scholarly journals Transposon Mutagenesis Screen of Klebsiella pneumoniae Identifies Multiple Genes Important for Resisting Antimicrobial Activities of Neutrophils in Mice

2020 ◽  
Vol 88 (4) ◽  
Author(s):  
Michelle K. Paczosa ◽  
Rebecca J. Silver ◽  
Anne L. McCabe ◽  
Albert K. Tai ◽  
Colin H. McLeish ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Bacterial Pathogen ◽  
Reactive Oxygen ◽  
Antimicrobial Activities ◽  
Virulence Determinants ◽  
Nitrogen Species ◽  
Content Type ◽  
Transposon Insertion ◽  
Tract Infections

ABSTRACT Klebsiella pneumoniae is a Gram-negative bacterial pathogen that causes a range of infections, including pneumonias, urinary tract infections, and septicemia, in otherwise healthy and immunocompromised patients. K. pneumoniae has become an increasing concern due to the rise and spread of antibiotic-resistant and hypervirulent strains. However, its virulence determinants remain understudied. To identify novel K. pneumoniae virulence factors needed to cause pneumonia, a high-throughput screen was performed with an arrayed library of over 13,000 K. pneumoniae transposon insertion mutants in the lungs of wild-type (WT) and neutropenic mice using transposon sequencing (Tn-seq). Insertions in 166 genes resulted in K. pneumoniae mutants that were significantly less fit in the lungs of WT mice than in those of neutropenic mice. Of these, mutants with insertions in 51 genes still had significant defects in neutropenic mice, while mutants with insertions in 52 genes recovered significantly. In vitro screens using a minilibrary of K. pneumoniae transposon mutants identified putative functions for a subset of these genes, including in capsule content and resistance to reactive oxygen and nitrogen species. Lung infections in mice confirmed roles in K. pneumoniae virulence for the ΔdedA, ΔdsbC, ΔgntR, Δwzm-wzt, ΔyaaA, and ΔycgE mutants, all of which were defective in either capsule content or growth in reactive oxygen or nitrogen species. The fitness of the ΔdedA, ΔdsbC, ΔgntR, ΔyaaA, and ΔycgE mutants was higher in neutropenic mouse lungs, indicating that these genes encode proteins that protect K. pneumoniae against neutrophil-related effector functions.

scholarly journals Comparative Effectiveness of Aminoglycosides, Polymyxin B, and Tigecycline for Clearance of Carbapenem-Resistant Klebsiella pneumoniae from Urine

2011 ◽  
Vol 55 (12) ◽  
pp. 5893-5899 ◽  
Author(s):  
Michael J. Satlin ◽  
Christine J. Kubin ◽  
Jill S. Blumenthal ◽  
Andrew B. Cohen ◽  
E. Yoko Furuya ◽  
...  
Keyword(s):  
New York ◽  
Klebsiella Pneumoniae ◽  
Urine Culture ◽  
Active Agent ◽  
Polymyxin B ◽  
Content Type ◽  
Carbapenem Resistant ◽  
Tract Infections

ABSTRACTCarbapenem-resistantKlebsiella pneumoniae(CRKP) is an increasingly common cause of health care-associated urinary tract infections. Antimicrobials within vitroactivity against CRKP are typically limited to polymyxins, tigecycline, and often, aminoglycosides. We conducted a retrospective cohort study of cases of CRKP bacteriuria at New York-Presbyterian Hospital from January 2005 through June 2010 to compare microbiologic clearance rates based on the use of polymyxin B, tigecycline, or an aminoglycoside. We constructed three active antimicrobial cohorts based on the active agent used and an untreated cohort of cases that did not receive antimicrobial therapy with Gram-negative activity. Microbiologic clearance was defined as having a follow-up urine culture that did not yield CRKP. Cases without an appropriate follow-up culture or that received multiple active agents or less than 3 days of the active agent were excluded. Eighty-seven cases were included in the active antimicrobial cohorts, and 69 were included in the untreated cohort. The microbiologic clearance rate was 88% in the aminoglycoside cohort (n= 41), compared to 64% in the polymyxin B (P= 0.02;n= 25), 43% in the tigecycline (P< 0.001;n= 21), and 36% in the untreated (P< 0.001;n= 69) cohorts. Using multivariate analysis, the odds of clearance were lower for the polymyxin B (odds ratio [OR], 0.10;P= 0.003), tigecycline (OR, 0.08;P= 0.001), and untreated (OR, 0.14;P= 0.003) cohorts than for the aminoglycoside cohort. Treatment with an aminoglycoside, when activein vitro, was associated with a significantly higher rate of microbiologic clearance of CRKP bacteriuria than treatment with either polymyxin B or tigecycline.

