Wladimir Sougakoff

Detection by GenoType MTBDRsl Test of Complex Mechanisms of Resistance to Second-Line Drugs and Ethambutol in Multidrug-Resistant Mycobacterium tuberculosis Complex Isolates

ABSTRACT The GenoType MTBDRsl test rapidly detects resistance to ethambutol, fluoroquinolones, and second-line aminoglycosides (amikacin and kanamycin) and cyclic peptide (capreomycin) in Mycobacterium tuberculosis. A set of 41 multidrug-resistant (MDR) M. tuberculosis strains, 8 extensively drug-resistant (XDR) M. tuberculosis strains, and 3 non-MDR M. tuberculosis strains were tested by the MTBDRsl test and by DNA sequencing of the resistance-determining regions in gyrA and gyrB (fluoroquinolones [FQ]), rpsL (streptomycin), rrs and tlyA (aminoglycosides and/or cyclic peptide), and embB (ethambutol). The sensitivity and specificity of the MTBDRsl test were as follows: 87% and 96%, respectively, for fluoroquinolones; 100% for both for amikacin; 77% and 100%, respectively, for kanamycin, 80% and 98%, respectively, for capreomycin; and 57% and 92%, respectively, for ethambutol. Analysis of the discrepant results indicated that three FQ-resistant strains (including one XDR strain) with mutations in gyrB were missed by the MTBDRsl test and that one FQ-susceptible strain, identified as resistant by the MTBDRsl test, had a double mutation (T80A-A90G) in GyrA that did not confer resistance to FQ. Five strains (including two XDR strains) without mutations in rrs were monoresistant to aminoglycosides or cyclic peptide and were missed by the MTBDRsl test. Finally, 12/28 ethambutol-resistant strains had no mutation at codon 306 in embB, while 2/24 ethambutol-susceptible strains had such a mutation. In conclusion, the MTBDRsl test efficiently detects the most common mutations involved in resistance to fluoroquinolones, aminoglycosides/cyclic peptide, and ethambutol and accurately assesses susceptibility to amikacin. However, due to mutations not included in the test (particularly in gyrB) or resistance mechanisms not yet characterized (particularly those related to ethambutol resistance and to monoresistance to aminoglycosides or cyclic peptide), the wild-type results yielded by the MTBDRsl test should be confirmed by drug susceptibility testing.

Cloning and sequence analysis of the gene for a carbapenem-hydrolyzing class A beta-lactamase, Sme-1, from Serratia marcescens S6.

Serratia marcescens S6 produces a pI 9.7 carbapenem-hydrolyzing beta-lactamase that is probably encoded by the chromosome (Y. Yang, P. Wu, and D. M. Livermore, Antimicrob. Agents Chemother. 34:755-758, 1990). A total of 11.3 kb of genomic DNA from this strain was cloned into plasmid pACYC184 in Escherichia coli. After further subclonings, the carbapenem-hydrolyzing beta-lactamase gene (blaSme-1) was sequenced (EMBL accession number Z28968). The gene corresponded to an 882-bp open reading frame which encoded a 294-amino-acid polypeptide. This open reading frame was preceded by a -10 and a -35 region consistent with a putative promoter sequence of members of the family Enterobacteriaceae. This promoter was active in E. coli and S. marcescens, as demonstrated by primer extension analysis. N-terminal sequencing showed that the Sme-1 enzyme had a 27-amino-acid leader peptide and enabled calculation of the molecular mass of the mature protein (29.3 kDa). Sequence alignment revealed that Sme-1 is a class A serine beta-lactamase and not a class B metalloenzyme. The earlier view that the enzyme was zinc dependent was discounted. Among class A beta-lactamases, Sme-1 had the greatest amino acid identity (70%) with the pI 6.9 carbapenem-hydrolyzing beta-lactamase, NMC-A, from Enterobacter cloacae NOR-1. Comparison of these two protein sequences suggested a role for specific residues in carbapenem hydrolysis. The relatedness of Sme-1 to other class A beta-lactamases such as the TEM and SHV types was remote. This work details the sequence of the second carbapenem-hydrolyzing class A beta-lactamase from an enterobacterial species and the first in the genus Serratia. Images

Genetic Basis for Natural and Acquired Resistance to the Diarylquinoline R207910 in Mycobacteria

ABSTRACT The atpE gene encoding the subunit c of the ATP synthase of Mycobacterium tuberculosis, the target of the new diarylquinoline drug R207910, has been sequenced from in vitro mutants resistant to the drug. The previously reported mutation A63P and a new mutation, I66M, were found. The genetic diversity of atpE in 13 mycobacterial species was also investigated, revealing that the region involved in resistance to R207910 is conserved, except in Mycobacterium xenopi in which the highly conserved residue Ala63 is replaced by Met, a modification that may be associated with the natural resistance of M. xenopi to R207910.

Performance of the genotype MTBDR line probe assay for detection of resistance to rifampin and isoniazid in strains of Mycobacterium tuberculosis with low- and high-level resistance.

