scholarly journals Screening of a Leptospira biflexa Mutant Library To Identify Genes Involved in Ethidium Bromide Tolerance

2014 ◽  
Vol 80 (19) ◽  
pp. 6091-6103 ◽  
Author(s):  
Helena Pětrošová ◽  
Mathieu Picardeau
Keyword(s):  
Antibiotic Resistance ◽  
Ethidium Bromide ◽  
Antimicrobial Agents ◽  
Genetic Manipulation ◽  
Content Type ◽  
Transposon Insertion ◽  
Damage Repair ◽  
Transposon Mutants ◽  
Living Species ◽  
Increased Sensitivity

ABSTRACTLeptospiraspp. are spirochete bacteria comprising both pathogenic and free-living species. The saprophyteL. biflexais a model bacterium for studying leptospiral biology due to relative ease of culturing and genetic manipulation. In this study, we constructed a library of 4,996 random transposon mutants inL. biflexa. We screened the library for increased susceptibility to the DNA intercalating agent, ethidium bromide (EtBr), in order to identify genetic determinants that reduceL. biflexasusceptibility to antimicrobial agents. By phenotypic screening, using subinhibitory EtBr concentrations, we identified 29 genes that, when disrupted via transposon insertion, led to increased sensitivity of the bacteria to EtBr. At the functional level, these genes could be categorized by function as follows: regulation and signaling (n= 11), transport (n= 6), membrane structure (n= 5), stress response (n= 2), DNA damage repair (n= 1), and other processes (n= 3), while 1 gene had no predicted function. Genes involved in transport (including efflux pumps) and regulation (two-component systems, anti-sigma factor antagonists, etc.) were overrepresented, demonstrating that these genes are major contributors to EtBr tolerance. This finding suggests that transport genes which would prevent EtBr to enter the cell cytoplasm are critical for EtBr resistance. We identified genes required for the growth ofL. biflexain the presence of sublethal EtBr concentration and characterized their potential as antibiotic resistance determinants. This study will help to delineate mechanisms of adaptation to toxic compounds, as well as potential mechanisms of antibiotic resistance development in pathogenicL. interrogans.

scholarly journals Daptomycin Resistance and Tolerance Due to Loss of Function inStaphylococcus aureusdsp1andasp23

2018 ◽  
Vol 63 (1) ◽  
Author(s):  
Elaine M. Barros ◽  
Melissa J. Martin ◽  
Elizabeth M. Selleck ◽  
François Lebreton ◽  
Jorge Luiz M. Sampaio ◽  
...  
Keyword(s):  
Cell Wall ◽  
Antimicrobial Peptides ◽  
Multidrug Resistant ◽  
Loss Of Function ◽  
Cationic Antimicrobial Peptides ◽  
Content Type ◽  
Transposon Insertion ◽  
Transposon Mutants ◽  
A Cell ◽  
Transposon Insertions

ABSTRACTLipopeptide daptomycin is a last-line cell-membrane-targeting antibiotic to treat multidrug-resistantStaphylococcus aureus. Alarmingly, daptomycin-resistantS. aureusisolates have emerged. The mechanisms underlying daptomycin resistance are diverse and share similarities with resistances to cationic antimicrobial peptides and other lipopeptides, but they remain to be fully elucidated. We selected mutants with increased resistance to daptomycin from a library of transposon insertions in sequent type 8 (ST8)S. aureusHG003. Insertions conferring increased daptomycin resistance were localized to two genes, one coding for a hypothetical lipoprotein (SAOUHSC_00362, Dsp1), and the other for an alkaline shock protein (SAOUHSC_02441, Asp23). Markerless loss-of-function mutants were then generated for comparison. All transposon mutants and knockout strains exhibited increased daptomycin resistance compared to those of wild-type and complemented strains. Null and transposon insertion mutants also exhibited increased resistance to cationic antimicrobial peptides. Interestingly, theΔdsp1mutant also showed increased resistance to vancomycin, a cell-wall-targeting drug with a different mode of action. Null mutations in bothdsp1andasp23resulted in increased tolerance as reflected by reduced killing to both daptomycin and vancomycin, as well as an increased tolerance to surfactant (Triton X-100). Neither mutant exhibited increased resistance to lysostaphin, a cell-wall-targeting endopeptidase. These findings identified two genes core to theS. aureusspecies that make previously uncharacterized contributions to antimicrobial resistance and tolerance inS. aureus.

