Cytotoxic Necrotizing Factor Research Articles

High Affinity Binding of Escherichia coli Cytotoxic Necrotizing Factor 1 (CNF1) to Lu/BCAM Adhesion Glycoprotein

The protein toxin Cytotoxic Necrotizing Factor 1 (CNF1) is a major virulence factor of pathogenic Escherichia coli strains. It belongs to a family of single chain AB-toxins, which enter mammalian cells by receptor-mediated endocytosis. Recently, we identified the Lutheran (Lu) adhesion glycoprotein/basal cell adhesion molecule (BCAM) as a cellular receptor for CNF1. Here, we identified the Ig-like domain 2 of Lu/BCAM as main interaction site of the toxin by direct protein-protein interaction and competition studies. Using surface plasmon resonance, we showed a high affinity CNF-Lu/BCAM interaction with a KD of 2.8 nM. Furthermore, we performed small-angle X-ray scattering to define the molecular envelope of the Lu/BCAM-CNF1 complex, suggesting a 6:1 ratio of Lu/BCAM to CNF1 in the receptor-toxin complex. This study leads to a deeper understanding of the interaction between CNF1 and Lu/BCAM, and presents novel opportunities for the development of future anti-toxin strategies.

Cytotoxic Escherichia coli strains encoding colibactin and cytotoxic necrotizing factor (CNF) colonize laboratory macaques

BackgroundMany Escherichia coli strains are considered to be a component of the normal flora found in the human and animal intestinal tracts. While most E. coli strains are commensal, some strains encode virulence factors that enable the bacteria to cause intestinal and extra-intestinal clinically-relevant infections. Colibactin, encoded by a genomic island (pks island), and cytotoxic necrotizing factor (CNF), encoded by the cnf gene, are genotoxic and can modulate cellular differentiation, apoptosis and proliferation. Some commensal and pathogenic pks+ and cnf+ E. coli strains have been associated with inflammation and cancer in humans and animals.ResultsIn the present study, E. coli strains encoding colibactin and CNF were identified in macaque samples. We performed bacterial cultures utilizing rectal swabs and extra-intestinal samples from clinically normal macaques. A total of 239 E. coli strains were isolated from 266 macaques. The strains were identified biochemically and selected isolates were serotyped as O88:H4, O25:H4, O7:H7, OM:H14, and OM:H16. Specific PCR for pks and cnf1 gene amplification, and phylogenetic group identification were performed on all E. coli strains. Among the 239 isolates, 41 (17.2%) were pks+/cnf1−, 19 (7.9%) were pks−/cnf1+, and 31 (13.0%) were pks+/cnf1+. One hundred forty-eight (61.9%) E. coli isolates were negative for both genes (pks−/cnf1−). In total, 72 (30.1%) were positive for pks genes, and 50 (20.9%) were positive for cnf1. No cnf2+ isolates were detected. Both pks+ and cnf1+ E. coli strains belonged mainly to phylogenetic group B2, including B21. Colibactin and CNF cytotoxic activities were observed using a HeLa cell cytotoxicity assay in representative isolates. Whole genome sequencing of 10 representative E. coli strains confirmed the presence of virulence factors and antibiotic resistance genes in rhesus macaque E. coli isolates.ConclusionsOur findings indicate that colibactin- and CNF-encoding E. coli colonize laboratory macaques and can potentially cause clinical and subclinical diseases that impact macaque models.

High yield purification and first structural characterization of the full-length bacterial toxin CNF1.

