scholarly journals Peptidoglycan sensing prevents quiescence and promotes quorum-independent growth of uropathogenic Escherichia coli

2019 ◽  
Author(s):  
Eric C. DiBiasio ◽  
Hilary J. Ranson ◽  
James R. Johnson ◽  
David C. Rowley ◽  
Paul S. Cohen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Structural Integrity ◽  
Recurrent Infection ◽  
Multidrug Resistant ◽  
Quiescent State ◽  
E Coli ◽  
Tract Infections ◽  
Patients Experience

AbstractThe layer of peptidoglycan surrounding bacteria provides structural integrity for the bacterial cell wall. Many organisms, including human cells and diverse bacteria, detect peptidoglycan fragments that are released as bacteria grow. Uropathogenic Escherichia coli (UPEC) strains are the leading cause of human urinary tract infections (UTIs) and many patients experience recurrent infection after successful antibiotic treatment. The source of recurrent infections may be persistent bacterial reservoirs in vivo that are in a quiescent state and, thus, are not susceptible to antibiotics. Here, we show that multiple UPEC strains require a quorum to proliferate in vitro with glucose as the sole carbon source; at low density, the bacteria remain viable but enter a quiescent, non-proliferative state. Of all clinical UPEC isolates tested to date, 35% (51/145) enter this quiescent state, including archetypal strains CFT073 (from classic endemic lineage ST73) and JJ1886 (from recently emerged, multidrug-resistant pandemic lineage ST131). We further show that quorum-dependent UPEC quiescence is prevented and reversed by small molecules, called proliferants, that stimulate growth, such as L-lysine, L-methionine, and peptidoglycan (PG) stem peptides, including an isolated PG pentapeptide from Staphylococcus aureus. Together, our results indicate that (i) uptake of L-lysine and (ii) PG peptide sensing by UPEC modulate the quorum-regulated decision to proliferate and further demonstrate that PG fragments are important for intra- and interspecies signaling in pathogenic E. coli.

scholarly journals Peptidoglycan Sensing Prevents Quiescence and Promotes Quorum-Independent Colony Growth of Uropathogenic Escherichia coli

2020 ◽  
Vol 202 (20) ◽  
Author(s):  
Eric C. DiBiasio ◽  
Hilary J. Ranson ◽  
James R. Johnson ◽  
David C. Rowley ◽  
Paul S. Cohen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Urinary Tract Infections ◽  
Recurrent Infection ◽  
Colony Growth ◽  
Quiescent State ◽  
Content Type ◽  
Tract Infections

ABSTRACT Uropathogenic Escherichia coli (UPEC) is the leading cause of human urinary tract infections (UTIs), and many patients experience recurrent infection after successful antibiotic treatment. The source of recurrent infections may be persistent bacterial reservoirs in vivo that are in a quiescent state and thus are not susceptible to antibiotics. Here, we show that multiple UPEC strains require a quorum to proliferate in vitro with glucose as the carbon source. At low cell density, the bacteria remain viable but enter a quiescent, nonproliferative state. Of the clinical UPEC isolates tested to date, 35% (51/145) enter this quiescent state, including isolates from the recently emerged, multidrug-resistant pandemic lineage ST131 (i.e., strain JJ1886) and isolates from the classic endemic lineage ST73 (i.e., strain CFT073). Moreover, quorum-dependent UPEC quiescence is prevented and reversed by small-molecule proliferants that stimulate colony formation. These proliferation cues include d-amino acid-containing peptidoglycan (PG) tetra- and pentapeptides, as well as high local concentrations of l-lysine and l-methionine. Peptidoglycan fragments originate from the peptidoglycan layer that supports the bacterial cell wall but are released as bacteria grow. These fragments are detected by a variety of organisms, including human cells, other diverse bacteria, and, as we show here for the first time, UPEC. Together, these results show that for UPEC, (i) sensing of PG stem peptide and uptake of l-lysine modulate the quorum-regulated decision to proliferate and (ii) quiescence can be prevented by both intra- and interspecies PG peptide signaling. IMPORTANCE Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTIs). During pathogenesis, UPEC cells adhere to and infiltrate bladder epithelial cells, where they may form intracellular bacterial communities (IBCs) or enter a nongrowing or slowly growing quiescent state. Here, we show in vitro that UPEC strains at low population density enter a reversible, quiescent state by halting division. Quiescent cells resume proliferation in response to sensing a quorum and detecting external signals, or cues, including peptidoglycan tetra- and pentapeptides.

