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. Author manuscript; available in PMC: 2018 Nov 6.
Published in final edited form as: J Microbiol. 2017 Jan 26;55(2):153–159. doi: 10.1007/s12275-017-6509-1

Phenotypic and genotypic correlates of daptomycin-resistant methicillin-susceptible Staphylococcus aureus clinical isolates

Kyoung-Mi Kang 1, Nagendra N Mishra 2,3, Kun Taek Park 4, Gi-Yong Lee 1, Yong Ho Park 4, Arnold S Bayer 2,3, Soo-Jin Yang 1,*
PMCID: PMC6218394  NIHMSID: NIHMS994357  PMID: 28120188

Abstract

Daptomycin (DAP) has potent activity in vitro and in vivo against both methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) strains. DAP-resistance (DAP-R) in S. aureus has been mainly observed in MRSA strains, and has been linked to single nucleotide polymorphisms (SNPs) within the mprF gene leading to altered cell membrane (CM) phospholipid (PL) profiles, enhanced positive surface charge, and changes in CM fluidity. The current study was designed to delineate whether these same genotypic and phenotypic perturbations are demonstrated in clinically-derived DAP-R MSSA strains. We used three isogenic DAP-susceptible (DAP-S)/DAP-R strain-pairs and compared: (i) presence of mprF SNPs, (ii) temporal expression profiles of the two key determinants (mprF and dltABCD) of net positive surface charge, (iii) increased production of mprF-dependent lysinylated-phosphatidylglycerol (L-PG), (iv) positive surface charge assays, and (v) susceptibility to cationic host defense peptides (HDPs) of neutrophil and platelet origins. Similar to prior data in MRSA, DAP-R (vs DAP-S) MSSA strains exhibited hallmark hot-spot SNPs in mprF, enhanced and dysregulated expression of both mprF and dltA, L-PG overproduction, HDP resistance and enhanced positive surface charge profiles. However, in contrast to most DAP-R MRSA strains, there were no changes in CM fluidity seen. Thus, charge repulsion via mprF- and dlt-mediated enhancement of positive surface charge may be the main mechanism to explain DAP-R in MSSA strains.

Keywords: Staphylococcus aureus, daptomycin resistance, mprF, single nucleotide polymorphism (SNP), host defense antimicrobial peptide

Introduction

Staphylococcus aureus causes a wide array of local and systemic infections in humans and animals, and is among the most prevalent community-acquired and nosocomial pathogens. The ability of S. aureus to develop multi-antibiotic resistance, including to methicillin, vancomycin (VAN), and daptomycin (DAP) is raising significant public health concerns. Moreover, these latter two drugs are often antibiotics of last resort for many S. aureus infections.

