AAC Accepted Manuscript Posted Online 31 October 2016 Antimicrob. Agents Chemother. doi:10.1128/AAC.01162-16 © Crown copyright 2016.
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Mechanisms of increased resistance to chlorhexidine and cross-resistance to colistin following
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exposure of Klebsiella pneumoniae clinical isolates to chlorhexidine
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Matthew E Wand*, Lucy J Bock, Laura C Bonney, and J Mark Sutton*
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Public Health England, National Infection Service, Porton Down, Salisbury, Wiltshire, SP4 0JG,
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UK.
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Running Head: Mechanisms of chlorhexidine resistance in Klebsiella pneumoniae
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*To whom correspondence should be addressed: Dr Mark Sutton or Dr Matthew Wand,
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Technology Development Group, Public Health England, Microbiology Services Division,
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Porton Down, Salisbury, Wiltshire, UK, SP4 0JG. Tel.: +44 (0) 1980 612316; Fax: +44 (0) 1980
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612622; e-mail: [email protected] or [email protected].
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M.E. Wand and L.J. Bock contributed equally to this work
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Word count abstract: 248
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Word count text: 4374
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Abstract. Klebsiella pneumoniae is an opportunistic pathogen which is often difficult to treat
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due to its multidrug resistance (MDR). We have previously shown that K. pneumoniae strains are
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able to “adapt to” (become more resistant) to the widely used bisbiguanide antiseptic
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chlorhexidine. Here we investigated the mechanisms responsible and the phenotypic
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consequences for chlorhexidine adaptation with particular reference to antibiotic cross-
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resistance. In five of six strains adaptation to chlorhexidine also led to resistance to the last resort
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antibiotic colistin. Here we show that chlorhexidine adaptation is associated with mutations in
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the two component regulator phoPQ and a putative tet-repressor gene (smvR), adjacent to the
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MFS family efflux pump smvA. Up-regulation of smvA (10-27 fold) was confirmed in smvR
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mutant strains and this effect and the associated phenotype was suppressed when a wild type
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copy of smvR was introduced on plasmid pACYC. Up-regulation of phoPQ (5-15 fold) and
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phoPQ-regulated genes, pmrD (6-19 fold) and pmrK (18-64 fold), were confirmed in phoPQ
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mutant strains. In contrast, adaptation of K. pneumoniae to colistin did not result in increased
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chlorhexidine resistance despite the presence of mutations in phoQ and elevated phoPQ, pmrD
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and pmrK transcript levels. Insertion of a plasmid containing phoPQ from chlorhexidine adapted
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strains into wild-type K. pneumoniae resulted in elevated expression levels of phoPQ, pmrD and
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pmrK and increased resistance to colistin but not chlorhexidine. The potential risk of colistin
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resistance emerging in K. pneumoniae as a consequence of exposure to chlorhexidine has
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important clinical implications in infection prevention procedures.
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Introduction
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Emergence of multidrug resistant (MDR) pathogens poses a significant challenge to healthcare
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delivery. Gram-negative pathogens, including Klebsiella pneumoniae, from the so-called
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“ESKAPEE” group are of particular concern due to their high levels of antibiotic resistance. The
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emergence and rapid dissemination of particular strains of carbapenemase-producing
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Enterobacteriacae (1, 2) has left an overreliance on last resort antibiotics e.g. colistin. It has also
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highlighted the need for infection control to support clinical practice, by reducing the potential
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for establishment of antibiotic-resistant infections.
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A range of disinfectants and antiseptics are commonly used in clinical practice and underpin
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current healthcare provision. There is ongoing debate about the presence or emergence of
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resistance to biocides in clinical populations, and the potential for this to translate into cross-
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resistance to antibiotics. There remain relatively few examples where this has been identified and
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fewer still where there is strong evidence for this happening in the clinic (3, 4). Whilst resistance
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to many biocides has been reported for multiple organisms; there is a distinct lack of
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understanding of the mechanisms responsible for this resistance.
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Chlorhexidine is a bisbiguanide antiseptic which is cationic in nature and functions by membrane
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disruption (5). It forms a bridge between pairs of adjacent phospholipid headgroups and
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displaces the associated divalent cations (Mg2+ and Ca2+) (6). This results in a reduction of
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membrane fluidity and osmoregulation, as well as changes in metabolic capability of the cell
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membrane associated enzymes (7). At higher concentrations, the interaction between
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chlorhexidine and the cellular membrane causes the membrane to lose its structural integrity and
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adopt a liquid crystalline state, which leads to a rapid loss of cellular contents (8).
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A review of products containing chlorhexidine which are commonly sold to the National Health
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Service (NHS) in England, identified a wide range of active concentrations of chlorhexidine,
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ranging from 0.02% in catheter maintenance solutions, through 0.2% for mouthwash, 0.5% in
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chlorhexidine-impregnated wound dressings and 2 and 4% (often in alcohol) solutions for skin
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antisepsis. Therefore, the potential for exposed bacteria to develop resistance to chlorhexidine is
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increased due to the variable selection pressure. Increased bacterial chlorhexidine resistance has
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implications for a wide-range of applications including treatment of wounds.
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In this study we investigated whether adaptation of clinical K. pneumoniae isolates to
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chlorhexidine caused cross resistance to other biocides and antibiotics, and whether adapted
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strains maintained fitness and virulence. The underlying mechanisms of increased resistance to
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chlorhexidine in K. pneumoniae were also investigated, particularly in connection with the
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observed cross resistance to colistin.
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Materials and Methods
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Bacterial strains and culture conditions
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The K. pneumoniae isolates used in this study are clinical strains with a variety of antibiotic
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resistance markers e.g. blaNDM-1, blaSHV-18 and have been described previously (9-11). K.
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pneumoniae was adapted to chlorhexidine as described in a previous study (11). Additional
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adaptation experiments to chlorhexidine or colistin were also performed. Briefly, strains were
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cultured at quarter MIC concentration of chlorhexidine or colistin and passaged every two days
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into fresh media containing double the previous concentration of chlorhexidine/colistin. This
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continued for a period of six passages. Stability was measured by passaging strains ten times in
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the absence of selective pressure (chlorhexidine/colistin). All strains were grown in tryptic soy
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broth (TSB) with aeration or on tryptic soy agar (TSA) plates at 37°C unless otherwise stated.
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Chlorhexidine digluconate (Sigma) was used throughout and was diluted in sterile water unless
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otherwise stated.
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Determination of MIC/MBC
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The MICs of various antibiotics and disinfectants/antiseptics for K. pneumoniae isolates were
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determined using a broth microdilution method with a starting inoculum of 1x105 cfu/mL. The
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OD600 was measured after 20 h of static incubation at 37°C and the MIC was defined as the
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lowest concentration of antibiotic/disinfectant at which no bacterial growth was observed. A
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change in MIC was considered significant if at least a 4-fold increase/decrease was observed in
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three replicate experiments. The efflux pump inhibitors (EPIs) phenylalanine-arginine-β-
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naphthylamide (PaβN) and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) were added at
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concentrations of 25 and 10 mg/L respectively where required. For MBC testing, 10 µl of 5
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suspension was removed from each well of the MIC microtitre plate where no bacterial growth was observed along with the two wells immediately below the MIC where growth was observed.
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This was spotted on to TSA plates and incubated at 37°C for 24 h. MBC was defined as the
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lowest concentration of antibiotic/disinfectant at which no bacterial growth was observed in three
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replicate experiments.
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Galleria mellonella killing assays
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Wax moth larvae (G. mellonella) were purchased from Livefood UJ Ltd (Rooks Bridge,
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Somerset, UK) and were maintained on wood chips in the dark at 14°C. They were stored for not
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longer than two weeks. Bacterial infection of G. mellonella is as essentially described by Wand
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et al (12). Data were analysed using the Mantel-Cox method using Prism Software Version 6
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(Graphpad, San Diego, CA, USA).
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Whole genome sequencing of K. pneumoniae strains
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Genomic DNA was purified using a Wizard Genomic DNA purification kit (Promega). DNA
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was tagged and multiplexed with the Nextera XT DNA kit (Illumina). Whole genome
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sequencing of K. pneumoniae isolates was performed by PHE-GSDU on an Illumina (HiSeq
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2500) with paired end read lengths of 150 bp. A minimum 150 Mb of Q30 quality data was
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obtained for each isolate. FastQ files were quality trimmed using Trimmomatic (13). SPAdes
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3.1.1 was used to produce draft chromosomal assemblies and contigs less than 1 kb were filtered
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out (14). FastQ reads from chlorhexidine exposed isolates were subsequently mapped to their
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respective wild-type pre-exposure chromosomal sequence using BWA 0.7.5 (15). Bam format
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files were generated using Samtools (16) and VCF files constructed using GATK2 Unified
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Genotyper (version 0.0.7) (17). These were further filtered using a filtering criteria of Mapping 6
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quality >30, Genotype quality >40, Variant ratio >0.9, Read depth >10 to identify high
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confidence SNPs. All the above sequencing analysis was performed using PHE Galaxy (18).
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BAM files were visualised in Integrative Genomics Viewer (IGV version 2.3.55) (Broad
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Institute). Changes in phoPQ and smvR regions were verified using Sanger sequencing
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(Beckmann Genomics, Takeley, UK) and analysed using DNASTAR Lasergene 10.
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Plasmid complementation in K. pneumoniae.
