Serological investigation and isolation of Salmonella abortus equi in horses in Xinjiang.

BACKGROUND: Salmonella enterica subspecies enterica serovar abortus equi (S. abortus equi) is one of the main pathogens that causes abortion in pregnant horses and donkeys, which was highly infectious and greatly restricts the healthy development of the horse industry. OBJECTIVES: In order to investigate the prevalence and biological characteristics of S. abortus equi in different regions and breeds of horses in Xinjiang. METHODS: This study conducted ELISA detection of S. abortus equi antibodies on serum samples of 971 horses collected from three large-scale horse farms and five free-range horse farms in Yili Prefecture and Bayingol Mongolian Autonomous Prefecture of Xinjiang from 2020 to 2023. On this basis, bacterial isolation, culture, identification, and drug sensitivity tests were conducted on 42 samples of aborted foal tissues and 23 mare vaginal swabs. RESULTS: The results showed that the positive rate of S. abortus equi antibody was as high as 20.91% in 971 horse serum samples. Among them, the positive rate in the Ili region (29.09%) was significantly higher than that in the Bayingole region (11.24%), and the positive rate in mares (22.45%) was higher than that in stallions (14.05%). In terms of horse breeds, the positive rates of self-propagating thoroughbred horses, half-bred horses, Ili horses and Yanqi horses were 43.22%, 28.81%, 14.72% and 11.24% respectively. In addition, S. abortus equi was more susceptible to juvenile and elderly horses, with positive rates of 70.00%and 41.86%, respectively, both of which were significantly higher than young (10.97%) and adult (19.79%) horses. Further, 9 strains of S. abortus equi were obtained through bacterial isolation, culture and identification, which were resistant to five antibiotics (Clarithromycin, Clindamycin, penicillin, Sulfamethoxazole and Rifampicin), and sensitive to 13 antimicrobial agents (Amoxicillin, Ciprofloxacin and Gentamicin, et al.). CONCLUSION: There was a high infection rate of S. abortus equi in Ili Prefecture and self-propagating thoroughbred horses, and juvenile or old mares were more susceptible, which will provide scientific basis for the prevention of S. abortus equi infection in different regions and breeds of horses in Xinjiang.

Inhibition of bacterial swimming by heparin binding of flagellin FliC from Escherichia coli strain Nissle 1917.

Escherichia coli Nissle1917 (EcN) is a non-pathogenic probiotic strain widely used to maintain gut health, treat gastrointestinal disorders, and modulate the gut microbiome due to its anti-inflammatory and competitive exclusion effects against pathogenic bacteria. Heparin, abundant on intestinal mucosal surfaces, is a highly sulfated glycosaminoglycan primarily produced by mast cells. Currently, the interaction between EcN surface protein and heparin has remained elusive. In this study, the flagellin FliC responsible for EcN's movement was separated and characterized as a heparin binding protein by mass spectrometry (MS) analysis. The recombinant FliC protein, expressed by plasmid pET28a( +)-fliC, was further prepared to confirm the interaction between FliC and heparin. The results showed that heparin-Sepharose's ability to bind FliC was 48-fold higher than its ability to bind the negative control, bovine serum albumin (BSA). Neither the knockout of gene fliC nor the addition of heparin affects the growth of EcN, but both significantly inhibit the swimming of EcN. Adding 10 mg/ml heparin reduced the swimming diameter of the wild type and the complemented strain to 29-41% of the original, but that did not affect the swimming ability of the knockout strains. These results demonstrate that heparin interacts with EcN flagellin FliC and inhibits bacteria swimming. Exploring this interaction could improve our understanding of the relationship between hosts and microorganisms and provide a potential basis for disease treatment.

ANTIBIOTIC RESISTANCE OF SALMONELLA TYPHI CARRIER ASSOCIATED WITH GALL BLADDER CHRONIC INFECTION IN AL-NAJAF PROVINCE.

