Modeling the Temperature Effect on the Growth of Uropathogenic Escherichia coli in Sous-Vide Chicken Breast.

Uropathogenic Escherichia coli (UPEC) is known to cause 65-75% of human urinary tract infection (UTI) cases. Poultry meat is a reservoir of UPEC, which is suspected to cause foodborne UTIs. In the present study, we aimed to determine the growth potential of UPEC in ready-to-eat chicken breasts prepared by sous-vide processing. Four reference strains isolated from the urine of UTI patients (Bioresource Collection and Research Center [BCRC] 10,675, 15,480, 15,483, and 17,383) were tested by polymerase chain reaction assay for related genes to identify their phylogenetic type and UPEC specificity. A cocktail of these UPEC strains was inoculated into sous-vide cooked chicken breast at 103-4 colony-forming unit (CFU)/g and stored at 4°C, 10°C, 15°C, 20°C, 30°C, and 40°C. Changes in the populations of UPEC during storage were analyzed by a one-step kinetic analysis method using the U.S. Department of Agriculture [USDA] Integrated Pathogen Modeling Program-Global Fit [IPMP-Global Fit]. The results showed that the combination of the no lag phase primary model and the Huang square-root secondary model fitted well with the growth curves to obtain the appropriate kinetic parameters. This combination for predicting UPEC growth kinetics was further validated using it to study additional growth curves at 25°C and 37°C, which showed that the root mean square error, bias factor, and accuracy factor were 0.49-0.59 (log CFU/g), 0.941-0.984, and 1.056-1.063, respectively. In conclusion, the models developed in this study are acceptable and can be used to predict the growth of UPEC in sous-vide chicken breast.

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.

Virulence characterization and clonal analysis of uropathogenic Escherichia coli metallo-beta-lactamase-producing isolates.

BACKGROUND: Uropathogenic Escherichia coli (UPEC) is a major cause of urinary tract infection (UTI); however, treatment of UTI has been challenging due to increased antimicrobial resistance (AMR). One of the most important types of AMR is carbapenem resistance (CR). CR bacteria are known as an important threat to global public health today. Class B metallo-beta-lactamases (MBLs) are one of the major factors for resistance against carbapenems. We aimed to investigate the characteristics of UPEC isolates producing MBL. METHODS: A cross-sectional study was conducted from October 2018 to December 2019 in Ahvaz; Iran. UPEC isolates were identified by biochemical and molecular methods. Metallo-beta-lactamase-producing isolates were detected using modified carbapenem inactivation method (mCIM) and EDTA-CIM (eCIM) tests. MBL genes, phylogenetic group, and virulence genes profile of carbapenem resistant isolates were determined. Conjugation assay and plasmid profiling were conducted to evaluate the ability of transferring of CR to other E. coli isolates. Clonal similarity of isolates were assessed using Enterobacterial intergenic repetitive element sequence (ERIC)-PCR. RESULTS: Among 406 UPEC isolates, 12 (2.95%) carbapenem-resistant were detected of which 11 were phenotypically MBL-producing strains. Four isolates were resistant to all investigated antimicrobial agents and were considered possible pandrug-resistant (PDR). blaNDM, blaOXA-48, blaIMP-1, and blaIMP-2 genes were found in 9, 5, 1, and 1 isolates, respectively. Among 30 virulence genes investigated, the traT, fyuA followed by fimH, and iutA with the frequency of 8 (66.7%), 8 (66.7%), 7 (58.3%), and 7 (58.3%) were the most identified genes, respectively. Siderophore production was the main virulence trait among carbapenem-resistant UPEC isolates. Except for two, all other isolates showed weak to moderate virulence index. In all recovered isolates, CR was readily transmitted via plasmids to other isolates during conjugation experiments. CONCLUSION: MBL and carbapenemase genes, especially blaNDM and blaOXA-48 are spreading rapidly among bacteria, which can be a threat to global public health. Therefore monitoring the emergence and dissemination of new AMR is necessary to continuously refine guidelines for empiric antimicrobial therapy. Understanding the mechanisms of resistance and virulence in this group of bacteria can play an effective role in providing new therapeutic methods.

Two Novel Mutations Associated with Polymyxin-B Resistance in a Pandemic Lineage of Uropathogenic Escherichia coli of the Sequence Type 69.

