Involvement of an Efflux System in High-Level Fluoroquinolone Resistance ofShigella dysenteriae

Twenty-eight human isolates of Escherichia coli from Argentina and Spain and eight veterinary isolates received from the Ministry of Agriculture Fisheries and Foods in the United Kingdom required 2 to > 128 micrograms of ciprofloxacin per ml for inhibition. Fragments of gyrA and parC encompassing the quinolone resistance-determining region were amplified by PCR, and the DNA sequences of the fragments were determined. All isolates contained a mutation in gyrA of a serine at position 83 (Ser83) to an Leu, and 26 isolates also contained a mutation of Asp87 to one of four amino acids: Asn (n = 14), Tyr (n = 6), Gly (n = 5), or His (n = 1). Twenty-four isolates contained a single mutation in parC, either a Ser80 to Ile (n = 17) or Arg (n = 2) or a Glu84 to Lys (n = 3). The role of a mutation in gyrB was investigated by introducing wild-type gyrB (pBP548) into all isolates; for three transformants MICs of ciprofloxacin were reduced; however, sequencing of PCR-derived fragments containing the gyrB quinolone resistance-determining region revealed no changes. The analogous region of parE was analyzed in 34 of 36 isolates by single-strand conformational polymorphism analysis and sequencing; however, no amino acid substitutions were discovered. The outer membrane protein and lipopolysaccharide profiles of all isolates were compared with those of reference strains, and the concentration of ciprofloxacin accumulated (with or without 100 microM carbony cyanide m-chlorophenylhydrazone [CCCP] was determined. Twenty-two isolates accumulated significantly lower concentrations of ciprofloxacin than the wild-type E. coli isolate; nine isolates accumulated less then half the concentration. The addition of CCCP increased the concentration of ciprofloxacin accumulated, and in all but one isolate the percent increase was greater than that in the control strains. The data indicate that high-level fluoroquinolone resistance in E. coli involves the acquisition of mutations at multiple loci.

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Resistance to β-Lactam Antibiotics inPseudomonas aeruginosa Due to Interplay between the MexAB-OprM Efflux Pump and β-Lactamase

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.5.1301 ◽

1999 ◽

Vol 43 (5) ◽

pp. 1301-1303 ◽

Cited By ~ 49

Author(s):

Taiji Nakae ◽

Akira Nakajima ◽

Toshihisa Ono ◽

Kohjiro Saito ◽

Hiroshi Yoneyama

Keyword(s):

Pseudomonas Aeruginosa ◽

Efflux Pump ◽

Efflux System ◽

High Level

ABSTRACT We evaluated the roles of the MexAB-OprM efflux pump and β-lactamase in β-lactam resistance in Pseudomonas aeruginosa by constructing OprM-deficient, OprM basal level, and OprM fully expressed mutants from β-lactamase-negative, -inducible, and -overexpressed strains. We conclude that, with the notable exception of imipenem, the MexAB-OprM pump contributes significantly to β-lactam resistance in both β-lactamase-negative and β-lactamase-inducible strains, while the contribution of the MexAB-OprM efflux system is negligible in strains with overexpressed β-lactamase. Overexpression of the efflux pump alone contributes to the high level of β-lactam resistance in the absence of β-lactamase.

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In Vitro Derivation of Fluoroquinolone-Resistant Mutants from Multiple Lineages of Haemophilus influenzae and Identification of Mutations Associated with Fluoroquinolone Resistance

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01500-19 ◽

2019 ◽

Vol 64 (2) ◽

Author(s):

Hiroyuki Honda ◽

Toyotaka Sato ◽

Masaaki Shinagawa ◽

Yukari Fukushima ◽

Chie Nakajima ◽

...

Keyword(s):

