immunoassay method to check the flagellin mediated binding of stenotrophomonas maltophilia to polystyrene

Plate count and spectrophotometric methods have been used to asses the ability of an organism to attach to different surfaces and form biofilms. In the present study we report a highly sensitive, specific and quick method to check the role of flagellin in bacterial adhesion to polystyrene. Flagellin from Stenotrophomonas maltophilia showed high affinity for polystyrene (P < 0.05), which decreased on pretreatment offlagellin with anti-flagellin in a dilution dependent manner. In an enzyme immunoassay format a positive correlation was detected between the anti-flagellin dilutions and flagellin attachment to polystyrene (correlation coefficient +0.860155). These evidences conclusively prove the involvement of flagella in the adhesion ofS. maltophilia to polystyrene surface and enzyme immunoassay, a quick and reliable method to check this phenomenon.     

Immunological and biological relationship among flagellin of <Emphasis Type="Italic">Pseudomonas aeruginosa,Burkholderia cepacia</Emphasis>, and <Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis>

The flagellar protein (flagellin) was isolated and purified from strains of Pseudomonas aeruginosa, Burkholderia cepacia and Stenotrophomonas maltophilia. A significant difference was observed in the molecular weight of different flagellin preparations obtained from these bacterial isolates. Antiserum prepared against S. maltophilia flagellin did not react with flagellin of P. aeruginosa or/and B. cepacia on Immunoblot or in indirect ELISA. In addition the anti-flagellin did not agglutinate P. aeruginosa and B. cepacia. No inhibition of motility of P. aeruginosa and B. cepacia was observed in presence of antiserum; though the latter inhibited the motility of S. maltophilia. The results of the present study prove that no specific relationship existed among all the studied flagellar proteins obtained from closely related bacteria.     

Functional Activity of Antibodies Directed towards Flagellin Proteins of Non-Typhoidal Salmonella

Non-typhoidal Salmonella (NTS) serovars Typhimurium and Enteritidis are major causes of invasive bacterial infections in children under 5 years old in sub-Saharan Africa, with case fatality rates of ~20%. There are no licensed NTS vaccines for humans. Vaccines that induce antibodies against a Salmonella Typhi surface antigen, Vi polysaccharide, significantly protect humans against typhoid fever, establishing that immune responses to Salmonella surface antigens can be protective. Flagella proteins, abundant surface antigens in Salmonella serovars that cause human disease, are also powerful immunogens, but the functional capacity of elicited anti-flagellar antibodies and their role in facilitating bacterial clearance has been unclear. We examined the ability of anti-flagellar antibodies to mediate microbial killing by immune system components in-vitro and assessed their role in protecting mice against invasive Salmonella infection. Polyclonal (hyperimmune sera) and monoclonal antibodies raised against phase 1 flagellin proteins of S. Enteritidis and S. Typhimurium facilitated bacterial uptake and killing of the homologous serovar pathogen by phagocytes. Polyclonal anti-flagellar antibodies accompanied by complement also achieved direct bacterial killing. Serum bactericidal activity was restricted to Salmonella serovars expressing the same flagellin used as immunogen. Notably, individual anti-flagellin monoclonal antibodies with complement were not bactericidal, but this biological activity was restored when different monoclonal anti-flagellin antibodies were combined. Passive transfer immunization with a monoclonal IgG antibody specific for phase 1 flagellin from S. Typhimurium protected mice against lethal challenge with a representative African invasive S. Typhimurium strain. These findings have relevance for the use of flagellin proteins in NTS vaccines, and confirm the role of anti-flagellin antibodies as mediators of protective immunity.     

The repeatability of feed intake and feed efficiency in beef cattle offered high-concentrate,grass silage and pasture-based diets

