Presence of fimH and iss type 1, 2 and 3 genes in uropathogenic Escherichia coli isolates recovered from an apex medical institute in North India.

PURPOSE: To detect the presence of fimH and iss type 1, 2 and 3 genes in uropathogenic Escherichia coli (UPEC) isolates recovered from patients coming to the out patient department (OPD) of our hospital. METHODS: E. coli isolates recovered from patients who had symptoms of urinary tract infection (UTI) were processed for the presence of fimH and iss genes. DNA was extracted using an in house method after which conventional PCR using forward and reverse primers targeting the four genes was carried out. The amplified products were electrophoresed and visualized in a gel documentation imager. Relevant demographic details of the patients were recorded on a pre-designed pro-forma and antimicrobial susceptibility testing of the isolates was done by disc diffusion method. RESULTS: fimH was present in 87.5% of UPEC isolates whereas iss type 1 was seen in 7.3%, type 2 in 4.2% and iss type 3 in 71.9% isolates. Age of the patients ranged from 3 months to 82 â€‹yrs (mean 43.5 SD â€‹± â€‹18.20). UTI was more common in females (60.2%) as compared to males patients (39.8%). Dysuria (66.7%) was the most common symptom in the studied subjects and diabetes mellitus (42.6%) the most common co-morbidity. A total of 56.5% patients gave a history of prior antibiotic intake. The UPEC isolates were resistant to most of the antibiotics tested. However all the isolates were sensitive to polymyxin B and colistin. Fosfomycin resistance was seen in 9.5% of the UPEC isolates harbouring fimH gene. CONCLUSION: This is the first study that highlights the presence of iss type 3 gene in UPEC isolates along with the fimH and iss type 1 and 2 genes. The results of this study can serve as a stepping stone for future in depth research into the significance of the iss genes in causing UTI.

Screening of antigenic epitopes related to the adhesion of the avian Escherichia coli Type 1 Fimbrial Agglutinin Domain.

BACKGROUND: Avian Escherichia coli (E.coli) type 1 fimbriae adhere to avian tracheal epithelial cells through the FimH protein. However, the adhesion-related antigen is still unknown. The purpose of this study was to analyze the antigenicity of the type 1 fimbrial FimH protein of wild-type avian E. coli, screen antigen epitopes, and prepare monoclonal antibodies (mAbs) that can block the adhesion of avian E. coli. RESULTS: In this study, the nucleic acid homologies of MG2 (O11), TS12 (O18), and YR5 (O78) with K12 were 97.7%, 99.6%, and 97.7%, respectively, and the amino acid sequence similarity reached 98.7%, 99.3%, and 98.0%, respectively. The epitopes and hydrophilicities of the FimH proteins of these three strains were similar. The more obvious lectin domain epitopes were located at FimH protein positions 111-124 and 154-162. The mAbs 7C2 and 7D8 against these two epitopes were prepared. An adhesion inhibition test showed that 7C2 and 7D8 blocked bacterial adhesion to avian tracheal epithelial cells. The mAb 7C2 against the 111-124 epitope inhibited O78 strain adhesion by 93%, and the mAb 7D8 against the 154-162 epitope inhibited O78 strain adhesion by 49%, indicating that these two epitopes are closely related to the adhesion of type 1 fimbriae. However, only the 111-124 epitope-recognizing mAb 7C2 inhibited bacterial agglutination of erythrocytes, indicating that host cell receptor binding and erythrocyte agglutination are not mediated by the same spatial locations within the FimH protein. CONCLUSIONS: The results demonstrate that the mAbs 7C2 and 7D8 against FimH protein positions 111-124 and 154-162 could inhibit the adhesion of E.coli to the chicken trachea.

Cytokine profile in HTB-4 cells with respect to fimH, papC and hlyA expression in uropathogenic Escherichia coli invasion.

