Genome-wide analysis of fitness-factors in uropathogenic Escherichia coli during growth in laboratory media and during urinary tract infections.

Uropathogenic Escherichia coli (UPEC) UTI89 is a well-characterized strain, which has mainly been used to study UPEC virulence during urinary tract infection (UTI). However, little is known on UTI89 key fitness-factors during growth in lab media and during UTI. Here, we used a transposon-insertion-sequencing approach (TraDIS) to reveal the UTI89 essential-genes for in vitro growth and fitness-gene-sets for growth in Luria broth (LB) and EZ-MOPS medium without glucose, as well as for human bacteriuria and mouse cystitis. A total of 293 essential genes for growth were identified and the set of fitness-genes was shown to differ depending on the growth media. A modified, previously validated UTI murine model, with administration of glucose prior to infection was applied. Selected fitness-genes for growth in urine and mouse-bladder colonization were validated using deletion-mutants. Novel fitness-genes, such as tusA, corA and rfaG; involved in sulphur-acquisition, magnesium-uptake, and LPS-biosynthesis, were proved to be important during UTI. Moreover, rfaG was confirmed as relevant in both niches, and therefore it may represent a target for novel UTI-treatment/prevention strategies.

Clonal Relatedness, Phylotyping and Antimicrobial Susceptibility of Extended- spectrum-beta-lactamase Producing Uropathogenic Escherichia coli Isolates from Outpatients and Inpatients.

OBJECTIVES: Antibiotic resistance, phylogenetic groups and Pulsed-Field Gel Electrophoresis (PFGE) patterns were evaluated in urinary tract infection (UTI) Escherichia coli (E. coli) isolates from outpatients and inpatients. METHODS: In this study, antibiotic resistance to E. coli isolated from non-hospitalized and hospitalized patients (153 outpatients and 147 inpatients ) was evaluated in Shiraz County, Iran. Phylogenetic groups and Pulse Field Gel Electrophoresis (PFGE) patterns of 143 ESBLs-producing E. coli were also assessed. RESULTS: The prevalence of ESBL-producing E. coli was shown to be 46.4% and 49% in the outpatient and inpatient UTI E. coli isolates, respectively. Most ESBL-producers were detected on patients hospitalized in clinical surgery units (66.7%) and intensive care units (62.5%). Phylogenetic group D was the dominant group in both the outpatient and inpatient isolates (67.6% and 61.1%, respectively) and also in internal, clinical surgery and ICU units. PFGE results showed more relatedness (>80% similarity) among inpatient isolates. PFGE analysis of 49 ESBL-producing inpatient E.coli in hospital units revealed 17 different pulsotypes, consisting of 11 clones and 6 single patterns. There were no clonal patterns in outpatient isolates, and similarity among the outpatient isolates and also between inpatient and outpatient isolates was less than 80% (75% and 66%, respectively). CONCLUSION: The results showed extreme genomic diversity among the ESBL-producing E. coli isolates in terms of the community and multiclonal dissemination of ESBL-producing E. coli isolated from hospital units.

Compounds targeting YadC of uropathogenic Escherichia coli and its host receptor annexin A2 decrease bacterial colonization in bladder.

