Association of Virulence Markers With Resistance to Oral Antibiotics in Escherichia coli Isolates Causing Uncomplicated Community-Acquired Cystitis.

INTRODUCTION: Uropathogenic Escherichia coli (UPEC) strains equipped with putative virulence factors (VFs) are known to cause approximately 90% of lower urinary tract infections (UTIs) or cystitis affecting individuals of all age groups. Only limited laboratory-based data on the correlation of antimicrobial resistant patterns and VFs of UPEC are available. MATERIALS AND METHODS: A total of 100 non-duplicate E. coli isolates associated with community-acquired UTIs in sexually active women were analysed for antimicrobial susceptibility patterns and putative virulence-associated genes. Antimicrobial susceptibility testing (AST) was carried out by the Kirby-Bauer disk diffusion method, and results were interpreted as per Clinical and Laboratory Standards Institute (CLSI) guidelines. The isolates non-susceptible to ≥1 agent in ≥3 different antimicrobial categories were considered multidrug-resistant (MDR). Multiplex polymerase chain reaction assay was performed on each E. coli isolate to characterize putative virulence genes (VGs) such as papA, malX, PAI, ibeA, fimH, fyuA, sfa/focDE, papGIII, iutA, papGI, kpsMTII, hlyA, papGII, traT, afa/draBC, cnf1, vat, and yfcV. Results: Capsule synthesis gene kpsMTII (59%)was the most predominant VG present, followed by serum resistance-associated transfer protein gene traT (58%) and adhesin gene fimH (57%); however, adhesin gene papGI (2%) was the least present. The prevalence of antimicrobial resistance was relatively high for commonly used oral antimicrobials of UTI treatment, such as trimethoprim-sulfamethoxazole (68%) and fluoroquinolones (63%). The majority of isolates were MDR (78%) and resistant to extended-spectrum cephalosporins (63.5%). Isolates resistant to norfloxacin and trimethoprim-sulfamethoxazole were also resistant to almost all available oral antimicrobials. Isolates resistant to extended-spectrum cephalosporins showed increased resistance to aztreonam and trimethoprim-sulfamethoxazole (84.6% each) and fluoroquinolones (ciprofloxacin and norfloxacin; 81.5% each). Fosfomycin and nitrofurantoin were the most sensitive antimicrobials for all these resistant isolates. In a multivariate analysis, it was found that MDR isolates were associated with many of the VGs; fimH (65.4%) being the most frequent followed by traT (64.1%). traT (66.2%) and iutA (60.3%) were most commonly present in E. coli isolates resistant to trimethoprim-sulfamethoxazole, while66.7% norfloxacin-resistant isolates have them. Isolates resistant to extended-spectrum cephalosporins were most commonly associated with fimH and traT (66.2% each). However, E. coli isolates positive for sfa/focDE and vat were more sensitive to norfloxacin and trimethoprim-sulfamethoxazole and were non-MDR strains predominantly (p < 0.05). Only two VGs (fimH and traT) were significantly associated with MDR strains. DISCUSSION: The results of the present study clearly show the association of VFs with some of the commonly used oral antibiotics emphasizing the need for further molecular studies and surveillance programs to monitor drug-resistant UPEC so as to form optimized diagnostic stewardship and appropriate regimen for patient treatment. The reason behind this phenomenon of association has not been studied in much detail here but it can be assumed that genes responsible for drug resistance may share neighbouring loci with VGs on the mobile genetic elements (e.g., plasmid), which transfer together from one bacterium to another.

Age Group-Specific Assessment of Changing Seroepidemiology of Hepatitis A Virus Infection in North India.

Introduction Routine immunization against hepatitis A virus (HAV) infection has not been warranted in India, but an epidemiological shift from hyperendemicity to intermediate endemicity has been detected in recent years. The present study was planned to gather the age group-specific seroprevalence data of hepatitis A IgG antibodies in various age groups and evaluate any early trends of seroepidemiological shift. Method This was a hospital-based cross-sectional study. The detection of IgG antibodies for hepatitis A was done using an HAV Ab kit (Dia.Pro, Milan, Italy) in sera of individuals from >1 to 80 years of age and consenting to participate. Data on sociodemographic factors and potentially predisposing factors of HAV was collected on a predesigned questionnaire. At the time of final analysis, patients were divided into three groups children one to <18 years, adults ≥18 to <60 years, and old ≥60 to 80 years for comparative analysis. Result A total of 1,250 patients were included in the final analysis (129 children, 928 adults, and 193 old). The male/female ratio of the study participants was 1.4:1. The majority (85%) of them came from rural and semi-urban areas. They generally had lower socioeconomic status (SES) with poor literacy rates. Most of the enrolled cases (n=800/1,250, 64%) reported the use of groundwater, and 58.7% (n=734/1,250) consume water without any purification. Of the study participants, 90.8% reported the use of toilets for defecation, and 96.7% of the cases use soap for handwashing after defecation. The majority of adult (90%) and old age (99%) participants were seropositive for anti-HAV IgG antibodies as compared to children (80%). No significant differences were observed in the seropositivity rates and the SES class of the study participants. Conclusion About 20% of children did not have anti-HAV IgG antibodies in the present study, indicating that they are not exposed to HAV. This could be because of their better living conditions such as the availability of safe drinking water and improved sanitation and hygiene. We support the current guidelines of the Indian Academy of Pediatrics (IAP), which recommends immunization for hepatitis A vaccination at 12 months of age. Adult vaccination is not needed in North India.