scholarly journals Cell Shape and Antibiotic Resistance Are Maintained by the Activity of Multiple FtsW and RodA Enzymes in Listeria monocytogenes

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Jeanine Rismondo ◽  
Sven Halbedel ◽  
Angelika Gründling
Keyword(s):  
Cell Wall ◽  
Antibiotic Resistance ◽  
Immune System ◽  
Cell Division ◽  
Listeria Monocytogenes ◽  
Antimicrobial Agents ◽  
Protein Complexes ◽  
Inducible Promoter ◽  
Human Pathogen ◽  
Content Type

ABSTRACT Rod-shaped bacteria have two modes of peptidoglycan synthesis: lateral synthesis and synthesis at the cell division site. These two processes are controlled by two macromolecular protein complexes, the elongasome and divisome. Recently, it has been shown that the Bacillus subtilis RodA protein, which forms part of the elongasome, has peptidoglycan glycosyltransferase activity. The cell division-specific RodA homolog FtsW fulfils a similar role at the divisome. The human pathogen Listeria monocytogenes carries genes that encode up to six FtsW/RodA homologs; however, their functions have not yet been investigated. Analysis of deletion and depletion strains led to the identification of the essential cell division-specific FtsW protein, FtsW1. Interestingly, L. monocytogenes carries a gene that encodes a second FtsW protein, FtsW2, which can compensate for the lack of FtsW1, when expressed from an inducible promoter. L. monocytogenes also possesses three RodA homologs, RodA1, RodA2, and RodA3, and their combined absence is lethal. Cells of a rodA1 rodA3 double mutant are shorter and have increased antibiotic and lysozyme sensitivity, probably due to a weakened cell wall. Results from promoter activity assays revealed that expression of rodA3 and ftsW2 is induced in the presence of antibiotics targeting penicillin binding proteins. Consistent with this, a rodA3 mutant was more susceptible to the β-lactam antibiotic cefuroxime. Interestingly, overexpression of RodA3 also led to increased cefuroxime sensitivity. Our study highlights that L. monocytogenes genes encode a multitude of functional FtsW and RodA enzymes to produce its rigid cell wall and that their expression needs to be tightly regulated to maintain growth, cell division, and antibiotic resistance. IMPORTANCE The human pathogen Listeria monocytogenes is usually treated with high doses of β-lactam antibiotics, often combined with gentamicin. However, these antibiotics only act bacteriostatically on L. monocytogenes, and the immune system is needed to clear the infection. Therefore, individuals with a compromised immune system are at risk to develop a severe form of Listeria infection, which can be fatal in up to 30% of cases. The development of new strategies to treat Listeria infections is necessary. Here we show that the expression of some of the FtsW and RodA enzymes of L. monocytogenes is induced by the presence of β-lactam antibiotics, and the combined absence of these enzymes makes bacteria more susceptible to this class of antibiotics. The development of antimicrobial agents that inhibit the activity or production of FtsW and RodA enzymes might therefore help to improve the treatment of Listeria infections and thereby lead to a reduction in mortality.

scholarly journals Listeria monocytogenes Mutants with Altered Growth Phenotypes at Refrigeration Temperature and High Salt Concentrations

2011 ◽  
Vol 78 (4) ◽  
pp. 1265-1272 ◽  
Author(s):  
Laurel S. Burall ◽  
Pongpan Laksanalamai ◽  
Atin R. Datta
Keyword(s):  
Listeria Monocytogenes ◽  
Substantial Reduction ◽  
Brain Heart Infusion ◽  
High Salt ◽  
Salt Adaptation ◽  
Content Type ◽  
Transposon Insertion ◽  
Food Isolate ◽  
Transposon Mutants ◽  
Growth Profiles