OBJECTIVE: The aim: This study was undertaken to identify antibiotic resistance of Salmonella Typhi in specimens of gall bladder tissue after cholecystectomy. PATIENTS AND METHODS: Materials and methods: Salmonella Typhi identification from the isolates have been depended on morphology of the colony and biochemical tests as a first step in identification while final identification has been achieved by the automated VITEK-2 compact system then PCR technique. RESULTS: Results: Depending to finding via the VITEK tests and PCR technique and thirty-five Salmonella Typhi sample have been obtained. This research shown that about 35 (70%) positive result contains, 12 (34.3%) isolates was positive from stool and 23(65.7%) from gall bladder tissue. The results revealed difference in S. Typhi resistance to some antibiotics, where S. Typhi has wide-ranging sensitivity: 35 (100%) to Cefepime, Cefixime and Ciprofloxacin and revealed great sensitivity 22 (62.8%) to Ampicillin. S. Typhi isolates proved extremely resistant 19 (54.2%) and 25 (71.4%) to Trimethoprim/Sulphamethoxazole and Chloramphenicol respectively. Increment in the rate of Salmonella that has multidrug resistance to chloramphenicol, ampicillin, furazolidonecotrimoxazole, streptomycin, and tetracycline is a developing problem and worldwide worry matte. CONCLUSION: Conclusions: Resistant forms of Salmonella enteric serotype Typhi were detected with increment in the rate of multidrug resistance to chloramphenicol, ampicillin, and tetracycline so currently, Cefepime, Cefixime and Ciprofloxacin and revealed great sensitivity and have become the mainstay of treatment. Challenging difficult which rises in this study is the extend of Multidrug resistant strain (MDR) of S. Typhi.

Identification of Two New Sequences of Flagellin-Encoding Gene in Escherichia coli Using PCR and Sequencing-Based Methods.

Escherichia coli has traditionally been serotyped using antisera against the O and H antigens. However, a proportion of E. coli isolates are nonmotile and, in addition, some isolates do not react with the currently available H-typing sera. Alternative molecular methods have been developed based on the detection of genes encoding for H antigens. In this study, we studied 13 serologically nontypable H antigen E. coli strains using polymerase chain reaction (PCR) and sequencing-based methods. We found two new sequences of flagellin-encoding gene, for each of which a specific antiserum was produced to confirm their expression. Sequencing of the flagellin gene offers a rapid determination of E. coli H antigens and could be used to detect potential novel flagellar antigens.

Motility and the genotype diversity of the flagellin genes fliC and fliD among Clostridioides difficile ribotypes.

OBJECTIVE: The motility and genotype of the flagellin fliC and fliD genes were investigated in 82 Clostridioides difficile isolates belonging to the ribotypes (RTs): 027 (n = 41), 176 (n = 17), 023 (n = 8), 017 (n = 6) and 046 (n = 10). The reference C. difficile strains 630 and M120 were included as controls for the motility assay. METHODS: A Multiple Locus Variable-number Tandem Repeat Analysis (MLVA) was used to exclude the genetic relatedness of C. difficile isolates belonging to the same RT. The variability of the fliC and fliD genes was determined by PCR-restriction fragment length polymorphism (RFLP) analysis and Sanger sequencing. The motility assay was carried out with 0.175% BHI agar tubes and BHI solid media plates with 0.4% agar. RESULTS: The highest motility was observed in C. difficile RT023 isolates (p < 0.01), followed by RTs 027 and 176. C. difficile isolates of RTs 017 and 046 were less motile than RTs 027, 176 and 023 (p < 0.01). The fliC and fliD genes were present in all clinical isolates irrespective of the motility results. In the fliC gene analysis, four different RFLP groups were identified (I, II, VII, X). The fliC group VII was identified in two RTs (027 and 176), whereas the remaining three groups (I, II and X) belonged to a single RT 046, 017 and 023, respectively. The fliD gene analysis identified four new RFLP groups (a, b, c and d). CONCLUSIONS: C. difficile RT023 is highly motile and its motility is comparable to the hypervirulent RT027 and its genetic relative RT176.

