scholarly journals Alterations in Two-Component Regulatory Systems of phoPQ and pmrAB Are Associated with Polymyxin B Resistance in Clinical Isolates of Pseudomonas aeruginosa

2009 ◽  
Vol 53 (12) ◽  
pp. 5150-5154 ◽  
Author(s):  
Kaddy Barrow ◽  
Dong H. Kwon
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Polymyxin B ◽  
Clinical Isolates ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Strain Pao1 ◽  
Truncated Protein ◽  
Regulatory Systems ◽  
Two Component

ABSTRACT Polymyxins are often the only option to treat acquired multidrug-resistant Pseudomonas aeruginosa. Polymyxin susceptibility in P. aeruginosa PAO1 is associated with the lipopolysaccharide structure that is determined by arnBCADTEF and modulated by phoPQ and pmrAB. We examined five clonally unrelated clinical isolates of polymyxin B-resistant P. aeruginosa to investigate the molecular basis of polymyxin resistance. All isolates grew with 4 μg/ml polymyxin B (MIC, 8 μg/ml), whereas P. aeruginosa PAO1 grew with 0.25 μg/ml polymyxin B (MIC, 0.5 μg/ml). The resistant isolates were converted to susceptible ones (the MICs fell from 8 to 0.5 μg/ml) following the introduction of phoPQ (four isolates) and pmrAB (one isolate), which had been cloned from strain PAO1. DNA sequence analysis revealed that a single-nucleotide substitution in three isolates replaced a single amino acid of PhoQ, the deletion of 17 nucleotides in one isolate truncated the protein of PhoQ, and two nucleotide substitutions in one isolate replaced two amino acids of PmrB. The involvement of these amino acid substitutions or the truncated protein of PhoQ and PmrB in polymyxin B resistance was confirmed using strain PAO1 lacking phoPQ or pmrAB that was transformed by phoPQ or pmrAB containing the amino acid substitutions or the truncated protein. The resistant clinical isolates were sensitized by the inactivation of arnBCADTEF (the MICs fell from 8 to 0.5 μg/ml). These results suggest that polymyxin B resistance among clinical isolates of P. aeruginosa is associated with alterations in two-component regulatory systems of phoPQ or pmrAB.

scholarly journals Gain-of-Function Mutations inUPC2Are a Frequent Cause ofERG11Upregulation in Azole-Resistant Clinical Isolates of Candida albicans

2012 ◽  
Vol 11 (10) ◽  
pp. 1289-1299 ◽  
Author(s):  
Stephanie A. Flowers ◽  
Katherine S. Barker ◽  
Elizabeth L. Berkow ◽  
Geoffrey Toner ◽  
Sean G. Chadwick ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Amino Acid ◽  
Clinical Isolates ◽  
Transcriptional Analysis ◽  
Single Amino Acid ◽  
Ergosterol Biosynthesis ◽  
Amino Acid Substitutions ◽  
Gain Of Function ◽  
Content Type ◽  
Zinc Cluster

ABSTRACTInCandida albicans, Upc2 is a zinc-cluster transcription factor that targets genes, including those of the ergosterol biosynthesis pathway. To date, three documentedUPC2gain-of-function (GOF) mutations have been recovered from fluconazole-resistant clinical isolates that contribute to an increase inERG11expression and decreased fluconazole susceptibility. In a group of 63 isolates with reduced susceptibility to fluconazole, we found that 47 overexpressedERG11by at least 2-fold over the average expression levels in 3 unrelated fluconazole-susceptible strains. Of those 47 isolates, 29 contained a mutation inUPC2, whereas the remaining 18 isolates did not. Among the isolates containing mutations inUPC2, we recovered eight distinct mutations resulting in putative single amino acid substitutions: G648D, G648S, A643T, A643V, Y642F, G304R, A646V, and W478C. Seven of these resulted in increasedERG11expression, increased cellular ergosterol, and decreased susceptibility to fluconazole compared to the results for the wild-type strain. Genome-wide transcriptional analysis was performed for the four strongest Upc2 amino acid substitutions (A643V, G648D, G648S, and Y642F). Genes commonly upregulated by all four mutations included those involved in ergosterol biosynthesis, in oxidoreductase activity, the major facilitator efflux pump encoded by theMDR1gene, and the uncharacterized ATP binding cassette transporterCDR11. These findings demonstrate that gain-of-function mutations inUPC2are more prevalent among clinical isolates than previously thought and make a significant contribution to azole antifungal resistance, but the findings do not account forERG11overexpression in all such isolates ofC. albicans.

scholarly journals Exploring the Role of the Ω-Loop in the Evolution of Ceftazidime Resistance in the PenA β-Lactamase from Burkholderia multivorans, an Important Cystic Fibrosis Pathogen