The Cytotoxic Necrotizing Factor 1 (CNF1) is a bacterial toxin secreted by certain Escherichia coli strains causing severe pathologies, making it a protein of pivotal interest in toxicology. In parallel, the CNF1 capability to influence important neuronal processes, like neuronal arborization, astrocytic support, and efficient ATP production, has been efficiently used in the treatment of neurological diseases, making it a promising candidate for therapy. Nonetheless, there are still some unsolved issues about the CNF1 mechanism of action and structuration probably caused by the difficulty to achieve sufficient amounts of the full-length protein for further studies. Here, we propose an efficient strategy for the production and purification of this toxin as a his-tagged recombinant protein from E. coli extracts (CNF1-H8). CNF1-H8 was expressed at the low temperature of 15°C to diminish its characteristic degradation. Then, its purification was achieved using an immobilized metal affinity chromatography (IMAC) and a size exclusion chromatography so as to collect up to 8 mg of protein per liter of culture in a highly pure form. Routine dynamic light scattering (DLS) experiments showed that the recombinant protein preparations were homogeneous and preserved this state for a long time. Furthermore, CNF1-H8 functionality was confirmed by testing its activity on purified RhoA and on HEp-2 cultured cells. Finally, a first structural characterization of the full-length toxin in terms of secondary structure and thermal stability was performed by circular dichroism (CD). These studies demonstrate that our system can be used to produce high quantities of pure recombinant protein for a detailed structural analysis. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:150-159, 2018.

Cytotoxic necrotizing factor 1 promotes prostate cancer progression through activating the Cdc42-PAK1 axis.

Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections and plays a role in prostatic carcinogenesis and prostate cancer (PCa) progression. However, the mechanisms through which UPEC promotes PCa development and progression are unclear. Cytotoxic necrotizing factor 1 (CNF1) is one of the most important UPEC toxins and its role in PCa progression has never been studied. We found that UPEC-secreted CNF1 promoted the migration and invasion of PCa cells and PCa metastasis. In vitro studies showed that CNF1 promotes pro-migratory and pro-invasive activity through entering PCa cells and activating Cdc42, which subsequently induced PAK1 phosphorylation and up-regulation of MMP-9 expression. CNF1 also promoted pulmonary metastasis in a xenograft mouse model through these mechanisms. PAK1 phosphorylation correlated with advanced grades of PCa in human clinical PCa tissues. These results suggest that CNF1 derived from UPEC plays an important role in PCa progression through activating a Cdc42-PAK1 signal axis and up-regulating the expression of MMP-9. Therefore, surveillance for and treatment of cnf1-carrying UPEC strains may diminish PCa progression and thus have an important clinical therapeutic impact. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Cytotoxic Necrotizing Factor-1 (CNF1) does not promote E. coli infection in a murine model of ascending pyelonephritis

BackgroundUrinary tract infections (UTI) are among the most common and costly infections in both hospitalized and ambulatory patients. Uropathogenic E. coli (UPEC) represent the majority of UTI isolates and are a diverse group of bacteria that utilize a variety of virulence factors to establish infection of the genitourinary tract. The virulence factor cytotoxic necrotizing factor-1 (CNF1) is frequently expressed in clinical UPEC isolates. To date, there have been conflicting reports on the role of CNF1 in the pathogenesis of E. coli urinary tract infections.ResultsWe examined the importance of CNF1 in a murine ascending kidney infection/ pyelonephritis model by performing comparative studies between a clinical UPEC isolate strain and a CNF1-deletion mutant. We found no alterations in bacterial burden with the loss of CNF1, whereas loss of the virulence factor fimH decreased bacterial burdens. In addition, we found no evidence that CNF1 contributed to the recruitment of inflammatory infiltrates in the kidney or bladder in vivo.ConclusionsWhile further examination of CNF-1 may reveal a role in UTI pathogenesis, our data casts doubt on the role of CNF-1 in the pathogenesis of UPEC UTI. As with other infections, different models and approaches are needed to elucidate the contribution of CNF1 to E. coli UTI.

Heterogeneous Family of Cyclomodulins: Smart Weapons That Allow Bacteria to Hijack the Eukaryotic Cell Cycle and Promote Infections.

Some bacterial pathogens modulate signaling pathways of eukaryotic cells in order to subvert the host response for their own benefit, leading to successful colonization and invasion. Pathogenic bacteria produce multiple compounds that generate favorable conditions to their survival and growth during infection in eukaryotic hosts. Many bacterial toxins can alter the cell cycle progression of host cells, impairing essential cellular functions and impeding host cell division. This review summarizes current knowledge regarding cyclomodulins, a heterogeneous family of bacterial effectors that induce eukaryotic cell cycle alterations. We discuss the mechanisms of actions of cyclomodulins according to their biochemical properties, providing examples of various cyclomodulins such as cycle inhibiting factor, γ-glutamyltranspeptidase, cytolethal distending toxins, shiga toxin, subtilase toxin, anthrax toxin, cholera toxin, adenylate cyclase toxins, vacuolating cytotoxin, cytotoxic necrotizing factor, Panton-Valentine leukocidin, phenol soluble modulins, and mycolactone. Special attention is paid to the benefit provided by cyclomodulins to bacteria during colonization of the host.

