scholarly journals Identification and Characterization of Two Klebsiella pneumoniaelpxL Lipid A Late Acyltransferases and Their Role in Virulence

2017 ◽  
Vol 85 (9) ◽  
Author(s):  
Grant Mills ◽  
Amy Dumigan ◽  
Timothy Kidd ◽  
Laura Hobley ◽  
José A. Bengoechea
Keyword(s):  
Lipid A ◽  
Galleria Mellonella ◽  
Inflammatory Responses ◽  
Two Component System ◽  
Content Type ◽  
E Coli ◽  
Wide Range ◽  
Candidate Target ◽  
Tract Infections

ABSTRACT Klebsiella pneumoniae causes a wide range of infections, from urinary tract infections to pneumonia. The lipopolysaccharide is a virulence factor of this pathogen, although there are gaps in our understanding of its biosynthesis. Here we report on the characterization of K. pneumoniae lpxL, which encodes one of the enzymes responsible for the late secondary acylation of immature lipid A molecules. Analysis of the available K. pneumoniae genomes revealed that this pathogen's genome encodes two orthologues of Escherichia coli LpxL. Using genetic methods and mass spectrometry, we demonstrate that LpxL1 catalyzes the addition of laureate and LpxL2 catalyzes the addition of myristate. Both enzymes acylated E. coli lipid A, whereas only LpxL2 mediated K. pneumoniae lipid A acylation. We show that LpxL1 is negatively regulated by the two-component system PhoPQ. The lipid A produced by the lpxL2 mutant lacked the 2-hydroxymyristate, palmitate, and 4-aminoarabinose decorations found in the lipid A synthesized by the wild type. The lack of 2-hydroxymyristate was expected since LpxO modifies the myristate transferred by LpxL2 to the lipid A. The absence of the other two decorations is most likely caused by the downregulation of phoPQ and pmrAB expression. LpxL2-dependent lipid A acylation protects Klebsiella from polymyxins, mediates resistance to phagocytosis, limits the activation of inflammatory responses by macrophages, and is required for pathogen survival in the wax moth (Galleria mellonella). Our findings indicate that the LpxL2 contribution to virulence is dependent on LpxO-mediated hydroxylation of the LpxL2-transferred myristate. Our studies suggest that LpxL2 might be a candidate target in the development of anti-K. pneumoniae drugs.

scholarly journals 2-Hydroxylation ofAcinetobacter baumanniiLipid A Contributes to Virulence

2019 ◽  
Vol 87 (4) ◽  
Author(s):  
Toby L. Bartholomew ◽  
Timothy J. Kidd ◽  
Joana Sá Pessoa ◽  
Raquel Conde Álvarez ◽  
José A. Bengoechea
Keyword(s):  
Protein Kinase ◽  
Lipid A ◽  
Galleria Mellonella ◽  
Inflammatory Responses ◽  
Polymyxin B ◽  
Interleukin 10 ◽  
Multidrug Resistant ◽  
Mitogen Activated Protein Kinase ◽  
Content Type ◽  
Wide Range

ABSTRACTAcinetobacter baumanniicauses a wide range of nosocomial infections. This pathogen is considered a threat to human health due to the increasingly frequent isolation of multidrug-resistant strains. There is a major gap in knowledge on the infection biology ofA. baumannii, and only a few virulence factors have been characterized, including lipopolysaccharide. The lipid A expressed byA. baumanniiis hepta-acylated and contains 2-hydroxylaurate. The late acyltransferases controlling the acylation of lipid A have been already characterized. Here, we report the characterization ofA. baumanniiLpxO, which encodes the enzyme responsible for the 2-hydroxylation of lipid A. By genetic methods and mass spectrometry, we demonstrate that LpxO catalyzes the 2-hydroxylation of the laurate transferred byA. baumanniiLpxL. LpxO-dependent lipid A 2-hydroxylation protectsA. baumanniifrom polymyxin B, colistin, and human β-defensin 3. LpxO contributes to the survival ofA. baumanniiin human whole blood and is required for pathogen survival in the waxmothGalleria mellonella. LpxO also protectsAcinetobacterfromG. mellonellaantimicrobial peptides and limits their expression. Further demonstrating the importance of LpxO-dependent modification in immune evasion, 2-hydroxylation of lipid A limits the activation of the mitogen-activated protein kinase Jun N-terminal protein kinase to attenuate inflammatory responses. In addition, LpxO-controlled lipid A modification mediates the production of the anti-inflammatory cytokine interleukin-10 (IL-10) via the activation of the transcriptional factor CREB. IL-10 in turn limits the production of inflammatory cytokines followingA. baumanniiinfection. Altogether, our studies suggest that LpxO is a candidate for the development of anti-A. baumanniidrugs.