Differential Impact of Hexuronate Regulators ExuR and UxuR on the Escherichia coli Proteome.

ExuR and UxuR are paralogous proteins belonging to the GntR family of transcriptional regulators. Both are known to control hexuronic acid metabolism in a variety of Gammaproteobacteria but the relative impact of each of them is still unclear. Here, we apply 2D difference electrophoresis followed by mass-spectrometry to characterise the changes in the Escherichia coli proteome in response to a uxuR or exuR deletion. Our data clearly show that the effects are different: deletion of uxuR resulted in strongly enhanced expression of D-mannonate dehydratase UxuA and flagellar protein FliC, and in a reduced amount of outer membrane porin OmpF, while the absence of ExuR did not significantly alter the spectrum of detected proteins. Consequently, the physiological roles of proteins predicted as homologs seem to be far from identical. Effects of uxuR deletion were largely dependent on the cultivation conditions: during growth with glucose, UxuA and FliC were dramatically altered, while during growth with glucuronate, activation of both was not so prominent. During the growth with glucose, maximal activation was detected for FliC. This was further confirmed by expression analysis and physiological tests, thus suggesting the involvement of UxuR in the regulation of bacterial motility and biofilm formation.

Multiple Copies of flhDC in Paraburkholderia unamae Regulate Flagellar Gene Expression, Motility, and Biofilm Formation.

FlhDC is a heterohexameric complex that acts as a master regulator of flagellar biosynthesis genes in numerous bacteria. Previous studies have identified a single flhDC operon encoding this complex. However, we found that two flhDC loci are present throughout Paraburkholderia, and two additional flhC copies are also present in Paraburkholderia unamae. Systematic deletion analysis in P. unamae of the different flhDC copies showed that one of the operons, flhDC1, plays the predominant role, with deletion of its genes resulting in a severe inhibition of motility and biofilm formation. Expression analysis using promoter-lacZ fusions and real-time quantitative PCR support the primary role of flhDC1 in flagellar gene regulation, with flhDC2 a secondary contributor. Phylogenetic analysis shows the presence of the flhDC1 and flhDC2 operons throughout Paraburkholderia. In contrast, Burkholderia and other bacteria only carry the copy syntenous with flhDC2. The variations in impact each copy of flhDC has on downstream processes indicate that regulation of FlhDC in P. unamae, and likely other Paraburkholderia species, is regulated at least in part by the presence of multiple copies of these genes. IMPORTANCE Motility is important in the colonization of plant roots by beneficial and pathogenic bacteria, with flagella playing essential roles in host cell adhesion, entrance, and biofilm formation. Flagellar biosynthesis is energetically expensive. Its complex regulation by the FlhDC master regulator is well studied in peritrichous flagella expressing enterics. We report the unique presence throughout Paraburkholderia of multiple copies of flhDC. In P. unamae, the flhDC1 copy showed higher expression and a greater effect on swim motility, flagellar development, and regulation of downstream genes, than the flhDC2 copy that is syntenous to flhDC in Escherichia coli and pathogenic Burkholderia spp. The flhDC genes have evolved differently in these plant-growth-promoting bacteria, giving an additional layer of complexity in gene regulation by FlhDC.

FliC of Vibrio splendidus-related strain involved in adhesion to Apostichopus japonicus.

Vibrio splendidus-related strains are important opportunistic marine pathogens, and they can infect many important marine animals, including the sea cucumber Apostichopus japonicus. In this study, one gene coding flagellin was cloned and a V. splendidus-related strain AJ01/GFPFliC with the overexpression of fliC gene was constructed to explore the function of FliC. AJ01/GFPFliC showed a 3-4 h delay in the initial growth phase and then its growth was faster than that of the wild type strain AJ01. The abilities of swarming motility and biofilm formation ability of AJ01/GFPFliC were also higher than that of AJ01. The adhesion rate of AJ01/GFPFliC to the slide and the coelomocytes of A. japonicus increased from 1% to 5%, and 25% to 40%, respectively, and the adhered AJ01/GFPFliC cells in intestinal tissue of A. japonicus reached 8.0 × 106 CFU/g, which was 2.5-fold higher than that of the control strain AJ01/GFP. Concluded from all the data suggested that FliC was an adhesion factor of V. splendidus-related strain AJ01 that could also contribute to bacterial swarming motility and biofilm formation.

