Identification phenotypic and genotypic characterization of biofilm formation in Escherichia coli isolated from urinary tract infections and their antibiotics resistance.
ABSTRACT: OBJECTIVE:Urinary tract infections (UTIs) are the most common infectious diseases, and Escherichia coli is the most common pathogen isolated from patients with UTIs. The products of sfa, afa and foc genes are important for binding of the bacterium to urinary tract epithelium. Our aim was to investigate these genes in E. colis isolated from patients with UTIS. The frequencies of the genes were determined using PCR. Biofilm formation and antibiotic resistance rates were determined using microtiter plate and disk diffusion methods, respectively. The P?
Project description:INTRODUCTION:Uropathogenic E. coli is the leading cause of Urinary tract infections (UTIs), contributing to 80-90% of all community-acquired and 30-50% of all hospital-acquired UTIs. Biofilm forming Uropathogenic E. coli are associated with persistent and chronic inflammation leading to complicated and or recurrent UTIs. Biofilms provide an environment for poor antibiotic penetration and horizontal transfer of virulence genes which favors the development of Multidrug-resistant organisms (MDRO). Understanding biofilm formation and antimicrobial resistance determinants of Uropathogenic E. coli strains will provide insight into the development of treatment options for biofilm-associated UTIs. The aim of this study was to determine the biofilm forming capability, presence of virulence genes and antimicrobial susceptibility pattern of Uropathogenic E. coli isolates in Uganda. METHODS:This was a cross-sectional study carried in the Clinical Microbiology and Molecular biology laboratories at the Department of Medical Microbiology, Makerere University College of Health Sciences. We randomly selected 200 Uropathogenic E. coli clinical isolates among the stored isolates collected between January 2018 and December 2018 that had significant bacteriuria (>?105?CFU). All isolates were subjected to biofilm detection using the Congo Red Agar method and Antimicrobial susceptibility testing was performed using the Kirby disk diffusion method. The isolates were later subjected PCR for the detection of Urovirulence genes namely; Pap, Fim, Sfa, Afa, Hly and Cnf, using commercially designed primers. RESULTS:In this study, 62.5% (125/200) were positive biofilm formers and 78% (156/200) of these were multi-drug resistant (MDR). The isolates were most resistant to Trimethoprim sulphamethoxazole and Amoxicillin (93%) followed by gentamycin (87%) and the least was imipenem (0.5%). Fim was the most prevalent Urovirulence gene (53.5%) followed by Pap (21%), Sfa (13%), Afa (8%), Cnf (5.5%) and Hyl (0%). CONCLUSIONS:We demonstrate a high prevalence of biofilm-forming Uropathogenic E. coli strains that are highly associated with the MDR phenotype. We recommend routine surveillance of antimicrobial resistance and biofilm formation to understand the antibiotics suitable in the management of biofilm-associated UTIs.
Project description:Extended Spectrum ?-Lactamases (ESBLs) confer resistance to third-generation cephalosporins and CTX-M types have emerged as the most prominent ESBLs worldwide. This study was designed to determine the prevalence of CTX-M positive ESBL-producing urinary E. coli isolates from HIV patients and to establish the association of multidrug resistance, phylogeny, and virulence profile with CTX-M production. A total of 57 ESBL producers identified among 76 E. coli strains isolated from HIV patients from South India were screened for bla CTX-M, AmpC production, multidrug resistance, and nine virulence associated genes (VAGs), fimH, pap, afa/dra, sfa/foc, iutA, fyuA, iroN, usp, and kpsMII. The majority (70.2%) of the ESBL producers harbored bla CTX-M and were AmpC coproducers. Among the CTX-M producers, 47.5% were found to be UPEC, 10% harbored as many as 7 VAGs, and 45% possessed kpsMII. Multidrug resistance (CIP(R)SXT(R)GEN(R)) was significantly more common among the CTX-M producers compared to the nonproducers (70% versus 41.2%). However, 71.4% of the multidrug resistant CTX-M producers exhibited susceptibility to nitrofurantoin thereby making it an effective alternative to cephalosporins/fluoroquinolones. The emergence of CTX-M-producing highly virulent, multidrug resistant uropathogenic E. coli is of significant public health concern in countries like India with a high burden of HIV/AIDS.
