Palmer, Kelli L.

Permanent URI for this collectionhttps://hdl.handle.net/10735.1/4295

Kelli Palmer is an Assistant Professor in the Department of Molecular and Cell Biology. Dr. Palmer uses genomic, transcriptomic, and biochemical approaches to study antibiotic resistance in pathogenic bacteria. Her research focuses on microorganisms contributing to significant mortality and cost burdens in the health care industry.

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A Type I Restriction-Modification System Associated with Enterococcus Faecium Subspecies Separation
(Amer Soc Microbiology, 2019-01-09) Huo, Wenwen; Adams, Hannah M.; Trejo, Cristian; Badia, Rohit; Palmer, Kelli L.; 0000-0002-7343-9271 (Palmer, KL); Huo, Wenwen; Adams, Hannah M.; Trejo, Cristian; Badia, Rohit; Palmer, Kelli L.
The gastrointestinal colonizer Enterococcus faecium is a leading cause of hospital-acquired infections. Multidrug-resistant (MDR) E. faecium isolates 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 isolates belong to clade A, while clade B consists of drug-susceptible fecal commensal E. faecium isolates. Isolates from clade A are further grouped into two subclades, 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 a bioinformatics analysis coupled with second-generation and third-generation deep-sequencing platforms to characterize the methylomes 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 the 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. IMPORTANCE Enterococcus 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 this 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 isolates.
Bacteriophage Resistance Alters Antibiotic-Mediated Intestinal Expansion of Enterococci
(American Society for Microbiology, 2019-05-21) Chatterjee, A.; Johnson, C. N.; Luong, P.; Hullahalli, Karthik; McBride, S. W.; Schubert, A. M.; Palmer, Kelli L.; Carlson, P. E.; Duerkop, B. A.; 0000-0002-7343-9271 (Palmer, KL); Hullahalli, Karthik; Palmer, Kelli L.
Enterococcus faecalis is a human intestinal pathobiont with intrinsic and acquired resistance to many antibiotics, including vancomycin. Nature provides a diverse and virtually untapped repertoire of bacterial viruses, or bacteriophages (phages), that could be harnessed to combat multidrug-resistant enterococcal infections. Bacterial phage resistance represents a potential barrier to the implementation of phage therapy, emphasizing the importance of investigating the molecular mechanisms underlying the emergence of phage resistance. Using a cohort of 19 environmental lytic phages with tropism against E. faecalis, we found that these phages require the enterococcal polysaccharide antigen (Epa) for productive infection. Epa is a surface-exposed heteroglycan synthesized by enzymes encoded by both conserved and strain-specific genes. We discovered that exposure to phage selective pressure favors mutation in nonconserved epa genes both in culture and in a mouse model of intestinal colonization. Despite gaining phage resistance, epa mutant strains exhibited a loss of resistance to cell wall-targeting antibiotics. Finally, we show that an E. faecalis epa mutant strain is deficient in intestinal colonization, cannot expand its population upon antibiotic-driven intestinal dysbiosis, and fails to be efficiently transmitted to juvenile mice following birth. This study demonstrates that phage therapy could be used in combination with antibiotics to target enterococci within a dysbiotic microbiota. Enterococci that evade phage therapy by developing resistance may be less fit at colonizing the intestine and sensitized to vancomycin, preventing their overgrowth during antibiotic treatment. Copyright © 2019 American Society for Microbiology. All Rights Reserved.
Direct Detection of Tissue-Resident Bacteria and Chronic Inflammation in the Bladder Wall of Postmenopausal Women with Recurrent Urinary Tract Infection
(Academic Press) De Nisco, N. J.; Neugent, Michael; Mull, J.; Chen, L.; Kuprasertkul, A.; de Souza Santos, M.; Palmer, Kelli L.; Zimmern, P.; Orth, K.; 0000-0002-7343-9271 (Palmer, KL); Neugent, Michael; Palmer, Kelli L.