Virulence Genotyping and Multidrug Resistance Pattern of Escherichia coli Isolated From Community-Acquired and Hospital-Acquired Urinary Tract Infections.

Introduction Uropathogenic Escherichia coli(UPEC) strains consist of a plethora of putative virulence factors (VFs), which help them to establish infection in the urinary tract. We compared genotypic profiles of Escherichia coli (E. coli) strains associated with community-acquired (CA) urinary tract infection (UTI; n=100) and hospital-acquired (HA) UTI (n=50) in the present study in order to identify specific virulence determinants, if any, associated with either form of UTI and its association with antibiotic resistance pattern of the isolates. Materials and methods E. coli strains were analyzed for antimicrobial susceptibility patterns, phylogroups, and 10 putative virulence-associated genes. The bacterial culture and identification were done using standard conventional methods. Tests for antimicrobial susceptibility and phenotypic detection for extende- spectrum beta-lactamases (ESBL) were done by using the Kirby Bauer disc diffusion method, and results were interpreted as per Clinical & Laboratory Standards Institute (CLSI) guidelines. The phylotype (A, B1, B2, and D) of each E. coli isolate was determined by a triplex polymerase chain reaction (PCR) based phylotyping method. They were further analyzed for the presence of 10 putative virulence genes (VGs), including adhesins papA (P fimbrial structural subunit), papG alleles I, II (P fimbrial adhesin variants), fimH (type 1 fimbriae), toxins hlyA (hemolysin) siderophores chuA (heme-binding protein); yfcV (encodes a major subunit of a putative chaperone-usher fimbria) capsule synthesis specific for group II (K1, K5, K12, etc.) kpsMII; serum resistance-associated traT, and upaH by multiplex PCR. Results HA E. coli isolates were significantly more drug-resistant than CA isolates; carbapenem (80% vs. 16%), ceftazidime (92% vs. 63%). The majority (52%) of E.coli isolates associated with HA UTI belong to commensal phylogroup A and B1, whereas the majority (66%) in CA were from pathotypic phylogroups, i.e., B2 & D. Most of VFs were frequently present amongst CA group except for traT and yfc, kpsMTII, hlyA, chuA, and upaH were significantly associated with CA E.coli isolates while yfc was significantly present in HA E.coli isolates. Though adhesin genes such as papA, papGI, papGII, fimH were frequently found in the CA group, they were not significantly associated. The average virulence score was higher for CA UTI isolates (4.25) than for the HA strains (3.9). Multidrug resistance (MDR) was present in every HA E.coli isolate, and fimH, traT, and yfc genes showed significant association with MDR strains. Conclusion On detailed analysis, we found that HA E. coli isolates had a high frequency of MDR and comparatively reduced VFs content. Thus, it can be assumed that a strain with lesser virulence is able to cause HA UTIs, as compared to CA UTIs, which probably indicates that the host's immune status/general condition can be an important determinant in acquiring infection rather than virulence potential of the pathogen alone.

Behavioral and Psychosocial Risk Factors Associated with First and Recurrent Cystitis in Indian Women: A Case-control Study.

BACKGROUND: The risk factors for urinary tract infections (UTIs) from developed countries are not applicable to women from developing world. OBJECTIVE: To analyze the behavioral practices and psychosocial aspects pertinent to women in our region and assess their association with acute first time or recurrent UTI. MATERIALS AND METHODS: Sexually active premenopausal women with their first (145) and recurrent (77) cystitis with Escherichia coli as cases and women with no prior history of UTI as healthy controls (257) were enrolled at a tertiary care hospital in India, between June 2011 and February 2013. Questionnaire-based data was collected from each participant through a structured face-to-face interview. RESULTS: Using univariate and multivariate regression models, independent risk factors for the first episode of cystitis when compared with healthy controls were (presented in odds ratios [ORs] with its 95% confidence interval [CI]): Anal sex (OR = 3.68, 95% CI = 1.59-8.52), time interval between last sexual intercourse and current episode of UTI was <5 days (OR = 2.27, 95% CI = 1.22-4.23), use of cloth during menstrual cycle (OR = 2.36, 95% CI = 1.31-4.26), >250 ml of tea consumption per day (OR = 4.73, 95% CI = 2.67-8.38), presence of vaginal infection (OR = 3.23, 95% CI = 1.85-5.62) and wiping back to front (OR = 2.52, 95% CI = 1.45-4.38). Along with the latter three, history of UTI in a first-degree female relative (OR = 10.88, 95% CI = 2.41-49.07), constipation (OR = 4.85, 95% CI = 1.97-11.92) and stress incontinence (OR = 2.45, 95% CI = 1.18-5.06) were additional independent risk factors for recurrent cystitis in comparison to healthy controls. CONCLUSION: Most of the risk factors for initial infection are potentially modifiable but sufficient to also pose risk for recurrence. Many of the findings reflect the cultural and ethnic practices in our country.

