gyrA and gyrB Mutations Are Implicated in Cross-Resistance to Ciprofloxacin and Moxifloxacin in Clostridium difficile

ABSTRACT A total of 198 nonrepetitive clinical strains of Clostridium difficile isolated from different French hospitals in 1991 (n = 100) and 1997 (n = 98) were screened for decreased susceptibility to fluoroquinolones by plating onto Wilkins-Chalgren agar containing 16 μg of ciprofloxacin per ml. The frequency of decreased susceptibility was 7% (14 of 198) and was identical for the years 1991 and 1997. Serogroups C, H, D, A9, and K accounted for five, four, two, one, and one of the resistant strains, respectively, one strain being nontypeable. Arbitrarily primed PCR typing showed that all resistant strains had unique patterns except two serotype C strains, which could not be clearly distinguished. All isolates with decreased susceptibility carried a mutation either in gyrA (eight mutations, amino acid changes Asp71→Val in one, Thr82→Ile in six, and Ala118→Thr in one) or in gyrB (six mutations, amino acid changes Asp426→Asn in five and Arg447→Leu in one). These changes are similar to those already described in other species except for Asp71→Val, which is novel, and Ala118→Thr, which is exceptional. Attempts to detect the topoisomerase IV parC gene by PCR amplification with universal parC primers or DNA-DNA hybridization under low-stringency conditions were unsuccessful. The susceptibilities of all resistant strains to ciprofloxacin and ethidium bromide were not affected by the addition of reserpine at 20 μg/ml. In conclusion, decreased susceptibility to fluoroquinolones in C. difficile is rare in France and is associated with the occurrence of a gyrA or gyrB mutation.

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rpoB Mutations in Multidrug-Resistant Strains of Mycobacterium tuberculosis Isolated in Italy

Journal of Clinical Microbiology ◽

10.1128/jcm.37.4.1197-1199.1999 ◽

1999 ◽

Vol 37 (4) ◽

pp. 1197-1199 ◽

Cited By ~ 46

Author(s):

G. Pozzi ◽

M. Meloni ◽

E. Iona ◽

G. Orrù ◽

O. F. Thoresen ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Amino Acid ◽

Amino Acid Change ◽

Multidrug Resistant ◽

Single Base Substitution ◽

Base Substitution ◽

Single Base ◽

Clinical Strains ◽

Resistant Strains ◽

Rifampin Resistance

Mutations of rpoB associated with rifampin resistance were studied in 37 multidrug-resistant (MDR) clinical strains ofMycobacterium tuberculosis isolated in Italy. At least one mutated codon was found in each MDR strain. It was always a single-base substitution leading to an amino acid change. Nine differentrpoB alleles, three of which had not been reported before, were found. The relative frequencies of specific mutations in this sample were different from those previously reported from different geographical areas, since 22 strains (59.5%) carried the mutated codon TTG in position 531 (Ser→Leu) and 11 (29.7%) had GAC in position 526 (His→Asp).

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Biological Characteristics and Molecular Mechanism of Fludioxonil Resistance in Botrytis cinerea From Henan Province of China

Plant Disease ◽

10.1094/pdis-08-19-1722-re ◽

2020 ◽

Vol 104 (4) ◽

pp. 1041-1047 ◽

Cited By ~ 3

Author(s):

Feng Zhou ◽

Hai-Yan Hu ◽

Yu-Lu Song ◽

Yu-Qing Gao ◽

Qi-Li Liu ◽

...