BACKGROUND: Uropathogenic Escherichia coli (UPEC) are frequent pathogens worldwide, impacting on the morbidity and economic costs associated with antimicrobial treatment. OBJECTIVES: We report two novel mutations associated with polymyxin-B resistance in an UPEC isolate collected in 2019. METHODS: Isolate was submitted to antimicrobial susceptibility testing including broth microdilution for polymyxin B. Whole genome was sequenced and analyzed. RESULTS: Polymyxin-B total inhibition occurred at 16 mg/L (resistant). UPEC isolate was assigned to the phylogroup D, serotype O117:H4, and Sequence Type 69. mcr genes were not detected, but two novel mutations in the pmrA/basS (A80S) and pmrB/basR (D149N) genes were identified. CONCLUSIONS: The occurrence of non-mcr polymyxin resistance in E. coli from extraintestinal infections underscores the need of a continuous surveillance of this evolving pathogen.

Virulence genes and phylogenetic groups of uropathogenic Escherichia coli isolates from patients with urinary tract infection and uninfected control subjects: a case-control study.

BACKGROUND: Urinary Tract Infection (UTI) is one of the most common bacterial infectious diseases which causes considerable morbidity and costly health problems. Uropathogenic Escherichia coli (UPEC), the most common pathogen causing UTI, is a highly heterogeneous group of extraintestinal pathogenic E. coli (ExPEC) which may carry a variety of virulence factors and belonging to different phylogenetic backgrounds. The current study aimed to investigate the frequency and association between various virulence factors (VFs) and phylogenetic groups of UPEC and commensal isolates. METHODS: UPEC and commensal E. coli strains isolated from UTI and feces of healthy humans were compared for the presence of VFs and phylogenetic groups. Association between virulence genes was investigated and cluster analysis was employed. RESULTS: According to the results, among a 30 virulence markers tested, the pathogenicity-associated island (PAI), papAH, papEF, fimH, fyuA, and traT genes prevalence were statistically significant in UPEC isolates. A strong association was found between the B2 and D phylogenetic groups and clinical isolates of UPEC; while, commensal isolates were mostly associated with phylogenetic group A. The aggregated VFs scores were more than twice higher in the UPEC isolates in comparison with the commensal isolates. Interestingly, the B2 group in both UPEC and commensal isolates had the highest VF scores. A strong positive association was found between several virulence genes. The clustering results demonstrated that UPEC or commensal E. coli isolates were highly heterogeneous due to different composition of their virulence gene pool and pathogenicity islands. CONCLUSION: Genetic structure and VFs of UPEC strains vary from region to region; therefore, to control the UTI, the epidemiological aspects and characterization of the UPEC isolates need to be investigated in different regions. Since UPEC isolates are generally originate from the commensal strains, it may be feasible to reduce the UTI burden by interfering the intestinal colonization, particularly in the highly pathogenic clonal lineages such as B2.

The Phylogenetic Structure of Reptile, Avian and Uropathogenic Escherichia coli with Particular Reference to Extraintestinal Pathotypes.

The impact of the Gram-negative bacterium Escherichia coli (E. coli) on the microbiomic and pathogenic phenomena occurring in humans and other warm-blooded animals is relatively well-recognized. At the same time, there are scant data concerning the role of E. coli strains in the health and disease of cold-blooded animals. It is presently known that reptiles are common asymptomatic carriers of another human pathogen, Salmonella, which, when transferred to humans, may cause a disease referred to as reptile-associated salmonellosis (RAS). We therefore hypothesized that reptiles may also be carriers of specific E. coli strains (reptilian Escherichia coli, RepEC) which may differ in their genetic composition from the human uropathogenic strain (UPEC) and avian pathogenic E. coli (APEC). Therefore, we isolated RepECs (n = 24) from reptile feces and compared isolated strains' pathogenic potentials and phylogenic relations with the aforementioned UPEC (n = 24) and APEC (n = 24) strains. To this end, we conducted an array of molecular analyses, including determination of the phylogenetic groups of E. coli, virulence genotyping, Pulsed-Field Gel Electrophoresis-Restriction Analysis (RA-PFGE) and genetic population structure analysis using Multi-Locus Sequence Typing (MLST). The majority of the tested RepEC strains belonged to nonpathogenic phylogroups, with an important exception of one strain, which belonged to the pathogenic group B2, typical of extraintestinal pathogenic E. coli. This strain was part of the globally disseminated ST131 lineage. Unlike RepEC strains and in line with previous studies, a high percentage of UPEC strains belonged to the phylogroup B2, and the percentage distribution of phylogroups among the tested APEC strains was relatively homogenous, with most coming from the following nonpathogenic groups: C, A and B1. The RA-PFGE displayed a high genetic diversity among all the tested E. coli groups. In the case of RepEC strains, the frequency of occurrence of virulence genes (VGs) was lower than in the UPEC and APEC strains. The presented study is one of the first attempting to compare the phylogenetic structures of E. coli populations isolated from three groups of vertebrates: reptiles, birds and mammals (humans).

