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1.  Molecular Characterization of Uropathogenic Escherichia coli: Nalidixic Acid and Ciprofloxacin Resistance, Virulent Factors and Phylogenetic Background 
Background and Objective: A proficient pathogen should be virulent, resistant to antibiotics, and epidemic. However, the interplay between resistance and virulence is poorly understood. Perhaps, the most commonly accepted view is that resistance to quinolones is linked to a loss of virulence factors. However, the low virulent phylogenetic groups may be more prone to acquire resistance to quinolones. The aim of this study was to identify and characterise the Nalidixic Acid (NA) and ciprofloxacin (CIP) resistant uropathogenic Escherichia coli (UPEC) isolates with respect to virulence and phylogenetic background, from hospital settings in Kolkata, an eastern region in India. Research based on these bacterial populations will help in understanding the molecular mechanisms underlying the association between resistance and virulence, that in turn, may help in managing the future disseminations of UTIs in their entirety.
Material and Methods: One hundred and ten E. coli isolates were screened against NA and CIP using Kirby-Bauer disk diffusion technique, following CLSI guidelines. Prevalence of virulent factor genes and distribution of phylogenetic groups amongst the isolates was determined by PCR, using gene specific primers against the different virulent factors and DNA markers (chuA, yjaA and DNA fragment, TSPE4.C2) respectively. Statistical analysis of the data was performed using SPSS software.
Results: Resistance to both NA and CIP was reported in 75.5 % of the isolates which were analysed. The virulent determinants, papC, pap GII, papEF, afa, cnf1, hlyA and iroN were significantly predominant in the drug susceptible than the resistant isolates. A significant reduction of phylogroup B2 in NA (85.7% versus 64.6%, χ2P<0.001) and CIP (85.2 % versus 61.4%, χ2P<0.001) resistant UPEC isolates, followed by increase in predominance of non-B2 phylotypes (group D and group B1), were observed.
Conclusion: This is the first report from India that has indicated possible evidence on horizontal gene transfer from pathogenic to commensal strains and selection of the latter, on extensive usage of this group of antimicrobials in hospital settings, where these drugs were routinely prescribed for treating urinary tract infection. Therefore, this information necessitates surveillance programs and administration of effective strategies, to put an end to random prescription policies involving this group of antimicrobials.
PMCID: PMC3919340  PMID: 24551624
Ciprofloxacin; Drug resistance; Virulence; Phylogeny
2.  Virulence Factors and O-Serogroups Profiles of Uropathogenic Escherichia Coli Isolated from Iranian Pediatric Patients 
Uropathogenic Escherichia coli O- Serogroups with their virulence factors are the most prevalent causes of UTIs.
The present investigation was performed to study the virulence factors and O-Serogroups profiles of UPEC isolated from Iranian pediatric patients.
Patients and Methods:
This cross sectional investigation was performed on 100 urine samples collected from hospitalized pediatrics of Baqiyatallah Hospital, Tehran, Iran. Midstream urine was collected to decrease potential bacterial, cellular and artifactual contamination. All samples were cultured and those with positive results were subjected to polymerase chain reactions to detect pap, cnf1, afa, sfa and hlyA genes and various O- Serogroups.
We found that 37.5% of boys and 75% of girls had positive results for Escherichia coli. We also found that O1 (19.33%), O2 (13.33%), O6 (13.33%), O4 (11.66%), and O18 (11.66 %) were the most commonly detected Serogroups. Totally, the serogroup of 5% of all strains were not detected. In addition, all of these O- Serogroups were pap+, cnf1+, hlyA+, and afa+. Totally, pap (70 %), cnf1 (56.66 %), and hlyA (43.33 %) were the most commonly detected virulence genes in the both studied groups of children. The sfa (30 %) and afa (26.66 %) genes had the lowest incidence rates.
Special health care should be performed on UTIs management in Iranian pediatric patients. Extended researches should be performed to evaluate relation between other O-Serogroups and virulent genes.
PMCID: PMC3965878  PMID: 24719745
Uropathogenic Escherichia Coli; Pediatrics; Iran
3.  Frequency distribution of virulence factors in uropathogenic Escherichia coli isolated from Kermanshah in 2011-2012 
Uropathogenic Escherichia coli (UPEC) can cause urinary tract infection (UTI). To prevent urine flow lavage, UPEC has acquired several virulence factors called adhesins. These adhesins are expressed and controlled by different genes.
This study was aimed to determine some of the most important genes that control virulence factors of UPEC (pyelonephritis associated pili [pap], S fimbrial adhesion [sfa] and A fimbrial adhesion [afa] genes), which code for adhesins and phenotypic factors.
Materials and Methods:
In total, 205 UPEC isolates from in- and out-patients with UTI were obtained. Polymerase chain reaction was used for gene amplification. One drop of bacterial suspension, one of red blood cells and one of peripheral blood smear were mixed for hemagglutination (HA). Formation of a clump was considered to be positive. Bacteria were grown on blood agar to determine hemolysis. Surface hydrophobicity was determined using the SAT test.
Frequencies of pap, afa and sfa were 42 (20.5%), 17 (8.3%) and 44 (21.5%), respectively. Frequencies of HA, hemolysis and hydrophobicity were 138 (67.3%), 56 (27.3%) and 39 (19%), respectively. Among HA-positive bacteria, 103 (74.6%) were mannose resistant. Our results highlight higher frequency of HA than that of other virulence factors, indicating a crucial role of this virulence factor in UPEC.
We concluded that major differences exist in the prevalence of virulence factors among different UPEC isolated from different countries. The association observed between pathogenicity and virulence factors may promote UPEC survival and growth within the urinary tract. Detecting these genes as the primary controllers of UPEC virulence factors may aid in better management of related infections.