Effect of flagellin on inhibition of infectious mechanisms by activating opsonization and salmonella flagellum disruption.

Some serovars of salmonella cause huge global diseases such as enteric fever and invasive non typhoidal Salmonella disease. Flagellin as a key antigenic component of salmonella, can induce humoral and cellular immunity responses. In this research, we performed an opsonophagocytic killing assay (OPKA) as an important mechanism of the host-defense system, for salmonella to study the activity of anti-sera of native FliC, truncated modified recombinant FliC (tmFliC) and full length recombinant FliC proteins (flFliC). Also, the potency of antibodies for inhibiting bacterial movement was evaluated by traditional and newly-designed motility inhibition assay methods. Results showed both recombinant FliC anti-sera and native FliC (nFliC) anti-serum had the ability to opsonize Salmonella typhimurim, which led to bacterial clearance by mice macrophages. Also, inhibition of bacterial motility was observed for all anti-sera. Anti-nFliC and anti-flFliC sera showed higher effects on Salmonella typhimurim motility than that of tmFliC. In traditional method, about 88%, 86% and 80% inhibition were observed by using 5% nFliC, anti-flFliC and anti-tmFliC sera, respectively. In the newly-designed method using SIM (Sulfide indole motility) medium, results confirmed the traditional method for motility inhibition. Our findings suggest that salmonella fliC as a protective antigen may disrupt the flagellum apparatus activity.

Parenteral immunization of Salmonella Typhimurium ghosts with surface-displayed Escherichia coli flagellin enhancesTLR-5 mediated activation of immune responses that protect the chicken against Salmonella infection.

The present study investigates the enhancement of immunogenicity and protection efficacy of Salmonella Typhimurium ghosts surface-displayed with FliC against chicken salmonellosis. The membrane-anchored FliC is a potential TLR-5 agonist, delivers an essential adjuvant effect for the ghost vaccine candidate. The present ghost plasmid pJHL184 construct carries a convergent dual promoter system that has the temperature-dependent induction of the phage lysis gene E and the target antigen FliC at the same time. Under permissible conditions of temperatures, less than 30 °C at the presence of 20 mM l-arabinose effectively suppresses expression of the lysis gene. Once the temperature is up-lifted to 42 °C without arabinose, cause the generation of ST ghosts expelling the cytoplasmic content. The addition of FliC adjuvant significantly enhanced the IgY response, cell-mediated immune responses, regulatory cytokine induction and subsequently enhanced protection against Salmonella challenge. Further, intramuscular immunization with ST ghosts displaying FliC induced particularly high CD8+ response demarcating its proficiency to elicit Type I immune responses. Further, ST ghosts displaying FliC caused an increase in both CD4+ and CD8+ response compared to the PBS control suggesting its capability to engage both cell-mediated and humoral immune responses essential for the elimination of Salmonella. Upon the virulent challenge, we could observe a significant reduction in challenged bacterial load on spleen, liver and cecum tissues in the ST ghosts surface-displaying FliC adjuvant. Our study suggests the biological incorporation of FliC on ST ghosts enhances vaccine immunogenic potency and acts as a safe and effective prevention strategy against chicken salmonellosis.

Generation of Markerless Deletions in the Nosocomial Pathogen Clostridium difficile by Induction of DNA Double-Strand Breaks.

