Genomic and Transcriptomic Analyses of Colistin-Resistant Clinical Isolates of Klebsiella pneumoniae Reveal Multiple Pathways of Resistance

ABSTRACTThe emergence of multidrug-resistant (MDR)Klebsiella pneumoniaehas resulted in a more frequent reliance on treatment using colistin. However, resistance to colistin (Colr) is increasingly reported from clinical settings. The genetic mechanisms that lead to ColrinK. pneumoniaeare not fully characterized. Using a combination of genome sequencing and transcriptional profiling by RNA sequencing (RNA-Seq) analysis, distinct genetic mechanisms were found among nine Colrclinical isolates. Colrwas related to mutations in three different genes inK. pneumoniaestrains, with distinct impacts on gene expression. Upregulation of thepmrHoperon encoding 4-amino-4-deoxy-l-arabinose (Ara4N) modification of lipid A was found in all Colrstrains. Alteration of themgrBgene was observed in six strains. One strain had a mutation inphoQ. Common among these seven strains was elevated expression ofphoPQand unaltered expression ofpmrCAB, which is involved in phosphoethanolamine addition to lipopolysaccharide (LPS). In two strains, separate mutations were found in a previously uncharacterized histidine kinase gene that is part of a two-component regulatory system (TCRS) now designatedcrrAB. In these strains, expression ofpmrCAB,crrAB, and an adjacent glycosyltransferase gene, but not that ofphoPQ, was elevated. Complementation with the wild-type allele restored colistin susceptibility in both strains. ThecrrABgenes are present in mostK. pneumoniaegenomes, but not inEscherichia coli. Additional upregulated genes in all strains include those involved in cation transport and maintenance of membrane integrity. Because thecrrABgenes are present in only some strains, Colrmechanisms may be dependent on the genetic background.

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Analysis of the Networks Controlling the Antimicrobial-Peptide-Dependent Induction of Klebsiella pneumoniae Virulence Factors

Infection and Immunity ◽

10.1128/iai.05226-11 ◽

2011 ◽

Vol 79 (9) ◽

pp. 3718-3732 ◽

Cited By ~ 66

Author(s):

Enrique Llobet ◽

Miguel A. Campos ◽

Paloma Giménez ◽

David Moranta ◽

José A. Bengoechea

Keyword(s):

Klebsiella Pneumoniae ◽

Defense Mechanisms ◽

Lipid A ◽

Multidrug Resistant ◽

Cross Resistance ◽

New Approach ◽

Content Type ◽

Signaling Systems ◽

Transcriptional Changes ◽

Initial Target

ABSTRACTAntimicrobial peptides (APs) impose a threat to the survival of pathogens, and it is reasonable to postulate that bacteria have developed strategies to counteract them. Polymyxins are becoming the last resort to treat infections caused by multidrug-resistant Gram-negative bacteria and, similar to APs, they interact with the anionic lipopolysaccharide. Given that polymyxins and APs share the initial target, it is possible that bacterial defense mechanisms against polymyxins will be also effective against host APs. We sought to determine whether exposure to polymyxin will increaseKlebsiella pneumoniaeresistance to host APs. Indeed, exposure ofK. pneumoniaeto polymyxin induces cross-resistance not only to polymyxin itself but also to APs present in the airways. Polymyxin treatment upregulates the expression of the capsule polysaccharide operon and the loci required to modify the lipid A with aminoarabinose and palmitate with a concomitant increase in capsule and lipid A species containing such modifications. Moreover, these surface changes contribute to APs resistance and also to polymyxin-induced cross-resistance to APs. Bacterial loads of lipid A mutants in trachea and lungs of intranasally infected mice were lower than those of wild-type strain. PhoPQ, PmrAB, and the Rcs system govern polymyxin-induced transcriptional changes, and there is a cross talk between PhoPQ and the Rcs system. Our findings support the notion thatKlebsiellaactivates a defense program against APs that is controlled by three signaling systems. Therapeutic strategies directed to prevent the activation of this program could be a new approach worth exploring to facilitate the clearance of the pathogen from the airways.

