[Show abstract][Hide abstract] ABSTRACT: In Gram-negative bacteria, acquired 16S rRNA methyltransferases ArmA and NpmA confer high-level resistance to all clinically useful aminoglycosides by modifying, respectively, G1405 and A1408 in the A-site. These enzymes must coexist with several endogenous methyltransferases that are essential for fine-tuning of the decoding center, such as RsmH and RsmI in Escherichia coli, which methylate C1402 and RsmF C1407. The resistance methyltransferases have a contrasting distribution-ArmA has spread worldwide, whereas a single clinical isolate producing NpmA has been reported. The rate of dissemination of resistance depends on the fitness cost associated with its expression. We have compared ArmA and NpmA in isogenic Escherichia coli harboring the corresponding structural genes and their inactive point mutants cloned under the control of their native constitutive promoter in the stable plasmid pGB2. Growth rate determination and competition experiments showed that ArmA had a fitness cost due to methylation of G1405, whereas NpmA conferred only a slight disadvantage to the host due to production of the enzyme. MALDI MS indicated that ArmA impeded one of the methylations at C1402 by RsmI, and not at C1407 as previously proposed, whereas NpmA blocked the activity of RsmF at C1407. A dual luciferase assay showed that methylation at G1405 and A1408 and lack of methylation at C1407 affect translation accuracy. These results indicate that resistance methyltransferases impair endogenous methylation with different consequences on cell fitness.
[Show abstract][Hide abstract] ABSTRACT: Whereas fluoroquinolone resistance mainly results from target modifications in gram-positive bacteria, it is primarily due to active efflux in Listeria monocytogenes. The aim of this study was to dissect a novel molecular mechanism of fluoroquinolone resistance in this important human pathogen. Isogenic L. monocytogenes clinical isolates BM4715 and BM4716, respectively susceptible and resistant to fluoroquinolones, were studied. MICs of norfloxacin and ciprofloxacin were determined in the presence or in the absence of reserpine (10 mg/L). Strain BM4715 was susceptible to norfloxacin (MIC, 4 mg/L) and ciprofloxacin (MIC, 0.5 mg/L) whereas BM4716 was highly resistant to both drugs (MICs 128 and 32 mg/L, respectively). Reserpine was responsible for a 16-fold decrease in both norfloxacin and ciprofloxacin MICs against BM4716 suggesting efflux associated resistance. Whole-genome sequencing of the strains followed by comparative genomic analysis revealed a single point mutation in the gene for a transcriptional regulator, designated fepR (for fluoroquinolone efflux protein regulator) belonging to the TetR family. The frame-shift mutation was responsible for the introduction of a premature stop codon resulting in an inactive truncated protein. Just downstream from fepR, the structural gene for an efflux pump of the MATE family (named FepA) was identified. Gene expression was quantified by qRT-PCR and demonstrated that fepA expression was more than 64-fold higher in BM4716 than in BM4715. The clean deletion of the fepR gene from BM4715 was responsible for an overexpression of fepA with resistance to norfloxacin and ciprofloxacin, confirming the role of FepR as a local repressor of fepA. In conclusion, we demonstrated that overexpression of the new MATE efflux pump FepA is responsible for fluoroquinolone resistance in L. monocytogenes and secondary to inactivation of the FepR repressor.
PLoS ONE 01/2014; 9(9):e106340. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Multidrug-resistant clinical isolate Klebsiella pneumoniae BM4686 was highly resistant to 4,6-disubstituted 2-deoxystreptamines and to fortimicin. Resistance was due to the presence, on the 40-kb non-self-transferable plasmid pIP849, of the rmtF gene which was cotranscribed with the upstream aac(6')-Ib gene. The deduced RmtF protein had 25 to 46% identity with members of the N7 G1405 family of aminoglycoside resistance 16S rRNA methyltransferases.