BACKGROUND: Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTIs), and fimbrial tip adhesins, play important roles in UPEC colonization. Few fimbrial tip adhesins and their receptors on host cells, which have the potential to be the therapeutic targets, have been identified. METHODS: the UPEC wild-type strain CFT073, ΔyadC and the complemented strain were used to perform assays in vitro and in vivo. The effects of D-xylose targeting YadC on UPEC colonization were evaluated. A YadC receptor was identified by far-western blotting, LC-MS/MS and co-immunoprecipitation. The effects of compounds targeting the receptor on UPEC colonization were tested. FINDINGS: YadC was investigated for its mediation of UPEC adhesion and invasion to bladder epithelial cells in vitro; and its promotion of UPEC colonization in bladder in vivo. D-xylose, targeting YadC, showed prophylactic and therapeutic effects on UPEC colonization. Annexin A2 (ANXA2) was identified as a YadC receptor, involved in UPEC infection. ANXA2 inhibitors attenuated UPEC infections. The yadC gene was widely present in UPEC clinical isolates and phylogenetic analysis of yadC was performed. INTERPRETATION: YadC and its receptor ANXA2 play important roles in UPEC colonization in bladder, leading to novel treatment strategies targeting YadC or ANXA2 for acute UTIs. FUND: This study was supported by grants from the National Natural Science Foundation of China (NSFC) Programs (31670071 and 31970133), the National Key Technologies R&D Program, Intergovernmental international innovation cooperation (2018YFE0102000), Tianjin Science and Technology Commissioner Project (18JCZDJC36000), the Science & Technology Development Fund of Tianjin Education Commission for Higher Education (2017ZD12). The Science Foundation of Tianjin Medical University (2016KY2M08).

Accessory Traits and Phylogenetic Background Predict Escherichia coli Extraintestinal Virulence Better Than Does Ecological Source.

Background: The distinguishing characteristics of extraintestinal pathogenic Escherichia coli (ExPEC) strains are incompletely defined. Methods: We characterized 292 diverse-source human Escherichia coli isolates (116 from fecal specimens, 79 from urine specimens [of which 39 were from patients with cystitis and 40 were from patients with pyelonephritis], and 97 from blood specimens) for phylogenetic group, sequence type complex (STc), and 49 putative extraintestinal pathogenic E. coli (ExPEC)-associated virulence genes. We then assessed these traits and ecological source as predictors of illness severity in a murine sepsis model. Results: The study isolates exhibited a broad range of virulence in mice. Most of the studied bacterial characteristics corresponded significantly with experimental virulence, as did ecological source and established molecular definitions of ExPEC and uropathogenic E. coli (UPEC). Multivariable modeling identified the following bacterial traits as independent predictors of illness severity both overall and among the fecal and clinical (ie, urine and blood) isolates separately: fyuA (yersiniabactin receptor), kpsM K1 (K1 capsule), and kpsM II (group 2 capsules). Molecular UPEC status predicted virulence independently only among fecal isolates. Neither ecological source (ie, clinical vs fecal) nor molecular ExPEC status added predictive power to these traits, which accounted collectively for up to 49% of the observed variation in virulence. Conclusions: Among human-source E. coli isolates, specific accessory traits and phylogenetic/clonal backgrounds predict experimental virulence in a murine sepsis model better than does ecological source.

Molecular characterisation of uropathogenic Escherichia coli isolates at a tertiary care hospital in South India.

Uropathogenic Escherichia coli (UPEC) express a multitude of virulence factors (VFs) to break the inertia of the mucosal barrier of the urinary tract. The aim of the present study was undertaken to characterised the UPEC strains and to correlate carriage of specific virulence markers with different phylogroups and also to correlate these findings with clinical outcome of patients. A total of 156 non-repeated, clinically significant UPEC isolates were studied. Virulent genes were determined by two set of multiplex polymerase chain reaction (PCR). Phylogenetic analysis was performed by triplex PCR methods. Antibiograms and patient's clinical outcomes were collected in a structured pro forma. Of the 156 patients infected by UPEC strains with significant bacterial counts the most common predisposing factors were diabetes (45.5%) followed by carcinoma (7%). On analysis of the VF genes of the isolates, a majority of strains (140; 90%) were possessing the fimH gene followed by iutA (98; 63%), papC (76; 49%), cnf1 (46; 29.5%), hlyA (45; 29%) and neuC (8; 5%), respectively. On phylogenetic analysis, 27 (17%) isolates were belong to phylogroup A, 16 (10%) strains to Group B1, 59 (38%) were from Group B2 and 54 (35%) were from Group D. High prevalence of antibiotic resistance was observed among the isolates. The incidence of papC, cnf1 and hlyA was significantly higher (P < 0.05) among the isolates from relapse patients. Our findings indicate that virulent as well as commensal strains are capable of causing urinary tract infection. Virulence genes as well as patients-related factors are equally responsible for the development of infections and also that virulence genes may help such isolates to persist even with appropriate chemotherapy and be responsible for recurrent infections.