2016 ◽  
Vol 61 (2) ◽  
Author(s):  
Krisztina M. Papp-Wallace ◽  
Scott A. Becka ◽  
Magdalena A. Taracila ◽  
Elise T. Zeiser ◽  
Julian A. Gatta ◽  
...  
Keyword(s):  
Amino Acid ◽  
Burkholderia Cepacia ◽  
Clinical Isolates ◽  
Dynamics Simulation ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Biochemical Basis ◽  
Content Type ◽  
Burkholderia Multivorans

ABSTRACT The unwelcome evolution of resistance to the advanced generation cephalosporin antibiotic, ceftazidime is hindering the effective therapy of Burkholderia cepacia complex (BCC) infections. Regrettably, BCC organisms are highly resistant to most antibiotics, including polymyxins; ceftazidime and trimethoprim-sulfamethoxazole are the most effective treatment options. Unfortunately, resistance to ceftazidime is increasing and posing a health threat to populations susceptible to BCC infection. We found that up to 36% of 146 tested BCC clinical isolates were nonsusceptible to ceftazidime (MICs ≥ 8 μg/ml). To date, the biochemical basis for ceftazidime resistance in BCC is largely undefined. In this study, we investigated the role of the Ω-loop in mediating ceftazidime resistance in the PenA β-lactamase from Burkholderia multivorans, a species within the BCC. Single amino acid substitutions were engineered at selected positions (R164, T167, L169, and D179) in the PenA β-lactamase. Cell-based susceptibility testing revealed that 21 of 75 PenA variants engineered in this study were resistant to ceftazidime, with MICs of >8 μg/ml. Under steady-state conditions, each of the selected variants (R164S, T167G, L169A, and D179N) demonstrated a substrate preference for ceftazidime compared to wild-type PenA (32- to 320-fold difference). Notably, the L169A variant hydrolyzed ceftazidime significantly faster than PenA and possessed an ∼65-fold-lower apparent Ki (Ki app) than that of PenA. To understand why these amino acid substitutions result in enhanced ceftazidime binding and/or turnover, we employed molecular dynamics simulation (MDS). The MDS suggested that the L169A variant starts with the most energetically favorable conformation (−28.1 kcal/mol), whereas PenA possessed the most unfavorable initial conformation (136.07 kcal/mol). In addition, we observed that the spatial arrangement of E166, N170, and the hydrolytic water molecules may be critical for enhanced ceftazidime hydrolysis by the L169A variant. Importantly, we found that two clinical isolates of B. multivorans possessed L169 amino acid substitutions (L169F and L169P) in PenA and were highly resistant to ceftazidime (MICs ≥ 512 μg/ml). In conclusion, substitutions in the Ω-loop alter the positioning of the hydrolytic machinery as well as allow for a larger opening of the active site to accommodate the bulky R1 and R2 side chains of ceftazidime, resulting in resistance. This analysis provides insights into the emerging phenotype of ceftazidime-resistant BCC and explains the evolution of amino acid substitutions in the Ω-loop of PenA of this significant clinical pathogen.

scholarly journals C-Terminal Region of Pseudomonas aeruginosa Outer Membrane Porin OprD Modulates Susceptibility to Meropenem

2001 ◽  
Vol 45 (6) ◽  
pp. 1780-1787 ◽  
Author(s):  
Simone F. Epp ◽  
Thilo Köhler ◽  
Patrick Plésiat ◽  
Mehri Michéa-Hamzehpour ◽  
Joachim Frey ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Basic Amino Acid ◽  
Clinical Isolates ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Strain Pao1 ◽  
External Loop ◽  
Outer Membrane Porin ◽  
Major Alteration

ABSTRACT We investigated the unusual susceptibility to meropenem observed for seven imipenem-resistant clinical isolates of Pseudomonas aeruginosa. These strains were genetically closely related, expressed OprD, as determined by Western blot analyses, and were resistant to imipenem (>5 μg/ml) but susceptible to meropenem (<1 μg/ml). The oprD genes from two isolates were entirely sequenced, and their deduced protein sequences showed 93% identity with that of OprD of strain PAO1. The major alteration consisted of the replacement of a stretch of 12 amino acids, located in putative external loop L7 of OprD, by a divergent sequence of 10 amino acid residues. The oprD gene variants and the wild-typeoprD gene were cloned and expressed in a definedoprD mutant. The meropenem MICs for strains carrying theoprD genes from clinical isolates were four times lower than that for the strain carrying the wild-type oprDgene. Imipenem activities, however, were comparable for all strains. Furthermore, meropenem hypersusceptibility was obtained with a hybrid OprD porin that consisted of the PAO1 oprD gene containing loop L7 from a clinical isolate. These results show that the C-terminal portion of OprD, in particular, loop L7, was responsible for the unusual meropenem hypersusceptibility. Competition experiments suggested that the observed OprD modifications in the clinical isolates did not affect antagonism between imipenem and the basic amino acidl-lysine. We further propose that shortening of putative loop L7 of the OprD porin by 2 amino acid residues sufficiently opens the porin channel to allow optimal penetration of meropenem and increase its activity. In contrast, this alteration would not affect susceptibility to a smaller carbapenem molecule, such as imipenem.