Molecular, phylogenetic and antibiotic resistance analysis of enteroaggregative escherichia coli/uropathogenic Escherichia coli hybrid genotypes causing urinary tract infections.

Background: Horizontal gene transfer of virulence genes (VGs) from different Escherichia coli pathotypes results in the evolution of hybrid strains. Hybrid genotypes of enteroaggregative E. coli and uropathogenic E. coli (EAEC/UPEC) have been reported in sporadic infections and outbreaks of extraintestinal origin. Yet, their association with routine infections is still underrated. Materials and Methods: In this study, we analysed 163 isolates of E. coli from cases of urinary tract infection seeking hybrid (EAEC/UPEC) strains. Using multiplex polymerase chain reaction, we investigated VGs (adhesive and toxin genes) of UPEC along with EAEC marker genes (aap and agg R), ast A (toxin genes) and serine protease autotransporters of Enterobacteriaceae, pet (plasmid-encoded toxin) and pic (mucinase gene). Those UPEC strains which had characteristic defining genes of EAEC (agg R/aap or their combination) were considered UPEC/EAEC hybrids. Results: Molecular predictors of EAEC (aap and aggR) were detected in 20.2% (33/163) of the strains. The pap C was also detected in 36% of the EAEC/UPEC hybrid strains. Phylogenetic analysis revealed that hybrid strains belonged to Group D (60.6%). Nearly 73.8% of UPEC and 75.7% of UPEC/EAEC hybrid strains were multidrug-resistant. Among UPEC isolates, 72.3% and in hybrid UPEC/EAEC, 78.7% isolates were able to produce biofilm. Conclusions: Our results indicated a closer relationship among EAEC and UPEC, which suggested that some EAEC strains can be potential uropathogens. Ours is a first study documenting the existence of EAEC pathotypes VGs in UPEC strains of nosocomial origin; further studies are required to understand the diarrhoeagenic potential of these hybrids.

Comprehensive phenotypic and genotypic characterization and comparison of virulence, biofilm, and antimicrobial resistance in urinary Escherichia coli isolated from canines.

Urinary tract infections (UTIs) affect nearly half of women and an estimated 14 % of the canine companion animal population at least once in their lifetime. As with humans, Escherichia coli is the most commonly isolated bacteria from canine UTIs and infections are dominated by specific phylogenetic groups with notable virulence attributes. In this study, we evaluated uropathogenic E. coli (UPEC) (n = 69) isolated from canine UTIs phenotypically and genotypically for virulence factors, biofilm formation and antimicrobial resistance profiles. Biofilm formation in UPEC strains was positively associated with common virulence factors including papG (p = 0.006), fimH (p < 0.0001), sfaS (p = 0.004), focA (p = 0.004), cnf-1 (p = 0.009) and hlyA (p = 0.006). There was a negative association between biofilm formation and phenotypic antimicrobial resistance for ampicillin (p < 0.0004), ciprofloxacin (p < 0.0001), and trimethoprim-sulfamethoxazole (p < 0.02), as well as multidrug resistance (isolates resistant to ≥ 3 classes of antimicrobials) (p < 0.0002), and the presence of extended spectrum beta-lactamase (ESBL)-producing genes (p < 0.05). In conclusion, UPECs isolated from clinical cases of canine UTIs show a broad negative association between antimicrobial resistance and biofilm formation, and this observation is supported both by phenotypic and genotypic endpoints. As the biofilm formation may result in antimicrobial tolerance, this could be a secondary evasive tactic of UPEC lacking traditional antimicrobial resistance traits. This observation is important for veterinary practitioners to consider when treating puzzling chronic intractable and/or recurrent cases of UTI that appear to be susceptible to antimicrobial therapy via traditional antimicrobial susceptibility testing (AST) methods.

