Bacterial Microcompartment-Mediated Ethanolamine Metabolism inEscherichia coliUrinary Tract Infection

ABSTRACTUrinary tract infections (UTIs) are common and in general are caused by intestinal uropathogenicEscherichia coli(UPEC) ascending via the urethra. Microcompartment-mediated catabolism of ethanolamine, a host cell breakdown product, fuels the competitive overgrowth of intestinalE. coli, both pathogenic enterohemorrhagicE. coliand commensal strains. During a UTI, urease-negativeE. colibacteria thrive, despite the comparative nutrient limitation in urine. The role of ethanolamine as a potential nutrient source during UTIs is understudied. We evaluated the role of the metabolism of ethanolamine as a potential nitrogen and carbon source for UPEC in the urinary tract. We analyzed infected urine samples by culture, high-performance liquid chromatography, reverse transcription-quantitative PCR, and genomic sequencing. The ethanolamine concentration in urine was comparable to the concentration of the most abundant reported urinary amino acid,d-serine. Transcription of theeutoperon was detected in the majority of urine samples containingE. coliscreened. All sequenced UPEC strains had conservedeutoperons, while metabolic genotypes previously associated with UTI (dsdCXA,metE) were mainly limited to phylogroup B2.In vitroethanolamine was found to be utilized as a sole source of nitrogen by UPEC strains. The metabolism of ethanolamine in artificial urine medium (AUM) induced metabolosome formation and provided a growth advantage at the physiological levels found in urine. Interestingly,eutE(which encodes acetaldehyde dehydrogenase) was required for UPEC strains to utilize ethanolamine to gain a growth advantage in AUM, suggesting that ethanolamine is also utilized as a carbon source. These data suggest that urinary ethanolamine is a significant additional carbon and nitrogen source for infectingE. colistrains.

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Determination of nitrofurantoin and fosfomycin susceptibility among urinary Escherichia coli isolates

International Journal of Research in Medical Sciences ◽

10.18203/2320-6012.ijrms20203676 ◽

2020 ◽

Vol 8 (9) ◽

pp. 3274

Author(s):

Rachana Kanaujia ◽

Amit Kumar ◽

Malay Bajpai

Keyword(s):

Escherichia Coli ◽

Urinary Tract ◽

Urinary Tract Infections ◽

Urine Samples ◽

E Coli ◽

Tract Infections ◽

Therapeutic Alternatives ◽

Micro Organisms ◽

Staphylococcus Spp

Background: Urinary tract infections (UTIs) are one of the most common infections. For treatment of UTIs, there are limited antibiotics due to increased resistance among uropathogens. Two older antibiotics; Nitrofurantoin and Fosfomycin have become novel oral therapeutic options against uropathogens. Aim of the study was to identify UTI causing micro-organisms and evaluate in-vitro activity of nitrofurantoin and fosfomycin against most common isolated organism (E. coli).Methods: Results of urine samples culture and susceptibility testing over a period of 1 year were analysed and included in this study.Results: Micro-organisms were isolated from 568 urine samples. Most commonly isolated organism was Escherichia coli (40.50%), followed by Klebsiella spp. (20.07%) and Staphylococcus spp. (17.07%). Susceptibility of E. coli to nitrofurantoin and fosfomycin was 91.74% and 65.65% respectively. Conclusion: Good activity of nitrofurantoin and fosfomycin against E. coli indicates that these two drugs are potential therapeutic alternatives for urinary tract infections.

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Escherichia coli CFT073 Fitness Factors during Urinary Tract Infection: Identification Using an Ordered Transposon Library

Applied and Environmental Microbiology ◽

10.1128/aem.00691-20 ◽

2020 ◽

Vol 86 (13) ◽

Cited By ~ 1

Author(s):

Allyson E. Shea ◽

Juan Marzoa ◽

Stephanie D. Himpsl ◽

Sara N. Smith ◽

Lili Zhao ◽

...