Background: In this study, uropathogenic Escherichia coli (UPEC) from pregnant and nonpregnant patients were characterized with respect to antimicrobial resistance (AMR) and expression of their virulence factors and cytokines elicited upon infection in urothelial (HTB-4) cells in vitro to frame proper therapeutics. Methods: Antibiotic sensitivity and adherence to HTB-4 cells were tested and PCR and real-time PCR were performed. Results: UPEC from nonpregnant patients showed the most resistance with a significant correlation between the expression of hlyA and TGF-ß and papC and GCSF. The expression of fimH and IFN-γ, fimH and IL-1ß, and fimH and IL-17A, respectively, were significantly correlated in UPEC from pregnant patients. Conclusion: Cytokine expression profiles were correlated with the expression of virulence genes in UPEC that was isolated from different populations, and should be accounted for along with AMR analysis.

Molecular typing of mcr-1 Escherichia coli isolates from pigs and farm environment based on fumC and fimH alleles.

Background: The dissemination of polymyxin resistance represents a significant threat to public health. Materials & methods: Sequence-based typing was performed by 53 mcr-1 Escherichia coli isolates using fumC/fimH (CH) genes to characterize clones spreading from pig farming. Furthermore, 12 isolates had their whole genome sequenced for phylogenetic study. Results: The isolates were classified into 22 distinct CH types, and two novel CH types (CH41-1578 and CH4-1579) and one sequence type (ST12652) was also described. According to phylogenetic study, both multilocus sequence typing and CH methods grouped the isolates similarly. Conclusion: Our findings suggest that the dissemination of the mcr-1 gene in pig farming has occurred mainly by horizontal gene transfer, and CH typing proved to be a good tool to characterize E. coli clones.

Escherichia coli Aggregates Mediated by Native or Synthetic Adhesins Exhibit Both Core and Adhesin-Specific Transcriptional Responses.

Bacteria can rapidly tune their physiology and metabolism to adapt to environmental fluctuations. In particular, they can adapt their lifestyle to the close proximity of other bacteria or the presence of different surfaces. However, whether these interactions trigger transcriptomic responses is poorly understood. We used a specific setup of E. coli strains expressing native or synthetic adhesins mediating bacterial aggregation to study the transcriptomic changes of aggregated compared to nonaggregated bacteria. Our results show that, following aggregation, bacteria exhibit a core response independent of the adhesin type, with differential expression of 56.9% of the coding genome, including genes involved in stress response and anaerobic lifestyle. Moreover, when aggregates were formed via a naturally expressed E. coli adhesin (antigen 43), the transcriptomic response of the bacteria was more exaggerated than that of aggregates formed via a synthetic adhesin. This suggests that the response to aggregation induced by native E. coli adhesins could have been finely tuned during bacterial evolution. Our study therefore provides insights into the effect of self-interaction in bacteria and allows a better understanding of why bacterial aggregates exhibit increased stress tolerance. IMPORTANCE The formation of bacterial aggregates has an important role in both clinical and ecological contexts. Although these structures have been previously shown to be more resistant to stressful conditions, the genetic basis of this stress tolerance associated with the aggregate lifestyle is poorly understood. Surface sensing mediated by different adhesins can result in various changes in bacterial physiology. However, whether adhesin-adhesin interactions, as well as the type of adhesin mediating aggregation, affect bacterial cell physiology is unknown. By sequencing the transcriptomes of aggregated and nonaggregated cells expressing native or synthetic adhesins, we characterized the effects of aggregation and adhesin type on E. coli physiology.

Investigation of the biofilm formation in extra-intestinal pathogenic Escherichia coli ST131 strains and its correlation with the presence of fimH, afa, and kpsMSTII genes.