ABSTRACTListeria monocytogenescan survive and grow in refrigerated temperatures and high-salt environments. In an effort to better understand the associated mechanisms, a library of ∼ 5,200 transposon mutants of LS411, a food isolate from the Jalisco cheese outbreak, were screened for their ability to grow in brain heart infusion (BHI) broth at 5°C or in the presence of 7% NaCl and two mutants with altered growth profiles were identified. The LS522 mutant has a transposon insertion betweensecA2andiapand showed a significant reduction in growth in BHI broth at 5°C and in the presence of 7% NaCl. Reverse transcriptase quantitative PCR (RT-qPCR) revealed a substantial reduction in the expression ofiap. Additionally, a hypothetical gene (met), containing a putativeS-adenosylmethionine-dependent methyltransferase domain, downstream ofiaphad downregulated expression. In-frame deletion mutants ofiapandmetwere created in LS411. The LS560 (LS411Δiap) mutant showed reduced growth at 5°C and in the presence of 7% salt, confirming its role in cold and salt growth attenuation. Surprisingly, the LS655 (LS411 Δmet) mutant showed slightly increased growth during refrigeration, though no alteration was seen in salt growth relative to the wild-type strain. The LS527 mutant, containing an insertion 36 bp upstream of thegbuoperon, showed reduced expression of thegbutranscript by RT-qPCR and also showed growth reduction at 5°C and in the presence of 7% salt. This attenuation was severely exacerbated when the mutant was grown under the combined stresses. Analysis of thegbuoperon deletion mutant showed decreased growth in 7% salt and refrigeration, supporting the previously characterized role for this gene in cold and salt adaptation. These studies indicate the potential for an intricate relationship between environmental stress regulation and virulence inL. monocytogenes.

scholarly journals Genome-Wide Transposon Mutagenesis in Pathogenic Leptospira Species

2008 ◽  
Vol 77 (2) ◽  
pp. 810-816 ◽  
Author(s):  
Gerald L. Murray ◽  
Viviane Morel ◽  
Gustavo M. Cerqueira ◽  
Julio Croda ◽  
Amporn Srikram ◽  
...  
Keyword(s):  
Genetic Manipulation ◽  
Transposon Mutagenesis ◽  
Hamster Model ◽  
Pathogenic Leptospira ◽  
Protein Coding ◽  
Transposon Insertion ◽  
Random Fashion ◽  
Genes Encoding ◽  
Transposon Mutants ◽  
Transposon Insertions

ABSTRACT Leptospira interrogans is the most common cause of leptospirosis in humans and animals. Genetic analysis of L. interrogans has been severely hindered by a lack of tools for genetic manipulation. Recently we developed the mariner-based transposon Himar1 to generate the first defined mutants in L. interrogans. In this study, a total of 929 independent transposon mutants were obtained and the location of insertion determined. Of these mutants, 721 were located in the protein coding regions of 551 different genes. While sequence analysis of transposon insertion sites indicated that transposition occurred in an essentially random fashion in the genome, 25 unique transposon mutants were found to exhibit insertions into genes encoding 16S or 23S rRNAs, suggesting these genes are insertional hot spots in the L. interrogans genome. In contrast, loci containing notionally essential genes involved in lipopolysaccharide and heme biosynthesis showed few transposon insertions. The effect of gene disruption on the virulence of a selected set of defined mutants was investigated using the hamster model of leptospirosis. Two attenuated mutants with disruptions in hypothetical genes were identified, thus validating the use of transposon mutagenesis for the identification of novel virulence factors in L. interrogans. This library provides a valuable resource for the study of gene function in L. interrogans. Combined with the genome sequences of L. interrogans, this provides an opportunity to investigate genes that contribute to pathogenesis and will provide a better understanding of the biology of L. interrogans.

scholarly journals Broad-Spectrum Adaptive Antibiotic Resistance Associated with Pseudomonas aeruginosa Mucin-Dependent Surfing Motility