Keyword(s):

Escherichia Coli ◽

Urinary Tract ◽

Urinary Tract Infections ◽

Human Urine ◽

Transposon Mutagenesis ◽

Content Type ◽

E Coli ◽

Tract Infections

ABSTRACT Urinary tract infections (UTI), the second most diagnosed infectious disease worldwide, are caused primarily by uropathogenic Escherichia coli (UPEC), placing a significant financial burden on the health care system. High-throughput transposon mutagenesis combined with genome-targeted sequencing is a powerful technique to interrogate genomes for fitness genes. Genome-wide analysis of E. coli requires random libraries of at least 50,000 mutants to achieve 99.99% saturation; however, the traditional murine model of ascending UTI does not permit testing of large mutant pools due to a bottleneck during infection. To address this, an E. coli CFT073 transposon mutant ordered library of 9,216 mutants was created and insertion sites were identified. A single transposon mutant was selected for each gene to assemble a condensed library consisting of 2,913 unique nonessential mutants. Using a modified UTI model in BALB/c mice, we identified 36 genes important for colonizing the bladder, including purB, yihE, and carB. Screening of the condensed library in vitro identified yigP and ubiG to be essential for growth in human urine. Additionally, we developed a novel quantitative PCR (qPCR) technique to identify genes with fitness defects within defined subgroups of related genes (e.g., genes encoding fimbriae, toxins, etc.) following UTI. The number of mutants within these subgroups circumvents bottleneck restriction and facilitates validation of multiple mutants to generate individual competitive indices. Collectively, this study investigates the bottleneck effects during UTI, provides two techniques for evading those effects that can be applied to other disease models, and contributes a genetic tool in prototype strain CFT073 to the field. IMPORTANCE Uropathogenic Escherichia coli strains cause most uncomplicated urinary tract infections (UTI), one of the most common infectious diseases worldwide. Random transposon mutagenesis techniques have been utilized to identify essential bacterial genes during infection; however, this has been met with limitations when applied to the murine UTI model. Conventional high-throughput transposon mutagenesis screens are not feasible because of inoculum size restrictions due to a bottleneck during infection. Our study utilizes a condensed ordered transposon library, limiting the number of mutants while maintaining the largest possible genome coverage. Screening of this library in vivo, and in human urine in vitro, identified numerous candidate fitness factors. Additionally, we have developed a novel technique using qPCR to quantify bacterial outputs following infection with small subgroups of transposon mutants. Molecular approaches developed in this study will serve as useful tools to probe in vivo models that are restricted by anatomical, physiological, or genetic bottleneck limitations.

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Bacterial Profile of Urinary Tract Infections: Evaluation of Biofilm Formation and Antibiotic Resistance Pattern of Uropathogenic Escherichia coli

Journal of Pure and Applied Microbiology ◽

10.22207/jpam.14.4.33 ◽

2020 ◽

Vol 14 (4) ◽

pp. 2577-2584

Author(s):

Tariq Ahmad Shah ◽

P. Preethishree ◽

Ashwini ◽

Vidya Pai

Keyword(s):

Escherichia Coli ◽

Antibiotic Resistance ◽

Urinary Tract ◽

Biofilm Formation ◽

Urine Samples ◽

Diffusion Method ◽

E Coli ◽

Biofilm Production ◽

Tract Infections

Urinary tract infection (UTI) is one of the most common complaints in the outpatient clinic and a major health problem owing to the emergence of antibiotic resistance and biofilm formation. The objective of this study was to isolate and identify the causative bacterial agent of UTI and detect in vitro biofilm formation by Escherichia coli and investigate its correlation with antibiotic resistance. Urine samples from 519 patients with suspected UTIs were collected and processed by conventional microbiological procedures. Antimicrobial susceptibility testing for E. coli isolates was performed on Mueller Hinton agar (MHA) plates using the Kirby-Bauer disk diffusion method. Biofilm production was evaluated using the tissue culture plate method. Of 519 urine samples, 115 (22.1%) showed significant bacteriuria. The most common isolate was E. coli (n=57, 49.6%), followed by Klebsiella spp. (n=23, 20%). All E. coli isolates were evaluated for their ability to form biofilms in vitro. Of 57 isolates, 50 (87.7%) were biofilm producers and 7 (12.3%) were non-biofilm producers. Antibiogram of E. coli isolates revealed the highest resistance to ampicillin (96.5%) and nitrofurantoin (91.2%), followed by amoxyclav (82.5%), ceftazidime (73.7%), cefepime (71.9%), and tetracycline (71.9%). A significant association (p<0.05) was observed between biofilm formation and resistance to amoxyclav, ceftazidime, cefepime, imipenem, and nitrofurantoin. A significant correlation was noted between biofilm production and antibiotic resistance. Hence, screening of all isolates of uropathogenic E. coli for biofilm production and studying their antibiogram would allow appropriate choice of antibiotic therapy.