Keyword(s):

Amino Acid ◽

Botrytis Cinerea ◽

Stop Codon ◽

Field Isolate ◽

Point Mutations ◽

Signal Transduction Pathway ◽

Resistant Isolate ◽

Cross Resistance ◽

Tomato Production ◽

Resistant Strains

The gray mold caused by Botrytis cinerea has a significant impact on tomato production throughout the world. Although the synthetic fungicide fludioxonil can effectively control B. cinerea, there have been several reports of resistance to this fungicide. This study indicated that all of the fludioxonil-resistant strains tested, including one field-resistant isolate and four laboratory strains, had reduced fitness relative to sensitive isolates. In addition to having reduced growth, sporulation, and pathogenicity, the resistant strains were more sensitive to osmotic stress and had significantly (P < 0.05) higher peroxidase activity. BOs1, a kinase in the high-osmolarity glycerol stress response signal transduction pathway, is believed to harbor mutations related to fludioxonil resistance. Sequence analysis of their BOs1 sequences indicated that the fludioxonil-resistant field isolate, XXtom1806, had four point mutations resulting in four amino acid changes (I365S, S531G, T565N, and T1267A) and three amino acids (I365S, S531G, and T565N) in the histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis receptors, and phosphatases domain, which associated with fludioxonil binding. Similarly, two of the laboratory strains, XXtom-Lab1 and XXtom-Lab4, had three (Q846S, I1126S, and G415D) and two (P1051S and V1241M) point mutations, respectively. A third strain, XXtom-lab3, had a 52-bp insertion that included a stop codon at amino acid 256. Interestingly, the BOs1 sequence of the fourth laboratory strain, XXtom-lab5, was identical to those of the sensitive isolates, indicating that an alternative resistance mechanism exists. The study also found evidence of positive cross-resistance between fludioxonil and the dicarboximide fungicides procymidone and iprodione, but no cross-resistance was detected with any other fungicides tested, including boscalid, carbendazim, tebuconazole, and fluazinam.

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Antibiotic usage promotes the evolution of resistance against gepotidacin, a novel multi-targeting drug

10.1101/495630 ◽

2018 ◽

Cited By ~ 1

Author(s):

Petra Szili ◽

Gabor Draskovits ◽

Tamas Revesz ◽

Ferenc Bogar ◽

David Balogh ◽

...

Keyword(s):

Antibiotic Usage ◽

Cross Resistance ◽

Stepping Stones ◽

Topoisomerase Iv ◽

Bacterial Dna ◽

Resistance Evolution ◽

Evolution Of Resistance ◽

Resistant Strains ◽

Clinically Significant ◽

Susceptible Organism

Multi-targeting antibiotics, i.e. single compounds capable to inhibit two or more bacterial targets offer a promising therapeutic strategy, but information on resistance evolution against such drugs is scarce. Gepotidacin is an antibiotic candidate that selectively inhibits both bacterial DNA gyrase and topoisomerase IV. In a susceptible organism, Klebsiella pneumoniae, a combination of two specific mutations in these target proteins provide an over 2000-fold increment in resistance, while individually none of these mutations affect resistance significantly. Alarmingly, gepotidacin-resistant strains are found to be as virulent as the wild-type K. pneumoniae strain in a murine model, and extensive cross-resistance was demonstrated between gepotidacin and ciprofloxacin, a fluoroquinolone antibiotic widely employed in clinical practice. This suggests that numerous fluoroquinolone-resistant pathogenic isolates carry mutations which would promote the evolution of clinically significant resistance against gepotidacin in the future. We conclude that prolonged antibiotic usage could select for mutations that serve as stepping-stones towards resistance against antimicrobial compounds still under development. More generally, our research indicates that even balanced multi-targeting antibiotics are prone to resistance evolution.

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Activities of Newer Fluoroquinolones against Streptococcus pneumoniae Clinical Isolates Including Those with Mutations in the gyrA, parC, and parELoci

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.2.329 ◽

1999 ◽

Vol 43 (2) ◽

pp. 329-334 ◽

Cited By ~ 79

Author(s):

J. H. Jorgensen ◽

L. M. Weigel ◽

M. J. Ferraro ◽

J. M. Swenson ◽

F. C. Tenover

Keyword(s):

