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Article

Genomic Insights into Staphylococcus aureus Isolates Exhibiting Diminished Daptomycin Susceptibility

by
Natalia Gómez-Casanova
1,2,*,
Mª Nieves Gutiérrez-Zufiaurre
1,3,
Ana Mª Blázquez de Castro
1,3 and
Juan Luis Muñoz-Bellido
1,2,3
1
Research Group on Clinical Microbiology and Parasitology and Antimicrobial Resistance (IIMD-16), Instituto de Investigación Biomédica de Salamanca (IBSAL), Universidad de Salamanca, CSIC, Hospital Universitario de Salamanca, 37007 Salamanca, Spain
2
Department of Biomedical and Diagnostic Sciences, Universidad de Salamanca, 37007 Salamanca, Spain
3
Department of Microbiology and Parasitology, Hospital Universitario de Salamanca, 37007 Salamanca, Spain
*
Author to whom correspondence should be addressed.
Pathogens 2024, 13(3), 206; https://doi.org/10.3390/pathogens13030206
Submission received: 25 January 2024 / Revised: 20 February 2024 / Accepted: 22 February 2024 / Published: 26 February 2024
(This article belongs to the Section Bacterial Pathogens)
Browse Figure
Versions Notes

Abstract

:
Daptomycin is one of the last therapeutic resources for multidrug-resistant gram-positive bacteria. Despite its structural similarities with glycopeptides, its mechanisms of action and resistance are different and in some respects are not completely understood. Mutations in several genes have been associated with daptomycin resistance, especially in mprF, walkR, rpoB and rpoC, but their role and importance remain to be elucidated. We have studied mutations in 11 genes, which have been previously associated with daptomycin non-susceptibility, in nine daptomycin-non-susceptible Staphylococcus aureus clinical isolates (daptomycin MIC: >1 mg/L). Susceptibility to daptomycin, vancomycin, linezolid, oxacillin, telavancin and dalbavancin was studied. walkR, agrA, cls1, cls2, fakA, pnpA, clpP, prs, rpoB, rpoC and mprF were amplified by PCR and sequenced. The sequences were compared with the S. aureus ATCC 25923 complete genome (GenBank gi: 685631213) by using BLAST® software. We did not find any changes in walkR, pnpA, prs and clpP. All isolates excepting isolate MSa5 showed a high number of significant mutations (between 13 and 25 amino acid changes) in mprF. Most isolates also showed mutations in the rpo genes, the cls genes and fakA. Daptomycin non-susceptibility in S. aureus clinical isolates seems to be reached through different mutation combinations when compared to S. aureus ATCC 25293. Especially mprF and cls1 showed very high polymorphism in most isolates. Meanwhile, one isolate, MSa5, showed only single mutation in mprF (P314T).