Urinary tract infections (UTIs) are the most commonly reported infections in adult women and have high rates of recurrence, especially in postmenopausal women. Recurrent UTI (RUTI) greatly reduces quality of life, places a significant burden on the healthcare system, and contributes to antimicrobial resistance. Because treatment of RUTI by long-term antibiotic therapy is often ineffective or poorly tolerated in elderly women, new therapies must be developed. The molecular basis of RUTI, especially in postmenopausal women, has remained unclear because modeling RUTI in mice is difficult, and human data are limited. Invasion of the urothelium and induction of host inflammation are hypothesized to be key mechanisms by which bacterial pathogens cause RUTI. To further our understanding of RUTI in humans, we performed a systematic analysis of urine and bladder biopsy samples from postmenopausal women undergoing cystoscopy with fulguration of trigonitis in the advanced management of antibiotic-refractory RUTI. We provide direct evidence that bacteria reside in the bladder wall of postmenopausal RUTI patients and that diverse bacterial species can be isolated from the bladder tissue. Histopathological scoring revealed significant edema and alterations of urothelial architecture in RUTI patient biopsies. Lymphocytes, including plasma B-cells, were detected within the mesenchyme, urothelium, and follicular aggregates in the majority of patients, indicating that the local adaptive immune response is active during human RUTI. These data provide conclusive evidence that bacteria invade the human urothelium and suggest that diverse bacterial species and the adaptive immune response play important roles in RUTI in humans. © 2019 Elsevier Ltd
Multifaceted Roles of Environmental Factors toward Dental Implant Performance: Observations from Clinical Retrievals and In Vitro Testing
(Elsevier Inc.) Sridhar, Sathyanarayanan; Wang, Frederick; Wilson, T. G., Jr.; Valderrama, P.; Palmer, Kelli; Rodrigues, Danieli C.; Sridhar, Sathyanarayanan; Wang, Frederick; Palmer, Kelli; Rodrigues, Danieli C.
Objective: Oral bacteria and periodontal pathogen have been predominantly linked with early- and late- stage failures of titanium (Ti) dental implants (DI) respectively. This study is based on the hypothesis that bacterial colonization can damage the surface oxide (TiO₂) layer. Early-failed DI were compared with DI post-in vitro immersion in early colonizing oral bacteria; late failed DI were weighed against DI immersed in late colonizing anaerobic pathogens. Methods: Retrieval analysis: Seven early- stage failed implants with five of them connected to healing abutments (HAs), and ten late- stage failed retrievals were subjected to surface analysis. Bacteria immersion test: Three dental implants each were immersed in polycultures containing (i) early colonizers (Streptococcus mutans, S. salivarius, S. sanguinis) (ii) late colonizers (Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans). The implants were immersed for 30 days to simulate the healing period and bacterial biofilm adhesion. Optical microscope, x-ray photoelectron spectroscopy (XPS), and electrochemical test were performed to analyze the surface- morphology, chemistry, and potential respectively. Results: Early colonizers inflicted surface morphological damage (discoloration and pitting). Even though, XPS detected thinner oxide layer in 2/3 early retrievals, XPS and electrochemical tests illustrated that the TiO₂ layer was intact in HAs, and in DI post- immersion. Late colonizers also caused similar morphological damage (discoloration and pitting), while mechanical wear was evident with scratches, cracks, and mechanical fracture observed in late-stage retrievals. XPS indicated thinner oxide layer in late-stage retrievals (3/4), and in DI post-immersion in late colonizers. This was reflected in electrochemical test results post-immersion but not in the late-stage retrievals, which suggested an intact surface with corrosion resistance. Significance: This study concluded that bacteria could negatively affect implant surface with late colonizers demonstrating more pronounced damage on the surface morphology and chemistry. ©2018 The Academy of Dental Materials
EfrEF and the Transcription Regulator ChIR Are Required for Chlorhexidine Stress Response in Enterococcus faecalis V583
(American Society for Microbiology) Li, Farry J.; Palmer, Kelli L.; Li, Farry J.; Palmer, Kelli L.