Streptococcus Pneumoniae ◽

Amino Acid ◽

Amino Acid Sequences ◽

Clinical Isolates ◽

Single Amino Acid ◽

Single Mutation ◽

Topoisomerase Iv ◽

In Vitro Susceptibility ◽

Resistant Strains ◽

Level Resistance

ABSTRACT Resistance to fluoroquinolone (FQ) antibiotics inStreptococcus pneumoniae has been attributed primarily to specific mutations in the genes for DNA gyrase (gyrA andgyrB) and topoisomerase IV (parC andparE). Resistance to some FQs can result from a single mutation in one or more of the genes encoding these essential enzymes. A group of 160 clinical isolates of pneumococci was examined in this study, including 36 ofloxacin-resistant isolates (MICs, ≥8 μg/ml) recovered from patients in North America, France, and Belgium. The susceptibilities of all isolates to clinafloxacin, grepafloxacin, levofloxacin, sparfloxacin, and trovafloxacin were examined by the National Committee for Clinical Laboratory Standards reference broth microdilution and disk diffusion susceptibility testing methods. Among the ofloxacin-resistant strains, 32 of 36 were also categorized as resistant to levofloxacin, 35 were resistant to sparfloxacin, 29 were resistant to grepafloxacin, and 19 were resistant to trovafloxacin. In vitro susceptibility to clinafloxacin appeared to be least affected by resistance to the other FQs. Eight isolates with high- and low-level resistance to the newer FQs were selected for DNA sequence analysis of the quinolone resistance-determining regions (QRDRs) ofgyrA, gyrB, parC, andparE. The DNA and the inferred amino acid sequences of the resistant strains were compared with the analogous sequences of reference strain S. pneumoniae ATCC 49619 and FQ-susceptible laboratory strain R6. Reduced susceptibilities to grepafloxacin and sparfloxacin (MICs, 1 to 2 μg/ml) and trovafloxacin (MICs, 0.5 to 1 μg/ml) were associated with either a mutation inparC that led to a single amino acid substitution (Ser-79 to Phe or Tyr) or double mutations that involved the genes for both GyrA (Ser-81 to Phe) and ParE (Asp-435 to Asn). High-level resistance to all of the compounds except clinafloxacin was associated with two or more amino acid substitutions involving both GyrA (Ser-81 to Phe) and ParC (Ser-79 to Phe or Ser-80 to Pro and Asp-83 to Tyr). No mutations were observed in the gyrB sequences of resistant strains. These data indicate that mutations in pneumococcal gyrA,parC, and parE genes all contribute to decreased susceptibility to the newer FQs, and genetic analysis of the QRDR of a single gene, either gyrA or parC, is not predictive of pneumococcal resistance to these agents.

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ETHIDIUM BROMIDE RESISTANCE AND PETITE INDUCTION IN SACCHAROMYCES CEREVISIAE

Canadian Journal of Genetics and Cytology ◽

10.1139/g72-084 ◽

1972 ◽

Vol 14 (3) ◽

pp. 681-689 ◽

Cited By ~ 18

Author(s):

N. T. Bech-Hansen ◽

G. H. Rank

Keyword(s):

Saccharomyces Cerevisiae ◽

Ethidium Bromide ◽

Mitochondrial Membrane ◽

Cetyltrimethylammonium Bromide ◽

Glucose Repression ◽

Sensitive Strain ◽

The Other ◽

Cross Resistance ◽

Resistant Strains ◽

Resistant Mutants

Six ethidium bromide (EB) resistant mutants were isolated and characterized. The resistance in three isolates was stable, nuclearly inherited and sensitive to glucose repression. Unstable EB dependent resistance was present in the other isolates. The mutants with stable and one of those with unstable EB resistance showed cross resistance to the cationic detergent, cetyltrimethylammonium bromide. Both anaerobiosis and oligomycin inhibited EB induction of petites in the EB sensitive strain and in the EB-sensitized resistant strains. The observations are discussed in relationship to a mechanism of EB resistance and petite induction at the level of the mitochondrial membrane.