DAP is a cyclic lipopeptide antibiotic with potent bactericidal activity against a wide range of Gram-positive pathogens, including MRSA and VAN-intermediate susceptibility (VISA) strains. However, there have been numerous recent reports of clinical S. aureus strains that have evolved DAP-resistance (DAP-R) in vivo during DAP treatment (Kaatz et al., 2006; Jones et al., 2008; Marco et al., 2008; Murthy et al., 2008; Bayer et al., 2014, 2015). Previous studies have linked several characteristic phenotypes with DAP resistance in S. aureus: i) enhanced positive surface charge (Jones et al., 2008; Yang et al., 2010); ii) altered cell membrane (CM) phospholipid profiles, especially increased content of positively-charged phospholipids (lysyl-phosphatidylglycerol [L-PG]) (Mishra et al., 2012b, 2014); iii) CM fluidity/rigidity. These phenotypes were often accompanied by a single or multiple genotypic changes such as upregulation of mprF and/or dlt-ABCD transcription (Yang et al., 2009a, 2009b; Bayer et al., 2016) and single nucleotide polymorphisms (SNPs) within the mprF open reading frame (ORF) (Murthy et al., 2008; Bayer et al., 2013, 2014, 2015, 2016; Yang et al., 2013a). The mprF gene product, MprF, is a L-PG synthase which adds positively-charged lysine to the negatively-charged phosphatidylglyderol (PG) molecule within the staphylococcal CM (Peschel et al., 2001; Staubitz et al., 2004). It has been shown that the mprF SNPs were correlated with excess synthesis of L-PG in S. aureus CM and enhanced surface positive charge (Yang et al., 2013a; Bayer et al., 2014, 2015, 2016). More recently, it has been reported that all the mprF SNPs observed in the vast majority of DAP-R S. aureus strains are clustered within either one of the two ‘hot spot’ MprF domains: the central bifunctional domain or the C-terminal L-PG synthase domain (Ernst et al., 2009; Bayer et al., 2015). However, these latter data have mainly emerged from studies of DAP-R MRSA strains. Hence, to investigate phenotypic and genotypic correlates of DAP resistance in MSSA strains, we used three DAP-S/DAP-R isogenic clinical MSSA strain pairs to assess (i) presence of mprF SNPs in such DAP-R strains, (ii) the relationship of the SNPs to DAP-R phenotype, (iii) temporal transcriptional expression profiles of mprF and dltABCD genes, (iv) CM phospholipid profiles and fluidity, (v) cross-resistance to three cationic HDPs (LL-37, human cathelicidin; hNP-1, human neutrophil peptide-1; and tPMPs, thrombin-induced platelet microbicidal proteins), and (vi) positive surface charge metrics.

Materials and Methods

Bacterial strains

The three DAP-S/DAP-R MSSA study strain pairs used in the current study were clinical bloodstream isolates randomly selected and obtained courtesy of the Cubist Strain Collection: each strain pair included an initial pre-DAP treatment isolate and a post-DAP treatment isolate from the same patient that became DAP non-susceptible (Table 1). All the three MSSA strain pairs were shown to be identical on pulsed-field gel electrophoresis (PFGE) analyses (data not shown). To further confirm the isogenicity of each strain pair, these isolates were genotyped by spa, inferred clonal complex and agr typing (Table 1).

Table 1.

Bacterial strains examined in the current studies

Strain spa type CC typea agr type DAP MICs (μg/ml) OX MICs (μg/ml) VAN MICs (μg/ml) mprF SNPs
Nucleotide position Amino acid change
C11 454 121 IV 0.38 0.75 1 CCT → CTT (941) P314L
C12 454 121 IV 3 0.5 2
C28 2 5 II 0.12 1.0 1 CCT → CTT (941) P314L
C29 2 5 II 2 0.38 1
C44 410 5 II 0.38 1.0 1 TCA → TTA (1010) S337L
C45 410 5 II 4 0.75 2
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OX, oxacillin; DAP, daptomycin; VAN, vancomycin; SNPs, single nucleotide polymorphisms.

a

Clonal complex (CC) types were inferred by comparing MLST data base.

All S. aureus strains were grown in either Tryptic Soy Broth (TSB; Difco Laboratories) or Meuller-Hinton broth (MH broth; Difco Laboratories) depending on the individual assays. Liquid cultures were grown in Erlenmeyer flasks at 37°C with shaking (250 rpm) in a volume that was no greater than 10% of the flask volume.

The MICs of the study strains to DAP, oxacillin (OX), and vancomycin (VAN) were determined by standard Etest (AB Biodisk) on MH agar plates, according to manufacturer’s recommendation. DAP resistance was defined as an Etest MIC of ≥ 2 μg/ml (Boucher and Sakoulas, 2007). All MIC determinations were performed three times.

DNA isolation and mprF sequencing

Genomic DNA was isolated from the S. aureus strains using the method described previously (Dyer and Iandolo, 1983). PCR amplification of the mprF ORFs was performed as described previously, using the mprF-specific primer pair (Yang et al., 2009a). DNA sequencing of the mprF ORFs was kindly performed at BIONEER, Daejeon, Korea. Multiple mprF sequence alignments were done using the BoxShade Server (http://www.ch.embnet.org/software/BOX_form.html).