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Antimicrobial Resistance in Urinary Tract Pathogens in Canada from 2007 to 2009: CANWARD Surveillance Study

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00066-11 ◽

2011 ◽

Vol 55 (7) ◽

pp. 3169-3175 ◽

Cited By ~ 55

Author(s):

James A. Karlowsky ◽

Philippe R. S. Lagacé-Wiens ◽

Patricia J. Simner ◽

Melanie R. DeCorby ◽

Heather J. Adam ◽

...

Keyword(s):

Urinary Tract ◽

Surveillance Study ◽

Multivariate Logistic Regression Model ◽

Content Type ◽

E Coli ◽

Oral Agents ◽

Amoxicillin Clavulanate ◽

Tract Infections ◽

Resistance Rates

ABSTRACTFrom January 2007 to December 2009, an annual Canadian national surveillance study (CANWARD) tested 2,943 urinary culture pathogens for antimicrobial susceptibilities according to Clinical and Laboratory Standards Institute guidelines. The most frequently isolated urinary pathogens were as follows (number of isolates, percentage of all isolates):Escherichia coli(1,581, 54%), enterococci (410, 14%),Klebsiella pneumoniae(274, 9%),Proteus mirabilis(122, 4%),Pseudomonas aeruginosa(100, 3%), andStaphylococcus aureus(80, 3%). The rates of susceptibility to trimethoprim-sulfamethoxazole (SXT) were 78, 86, 84, and 93%, respectively, forE. coli,K. pneumoniae,P. mirabilis, andS. aureus. The rates of susceptibility to nitrofurantoin were 96, 97, 33, and 100%, respectively, forE. coli, enterococci,K. pneumoniae, andS. aureus. The rates of susceptibility to ciprofloxacin were 81, 40, 86, 81, 66, and 41%, respectively, forE. coli, enterococci,K. pneumoniae,P. mirabilis,P. aeruginosa, andS. aureus. Statistical analysis of resistance rates (resistant plus intermediate isolates) by year forE. coliover the 3-year study period demonstrated that increased resistance rates occurred only for amoxicillin-clavulanate (from 1.8 to 6.6%;P< 0.001) and for SXT (from 18.6 to 24.3%;P= 0.02). For isolates ofE. coli, in a multivariate logistic regression model, hospital location was independently associated with resistance to ciprofloxacin (P= 0.026) with higher rates of resistance observed in inpatient areas (medical, surgical, and intensive care unit wards). Increased age was also associated with resistance to ciprofloxacin (P< 0.001) and with resistance to two or more commonly prescribed oral agents (amoxicillin-clavulanate, ciprofloxacin, nitrofurantoin, and SXT) (P= 0.005). We conclude that frequently prescribed empirical agents for urinary tract infections, such as SXT and ciprofloxacin, demonstrate loweredin vitrosusceptibilities when tested against recent clinical isolates.

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Role of Klebsiella pneumoniae Type 1 and Type 3 Fimbriae in Colonizing Silicone Tubes Implanted into the Bladders of Mice as a Model of Catheter-Associated Urinary Tract Infections

Infection and Immunity ◽

10.1128/iai.00348-13 ◽

2013 ◽

Vol 81 (8) ◽

pp. 3009-3017 ◽

Cited By ~ 57

Author(s):

Caitlin N. Murphy ◽

Martin S. Mortensen ◽

Karen A. Krogfelt ◽

Steven Clegg

Keyword(s):

Urinary Tract ◽

Klebsiella Pneumoniae ◽

Urinary Tract Infections ◽

Wild Type ◽

Content Type ◽

Tract Infections ◽

Type 3

ABSTRACTCatheter-associated urinary tract infections are biofilm-mediated infections that cause a significant economic and health burden in nosocomial environments. Using a newly developed murine model of this type of infection, we investigated the role of fimbriae in implant-associated urinary tract infections by the Gram-negative bacteriumKlebsiella pneumoniae, which is a proficient biofilm former and a commonly isolated nosocomial pathogen. Studies have shown that type 1 and type 3 fimbriae are involved in attachment and biofilm formationin vitro, and these fimbrial types are suspected to be important virulence factors during infection. To test this hypothesis, the virulence of fimbrial mutants was assessed in independent challenges in which mouse bladders were inoculated with the wild type or a fimbrial mutant and in coinfection studies in which the wild type and fimbrial mutants were inoculated together to assess the results of a direct competition in the urinary tract. Using these experiments, we were able to show that both fimbrial types serve to enhance colonization and persistence. Additionally, a double mutant had an additive colonization defect under some conditions, indicating that both fimbrial types have unique roles in the attachment and persistence in the bladder and on the implant itself. All of these mutants were outcompeted by the wild type in coinfection experiments. Using these methods, we are able to show that type 1 and type 3 fimbriae are important colonization factors in the murine urinary tract when an implanted silicone tube is present.