2018 ◽  
Vol 62 (9) ◽  
Author(s):  
Evelyn Sun ◽  
Erin E. Gill ◽  
Reza Falsafi ◽  
Amy Yeung ◽  
Sijie Liu ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antibiotic Resistance ◽  
Broad Spectrum ◽  
Rna Seq ◽  
Content Type ◽  
Adaptive Resistance ◽  
Mucosal Surfaces ◽  
Broad Spectrum Resistance ◽  
Dependent Inhibition ◽  
Transposon Mutants

ABSTRACT Surfing motility is a novel form of surface adaptation exhibited by the nosocomial pathogen Pseudomonas aeruginosa in the presence of the glycoprotein mucin, which is found in high abundance at mucosal surfaces, especially those of the lungs of cystic fibrosis and bronchiectasis patients. Here, we investigated the adaptive antibiotic resistance of P. aeruginosa under conditions in which surfing occurs compared that in to cells undergoing swimming. P. aeruginosa surfing cells were significantly more resistant to several classes of antibiotics, including aminoglycosides, carbapenems, polymyxins, and fluoroquinolones. This was confirmed by incorporation of antibiotics into growth medium, which revealed a concentration-dependent inhibition of surfing motility that occurred at concentrations much higher than those needed to inhibit swimming. To investigate the basis of resistance, transcriptome sequencing (RNA-Seq) was performed and revealed that surfing influenced the expression of numerous genes. Included among genes dysregulated under surfing conditions were multiple genes from the Pseudomonas resistome; these genes are known to affect antibiotic resistance when mutated. Screening transposon mutants in these surfing-dysregulated resistome genes revealed that several of these mutants exhibited changes in susceptibility to one or more antibiotics under surfing conditions, consistent with a contribution to the observed adaptive resistance. In particular, several mutants in resistome genes, including armR, recG, atpB, clpS, nuoB, and certain hypothetical genes, such as PA5130, PA3576, and PA4292, showed contributions to broad-spectrum resistance under surfing conditions and could be complemented by their respective cloned genes. Therefore, we propose that surfing adaption led to extensive multidrug adaptive resistance as a result of the collective dysregulation of diverse genes.

scholarly journals Antagonism between Front-Line Antibiotics Clarithromycin and Amikacin in the Treatment of Mycobacterium abscessus Infections Is Mediated by the whiB7 Gene

2017 ◽  
Vol 61 (11) ◽  
Author(s):  
Mark Pryjma ◽  
Ján Burian ◽  
Kevin Kuchinski ◽  
Charles J. Thompson
Keyword(s):  
Cystic Fibrosis ◽  
Antibiotic Resistance ◽  
Transcriptional Activation ◽  
Mycobacterium Abscessus ◽  
Front Line ◽  
Content Type ◽  
Increased Sensitivity ◽  
Induction Of Resistance ◽  
Antagonistic Interactions

ABSTRACT Combinations of antibiotics, each individually effective against Mycobacterium abscessus, are routinely coadministered based on the concept that this minimizes the spread of antibiotic resistance. However, our in vitro data contradict this assumption and instead document antagonistic interactions between two antibiotics (clarithromycin and amikacin) used to treat M. abscessus infections. Clinically relevant concentrations of clarithromycin induced increased resistance to both amikacin and itself. The induction of resistance was dependent on whiB7, a transcriptional activator of intrinsic antibiotic resistance that is induced by exposure to many different antibiotics. In M. abscessus, the deletion of whiB7 (MAB_3508c) resulted in increased sensitivity to a broad range of antibiotics. WhiB7 was required for transcriptional activation of genes that confer resistance to three commonly used anti-M. abscessus drugs: clarithromycin, amikacin, and tigecycline. The whiB7-dependent gene that conferred macrolide resistance was identified as erm(41) (MAB_2297), which encodes a ribosomal methyltransferase. The whiB7-dependent gene contributing to amikacin resistance was eis2 (MAB_4532c), which encodes a Gcn5-related N-acetyltransferase (GNAT). Transcription of whiB7 and the resistance genes in its regulon was inducible by subinhibitory concentrations of clarithromycin but not by amikacin. Thus, exposure to clarithromycin, or likely any whiB7-inducing antibiotic, may antagonize the activities of amikacin and other drugs. This has important implications for the management of M. abscessus infections, both in cystic fibrosis (CF) and non-CF patients.