Significant association between genes encoding virulence factors with antibiotic resistance and phylogenetic groups in community acquired uropathogenic Escherichia coli isolates.

BACKGROUND: Antibiotic resistance is an increasing phenomenon in many bacterial pathogens including uropathogenic Escherichia coli. Hypothetical anti-virulent agents could be a solution, but first clear virulence associated gene-pool of antibiotic resistant isolates have to be determined. The aim of this study is to investigate the significant associations between genes encoding VFs with antibiotic resistance and phylogenetic groups in UPEC isolates. RESULTS: The majority of 248 UPEC isolates belonged to phylogenetic group B2 (67.3%). The maximum and minimum resistance was attributed to amoxicillin (90.3%) and both fosfomycin and imipenem (1.6%) respectively. 11.3% of isolates were resistant to all antibiotic agents except that of imipenem, nitrofurantoin and fosfomycin. These highly resistant isolates were placed only in group B2 and D. The most prevalent virulence gene was ompA (93.5%). The hlyA was the only virulence gene that was significantly more prevalent in the highly resistant isolates. The ompA, malX and hlyA genes were obviously more abundant in the antibiotic resistant isolates in comparison to susceptible isolates. The papC gene was associated with amoxicillin resistance (p-value = 0.006, odds ratio: 26.00). CONCLUSIONS: Increased resistance to first line drugs prescribed for UTIs were detected in CA-UPEC isolates in our study.. Minimal resistance was observed against nitrofurantoin, fosfomycin and imipenem. Therefore, they are introduced for application in empirical therapy of UTIs. Fosfomycin may be the most effective antibiotic agent against highly resistant UPEC isolates. The presence of the ompA, malX and hlyA genes were significantly associated with resistance to different antibiotic agents. We assume that the ability of UPEC isolates to upgrade their antibiotic resistance capacity may occurs in compliance with the preliminary existence of specific virulence associated genes. But, more investigation with higher number of bacterial isolates, further virulence associated genes and comparison of gene pools from CA-UPEC isolates with HA-UPEC are proposed to confirm these finding and discovering new aspects of this association.

The novel E. coli cell division protein, YtfB, plays a role in eukaryotic cell adhesion.

Characterisation of protein function based solely on homology searches may overlook functions under specific environmental conditions, or the possibility of a protein having multiple roles. In this study we investigated the role of YtfB, a protein originally identified in a genome-wide screen to cause inhibition of cell division, and has demonstrated to localise to the Escherichia coli division site with some degree of glycan specificity. Interestingly, YtfB also shows homology to the virulence factor OapA from Haemophilus influenzae, which is important for adherence to epithelial cells, indicating the potential of additional function(s) for YtfB. Here we show that E. coli YtfB binds to N'acetylglucosamine and mannobiose glycans with high affinity. The loss of ytfB results in a reduction in the ability of the uropathogenic E. coli strain UTI89 to adhere to human kidney cells, but not to bladder cells, suggesting a specific role in the initial adherence stage of ascending urinary tract infections. Taken together, our results suggest a role for YtfB in adhesion to specific eukaryotic cells, which may be additional, or complementary, to its role in cell division. This study highlights the importance of understanding the possible multiple functions of proteins based on homology, which may be specific to different environmental conditions.

Multilocus sequence typing of Escherichia coli isolates from urinary tract infection patients and from fecal samples of healthy subjects in a college community.

Community-acquired urinary tract infections (UTIs) are one of the most common bacterial infections worldwide. Escherichia coli is the most common cause of community-acquired UTI. In general, UTI results from E. coli in the intestine that enters the bladder via the urethra. However, whether these E. coli strains that cause UTI represent members of the intestinal commensal E. coli or a distinct subgroup of pathogenic E. coli remains unestablished. Here, we analyzed E. coli isolates from fecal samples of healthy volunteers and urine samples of UTI patients obtained from a university-affiliated health center. The E. coli isolates were genotyped by multilocus sequence typing (MLST). From May to October 2018, we analyzed 89 E. coli isolates from 76 (75%) rectal swabs from 113 unique healthy volunteers. We also analyzed 106 (27%) E. coli isolates from 398 unique urine samples collected between August and October 2018. Fecal and urine E. coli isolates each contained 31 distinct sequence types (STs). Nine STs were shared by fecal and urine E. coli isolates, which accounted for approximately 50% of urine isolates typed by MLST. Among the shared genotypes, ST10 and ST131 were significantly more frequently found in fecal samples, whereas ST95 and ST127 were significantly more frequently recovered from UTI samples. ST73 was found only among urine samples. These E. coli genotypes clustered and fluctuated over time. These observations suggest that E. coli genotypes found to cause UTI transiently colonize the intestine and that their primary reservoir may reside outside of the human intestine.