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phoPQ and smvR genes from both wild type and chlorhexidine adapted strains were amplified
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using primers listed in Table S1. They were digested with ClaI and XbaI and ligated into
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pACYC-184 cloning vector (NEB). Plasmids were checked for correct sequence using Sanger
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sequencing. Resultant plasmids were electroporated into K. pneumoniae strains using a
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MicroPulser (Biorad) and the following settings: 2.5 kV, 25 µF, 200 Ω.and a 0.2 mm gap
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cuvette. Subsequent plasmid insertion was tested using primers pACYC seqF
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(CGTTTTCAGAGCAAGAGATTAC) and pACYC seqR
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(GCATTGTTAGATTTCATACACG).
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Results
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Phenotypic assessment of K. pneumoniae isolates following chlorhexidine adaptation.
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Previous work has shown that K. pneumoniae strains are able to adapt to increasing
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concentrations of chlorhexidine and this leads to increased MIC/MBC (minimum bactericidal
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concentration) levels to chlorhexidine digluconate (CHD) and chlorhexidine containing clinical
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products (11). To determine whether adaptation to chlorhexidine also led to development of
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increased resistance to other antiseptics and cross-resistance to frontline antibiotics, MICs were
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performed against a range of biocides for wild-type (WT) and chlorhexidine-adapted (CA)
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strains (Table 1). Following chlorhexidine adaptation MIC values for colistin increased from 2-4
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mg/L to >64 mg/L in five out of the six strains. The only strain which did not show increased
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resistance to colistin following chlorhexidine adaptation was M109 CA. This strain also had the
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lowest MIC for chlorhexidine (32 mg/L compared to a range between 128 and 512 mg/L for the
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other strains). Resistance to colistin is often accompanied by changes in membrane composition,
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which can be detected by altered MIC levels to azithromycin, teicoplanin and cefepime. Apart
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from MGH 78578 CA, where the MIC of cefepime changed from >64 mg/L to 0.5 mg/L
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following chlorhexidine exposure, no significant differences were observed. For other antibiotics
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tested there was also no general change in susceptibility although there were individual strain
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differences e.g. for strain NCTC 13443 there was a significant reduction in the MIC levels to
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meropenem and chloramphenicol after chlorhexidine adaptation (Table S2). There was also no
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significant change (>2-fold) in MIC levels to other antiseptics after chlorhexidine adaptation for
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any of the strains tested.
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Due to increased disinfectant/antiseptic resistance often being associated with increased efflux,
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the MICs for chlorhexidine were re-tested in the presence of known efflux pump inhibitors 8
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CCCP and PaβN. In all of the WT strains, the addition of CCCP gave a significant reduction in
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MIC, with values reduced from 8-32 mg/L, to between 0.5 and 4 mg/L (8-16 fold reduction). The
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addition of CCCP also reduced the MIC to chlorhexidine in all of the chlorhexidine-adapted
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strains, with an MIC reduction of between 32 (M109 CA) and 512 fold (MGH 78578 CA and
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NCTC 13368 CA respectively). In each case, the MIC of the WT and chlorhexidine adapted
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strains, in the presence of CCCP was either identical or within a 2-fold dilution (Table 1). The
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addition of CCCP also increased the susceptibility to colistin in both chlorhexidine-adapted and
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WT strains. For PaβN, no reduction was seen in the MICs for the WT or chlorhexidine-adapted
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strains (data not shown).
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The fitness of strains following chlorhexidine adaptation was assessed. Following chlorhexidine
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adaptation, virulence in Galleria mellonella was reduced in several strains (NCTC 13443 CA,
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MGH 78578 CA, M109 CA, NCTC 13368 CA) but not for M3 CA and NCTC 13439 CA (Fig.
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S1). To investigate whether the observed loss of virulence was due to a growth defect, growth
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curves were assessed. A reduction in growth rate was observed for some isolates, but this did not
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always correlate with loss of virulence (data not shown).
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Whole genome sequencing analysis of chlorhexidine-adapted strains
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To understand what mechanisms are responsible for increased tolerance to chlorhexidine all
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chlorhexidine adapted strains and their respective parental counterparts were whole genome
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sequenced. Several genetic changes (both InDels and non-synonymous SNPs) were identified in
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the chlorhexidine adapted strains (Table 2 and Table S3). Four out of the six strains had non-
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synonymous SNPs in the two component regulator system phoPQ and four out of the six strains
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showed mutations in a putative Tet-repressor gene (hereafter called smvR (Regulator of smvA))
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adjacent to and divergently transcribed from a putative homolog of the methyl viologen
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resistance smvA gene. In three of these four strains a truncated Tet-repressor protein was
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observed, either through a missense mutation (M3 CA), deletion of the C-terminus (M109 CA)
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or a non-synonymous SNP leading to the formation of a premature STOP codon (NCTC 13443
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CA). In the fourth strain (NCTC 13439 CA) the entire smvR gene was deleted along with
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downstream sequences, which included the nitrate extrusion protein (narU) and genes encoding
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proteins involved in nitrate reductase (narZ and narY). In two strains (M3 CA and NCTC 13443
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CA), mutations in both smvR and phoPQ were identified.
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In all adapted strains, genetic changes were identified in genes in addition to phoPQ and/or
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smvR. Mutations were observed in genes associated with the outer membrane or LPS e.g. lptD in
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NCTC 13443 CA. Strain MGH 78578 CA contained a mutation in the DNA mismatch repair
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gene mutS which explains the increased frequency of mutations after chlorhexidine adaptation in
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this strain. Two strains also contained alterations in the outer membrane protein MipA (MltA-
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interacting protein). All other mutations were only present in one strain out of six following
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chlorhexidine adaptation. There was also evidence for plasmid loss following chlorhexidine
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adaptation which explains the loss of antibiotic resistance in certain strains e.g. meropenem in
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NCTC 13443 CA.
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Chlorhexidine adaptation experiments were repeated independently in the same strains and again
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mutations in smvR and phoPQ were observed (data not shown). Therefore, since all
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chlorhexidine adapted strains had mutations in phoPQ and/or smvR, further analysis concentrated
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on these genes.
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Role of SmvR and PhoPQ in resistance to chlorhexidine.
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To define the role of smvR in increasing resistance to chlorhexidine and colistin, pACYC-184
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plasmids containing either the WT or the CA smvR versions from NCTC 13443 and M3 (see
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Table 2 for individual mutations) were introduced into strain M109 CA (which has a deletion in
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the C-terminus of smvR). Introduction of the wildtype versions of smvR from either NCTC
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13443 or M3 resulted in a decrease in MIC levels to CHD compared to the wildtype value (from
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32 to 8 mg/L), whereas the introduction of the CA smvR from either NCTC 13443 or M3 had no
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effect on the MIC level to CHD (Table 3) indicating that, in M109 CA, the deletion of smvR
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increased resistance to chlorhexidine. No change in MIC levels to colistin was observed
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following introduction of either CA or WT smvR versions from NCTC 13443 or M3 into M109
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CA. To confirm the role of smvA/R in elevated resistance to chlorhexidine, we examined 10 pre-
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antibiotic era K. pneumoniae Murray strains which do not possess a homologue to these genes
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(10). These strains were highly susceptible to chlorhexidine with MIC levels ranging from 1-
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64 mg/L), but the susceptibility to chlorhexidine remained the same when compared to pre-
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exposure strains (Table 4). When whole genome analysis was carried out on these isolates, strain
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M3 CST was shown to have an identical mutation (PhoQ L348Q) to that found in strain MGH
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78578 CA and no other mutations.
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To understand whether colistin resistant K. pneumoniae strains had similar phoPQ expression
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profiles to chlorhexidine adapted strains we analysed four strains with different colistin resistant
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mutations. These included strains 16 CST (PhoQ T244N), M3 CST (PhoQ L348Q), 51851
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(∆mgrB) and NCTC 13438 CST (PmrB D150Y). All these strains had MIC levels to CST of 64
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mg/L and CHD (16-32 mg/L) respectively. Transcriptional analysis showed that these strains had
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elevated levels of pmrK (20-60 fold) and with the exception of 13438 CST elevated levels of
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phoP (5-10 fold), phoQ (4-9 fold), pmrD (5-11 fold) and pagP (4-7 fold). Strains which had
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mutations in phoQ had very similar elevated expression levels compared to chlorhexidine
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adapted strains containing mutations in phoP or phoQ (Table 4).
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Discussion
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This study has shown that adaptation of clinical K. pneumoniae isolates to chlorhexidine
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exposure can not only lead to stable resistance to chlorhexidine but also cross-resistance to
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colistin. This has important clinical implications for the treatment of MDR (particularly
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carbapenem-resistant) K. pneumoniae infections and outbreaks, given their increasingly
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prevalence in hospitals (19). Many carbapenem-resistant K. pneumoniae isolates are susceptible
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to very few antibiotics notably colistin; treatment often involves combination therapy including
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colistin (20). Therefore, any potential loss of colistin efficacy has implications for treatment of
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these infections. Whilst chlorhexidine has been successfully used as part of a multifaceted
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intervention to reduce the prevalence of carbapenem-resistant K. pneumoniae in hospitals (21)
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the observation that exposure to chlorhexidine leads to colistin resistance means eradication of
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potentially colistin and carbapenem-resistant isolates is very problematic. Since the isolate has
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also acquired increased resistance to chlorhexidine this also makes prevention of colonisation
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with these isolates more difficult, which has the potential to either prolong existing outbreaks or
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lead to new outbreaks.