Antimicrobial Agents and Chemotherapy 04/2012; 56(7):3960-2. · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Streptococcus suis is an emerging zoonotic pathogen. With the lack of an effective vaccine, antibiotics remain the main tool to fight infections caused by this pathogen. We have previously observed a reserpine-sensitive fluoroquinolone (FQ) efflux phenotype in this species. Here, SatAB and SmrA, two pumps belonging to the ATP binding cassette (ABC) and the major facilitator superfamily (MFS), respectively, have been analyzed in the fluoroquinolone-resistant clinical isolate BB1013. Genes encoding these pumps were overexpressed either constitutively or in the presence of ciprofloxacin in this strain. These genes could not be cloned in plasmids in Escherichia coli despite strong expression repression. Finally, site-directed insertion of smrA and satAB in the amy locus of the Bacillus subtilis chromosome using ligated PCR amplicons allowed for the functional expression and study of both pumps. Results showed that SatAB is a narrow-spectrum fluoroquinolone exporter (norfloxacin and ciprofloxacin), susceptible to reserpine, whereas SmrA was not involved in fluoroquinolone resistance. Chromosomal integration in Bacillus is a novel method for studying efflux pumps from Gram-positive bacteria, which enabled us to demonstrate the possible role of SatAB, and not SmrA, in fluoroquinolone efflux in S. suis.
Antimicrobial Agents and Chemotherapy 09/2011; 55(12):5850-60. · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aminoglycosides are ribosome-targeting antibiotics and a major drug group of choice in the treatment of serious enterococcal infections. Here we show that aminoglycoside resistance in Enterococcus faecium strain CIP 54-32 is conferred by the chromosomal gene efmM, encoding the E. faecium methyltransferase, as well as by the previously characterized aac(6')-Ii that encodes a 6'-N-aminoglycoside acetyltransferase. Inactivation of efmM in E. faecium increases susceptibility to the aminoglycosides kanamycin and tobramycin, and, conversely, expression of a recombinant version of efmM in Escherichia coli confers resistance to these drugs. The EfmM protein shows significant sequence similarity to E. coli RsmF (previously called YebU), which is a 5-methylcytidine (m⁵C) methyltransferase modifying 16S rRNA nucleotide C1407. The target for EfmM is shown by mass spectrometry to be a neighboring 16S rRNA nucleotide at C1404. EfmM uses the methyl group donor S-adenosyl-L-methionine to catalyze formation of m⁵C1404 on the 30S ribosomal subunit, whereas naked 16S rRNA and the 70S ribosome are not substrates. Addition of the 5-methyl to C1404 sterically hinders aminoglycoside binding. Crystallographic structure determination of EfmM at 2.28 Å resolution reveals an N-terminal domain connected to a central methyltransferase domain that is linked by a flexible lysine-rich region to two C-terminal subdomains. Mutagenesis of the methyltransferase domain established that two cysteines at specific tertiary locations are required for catalysis. The tertiary structure of EfmM is highly similar to that of RsmF, consistent with m⁵C formation at adjacent sites on the 30S subunit, while distinctive structural features account for the enzymes' respective specificities for nucleotides C1404 and C1407.
[Show abstract][Hide abstract] ABSTRACT: Pseudomonas aeruginosa is a major human opportunistic pathogen, especially for patients in intensive care units or with cystic fibrosis. Multidrug resistance is a common feature of this species. In a previous study we detected the ant(4')-IIb gene in six multiresistant clinical isolates of P. aeruginosa, and determination of the environment of the gene led to characterization of Tn6061. This 26 586 bp element, a member of the Tn3 family of transposons, carried 10 genes conferring resistance to six drug classes. The ant(4')-IIb sequence was flanked by directly repeated copies of ISCR6 in all but one of the strains studied, consistent with ISCR6-mediated gene acquisition. Tn6061 was chromosomally located in six strains and plasmid-borne in the remaining isolate, suggesting horizontal acquisition. Duplication-insertion of IS6100, that ended Tn6061, was responsible for large chromosomal inversions. Acquisition of Tn6061 and chromosomal inversions are further examples of intricate mechanisms that contribute to the genome plasticity of P. aeruginosa.