Clostridium difficile is an important nosocomial pathogen associated with potentially fatal disease induced by the use of antibiotics. Genetic characterization of such clinically important bacteria is often hampered by lack of availability of suitable tools. Here, we describe the use of I-SceI to induce DNA double-strand breaks, which increase the frequency of allelic exchange and enable the generation of markerless deletions in C. difficile The usefulness of the system is illustrated by the deletion of genes encoding putative AddAB homologues. The ΔaddAB mutants are sensitive to ultraviolet light and the antibiotic metronidazole, indicating a role in homologous recombination and the repair of DNA breaks. Despite the impairment in recombination, the mutants are still proficient for induction of the SOS response. In addition, deletion of the fliC gene, and subsequent complementation, reveals the importance of potential regulatory elements required for expression of a downstream gene encoding the flagellin glycosyltransferase.IMPORTANCE Most sequenced bacterial genomes contain genes encoding proteins of unknown or hypothetical function. To identify a phenotype for mutations in such genes, deletion is the preferred method for mutagenesis because it reduces the likelihood of polar effects, although it does not eliminate the possibility. Allelic exchange to produce deletions is dependent on the length of homologous regions used to generate merodiploids. Shorter regions of homology resolve at lower frequencies. The work presented here demonstrates the utility of inducing DNA double-strand breaks to increase the frequency of merodiploid resolution in Clostridium difficile Using this approach, we reveal the roles of two genes, encoding homologues of AddAB, in survival following DNA damage. The method is readily applicable to the production of deletions in C. difficile and expands the toolbox available for genetic analysis of this important anaerobic pathogen.

Introducing a cost-effective method for purification of bioactive flagellin from several flagellated gram-negative bacteria.

The objective of this study was to introduce a simple and cheap method for purification of flagellin. So, flagellin proteins of Salmonella typhimurium (S. typhimurium), Escherichia coli (E. coli), Citrobacter freundii (C. freundii) and Salmonella typhi (S. typhi) were purified by a modified simple method. Bacterial cultures were precipitated by centrifugation. Precipitates were washed twice and flagellin proteins were detached by shaking vigorously (in PBS pH = 2), and then flagellin proteins were precipitated by ammonium sulfate saturation. Evaluation of purification efficiency and concentration were examined by SDS-PAGE and Bradford assay. Polyclonal antibodies were produced against S. typhimurium FliC and cross-reactivity of anti-S. typhimurium was assessed against other flagellins. Bioactivity of flagellins was evaluated by cell proliferation and IL-8 protein expression assay in HEK293 cells, and also, IL-6 and TNF-α genes expression in chicken cells. Results showed a single band for flagellin proteins of all bacteria on %10 SDS-PAGE, which concentration ranged from 150 to 400 µg/mL. All flagellin proteins increased cell proliferation, and IL-8 levels were increased after treatment by flagellins and levels of IL-6 and TNF-α were increased after treatment with S. typhimurium FliC. All flagellin proteins showed cross-reactivity with antibodies. Findings showed that application of our method, not only reduced time and cost, but also, the purified flagellin proteins had acceptable bioactivity.

Nonconservation of TLR5 activation site in Edwardsiella tarda flagellin decreases expression of interleukin-1ß and NF-κB genes in Japanese flounder, Paralichthys olivaceus.