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Adaptive and Mutational Responses to Peptide Dendrimer Antimicrobials in Pseudomonas aeruginosa

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02040-19 ◽

2020 ◽

Vol 64 (4) ◽

Cited By ~ 3

Author(s):

Fatma Ben Jeddou ◽

Léna Falconnet ◽

Alexandre Luscher ◽

Thissa Siriwardena ◽

Jean-Louis Reymond ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Lipid A ◽

Polymyxin B ◽

Multidrug Resistant ◽

Sensor Kinase ◽

Loss Of Function ◽

Adaptive Responses ◽

Kinase Gene ◽

Polyamine Biosynthesis ◽

Content Type

ABSTRACT Colistin (polymyxin E) is a last-resort antibiotic against multidrug-resistant isolates of Pseudomonas aeruginosa. However, the nephro-toxicity of colistin limits its use, spurring the interest in novel antimicrobial peptides (AMP). Here, we show that the synthetic AMP-dendrimer G3KL (MW 4,531.38 Da, 15 positive charges, MIC = 8 mg/liter) showed faster killing than polymyxin B (Pmx-B) with no detectable resistance selection in P. aeruginosa strain PA14. Spontaneous mutants selected on Pmx-B, harboring loss of function mutations in the PhoQ sensor kinase gene, showed increased Pmx-B MICs and arnB operon expression (4-amino-l-arabinose addition to lipid A), but remained susceptible to dendrimers. Two mutants carrying a missense mutation in the periplasmic loop of the PmrB sensor kinase showed increased MICs for Pmx-B (8-fold) and G3KL (4-fold) but not for the dendrimer T7 (MW 4,885.64 Da, 16 positive charges, MIC = 8 mg/liter). The pmrB mutants showed increased expression of the arnB operon as well as of the speD2-speE2-PA4775 operon, located upstream of pmrAB, and involved in polyamine biosynthesis. Exogenous supplementation with the polyamines spermine and norspermine increased G3KL and T7 MICs in a phoQ mutant background but not in the PA14 wild type. This suggests that both addition of 4-amino-l-arabinose and secretion of polyamines are required to reduce susceptibility to dendrimers, probably neutralizing the negative charges present on the lipid A and the 2-keto-3-deoxyoctulosonic acid (KDO) sugars of the lipopolysaccharide (LPS), respectively. We further show by transcriptome analysis that the dendrimers G3KL and T7 induce adaptive responses through the CprRS two-component system in PA14.

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RND-Type Efflux Pumps in Multidrug-Resistant Clinical Isolates of Acinetobacter baumannii: Major Role for AdeABC Overexpression and AdeRS Mutations

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02556-12 ◽

2013 ◽

Vol 57 (7) ◽

pp. 2989-2995 ◽

Cited By ~ 113

Author(s):

Eun-Jeong Yoon ◽

Patrice Courvalin ◽

Catherine Grillot-Courvalin

Keyword(s):

Acinetobacter Baumannii ◽

Efflux Pump ◽

Regulatory System ◽

Multidrug Resistant ◽

Clinical Isolates ◽

Clinical Settings ◽

Two Component System ◽

Content Type ◽

High Incidence ◽

Two Component

ABSTRACTIncreased expression of chromosomal genes for resistance-nodulation-cell division (RND)-type efflux systems plays a major role in the multidrug resistance (MDR) ofAcinetobacter baumannii. However, the relative contributions of the three most prevalent pumps, AdeABC, AdeFGH, and AdeIJK, have not been evaluated in clinical settings. We have screened 14 MDR clinical isolates shown to be distinct on the basis of multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) for the presence and overexpression of the three Ade efflux systems and analyzed the sequences of the regulators AdeRS, a two-component system, for AdeABC and AdeL, a LysR-type regulator, for AdeFGH. GeneadeBwas detected in 13 of 14 isolates, andadeGand the intrinsicadeJgene were detected in all strains. Significant overexpression ofadeBwas observed in 10 strains, whereas only 7 had moderately increased levels of expression of AdeFGH, and none overexpressed AdeIJK. Thirteen strains had reduced susceptibility to tigecycline, but there was no correlation between tigecycline MICs and the levels of AdeABC expression, suggesting the presence of other mechanisms for tigecycline resistance. No mutations were found in the highly conserved LysR regulator of the nine strains expressing AdeFGH. In contrast, functional mutations were found in conserved domains of AdeRS in all the strains that overexpressed AdeABC with two mutational hot spots, one in AdeS near histidine 149 suggesting convergent evolution and the other in the DNA binding domain of AdeR compatible with horizontal gene transfer. This report outlines the high incidence of AdeABC efflux pump overexpression in MDRA. baumanniias a result of a variety of single mutations in the corresponding two-component regulatory system.