scholarly journals The Repeat-In-Toxin Family Member TosA Mediates Adherence of Uropathogenic Escherichia coli and Survival during Bacteremia

2011 ◽  
Vol 80 (2) ◽  
pp. 493-505 ◽  
Author(s):  
Patrick D. Vigil ◽  
Travis J. Wiles ◽  
Michael D. Engstrom ◽  
Lev Prasov ◽  
Matthew A. Mulvey ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Virulence Factors ◽  
Upper Urinary Tract ◽  
Host Cells ◽  
Content Type ◽  
E Coli ◽  
Wide Range ◽  
Novel Target ◽  
Tract Infections

ABSTRACTUropathogenicEscherichia coli(UPEC) is responsible for the majority of uncomplicated urinary tract infections (UTI) and represents the most common bacterial infection in adults. UPEC utilizes a wide range of virulence factors to colonize the host, including the novel repeat-in-toxin (RTX) protein TosA, which is specifically expressed in the host urinary tract and contributes significantly to the virulence and survival of UPEC.tosA, found in strains within the B2 phylogenetic subgroup ofE. coli, serves as a marker for strains that also contain a large number of well-characterized UPEC virulence factors. The presence oftosAin anE. coliisolate predicts successful colonization of the murine model of ascending UTI, regardless of the source of the isolate. Here, a detailed analysis of the function oftosArevealed that this gene is transcriptionally linked to genes encoding a conserved type 1 secretion system similar to other RTX family members. TosA localized to the cell surface and was found to mediate (i) adherence to host cells derived from the upper urinary tract and (ii) survival in disseminated infections and (iii) to enhance lethality during sepsis (as assessed in two different animal models of infection). An experimental vaccine, using purified TosA, protected vaccinated animals against urosepsis. From this work, it was concluded that TosA belongs to a novel group of RTX proteins that mediate adherence and host damage during UTI and urosepsis and could be a novel target for the development of therapeutics to treat ascending UTIs.

scholarly journals Epigenetic Mechanisms Regulate Innate Immunity against Uropathogenic and Commensal-Like Escherichia coli in the Surrogate Insect Model Galleria mellonella

2017 ◽  
Vol 85 (10) ◽  
Author(s):  
Miriam Heitmueller ◽  
André Billion ◽  
Ulrich Dobrindt ◽  
Andreas Vilcinskas ◽  
Krishnendu Mukherjee
Keyword(s):  
Escherichia Coli ◽  
Innate Immunity ◽  
Urinary Tract ◽  
Histone Acetylation ◽  
Galleria Mellonella ◽  
Epigenetic Mechanisms ◽  
Upec Strain ◽  
Content Type ◽  
E Coli ◽  
Tract Infections

ABSTRACT Innate-immunity-related genes in humans are activated during urinary tract infections (UTIs) caused by pathogenic strains of Escherichia coli but are suppressed by commensals. Epigenetic mechanisms play a pivotal role in the regulation of gene expression in response to environmental stimuli. To determine whether epigenetic mechanisms can explain the different behaviors of pathogenic and commensal bacteria, we infected larvae of the greater wax moth, Galleria mellonella, a widely used model insect host, with a uropathogenic E. coli (UPEC) strain that causes symptomatic UTIs in humans or a commensal-like strain that causes asymptomatic bacteriuria (ABU). Infection with the UPEC strain (CFT073) was more lethal to larvae than infection with the attenuated ABU strain (83972) due to the recognition of each strain by different Toll-like receptors, ultimately leading to differential DNA/RNA methylation and histone acetylation. We used next-generation sequencing and reverse transcription (RT)-PCR to correlate epigenetic changes with the induction of innate-immunity-related genes. Transcriptomic analysis of G. mellonella larvae infected with E. coli strains CFT073 and 83972 revealed strain-specific variations in the class and expression levels of genes encoding antimicrobial peptides, cytokines, and enzymes controlling DNA methylation and histone acetylation. Our results provide evidence for the differential epigenetic regulation of transcriptional reprogramming by UPEC and ABU strains of E. coli in G. mellonella larvae, which may be relevant to understanding the different behaviors of these bacterial strains in the human urinary tract.