Flagellin is the subunit protein that composes bacterial flagella and is recognized by toll-like receptor 5 (TLR5) as a ligand. Flagellin protein (e.g., FliC and FlaA) contains the D1, D2, and D3 domains; the D1 domain is important for recognition by TLR5 for activation of the innate immune system. In teleosts, there are two types of TLR5, the membrane form (TLR5M) and soluble form (TLR5S), the latter of which is not present in mammals. In this study, the potential of flagellin from Edwardsiella tarda (EtFliC) to induce inflammation-related genes interleukin (IL)-1ß and NF-κB-p65 through TLR5S in Japanese flounder (Paralichthys olivaceus) was elucidated. A transient overexpression system was developed in flounder natural embryonic (HINAE) cells using constructs encoding two flagellin genes derived from E. tarda (pEtFliC) and Escherichia coli (pEcoFliC) and the flounder TLR5S gene (pPoTLR5S). Expression of inflammation-related genes in EtFliC- and PoTLR5S-overexpressing HINAE cells was significantly lower than in EcoFliC- and PoTLR5S-overexpressing cells. To clarify the difference between EtFliC and EcoFliC potency, the amino acid sequence of EtFliC was compared with that of other bacterial flagellin. The 91st arginine residue, known as the mammalian TLR5 activation site, was conserved in the flagellin of E. coli and other bacteria but not in EtFliC. To reveal the importance of the 91st arginine residue in FliC, a pEtFliC construct in which the 91st asparagine was mutated to arginine (pEtFliC_N91R) was generated. Expression of the IL-1ß and NF-κB-p65 genes in the HINAE cells co-transfected with pEtFliC_N91R and pPoTLR5S was significantly higher than that in cells co-transfected with pEtFliC and pPoTLR5S. The results suggested that the 91st arginine residue of bacterial flagellin is involved in inflammatory response through TLR5S in teleosts. Thus, EtFliC improved by site-directed mutagenesis could be an effective adjuvant against E. tarda infection in Japanese flounder.

A fluorescence quenching based gene assay for Escherichia coli O157:H7 using graphene quantum dots and gold nanoparticles.

A fluorometric assay is described for highly sensitive quantification of Escherichia coli O157:H7. Reporter oligos were immobilized on graphene quantum dots (GQDs), and quencher oligos were immobilized on gold nanoparticles (AuNPs). Target DNA was co-hybridized with reporter oligos on the GQDs and quencher oligos on AuNPs. This triggers quenching of fluorescence (with excitation/emission peaks at 400 nm/530 nm). On introducing target into the system, fluorescence is quenched by up to 95% by 100 nM concentrations of target oligos having 20 bp. The response to the fliC gene of E. coli O157:H7 increases with the logarithm of the concentration in the range from 0.1 nM to 150 nM. The limit of detection is 1.1 ± 0.6 nM for n = 3. The selectivity and specificity of the assay was confirmed by evaluating the various oligos sequences and PCR product (fliC gene) amplified from genomic DNA of the food samples spiked with E. coli O157:H7. Graphical abstractSchematic representation of fluorometric assay for highly sensitive quantification of Escherichia coli O157:H7 based on fluorescence quenching gene assay for fliC gene of E. coli O157:H7.

Expression of Salmonella typhimurium and Escherichia coli flagellin protein and its functional characterization as an adjuvant.

BACKGROUND: Flagellin is the major structural protein monomer of bacterial flagella. Flagellin through binding to its receptor and activation of antigen presenting cells stimulates the innate and adaptive immune responses. Flagellin is used as an effective systemic or mucosal adjuvant to stimulate the immune system. Recently, the therapeutic and protective role of flagellin in some infectious diseases and cancers has been investigated. In this study, we cloned the fliC genes from Salmonella typhimurium and Escherichia coli into pET-28a vector and investigated their expression in the prokaryotic system. METHODS: The fliC genes of S. typhimurium and E. coli were amplified by PCR with a specific oligonucleotide primer set. thse were cloned into the pET-28a vector and the recombinant pET-28a-fliC plasmids were successfully transformed into the E. coli strain BL-21(DE3). The expression of flagellin proteins in the prokaryotic cells were evaluated. Finally, Transcription of TNF-α mRNA was confirmed using Real-time PCR. RESULTS: The expression of proteins in the prokaryotic cells were approved by SDS-PAGE and western blotting method. Further, the functional characterization of flagellin proteins were evaluated using their ability to induce increased m-RNA expression of pro-inflammatory cytokine. CONCLUSIONS: The flagellin proteins were expressed in the prokaryotic system. These proteins can be used to link target antigens as an effective adjuvant for future DNA vaccine studies. Purified recombinant proteins in this study can also be used for therapeutic and prophylactic purposes.

Determination immunogenic property of truncated MrpH.FliC as a vaccine candidate against urinary tract infections caused by Proteus mirabilis.

