All the bacterial strains and plasmids used in this study are listed in Table 1. Strains were cultured in BBL™ Muller Hinton II (Cation-Adjusted) Broth (MHB) or Trypic Soy Broth (TSB) (Becton Dickinson, Franklin Lakes, NJ). For solid media, broth was supplemented with 1.7% agar (Alfa Aesar, Waltham, MA). Where needed, strains were cured of their plasmid as described in Udo et al. (1997). Briefly, a culture was grown overnight shaking at 37°C and then diluted 1:500 in TSB. This culture was grown at 42°C for 48 h and dilutions were then plated on TSA and grown at 37°C to obtain single colonies. Plates containing isolated colonies were replica plated to MHA and MHA supplemented with 20 μg/mL of cadmium chloride and then grown at 37°C. Cadmium sensitive colonies were double purified and screened with the pC02 PCR screen described below to test for the presence of the large plasmid(s). Large plasmid cured strains were made resistant to rifampin and novobiocin as previously described by selecting mutant strains that were resistant to rifampin (Sigma-Aldrich, St Louis, MO) followed by subsequent passage in increasing concentrations of novobiocin (Sigma) (Udo et al., 1997).

The strains isolated from Ft. Benning were globally characterized to define pulse field type, gene presence and antibiotic resistance patterns as previously described (Schlett et al., 2014).

DNA extraction for Pacbio sequencing was performed from overnight liquid cultures using phenol-chloroform extraction with the Easy-DNA kit (Invitrogen, Waltham, MA). The DNA samples were sent to the University of Maryland Institute for Genome Sciences and sequenced with Pacbio RSII (Pacific Biosciences, Menlo Park, CA).

DNA extraction for Illumina sequencing was performed from overnight liquid cultures using the Wizard Kit (Promega, Madison, WI) and libraries were produced using the Nextera XT DNA Library Preparation Kit (Illumina, Inc., San Diego, CA) according to the manufacturer's instructions. Libraries were multiplexed and sequenced using an Illumina MiSeq 600-cycle kit and 2 × 300 base pair read lengths. Sequence read quality was analyzed with FastQC (Andrews, 2016) and low-quality bases were trimmed with Sickle (Joshi and Fass, 2011). Illumina and Pacbio sequence reads were assembled using SPAdes (Bankevich et al., 2012) and plasmidSPAdes (Antipov et al., 2016). The resulting contig sequences were compared to each other, to published reference genomes, and to PCR, Sanger sequencing, and agarose gel electrophoresis results of restriction enzyme digested and non-digested DNA in order to correctly assign contigs as chromosomal or plasmid as well as to look for assembly artifacts.

In order to determine the closest published reference for single nucleotide variation (SNV) analysis, the longest contig from each assembly was aligned against the National Center for Biotechnology Information (NCBI) nucleotide database using BLAST (Altschul et al., 1990). Based on these results, S. aureus USA300 strain TCH1516 (NCBI accession number CP000730) was selected as the reference. SNV data were analyzed using methods previously described (Millar et al., 2017). Briefly, the Bacterial and Archaeal Genome Analyzer (Williams, 2016) a wrapper for proven third-party bioinformatics tools, was used. Sequence reads were mapped to the reference using BWA (Li and Durbin, 2009), and variant calls and filtering were performed with GATK (McKenna et al., 2010). Genomic regions that contained insertions/deletions (indels), potential chromosomal rearrangements, and sequence repeats known to increase the likelihood of false-positive variant calls were excluded from the SNV set. A multiple sequence alignment (MSA) was created from nucleotide substitutions, small deletions called by GATK, and putative large deletions detected in the BWA sequence alignments where no reads mapped. A maximum likelihood tree was constructed from the nucleic acid MSA using PhyML (version 3.2) (Guindon et al., 2010) tree search with the GTR substitution model. Areas in the MSA containing structural variants (indels) were masked in the PHYLIP representation of the MSA as undetermined nucleotides and excluded from consideration in phylogenetic tree construction. A full list of indels and SNVs are available as Supplemental Tables 1, 2, respectively.

Gene content comparisons among strains were conducted using RAST for gene annotation and visualization (Aziz et al., 2008; Overbeek et al., 2014; Brettin et al., 2015).