scholarly journals Development of Broth Microdilution MIC and Disk Diffusion Antimicrobial Susceptibility Test Quality Control Ranges for the Combination of Cefepime and the Novel β-Lactamase Inhibitor Enmetazobactam

2019 ◽  
Vol 57 (8) ◽  
Author(s):  
Adam Belley ◽  
Michael D. Huband ◽  
Kelley A. Fedler ◽  
Amy A. Watters ◽  
Robert K. Flamm ◽  
...  
Keyword(s):  
Quality Control ◽  
Performance Criteria ◽  
Disk Diffusion ◽  
Broth Microdilution ◽  
Content Type ◽  
E Coli ◽  
Wide Range ◽  
Tract Infections ◽  
Reference Strains ◽  
Lactamase Inhibitor

ABSTRACTThird-generation cephalosporin resistance amongEnterobacteriaceae, mediated by the spread of extended-spectrum β-lactamases (ESBLs), is a very serious medical concern with limited therapeutic options. Enmetazobactam (formerly AAI101) is a novel penicillanic sulfone β-lactamase inhibitor active against a wide range of ESBLs. The combination of enmetazobactam and cefepime has entered phase 3 development in patients with complicated urinary tract infections. Using the Clinical and Laboratory Standards Institute (CLSI) M23 tier 2 study design, broth microdilution MIC and disk diffusion quality control (QC) ranges were determined for cefepime-enmetazobactam. Enmetazobactam was tested at a fixed concentration of 8 μg/ml in the MIC assay, and a cefepime-enmetazobactam disk mass of 30/20 μg was used in the disk diffusion assay.Escherichia coliATCC 25922,E. coliATCC 35218,E. coliNCTC 13353,Klebsiella pneumoniaeATCC 700603, andPseudomonas aeruginosaATCC 27853 were chosen as reference strains. The CTX-M-15-producingE. coliNCTC 13353 isolate is recommended for routine testing to control for inhibition of ESBL activity by enmetazobactam. Broth microdilution MIC QC ranges spanned 3 to 4 doubling dilutions and contained 99.6% to 100.0% of obtained MIC values for the five reference strains. Disk diffusion yielded inhibition zone diameter QC ranges that spanned 7 mm and encompassed 97.1% to 100.0% of the obtained values. Quality control ranges were approved by the CLSI in 2017 (broth microdilution MIC) and 2019 (disk diffusion). The established QC ranges will ensure that appropriate assay performance criteria are attained using CLSI reference methodology when determining the susceptibility of clinical isolates to cefepime-enmetazobactam.

scholarly journals The development and characterization of an E. coli O25B bioconjugate vaccine

2021 ◽  
Author(s):  
Michael Kowarik ◽  
Michael Wetter ◽  
Micha A. Haeuptle ◽  
Martin Braun ◽  
Michael Steffen ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Multidrug Resistant ◽  
Sequence Type ◽  
Detailed Characterization ◽  
E Coli ◽  
Wide Range ◽  
Physicochemical Methods ◽  
Tract Infections ◽  
Opening Up

AbstractExtraintestinal pathogenic Escherichia coli (ExPEC) cause a wide range of clinical diseases such as bacteremia and urinary tract infections. The increase of multidrug resistant ExPEC strains is becoming a major concern for the treatment of these infections and E. coli has been identified as a critical priority pathogen by the WHO. Therefore, the development of vaccines has become increasingly important, with the surface lipopolysaccharide constituting a promising vaccine target. This study presents genetic and structural analysis of clinical urine isolates from Switzerland belonging to the serotype O25. Approximately 75% of these isolates were shown to correspond to the substructure O25B only recently described in an emerging clone of E. coli sequence type 131. To address the high occurrence of O25B in clinical isolates, an O25B glycoconjugate vaccine was prepared using an E. coli glycosylation system. The O antigen cluster was integrated into the genome of E. coli W3110, thereby generating an E. coli strain able to synthesize the O25B polysaccharide on a carrier lipid. The polysaccharide was enzymatically conjugated to specific asparagine side chains of the carrier protein exotoxin A (EPA) of Pseudomonas aeruginosa by the PglB oligosaccharyltransferase from Campylobacter jejuni. Detailed characterization of the O25B-EPA conjugate by use of physicochemical methods including NMR and GC-MS confirmed the O25B polysaccharide structure in the conjugate, opening up the possibility to develop a multivalent E. coli conjugate vaccine containing O25B-EPA.

