Type 1 fimbrial phase variation in multidrug-resistant asymptomatic uropathogenic Escherichia coli clinical isolates upon adherence to HTB-4 cells.

The adherence of bladder uroepithelial cells, subsequent expression, and regulation of type 1 fimbrial genes (key mediator of attachment) in clinical multidrug-resistant uropathogenic Escherichia coli (MDR-UPECs) isolated from individuals with asymptomatic bacteriuria (ABU) remain unexplored till date. Therefore, this study aimed to investigate the underlying molecular mechanisms associated with the adherence of clinical MDR-ABU-UPECs to human a uroepithelial cell line (HTB-4), both in the absence and presence of D-Mannose. These investigations focused on phase variation, expression, and regulation of type 1 fimbriae and were compared to a prototype ABU-strain (E. coli 83972) and symptomatic MDR-UPECs. Discordant to the ABU prototype strain, MDR-ABU-UPECs exhibited remarkable adhesive capacity that was significantly reduced after D-mannose exposure, fairly like the MDR symptomatic UPECs. The type 1 fimbrial phase variation, determined by the fim switch analysis, asserted the statistically significant incidence of "both OFF and ON" orientation among the adherent MDR-ABU-UPECs with a significant reduction in phase-ON colonies post-D-mannose exposure, akin to the symptomatic ones. This was indicative of an operative and alternating type 1 fimbrial phase switch. The q-PCR assay revealed a coordinated action of the regulatory factors; H-NS, IHF, and Lrp on the expression of FimB and FimE recombinases, which further controlled the function of fimH and fimA genes in ABU-UPECs, similar to symptomatic strains. Therefore, this study is the first of its kind to provide an insight into the regulatory crosstalk of different cellular factors guiding the adhesion of ABU-UPECs to the host. Additionally, it also advocated for the need to accurately characterize ABU-UPECs.

Comprehensive analysis of multiple cytokines to stratify uropathogenic Escherichia coli pathogenesis in mouse model of urinary tract infection.

PURPOSE: Urinary tract infection (UTI) is one of the most common human bacterial infections primarily caused by uropathogenic E. coli (UPEC). Empiric treatment in UTI cause emergence of multidrug resistance and limit treatment options. Understanding UTI at the molecular level with respect to the causative pathogen as well as subsequent host response pose an absolute necessity towards appropriate clinical management. This study aimed to investigate host cytokine response in mouse UTI model with respect to bacterial colonization and associated virulence gene expression upon infection. METHOD: Mouse UTI model was established with two clinical UPEC isolates E. coli NP105 and E. coli P025. UPEC colonization in bladder and kidney was evaluated by bacterial culture (CFU/ml). Histopathology of the tissues were examined by hematoxylin and eosin staining. PCR and real time PCR were used to detect the incidence and expression of respective bacterial genes. Cytokine concentrations in tissues and sera were evaluated using ELISA. GraphPad prism version 8.0.2 was used for statistical interpretation. RESULT: Highest bacterial colonization was observed on 7th and 9th day post infection (p.i). in bladder and kidney of mouse infected with E. coli P025 and E. coli NP105 respectively with a distinct difference in relative expression of fimH and papC adhesin genes in vivo. IL-1ß level in tissues and sera of E. coli NP105 and E. coli P025 infected mouse was significantly different but the IL-17A, GCSF, TGF-ß levels were comparable. CONCLUSION: These findings show a role of IL1ß to stratify pathogenicity of UPEC in mouse UTI model.

Occurrence of Imipenem-Resistant Uropathogenic Escherichia coli in Pregnant Women: An Insight into Their Virulence Profile and Clonal Structure.