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Genome analysis of chlorhexidine adapted strains identified mutations in phoPQ and/or smvR.
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SmvR is a putative Tet-repressor family protein, which is adjacent to and transcribed divergently
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from the smvA gene. Adaptation to chlorhexidine resulted in disruption of smvR either by
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complete deletion or production of a truncated version. SmvA is an efflux pump of the Major
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Facilitator Superfamily (MFS) and has been implicated in methyl viologen resistance and the
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efflux of acriflavine and other quaternary ammonium compounds (QACs) in S. enterica serovar
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Typhimurium (22, 23). Upregulation of smvA was confirmed in all strains with a non-functional
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SmvR suggesting that it acts as a repressor of smvA and that increased expression of smvA leads 14
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to increased chlorhexidine resistance. Divergent expression from the same promoter is
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commonly observed in genes which are negatively regulated by Tet-repressors (24). Examples of
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TetR homologues involved in resistance to biocides include QacR in Staphylococcus aureus and
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EnvR and NemR in E. coli (25-27).
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The observation that K. pneumoniae (Murray) isolates which do not possess homologues to
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smvA/R are highly susceptible to chlorhexidine again suggests that smvA is an important efflux
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pump in chlorhexidine resistance. E. coli does not possess a homologue to smvA and is
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significantly more susceptible than Klebsiella to chlorhexidine (28). However, potential
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homologues of smvA/R are found in most species of Enterobacteriaceae as well as other Gram-
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negative MDR pathogens e.g. P. aeruginosa and A. baumannii. This potentially means that
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SmvA homologues mediate reduced susceptibility to polycationic antiseptics in other bacteria
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and that a similar mechanism of adaptation, with deletion of smvR, may be observed in other
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clinically-important pathogens. In A. baumannii, deletion of another Tet-repressor (adeN) which
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is adjacent to an efflux pump operon (adeIJK) resulted in acquisition of increased resistance to
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Triclosan (29).
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The addition of the EPI CCCP increased susceptibility to chlorhexidine, both in the wild-type
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and chlorhexidine-adapted strains, suggesting that efflux pumps known to be affected by this
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uncoupling agent including MFS-type pumps such as smvA, might be involved. In colistin-
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resistant, chlorhexidine adapted strains, CCCP also increased the susceptibility to colistin. This is
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consistent with other studies suggesting a role for efflux in colistin resistance in a range of Gram-
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negative pathogens (30). CCCP, due to its function as a general uncoupler of proton motive
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force, may affect metabolic activity, which has been linked to increased susceptibility to colistin
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in A. baumannii (31) and chlorhexidine in P. aeruginosa (32). Indeed growth of both WT and
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CA strains was diminished following addition of CCCP.
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The cross-resistance to colistin following chlorhexidine adaptation is likely due to upregulation
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of the operon containing pmrK. This operon functions to modify LPS with cationic substitution
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of the phosphate groups with 4-amino-4-deoxy-L-arabinose (L-Ara4N). This reduces the lipid A
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net negative charge and causes a reduction in the binding affinity of colistin (33). PhoPQ has
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been linked to modification of LPS and outer membrane content (34-36) and regulates genes
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involved in colistin resistance including pmrD and pmrAB (37, 38). Mutations in PhoPQ in K.
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pneumoniae have already been shown to be associated with colistin and cationic peptide
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resistance and lipid A modification (37, 39-41).qPCR analysis of strains with phoPQ mutations
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showed a significant upregulation in expression levels of phoPQ (at least 5 fold). For these
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strains, genes regulated directly or indirectly by phoPQ (pmrD and pmrK) were also upregulated.
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This upregulation was consistent for K. pneumoniae strains which are resistant to both colistin
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and chlorhexidine or colistin alone. However, there are examples of mutations in PhoQ leading
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to elevated phoPQ expression but not development of colistin resistance (42). In this study one
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chlorhexidine adapted strain (NCTC 13439 CA) had elevated colistin resistance but no detected
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mutations in phoPQ and did not show upregulation of their expression. However, this strain did
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have upregulated pmrD and pmrK expression levels, which suggested that these genes were
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activated independently of phoPQ. The expression levels of pmrK are regulated by another two
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component regulator implicated in colistin resistance, pmrAB. Whilst expression levels of pmrAB
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may be regulated by PhoPQ (through PmrD) pmrAB is also transcribed, independently of
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PhoPQ, from a constitutive promoter within the upstream pagB gene (34). Expression of pmrD
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may be subject to regulation by a second, unknown system based on the observation in E. coli, 16
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that expression of pmrD was not abolished following inactivation of phoPQ (43). In K.
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pneumoniae, upregulation of pmrD following adaptation to colistin was not always linked to
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upregulation of phoQ (44). The presence of a mutation in the RarA regulator in NCTC 13439
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CA is consistent with up-regulation of rarA in K. pneumoniae enhancing growth on polymyxin B
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(45). The mutated amino acid (W37R) is predicted to directly interact with DNA, according to
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the crystal structure of the related regulator MarA (46).
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Results demonstrate that PhoPQ mutations arise through chlorhexidine adaptation and can be
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clearly linked to colistin resistance. However, the data suggests that additional factors are
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important in mediating resistance to chlorhexidine and these may operate independently of
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PhoPQ. qPCR analysis using colistin-adapted strains with specific mutations in PhoQ (T244N
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and L348Q) showed very similar expression levels of phoPQ to chlorhexidine adapted strains
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containing PhoPQ mutations (NCTC 13368 CA, NCTC 13443 CA, M3 CA and MGH 78578
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CA) suggesting that increased expression levels of phoPQ, pmrD and pmrK, whilst important for
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colistin resistance are not sufficient for increased resistance to chlorhexidine. This observation
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was re-enforced by colistin resistance but not chlorhexidine resistance being transferred on
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pACYC phoQ A20P, which again caused upregulation of phoQ, pmrD and pmrK relative to
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pACYC phoPQ WT. An identical PhoQ mutation (L348Q) was present in both strains M3 CST
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and MGH 78578 CA suggesting that this mutation alone is not sufficient to lead to increased
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chlorhexidine resistance but is enough to generate colistin resistance. Interestingly, upregulated
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pagP expression was observed in colistin adapted strains where upregulated phoPQ expression
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was also observed (KP16 CST, M3 CST, 51851). For chlorhexidine-adapted strains, those with
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mutations in phoPQ but not smvR showed no pagP upregulation. pagP expression does not
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appear to be dependent upon specific allelic changes since both MGH 78578 CA and M3 CST 17
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have mutations in phoQ (L348Q) but only M3 CST showed upregulated pagP expression. These
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observations could imply that upregulated pagP expression is antagonistic to chlorhexidine
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resistance. M3 CA and NCTC 13443 CA have elevated pagP levels but also have mutations in
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smvR in addition to mutations in phoPQ. We have demonstrated that smvR has a role in
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chlorhexidine resistance and it is plausible that mutations in smvR “mask” any negative effects
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on chlorhexidine resistance caused by elevated pagP expression. PagP causes palmitoylation of
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lipid A which has been found to not contribute to colistin resistance in other pathogens (47), but
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its role in chlorhexidine resistance has not been investigated. It is possible that increased
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palmitoylation of lipid A reduces the frequency of other lipid A modifications which are
369
important in chlorhexidine resistance. Future work will focus on understanding the role of lipid
370
A modification in chlorhexidine resistance including the role of PhoPQ, PagP and MarA.
371
It is also plausible that mutations in phoPQ are secondary-site or compensatory mutations, which
372
enable the resistant strain to recover its fitness and virulence following the initial development of
373
biocide resistance (48). PhoPQ is a global regulator and regulates a number of genes important in
374
fitness and virulence (49-51). Again, further work is needed to investigate this.
375
Overall this study has identified a novel resistance mechanism to chlorhexidine (smvA/R) that
376
may potentially operate in a number of different species. Clearly increased smvA expression is
377
important for chlorhexidine adaptation in K. pneumoniae but it is not the only mechanism and
378
may operate in conjunction with other regulatory processes. Chlorhexidine-adaptation is also
379
associated with the generation of mutations in PhoPQ, which affect a number of known
380
regulatory targets (notably pmrD and pmrK). Upregulation of these genes also correlates with the
381
presence of colistin resistance. That increased colistin and chlorhexidine resistance may occur in
382
clinical isolates without significant loss of fitness/virulence highlights the potential challenges 18
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360
associated with critical infection control procedures and the use of chlorhexidine as an antiseptic
384
to control healthcare–associated infections.
385
Funding information
386
This project was funded by Public Health England GIA grant project 109506. The views
387
expressed are those of the authors and not necessarily those of the funding body.
388
Acknowledgements.
389
The authors would like to thank Dr Steven Pullan for his assistance in the analysis of the whole
390
genome sequence data. The authors declare no conflicts of interest.