scholarly journals Urinary Excretion and Bactericidal Activities of a Single Oral Dose of 400 Milligrams of Fleroxacin versus a Single Oral Dose of 800 Milligrams of Pefloxacin in Healthy Volunteers

1998 ◽  
Vol 42 (7) ◽  
pp. 1659-1665 ◽  
Author(s):  
Kurt G. Naber ◽  
Ursula Theuretzbacher ◽  
Martina Kinzig ◽  
Orlin Savov ◽  
Fritz Sörgel
Keyword(s):  
Oral Dose ◽  
Healthy Volunteers ◽  
Single Oral Dose ◽  
E Coli ◽  
Wide Range ◽  
The Mean ◽  
Bactericidal Activities ◽  
Tract Infections ◽  
Time Kill ◽  
Randomized Crossover Study

ABSTRACT Twelve healthy volunteers participated in this randomized crossover study to compare the concentrations and recovery levels of fleroxacin and pefloxacin in urine and to assess their bactericidal activities against 12 strains of urinary pathogens with different susceptibilities over a wide range of MICs. The volunteers received a single oral dose of 400 mg of fleroxacin or 800 mg of pefloxacin. The mean cumulative renal excretion of unchanged fleroxacin,N-demethyl-fleroxacin, and N-oxide-fleroxacin accounted for 67, 7, and 6% of the total dose, respectively. The total urinary recovery of pefloxacin and the active metabolite norfloxacin was 34%. In the time-kill and the urinary bactericidal titer (UBT) studies, only the subjects’ urine not supplemented with broth was used. With most tested organisms and both quinolones it took more than 8 h to achieve a reduction in CFU of 99.9% (3 log units). Overall, there was a good correlation between UBTs and MICs for the strains. Against Escherichia coli ATCC 25922 the median UBTs were similar for both antibiotics and at least 1:8 for 96 h; against the E. coli strain for which the MIC was 0.5 μg/ml the UBT was at least 1:4 for 48 h. The UBTs of both drugs against Klebsiella pneumoniae were at least 1:16 for 72 h. The UBTs for Staphylococcus aureus (the MIC for which was 16 μg/ml) of both antibiotics were low, and in some of the samples, no bactericidal titers were observed. UBTs for Proteus mirabilis of pefloxacin are significantly higher than those of fleroxacin. For Pseudomonas aeruginosa the median UBTs were present for the 24-to-48-h interval. The same is true forEnterococcus faecalis. Against Staphylococcus saprophyticus, UBTs were present for at least 48 h with both quinolones. Overall, a single oral dose of 400 mg of fleroxacin exhibits UBTs comparable to those of 800 mg of pefloxacin. Therefore, it may be expected that half of the dose of fleroxacin gives comparable results in the treatment of urinary tract infections; this should be substantiated in comparative clinical trials.

scholarly journals Vibrio cholerae Triggers SOS and Mutagenesis in Response to a Wide Range of Antibiotics: a Route towards Multiresistance

2011 ◽  
Vol 55 (5) ◽  
pp. 2438-2441 ◽  
Author(s):  
Zeynep Baharoglu ◽  
Didier Mazel
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Vibrio Cholerae ◽  
Fluorescent Protein ◽  
Resistance Development ◽  
Content Type ◽  
E Coli ◽  
Wide Range ◽  
Green Fluorescent ◽  
Regulated Promoters

ABSTRACTAntibiotic resistance development has been linked to the bacterial SOS stress response. InEscherichia coli, fluoroquinolones are known to induce SOS, whereas other antibiotics, such as aminoglycosides, tetracycline, and chloramphenicol, do not. Here we address whether various antibiotics induce SOS inVibrio cholerae. Reporter green fluorescent protein (GFP) fusions were used to measure the response of SOS-regulated promoters to subinhibitory concentrations of antibiotics. We show that unlike the situation withE. coli, all these antibiotics induce SOS inV. cholerae.

scholarly journals Occurrence of Antimicrobial-Resistant Escherichia coli and Salmonella enterica in the Beef Cattle Production and Processing Continuum