Prevalence of Genes Encoding Outer Membrane Virulence Factors Among Fecal Escherichia coli Isolates

Objective: Escherichia coli is commensal bacterium of human intestine. The gut is a common pool of E. coli isolates causing urinary tract infections (UTIs). Some of fecal E. coli (FeEC) by the possession of certain virulence factors is able to cause diseases in human and other mammalian models. To evaluate the health threats coordinated with a given fecal source of E. coli strains, we determined the frequency of genes expressing virulence determinants in fecal E. coli isolates collected from human feces in Zabol, southeast of Iran. Methods: Escherichia coli isolates (n = 94) were separated from the feces of patients attending teaching hospitals, and screened for various virulence genes: fimH, his, hlyA, ompT, irp2, iucD, iroN, and cnf1 by using the multiplex polymerase chain reaction (PCR) method. Results: The prevalence of virulence genes was as follows: adhesins (fimH, 98% and iha, 26%), alpha-hemolysins (hlyA, 10%), outer membrane protease (ompT, 67%), aerobactin (iucD, 67%), iron-repressible protein (irp2, 91%) and salmochelin (iroN, 33%) and cytotoxic necrotizing factor 1 (cnf1). According to the diversity of different virulence genes, the examined isolates exhibited 29 different patterns. Conclusion: Our results demonstrated that most of the assessed isolates harbored several virulence factors. Our findings propose possibility of human feces serving as a source for pathogenic organisms, supporting the notion that fecal materials of humans play a role in the epidemiological chain of extra-intestinal pathogenic E. coli. This is the first report of the frequency of virulence factors among E. coli isolates collected from human feces in Iran.

Human Escherichia coli isolates from hemocultures: Septicemia linked to urogenital tract infections is caused by isolates harboring more virulence genes than bacteraemia linked to other conditions

Escherichia coli is the most common cause of bloodstream infections and community-acquired sepsis. The main aim of this study was to determine virulence characteristics of E. coli isolates from hemocultures of patients with a primary disease of urogenital tract, digestive system, a neoplastic blood disease, or other conditions. Results from a set of 314 E. coli isolates from hemocultures were compared to data from a previously published analysis of 1283 fecal commensal E. coli isolates. Genetic profiling of the 314 E. coli isolates involved determination of phylogenetic group (A, B1, B2, D, C, E, and F), identification of 21 virulence factors, as well as 30 bacteriocin-encoding determinants. Pulsed-field gel electrophoresis was used to analyze clonal character of the hemoculture-derived isolates. The E. coli isolates from hemocultures belonged mainly to phylogenetic groups B2 (59.9%) and D (21.0%), and less frequently to phylogroups A (10.2%) and B1 (5.7%). Commonly detected virulence factors included adhesins (fimA 92.0%, pap 47.1%, and sfa 26.8%), and iron-uptake encoding genes (fyuA 87.9%, fepC 79.6%, aer 70.7%, iucC 68.2%, and ireA 13.7%), followed by colibactin (pks island 31.5%), and cytotoxic necrotizing factor (cnf1 11.1%). A higher frequency of microcin producers (and microcin M determinant) and a lower frequency of colicin Ib and microcin B17 was found in hemoculture-derived isolates compared to commensal fecal isolates. E. coli isolates from hemocultures harbored more virulence genes compared to fecal E. coli isolates. In addition, hemoculture E. coli isolates from patients with primary diagnosis related to urogenital tract were clearly different and more virulence genes were detected in these isolates compared to both fecal isolates and hemoculture-derived isolates from patients with blood and gastrointestinal diseases.

Progression of motor deficits in glioma-bearing mice: impact of CNF1 therapy at symptomatic stages.