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

2005 ◽  
Vol 49 (6) ◽  
pp. 2343-2351 ◽  
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.

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

2020 ◽  
Vol 86 (13) ◽  
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.

scholarly journals Characterization of Stg Fimbriae from an Avian Pathogenic Escherichia coli O78:K80 Strain and Assessment of Their Contribution to Colonization of the Chicken Respiratory Tract

2006 ◽  
Vol 188 (18) ◽  
pp. 6449-6459 ◽  
Author(s):  
Maria H. Lymberopoulos ◽  
Sébastien Houle ◽  
France Daigle ◽  
Simon Léveillé ◽  
Annie Brée ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Distinct Group ◽  
Immunogold Electron Microscopy ◽  
Carbohydrate Source ◽  
E Coli ◽  
Tract Infections ◽  
K 12

ABSTRACT In a previous study, ecs-3, a sequence from avian pathogenic Escherichia coli (APEC) O78:K80 strain χ7122, was found to be expressed in vivo in infected chicken tissues. The region encompassing ecs-3 carries a fimbrial gene cluster that is a putative ortholog of the stg fimbrial gene cluster of Salmonella enterica serovar Typhi. This APEC fimbrial gene cluster, which we have termed stg, is a member of a distinct group of related fimbriae that are located in the glmS-pstS intergenic region of certain E. coli and S. enterica strains. Under the control of the pBAD promoter, the production of Stg fimbriae was demonstrated by Western blotting and immunogold electron microscopy with E. coli K-12. Transcriptional fusions suggest that stg expression is influenced by the carbohydrate source and decreased by the addition of iron and that Fur plays a role in the regulation of stg expression. stg sequences were associated with APEC O78 isolates, and stg was phylogenetically distributed among E. coli reference strains and clinical isolates from human urinary tract infections. Stg fimbriae contributed to the adherence of a nonfimbriated E. coli K-12 strain to avian lung sections and human epithelial cells in vitro. Coinfection experiments with APEC strain χ7122 and an isogenic Δstg mutant demonstrated that compared to the wild-type parent, the Δstg mutant was less able to colonize air sacs, equally able to colonize lungs, and able to more effectively colonize tracheas of infected chickens. Stg fimbriae, together with other adhesins, may therefore contribute to the colonization of avian respiratory tissues by certain APEC strains.

scholarly journals Synergy between Florfenicol and Aminoglycosides against Multidrug-Resistant Escherichia coli Isolates from Livestock

Antibiotics ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 185 ◽  
Author(s):  
Shukho Kim ◽  
Jung Hwa Woo ◽  
So Hyun Jun ◽  
Dong Chan Moon ◽  
Suk-Kyung Lim ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Synergistic Effect ◽  
Antimicrobial Agents ◽  
Alternative Therapy ◽  
Multidrug Resistant ◽  
Combination Regimen ◽  
E Coli ◽  
Effective Combination

The increasing prevalence of antimicrobial resistance and the laborious development of novel antimicrobial agents have limited the options for effective antimicrobial therapy. The combination of previously used antimicrobial agents represents an alternative therapy for multidrug-resistant (MDR) pathogens. The objective of this study was to investigate the synergistic effect of a florfenicol (FFL)-based combination with other antimicrobial agents against MDR Escherichia coli isolates from livestock using checkerboard assays and murine infection models. The FFL/amikacin (AMK) and FFL/gentamicin (GEN) combinations showed synergy against 10/11 and 6/11 MDR E. coli isolates in vitro, respectively. The combination of FFL with aminoglycosides (AMK or GEN) exhibited a better synergistic effect against MDR E. coli isolates than the cephalothin (CEF)/GEN or FFL/CEF combinations. The combination of FFL with AMK or GEN could reduce the emergence of resistant mutants in vitro. The FFL/AMK combination showed a higher survival rate of mice infected with MDR E. coli isolates than FFL or AMK alone. In summary, the combination of FFL with aminoglycosides (AMK or GEN) is highly effective against MDR E. coli isolates both in vitro and in vivo. Our findings may contribute to the discovery of an effective combination regimen against MDR E. coli infections in veterinary medicine.