Selective depletion of uropathogenic E. coli from the gut by a FimH antagonist.

Urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC) affect 150 million people annually. Despite effective antibiotic therapy, 30-50% of patients experience recurrent UTIs. In addition, the growing prevalence of UPEC that are resistant to last-line antibiotic treatments, and more recently to carbapenems and colistin, make UTI a prime example of the antibiotic-resistance crisis and emphasize the need for new approaches to treat and prevent bacterial infections. UPEC strains establish reservoirs in the gut from which they are shed in the faeces, and can colonize the periurethral area or vagina and subsequently ascend through the urethra to the urinary tract, where they cause UTIs. UPEC isolates encode up to 16 distinct chaperone-usher pathway pili, and each pilus type may enable colonization of a habitat in the host or environment. For example, the type 1 pilus adhesin FimH binds mannose on the bladder surface, and mediates colonization of the bladder. However, little is known about the mechanisms underlying UPEC persistence in the gut. Here, using a mouse model, we show that F17-like and type 1 pili promote intestinal colonization and show distinct binding to epithelial cells distributed along colonic crypts. Phylogenomic and structural analyses reveal that F17-like pili are closely related to pilus types carried by intestinal pathogens, but are restricted to extra-intestinal pathogenic E. coli. Moreover, we show that targeting FimH with M4284, a high-affinity inhibitory mannoside, reduces intestinal colonization of genetically diverse UPEC isolates, while simultaneously treating UTI, without notably disrupting the structural configuration of the gut microbiota. By selectively depleting intestinal UPEC reservoirs, mannosides could markedly reduce the rate of UTIs and recurrent UTIs.

Virulence factors, antibiotic resistance phenotypes and O-serogroups of Escherichia coli strains isolated from community-acquired urinary tract infection patients in Mexico.

BACKGROUND/PURPOSE: Uropathogenic Escherichia coli (UPEC) strains isolated from patients with community-acquired urinary tract infections (UTIs) were assessed to determine the prevalence of virulence genes, antibiotic resistance, and the O-serogroup of the strains. METHODS: Consenting patients with community-acquired UTI were enrolled at Unidad Médica Familiar Number 64 (Instituto Mexicano del Seguro Social, Estado de Mexico, Mexico) and 321 urine samples were collected. Polymerase chain reaction (PCR) was used to assess 24 virulence genes and 14 O-serogroups. The Kirby-Bauer method was used to evaluate the antibiotic susceptibility of the isolated strains to 12 commonly used antibiotics. RESULTS: A total of 194 strains were identified as E. coli using standard biochemical tests, followed by PCR amplification of 16S ribosomal RNA gene. Only 58.2% of the strains belonged to the assessed 14 O-serogroups. The serogroups O25, O15, O8, and O75 were present in 20.6%, 17%, 6.1%, and 4.6% of strains, respectively. The most frequently occurring virulence genes among UPEC strains included kpsMT (92.2% strains), usp (87.1%), irp2 (79.3%), iha (64.9%), fim (61.3%), set (36%), astA (33.5%), pap (24.7%), and papGII (21.1%). In addition, 97% of the strains were multi-drug resistant (coresistance to 3-11 antibiotics). CONCLUSION: The isolated UPEC strains predominantly belonged to three serogroups (O25, O15, and O8), harboured numerous virulence genes, and are multiresistant to antibiotics. The findings of this study could be used to orient UTI treatment strategies and in epidemiological studies in Mexico.

Characterization of Ciprofloxacin-Resistant and Ciprofloxacin-Susceptible Uropathogenic Escherichia coli Obtained from Patients with Gynecological Cancer.

