Mechanisms and phenotypic consequences of acquisition of tigecycline resistance by Stenotrophomonas maltophilia

2019 ◽  
Vol 74 (11) ◽  
pp. 3221-3230 ◽  
Author(s):  
Paula Blanco ◽  
Fernando Corona ◽  
José Luis Martinez
Keyword(s):  
Stenotrophomonas Maltophilia ◽  
Efflux Pump ◽  
Maximum Growth ◽  
Opportunistic Pathogen ◽  
Resistance Mechanisms ◽  
Fitness Cost ◽  
Negative Regulator ◽  
Cross Resistance ◽  
Bacterial Fitness ◽  
High Level

Abstract Objectives To elucidate the potential mutation-driven mechanisms involved in the acquisition of tigecycline resistance by the opportunistic pathogen Stenotrophomonas maltophilia. The mutational trajectories and their effects on bacterial fitness, as well as cross-resistance and/or collateral susceptibility to other antibiotics, were also addressed. Methods S. maltophilia populations were submitted to experimental evolution in the presence of increasing concentrations of tigecycline for 30 days. The genetic mechanisms involved in the acquisition of tigecycline resistance were determined by WGS. Resistance was evaluated by performing MIC assays. Fitness of the evolved populations and individual clones was assessed by measurement of the maximum growth rates. Results All the tigecycline-evolved populations attained high-level resistance to tigecycline following different mutational trajectories, yet with some common elements. Among the mechanisms involved in low susceptibility to tigecycline, mutations in the SmeDEF efflux pump negative regulator smeT, changes in proteins involved in the biogenesis of the ribosome and modifications in the LPS biosynthesis pathway seem to play a major role. Besides tigecycline resistance, the evolved populations presented cross-resistance to other antibiotics, such as aztreonam and quinolones, and they were hypersusceptible to fosfomycin, suggesting a possible combination treatment. Further, we found that the selected resistance mechanisms impose a relevant fitness cost when bacteria grow in the absence of antibiotic. Conclusions Mutational resistance to tigecycline was easily selected during exposure to this antibiotic. However, the fitness cost may compromise the maintenance of S. maltophilia tigecycline-resistant populations in the absence of antibiotic.

scholarly journals Antimicrobial Peptide Exposure Selects for Resistant and Fit Stenotrophomonas maltophilia Mutants That Show Cross-Resistance to Antibiotics

mSphere ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Paula Blanco ◽  
Karin Hjort ◽  
José L. Martínez ◽  
Dan I. Andersson
Keyword(s):  
Stenotrophomonas Maltophilia ◽  
Opportunistic Pathogen ◽  
Therapeutic Agents ◽  
Cross Resistance ◽  
Content Type ◽  
Essential Components ◽  
Wide Range ◽  
Genetic Mechanisms ◽  
Bacterial Fitness ◽  
High Level

ABSTRACT Antimicrobial peptides (AMPs) are essential components of the innate immune system and have been proposed as promising therapeutic agents against drug-resistant microbes. AMPs possess a rapid bactericidal mode of action and can interact with different targets, but bacteria can also avoid their effect through a variety of resistance mechanisms. Apart from hampering treatment by the AMP itself, or that by other antibiotics in the case of cross-resistance, AMP resistance might also confer cross-resistance to innate human peptides and impair the anti-infective capability of the human host. A better understanding of how resistance to AMPs is acquired and the genetic mechanisms involved is needed before using these compounds as therapeutic agents. Using experimental evolution and whole-genome sequencing, we determined the genetic causes and the effect of acquired de novo resistance to three different AMPs in the opportunistic pathogen Stenotrophomonas maltophilia, a bacterium that is intrinsically resistant to a wide range of antibiotics. Our results show that AMP exposure selects for high-level resistance, generally without any reduction in bacterial fitness, conferred by mutations in different genes encoding enzymes, transporters, transcriptional regulators, and other functions. Cross-resistance to AMPs and to other antibiotic classes not used for selection, as well as collateral sensitivity, was observed for many of the evolved populations. The relative ease by which high-level AMP resistance is acquired, combined with the occurrence of cross-resistance to conventional antibiotics and the maintained bacterial fitness of the analyzed mutants, highlights the need for careful studies of S. maltophilia resistance evolution to clinically valuable AMPs. IMPORTANCE Stenotrophomonas maltophilia is an increasingly relevant multidrug-resistant (MDR) bacterium found, for example, in people with cystic fibrosis and associated with other respiratory infections and underlying pathologies. The infections caused by this nosocomial pathogen are difficult to treat due to the intrinsic resistance of this bacterium against a broad number of antibiotics. Therefore, new treatment options are needed, and considering the growing interest in using AMPs as alternative therapeutic compounds and the restricted number of antibiotics active against S. maltophilia, we addressed the potential for development of AMP resistance, the genetic mechanisms involved, and the physiological effects that acquisition of AMP resistance has on this opportunistic pathogen.