scholarly journals Fosfomycin Susceptibility in Multidrug Resistant Urinary Escherichia coli Isolates

2021 ◽  
Vol 10 (7) ◽  
pp. 414-418
Author(s):  
Greeshma Hareendranath
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Multidrug Resistant ◽  
Diffusion Method ◽  
Antibiofilm Activity ◽  
E Coli ◽  
Development Of Resistance ◽  
Microbiological Methods ◽  
Tract Infections

BACKGROUND Escherichia coli is one of the most important causes of urinary tract infections (UTIs). Increased antibiotic resistance may limit the therapeutic options for the treatment of these infections. Fosfomycin trometamol is a phosphonic acid derivative, which acts primarily by interfering with bacterial peptidoglycan synthesis with broad spectrum of activity against agents causing urinary tract infection with good antibiofilm activity and limited reports of resistance and hence is increasingly called upon for the treatment of multi drug resistant (MDR) organisms causing UTI. There are limited studies from India regarding the efficacy of this drug; so, the study was conducted to determine the in vitro efficacy of fosfomycin against uropathogenic MDR E. coli. METHODS This was a prospective study done in the Department of Microbiology, Government T.D. Medical College, Alappuzha, over a period of 1 year from April 2018 to March 2019. A total of 150 MDR urine samples were processed by routine microbiological methods and after identification of E. coli urinary isolates, antibiotic susceptibility testing was performed and results were interpreted following the Clinical and Laboratory Standards Institute guidelines (CLSI). Fosfomycin sensitivity was tested by the Kirby-Bauer disc diffusion method. RESULTS Among the 150 MDR urinary E. coli isolates, 148 (98 %) were sensitive to fosfomycin in our study. The susceptibility rate of fosfomycin was clearly higher than other commonly used drugs for UTI. All extended-spectrum beta-lactamases (ESBL) producing E. coli were sensitive to this drug. The susceptibility for nitrofurantoin was fair, whereas for ampicillin, norfloxacin, cefotaxime and trimethoprim / sulphamethoxazole was found poor. Relatively better rates of resistance were observed for parenteral antibiotics. CONCLUSIONS With an enormous increase in the bacterial pathogens resistant to first-line antibiotics, there has been a revival in the use of fosfomycin. The convenience of a single dose regimen, a good activity proven invitro, and minimal propensity for development of resistance pathogens makes fosfomycin an attractive regimen for the treatment of uncomplicated community and hospital acquired UTIs. In this regard, with the existing limited options for treating MDR organisms, fosfomycin finds its utility acting as an effective and promising option in the treatment of UTIs due to MDR pathogens in the future.

scholarly journals Bactericidal Monoclonal Antibodies Specific to the Lipopolysaccharide O Antigen from Multidrug-Resistant Escherichia coli Clone ST131-O25b:H4 Elicit Protection in Mice

2015 ◽  
Vol 59 (6) ◽  
pp. 3109-3116 ◽  
Author(s):  
Valéria Szijártó ◽  
Luis M. Guachalla ◽  
Zehra C. Visram ◽  
Katharina Hartl ◽  
Cecília Varga ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Monoclonal Antibodies ◽  
Human Serum ◽  
Significant Proportion ◽  
Multidrug Resistant ◽  
Bacterial Killing ◽  
Content Type ◽  
E Coli

ABSTRACTTheEscherichia colisequence type 131 (ST131)-O25b:H4 clone has spread worldwide and become responsible for a significant proportion of multidrug-resistant extraintestinal infections. We generated humanized monoclonal antibodies (MAbs) that target the lipopolysaccharide O25b antigen conserved within this lineage. These MAbs bound to the surface of live bacterial cells irrespective of the capsular type expressed. In a serum bactericidal assayin vitro, MAbs induced >95% bacterial killing in the presence of human serum as the complement source. Protective efficacy at low antibody doses was observed in a murine model of bacteremia. The mode of actionin vivowas investigated by using aglycosylated derivatives of the protective MAbs. The significant binding to liveE. colicells and thein vitroandin vivoefficacy were corroborated in assays using bacteria grown in human serum to mimic relevant clinical conditions. Given the dry pipeline of novel antibiotics against multidrug-resistant Gram-negative pathogens, passive immunization with bactericidal antibodies offers a therapeutic alternative to control infections caused byE. coliST131-O25b:H4.

