scholarly journals Efflux Pump-Mediated Quinolone Resistance inStaphylococcus aureus Strains Wild Type for gyrA,gyrB, grlA, and norA

1999 ◽  
Vol 43 (2) ◽  
pp. 354-356 ◽  
Author(s):  
Juan Luis Muñoz-Bellido ◽  
M. A. Alonso Manzanares ◽  
J. A. Martínez Andrés ◽  
M. N. Gutiérrez Zufiaurre ◽  
G. Ortiz ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Promoter Region ◽  
Efflux Pump ◽  
Quinolone Resistance ◽  
Wild Type ◽  
Clinical Strains

ABSTRACT Fluoroquinolone efflux was studied in 47 Staphylococcus aureus clinical strains with MICs of ciprofloxacin (CFX) of ≤2 μg/ml. Forty-three strains were wild type for gyrA,gyrB, and grlA quinolone resistance-determining regions and for norA and its promoter region. Forty of these strains (MICs of CFX, 0.1 to 0.2 μg/ml) did not show efflux of fluoroquinolones. Three strains (MICs of CFX, 1 to 2 μg/ml) showed efflux. These results suggest that efflux can appear in S. aureus clinical strains in the absence of mutations innorA and its promoter.

scholarly journals Type II Topoisomerase Mutations in Ciprofloxacin-Resistant Strains of Pseudomonas aeruginosa

1999 ◽  
Vol 43 (1) ◽  
pp. 62-66 ◽  
Author(s):  
Hyam Mouneimné ◽  
Jérome Robert ◽  
Vincent Jarlier ◽  
Emmanuelle Cambau
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Novel Mutation ◽  
Geometric Mean ◽  
Quinolone Resistance ◽  
Single Mutation ◽  
Type Ii ◽  
Wild Type ◽  
Clinical Strains ◽  
Major Mechanism ◽  
Resistant Strains

ABSTRACT We determined the sequences of the quinolone resistance-determining regions of gyrA, gyrB, and parCgenes for 30 clinical strains of Pseudomonas aeruginosaresistant to ciprofloxacin that were previously complemented by wild-type gyrA and gyrB plasmid-borne alleles and studied for their coresistance to imipenem (E. Cambau, E. Perani, C. Dib, C. Petinon, J. Trias, and V. Jarlier, Antimicrob. Agents Chemother. 39:2248–2252, 1995). In the present study, we found mutations in type II topoisomerase genes for all strains. Twenty-eight strains had a missense mutation in gyrA (codon 83 or 87). Ten of them had an additional mutation in parC(codon 80 or 84), including a novel mutation of Ser-80 to Trp, but all were fully complemented by a plasmid-borne wild-type gyrAallele. The remaining two strains harbored the first gyrBmutation described in P. aeruginosa, leading to the substitution of phenylalanine for serine 464. The strains which had two mutations in type II topoisomerase genes (i.e.,gyrA and parC) were significantly more resistant to fluoroquinolones than those with a single mutation ingyrA or gyrB (geometric mean MICs of ciprofloxacin, 39.4 versus 10.9 μg/ml, P < 0.01; geometric mean MICs of sparfloxacin, 64.0 versus 22.6,P < 0.01). No mutant with a parC mutation alone was observed, which favors DNA gyrase being the primary target for fluoroquinolones. These results demonstrate that gyrAmutations are the major mechanism of resistance to fluoroquinolones for clinical strains of P. aeruginosa and that additional mutations in parC lead to a higher level of quinolone resistance.

scholarly journals Alterations in the DNA topoisomerase IV grlA gene responsible for quinolone resistance in Staphylococcus aureus.

1996 ◽  
Vol 40 (5) ◽  
pp. 1157-1163 ◽  
Author(s):  
J Yamagishi ◽  
T Kojima ◽  
Y Oyamada ◽  
K Fujimoto ◽  
H Hattori ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Gene Dosage ◽  
Dna Gyrase ◽  
Quinolone Resistance ◽  
Wild Type Allele ◽  
Wild Type ◽  
Topoisomerase Iv ◽  
Dna Topoisomerase ◽  
Transformation Experiment ◽  
Dna Fragment