Quinoline resistance mechanisms in Plasmodium falciparum: the debate goes on

Parasitology ◽  
1997 ◽  
Vol 114 (7) ◽  
pp. 125-136 ◽  
Author(s):  
S. A. WARD ◽  
P. G. BRAY ◽  
S. R. HAWLEY
Keyword(s):  
Efflux Pump ◽  
Resistance Mechanism ◽  
Resistance Mechanisms ◽  
Food Vacuole ◽  
Drug Binding ◽  
Drug Uptake ◽  
Cross Resistance ◽  
Parasite Resistance ◽  
Therapeutic Success ◽  
Drug Binding Site

Despite considerable therapeutic success with the antimalarial 4-aminoquinolines such as chloroquine, there is serious doubt about the future of this drug class due mainly to the development and spread of parasite resistance throughout endemic areas. In this article we review the possible biochemical and molecular basis of resistance. Based on our current understanding we have considered the possibility of developing strategies which may allow the aminoquinolines to once again be used effectively against P. falciparum. Our conclusions are that drug resistance is the result of a reduced rate of drug uptake which in turn reduces the amount of drug available to bind the target. The basis for this reduced accumulation could be an altered pH gradient making the food vacuole more alkaline or the parasite cytosol more acidic, an efflux pump removing drug directly from the membrane or any other process which will reduce the rate of drug uptake. Central to the effectiveness of this resistance mechanism is the transient availability of a high affinity, low capacity drug binding site (possibly haem) within the parasite. Resistance reversers such as verapamil influence the apparent Ka for this drug binding phenomenon via an increased drug uptake rate. We demonstrate that by chemical modification of the aminoquinolines, producing predictable alterations in their physicochemical properties, that it is possible to minimise the verapamil sensitive component of resistance and reduce significantly cross-resistance patterns without loss in absolute activity. Based on these views we suggest that the aminoquinoline antimalarials still have a role to play in the cheap, safe and effective chemotherapy of falciparum malaria.

scholarly journals Mutations that increase expression of the EmrAB-TolC efflux pump confer increased resistance to nitroxoline in Escherichia coli

2019 ◽  
Author(s):  
Fabiola Puértolas-Balint ◽  
Omar Warsi ◽  
Marius Linkevicius ◽  
Po-Cheng Tang ◽  
Dan I Andersson
Keyword(s):  
Escherichia Coli ◽  
Target Genes ◽  
Efflux Pump ◽  
Resistance Mechanisms ◽  
Cross Resistance ◽  
Resistance Mutations ◽  
Double Knockout ◽  
Low Level ◽  
Resistant Mutants ◽  
Level Resistance

Abstract Objectives To determine the mechanism of resistance to the antibiotic nitroxoline in Escherichia coli. Methods Spontaneous nitroxoline-resistant mutants were selected at different concentrations of nitroxoline. WGS and strain reconstruction were used to define the genetic basis for the resistance. The mechanistic basis of resistance was determined by quantitative PCR (qPCR) and by overexpression of target genes. Fitness costs of the resistance mutations and cross-resistance to other antibiotics were also determined. Results Mutations in the transcriptional repressor emrR conferred low-level resistance to nitroxoline [nitroxoline MIC (MICNOX) = 16 mg/L] by increasing the expression of the emrA and emrB genes of the EmrAB-TolC efflux pump. These resistant mutants showed no fitness reduction and displayed cross-resistance to nalidixic acid. Second-step mutants with higher-level resistance (MICNOX = 32–64 mg/L) had mutations in the emrR gene, together with either a 50 kb amplification, a mutation in the gene marA, or an IS upstream of the lon gene. The latter mutations resulted in higher-level nitroxoline resistance due to increased expression of the tolC gene, which was confirmed by overexpressing tolC from an inducible plasmid in a low-level resistance mutant. Furthermore, the emrR mutations conferred a small increase in resistance to nitrofurantoin only when combined with an nfsAB double-knockout mutation. However, nitrofurantoin-resistant nfsAB mutants showed no cross-resistance to nitroxoline. Conclusions Mutations in different genes causing increased expression of the EmrAB-TolC pump lead to an increased resistance to nitroxoline. The structurally similar antibiotics nitroxoline and nitrofurantoin appear to have different modes of action and resistance mechanisms.