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Draft Genome Assemblies of Clinical Isolates of Klebsiella pneumoniae V9011662 and Enterobacter hormaechei Entb306

Microbiology Resource Announcements ◽

10.1128/mra.00091-19 ◽

2019 ◽

Vol 8 (15) ◽

Author(s):

Lucas B. Harrison ◽

Anna Selmecki ◽

Nancy D. Hanson

Keyword(s):

Antibiotic Resistance ◽

Klebsiella Pneumoniae ◽

Clinical Isolate ◽

Draft Genome ◽

Multidrug Resistant ◽

Clinical Isolates ◽

Content Type ◽

Enterobacter Hormaechei ◽

Genome Assemblies

Enterobacter hormaechei and Klebsiella pneumoniae are pathogenic Enterobacteriaceae that have been associated with the spread of antibiotic resistance. Here, we report draft genome assemblies of an Enterobacter hormaechei clinical isolate and a multidrug-resistant clinical isolate of Klebsiella pneumoniae.

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Phosphoethanolamine Modification of Lipid A in Colistin-Resistant Variants of Acinetobacter baumannii Mediated by the pmrAB Two-Component Regulatory System

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00079-11 ◽

2011 ◽

Vol 55 (7) ◽

pp. 3370-3379 ◽

Cited By ~ 223

Author(s):

Alejandro Beceiro ◽

Enrique Llobet ◽

Jesús Aranda ◽

José Antonio Bengoechea ◽

Michel Doumith ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Clinical Isolate ◽

Lipid A ◽

Gene Knockout ◽

Regulatory System ◽

Clinical Isolates ◽

Strain Atcc ◽

Colistin Resistance ◽

Content Type ◽

Knockout Mutants

ABSTRACTColistin resistance is rare inAcinetobacter baumannii, and little is known about its mechanism. We investigated the role of PmrCAB in this trait, using (i) resistant and susceptible clinical strains, (ii) laboratory-selected mutants of the type strain ATCC 19606 and of the clinical isolate ABRIM, and (iii) a susceptible/resistant pair of isogenic clinical isolates, Ab15/133 and Ab15/132, isolated from the same patient.pmrABsequences in all the colistin-susceptible isolates were identical to reference sequences, whereas resistant clinical isolates harbored one or two amino acid replacements variously located in PmrB. Single substitutions in PmrB were also found in resistant mutants of strains ATCC 19606 and ABRIM and in the resistant clinical isolate Ab15/132. No mutations in PmrA or PmrC were found. Reverse transcriptase (RT)-PCR identified increased expression ofpmrA(4- to 13-fold),pmrB(2- to 7-fold), andpmrC(1- to 3-fold) in resistant versus susceptible organisms. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry showed the addition of phosphoethanolamine to the hepta-acylated form of lipid A in the resistant variants and in strain ATCC 19606 grown under low-Mg2+induction conditions.pmrBgene knockout mutants of the colistin-resistant ATCC 19606 derivative showed >100-fold increased susceptibility to colistin and 5-fold decreased expression ofpmrC; they also lacked the addition of phosphoethanolamine to lipid A. We conclude that the development of a moderate level of colistin resistance inA. baumanniirequires distinct genetic events, including (i) at least one point mutation inpmrB, (ii) upregulation ofpmrAB, and (iii) expression ofpmrC, which lead to addition of phosphoethanolamine to lipid A.

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Heteroresistance to Colistin in Klebsiella pneumoniae Associated with Alterations in the PhoPQ Regulatory System

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05055-14 ◽

2015 ◽

Vol 59 (5) ◽

pp. 2780-2784 ◽

Cited By ~ 76

Author(s):

Aurélie Jayol ◽

Patrice Nordmann ◽

Adrian Brink ◽

Laurent Poirel

Keyword(s):