Enterococcus faecalis is an opportunistic pathogen and leading cause of health care-associated infections. Daily chlorhexidine gluconate (CHG) bathing of patients is generally regarded as an effective strategy to reduce the occurrence of health care-associated infections. It is likely that E. faecalis is frequently exposed to inhibitory and subinhibitory concentrations of CHG in clinical settings. The goal of this study was to investigate how the vancomycin-resistant strain E. faecalis V583 transcriptionally responds to and tolerates stress from CHG. We used transcriptome (microarray) analysis to identify genes upregulated by E. faecalis V583 in response to CHG. The genes efrE (EF2226) and efrF (EF2227), encoding a heterodimeric ABC transport system, were the most highly upregulated genes. efrEF expression was induced by CHG at concentrations several 2-fold dilutions below the MIC. Deletion of efrEF increased E. faecalis V583 susceptibility to CHG. We found that ChlR, a MerR-like regulator encoded by a sequence upstream of efrEF, mediated the CHG-dependent upregulation of efrEF, and deletion of chlR also increased chlorhexidine susceptibility. Overall, our study gives insight into E. faecalis stress responses to a commonly used antiseptic.
An Attenuated CRISPR-Cas System In Enterococcus faecalis Permits DNA Acquisition
(Amer Soc Microbiology) Hullahalli, Karthik; Rodrigues, Marinelle; Uyen Thy Nguyen; Palmer, Kelli L.; 0000-0002-7343-9271 (Palmer, KL); Hullahalli, Karthik; Rodrigues, Marinelle; Uyen Thy Nguyen; Palmer, Kelli L.
Antibiotic-resistant bacteria are critical public health concerns. Among the prime causative factors for the spread of antibiotic resistance is horizontal gene transfer (HGT). A useful model organism for investigating the relationship between HGT and antibiotic resistance is the opportunistic pathogen Fnterococcus faecolis, since the species possesses highly conjugative plasmids that readily disseminate antibiotic resistance genes and virulence factors in nature. Unlike many commensal E. faecalis strains, the genomes of multidrug-resistant (MDR) E. faecalis clinical isolates are enriched for mobile genetic elements (MGEs) and lack clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) genome defense systems. CRISPRCas systems cleave foreign DNA in a programmable, sequence-specific manner and are disadvantageous for MGE-derived genome expansion. An unexplored facet of CRISPR biology in F. faecolis is that MGEs that are targeted by native CRISPR-Cas systems can be maintained transiently. Here, we investigate the basis for this "CRISPR tolerance." We observe that E. faecalis can maintain self-targeting constructs that direct Cas9 to cleave the chromosome, but at a fitness cost. Interestingly. DNA repair genes were not upregulated during self-targeting, but integrated prophages were strongly induced. We determined that low cas9 expression contributes to this transient nonlethality and used this knowledge to develop a robust CRISPR- assisted genome-editing scheme. Our results suggest that E. faecatis has maximized the potential for DNA acquisition by attenuating its CRISPR machinery, thereby facilitating the acquisition of potentially beneficial MGEs that may otherwise be restricted by genome defense. IMPORTANCE CRISPR-Cas has provided a powerful toolkit to manipulate bacteria, resulting in improved genetic manipulations and novel antimicrobials. These powerful applications rely on the premise that CRISPR-Cas chromosome targeting, which leads to double-stranded DNA breaks, is lethal. In this study, we show that chromosomal CRISPR targeting in Enterococcus faecalis is transiently nonlethal. We uncover novel phenotypes associated with this "CRISPR tolerance" and, after determining its genetic basis, develop a genome-editing platform in E. faecafis with negligible off target effects. Our findings reveal a novel strategy exploited by a bacterial pathogen to cope with CRISPR- induced conflicts to more readily accept DNA, and our robust CRISPR editing platform will help simplify genetic modifications in this organism.
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.
Modulators of Enterococcus Faecalis Cell Envelope Integrity and Antimicrobial Resistance Influence Stable Colonization of the Mammalian Gastrointestinal Tract
(Amer Soc Microbiology, 2018-10-22) Banla, Ismael L.; Kommineni, Sushma; Hayward, Michael; Rodrigues, Marinelle; Palmer, Kelli L.; Salzman, Nita H.; Kristich, Christopher J.; 0000-0002-7343-9271 (Palmer, KL); Rodrigues, Marinelle; Palmer, Kelli L.