1. Introduction

Staphylococcus aureus has shown a high capacity for developing resistance to each newer antistaphylococcal antibiotic developed. Antibiotic resistance in S. aureus may result in higher morbidity, mortality, lengths of hospitalization and health expenditures [1]. The cyclic anionic lipopeptide antibiotic daptomycin (DAP) [2] is becoming a main resource in therapy against multidrug-resistant S. aureus, especially methicillin-resistant S. aureus (MRSA), due to its activity against glycopeptide and linezolid-resistant MRSA [3,4]. The mechanism of action of DAP differs from conventional glycopeptides (vancomycin, teicoplanin) and is more like the cationic antimicrobial peptides produced by the immune system. Vancomycin and teicoplanin impair bacterial cell wall synthesis. They bind to the terminal dipeptide D-alanyl-D-alanine (D-Ala-D-Ala) within the disaccharide pentapeptide attached to lipid II and the forming PGN chain. This way, transglycosylation and transpeptidation are prevented, thus hindering proper wall synthesis [5,6]. In addition, vancomycin has other secondary actions, probably less important in the lysis of the microorganism, such as inhibition of RNA synthesis and alteration of wall permeability.
The action of DAP is based on binding to the bacterial cell membrane through its hydrophobic end in the presence of calcium ions [7,8,9]. This binding occurs both in the exponential growth phase and the stationary phase, causing membrane depolarization due to the loss of potassium ions from the cytoplasm [10]. This process leads to the disruption of multiple functions of the bacterial cell membrane, such as the synthesis of proteins, DNA and RNA without the need for the antimicrobial to penetrate the cytoplasm. Unlike other antimicrobials, the mechanism of action does not involve the lysis of bacterial cells [11] but rather bacterial cell death due to alterations in cellular homeostasis [7,12].
Intrinsic resistance to DAP by the hydrolytic cleavage of ester bonds inside the DAP molecule has been described, in absence of contact with this or other antibiotics,, among gram-positive bacteria with a low percentage of GC, such as staphylococci [13].
Streptomyces strains highly resistant to daptomycin (MIC ≥ 256 µg/mL) have been found to inactivate the antibiotic through two primary modes of daptomycin inactivation: ring hydrolysis, leading to linearization of the cyclic compound, and deacylation of the lipid tail [14]. Additionally, Paenibacillus lautus has been shown to inactivate daptomycin. The production of the inactivating activity in P. lautus is inducible by exposure to daptomycin, and some data suggest it might be caused by metalloesterases [14].
This “natural” resistance mechanism has been suggested to be associated with the natural production of DAP-like molecules by different microorganisms. Though this type of resistance has not been shown in clinically important bacteria yet, resistance genes coding for these DAP-inactivating enzymes might be a potential source of resistance if they could be captured by pathogenic bacteria, as has happened before for other resistance determinants, such as CTX-M enzymes, which originated in Kluyvera spp. and are now present in most clinically important enterobacteria [15].
Nevertheless, DAP non-susceptibility observed in isolates obtained both in vitro and from therapeutic failures in S. aureus seems to be mainly associated with electrostatic repulsion of the DAP–calcium complex from the cell surface due to an increase in the positive charge of the bacterial surface [16].
DAP-non-susceptible isolates obtained both in vitro and from therapeutic failures usually harbor mutations in genes associated with cytoplasmic membrane (mprF) and cell wall homeostasis (yycFG, also known as walK) and mutations in RNA polymerase subunits rpoB/rpoC [7,8]. DAP-non-susceptible isolates have been described, both in vitro and in vivo, lacking mutations in some of these genes but not in all of them [7,9,10,11]. Other authors have shown that DAP non-susceptibility may also involve the upregulation of genes associated with cell wall synthesis and turnover, such as the two-component regulator vraSR [12]. Otherwise, whole-genome-sequencing studies have shown that DAP-non-susceptible isolates may harbor mutations in other genes, such as agrA, pnpA, pgsA, prs, cls2 and clpP, some of them associated with the cytoplasmic membrane and cell wall metabolism [10,11].
Therefore, the present study had the following aims: the identification of S. aureus strains (methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA)) non-susceptible to DAP, isolated from three different hospitals in Spain; testing sensitivity to different drugs such as oxacillin, vancomycin, linezolid, dalbavancin and telavancin; and the amplification and sequencing of the main genes associated with DAP resistance.

2. Materials and Methods

2.1. Selection of Bacterial Strains

Nine S. aureus clinical isolates identified as DAP-non-susceptible S. aureus (Minimum Inhibitory Concentration, MIC > 1 mg/L) by microdilution conventional susceptibility systems (WalkAway, Perkin Elmer, Shelton, CT, USA) were included in this study. Seven of them had been isolated in hospitals in the province of Badajoz (Southwest of Spain) and two in the Hospital Universitario de Salamanca (Middle West of Spain). All isolates except MSa9 were from patients who had received daptomycin treatment, although data on duration of treatment and dosage were not accessible. To our knowledge, the patient from whom the MSa9 isolate was obtained did not receive treatment with daptomycin at any time.
Microorganisms were identified by MALDI-TOF mass spectrometry (MicroflexTM, Bruker Daltonics, Bremen, Germany). All the isolates used were preserved and stored at −80 °C in glycerol broth with 10% skimmed milk.