The objective of this work was to assess the genetic characteristics of uropathogenic Escherichia coli, ciprofloxacin resistance or susceptibility, obtained from patients with gynecological cancer and urinary tract infection (UTI). Seventy-seven E. coli ciprofloxacin-resistant isolates and 38 ciprofloxacin-susceptible were analyzed by polymerase chain reaction (PCR) to determine the phylogenetic groups, virulence factors as iucC, fyuA, hlyC, cnf1 genes, and pks pathogenicity island. The presence of genes related to ciprofloxacin resistance such as qnrA, qnrB, qnrS, aac(6')-Ib-cr, and qepA, and the sequencing of DNA gyrase genes and topoisomerase IV were determined. The genetic profile of the isolates was determined by pulsed-field gel electrophoresis (PFGE). Statistical analysis was performed using Fisher's exact test and Chi-square test. Phylogenetic group B2 was the most prevalent although a great genetic diversity was observed by PFGE. Only genes associated to siderophores were found in ciprofloxacin-resistant isolates; however, in ciprofloxacin-susceptible isolates, genes related to siderophores and toxin, were detected. Additionally qnrB was detected in both populations, ciprofloxacin resistant and susceptible. DNA mutations in gyrA were Ser-83-Leu and Asp-87-Asn and in parC were Ser-80-Ile and Glu-84-Val, Glu-84-Lys. In conclusion, it was observed a high prevalence of qnrB in the population studied; in addition, it was the first time the pks island was observed only in ciprofloxacin-susceptible isolates.

UPEC strain characterization isolated from Mexican patients with recurrent urinary infections.

INTRODUCTION: Any microorganism is capable of causing urinary tract infections (UTI). However, Uropathogenic Escherichia coli (UPEC) is responsible for the majority of UTI. A variety of virulence genes have been identified in UPEC. Diverse epidemiological studies support that specific subsets of genes are characteristic of each E. coli uropathogenic subtype involved in the development of cystitis, pyelonephritis, and urosepsis. METHODOLOGY: Twenty-three E. coli strains isolated from women with cystitis, 25 from men with prostatitis were characterized according to serotype, virulence genes, PFGE profile and susceptibility antimicrobials. RESULTS: E. coli O25:H4-ST131 was more frequently isolated from cystitis than prostatitis. The majority of cystitis strains presented almost all the virulence genes, contrary to 60% of prostatitis strains. The strains characterized by serology, PFGE and MLST and virulence profile showed that cystitis strains shared a lineage with their own genomic fingerprint, indicating that these strains derived from a common ancestor. The prostatitis strains showed serotypes diverse and much higher degree of genetic diversity, indicating that they are an unrelated group.  More than 50% of isolates were resistant to at least 7 of the antimicrobials tested. Antibiotic-tolerant cells were observed, presenting with more frequency in response chloramphenicol, tetracycline, trimethoprim-sulfometoxazole and dicloxicillin. CONCLUSIONS: Persister cells can be detected from the beginning of the infection. The importance of these persister cells that exhibit multidrug tolerance is that a single surviving bacterium can initiate again an infection making it clinical relevant in the chronic urinary tract infection.

Association of O-Antigen Serotype with the Magnitude of Initial Systemic Cytokine Responses and Persistence in the Urinary Tract.

