Bruno Périchon

Efflux-Mediated Antibiotic Resistance in Acinetobacter spp.

ABSTRACT Among Acinetobacter spp., A. baumannii is the most frequently implicated in nosocomial infections, in particular in intensive care units. It was initially thought that multidrug resistance (MDR) in this species was due mainly to horizontal acquisition of resistance genes. However, it has recently become obvious that increased expression of chromosomal genes for efflux systems plays a major role in MDR. Among the five superfamilies of pumps, resistance-nodulation-division (RND) systems are the most prevalent in multiply resistant A. baumannii. RND pumps typically exhibit a wide substrate range that can include antibiotics, dyes, biocides, detergents, and antiseptics. Overexpression of AdeABC, secondary to mutations in the adeRS genes encoding a two-component regulatory system, constitutes a major mechanism of multiresistance in A. baumannii. AdeIJK, intrinsic to this species, is responsible for natural resistance, but since overexpression above a certain threshold is toxic for the host, its contribution to acquired resistance is minimal. The recently described AdeFGH, probably regulated by a LysR-type transcriptional regulator, also confers multidrug resistance when overexpressed. Non-RND efflux systems, such as CraA, AmvA, AbeM, and AbeS, have also been characterized for A. baumannii, as have AdeXYZ and AdeDE for other Acinetobacter spp. Finally, acquired narrow-spectrum efflux pumps, such as the major facilitator superfamily (MFS) members TetA, TetB, CmlA, and FloR and the small multidrug resistance (SMR) member QacE in Acinetobacter spp., have been detected and are mainly encoded by mobile genetic elements.

Transferable Resistance to Aminoglycosides by Methylation of G1405 in 16S rRNA and to Hydrophilic Fluoroquinolones by QepA-Mediated Efflux in Escherichia coli

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.

VanA-Type Vancomycin-Resistant Staphylococcus aureus

ABSTRACT Since 2002, nine methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) strains that are also resistant to vancomycin (VRSA) have been reported in the United States, including seven clinical isolates from Michigan. The strains harbor a plasmid-borne Tn1546 element following conjugation from a glycopeptide-resistant Enterococcus strain. In the second step, Tn1546 transposed to a resident plasmid in five strains; the acquired plasmid behaved as a suicide gene delivery vector, and the incoming DNA had been rescued by illegitimate recombination. Surprisingly, combination of a glycopeptide with a β-lactam has a strong synergistic effect against VRSA, both in vitro and in an animal model, despite resistance of the strains to both drug classes when administered separately. This results from the fact that the late peptidoglycan precursors ending in d-alanine-d-lactate (d-Ala-d-Lac) that are mainly synthesized in the presence of glycopeptide inducers are not substrates for PBP2′, which is the only transpeptidase that remains active in the presence of oxacillin. One VRSA strain is partially dependent on vancomycin for growth due to a mutation in the host d-Ala:d-Ala ligase, thus having to rely on the inducible resistance pathway for cell wall synthesis. Competition growth experiments in the absence of inducer between the MRSA recipient and isogenic VRSA transconjugant revealed a disadvantage for the transconjugant, accounting, in part, for the low level of dissemination of the VRSA clinical isolates. The association of multiple molecular and environmental factors has been implicated in the regional emergence of VRSA in Michigan.

Detection of the van Alphabet and Identification of Enterococci and Staphylococci at the Species Level by Multiplex PCR

ABSTRACT A multiplex PCR assay was developed for detection of the six types of glycopeptide resistance characterized in enterococci and for identification of Enterococcus faecium, Enterococcus faecalis, Staphylococcus aureus, and Staphylococcus epidermidis at the species level. Primers targeting the genes vanA, vanB, vanC, vanD, vanE, vanG, and ddl of E. faecium and E. faecalis and nuc of S. aureus and a chromosomal portion specific to S. epidermidis were designed to allow amplification of fragments with various sizes. This specific and sensitive technique allows detection of glycopeptide-resistant strains, in particular methicillin-resistant S. aureus, that may escape phenotype-based automated rapid methods.