2.2. Antibiotic Susceptibility

Susceptibility to DAP, vancomycin (VAN), linezolid (LZD) and oxacillin (OXA) were tested by using a broth microdilution method (MicroScan WalkAway, Perkin Elmer, Waltham, MA, USA), according to manufacturer instructions. Telavancin (TLV) and dalbavancin (DLV) were studied by E-test (Liofilchem, Roseto degli Abruzzi, Italy). The microdilution sensitivity study was carried out using an automated commercial method (MicroScan WalkAway, Perkin Elmer, USA) in which antimicrobials are lyophilized so that a predetermined concentration is reached by adding a set amount of culture medium. The microorganism is inoculated in cation-adjusted Mueller Hinton broth supplemented with 2% NaCl for oxacillin sensitivity and 50 microg/mL calcium for DAP. An inoculum of the microorganism equivalent to 0.5 of the McFarland (McF) standard is inoculated and incubated at 37 °C for 20–24 h. The reading is carried out in an automated way in MicroScan equipment.
For the E-test study, a Mueller Hinton agar plate was inoculated with an inoculum equivalent to 0.5 McF; strips of the different antimicrobials were deposited on the inoculated agar, incubated at 37 °C for 24 h and read visually. For the DAP E-tests, it was not necessary to supplement the agar with Ca2+, as the DAP strips had it incorporated.
Methicillin resistance, in strains with E-test sensitivity to oxacillin, was verified by PCR specific to the mecA and mecC genes (the primers used appear in Table 1). The isolates were subjected to the E-test according to the manufacturer’s instructions. The susceptibility criteria for VAN, LZD, DLV, TLV and OXA were those proposed by the CLSI (VAN: <2 mg/L, susceptible; 4–8 mg/L, intermediate; >16 mg/L, resistant. LZD: <4 mg/L, susceptible; >8 mg/L, resistant. DLV: <0.25 mg/L, susceptible; intermediate and resistant categories not defined. TLV: <0.12 mg/L, susceptible; intermediate and resistant categories not defined. OXA: <0.5 mg/L, susceptible; >1 mg/L, resistant) [13]. The only categories considered for DAP were “susceptible” and “non-susceptible”, as CLSI reports only the sensitivity criterion (MIC < 1 mg/L).

2.3. DNA Extraction and Amplification of Genes Associated with DAP Non-Susceptibility in S. aureus

A suspension with turbidity equivalent to 1 McF was obtained from colonies grown for 24 h at 37 °C on blood agar. Then, 500 μL of the suspension was transferred to the automated nucleic acid extraction system NucliSENS® easyMAG (bioMérieux, Marcy-l’Étoile, France). Nucleic acid extraction was performed according to manufacturer instructions. DNA was preserved at −80 °C. A series of genes previously associated with DAP non-susceptibility [7,14] was amplified and sequenced. Amplification was carried out in an Eppendorf Master Cycler® thermal cycler (Eppendorf, Hamburg, Germany). All primers used in this study were designed specifically (Table 1) based on the S. aureus ATCC 25,923 complete genome sequence deposited in GenBank (gi: 685631213) using the Primer Blast® application (NCBI, Bethesda, MA, USA). The primers were manufactured by Sigma-Genosys (The Woodlands, TX, USA), and the MasterMix® PCR amplification mix (Promega, Madison, WI, USA), a premixed 2X solution of Taq DNA Polymerase, dNTPs and reaction buffer, was used for PCR. PCR conditions were as follows: 95 °C × 5 min (denaturation); 35 cycles of: 95 °C × 30 s, 1 min at the temperature reported in the supplementary section (Table S1) specific to each primer (hybridization), 72 °C × 1 min (elongation), and finally, 72 °C × 5 min (elongation).