19
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383
391
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392
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534
Table 1 MIC (mg/L) values of various antibiotics and disinfectants for chlorhexidine-adapted strains. Disinfectants used were
542
chlorhexidine digluconate (CHD), benzalkonium chloride (BCl), Octenidine Dihydrochloride (Oct), hexadecylpyridinium chloride monohydrate
543
(HDPCM) and ethanol (EtOH). Antibiotics used were colistin (CST), azithromycin (AZM), cefepime (FEP) and teicoplanin (TEC). All values
544
are given in mg/L. All MICs are shown as the range from at least three independent experiments. + CCCP indicates the addition of the efflux
545
pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone. Additional antibiotics are shown in Table S2. Strain M109 WT M109 CA NCTC 13439 WT NCTC 13439 CA M3 WT M3 CA NCTC 13443 WT NCTC 13443 CA NCTC 13368 WT NCTC 13368 CA MGH 78578 WT MGH 78578 CA
546
a
CHD 8 32-64a 8-16 256a 8-16 32-64a 8-16 256-512a 32 256a 8-16 256-512a
CHD + CCCP 0.5-1 0.5-1 2-4 1-2 1-2 0.5-2 1-2 1-2 2-4 1-2 1-2 0.5-2
BCl
Oct
HDPCM
EtOH (%)
CST
16 8-16 16 16 8-16 8-16 8-16 8-16 32 16 8-16 8-16
4 2-4 2-4 2-4 2-4 2-4 4 2 4-8 4-8 4 4
4-8 4-8 16 8-16 8 8-16 8-16 8-16 32-64 16 8-16 8
3.125 6.25 6.25 6.25 6.25 3.125 3.125 3.125 6.25 6.25 6.25 3.125
2 2-4 4 >64a 2-4 >64a 2 >64a 2-4 >64a 2-4 >64a
CST + CCCP 2 0.5-1 2 1 2 1-2 2 2 2-4 2-4 2-4 1-2
AZM
FEP
TEC
8-16 8-16 32 32 16-32 8-16 64 16-32 64 64 32 32-64
0.06-0.125 0.06-0.125 >64 >64 >64 >64 >64 >64 64 64 >64 0.5a
>64 >64 >64 >64 >64 >64 >64 >64 >64 >64 >64 >64
≥ 4-fold increase or decrease in MIC for chlorhexidine-adapted strains (CA) relative to non-adapted strains (WT)
547 548
27
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541
550
Table 2 Chromosomal genetic changes after exposure to chlorhexidine Strain and gene name (if known) M109 wcaJ yfiN smvR NCTC 13439* mipA rarA smvR narU narZ narH narJ M3 phoP ackA smvR NCTC 13443* sufD phoQ lptD smvR NCTC 13368 28
Type of Change
Change
MGH 78578 equivalent based on (NC_009648.1)
Function
SNP SNP Deletion
Q399STOP A173V 400 bp Del
KPN_RS15690 KPN_RS10110
CPS biosynthesis glycosyltransferase Diguanylate cyclase TetR family transcriptional regulator
SNP SNP Deletion Deletion Deletion Deletion Deletion
Q98STOP W37R Complete Del Complete Del Complete Del Complete Del First 130 aa Del
KPN_RS06390 KPN_RS15910 KPN_RS10110 KPN_RS10115 KPN_RS10120 KPN_RS10125 KPN_RS10130
MltA-interacting protein AraC family transcriptional regulator TetR family transcriptional regulator nitrite extrusion protein 2 nitrate reductase A subunit alpha nitrate reductase A subunit beta nitrate reductase molybdenum cofactor assembly chaperone
SNP SNP Deletion of 5 bp after nucleotide 22 Deletion (G) after nucleotide 445
E82K S274F Truncation of 72 aa, (normally 191 aa) Truncation of 174 aa, (normally 184 aa)
KPN_RS06075 KPN_RS14420 KPN_RS10110
PhoP family transcriptional regulator Acetate kinase TetR family transcriptional regulator
KPN_RS17785
isopentenyl-diphosphate Delta-isomerase
SNP (synonymous) SNP SNP SNP Insertion (T) after nucleotide 375
A20P Y625N W125STOP Truncation of 125 aa, (normally 235 aa)
KPN_RS11525 KPN_RS06070 KPN_RS00270 KPN_RS10110 KPN_RS14015
Fe-S cluster assembly protein Two component sensor protein LPS assembly outer membrane complex protein TetR family transcriptional regulator Membrane protein, putative permease
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549
SNP SNP SNP SNP
E253A V122F V27E Y98C
SNP
-
SNP
mutS surA phoQ -
SNP SNP SNP SNP SNP SNP Insertion (A)
LuxR family transcriptional regulator 30S Ribosome protein S1 Part of outer membrane protein complex PhoP family transcriptional regulator
G121S
KPN_RS01550
A276V
KPN_RS11605
T115P R46C L74P L348Q N30S N32S In repeat region AAGCTAA Truncation of 136aa (normally 170aa) Truncation of 172 aa, (normally 329 aa) Elongation of 5 aa, (normally 1490 aa) Truncation of 649 aa, (normally 1332 aa) Extra Glycine
KPN_RS16580 KPN_RS24340 KPN_RS00265 KPN_RS06070 KPN_RS04685 KPN_RS20410 -
Phosphonate ABC transporter substrate-binding protein 5-methyltetrahydropteroyltriglutamate homocysteine methyltransferase DNA mismatch repair protein LuxR family transcriptional regulator Peptidyl-prolyl cis-trans isomerase Two component sensor protein Aldose dehydrogenase Transcription accessory protein -
Insertion (C) after KPN_RS08585 nucleotide 122 Insertion (G) after KPN_RS10945 nucleotide 131 Insertion (T) after KPN_RS15800 nucleotide 4423 Insertion (GG) after KPN_RS25360 nucleotide 1920 nuoC/D Insertion (GGG) after KPN_RS14365 nucleotide 1382 mipA Insertion (G) after Truncation of 72 aa, KPN_RS06390 nucleotide 195 (normally 275 aa) nikE Insertion (C) after Truncation of 81 aa, KPN_RS20815 nucleotide 87 (normally 263 aa) *Evidence of plasmid loss in these strains following chlorhexidine adaptation comA
551
KPN_RS05050 KPN_RS15655 KPN_RS06075
552
29
Competence protein nitrate ABC transporter substrate-binding protein Hypothetical protein helicase NADH-quinone oxidoreductase subunit C/D MltA-interacting protein MipA ATP-binding protein of nickel transport system
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acoK rpsA yfiO (bamD) phoP MGH 78578* -
554
Table 3 MIC values of chlorhexidine (CHD) and colistin (CST) after plasmid complementation. Levels of resistance to chlorhexidine
555
(CHD) and colistin (CST) were measured after conjugation of plasmids into strains listed. All values are given in mg/L. All MICs are shown as
556
the range from at least three independent experiments. Strain
M109 CA
M109 WT 25 557
30
Plasmid pACYC-184 alone pACYC M3 smvR WT pACYC M3 smvR CHD pACYC 13443 smvR WT pACYC 13443 smvR CHD pACYC phoPQ WT pACYC phoQ A20P pACYC-184 alone pACYC phoPQ WT pACYC phoQ A20P pACYC-184 alone pACYC phoPQ WT pACYC phoQ A20P
Description empty vector smvR from strain M3 smvR from strain M3 CA smvR from strain NCTC 13443 smvR from strain NCTC 13443 CA phoPQ from strain NCTC 13443 phoPQ from strain NCTC 13443 CA empty vector phoPQ from strain NCTC 13443 phoPQ from strain NCTC 13443 CA empty vector phoPQ from strain NCTC 13443 phoPQ from strain NCTC 13443 CA
CHD MIC 32 8 32 8 32 32-64 32-64 8-16 8-16 8-16 8-16 8-16 8-16
CST MBC 32 8 32 8 32 32-64 64 8-16 8-32 16-32 8-16 16-32 16-64
MIC 2-4 2 2-4 2 2-4 1-2 64 0.5-1 0.5-1 32-64 0.5 0.5-1 32-64
MBC 2-4 2 2-4 2 2-4 2 64->64 2-4 2-4 64 2-4 1-4 32->64
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553
Table 4 Expression levels of select genes following chlorhexidine (CA) or colistin (CST) adaptation. Numbers represent fold upregulation
559
with respect to wildtype levels except where indicated. Relevant mutations and fold increase in CHD and CST following adaptation are also
560
shown.
561
Significant fold upregulation compared to wt or pACYC phoPQ WT*
Strain phoP M109 CA NCTC 13439 CA M3 CA NCTC 13443 CA NCTC 13368 CA MGH 78578 CA M109 WT pACYC phoQ A20P* KP16 CST M3 CST 51851 vs 46704 NCTC 13438 CST
562 563 564 31
phoQ
smvA 10 19 18 27
smvR
7 6
pmrK
pmrD
12 64 26 18 24
3 19 6 6 6
15 5 8 6
9 5 7 8 3
7
5 8 10
4 5 9
20 38 60 55
mutation pagP
Fold increase in MIC over wt CHD CST 4 none 16 >32 8 >32 16 >32 16 >32 16 >32
7 2
ΔsmvR CT ΔsmvR PhoP E82K, ΔsmvR PhoQ A20P, ΔsmvR CT PhoP Y98C PhoQ L348Q
2
4
phoQ A20P on plasmid
none
64
5 7 11
7 4 6
PhoQ T244N PhoQ L348Q MgrB Q30STOP PmrB D150Y
none none none none
>64 >64 >64 >64
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558
1
Mechanisms of increased resistance to chlorhexidine and cross-resistance to colistin following
2
exposure of Klebsiella pneumoniae clinical isolates to chlorhexidine
3
5
Matthew E Wand*, Lucy J Bock, Laura C Bonney, and J Mark Sutton*
6 7
Public Health England, National Infection Service, Porton Down, Salisbury, Wiltshire, SP4 0JG,
8
UK.