[Show abstract][Hide abstract] ABSTRACT: We report the emergence of Salmonella enterica isolates of serotype Concord (and its monophasic variant 6,7:l,v:-) producing the extended-spectrum beta-lactamases (ESBLs) SHV-12 and CTX-M-15 in France and Norway between 2001 and 2006 (43 in France and 26 in Norway). The majority of these isolates were from adopted children from Ethiopia, most of whom were healthy carriers. Several symptomatic secondary cases were found in the adoptive families and health care facilities in France. Serotype Concord isolates collected before 2003 produced SHV-12 encoded on a 340-kb conjugative plasmid of replicon IncI1. Isolates collected after 2003 produced CTX-M-15. We detected two conjugative plasmids carrying bla(CTX-M-15). One plasmid, approximately 300 kb in size, was positive for the IncHI2 replicon and the plasmid-mediated quinolone resistance gene qnrA1. The other plasmid, from one of the earliest CTX-M-15-producing isolates collected, was a fusion plasmid with IncY and IncA/C(2) replicons and was 200 kb in size. However, we showed, using Southern hybridization of I-CeuI-digested chromosomal DNA and S1 nuclease analysis of plasmid DNA, that most isolates had a bla(CTX-M-15) gene located on chromosomal DNA. Analysis of the flanking regions of the chromosomally located bla(CTX-M-15) gene by cloning revealed an ISEcp1 truncated by an intact IS26 upstream from the bla(CTX-M-15) gene and a truncated orf477 gene downstream from bla(CTX-M-15). We found regions beyond the IS26 and the orf477 genes that were derived from IncA/C(2) plasmids, suggesting the chromosomal integration of part of the bla(CTX-M-15)-carrying IncY and IncA/C(2) fusion plasmid from early CTX-M-15-producing isolates.
Antimicrobial Agents and Chemotherapy 04/2009; 53(5):1808-16. · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aminoglycosides are used extensively for the treatment of severe infections due to Gram-negative bacteria. However, certain species have become highly resistant after acquisition of genes for methyltransferases which catalyze post-transcriptional methylation of N7-G1405 in 16 S rRNA of 30 S ribosomal subunits. Inactivation of this enzymatic activity is therefore an important challenge for development of an effective therapy. The present work describes the crystallographic structures of methyltransferases RmtB and ArmA from clinical isolates. Together with biochemical experiments, the 3D structures indicate that the N-terminal domain specific for this family of methyltransferases is required for enzymatic activity. Site-directed mutagenesis has enabled important residues for catalysis and RNA binding to be identified. These high-resolution structures should underpin the design of potential inhibitors of these enzymes, which could be used to restore the activity of aminoglycosides against resistant pathogens.
Journal of Molecular Biology 04/2009; 388(3):570-82. · 3.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Self-transferable IncFI plasmid pIP1206, isolated from an Escherichia coli clinical isolate, carries two new resistance determinants: qepA, which confers resistance to hydrophylic fluoroquinolones by efflux, and rmtB, which specifies a 16S rRNA methylase conferring high-level aminoglycoside resistance. Analysis of the 168,113-bp sequence (51% G+C) revealed that pIP1206 was composed of several subregions separated by copies of insertion sequences. Of 151 open reading frames, 56 (37%) were also present in pRSB107, isolated from a bacterium in a sewage treatment plant. pIP1206 contained four replication regions (RepFIA, RepFIB, and two partial RepFII regions) and a transfer region 91% identical with that of pAPEC-O1-ColBM, a plasmid isolated from an avian pathogenic E. coli. A putative oriT region was found upstream from the transfer region. The antibiotic resistance genes tet(A), catA1, bla(TEM-1), rmtB, and qepA were clustered in a 33.5-kb fragment delineated by two IS26 elements that also carried a class 1 integron, including the sulI, qacEDelta1, aad4, and dfrA17 genes and Tn10, Tn21, and Tn3-like transposons. The plasmid also possessed a raffinose operon, an arginine deiminase pathway, a putative iron acquisition gene cluster, an S-methylmethionine metabolism operon, two virulence-associated genes, and a type I DNA restriction-modification (R-M) system. Three toxin/antitoxin systems and the R-M system ensured stabilization of the plasmid in the host bacteria. These data suggest that the mosaic structure of pIP1206 could have resulted from recombination between pRSB107 and a pAPEC-O1-ColBM-like plasmid, combined with structural rearrangements associated with acquisition of additional DNA by recombination and of mobile genetic elements by transposition.