scholarly journals Contribution of Clinically Derived Mutations inERG11to Azole Resistance in Candida albicans

2014 ◽  
Vol 59 (1) ◽  
pp. 450-460 ◽  
Author(s):  
Stephanie A. Flowers ◽  
Brendan Colón ◽  
Sarah G. Whaley ◽  
Mary A. Schuler ◽  
P. David Rogers
Keyword(s):  
Candida Albicans ◽  
Amino Acid ◽  
Amino Acid Substitution ◽  
Single Amino Acid Substitution ◽  
Clinical Isolates ◽  
Single Amino Acid ◽  
Azole Resistance ◽  
Amino Acid Substitutions ◽  
Content Type ◽  
Proximal Surface

ABSTRACTInCandida albicans, theERG11gene encodes lanosterol demethylase, the target of the azole antifungals. Mutations inERG11that result in an amino acid substitution alter the abilities of the azoles to bind to and inhibit Erg11, resulting in resistance. AlthoughERG11mutations have been observed in clinical isolates, the specific contributions of individualERG11mutations to azole resistance inC. albicanshave not been widely explored. We sequencedERG11in 63 fluconazole (FLC)-resistant clinical isolates. Fifty-five isolates carried at least one mutation inERG11, and we observed 26 distinct positions in which amino acid substitutions occurred. We mapped the 26 distinct variant positions in these alleles to four regions in the predicted structure for Erg11, including its predicted catalytic site, extended fungus-specific external loop, proximal surface, and proximal surface-to-heme region. In total, 31 distinctERG11alleles were recovered, with 10ERG11alleles containing a single amino acid substitution. We then characterized 19 distinctERG11alleles by introducing them into the wild-type azole-susceptibleC. albicansSC5314 strain and testing them for susceptibilities to FLC, itraconazole (ITC), and voriconazole (VRC). The strains that were homozygous for the single amino acid substitutions Y132F, K143R, F145L, S405F, D446E, G448E, F449V, G450E, and G464S had a ≥4-fold increase in FLC MIC. The strains that were homozygous for several double amino acid substitutions had decreased azole susceptibilities beyond those conferred by any single amino acid substitution. These findings indicate that mutations inERG11are prevalent among azole-resistant clinical isolates and that most mutations result in appreciable changes in FLC and VRC susceptibilities.

scholarly journals Contribution of the PhoP-PhoQ and PmrA-PmrB Two-Component Regulatory Systems to Mg2+-Induced Gene Regulation in Pseudomonas aeruginosa

2006 ◽  
Vol 188 (11) ◽  
pp. 3995-4006 ◽  
Author(s):  
Joseph B. McPhee ◽  
Manjeet Bains ◽  
Geoff Winsor ◽  
Shawn Lewenza ◽  
Agnieszka Kwasnicka ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Transcriptional Profiling ◽  
Polymyxin B ◽  
Dependent Manner ◽  
Binding Assays ◽  
Response Regulators ◽  
Binding Motifs ◽  
Regulatory Systems ◽  
Two Component ◽  
Genes Encoding

ABSTRACT When grown in divalent cation-limited medium, Pseudomonas aeruginosa becomes resistant to cationic antimicrobial peptides and polymyxin B. This resistance is regulated by the PhoP-PhoQ and PmrA-PmrB two-component regulatory systems. To further characterize Mg2+ regulation in P. aeruginosa, microarray transcriptional profiling was conducted to compare wild-type P. aeruginosa grown under Mg2+-limited and Mg2+-replete conditions to isogenic phoP and pmrA mutants grown under Mg2+-limited conditions. Under Mg2+-limited conditions (0.02 mM Mg2+), approximately 3% of the P. aeruginosa genes were differentially expressed compared to the expression in bacteria grown under Mg2+-replete conditions (2 mM Mg2+). Only a modest subset of the Mg2+-regulated genes were regulated through either PhoP or PmrA. To determine which genes were directly regulated, a bioinformatic search for conserved binding motifs was combined with confirmatory reverse transcriptase PCR and gel shift promoter binding assays, and the results indicated that very few genes were directly regulated by these response regulators. It was found that in addition to the previously known oprH-phoP-phoQ operon and the pmrHFIJKLM-ugd operon, the PA0921 and PA1343 genes, encoding small basic proteins, were regulated by Mg2+ in a PhoP-dependent manner. The number of known PmrA-regulated genes was expanded to include the PA1559-PA1560, PA4782-PA4781, and feoAB operons, in addition to the previously known PA4773-PA4775-pmrAB and pmrHFIJKLM-ugd operons.