Breeding values for feed intake and feed efficiency in beef cattle are generally derived indoors on high-concentrate (HC) diets. Within temperate regions of north-western Europe, however, the majority of a growing beef animal’s lifetime dietary intake comes from grazed grass and grass silage. Using 97 growing beef cattle, the objective of the current study was to assess the repeatability of both feed intake and feed efficiency across 3 successive dietary test periods comprising grass silage plus concentrates (S+C), grazed grass (GRZ) and a HC diet. Individual DM intake (DMI), DMI/kg BW and feed efficiency-related parameters, residual feed intake (RFI) and gain to feed ratio (G : F) were assessed. There was a significant correlation for DMI between the S+C and GRZ periods (r = 0.32; P < 0.01) as well as between the S+C and HC periods (r = 0.41; P < 0.001), whereas there was no association for DMI between the GRZ and HC periods. There was a significant correlation for DMI/kg BW between the S+C and GRZ periods (r = 0.33; P < 0.01) and between the S+C and HC periods (r = 0.40; P < 0.001), but there was no association for the trait between the GRZ and HC periods. There was a significant correlation for RFI between the S+C and GRZ periods (r = 0.25; P < 0.05) as well as between S+C and HC periods (r = 0.25; P < 0.05), whereas there was no association for RFI between the GRZ and HC periods. Gain to feed ratio was not correlated between any of the test periods. A secondary aspect of the study demonstrated that traits recorded in the GRZ period relating to grazing bite rate, the number of daily grazing bouts and ruminating bouts were associated with DMI (r = 0.28 to 0.42; P < 0.05 - 0.001), DMI/kg BW (r = 0.36 to 0.45; P < 0.01 - 0.001) and RFI (r = 0.31 to 0.42; P < 0.05 - 0.001). Additionally, the number of ruminating boli produced per day and per ruminating bout were associated with G : F (r = 0.28 and 0.26, respectively; P < 0.05). Results from this study demonstrate that evaluating animals for both feed intake and feed efficiency indoors on HC diets may not reflect their phenotypic performance when consuming conserved forage-based diets indoors or when grazing pasture.     

Identification of the Flagellin Glycosylation System in Burkholderia cenocepacia and the Contribution of Glycosylated Flagellin to Evasion of Human Innate Immune Responses

Burkholderia cenocepacia is an opportunistic pathogen threatening patients with cystic fibrosis. Flagella are required for biofilm formation, as well as adhesion to and invasion of epithelial cells. Recognition of flagellin via the Toll-like receptor 5 (TLR5) contributes to exacerbate B. cenocepacia-induced lung epithelial inflammatory responses. In this study, we report that B. cenocepacia flagellin is glycosylated on at least 10 different sites with a single sugar, 4,6-dideoxy-4-(3-hydroxybutanoylamino)-d-glucose. We have identified key genes that are required for flagellin glycosylation, including a predicted glycosyltransferase gene that is linked to the flagellin biosynthesis cluster and a putative acetyltransferase gene located within the O-antigen lipopolysaccharide cluster. Another O-antigen cluster gene, rmlB, which is required for flagellin glycan and O-antigen biosynthesis, was essential for bacterial viability, uncovering a novel target against Burkholderia infections. Using glycosylated and nonglycosylated purified flagellin and a cell reporter system to assess TLR5-mediated responses, we also show that the presence of glycan in flagellin significantly impairs the inflammatory response of epithelial cells. We therefore suggest that flagellin glycosylation reduces recognition of flagellin by host TLR5, providing an evasive strategy to infecting bacteria.     

Phosphoglycerate mutase affects Stenotrophomonas maltophilia attachment to biotic and abiotic surfaces

Stenotrophomonas maltophilia biofilm formation is of increasing medical concern, particularly for lung infections. However, the molecular mechanisms facilitating the biofilm lifestyle in S. maltophilia are poorly understood. We generated and screened a transposon mutant library for mutations that lead to altered biofilm formation compared to wild type. One of these mutations, in the gene for glycolytic enzyme phosphoglycerate mutase (gpmA), resulted in impaired attachment on abiotic and biotic surfaces. As adherence to a surface is the initial step in biofilm developmental processes, our results reveal a unique factor that could affect S. maltophilia biofilm initiation and, possibly, subsequent development.     

The complete genome, comparative and functional analysis of Stenotrophomonas maltophilia reveals an organism heavily shielded by drug resistance determinants

Background

Stenotrophomonas maltophilia is a nosocomial opportunistic pathogen of the Xanthomonadaceae. The organism has been isolated from both clinical and soil environments in addition to the sputum of cystic fibrosis patients and the immunocompromised. Whilst relatively distant phylogenetically, the closest sequenced relatives of S. maltophilia are the plant pathogenic xanthomonads.

Results

The genome of the bacteremia-associated isolate S. maltophilia K279a is 4,851,126 bp and of high G+C content. The sequence reveals an organism with a remarkable capacity for drug and heavy metal resistance. In addition to a number of genes conferring resistance to antimicrobial drugs of different classes via alternative mechanisms, nine resistance-nodulation-division (RND)-type putative antimicrobial efflux systems are present. Functional genomic analysis confirms a role in drug resistance for several of the novel RND efflux pumps. S. maltophilia possesses potentially mobile regions of DNA and encodes a number of pili and fimbriae likely to be involved in adhesion and biofilm formation that may also contribute to increased antimicrobial drug resistance.

