Analysis of AcrB in Klebsiella pneumoniae reveals natural variants promoting enhanced multidrug resistance

ABSTRACTTranscriptional regulators, such as SoxS, RamA, MarA, and Rob, which upregulate the AcrAB efflux pump, have been shown to be associated with multidrug resistance in clinically relevant Gram-negative bacteria. In addition to the multidrug resistance phenotype, these regulators have also been shown to play a role in the cellular metabolism and possibly the virulence potential of microbial cells. As such, the increased expression of these proteins is likely to cause pleiotropic phenotypes.Klebsiella pneumoniaeis a major nosocomial pathogen which can express the SoxS, MarA, Rob, and RamA proteins, and the accompanying paper shows that the increased transcription oframAis associated with tigecycline resistance (M. Veleba and T. Schneiders, Antimicrob. Agents Chemother. 56:4466–4467, 2012). Bioinformatic analyses of the availableKlebsiellagenome sequences show that an additional AraC-type regulator is encoded chromosomally. In this work, we characterize this novel AraC-type regulator, hereby called RarA (Regulator of antibiotic resistance A), which is encoded inK. pneumoniae,Enterobactersp. 638,Serratia proteamaculans568, andEnterobacter cloacae. We show that the overexpression ofrarAresults in a multidrug resistance phenotype which requires a functional AcrAB efflux pump but is independent of the other AraC regulators. Quantitative real-time PCR experiments show thatrarA(MGH 78578 KPN_02968) and its neighboring efflux pump operonoqxAB(KPN_02969_02970) are consistently upregulated in clinical isolates collected from various geographical locations (Chile, Turkey, and Germany). Our results suggest thatrarAoverexpression upregulates theoqxABefflux pump. Additionally, it appears thatoqxR, encoding a GntR-type regulator adjacent to theoqxABoperon, is able to downregulate the expression of theoqxABefflux pump, where OqxR complementation resulted in reductions to olaquindox MICs.

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Comparative Genomics of Klebsiella pneumoniae Strains with Different Antibiotic Resistance Profiles

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00052-11 ◽

2011 ◽

Vol 55 (9) ◽

pp. 4267-4276 ◽

Cited By ~ 48

Author(s):

Vinod Kumar ◽

Peng Sun ◽

Jessica Vamathevan ◽

Yong Li ◽

Karen Ingraham ◽

...

Keyword(s):

Antibiotic Resistance ◽

Multidrug Resistance ◽

Klebsiella Pneumoniae ◽

Resistance Genes ◽

Antibiotic Resistance Genes ◽

Susceptible Strain ◽

Clinical Isolates ◽

Whole Genome ◽

Content Type ◽

Extended Spectrum

ABSTRACTThere is a global emergence of multidrug-resistant (MDR) strains ofKlebsiella pneumoniae, a Gram-negative enteric bacterium that causes nosocomial and urinary tract infections. While the epidemiology ofK. pneumoniaestrains and occurrences of specific antibiotic resistance genes, such as plasmid-borne extended-spectrum β-lactamases (ESBLs), have been extensively studied, only four complete genomes ofK. pneumoniaeare available. To better understand the multidrug resistance factors inK. pneumoniae, we determined by pyrosequencing the nearly complete genome DNA sequences of two strains with disparate antibiotic resistance profiles, broadly drug-susceptible strain JH1 and strain 1162281, which is resistant to multiple clinically used antibiotics, including extended-spectrum β-lactams, fluoroquinolones, aminoglycosides, trimethoprim, and sulfamethoxazoles. Comparative genomic analysis of JH1, 1162281, and other publishedK. pneumoniaegenomes revealed a core set of 3,631 conserved orthologous proteins, which were used for reconstruction of whole-genome phylogenetic trees. The close evolutionary relationship between JH1 and 1162281 relative to otherK. pneumoniaestrains suggests that a large component of the genetic and phenotypic diversity of clinical isolates is due to horizontal gene transfer. Using curated lists of over 400 antibiotic resistance genes, we identified all of the elements that differentiated the antibiotic profile of MDR strain 1162281 from that of susceptible strain JH1, such as the presence of additional efflux pumps, ESBLs, and multiple mechanisms of fluoroquinolone resistance. Our study adds new and significant DNA sequence data onK. pneumoniaestrains and demonstrates the value of whole-genome sequencing in characterizing multidrug resistance in clinical isolates.