PMCID: PMC4137634  PMID: 25143887
Hemagglutination; hemolysin; hydrophobicity; uropathogenic Escherichia coli; virulence factors
4.  Characterisation of Escherichia coli isolates from the blood of haematological adult patients with bacteraemia: translocation from gut to blood requires the cooperation of multiple virulence factors 
The aim of the study was to investigate whether there are unique pathotypes of Escherichia coli capable of transmission from the gastrointestinal tract to the vascular bed. The study included E. coli strains isolated from clinical materials collected from 115 patients suffering from haematologic malignancies diagnosed with bacteraemia. The genotyping techniques established that 89 E. coli isolates from the blood had the same genotype as the E. coli from the patient’s bowel. The presence of 21 genes encoding virulence factors typical of various E. coli pathotypes and their relationship with the phylogenetic group was established. One-dimensional analysis showed that the focG gene occurred more frequently in the control bowel group, while the ampicillin-resistant afa/dr E. coli were associated with bacteraemia. Blood isolates with the highest occurrence of virulence factors belonged to pathogenic group B2 and non-pathogenic group A. The co-occurrence of multiple genes encoding papC, sfa, usp and cnf1 virulence factors probably predisposes E. coli to translocation from the gastrointestinal tract to the vascular bed in the group of patients with haematologic malignancies. Based on clustering analysis, dominance of the most virulent strains assigned to the cluster with seven virulence factors encoded by the following genes, papC, sfaD/E, cnf1, usp, agn43, hlyA and iutA, was found. The obtained results enforce the previously proposed concept of bowel–blood translocation and further expand our hypothesis by defining the unique virulence characteristics of E. coli isolates, which predispose them to bowel colonisation or translocation and bacteraemia in this group of patients.
PMCID: PMC4426128  PMID: 25655758
5.  Virulence factors and genetic variability of uropathogenic Escherichia coli isolated from dogs and cats in Italy 
Journal of Veterinary Science  2011;12(1):49-55.
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.
PMCID: PMC3053467  PMID: 21368563
cats; dogs; Escherichia coli; genetic variability; virulence factors
6.  Identification of Virulence Factors Genes in Escherichia coli Isolates from Women with Urinary Tract Infection in Mexico 
BioMed Research International  2014;2014:959206.
E coli isolates (108) from Mexican women, clinically diagnosed with urinary tract infection, were screened to identify virulence genes, phylogenetic groups, and antibiotic resistance. Isolates were identified by MicroScan4 system; additionally, the minimum inhibitory concentration (MIC) was assessed. The phylogenetic groups and 16 virulence genes encoding adhesins, toxins, siderophores, lipopolysaccharide (LPS), and invasins were identified by PCR. Phylogenetic groups distribution was as follows: B1 9.3%, A 30.6%, B2 55.6%, and D 4.6%. Virulence genes prevalence was ecp 98.1%, fimH 86.1%, traT 77.8%, sfa/focDE 74.1%, papC 62%, iutA 48.1%, fyuA 44.4%, focG 2.8%, sfaS 1.9%, hlyA 7.4%, cnf-1 6.5%, cdt-B 0.9%, cvaC 2.8%, ibeA 2.8%, and rfc 0.9%. Regarding antimicrobial resistance it was above 50% to ampicillin/sulbactam, ampicillin, piperacillin, trimethoprim/sulfamethoxazole, ciprofloxacin, and levofloxacin. Uropathogenic E. coli clustered mainly in the pathogenic phylogenetic group B2. The isolates showed a high presence of siderophores and adhesion genes and a low presence of genes encoding toxins. The high frequency of papC gene suggests that these isolates have the ability to colonize the kidneys. High resistance to drugs considered as first choice treatment such as trimethoprim/sulfamethoxazole and fluoroquinolones was consistently observed.
PMCID: PMC4026957  PMID: 24895634
7.  Comparison of Adhesin Genes and Antimicrobial Susceptibilities between Uropathogenic and Intestinal Commensal Escherichia coli Strains 
PLoS ONE  2013;8(4):e61169.
The presence of adhesins is arguably an important determinant of pathogenicity for Uropathogenic Escherichia coli (UPEC). Antimicrobial susceptibilities were tested by agar dilution method, fifteen adhesin genes were detected by polymerase chain reaction, and multilocus sequence typing (MLST) was analyzed in 70 UPEC isolates and 41 commensal E. coli strains. Extended-spectrum β-lactamase (ESBL) was determined with confirmatory test. The prevalence of ESBL-producers in UPEC (53%, 37/70) was higher than the commensal intestinal isolates (7%, 3/41), and 97% (36/37) of the ESBL-producing UPEC harbored blaCTX-M genes. afa was present in 36% (10/28) UPEC isolates from recurrent lower urinary tract infection (UTI), and none in the acute pyelonephritis, acute uncomplicated cystitis or commensal strains (P<0.0001). papG was detected in 28% (20/70) of UPEC isolates, while 5% (2/41) of the commensal strains were papG positive (P = 0.0025), and the prevalence of papG was significantly higher in acute pyelonephritis group (71%) than the other two UTI groups (P<0.0001). The prevalence of flu, yqi, yadN and ygiL was significantly higher in UPEC isolates than in the commensal strains. ESBL-producing UPEC showed a lower prevalence of adhesin genes compared with non-ESBL-producing strains. The MLST profiles were different between UPEC and commensal strains, with ST131 (19%, 13/70) and ST10 (20%, 8/41) being the most common MLSTs, respectively. This study demonstrated that several adhesin genes were more prevalent in UPEC isolates than in commensal E. coli, and afa may be associated with recurrent lower UTI whereas papG is more frequently associated with acute pyelonephritis.
PMCID: PMC3621879  PMID: 23593422
8.  Virulence Potential and Genomic Mapping of the Worldwide Clone Escherichia coli ST131 
PLoS ONE  2012;7(3):e34294.