UNLABELLED: Urinary tract infection (UTI) is one of the most common ailments requiring both short-term and prophylactic antibiotic therapies. Progression of infection from the bladder to the kidney is associated with more severe clinical symptoms (e.g., fever and vomiting) as well as with dangerous disease sequelae (e.g., renal scaring and sepsis). Host-pathogen interactions that promote bacterial ascent to the kidney are not completely understood. Prior studies indicate that the magnitude of proinflammatory cytokine elicitation in vitro by clinical isolates of uropathogenic Escherichia coli (UPEC) inversely correlates with the severity of clinical disease. Therefore, we hypothesize that the magnitude of initial proinflammatory responses during infection defines the course and severity of disease. Clinical UPEC isolates obtained from patients with a nonfebrile UTI elicited high systemic proinflammatory responses early during experimental UTI in a murine model and were attenuated in bladder and kidney persistence. Conversely, UPEC isolates obtained from patients with febrile UTI elicited low systemic proinflammatory responses early during experimental UTI and exhibited prolonged persistence in the bladder and kidney. Soluble factors in the supernatant from saturated cultures as well as the lipopolysaccharide (LPS) serotype correlated with the magnitude of proinflammatory responses in vitro. Our data suggest that the structure of the O-antigen sugar moiety of the LPS may determine the strength of cytokine induction by epithelial cells. Moreover, the course and severity of disease appear to be the consequence of the magnitude of initial cytokines produced by the bladder epithelium during infection. IMPORTANCE: The specific host-pathogen interactions that determine the extent and course of disease are not completely understood. Our studies demonstrate that modest changes in the magnitude of cytokine production observed using in vitro models of infection translate into significant ramifications for bacterial persistence and disease severity. While many studies have demonstrated that modifications of the LPS lipid A moiety modulate the extent of Toll-like receptor 4 (TLR4) activation, our studies implicate the O-antigen sugar moiety as another potential rheostat for the modulation of proinflammatory cytokine production.

Relationship between phylogenetic groups, antibiotic resistance and patient characteristics in terms of adhesin genes in cystitis and pyelonephritis isolates of Escherichia coli.

Extraintestinal pathogenic Escherichia coli (E. coli) is considered as the main causative agent of urinary tract infections worldwide. The relationship between antimicrobial resistance, phylogenetic groups, patient characteristics and adhesin virulence genes are complex and not fully understood. In this study, among 146 urinary isolates of E. coli, phylogenetic groups and various adhesin virulence genes were examined with multiplex Polymerase Chain Reaction methods. Patient characteristics divided into sex, cystitis and pyelonephritis; community-acquired and hospital-acquired; complicated and uncomplicated infection. Antimicrobial resistance was also determined. The papAH gene was seen more often in pyelonephritis than cystitis and female than male patients. iha gene was more frequent in hospital-acquired infections than in community-acquired infections. sfa/focDE was more frequent in ampicillin, amikacin, gentamicin, nalidixic acid, norfloxacin, cefuroxime, ceftriaxone, cefazolin, cefotaxime, ciprofloxacin and trimethoprim/sulfamethoxazole susceptible and extended-spectrum ß-lactamase (ESBL) and multi-drug resistance (MDR) negative isolates. focG was seen more often in nalidixic acid, norfloxacin, cefuroxime, ceftriaxone, ciprofloxacin susceptible and MDR negative isolates. fimH and papAH were more commonly observed in amoxicillin/clavulanic acid and cefotaxime susceptible isolates, respectively. iha and afa/draBC genes were more frequent in resistant isolates than the susceptible ones; for iha, in ampicillin, amoxicillin/clavulanic acid, nalidixic acid, cefuroxime, ceftriaxone resistant and ESBL and MDR positive isolates; for afa/draBC, in cefotaxime, cefuroxime, ciprofloxacin, trimethoprim/sulfamethoxazole resistant and ESBL and MDR positive isolates, this trend was observed. ST 131 E. coli virulence gene pattern has a direct effect on resistance profile. Isolates belong to that clonal group has MDR and commonly harbour afa/draBC and iha genes. Our findings may provide new insights into the relationships between pathogenesis, patient characteristics and resistance of E. coli UTI.

Interactions of uroseptic Escherichia coli with renal (A-498) and gastrointestinal (HT-29) cell lines.