Proteus mirabilis is common cause of urinary tract infections (UTIs) especially in complicated UTIs which are resistant to antibiotic therapy, Consequently, an ideal vaccine is inevitably required. The N-terminal domain of MrpH (Truncated form of MrpH) lies between the most critical antigens of P. mirabilis to consider as vaccine candidate. FliC of Salmonella typhimurium induces several pathways of immunity system, which leads to produce antibody and cytokines. In this study, adjuvant properties of FliC and efficacy of truncated MrpH as important antigen, in tMrpH.FliC were determined in in vitro and in vivo circumstances. Three proteins including: FliC, MrpH and tMrpH.FliC were injected to mice and subsequently sera and supernatant of cell culture were collected to evaluate different immune responses. According to our findings, tMrpH.FliC could stimulate both humoral and cellular immune responses, so that serum IgG, urine IgA, IL.4, IFN-γ and IL.17 were increased significantly in comparison to MrpH and FliC alone, this augmentation was considerable. Results showed significant decrease of bacterial load in all of the challenged groups compared to the control group, although this protective effect was the highest in mice vaccinated with tMrpH.FliC. Our results showed truncated MrpH, without an unwanted domain is an ideal vaccine target and FliC, as adjuvant, increases its immunogenic property. Thus, fusion protein tMrpH.FliC can be considered as promising vaccine against P. mirabilis.

Plasmid AZOBR_p1-borne fabG gene for putative 3-oxoacyl-[acyl-carrier protein] reductase is essential for proper assembly and work of the dual flagellar system in the alphaproteobacterium Azospirillum brasilense Sp245.

Azospirillum brasilense can swim and swarm owing to the activity of a constitutive polar flagellum (Fla) and inducible lateral flagella (Laf), respectively. Experimental data on the regulation of the Fla and Laf assembly in azospirilla are scarce. Here, the coding sequence (CDS) AZOBR_p1160043 (fabG1) for a putative 3-oxoacyl-[acyl-carrier protein (ACP)] reductase was found essential for the construction of both types of flagella. In an immotile leaky Fla- Laf- fabG1::Omegon-Km mutant, Sp245.1610, defects in flagellation and motility were fully complemented by expressing the CDS AZOBR_p1160043 from plasmid pRK415. When pRK415 with the cloned CDS AZOBR_p1160045 (fliC) for a putative 65.2 kDa Sp245 Fla flagellin was transferred into the Sp245.1610 cells, the bacteria also became able to assemble a motile single flagellum. Some cells, however, had unusual swimming behavior, probably because of the side location of the organelle. Although the assembly of Laf was not restored in Sp245.1610 (pRK415-p1160045), this strain was somewhat capable of swarming motility. We propose that the putative 3-oxoacyl-[ACP] reductase encoded by the CDS AZOBR_p1160043 plays a role in correct flagellar location in the cell envelope and (or) in flagellar modification(s), which are also required for the inducible construction of Laf and for proper swimming and swarming motility of A. brasilense Sp245.

Variation of mucin adhesion, cell surface characteristics, and molecular mechanisms among Lactobacillus plantarum isolated from different habitats.

The adhesion ability to mucin varied greatly among 18 Lactobacillus plantarum isolates depending on their isolation habitats. Such ability remained at high level even though they were sequentially exposed to the gastrointestinal (GI) stresses. The majority of L. plantarum isolated from shrimp intestine and about half of food isolates exhibited adhesion ability (51.06-55.04%) about the same as the well-known adhesive L. plantarum 299v. Interestingly, five infant isolates of CIF17A2, CIF17A4, CIF17A5, CIF17AN2, and CIF17AN8 exhibited extremely high adhesion ranging from 62.69 to 72.06%. Such highly adhesive property correlating to distinctively high cell surface hydrophobicity was significantly weaken after pretreatment with LiCl and guanidine-HCl confirming the entailment of protein moiety. Regarding the draft genome information, all molecular structures of major cell wall-anchored proteins involved in the adhesion based on L. plantarum WCSF1, including lp_0964, lp_1643, lp_3114, lp_2486, lp_3127, and lp_3059 orthologues were detected in all isolates. Exceptionally, the gene-trait matching between yeast agglutination assay and the relevant mannose-specific adhesin (lp_1229) encoding gene confirmed the Msa absence in five infant isolates expressed distinctively high adhesion. Interestingly, the predicted flagellin encoding genes (fliC) firstly revealed in lp_1643, lp_2486, and lp_3114 orthologues may potentially contribute to such highly adhesive property of these isolates.