Recently, the worldwide propagation of clonal CTX-M-15-producing Escherichia coli isolates, namely ST131 and O25b:H4, has been reported. Like the majority of extra-intestinal pathogenic E. coli isolates, the pandemic clone ST131 belongs to phylogenetic group B2, and has recently been shown to be highly virulent in a mouse model, even though it lacks several genes encoding key virulence factors (Pap, Cnf1 and HlyA). Using two animal models, Caenorhabditis elegans and zebrafish embryos, we assessed the virulence of three E. coli ST131 strains (2 CTX-M-15- producing urine and 1 non-ESBL-producing faecal isolate), comparing them with five non-ST131 B2 and a group A uropathogenic E. coli (UPEC). In C. elegans, the three ST131 strains showed intermediate virulence between the non virulent group A isolate and the virulent non-ST131 B2 strains. In zebrafish, the CTX-M-15-producing ST131 UPEC isolates were also less virulent than the non-ST131 B2 strains, suggesting that the production of CTX-M-15 is not correlated with enhanced virulence. Amongst the non-ST131 B2 group isolates, variation in pathogenic potential in zebrafish embryos was observed ranging from intermediate to highly virulent. Interestingly, the ST131 strains were equally persistent in surviving embryos as the non-ST131-group B2 strains, suggesting similar mechanisms may account for development of persistent infection. Optical maps of the genome of the ST131 strains were compared with those of 24 reference E. coli strains. Although small differences were seen within the ST131 strains, the tree built on the optical maps showed that these strains belonged to a specific cluster (86% similarity) with only 45% similarity with the other group B2 strains and 25% with strains of group A and D. Thus, the ST131 clone has a genetic composition that differs from other group B2 strains, and appears to be less virulent than previously suspected.
PMCID: PMC3311635  PMID: 22457832
9.  Presence of Putative Repeat-in-Toxin Gene tosA in Escherichia coli Predicts Successful Colonization of the Urinary Tract 
mBio  2011;2(3):e00066-11.
Uropathogenic Escherichia coli (UPEC) strains, which cause the majority of uncomplicated urinary tract infections (UTIs), carry a unique assortment of virulence or fitness genes. However, no single defining set of virulence or fitness genes has been found in all strains of UPEC, making the differentiation between UPEC and fecal commensal strains of E. coli difficult without the use of animal models of infection or phylogenetic grouping. In the present study, we consider three broad categories of virulence factors simultaneously to better define a combination of virulence factors that predicts success in the urinary tract. A total of 314 strains of E. coli, representing isolates from fecal samples, asymptomatic bacteriuria, complicated UTIs, and uncomplicated bladder and kidney infections, were assessed by multiplex PCR for the presence of 15 virulence or fitness genes encoding adhesins, toxins, and iron acquisition systems. The results confirm previous reports of gene prevalence among isolates from different clinical settings and identify several new patterns of gene associations. One gene, tosA, a putative repeat-in-toxin (RTX) homolog, is present in 11% of fecal strains but 25% of urinary isolates. Whereas tosA-positive strains carry an unusually high number (11.2) of the 15 virulence or fitness genes, tosA-negative strains have an average of only 5.4 virulence or fitness genes. The presence of tosA was predictive of successful colonization of a murine model of infection, even among fecal isolates, and can be used as a marker of pathogenic strains of UPEC within a distinct subset of the B2 lineage.
Escherichia coli is the primary cause of urinary tract infections, the most common bacterial infection of humans. Virulence of a uropathogenic strain is typically defined by the clinical source of the isolate, the ability to colonize the bladder and kidneys in a murine model, the phylogenetic group of the bacterium, and virulence gene content. Here we describe a novel single gene, the repeat-in-toxin gene tosA, the presence of which predicts virulence of E. coli isolates regardless of source. Rapid identification of uropathogenic strains of E. coli may aid in the development of therapeutic and preventive therapies.
PMCID: PMC3088117  PMID: 21540363
10.  Chinese Herb-Resistance and Adherence to Human Uroepithelial Cells of Uropathogenic Escherichia Coli 
In order to define the virulence factors between Chinese herb-resistant uropathogenic E. coli and susceptible strains, the UPEC isolates were classified into two groups according to its resistance to Chinese herbs.
Materials and Methods
The susceptibility profile of strains was determined by disk diffusion method. PCR systems were used to detect genes encoding papC, Aer, hly and cnf1. Isolated human urothelial cells were incubated in vitro and investigated with light microscope immunohistochemistry. Adhesion of E. coli to urothelial cells was studied in vitro.
The results showed that, among the 105 UPEC isolates, 18 were resistant to the herbal concoction. Cnf1 and papC occurred in ≥66.7%, of herb-resistant isolates, while, hly and Aer occurred in 22.2% and 27.8% of strains respectively. Only one gene (Cnf1) occurred in >40%, of Herb-susceptible isolates. Other genes were also found in susceptible isolates: papC (20.7%), hly (11.5%), and Aer (6.9%). Light microscopy and immunochemical investigations demonstrated the normal pelvic transitional epithelial cells cultured. The adherence of strains in both groups increased in 30 min., and reached its peak at 60, (Susceptible E. coli) or 120 min., (Resistant E. coli). The adhesion of the susceptible bacteria to human uroepithelial cells was significantly lower compared with that of the resistant E. coli (p<0.05).
These findings revealed that, Chinese herb-resistant uropathogenic E. coli isolates that are hemolytic, and have Aer, papC, hly, Cnf1 genes are more able to be uropathogenic and adherent.
PMCID: PMC3957250  PMID: 24653562
Escherichia coli; Adherence; Virulence genes; Human uroepithelial cells; Chinese herb-resistance
11.  Similarity and Divergence of Phylogenies, Antimicrobial Susceptibilities, and Virulence Factor Profiles of Escherichia coli Isolates Causing Recurrent Urinary Tract Infections That Persist or Result from Reinfection 
Journal of Clinical Microbiology  2012;50(12):4002-4007.