Haemophilus Influenzae ◽

Clinical Setting ◽

Pathogenic Bacterium ◽

Fluoroquinolone Resistance ◽

Novel Mutations ◽

Content Type ◽

Resistant Mutants ◽

High Level ◽

Current Risk

ABSTRACT Haemophilus influenzae is a pathogenic bacterium that causes respiratory and otolaryngological infections. The increasing prevalence of β-lactamase–negative high-level ampicillin-resistant H. influenzae (high-BLNAR) is a clinical concern. Fluoroquinolones are alternative agents to β-lactams. However, the emergence and increasing prevalence of fluoroquinolone-resistant H. influenzae have been reported. The current risk of fluoroquinolone resistance in H. influenzae (especially in high-BLNAR) has not yet been evaluated. Here, we examined the development of fluoroquinolone resistance in fluoroquinolone-susceptible clinical H. influenzae isolates in vitro during passaging in the presence of moxifloxacin (from 0.03 to 128 mg/liter). Twenty-nine isolates were examined. Seventeen isolates (58.6%) showed reduced moxifloxacin susceptibility, and 10 of these 17 isolates (34.5% of all isolates) exceeded the Clinical and Laboratory Standards Institute breakpoint for moxifloxacin (MIC of >1 mg/liter) after repeat cultivation on moxifloxacin-containing agar. Seven of these ten isolates were high-BLNAR and represented multiple lineages. We identified 56 novel mutations in 45 genes induced during the development of fluoroquinolone resistance, except the defined quinolone resistance-determining regions (Ser84Leu and Asp88Tyr/Gly/Asn in GyrA and Gly82Asp, Ser84Arg, and Glu88Lys in ParC). Glu153Leu and ΔGlu606 in GyrA, Ser467Tyr and Glu469Asp in GyrB, and ompP2 mutations were novel mutations contributing to fluoroquinolone resistance in H. influenzae. In conclusion, H. influenzae clinical isolates from multiple lineages can acquire fluoroquinolone resistance by multiple novel mutations. The higher rate of derivation of fluoroquinolone-resistant H. influenzae from high-BLNAR than β-lactamase-negative ampicillin-susceptible isolates (P = 0.01) raises the possibility of the emergence and spread of fluoroquinolone-resistant high-BLNAR in the clinical setting.

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Development of fluoroquinolone resistance in Enterococcus faecalis and role of mutations in the DNA gyrase gyrA gene.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.40.11.2558 ◽

1996 ◽

Vol 40 (11) ◽

pp. 2558-2561 ◽

Cited By ~ 30

Author(s):

J Tankovic ◽

F Mahjoubi ◽

P Courvalin ◽

J Duval ◽

R Leclerco

Keyword(s):

Enterococcus Faecalis ◽

Resistant Isolate ◽

Fluoroquinolone Resistance ◽

Gyra Gene ◽

Single Clone ◽

Total Dna ◽

Resistant Strains ◽

French Hospital ◽

High Level

We have analyzed the development of fluoroquinolone resistance between 1986 and 1993 among clinical isolates of Enterococcus faecalis from a French hospital. One hundred randomly selected isolates per year were screened for resistance to ciprofloxacin (MIC > 2 micrograms/ml) and for high-level resistance to gentamicin (MIC > 1,000 micrograms/ml). The percentages of ciprofloxacin-resistant strains for these years were as follows: 1986, 0; 1987, 1; 1988 to 1989, 2; 1990, 6; 1991, 16; 1992, 24; and 1993, 14. Eighty-three percent of the ciprofloxacin-resistant isolates were coresistant to high levels of gentamicin. Forty-eight high-level gentamicin-resistant E. faecalis strains, which were resistant (24 strains) or susceptible (24 strains) to ciprofloxacin, were examined by pulsed-field gel electrophoresis (PFGE) of SmaI-digested total DNA. Numerous PFGE types were observed among the ciprofloxacin-susceptible isolates, whereas one type was largely predominant among the ciprofloxacin-resistant strains, which suggests that the increase in fluoroquinolone resistance was due to the spread of a single clone. A 241-bp fragment of gyrA, corresponding to the quinolone resistance-determining region, was amplified and sequenced for seven ciprofloxacin-resistant isolates. Six strains had high levels of resistance (MICs, 32 to 64 micrograms/ml) and had a mutation at position 83 (Escherichia coli coordinates) from Ser to Arg (three strains) or to Ile (two strains) or at position 87 from Glu to Gly (one strain), whereas the low-level-resistant isolate (MIC, 8 micrograms/ml) had no mutations.

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In Vivo Development of High-Level Fluoroquinolone Resistance in Streptococcus pneumoniae in Chronic Obstructive Pulmonary Disease

Clinical Infectious Diseases ◽

10.1086/432062 ◽

2005 ◽

Vol 41 (4) ◽

pp. 560-564 ◽

Cited By ~ 22

Author(s):

E. Purez-Trallero ◽

J. M. Marimon ◽

A. Gonzalez ◽

M. Ercibengoa ◽

J. Larruskain

Keyword(s):

Chronic Obstructive Pulmonary Disease ◽

Streptococcus Pneumoniae ◽

Pulmonary Disease ◽

Fluoroquinolone Resistance ◽

Chronic Obstructive ◽

Obstructive Pulmonary Disease ◽

High Level

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Genetic Characterization of Highly Fluoroquinolone-Resistant Clinical Escherichia coli Strains from China: Role ofacrR Mutations