scholarly journals Genetic and Molecular Characterization of β-Lactamase-Negative Ampicillin-Resistant Haemophilus influenzae with Unusually High Resistance to Ampicillin

2004 ◽  
Vol 48 (5) ◽  
pp. 1630-1639 ◽  
Author(s):  
Frank S. Kaczmarek ◽  
Thomas D. Gootz ◽  
Fadia Dib-Hajj ◽  
Wenchi Shang ◽  
Shawn Hallowell ◽  
...  
Keyword(s):  
North America ◽  
Haemophilus Influenzae ◽  
Efflux Pump ◽  
Geometric Mean ◽  
Resistance Mechanisms ◽  
Clinical Isolates ◽  
Ampicillin Resistance ◽  
Group I ◽  
Acrab Efflux Pump ◽  
High Level

ABSTRACT Previous studies with beta-lactamase-negative, ampicillin-resistant (BLNAR) Haemophilus influenzae from Japan, France, and North America indicate that mutations in ftsI encoding PBP3 confer ampicillin MICs of 1 to 4 μg/ml. Several BLNAR strains with ampicillin MICs of 4 to 16 μg/ml recently isolated from North America were studied. Pulsed-field gel electrophoresis identified 12 unique BLNAR strains; sequencing of their ftsI transpeptidase domains identified 1 group I and 11 group II mutants, as designated previously (K. Ubukata, Y. Shibasaki, K. Yamamoto, N. Chiba, K. Hasegawa, Y. Takeuchi, K. Sunakawa, M. Inoue, and M. Konno, Antimicrob. Agents Chemother. 45:1693-1699, 2001). Geometric mean ampicillin MICs for several clinical isolates were 8 to 10.56 μg/ml. Replacement of the ftsI gene in H. influenzae Rd with the intact ftsI from several clinical isolates resulted in integrants with typical BLNAR geometric mean ampicillin MICs of 1.7 to 2.2 μg/ml. Cloning and purification of His-tagged PBP3 from three clinical BLNAR strains showed significantly reduced Bocillin binding compared to that of PBP3 from strain Rd. Based on these data, changes in PBP3 alone could not account for the high ampicillin MICs observed for these BLNAR isolates. In an effort to determine the presence of additional mechanism(s) of ampicillin resistance, sequencing of the transpeptidase regions of pbp1a, -1b, and -2 was performed. While numerous changes were observed compared to the sequences from Rd, no consistent pattern correlating with high-level ampicillin resistance was apparent. Additional analysis of the resistant BLNAR strains revealed frame shift insertions in acrR for all four high-level, ampicillin-resistant isolates. acrR was intact for all eight low-level ampicillin-resistant and four ampicillin-susceptible strains tested. A knockout of acrB made in one clinical isolate (initial mean ampicillin MIC of 10.3 μg/ml) lowered the ampicillin MIC to 3.67 μg/ml, typical for BLNAR strains. These studies illustrate that BLNAR strains with high ampicillin MICs exist that have combined resistance mechanisms in PBP3 and in the AcrAB efflux pump.

scholarly journals In vitro dynamics and mechanisms of resistance development to imipenem and imipenem/relebactam in Pseudomonas aeruginosa

2020 ◽  
Vol 75 (9) ◽  
pp. 2508-2515 ◽  
Author(s):  
María A Gomis-Font ◽  
Gabriel Cabot ◽  
Irina Sánchez-Diener ◽  
Pablo A Fraile-Ribot ◽  
Carlos Juan ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Efflux Pump ◽  
Resistance Mechanisms ◽  
Infection Model ◽  
Mechanisms Of Resistance ◽  
Resistance Development ◽  
C Elegans ◽  
Imipenem Resistance ◽  
High Level