Conclusion

The panoply of antimicrobial drug resistance genes and mobile genetic elements found suggests that the organism can act as a reservoir of antimicrobial drug resistance determinants in a clinical environment, which is an issue of considerable concern.     

Hydrophobin coating prevents Staphylococcus epidermidis biofilm formation on different surfaces

Staphylococcus epidermidis is a significant nosocomial pathogen in predisposed hosts because of its capability of forming a biofilm on indwelling medical devices. The initial stage of biofilm formation has a key role in S. epidermidis abiotic surface colonization. Recently, many strategies have been developed to create new anti-biofilm surfaces able to control bacterial adhesion mechanisms. In this work, the self-assembled amphiphilic layers formed by two fungal hydrophobins (Vmh2 and Pac3) have proven to be able to reduce the biofilm formed by different strains of S. epidermidis on polystyrene surfaces. The reduction in the biofilm thickness on the coated surfaces and the preservation of cell vitality have been demonstrated through confocal laser scanning microscope analysis. Moreover, the anti-biofilm efficiency of the self-assembled layers on different medically relevant materials has also been demonstrated using a CDC biofilm reactor.     

Adhesion to and biofilm formation on IB3-1 bronchial cells by <Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis> isolates from cystic fibrosis patients

Background  

Stenotrophomonas maltophilia has recently gained considerable attention as an important emerging pathogen in cystic fibrosis (CF) patients. However, the role of this microorganism in the pathophysiology of CF lung disease remains largely unexplored. In the present study for the first time we assessed the ability of S. maltophilia CF isolates to adhere to and form biofilm in experimental infection experiments using the CF-derived bronchial epithelial IB3-1cell line. The role of flagella on the adhesiveness of S. maltophilia to IB3-1 cell monolayers was also assessed by using fliI mutant derivative strains.     

Biofilm production of clinical isolates ofStenotrophomonas maltophilia altered by sodium phosphate buffer supplementation of the medium

Biofilm plays an important role in the pathogenicity of the bacteriumStenotrophomonas maltophilia. When sodium phosphate buffer (SPB) supplemented the Luria-Bertani media of 11 clinical isolates of this bacterium, mean growth at 30 h was little changed compared to non-supplemented controls in 45% of the isolates, decreasing by 4%. Increase in mean biofilm formation in that low growth change sub-sample was statistically significant ( $\underline t = 5.0$ , dt 4,p < 0.008). For a further 36% of isolates, growth at 30 h with SPB supplementation was decreased by 8.5% of controls. Decrease in biofilm formation was seen in that sub-sample compared to controls ( $\underline t = 4.0$ , df 3,p < 0.03). The significant phenotypical variety of the clinical presentation of this pathogen may allow population adaptability to varying metabolic provision, improving its pathogenicity.     

Interference of sanitizers,NaCl and curing salts on Listeria monocytogenes adhesion and subsequent biofilm formation

Listeria monocytogenes, a well-known foodborne pathogen and the causative agent of listeriosis, has the ability to persist in food processing environments due to its high adhesion ability in different surfaces, playing an important role in the food industry. The aim of this study was to assess how the main stressing conditions, usually observed in meat processing facilities (sanitizers, NaCl, curing salts), interfere in L. monocytogenes adhesion and biofilm formation. The isolates, representatives of different L. monocytogenes lineages (n = 6) were subjected to four different sanitizers (S1: quaternary ammonium; S2: peracetic acid, hydrogen peroxide and glacial acetic acid, S3: biguanide polyhexamethylene hydrochloride, S4: hydrogen peroxide) to verify adhesion ability and susceptibility based on minimum inhibitory concentration (MIC). In addition, the isolates adhesion and biofilm were assessed up to 72 h under different conditions: sanitizers (MIC values), curing salts and NaCl (both at 5, 7·5, 10%), at different temperatures (4, 12 and 37°C). Despite the effectiveness of sanitizers, isolates presented higher biofilm development when compared to controls in the presence of quaternary ammonium (S1, 1: 1,024) at 4°C, over the tested time (P < 0·05). Furthermore, different responses were observed for the different L. monocytogenes strains tested, providing a better understanding of the persistence of this pathogen in the food processing facilities.     