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In Vivo Phase Variation of Escherichia coli Type 1 Fimbrial Genes in Women with Urinary Tract Infection

Infection and Immunity ◽

10.1128/iai.66.7.3303-3310.1998 ◽

1998 ◽

Vol 66 (7) ◽

pp. 3303-3310 ◽

Cited By ~ 85

Author(s):

Jean K. Lim ◽

Nereus W. Gunther ◽

Hui Zhao ◽

David E. Johnson ◽

Susan K. Keay ◽

...

Keyword(s):

Escherichia Coli ◽

Urinary Tract ◽

Acute Pyelonephritis ◽

Urine Samples ◽

E Coli ◽

Type 1 Fimbriae ◽

Tract Infections

ABSTRACT Type 1 fimbriae, expressed by most Escherichia colistrains, are thought to attach to human uroepithelium as an initial step in the pathogenesis of urinary tract infections (UTI). Numerous reports using both in vitro and murine models support this role for type 1 fimbriae in colonization. Unfortunately, only a limited number of studies have directly examined the expression of fimbriae in vivo. To determine whether type 1 fimbrial genes are transcribed during an acute UTI, we employed a modification of an established method. The orientation (ON or OFF) of the invertible promoter element, which drives transcription of type 1 fimbrial genes, was determined by PCR amplification using primers that flank the invertible element, followed by SnaBI digestion. The orientation of the type 1 fimbrial switch was determined under three experimental conditions. First,E. coli strains from different clinical sources (acute pyelonephritis patients, cystitis patients, and fecal controls) were tested under different in vitro culture conditions (agar versus broth; aerated versus static). The genes in the more-virulent strains (those causing acute pyelonephritis) demonstrated a resistance, in aerated broth, to switching from OFF to ON, while those in fecal strains readily switched from OFF to ON. Second, bladder and kidney tissue from CBA mice transurethrally inoculated with E. coli CFT073 (an established murine model of ascending UTI) was assayed. The switches directly amplified from infected bladder and kidney tissues were estimated to be 33 and 39% ON, respectively, by using a standard curve. Finally, bacteria present in urine samples collected from women with cystitis were tested for type 1 fimbria switch orientation. For all 11 cases, an average of only 4% of the switches in the bacteria in the urine were ON. In 7 of the 11 cases, we found that all of the visible type 1 fimbrial switches were in the OFF position (upper limit of detection of assay, 98% OFF). Strains recovered from these urine samples, however, were shown after culture in vitro to be capable of switching the fimbrial gene to the ON position and expressing mannose-sensitive hemagglutinin. The results from experimental infections and cases of cystitis in women suggest that type 1 fimbrial genes are transcribed both in the bladder and in the kidney. However, those bacteria found in the urine and not attached to the uroepithelium are not transcriptionally active for type 1 fimbrial genes.

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Cefoxitin as an Alternative to Carbapenems in a Murine Model of Urinary Tract Infection Due to Escherichia coli Harboring CTX-M-15-Type Extended-Spectrum β-Lactamase

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.06233-11 ◽

2012 ◽

Vol 56 (3) ◽

pp. 1376-1381 ◽

Cited By ~ 32

Author(s):

Raphaël Lepeule ◽

Etienne Ruppé ◽

Patrick Le ◽

Laurent Massias ◽

Françoise Chau ◽

...