Uropathogenic Escherichia coli (UPEC) is the predominant pathogen in Urinary Tract Infection (UTI) in pregnant and non-pregnant women. Limited studies were initiated to explore UPEC from pregnant women with respect to imipenem resistance, pathogenicity, and their clonal lineage. In this study, imipenem resistance, phylogenetic background, virulence-associated genes, and clonal characteristics in UPECs isolated from pregnant and non-pregnant cohorts were investigated. E. coli was identified biochemically from urine culture-positive samples from pregnant and non-pregnant women. Carbapenem (meropenem, ertapenem, imipenem) susceptibility was determined by Kirby-Bauer disk diffusion test. The pathogenic determinants were identified by PCR. MEGA 11 was used to interpret clonal lineages from MLST. GraphPad Prism 8.0 and SPSS 26.0 were used for statistical interpretation. Results indicated highest resistance against imipenem compared to meropenem and ertapenem in UPECs isolated from pregnant (UPECp; 63.89%) and non-pregnant (UPECnp; 87.88%) women. Although phylogroup E was predominant in both imipenem-resistant isolates, acquisition of virulence factors was higher among UPECnp than UPECp. Akin to this observation, the presence of PAI III536 and PAI IV536 was statistically significant (p < 0.05) in the former. MLST analysis revealed similar clonal lineages between UPECnp and UPECp, which showed an overall occurrence of ST405 followed by ST101, ST410, ST131, and ST1195 in UPECnp and ST167 in UPECp, respectively, with frequent occurrence of CC131, CC405. Therefore, imipenem-resistant UPECp although discrete with respect to their virulence determinants when compared to UPECnp shared similar STs and CCs, which implied common evolutionary history. Thus, empiric treatment must be restricted in UTIs to especially protect maternal and fetal health.

Cytokine profile in HTB-4 cells with respect to fimH, papC and hlyA expression in uropathogenic Escherichia coli invasion.

Background: In this study, uropathogenic Escherichia coli (UPEC) from pregnant and nonpregnant patients were characterized with respect to antimicrobial resistance (AMR) and expression of their virulence factors and cytokines elicited upon infection in urothelial (HTB-4) cells in vitro to frame proper therapeutics. Methods: Antibiotic sensitivity and adherence to HTB-4 cells were tested and PCR and real-time PCR were performed. Results: UPEC from nonpregnant patients showed the most resistance with a significant correlation between the expression of hlyA and TGF-ß and papC and GCSF. The expression of fimH and IFN-γ, fimH and IL-1ß, and fimH and IL-17A, respectively, were significantly correlated in UPEC from pregnant patients. Conclusion: Cytokine expression profiles were correlated with the expression of virulence genes in UPEC that was isolated from different populations, and should be accounted for along with AMR analysis.

Combination of bactericidal antibiotics and inhibitors of Universal stress protein A (UspA): a potential therapeutic alternative against multidrug resistant Escherichia coli in urinary tract infections.

The increasing incidence of multidrug resistant uropathogenic E. coli (MDR-UPEC), the most common opportunistic pathogen in urinary tract infections (UTI) pose a global health problem and demands searching for alternative therapeutics. Antibiotics generate oxidative stress in bacteria which results in overexpression of the universal stress protein, UspA that helps in bacterial survival. An in silico study showed that two compounds ZINC000104153710, and ZINC000000217308 effectively bound bacterial UspA. This study aimed to determine the activity of ZINC000104153710, and ZINC000000217308 against bacterial UspA function in MDR-UPEC in vitro. Twenty-five highly MDR-UPEC were screened against ZINC000104153710, and, ZINC000000217308 either alone or in combination with the bactericidal antibiotics; ciprofloxacin (CIP), ceftazidime(CAZ), gentamicin(GEN) respectively by determining minimum inhibitory concentrations (MICs) using a broth microdilution assay. Additionally, the effect of ZINC000104153710, and ZINC000000217308 in the absence and presence of antibiotics on the bacteria was monitored by bacterial growth curve assays, ROS production, structure of the organism by scanning electron microscopy (FESEM) and quantitating UspA using a western blot technique. A 2-8 fold reduction in MIC values against ZINC000104153710, and ZINC000000217308 was observed against all 25 MDR-UPEC isolates in the presence of antibiotics with no alteration in intracellular ROS production. Discrete changes in cell morphology was evident in bacteria treated with ZINC000104153710 or ZINC000000217308 and antibiotics individually by FESEM compared with untreated control. Reduction in the level of UspA protein in bacteria treated with combination of ZINC000104153710 or ZINC000000217308 with individual antibiotics established their ability to inhibit UspA whose expression was elevated in presence of antibiotics alone. Therefore this study validated ZINC000104153710, and ZINC000000217308 as potent inhibitors of bacterial UspA function and indicated their potential as alternative therapeutics to combat the MDR-UPEC.

Epidemiologic and molecular characterization of ß-lactamase-producing multidrug-resistant uropathogenic Escherichia coli isolated from asymptomatic hospitalized patients.