Overexpression of Resistance-Nodulation-Cell Division Pump AdeFGH Confers Multidrug Resistance in Acinetobacter baumannii

ABSTRACT Acinetobacter baumannii is a major nosocomial pathogen which frequently develops multidrug resistance by acquisition of antibiotic resistance genes and overexpression of intrinsic efflux systems, such as the RND efflux pumps AdeABC and AdeIJK. A third RND system was characterized by studying spontaneous mutants BM4663 and BM4664, which were selected in the presence of chloramphenicol and norfloxacin, respectively, from the AdeABC- and AdeIJK-defective derivative A. baumannii BM4652. They exhibited enhanced resistance to fluoroquinolones, tetracycline-tigecycline, chloramphenicol, clindamycin, trimethoprim, sulfamethoxazole, sodium dodecyl sulfate, and dyes such as ethidium bromide, safranin O, and acridine orange. Comparison of transcriptomes of mutants with that of their parental strain, using a microarray technology, demonstrated the overexpression of three genes that encoded an RND efflux system, named AdeFGH. Inactivation of AdeFGH in BM4664 restored an antibiotic susceptibility profile identical to that of BM4652, indicating that AdeFGH was cryptic in BM4652 and responsible for multidrug resistance in its mutants. RNA analysis demonstrated that the three genes were cotranscribed. The adeFGH operon was found in 36 out of 40 A. baumannii clinical isolates, but none of the 22 isolates tested overexpressed the pump genes. Spontaneous MDR mutant BM4684, overexpressing adeFGH, was obtained from clinical isolate BM4587, indicating that adeFGH can be overexpressed in a strain harboring adeABC-adeIJK. An open reading frame, coding a LysR-type transcriptional regulator, named adeL, was located upstream from the adeFGH operon and transcribed in the opposite direction. Mutations in adeL were found in the three adeFGH-overexpressing mutants, suggesting that they were responsible for overexpression of AdeFGH.

Screening and Quantification of the Expression of Antibiotic Resistance Genes in Acinetobacter baumannii with a Microarray

ABSTRACT An oligonucleotide-based DNA microarray was developed to evaluate expression of genes for efflux pumps in Acinetobacter baumannii and to detect acquired antibiotic resistance determinants. The microarray contained probes for 205 genes, including those for 47 efflux systems, 55 resistance determinants, and 35 housekeeping genes. The microarray was validated by comparative analysis of mutants overexpressing or deficient in the pumps relative to the parental strain. The performance of the microarray was also evaluated using in vitro single-step mutants obtained on various antibiotics. Overexpression, confirmed by quantitative reverse transcriptase PCR, of RND efflux pumps AdeABC, due to a G30D substitution in AdeS in a multidrug-resistant (MDR) strain obtained on gentamicin, and AdeIJK, in two mutants obtained on cefotaxime or tetracycline, was detected. A new efflux pump, AdeFGH, was found to be overexpressed in a mutant obtained on chloramphenicol. Study of MDR clinical isolates, including the AYE strain, whose entire sequence has been determined, indicated overexpression of AdeABC and of the chromosomally encoded cephalosporinase as well as the presence of several acquired resistance genes. The overexpressed and acquired determinants detected by the microarray could account for nearly the entire MDR phenotype of the isolates. The microarray is potentially useful for detection of resistance in A. baumannii and should allow detection of new efflux systems associated with antibiotic resistance.

Sequence of Conjugative Plasmid pIP1206 Mediating Resistance to Aminoglycosides by 16S rRNA Methylation and to Hydrophilic Fluoroquinolones by Efflux

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, blaTEM-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, qacEΔ1, 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.