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.5.1515-1521.2001 ◽

2001 ◽

Vol 45 (5) ◽

pp. 1515-1521 ◽

Cited By ~ 181

Author(s):

Hui Wang ◽

Joann L. Dzink-Fox ◽

Minjun Chen ◽

Stuart B. Levy

Keyword(s):

Escherichia Coli ◽

Blot Analysis ◽

Genetic Basis ◽

Efflux Pump ◽

Pcr Amplification ◽

Fluoroquinolone Resistance ◽

Multidrug Efflux ◽

Multidrug Efflux Pump ◽

E Coli ◽

High Level

ABSTRACT The genetic basis for fluoroquinolone resistance was examined in 30 high-level fluoroquinolone-resistant Escherichia coliclinical isolates from Beijing, China. Each strain also demonstrated resistance to a variety of other antibiotics. PCR sequence analysis of the quinolone resistance-determining region of the topoisomerase genes (gyrA/B, parC) revealed three to five mutations known to be associated with fluoroquinolone resistance. Western blot analysis failed to demonstrate overexpression of MarA, and Northern blot analysis did not detect overexpression of soxS RNA in any of the clinical strains. The AcrA protein of the AcrAB multidrug efflux pump was overexpressed in 19 of 30 strains of E. colitested, and all 19 strains were tolerant to organic solvents. PCR amplification of the complete acrR (regulator/repressor) gene of eight isolates revealed amino acid changes in four isolates, a 9-bp deletion in another, and a 22-bp duplication in a sixth strain. Complementation with a plasmid-borne wild-type acrR gene reduced the level of AcrA in the mutants and partially restored antibiotic susceptibility 1.5- to 6-fold. This study shows that mutations in acrR are an additional genetic basis for fluoroquinolone resistance.

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Antimicrobial susceptibility and mechanisms of high-level macrolide resistance in clinical isolates of Moraxella nonliquefaciens

Journal of Medical Microbiology ◽

10.1099/jmm.0.061788-0 ◽

2014 ◽

Vol 63 (2) ◽

pp. 242-247 ◽

Cited By ~ 6

Author(s):

Shotaro Nonaka ◽

Kosuke Matsuzaki ◽

Tomoya Kazama ◽

Hiroyuki Nishiyama ◽

Yoko Ida ◽

...

Keyword(s):

Antimicrobial Susceptibility ◽

Ribosomal Proteins ◽

Macrolide Resistance ◽

Clinical Isolates ◽

23S Rrna ◽

Rrna Gene ◽

Efflux System ◽

Strong Activity ◽

23S Rrna Gene ◽

High Level

We investigated antimicrobial susceptibility and the molecular mechanism involved in conferring high-level macrolide resistance in 47 clinical isolates of Moraxella nonliquefaciens from Japan. Antimicrobial susceptibility was determined using Etest and agar dilution methods. Thirty-two erythromycin-non-susceptible strains were evaluated for the possibility of clonal spreading, using PFGE. To analyse the mechanism related to macrolide resistance, mutations in the 23S rRNA gene and the ribosomal proteins, and the presence of methylase genes were investigated by PCR and sequencing. The efflux system was examined using appropriate inhibitors. Penicillin, ampicillin, amoxicillin, cefixime, levofloxacin and antimicrobials containing β-lactamase inhibitors showed strong activity against 47 M. nonliquefaciens isolates. Thirty-two (68.1 %) of the 47 isolates showed high-level MICs to macrolides (MIC ≥128 mg l−1) and shared the A2058T mutation in the 23S rRNA gene. The geometric mean MIC to macrolides of A2058T-mutated strains was significantly higher than that of WT strains (P<0.0001). Thirty-two isolates with high-level macrolide MICs clustered into 30 patterns on the basis of the PFGE dendrogram, indicating that the macrolide-resistant strains were not clonal. In contrast, no common mutations of the ribosomal proteins or methylase genes, or overproduction of the efflux system were observed in A2058T-mutated strains. Moreover, of the 47 M. nonliquefaciens strains, 43 (91.5 %) were bro-1 and 4 (8.5 %) were bro-2 positive. Our results suggest that most M. nonliquefaciens clinical isolates show high-level macrolide resistance conferred by the A2058T mutation in the 23S rRNA gene. This study represents the first characterization of M. nonliquefaciens.

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