In order to obtain a better molecular understanding of recurrent urinary tract infection (RUTI), we collected 75 cases with repeatedly occurring uncomplicated UTI. The genetic relationships among uropathogenic Escherichia coli (UPEC) isolates were analyzed by pulsed-field gel electrophoresis. While 39 (52%) of the RUTI cases were defined as “persistence” of the same strain as the primary infecting strain, 36 (48%) were characterized by “reinfection” with a new strain that is different from the primary strain. We then examined the antimicrobial susceptibilities and phylogenetic backgrounds of 39 persistence and 86 reinfection UPEC isolates, and screened 44 virulence factor (VF) genes. We found that isolates had significant differences in the following: placement in phylogenetic group B2 (41% versus 21%; P = 0.0193) and the presence of adhesin genes iha (49% versus 28%; P = 0.0233) and papG allele I′ (51% versus 24%; P = 0.003), iron uptake genes fyuA (85% versus 58%; P = 0.0037), irp-2 (87% versus 65%; P = 0.0109), and iutA (87% versus 58%; P = 0.0014), and an aggregate VF score (median, 11 versus 9; P = 0.0030). In addition, 41% of persistence strains harbored three adhesin genes simultaneously, whereas 22% of reinfection isolates did (P = 0.0289). Moreover, 59% versus 29% (P = 0.0014) of persistence and reinfection isolates contained seven types of iron uptake genes. Taken together, the antimicrobial susceptibilities of UPEC isolates had little effect on the RUTI. Compared with reinfection strains, persistence UPEC isolates exhibited higher VF scores and carried more VF genes than may be involved in the development and progression of RUTI.
PMCID: PMC3502954  PMID: 23035197
12.  Phylogenetic grouping and pathotypic comparison of urine and fecal Escherichia coli isolates from children with urinary tract infection 
Brazilian Journal of Microbiology  2014;45(2):509-514.
The aim of this study was to investigate the phylogenetic background and to assess hlyD (involved in the secretion of haemolysin A) and intI1 (encoding a class 1 integrase) in Escherichia coli isolates derived from urinary and fecal specimens. A total of 200 E. coli isolates was collected from patients presenting with urinary tract infection (UTI) during September 2009 to September 2010 and screened for hlyD and intI1 genes by polymerase chain reaction (PCR). Phylogenetic analysis showed that E. coli is composed of four main phylogenetic groups (A, B1, B2 and D) and that uropathogenic E. coli (UPEC) isolates mainly belong to groups B2 (54%) and D (34%) whereas group A (44%) and D (26%) are predominant among commensal E. coli isolates. In this study, hlyD was present in 26% of UPEC and 2% of commensal E. coli isolates. However, hemolytic activity was detected for 42% of UPEC and 6% of commensal E. coli isolates (p < 0.05). intI1 gene was more frequently expressed in UPEC (24%) in comparison with commensal E. coli isolates (12%). Resistance to aztreonam, co-trimoxazole and cefpodoxime were frequently found among UPEC isolates whereas commensal E. coli isolates were commonly resistant to co-trimoxazole, nalidixic acid and cefotaxime. Concluding, a considerable difference between UPEC and commensal E. coli isolates was observed regarding their phylogenetic groups, presence of class 1 integron and hlyD gene, hemolysin activity and resistance pattern. The detection of class 1 integrons and hlyD gene was higher among UPEC compared with commensal E. coli isolates. These findings may contribute for a better understanding of the factors involved in the pathogenesis of UPEC.
PMCID: PMC4166276  PMID: 25242935
Escherichia coli; urinary tract infection (UTI); phylogenetic typing groups; hlyD; intI1
13.  Characterization of Escherichia coli Isolates from Hospital Inpatients or Outpatients with Urinary Tract Infection 
Journal of Clinical Microbiology  2014;52(2):407-418.
Uropathogenic Escherichia coli (UPEC) is the most common cause of community- and hospital-acquired urinary tract infections (UTIs). Isolates from uncomplicated community-acquired UTIs express a variety of virulence traits that promote the efficient colonization of the urinary tract. In contrast, nosocomial UTIs can be caused by E. coli strains that differ in their virulence traits from the community-acquired UTI isolates. UPEC virulence markers are used to distinguish these facultative extraintestinal pathogens, which belong to the intestinal flora of many healthy individuals, from intestinal pathogenic E. coli (IPEC). IPEC is a diarrheagenic pathogen with a characteristic virulence gene set that is absent in UPEC. Here, we characterized 265 isolates from patients with UTIs during inpatient or outpatient treatment at a hospital regarding their phylogenies and IPEC or UPEC virulence traits. Interestingly, 28 of these isolates (10.6%) carried typical IPEC virulence genes that are characteristic of enteroaggregative E. coli (EAEC), Shiga toxin-producing E. coli (STEC), and atypical enteropathogenic E. coli (aEPEC), although IPEC is not considered a uropathogen. Twenty-three isolates harbored the astA gene coding for the EAEC heat-stable enterotoxin 1 (EAST1), and most of them carried virulence genes that are characteristic of UPEC and/or EAEC. Our results indicate that UPEC isolates from hospital patients differ from archetypal community-acquired isolates from uncomplicated UTIs by their spectrum of virulence traits. They represent a diverse group, including EAEC, as well as other IPEC pathotypes, which in addition contain typical UPEC virulence genes. The combination of typical extraintestinal pathogenic E. coli (ExPEC) and IPEC virulence determinants in some isolates demonstrates the marked genome plasticity of E. coli and calls for a reevaluation of the strict pathotype classification of EAEC.
PMCID: PMC3911323  PMID: 24478469
14.  Comparison of extended spectrum β-lactamases-producing Escherichia coli with non-ESBLs-producing E.coli: drug-resistance and virulence 
The virulent factors of Escherichia coli (E.coli) play an important role in the process of pathopoiesis. The study aimed to compare drug-resistant genes and virulence genes between extended spectrum β-lactamases (ESBLs)-producing E.coli and non-ESBLs-producing E.coli to provide a reference for physicians in management of hospital infection.