Uropathogenic Escherichia coli (UPECs) are the predominant cause of asymptomatic bacteriuria (ABU) and symptomatic UTI. In this study, multidrug-resistant (MDR) ABU-UPECs from hospitalized patients of Kolkata, India, were characterized with respect to their ESBL phenotype, acquisition of ß-lactamase genes, mobile genetic elements (MGEs), phylotype property, ERIC-PCR profile, sequence types (STs), clonal complexes (CCs) and evolutionary and quantitative relationships and compared to the symptomatic ones to understand their epidemiology and evolutionary origin. Statistically significant incidence of ESBL producers, ß-lactamase genes, MGEs and novel phylotype property (NPP) among ABU-UPECs similar to the symptomatic ones indicated the probable incidence of chromosomal plasticity on resistance gene acquisition through MGEs due to indiscriminate drug usage. ERIC-PCR typing and MLST analysis showed clonal heterogeneity and predominance of ST940 (CC448) among asymptomatic isolates akin to symptomatic ones along with the evidence of zoonotic transmissions. Minimum spanning tree analysis showed a close association between ABU-UPEC with known and unidentified STs having NPPs with isolates that belonged to phylogroups clade I, D, and B2. This is the first study that reported the occurrence of MGEs and NPPs among ABU-UPECs with the predominance of ESBL production which displayed the deleterious effect of MDR among this pathogen demanding alternative therapeutic interventions. Moreover, this study for the first time attempted to introduce a new approach to ascertain the phylotype property of unassigned UPECs. Withal, increased recognition, proper understanding and characterization of ABU-UPECs with the implementation of appropriate therapeutic measures against them when necessary are the need of the era which otherwise might lead to serious complications in the vulnerable population.

Impact of Mutation on the Structural Stability and the Conformational Landscape of Inhibitor-Resistant TEM ß-Lactamase: A High-Performance Molecular Dynamics Simulation Study.

Gain-of-function mutations and structural adjustment toward ß-lactamase inhibitors in the TEM-type ß-lactamases among the uropathogenic E. coli (UPEC) culminate in treatment complications and demands detailed investigation. In this study, uncharacterized amino acid substitutions, M69L/I84V/W165G/V184A/V262I/N276S, in inhibitor-resistant TEM (IRT) ß-lactamase isolated from clinical UPEC were subjected to extensive molecular dynamics (EMD) simulations for 100 ns to estimate parameters such as root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), the radius of gyration (Rg), contour plot (Rg/RMSD), secondary structure element (SSE), etc. Residue interaction networks, principal component analysis (PCA), and correlation heatmaps were generated to predict the relation between functionally important atomic motions to uncover the structural stability of the mutants. To avoid the false positive conclusion of the simulation study, we performed three identically parameterize replicas of 100 ns each. Alterations in hydrophobic interactions resulted in conformation changes exhibited as comparable residue interaction networks. Besides, PCA and porcupine plot analysis based on the ensemble of structure from molecular dynamics trajectories revealed the collective atomic motions of the IRT variants that impart structural flexibility to their active site loop. This study conducted on IRT mutants that delineate intricate protein motions contributes to their stability and folding, which is an absolute necessity for designing candidate molecules owing to the clinical threat of emerging resistance against potent ß-lactam antibiotics.

Uropathogenic Escherichia coli in India-an Overview on Recent Research Advancements and Trends.

Urinary tract infection (UTI), a prevalent disease in India, also ranks among the most common infections in developing countries. The rapid emergence of antibiotic-resistant uropathogenic Escherichia coli (UPECs), the leading etiologic agent of UTI, in the last few years, led to an upsurge in the health care cost. This caused a considerable economic burden, especially in low-middle income country, India. This review aimed to provide an explicit overview of the recent advancements in E. coli-mediated UTI in India by incorporation of valuable information from the works published in PubMed and Google Scholar in the last six years (2015 to August, 2020). The literature survey demonstrated UPECs as the most predominant uropathogen in India, especially among females, causing both asymptomatic bacteriuria (ABU) and symptomatic UTI. An overall increasing national trend in resistance to penicillins, cephalosporins, aminoglycosides, fluoroquinolones, and sulfonamides was perceived irrespective of ABU and symptomatic UPECs during the aforementioned study period. High incidences of multidrug resistance, extended-spectrum ß-lactamases, metallo ß-lactamases, and AmpCs in UPECs were reported. Notable information on the pathogenic profiles, phylogroups, pathogenicity islands, and evidence of pathoadaptive FimH mutations was described. Alternative therapeutics and potential drug targets against UPECs were also reconnoitered. Therefore, the nationwide widespread occurrences of highly virulent MDR UPEC together with the limited availability of therapeutics highlighted the urgent need for promotion and invention of alternative therapeutics, search for which had already been started. Moreover, investigation of several mechanisms of UPEC infection and the search for potential drug targets might help to design newer therapeutics.