The Gram-positive bacterium Enterococcus faecalis is both a colonizer of the gastrointestinal tract (GIT) and an agent of serious nosocomial infections. Although it is typically required for pathogenesis, GIT colonization by E. faecalis is poorly understood. E. faecalis tolerates high concentrations of GIT antimicrobials, like cholate and lysozyme, leading us to hypothesize that resistance to intestinal antimicrobials is essential for long-term GIT colonization. Analyses of E. faecalis mutants exhibiting defects in antimicrobial resistance revealed that IreK, a determinant of envelope integrity and antimicrobial resistance, is required for long-term GIT colonization. IreK is a member of the PASTA kinase protein family, bacterial transmembrane signaling proteins implicated in the regulation of cell wall homeostasis. Among several determinants of cholate and lysozyme resistance in E. faecalis, IreK was the only one found to be required for intestinal colonization, emphasizing the importance of this protein to enterococcal adaptation to the GIT. By studying.ireK suppressor mutants that recovered the ability to colonize the GIT, we identified two conserved enterococcal proteins (OG1RF_11271 and OG1RF_11272) that function antagonistically to IreK and interfere with cell envelope integrity, antimicrobial resistance, and GIT colonization. Our data suggest that IreK, through its kinase activity, inhibits the actions of these proteins. IreK, OG1RF_11271, and OG1RF_11272 are found in all enterococci, suggesting that their effect on GIT colonization is universal across enterococci. Thus, we have defined conserved genes in the enterococcal core genome that influence GIT colonization through their effect on enterococcal envelope integrity and antimicrobial resistance.
Exploiting CRISPR-Cas to Manipulate Enterococcus Faecalis Populations
(eLife Sciences Publications Ltd, 2018-08-20) Hullahalli, Karthik; Rodrigues, Marinelle; Palmer, Kelli L.; 0000-0002-5509-605X (Rodrigues, M); 0000-0002-7343-9271 (Palmer, KL); Rodrigues, Marinelle; Palmer, Kelli L.
CRISPR-Cas provides a barrier to horizontal gene transfer in prokaryotes. It was previously observed that functional CRISPR-Cas systems are absent from multidrug-resistant (MDR) Enterococcus faecalis, which only possess an orphan CRISPR locus, termed CRISPR2, lacking cas genes. Here, we investigate how the interplay between CRISPR-Cas genome defense and antibiotic selection for mobile genetic elements shapes in vitro E. faecalis populations. We demonstrate that CRISPR2 can be reactivated for genome defense in MDR strains. Interestingly, we observe that E. faecalis transiently maintains CRISPR targets despite active CRISPR-Cas systems. Subsequently, if selection for the CRISPR target is present, toxic CRISPR spacers are lost over time, while in the absence of selection, CRISPR targets are lost over time. We find that forced maintenance of CRISPR targets induces a fitness cost that can be exploited to alter heterogeneous E. faecalis populations.
Streptococcus mitis and S. oralis Lack a Requirement for CdsA, the Enzyme Required for Synthesis of Major Membrane Phospholipids in Bacteria
(Amer Soc Microbiology, 2018-06-01) Adams, Hannah M.; Joyce, Luke R.; Guan, Ziqiang; Akins, Ronda L.; Palmer, Kelli L.; 0000-0002-7343-9271 (Palmer, KL); Adams, Hannah M.; Joyce, Luke R.; Akins, Ronda L.; Palmer, Kelli L.
Synthesis and integrity of the cytoplasmic membrane are fundamental to cellular life. Experimental evolution studies have hinted at unique physiology in the Gram-positive bacteria Streptococcus mitis and S. oralis. These organisms commonly cause bacteremia and infectious endocarditis (IE) but are rarely investigated in mechanistic studies of physiology and evolution. Unlike in other Gram-positive pathogens, high-level (MIC ≥ 256 mu g/ml) daptomycin resistance rapidly emerges in S. mitis and S. oralis after a single drug exposure. In this study, we found that inactivating mutations in cdsA are associated with high-level daptomycin resistance in S. mitis and S. oralis IE isolates. This is surprising given that cdsA is an essential gene for life in commonly studied model organisms. CdsA is the enzyme responsible for the synthesis of CDP-diacylglycerol, a key intermediate for the biosynthesis of all major phospholipids in prokaryotes and most anionic phospholipids in eukaryotes. Lipidomic analysis by liquid chromatography-mass spectrometry (LC-MS) showed that daptomycin-resistant strains have an accumulation of phosphatidic acid and completely lack phosphatidylglycerol and cardiolipin, two major anionic phospholipids in wild-type strains, confirming the loss of function of CdsA in the daptomycin-resistant strains. To our knowledge, these daptomycin-resistant streptococci represent the first model organisms whose viability is CdsA independent. The distinct membrane compositions resulting from the inactivation of cdsA not only provide novel insights into the mechanisms of daptomycin resistance but also offer unique opportunities to study the physiological functions of major anionic phospholipids in bacteria.
