J Antimicrob Chemother doi:10.1093/jac/dkz513 Downloaded from https://academic.oup.com/jac/advance-article-abstract/doi/10.1093/jac/dkz513/5686405 by Health Canada /PHAC user on 22 January 2020 Constitutive expression of the cryptic vanGCd operon promotes vancomycin resistance in Clostridioides difficile clinical isolates Wan-Jou Shen1, Aditi Deshpande1, Kirk E. Hevener2, Bradley T. Endres3, Kevin W. Garey3, Kelli L. Palmer4 and Julian G. Hurdle1* 1Center for Infectious and Inflammatory Diseases, Institute of Biosciences and Technology, Texas A&M Health Science Center, Houston, TX 77030, USA; 2Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center, Memphis, TN 38105, USA; 3Department of Pharmacy Practice and Translational Research, College of Pharmacy, University of Houston, Houston, TX 77204, USA; 4Department of Biological Sciences, University of Texas at Dallas, Richardson, TX 75080, USA *Corresponding author. E-mail: [email protected] Received 24 July 2019; returned 14 October 2019; revised 1 November 2019; accepted 9 November 2019 Objectives: To describe, for the first time (to the best of our knowledge), the genetic mechanisms of vancomycin resistance in clinical isolates of Clostridioides difficile ribotype 027. Methods: Clinical isolates and laboratory mutants were analysed: genomically to identify resistance mutations; by transcriptional analysis of vanGCd, the vancomycin resistance operon encoding lipid II D-alanine-D-serine that is less bound by vancomycin than native lipid II D-alanine-D-alanine; by imaging of vancomycin binding to cell walls; and for changes in vancomycin bactericidal activity and autolysis. Results: Vancomycin-resistant laboratory mutants and clinical isolates acquired mutations to the vanSR two- component system that regulates vanGCd. The substitutions impaired VanSR’s function, resulting in constitutive transcription of vanGCd. Resistance was reversed by silencing vanG, encoding D-alanine-D-serine ligase in the vanGCd operon. In resistant cells, vancomycin was less bound to the cell wall septum, the site where vancomycin interacts with lipid II. Vancomycin’s bactericidal activity was reduced against clinical isolates and laboratory mutants (64 and 1024 mg/L, respectively) compared with WT strains (4 mg/L). Truncation of the potassium transporter TrkA occurred in laboratory mutants, which were refractory to autolysis, accounting for their survival in high drug concentrations. Conclusions: Ribotype 027 evolved first-step resistance to vancomycin by constitutively expressing vanGCd, which is otherwise silent. Experimental evolutions and bactericidal assays show that ribotype 027 can acquire mutations to drastically enhance its tolerance to vancomycin. Thus, further epidemiological studies are war- ranted to examine the extent to which vancomycin resistance impacts clinical outcomes and the potential for these strains to evolve higher-level resistance, which would be devastating. Introduction will inadvertently raise the selection pressure for the spread of Clostridioides difficile infection (CDI) is the most common hospital- vancomycin-resistant strains. acquired infection in the USA and the leading cause of death due Vancomycin inhibits cell wall biosynthesis in dividing cells by 1,2 to gastroenteritis. Treatment response rates in patients with binding to the C-terminal peptide of D-alanine-D-alanine (D-Ala-D- CDI, and in particular infections with the recent epidemic NAP1/ Ala) in the cell wall precursor lipid II and in the maturing cell 027/BI, have decreased significantly, while the incidence of CDI wall.9,10 C. difficile with reduced susceptibility to vancomycin has increased, making it an urgent public health threat.3,4 For has emerged11–14 and is defined by a breakpoint of >2 mg/L from >30 years, metronidazole and vancomycin have been the main EUCAST. For example, vancomycin-resistant C. difficile were treatments for CDI.5,6 Fidaxomicin was approved for CDI in 2010, reported from US surveillances by Tickler et al.11 (39.1% of but its clinical use has mainly been limited to recurrent infections.7 143 027/BI isolates from 2011 to 2013) and Snydman et al.12 In the recent IDSA/SHEA 2017 guidelines for CDI management, (17.9% of 925 isolates from 2011 to 2012). Vancomycin concen- metronidazole is no longer recommended as a first-line drug for tration in stools is expected to be about 500–2000 mg/L [i.e. 500– adult CDI.8 Vancomycin usage is therefore likely to increase, which 2000% MIC for most strains (1 mg/L)] and is thought to mirror VC The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: [email protected]. 