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Influence of Transcriptional Activator RamA on Expression of Multidrug Efflux Pump AcrAB and Tigecycline Susceptibility in Klebsiella pneumoniae

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.3.1017-1022.2005 ◽

2005 ◽

Vol 49 (3) ◽

pp. 1017-1022 ◽

Cited By ~ 137

Author(s):

Alexey Ruzin ◽

Melissa A. Visalli ◽

David Keeney ◽

Patricia A. Bradford

Keyword(s):

Multidrug Resistance ◽

Klebsiella Pneumoniae ◽

Antibacterial Agent ◽

Northern Blot Analysis ◽

Efflux Pump ◽

Multidrug Efflux ◽

Multidrug Efflux Pump ◽

Transposon Insertion ◽

Constitutive Overexpression ◽

Parental Phenotype

ABSTRACT Tigecycline is an expanded broad-spectrum antibacterial agent that is active against many clinically relevant species of bacterial pathogens, including Klebsiella pneumoniae. The majority of K. pneumoniae isolates are fully susceptible to tigecycline; however, a few strains that have decreased susceptibility have been isolated. One isolate, G340 (for which the tigecycline MIC is 4 μg/ml and which displays a multidrug resistance [MDR] phenotype), was selected for analysis of the mechanism for this decreased susceptibility by use of transposon mutagenesis with IS903φkan. A tigecycline-susceptible mutant of G340, GC7535, was obtained (tigecycline MIC, 0.25 μg/ml). Analysis of the transposon insertion mapped it to ramA, a gene that was previously identified to be involved in MDR in K. pneumoniae. For GC7535, the disruption of ramA led to a 16-fold decrease in the MIC of tigecycline and also a suppression of MDR. Trans-complementation with plasmid-borne ramA restored the original parental phenotype of decreased susceptibility to tigecycline. Northern blot analysis revealed a constitutive overexpression of ramA that correlated with an increased expression of the AcrAB transporter in G340 compared to that in tigecycline-susceptible strains. Laboratory mutants of K. pneumoniae with decreased susceptibility to tigecycline could be selected at a frequency of approximately 4 × 10−8. These results suggest that ramA is associated with decreased tigecycline susceptibility in K. pneumoniae due to its role in the expression of the AcrAB multidrug efflux pump.

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Membrane Efflux and Influx Modulate both Multidrug Resistance and Virulence of Klebsiella pneumoniae in a Caenorhabditis elegans Model

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01607-09 ◽

2010 ◽

Vol 54 (10) ◽

pp. 4373-4378 ◽

Cited By ~ 39

Author(s):

Suzanne Bialek ◽

Jean-Philippe Lavigne ◽

Jacqueline Chevalier ◽

Estelle Marcon ◽

Véronique Leflon-Guibout ◽

...

Keyword(s):

Antibiotic Resistance ◽

Multidrug Resistance ◽

Caenorhabditis Elegans ◽

Klebsiella Pneumoniae ◽

Resistance Mechanisms ◽

Cross Resistance ◽

Efflux System ◽

Virulence Potential ◽

Key Factor ◽

Potential Impact

ABSTRACT Cross-resistance to cefoxitin (FOX), chloramphenicol (CMP), and quinolones (nalidixic acid [NAL]) related to a putative efflux system overexpression has recently been reported for Klebsiella pneumoniae. The potential impact of this multidrug resistance (MDR) on the virulence of K. pneumoniae was evaluated in the Caenorhabditis elegans model. For 2 of the 3 MDR clinical isolates studied, a significant increase in acrB transcription was found in comparison with their antibiotic-susceptible revertants. ATCC 138821 and MDR, revertant, and derivative strains with altered porin expression were studied. Strains proved or suspected to overexpress an efflux system were significantly more virulent than the ATCC and revertant strains (time to kill 50% of nematodes [LT50] in days: 3.4 to 3.8 ± 0.2 versus 4.1 to 4.4 ± 0.3, P < 0.001). Inversely, strains with altered porin expression were significantly less virulent, independently of the expression level of efflux system (LT50 = 5.4 to 5.6 ± 0.2, P < 0.001). Altered porin expression did not change MICs of CMP and NAL but did those of FOX (4 to 16× MIC) and ertapenem (16 to 64× MIC). The strains with a normally or an overexpressed efflux system that received the β-lactamase CTX-M-15 became more widely resistant without modification of their virulence potential, suggesting that balance between resistance and virulence is dependent on the type of resistance mechanisms. In conclusion, this study shows that the expression of both efflux systems and porins is a key factor not only for antibiotic resistance but also virulence potential in K. pneumoniae.