9 10
Running Head: Mechanisms of chlorhexidine resistance in Klebsiella pneumoniae
11 12
*To whom correspondence should be addressed: Dr Mark Sutton or Dr Matthew Wand,
13
Technology Development Group, Public Health England, Microbiology Services Division,
14
Porton Down, Salisbury, Wiltshire, UK, SP4 0JG. Tel.: +44 (0) 1980 612316; Fax: +44 (0) 1980
15
612622; e-mail: [email protected] or [email protected].
16
M.E. Wand and L.J. Bock contributed equally to this work
17 18 19
Word count abstract: 248
20
Word count text: 4374
1
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4
Abstract. Klebsiella pneumoniae is an opportunistic pathogen which is often difficult to treat
22
due to its multidrug resistance (MDR). We have previously shown that K. pneumoniae strains are
23
able to “adapt to” (become more resistant) to the widely used bisbiguanide antiseptic
24
chlorhexidine. Here we investigated the mechanisms responsible and the phenotypic
25
consequences for chlorhexidine adaptation with particular reference to antibiotic cross-
26
resistance. In five of six strains adaptation to chlorhexidine also led to resistance to the last resort
27
antibiotic colistin. Here we show that chlorhexidine adaptation is associated with mutations in
28
the two component regulator phoPQ and a putative tet-repressor gene (smvR), adjacent to the
29
MFS family efflux pump smvA. Up-regulation of smvA (10-27 fold) was confirmed in smvR
30
mutant strains and this effect and the associated phenotype was suppressed when a wild type
31
copy of smvR was introduced on plasmid pACYC. Up-regulation of phoPQ (5-15 fold) and
32
phoPQ-regulated genes, pmrD (6-19 fold) and pmrK (18-64 fold), were confirmed in phoPQ
33
mutant strains. In contrast, adaptation of K. pneumoniae to colistin did not result in increased
34
chlorhexidine resistance despite the presence of mutations in phoQ and elevated phoPQ, pmrD
35
and pmrK transcript levels. Insertion of a plasmid containing phoPQ from chlorhexidine adapted
36
strains into wild-type K. pneumoniae resulted in elevated expression levels of phoPQ, pmrD and
37
pmrK and increased resistance to colistin but not chlorhexidine. The potential risk of colistin
38
resistance emerging in K. pneumoniae as a consequence of exposure to chlorhexidine has
39
important clinical implications in infection prevention procedures.
40 41
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Introduction
43
Emergence of multidrug resistant (MDR) pathogens poses a significant challenge to healthcare
44
delivery. Gram-negative pathogens, including Klebsiella pneumoniae, from the so-called
45
“ESKAPEE” group are of particular concern due to their high levels of antibiotic resistance. The
46
emergence and rapid dissemination of particular strains of carbapenemase-producing
47
Enterobacteriacae (1, 2) has left an overreliance on last resort antibiotics e.g. colistin. It has also
48
highlighted the need for infection control to support clinical practice, by reducing the potential
49
for establishment of antibiotic-resistant infections.
50
A range of disinfectants and antiseptics are commonly used in clinical practice and underpin
51
current healthcare provision. There is ongoing debate about the presence or emergence of
52
resistance to biocides in clinical populations, and the potential for this to translate into cross-
53
resistance to antibiotics. There remain relatively few examples where this has been identified and
54
fewer still where there is strong evidence for this happening in the clinic (3, 4). Whilst resistance
55
to many biocides has been reported for multiple organisms; there is a distinct lack of
56
understanding of the mechanisms responsible for this resistance.
57
Chlorhexidine is a bisbiguanide antiseptic which is cationic in nature and functions by membrane
58
disruption (5). It forms a bridge between pairs of adjacent phospholipid headgroups and
59
displaces the associated divalent cations (Mg2+ and Ca2+) (6). This results in a reduction of
60
membrane fluidity and osmoregulation, as well as changes in metabolic capability of the cell
61
membrane associated enzymes (7). At higher concentrations, the interaction between
62
chlorhexidine and the cellular membrane causes the membrane to lose its structural integrity and
63
adopt a liquid crystalline state, which leads to a rapid loss of cellular contents (8).
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42
A review of products containing chlorhexidine which are commonly sold to the National Health
65
Service (NHS) in England, identified a wide range of active concentrations of chlorhexidine,
66
ranging from 0.02% in catheter maintenance solutions, through 0.2% for mouthwash, 0.5% in
67
chlorhexidine-impregnated wound dressings and 2 and 4% (often in alcohol) solutions for skin
68
antisepsis. Therefore, the potential for exposed bacteria to develop resistance to chlorhexidine is
69
increased due to the variable selection pressure. Increased bacterial chlorhexidine resistance has
70
implications for a wide-range of applications including treatment of wounds.
71
In this study we investigated whether adaptation of clinical K. pneumoniae isolates to
72
chlorhexidine caused cross resistance to other biocides and antibiotics, and whether adapted
73
strains maintained fitness and virulence. The underlying mechanisms of increased resistance to
74
chlorhexidine in K. pneumoniae were also investigated, particularly in connection with the
75
observed cross resistance to colistin.
76
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Materials and Methods
78
Bacterial strains and culture conditions
79
The K. pneumoniae isolates used in this study are clinical strains with a variety of antibiotic
80
resistance markers e.g. blaNDM-1, blaSHV-18 and have been described previously (9-11). K.
81
pneumoniae was adapted to chlorhexidine as described in a previous study (11). Additional
82
adaptation experiments to chlorhexidine or colistin were also performed. Briefly, strains were
83
cultured at quarter MIC concentration of chlorhexidine or colistin and passaged every two days
84
into fresh media containing double the previous concentration of chlorhexidine/colistin. This
85
continued for a period of six passages. Stability was measured by passaging strains ten times in
86
the absence of selective pressure (chlorhexidine/colistin). All strains were grown in tryptic soy
87
broth (TSB) with aeration or on tryptic soy agar (TSA) plates at 37°C unless otherwise stated.
88
Chlorhexidine digluconate (Sigma) was used throughout and was diluted in sterile water unless
89
otherwise stated.
90
Determination of MIC/MBC
91
The MICs of various antibiotics and disinfectants/antiseptics for K. pneumoniae isolates were
92
determined using a broth microdilution method with a starting inoculum of 1x105 cfu/mL. The
93
OD600 was measured after 20 h of static incubation at 37°C and the MIC was defined as the
94
lowest concentration of antibiotic/disinfectant at which no bacterial growth was observed. A
95
change in MIC was considered significant if at least a 4-fold increase/decrease was observed in
96
three replicate experiments. The efflux pump inhibitors (EPIs) phenylalanine-arginine-β-
97
naphthylamide (PaβN) and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) were added at
98
concentrations of 25 and 10 mg/L respectively where required. For MBC testing, 10 µl of 5
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99
suspension was removed from each well of the MIC microtitre plate where no bacterial growth was observed along with the two wells immediately below the MIC where growth was observed.
101
This was spotted on to TSA plates and incubated at 37°C for 24 h. MBC was defined as the
102
lowest concentration of antibiotic/disinfectant at which no bacterial growth was observed in three
103
replicate experiments.
104
Galleria mellonella killing assays
105
Wax moth larvae (G. mellonella) were purchased from Livefood UJ Ltd (Rooks Bridge,
106
Somerset, UK) and were maintained on wood chips in the dark at 14°C. They were stored for not
107
longer than two weeks. Bacterial infection of G. mellonella is as essentially described by Wand
108
et al (12). Data were analysed using the Mantel-Cox method using Prism Software Version 6
109
(Graphpad, San Diego, CA, USA).
110
Whole genome sequencing of K. pneumoniae strains
111
Genomic DNA was purified using a Wizard Genomic DNA purification kit (Promega). DNA
112
was tagged and multiplexed with the Nextera XT DNA kit (Illumina). Whole genome
113
sequencing of K. pneumoniae isolates was performed by PHE-GSDU on an Illumina (HiSeq
114
2500) with paired end read lengths of 150 bp. A minimum 150 Mb of Q30 quality data was
115
obtained for each isolate. FastQ files were quality trimmed using Trimmomatic (13). SPAdes
116
3.1.1 was used to produce draft chromosomal assemblies and contigs less than 1 kb were filtered
117
out (14). FastQ reads from chlorhexidine exposed isolates were subsequently mapped to their
118
respective wild-type pre-exposure chromosomal sequence using BWA 0.7.5 (15). Bam format
119
files were generated using Samtools (16) and VCF files constructed using GATK2 Unified
120
Genotyper (version 0.0.7) (17). These were further filtered using a filtering criteria of Mapping 6
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100
quality >30, Genotype quality >40, Variant ratio >0.9, Read depth >10 to identify high
122
confidence SNPs. All the above sequencing analysis was performed using PHE Galaxy (18).
123
BAM files were visualised in Integrative Genomics Viewer (IGV version 2.3.55) (Broad
124
Institute). Changes in phoPQ and smvR regions were verified using Sanger sequencing
125
(Beckmann Genomics, Takeley, UK) and analysed using DNASTAR Lasergene 10.
126
Plasmid complementation in K. pneumoniae.