Keyword(s):

Escherichia Coli ◽

Urinary Tract ◽

Murine Model ◽

Content Type ◽

E Coli ◽

Extended Spectrum ◽

Drug Concentrations ◽

Tract Infections

ABSTRACTWe investigated the efficiency of the cephamycin cefoxitin as an alternative to carbapenems for the treatment of urinary tract infections (UTIs) due toEscherichia coliproducing CTX-M-type extended-spectrum β-lactamases. The susceptible, UTI-inducingE. coliCFT073-RR strain and its transconjugant CFT073-RR Tc (pblaCTX-M-15), harboring ablaCTX-M-15carrying-plasmid, were used for all experiments. MICs of cefoxitin (FOX), ceftriaxone (CRO), imipenem (IMP), and ertapenem (ETP) for CFT073-RR and CFT073-RR Tc (pblaCTX-M-15) were 4 and 4, 0.125 and 512, 0.5 and 0.5, and 0.016 and 0.032 μg/ml, respectively. Bactericidal activity was similarly achievedin vitroagainst the two strains after 3 h of exposure to concentrations of FOX, IMI, and ETP that were 2 times the MIC, whereas CRO was not bactericidal against CFT073-RR Tc (pblaCTX-M-15). The frequencies of spontaneous mutants of the 2 strains were not higher for FOX than for IMP or ETP. In the murine model of UTIs, mice infected for 5 days were treated over 24 h. Therapeutic regimens in mice (200 mg/kg of body weight every 3 h or 4 h for FOX, 70 mg/kg every 6 h for CRO, 100 mg/kg every 2 h for IMP, and 100 mg/kg every 4 h for ETP) were chosen in order to reproduce the percentage of time that free-drug concentrations above the MIC are obtained in humans with standard regimens. All antibiotic regimens produced a significant reduction in bacterial counts (greater than 2 log10CFU) in kidneys and bladders for both strains (P< 0.001) without selecting resistant mutantsin vivo, but the reduction obtained with CRO against CFT073-RR Tc (pblaCTX-M-15) in kidneys was significantly lower than that obtained with FOX. In conclusion, FOX appears to be an effective therapeutic alternative to carbapenems for the treatment of UTIs due to CTX-M-producingE. coli.

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In Vitro Coliform Resistance to Bioactive Compounds in Urinary Infection, Assessed in a Lab Catheterization Model

Applied Sciences ◽

10.3390/app11094315 ◽

2021 ◽

Vol 11 (9) ◽

pp. 4315

Author(s):

Emanuel Vamanu ◽

Laura Dorina Dinu ◽

Cristina Mihaela Luntraru ◽

Alexandru Suciu

Keyword(s):

Urinary Tract ◽

Bioactive Compounds ◽

Urinary Tract Infections ◽

Antimicrobial Effect ◽

Biologically Active ◽

Bacterial Strains ◽

E Coli ◽

Functional Product ◽

Tract Infections

Bioactive compounds and phenolic compounds are viable alternatives to antibiotics in recurrent urinary tract infections. This study aimed to use a natural functional product, based on the bioactive compounds’ composition, to inhibit the uropathogenic strains of Escherichia coli. E. coli ATCC 25922 was used to characterize the IVCM (new in vitro catheterization model). As support for reducing bacterial proliferation, the cytotoxicity against a strain of Candida albicans was also determined (over 75% at 1 mg/mL). The results were correlated with the analysis of the distribution of biologically active compounds (trans-ferulic acid-268.44 ± 0.001 mg/100 g extract and an equal quantity of Trans-p-coumaric acid and rosmarinic acid). A pronounced inhibitory effect against the uropathogenic strain E. coli 317 (4 log copy no./mL after 72 h) was determined. The results showed a targeted response to the product for tested bacterial strains. The importance of research resulted from the easy and fast characterization of the functional product with antimicrobial effect against uropathogenic strains of E. coli. This study demonstrated that the proposed in vitro model was a valuable tool for assessing urinary tract infections with E. coli.

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Single Clinical Isolates from Acute Uncomplicated Urinary Tract Infections Are Representative of Dominant In Situ Populations

mBio ◽

10.1128/mbio.01064-13 ◽

2014 ◽

Vol 5 (2) ◽

Cited By ~ 18

Author(s):

Dana Willner ◽

Serene Low ◽

Jason A. Steen ◽

Narelle George ◽

Graeme R. Nimmo ◽

...