Sensitivity of nonfermentative gram-negative bacteria to essential oils of different origin

The sensitivity of MDR (multi drug resistant) strains of Stenotrophomonas maltophilia, Acinetobacter baumannii, and Pseudomonas aeruginosa to essential oils and their individual components was studied; bacteriostatic and bactericidal concentrations of 16 substances were determined. Crimean rose oil exhibited the highest activity, with the minimum inhibitory concentration of 1.95 μL/ mL. Growth of bacterial batch cultures in the presence of subinhibitory concentrations of essential oils or their individual components was studied. Kinetic models analysis revealed positive correlations of growth characteristics of the studied bacteria the effects of essential oils (p-level < 0.05). Correlations between lag phase duration and the death of bacterial cultures and correlations between the concentration of linalool (a component of essential oils) and the degree of growth suppression for S. maltophilia and A. baumannii were revealed.     

Effect of monospecific and mixed Cunninghamia lanceolata plantations on microbial community and two functional genes involved in nitrogen cycling

Conversion of native broadleaf forest (NF) and introduction of broadleaf species into monospecific Cunninghamia lanceolata plantations are silvicultural practices driven by the increasing demand for timber production. This study was conducted to evaluate the impacts of successive planting of C. lanceolata and mixed plantations (C. lanceolata-Michelia macclurei, CFM; C. lanceolata-Alnus cremastogyne, CFA; C. lanceolata-Kalopanax septemlobus, CFK) on microbial community diversity. Microbial biomass (MBC) was assessed using chloroform fumigation-extraction. Using denaturing gradient gel electrophoresis (DGGE), we examined the biodiversity within eubacterial (16S rRNA gene) and fungal (28S rRNA gene) species and two genes involved in N cycling: nifH and amoA. Microbial community diversities and microbial biomass decreased as NF was substituted by successive plantings of C. lanceolata plantations, whereas the trend reversed after introducing the broadleaf, M. macclurei, into pure C. lanceolata plantations. A strong positive correlation between MBC changes and total organic C (TOC), total organic N (TON), available N and extractable C (C_ext) were seen, which suggests that MBC was tightly coupled with the content of soil organic matter. The Shannon index showed that bacterial diversity and two functional genes (nifH and amoA) diversities associated with monospecific C. lanceolata plantations were lower than that of NF or mixed C. lanceolata plantations, such as CFM and CFA, whereas the opposite was seen for fungal diversity. Bacterial diversity was positively correlated with pH, TOC, TON, C_ext and NH ₄ ⁺ -N; while fungal diversity was positively correlated with C/N ratio and negatively correlated with pH. Both nitrogen fixing and ammonia oxidizing bacterial diversities were positively correlated with pH. Thus, soil pH was not only significantly positively correlated with bacterial diversity (r?=?0.502, P?<?0.05), nifH gene diversity (r?=?0.564, P?<?0.01) and amoA gene diversity (r?=?0.659, P?<?0.001), but also negatively correlated with fungal diversity (r?=?? 0.505, P?<?0.05), which seemed to be responsible for the discrimination of the soil microbial communities among these plantations. These findings suggest that different silvicultural practices have significant impacts on the soil microbial community through influences on soil chemical properties.     

Native flagellin does not protect mice against an experimental Proteus mirabilis ascending urinary tract infection and neutralizes the protective effect of MrpA fimbrial protein

Proteus mirabilis expresses several virulence factors including MR/P fimbriae and flagella. Bacterial flagellin has frequently shown interesting adjuvant and protective properties in vaccine formulations. However, native P. mirabilis flagellin has not been analyzed so far. Native P. mirabilis flagellin was evaluated as a protective antigen and as an adjuvant in co-immunizations with MrpA (structural subunit of MR/P fimbriae) using an ascending UTI model in the mouse. Four groups of mice were intranasally treated with either MrpA, native flagellin, both proteins and PBS. Urine and blood samples were collected before and after immunization for specific antibodies determination. Cytokine production was assessed in immunized mice splenocytes cultures. Mice were challenged with P. mirabilis, and bacteria quantified in kidneys and bladders. MrpA immunization induced serum and urine specific anti-MrpA antibodies while MrpA coadministered with native flagellin did not. None of the animals developed significant anti-flagellin antibodies. Only MrpA-immunized mice showed a significant decrease of P. mirabilis in bladders and kidneys. Instead, infection levels in MrpA-flagellin or flagellin-treated mice showed no significant differences with the control group. IL-10 was significantly induced in splenocytes of mice that received native flagellin or MrpA-flagellin. Native P. mirabilis flagellin did not protect mice against an ascending UTI. Moreover, it showed an immunomodulatory effect, neutralizing the protective role of MrpA. P. mirabilis flagellin exhibits particular immunological properties compared to other bacterial flagellins.     