Susceptibilities to host defense peptides

Purified LL-37 (found in human epithelial cells and neutrophils) and human neutrophil peptide-1 (hNP-1) were purchased from Peptide International. Thrombin-induced platelet microbicidal protein preparations (tPMPs) were obtained from fresh rabbit platelets as previously described (Yeaman et al., 1992, 1994). This preparation is a mixture of the two predominant tPMPs seen in humans, tPMP-1 and tPMP-2, with a dominance of tPMP-1.

Since standard MIC testing in nutrient broth may underestimate cationic antimicrobial peptide activities (Yeaman et al., 1992; Xiong et al., 2005), in vitro susceptibility assays were performed with LL-37, hNP-1, and tPMPs as described previously using a two-hour microdilution method in Eagle’s Minimal Essential Medium (Xiong et al., 2005; Jones et al., 2008). These assays were performed with LL-37 (1 μg/ml), hNP-1 (20 μg/ml), and tPMPs (2 μg/ml bioequivalents) using initial inocula of 5 × 103 CFU S. aureus cells (Xiong et al., 2005; Jones et al., 2008). These peptide concentrations were selected based on extensive pilot studies showing their inability to completely eradicate the starting inocula of the parental DAP-S strains over the two-hr time point. Data were calculated and expressed as the percent of survival (± SD) of peptide-exposed vs. peptide-unexposed S. aureus cells. A minimum of three independent studies in triplicate samples was performed for each peptide on distinct days.

Cell membrane phospholipid (PL) composition

The three major staphylococcal CM phospholipids are phosphatidylglycerol (PG), cardiolipin (CL), and lysyl-phosphatidylglycerol (L-PG). To assess the relative proportions of these three PLs in the study strains, CM PLs were extracted from the S. aureus strains as described previously (Mukhopadhyay et al., 2007; Mishra et al., 2009). These PLs were separated and identified via two-dimensional thin-layer chromatography (2D-TLC), then quantified by spectrophoto-metric analysis as described before (Mukhopadhyay et al., 2007; Mishra et al., 2009). A minimum of three independent experiments in triplicate samples were performed to analyze the proportion of PL content.

CM fluidity

The comparative CM fluidity of the three MSSA strain-pairs were determined by fluorescence polarization spectroscopy using the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) as described previously (Bayer et al., 2000; Mishra et al., 2009). Data were expressed as the polarization index (PI), in which there is an inverse relationship between the PI and CM fluidity (i.e. higher PI equates less fluid cell membranes) (Xiong et al., 2005; Jones et al., 2008). These assays were repeated at least five times independently.

Positive surface charge

The cytochrome c binding assay was performed to measure the relative net positive surface charge of the strain-pairs as described previously (Yang et al., 2009a, 2010, 2012). Briefly, S. aureus cells were grown overnight in TSB, washed with 20 mM MOPS buffer (pH 7.0) four times, and resuspended in the MOPS buffer at OD600 of 1.0. Next, cells were incubated with 0.5 mg/ml cytochrome c for 15 min and the amount of cytochrome c remaining in the supernatant was measured by determining absorbance at 530 nm (the absorption maximum if the prosthetic group) (Peschel et al., 1999). The more unbound cytochrome c that was detected in the super-natant, the more net positively charged the bacterial surface. At least three independent runs in triplicate samples were performed on separate days.

RNA isolation and qRT-PCR analysis

For RNA isolation, fresh overnight cultures of S. aureus strains were used to inoculate TSB to OD600 of 0.1. Cells were harvested during both exponential growth (2.5 h) and stationary phase (12 h). Total RNA was isolated from the cell pellets by using the RNeasy kit (Qiagen) and the FASTPREP FP120 instrument (BIO 101), according to the manufacturer’s recommended protocols.