The minimum inhibitory concentration (MIC) of chlorhexidine for each strain was tested as previously described (Johnson et al., 2015). Briefly, ~1.5 × 10⁵ colony forming units (CFU) were inoculated into MHB containing increasing concentrations of purified (≥99.5%) chlorhexidine (Sigma). The cultures were grown at 37°C degrees overnight, shaking at 220 rpm. The MIC was determined by visual analysis of the culture's turbidity. Graphs were generated with Prism Software (GraphPad Software Inc., La Jolla, CA) with three independent replicates.

S. aureus strains were grown overnight in MHB and 1.5 mL of each sample was centrifuged to pellet the cells. Cell pellets were treated with 0.1 mg of lysostaphin for 30–45 min at 37°C and plasmid DNA was purified using the QIAprep Spin Miniprep Kit (Qiagen, Germantown, MD). Chromosomal DNA was purified using the Wizard Genomic DNA Purification Kit (Promega) after lysostaphin treatment of cells for 30–45 min.

The primer sequences used to PCR amplify the backbone of pC02 are listed in Supplemental Table 3. Initially, primers were designed to be evenly spaced around the pC02 plasmid but the original primer set that annealed in the 40,000 kb region yielded multiple products. Thus, these primers were redesigned, shifted upstream and became primer-pair 7. The PCRs were performed in 25 μL reactions containing nuclease-free water, plasmid DNA, GoTaq Green Master Mix (Promega), and 0.3 μM each of the forward and reverse primers (Integrated DNA Technologies, Coralville, IA). PCR cycling conditions were as follows: incubation at 95°C for 180 s, 28 cycles of 95°C for 30 s, 53°C for 30 s and 72°C for 160 s followed by a final extension of 72°C for 5 min. The reaction amplicons were separated and visualized on 1% agarose gels.

For strains that were not subjected to WGS, the qacA/B gene was amplified and a portion was sequenced as previously described (Johnson et al., 2015). Utilized primers are listed in Supplemental Table 3. The plasmid map image (Figure 1) was generated with AngularPlasmid http://angularplasmid.vixis.com (AngularPlasmid, 2015).

MLST was performed as previously described (Enright et al., 2000). The trimmed sequences of arc, aroE, glpF, gmk, pta, tpi, yqi, were submitted to the MLST website: http://saureus.mlst.net/ (Imperial College in London, United Kingdom) for typing.

Filter-mating experiments were completed similar to a previous description (Forbes and Schaberg, 1983). Briefly, for S. aureus, recipient and donor cells were grown overnight in MHB and then subcultured 1:50 in MHB the next day. The cells were grown to an OD₆₀₀ of 0.3–0.4 and 0.25 mL of both the recipient and donor cultures were then added to 2.5 mL of prewarmed MHB. The cells were then collected on a 0.45 μm filter and placed cell-side down on prewarmed Muller Hinton II (Cation-Adjusted) Agar (MHA). The mating mixtures were incubated overnight at 37°C and the following morning, filters were removed from the agar and vortexed vigorously for 1 min in 1.5 mL of PBS to release the cells from the filter. 10 μL of the cell suspension was taken to determine the recipient CFU and the remaining cells were plated onto selective media. Recipient CFU were enumerated on MHA supplemented with 5 μg/mL of novobiocin sodium salt (Sigma) and 10 μg/mL of rifampicin (Sigma). The transconjugants were identified after 48–72 h of growth on MHA supplemented with 5 μg/mL novobiocin, 10 μg/mL of rifampicin and 20 μg/mL of cadmium chloride. For experiments where Staphylococcus epidermidis was the recipient, numbers of recipient cells were determined by plating on TSA supplemented with 5 μg/mL novobiocin sodium salt and 10 μg/mL rifampicin and transconjugants were enumerated after 48 h of growth on TSA supplemented with 5 μg/mL of novobiocin sodium salt, 10 μg/mL of rifampicin and 10 μg/mL of erythromycin. For conjugation experiments where Enterococcus faecalis was the recipient, MHB/MHA were substituted with TSB/Trypic Soy Agar (TSA), respectively, in order to obtain similar growth between S. aureus and E. faecalis. Numbers of recipient cells were determined by plating on TSA supplemented with 10 μg/mL fusidic acid sodium salt (Sigma) and 10 μg/mL rifampicin. The transconjugants were identified after 48 h of growth on TSA supplemented with 10 μg/mL of fusidic acid sodium salt, 10 μg/mL of rifampicin and 10 μg/mL of erythromycin (Sigma). In all cases, the conjugation frequency was determined by dividing the number of transconjugants by the number of recipient cells. The reported frequencies are the average of at least three independent experiments. To confirm that transconjugants actually contained the pC02 plasmid, a subset of the isolates were restreaked onto their appropriate selective media, and then screened by colony PCR using primer-pair 7 of the pC02 screen.