scholarly journals Efficacy of Human Exposures of Gepotidacin (GSK2140944) againstEscherichia coliin a Rat Pyelonephritis Model

2019 ◽  
Vol 63 (7) ◽  
Author(s):  
Jennifer L. Hoover ◽  
Christine M. Singley ◽  
Philippa Elefante ◽  
Stephen Rittenhouse
Keyword(s):  
Time Course ◽  
Limit Of Detection ◽  
Multidrug Resistant ◽  
Mass Spectrometry Analysis ◽  
Maximal Response ◽  
Content Type ◽  
E Coli ◽  
Tract Infections

ABSTRACTGepotidacin is a first-in-class triazaacenaphthylene antibacterial that inhibits bacterial type II topoisomerases and hasin vitroactivity against a range of bacterial pathogens, includingEscherichia coli. Urinary tract infections often progress to pyelonephritis and are a worldwide problem due to the prevalence of multidrug-resistantE. colistrains. This study evaluated thein vivoefficacy of gepotidacin against four strains of multidrug-resistantE. coliin a rat pyelonephritis model. Infected rats received controlled intravenous infusions of gepotidacin every 12 h for 4 days that recreated human systemic exposures from oral gepotidacin (800 or 1,500 mg twice daily for 4 days). Liquid chromatography-tandem mass spectrometry analysis of blood samples and kidney homogenates showed that gepotidacin levels were 6- to 7-fold higher in kidneys than in blood. Across experiments with 4-day gepotidacin treatments, bacterial CFU in kidneys were reduced by 2.9 to 4.9 log10compared to pretreatment levels, and bladder CFU were reduced to the lower limit of detection (1.2 log10). The efficacies of 800- and 1,500-mg gepotidacin exposures were statistically similar. A time-course experiment indicated that a period of more than 24 h of gepotidacin treatment was required for efficacy and that 4 days were needed for maximal response. Overall, these results demonstrate that the recreated human exposures of gepotidacin studied were effective in an animal model of pyelonephritis caused by multidrug-resistantE. coliand that further evaluation for clinical use is warranted.

scholarly journals Dissecting and Evaluating the Therapeutic Targets of Coptis Chinensis Franch in the Treatment of Urinary Tract Infections Induced by Escherichia coli

2022 ◽  
Vol 12 ◽  
Author(s):  
Zhenglin Chang ◽  
Jinhu Zhang ◽  
Min Lei ◽  
Zheng Jiang ◽  
Xiangkun Wu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Urinary Tract Infections ◽  
Host Cells ◽  
Network Pharmacology ◽  
Coptis Chinensis ◽  
E Coli ◽  
Tract Infections

Coptis chinensis Franch (CCF) is extensively used in the treatment of inflammatory-related diseases. Accumulating studies have previously demonstrated the anti-inflammatory properties of CCF, yet data on its exact targets against urinary tract infections (UTIs) remain largely unknown. Therefore, the present study decodes the potential targets of action of CCF against UTIs by network pharmacology combined with experiment evaluations. Based on the pharmacology network analysis, the current study yielded six core ingredients: quercetin, palmatine (R)-canadine, berlambine, berberine, and berberrubine. The protein–protein interaction network (PPI) was generated by the string database, and then, four targets (IL6, FOS, MYC, and EGFR) were perceived as the major CCF targets using the CytoNCA plug-in. The results of molecular docking showed that the six core constituents of CCF had strong binding affinities toward the four key targets of UTIs after docking into the crystal structure. The enrichment analysis indicated that the possible regulatory mechanisms of CCF against UTIs were based on the modules of inflammation, immune responses, and apoptosis among others. Experimentally, the Escherichia coli (E. coli) strain CFT073 was applied to establish in vivo and in vitro models. In vivo results revealed that the key targets, IL6 and FOS, are significantly upregulated in rat bladder tissues of UTIs, whereas the expression of MYC and EGFR remained steady. Last, in vitro results further confirmed the therapeutic potential of CCF by reducing the expression of IL6 and FOS. In conclusion, IL6 and FOS were generally upregulated in the progression of E. coli–induced UTIs, whereas the CCF intervention exerted a preventive role in host cells stimulated by E. coli, partially due to inhibiting the expression of IL6 and FOS.

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

1998 ◽  
Vol 66 (7) ◽  
pp. 3303-3310 ◽  
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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