A 4.2-kb DNA fragment conferring quinolone resistance was cloned from a quinolone-resistant clinical isolate of Staphylococcus aureus and was shown to possess a part of the grlB gene and a mutated grlA gene. S-80-->F and E-84-->K mutations in the grlA gene product were responsible for the quinolone resistance. The mutated grlA genes responsible for quinolone resistance were dominant over the wild-type allele, irrespective of gene dosage in a transformation experiment with the grlA gene alone. However, dominance by mutated grlA genes depended on gene dosage when bacteria were transformed with the grlA and grlB genes in combination. Quinolone-resistant gyrA mutants were easily isolated from a strain, S. aureus RN4220, carrying a plasmid with the mutated grlA gene, though this was not the case for other S. aureus strains lacking the plasmid. The elimination of this plasmid from such quinolone-resistant gyrA mutants resulted in marked increases in quinolone susceptibility. These results suggest that both DNA gyrase and DNA topoisomerase IV may be targets of quinolones and that the quinolone susceptibility of organisms may be determined by which of these enzymes is most quinolone sensitive.

scholarly journals Characterization of NorR Protein, a Multifunctional Regulator of norA Expression in Staphylococcus aureus

2003 ◽  
Vol 185 (10) ◽  
pp. 3127-3138 ◽  
Author(s):  
Que Chi Truong-Bolduc ◽  
Xiamei Zhang ◽  
David C. Hooper
Keyword(s):  
Gene Expression ◽  
Staphylococcus Aureus ◽  
Efflux Pump ◽  
Protein A ◽  
Protein Overexpression ◽  
Wild Type ◽  
Liquid Media ◽  
Cell Surface Properties ◽  
Regulatory Systems

ABSTRACT We characterized a Staphylococcus aureus norA gene expression regulator, NorR, initially identified from its binding to the norA promoter. The norR gene was 444 bp in length, located ∼7 kb upstream from the norA gene, and encoded a predicted 17.6-kDa protein. Overexpression of norR in wild-type S. aureus strain ISP794 led to a fourfold decrease in sensitivity to quinolones and ethidium bromide and an increase in the level of norA transcripts, suggesting that NorR acts as a positive regulator of norA expression. Overexpression of norR in sarA and agr mutants did not alter quinolone sensitivity or levels of norA transcription, indicating that the presence of these two global regulatory systems is necessary for NorR to affect the expression of norA. Insertion and disruption of norR in ISP794 increased resistance to quinolones by 4- to 16-fold but had no effect on norA transcription, suggesting that NorR acts as a repressor for another unidentified efflux pump or pumps. These mutants also exhibited an exaggerated clumping phenotype in liquid media, which was complemented fully by a plasmid-encoded norR gene. Collectively, these results indicate that NorR is a multifunctional regulator, affecting cell surface properties as well as the expression of NorA and likely other multidrug resistance efflux pumps.

scholarly journals In Vitro Activities of Novel Nonfluorinated Quinolones PGE 9262932 and PGE 9509924 against Clinical Isolates of Staphylococcus aureus and Streptococcus pneumoniae with Defined Mutations in DNA Gyrase and Topoisomerase IV

2002 ◽  
Vol 46 (6) ◽  
pp. 1651-1657 ◽  
Author(s):  
Mark E. Jones ◽  
Ian A. Critchley ◽  
James A. Karlowsky ◽  
Renée S. Blosser-Middleton ◽  
Franz-Josef Schmitz ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Streptococcus Pneumoniae ◽  
Dna Gyrase ◽  
Quinolone Resistance ◽  
Clinical Isolates ◽  
Wild Type ◽  
Topoisomerase Iv ◽  
Selection Of

