Biology subjects

Palmer, K. L.

Publications and source records attributed to Palmer, K. L..

5 recordsLinked to original sources

bioRxiv2018

Factors mediating synergism between vancomycin and chlorhexidine in VanA-type vancomycin-resistant Enterococcus faecium

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. Overall, our work identified factors that alter chlorhexidine-induced vancomycin resensitization in a model VanA-type VRE strain.

microbiology↗

bioRxiv2018

A Type I Restriction-Modification System Associated with Enterococcus faecium Subspecies Separation

The gastrointestinal colonizer Enterococcus faecium is a leading cause of hospital-acquired infections. Multidrug-resistant (MDR) E. faecium are particularly concerning for infection treatment. Previous comparative genomic studies revealed that subspecies referred to as Clade A and Clade B exist within E. faecium. MDR E. faecium belong to Clade A, while Clade B consists of drug-susceptible fecal commensal E. faecium. Isolates from Clade A are further grouped into two sub-clades, A1 and A2. In general, Clade A1 isolates are hospital epidemic isolates whereas Clade A2 isolates are isolates from animals and sporadic human infections. Such phylogenetic separation indicates that reduced gene exchange occurs between the clades. We hypothesize that endogenous barriers to gene exchange exist between E. faecium clades. Restriction-modification (R-M) systems are such barriers in other microbes. We utilized bioinformatics analysis coupled with second generation and third generation deep sequencing platforms to characterize the methylome of two representative E. faecium strains, one from Clade A1 and one from Clade B. We identified a Type I R-M system that is Clade A1-specific, is active for DNA methylation, and significantly reduces transformability of Clade A1 E. faecium. Based on our results, we conclude that R-M systems act as barriers to horizontal gene exchange in E. faecium and propose that R-M systems contribute to E. faecium subspecies separation.\n\nIMPORTANCEEnterococcus faecium is a leading cause of hospital-acquired infections around the world. Rising antibiotic resistance in certain E. faecium lineages leaves fewer treatment options. The overarching aim of the attached work was to determine whether restriction-modification (R-M) systems contribute to the structure of the E. faecium species, wherein hospital-epidemic and non-hospital-epidemic isolates have distinct evolutionary histories and highly resolved clade structures. R-M provides bacteria with a type of innate immunity to horizontal gene transfer (HGT). We identified a Type I R-M system that is enriched in the hospital-epidemic clade and determined that it is active for DNA modification activity and significantly impacts HGT. Overall, this work is important because it provides a mechanism for the observed clade structure of E. faecium as well as a mechanism for facilitated gene exchange among hospital-epidemic E. faecium.

microbiology↗

bioRxiv2018

CRISPR-Cas blocks antibiotic resistance plasmid transfer between Enterococcus faecalis strains in the gastrointestinal tract

CRISPR-Cas systems are barriers to horizontal gene transfer (HGT) in bacteria. Little is known about CRISPR-Cas interactions with conjugative plasmids, and studies investigating CRISPR-Cas/plasmid interactions in in vivo models relevant to infectious disease are lacking. These are significant gaps in knowledge because conjugative plasmids disseminate antibiotic resistance genes among pathogens in vivo, and it is essential to identify strategies to reduce the spread of these elements. We use enterococci as models to understand the interactions of CRISPR-Cas with conjugative plasmids. Enterococcus faecalis is a native colonizer of the mammalian intestine and harbors pheromone-responsive plasmids (PRPs). PRPs mediate inter- and intraspecies transfer of antibiotic resistance genes. We assessed E. faecalis CRISPR-Cas anti-PRP activity in the mouse intestine and under varying in vitro conditions. We observed striking differences in CRISPR-Cas efficiency in vitro versus in vivo. With few exceptions, CRISPR-Cas blocked intestinal PRP dissemination, while in vitro, the PRP frequently escaped CRISPR-Cas defense. Our results further the understanding of CRISPR-Cas biology by demonstrating that standard in vitro experiments do not adequately model the in vivo anti-plasmid activity of CRISPR-Cas. Additionally, our work identifies several variables that impact the apparent in vitro anti-plasmid activity of CRISPR-Cas, including planktonic versus biofilm settings, different donor/recipient ratios, production of a plasmid-encoded bacteriocin, and the time point at which matings are sampled. Our results are clinically significant because they demonstrate that barriers to HGT encoded by normal human microbiota can have significant impacts on in vivo antibiotic resistance dissemination.\n\nImportanceCRISPR-Cas is a type of immune system encoded by bacteria that is hypothesized to be a natural impediment to the spread of antibiotic resistance genes. In this study, we directly assessed the impact of CRISPR-Cas on antibiotic resistance dissemination in the mammalian intestine and under varying in vitro conditions. We observed a robust effect of CRISPR-Cas on in vivo but not in vitro dissemination of antibiotic resistance plasmids in the native mammalian intestinal colonizer Enterococcus faecalis. We conclude that standard laboratory experiments currently do not appropriately model the in vivo conditions where antibiotic resistance dissemination occurs between E. faecalis strains. Moreover, our results demonstrate that CRISPR-Cas encoded by native members of the mammalian intestinal microbiota can block the spread of antibiotic resistance plasmids.