scholarly journals Evaluation of CpxRA as a Therapeutic Target for UropathogenicEscherichia coliInfections

2018 ◽  
Vol 86 (3) ◽  
pp. e00798-17 ◽  
Author(s):  
Lana Dbeibo ◽  
Julia J. van Rensburg ◽  
Sara N. Smith ◽  
Kate R. Fortney ◽  
Dharanesh Gangaiah ◽  
...  
Keyword(s):  
Phosphatase Activity ◽  
Virulence Determinants ◽  
Membrane Repair ◽  
Single Strain ◽  
Content Type ◽  
Phosphatase Inhibitors ◽  
Genes Encoding ◽  
Tract Infections

ABSTRACTCpxRA is an envelope stress response system found in all members of the familyEnterobacteriaceae; CpxA has kinase activity for CpxR and phosphatase activity for phospho-CpxR, a transcription factor. CpxR also accepts phosphate groups from acetyl phosphate, a glucose metabolite. Activation of CpxR increases the transcription of genes encoding membrane repair and downregulates virulence determinants. We hypothesized that activation of CpxR could serve as an antimicrobial/antivirulence strategy and discovered compounds that activate CpxR inEscherichia coliby inhibiting CpxA phosphatase activity. As a prelude to testing such compoundsin vivo, here we constructedcpxA(in the presence of glucose, CpxR is activated because of a lack of CpxA phosphatase) andcpxR(system absent) deletion mutants of uropathogenicE. coli(UPEC) CFT073. By RNA sequencing, few transcriptional differences were noted between thecpxRmutant and its parent, but in thecpxAmutant, several UPEC virulence determinants were downregulated, including thefimandpapoperons, and it exhibited reduced mannose-sensitive hemagglutination of guinea pig red blood cellsin vitro. In competition experiments with mice, both mutants were less fit than the parent in the urine, bladder, and kidney; these fitness defects were complemented intrans. Unexpectedly, in single-strain challenges, only thecpxAmutant was attenuated for virulence in the kidney but not in the bladder or urine. For thecpxAmutant, this may be due to the preferential use of amino acids over glucose as a carbon source in the bladder and urine by UPEC. These studies suggest that CpxA phosphatase inhibitors may have some utility for treating complex urinary tract infections.

scholarly journals Therapeutic Efficacy of Novel Antimicrobial Peptide AA139-Nanomedicines in a Multidrug-Resistant Klebsiella pneumoniae Pneumonia-Septicemia Model in Rats

2020 ◽  
Vol 64 (9) ◽  
Author(s):  
Hessel van der Weide ◽  
Unai Cossío ◽  
Raquel Gracia ◽  
Yvonne M. te Welscher ◽  
Marian T. ten Kate ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Therapeutic Efficacy ◽  
Antimicrobial Agents ◽  
Polymeric Nanoparticles ◽  
Multidrug Resistant ◽  
Antimicrobial Activities ◽  
Bacterial Killing ◽  
Gram Negative ◽  
Content Type