127
phoPQ and smvR genes from both wild type and chlorhexidine adapted strains were amplified
128
using primers listed in Table S1. They were digested with ClaI and XbaI and ligated into
129
pACYC-184 cloning vector (NEB). Plasmids were checked for correct sequence using Sanger
130
sequencing. Resultant plasmids were electroporated into K. pneumoniae strains using a
131
MicroPulser (Biorad) and the following settings: 2.5 kV, 25 µF, 200 Ω.and a 0.2 mm gap
132
cuvette. Subsequent plasmid insertion was tested using primers pACYC seqF
133
(CGTTTTCAGAGCAAGAGATTAC) and pACYC seqR
134
(GCATTGTTAGATTTCATACACG).
135 136 137 138 139 140
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121
Results
142
Phenotypic assessment of K. pneumoniae isolates following chlorhexidine adaptation.
143
Previous work has shown that K. pneumoniae strains are able to adapt to increasing
144
concentrations of chlorhexidine and this leads to increased MIC/MBC (minimum bactericidal
145
concentration) levels to chlorhexidine digluconate (CHD) and chlorhexidine containing clinical
146
products (11). To determine whether adaptation to chlorhexidine also led to development of
147
increased resistance to other antiseptics and cross-resistance to frontline antibiotics, MICs were
148
performed against a range of biocides for wild-type (WT) and chlorhexidine-adapted (CA)
149
strains (Table 1). Following chlorhexidine adaptation MIC values for colistin increased from 2-4
150
mg/L to >64 mg/L in five out of the six strains. The only strain which did not show increased
151
resistance to colistin following chlorhexidine adaptation was M109 CA. This strain also had the
152
lowest MIC for chlorhexidine (32 mg/L compared to a range between 128 and 512 mg/L for the
153
other strains). Resistance to colistin is often accompanied by changes in membrane composition,
154
which can be detected by altered MIC levels to azithromycin, teicoplanin and cefepime. Apart
155
from MGH 78578 CA, where the MIC of cefepime changed from >64 mg/L to 0.5 mg/L
156
following chlorhexidine exposure, no significant differences were observed. For other antibiotics
157
tested there was also no general change in susceptibility although there were individual strain
158
differences e.g. for strain NCTC 13443 there was a significant reduction in the MIC levels to
159
meropenem and chloramphenicol after chlorhexidine adaptation (Table S2). There was also no
160
significant change (>2-fold) in MIC levels to other antiseptics after chlorhexidine adaptation for
161
any of the strains tested.
162
Due to increased disinfectant/antiseptic resistance often being associated with increased efflux,
163
the MICs for chlorhexidine were re-tested in the presence of known efflux pump inhibitors 8
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CCCP and PaβN. In all of the WT strains, the addition of CCCP gave a significant reduction in
165
MIC, with values reduced from 8-32 mg/L, to between 0.5 and 4 mg/L (8-16 fold reduction). The
166
addition of CCCP also reduced the MIC to chlorhexidine in all of the chlorhexidine-adapted
167
strains, with an MIC reduction of between 32 (M109 CA) and 512 fold (MGH 78578 CA and
168
NCTC 13368 CA respectively). In each case, the MIC of the WT and chlorhexidine adapted
169
strains, in the presence of CCCP was either identical or within a 2-fold dilution (Table 1). The
170
addition of CCCP also increased the susceptibility to colistin in both chlorhexidine-adapted and
171
WT strains. For PaβN, no reduction was seen in the MICs for the WT or chlorhexidine-adapted
172
strains (data not shown).
173
The fitness of strains following chlorhexidine adaptation was assessed. Following chlorhexidine
174
adaptation, virulence in Galleria mellonella was reduced in several strains (NCTC 13443 CA,
175
MGH 78578 CA, M109 CA, NCTC 13368 CA) but not for M3 CA and NCTC 13439 CA (Fig.
176
S1). To investigate whether the observed loss of virulence was due to a growth defect, growth
177
curves were assessed. A reduction in growth rate was observed for some isolates, but this did not
178
always correlate with loss of virulence (data not shown).
179
Whole genome sequencing analysis of chlorhexidine-adapted strains
180
To understand what mechanisms are responsible for increased tolerance to chlorhexidine all
181
chlorhexidine adapted strains and their respective parental counterparts were whole genome
182
sequenced. Several genetic changes (both InDels and non-synonymous SNPs) were identified in
183
the chlorhexidine adapted strains (Table 2 and Table S3). Four out of the six strains had non-
184
synonymous SNPs in the two component regulator system phoPQ and four out of the six strains
185
showed mutations in a putative Tet-repressor gene (hereafter called smvR (Regulator of smvA))
9
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164
adjacent to and divergently transcribed from a putative homolog of the methyl viologen
187
resistance smvA gene. In three of these four strains a truncated Tet-repressor protein was
188
observed, either through a missense mutation (M3 CA), deletion of the C-terminus (M109 CA)
189
or a non-synonymous SNP leading to the formation of a premature STOP codon (NCTC 13443
190
CA). In the fourth strain (NCTC 13439 CA) the entire smvR gene was deleted along with
191
downstream sequences, which included the nitrate extrusion protein (narU) and genes encoding
192
proteins involved in nitrate reductase (narZ and narY). In two strains (M3 CA and NCTC 13443
193
CA), mutations in both smvR and phoPQ were identified.
194
In all adapted strains, genetic changes were identified in genes in addition to phoPQ and/or
195
smvR. Mutations were observed in genes associated with the outer membrane or LPS e.g. lptD in
196
NCTC 13443 CA. Strain MGH 78578 CA contained a mutation in the DNA mismatch repair
197
gene mutS which explains the increased frequency of mutations after chlorhexidine adaptation in
198
this strain. Two strains also contained alterations in the outer membrane protein MipA (MltA-
199
interacting protein). All other mutations were only present in one strain out of six following
200
chlorhexidine adaptation. There was also evidence for plasmid loss following chlorhexidine
201
adaptation which explains the loss of antibiotic resistance in certain strains e.g. meropenem in
202
NCTC 13443 CA.
203
Chlorhexidine adaptation experiments were repeated independently in the same strains and again
204
mutations in smvR and phoPQ were observed (data not shown). Therefore, since all
205
chlorhexidine adapted strains had mutations in phoPQ and/or smvR, further analysis concentrated
206
on these genes.
207
Role of SmvR and PhoPQ in resistance to chlorhexidine.
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186
To define the role of smvR in increasing resistance to chlorhexidine and colistin, pACYC-184
209
plasmids containing either the WT or the CA smvR versions from NCTC 13443 and M3 (see
210
Table 2 for individual mutations) were introduced into strain M109 CA (which has a deletion in
211
the C-terminus of smvR). Introduction of the wildtype versions of smvR from either NCTC
212
13443 or M3 resulted in a decrease in MIC levels to CHD compared to the wildtype value (from
213
32 to 8 mg/L), whereas the introduction of the CA smvR from either NCTC 13443 or M3 had no
214
effect on the MIC level to CHD (Table 3) indicating that, in M109 CA, the deletion of smvR
215
increased resistance to chlorhexidine. No change in MIC levels to colistin was observed
216
following introduction of either CA or WT smvR versions from NCTC 13443 or M3 into M109
217
CA. To confirm the role of smvA/R in elevated resistance to chlorhexidine, we examined 10 pre-
218
antibiotic era K. pneumoniae Murray strains which do not possess a homologue to these genes
219
(10). These strains were highly susceptible to chlorhexidine with MIC levels ranging from 1-
220
64 mg/L), but the susceptibility to chlorhexidine remained the same when compared to pre-
256
exposure strains (Table 4). When whole genome analysis was carried out on these isolates, strain
257
M3 CST was shown to have an identical mutation (PhoQ L348Q) to that found in strain MGH
258
78578 CA and no other mutations.
259
To understand whether colistin resistant K. pneumoniae strains had similar phoPQ expression
260
profiles to chlorhexidine adapted strains we analysed four strains with different colistin resistant
261
mutations. These included strains 16 CST (PhoQ T244N), M3 CST (PhoQ L348Q), 51851
262
(∆mgrB) and NCTC 13438 CST (PmrB D150Y). All these strains had MIC levels to CST of 64
263
mg/L and CHD (16-32 mg/L) respectively. Transcriptional analysis showed that these strains had
264
elevated levels of pmrK (20-60 fold) and with the exception of 13438 CST elevated levels of
265
phoP (5-10 fold), phoQ (4-9 fold), pmrD (5-11 fold) and pagP (4-7 fold). Strains which had
266
mutations in phoQ had very similar elevated expression levels compared to chlorhexidine
267
adapted strains containing mutations in phoP or phoQ (Table 4).
268
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252
Discussion
270
This study has shown that adaptation of clinical K. pneumoniae isolates to chlorhexidine
271
exposure can not only lead to stable resistance to chlorhexidine but also cross-resistance to
272
colistin. This has important clinical implications for the treatment of MDR (particularly
273
carbapenem-resistant) K. pneumoniae infections and outbreaks, given their increasingly
274
prevalence in hospitals (19). Many carbapenem-resistant K. pneumoniae isolates are susceptible
275
to very few antibiotics notably colistin; treatment often involves combination therapy including
276
colistin (20). Therefore, any potential loss of colistin efficacy has implications for treatment of
277
these infections. Whilst chlorhexidine has been successfully used as part of a multifaceted
278
intervention to reduce the prevalence of carbapenem-resistant K. pneumoniae in hospitals (21)
279
the observation that exposure to chlorhexidine leads to colistin resistance means eradication of
280
potentially colistin and carbapenem-resistant isolates is very problematic. Since the isolate has
281
also acquired increased resistance to chlorhexidine this also makes prevention of colonisation
282
with these isolates more difficult, which has the potential to either prolong existing outbreaks or
283
lead to new outbreaks.