The possibility of movement of pC02 by phage was tested in several possible ways. First, the filter-mating protocol was conducted with the following modifications: 0.22 μm filter-sterilized supernatants (0.250 mL) from the C02 donor were mixed with 0.250 mL of the C02-RN recipient cells in 2.5 mL of MHB. This mixture was incubated at 30°C for 90 min, and then cells were collected on a 0.45 μm filter. Following collection on the 0.45 μm filter, the assay proceeded as described for the other filter-mating experiments.

Secondly, the necessity of cell-to-cell contact for conjugation was tested using a modified filter-mating assay. Briefly, C02 and C02-RN were grown to an OD₆₀₀ of 0.3 to 0.4. 0.25 mL of both the C02-RN recipient and C02 donor cultures were individually added to separate culture flasks containing 2.5 mL of prewarmed MHB. The donor and recipient cells were individually collected on separate 0.45 μm filters. The recipient cell filter was placed cell-side-down on MHA and the donor cell filter was placed on top of the recipient filter with the cells adjacent to the back of the recipient filter. At the end of the mating assay, both of the filters were removed and vortexed together in 1.5 mL of PBS and processed as described above. Finally, to examine the possibility of any endogenous phage release, donor cells were also pretreated with mitomycin C (Sigma) to induce any possible phage. Overnight cultures of C02 were diluted 1:100 in 10 mL of TSB and grown at 37°C to an OD₆₀₀ of 0.6–0.8. 1.0–2.5 μg/mL of mitomycin C was then added to the cultures and induction was allowed to proceed at 30°C for 6–8 h before cultures were placed at 4°C for 16 h. Afterwards, the cultures were filter sterilized through a 0.45 μm filter and then serial diluted in phage buffer (Novick, 1963). Resident phage were titrated by serial dilution with RN4220 on TSA plates with TSB soft agar (0.5%). No plaques were ever obtained.

A previous molecular epidemiologic study used WGS to identify a clonal HA-MRSA outbreak strain that contained a qacA-positive plasmid that may have given the strain a fitness advantage within the hospital environment (Senn et al., 2016). However, there is a lack of genomic information available for CA-MRSA strains that carry qacA. To characterize both colonizing and clinically relevant qacA-positive S. aureus strains, we initially examined 11 qacA/B positive strains and 8 qacA/B-negative control strains (Tables 1, 2) that were all obtained from a chlorhexidine-based SSTI prevention trial (Ellis et al., 2014; Schlett et al., 2014); the previously characterized C01 (qacA-) and C02 (qacA+) strains (Johnson et al., 2015) were included for comparison (Table 1). These S. aureus strains were collected from all three study groups (chlorhexidine, standard and enhanced standard-see Table 1 footnote d) from July 2010 through October 2011. All of the strains were USA300 with the exception of one Methicillin-Sensitive Staphylococcus aureus (MSSA) strain (2148.N), which had a pulse field-type that was indistinguishable. A majority of the strains were from two MLST lineages (ST8, ST72) and one strain (5107.N) had a previously uncharacterized sequence type due to a novel aroE allele. Antibiotic susceptibility testing revealed erythromycin resistance was common in the strains, occurring in 16/18 (88%) strains. Additionally, one strain (1960.N02) was also resistant to clindamycin. All tested strains were susceptible to sulfamethoxazole. Moreover, mupA, which is required for high-level resistance to mupirocin, was identified in four strains that contained qacA/B.

To specifically examine the qacA/B-positive strains for the presence of pC02, we developed a PCR based screen. A total of 10 primer-pairs were designed to span the entire pC02 sequence (Figure 1). Of these, the primers in primer-pair 7 annealed to qacA/B and to the pC02 backbone. Additionally, an internal qacA/B primer pair was also included in the screen. A total of 3/11 qacA/B-positive strains, including the C02 control, yielded positive PCR amplicons for all 10 primer-pairs within the pC02 screen, suggesting that these strains carry pC02 or a pC02-like plasmid (Table 2). In addition to the pC02-like amplicon pattern, among qacA/B-positive strains we also identified two additional amplicon patterns; 4/11 strains amplified with primer pairs 6, 8 and the internal qacA/B primers while 4/11 amplified with primer pairs 6, 7, 8 and the internal qacA/B primers. Among the qacA-negative strains, two major patterns were observed; five strains showed a pattern that was indistinguishable from the C01 isolate (amplicons with primer pairs 6 and 8, termed pC01-like) and two strains yielded no amplification products.