Mapping the Transcriptional and Fitness Landscapes of a Pathogenic E. coli Strain: The Effects of Organic Acid Stress under Aerobic and Anaerobic Conditions.

Several methods are available to probe cellular responses to external stresses at the whole genome level. RNAseq can be used to measure changes in expression of all genes following exposure to stress, but gives no information about the contribution of these genes to an organism's ability to survive the stress. The relative contribution of each non-essential gene in the genome to the fitness of the organism under stress can be obtained using methods that use sequencing to estimate the frequencies of members of a dense transposon library grown under different conditions, for example by transposon-directed insertion sequencing (TraDIS). These two methods thus probe different aspects of the underlying biology of the organism. We were interested to determine the extent to which the data from these two methods converge on related genes and pathways. To do this, we looked at a combination of biologically meaningful stresses. The human gut contains different organic short-chain fatty acids (SCFAs) produced by fermentation of carbon compounds, and Escherichia coli is exposed to these in its passage through the gut. Their effect is likely to depend on both the ambient pH and the level of oxygen present. We, therefore, generated RNAseq and TraDIS data on a uropathogenic E. coli strain grown at either pH 7 or pH 5.5 in the presence or absence of three SCFAs (acetic, propionic and butyric), either aerobically or anaerobically. Our analysis identifies both known and novel pathways as being likely to be important under these conditions. There is no simple correlation between gene expression and fitness, but we found a significant overlap in KEGG pathways that are predicted to be enriched following analysis of the data from the two methods, and the majority of these showed a fitness signature that would be predicted from the gene expression data, assuming expression to be adaptive. Genes which are not in the E. coli core genome were found to be particularly likely to show a positive correlation between level of expression and contribution to fitness.

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).

Complex Multilevel Control of Hemolysin Production by Uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) is the major cause of urinary tract infections. Nearly half of all UPEC strains secrete hemolysin, a cytotoxic pore-forming toxin. Here, we show that the prevalence of the hemolysin toxin gene (hlyA) is highly variable among the most common 83 E. coli sequence types (STs) represented on the EnteroBase genome database. To explore this diversity in the context of a defined monophyletic lineage, we contextualized sequence variation of the hlyCABD operon within the genealogy of the globally disseminated multidrug-resistant ST131 clone. We show that sequence changes in hlyCABD and its newly defined 1.616-kb-long leader sequence correspond to phylogenetic designation, and that ST131 strains with the strongest hemolytic activity belong to the most extensive multidrug-resistant sublineage (clade C2). To define the set of genes involved in hemolysin production, the clade C2 strain S65EC was completely sequenced and subjected to a genome-wide screen by combining saturated transposon mutagenesis and transposon-directed insertion site sequencing with the capacity to lyse red blood cells. Using this approach, and subsequent targeted mutagenesis and complementation, 13 genes were confirmed to be specifically required for production of active hemolysin. New hemolysin-controlling elements included discrete sets of genes involved in lipopolysaccharide (LPS) inner core biosynthesis (waaC, waaF, waaG, and rfaE) and cytoplasmic chaperone activity (dnaK and dnaJ), and we show these are required for hemolysin secretion. Overall, this work provides a unique description of hemolysin sequence diversity in a single clonal lineage and describes a complex multilevel system of regulatory control for this important toxin.IMPORTANCE Uropathogenic E. coli (UPEC) is the major cause of urinary tract infections and a frequent cause of sepsis. Nearly half of all UPEC strains produce the potent cytotoxin hemolysin, and its expression is associated with enhanced virulence. In this study, we explored hemolysin variation within the globally dominant UPEC ST131 clone, finding that strains from the ST131 sublineage with the greatest multidrug resistance also possess the strongest hemolytic activity. We also employed an innovative forward genetic screen to define the set of genes required for hemolysin production. Using this approach, and subsequent targeted mutagenesis and complementation, we identified new hemolysin-controlling elements involved in LPS inner core biosynthesis and cytoplasmic chaperone activity, and we show that mechanistically they are required for hemolysin secretion. These original discoveries substantially enhance our understanding of hemolysin regulation, secretion and function.