Keyword(s):

Escherichia Coli ◽

Urinary Tract ◽

Urinary Tract Infections ◽

Clinical Isolates ◽

Content Type ◽

E Coli ◽

Strain Diversity ◽

Amplicon Pyrosequencing ◽

Culture Independent ◽

Tract Infections

ABSTRACTUrinary tract infections (UTIs) are one of the most commonly acquired bacterial infections in humans, and uropathogenicEscherichia colistrains are responsible for over 80% of all cases. The standard method for identification of uropathogens in clinical laboratories is cultivation, primarily using solid growth media under aerobic conditions, coupled with morphological and biochemical tests of typically a single isolate colony. However, these methods detect only culturable microorganisms, and characterization is phenotypic in nature. Here, we explored the genotypic identity of communities in acute uncomplicated UTIs from 50 individuals by using culture-independent amplicon pyrosequencing and whole-genome and metagenomic shotgun sequencing. Genus-level characterization of the UTI communities was achieved using the 16S rRNA gene (V8 region). Overall UTI community richness was very low in comparison to other human microbiomes. We strain-typedEscherichia-dominated UTIs using amplicon pyrosequencing of the fimbrial adhesin gene,fimH. There were nine highly abundantfimHtypes, and each UTI sample was dominated by a single type. Molecular analysis of the corresponding clinical isolates revealed that in the majority of cases the isolate was representative of the dominant taxon in the community at both the genus and the strain level. Shotgun sequencing was performed on a subset of eightE. coliurine UTI and isolate pairs. The majority of UTI microbial metagenomic sequences mapped to isolate genomes, confirming the results obtained using phylogenetic markers. We conclude that for the majority of acute uncomplicatedE. coli-mediated UTIs, single cultured isolates are diagnostic of the infection.IMPORTANCEIn clinical practice, the diagnosis and treatment of acute uncomplicated urinary tract infection (UTI) are based on analysis of a single bacterial isolate cultured from urine, and it is assumed that this isolate represents the dominant UTI pathogen. However, these methods detect only culturable bacteria, and the existence of multiple pathogens as well as strain diversity within a single infection is not examined. Here, we explored bacteria present in acute uncomplicated UTIs using culture-independent sequence-based methods.Escherichia coliwas the most common organism identified, and analysis ofE. colidominant UTI samples and their paired clinical isolates revealed that in the majority of infections the cultured isolate was representative of the dominant taxon at both the genus and the strain level. Our data demonstrate that in most cases single cultured isolates are diagnostic of UTI and are consistent with the notion of bottlenecks that limit strain diversity during UTI pathogenesis.

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Biological Cost of Single and Multiple Norfloxacin Resistance Mutations in Escherichia coli Implicated in Urinary Tract Infections

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.6.2343-2351.2005 ◽

2005 ◽

Vol 49 (6) ◽

pp. 2343-2351 ◽

Cited By ~ 91

Author(s):

Patricia Komp Lindgren ◽

Linda L. Marcusson ◽

Dorthe Sandvang ◽

Niels Frimodt-Møller ◽

Diarmaid Hughes

Keyword(s):

Escherichia Coli ◽

Urinary Tract ◽

Infection Model ◽

Resistance Mutations ◽

Topoisomerase Iv ◽

E Coli ◽

Tract Infections ◽

Subsequent Selection

ABSTRACT Resistance to fluoroquinolones in urinary tract infection (UTIs) caused by Escherichia coli is associated with multiple mutations, typically those that alter DNA gyrase and DNA topoisomerase IV and those that regulate AcrAB-TolC-mediated efflux. We asked whether a fitness cost is associated with the accumulation of these multiple mutations. Mutants of the susceptible E. coli UTI isolate Nu14 were selected through three to five successive steps with norfloxacin. Each selection was performed with the MIC of the selected strain. After each selection the MIC was measured; and the regions of gyrA, gyrB, parC, and parE, previously associated with resistance mutations, and all of marOR and acrR were sequenced. The first selection step yielded mutations in gyrA, gyrB, and marOR. Subsequent selection steps yielded mutations in gyrA, parE, and marOR but not in gyrB, parC, or acrR. Resistance-associated mutations were identified in almost all isolates after selection steps 1 and 2 but in less than 50% of isolates after subsequent selection steps. Selected strains were competed in vitro, in urine, and in a mouse UTI infection model against the starting strain, Nu14. First-step mutations were not associated with significant fitness costs. However, the accumulation of three or more resistance-associated mutations was usually associated with a large reduction in biological fitness, both in vitro and in vivo. Interestingly, in some lineages a partial restoration of fitness was associated with the accumulation of additional mutations in late selection steps. We suggest that the relative biological costs of multiple mutations may influence the evolution of E. coli strains that develop resistance to fluoroquinolones.

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