scholarly journals A Novel VIM-Type Metallo-β-Lactamase Variant, VIM-60, with Increased Hydrolyzing Activity against Fourth-Generation Cephalosporins in Pseudomonas aeruginosa Clinical Isolates in Japan

2019 ◽  
Vol 63 (6) ◽  
Author(s):  
Tomomi Hishinuma ◽  
Tatsuya Tada ◽  
Hiroki Uchida ◽  
Masahiro Shimojima ◽  
Teruo Kirikae
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Multidrug Resistant ◽  
Clinical Isolates ◽  
Fourth Generation ◽  
Amino Acid Substitutions ◽  
Content Type ◽  
Catalytic Activities ◽  
Genetic Context

ABSTRACT A novel VIM-type metallo-β-lactamase variant, VIM-60, was identified in multidrug-resistant Pseudomonas aeruginosa clinical isolates in Japan. Compared with VIM-2, VIM-60 had two amino acid substitutions (Arg228Leu and His252Arg) and higher catalytic activities against fourth-generation cephalosporins. The genetic context for blaVIM-60 was intI1-blaVIM-60-aadA1-aacA31-qacEdeltaI-sulI on the chromosome.

scholarly journals Terbinafine Resistance of Trichophyton Clinical Isolates Caused by Specific Point Mutations in the Squalene Epoxidase Gene

2017 ◽  
Vol 61 (7) ◽  
Author(s):  
Tsuyoshi Yamada ◽  
Mari Maeda ◽  
Mohamed Mahdi Alshahni ◽  
Reiko Tanaka ◽  
Takashi Yaguchi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Single Point ◽  
Point Mutations ◽  
Trichophyton Mentagrophytes ◽  
Antifungal Drugs ◽  
Clinical Isolates ◽  
Single Amino Acid ◽  
Squalene Epoxidase ◽  
Amino Acid Substitutions ◽  
Content Type

ABSTRACT Terbinafine is one of the allylamine antifungal agents whose target is squalene epoxidase (SQLE). This agent has been extensively used in the therapy of dermatophyte infections. The incidence of patients with tinea pedis or unguium tolerant to terbinafine treatment prompted us to screen the terbinafine resistance of all Trichophyton clinical isolates from the laboratory of the Centre Hospitalier Universitaire Vaudois collected over a 3-year period and to identify their mechanism of resistance. Among 2,056 tested isolates, 17 (≈1%) showed reduced terbinafine susceptibility, and all of these were found to harbor SQLE gene alleles with different single point mutations, leading to single amino acid substitutions at one of four positions (Leu393, Phe397, Phe415, and His440) of the SQLE protein. Point mutations leading to the corresponding amino acid substitutions were introduced into the endogenous SQLE gene of a terbinafine-sensitive Arthroderma vanbreuseghemii (formerly Trichophyton mentagrophytes) strain. All of the generated A. vanbreuseghemii transformants expressing mutated SQLE proteins exhibited obvious terbinafine-resistant phenotypes compared to the phenotypes of the parent strain and of transformants expressing wild-type SQLE proteins. Nearly identical phenotypes were also observed in A. vanbreuseghemii transformants expressing mutant forms of Trichophyton rubrum SQLE proteins. Considering that the genome size of dermatophytes is about 22 Mb, the frequency of terbinafine-resistant clinical isolates was strikingly high. Increased exposure to antifungal drugs could favor the generation of resistant strains.

scholarly journals Mutations in the gyrA and parC genes in fluoroquinolone-resistant clinical isolates of Pseudomonas aeruginosa.

1997 ◽  
Vol 41 (10) ◽  
pp. 2289-2291 ◽  
Author(s):  
M Nakano ◽  
T Deguchi ◽  
T Kawamura ◽  
M Yasuda ◽  
M Kimura ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Amino Acid Change ◽  
Susceptible Strain ◽  
Clinical Isolates ◽  
Single Amino Acid ◽  
Strain Atcc ◽  
Single Amino Acid Change ◽  
Double Amino Acid

We determined partial sequences of the gyrA and parC genes of the fluoroquinolone-susceptible strain ATCC 27853 and 22 clinical isolates of Pseudomonas aeruginosa. While a single amino acid change in GyrA with or without a change in ParC was found in 14 isolates with decreased susceptibility to fluoroquinolones, 3 higher-level fluoroquinolone-resistant isolates had a double amino acid change in GyrA and a single amino acid change in ParC.

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