Antimicrobial Agents and Chemotherapy 08/2008; 52(7):2581-92. · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Brachyspira pilosicoli BM4442, isolated from the feces of a patient with diarrhea at the Hospital Saint-Michel in Paris, was resistant to oxacillin (MIC > 256 microg/ml) but remained susceptible to cephalosporins and to the combination of amoxicillin and clavulanic acid. Cloning and sequencing of the corresponding resistance determinant revealed a coding sequence of 807 bp encoding a new class D beta-lactamase named OXA-63. The bla OXA-63 gene was chromosomally located and not part of a transposon or of an integron. OXA-63 shared 54% identity with FUS-1 (OXA-85), an oxacillinase from Fusobacterium nucleatum subsp. polymorphum, and 25 to 44% identity with other class D beta-lactamases (DBLs) and contained all the conserved structural motifs of DBLs. Escherichia coli carrying the bla OXA-63 gene exhibited resistance to benzylpenicillin and amoxicillin but remained susceptible to amoxicillin in combination with clavulanic acid. Mature OXA-63 consisted of a 31.5-kDa polypeptide and appeared to be dimeric. Kinetic analysis revealed that OXA-63 exhibited a narrow substrate profile, hydrolyzing oxacillin, benzylpenicillin, and ampicillin with catalytic efficiencies of 980, 250, and 150 mM(-1) s(-1), respectively. The enzyme did not apparently interact with oxyimino-cephalosporins, imipenem, or aztreonam. Unlike FUS-1 and other DBLs, OXA-63 was strongly inhibited by clavulanic acid (50% inhibitory concentration [IC50] of 2 microM) and tazobactam (IC50 of 0.16 microM) and exhibited low susceptibility to NaCl (IC50 of >2 M). OXA-63 is the first DBL described for the anaerobic spirochete B. pilosicoli.
Antimicrobial Agents and Chemotherapy 05/2008; 52(4):1264-8. · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Plasmid pIP1206 was detected in Escherichia coli strain 1540 during the screening of clinical isolates of Enterobacteriaceae for high-level resistance to aminoglycosides. The sequence of this IncFI conjugative plasmid of ca. 100 kb was partially determined. pIP1206 carried the rmtB gene for a ribosome methyltransferase that was shown to modify the N7 position of nucleotide G1405, located in the A site of 16S rRNA. It also contained the qepA (quinolone efflux pump) gene that encodes a 14-transmembrane-segment putative efflux pump belonging to the major facilitator superfamily of proton-dependent transporters. Disruption of membrane proton potential by the efflux pump inhibitor carbonyl cyanide m-chlorophenylhydrazone in a transconjugant harboring the qepA gene resulted in elevation of norfloxacin accumulation. The transporter conferred resistance to the hydrophilic quinolones norfloxacin and ciprofloxacin.
Antimicrobial Agents and Chemotherapy 08/2007; 51(7):2464-9. · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 16S rRNA methylase-mediated high-level resistance to aminoglycosides has been reported recently in clinical isolates of Gram-negative bacilli only from a limited number of countries. This study was conducted to investigate the occurrence of this type of resistance in clinical isolates of Enterobacteriaceae from two Belgian hospitals and the characteristics of the strains.