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In vitro activity of sitafloxacin against clinical strains of Streptococcus pneumoniae with defined amino acid substitutions in QRDRs of gyrase A and topoisomerase IV

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkl427 ◽

2006 ◽

Vol 58 (6) ◽

pp. 1279-1282 ◽

Cited By ~ 10

Author(s):

M. Touyama ◽

F. Higa ◽

C. Nakasone ◽

T. Shinzato ◽

M. Akamine ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Amino Acid ◽

Vitro Activity ◽

Amino Acid Substitutions ◽

In Vitro Activity ◽

Topoisomerase Iv ◽

Clinical Strains ◽

Gyrase A

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Pseudomonas syringae pv. phaseolicola can be separated into two genetic lineages distinguished by the possession of the phaseolotoxin biosynthetic cluster

Microbiology ◽

10.1099/mic.0.26635-0 ◽

2004 ◽

Vol 150 (2) ◽

pp. 473-482 ◽

Cited By ~ 20

Author(s):

José A. Oguiza ◽

Arantza Rico ◽

Luis A. Rivas ◽

Laurent Sutra ◽

Alan Vivian ◽

...

Keyword(s):

Pseudomonas Syringae ◽

Dna Sequences ◽

Dna Hybridization ◽

Pcr Amplification ◽

Pathogenicity Island ◽

Large Plasmid ◽

Genetic Lineages ◽

Genomic Divergence ◽

Arbitrarily Primed Pcr ◽

Genomic Insertions

The bean (Phaseolus spp.) plant pathogen Pseudomonas syringae pv. phaseolicola is characterized by the ability to produce phaseolotoxin (Tox+). We recently reported that the majority of the Spanish P. syringae pv. phaseolicola population is unable to synthesize this toxin (Tox−). These Tox− isolates appear to lack the entire DNA region for the biosynthesis of phaseolotoxin (argK-tox gene cluster), as shown by PCR amplification and DNA hybridization using DNA sequences specific for separated genes of this cluster. Tox+ and Tox− isolates also showed genomic divergence that included differences in ERIC-PCR and arbitrarily primed-PCR profiles. Tox+ isolates showed distinct patterns of IS801 genomic insertions and contained a chromosomal IS801 insertion that was absent from Tox− isolates. Using a heteroduplex mobility assay, sequence differences were observed only among the intergenic transcribed spacer of the five rDNA operons of the Tox− isolates. The techniques used allowed the unequivocal differentiation of isolates of P. syringae pv. phaseolicola from the closely related soybean (Glycine max) pathogen, P. syringae pv. glycinea. Finally, a pathogenicity island that is essential for the pathogenicity of P. syringae pv. phaseolicola on beans appears to be conserved among Tox+, but not among Tox− isolates, which also lacked the characteristic large plasmid that carries this pathogenicity island. It is proposed that the results presented here justify the separation of the Tox+ and Tox− P. syringae pv. phaseolicola isolates into two distinct genetic lineages, designated Pph1 and Pph2, respectively, that show relevant genomic differences that include the pathogenicity gene complement.

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Corticosteroid Catabolism by Klebsiella pneumoniae as a Possible Mechanism for Increased Pneumonia Risk

Current Pharmaceutical Biotechnology ◽

10.2174/1389201020666190313153841 ◽

2019 ◽

Vol 20 (4) ◽

pp. 309-316 ◽

Cited By ~ 1

Author(s):

Pritam Chattopadhyay ◽

Goutam Banerjee

Keyword(s):