ABSTRACT Two 8-methoxy nonfluorinated quinolones (NFQs), PGE 9262932 and PGE 9509924, were tested against contemporary clinical isolates of Staphylococcus aureus (n = 122) and Streptococcus pneumoniae (n = 69) with genetically defined quinolone resistance-determining regions (QRDRs). For S. aureus isolates with wild-type (WT) sequences at the QRDRs, the NFQs demonstrated activities 4- to 32-fold more potent (MICs at which 90% of isolates are inhibited [MIC90s], 0.03 μg/ml) than those of moxifloxacin (MIC90, 0.12 μg/ml), gatifloxacin (MIC90, 0.25 μg/ml), levofloxacin (MIC90, 0.25 μg/ml), and ciprofloxacin (MIC90, 1 μg/ml). Against S. pneumoniae isolates with WT sequences at gyrA and parC, the NFQs PGE 9262932 (MIC90, 0.03 μg/ml) and PGE 9509924 (MIC90, 0.12 μg/ml) were 8- to 64-fold and 2- to 16-fold more potent, respectively, than moxifloxacin (MIC90, 0.25 μg/ml), gatifloxacin (MIC90, 0.5 μg/ml), levofloxacin (MIC90, 2 μg/ml), and ciprofloxacin (MIC90, 2 μg/ml). The MICs of all agents were elevated for S. aureus isolates with alterations in GyrA (Glu88Lys or Ser84Leu) and GrlA (Ser80Phe) and S. pneumoniae isolates with alterations in GyrA (Ser81Phe or Ser81Tyr) and ParC (Ser79Phe or Lys137Asn). Fluoroquinolone MICs for S. aureus strains with double alterations in GyrA combined with double alterations in GrlA were ≥32 μg/ml, whereas the MICs of the NFQs for strains with these double alterations were 4 to 8 μg/ml. The PGE 9262932 and PGE 9509924 MICs for the S. pneumoniae isolates did not exceed 0.5 and 1 μg/ml, respectively, even for isolates with GyrA (Ser81Phe) and ParC (Ser79Phe) alterations, for which levofloxacin MICs were >16 μg/ml. No difference in the frequency of selection of mutations (<10−8 at four times the MIC) in wild-type or first-step mutant isolates of S. aureus or S. pneumoniae was detected for the two NFQs. On the basis of their in vitro activities, these NFQ agents show potential for the treatment of infections caused by isolates resistant to currently available fluoroquinolones.

scholarly journals DX-619, a Novel Des-Fluoro(6) Quinolone Manifesting Low Frequency of Selection of Resistant Staphylococcus aureus Mutants: Quinolone Resistance beyond Modification of Type II Topoisomerases

2005 ◽  
Vol 49 (12) ◽  
pp. 5051-5057 ◽  
Author(s):  
Jacob Strahilevitz ◽  
Que Chi Truong-Bolduc ◽  
David C. Hooper
Keyword(s):  
Staphylococcus Aureus ◽  
Efflux Pump ◽  
Low Frequency ◽  
Single Step ◽  
Type Ii ◽  
Wild Type ◽  
Topoisomerase Iv ◽  
Dual Targeting ◽  
Selection Of ◽  
Stimulation Of

ABSTRACT DX-619, a novel des-fluoro(6) quinolone, was 16- to 32-fold, twofold, and four- to eightfold more potent than ciprofloxacin, gemifloxacin, and garenoxacin, respectively, against wild-type Staphylococcus aureus. DX-619 manifested equal fourfold increases in MIC against a common parC mutant and a common gyrA mutant and selected for mutants at up to two- to fourfold its MIC, consistent with dual-targeting properties. Of the four independent single-step mutants selected, two had new single mutations in parC (V87F and R17H), and two shared a new gyrA mutation (A26V), one with an additional deletion mutation in parE (Δ215-7). By allelic exchange, the ParC but not the GyrA or ParE mutation was shown to be fully responsible for the resistance phenotypes, suggesting an as yet undefined mechanism of resistance operating in conjunction with type II topoisomerase mutations contributed to resistance to DX-619. Studies with purified topoisomerase IV and gyrase from S. aureus also showed that DX-619 had similar activity against topoisomerase IV and gyrase (50% stimulation of cleavage complexes concentration, 1.25 and 0.62 to 1.25 μg/ml, respectively). Susceptibility studies with DX-619 and an array of efflux pump substrates with and without reserpine, an inhibitor of efflux pumps, suggested that resistance in DX-619-selected mutants is affected by mechanisms other than mutations in topoisomerases or known reserpine-inhibitable pumps in S. aureus and thus are likely novel.

scholarly journals Novel Ciprofloxacin-Resistant, Nalidixic Acid-Susceptible Mutant of Staphylococcus aureus

2002 ◽  
Vol 46 (7) ◽  
pp. 2276-2278 ◽  
Author(s):  
Laura J. V. Piddock ◽  
Yu Fang Jin ◽  
Mark A. Webber ◽  
Martin J. Everett
Keyword(s):  
Staphylococcus Aureus ◽  
Nalidixic Acid ◽  
Promoter Region ◽  
Efflux Pump ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump

ABSTRACT A ciprofloxacin-resistant, nalidixic acid-susceptible mutant of Staphylococcus aureus (F145) contained no mutations within gyrA, gyrB, grlA, and grlB or within norA or its promoter region. MICs and accumulation studies suggest the role of a novel multidrug efflux pump.

scholarly journals A Novel MATE Family Efflux Pump Contributes to the Reduced Susceptibility of Laboratory-Derived Staphylococcus aureus Mutants to Tigecycline