We investigated the ability of Escherichia coli isolated from septic patients with urinary tract infection (UTI) to translocate through the gastrointestinal (GI) tract of the same patients using cell-culture models. Forty-seven hospitalized patients with urosepsis were included in this study. E. coli was isolated from their urine and blood (total 94 isolates) and investigated for genetic relatedness and interaction with the cell lines A-498 and HT-29. An initial comparison of the strains isolated from urine and blood showed that 44 out of 47 patients (94 %) had identical strains in their blood and urine. The blood isolates adhered to both cell lines, although their rate of adherence to A-498 cells was significantly higher than that to HT-29 cells (5.8±3.8 per cell vs 2.8±1.9; P<0.0001). The rate of translocation in A-498 cells was also significantly higher after 120 min (8.7×10(5) vs 2.9×10(5); P = 0.0006). Three non-identical blood isolates were unable to translocate in HT-29 cells, indicating that host immune factors might be more important than bacterial ability to translocate the GI epithelium in these patients. Our data showed that blood isolates from uroseptic patients are able to adhere to and translocate through both cell lines. This suggests that E. coli in patients with UTI may translocate from either the GI tract or the urinary tract, hence questioning the assumption that the urinary tract is the only source of septicaemia in these patients.

Virulence determinants in Escherichia coli associated with recurrent cystitis in sexually active women.

More than a quarter of women who experience acute cystitis develop recurrence but information on specific urovirulent genetic profile of uropathogenic Escherichia coli associated with recurrent cystitis is still limited. In this prospective cohort study, index episode E. coli from a cohort of 46 sexually active women with acute cystitis who reported recurrence during followup were grouped into repeat infection (RI) and single infection (SI) isolates, based on enterobacterial repetitive intergenic consensus (ERIC) PCR profile comparison with subsequent E. coli isolated from same women. PCR for phylogrouping and 15 virulence genes along with test for biofilm formation were done. Virulence score was calculated for each isolate as number of virulence genes detected. Among 46 index E. coli, 22 were RI, and 24 were SI isolates. RI isolates had phylogroup B2 as majority (54.5%) which is typically described as more virulent phylogroup and virulence score for RI isolates was also significantly higher compared to SI isolates. Virulence gene malX (p = 0.03) was significantly associated with RI isolates. 68.2% RI isolates were strong to moderate biofilm producers in comparison to 33.3% SI isolates, an important survival strategy to reside in bladder and or vagina. Overall, E. coli associated with recurrent cystitis appear to be more virulent and malX seems to have a role in causing repeat infection.

Draft genome sequences of five recent human uropathogenic Escherichia coli isolates.

This study reports the release of draft genome sequences of five isolates of uropathogenic Escherichia coli (UPEC), isolated from patients suffering from uncomplicated cystitis in 2012 in Ann Arbor, Michigan. Phylogenetic analyses revealed that these strains belonged to E. coli phylogroups B2 and D and are closely related to known UPEC strains. Comparative genomic analysis revealed that more conserved proteins were shared between these recent isolates and UPEC strains causing cystitis than those causing pyelonephritis. Additional genomic comparisons identified that three isolates encode a type III secretion system (T3SS) and a putative T3SS effector gene cluster along with an invasin-like outer membrane protein. The presence of T3SS genes is a rare occurrence among UPEC strains. These genomes further substantiate the heterogeneity of the gene pool of UPEC and provide a foundation for comparative genomic studies using recent clinical isolates.

Escherichia coli-mediated impairment of ureteric contractility is uropathogenic E. coli specific.

BACKGROUND: Ureters are fundamental for keeping kidneys free from uropathogenic Escherichia coli (UPEC), but we have shown that 2 strains (J96 and 536) can subvert this role and reduce ureteric contractility. To determine whether this is (1) a widespread feature of UPEC, (2) exhibited only by UPEC, and (3) dependent upon type 1 fimbriae, we analyzed strains representing epidemiologically important multilocus sequence types ST131, ST73, and ST95 and non-UPEC E. coli. METHODS: Contractility and calcium transients in intact rat ureters were compared between strains. Mannose and fim mutants were used to investigate the role of type 1 fimbriae. RESULTS: Non-UPEC had no significant effect on contractility, with a mean decrease after 8 hours of 8.8%, compared with 8.8% in controls. UPEC effects on contractility were strain specific, with decreases from 9.47% to 96.7%. Mannose inhibited the effects of the most potent strains (CFT073 and UTI89) but had variable effects among other UPEC strains. Mutation and complementation studies showed that the effects of the UTI89 cystitis isolate were fimH dependent. CONCLUSIONS: We find that (1) non-UPEC do not affect ureteric contractility, (2) impairment of contractility is a common feature of UPEC, and (3) the mechanism varies between strains, but for the most potent UPEC type 1 fimbriae are involved.