Glioblastoma (GBM) is the most aggressive type of brain tumor. In this context, animal models represent excellent tools for the early detection and longitudinal mapping of neuronal dysfunction, that are critical in the preclinical validation of new therapeutic strategies. In a mouse glioma model, we developed sensitive behavioral readouts that allow early recognizing and following neurological symptoms. We injected GL261 cells into the primary motor cortex of syngenic mice and we used a battery of behavioral tests to longitudinally monitor the dysfunction induced by tumor growth. Grip strength test revealed an early onset of functional deficit associated to the glioma growth, with a significant forelimb weakness appearing 9 days after tumor inoculation. A later deficit was observed in the rotarod and in the grid walk tasks. Using this model, we found reduced tumor growth and maintenance of behavioral functions following treatment with Cytotoxic Necrotizing Factor 1 (CNF1) at a symptomatic stage. Our data provide a detailed and precise examination of how tumor growth reverberates on the behavioral functions of glioma-bearing mice, providing normative data for the study of therapeutic strategies for glioma treatment. The reduced tumor volume and robust functional sparing observed in CNF1-treated, glioma-bearing mice strengthen the notion that CNF1 delivery is a promising strategy for glioma therapy.

Distribution of Uropathogenic Virulence Genes in Escherichia coli Isolated from Children with Urinary Tract Infection in Sanandaj, Iran

Background and Objectives: Uropathogenic Escherichia coli (UPEC) strains are the most common cause of urinary tract infections (UTIs) in children. UPEC isolates express a range of virulence traits promoting effective colonization of urinary tract. The aim of this study was to determine antibiotic susceptibility and virulence determinants of UPEC isolated from children. Methods: This cross-sectional study was performed on 32 E. coli strains recovered from urine samples of children with UTI aged 0 to 12 years in spring 2015 (between April and June) in Sanandaj, Iran. The isolates were examined by PCR for the presence of virulence genes encoding haemolysin (hly), cytotoxic necrotizing factor type 1 (cnf1), P-fimbriae (Pap), and afimbrial adhesin (afa). Sensitivity to antibiotics was determined using the disk diffusion method. Results: The prevalence of genes encoding adhesins was 25% for pap, and 15.6% for afa. The hly and cnf genes encoding toxins were amplified in 15.6% and 25% of isolates, respectively. The strains isolated from hospitalized patients displayed a greater number of virulence genes compared to the isolates from outpatients. Different patterns of virulence genes were identified. Nitrofurantoin and trimethoprim/sulfamethoxazole were the most and least effective antibiotics with susceptibility rates of 96.9% and 21.9%, respectively. Conclusions: These data show the need for monitoring of drug resistance and its consideration in the treatment of E. coli infections. Investigation of bacterial pathogenicity associated with UTI may help have better medical intervention and management of UTI.

The bacterial toxin CNF1 as a tool to induce retinal degeneration reminiscent of retinitis pigmentosa

Retinitis pigmentosa (RP) comprises a group of inherited pathologies characterized by progressive photoreceptor degeneration. In rodent models of RP, expression of defective genes and retinal degeneration usually manifest during the first weeks of postnatal life, making it difficult to distinguish consequences of primary genetic defects from abnormalities in retinal development. Moreover, mouse eyes are small and not always adequate to test pharmacological and surgical treatments. An inducible paradigm of retinal degeneration potentially extensible to large animals is therefore desirable. Starting from the serendipitous observation that intraocular injections of a Rho GTPase activator, the bacterial toxin Cytotoxic Necrotizing Factor 1 (CNF1), lead to retinal degeneration, we implemented an inducible model recapitulating most of the key features of Retinitis Pigmentosa. The model also unmasks an intrinsic vulnerability of photoreceptors to the mechanism of CNF1 action, indicating still unexplored molecular pathways potentially leading to the death of these cells in inherited forms of retinal degeneration.