Given the functional data that indicate differences in the ability of QacA and QacB to mediate reduced susceptibility to chlorhexidine (Paulsen et al., 1996), we sequenced the qacA/B amplicon to distinguish qacA and qacB. Of the seven identified single nucleotide variants (SNVs) that discriminate qacA from qacB, a single SNV at residue 323 is responsible for an amino acid substitution that confers the substrate specificity for the efflux pumps (Paulsen et al., 1996). Sequencing analysis from the amplicon produced from the internal qacA/B primers (Supplemental Table 3) revealed that all of the strains encoded the qacA sequence at this key residue (data not shown). En masse, the data from this screen suggested that several different qacA-containing plasmid types were present in this study population and that pC02 or a pC02-like plasmid was present in clinical isolates that were obtained from multiple individuals.

Given that the presence of qacA has been shown in some, but not all, cases to be associated with decreased susceptibility to chlorhexidine (Johnson et al., 2013; McDanel et al., 2013) and given that our sequencing results indicated that all of the qac-positive strains carried qacA, we next sought to determine whether these strains showed decreased susceptibility to chlorhexidine. Broth dilution assays using pure chlorhexidine revealed a 3–4 fold decrease in susceptibility to chlorhexidine in all strains that harbored qacA (Table 2). When the MIC values were plotted based on plasmid type (Table 2), all strains carrying qacA displayed a statistically significant decrease in susceptibility to chlorhexidine as compared to strains lacking qacA (Figure 2). Furthermore, the plasmid-pattern group (positive for primer-pair 6,7,8) displayed significantly higher resistance to chlorhexidine than all other strains that contained qacA (Figure 2). Taken together, these results indicate that S. aureus strains that carry qacA show a decreased susceptibility to chlorhexidine.

While MLST and PFGE profiles can provide fast preliminary information concerning strain relatedness, these techniques do not accurately determine strain clonality or provide detailed genomic information regarding the chromosome or mobile genetic elements, such as plasmids, which often carry resistance determinants and virulence genes (McCarthy and Lindsay, 2012). To further characterize the obtained S. aureus strains, eight strains were selected for WGS: two qacA negative strains, and two strains from every qacA positive plasmid-pattern group (Table 2). In addition, C01 and C02, which were both previously sequenced using PacBio technology (Johnson et al., 2015), were included for resequencing using MiSeq. To improve the accuracy of the assembled genomes, each strain was ultimately sequenced using both MiSeq and PacBio technologies. The average (min-max) read depth obtained with the MiSeq was 110 (range 67–167), whereas for the PacBio data the average (min-max) read depth was 17 (range 11–24). Analysis of the co-assembled Illumina short read and PacBio long read sequencing data revealed that each of the strains sequenced in this study had a similarly-sized chromosome, ranging in size from ~2.7 to 2.9 million base pairs. Additionally, each strain carried at least one, and in many cases, several plasmids of varying sizes. The various replicons and their sizes are presented in Table 3.

Overall, the 10 chromosomes sequenced in this study were found to be very similar to one another and to the reference strain USA300_TCH1516 (Highlander et al., 2007). However, a few interesting differences were observed. For example, gross inspection of chromosomal sequence alignments indicated three main areas of significant sequence variation with respect to the reference: a region in and around the gene encoding the replication initiation protein; a nearby region encoding a cassette chromosome recombinase A, a Zn-dependent hydroxyacylglutathione hydrolase/polysulfide binding protein, and some hypothetical proteins; and perhaps most significantly, a roughly 13 kilobase (kb) super-antigen encoding pathogenicity island (PAI; Supplemental Figure 1). This PAI, which is found in S. aureus USA300_TCH1516, was found to be absent in three of the 10 strains sequenced in this study. In strain USA300_TCH1516 and in seven of the strains described herein, this PAI encodes 17 genes, including genes for superantigen enterotoxin SEK, superantigen enterotoxin SEL, and secreted protein Ear. To verify that the apparent lack of this PAI in strains C02, 3020.C01, and 1969.N was not due to a missassembly, we performed BLAST against chromosomal and plasmid contigs as well as mapping of raw reads to the PAI sequence; in all cases the absence of the PAI was verified.

