High-Level Ciprofloxacin Resistance from Point Mutations in gyrA and parC Confined to Global Hospital-Adapted Clonal Lineage CC17 of Enterococcus faecium

Abstract

To substantiate a common genetic background of ciprofloxacin-resistant Enterococcus faecium, 32 ciprofloxacin-resistant (Cip r ) and 31 ciprofloxacin-susceptible (Cip s ) isolates from outbreaks, clinical infections, surveillances, and animals from 10 different countries were genotyped by multilocus sequence typing. Additionally, susceptibilities to ampicillin and vancomycin and the presence of esp were determined and the quinolone resistance-determining regions of parC, gyrA, parB, and gyrE were sequenced. High-level Cip r (MIC > 64 g/ml) due to point mutations in the quinolone resistance-determining region was unique to a distinct hospital-adapted genetic complex in E. faecium, previously designated CC17. Low-level Cip r (MIC ‫؍‬ 4 g/ml) in non-CC17 strains is not attributable to point mutations in any subunit of the topoisomerase genes, and the mechanism of resistance remains unclear. Acquisition of mutations in parC and gyrA, leading to high-level Cip r , is, in addition to ampicillin resistance and the presence of a putative pathogenicity island, another cumulative step in hospital adaptation of CC17.Over the last two decades, enterococci have become increasingly important as nosocomial pathogens (3). These organisms are intrinsically resistant to a large number of antimicrobials and have the ability to easily acquire new resistance traits (40, 50). The emergence of nosocomial infections caused by ampicillin-, high-level aminoglycoside-, and glycopeptide-resistant Enterococcus faecium has caused clinical concern due to intraand interhospital spread and limited therapeutic options (40, 50). Glycopeptide-resistant enterococci (vancomycin-resistant enterococci [VRE]) are nowadays endemic to the United States, with ϳ30% of enterococcal infections caused by VRE (38, 43); in Europe, the epidemiology of VRE is now changing from a near absence of VRE in hospital-acquired infections at the turn of the century to a situation in which nosocomial epidemics and infections are increasingly reported (3, 13, 17). We recently described a hospital-adapted genetic subtype of E. faecium associated with epidemics and clinical infections, which has spread globally (20,32,62,65). This subpopulation belongs to a distinct genetic lineage labeled clonal complex 17 (CC17) (62) and is associated with the presence of the variant esp gene as part of a pathogenicity island (31) and resistance to ampicillin. Recently, resistance to ciprofloxacin appeared to be associated with ampicillin resistance in genotypically related E. faecium isolates from Norway (25,55,56) and Spain (7).To substantiate a common genetic background of quinoloneresistant E. faecium and association with the hospital-adapted CC17, we studied the genetic relatedness of 32 ciprofloxacinresistant and 31 ciprofloxacin-susceptible E. faecium isolates from various human and animal origins by multilocus sequence typing (MLST) and determined susceptibility to vancomycin and ampicillin and the presence of esp. Finally, we sequenced the quinolone resistance-determining regions (...

Antimicrobial Resistance and Multilocus Sequence Typing of Vancomycin-ResistantEnterococcus faeciumIsolated from the Chungcheong Area

“…97% of those isolates showed ciprofloxacin resistance according to antibiotic susceptibility testing. Ser80→Arg mutation of parC and Ser83→Arg mutation of gyrA were most commonly found in 17 of 37 isolates (45.9%), and these mutations are known as most frequently observed mutations in QRDR [13].…”

“…It is thought that genetically similar VREs are prevalent in this study. The genetic background of quinolone resistant E. faecium and its relation with CC17 were proved in Holland in 2005 [13]. MLST revealed that most of isolates belonged to CC17 and consequently investigation on CC17 as possible genetic aberrations causing quinolone resistance was made.…”

“…Primers for the detection of parC and gyrA gene were utilized as follows: parC-F (5'-TTCCCGTGCATTTCGATCAGTACT-TC-3') and parC-R (5'-CGTATGACAAAGGATTCGGTAAA-TC-3') and gyrA-F (CGGGATGAACGAATTGGGTGTGA-3') and gyrA-R (5'-AATTTTACTCATACGTGCTTCGG-3') [13]. …”

Current Trends of Emergence and Spread of Vancomycin-Resistant Enterococci

“…and the necessity of specific mutations in one or both A subunit genes to confer what is specified as high-level ciprofloxacin resistance (Onodera et al, 2002;Oyamada et al, 2006a;Oyamada et al, 2006b). Molecular studies with high-level ciprofloxacin-resistant clinical isolates revealed mutations in both A subunits associated with different levels of ciprofloxacin resistance, whereas mutations in gyrB and parE alleles were only infrequently found Leavis et al, 2006;Valdezate et al, 2009;Werner et al, 2010a).…”

Performance of three chromogenic VRE screening agars, two Etest® vancomycin protocols, and different microdilution methods in detecting vanB genotype Enterococcus faecium with varying vancomycin MICs

“…Classification into susceptible/intermediate/resistant (S-I-R) differs between European Committee on Antimicrobial Susceptibility Testing (EUCAST) and Clinical Laboratory Standard Institutes (CLSI) (CLSI, 2011); EUCAST defines MICs for vancomycin with S ≤ 4 mg/L and R > 4 mg/L (no intermediate range) and CLSI defines S ≤ 4 mg/L, I = 8-16 mg/L, and R ≥ 32 mg/L. We used the EUCAST clinical breakpoints when available; for other antibiotics, we applied breakpoints derived from CLSI, DIN, and based on other criteria, e.g., for high-level ciprofloxacin resistance defined as an MIC > 16 mg/L ( Leavis et al, 2006 andWerner et al, 2010). Strains were classified as resistant with MICs (in milligrams per liter) as follows: penicillin/ampicillin > 8, teicoplanin > 2, erythromycin > 4, linezolid > 4, tetracycline > 4, rifampicin > 0.5, chloramphenicol > 16, tigecycline > 0.5, daptomycin > 4, gentamicin (high-level) > 128, streptomycin (high-level) > 512, quinupristin/dalfopristin > 4.…”