284
Genome analysis of chlorhexidine adapted strains identified mutations in phoPQ and/or smvR.
285
SmvR is a putative Tet-repressor family protein, which is adjacent to and transcribed divergently
286
from the smvA gene. Adaptation to chlorhexidine resulted in disruption of smvR either by
287
complete deletion or production of a truncated version. SmvA is an efflux pump of the Major
288
Facilitator Superfamily (MFS) and has been implicated in methyl viologen resistance and the
289
efflux of acriflavine and other quaternary ammonium compounds (QACs) in S. enterica serovar
290
Typhimurium (22, 23). Upregulation of smvA was confirmed in all strains with a non-functional
291
SmvR suggesting that it acts as a repressor of smvA and that increased expression of smvA leads 14
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269
to increased chlorhexidine resistance. Divergent expression from the same promoter is
293
commonly observed in genes which are negatively regulated by Tet-repressors (24). Examples of
294
TetR homologues involved in resistance to biocides include QacR in Staphylococcus aureus and
295
EnvR and NemR in E. coli (25-27).
296
The observation that K. pneumoniae (Murray) isolates which do not possess homologues to
297
smvA/R are highly susceptible to chlorhexidine again suggests that smvA is an important efflux
298
pump in chlorhexidine resistance. E. coli does not possess a homologue to smvA and is
299
significantly more susceptible than Klebsiella to chlorhexidine (28). However, potential
300
homologues of smvA/R are found in most species of Enterobacteriaceae as well as other Gram-
301
negative MDR pathogens e.g. P. aeruginosa and A. baumannii. This potentially means that
302
SmvA homologues mediate reduced susceptibility to polycationic antiseptics in other bacteria
303
and that a similar mechanism of adaptation, with deletion of smvR, may be observed in other
304
clinically-important pathogens. In A. baumannii, deletion of another Tet-repressor (adeN) which
305
is adjacent to an efflux pump operon (adeIJK) resulted in acquisition of increased resistance to
306
Triclosan (29).
307
The addition of the EPI CCCP increased susceptibility to chlorhexidine, both in the wild-type
308
and chlorhexidine-adapted strains, suggesting that efflux pumps known to be affected by this
309
uncoupling agent including MFS-type pumps such as smvA, might be involved. In colistin-
310
resistant, chlorhexidine adapted strains, CCCP also increased the susceptibility to colistin. This is
311
consistent with other studies suggesting a role for efflux in colistin resistance in a range of Gram-
312
negative pathogens (30). CCCP, due to its function as a general uncoupler of proton motive
313
force, may affect metabolic activity, which has been linked to increased susceptibility to colistin
15
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292
in A. baumannii (31) and chlorhexidine in P. aeruginosa (32). Indeed growth of both WT and
315
CA strains was diminished following addition of CCCP.
316
The cross-resistance to colistin following chlorhexidine adaptation is likely due to upregulation
317
of the operon containing pmrK. This operon functions to modify LPS with cationic substitution
318
of the phosphate groups with 4-amino-4-deoxy-L-arabinose (L-Ara4N). This reduces the lipid A
319
net negative charge and causes a reduction in the binding affinity of colistin (33). PhoPQ has
320
been linked to modification of LPS and outer membrane content (34-36) and regulates genes
321
involved in colistin resistance including pmrD and pmrAB (37, 38). Mutations in PhoPQ in K.
322
pneumoniae have already been shown to be associated with colistin and cationic peptide
323
resistance and lipid A modification (37, 39-41).qPCR analysis of strains with phoPQ mutations
324
showed a significant upregulation in expression levels of phoPQ (at least 5 fold). For these
325
strains, genes regulated directly or indirectly by phoPQ (pmrD and pmrK) were also upregulated.
326
This upregulation was consistent for K. pneumoniae strains which are resistant to both colistin
327
and chlorhexidine or colistin alone. However, there are examples of mutations in PhoQ leading
328
to elevated phoPQ expression but not development of colistin resistance (42). In this study one
329
chlorhexidine adapted strain (NCTC 13439 CA) had elevated colistin resistance but no detected
330
mutations in phoPQ and did not show upregulation of their expression. However, this strain did
331
have upregulated pmrD and pmrK expression levels, which suggested that these genes were
332
activated independently of phoPQ. The expression levels of pmrK are regulated by another two
333
component regulator implicated in colistin resistance, pmrAB. Whilst expression levels of pmrAB
334
may be regulated by PhoPQ (through PmrD) pmrAB is also transcribed, independently of
335
PhoPQ, from a constitutive promoter within the upstream pagB gene (34). Expression of pmrD
336
may be subject to regulation by a second, unknown system based on the observation in E. coli, 16
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314
that expression of pmrD was not abolished following inactivation of phoPQ (43). In K.
338
pneumoniae, upregulation of pmrD following adaptation to colistin was not always linked to
339
upregulation of phoQ (44). The presence of a mutation in the RarA regulator in NCTC 13439
340
CA is consistent with up-regulation of rarA in K. pneumoniae enhancing growth on polymyxin B
341
(45). The mutated amino acid (W37R) is predicted to directly interact with DNA, according to
342
the crystal structure of the related regulator MarA (46).
343
Results demonstrate that PhoPQ mutations arise through chlorhexidine adaptation and can be
344
clearly linked to colistin resistance. However, the data suggests that additional factors are
345
important in mediating resistance to chlorhexidine and these may operate independently of
346
PhoPQ. qPCR analysis using colistin-adapted strains with specific mutations in PhoQ (T244N
347
and L348Q) showed very similar expression levels of phoPQ to chlorhexidine adapted strains
348
containing PhoPQ mutations (NCTC 13368 CA, NCTC 13443 CA, M3 CA and MGH 78578
349
CA) suggesting that increased expression levels of phoPQ, pmrD and pmrK, whilst important for
350
colistin resistance are not sufficient for increased resistance to chlorhexidine. This observation
351
was re-enforced by colistin resistance but not chlorhexidine resistance being transferred on
352
pACYC phoQ A20P, which again caused upregulation of phoQ, pmrD and pmrK relative to
353
pACYC phoPQ WT. An identical PhoQ mutation (L348Q) was present in both strains M3 CST
354
and MGH 78578 CA suggesting that this mutation alone is not sufficient to lead to increased
355
chlorhexidine resistance but is enough to generate colistin resistance. Interestingly, upregulated
356
pagP expression was observed in colistin adapted strains where upregulated phoPQ expression
357
was also observed (KP16 CST, M3 CST, 51851). For chlorhexidine-adapted strains, those with
358
mutations in phoPQ but not smvR showed no pagP upregulation. pagP expression does not
359
appear to be dependent upon specific allelic changes since both MGH 78578 CA and M3 CST 17
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337
have mutations in phoQ (L348Q) but only M3 CST showed upregulated pagP expression. These
361
observations could imply that upregulated pagP expression is antagonistic to chlorhexidine
362
resistance. M3 CA and NCTC 13443 CA have elevated pagP levels but also have mutations in
363
smvR in addition to mutations in phoPQ. We have demonstrated that smvR has a role in
364
chlorhexidine resistance and it is plausible that mutations in smvR “mask” any negative effects
365
on chlorhexidine resistance caused by elevated pagP expression. PagP causes palmitoylation of
366
lipid A which has been found to not contribute to colistin resistance in other pathogens (47), but
367
its role in chlorhexidine resistance has not been investigated. It is possible that increased
368
palmitoylation of lipid A reduces the frequency of other lipid A modifications which are
369
important in chlorhexidine resistance. Future work will focus on understanding the role of lipid
370
A modification in chlorhexidine resistance including the role of PhoPQ, PagP and MarA.
371
It is also plausible that mutations in phoPQ are secondary-site or compensatory mutations, which
372
enable the resistant strain to recover its fitness and virulence following the initial development of
373
biocide resistance (48). PhoPQ is a global regulator and regulates a number of genes important in
374
fitness and virulence (49-51). Again, further work is needed to investigate this.
375
Overall this study has identified a novel resistance mechanism to chlorhexidine (smvA/R) that
376
may potentially operate in a number of different species. Clearly increased smvA expression is
377
important for chlorhexidine adaptation in K. pneumoniae but it is not the only mechanism and
378
may operate in conjunction with other regulatory processes. Chlorhexidine-adaptation is also
379
associated with the generation of mutations in PhoPQ, which affect a number of known
380
regulatory targets (notably pmrD and pmrK). Upregulation of these genes also correlates with the
381
presence of colistin resistance. That increased colistin and chlorhexidine resistance may occur in
382
clinical isolates without significant loss of fitness/virulence highlights the potential challenges 18
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360
associated with critical infection control procedures and the use of chlorhexidine as an antiseptic
384
to control healthcare–associated infections.
385
Funding information
386
This project was funded by Public Health England GIA grant project 109506. The views
387
expressed are those of the authors and not necessarily those of the funding body.
388
Acknowledgements.
389
The authors would like to thank Dr Steven Pullan for his assistance in the analysis of the whole
390
genome sequence data. The authors declare no conflicts of interest.
