Browsing by Author "Bhardwaj, Pooja"
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Item Chlorhexidine Induces Vana-Type Vancomycin Resistance Genes in Enterococci(American Society for Microbiology, 2016-03/25) Bhardwaj, Pooja; Ziegler, Elizabeth; Palmer, Kelli L.; 0000-0002-7343-9271 (Palmer, KL); Bhardwaj, Pooja; Ziegler, Elizabeth; Palmer, Kelli L.Chlorhexidine is a bisbiguanide antiseptic used for infection control. Vancomycin-resistant E. faecium (VREfm) is among the leading causes of hospital-acquired infections. VREfm may be exposed to chlorhexidine at supra- and subinhibitory concentrations as a result of chlorhexidine bathing and chlorhexidine-impregnated central venous catheter use. We used RNA sequencing to investigate how VREfm responds to chlorhexidine gluconate exposure. Among the 35 genes upregulated ≥10-fold after 15 min of exposure to the MIC of chlorhexidine gluconate were those encoding VanA-type vancomycin resistance (vanHAX) and those associated with reduced daptomycin susceptibility (liaXYZ). We confirmed that vanA upregulation was not strain or species specific by querying other VanA-type VRE. VanB-type genes were not induced. The vanH promoter was found to be responsive to subinhibitory chlorhexidine gluconate in VREfm, as was production of the VanX protein. Using vanH reporter experiments with Bacillus subtilis and deletion analysis in VREfm, we found that this phenomenon is VanR dependent. Deletion of vanR did not result in increased chlorhexidine susceptibility, demonstrating that vanHAX induction is not protective against chlorhexidine. As expected, VanA-type VRE is more susceptible to ceftriaxone in the presence of sub-MIC chlorhexidine. Unexpectedly, VREfm is also more susceptible to vancomycin in the presence of subinhibitory chlorhexidine, suggesting that chlorhexidine-induced gene expression changes lead to additional alterations in cell wall synthesis. We conclude that chlorhexidine induces expression of VanA-type vancomycin resistance genes and genes associated with daptomycin nonsusceptibility. Overall, our results indicate that the impacts of subinhibitory chlorhexidine exposure on hospital-associated pathogens should be further investigated in laboratory studies.Item Comparative Analysis of the Orphan Crispr2 Locus in 242 Enterococcus Faecalis Strains(Public Library of Science, 2015-09-23) Hullahalli, Karthik; Rodrigues, Marinelle; Schmidt, Brendan D.; Li, Xiang; Bhardwaj, Pooja; Palmer, Kelli L.; Hullahalli, Karthik; Rodrigues, Marinelle; Schmidt, Brendan D.; Li, Xiang; Bhardwaj, Pooja; Palmer, Kelli L.Clustered, Regularly Interspaced Short Palindromic Repeats and their associated Cas proteins (CRISPR-Cas) provide prokaryotes with a mechanism for defense against mobile genetic elements (MGEs). A CRISPR locus is a molecular memory of MGE encounters. It contains an array of short sequences, called spacers, that generally have sequence identity to MGEs. Three different CRISPR loci have been identified among strains of the opportunistic pathogen Enterococcus faecalis. CRISPR1 and CRISPR3 are associated with the cas genes necessary for blocking MGEs, but these loci are present in only a subset of E. faecalis strains. The orphan CRISPR2 lacks cas genes and is ubiquitous in E. faecalis, although its spacer content varies from strain to strain. Because CRISPR2 is a variable locus occurring in all E. faecalis, comparative analysis of CRISPR2 sequences may provide information about the clonality of E. faecalis strains. We examined CRISPR2 sequences from 228 E. faecalis genomes in relationship to subspecies phylogenetic lineages (sequence types; STs) determined by multilocus sequence typing (MLST), and to a genome phylogeny generated for a representative 71 genomes. We found that specific CRISPR2 sequences are associated with specific STs and with specific branches on the genome tree. To explore possible applications of CRISPR2 analysis, we evaluated 14 E. faecalis bloodstream isolates using CRISPR2 analysis and MLST. CRISPR2 analysis identified two groups of clonal strains among the 14 isolates, an assessment that was confirmed by MLST. CRISPR2 analysis was also used to accurately predict the ST of a subset of isolates. We conclude that CRISPR2 analysis, while not a replacement for MLST, is an inexpensive method to assess clonality among E. faecalis isolates, and can be used in conjunction with MLST to identify recombination events occurring between STs.;Item Dicationic Imidazolium-Based Ionic Liquids: A New Strategy for Non-Toxic and Antimicrobial Materials(Royal Soc Chemistry) Gindri, Izabelle M.; Siddiqui, Danyal A.; Bhardwaj, Pooja; Rodriguez, Lucas C.; Palmer, Kelli