Uropathogenic Escherichia coli isolates from pregnant women in different countries.

Urinary tract infection (UTI) is common during pregnancy and can be associated with negative outcomes for both the mother and fetus. Increased risk of infection among these patients has been attributed to physiological changes, and less focus has been placed on Escherichia coli, the most frequent causative agent. We investigated the virulence properties of isolates causing UTI in pregnant women in Sweden, Uganda, and Vietnam, as well as nonpregnant women in Sweden. Although phylogenetic group B2 was the most prevalent group, more Ugandan isolates belonged to group B1, associated with commensal strains, than isolates from other countries. Adherence to and invasion of urothelial cells, key events in the infection process, were low among group B1 isolates from pregnant Swedish women compared to those from nonpregnant patients. Similar levels of adherence and invasion were seen in isolates from pregnant women in Uganda and Vietnam. More biofilm was formed by group B2 isolates than by those belonging to group B1 and by Ugandan group B2 isolates than by those from pregnant Swedish and Vietnamese women. The antigen 43a-encoding gene, fluA(CFT073), was most prevalent among Ugandan isolates. Expression of the biofilm components, curli and cellulose, was low among all isolates. Multidrug resistance was more common among isolates from Uganda and Vietnam than among those from Swedish patients. We suggest that while bacterial virulence properties play an important role in UTI during pregnancy, physiological changes in the host may contribute more to the incidence of infection caused by less virulent E. coli.

Fimbrial profiles predict virulence of uropathogenic Escherichia coli strains: contribution of ygi and yad fimbriae.

Escherichia coli, a cause of ∼90% of urinary tract infections (UTI), utilizes fimbrial adhesins to colonize the uroepithelium. Pyelonephritis isolate E. coli CFT073 carries 12 fimbrial operons, 5 of which have never been studied. Using multiplex PCR, the prevalence of these 12 and 3 additional fimbrial types was determined for a collection of 303 E. coli isolates (57 human commensal, 32 animal commensal, 54 asymptomatic bacteriuria, 45 complicated UTI, 38 uncomplicated cystitis, and 77 pyelonephritis). The number of fimbrial types per E. coli isolate was distributed bimodally: those with low (3.2 ± 1.1) and those with high (8.3 ± 1.3) numbers of fimbrial types (means ± standard errors of the means). The fimbrial genes ygiL, yadN, yfcV, and c2395 were significantly more prevalent among urine isolates than human commensal isolates. The effect of deletion of Ygi and Yad fimbrial operons on growth, motility, biofilm formation, adherence to immortalized human epithelial cells, and pathogenesis in the mouse model of UTI was examined. Yad fimbriae were necessary for wild-type levels of adherence to a bladder epithelial cell line and for biofilm formation. Deletion of these fimbrial genes increased motility. Ygi fimbriae were necessary for wild-type levels of adherence to a human embryonic kidney cell line, biofilm formation, and in vivo fitness in the urine and kidneys. Complementation of each fimbrial mutant restored wild-type levels of motility, biofilm formation, adherence and, for ygi, in vivo fitness. A double deletion strain, Δygi Δyad, was attenuated in the urine, bladder, and kidneys in the mouse model, demonstrating that these fimbriae contribute to uropathogenesis.

Virulence factors and genetic variability of uropathogenic Escherichia coli isolated from dogs and cats in Italy.