We screened for high-level gentamicin, tobramycin and amikacin resistance in clinical isolates of Enterobacteriaceae consecutively collected between 2000 and 2005 at two laboratories by PCR for the armA, rmtA and rmtB 16S rRNA methylase genes. The beta-lactamase presence in the strains was also determined by phenotypic and genotypic methods.
Overall armA genes were detected in 18 Klebsiella pneumoniae, Escherichia coli, Enterobacter aerogenes, Enterobacter cloacae and Citrobacter amalonaticus whereas rmtB was detected in a single E. coli isolate. The rmtA gene was not found. All 16S rRNA methylase-bearing strains produced extended-spectrum beta-lactamases (ESBLs), predominantly type CTX-M-3, as well as various types of beta-lactamases. In the majority of the strains, the armA gene was carried by conjugative plasmids of the IncL/M incompatibility group whereas rmtB was borne by an IncFI plasmid.
This is the first report of the emergence of 16S rRNA methylases in Enterobacteriaceae in Belgium. The rapid spread of multidrug-resistant isolates producing both ESBLs and 16S rRNA methylases raises clinical concern and may become a major therapeutic threat in the future.
Journal of Antimicrobial Chemotherapy 04/2007; 59(3):459-64. · 5.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Since the beginning of the 1990s, the number of human cases of plague has been rising, and outbreaks are reappearing in various countries after decades of quiescence. Plague is there- fore categorized as a reemerging disease. Until recently, Yer- sinia pestis was considered as uniformly susceptible to agents that are active against gram-negative bacteria. The isolation in Madagascar of two multidrug-resistant strains of Y. pestis, one resistant to all of the antimicrobial agents recommended for treatment and prophylaxis of plague and the other resistant to a smaller array of drugs, is worrisome. The demonstration that horizontal gene transfer in the flea midgut may be the source of antibiotic-resistant Y. pestis strains is of great concern and indicates that such a clinically ominous event may occur again. There is also concern that a biological attack with Y. pestis might employ a natural or engineered antimicrobial-resistant strain. Surveillance of antibiotic resistance in Y. pestis should therefore become systematic worldwide.
Antimicrobial Agents and Chemotherapy 11/2006; 50(10):3233-6. · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aminoglycosides are a medically important class of antibiotics used to treat serious infections. Methylation of the ribosomal target is an emerging mechanism that produces a high level of resistance to all clinically available aminoglycosides for systemic therapy except streptomycin. ArmA was the first methyltransferase using this mechanism to be discovered in a clinical isolate. We demonstrate that ArmA methylates the N7 position of nucleotide G1405 in 16S rRNA. Methylation at this position is presumed to mediate cellular resistance by blocking aminoglycoside binding by ribosomes. To test this hypothesis, we measured the binding of gentamicin by 30S subunits. Under our conditions, we did not observe binding by ribosomes methylated by ArmA. Furthermore, the ArmA methylation reaction is specific for the 30S ribosomal subunit; neither 16S rRNA alone nor the 70S ribosome is a substrate for this reaction under our experimental conditions, implicating ribosomal proteins in substrate recognition. The biochemical characteristics of ArmA place it in the Agr family of methyltransferases, whose members are predominantly anti-suicide genes from Actinomycetes aminoglycoside producers. The discrepancy between the 30% GC content of armA and the >60% GC content of Actinomycetes, however, calls into question the origin of armA. We demonstrate that surprisingly, the natural promoter of armA from gram-negative Klebsiella pneumoniae was active in gram-positive Bacillus subtilis, suggesting that armA originated from a low-GC, gram-positive aminoglycoside-producing organism.