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

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

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.

Role of special pathogenicity versus prevalence theory in pathogenesis of acute cystitis caused by Escherichia coli.

Escherichia coli is the most common pathogen causing acute cystitis in sexually active women. Human faeces are generally considered the primary reservoir for infection and the faecal-perineal-urethral pathway is the accepted route of infection. Two theories have been proposed for the pathogenesis of acute cystitis: (1) special pathogenicity, where uropathogenic E. coli (UPEC) encoding special virulence factors causes infection; and (2) prevalence, wherein ordinary faecal E. coli causes infection by simple mass action. The aim of this study was to compare concurrent urinary E. coli isolates from women with acute cystitis with their own dominant faecal, vaginal E. coli isolates; thus, these patients served as their own control. E. coli isolates from 80 women were analysed by phylotyping, virulence profiling (for 15 putative virulence genes) and enterobacterial repetitive intergenic consensus (ERIC) PCR. A virulence score was calculated for each isolate based on the number of virulence genes detected. Four host ecological groups of E. coli were created on the basis of ERIC PCR: group UVF, where vaginal and faecal isolates yielded the infecting urine clone; group UV, where only vaginal isolates yielded the infecting urine clone; group UF, where faecal isolates yielded the infecting urine clone; and group U, where the infecting urine clone was distinct. In the majority of cases the infecting E. coli clone from urine was also the dominant faecal clone (56.3%; groups UVF and UF possessing high virulence scores of 4.6 and 3.9, respectively), indicating that both mechanisms play a role in pathogenesis. Non-dominant yet virulent faecal clones or an external source of E. coli seems a possibility in the UV group (13.7%, VF score 4.8). In 30% of patients (U group) the infecting urine clone was non-dominant and possessed a low virulence score (2.7); suggesting a possible role for host factors in establishing infection.

Transgenic tomato line expressing modified Bacillus thuringiensis cry1Ab gene showing complete resistance to two lepidopteran pests.

The modified truncated Bt-cry1Ab gene of Bacillus thuringiensis has been used for the development and selection of over expressing transgenic events in a commercially important variety of tomato (Solanum lycopersicum L.) by Agrobacterium-mediated leaf-disc transformation procedure. The integration and inheritance of cry1Ab gene in T0 transgenic plants and their progenies were determined by PCR, RT-PCR and Southern blot hybridization analysis. The toxin expression was monitored by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). The transgenic line Ab25 E, expressing 0.47 ± 0.01% Cry1Ab toxin of total soluble protein (TSP) was finally selected in the T4 generation from the segregating population, showing 100% mortality to the second instar larvae of H. armigera and S. litura and minimal damages to leaves and fruits. Southern blot analysis data revealed single copy introgression of cry1Ab gene in highly-expressing Ab25 E transgenic line and expression of Cry1Ab toxin of molecular mass ~65 kDa was evident in Western blot analyses in transgenic plants of T4, T5 and T6 generation. Receptor binding assay performed with partially purified Cry1Ab protein from Ab25 E transgenic tomato line, confirmed efficient protein-protein interaction of Cry1Ab toxin with receptor(s) of both the insects. The higher level of Cry1Ab toxin (≈ 0.47 ± 0.01% TSP) did not affect the normal in vitro regeneration, plant development and fruit yield in this transgenic line. This high expressing Cry1Ab homozygous transgenic line can be a useful candidate in tomato breeding programmes for introgression of important agronomical traits.

Genotypic characteristics and biofilm formation among Escherichia coli isolates from Indian women with acute cystitis.

BACKGROUND: The purpose of present study was to characterize uropathogenic Escherichia coli (UPEC) associated with acute cystitis in Indian women. METHODS: In this prospective descriptive study we investigated phylogenetic background and virulence genotypes for 15 genes by multiplex PCR and in vitro biofilm formation ability of 172 E. coli strains and explored possible association amongst them. RESULTS: Most isolates (81, 47.1%) belonged to group B2 and A (50, 29.1%); few were from groups B1 (22, 12.7%) and D (19, 11.1%). The mean virulence scores of phylogroups A, B1, B2 and D were 3.3, 4.0, 4.6 and 4.9, respectively. We found higher prevalence of fimH, traT iutA, kpsMII, papG allele II and fyuA genes indicating putative role of adhesins, iron acquisition systems and protectins in causing bladder infection. Total 145 (84.3%) isolates produced biofilm; majority were weak biofilm producers and there was no significant difference in intensity and biofilm formation ability amongst various phylogroups or virulence scores. The phylogenetic distribution was different from western studies, including a lower prevalence of group B2 (dominant but not a majority), followed by group A, and some traditionally group B2-associated virulence genes were more prevalent in phylogroup A isolates. CONCLUSIONS: Distribution of virulence genotypes and phylotypes can vary with geographical location and the isolates from western countries cannot be treated as prototypes for Asian cystitis isolates.