Distribution of virulence determinants among antimicrobial-resistant and antimicrobial-susceptible Escherichia coli implicated in urinary tract infections

Introduction: Uropathogenic Escherichia coli (UPEC) rely on the correlation of virulence expression with antimicrobial resistance to persist and cause severe urinary tract infections (UTIs). Objectives: We assessed the virulence pattern and prevalence among UPEC strains susceptible and resistant to multiple antimicrobial classes. Methods: A total of 174 non-duplicate UPEC strains from patients with clinically significant UTIs were analysed for susceptibility to aminoglycoside, antifolate, cephalosporin, nitrofuran and quinolone antibiotics for the production of extended-spectrum β-lactamases and for the presence of six virulence determinants encoding adhesins (afimbrial, Type 1 fimbriae, P and S-fimbriae) and toxins (cytotoxic necrotising factor and haemolysin). Results: Relatively high resistance rates to nalidixic acid, ciprofloxacin, cephalothin and trimethoprim-sulfamethoxazole (82%, 78%, 62% and 59%, respectively) were observed. Fourteen distinct patterns were identified for the virulence determinants such as afaBC, cnfI, fimH, hylA, papEF and sfaDE. The toxin gene, cnfI (75.3%), was the second most prevalent marker to the adhesin, fimH (97.1%). The significant association of sfaDE/hylA (P < 0.01) among antimicrobial resistant and susceptible strains was also observed notwithstanding an overall greater occurrence of virulence factors among the latter. Conclusions: This study provides a snapshot of UPEC complexity in Jamaica and highlights the significant clonal heterogeneity among strains. Such outcomes emphasise the need for evidence-based strategies in the effective management and control of UTIs.

Therapeutic effects of the Rho GTPase modulator CNF1 in a model of Parkinson’s disease

Recent evidence suggests an early involvement of dopaminergic (DA) processes and terminals in Parkinson’s disease (PD). The arborization of neurons depends on the actin cytoskeleton, which in turn is regulated by small GTPases of the Rho family, encompassing Rho, Rac and Cdc42 subfamilies. Indeed, some reports point to a role for Rac and Cdc42 signaling in the pathophysiology of inherited parkinsonisms. We thus investigated the potential therapeutic effect of the modulation of cerebral Rho GTPases in PD. Cytotoxic necrotizing factor 1 (CNF1), a 114 kDa protein toxin produced by Escherichia coli, permanently activates RhoA, Rac1 and Cdc42 in intact cells. We report that the modulation of Rho GTPases by CNF1 results in hypertrophy of DA cell processes of cultured substantia nigra neurons, including increase in length, branching and varicosity. In vivo, the treatment corrects long-standing motor and biochemical asymmetries and restores degenerated nigrostriatal DA tissue after 6-hydroxydopamine lesion. We conclude that the pharmacological modulation of Rho GTPases shows neurorestorative potential and represents a promising avenue in the treatment PD. The study also suggests that naturally occurring molecules acting on Rho GTPase signaling, such as some bacterial protein toxins, might play a role in the development of PD.

Electrophysiology of glioma: a Rho GTPase-activating protein reduces tumor growth and spares neuron structure and function.

Glioblastomas are the most aggressive type of brain tumor. A successful treatment should aim at halting tumor growth and protecting neuronal cells to prevent functional deficits and cognitive deterioration. Here, we exploited a Rho GTPase-activating bacterial protein toxin, cytotoxic necrotizing factor 1 (CNF1), to interfere with glioma cell growth in vitro and vivo. We also investigated whether this toxin spares neuron structure and function in peritumoral areas. We performed a microarray transcriptomic and in-depth proteomic analysis to characterize the molecular changes triggered by CNF1 in glioma cells. We also examined tumor cell senescence and growth in vehicle- and CNF1-treated glioma-bearing mice. Electrophysiological and morphological techniques were used to investigate neuronal alterations in peritumoral cortical areas. Administration of CNF1 triggered molecular and morphological hallmarks of senescence in mouse and human glioma cells in vitro. CNF1 treatment in vivo induced glioma cell senescence and potently reduced tumor volumes. In peritumoral areas of glioma-bearing mice, neurons showed a shrunken dendritic arbor and severe functional alterations such as increased spontaneous activity and reduced visual responsiveness. CNF1 treatment enhanced dendritic length and improved several physiological properties of pyramidal neurons, demonstrating functional preservation of the cortical network. Our findings demonstrate that CNF1 reduces glioma volume while at the same time maintaining the physiological and structural properties of peritumoral neurons. These data indicate a promising strategy for the development of more effective antiglioma therapies.

Immunoadjuvant Properties of the Rho Activating Factor CNF1 in Prophylactic and Curative Vaccination against Leishmania infantum.