From October 2010 to August 2011, 96 drug-resistant strains of E.coli isolated were collected from the specimens in Qingdao Municipal Hospital, Qingdao, China. These bacteria strains were divided into a ESBLs-producing group and a non-ESBLs-producing group. Drug sensitivity tests were performed using the Kirby-Bauer (K-B) method. Disinfectant gene, qacEΔ1-sull and 8 virulence genes (CNF2, hlyA, eaeA, VT1, est, bfpA, elt, and CNF1) were tested by polymerase chain reaction (PCR).
Among the 96 E.coli isolates, the ESBLs-producing E.coli comprised 46 (47.9%) strains and the non-ESBLs-producing E.coli consisted of 50 (52.1%) strains. The detection rates of multiple drug-resistant strain, qacEΔ1-sull, CNF2, hlyA, eaeA,VT1, est, bfpA, elt, and CNF1 in 46 ESBLs-producing E.coli isolates were 89.1%, 76.1%, 6.5%, 69.6%, 69.6%, 89.1%, 10.9%, 26.1%, 8.7%, and 19.6%, respectively. In the non-ESBLs-producing E.coli strains, the positive rates of multiple drug-resistant strain, qacEΔ1-sull, CNF2, hlyA, eaeA, VT1, est, bfpA, elt, and CNF1 were 62.0%, 80.0%, 16.0%, 28.0%, 64.0%, 38.0%, 6.0%, 34.0%, 10.0%, and 24.0%, respectively. The difference in the detection rates of multiple drug-resistant strain, hlyA and VT1 between the ESBLs-producing E.coli strains and the non-ESBLs-producing E.coli strains was statistically significant (P<0.05).
The positive rate of multiple drug-resistant strains is higher in the ESBLs-producing strains than in the non-ESBLs-producing strains. The expression of some virulence genes hlyA and VT1 varies between the ESBLs-producing strains and the non-ESBLs-producing strains. Increased awareness of clinicians and enhanced testing by laboratories are required to reduce treatment failures and prevent the spread of multiple drug-resistant strains.
PMCID: PMC4129785  PMID: 25215065
ESBLs-producing Escherichia coli; Non-ESBLs-producing E.coli; Drug-resistant genes; Virulence genes; Multiple drug-resistant
15.  Defining Genomic Islands and Uropathogen-Specific Genes in Uropathogenic Escherichia coli▿  
Journal of Bacteriology  2007;189(9):3532-3546.
Uropathogenic Escherichia coli (UPEC) strains are responsible for the majority of uncomplicated urinary tract infections, which can present clinically as cystitis or pyelonephritis. UPEC strain CFT073, isolated from the blood of a patient with acute pyelonephritis, was most cytotoxic and most virulent in mice among our strain collection. Based on the genome sequence of CFT073, microarrays were utilized in comparative genomic hybridization (CGH) analysis of a panel of uropathogenic and fecal/commensal E. coli isolates. Genomic DNA from seven UPEC (three pyelonephritis and four cystitis) isolates and three fecal/commensal strains, including K-12 MG1655, was hybridized to the CFT073 microarray. The CFT073 genome contains 5,379 genes; CGH analysis revealed that 2,820 (52.4%) of these genes were common to all 11 E. coli strains, yet only 173 UPEC-specific genes were found by CGH to be present in all UPEC strains but in none of the fecal/commensal strains. When the sequences of three additional sequenced UPEC strains (UTI89, 536, and F11) and a commensal strain (HS) were added to the analysis, 131 genes present in all UPEC strains but in no fecal/commensal strains were identified. Seven previously unrecognized genomic islands (>30 kb) were delineated by CGH in addition to the three known pathogenicity islands. These genomic islands comprise 672 kb of the 5,231-kb (12.8%) genome, demonstrating the importance of horizontal transfer for UPEC and the mosaic structure of the genome. UPEC strains contain a greater number of iron acquisition systems than do fecal/commensal strains, which is reflective of the adaptation to the iron-limiting urinary tract environment. Each strain displayed distinct differences in the number and type of known virulence factors. The large number of hypothetical genes in the CFT073 genome, especially those shown to be UPEC specific, strongly suggests that many urovirulence factors remain uncharacterized.
PMCID: PMC1855899  PMID: 17351047
16.  Phylogenetic Analysis and Prevalence of Urosepsis Strains of Escherichia coli Bearing Pathogenicity Island-Like Domains 
Infection and Immunity  2002;70(6):3216-3226.
We characterized 100 Escherichia coli urosepsis isolates from adult patients according to host compromise status by means of ribotyping, PCR phylogenetic grouping, and PCR detection of papG alleles and the virulence-related genes sfa/foc, fyuA, irp-2, aer, hly, cnf-1 and hra. We also tested these strains for copies of pap and hly and their direct physical linkage with other virulence genes in an attempt to look for pathogenicity islands (PAIs) described for the archetypal uropathogenic strains J96, CFT073, and 536. Most of the isolates belonged to E. coli phylogenetic groups B2 and D and bore papG allele II, aer, and fyuA/irp-2. papG allele II-bearing strains were more common in noncompromised patients, while papG allele-negative strains were significantly more frequent in compromised patients. Fifteen ribotypes were identified. The three archetypal strains harbored different ribotypes, and only one-third of our urosepsis strains were genetically related to one of the archetypal strains. Three and 18 strains harbored three and two copies of pap, respectively, and 5 strains harbored two copies of hly. papGIII was physically linked to hly, cnf-1, and hra (reported to be PAI IIJ96-like genetic elements) in 14% of the strains. The PAI IIJ96-like domain was inserted within pheR tRNA in 11 strains and near leuX tRNA in 3 strains. Moreover, the colocalized genes cnf-1, hra, and hly were physically linked to papGII in four strains and to no pap gene in three strains. papGII and hly (reported to be PAI ICFT073-like genetic elements) were physically linked in 16 strains, pointing to a PAI ICFT073-like domain. Three strains contained both a PAI IIJ96-like domain and a PAI ICFTO73-like domain. Forty-two strains harbored papGII but not hly, in keeping with the presence of a PAI IICFT073-like domain. Only one strain harbored a PAI I536-like domain (hly only), and none harbored a PAI IJ96-like domain (papGI plus hly) or a PAI II536-like domain (papGIII plus hly). This study provides new data on the prevalence and variability of physical genetic linkage between pap and certain virulence-associated genes that are consistent with their colocalization on archetypal PAIs.