Escherichia coli sequence type 131 (ST131) is a multidrug-resistant strain with the global dissemination. Biofilm formation-related factors include the most important virulence factors in extra-intestinal pathogenic E. coli (ExPEC) ST131 strains causing infections with treatment-limited subjects. This study aims to investigate the biofilm formation ability and its correlation with the presence of fimH, afa, and kpsMSTII genes in clinical isolates of ExPEC ST131. In this regard, the prevalence and characteristics of these strains collected and evaluated. The results revealed strong, moderate, and weak attachment abilities related to biofilm formation attributes in 45%, 20%, and 35% of strains, respectively. In the meantime, the frequency of the fimH, afa, and kpsMSTII genes among the isolates was observed as follows: fimH positive: 65%; afa positive: 55%; and kpsMSTII positive: 85%. The results convey a significant different of biofilm formation ability between clinical E. coli ST131 and non-ST131 isolates. Furthermore, while 45% of ST131 isolates produced strong biofilms, only 2% of non-ST131 isolates showed the ability to form strong biofilms. The attending of fimH, afa, and kpsMSTII genes in the majority of ST131 strains demonstrated a key role leading to biofilm formation. These findings suggested the application of fimH, afa, and kpsMSTII gene suppressors for treating biofilm infections caused by drug-resistant ST131 strains.

Design of a chimeric protein composed of FimH, FyuA and CNF-1 virulence factors from uropathogenic Escherichia coli and evaluation its biological activity and immunogenicity in vitro and in vivo.

Urinary tract infections (UTIs) caused by Uropathogenic Escherichia coli (UPEC) are among the most prevalent bacterial infections in humans. Antibiotic resistance among UPEC isolates is increasing, and designing an effective vaccine can prevent or reduce these infections. FimH adhesin, iron scavenger receptor FyuA, and cytotoxic necrotizing factor -1 (CNF-1) are among the most important virulence factors of UPEC strains. Thus, a novel multi-epitope protein composed of FimH, FyuA, and CNF-1 was designed to evaluate its biological activity and immunogenicity in vitro and in vivo, respectively. The final vaccine design had seven domains, including the N-terminal domain of FimH, four domains of FyuA, and two domains of CNF-1, as determined by immunoinformatics analysis. The results of tertiary structure prediction showed that the chimeric protein had a C-score of -0.25 and Z-score of -1.94. Molecular docking indicated that thirty six ligand residues of the chimeric protein interacted with 53 receptor residues of TLR-4 by hydrogen bonds and hydrophobic interactions. Analysis of protein expression by SDS-PAGE showed an approximately 44 kDa band with different concentrations of IPTG which were confirmed by Western blot. According to ELISA results, the level of IL-8 produced by stimulated Ht29 cells with the chimeric protein was significantly higher than the stimulated Ht29 cells with CNF-1 alone and un-stimulated Ht29 cells. Rabbits subcutaneously immunized with the chimeric protein admixed with Freund adjuvant induced higher level of serum IgG on day 14 after the first vaccination than control rabbits. Furthermore, the booster dose of the chimeric protein significantly enhanced the IgG levels as compared to day 14 and also controls. As, the chimeric protein has suitable B-cell epitopes and MHC-I and MHC-II binding epitopes to stimulate humoral and cellular immunity, it could be a promising vaccine candidate against UTIs caused by UPEC. Evaluating the multi-epitope protein in inducing humoral and cellular immune responses, as well as protection, is ongoing in the mice models.

The EcpD Tip Adhesin of the Escherichia coli Common Pilus Mediates Binding of Enteropathogenic E. coli to Extracellular Matrix Proteins.

The attachment of enteropathogenic Escherichia coli (EPEC) to intestinal epithelial cells is facilitated by several adhesins; however, the individual host-cell receptors for pili-mediated adherence have not been fully characterized. In this study, we evaluated the hypothesis that the E. coli common pilus (ECP) tip adhesin protein EcpD mediates attachment of EPEC to several extracellular matrix (ECM) glycoproteins (fibronectin, laminin, collagens I and IV, and mucin). We found that the ΔecpA mutant, which lacks production of the EcpA filament but retains EcpD on the surface, adhered to these glycoproteins below the wild-type levels, while the ΔecpD mutant, which does not display EcpA or EcpD, bound significantly less to these host glycoproteins. In agreement, a purified recombinant EcpD subunit bound significantly more than EcpA to laminin, fibronectin, collagens I and IV, and mucin in a dose-dependent manner. These are compelling data that strongly suggest that ECP-producing EPEC may bind to host ECM glycoproteins and mucins through the tip adhesin protein EcpD. This study highlights the versatility of EPEC to bind to different host proteins and suggests that the interaction of ECP with the host's ECM glycoproteins may facilitate colonization of the intestinal mucosal epithelium.