There is a need to develop new effective immunoadjuvants for prophylactic or therapeutic vaccines against intracellular pathogens. The activation of Rho GTPases by bacterial cytotoxic necrotizing factor 1 (CNF1) elicits humoral protective responses against protein antigens. Here, we set out to investigate whether CNF1 activity initiates humoral immunity against co-administered parasite antigens and anti-microbial immune signaling. We report that co-administration of wild-type (WT) CNF1 with Leishmania (L.) promastigote antigens at the nasal mucosa triggered prophylactic and curative vaccine responses against this parasite. Vaccination of the mucosa with promastigote lysate antigens combined with WT CNF1 conferred protection against high inoculum L. infantum infection, which reached 82% in the spleen. Immune parameter analysis by antigen recall indicated robust T-helper (Th)1 polarization of immune memory cells, with high IL-2 and IFN-γ production combined with decreased IL-4 production. Additionally, we explored the curative effect of WT CNF1 on previously infected animals. We observed that PL combined with WT CNF1, but not the inactive C866S mutant CNF1 (mCNF1), induced a 58% decrease in the parasite burden in the spleen.

The effects of cytotoxic necrotizing factor 1 expression in the uptake of Escherichia coli K1 by macrophages and the onset of meningitis in newborn mice

ABSTRACTMacrophages are a permissive niche for E. coli K1 multiplication for which the interaction of the bacterial outer membrane protein A and its cognate receptor CD64 are critical. Using in vitro immunofluorescence and live microscopy with ex vivo macrophage cultures from RFP-Lifeact mice, we show that cytotoxic necrotizing factor 1 (CNF1) secreted by E. coli K1 sequesters cellular actin toward microspike formation, thereby limiting actin availability for OmpA-mediated bacterial invasion. Surprisingly, the observed effects of CNF1 occur despite the absence of 67-kDa laminin receptor in macrophages. Concomitantly, the CNF1 deletion mutant of E. coli K1 (Δcnf1) invades macrophages and the brains of newborn mice in greater numbers compared to wild-type. However, the Δcnf1 strain induces less severe pathology in the brain. These results suggest a novel role for CNF1 in limiting E. coli K1 entry into macrophages while exacerbating disease severity in the brains of newborn mice.

Uropathogenic Escherichia coli-Associated Exotoxins.

Escherichia coli are a common cause of infectious disease outside of the gastrointestinal tract. Several independently evolved E. coli clades are common causes of urinary tract and bloodstream infections. There is ample epidemiological and in vitro evidence that several different protein toxins common to many, but not all, of these strains are likely to aid the colonization and immune-evasion ability of these bacteria. This review discusses our current knowledge and areas of ignorance concerning the contribution of the hemolysin; cytotoxic-necrotizing factor-1; and the autotransporters, Sat, Pic, and Vat, to extraintestinal human disease.

Virulence Determinants among Extended-Spectrum B-Lactamases Producers of Uropathogenic Escherichia Coli Isolates in Zagagig University Hospitals, Egypt

Background: Uropathogenic E. coli (UPEC), the major causative agent of urinary tract infections (UTI) worldwide have variant virulent mechanisms. UPEC resistance to commonly used antibiotics represents a major health problem all over the world. The empiric antibacterial therapy of the infections should be based on a local experience of the susceptibility and the resistance profile. Our objectives are to detect the presence of and possible relation between virulence genes and ESBL production in E. coli strains isolated from patients with UTI in Zagazig, Egypt. Methodology: 75 E. coli strains isolated from patients with UTI were screened for virulence genes: fimH (type 1 fimbriae), pap (pyelonephritis-associated pili), sfa/foc (S and F1C fimbriae), afa (afimbrial adhesins), hly (hemolysin), cnf1 (cytotoxic necrotizing factor), aer (aerobactin), traT (transfer gene foe serum resistance) by polymerase chain reaction (PCR) and for antimicrobial resistance by disc diffusion method according to CLSI criteria along with extended spectrum B lactamases (ESBL) detection by double disc synergy (DDS) method. Results: The fimH gene was the most common virulence gene and was detected in 85.3% of the UTI isolates. The trat gene was present in 69.3% (52/75) and the pap gene in 52% of isolates. The aer gene was detected in 40% while the sfa gene and afa gene were shown in 29.3% and 21.3% of isolates respectively. Among the genes coding for toxin, hly was found in 9.3%, while cnf was present in only 8% of isolates. Eight strains genes lacked any tested virulence markers accounting (10.7%). Out of 75 strains, 41 (54.7%) were ESBL producers. Statistically significant relationships were found between fimH, tratT and pap with ESBL production (p values of <0.05, <0.0001 & <0.0001 respectively). Conclusions : There was a significant association between some virulence determinants of UPEC and ESBL production, these determinants may contribute to a better medical intervention. Also we need the used treatment regimens to be revised to reach better therapeutic effects .