scholarly journals Identification of Mycobacterial Genes Involved in Antibiotic Sensitivity: Implications for the Treatment of Tuberculosis with β-Lactam-Containing Regimens

2017 ◽  
Vol 61 (7) ◽  
Author(s):  
Gopinath Viswanathan ◽  
Sangya Yadav ◽  
Tirumalai R. Raghunand
Keyword(s):  
Antibiotic Resistance ◽  
Mycobacterium Tuberculosis ◽  
Bactericidal Activity ◽  
Mycobacterium Smegmatis ◽  
Antibiotic Sensitivity ◽  
Mutant Library ◽  
Content Type ◽  
Factors Associated ◽  
Library Screen ◽  
Transposon Mutants

ABSTRACT In a Mycobacterium smegmatis mutant library screen, transposon mutants with insertions in fhaA, dprE2, rpsT, and parA displayed hypersusceptibility to antibiotics, including the β-lactams meropenem, ampicillin, amoxicillin, and cefotaxime. Sub-MIC levels of octoclothepin, a psychotic drug inhibiting ParA, phenocopied the parA insertion and enhanced the bactericidal activity of meropenem against Mycobacterium tuberculosis in combination with clavulanate. Our study identifies novel factors associated with antibiotic resistance, with implications in repurposing β-lactams for tuberculosis treatment.

scholarly journals Utility of the Clostridial Site-Specific Recombinase TnpX To Clone Toxic-Product-Encoding Genes and Selectively Remove Genomic DNA Fragments

2014 ◽  
Vol 80 (12) ◽  
pp. 3597-3603 ◽  
Author(s):  
Vicki Adams ◽  
Radhika Bantwal ◽  
Lauren Stevenson ◽  
Jackie K. Cheung ◽  
Milena M. Awad ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Genetic Manipulation ◽  
Antibiotic Resistance Genes ◽  
Dna Fragments ◽  
Site Specific ◽  
Content Type ◽  
E Coli ◽  
Marker Recycling ◽  
A Site

ABSTRACTTnpX is a site-specific recombinase responsible for the excision and insertion of the transposons Tn4451and Tn4453inClostridium perfringensandClostridium difficile, respectively. Here, we exploit phenotypic features of TnpX to facilitate genetic mutagenesis and complementation studies. Genetic manipulation of bacteria often relies on the use of antibiotic resistance genes; however, a limited number are available for use in the clostridia. The ability of TnpX to recognize and excise specific DNA fragments was exploited here as the basis of an antibiotic resistance marker recycling system, specifically to remove antibiotic resistance genes from plasmids inEscherichia coliand from marked chromosomalC. perfringensmutants. This methodology enabled the construction of aC. perfringensplc virRdouble mutant by allowing the removal and subsequent reuse of the same resistance gene to construct a second mutation. Genetic complementation can be challenging when the gene of interest encodes a product toxic toE. coli. We show that TnpX represses expression from its own promoter, PattCI, which can be exploited to facilitate the cloning of recalcitrant genes inE. colifor subsequent expression in the heterologous hostC. perfringens. Importantly, this technology expands the repertoire of tools available for the genetic manipulation of the clostridia.

scholarly journals High-Temperature Protein G Is an Essential Virulence Factor of Leptospira interrogans

2013 ◽  
Vol 82 (3) ◽  
pp. 1123-1131 ◽  
Author(s):  
Amy M. King ◽  
Gabriela Pretre ◽  
Thanatchaporn Bartpho ◽  
Rasana W. Sermswan ◽  
Claudia Toma ◽  
...  
Keyword(s):  
Virulence Factor ◽  
Stress Responses ◽  
Vaccine Development ◽  
Bacterial Species ◽  
Insertion Mutant ◽  
Leptospira Interrogans ◽  
Hamster Model ◽  
Content Type ◽  
Transposon Insertion ◽  
Increased Sensitivity