Distribution of papG alleles among uropathogenic Escherichia coli from reproductive age women.

BACKGROUND: Extraintestinal Escherichia coli (E. coli) causing urinary tract infections (UTIs), and often referred to as uropathogenic E. coli (UPEC), are a major contributor to the morbidity of UTIs and associated healthcare costs. UPEC possess several virulence factors (VFs) for infecting and injuring the host. We studied the papG allele distribution, and its association with other VF genes and phylogenetic groups, amongst 836 UPEC and fecal isolates from reproductive age women. RESULTS: The papGII gene was highly prevalent amongst pyelonephritis isolates (68%), whilst the majority, albeit smaller proportion, of cystitis isolates (31%) harboured the papGIII gene. Among the pyelonephritis and cystitis isolates, papG positive isolates on average had higher VF gene scores, and were more likely to belong to phylogenetic group B2, than their negative counterparts. This was mostly due to the contribution of papGII isolates, which on average contained more VF genes than their papGIII counterparts, irrespective of the uro-clinical syndrome. However, the papGII isolates from the pyelonephritis cohort had higher VF gene scores than the cystitis ones, suggesting presence of possible papGII clones with differing inferred virulence potential. Furthermore, papGII isolates were more likely to possess an intact pap gene operon than their papGIII counterparts. Also of note was the high proportion of isolates with the papGI allele which was not associated with other pap operon genes; and this finding has not been described before. CONCLUSIONS: The association of the papGII gene with several VF genes compared to the papGIII gene, appears to explain the abundance of these genes in pyelonephritis and cystitis isolates, respectively.

Type 1 piliated uropathogenic Escherichia coli hijack the host immune response by binding to CD14.

A key attribute of persistent or recurring bacterial infections is the ability of the pathogen to evade the host's immune response. Many Enterobacteriaceae express type 1 pili, a pre-adapted virulence trait, to invade host epithelial cells and establish persistent infections. However, the molecular mechanisms and strategies by which bacteria actively circumvent the immune response of the host remain poorly understood. Here, we identified CD14, the major co-receptor for lipopolysaccharide detection, on mouse dendritic cells (DCs) as a binding partner of FimH, the protein located at the tip of the type 1 pilus of Escherichia coli. The FimH amino acids involved in CD14 binding are highly conserved across pathogenic and non-pathogenic strains. Binding of the pathogenic strain CFT073 to CD14 reduced DC migration by overactivation of integrins and blunted expression of co-stimulatory molecules by overactivating the NFAT (nuclear factor of activated T-cells) pathway, both rate-limiting factors of T cell activation. This response was binary at the single-cell level, but averaged in larger populations exposed to both piliated and non-piliated pathogens, presumably via the exchange of immunomodulatory cytokines. While defining an active molecular mechanism of immune evasion by pathogens, the interaction between FimH and CD14 represents a potential target to interfere with persistent and recurrent infections, such as urinary tract infections or Crohn's disease.

A Novel Adult Murine Model of Typical Enteroaggregative Escherichia coli Infection Reveals Microbiota Dysbiosis, Mucus Secretion, and AAF/II-Mediated Expression and Localization of ß-Catenin and Expression of MUC1 in Ileum.