Reactive oxygen species and uspA overexpession: an alternative bacterial response toward selection and maintenance of multidrug resistance in clinical isolates of uropathogenic E. coli.

Emergence of multidrug resistance (MDR) in uropathogenic E. coli (UPEC) demands alternative therapeutic interventions. Bactericidal antibiotics at their sub-inhibitory concentration stimulate production of reactive oxygen species (ROS) that results in oxidative stress, generates mutations, and alters transcription of different genes. Sub-inhibitory concentration of antibiotics facilitates selection of highly resistant population. Universal stress protein A (uspA) overexpression in MDR-UPEC at sub-inhibitory bactericidal antibiotics concentration was investigated to explore alternative survival strategy against them. Fifty clinical UPEC isolates were screened. Minimum inhibitory concentration (MIC) against three different bactericidal antibiotics (ciprofloxacin, CIP; ceftazidime, CAZ; gentamycin, GEN) was determined by broth dilution method; ROS production by DCFDA and overexpression of uspA by real-time PCR were determined at the sub-inhibitory concentration of antibiotics. DNA ladder formation and SEM studies were performed with drug untreated and treated samples. Statistical analysis was done by Student's t test and Pearson's correlation analysis; 25 out of 50 UPEC exhibited high MIC against CIP (> 200 µg/ml), CAZ (> 500 µg/ml), GEN (> 500 µg/ml), with varied ROS production (p ≤ 0.001) in treated than untreated controls. DNA ladder formation confirmed ROS production in drug-treated samples. SEM analysis revealed unaltered cell morphology in both untreated and drug-treated bacteria. uspA was universally overexpressed in all 25 UPEC. A significant correlation (p ≤ 0.001) between ROS production and uspA overexpression was observed in 19 out of 25 MDR isolates at sub-inhibitory doses of the bactericidal antibiotics. Therefore, this study highlights an alternative strategy that the MDR isolates may acquire when exposed to sub-inhibitory drug concentration for their survival.

A spectroscopic and computational intervention of interaction of lysozyme with 6-mercaptopurine.

In the present work, biophysical insight into the binding interactions of the protein, hen egg white (HEW) lysozyme (Lyz) with an anticancer drug, 6-mercaptopurine (6-MP)' was investigated by using a combination of spectroscopic and computational tools. 6-MP, a synthetic analog of natural purines, is a well-known anticancer drug and antiviral agent that inhibits the synthesis of RNA, DNA, and proteins. Lysozyme is a single-chain protein that can combine with endogenous and exogenous substances to exert its antiviral, antibacterial, and antitumor effects. The intrinsic fluorescence of lysozyme was quenched with the increased addition of 6-MP. The quenching mechanism was found to be static in nature as shown by the fluorescence lifetime and excitation spectrum measurements. The conformational changes of Lyz in the presence of 6-MP were monitored both at the ensemble and single-molecule level by using synchronous fluorescence spectroscopy, circular dichroism (CD), and fluorescence correlation spectroscopy (FCS). Molecular docking results predicted the probable binding sites for 6-MP on Lyz. The experimental findings are in good agreement with the results obtained by the molecular dynamics (MD) simulation study. Graphical abstract.

Conjugal transfer of PMQR from uropathogenic E.coli under high ciprofloxacin selection pressure generates gyrA mutation.