ABSTRACT We assessed the performance of the Genotype MTBDR line probe assay that offers the simultaneous identification of Mycobacterium tuberculosis and its resistance to rifampin (RIF) and isoniazid (INH) by detecting the most commonly found mutations in the rpoB and katG genes. One hundred thirteen M. tuberculosis isolates were tested. The nucleotide sequences of the katG and inhA genes and the mabA-inhA promoter region were also determined. The MTBDR assay detected 100% and 67% (n = 64) of the strains resistant to RIF and INH, respectively. Among the latter, 62 strains carried a Ser315Thr mutation in katG, 59 of them displaying a high level of resistance to INH. Two strains with a low level of INH resistance had a Ser315Asn mutation. No mutation was found by the MTBDR assay for 31 INH-resistant strains (33%), of which 24 showed a low level of resistance. By DNA sequencing, we found among them various mutations in the KatG protein for 7 strains, a C→T mutation in position −15 of the mabA-inhA promoter in 17 strains, and a Ser94Ala mutation in InhA for 7 strains. In conclusion, the MTBDR assay, which fits easily in the workflow of a routine laboratory, enabled the detection of 100% of the RIF-resistant strains and 89% of the INH-resistant strains with a high level of resistance but only 17% of the strains characterized by a low level of INH resistance, indicating that the test can be used as a rapid method to detect in the same experiment the rifampin-resistant and the high-level isoniazid-resistant strains of M. tuberculosis.

Genetic and Structural Insights into the Dissemination Potential of the Extremely Broad-Spectrum Class A β-Lactamase KPC-2 Identified in an Escherichia coli Strain and an Enterobacter cloacae Strain Isolated from the Same Patient in France

ABSTRACT Two clinical strains of Escherichia coli (2138) and Enterobacter cloacae (7506) isolated from the same patient in France and showing resistance to extended-spectrum cephalosporins and low susceptibility to imipenem were investigated. Both strains harbored the plasmid-contained blaTEM-1 and blaKPC-2 genes. blaKLUC-2, encoding a mutant of the chromosomal β-lactamase of Kluyvera cryocrescens, was also identified at a plasmid location in E. cloacae 7506, suggesting the ISEcp1-assisted escape of blaKLUC from the chromosome. Determination of the KPC-2 structure at 1.6 Å revealed that the binding site was occupied by the C-terminal (C-ter) residues coming from a symmetric KPC-2 monomer, with the ultimate C-ter Glu interacting with Ser130, Lys234, Thr235, and Thr237 in the active site. This mode of binding can be paralleled to the inhibition of the TEM-1 β-lactamase by the inhibitory protein BLIP. Determination of the 1.23-Å structure of a KPC-2 mutant in which the five C-ter residues were deleted revealed that the catalytic site was filled by a citrate molecule. Structure analysis and docking simulations with cefotaxime and imipenem provided further insights into the molecular basis of the extremely broad spectrum of KPC-2, which behaves as a cefotaximase with significant activity against carbapenems. In particular, residues 104, 105, 132, and 167 draw a binding cavity capable of accommodating both the aminothiazole moiety of cefotaxime and the 6α-hydroxyethyl group of imipenem, with the binding of the former drug being also favored by a significant degree of freedom at the level of the loop at positions 96 to 105 and by an enlargement of the binding site at the end of strand β3.

Identification of Mycobacterial Species by PCR Sequencing of Quinolone Resistance-Determining Regions of DNA Gyrase Genes

ABSTRACT The determination of the amino acid sequence of quinolone resistance-determining regions (QRDRs) in the A and B subunits of DNA gyrase is the molecular test for the detection of fluoroquinolone resistance in mycobacteria. We looked to see if the assignment of mycobacterial species could be obtained simultaneously by analysis of the corresponding nucleotide sequences. PCR sequencing of gyrA and gyrB QRDRs was performed for 133 reference and clinical strains of 21 mycobacterial species commonly isolated in clinical laboratories. Nucleotide sequences of gyrA and gyrB QRDRs were species specific, regardless of fluoroquinolone susceptibility.

Molecular Investigation of Resistance to the Antituberculous Drug Ethionamide in Multidrug-Resistant Clinical Isolates of Mycobacterium tuberculosis