microbiology↗

bioRxiv2017

Evolutionary outcomes of plasmid-CRISPR conflicts in an opportunistic pathogen

Enterococcus faecalis is a Gram-positive bacterium that natively colonizes the human gastrointestinal tract and opportunistically causes life-threatening infections. Multidrug-resistant (MDR) E. faecalis strains have emerged that are replete with mobile genetic elements (MGEs). Non-MDR E. faecalis strains frequently possess CRISPR-Cas systems, which reduce the frequency of mobile genetic element (MGE) acquisition. We demonstrated in previous studies that E. faecalis populations can transiently maintain both a functional CRISPR-Cas system and a CRISPR-Cas target. In this study, we used serial passage and deep sequencing to analyze these populations. In the presence of antibiotic selection for the plasmid, mutants with compromised CRISPR-Cas defense and enhanced ability to acquire a second antibiotic resistance plasmid emerged. Conversely, in the absence of selection, the plasmid was lost from wild-type E. faecalis populations, but not E. faecalis populations that lacked the cas9 gene. Our results indicate that E. faecalis CRISPR-Cas can become compromised under antibiotic selection, generating populations with enhanced abilities to undergo horizontal gene transfer. ImportanceEnterococcus faecalis is a leading cause of hospital-acquired infections and disseminator of antibiotic resistance plasmids among Gram-positive bacteria. We have previously shown that E. faecalis strains with an active CRISPR-Cas system can prevent plasmid acquisition and thus limit the transmission of antibiotic resistance determinants. Yet, CRISPR-Cas was not a perfect barrier. In this study, we observed populations of E. faecalis with transient co-existence of CRISPR-Cas and one of its plasmid targets. Our experimental data demonstrate that antibiotic selection results in compromised E. faecalis CRISPR-Cas function, thereby facilitating the acquisition of additional resistance plasmids by E. faecalis.

microbiology↗

bioRxiv2017

Reduced chlorhexidine and daptomycin susceptibility arises in vancomycin-resistant Enterococcus faecium after serial chlorhexidine exposure

Vancomycin-resistant Enterococcus faecium (VREfm) are critical public health concerns because they are among the leading causes of hospital-acquired bloodstream infections. Chlorhexidine (CHX) is a bisbiguanide cationic antiseptic that is routinely used for patient bathing and other infection control practices. VREfm are likely frequently exposed to CHX; however, the long-term effects of CHX exposure have not been studied in enterococci. In this study, we serially exposed VREfm to increasing concentrations of CHX for a period of 21 days in two independent experimental evolution trials. Reduced CHX susceptibility emerged (4-fold shift in CHX MIC). Sub-populations with reduced daptomycin (DAP) susceptibility were detected, which were further analyzed by genome sequencing and lipidomic analysis. Across the trials, we identified adaptive changes in genes with predicted or experimentally confirmed roles in chlorhexidine susceptibility (efrE), global nutritional stress response (relA), nucleotide metabolism (cmk), phosphate acquisition (phoU), and glycolipid biosynthesis (bgsB), among others. Moreover, significant alterations in membrane phospholipids were identified. Our results are clinically significant because they identify a link between serial sub-inhibitory CHX exposure and reduced DAP susceptibility. In addition, the CHX-induced genetic and lipidomic changes described in this study offer new insights into the mechanisms underlying the emergence of antibiotic resistance in VREfm.

microbiology↗