Typical enteroaggregative Escherichia coli (tEAEC) is a diarrheagenic E. coli pathotype associated with pediatric and traveler's diarrhea. Even without diarrhea, EAEC infections in children also lead to increased gut inflammation and growth shortfalls. EAEC strain's defining phenotype is the aggregative adherence pattern on epithelial cells attributable to the aggregative adherence fimbriae (AAF). EAEC only causes diarrhea in humans; therefore, not much is known of the exact intestinal region of infection and damage or its interactions with intestinal enterocytes in vivo and in situ. This study aimed to develop a new tEAEC mouse model of infection, characterize the microbiota of infected mice, and evaluate in situ the expression of host adherence and surface molecules triggering EAEC infection and the role of the EAEC AAF-II in adherence. Six-week-old C57BL/6 mice, without previous antibiotic treatment, were orally challenged with EAEC 042 strain or EAEC 042 AAF-II mutant (ΔAAF/II) strain, or DAEC-MXR strain (diffusely adherent E. coli clinical isolate), and with saline solution (control group). Paraffin sections of the colon and ileum were stained with H&E and periodic acid-Schiff. ZO-1, ß-catenin, MUC1, and bacteria were analyzed by immunofluorescence. EAEC-infected mice, in comparison with DAEC-MXR-infected and control mice, significantly lost weight during the first 3 days. After 7 days post-infection, mucus production was increased in the colon and ileum, ZO-1 localization remained unaltered, and morphological alterations were more pronounced in the ileum since increased expression and apical localization of ß-catenin in ileal enterocytes were observed. EAEC-infected mice developed dysbiosis 21 days post-infection. At 4 days post-infection, EAEC strain 042 formed a biofilm on ileal villi and increased the expression and apical localization of ß-catenin in ileal enterocytes; these effects were not seen in animals infected with the 042 ΔAAF/II strain. At 3 days post-infection, MUC1 expression on ileal enterocytes was mainly detectable among infected mice and colocalized with 042 strains on the enterocyte surface. We developed a novel mouse model of EAEC infection, which mimics human infection, not an illness, revealing that EAEC 042 exerts its pathogenic effects in the mouse ileum and causes dysbiosis. This model is a unique tool to unveil early molecular mechanisms of EAEC infection in vivo and in situ.

Ucl fimbriae regulation and glycan receptor specificity contribute to gut colonisation by extra-intestinal pathogenic Escherichia coli.

Extra-intestinal pathogenic Escherichia coli (ExPEC) belong to a critical priority group of antibiotic resistant pathogens. ExPEC establish gut reservoirs that seed infection of the urinary tract and bloodstream, but the mechanisms of gut colonisation remain to be properly understood. Ucl fimbriae are attachment organelles that facilitate ExPEC adherence. Here, we investigated cellular receptors for Ucl fimbriae and Ucl expression to define molecular mechanisms of Ucl-mediated ExPEC colonisation of the gut. We demonstrate differential expression of Ucl fimbriae in ExPEC sequence types associated with disseminated infection. Genome editing of strains from two common sequence types, F11 (ST127) and UTI89 (ST95), identified a single nucleotide polymorphism in the ucl promoter that changes fimbriae expression via activation by the global stress-response regulator OxyR, leading to altered gut colonisation. Structure-function analysis of the Ucl fimbriae tip-adhesin (UclD) identified high-affinity glycan receptor targets, with highest affinity for sialyllacto-N-fucopentose VI, a structure likely to be expressed on the gut epithelium. Comparison of the UclD adhesin to the homologous UcaD tip-adhesin from Proteus mirabilis revealed that although they possess a similar tertiary structure, apart from lacto-N-fucopentose VI that bound to both adhesins at low-micromolar affinity, they recognize different fucose- and glucose-containing oligosaccharides. Competitive surface plasmon resonance analysis together with co-structural investigation of UcaD in complex with monosaccharides revealed a broad-specificity glycan binding pocket shared between UcaD and UclD that could accommodate these interactions. Overall, our study describes a mechanism of adaptation that augments establishment of an ExPEC gut reservoir to seed disseminated infections, providing a pathway for the development of targeted anti-adhesion therapeutics.

Recombinant FimH Adhesin Demonstrates How the Allosteric Catch Bond Mechanism Can Support Fast and Strong Bacterial Attachment in the Absence of Shear.