Abstract Objectives We analysed the dynamics and mechanisms of resistance development to imipenem alone or combined with relebactam in Pseudomonas aeruginosa WT (PAO1) and mutator (PAOMS; ΔmutS) strains. Methods PAO1 or PAOMS strains were incubated for 24 h in Mueller–Hinton Broth with 0.125–64 mg/L of imipenem ± relebactam 4 mg/L. Tubes from the highest antibiotic concentration showing growth were reinoculated in fresh medium containing concentrations up to 64 mg/L of imipenem ± relebactam for 7 days. Two colonies per strain, replicate experiment and antibiotic from early (Day 1) and late (Day 7) cultures were characterized by determining the susceptibility profiles, WGS and determination of the expression of ampC and efflux-pump-coding genes. Virulence was studied in a Caenorhabditis elegans infection model. Results Relebactam reduced imipenem resistance development for both strains, although resistance emerged much faster for PAOMS. WGS indicated that imipenem resistance was associated with mutations in the porin OprD and regulators of ampC, while the mutations in imipenem/relebactam-resistant mutants were located in oprD and regulatoras of MexAB-OprM. High-level imipenem/relebactam resistance was only documented in the PAOMS strain and was associated with an additional specific (T680A) mutation located in the catalytic pocket of ponA (PBP1a) and with reduced virulence in the C. elegans model. Conclusions Imipenem/relebactam could be a useful alternative for the treatment of MDR P. aeruginosa infections, potentially reducing resistance development during treatment. Moreover, this work deciphers the potential resistance mechanisms that may emerge upon the introduction of this novel combination into clinical practice.

scholarly journals Patterns of resistance to AHAS inhibitors in Limnocharis flava from Malaysia

2017 ◽  
Vol 54 (No. 1) ◽  
pp. 48-59
Author(s):  
Zakaria Norazua ◽  
Ahmad-Hamdani Muhammad Saiful ◽  
Juraimi Abdul Shukor
Keyword(s):  
Dose Response ◽  
Resistance Mechanisms ◽  
Rice Fields ◽  
Cross Resistance ◽  
Multiple Resistance ◽  
Low Level ◽  
Metsulfuron Methyl ◽  
Synthetic Auxin ◽  
Target Site Resistance ◽  
High Level

Limnocharis flava (L.) Buchenau is among the most problematic rice weeds in Malaysia and is also reported to have developed multiple resistance to AHAS inhibitor bensulfuron-methyl and synthetic auxin 2,4-D. In this study, resistance across different AHAS inhibitors was characterised in a L. flava population infesting rice fields in Pulau Pinang, Malaysia. Dose-response experiments were conducted to determine the level of resistance to sulfonylureas, imidazolinone, triazolopyrimidine, and pyrimidinyl-thiobenzoate. Cross-resistance across different AHAS inhibitors was observed in the resistant L. flava population, exhibiting a high level of resistance to bensulfuron-methyl, while exhibiting a moderate level of resistance to metsulfuron-methyl and a low level of resistance to pyrazosulfuron-ethyl and pyribenzoxim. However, all resistant L. flava individuals were still sensitive to imazethapyr, penoxsulam, and bispyribac-sodium. Based on the results, it is likely that resistance to AHAS inhibitors in L. flava is conferred by target-site resistance mechanisms.

scholarly journals High Levels of Intrinsic Tetracycline Resistance inMycobacterium abscessusAre Conferred by a Tetracycline-Modifying Monooxygenase

2018 ◽  
Vol 62 (6) ◽  
Author(s):  
Paulami Rudra ◽  
Kelley Hurst-Hess ◽  
Pascal Lappierre ◽  
Pallavi Ghosh
Keyword(s):  
Bacterial Infections ◽  
Efflux Pump ◽  
Tetracycline Resistance ◽  
Resistance Mechanisms ◽  
Mycobacterium Abscessus ◽  
Repeat Sequence ◽  
Content Type ◽  
High Level