Incidence of high fluoroquinolone resistance has been rising rapidly worldwide. Resistance against fluoroquinolones can be either chromosomal or plasmid mediated. Plasmid mediated quinolone resistant(PMQR) genes impart low level of resistance against fluoroquinolones but provides favorable background for selection of additional chromosomally encoded resistance mechanisms. In the natural habitat, conjugal transfer of PMQR genes provides a vehicle for dissemination of fluoroquinolone resistance. Our study indicated successful transmission of PMQR determinants(aac(6')-Ib-cr, qnrA, qnrB, qnrS, oqxB) from ciprofloxacin resistant clinical uropathogenic E.coli(UPEC) isolates to susceptible E.coliJ53Azide-resistant strain in vitro in presence of high ciprofloxacin (5 µg/ml) selection pressure generating transconjugants that exhibited varied MIC(25-800µg/ml) towards the drug with acquired mutations Ser83Leu and Asp87Asn in the quinolone resistant determining regions(QRDR) in gyrA. Therefore this is a first study of its kind that identified high rate of gyrA mutations among transconjugants selected under high ciprofloxacin pressure resulting from bacterial conjugation a common phenomenon in natural habitat along with PMQR gene transmission which imposes a major public health concern.

Incidence of multidrug resistance, pathogenicity island markers, and pathoadaptive FimH mutations in uropathogenic Escherichia coli isolated from asymptomatic hospitalized patients.

Asymptomatic uropathogenic Escherichia coli (UPECs) are the leading cause of asymptomatic bacteriuria (ABU) in humans. So this study aimed to identify and characterize ABU UPECs from hospitalized patients of Kolkata, India, with respect to their antibiogram profile, phylogeny, pathogenicity islands, and virulence factor gene acquisition and FimH mutations in comparison to symptomatic UPECs. E. coli was detected biochemically in 44.44% (20/45) and 32.26% (20/62) of urine culture-positive asymptomatic and symptomatic hospitalized individuals respectively. Ninety-five percent of the asymptomatic isolates were multidrug resistant (MDR) compared to the symptomatic isolates (100%). Significant predominance of unknown phylogroup, pathogenicity island markers (PAI IV536, PAI I CFT073), and distribution patterns of different virulence factor genes respectively was evident among both groups. A significant correlation was observed between both groups of isolates with respect to their antibiotic resistances (except imipenem, amikacin, and nitrofurantoin), prevalence of phylogenetic groups and PAIs, and virulence factor gene (fimH, papC, papEF, papGII, iucD, and cnf1) acquisition. Pathoadaptive FimH adhesin mutations, especially hot spot mutation V27A, were detected in 80% asymptomatic isolates mostly reported in symptomatic ones worldwide. Moreover, this is the first study from India that reported incidence of "Unknown" phylogroup, pathogenicity island markers, and potentially pathoadaptive FimH mutations in asymptomatic UPECs isolated from hospitalized patients which further indicated that these ABU E. coli might have originated from their symptomatic counterparts due to unbridled use of unprescribed antibiotics. Therefore, this study demands antibiotic de-escalation along with regular and intricate monitoring at the molecular level for efficient management of ABU that addresses a major public health concern.

Characterization and Bio-Typing of Multidrug Resistance Plasmids From Uropathogenic Escherichia coli Isolated From Clinical Setting.

Urinary tract infection is primarily caused by Escherichia coli. Multidrug resistance and their rapid dissemination in this pathogenic microbe complicate therapeutic strategies and threaten public health. Conjugation systems responsible for interbacterial transmission of antibiotic resistance are plasmid-encoded and can be classified as the P, F, and I types. Specific pili types and pili associated proteins were related to the transfer among this gram-negative organism and were thought to depend on contacts created by these structures at the time of DNA transport. In this study, conjugation system types of the plasmids that harbor multidrug resistant genes (aac-1b-cr, oqxAB, qnrB, qnrS, bla TEM, bla OXA) amongst 19 E. coli uropathogenic isolates were characterized under ciprofloxacin/ceftazidime selection individually by pili and pili associated gene types. Investigations indicated incidence of single plasmid of multiple replicon type amongst the transconjugants. bla TEM, bla CTX-M, bla OXA, aac-1b-cr, oqxAB, qnrB, qnrS genes in varied combination were observed to be successfully co-transmitted against ceftazidme/ciprofloxacin selection. Seven primer pair sets were selected that encodes pili and pili associated genes (traF, trwJ, traE, trhE, traG, pilM, pilx4) by nucleotide database search tools using annotated plasmids of different incompatibility types to assign the conjugation system type of the transmissible resistant plasmids by PCR. traF was predominant irrespective of drug selection that indicated F-type conjugation system was responsible for transmission of resistant plasmids which results in the rapid dissemination of antibiotic resistance in the isolates screened. Therefore this is a first report of its kind that investigated pili and pili associated genes to bio-type multidrug resistant plasmids and their transmission in clinical settings amongst uropathogenic E. coli circulated in the eastern part of India.