Evaluation of Mammalian and Bacterial Cell Activity on Titanium Surface Coated with Dicationic Imidazolium-Based Ionic Liquids
(Royal Soc Chemistry, 2016-04-04) Gindri, Izabelle M.; Palmer, Kelli L.; Siddiqui, Danyal A.; Aghyarian, Shant; Frizzo, Clarissa P.; Martins, Marcos A. P.; Rodrigues, Danieli C.; 0000-0002-7343-9271 (Palmer, KL); Gindri, Izabelle M.; Palmer, Kelli L.; Siddiqui, Danyal A.; Aghyarian, Shant; Rodrigues, Danieli C.
This work presents a new strategy to protect titanium surfaces against bacterial colonization and biofilm formation using dicationic imidazolium-based ionic liquid coatings. Ionic liquids (ILs) were designed as multi-functional coatings and their compatibility with human gingival fibroblasts (HGF-1) and preosteoblast (MC3T3-E1) cells was investigated. Results demonstrated that IL coatings were stable and present on titanium surfaces after 7 days of immersion and showed that using phenylalanine as the anionic moiety allowed for cell proliferation and differentiation on titanium surface while also providing strong antimicrobial and anti-biofilm activity against bacterial strains relevant to the oral environment (Streptococcus sp.). Strains such as Streptococcus mutans, S. sanguinis, S salivarius, S. gordonii and S. uberis are known to colonize the surface of dental implants in the early stages after implantation (early colonizers), compromising the success of these devices. The "race for the surface" between cells and bacteria was established by correlating results obtained from cell proliferation (epithelial and osteoblast) and differentiation (osteoblast) studies with that of antimicrobial activity against early bacterial colonizers.
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.
Mutations Associated with Reduced Surotomycin Susceptibility in Clostridium Difficile and Enterococcus Species
(American Society for Microbiology, 2015-05-04) Adams, Hannah M.; Li, X.; Mascio, C.; Chesnel, Laurent; Palmer, Kelli L.; Adams, Hannah M.; Li, X. Chesnel, Laurent; Palmer, Kelli L.
Clostridium difficile infection (CDI) is an urgent public health concern causing considerable clinical and economic burdens. CDI can be treated with antibiotics, but recurrence of the disease following successful treatment of the initial episode often occurs. Surotomycin is a rapidly bactericidal cyclic lipopeptide antibiotic that is in clinical trials for CDI treatment and that has demonstrated superiority over vancomycin in preventing CDI relapse. Surotomycin is a structural analogue of the membrane-active antibiotic daptomycin. Previously, we utilized in vitro serial passage experiments to derive C. difficile strains with reduced surotomycin susceptibilities. The parent strains used included ATCC 700057 and clinical isolates from the restriction endonu-clease analysis (REA) groups BI and K. Serial passage experiments were also performed with vancomycin-resistant and vancomycin-susceptible Enterococcus faecium and Enterococcus faecalis. The goal of this study is to identify mutations associated with reduced surotomycin susceptibility in C. difficile and enterococci. Illumina sequence data generated for the parent strains and serial passage isolates were compared. We identified nonsynonymous mutations in genes coding for cardiolipin synthase in C. difficile ATCC 700057, enoyl-(acyl carrier protein) reductase II (FabK) and cell division protein FtsH2 in C. difficile REA type BI, and a PadR family transcriptional regulator in C. difficile REA type K. Among the 4 enterococcal strain pairs, 20 mutations were identified, and those mutations overlap those associated with daptomycin resistance. These data give insight into the mechanism of action of surotomycin against C. difficile, possible mechanisms for resistance emergence during clinical use, and the potential impacts of surotomycin therapy on intestinal enterococci.
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.;
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.

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