2.4. Visualization of Amplicons and Purification of PCR Product

The products were subjected to electrophoresis in 2% agarose gel together with a size control (DNA Ladder 100 bp, Promega, Madison, WI, USA). Blue/Orange 6x Loading Dye (Promega, Madison, WI, USA) was used as a loading buffer, and RedSafe® Nucleic Acid Staining Solution (Invitrogen, Carlsbad, CA, USA) was used as a fluorescent dye for DNA detection.
As a general rule, amplified DNA was cleaned for sequencing by using Exo-SAP It® (Chang Biosciences, Tianjin, China). When DNA was purified from the gel, the MEGAquick-Spin Total Fragment DNA Purification kit (Intron Biotechnology, Gyeonggi-do, Republic of Korea) was used.
Amplicons were sequenced by the Sanger method in a 3500 Genetic Analyzer sequencer (Applied Biosystems, San Francisco, CA, USA) and visualized by using the software Chromas 2.5.1.0. Sequences were compared with the S. aureus ATCC 25,923 complete genome sequence deposited in GenBank (gi: 685631213). S. aureus ATCC 25,923 is commonly used as a control in standard laboratory tests. Comparison and analysis were carried out by using the BLAST® software freely available on the NIH website.

3. Results

3.1. Antibiotic Susceptibility

DAP MICs ranged between 1 and 4 mg/L (Table 1). All the isolates were susceptible to VAN, LZD, TLV and DLV (Table 1). The MIC of DLV for the MSa3 isolate was higher (0.1 mg/L), though still in the susceptible range (break point: 0.25 mg/L). Three out of nine isolates (33.3%) were phenotypically MRSA (OXA MIC ≥ 4 mg/L) and four MSSA (44.44%) (OXA MIC < 2 mg/L). Two isolates (MSa4 and MSa7) were OS-MRSA (OXA MICs = 2 mg/L, PCR positive for mecA) (primers in Supplementary Section Table S1).

3.2. Mutation Profiles Associated with Non-Susceptibility

All the isolates, excepting MSa5, showed significant mutations in the genes rpoB, mprF, rpoC, cls1, cls2 and fakA, while MSa5 showed significant mutations only in the rpoB and mprF genes (Figure 1) (Table 2, Table 3 and Table 4). We did not detect any significant mutations in the walk, agrA, pnpA, clpP and prS genes. The genes with the highest number of mutations were mprF (31–35 amino acid changes) and cls1 (6–16 amino acid changes) (Figure 1).
The mprF gene presented a high number of mutations in all isolates (Table 2), excepting MSa5, which only had one mutation in this region: P314T. Mutations in this position also appeared in other clinical isolates, such as MSa1 (P314T) and MSa6 (P314L). Forty-three different mutations were identified in all in the mprF genes. Most isolates showed different combinations of these mutations. Most changes appeared in the central area of the genes, although we found changes throughout the genes. MSa6 had the highest number of mutations in mprF, exactly 35, followed by the strains MSa1, MSa4 and MSa8, with 34 changes.
As in mprF, we found a high number of changes in the genes that encoded for the cardiolipin synthase, especially in cls1. Only MSa5 showed no mutations in cls1 (Table 3). Though a number of mutations appeared in more than one isolate, no isolates showed the same mutation profile. Only one mutation (I238V) appeared in all the isolates showing mutations in this gene. MSa6 was the isolate with the highest number of mutations in this gene (16 mutations). cls2 only showed four different mutations (Table 3), half of which were shared by all the isolates, excepting MSa5, which did not show any mutation in this gene.
All the isolates, including MSa5, showed mutations in rpoB; however, all the isolates excepting MSa5 had mutations in rpoC (Table 4).
fakA, a protein also referred to as mw1109 in previous studies, is a fatty acid kinase that has been associated with unsensitivity to DAP in mutants selected in vitro [10]. All the isolates tested in this study, excepting the clinical isolate MSa5, showed mutations in this gene (Table 4). Mutation profiles in fakA were more homogeneous than in mprF and cls1. Only MSa6 showed a mutation profile that was neatly different.
We did not identify changes in walkR in any of our nine clinical isolates. Other genes also associated occasionally with reduced susceptibility to DAP, such as agrA, clpP, pnpA and prs, did not show any significant mutations in this study.
In summary, we found two different mutation profiles. A group of eight isolates had changes in six genes (mprF, rpoB, rpoC, cls1, cls2 and fakA), while one isolate had changes in only two genes (mprF and rpoB). mprF and cls1 showed high numbers of mutations/isolate in most isolates, while for the other genes, the number of mutations/isolate was much shorter. In the absence of the Multilocus Sequence Typing (MLST) analysis, we could not rule out that the identified substitutions could potentially be associated with a specific genetic lineage rather than daptomycin resistance.