Incidence and risk of co-transmission of plasmid-mediated quinolone resistance and extended-spectrum ß-lactamase genes in fluoroquinolone-resistant uropathogenic Escherichia coli: a first study from Kolkata, India.

OBJECTIVES: Co-resistance to fluoroquinolones and ß-lactams results in treatment complications for uropathogenic Escherichia coli (UPEC) infections. This study aimed to detect the coexistence and co-transmission of plasmid-mediated quinolone resistance (PMQR) and extended-spectrum ß-lactamase (ESBL) genes in UPEC from Kolkata, India. METHODS: Escherichia coli was detected biochemically from culture-positive urine samples. Antimicrobial resistance and ESBL production were confirmed by disk diffusion assay. Transfer of PMQR and ESBL genes was performed using azide-resistant E. coli J53 as recipient. PCR was conducted to identify PMQR and ESBL genes, plasmid incompatibility types, insertion sequences, integrons and ERIC-PCR patterns. RESULTS: PMQR determinants were detected in 50.0% (35/70) of ciprofloxacin-resistant isolates, with ESBL production in 42.9% (15/35) and a ß-lactamase inhibitor-resistant phenotype in 51.4% (18/35). The highest co-occurrence (37.1%; 13/35) and co-transmission of aac(6')-Ib-cr with blaTEM, blaCTX-M and blaOXA was observed. Among the conjugal plasmids, replicon types FrepB/FrepB+F1B were predominant, with rare incidences of A/C, N, X, I1, FIIS, L/M and H1. Distribution of integrons and ISEcp1 and IS26, either alone or in combination, irrespective of PMQR and ESBL gene types was observed. Discrete ERIC-PCR profiles indicated that acquisition of PMQR and ESBLs and their dissemination may be attributed to horizontal gene transfer. CONCLUSION: This study demonstrates for the first time the risk of co-transmission of fluoroquinolone and ß-lactam resistance amongst UPEC from Kolkata, posing a major public-health threat and limiting treatment options. Monitoring at the molecular level is necessary to design appropriate prescription policies to combat the alarming rise in drug resistance amongst these uropathogens.

Phylogenetic background of E. coli isolated from asymptomatic pregnant women from Kolkata, India.

INTRODUCTION: Asymptomatic bacteriuria (ABU) in pregnancy generates medical complications. E. coli is the common etiologic agent responsible for ABU-associated infections. This study aimed to identify the phylogenetic background and drug resistance in asymptomatic E. coli from a pregnant population. METHODOLOGY: E. coli was confirmed biochemically from culture-positive urine samples collected from asymptomatic pregnant women. Phylogenetic typing was done by polymerase chain reaction (PCR). The isolates were subjected to antibiotic susceptibility testing and extended-spectrum beta-lactamase (ESBL) production. Statistical significance was determined using SPSS 17.0 software. RESULTS: Bacteriuria was observed in 113 (22.6%) of 500 asymptomatic pregnant females. E. coli was reported in 44 (38.9%) of 113 isolates. The mean age-wise distribution was 25.14 ± 4.63. Although pathogenic phylogroup B2 was predominant (54.5%), incidence of non-pathogenic phylogroup B1 (27.3%) was found to be statistically significant (p ≤ 0.001), and B1 and B2 were correlated with respect to total ABU population. Antibiotic sensitivity against ampicillin (34.1%), ceftazidime (50%), cefotaxime (47.7%), ciprofloxacin (47.7%), amikacin (86.4%), nitrofurantion (79.5%), and co-trimoxazole (36.4%) was observed. Multidrug resistance (MDR) and ESBL production was reported in 26 (59.1%) of 44 and 18 (69.2%) of the 26 MDR isolates, respectively. A significant distribution of phylogroup B1 (p = 0.03) with drug resistance was also observed. CONCLUSIONS: This is the first study that reported significant incidence of non-pathogenic phylogroup B1 in asymptomatic E. coli with high incidence of MDR isolated from pregnant women in Kolkata, India.  These varied resistance patterns present major therapeutic and infection control challenges during pregnancy.