The FimH protein of Escherichia coli is a model two-domain adhesin that is able to mediate an allosteric catch bond mechanism of bacterial cell attachment, where the mannose-binding lectin domain switches from an 'inactive' conformation with fast binding to mannose to an 'active' conformation with slow detachment from mannose. Because mechanical tensile force favors separation of the domains and, thus, FimH activation, it has been thought that the catch bonds can only be manifested in a fluidic shear-dependent mode of adhesion. Here, we used recombinant FimH variants with a weakened inter-domain interaction and show that a fast and sustained allosteric activation of FimH can also occur under static, non-shear conditions. Moreover, it appears that lectin domain conformational activation happens intrinsically at a constant rate, independently from its ability to interact with the pilin domain or mannose. However, the latter two factors control the rate of FimH deactivation. Thus, the allosteric catch bond mechanism can be a much broader phenomenon involved in both fast and strong cell-pathogen attachments under a broad range of hydrodynamic conditions. This concept that allostery can enable more effective receptor-ligand interactions is fundamentally different from the conventional wisdom that allostery provides a mechanism to turn binding off under specific conditions.

A Newly Identified Group of P-like (PL) Fimbria Genes from Extraintestinal Pathogenic Escherichia coli (ExPEC) Encode Distinct Adhesin Subunits and Mediate Adherence to Host Cells.

Fimbrial adhesins promote bacterial adherence and biofilm formation. Sequencing of avian pathogenic Escherichia coli (APEC) strain QT598 identified new fimbriae belonging to the π group, which we named PL (P-like) fimbriae since the genetic organization and sequence are similar to those of P and related fimbriae. Genes encoding PL fimbriae located on IncF plasmids are present in diverse E. coli isolates from poultry, human systemic infections, and other sources. As with P fimbriae, PL fimbriae exhibit divergence in adhesin-encoding genes and could be divided into 5 classes based on sequence differences in the PlfG adhesin. plf genes from two predominant PlfG adhesin classes, PlfG class I (PlfGI) and PlfGII, were cloned. PL fimbriae were visualized by electron microscopy, associated with increased biofilm, demonstrated distinct hemagglutination profiles, and promoted adherence to human bladder and kidney epithelial cells. The genes encoding hybrid fimbriae were comprised of genes from plfQT598, wherein plfG was replaced by papG; the adhesin-encoding genes were also functional and mediated adherence to epithelial cells, demonstrating compatibility between the components of these two types of fimbriae. Deletion of plf genes did not reduce colonization of the mouse urinary tract in a single-strain infection model. In contrast, loss of plf genes significantly reduced competitive colonization in the mouse kidneys. Furthermore, plf gene expression was increased over 40-fold in the bladder compared to during in vitro culture. Overall, PL fimbriae represent a new group of fimbriae demonstrating both functional differences from and similarities to P fimbriae, which mediated adherence to host cells and improved competitive colonization of the mouse kidney. IMPORTANCE Fimbriae are important colonization factors in many bacterial species. The identification of a new type of fimbriae encoded on some IncF plasmids in E. coli was investigated. Genomic sequences demonstrated these fimbrial gene clusters have genetic diversity, particularly in the adhesin-encoding plfG gene. Functional studies demonstrated differences in hemagglutination specificity, although both types of Plf adhesin under study mediated adherence to human urinary epithelial cells. A plf mutant also showed decreased colonization of the kidneys in a mouse competitive infection model. PL fimbriae may represent previously unrecognized adhesins that could contribute to host specificity and tissue tropism of some E. coli strains.

Escherichia coli FimH adhesins act synergistically with PapGII adhesins for enhancing establishment and maintenance of kidney infection.