Amino Acid ◽

Klebsiella Pneumoniae ◽

Kegg Pathway ◽

Degradation Pathway ◽

Amino Acid Sequences ◽

Biological Mechanism ◽

Clinical Strains ◽

Catabolism Pathway ◽

Steroid Catabolism ◽

Nutrition Source

Background: Several strains of Klebsiella pneumoniae are responsible for causing pneumonia in lung and thereby causing death in immune-suppressed patients. In recent year, few investigations have reported the enhancement of K. pneumoniae population in patients using corticosteroid containing inhaler. Objectives: The biological mechanism(s) behind this increased incidence has not been elucidated. Therefore, the objective of this investigating was to explore the relation between Klebsiella pneumoniae and increment in carbapenamase producing Enterobacteriaceae score (ICS). Methods: The available genomes of K. pneumoniae and the amino acid sequences of steroid catabolism pathway enzymes were taken from NCBI database and KEGG pathway tagged with UniPort database, respectively. We have used different BLAST algorithms (tBLASTn, BLASTp, psiBLAST, and delBLAST) to identify enzymes (by their amino acid sequence) involved in steroid catabolism. Results: A total of 13 enzymes (taken from different bacterial candidates) responsible for corticosteroid degradation have been identified in the genome of K. pneumoniae. Finally, 8 enzymes (K. pneumoniae specific) were detected in four clinical strains of K. pneumoniae. This investigation intimates that this ability to catabolize corticosteroids could potentially be one mechanism behind the increased pneumonia incidence. Conclusion: The presence of corticosteroid catabolism enzymes in K. pneumoniae enhances the ability to utilize corticosteroid for their own nutrition source. This is the first report to demonstrate the corticosteroid degradation pathway in clinical strains of K. pneumoniae.

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A Simple Method for Assessment of MDR Bacteria for Over-Expressed Efflux Pumps

The Open Microbiology Journal ◽

10.2174/1874285801307010072 ◽

2013 ◽

Vol 7 (1) ◽

pp. 72-82 ◽

Cited By ~ 39

Author(s):

Marta Martins ◽

Matthew P McCusker ◽

Miguel Viveiros ◽

Isabel Couto ◽

Séamus Fanning ◽

...

Keyword(s):

Ethidium Bromide ◽

Efflux Pump ◽

Efflux Pumps ◽

Outer Cell ◽

Gram Negative Bacteria ◽

Drug Resistant ◽

Topoisomerase Iv ◽

Simple Method ◽

Gram Negative ◽

Over Expression

It is known that bacteria showing a multi-drug resistance phenotype use several mechanisms to overcome the action of antibiotics. As a result, this phenotype can be a result of several mechanisms or a combination of thereof. The main mechanisms of antibiotic resistance are: mutations in target genes (such as DNA gyrase and topoisomerase IV); over-expression of efflux pumps; changes in the cell envelope; down regulation of membrane porins, and modified lipopolysaccharide component of the outer cell membrane (in the case of Gram-negative bacteria). In addition, adaptation to the environment, such as quorum sensing and biofilm formation can also contribute to bacterial persistence. Due to the rapid emergence and spread of bacterial isolates showing resistance to several classes of antibiotics, methods that can rapidly and efficiently identify isolates whose resistance is due to active efflux have been developed. However, there is still a need for faster and more accurate methodologies. Conventional methods that evaluate bacterial efflux pump activity in liquid systems are available. However, these methods usually use common efflux pump substrates, such as ethidium bromide or radioactive antibiotics and therefore, require specialized instrumentation, which is not available in all laboratories. In this review, we will report the results obtained with the Ethidium Bromide-agar Cartwheel method. This is an easy, instrument-free, agar based method that has been modified to afford the simultaneous evaluation of as many as twelve bacterial strains. Due to its simplicity it can be applied to large collections of bacteria to rapidly screen for multi-drug resistant isolates that show an over-expression of their efflux systems. The principle of the method is simple and relies on the ability of the bacteria to expel a fluorescent molecule that is substrate for most efflux pumps, ethidium bromide. In this approach, the higher the concentration of ethidium bromide required to produce fluorescence of the bacterial mass, the greater the efflux capacity of the bacterial cells. We have tested and applied this method to a large number of Gram-positive and Gram-negative bacteria to detect efflux activity among these multi-drug resistant isolates. The presumptive efflux activity detected by the Ethidium Bromide-agar Cartwheel method was subsequently confirmed by the determination of the minimum inhibitory concentration for several antibiotics in the presence and absence of known efflux pump inhibitors.

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