To examine relatedness of the strains, single nucleotide variation (SNV)-based phylogenies were constructed. Applying the GATK standard “hard” filter, and after filtering variants that occur in regions of genome repeats and putative chromosomal rearrangements, a total of 785 SNVs were identified (Supplemental Table 2). One of the nasal carriage isolates, 2148.N, was an outlier from the remainder of the strains and was subsequently removed from the phylogenetic tree that is presented in Figure 3; the original tree is presented in Supplemental Figure 2. Of note, the other two sequenced nasal carriage isolates clustered in a single clade with several SSTI isolates. Additionally of note, C02 and 3020.C01 were essentially clonal with the nasal carriage isolate 1969.N, perhaps reflecting the origin of these three isolates from the same battalion and company within the span of 2 months (Table 1). These three strains were also the only three sequenced strains in this study that were found to lack a PAI. Between C02 and 3020.C01, only six SNVs were identified, five of which were located in coding regions. Of those five, three were synonymous SNVs. Between 3020.C01 and 1969.N, eleven SNVs were identified, nine of which were in coding regions; of those nine, seven were synonymous. Between 1969.N and C02, seven SNVs were identified, six of which were located in coding regions; of those six, two were synonymous. Table 4 represents the SNVs by position (with respect to the coordinates of the reference genome S. aureus USA300_TCH1516; NC_010079.1) for each of the three isolates that were found to be clonal. The bulk of the SNVs were identified in a gene encoding an enoyl-ACP reductase. Of note, all of the SNVs in this gene were found to be synonymous substitutions. We hypothesize that that this may be due to the essential nature of the product of this gene, which is used to complete the fatty acid chain elongation cycle and is necessary for a properly functioning cell membrane (Chaudhuri et al., 2009). Specifically, it catalyzes the stereospecific reduction of a double bond between the C2 and C3 positions of the growing fatty acid chain (Priyadarshi et al., 2010).

Consistent with the very similar chromosomal sequences for C02, 3020.C01, and 1969.N, these three strains also had similar plasmid profiles, each having one roughly 3 kb plasmid and one roughly 61 kb plasmid; all three strains were qacA-positive by PCR (Table 2). Comparison of the WGS data for all of the strains in this study, showed roughly three categories of plasmid (Table 3) (1) an ~3 kb plasmid, which was identified in nine of ten strains and found to have 65–100% nucleotide identity among the ten strains; (2) an ~27–32 kb plasmid, which was identified in four of ten strains and found to have 69–99% nucleotide identity; and (3) an ~61kb plasmid, which was identified in three of ten strains and found to have 99.78–99.94% nucleotide identity. Among the various plasmid size categories, the 3 kb plasmids were all cryptic plasmids with the exception of p2148.N, which harbored a predicted cadmium transporter. The 3kb plasmids all contained Rep_1 rolling circle replication initiation proteins (Kwong et al., 2017). Conversely, the large plasmids all carried theta replicating RepA_N domain replication initiation proteins. In addition to a RepA_N protein, p1626.C01c and p1971.C01c also harbored a Rep_2 and Rep_1 rolling circle replication initiation protein, respectively. The multiple replication proteins suggest a recent plasmid integration event with a small rolling circle replicating plasmid. The 27 kb plasmids were nearly identical and contained genes for macrolide, β-lactams and cadmium resistance. In addition to the resistance genes carried on the 27 kb plasmids, the 32 kb plasmid (p2148.C01b) harbored a 6.5 kb region containing qacA and an IS5/IS1182 family transposase. The 61kb pC02-like plasmids carried qacA and resistance genes for cadmium, macrolides and β-lactams. When the plasmids were grouped and analyzed based on qacA status and not size, we found that the plasmids that carried qacA ranged in size from 21 to 61 kb. As a whole, the large plasmids contained numerous insertion elements, including IS6, IS257, and IS5 transposases. Moreover, enzymes required for DNA mobilization, including invertases and resolvases, were found throughout the large plasmids. Indeed, examination of the plasmid population as a whole, suggested modularity in the staphylococcal plasmids where some segments were highly conserved between the various plasmid groups. Additionally, putative metabolic genes were identified in several plasmids, for example, p5118.Nb harbored two enzymes predicted to play a role in the ribulose monophosphate pathway for formaldehyde detoxification (Orita et al., 2006). En masse, these findings highlight the constant genetic flux that occurs between these extrachromosomal genetic molecules. Overall the genetic diversity seen across these plasmids likely contributes to the ability of Staphylococcus to adapt to a wide variety of environments and stresses.