1of9 Shen et al. the drug’s concentration at the colonic site of infection.12,15,16 It is Selection of mutants via serial passage therefore anticipated that colonic concentrations of vancomycin Overnight R20291 colonies were resuspended in BHI broth (1 mL) to an 5 6 Downloaded from https://academic.oup.com/jac/advance-article-abstract/doi/10.1093/jac/dkz513/5686405 by Health Canada /PHAC user on 22 January 2020 will equally inhibit the growth of vancomycin-resistant strains OD600 of 0.8–1.0. At each passage, an aliquot of 0.01 mL (10 –10 cfu/mL) even at higher MICs. However, this view is undermined by was streaked onto BHI agars containing 0.25% to 8% MIC of vancomycin. the fact that exact concentrations of vancomycin along the After 48 h of incubation, all colonies from the highest concentration of length of the colon are unknown. Furthermore, the genetic basis vancomycin were resuspended into fresh broth to create a mixed popula- for resistance to vancomycin in clinical C. difficile is unknown, tion that was then passaged onto higher drug concentrations. There was but could be the deciding factor on whether mutants with no calculation of mutation frequencies at each passage step. The remain- reduced susceptibility survive in colonic concentrations of ing suspension was stored as glycerol stocks. vancomycin. Indeed, such is the case with VISA that survive in vancomycin concentrations 32-fold higher than the MICs.17,18 Measurement of vancomycin’s bactericidal activity and About 85% of C. difficile carry a functional vancomycin resist- autolysis ance vanG gene cluster (designated as vanGCd), which is closely Bactericidal activity related to the vanG operon in Enterococcus faecalis.19 Induction of this operon by vancomycin results in the production of an Logarithmic cultures (OD600 of 0.2) were exposed to 0–1024 mg/L vanco- mycin and, after 24 h of incubation, viable counts were determined by serial alternative lipid II bearing a D-alanine-D-serine (D-Ala-D-Ser) ter- dilution. Agars with 15% (w/v) charcoal were controls to test the antibiotic minus that is 7-fold less bound by vancomycin compared with 20,21 carry-over effect on viable counting. the D-Ala-D-Ala terminus. Expression of the operon in WT vancomycin-susceptible C. difficile leads to production of lipid II Autolysis D-Ala-D-Ser. However, vancomycin-induced expression of vanGCd 25 does not confer resistance in C. difficile,incontrasttoE. faecalis, This was performed on harvested mid-log cells (OD600 of 0.2) resus- 19,22 pended in 50 mM potassium buffer with 0.01% Triton X-100. Optical read- where induction of vanG causes resistance. C. difficile vanGCd is therefore considered cryptic. Induction of vanG genes is thought to ings were recorded in a Biotek 2 plate reader under anaerobic conditions. occur when the membrane-bound sensor kinase (VanSCd)detects vancomycin and, after undergoing ATP-dependent autophosphor- Imaging of changes in vancomycin cell wall binding ylation, transfers its phosphoryl group to the cytoplasmic response 23 Vancomycin binding was visualized by staining with fluorescently labelled regulator VanR. Phosphorylated VanR (VanRP) then binds up- vancomycin (BODIPYVR FL vancomycin, from Thermo Fisher). Cultures with 23 VR stream of van genes to increase transcription. In the absence of an OD600 of 0.2 were mixed with 1 mg/L vancomycin and BODIPY FL vancomycin, the VanS phosphatase activity attenuates gene ex- vancomycin for 20 min at 37C. Next cells were centrifuged, washed three pression by dephosphorylating VanRP.21 Several studies report times with PBS and fixed with methanol at #20C for 20 min, before wash- that mutations to VanSR proteins cause constitutive expression or ing with PBS. DNA was stained with DAPI (Sigma) at 2 mg/L for 2 min. Cells increased inducibility of van operons in enterococci.21,24 These were aliquotted onto glass slides and air dried before fluorescence imaging mechanisms have not been reported in C. difficile. Herein, we de- with a DeltaVision Elite microscope. scribe the first, to the best of our knowledge, report of mutations to C. difficile VanSRCd that promote constitutive expression of vanGCd Genome sequencing in vancomycin-resistant clinical isolates of ribotype 027. This Accession numbers are listed in Table S1 (available as Supplementary data phenotype was also recapitulated in laboratory mutants. We show at JAC Online). that reduced susceptibility improves the in vitro survival of strains in vancomycin. General methodology Multiplexed DNA libraries were sequenced by paired-end 2%150 bp on an Materials and methods Illumina NextSeqTM500 platform at the Institute of Biosciences and Strains, growth and chemicals Technology and University of Texas Dallas Genome Center. CLC Genomics Workbench version 12 was used for de novo assembly of paired-end reads The 027/BI strain C. difficile R20291 and 027/BI clinical isolates were culti- of sequenced genomes. Assembled genomes were annotated with Rapid vated in brain heart infusion (BHI) broth or agar at 37C in a Whitley A35 an- Annotation using Subsystem Technology (RAST). Sequence variations were aerobic workstation (Don Whitley Scientific). Clinical strains in Table 1 were identified using the ‘Quality-based variant detection’ tool in CLC Genomics from hospitalized patients in the Texas Medical Center and a recent Israeli Workbench with default parameters (10-fold coverage of the reference 13 surveillance.