ABSTRACT Antimicrobial peptides (AMPs) have seen limited clinical use as antimicrobial agents, largely due to issues relating to toxicity, short biological half-life, and lack of efficacy against Gram-negative bacteria. However, the development of novel AMP-nanomedicines, i.e., AMPs entrapped in nanoparticles, has the potential to ameliorate these clinical problems. The authors investigated two novel nanomedicines based on AA139, an AMP currently in development for the treatment of multidrug-resistant Gram-negative infections. AA139 was entrapped in polymeric nanoparticles (PNPs) or lipid-core micelles (MCLs). The antimicrobial activity of AA139-PNP and AA139-MCL was determined in vitro. The biodistribution and limiting doses of AA139-nanomedicines were determined in uninfected rats via endotracheal aerosolization. The early bacterial killing activity of the AA139-nanomedicines in infected lungs was assessed in a rat model of pneumonia-septicemia caused by extended-spectrum β-lactamase-producing Klebsiella pneumoniae. In this model, the therapeutic efficacy was determined by once-daily (q24h) administration over 10 days. Both AA139-nanomedicines showed equivalent in vitro antimicrobial activities (similar to free AA139). In uninfected rats, they exhibited longer residence times in the lungs than free AA139 (∼20% longer for AA139-PNP and ∼80% longer for AA139-MCL), as well as reduced toxicity, enabling a higher limiting dose. In rats with pneumonia-septicemia, both AA139-nanomedicines showed significantly improved therapeutic efficacy in terms of an extended rat survival time, although survival of all rats was not achieved. These results demonstrate potential advantages that can be achieved using AMP-nanomedicines. AA139-PNP and AA139-MCL may be promising novel therapeutic agents for the treatment of patients suffering from multidrug-resistant Gram-negative pneumonia-septicemia.

scholarly journals 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

2013 ◽  
Vol 81 (8) ◽  
pp. 3009-3017 ◽  
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.

scholarly journals Genome-wide analysis of fitness-factors in uropathogenic Escherichia coli during growth in laboratory media and during urinary tract infections

2021 ◽  
Vol 7 (12) ◽  
Author(s):  
Vanesa García ◽  
Rasmus B. Grønnemose ◽  
Sergi Torres-Puig ◽  
Egle Kudirkiene ◽  
Mateo Piantelli ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Luria Broth ◽  
Essential Genes ◽  
Growth Media ◽  
Content Type ◽  
Transposon Insertion ◽  
Magnesium Uptake ◽  
Tract Infections

Uropathogenic Escherichia coli (UPEC) UTI89 is a well-characterized strain, which has mainly been used to study UPEC virulence during urinary tract infection (UTI). However, little is known on UTI89 key fitness-factors during growth in lab media and during UTI. Here, we used a transposon-insertion-sequencing approach (TraDIS) to reveal the UTI89 essential-genes for in vitro growth and fitness-gene-sets for growth in Luria broth (LB) and EZ-MOPS medium without glucose, as well as for human bacteriuria and mouse cystitis. A total of 293 essential genes for growth were identified and the set of fitness-genes was shown to differ depending on the growth media. A modified, previously validated UTI murine model, with administration of glucose prior to infection was applied. Selected fitness-genes for growth in urine and mouse-bladder colonization were validated using deletion-mutants. Novel fitness-genes, such as tusA, corA and rfaG; involved in sulphur-acquisition, magnesium-uptake, and LPS-biosynthesis, were proved to be important during UTI. Moreover, rfaG was confirmed as relevant in both niches, and therefore it may represent a target for novel UTI-treatment/prevention strategies.

scholarly journals Doripenem MICs andompK36Porin Genotypes of Sequence Type 258, KPC-Producing Klebsiella pneumoniae May Predict Responses to Carbapenem-Colistin Combination Therapy among Patients with Bacteremia

2014 ◽  
Vol 59 (3) ◽  
pp. 1797-1801 ◽  
Author(s):  
Ryan K. Shields ◽  
M. Hong Nguyen ◽  
Brian A. Potoski ◽  
Ellen G. Press ◽  
Liang Chen ◽  
...  
Keyword(s):  
Amino Acids ◽  
Combination Therapy ◽  
Klebsiella Pneumoniae ◽  
Sequence Type ◽  
Content Type