Previous sequence analysis of the qacA-containing pC02 plasmid identified a putative type IV secretion system (Johnson et al., 2015). To further investigate the pC02 components, the resequenced plasmid was re-annotated with the Rapid Annotation using Subsystem Technology (RAST) service (Aziz et al., 2008; Overbeek et al., 2014; Brettin et al., 2015). RAST revealed a putative TraG protein, which is commonly associated with conjugative plasmids (Firth et al., 1993; Morton et al., 1993). Although the exact function of TraG has yet to be identified in Gram-positive bacteria, it is believed to function as a coupling protein for the relaxase enzyme and the membrane bound secretion system (Cabezón et al., 1997). Additionally, RAST identified a putative conjugal ATPase, TraE, which is also known as VirB4. TraE helps supply the energy necessary for conjugative transfer (Walldén et al., 2012). The presence of two genes commonly associated with conjugation systems and the significance of possible conjugal transfer of qacA-containing plasmids in S. aureus led us to explore the conjugation potential of pC02. We questioned whether pC02 could be mobilized by conjugation from the parental C02 strain to a pC02 plasmid-cured derivative that was resistant to rifampin and novobiocin (C02-RN). Filter-mating assays with C02 (donor) and C02-RN (recipient) followed by selection of transconjugants on rifampin, novobiocin and cadmium (pC02 contains a cadmium resistance gene), revealed an average frequency of conjugation of 7.51 × 10⁻⁹ (Table 5). Moreover, the other S. aureus strains that harbored pC02 (3020.C01 and 1969.N) were able to transfer their plasmid to C02-RN at a similar frequency (Table 5). Transfer required cell-to-cell contact as transconjugants were never identified when the donor and recipient cells were separated by a filter.

To examine the host range of pC02 conjugation, five additional recipient strains were tested: two S. aureus strains, two S. epidermidis strains and one Enterococcus faecalis strain. For the additional S. aureus strains, we chose the common lab strain RN4220 as well as a qacA minus strain from our collection (1625.C01). These two S. aureus strains both yielded transconjugates at a similar frequency to that seen with C02-RN. The S. epidermidis strains included a lab passaged strain (DSM1581) as well as strain RP62a, which is a recent clinical isolate (Gill et al., 2005). Again, transfer of pC02 to the lab strain occurred at a similar frequency to that seen with C02-RN. However, transfer to the clinical isolate was a very rare event; only a single transconjugant was ever detected for the S. epidermidis RP62a assays. Cross genera conjugation via mating with E. faecalis OG1-RF (Dunny et al., 1978) was not detected (Table 5). This conjugal transfer of pC02 was unique as the other qacA-positive plasmids found in strains 1626.N, 5118.N, and 1971.C01 were unable to be mobilized by conjugation; the lack of mobilization is likely no surprise given the absence of identifiable putative transfer genes on any of these other plasmids.

To ensure that pC02 was being mobilized by conjugation and not by phage-mediated transduction, supernatants collected from donor strains were mixed with recipient cells and the appearance of transductants was assessed; supernatants from overnight and log phase donor cultures were examined. No transductants were ever identified, suggesting that pC02 mobilization does occur via conjugation. Furthermore, to check for endogenous phage within C02, mitomycin C induction at various concentrations produced no visible plaques at a limit of detection of 100 plaque forming units.

Given that qacA containing strains showed decreased susceptibility to chlorhexidine (Figure 2) and that pC02 was able to mobilize qacA to multiple recipient strains, we next asked whether conjugal transfer of pC02 was sufficient to affect chlorhexidine susceptibility of the recipient strains; we tested individual transconjugants from various matings for their susceptibility to chlorhexidine. Indeed, we found that all tested S. aureus pC02-containing transconjugants displayed an observable decrease in their susceptibility to chlorhexidine as compared to their respective parental strains (Figure 4A). This decrease in susceptibility was not only seen with the S. aureus transconjugants, but was also seen with S. epidermidis; the pC02-containing strain showed a 3-fold increase in chlorhexidine resistance as compared to the parental S. epidermidis strain (Figure 4B). Thus, transfer of pC02 and qacA was sufficient to impart decreased susceptibility to chlorhexidine in a susceptible strain background. En masse, our conjugation results show the first demonstration of conjugative transfer of qacA across S. aureus strains and to other Staphylococcal species; the downstream result of mobilization of qacA for the recipient cells was a decrease in susceptibility to chlorhexidine.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.