Quantitative real time PCR assay was carried out as detailed previously (Bertsche et al., 2011; Yang et al., 2013b). Briefly, 2 μg of DNase-treated RNA was reverse transcribed using the SuperScript III first-strand synthesis kit (Invitrogen) according to the manufacturer’s protocols. Quantification of cDNA levels was performed following the instructions of the Power SYBR green master mix kit (Applied Biosystems) on a LineGene 9620 fluorescent quantitative detection system (BIOER technology) in triplicate samples. The mprF, dltA, and gyrB genes were detected using respective specific primers as described before (Yang et al., 2009b, 2012). Fold changes in expression levels of mprF and dltA genes were determined in relation to the housekeeping gene, gyrB. The expression profiling was performed at both exponential and stationary growth phases. At least two independent runs were performed for each RNA samples.

Statistical analysis

The Kruskal-Wallis ANOVA test with the Tukey post hoc correction for multiple comparisons was utilized. Significance was determined at P value of < 0.05.

Results

MIC testing

Previous studies have suggested that VAN treatment in humans might be an important conditioning for subsequent increase of DAP MICs during DAP treatment (Cui et al., 2006; Pillai et al., 2007). Of note, two (C12 and C45) of the three DAP-R strains exhibited moderate increase in VAN MICs (Table 1). Susceptibilities to OX has also been moderately increased in all the three DAP-R strains.

mprF SNPs among DAP-R MSSA strains

As shown in Table 1, mprF sequencing analyses revealed that all three DAP-R MSSA strains had a nonsynonymous mutation in the mprF ORF compared with their respective DAP-S parental strains, causing distinct amino acid substitutions. The C12 and C29 DAP-R strains had a P314L substitution, while the C45 DAP-R strain had a S337L substitution. These two mprF SNPs were previously shown to be associated with DAP-R among MRSA isolates (Bayer et al., 2015, 2016). Based on previous topographical studies (Ernst et al., 2009; Bayer et al., 2015), these amino acid substitutions were located in the central bifunctional domain of the MprF protein.

HDP susceptibility profiles

As shown in Table 2, all three DAP-R MSSA strains exhibited an overall higher survival profile vs. their respective DAP-S parental strains against all HDPs tested. The C12 strain had most substantial differences in HDP killing profiles vs the DAP-S parental C11 strain for all three HDPs.

Table 2.

In vitro susceptibility to killing by cationic host defense peptides

Strain % Survival (mean ± SD) after 2-h exposure to:
10 μg/ml LL-37 20 μg/ml hNP-1 2 μg/ml tPMPs
C11 21.8 ± 11.2 34.2 ± 8.6 4.9 ± 3.2
C12 56.2 ± 22.6** 42.7 ± 6.4* 34.7 ± 15.4*
C28 5.8 ± 3.7 33.4 ± 9.8 43.5 ± 17.1
C29 40.2 ± 23.2* 37.6 ± 7.4 46.6 ± 21.7
C44 42.5 ± 19.7 49.8 ± 22.2 13.6 ± 6.7
C45 60.3 ± 25.4 59.3 ± 16.7 37.3 ± 14.1*
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*

P < 0.01;

**

P < 0.05 vs the DAP-S parental strains

Positive surface charge and CM phospholipid (PL) content

Cytochrome c binding assays revealed that all three DAP-R strains had significantly increased positive surface charge versus their respective DAP-S parental strains (Fig. 1).

Fig. 1. Relative positive surface charge by cytochrome c binding.

Fig. 1.

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The graph shows percent of cytochrome c bound after 15 min of incubation with S. aureus cells at room temperature. Each DAP-S parental S. aureus strain was normalized to 100%. Data represent the means and standard deviations from three independent experiments. *P < 0.01; **P < 0.05 vs DAP-S parental strains.

Since increases in L-PG synthesis and/or translocation usually result in enhanced positive surface charge, we compared the proportion of total L-PG within the overall PL content of the three DAP-S/DAP-R MSSA strain pairs. As noted in Table 3, all three DAP-R strains contained significantly more L-PG than their respective DAP-S parental strains. This outcome was associated with the significant reduction in PG content in the three DAP-R strains, although this only reached statistical significance in the C11-C12 strain-pair.

