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

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

Staphylococcus aureus VRSA-11B Is a Constitutive Vancomycin-Resistant Mutant of Vancomycin-Dependent VRSA-11A

International audienceABSTRACT Vancomycin-resistant Staphylococcus aureus VRSA-10 was isolated in 2009, whereas VRSA-11A and VRSA-11B were isolated from the same patient in 2010. Growth curves and determination of the nature of the peptidoglycan precursors and of the VanX d,d -dipeptidase activity in the absence and in the presence of vancomycin indicated that vancomycin resistance was inducible in VRSA-10, that VRSA-11A was partially dependent on glycopeptide for growth, and that VRSA-11B was constitutively resistant. Both VRSA-11A and -11B harbored an insertion sequence, IS Ef1 , at the same locus in the vanX-vanY intergenic region of Tn 1546 and an S 183 A mutation in the chromosomal d -alanyl: d -alanine ligase (Ddl). This substitution has been shown to be responsible for a drastic diminution of the affinity of the enzyme for d -Ala at subsite 1 in Escherichia coli DdlB. VRSA-11B exhibited an additional mutation, P 216 T, in the transcriptional regulator VanR, most probably associated with constitutive expression of vancomycin resistance. It is thus likely that VRSA-11B is a constitutive derivative of VRSA-11A selected during prolonged vancomycin therapy. Synthesis of peptidoglycan precursors ending in d -Ala- d -lactate was responsible for oxacillin susceptibility of VRSA-11A and VRSA-11B despite the presence of a wild-type mecA gene in both strains

Glycopeptide Resistance

International audienceGlycopeptides such as vancomycin and teicoplanin are active against clinically important Gram-positive pathogens. They act by binding, in a noncovalent fashion, to the C-terminal D-alanine-D-alanine dipeptide of the peptidoglycan precursors, preventing their incorporation into the growing wall and thus inhibiting cell-wall formation. Emergence of vancomycin resistance in enterococci was reported in 1986, approximately 30 years after the introduction of this antibiotic into clinical practice. Since then, vancomycin resistant enterococci have spread worldwide and are now one of the most common types of bacteria implicated in nosocomial infections in numerous countries. Glycopeptide resistance is due to acquisition of operons that encode enzymes responsible for synthesis of modified peptidoglycan precursors and for elimination of the precursors normally synthesized by the host. The origin of the resistance genes remains unclear. The mobility of certain van gene clusters by conjugation and transposition is expected to facilitate transfer of glycopeptide resistance from Enterococcus to more pathogenic bacteria such as staphylococci and streptococci. This mobility was confirmed in 2002 when the first clinical methicillin-resistant Staphylococcus aureus, highly resistant to glycopeptides by acquisition of a van operon from Enterococcus, was reported in the USA

VanA-Type Vancomycin-Resistant Staphylococcus aureus▿

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

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

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

Efflux-Mediated Antibiotic Resistance in Acinetobacter spp. ▿

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

Expression of the Resistance-Nodulation-Cell Division Pump AdeIJK in Acinetobacter baumannii Is Regulated by AdeN, a TetR-Type Regulator

International audienceABSTRACT 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 (C 582 ) 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

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

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