Isolation and characterization of biofilm-forming bacteria and associated extracellular polymeric substances from oral cavity

The current work deals with the studies on characterization of two biofilm-forming bacteria isolated from the oral cavity. The major constituent of biofilm other than bacterial cells is the extracellular polymeric substance (EPS) matrix, which is secreted by the bacterial cells themselves. Physical properties of biofilms such as attachment, mechanical strength, antibiotic resistance can be attributed to EPS matrix. Molecular phylogeny confirmed these two isolates as Pseudomonas aeruginosa and Bacillus subtilis. It was observed that cell attachment in both the strains was maximal when xylose was used as the sole carbon source. The EPS characterization result indicated the presence of a macromolecular complex constituting of carbohydrate, protein, lipids and nucleic acids. Test for biofilm formation in the presence of metal salts of iron and zinc showed moderate to high inhibition of biofilm formation. However, calcium, iron and copper have been found to enhance biofilm growth significantly. There was more than 50 % increase in biofilm growth by P. aeruginosa with an increase in calcium concentration up to 80 ppm (Two tailed t-test P?<?0.05), whereas ≥ 15 % increase in biofilm growth by B. subtilis was observed in the presence of 80 ppm of calcium. However, variations were significant (Two way ANOVA, P?<?0.01) between different metals in different concentrations. In this study, attempts have been made to examine the effect of different carbon sources and physiological conditions on biofilm growth.     

The Impact of spgM,rpfF, rmlA Gene Distribution on Biofilm Formation in Stenotrophomonas maltophilia

Background

Stenotrophomonas maltophilia is emerging as one of the most frequently found bacteria in chronic pulmonary infection. Biofilm is increasingly recognized as a contributing factor to disease pathogenesis. In the present study, a total of 37 isolates of S. maltophilia obtained from chronic pulmonary infection patients were evaluated to the relationship between biofilm production and the relative genes expression.

Methods

The clonal relatedness of isolates was determined by pulse-field gel electrophoresis. Biofilm formation assays were performed by crystal violet assay, and confirmed by Electron microscopy analysis and CLSM analysis. PCR was employed to learn gene distribution and expression.

Results

Twenty-four pulsotypes were designated for 37 S. maltophilia isolates, and these 24 pulsotypes exhibited various levels of biofilm production, 8 strong biofilm-producing S. maltophilia strains with OD492 value above 0.6, 14 middle biofilm-producing strains with OD492 average value of 0.4 and 2 weak biofilm-producing strains with OD492 average value of 0.19. CLSM analysis showed that the isolates from the early stage of chronic infection enable to form more highly structured and multilayered biofim than those in the late stage. The prevalence of spgM, rmlA, and rpfF genes was 83.3%, 87.5%, and 50.0% in 24 S. maltophilia strains, respectively, and the presence of rmlA, spgM or rpfF had a close relationship with biofilm formation but did not significantly affect the mean amount of biofilm. Significant mutations of spgM and rmlA were found in both strong and weak biofilm-producing strains.

Conclusion

Mutations in spgM and rmlA may be relevant to biofilm formation in the clinical isolates of S. maltophilia.     

Predicting the efficacy of the nitrification inhibitor dicyandiamide in pastoral soils

Aims

Identification of soil, environmental, or microbial properties linked with efficacy of the nitrification inhibitor dicyandiamide (DCD) in high and low-input pastoral farming system soils.

Methods

Soils were collected from under 25 pastures. Potential nitrification rate (PRN) was quantified in the presence and absence of DCD, and percentage efficacy of DCD in reducing PNR calculated. PNR and %DCD efficacy were statistically tested (REML analysis) for relationships to a suite of edaphic (33), environmental (5), and microbiological (8) variables. Microbiological properties included measurement of bacterial and archaeal ammonia monooxygenase genes (amoA qPCR) and soil DNA content.

Results

DCD reduced PRN by an average of 36 %. The percent DCD efficacy was not related to system intensity, soil type, nor PNR (all P?>?0.05). However the numbers of bacterial amoA genes (r?=?0.46; P?<?0.05), and ratios of bacterial:archaeal amoA (r?=??0.53; P?<?0.05), were strongly correlated to %DCD efficacy. In both high and low input systems, models best explaining variance in %DCD efficacy fitted AOA: AOB g soil^?1 as the first varaible (P?<?0.05).

Conclusions

Characterisation of soils based on ammonia oxidising communities may increase the ability to predict the % efficacy of DCD between sites and provide for more targeted application of this nitrification inhibitor.