2005 ◽  
Vol 49 (5) ◽  
pp. 1865-1871 ◽  
Author(s):  
Fionnuala McAleese ◽  
Peter Petersen ◽  
Alexey Ruzin ◽  
Paul M. Dunman ◽  
Ellen Murphy ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
In Vitro Selection ◽  
Efflux Pump ◽  
Transcriptional Profiling ◽  
Tetracycline Resistance ◽  
Serial Passage ◽  
Hypothetical Protein ◽  
Wild Type ◽  
Stem Loop

ABSTRACT Tigecycline, an expanded-broad-spectrum glycylcycline antibiotic is not affected by the classical tetracycline resistance determinants found in Staphylococcus aureus. The in vitro selection of mutants with reduced susceptibility to tigecycline was evaluated for two methicillin-resistant S. aureus strains by serial passage in increasing concentrations of tigecycline. Both strains showed a stepwise elevation in tigecycline MIC over a period of 16 days, resulting in an increase in tigecycline MIC of 16- and 32-fold for N315 and Mu3, respectively. Transcriptional profiling revealed that both mutants exhibited over 100-fold increased expression of a gene cluster, mepRAB (multidrug export protein), encoding a MarR-like transcriptional regulator (mepR), a novel MATE family efflux pump (mepA), and a hypothetical protein of unknown function (mepB). Sequencing of the mepR gene in the mutant strains identified changes that presumably inactivated the MepR protein, which suggested that MepR functions as a repressor of mepA. Overexpression of mepA in a wild-type background caused a decrease in susceptibility to tigecycline and other substrates for MATE-type efflux pumps, although it was not sufficient to confer high-level resistance to tigecycline. Complementation of the mepR defect by overexpressing a wild-type mepR gene reduced mepA transcription and lowered the tigecycline MIC in the mutants. Transcription of tet(M) also increased by over 40-fold in the Mu3 mutant. This was attributed to a deletion in the promoter region of the gene that removed a stem-loop responsible for transcriptional attenuation. However, overexpression of the tet(M) transcript in a tigecycline-susceptible strain was not enough to significantly increase the MIC of tigecycline. These results suggest that the overexpression of mepA but not tet(M) may contribute to decreased susceptibility of tigecycline in S. aureus.

scholarly journals A Mutation in the 5′ Untranslated Region Increases Stability of norA mRNA, Encoding a Multidrug Resistance Transporter of Staphylococcus aureus

2001 ◽  
Vol 183 (7) ◽  
pp. 2367-2371 ◽  
Author(s):  
Bénédicte Fournier ◽  
Que Chi Truong-Bolduc ◽  
Xiamei Zhang ◽  
David C. Hooper
Keyword(s):  
Staphylococcus Aureus ◽  
Untranslated Region ◽  
Efflux Pump ◽  
Initiation Site ◽  
Wild Type ◽  
Rnase Iii ◽  
Multidrug Efflux Pump ◽  
Stem Loop ◽  
Transcriptional Initiation ◽  
Multiple Drugs

ABSTRACT NorA, a multidrug efflux pump in Staphylococcus aureus, protects the cell from multiple drugs, including quinolones. TheflqB mutation (T→G) in the 5′ untranslated region upstream of norA causes norA overexpression of 4.9-fold in cis, as measured innorA::blaZ fusions. The transcriptional initiation site of norA was unchanged in mutant and wild-type strains, but the half-life of norAmRNA was increased 4.8-fold in the flqB mutant compared to the wild-type strain. Computer-generated folding of the first 68 nucleotides of the norA transcript predicts an additional stem-loop and changes in a putative RNase III cleavage site in theflqB mutant.

scholarly journals In Vitro Activity of the New Quinolone WCK 771 against Staphylococci

2004 ◽  
Vol 48 (9) ◽  
pp. 3338-3342 ◽  
Author(s):  
Michael R. Jacobs ◽  
Saralee Bajaksouzian ◽  
Anne Windau ◽  
Peter C. Appelbaum ◽  
Mahesh V. Patel ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Efflux Pump ◽  
High Density ◽  
Quinolone Resistance ◽  
Vitro Activity ◽  
In Vitro Activity ◽  
Resistant Strains