2014 ◽  
Vol 81 (2) ◽  
pp. 713-725 ◽  
Author(s):  
John W. Schmidt ◽  
Getahun E. Agga ◽  
Joseph M. Bosilevac ◽  
Dayna M. Brichta-Harhay ◽  
Steven D. Shackelford ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Salmonella Enterica ◽  
Generation Cephalosporin ◽  
Resistant Bacteria ◽  
Processing Plant ◽  
Cattle Production ◽  
Content Type ◽  
E Coli ◽  
Beef Processing ◽  
Tract Infections

ABSTRACTSpecific concerns have been raised that third-generation cephalosporin-resistant (3GCr)Escherichia coli, trimethoprim-sulfamethoxazole-resistant (COTr)E. coli, 3GCrSalmonella enterica, and nalidixic acid-resistant (NALr)S. entericamay be present in cattle production environments, persist through beef processing, and contaminate final products. The prevalences and concentrations of these organisms were determined in feces and hides (at feedlot and processing plant), pre-evisceration carcasses, and final carcasses from three lots of fed cattle (n= 184). The prevalences and concentrations were further determined for strip loins from 103 of the carcasses. 3GCrSalmonellawas detected on 7.6% of hides during processing and was not detected on the final carcasses or strip loins. NALrS. entericawas detected on only one hide. 3GCrE. coliand COTrE. coliwere detected on 100.0% of hides during processing. Concentrations of 3GCrE. coliand COTrE. colion hides were correlated with pre-evisceration carcass contamination. 3GCrE. coliand COTrE. coliwere each detected on only 0.5% of final carcasses and were not detected on strip loins. Five hundred and 42 isolates were screened for extraintestinal pathogenicE. coli(ExPEC) virulence-associated markers. Only two COTrE. coliisolates from hides were ExPEC, indicating that fed cattle products are not a significant source of ExPEC causing human urinary tract infections. The very low prevalences of these organisms on final carcasses and their absence on strip loins demonstrate that current sanitary dressing procedures and processing interventions are effective against antimicrobial-resistant bacteria.

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

mBio ◽  
2014 ◽  
Vol 5 (2) ◽  
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.

scholarly journals Amdinocillin (Mecillinam) Resistance Mutations in Clinical Isolates and Laboratory-Selected Mutants of Escherichia coli

2015 ◽  
Vol 59 (3) ◽  
pp. 1718-1727 ◽  
Author(s):  
Elisabeth Thulin ◽  
Martin Sundqvist ◽  
Dan I. Andersson
Keyword(s):  
Escherichia Coli ◽  
Clinical Isolates ◽  
Cysteine Biosynthesis ◽  
Resistance Mutations ◽  
Content Type ◽  
E Coli ◽  
Major Mechanism ◽  
Laboratory Selection ◽  
Lactam Antibiotic ◽  
Tract Infections

ABSTRACTAmdinocillin (mecillinam) is a β-lactam antibiotic that is used mainly for the treatment of uncomplicated urinary tract infections. The objectives of this study were to identify mutations that confer amdinocillin resistance on laboratory-isolated mutants and clinical isolates ofEscherichia coliand to determine why amdinocillin resistance remains rare clinically even though resistance is easily selected in the laboratory. Under laboratory selection, frequencies of mutation to amdinocillin resistance varied from 8 × 10−8to 2 × 10−5per cell, depending on the concentration of amdinocillin used during selection. Several genes have been demonstrated to give amdinocillin resistance, but here eight novel genes previously unknown to be involved in amdinocillin resistance were identified. These genes encode functions involved in the respiratory chain, the ribosome, cysteine biosynthesis, tRNA synthesis, and pyrophosphate metabolism. The clinical isolates exhibited significantly greater fitness than the laboratory-isolated mutants and a different mutation spectrum. ThecysBgene was mutated (inactivated) in all of the clinical isolates, in contrast to the laboratory-isolated mutants, where mainly other types of more costly mutations were found. Our results suggest that the frequency of mutation to amdinocillin resistance is high because of the large mutational target (at least 38 genes). However, the majority of these resistant mutants have a low growth rate, reducing the probability that they are stably maintained in the bladder. Inactivation of thecysBgene and a resulting loss of cysteine biosynthesis are the major mechanism of amdinocillin resistance in clinical isolates ofE. coli.

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