ABSTRACTTetracyclines have been one of the most successful classes of antibiotics. However, its extensive use has led to the emergence of widespread drug resistance, resulting in discontinuation of use against several bacterial infections. Prominent resistance mechanisms include drug efflux and the use of ribosome protection proteins. Infrequently, tetracyclines can be inactivated by the TetX class of enzymes, also referred to as tetracycline destructases. Low levels of tolerance to tetracycline inMycobacterium smegmatisandMycobacterium tuberculosishave been previously attributed to the WhiB7-dependent TetV/Tap efflux pump. However,Mycobacterium abscessusis ∼500-fold more resistant to tetracycline thanM. smegmatisandM. tuberculosis. In this report, we show that this high level of resistance to tetracycline and doxycycline inM. abscessusis conferred by a WhiB7-independent tetracycline-inactivating monooxygenase, MabTetX (MAB_1496c). The presence of sublethal doses of tetracycline and doxycycline results in a >200-fold induction of MabTetX, and an isogenic deletion strain is highly sensitive to both antibiotics. Further, purified MabTetX can rapidly monooxygenate both antibiotics. We also demonstrate that expression of MabTetX is repressed by MabTetRx, by binding to an inverted repeat sequence upstream of MabTetRx; the presence of either antibiotic relieves this repression. Moreover, anhydrotetracycline (ATc) can effectively inhibit MabTetX activityin vitroand decreases the MICs of both tetracycline and doxycyclinein vivo. Finally, we show that tigecycline, a glycylcycline tetracycline, not only is a poor substrate of MabTetX but also is incapable of inducing the expression of MabTetX. This is therefore the first demonstration of a tetracycline-inactivating enzyme in mycobacteria. It (i) elucidates the mechanism of tetracycline resistance inM. abscessus, (ii) demonstrates the use of an inhibitor that can potentially reclaim the use of tetracycline and doxycycline, and (iii) identifies two sequential bottlenecks—MabTetX and MabTetRx—for acquiring resistance to tigecycline, thereby reiterating its use againstM. abscessus.

scholarly journals Expression of the qepA1 gene is induced under antibiotic exposure

2021 ◽  
Author(s):  
Gerrit Brandis ◽  
Jonas Gockel ◽  
Linnéa Garoff ◽  
Lionel Guy ◽  
Diarmaid Hughes
Keyword(s):  
Gene Expression ◽  
Efflux Pump ◽  
Class I ◽  
Upstream Region ◽  
Antibiotic Exposure ◽  
Negative Effects ◽  
Integrase Gene ◽  
Bacterial Fitness ◽  
High Level

Abstract Background The qepA1 gene encodes an efflux pump that reduces susceptibility to ciprofloxacin. Little is known about the regulation of qepA1 expression. Objectives To assess the potential role of ciprofloxacin and other antibiotics in the regulation of qepA1 gene expression. To identify the promoter that drives qepA1 expression and other factors involved in expression regulation. To assess whether the identified features are universal among qepA alleles. Methods A translational qepA1-yfp fusion under the control of the qepA1 upstream region was cloned into the Escherichia coli chromosome. Expression of the fusion protein was measured in the presence of various antibiotics. Deletions within the upstream region were introduced to identify regions involved in gene expression and regulation. The qepA1 coding sequence and upstream region were compared with all available qepA sequences. Results Cellular stress caused by the presence of various antibiotics can induce qepA1 expression. The qepA1 gene is fused to a class I integron and gene expression is driven by the Pc promoter within the integrase gene. A segment within the integron belonging to a truncated dfrB4 gene is essential for the regulation of qepA1 expression. This genetic context is universal among all sequenced qepA alleles. Conclusions The fusion of the qepA1 gene to a class I integron has created a novel regulatory unit that enables qepA1 expression to be under the control of antibiotic exposure. This setup mitigates potential negative effects of QepA1 production on bacterial fitness by restricting high-level expression to environmental conditions in which QepA1 is beneficial.

scholarly journals Antibiotic resistance, virulence-associated genes analysis and molecular typing of Klebsiella pneumoniae strains recovered from clinical samples

AMB Express ◽  
2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Amir Mirzaie ◽  
Reza Ranjbar
Keyword(s):  
Antibiotic Resistance ◽  
Klebsiella Pneumoniae ◽  
Molecular Typing ◽  
Efflux Pump ◽  
Opportunistic Pathogen ◽  
Relevant Information ◽  
Multidrug Resistant ◽  
Clinical Samples ◽  
Microbiological Methods ◽  
High Level