BACKGROUND: FimH adhesin is proposed to enhance Escherichia coli kidney infection by acting with PapGII adhesin, but genetic epidemiology study and animal study have not been widely conducted to confirm this hypothesis. METHODS: We compared the prevalence of adhesin gene and their coexistent pattern between upper and lower urinary tract infection (UTI) strains. fimH mutant (EC114FM), papGII mutant (EC114PM) and fimH/papGII double mutant (EC114DM) were constructed from a pylonephritogenic strain (EC114). We compared among these strains for the infection ability in bladders and kidneys of female BALB/c mice challenged transurethrally with these bacteria and assessed 1, 3, and 7 days after inoculation. RESULTS: Strains carrying fimH-only genotype were significantly more prevalent in lower UTI (P < 0.001). Strains carrying the fimH/papGII, but not papGII-only, were significantly associated with upper UTI (P = 0.001). Incidence of kidney infection increased after inoculation with EC114 on days 1 and 3, at both low and high dose, as compared with EC114DM; and the effect was greater than the sum of individual effect of EC114PM and EC114FM. Geometric means of quantitative bacterial counts in the kidneys significantly decreased when challenged with EC114FM on days 3 and 7, EC114PM on day 3 and EC114DM on day 1 after inoculation at high dose, as compared with EC114 (all P < 0.05). CONCLUSIONS: We confirmed the advantage and synergistic action of FimH and PapGII for E. coli kidney infection and concluded that antagonists against FimH and PapGII adhesin may prevent kidney infection and enable its management.

Genome placement of alpha-haemolysin cluster is associated with alpha-haemolysin sequence variation, adhesin and iron acquisition factor profile of Escherichia coli.

Since the discovery of haemolysis, many studies focused on a deeper understanding of this phenotype in Escherichia coli and its association with other virulence genes, diseases and pathogenic attributes/functions in the host. Our virulence-associated factor profiling and genome-wide association analysis of genomes of haemolytic and nonhaemolytic E. coli unveiled high prevalence of adhesins, iron acquisition genes and toxins in haemolytic bacteria. In the case of fimbriae with high prevalence, we analysed sequence variation of FimH, EcpD and CsgA, and showed that different adhesin variants were present in the analysed groups, indicating altered adhesive capabilities of haemolytic and nonhaemolytic E. coli. Analysis of over 1000 haemolytic E. coli genomes revealed that they are pathotypically, genetically and antigenically diverse, but their adhesin and iron acquisition repertoire is associated with genome placement of hlyCABD cluster. Haemolytic E. coli with chromosome-encoded alpha-haemolysin had high frequency of P, S, Auf fimbriae and multiple iron acquisition systems such as aerobactin, yersiniabactin, salmochelin, Fec, Sit, Bfd and hemin uptake systems. Haemolytic E. coli with plasmid-encoded alpha-haemolysin had similar adhesin profile to nonpathogenic E. coli, with high prevalence of Stg, Yra, Ygi, Ycb, Ybg, Ycf, Sfm, F9 fimbriae, Paa, Lda, intimin and type 3 secretion system encoding genes. Analysis of HlyCABD sequence variation revealed presence of variants associated with genome placement and pathotype.

Structural and functional characterization of the FimH adhesin of uropathogenic Escherichia coli and its novel applications.

Type 1 fimbriae are responsible for bacterial pathogenicity and biofilm production, which are important virulence factors in uropathogenic Escherichia coli strains. Many articles are published on fimH, but each examined a specific aspect of this protein. The current review study aimed at focusing on structure and conformational changes and describing efforts to use this protein in novel potential treatments for urinary tract infections, typing methods, and expression systems. The current study was the first review that briefly and effectively examined issues related to fimH adhesin.

Targeting of Uropathogenic Escherichia coli papG gene using CRISPR-dot nanocomplex reduced virulence of UPEC.

Urinary tract infections (UTI) are the most common infectious diseases in the world. It is becoming increasingly tough to treat because of emergence of antibiotic resistance. So, there is an exigency to develop novel anti-virulence therapeutics to combat multi-drug resistance pathogenic strains. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) discovery has revolutionized the gene editing technology for targeted approach. The greatest obstacle for CRISPR/Cas9 is cargo delivery systems and both viral and plasmid methods have disadvantages. Here, we report a highly efficient novel CRISPR based gene editing strategy, CRISPR-dots for targeting virulence factor Fimbrial Adhesion (papG gene), the bacterial adhesion molecule. Carbon quantum dots (CQD) were used as a delivery vehicle for Cas9 and gRNA into CFT073, a UPEC strain. CQDs were covalently conjugated to cas9 and papG-targeted guide RNA (gRNA) forming a nanocomplex CRISPR-dots (Cri-dots) as confirmed by DLS and transmission electron microscopy. Cri-dots-papG significantly targeted papG as demonstrated by decrease in the expression of papG.Further papG deficient UPEC had significantly reduced adherence ability and biofilm forming ability as demonstrated by fluorescence microscopy and scanning electron microscopy. Also, papG deficient UPEC had reduced virulence as shown by significantly increased survival of Caenorhabditis elegans (C. elegans) worms compared to UPEC. Our findings suggest that targeting of papG gene using Cri-dots nanocomplexes significantly reduced the pathogenicity of UPEC. Thus, Cri-dots nanocomplex offer a novel anti-bacterial strategy against multi-drug resistant UPEC.