ABSTRACTLeptospira interrogansis a global zoonotic pathogen and is the causative agent of leptospirosis, an endemic disease of humans and animals worldwide. There is limited understanding of leptospiral pathogenesis; therefore, further elucidation of the mechanisms involved would aid in vaccine development and the prevention of infection. HtpG (high-temperatureproteinG) is the bacterial homolog to the highly conserved molecular chaperone Hsp90 and is important in the stress responses of many bacteria. The specific role of HtpG, especially in bacterial pathogenesis, remains largely unknown. Through the use of anL. interroganshtpGtransposon insertion mutant, this study demonstrates thatL. interrogansHtpG is essential for virulence in the hamster model of acute leptospirosis. Complementation of thehtpGmutant completely restored virulence. Surprisingly, thehtpGmutant did not appear to show sensitivity to heat or oxidative stress, phenotypes common inhtpGmutants in other bacterial species. Furthermore, the mutant did not show increased sensitivity to serum complement, reduced survival within macrophages, or altered protein or lipopolysaccharide expression. The underlying cause for attenuation thus remains unknown, but HtpG is a novel leptospiral virulence factor and one of only a very small number identified to date.

scholarly journals Benzoate- and Salicylate-Tolerant Strains of Escherichia coli K-12 Lose Antibiotic Resistance during Laboratory Evolution

2016 ◽  
Vol 83 (2) ◽  
Author(s):  
Kaitlin E. Creamer ◽  
Frederick S. Ditmars ◽  
Preston J. Basting ◽  
Karina S. Kunka ◽  
Issam N. Hamdallah ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Drug Resistance ◽  
Antibiotic Resistance ◽  
Multidrug Resistance ◽  
Gut Microbiome ◽  
Nucleotide Polymorphisms ◽  
Content Type ◽  
Large Deletions ◽  
Increased Sensitivity ◽  
K 12

ABSTRACT Escherichia coli K-12 W3110 grows in the presence of membrane-permeant organic acids that can depress cytoplasmic pH and accumulate in the cytoplasm. We conducted experimental evolution by daily diluting cultures in increasing concentrations of benzoic acid (up to 20 mM) buffered at external pH 6.5, a pH at which permeant acids concentrate in the cytoplasm. By 2,000 generations, clones isolated from evolving populations showed increasing tolerance to benzoate but were sensitive to chloramphenicol and tetracycline. Sixteen clones grew to stationary phase in 20 mM benzoate, whereas the ancestral strain W3110 peaked and declined. Similar growth occurred in 10 mM salicylate. Benzoate-evolved strains grew like W3110 in the absence of benzoate, in media buffered at pH 4.8, pH 7.0, or pH 9.0, or in 20 mM acetate or sorbate at pH 6.5. Genomes of 16 strains revealed over 100 mutations, including single-nucleotide polymorphisms (SNPs), large deletions, and insertion knockouts. Most strains acquired deletions in the benzoate-induced multiple antibiotic resistance (Mar) regulon or in associated regulators such as rob and cpxA, as well as the multidrug resistance (MDR) efflux pumps emrA, emrY, and mdtA. Strains also lost or downregulated the Gad acid fitness regulon. In 5 mM benzoate or in 2 mM salicylate (2-hydroxybenzoate), most strains showed increased sensitivity to the antibiotics chloramphenicol and tetracycline; some strains were more sensitive than a marA knockout strain. Thus, our benzoate-evolved strains may reveal additional unknown drug resistance components. Benzoate or salicylate selection pressure may cause general loss of MDR genes and regulators. IMPORTANCE Benzoate is a common food preservative, and salicylate is the primary active metabolite of aspirin. In the gut microbiome, genetic adaptation to salicylate may involve loss or downregulation of inducible multidrug resistance systems. This discovery implies that aspirin therapy may modulate the human gut microbiome to favor salicylate tolerance at the expense of drug resistance. Similar aspirin-associated loss of drug resistance might occur in bacterial pathogens found in arterial plaques.

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