PMCID: PMC128014  PMID: 12011017
17.  Virulence Characteristics and Genetic Affinities of Multiple Drug Resistant Uropathogenic Escherichia coli from a Semi Urban Locality in India 
PLoS ONE  2011;6(3):e18063.
Extraintestinal pathogenic Escherichia coli (ExPEC) are of significant health concern. The emergence of drug resistant E. coli with high virulence potential is alarming. Lack of sufficient data on transmission dynamics, virulence spectrum and antimicrobial resistance of certain pathogens such as the uropathogenic E. coli (UPEC) from countries with high infection burden, such as India, hinders the infection control and management efforts. In this study, we extensively genotyped and phenotyped a collection of 150 UPEC obtained from patients belonging to a semi-urban, industrialized setting near Pune, India. The isolates representing different clinical categories were analyzed in comparison with 50 commensal E. coli isolates from India as well as 50 ExPEC strains from Germany. Virulent strains were identified based on hemolysis, haemagglutination, cell surface hydrophobicity, serum bactericidal activity as well as with the help of O serotyping. We generated antimicrobial resistance profiles for all the clinical isolates and carried out phylogenetic analysis based on repetitive extragenic palindromic (rep)-PCR. E. coli from urinary tract infection cases expressed higher percentages of type I (45%) and P fimbriae (40%) when compared to fecal isolates (25% and 8% respectively). Hemolytic group comprised of 60% of UPEC and only 2% of E. coli from feces. Additionally, we found that serum resistance and cell surface hydrophobicity were not significantly (p = 0.16/p = 0.51) associated with UPEC from clinical cases. Moreover, clinical isolates exhibited highest resistance against amoxicillin (67.3%) and least against nitrofurantoin (57.3%). We also observed that 31.3% of UPEC were extended-spectrum beta-lactamase (ESBL) producers belonging to serotype O25, of which four were also positive for O25b subgroup that is linked to B2-O25b-ST131-CTX-M-15 virulent/multiresistant type. Furthermore, isolates from India and Germany (as well as global sources) were found to be genetically distinct with no evidence to espouse expansion of E. coli from India to the west or vice-versa.
PMCID: PMC3064663  PMID: 21464963
18.  Presence of pap-, sfa-, and afa-related sequences in necrotoxigenic Escherichia coli isolates from cattle: evidence for new variants of the AFA family. 
Necrotoxigenic Escherichia coli (NTEC) are associated with intestinal and extraintestinal diseases in animals and human beings and produce Cytotoxic Necrotizing Factor 1 (CNF1) or 2 (CNF2). Fourty-three NTEC1, 42 NTEC2, and 32 CNF-negative isolates from cattle were tested by colony DNA hybridization, by plasmid DNA hybridization and by PCR assays for the presence of DNA sequences homologous to the operons coding for fimbrial (PAP/PRS, SFA/FIC, and F17) and afimbrial (AFA/Dr) adhesins of extraintestinal E. coli. Most NTEC1 isolates hybridized with the PAP probes and either the SFA probe (37%) or the AFA probes (49%). Most NTEC2 isolates, in contrast, hybridized with the F17 probe (45%), the AFA probes (19%), or the F17 and AFA probes (22%). A probe-positive plasmid was identified in each of the 19 NTEC2 isolates studied. They all hybridized with the CNF2 toxin probe (Vir plasmids) and most of them with the F17 (6 plasmids) or AFA (7 plasmids) probes. PCR amplification was obtained with 6 of the 11 NTEC isolates tested for the papGII/prsG genes; with all 5 NTEC isolates tested for the sfa and related operons; but with none of the 18 NTEC isolates tested for the afa and related operons. pap-, sfa-, and afa-related sequences are thus present in NTEC isolates from cattle in addition to f17-related operons and may code for adhesins corresponding to specific colonization factors. f17- and afa-related sequences can be located on the Vir plasmids along with the cnf2 gene. Existence of new variants of the AFA/Dr family is evident from the negative results of this family-specific PCR assay.
PMCID: PMC1189403  PMID: 9242999
19.  The Repeat-In-Toxin Family Member TosA Mediates Adherence of Uropathogenic Escherichia coli and Survival during Bacteremia 
Infection and Immunity  2012;80(2):493-505.
Uropathogenic Escherichia coli (UPEC) is responsible for the majority of uncomplicated urinary tract infections (UTI) and represents the most common bacterial infection in adults. UPEC utilizes a wide range of virulence factors to colonize the host, including the novel repeat-in-toxin (RTX) protein TosA, which is specifically expressed in the host urinary tract and contributes significantly to the virulence and survival of UPEC. tosA, found in strains within the B2 phylogenetic subgroup of E. coli, serves as a marker for strains that also contain a large number of well-characterized UPEC virulence factors. The presence of tosA in an E. coli isolate predicts successful colonization of the murine model of ascending UTI, regardless of the source of the isolate. Here, a detailed analysis of the function of tosA revealed that this gene is transcriptionally linked to genes encoding a conserved type 1 secretion system similar to other RTX family members. TosA localized to the cell surface and was found to mediate (i) adherence to host cells derived from the upper urinary tract and (ii) survival in disseminated infections and (iii) to enhance lethality during sepsis (as assessed in two different animal models of infection). An experimental vaccine, using purified TosA, protected vaccinated animals against urosepsis. From this work, it was concluded that TosA belongs to a novel group of RTX proteins that mediate adherence and host damage during UTI and urosepsis and could be a novel target for the development of therapeutics to treat ascending UTIs.