In this study, the association between virulence genotypes and phylogenetic groups among Escherichia (E.) coli isolates obtained from pet dogs and cats with cystitis was detected, and fingerprinting methods were used to explore the relationship among strains. Forty uropathogenic E. coli (UPEC) isolated from dogs (n = 30) and cats (n = 10) in Italy were analysed by polymerase chain reaction (PCR) for the presence of virulence factors and their classification into phylogenetic groups. The same strains were characterized by repetitive extragenic palindromic (REP)- and enterobacterial repetitive intergenic consensus (ERIC)-PCR techniques. We found a high number of virulence factors such as fimbriae A, S fimbriae (sfa) and cytotoxic necrotizing factor 1 (cnf1) significantly associated with phylogenetic group B2. We demonstrated a high correlation between α-hemolysin A and pyelonephritis C, sfa, and cnf1 operons, confirming the presence of pathogenicity islands in these strains. In addition, UPEC belonging to group B2 harboured a greater number of virulence factors than strains from phylogenetic groups A, B1, and D. REP- and ERIC-PCR grouped the UPEC isolates into two major clusters, the former grouping E. coli strains belonging to phylogenetic group B2 and D, the latter grouping those belonging to groups A and B1. Given the significant genetic variability among the UPEC strains found in our study, it can be hypothesized that no specific genotype is responsible for cystitis in cats or dogs.

Comparative genomics of Escherichia coli strains causing urinary tract infections.

The virulence determinants of uropathogenic Escherichia coli have been studied extensively over the years, but relatively little is known about what differentiates isolates causing various types of urinary tract infections. In this study, we compared the genomic profiles of 45 strains from a range of different clinical backgrounds, i.e., urosepsis, pyelonephritis, cystitis, and asymptomatic bacteriuria (ABU), using comparative genomic hybridization analysis. A microarray based on 31 complete E. coli sequences was used. It emerged that there is little correlation between the genotypes of the strains and their disease categories but strong correlation between the genotype and the phylogenetic group association. Also, very few genetic differences may exist between isolates causing symptomatic and asymptomatic infections. Only relatively few genes that could potentially differentiate between the individual disease categories were identified. Among these were two genomic islands, namely, pathogenicity island (PAI)-CFT073-serU and PAI-CFT073-pheU, which were significantly more associated with the pyelonephritis and urosepsis isolates than with the ABU and cystitis isolates. These two islands harbor genes encoding virulence factors, such as P fimbriae (pyelonephritis-associated fimbriae) and an important immunomodulatory protein, TcpC. It seems that both urovirulence and growth fitness can be attributed to an assortment of genes rather than to a specific gene set. Taken together, urovirulence and fitness are the results of the interplay of a mixture of factors taken from a rich menu of genes.

Molecular characterization of uropathogenic and diarrheagenic Escherichia coli pathotypes.

In this study diarrheagenic and uropathogenic Escherichia coli (UPEC) strains were comparatively characterized according to serotype, hemolytic activity, protein polymorphism among housekeeping enzymes, phylogenetic group and urovirulence genes. Intra-serogroup/serotype variations were observed. Hemolytic activity was detected in 100%, 93%, 67% and 39% of UPEC, EAEC, EPEC and ETEC strains, respectively. The alpha-hemolytic phenotype was observed in all pathogenic groups while beta-hemolytic phenotype was less frequent. PCR phylotyping revealed higher prevalence of diarrheagenic E. coli in groups A and D while uropathogenic strains were mainly found in subgroup B2. Amplification assays revealed that 74%, 45% and 22% of UPEC, EAEC and EPEC strains, respectively, carried at least one of the urovirulence sequences. The molecular typing system revealed a pathotype-specific clonal group distribution and showed a closer relationship between the EAEC and UPEC. Additionally, the occurrence of urovirulence traits, especially those related to iron acquisition, was more frequent among EAEC and UPEC than among the other E. coli pathotypes. This observation is of special value considering that the EAEC pathotype constitutes an emerging group of enteropathogens, particularly, in developing countries, and information on their pathogenic and phylogenetic characteristics is still scarce.