Klebsiella Pneumoniae ◽

Regulatory System ◽

Multidrug Resistant ◽

Single Amino Acid ◽

Two Component System ◽

Partial Deletion ◽

Content Type ◽

Reading Frame ◽

Single Amino Acid Change ◽

Inactive Protein

ABSTRACTA multidrug-resistantKlebsiella pneumoniaeisolate exhibiting heteroresistance to colistin was investigated. The colistin-resistant subpopulation harbored a single amino acid change (Asp191Tyr) in protein PhoP, which is part of the PhoPQ two-component system that activatespmrHFIJKLMexpression responsible forl-aminoarabinose synthesis and polymyxin resistance. Complementation assays with a wild-typephoPgene restored full susceptibility to colistin. Then, analysis of the colistin-susceptible subpopulation showed a partial deletion (25 bp) in thephoPgene compared to that in the colistin-resistant subpopulation. That deletion disrupted the reading frame ofphoP, leading to a longer and inactive protein (255 versus 223 amino acids long). This is the first report showing the involvement of mutation(s) in PhoP in colistin resistance. Furthermore, this is the first study to decipher the mechanisms leading to colistin heteroresistance inK. pneumoniae.

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Susceptibility of Colistin-Resistant, Gram-Negative Bacteria to Antimicrobial Peptides and Ceragenins

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00292-17 ◽

2017 ◽

Vol 61 (8) ◽

Cited By ~ 22

Author(s):

Marjan M. Hashemi ◽

John Rovig ◽

Scott Weber ◽

Brian Hilton ◽

Mehdi M. Forouzan ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Antimicrobial Peptides ◽

Lipid A ◽

Resistance Mechanisms ◽

Clinical Isolates ◽

Cross Resistance ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Content Type

ABSTRACT The susceptibility of colistin-resistant clinical isolates of Klebsiella pneumoniae to ceragenins and antimicrobial peptides (AMPs) suggests that there is little to no cross-resistance between colistin and ceragenins/AMPs and that lipid A modifications are found in bacteria with modest changes in susceptibility to ceragenins and with high levels of resistance to colistin. These results suggest that there are differences in the resistance mechanisms to colistin and ceragenins/AMPs.

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Antibiofilm Peptides Increase the Susceptibility of Carbapenemase-Producing Klebsiella pneumoniae Clinical Isolates to β-Lactam Antibiotics

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00092-15 ◽

2015 ◽

Vol 59 (7) ◽

pp. 3906-3912 ◽

Cited By ~ 67

Author(s):

Suzana Meira Ribeiro ◽

César de la Fuente-Núñez ◽

Beverlie Baquir ◽

Célio Faria-Junior ◽

Octávio L. Franco ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Biofilm Formation ◽

Multidrug Resistant ◽

Flow Cell ◽

Clinical Isolates ◽

Antibiofilm Activity ◽

Planktonic Cells ◽

Content Type ◽

Promising Strategy ◽

Health Care Centers

ABSTRACTMultidrug-resistant carbapenemase-producingKlebsiella pneumoniae(KpC) strains are becoming a common cause of infections in health care centers. Furthermore,Klebsiellacan develop multicellular biofilms, which lead to elevated adaptive antibiotic resistance. Here, we describe the antimicrobial and antibiofilm activities of synthetic peptides DJK-5, DJK-6, and 1018 against five KpC isolates. Using static microplate assays, it was observed that the concentration required to prevent biofilm formation by these clinical isolates was below the MIC for planktonic cells. More-sophisticated flow cell experiments confirmed the antibiofilm activity of the peptides against 2-day-old biofilms of different KpC isolates, and in some cases, the peptides induced significant biofilm cell death. Clinically relevant combinations of DJK-6 and β-lactam antibiotics, including the carbapenem meropenem, also prevented planktonic growth and biofilm formation of KpC strain1825971. Interestingly, peptide DJK-6 was able to enhance, at least 16-fold, the ability of meropenem to eradicate preformed biofilms formed by this strain. Using peptide DJK-6 to potentiate the activity of β-lactams, including meropenem, represents a promising strategy to treat infections caused by KpC isolates.