Secretion of Alpha-Hemolysin by Escherichia coli Disrupts Tight Junctions in Ulcerative Colitis Patients.

Objectives:The potential of Escherichia coli (E. coli) isolated from inflammatory bowel disease (IBD) patients to damage the integrity of the intestinal epithelium was investigated.Methods:E. coli strains isolated from patients with ulcerative colitis (UC) and healthy controls were tested for virulence capacity by molecular techniques and cytotoxic assays and transepithelial electric resistance (TER). E. coli isolate p19A was selected, and deletion mutants were created for alpha-hemolysin (α-hemolysin) (hly) clusters and cytotoxic necrotizing factor type 1 (cnf1). Probiotic E. coli Nissle and pathogenic E. coli LF82 were used as controls.Results:E. coli strains from patients with active UC completely disrupted epithelial cell tight junctions shortly after inoculation. These strains belong to phylogenetic group B2 and are all α-hemolysin positive. In contrast, probiotic E. coli Nissle, pathogenic E. coli LF82, four E. coli from patients with inactive UC and three E. coli strains from healthy controls did not disrupt tight junctions. E. coli p19A WT as well as cnf1, and single loci of hly mutants from cluster I and II were all able to damage Caco-2 (Heterogeneous human epithelial colorectal adenocarcinoma) cell tight junctions. However, this phenotype was lost in a mutant with knockout (Δ) of both hly loci (P<0.001).Conclusions:UC-associated E. coli producing α-hemolysin can cause rapid loss of tight junction integrity in differentiated Caco-2 cell monolayers. This effect was abolished in a mutant unable to express α-hemolysin. These results suggest that high Hly expression may be a mechanism by which specific strains of E. coli pathobionts can contribute to epithelial barrier dysfunction and pathophysiology of disease in IBD.

Virulence versus fitness determinants in Escherichia coli isolated from asymptomatic bacteriuria in healthy nonpregnant women

Purpose:Escherichia coli isolated from asymptomatic bacteriuria (ABU) correlated genotypically and phenotypically with cystitis isolates may help in distinguishing urovirulence determinants from ‘fitness factors’, latter necessary only for survival of E. coli in urinary tract; for gaining insight into the pathogenesis of urinary tract infection. Materials and Methods: In this cross-sectional study, we compared genotypic (phylogroups and 15 putative virulence genes), and phenotypic profiles of ABU E. coli strains with our previously genotyped collection of cystitis isolates. Virulence score was calculated for each isolate as a number of virulence genes detected. Results: Significant differences were observed in the proportion of four phylogenetic groups (P = 0.009) amongst cystitis and ABU isolates. Average virulence score was higher for ABU isolates (6.6) than cystitis strains (4.2); and hlyA (P = 0.001), cytotoxic necrotising factor 1 (P = 0.00), fyuA (P = 0.00), ibeA (P = 0.00), kpsMII (P = 0.01), and malX/pathogenicity-associated island (P = 0.01) were more frequently present in ABU strains. Conclusions: The expression of adhesins, haemolysin, aerobactin, and capsule synthesis gene were similar in two groups suggesting their role as fitness factors. ABU isolates were better biofilm producers, reflecting its importance in silent persistence. Serum resistance gene which was more expressed in cystitis isolates may represent virulence determinant. Genetic makeup of E. coli does not change much rather genes helping in survival and colonisation are expressed equally in ABU and cystitis isolates as opposed to phenotypic attenuation of those that helps in invasion or inflammation in ABU isolates.