Evaluation of fliC-d based direct blood PCR assays for typhoid diagnosis.

BACKGROUND: Typhoid cases need to be diagnosed accurately for early antibiotic therapy and reducing mortality. Identification of Salmonella Typhi (S. Typhi) in blood culture is conclusive, but has poor sensitivity. Detection of S. Typhi by PCR from blood sample has shown promise. Real-time quantitative PCR (Q-PCR) has been widely used in diagnostics for its rapidity and reliability. In the present study, the performance of molecular methods like conventional PCR (C-PCR), nested PCR (N-PCR) and Q-PCR were investigated and compared by targeting S. Typhi specific flagellar fliC-d gene directly in blood samples for typhoid diagnosis. RESULTS: Analytical sensitivities and specificities of the PCR assays were determined under laboratory condition followed by diagnostic performances were demonstrated in 110 clinically diagnosed typhoid fever (CDTF) cases included as study subjects. The DNA detection limit of C-PCR was observed 3 × 10(4) copies/reaction; those of N-PCR and Q-PCR (cutoff Ct value, ≤37) were 3 copies/reaction. The C-PCR was not further evaluated since it showed negative results with all clinical samples due to low sensitivity. Low isolation rate (21.8 %, 24/110) of S. Typhi by blood culture did not reflect the true burden of typhoid fever among the study subjects. Hence diagnostic performances of N-PCR and Q-PCR were determined considering CDTF cases positive by any of the diagnostic assay methods (n = 81) as true positives. Laboratory confirmed non-typhoidal cases (n = 29) were included as true negatives. On comparison, although both the assays were 100 % specific; sensitivity (91.4 % vs. 81.5 %) and efficiency (93.6 % vs. 86.4 %) of Q-PCR were better, but statistically not significant (p > 0.1) than N-PCR. The positive and negative likelihood ratios of Q-PCR were ∞ and 0.09 which indicated the potential clinical utility of Q-PCR for typhoid diagnosis. Q-PCR was more rapid than N-PCR (2 h vs. 6 h) in obtaining test results. CONCLUSIONS: This study demonstrates for the first time that TaqMan-based Q-PCR assay performs more favorably than N-PCR for direct detection of S. Typhi DNA in blood samples. Direct and quantitative blood Q-PCR is a rapid and reliable method for diagnosis of typhoid fever.

Use of flagellin and cholera toxin as adjuvants in intranasal vaccination of mice to enhance protective immune responses against uropathogenic Escherichia coli antigens.

Urinary tract infections (UTIs) caused by Uropathogenic Escherichia coli (UPEC) are among the most common infections in human. Antibiotics are common therapy for UTIs, but increase in antibiotic resistance will complicate future treatment of the infections, making the development of an efficacious UTI vaccine more urgent. In this study, we have evaluated intranasally the efficacy of FliC and FimH antigens of UPEC in different vaccine formulations with and without cholera toxin (CT) adjuvant. Immunization of mice with FliC in fusion form or admixed with FimH elicited higher levels of serum, mucosal and cell-mediated responses than FimH alone. Furthermore, the use of CT in synergism with FliC resulted in the stimulation of a mixed Th1 and Th2 responses against FimH and FliC as antigen and maintained the antibody responses for at least 24 weeks following the last vaccine dose. Of the vaccine preparations, Fusion, Fusion + CT, and FimH admixed with FliC and CT showed the best protection against UPEC. These data indicated that intranasal administration of a FliC and CT adjuvant-based vaccine has the potential to provide protective responses against UPEC strains.