Genetically diverse uropathogenic Escherichia coli adopt a common transcriptional program in patients with UTIs.

Uropathogenic Escherichia coli (UPEC) is the major causative agent of uncomplicated urinary tract infections (UTIs). A common virulence genotype of UPEC strains responsible for UTIs is yet to be defined, due to the large variation of virulence factors observed in UPEC strains. We hypothesized that studying UPEC functional responses in patients might reveal universal UPEC features that enable pathogenesis. Here we identify a transcriptional program shared by genetically diverse UPEC strains isolated from 14 patients during uncomplicated UTIs. Strikingly, this in vivo gene expression program is marked by upregulation of translational machinery, providing a mechanism for the rapid growth within the host. Our analysis indicates that switching to a more specialized catabolism and scavenging lifestyle in the host allows for the increased translational output. Our study identifies a common transcriptional program underlying UTIs and illuminates the molecular underpinnings that likely facilitate the fast growth rate of UPEC in infected patients.

Virulence factors of uropathogenic Escherichia coli (UPEC) and correlation with antimicrobial resistance.

BACKGROUND: Escherichia coli has found to be the predominant uropathogen (50-90%) in uncomplicated, community acquired urinary tract infection (UTI). Uropathogenic Escherichia coli (UPEC) express a multitude of virulence factors, which enable the bacteria to establish UTI. The objective of this study was to evaluate the presence of different phenotypic virulence markers in UPEC isolates and determine their correlation with antibiotic resistance pattern. RESULTS: Out of 105 patients, 56 (53%) were females and 49 (47%) were males. The age of the patients in the study ranged from 18 years to 87 years and majority of the patients belonged to the age group 20-29 years. Virulence factor was observed in 65% (n = 69) of UPEC and 20% (n = 22) of control isolates (P = 0.0001). Haemolysin production was observed in 34(32.3%) of uroisolates and 12 (11.4%) of control strain. Similarly, 62% of UPEC and 1% of control produced biofilm (P = 0.0001). The expression of Mannose-resistant hemagglutinin (MRHA) and mannose-sensitive hemagglutinin (MSHA) in uroisolates were 52.3% (n = 55) and 5.7% (n = 6) respectively, whereas in faecal isolates, 8.5% (n = 9) expressed MRHA and none produced MSHA. Antimicrobial resistance showed a high degree of resistance towards ampicillin, cotrimoxazole and norfloxacin. The resistance was observed in significant higher degree in biofilm formers as compared to non-formers. MDR and ESBL was observed in 51 and 46% of test strains and 9 and 7.6% of control strains (P = 0.0001). CONCLUSION: A significant association between virulence factors of UPEC and antimicrobial resistance in UPEC was present. Routine testing of these factors and co-relation with AMR is recommended. These findings will certainly help understand the pathogenicity and proper management of UTI patients, thus decreasing the improper use of antibiotics.

Phylogenic classification and virulence genes profiles of uropathogenic E. coli and diarrhegenic E. coli strains isolated from community acquired infections.

The emergence of E.coli strains displaying patterns of virulence genes from different pathotypes shows that the current classification of E.coli pathotypes may be not enough, the study aimed to compare the phylogenetic groups and urovirulence genes of uropathogenic Escherichia coli (UPEC) and diarrheagenic E.coli (DEC) strains to extend the knowledge of E.coli classification into different pathotypes. A total of 173 UPEC and DEC strains were examined for phylogenetic typing and urovirulence genes by PCR amplifications. In contrast to most reports, phylogenetic group A was the most prevalent in both UPEC and DEC strains, followed by B2 group. Amplification assays revealed that 89.32% and 94.29% of UPEC and DEC strains, respectively, carried at least one of the urovirulence genes, 49.5% and 31.4% of UPEC and DEC strains, respectively, carried ≥ 2 of the urovirulence genes, fim H gene was the most prevalent (66.9% and 91.4%) in UPEC and DEC strains respectively. Twenty different patterns of virulence genes were identified in UPEC while 5 different patterns in DEC strains. Strains with combined virulence patterns of four or five genes were belonged to phylogenetic group B2. Our finding showed a closer relationship between the DEC and UPEC, so raised the suggestion that some DEC strains might be potential uropathogens. These findings also provide different insights into the phylogenetic classification of E. coli as pathogenic or commensals where group A can be an important pathogenic type as well as into the classification as intestinal or extra- intestinal virulence factors.