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Evolution of Colistin Resistance in the Klebsiella pneumoniae Complex Follows Multiple Evolutionary Trajectories with Variable Effects on Fitness and Virulence Characteristics

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01958-20 ◽

2020 ◽

Vol 65 (1) ◽

pp. e01958-20

Author(s):

Axel B. Janssen ◽

Dennis J. Doorduijn ◽

Grant Mills ◽

Malbert R. C. Rogers ◽

Marc J. M. Bonten ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Lipid A ◽

Multidrug Resistant ◽

Sensu Stricto ◽

Colistin Resistance ◽

Content Type ◽

Evolutionary Trajectories ◽

Resistant Strains ◽

Phosphate Groups

ABSTRACTThe increasing prevalence of multidrug-resistant Klebsiella pneumoniae has led to a resurgence in the use of colistin as a last-resort drug. Colistin is a cationic antibiotic that selectively acts on Gram-negative bacteria through electrostatic interactions with anionic phosphate groups of the lipid A moiety of lipopolysaccharides (LPSs). Colistin resistance in K. pneumoniae is mediated through loss of these phosphate groups, their modification by cationic groups, and by the hydroxylation of acyl groups of lipid A. Here, we study the in vitro evolutionary trajectories toward colistin resistance in four clinical K. pneumoniae complex strains and their impact on fitness and virulence characteristics. Through population sequencing during in vitro evolution, we found that colistin resistance develops through a combination of single nucleotide polymorphisms, insertions and deletions, and the integration of insertion sequence elements, affecting genes associated with LPS biosynthesis and modification and capsule structures. Colistin resistance decreased the maximum growth rate of one K. pneumoniaesensu stricto strain, but not those of the other three K. pneumoniae complex strains. Colistin-resistant strains had lipid A modified through hydroxylation, palmitoylation, and l-Ara4N addition. K. pneumoniaesensu stricto strains exhibited cross-resistance to LL-37, in contrast to the Klebsiella variicola subsp. variicola strain. Virulence, as determined in a Caenorhabditis elegans survival assay, was increased in two colistin-resistant strains. Our study suggests that nosocomial K. pneumoniae complex strains can rapidly develop colistin resistance through diverse evolutionary trajectories upon exposure to colistin. This effectively shortens the life span of this last-resort antibiotic for the treatment of infections with multidrug-resistant Klebsiella.

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The MisR Response Regulator Is Necessary for Intrinsic Cationic Antimicrobial Peptide and Aminoglycoside Resistance in Neisseria gonorrhoeae

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00823-16 ◽

2016 ◽

Vol 60 (8) ◽

pp. 4690-4700 ◽

Cited By ~ 13

Author(s):

Justin L. Kandler ◽

Concerta L. Holley ◽

Jennifer L. Reimche ◽

Vijaya Dhulipala ◽

Jacqueline T. Balthazar ◽

...

Keyword(s):

Neisseria Gonorrhoeae ◽

Lipid A ◽

Efflux Pump ◽

Response Regulator ◽

Membrane Integrity ◽

Regulatory System ◽

Phagocytic Cells ◽

Aminoglycoside Resistance ◽

Cationic Antimicrobial Peptide ◽

Content Type

ABSTRACTDuring infection, the sexually transmitted pathogenNeisseria gonorrhoeae(the gonococcus) encounters numerous host-derived antimicrobials, including cationic antimicrobial peptides (CAMPs) produced by epithelial and phagocytic cells. CAMPs have both direct and indirect killing mechanisms and help link the innate and adaptive immune responses during infection. Gonococcal CAMP resistance is likely important for avoidance of host nonoxidative killing systems expressed by polymorphonuclear granulocytes (e.g., neutrophils) and intracellular survival. Previously studied gonococcal CAMP resistance mechanisms include modification of lipid A with phosphoethanolamine by LptA and export of CAMPs by the MtrCDE efflux pump. In the related pathogenNeisseria meningitidis, a two-component regulatory system (2CRS) termed MisR-MisS has been shown to contribute to the capacity of the meningococcus to resist CAMP killing. We report that the gonococcal MisR response regulator but not the MisS sensor kinase is involved in constitutive and inducible CAMP resistance and is also required for intrinsic low-level resistance to aminoglycosides. The 4- to 8-fold increased susceptibility ofmisR-deficient gonococci to CAMPs and aminoglycosides was independent of phosphoethanolamine decoration of lipid A and the levels of the MtrCDE efflux pump and seemed to correlate with a general increase in membrane permeability. Transcriptional profiling and biochemical studies confirmed that expression oflptAandmtrCDEwas not impacted by the loss of MisR. However, several genes encoding proteins involved in membrane integrity and redox control gave evidence of being MisR regulated. We propose that MisR modulates the levels of gonococcal susceptibility to antimicrobials by influencing the expression of genes involved in determining membrane integrity.

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