AbstractKlebsiella pneumoniae is a multidrug-resistant (MDR) opportunistic pathogen that causes nosocomial infections. Virulence analysis and molecular typing as powerful approaches can provide relevant information on K. pneumoniae infection. In the current study, antibiotic resistance, virulence-associated genes analysis, as well as molecular typing of K. pneumoniae strains were investigated. Out of 505 clinical samples collected from hospitalized patients, 100 K. pneumoniae strains were isolated by standard microbiological methods and subjected to the phenotypic and genotyping analysis. The highest prevalence of resistance was observed against ciprofloxacin (75%), trimethoprim–sulfamethoxazole (73%) and nitrofurantoin (68%). Virulence associated genes including entB, traT, ybts, magA, iucC, htrA and rmpA were found in 80%, 62%, 75%, 5%, 30%, 72% and 48%, of the isolates, respectively. The prevalence of biofilm-associated genes including mrkA, fimH, and mrkD were equally 88% for all tested isolates. Moreover, the efflux pump genes including AcrAB, TolC and mdtK were observed in 41 (41%), 33 (33%) and 26 (26%) of the strains respectively. A significant statistical association was observed between MDR strains and high expression of efflux pump and biofilm genes. The K. pneumoniae strains were differentiated into 11 different genetic patterns using the repetitive element sequence-based PCR (rep-PCR) technique. High prevalence of resistance, presence of various virulence factors, high level of efflux pump, and biofilm gene expression in diverse clones of K. pneumoniae strains pose an important health issue in clinical settings.

Overexpression of SmeGH contributes to the acquired MDR of Stenotrophomonas maltophilia

2019 ◽  
Vol 74 (8) ◽  
pp. 2225-2229 ◽  
Author(s):  
Li-Hua Li ◽  
Man-San Zhang ◽  
Chao-Jung Wu ◽  
Yi-Tsung Lin ◽  
Tsuey-Ching Yang
Keyword(s):  
Stenotrophomonas Maltophilia ◽  
Treatment Options ◽  
Acquired Resistance ◽  
Resistance Mechanisms ◽  
Fluoroquinolone Resistance ◽  
Dilution Method ◽  
Agar Dilution Method ◽  
Real Time Quantitative Pcr ◽  
Resistant Mutants ◽  
High Level

Abstract Background Stenotrophomonas maltophilia displays high-level resistance to various antibiotics. Fluoroquinolone is among the few treatment options for S. maltophilia infection. Overexpression of SmeDEF, SmeVWX and SmQnr are the main mechanisms responsible for fluoroquinolone resistance in S. maltophilia. Objectives To reveal the unidentified fluoroquinolone resistance mechanisms in S. maltophilia. Methods Fluoroquinolone-resistant spontaneous mutants were selected by spreading KJΔDEFΔ5, a SmeDEF- and SmeVWX-null double mutant, on ciprofloxacin- or levofloxacin-containing medium. Antibiotic susceptibility was assessed by the agar dilution method. Outer membrane protein profiles of fluoroquinolone-resistant mutants were assayed by SDS-PAGE and significant protein was characterized by LC-MS/MS. The expression of tolCsm, smeH, smeK, smeN, smeP, smeZ and smQnr was investigated by real-time quantitative PCR. The contribution of SmeGH overexpression to antibiotic resistance was verified by ΔsmeH mutant construction and smeGH complementation assay. Results Most fluoroquinolone-resistant mutants displayed MDR. The TolCsm protein and smeH transcript were concomitantly overexpressed in some MDR mutants. smeH deletion increased the susceptibility of the MDR mutants to fluoroquinolone, macrolide, chloramphenicol and tetracycline, and the resistance compromise was partially reversed by complementation with a plasmid containing smeGH. SmeGH overexpression was found in some fluoroquinolone-resistant clinical S. maltophilia isolates whose SmeDEF, SmeVWX and SmQnr proteins were not or were lowly expressed. Conclusions Overexpression of SmeGH contributes to the acquired resistance of S. maltophilia to fluoroquinolone, macrolide, chloramphenicol and tetracycline.

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