Project description:The pathogenicity and clinical pertinence of diffusely adhering Escherichia coli expressing the Afa/Dr adhesins (Afa/Dr DAEC) in urinary tract infections (UTIs) and pregnancy complications are well established. In contrast, the implication of intestinal Afa/Dr DAEC in diarrhea is still under debate. These strains are age dependently involved in diarrhea in children, are apparently not involved in diarrhea in adults, and can also be asymptomatic intestinal microbiota strains in children and adult. This comprehensive review analyzes the epidemiology and diagnosis and highlights recent progress which has improved the understanding of Afa/Dr DAEC pathogenesis. Here, I summarize the roles of Afa/Dr DAEC virulence factors, including Afa/Dr adhesins, flagella, Sat toxin, and pks island products, in the development of specific mechanisms of pathogenicity. In intestinal epithelial polarized cells, the Afa/Dr adhesins trigger cell membrane receptor clustering and activation of the linked cell signaling pathways, promote structural and functional cell lesions and injuries in intestinal barrier, induce proinflammatory responses, create angiogenesis, instigate epithelial-mesenchymal transition-like events, and lead to pks-dependent DNA damage. UTI-associated Afa/Dr DAEC strains, following adhesin-membrane receptor cell interactions and activation of associated lipid raft-dependent cell signaling pathways, internalize in a microtubule-dependent manner within urinary tract epithelial cells, develop a particular intracellular lifestyle, and trigger a toxin-dependent cell detachment. In response to Afa/Dr DAEC infection, the host epithelial cells generate antibacterial defense responses. Finally, I discuss a hypothetical role of intestinal Afa/Dr DAEC strains that can act as "silent pathogens" with the capacity to emerge as "pathobionts" for the development of inflammatory bowel disease and intestinal carcinogenesis.
Project description:<h4>Background</h4>Urinary tract infections (UTIs) are the most common bacterial infections and are often caused by uropathogenic <i>Escherichia coli</i> (UPEC). We investigated the distribution of phylogenetic groups, adhesin genes, antimicrobial resistance, and biofilm formation in <i>E. coli</i> isolated from patients with UTIs.<h4>Methods</h4>In the present study, 208 UPEC isolated from Thai patients were classified into phylogenetic groups and adhesin genes were detected using multiplex PCR. Antimicrobial susceptibility testing was performed using agar disk diffusion. The Congo red agar method was used to determine the ability of the UPEC to form biofilm.<h4>Results</h4>The most prevalent UPEC strains in this study belonged to phylogenetic group B2 (58.7%), followed by group C (12.5%), group E (12.0%), and the other groups (16.8%). Among adhesin genes, the prevalence of <i>fimH</i> (91.8%) was highest, followed by <i>pap</i> (79.3%), <i>sfa</i> (12.0%), and <i>afa</i> (7.7%). The rates of resistance to fluoroquinolones, trimethoprim-sulfamethoxazole, and amoxicillin-clavulanate were 65%, 54.3%, and 36.5%, respectively. The presence of adhesin genes and antibiotic resistance were more frequent in groups B2 and C compared to the other groups. Of the 129 multidrug-resistant UPEC strains, 54% were biofilm producers. Our findings further indicated that biofilm production was significantly correlated with the <i>pap</i> adhesin gene (<i>p</i> ? 0.05).<h4>Conclusion</h4>These findings provide molecular epidemiologic data, antibiotic resistance profiles, and the potential for biofilm formation among UPEC strains that can inform further development of the appropriate prevention and control strategies for UTIs in this region.