Synergism between β-Lactams and Glycopeptides against VanA-Type Methicillin-Resistant Staphylococcus aureus and Heterologous Expression of the vanA Operon

ABSTRACT Vancomycin resistance of Staphylococcus aureus NY-VRSA and VRSA-5 is due to acquisition of a vanA operon located in a Tn1546-like element. The vanA gene cluster of NY-VRSA contained one copy of insertion sequences IS1251 and IS1216V relative to that of VRSA-5. As evidenced by the nature of the late peptidoglycan precursors and by quantification of d,d-peptidase activities, the vancomycin resistance genes were efficiently expressed in both strains. Study of the stability and inducibility of glycopeptide resistance suggested that low-level glycopeptide resistance of NY-VRSA was most probably due to plasmid instability combined with a long delay for resistance induction. The activity of combinations of vancomycin or teicoplanin with oxacillin against the four VanA-type S. aureus strains already reported was tested by single and double disk diffusion, E-test on agar alone or supplemented with antibiotics, the checkerboard technique, and by determining time-kill curves. A strong synergism against the four clinical isolates, with fractional inhibitory concentration indexes from 0.008 to 0.024, was reproducibly observed between the two antibiotics by all methods. These observations indicate that cell wall inhibitors of the β-lactam and glycopeptide classes exert strong and mutual antagonistic effects on resistance to each other against VanA-type methicillin-resistant S. aureus.

Expression of the resistance-nodulation-cell division pump AdeIJK in Acinetobacter baumannii is regulated by AdeN, a TetR-type regulator.

ABSTRACT Resistance-nodulation-division efflux system AdeIJK contributes to intrinsic resistance to various drug classes in Acinetobacter baumannii. By whole-genome sequencing, we have identified in clinical isolate BM4587 the adeN gene, located 813 kbp upstream from adeIJK, which encodes a TetR transcriptional regulator. In one-step mutant BM4666 overexpressing adeIJK, the deletion of cytosine 582 (C582) in the 3′ portion of this gene was responsible for a frameshift mutation resulting in the deletion of the seven C-terminal residues. trans-Complementation of this BM4587 derivative with a plasmid expressing adeN restored antibiotic susceptibility to the host associated with the loss of adeJ overexpression. The inactivation of adeN in BM4587 led to a diminished susceptibility to antibiotics that are substrates for AdeIJK and to a 5-fold increase in adeJ expression. Taken together, these results indicate that AdeN represses AdeIJK expression. Quantitative reverse transcription-PCR (qRT-PCR) demonstrated that AdeN is constitutively expressed in BM4587 and does not regulate its own expression. Deletion of cytosine 582 and a 394-bp deletion of the 3′ part of adeN were found in independent one-step adeIJK-overexpressing mutants selected from clinical isolates BM4667 and BM4651, respectively. The corresponding alterations were located in the α9 helix, which is known to be involved in dimerization, a process essential for the activity of TetR regulators. The adeN gene was detected in all of the 30 A. baumannii strains tested and in Acinetobacter calcoaceticus, Acinetobacter nosocomialis, and Acinetobacter pittii.

Heterologous Expression of the Enterococcal vanA Operon in Methicillin-Resistant Staphylococcus aureus

ABSTRACT Two methicillin- and vancomycin-resistant Staphylococcus aureus strains, MI-VRSA and PA-VRSA, and Enterococcus faecalis DMC83006B, considered to be the potential donor of glycopeptide resistance to MI-VRSA, were studied. MI-VRSA is highly resistant to both glycopeptides, whereas PA-VRSA displays low-level resistance to vancomycin and reduced susceptibility to teicoplanin. We have analyzed the expression of the vanA operon in the three clinical isolates. Determination of the relative amounts of late peptidoglycan precursors and quantification of the d,d-peptidase activities, in the absence or after induction by glycopeptides, revealed that the resistance genes were expressed at similarly high levels in the three strains. Glycopeptide resistance stability in the three strains was studied by replica plating. Resistance was lost at high frequency, ca. 50%, after overnight growth of PA-VRSA in the absence of antibiotics, whereas it was fully stable in MI-VRSA and E. faecalis DMC83006B. Induction of resistance by vancomycin was significantly delayed in PA-VRSA relative to MI-VRSA. Low-level glycopeptide resistance of S. aureus PA-VRSA is thus likely due to instability of the genetic element, plasmid or transposon, carrying the vanA operon associated with a longer lag phase before growth resumes after induction by vancomycin.