PMCID: PMC3264304  PMID: 22083710
20.  Extended Virulence Genotype of Pathogenic Escherichia coli Isolates Carrying the afa-8 Operon: Evidence of Similarities between Isolates from Humans and Animals with Extraintestinal Infections 
Journal of Clinical Microbiology  2003;41(1):218-226.
The afimbrial AfaE-VIII adhesin is common among Escherichia coli isolates from calves with intestinal and/or extraintestinal infections and from humans with sepsis or pyelonephritis. The virulence genotypes of 77 Escherichia coli afa-8 isolates from farm animals and humans were compared to determine whether any trait of commonality exists between isolates of the different host species. Over half of the extraintestinal afa-8 isolates were associated with pap and f17Ac adhesin genes and contained virulence genes (pap, hly, and cnf1) which are characteristic of human extraintestinal pathogenic E. coli (ExPEC). PapG, which occurs as three known variants (variants I to III), is encoded by the corresponding three alleles of papG. Among the pap-positive strains, new papG variants (papGrs) that differed from the isolates with genes for the three adhesin classes predominated over isolates with papG allele III, which in turn were more prevalent than those with allele II. The data showed the substantial prevalence of the enteroaggregative E. coli heat-stable enterotoxin gene (east1) among afa-8 isolates. Most of the afa-8 isolates harbored the high-pathogenicity island (HPI) present in pathogenic Yersinia; however, two-thirds of the HPI-positive strains shared a truncated HPI integrase gene. The presence of ExPEC-associated virulence factors (VFs) in extraintestinal isolates that carry genes typical of enteric strains and that express O antigens associated with intestinal E. coli is consistent with transfer of VFs and O-antigen determinants between ExPEC and enteric strains. The similarities between animal and human ExPEC strains support the hypothesis of overlapping populations, with members of certain clones or clonal groups including animal and human strains. The presence of multiple-antibiotic-resistant bovine afa-8 strains among such clones may represent a potential public health risk.
PMCID: PMC149575  PMID: 12517852
21.  Detection & characterization of necrotoxin producing Escherichia coli (NTEC) from patients with urinary tract infection (UTI) 
Background & objectives:
Urinary tract infections (UTI) are a serious health problem affecting millions of people each year. Although appreciable work on various aspects of UTI including aetiology per se has been done, information on the emerging pathogens like necrotoxigenic Escherichia coli (NTEC) is largely lacking in India. In the present study E. coli isolates from patients with urinary tract infection from northeastern India were investigated for detection and characterization of NTEC.
E. coli isolated and identified from urine samples of patients with UTI were serotyped. Antibiogram was determined by disc diffusion test. Plasmid profile was also determined. Virulence genes of NTEC (cnf1, cnf2, pap, aer, sfa, hly, afa) were detected by PCR assay. E.coli isolates carrying cnf gene (s) were identified as NTEC.
A total of 550 E. coli were isolated and tested for the presence of cnf genes. Of these, 84 (15.27%) belonged to NTEC. The cnf1 gene was present in 52 (61.9%) isolates, cnf2 in 23 (27.4%) and 9 (10.7%) carried both cnf1 and cnf2 genes. All the NTEC strains were found to harbour the pap and aer genes. Serogroup O4 was found to be the most common among the 12 serogroups identified amongst the NTEC isolates. Majority of the isolates (96.4%) were sensitive to furazolidone and were highly resistant to ampicillin. NTEC were found to harbour different numbers of plasmids (1 to 7). No association was observed between the number of plasmids and the antibiotic resistance of the isolates.
Interpretation & conclusions:
The results of the present study showed that about 15 per cent of E. coli isolates associated with UTI belonged to NTEC. More studies need to be done from other parts of the country.
PMCID: PMC4078504  PMID: 24927352
Antibiogram; Escherichia coli; NTEC; plasmid profile; serotyping; UTI; virulence genes
22.  Cytotoxic Necrotizing Factor 1 and Hemolysin from Uropathogenic Escherichia coli Elicit Different Host Responses in the Murine Bladder 
Infection and Immunity  2013;81(1):99-109.
Cytotoxic necrotizing factor 1 (CNF1) and hemolysin (HlyA1) are toxins produced by uropathogenic Escherichia coli (UPEC). We previously showed that these toxins contribute to the inflammation and tissue damage seen in a mouse model of ascending urinary tract infection. CNF1 constitutively activates small Rho GTPases by deamidation of a conserved glutamine residue, and HlyA1 forms pores in eukaryotic cell membranes. In this study, we used cDNA microarrays of bladder tissue isolated from mice infected intraurethrally with wild-type CP9, CP9cnf1, or CP9ΔhlyA to further evaluate the role that each toxin plays in the host response to UPEC. Regardless of the strain used, we found that UPEC itself elicited a significant change in host gene expression 24 h after inoculation. The largest numbers of upregulated genes were in the cytokine and chemokine signaling and Toll-like receptor signaling pathways. CNF1 exerted a strong positive influence on expression of genes involved in innate immunity and signal transduction and a negative impact on metabolism- and transport-associated genes. HlyA1 evoked an increase in expression of genes that encode innate immunity factors and a decrease in expression of genes involved in cytoskeletal and metabolic processes. Multiplex cytokine and myeloperoxidase assays corroborated our finding that a strong proinflammatory response was elicited by all strains tested. Bladders challenged intraurethrally with purified CNF1 displayed pathology similar to but significantly less intense than the pathology that we observed in CP9-challenged mice. Our data demonstrate substantial roles for CNF1 and HlyA1 in initiation of a strong proinflammatory response to UPEC in the bladder.