Project description:Nitrofurantoin has been used for decades for the treatment of urinary tract infections (UTIs), but clinically significant resistance in Escherichia coli is uncommon. Nitrofurantoin concentrations in the gastrointestinal tract tend to be low, which might facilitate selection of nitrofurantoin-resistant (NIT-R) strains in the gut flora. We subjected two nitrofurantoin-susceptible intestinal E. coli strains (ST540-p and ST2747-p) to increasing nitrofurantoin concentrations under aerobic and anaerobic conditions. Whole-genome sequencing was performed for both susceptible isolates and selected mutants that exhibited the highest nitrofurantoin resistance levels aerobically (ST540-a and ST2747-a) and anaerobically (ST540-an and ST2747-an). ST540-a/ST540-an and ST2747-a (aerobic MICs of >64 ?g/ml) harbored mutations in the known nitrofurantoin resistance determinants nfsA and/or nfsB, which encode oxygen-insensitive nitroreductases. ST2747-an showed reduced nitrofurantoin susceptibility (aerobic MIC of 32 ?g/ml) and exhibited remarkable growth deficits but did not harbor nfsA/nfsB mutations. We identified a 12-nucleotide deletion in ribE, encoding lumazine synthase, an essential enzyme involved in the biosynthesis of flavin mononucleotide (FMN), which is an important cofactor for NfsA and NfsB. Complementing ST2747-an with a functional wild-type lumazine synthase restored nitrofurantoin susceptibility. Six NIT-R E. coli isolates (NRCI-1 to NRCI-6) from stools of UTI patients treated with nitrofurantoin, cefuroxime, or a fluoroquinolone harbored mutations in nfsA and/or nfsB but not ribE. Sequencing of the ribE gene in six intestinal and three urinary E. coli strains showing reduced nitrofurantoin susceptibility (MICs of 16 to 48 ?g/ml) also did not identify any relevant mutations. NRCI-1, NRCI-2, and NRCI-5 exhibited up to 4-fold higher anaerobic MICs, compared to the mutants generated in vitro, presumably because of additional mutations in oxygen-sensitive nitroreductases.
Project description:Diffusely adhering Escherichia coli strains harboring Afa/Dr adhesins (Afa/Dr DAEC) have been associated with diarrhea and urinary tract infections (UTIs). The present work is the first extensive molecular study of a Afa/Dr DAEC strain using the representational difference analysis technique. We have searched for DNA sequences present in strain C1845, recovered from a diarrheagenic child, but absent from a nonpathogenic K-12 strain. Strain C1845 harbors part of a pathogenicity island (PAI(CFT073)) and several iron transport systems found in other E. coli pathovars. We did not find genes encoding factors known to subvert host cell proteins, such as type III secretion system or effector proteins. Several C1845-specific sequences are homologous to putative virulence genes or show no homology with known sequences, and we have analyzed their distribution among Afa/Dr and non-Afa/Dr clinical isolates and among strains from the E. coli Reference Collection. Three C1845-specific sequences (MO30, S109, and S111) have a high prevalence (77 to 80%) among Afa/Dr strains and a low prevalence (12 to 23%) among non-Afa/Dr strains. In addition, our results indicate that strain IH11128, an Afa/Dr DAEC strain recovered from a patient with a UTI, is genetically closely related to strain C1845.
Project description:<h4>Background</h4>Urinary tract infections (UTIs) affect up to 150 million individuals annually worldwide, mainly due to <i>Escherichia coli</i> (<i>E. coli</i>) and <i>Klebsiella</i>. The emergence and spread of multidrug-resistant (MDR) bacteria are increasing, representing one of the biggest threats for human health. The objective of our study was to describe antimicrobial patterns of resistance and identify risk factors associated with MDR uropathogens.<h4>Methods</h4>We conducted a cross-sectional study in 296 patients with community-acquired UTI who underwent clinical and microbiologic analysis, and clinical associations to MDR uropathogens were investigated. <i>Findings</i>. Microbiological analysis included <i>E. coli</i> (55%), ESBL-<i>E. coli</i> (26%), <i>Enterococcus</i> (6%), <i>Klebsiella</i> (5%), and others (8%). Higher frequencies of MDR bacteria were found among ESBL-<i>E. coli</i>, with resistance to ampicillin (100%), ceftriaxone (96%), gentamicin (57%), ciprofloxacin (89%), and TMP/SMX (53%). However, they were sensitive to fosfomycin (6.6%), nitrofurantoin (1.3%), and carbapenems (0%). Fosfomycin MIC90 for ESBL-<i>E. coli</i> was 5.78?<i>?</i>g/mL. The only clinical variable with significant association to ESBL producers was the presence of comorbidities: hypertension and type 2 diabetes mellitus with an OR (95%CI) of 2.5(1.3 - 4.9)(<i>p</i> < 0.01) and 2.8(1.2 - 6.7)(<i>p</i> < 0.05), respectively.<h4>Conclusions</h4>In the majority of cases, resistance rates to commonly prescribed antimicrobials in UTIs were high, except for fosfomycin, nitrofurantoin, and carbapenems. To provide appropriate treatment, both the identification of risk factors and the uropathogen would be important. An active surveillance in UTIs in the community is required since the proportion of ESBL producers is increasing.