Blockage of bacterial FimH prevents mucosal inflammation associated with Crohn's disease.

BACKGROUND: An Escherichia coli (E. coli) pathotype with invasive properties, first reported by Darfeuille-Michaud and termed adherent-invasive E. coli (AIEC), was shown to be prevalent in up to half the individuals with Crohn's Disease (CD), suggesting that these bacteria could be involved in the pathophysiology of CD. Among the genes related to AIEC pathogenicity, fim has the potential to generate an inflammatory reaction from the intestinal epithelial cells and macrophages, as it interacts with TLR4, inducing the production of inflammatory cytokines independently of LPS. Therefore, targeting the bacterial adhesion of FimH-expressing bacteria seems a promising therapeutic approach, consisting of disarming bacteria without killing them, representing a selective strategy to suppress a potentially critical trigger of intestinal inflammation, without disturbing the intestinal microbiota. RESULTS: We analyzed the metagenomic composition of the gut microbiome of 358 patients with CD from two different cohorts and characterized the presence of FimH-expressing bacteria. To assess the pathogenic role of FimH, we used human intestinal explants and tested a specific FimH blocker to prevent bacterial adhesion and associated inflammation. We observed a significant and disease activity-dependent enrichment of Enterobacteriaceae in the gut microbiome of patients with CD. Bacterial FimH expression was functionally confirmed in ileal biopsies from 65% of the patients with CD. Using human intestinal explants, we further show that FimH is essential for adhesion and to trigger inflammation. Finally, a specific FimH-blocker, TAK-018, inhibits bacterial adhesion to the intestinal epithelium and prevents inflammation, thus preserving mucosal integrity. CONCLUSIONS: We propose that TAK-018, which is safe and well tolerated in humans, is a promising candidate for the treatment of CD and in particular in preventing its recurrence. Video abstract.

Autotransporter-based surface expression and complementation of split TreA fragments utilized for the detection of antibodies against bovine leukemia virus.

Bovine Leukemia Virus (BLV) is an oncogenic virus which is the etiological agent of a neoplastic disease in infected cattle called enzootic bovine leukemia (EBL). The most common and sensitive diagnostic methods for EBL like enzyme-linked immunosorbent assay (ELISA) is time-consuming and requires manual handling which makes it unsuitable as an on-farm diagnostic test. Hence, there is a need for an alternative test with rapid detection and reduced manual labour. We have previously reported the use of E. coli periplasmic trehalase (TreA) in a split enzyme sensor diagnostic technology to detect immunoglobulins and antigen-specific antibodies. In the current study, a more sensitive detection was attempted by bacterial surface display of split TreA fragment by fusion with the autotransporter AIDA-I. The split TreA fragments fused to antigens require antigen-specific antibodies for complementation and to trigger trehalase activity. This surface complementation strategy was used to detect anti-BLV antibodies in clinical serum by incorporating the antigenic BLV capsid protein in the fusion proteins. To validate this assay, a panel of serum samples obtained from BLV positive and negative cattle were tested in comparison with ELISA results. Evaluation of this panel resulted in positive detection of all true positive samples. We further demonstrated that this assay can be enhanced by pre-adsorption of clinical serum samples using E. coli cells to increase the specificity and help reduce nonspecific binding. In conclusion, the p24 antigen specific BLV assay is a potential tool for simple and rapid diagnosis of BLV infection, which is compatible with both lab-based and a more user friendly on-farm format.