Table 3.

Comparative cell membrane phospholipid profiles and cell membrane fluidity of study strains

Strains % of total phospholipids ± SD CM fluidity
Total LPG PG CL
C11 18 ± 2.3 78 ± 3.6 5 ± 2.0 0.208 ± 0.05
C12 25 ± 4.4* 68 ± 6.3* 7 ± 4.4 0.193 ± 0.02
C28 15 ± 3.1 79 ± 7.1 6 ± 5.7 0.233 ± 0.03
C29 23 ± 5.8** 70 ± 10.5 7 ± 5.8 0.234 ± 0.04
C44 20 ± 4.1 74 ± 3.2 5 ± 2.4 0.223 ± 0.02
C45 26 ± 3.8** 68 ± 7.5 6 ± 5.0 0.202 ± 0.02
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Abbreviations: LPG, lysyl-phosphatydylglycerol; PG, phosphatidylglycerol; CL, cardiolipin.

*

P < 0.01;

**

P < 0.05 vs respective DAP-S parental strains.

CM fluidity

For all three pairs, CM fluidity analyses revealed no significant differences in the polarization indices (PI values), indicating that the CMs of the three DAP-R strains had no differences in their CM order profiles (Table 3).

Expression of mprF and dltABCD

Quantitative real-time PCR analysis of mprF transcripts during exponential and stationary growth phases revealed that C12 and C29 DAP-R strains demonstrated enhanced mprF expression vs their respective isogenic DAP-S strains during the both growth phases (Fig. 2A and B). Although not statistically significant, the C45 DAP-R strain had a moderate increase in mprF expression vs. its DAP-S parental strain only during exponential growth phase.

Fig. 2. Relative transcription level of mprF during exponential (A) and stationary (B) growth phase.

Fig. 2.

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RNA samples were isolated from exponential-and stationary-phase cultures of the strains and were subjected to qRTPCR to detect transcription of mprF and gyrA. EXPO = exponential growth phase; ST = stationary growth phase; Fold expression = compared to gyrB gene, with parental strain fold-expression set at “1”. *P < 0.01 vs DAP-S parental strains.

As shown in Fig. 3A, all the three DAP-R strains showed enhanced expression of dltA versus their respective DAP-S strains during exponential growth phase. In contrast, at stationary growth, when dltABCD espression is generally minimal (Yang et al., 2009a, 2010), only the C29 DAP-R strain exhibited ~2-fold increase in dltA expression as compared to its C28 DAP-S parental strain (Fig. 3B). Of note, the two DAP-R strains, C12 and C45, showed significant decrease in dltA expression vs. their respective DAP-S parental strains during stationary growth phase.

Fig. 3. Relative transcription level of dltA during exponential (A) and stationary (B) growth phase.

Fig. 3.

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RNA samples were isolated from exponential-and stationary-phase cultures of the strains and were subjected to RT-PCR to detect transcription of dltA and gyrA. EXPO, exponential growth phase; ST, stationary growth phase; Fold expression, compared to gyrB gene, with parental strain fold-expression set at “1”. *P < 0.01; **P < 0.05 vs DAP-S parental strains.

Discussion

It has been previously shown that the mprF SNPs correlated with perturbed growth phase-dependent mprF expression in a number of DAP-R MRSA isolates (Yang et al., 2009a, 2009b; Bayer et al., 2014, 2015). ThemprF dysregulations have been correlated with phenotypic ‘gains-in-function’ readouts of MprF, such as increased L-PG synthesis and elevated L-PG translocation to the outer CM leaflet, resulting in increased surface positive charge (Jones et al., 2008; Ernst et al., 2009, 2015; Yang et al., 2009a, 2009b). In addition, we previously found that upregulation in the expression of the gene responsible for d-alanylation of wall teichoic acid (dlt-ABCD) also accounted for enhancement in positive surface charge (Yang et al., 2009a).