Journal of Molecular Biology 07/2006; 359(2):358-64. · 3.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The armA (aminoglycoside resistance methylase) gene, which confers resistance to 4,6-disubstituted deoxystreptamines and fortimicin, was initially found in Klebsiella pneumoniae BM4536 on IncL/M plasmid pIP1204 of ca. 90 kb which also encodes the extended-spectrum beta-lactamase CTX-M-3. Thirty-four enterobacteria from various countries that were likely to produce a CTX-M enzyme since they were more resistant to cefotaxime than to ceftazidime were studied. The armA gene was detected in 12 clinical isolates of Citrobacter freundii, Enterobacter cloacae, Escherichia coli, K. pneumoniae, Salmonella enterica, and Shigella flexneri, in which it was always associated with bla(CTX-M-3) on an IncL/M plasmid. Conjugation, analysis of DNA sequences, PCR mapping, and plasmid conduction experiments indicated that the armA gene was part of composite transposon Tn1548 together with genes ant3"9, sul1, and dfrXII, which are responsible for resistance to streptomycin-spectinomycin, sulfonamides, and trimethoprim, respectively. The 16.6-kb genetic element was flanked by two copies of IS6 and migrated by replicative transposition. This observation accounts for the presence of armA on self-transferable plasmids of various incompatibility groups and its worldwide dissemination. It thus appears that posttranscriptional modification of 16S rRNA confers high-level resistance to all the clinically available aminoglycosides except streptomycin in gram-negative human and animal pathogens.
Antimicrobial Agents and Chemotherapy 08/2005; 49(7):2949-53. · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Twenty-four community isolates of Streptococcus pyogenes resistant to ciprofloxacin and susceptible to levofloxacin, gatifloxacin, and moxifloxacin were studied. Sequence determination of the quinolone resistance-determining regions in the gyrA and parC genes revealed a T/G mutation in parC leading to a Ser79Ala substitution in ParC. All isolates were of the emm type 6, and 18 and 2 of them were indistinguishable or closely related, respectively, on the basis of pulsed-field gel electrophoresis.
Journal of Clinical Microbiology 06/2005; 43(5):2492-3. · 4.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A recent study revealed a subfamily of N6-adenine (m6A) methyltransferases that comprises a few functionally studied eukaryotic members acting on mRNA and prokaryotic members acting on DNA as well as numerous uncharacterized open reading frames. Here, we report cloning and functional characterization of a prokaryotic member of this family encoded by transposon Tn1549 from Enterococcus spp.
Polish journal of microbiology / Polskie Towarzystwo Mikrobiologów = The Polish Society of Microbiologists 01/2005; 54(3):249-52. · 0.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A self-transferable plasmid of ca. 80 kb, pIP1204, conferred multiple-antibiotic resistance to Klebsiella pneumoniae BM4536, which was isolated from a urinary tract infection. Resistance to beta-lactams was due to the bla(TEM1) and bla(CTX-M) genes, resistance to trimethroprim was due to the dhfrXII gene, resistance to sulfonamides was due to the sul1 gene, resistance to streptomycin-spectinomycin was due to the ant3"9 gene, and resistance to nearly all remaining aminoglycosides was due to the aac3-II gene and a new gene designated armA (aminoglycoside resistance methylase). The cloning of armA into a plasmid in Escherichia coli conferred to the new host high-level resistance to 4,6-disubstituted deoxystreptamines and fortimicin. The deduced sequence of ArmA displayed from 37 to 47% similarity to those of 16S rRNA m(7)G methyltransferases from various actinomycetes, which confer resistance to aminoglycoside-producing strains. However, the low guanine-plus-cytosine content of armA (30%) does not favor an actinomycete origin for the gene. It therefore appears that posttranscriptional modification of 16S rRNA can confer high-level broad-range resistance to aminoglycosides in gram-negative human pathogens.
Antimicrobial Agents and Chemotherapy 09/2003; 47(8):2565-71. · 4.57 Impact Factor