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.

Molecular characterization was performed for 46 vancomycin-resistant E. faecium (VREfm) isolates and one vancomycin-sensitive comparator obtained during routine fecal surveillance of high-risk patients from a Dallas, Texas area hospital system. Hybrid assemblies of long (Oxford Nanopore Technology) and short (Illumina) sequence reads enabled the generation of 31 complete and 16 draft high quality genome sequences. The VREfm isolates possessed up to 12 plasmids each. A total of 251 closed plasmid sequences, assigned to 12 previously described and 9 newly defined rep family types, were obtained. Phylogenetic analysis clustered the Dallas isolates into genomic Clade A1, including 10 different sequence types (STs), the most prevalent ones being ST17 and ST18. VREfm isolates with the novel sequence type ST1703 were also identified. All but three of the VREfm isolates encoded vanA-type vancomycin resistance within Tn1546-like elements on a pRUM-like plasmid backbone. New variants of the Tn1546 were described that harbored a combination of 7 insertion sequences (IS) including 3 novel IS elements reported in this study (ISEfa16, ISEfa17 and ISEfa18). The pRUM-like plasmids carrying the Tn1546 were grouped into 4 sub-groups based on the replication and stability modules of the plasmid backbone. Overall, we conclude that the VREfm isolates analyzed in our collection are representative of other global VanA-type VREfm in that they belong to the Clade A1 lineage, but they possess novel arrangements of the Tn1546-like elements and the vanA operon, which have evolved independently of the pRUM-like backbone.

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...

Vancomycin is an antibiotic used to treat infections caused by multidrug-resistant Gram-positive bacteria. Vancomycin resistance is common in clinical isolates of the Gram-positive pathogen Enterococcus faecium .

Enterococcus faecalis is a leading cause of hospital-acquired infections. These infections are becoming more difficult to treat due to the increasing emergence of E. faecalis strains resistant to last resort antibiotics. Over the past decade, multiple groups have engineered the naturally occurring bacterial defense system CRISPR-Cas as a sequence-specific antimicrobial to combat antibiotic-resistant bacteria. We have previously established that the type II CRISPR-Cas system of E. faecalis can be reprogrammed as a CRISPR-Cas antimicrobial and delivered to antibiotic-resistant recipients on a conjugative pheromone-responsive plasmid. Using a co-culture system, we showed sequence-specific depletion of antibiotic resistance from E. faecalis model strains, both in vitro and in vivo. Although this and other studies have demonstrated the potential use for CRISPR-Cas as an antimicrobial, most have deployed the system against model bacterial strains. Thus, there is limited knowledge on how effective these potential therapies are against recently isolated and uncharacterized strains with limited laboratory passage, which we refer to here as wild strains. Here, we compare the efficacy of our previously established CRISPR-Cas antimicrobials against both E. faecalis model strains and wild E. faecalis fecal isolates. We demonstrate that these wild isolates can antagonize the CRISPR-Cas antimicrobial donor strain via competitive factors like cytolysin. Furthermore, we show that the wild isolates can effectively prevent delivery of the CRISPR-Cas antimicrobial plasmids, consequently avoiding CRISPR-Cas targeting. Our results emphasize the requisite to study CRISPR-Cas antimicrobials against wild strains to understand limitations and develop delivery systems that can endure competitive interspecies interactions in the gut microenvironment and effectively deliver CRISPR-Cas antimicrobials to their intended targets.

The human microbiota harbors diverse bacterial and bacteriophage (phage) communities. Bacteria evolve to overcome phage infection, thereby driving phage evolution to counter bacterial resistance. Understanding how phages select for genetic alterations in medically relevant bacteria is important as phages become established biologics for the treatment of multidrug‐resistant (MDR) bacterial infections. Before phages can be widely used as standalone or combination antibacterial therapies, we must obtain a deep understanding of the molecular mechanisms of phage infection and how host bacteria alter their genomes to become resistant. We performed coevolution experiments using a single Enterococcus faecalis strain and two distantly related phages to determine how phage pressure impacts the evolution of the E. faecalis genome. Whole‐genome sequencing of E. faecalis following continuous exposure to these two phages revealed mutations previously demonstrated to be essential for phage infection. We also identified mutations in genes previously unreported to be associated with phage infection in E. faecalis . Intriguingly, there was only one shared mutation in the E. faecalis genome that was selected by both phages tested, demonstrating that infection by two genetically distinct phages selects for diverse variants. This knowledge serves as the basis for the continued study of E. faecalis genome evolution during phage infection and can be used to inform the design of future therapeutics, such as phage cocktails, intended to target MDR E. faecalis .

The human microbiota harbors diverse bacterial and bacteriophage (phage) communities. Bacteria evolve to overcome phage infection, thereby driving phage evolution to counter bacterial resistance. Understanding how phages promote genetic alterations in medically relevant bacteria is important as phages continue to become established biologics for the treatment of multidrug-resistant (MDR) bacterial infections. Before phages are used as standalone or combination antibacterial therapies, we must obtain a deep understanding of the molecular mechanisms of phage infection and how host bacteria alter their genomes to become resistant. We performed coevolution experiments using a single Enterococcus faecalis strain and two distantly related phages, to determine how phage pressure impacts the evolution of the E. faecalis genome. Whole genome sequencing revealed mutations previously demonstrated to be essential for phage infection. We also identified mutations in several genes previously unreported to be associated with phage infection in E. faecalis. Intriguingly, there was only one shared mutation in the E. faecalis genome in response to each of the two phages tested, demonstrating that infection by genetically distinct phages results in different host responses. This study shows that infection of the same host by disparate phages leads to evolutionary trajectories that result in distinct genetic changes. This implies that bacteria respond to phage pressure through host responses that are tailored to specific phages. This work serves as the basis for the study of E. faecalis genome evolution during phage infection and will inform the design of future therapeutics, such as phage cocktails, intended to target MDR E. faecalis.IMPORTANCEStudies characterizing the genome evolution of bacterial pathogens following phage selective pressure are lacking. Phage therapy is experiencing a rebirth in Western medicine. Such studies are critical for understanding how bacteria subvert phage infection and how phages evolve to counter such mutations. This study utilizes comparative genomic analyses to demonstrate how a pathogenic strain of Enterococcus faecalis responds to infection by two genetically distant phages. We show that genetic alterations in the E. faecalis genome accumulate in a manner that is specific to the infecting phage with little to no overlap in shared fixed mutations. This suggests that bacterial genome evolution in response to phage infection is uniquely tied to phage genotype, and sets a precedence for investigations into how phages drive bacterial genome evolution relevant to phage therapeutic applications.

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