In the current investigation, we used three isogenic DAP-S/DAP-R MSSA clinical strain-pairs to evaluate and extend DAP-R mechanisms in MSSA isolates in terms of both phenotypic and genotypic correlates.

First, as has been shown previously in MRSA strains (Yang et al., 2010; Bayer et al., 2014, 2015), mprF ORF sequencing revealed that all the three DAP-R MSSA strains contained SNPs within the bifunctional domain of MprF (Ernst et al., 2009; Bayer et al., 2015). The two amino acid sequence substitutions within MprF (P314L and S337L) have been shown to be associated with enhanced L-PG synthesis in the context of DAP-R phenotype in MRSA strains (Bayer et al., 2015). As shown in Fig. 2A and B, C12 and C29 DAP-R strains had enhanced mprF expression versus their respective isogenic DAP-S parental strains during both growth phases. The C45 strain also had moderate, albeit non-significant, increases in mprF expression during exponential growth phase. The mprF promoter sequences of these three DAP-R strains were identical to those of their respective DAP-S parental stranis (data not shown), indicating the increase in mprF expression in the three DAP-R strains was not related to point mutations within the mprF promoter

Next, to assess the correlation among mprF SNPs, altered expression of mprF transcription, and L-PG synthesis, phospholipid (PL) compositional analyses were performed. As expected, the proportion of total L-PG among the overall PL content was significantly increased in the all three DAP-R MSSA strains (Table 3). The increased synthesis of cell membrane L-PG in DAP-R MSSA strains was well correlated with enhanced net positive surface charge in these strains (Fig. 1). These findings are generally in line with prior observations in MRSA strains (Bayer et al., 2014; Mishra et al., 2014; Bayer et al., 2015, 2016), suggesting that hot spot SNPs within the mprF ORF of DAP-R MSSA are gain-in-function mutations which translate into increased L-PG production and coincident enhancement of positive surface charge.

Second, altered dltA expression profiles were similar to those of mprF in all three DAP-R MSSA strains during exponential growth phase (Fig. 3A). Moreover, the DAP-R C29 strain displayed increased expression of dltA during stationary growth phase (Fig. 3B). It should be noted that although the DAP-R C29 strain exhibited significantly enhanced expression of both mprF and dltABCD during both growth phases, the extent of its increased surface positive charge was similar to the other two DAP-R MSSA strains. These data indicate that mprF and dltABCD expression profiles alone are not enough to explain the surface positive charge regulation in S. aureus. Present investigations are in progress to further elucidate genetic factors linked to the DAP-R phenotype, including graRS, yycFG, and rpoB (Friedman et al., 2006; Bertsche et al., 2011; Cheung et al., 2014).

Third, recent publications have suggested that DAP-R MRSA strains frequently display cross-resistance to HDPs and other cationic molecules (Peschel et al., 2001; Yang et al., 2010, 2013a; Mishra et al., 2011b, 2012a; Bayer et al., 2015). In agreement with these observations in MRSA strains, all three DAP-R MSSA strains in our study exhibited significantly enhanced resistance to killing by one or more of the HDPs tested vs their respective DAP-S parental strains (Table 2). It has been proposed that there are two major HDP resistance mechanisms in S. aureus: (i) enhanced positive surface charge leading to a charge-repulsive milieu (Jones et al., 2008; Yang et al., 2009a, 2009b) and (ii) alteration of CM biophysical order (fluidity/rigidity) to perturb the optimal interactions of the peptide-of-interest with the target CM (Mukhopadhyay et al., 2007; Mishra et al., 2009, 2011a). Data from the current study would suggest that mainly charge-mediated mechanisms are in-play among MSSA DAP-R strains.

Acknowledgements

We thank Aileen Rubio (Cubist Pharmaceuticals) for providing the DAP-S/DAP-R strain pairs. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2015R1D1A1A01057442) and the Chung-Ang University Research Grants in 2015. ASB was supported by a research grant from the National Institutes of Health (NIAID), 5RO1 A39108–18.

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