ABSTRACT The activity of WCK 771, an experimental quinolone developed to overcome quinolone resistance in staphylococci and other bacteria, was determined against quinolone-susceptible and -resistant Staphylococcus aureus and S. epidermidis. WCK 771 MICs for 50 and 90% of the strains tested (MIC50 and MIC90, respectively) were 0.008 and 0.015 μg/ml for S. aureus (n = 43) and 0.015 and 0.03 μg/ml for S. epidermidis (n = 44) for quinolone-susceptible isolates, compared to ciprofloxacin values of 0.12 and 0.25 μg/ml and 0.25 and 0.5 μg/ml, respectively. Values for levofloxacin were 0.12 and 0.25 μg/ml and 0.12 and 0.25 μg/ml, those for clinafloxacin were 0.015 and 0.03 μg/ml and 0.015 and 0.03 μg/ml, those for moxifloxacin were 0.03 and 0.06 μg/ml and 0.06 and 0.12 μg/ml, and those for gatifloxacin were 0.06 and 0.12 μg/ml and 0.12 and 0.25 μg/ml, respectively. The WCK 771 MIC50 and MIC90, respectively, were 0.5 and 1 μg/ml for both species of staphylococci (n = 73 for S. aureus, n = 70 for S. epidermidis) for isolates highly resistant to ciprofloxacin (MIC50 and MIC90, >32 and >32 μg/ml, respectively). Values for levofloxacin were 8 and 32 μg/ml and 8 and 32 μg/ml, those for clinafloxacin were 1 and 2 μg/ml and 0.5 and 2 μg/ml, those for moxifloxacin 4 and >4 μg/ml and 4 and >4 μg/ml, and those for gatifloxacin were 4 and >4 μg/ml and 2 and >4 μg/ml, respectively. WCK 771 and clinafloxacin demonstrated MICs of 1 μg/ml against three vancomycin-intermediate strains. WCK 771 showed concentration-independent killing for up to 24 h at 2, 4, and 8 times the MICs against quinolone-resistant staphylococci and was also bactericidal after 8 h for high-density inocula (108 CFU/ml) of quinolone-resistant strains at 5 μg/ml, whereas moxifloxacin at 7.5 μg/ml was bacteriostatic. WCK 771 was not a substrate of the NorA efflux pump as evident from the similar MICs against both an efflux-positive and an efflux-negative strain. Overall, WCK 771 was the most potent quinolone tested against the staphylococci tested, regardless of quinolone susceptibility.

scholarly journals Evaluation of Reduced Susceptibility to Quaternary Ammonium Compounds and Bisbiguanides in Clinical Isolates and Laboratory-Generated Mutants of Staphylococcus aureus

2013 ◽  
Vol 57 (8) ◽  
pp. 3488-3497 ◽  
Author(s):  
Leonardo Furi ◽  
Maria Laura Ciusa ◽  
Daniel Knight ◽  
Valeria Di Lorenzo ◽  
Nadia Tocci ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Promoter Region ◽  
Benzalkonium Chloride ◽  
Galleria Mellonella ◽  
Efflux Pump ◽  
Multidrug Resistant ◽  
Clinical Isolates ◽  
Content Type ◽  
Ammonium Compounds

ABSTRACTThe MICs and minimum bactericidal concentrations (MBCs) for the biocides benzalkonium chloride and chlorhexidine were determined against 1,602 clinical isolates ofStaphylococcus aureus. Both compounds showed unimodal MIC and MBC distributions (2 and 4 or 8 mg/liter, respectively) with no apparent subpopulation with reduced susceptibility. To investigate further, all isolates were screened forqacgenes, and 39 of these also had the promoter region of the NorA multidrug-resistant (MDR) efflux pump sequenced. The presence ofqacA,qacB,qacC, andqacGgenes increased the mode MIC, but not MBC, to benzalkonium chloride, while onlyqacAandqacBincreased the chlorhexidine mode MIC. Isolates with a wild-typenorApromoter or mutations in thenorApromoter had similar biocide MIC distributions; notably, not all clinical isolates withnorAmutations were resistant to fluoroquinolones.In vitroefflux mutants could be readily selected with ethidium bromide and acriflavine. Multiple passages were necessary to select mutants with biocides, but these mutants showed phenotypes comparable to those of mutants selected by dyes. All mutants showed changes in the promoter region ofnorA, but these were distinct from this region of the clinical isolates. Still, none of thein vitromutants displayed fitness defects in a killing assay inGalleria mellonellalarvae. In conclusion, our data provide an in-depth comparative overview on efflux inS. aureusmutants and clinical isolates, showing also that plasmid-encoded efflux pumps did not affect bactericidal activity of biocides. In addition, currentin vitrotests appear not to be suitable for predicting levels of resistance that are clinically relevant.

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