Vancomycin-resistant enterococci (VRE) are important nosocomial pathogens that cause life-threatening infections. To control hospital-associated infections, skin antisepsis and bathing utilizing chlorhexidine is recommended for VRE patients in acute care hospitals. Previously, we reported that exposure to inhibitory chlorhexidine levels induced the expression of vancomycin resistance genes in VanA-type Enterococcus faecium. However, vancomycin susceptibility actually increased for VanA-type E. faecium in the presence of chlorhexidine. Hence, a synergistic effect of the two antimicrobials was observed. In this study, we used multiple approaches to investigate the mechanism of synergism between chlorhexidine and vancomycin in the VanA-type VRE strain E. faecium 1,231,410. We generated clean deletions of 7 of 11 pbp, transpeptidase, and carboxypeptidase genes in this strain (ponA, pbpF, pbpZ, pbpA, ddcP, ldtfm, and vanY). Deletion of ddcP, encoding a membrane-bound carboxypeptidase, altered the synergism phenotype. Furthermore, using in vitro evolution, we isolated a spontaneous synergy escaper mutant and utilized whole genome sequencing to determine that a mutation in pstB, encoding an ATPase of phosphate-specific transporters, also altered synergism. Finally, addition of excess D-lactate, but not D-alanine, enhanced synergism to reduce vancomycin MIC levels. Overall, our work identified factors that alter chlorhexidine and vancomycin synergism in a model VanA-type VRE strain.
28Enterococci are Gram-positive gastrointestinal tract colonizers of humans and animals. 29Vancomycin-resistant enterococci (VRE) are important nosocomial pathogens and can cause 30 life-threatening infections. To control hospital-associated infections, skin antisepsis and bathing 31 utilizing chlorhexidine is recommended for VRE patients in acute care hospitals. Previously, we 32 reported that exposure to inhibitory chlorhexidine levels induced the expression of vancomycin 33 resistance genes in VanA-type Enterococcus faecium. However, vancomycin susceptibility 34 actually increased for VanA-type E. faecium in the presence of chlorhexidine. Hence, a 35 synergistic effect of the two antimicrobials was observed. In this study, we tested various 36 models to elucidate the mechanism(s) of synergism between chlorhexidine and vancomycin. 37We deleted each of the pbp genes from a model VanA-type VRE E. faecium strain. We found 38 that deletion of ddcP, a membrane-bound carboxypeptidase, resulted in partial loss of 39 synergism. Interestingly, addition of excess D-lactate, but not D-alanine, enhanced synergism. 40Furthermore, we isolated a synergy escaper mutant in E. faecium and utilized whole genome 41 sequencing to determine that a mutation in a gene encoding an ATPase of phosphate-specific 42 transporters (pstB) also resulted in loss of synergism. Our study is significant because 43 understanding the mechanisms for chlorhexidine-induced vancomycin resensitization in VRE 44 could lead to new combinatorial therapeutics to treat VRE infections. 45 46 47 48 49 50 51 52 Enterococcus faecium and E. faecalis are Gram-positive commensal bacteria inhabiting the 55 gastrointestinal tracts of humans and animals (1). A recently published evolutionary history of 56 the enterococci elucidated how these bacteria became the leading causes of hospital-57 associated infections (2). The ability to survive in harsh environmental conditions including 58 starvation and desiccation facilitated the emergence of hospital-adapted strains which are 59 resistant to the action of antibiotics and disinfectants. Hospital-adapted enterococcal strains 60 have limited treatment options and are typically characterized by high-level resistance to 61 vancomycin, a glycopeptide antibiotic which inhibits the process of peptidoglycan synthesis (3, 62 4). Vancomycin-resistant enterococci (VRE) synthesize peptidoglycan precursors for which 63 vancomycin has low affinity (5-8). Vancomycin resistance in hospital-adapted enterococcal 64isolates occurs through the horizontal acquisition of resistance genes (9, 10). For VanA-type 65 VRE, vancomycin resistance is conferred and controlled by the activities encoded by the 66 vanRS, vanHAX, and vanYZ genes. 68Patients in critical care units are frequently bathed or cleansed with chlorhexidine, a cationic cell 69 membrane-targeting antimicrobial, to reduce the occurrence of hospital-associated infections (11)(12)(13). Chlorhexidine interacts with the negatively charged phospholipids and proteins on the 71 cell membra...
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