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534
Table 1 MIC (mg/L) values of various antibiotics and disinfectants for chlorhexidine-adapted strains. Disinfectants used were
542
chlorhexidine digluconate (CHD), benzalkonium chloride (BCl), Octenidine Dihydrochloride (Oct), hexadecylpyridinium chloride monohydrate
543
(HDPCM) and ethanol (EtOH). Antibiotics used were colistin (CST), azithromycin (AZM), cefepime (FEP) and teicoplanin (TEC). All values
544
are given in mg/L. All MICs are shown as the range from at least three independent experiments. + CCCP indicates the addition of the efflux
545
pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone. Additional antibiotics are shown in Table S2. Strain M109 WT M109 CA NCTC 13439 WT NCTC 13439 CA M3 WT M3 CA NCTC 13443 WT NCTC 13443 CA NCTC 13368 WT NCTC 13368 CA MGH 78578 WT MGH 78578 CA
546
a
CHD 8 32-64a 8-16 256a 8-16 32-64a 8-16 256-512a 32 256a 8-16 256-512a
CHD + CCCP 0.5-1 0.5-1 2-4 1-2 1-2 0.5-2 1-2 1-2 2-4 1-2 1-2 0.5-2
BCl
Oct
HDPCM
EtOH (%)
CST
16 8-16 16 16 8-16 8-16 8-16 8-16 32 16 8-16 8-16
4 2-4 2-4 2-4 2-4 2-4 4 2 4-8 4-8 4 4
4-8 4-8 16 8-16 8 8-16 8-16 8-16 32-64 16 8-16 8
3.125 6.25 6.25 6.25 6.25 3.125 3.125 3.125 6.25 6.25 6.25 3.125
2 2-4 4 >64a 2-4 >64a 2 >64a 2-4 >64a 2-4 >64a
CST + CCCP 2 0.5-1 2 1 2 1-2 2 2 2-4 2-4 2-4 1-2
AZM
FEP
TEC
8-16 8-16 32 32 16-32 8-16 64 16-32 64 64 32 32-64
0.06-0.125 0.06-0.125 >64 >64 >64 >64 >64 >64 64 64 >64 0.5a
>64 >64 >64 >64 >64 >64 >64 >64 >64 >64 >64 >64
≥ 4-fold increase or decrease in MIC for chlorhexidine-adapted strains (CA) relative to non-adapted strains (WT)
547 548
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541
550
Table 2 Chromosomal genetic changes after exposure to chlorhexidine Strain and gene name (if known) M109 wcaJ yfiN smvR NCTC 13439* mipA rarA smvR narU narZ narH narJ M3 phoP ackA smvR NCTC 13443* sufD phoQ lptD smvR NCTC 13368 28
Type of Change
Change
MGH 78578 equivalent based on (NC_009648.1)
Function
SNP SNP Deletion
Q399STOP A173V 400 bp Del
KPN_RS15690 KPN_RS10110
CPS biosynthesis glycosyltransferase Diguanylate cyclase TetR family transcriptional regulator
SNP SNP Deletion Deletion Deletion Deletion Deletion
Q98STOP W37R Complete Del Complete Del Complete Del Complete Del First 130 aa Del
KPN_RS06390 KPN_RS15910 KPN_RS10110 KPN_RS10115 KPN_RS10120 KPN_RS10125 KPN_RS10130
MltA-interacting protein AraC family transcriptional regulator TetR family transcriptional regulator nitrite extrusion protein 2 nitrate reductase A subunit alpha nitrate reductase A subunit beta nitrate reductase molybdenum cofactor assembly chaperone
SNP SNP Deletion of 5 bp after nucleotide 22 Deletion (G) after nucleotide 445
E82K S274F Truncation of 72 aa, (normally 191 aa) Truncation of 174 aa, (normally 184 aa)
KPN_RS06075 KPN_RS14420 KPN_RS10110
PhoP family transcriptional regulator Acetate kinase TetR family transcriptional regulator
KPN_RS17785
isopentenyl-diphosphate Delta-isomerase
SNP (synonymous) SNP SNP SNP Insertion (T) after nucleotide 375
A20P Y625N W125STOP Truncation of 125 aa, (normally 235 aa)
KPN_RS11525 KPN_RS06070 KPN_RS00270 KPN_RS10110 KPN_RS14015
Fe-S cluster assembly protein Two component sensor protein LPS assembly outer membrane complex protein TetR family transcriptional regulator Membrane protein, putative permease
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549
SNP SNP SNP SNP
E253A V122F V27E Y98C
SNP
-
SNP
mutS surA phoQ -
SNP SNP SNP SNP SNP SNP Insertion (A)
LuxR family transcriptional regulator 30S Ribosome protein S1 Part of outer membrane protein complex PhoP family transcriptional regulator
G121S
KPN_RS01550
A276V
KPN_RS11605
T115P R46C L74P L348Q N30S N32S In repeat region AAGCTAA Truncation of 136aa (normally 170aa) Truncation of 172 aa, (normally 329 aa) Elongation of 5 aa, (normally 1490 aa) Truncation of 649 aa, (normally 1332 aa) Extra Glycine
KPN_RS16580 KPN_RS24340 KPN_RS00265 KPN_RS06070 KPN_RS04685 KPN_RS20410 -
Phosphonate ABC transporter substrate-binding protein 5-methyltetrahydropteroyltriglutamate homocysteine methyltransferase DNA mismatch repair protein LuxR family transcriptional regulator Peptidyl-prolyl cis-trans isomerase Two component sensor protein Aldose dehydrogenase Transcription accessory protein -
Insertion (C) after KPN_RS08585 nucleotide 122 Insertion (G) after KPN_RS10945 nucleotide 131 Insertion (T) after KPN_RS15800 nucleotide 4423 Insertion (GG) after KPN_RS25360 nucleotide 1920 nuoC/D Insertion (GGG) after KPN_RS14365 nucleotide 1382 mipA Insertion (G) after Truncation of 72 aa, KPN_RS06390 nucleotide 195 (normally 275 aa) nikE Insertion (C) after Truncation of 81 aa, KPN_RS20815 nucleotide 87 (normally 263 aa) *Evidence of plasmid loss in these strains following chlorhexidine adaptation comA
551
KPN_RS05050 KPN_RS15655 KPN_RS06075
552
29
Competence protein nitrate ABC transporter substrate-binding protein Hypothetical protein helicase NADH-quinone oxidoreductase subunit C/D MltA-interacting protein MipA ATP-binding protein of nickel transport system
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acoK rpsA yfiO (bamD) phoP MGH 78578* -
554
Table 3 MIC values of chlorhexidine (CHD) and colistin (CST) after plasmid complementation. Levels of resistance to chlorhexidine
555
(CHD) and colistin (CST) were measured after conjugation of plasmids into strains listed. All values are given in mg/L. All MICs are shown as
556
the range from at least three independent experiments. Strain
M109 CA
M109 WT 25 557
30
Plasmid pACYC-184 alone pACYC M3 smvR WT pACYC M3 smvR CHD pACYC 13443 smvR WT pACYC 13443 smvR CHD pACYC phoPQ WT pACYC phoQ A20P pACYC-184 alone pACYC phoPQ WT pACYC phoQ A20P pACYC-184 alone pACYC phoPQ WT pACYC phoQ A20P
Description empty vector smvR from strain M3 smvR from strain M3 CA smvR from strain NCTC 13443 smvR from strain NCTC 13443 CA phoPQ from strain NCTC 13443 phoPQ from strain NCTC 13443 CA empty vector phoPQ from strain NCTC 13443 phoPQ from strain NCTC 13443 CA empty vector phoPQ from strain NCTC 13443 phoPQ from strain NCTC 13443 CA
CHD MIC 32 8 32 8 32 32-64 32-64 8-16 8-16 8-16 8-16 8-16 8-16
CST MBC 32 8 32 8 32 32-64 64 8-16 8-32 16-32 8-16 16-32 16-64
MIC 2-4 2 2-4 2 2-4 1-2 64 0.5-1 0.5-1 32-64 0.5 0.5-1 32-64
MBC 2-4 2 2-4 2 2-4 2 64->64 2-4 2-4 64 2-4 1-4 32->64
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553
Table 4 Expression levels of select genes following chlorhexidine (CA) or colistin (CST) adaptation. Numbers represent fold upregulation
559
with respect to wildtype levels except where indicated. Relevant mutations and fold increase in CHD and CST following adaptation are also
560
shown.
561
Significant fold upregulation compared to wt or pACYC phoPQ WT*
Strain phoP M109 CA NCTC 13439 CA M3 CA NCTC 13443 CA NCTC 13368 CA MGH 78578 CA M109 WT pACYC phoQ A20P* KP16 CST M3 CST 51851 vs 46704 NCTC 13438 CST
562 563 564 31
phoQ
smvA 10 19 18 27
smvR
7 6
pmrK
pmrD
12 64 26 18 24
3 19 6 6 6
15 5 8 6
9 5 7 8 3
7
5 8 10
4 5 9
20 38 60 55
mutation pagP
Fold increase in MIC over wt CHD CST 4 none 16 >32 8 >32 16 >32 16 >32 16 >32
7 2
ΔsmvR CT ΔsmvR PhoP E82K, ΔsmvR PhoQ A20P, ΔsmvR CT PhoP Y98C PhoQ L348Q
2
4
phoQ A20P on plasmid
none
64
5 7 11
7 4 6
PhoQ T244N PhoQ L348Q MgrB Q30STOP PmrB D150Y
none none none none
>64 >64 >64 >64
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558