PMCID: PMC3536159  PMID: 23090961
23.  Dra/AfaE Adhesin of Uropathogenic Dr/Afa+ Escherichia coli Mediates Mortality in Pregnant Rats  
Infection and Immunity  2005;73(11):7597-7601.
Escherichia coli bearing adhesins of the Dr/Afa family frequently causes urogenital infections during pregnancy in humans and has been associated with mortality in pregnant rats. Two components of the adhesin, Dra/AfaE and Dra/AfaD, considered virulence factors, are responsible for bacterial binding and internalization. We hypothesize that gestational mortality caused by Dr/Afa+ E. coli is mediated by one of these two proteins, Dra/AfaE or Dra/AfaD. In this study, using afaE and/or afaD mutants, we investigated the role of the afaE and afaD genes in the mortality of pregnant rats from intrauterine infection. Sprague-Dawley rats, on the 17th day of pregnancy, were infected with the E. coli afaE+ afaD and afaE afaD+ mutants. The clinical E. coli strain (afaE+ afaD+) and the afaE afaD double mutant were used as positive and negative controls, respectively. The mortality rate was evaluated 24 h after infection. The highest maternal mortality was observed in the group infected with the afaE+ afaD+ strain, followed by the group infected with the afaE+ afaD strain. The mortality was dose dependent. The afaE afaD double mutant did not cause maternal mortality, even with the highest infection dose. The in vivo studies corresponded with the invasion assay, where the afaE+ strains were the most invasive (afaE+ afaD strain > afaE+ afaD+ strain), while the afaE mutant strains (afaE afaD+ and afaE afaD strains) seemed to be noninvasive. This study shows for the first time that the afaE gene coding for the AfaE subunit of Dr/Afa adhesin is involved in the lethal outcome of gestational infection in rats. This lethal effect associated with AfaE correlates with the invasiveness of afaE+ E. coli strains in vitro.
PMCID: PMC1273835  PMID: 16239563
24.  Extraintestinal Escherichia coli Carrying Virulence Genes in Coastal Marine Sediments▿ †  
Applied and Environmental Microbiology  2010;76(17):5659-5668.
Despite the recognized potential of long-term survival or even growth of fecal indicators bacteria (FIB) in marine sediments, this compartment is largely ignored by health protection authorities. We conducted a large-scale study over approximately 50 km of the Marche coasts (Adriatic Sea) at depths ranging from 2 to 5 m. Total and fecal coliforms (FC) were counted by culture-based methods. Escherichia coli was also quantified using fluorescence in situ hybridization targeting specific 16S rRNA sequences, which yielded significantly higher abundances than culture-based methods, suggesting the potential importance of viable but nonculturable E. coli cells. Fecal coliforms displayed high abundances at most sites and showed a prevalence of E. coli. FC isolates (n = 113) were identified by API 20E, additional biochemical tests, and internal transcribed spacer-PCR. E. coli strains, representing 96% of isolates, were then characterized for genomic relatedness and phylogenetic group (A, B1, B2, and D) of origin by randomly amplified polymorphic DNA and multiplex-PCR. The results indicated that E. coli displayed a wide genotypic diversity, also among isolates from the same station, and that 44 of the 109 E. coli isolates belonged to groups B2 and D. Further characterization of B2 and D isolates for the presence of 11 virulence factor genes (pap, sfa/foc, afa, eaeA, ibeA, traT, hlyA, stx1, stx2, aer, and fyuA) showed that 90% of B2 and 65% of D isolates were positive for at least one of these. Most of the variance of both E. coli abundance and assemblage composition (>62%) was explained by a combination of physical-chemical and trophic variables. These findings indicate that coastal sediments could represent a potential reservoir for commensal and pathogenic E. coli and that E. coli distribution in marine coastal sediments largely depends upon the physical and trophic status of the sediment. We conclude that future sampling designs aimed at monitoring the microbiological quality of marine coastal areas should not further neglect the analysis of the sediment and that monitoring of these environments can be improved by including molecular methods as a complement of culture-based techniques.
PMCID: PMC2935034  PMID: 20601507
25.  Phenotypical characterization and adhesin identification in Escherichia coli strains isolated from dogs with urinary tract infections 
Brazilian Journal of Microbiology  2012;43(1):375-381.
Pathogenic strains of Escherichia coli are the most common bacteria associated with urinary tract infections in both humans and companion animals. Standard biochemical tests may be useful in demonstrating detailed phenotypical characteristics of these strains. Thirteen strains of E. coli isolated from dogs with UTIs were submitted to biochemical tests, serotyping for O and H antigens and antimicrobial resistance testing. Furthermore, the presence of papC, sfa, and afa genes was evaluated by PCR, and genetic relationships were established using enterobacterial repetitive intergenic consensus PCR (ERIC-PCR). The antimicrobial that showed the highest resistance rate among the isolates was nalidixic acid (76.9%), followed by cephalotin (69.2%), sulfamethoxazole + trimethoprim (61.5%), tetracycline (61.5%), streptomycin (53.8%), ciprofloxacin (53.8%), ampicillin (46.2%), gentamicin (30.8%) and chloramphenicol (23.1%). No isolate was resistant either to meropenem or nitrofurantoin. Among the five clusters that were identified using ERIC-PCR, one cluster (A) had only one strain, which belonged to a serotype with zoonotic potential (O6:H31) and showed the genes papC+, sfa+, afa-. Strains with the genes papC-, sfa+, afa- were found in two other clusters (C and D), whereas all strains in clusters B and E possessed papC-, sfa-, afa- genes. Sucrose and raffinose phenotypic tests showed some ability in discriminating clusters A, B and C from clusters D and E.
PMCID: PMC3768953  PMID: 24031842
biomarkers; biotyping; ERIC-PCR; UPEC

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