Multidrug-Resistance and Extended Spectrum Beta-Lactamase Production in Uropathogenic E. Coli which were Isolated from Hospitalized Patients in Kolkata, India.

BACKGROUND AND OBJECTIVE: Urinary Tract Infections (UTIs) are mostly caused by Escherichia coli. The appropriate therapy demands a current knowledge on the antimicrobial susceptibility pattern amongst these pathogens, as an inappropriate use of antibiotics may lead to complications and treatment failure. The UTIs which are caused by multidrug resistant Extended-Spectrum Beta-Lactamase (ESBL) producing bacteria further pose a severe problem, as the treatment options are limited. The aim of this study was to identify the pattern of multi drug resistance amongst the uropathogenic E. coli (UPEC) isolates which were obtained from hospitalized patients. MATERIALS AND METHODS: Forty UPEC were isolated from 200 urine samples of hospitalized patients who were clinically suspected for UTIs. Antimicrobial susceptibility screening was performed by using 16 antibiotics, by the Kirby Bauer disk diffusion technique. The isolates which were resistant to the third generation cephalosporins were subjected to the ESBL confirmatory test by using drug and drug-inhibitor combination disks by following the CLSI guidelines. RESULTS: All the 40 isolates except three were multidrug resistant. They showed the highest sensitivities for nitrofurantoin (72.5%) and amikacin (70%). A high level of resistance was observed against ampicillin (97.5%), nalidixic acid and cefelexin (95%), amoxicillin (92.5%), cotrimoxazole (82.5%) and ciprofloxacin (80%) respectively. Thirty different antibiotic resistance patterns were observed against the different antibiotics. Twenty-eight out of the 40 isolates were resistant to the third generation cephalosporins. However, the phenotypic test for the ESBL confirmation indicated that eighteen out of the twenty-eight isolates were ESBL producers and that eleven different drug resistance patterns were observed amongst them. CONCLUSIONS: Therefore, this study accounts for the varied multidrug resistance pattern amongst the uropathogenic E. coli which were isolated from hospitalized patients in Kolkata, an eastern region of India. Nitrofurantoin and amikacin should be assigned as potent drugs to treat this infection in this region of the country. These varied resistance patterns present major therapeutic and infection control challenges and they suggest a heterogeneous population of the uropathogenic E. coli isolates which circulate in this sector of India.

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%, χ(2)P<0.001) and CIP (85.2 % versus 61.4%, χ(2)P<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.

Draft genome sequence of the sexually transmitted pathogen Trichomonas vaginalis.

We describe the genome sequence of the protist Trichomonas vaginalis, a sexually transmitted human pathogen. Repeats and transposable elements comprise about two-thirds of the approximately 160-megabase genome, reflecting a recent massive expansion of genetic material. This expansion, in conjunction with the shaping of metabolic pathways that likely transpired through lateral gene transfer from bacteria, and amplification of specific gene families implicated in pathogenesis and phagocytosis of host proteins may exemplify adaptations of the parasite during its transition to a urogenital environment. The genome sequence predicts previously unknown functions for the hydrogenosome, which support a common evolutionary origin of this unusual organelle with mitochondria.

Proteins of the glycine decarboxylase complex in the hydrogenosome of Trichomonas vaginalis.

Trichomonas vaginalis is a unicellular eukaryote that lacks mitochondria and contains a specialized organelle, the hydrogenosome, involved in carbohydrate metabolism and iron-sulfur cluster assembly. We report the identification of two glycine cleavage H proteins and a dihydrolipoamide dehydrogenase (L protein) of the glycine decarboxylase complex in T. vaginalis with predicted N-terminal hydrogenosomal presequences. Immunofluorescence analyses reveal that both H and L proteins are localized in hydrogenosomes, providing the first evidence for amino acid metabolism in this organelle. All three proteins were expressed in Escherichia coli and purified to homogeneity. The experimental Km of L protein for the two H proteins were 2.6 microM and 3.7 microM, consistent with both H proteins serving as substrates of L protein. Analyses using purified hydrogenosomes showed that endogenous H proteins exist as monomers and endogenous L protein as a homodimer in their native states. Phylogenetic analyses of L proteins revealed that the T. vaginalis homologue shares a common ancestry with dihydrolipoamide dehydrogenases from the firmicute bacteria, indicating its acquisition via a horizontal gene transfer event independent of the origins of mitochondria and hydrogenosomes.