ABSTRACT Ethionamide (ETH) needs to be activated by the mono-oxygenase EthA, which is regulated by EthR, in order to be active against Mycobacterium tuberculosis. The activated drug targets the enzyme InhA, which is involved in cell wall biosynthesis. Resistance to ETH has been reported to result from various mechanisms, including mutations altering EthA/EthR, InhA and its promoter, the NADH dehydrogenase encoded by ndh, and the MshA enzyme, involved in mycothiol biosynthesis. We searched for such mutations in 87 clinical isolates: 47 ETH-resistant (ETHr) isolates, 24 ETH-susceptible (ETHs) isolates, and 16 isolates susceptible to ETH but displaying an intermediate proportion of resistant cells (ETHSip; defined as ≥1% but <10% resistant cells). In 81% (38/47) of the ETHr isolates, we found mutations in ethA, ethR, or inhA or its promoter, which mostly corresponded to new alterations in ethA and ethR. The 9 ETHr isolates without a mutation in these three genes (9/47, 19%) had no mutation in ndh, and a single isolate had a mutation in mshA. Of the 16 ETHSip isolates, 7 had a mutation in ethA, 8 had no detectable mutation, and 1 had a mutation in mshA. Finally, of the 24 ETHs isolates, 23 had no mutation in the studied genes and 1 displayed a yet unknown mutation in the inhA promoter. Globally, the mechanism of resistance to ETH remained unknown for 19% of the ETHr isolates, highlighting the complexity of the mechanisms of ETH resistance in M. tuberculosis.

Emergence in Klebsiella pneumoniae of a Chromosome-Encoded SHV β-Lactamase That Compromises the Efficacy of Imipenem

ABSTRACT A Klebsiella pneumoniae isolate was identified that had reduced susceptibility to several expanded-spectrum cephalosporins and imipenem. That isolate produced a chromosome-encoded SHV-type β-lactamase, SHV-38, that had an alanine to valine substitution in position Ambler 146 compared to β-lactamase SHV-1. The kinetic parameters for purified β-lactamases SHV-38 and SHV-1 showed that the hydrolytic spectrum of SHV-38 included only ceftazidime and imipenem. This report is the first example of an SHV-type β-lactamase capable of hydrolyzing imipenem.

Novel class A beta-lactamase Sed-1 from Citrobacter sedlakii: genetic diversity of beta-lactamases within the Citrobacter genus.

ABSTRACT Citrobacter sedlakii 2596, a clinical strain resistant to aminopenicillins, carboxypenicillins, and early cephalosporins such as cephalothin, but remaining susceptible to acylureidopenicillins, carbapenems, and later cephalosporins such as cefotaxime, was isolated from the bile of a patient treated with β-lactam and quinolone antibiotics. The isolate produced an inducible class A β-lactamase of pI 8.6, named Sed-1, which was purified. Characterized by a molecular mass of 30 kDa, Sed-1 preferentially hydrolyzed benzylpenicillin, cephalothin, and cloxacillin. The corresponding gene,blaSed-1, was cloned and sequenced. Its deduced amino acid sequence shared more than 60% identity with the chromosome-encoded β-lactamases from Citrobacter koseri(formerly C. diversus) (84%), Klebsiella oxytoca (74%), Serratia fonticola (67%), andProteus vulgaris (63%) and 71% identity with the plasmid-mediated enzyme MEN-1. A gene coding for a LysR transcriptional regulator was found upstream from blaSed-1. This regulator, named SedR, displayed 90% identity with the AmpR sequence of the chromosomal β-lactamase from C. koseriand 63 and 50% identity with the AmpR sequences of P. vulgaris and Enterobacter cloacae, respectively. By using DNA-DNA hybridization, a blaSed-1-like gene was identified in two reference strains, C. sedlakii(CIP-105037) and Citrobacter rodentium (CIP-104675), but not in the 18 strains of C. koseri studied. Two DNA fragments were amplified and sequenced from the reference strains ofC. sedlakii CIP-105037 and C. rodentiumCIP-104675 using two primers specific forblaSed-1. They shared 98 and 80% identity withblaSed-1, respectively, confirming the diversity of the chromosomally encoded class A β-lactamases found inCitrobacter.

Structure of the imipenem-hydrolyzing class A β-­lactamase SME-1 from Serratia marcescens

The structure of the beta-lactamase SME-1 from Serratia marcescens, a class A enzyme characterized by its significant activity against imipenem, has been determined to 2.13 A resolution. The overall structure of SME-1 is similar to that of other class A beta-lactamases. In the active-site cavity, most of the residues found in SME-1 are conserved among class A beta-lactamases, except at positions 104, 105 and 237, where a tyrosine, a histidine and a serine are found, respectively, and at position 238, which is occupied by a cysteine forming a disulfide bridge with the other cysteine residue located at position 69. The crucial role played by this disulfide bridge in SME-1 was confirmed by site-directed mutagenesis of Cys69 to Ala, which resulted in a mutant unable to confer resistance to imipenem and all other beta-lactam antibiotics tested. Another striking structural feature found in SME-1 was the short distance separating the side chains of the active serine residue at position 70 and the strictly conserved glutamate at position 166, which is up to 1.4 A shorter in SME-1 compared with other class A beta-lactamases. Consequently, the SME-1 structure cannot accommodate the essential catalytic water molecule found between Ser70 and Glu166 in the other class A beta-lactamases described so far, suggesting that a significant conformational change may be necessary in SME-1 to properly position the hydrolytic water molecule involved in the hydrolysis of the acyl-enzyme intermediate.