L.; Frizzo, Clarissa P.; Martins, Marcos A. P.; Rodrigues, Danieli C.; Palmer, Kelli L.; Rodrigues, Danieli C.New dicationic imidazolium-based ionic liquids (ILs) were synthesized, characterized and tested in regards to cytotoxicity and antimicrobial activity. Insertion of a new cationic head and use of organic anions increased the biocompatibility of the ILs developed. IC₅₀ (concentration necessary to inhibit 50% of enzymatic activity) values obtained were considerably higher than those described for monocationic ILs, which indicates an improvement in cytocompatibility. Antimicrobial activity against bacterial species of clinical relevance in wounds and the oral environment was tested. The results showed that ILs were effective in inhibiting bacterial growth even below the minimum inhibitory concentration (MIC). It was observed that structural features that confer higher hydrophobicity to ILs decreased both the IC₅₀ and MIC simultaneously. However, it was possible to establish an equilibrium between those two effects, which gives the safe range of concentrations that ILs can be employed. The results demonstrated that the dicationic-imidazolium-based ILs synthesized may constitute a potent strategy for applications requiring non-toxic materials exhibiting antimicrobial activity.Item Reduced Chlorhexidine and Daptomycin Susceptibility in Vancomycin-Resistant Enterococcus Faecium after Serial Chlorhexidine Exposure(Amer Soc Microbiology, 2018-10-22) Bhardwaj, Pooja; Hans, Amrita; Ruikar, Kinnari; Guan, Ziqiang; Palmer, Kelli L.; 0000-0002-7343-9271 (Palmer, KL); Bhardwaj, Pooja; Hans, Amrita; Ruikar, Kinnari; Palmer, Kelli L.Vancomycin-resistant Enterococcus faecium strains (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). Subpopulations 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 for some populations with reduced DAP susceptibility. Our results are clinically significant because they identify a link between serial subinhibitory 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.Item Transcriptional and Adaptive Responses of Enterococcus Faecium to the Antiseptic Chlorhexidine(2017-08) Bhardwaj, Pooja; Palmer, Kelli L.Enterococcus faecium and E. faecalis are Gram-positive cocci that naturally colonize the gastrointestinal tracts of humans and animals. However, they are opportunistic pathogens that are particularly associated with central line-associated bloodstream infections (CLABSIs), wound infections, bacteremia, and endocarditis. Chlorhexidine (CHX), a bisbiguanide cationic antiseptic, is routinely used for patient bathing in critical care units and in various infection control practices to reduce CLABSI occurrence. Enterococci are likely frequently exposed to inhibitory as well as sub-inhibitory levels of CHX as a result of colonization of patients bathed with CHX or use of CHX-impregnated catheters. However, there is a critical gap in knowledge about the evolutionary outcomes of CHX bathing on bacteria associated with CLABSIs. Also, more generally, the mechanisms by which enterococci detect and respond to cell surface stress are poorly understood. The goal of my research is to investigate how vancomycin-resistant E. faecium detect and respond to CHX using RNA sequencing. In this dissertation, I describe the global transcriptional responses of E. faecium to CHX at the minimum inhibitory concentration. I find that CHX exposure induces expression of genes related to vancomycin and daptomycin (DAP) resistance, as well as transport systems and genes associated with extra-cytoplasmic stress. Due to the clinical significance of the upregulation of antibiotic resistance genes by a routinely used antiseptic, I use molecular and genetic approaches to understand the mechanism of induction of vancomycin resistance genes. In addition, I investigate evolutionary outcomes of CHX exposure on E. faecium utilizing in vitro serial passage experiments. I find that the bacterial populations with reduced susceptibility to CHX also have a significantly higher number of DAP-resistant mutants. I utilize whole-genome sequencing to investigate the mechanistic basis of CHX-induced daptomycin resistance in E. faecium. The findings of my dissertation have important clinical implications. Exposure to sub-inhibitory CHX can prime the enterococci for subsequent vancomycin exposure and impact treatment outcomes. If routine use of CHX can select for mutants with reduced susceptibility to CHX as well as cell surface-targeting antibiotics like DAP, this can contribute to treatment failures. Overall, the results of the dissertation highlight the importance of monitoring the effects of CHX exposure on E. faecium in healthcare settings.