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Tigecycline Resistance Can Occur Independently of theramAGene in Klebsiella pneumoniae

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.06224-11 ◽

2012 ◽

Vol 56 (8) ◽

pp. 4466-4467 ◽

Cited By ~ 29

Author(s):

Mark Veleba ◽

Thamarai Schneiders

Keyword(s):

Multidrug Resistance ◽

Klebsiella Pneumoniae ◽

Efflux Pump ◽

Content Type ◽

Regulatory Pathways ◽

Resistance Phenotype

ABSTRACTTigecycline resistance inKlebsiella pneumoniaeresults fromramAupregulation that causes the overexpression of the efflux pump, AcrAB-TolC. Tigecycline mutants, derived from Ecl8ΔramA, can exhibit a multidrug resistance phenotype due to increased transcription of themarA,rarA,acrAB, andoqxABgenes. These findings support the idea that tigecycline or multidrug resistance inK. pneumoniae, first, is not solely dependent on theramAgene, and second, can arise via alternative regulatory pathways inK. pneumoniae.

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Inhibitors of Antibiotic Efflux in Resistant Enterobacter aerogenes and Klebsiella pneumoniae Strains

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.3.1043-1046.2004 ◽

2004 ◽

Vol 48 (3) ◽

pp. 1043-1046 ◽

Cited By ~ 54

Author(s):

Jacqueline Chevalier ◽

Jérôme Bredin ◽

Abdallah Mahamoud ◽

Monique Malléa ◽

Jacques Barbe ◽

...

Keyword(s):

Multidrug Resistance ◽

Klebsiella Pneumoniae ◽

Efflux Pump ◽

Enterobacter Aerogenes ◽

Drug Susceptibility ◽

Multidrug Resistant ◽

Resistance Phenotype ◽

Pump Mechanism ◽

Energy Dependent ◽

Antibiotic Efflux

ABSTRACT In Enterobacter aerogenes and Klebsiella pneumoniae, efflux provides efficient extrusion of antibiotics and contributes to the multidrug resistance phenotype. One of the alkoxyquinoline derivatives studied here, 2,8-dimethyl-4-(2′-pyrrolidinoethyl)-oxyquinoline, restores noticeable drug susceptibility to resistant clinical strains. Analyses of energy-dependent chloramphenicol efflux indicate that this compound inhibits the efflux pump mechanism and improves the activity of structurally unrelated antibiotics on multidrug-resistant E. aerogenes and K. pneumoniae isolates.

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Natural variants modify Klebsiella pneumoniae carbapenemase (KPC) acyl-enzyme conformational dynamics to extend antibiotic resistance

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.016461 ◽

2020 ◽

pp. jbc.RA120.016461

Author(s):

Catherine L. Tooke ◽

Philip Hinchliffe ◽

Robert A. Bonomo ◽

Christopher J. Schofield ◽

Adrian J. Mulholland ◽

...

Keyword(s):

Antibiotic Resistance ◽

Klebsiella Pneumoniae ◽

Active Site ◽

Conformational Dynamics ◽

Point Mutations ◽

Klebsiella Pneumoniae Carbapenemase ◽

Natural Variants ◽

Activity Spectrum ◽

Dynamics Simulations ◽

Enzyme Intermediate

Class A serine β-lactamases (SBLs) are key antibiotic resistance determinants in Gram-negative bacteria. SBLs neutralize β-lactams via a hydrolytically labile covalent acyl-enzyme intermediate. Klebsiella pneumoniae carbapenemase (KPC) is a widespread SBL that hydrolyzes carbapenems, the most potent β-lactams; known KPC variants differ in turnover of expanded-spectrum oxyimino-cephalosporins (ESOCs), e.g. cefotaxime and ceftazidime. Here, we compare ESOC hydrolysis by the parent enzyme KPC-2 and its clinically observed double variant (P104R/V240G) KPC-4. Kinetic analyses show KPC-2 hydrolyzes cefotaxime more efficiently than the bulkier ceftazidime, with improved ESOC turnover by KPC-4 resulting from enhanced turnover (kcat), rather than binding (KM). High-resolution crystal structures of ESOC acyl-enzyme complexes with deacylation-deficient (E166Q) KPC-2 and KPC-4 mutants show that ceftazidime acylation causes rearrangement of three loops; the Ω-, 240- and 270-loops, that border the active site. However, these rearrangements are less pronounced in the KPC-4 than the KPC-2 ceftazidime acyl-enzyme, and are not observed in the KPC-2:cefotaxime acyl-enzyme. Molecular dynamics simulations of KPC:ceftazidime acyl-enyzmes reveal that the deacylation general base E166, located on the Ω-loop, adopts two distinct conformations in KPC-2, either pointing ‘in’ or ‘out’ of the active site; with only the ‘in’ form compatible with deacylation. The ‘out’ conformation was not sampled in the KPC-4 acyl-enzyme, indicating that efficient ESOC breakdown is dependent upon the ordering and conformation of the KPC Ω-loop. The results explain how point mutations expand the activity spectrum of the clinically important KPC SBLs to include ESOCs through their effects on the conformational dynamics of the acyl-enzyme intermediate.

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