Project description:The antibiotic nitrofurantoin is a furan flanked by a nitro group and a hydantoin ring. It is used to treat lower urinary tract infections (UTIs) that have a lifetime incidence of 50-60% in adult women. UTIs are typically caused by uropathogenic <i>Escherichia coli</i> (UPEC), which are increasingly expressing extended-spectrum beta-lactamases (ESBL), rendering them multi-drug resistant. Nitrofurantoin is a first-line treatment for gram-negative ESBL-positive UTI patients, given that resistance to it is still rare (0% to 4.4%). Multiplex PCR of ?-lactamase genes of the <i>blaCTX-M</i> groups 1, 2, 9 and 8/25 from ESBL-positive UTI patients treated at three referral hospitals in North Wales (UK) revealed the presence of a novel <i>CTX-M-14-like</i> gene harbouring the missense mutations T55A, A273P and R277C. While R277 is close to the active site, T55 and A273 are both located in external loops. Recombinant expression of CTX-M-14 and the mutated CTX-M-14 in the periplasm of <i>E. coli</i> revealed a significant increase in the Minimum Inhibitory Concentration (MIC) for nitrofurantoin from ?6 ?g/mL (CTX-M-14) to ?512 ?g/mL (mutated CTX-M-14). Consistent with this finding, the mutated CTX-M protein hydrolysed nitrofurantoin in a cell-free assay. Detection of a novel nitrofurantoin resistance gene indicates an emerging clinical problem in the treatment of gram-negative ESBL-positive UTI patients.
Project description:Candida albicans is one of the most common causes of hospital-acquired urinary tract infections (UTIs). However, azoles are poorly active against biofilms, echinocandins do not achieve clinically useful urinary concentrations, and amphotericin B exhibits severe toxicities. Thus, novel strategies are needed to prevent Candida UTIs, which are often associated with urinary catheter biofilms. We previously demonstrated that cranberry-derived proanthocyanidins (PACs) prevent C. albicans biofilm formation in an in vitro urinary model. To elucidate functional pathways unique to urinary biofilm development and PAC inhibition, we investigated the transcriptome of C. albicans in artificial urine (AU), with and without PACs. C. albicans biofilm and planktonic cells were cultivated with or without PACs. Genome-wide expression analysis was performed by RNA sequencing. Differentially expressed genes were determined using DESeq2 software; pathway analysis was performed using Cytoscape. Approximately 2,341 of 6,444 total genes were significantly expressed in biofilm relative to planktonic cells. Functional pathway analysis revealed that genes involved in filamentation, adhesion, drug response and transport were up-regulated in urinary biofilms. Genes involved in carbon and nitrogen metabolism and nutrient response were down-regulated. In PAC-treated urinary biofilms compared to untreated control biofilms, 557 of 6,444 genes had significant changes in gene expression. Genes downregulated in PAC-treated biofilms were implicated in iron starvation and adhesion pathways. Although urinary biofilms share key features with biofilms formed in other environments, many genes are uniquely expressed in urinary biofilms. Cranberry-derived PACs interfere with the expression of iron acquisition and adhesion genes within urinary biofilms.
Project description:<h4>Background</h4>Risk factors affecting early morality of patients with Escherichia coli bloodstream infection (BSI) were investigated including the host-pathogen-treatment tripartite components.<h4>Methods</h4>Six general hospitals in South Korea participated in this multicentre prospective observational study from May 2016 to April 2017 and a total of 1492 laboratory-confirmed E. coli BSI cases were studied. Cox regression was used to estimate risks of the primary endpoint, i.e., all-cause mortality within 30?days from the initial blood culture. Six multivariate analysis models were constructed in accordance to the clinical importance and intra- and inter-component multicollinearity.<h4>Findings</h4>Among the 1492 E. coli BSI cases, 9.5% (n?=?141) patients expired within 30?days. Six models of multivariate analysis indicated risk factors of critical illness, primary infection of peritoneum, and chronic liver disease including cirrhosis for host variables; of phylogenetic group B2, ST131-sublineage H30Rx, multidrug resistance, group 1 CTX-M extended-spectrum beta-lactamase production, and having either of fyuA, afa, and sfa/foc virulence genes for causative E. coli pathogen variables; and of delayed definitive therapy for antimicrobial treatment variables. In addition, as a protective factor, primary urinary tract infection was identified.<h4>Interpretation</h4>Despite decades' effort searching for the risk factors for E. coli BSI, systemic understanding covering the entire tripartite component is still lacking. This study detailed the organic impact of host-pathogen-treatment tripartite components for early mortality in patients with E. coli BSI.