Phylogeny, sequence-typing and virulence profile of uropathogenic Escherichia coli (UPEC) strains from Pakistan.

BACKGROUND: Escherichia coli lineage ST131 predominates across various spectra of extra-intestinal infections, including urinary tract infection (UTI). The distinctive resistance profile, diverse armamentarium of virulence factors and rapid global dissemination of ST131 E. coli makes it an intriguing pathogen. However, not much is known about the prevalence and genetic attributes of ST131 lineage in Pakistan. METHODS: We estimated prevalence and genetic attributes of E. coli ST131 isolates causing UTI among 155 randomly selected samples. Samples were analyzed for phylogenetic grouping, O-typing and fumC/fimH typing. Isolates were further tested for the ESBL and virulence factors using PCR. RESULTS: Overall, 59% of the UPEC isolates belonged to the phylogenetic group B2, followed by D = 28%, B1 = 8% and A = 5%. Among 18 different Sequence-types, ST131 was the dominant lineage (n = 71; 46%) out of which 72% of the isolates were assigned to the phylogenetic group B2, while 61% adhered to the serogroup O25b. FumC/fimH typing confirmed 49% of the ST131 as H30 sub-types. In this study, significant numbers of the identified ST131 isolates were MDR and 42% showed ESBL phenotypes, out of which 37% carried bla-CTX-M-15. Moreover, different virulence factors were detected in following percentages: fimH,155(100%), iutA 86 (55%), feoB 76 (49%), papC 75 (48%), papGII 70 (45%), kpsMTII 40 (26%), papEF 37 (24%), fyuA 37 (24%), usp 22 (14%), papA 20 (13%), sfa/foc20 (13%), hlyA 18 (12%), afa 15 (10%), cdtB 11 (7%), papGI 6 (4%), papGIII 6 (4%), kpsMTIII 4 (3%) and bmaE2 (1%). CONCLUSION: Conclusively, this study provides important insight into the genetic and virulence attributes of pandemic MDR ST131 strains involved in UTIs. It also highlights higher prevalence of ST131-O25b-H30 UPEC isolates in patients, which was previously unreported from this part of globe.

Serogroup distribution, diversity of exotoxin gene profiles, and phylogenetic grouping of CTX-M-1- producing uropathogenic Escherichia coli.

The emergence of CTX-M-1 producing Uropathogenic Escherichia coli (UPEC) has become a serious challenge. In addition to antimicrobial resistance, a number of virulence factors have been shown. Therefore, this study was designed to determine the prevalence of O- serogroups, phylogenetic groups, exotoxin genes, and antimicrobial resistance properties of CTX-M-1- producing UPEC. A total of 248 UPEC isolates were collected. The antibiotic resistance was performed, and PCR was used to detect the blaCTX-M1, exotoxins, serogroups and phylogroups of UPEC. Of 248 isolates, 95 (38.3%) harbored blaCTX-M-1. Of them, serogroups O1 and O25 were predominant, accounting for 20% and 13.7%, respectively. The hlyA was the dominant exotoxin gene (32.6%), followed by sat (28.4%), vat (22.1%), cnf (13.7%), picU (8.4%), and cdt (2.1%). The hlyA gene was significantly associated with pyelonephritis (P = 0.003). Moreover, almost half of the isolates (45.4%) belonged to phylogenetic group B2. Most of exotoxin genes were present in significantly higher proportions in group B2 isolates except cdt gene (P < 0.05). All of the isolates were susceptible to imipenem, nitrofurantoin, and fosfomycin. The CTX-M-1-producing UPEC strains causing nosocomial infections are more likely to harbor certain exotoxin genes, raising the possibility that this increase in virulence genes may result in an increased risk of complicated UTI.