ABSTRACTTreatment failures of a carbapenem-colistin regimen among patients with bacteremia due to sequence type 258 (ST258), KPC-2-producingKlebsiella pneumoniaewere significantly more likely if both agents were inactivein vitro, as defined by a colistin MIC of >2 μg/ml and the presence of either a majorompK36porin mutation (guanine and alanine insertions at amino acids 134 and 135 [ins aa 134–135 GD], IS5promoter insertion [P= 0.007]) or a doripenem MIC of >8 μg/ml (P= 0.01). MajorompK36mutations among KPC-K. pneumoniaestrains are important determinants of carbapenem-colistin responsesin vitroandin vivo.

scholarly journals Mycobacterium tuberculosis Alters the Metalloprotease Activity of the COP9 Signalosome

mBio ◽  
2014 ◽  
Vol 5 (4) ◽  
Author(s):  
Lia Danelishvili ◽  
Lmar Babrak ◽  
Sasha J. Rose ◽  
Jamie Everman ◽  
Luiz E. Bermudez
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Cop9 Signalosome ◽  
Virulence Determinants ◽  
Bacterial Survival ◽  
Phagocytic Cells ◽  
Content Type ◽  
Metalloprotease Activity ◽  
Macrophage Protein

ABSTRACT Inhibition of apoptotic death of macrophages by Mycobacterium tuberculosis represents an important mechanism of virulence that results in pathogen survival both in vitro and in vivo. To identify M. tuberculosis virulence determinants involved in the modulation of apoptosis, we previously screened a transposon bank of mutants in human macrophages, and an M. tuberculosis clone with a nonfunctional Rv3354 gene was identified as incompetent to suppress apoptosis. Here, we show that the Rv3354 gene encodes a protein kinase that is secreted within mononuclear phagocytic cells and is required for M. tuberculosis virulence. The Rv3354 effector targets the metalloprotease (JAMM) domain within subunit 5 of the COP9 signalosome (CSN5), resulting in suppression of apoptosis and in the destabilization of CSN function and regulatory cullin-RING ubiquitin E3 enzymatic activity. Our observation suggests that alteration of the metalloprotease activity of CSN by Rv3354 possibly prevents the ubiquitin-dependent proteolysis of M. tuberculosis-secreted proteins. IMPORTANCE Macrophage protein degradation is regulated by a protein complex called a signalosome. One of the signalosomes associated with activation of ubiquitin and protein labeling for degradation was found to interact with a secreted protein from M. tuberculosis, which binds to the complex and inactivates it. The interference with the ability to inactivate bacterial proteins secreted in the phagocyte cytosol may have crucial importance for bacterial survival within the phagocyte.

scholarly journals Tamm-Horsfall Protein Protects the Urinary Tract againstCandida albicans

2018 ◽  
Vol 86 (12) ◽  
Author(s):  
Alison Coady ◽  
Anissa R. Ramos ◽  
Joshua Olson ◽  
Victor Nizet ◽  
Kathryn A. Patras
Keyword(s):  
Urinary Tract ◽  
Fungal Pathogens ◽  
Treatment Strategies ◽  
Immune Defense ◽  
Renal Physiology ◽  
Bladder Epithelium ◽  
Indwelling Catheters ◽  
Content Type ◽  
Tract Infections

ABSTRACTUrinary tract infections (UTIs) caused by the human fungal pathogenCandida albicansand related species are prevalent in hospitalized patients, especially those on antibiotic therapy, with indwelling catheters, or with predisposing conditions such as diabetes or immunodeficiency. Understanding of key host defenses againstCandidaUTI is critical for developing effective treatment strategies. Tamm-Horsfall glycoprotein (THP) is the most abundant urine protein, with multiple roles in renal physiology and bladder protection. THP protects against bacterial UTI by blocking bacterial adherence to the bladder epithelium, but its role in defense against fungal pathogens is not yet described. Here we demonstrate that THP restricts colonization of the urinary tract byC. albicans. THP binds toC. albicanshyphae, but not the yeast form, in a manner dependent on fungal expression of the Als3 adhesion glycoprotein. THP directly blocksC. albicansadherence to bladder epithelial cellsin vitro, and THP-deficient mice display increased fungal burden in aC. albicansUTI model. This work outlines a previously unknown role for THP as an essential component for host immune defense against fungal urinary tract infection.

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