4. Discussion

DAP resistance seems to be based on complex mechanisms and has been associated with the presence of mutations in different genes, most of them involved in the metabolism and homeostasis of the cell membrane and, in some cases, of the bacterial wall [7,14,15]. A cause–effect relationship between specific mutations in specific genes and a specific increase in the MICs of DAP has hardly been described, and the repulsion hypothesis does not explain the resistance to DAP in all S. aureus isolates. Only some studies have shown that the replacement of the mutated mprF gene with a wild one reverses, at least partially, the MICs of DAP [16,18]. In the present study, the MIC interval of DAP ranged between 2 and 4 mg/L in all cases, except MSa2, which had an MIC of 1 mg/L. Although the non-sensitivity to daptomycin in S. aureus is MIC > 1 mg/L, due to its proximity to the range, MSa2 was not ruled out from this study.
Mutations in the rpoB and mprF genes have been reported in most staphylococcal clinical isolates showing DAP MIC increases [7,10,11]. mprF codes for the bifunctional enzyme MprF, which contributes to the positive charge of the cell membrane surface through the lysinylation of phosphatidylglycerol (PG) and the translocation of lysinylated PG (L-PG) from the inner to the outer leaflet of the cell membrane. The positively charged cell membrane surface resulting from this process helps repel the DAP molecule from the surface [19,20,21]. The association between mprF mutations and DAP non-susceptibility is supported by the finding that the inactivation of mutated mprF can reverse, at least partially, DAP non-susceptibility [22].
The mprF protein is composed of 14 transmembrane domains and a cytosolic C-terminal domain [11]. The first eight N-terminal transmembrane domains are involved in the translocation of L-PG to the outer CM. The next four transmembrane “central” domains are involved both in L-PG synthesis and flipping, while the cytosolic C-terminal domain is involved only in L-PG synthesis. Single-nucleotide polymorphism (SNP) in mprF genes has been reported in most staphylococcal clinical isolates showing DAP MIC increases [7,10,11] and is associated with gain of function. Nevertheless, some authors have shown that SNPs can also be found in 30% of DAP-susceptible isolates and that some DAP-susceptible isolates with MICs of 1 mg/L can show up to 30 amino acid changes in mprF [23], suggesting a high level of heterogeneity within mprF in some staphylococcal isolates. Nevertheless, the DAP-non-susceptible isolates studied by these authors [23] showed only one amino acid substitution in the mprF (L341S or L826F). Changes in these or in very near positions have been previously reported to be associated with DAP non-susceptibility (S829L, S295L, P314L, S337L, T345I, T345A, I420N) [7,8,11,17,24].
Unlike results reported by other authors, we found one SNP in mprF only in one isolate (MSa5). Mutations at the 314 position had been reported to be previously associated with DAP non-susceptibility, but as P314L [7] instead of P314T, as appears in MSa5. P314L was found in another strain (MSa6, Table 2). The other isolates had high numbers of mutations affecting the three areas. They had between six and eight mutations in the N-terminal “flippase” domains (mode: six mutations), seventeen and twenty mutations (mode: eighteen mutations) in the central, bifunctional domains and eight and nine mutations (mode: eight) in the phosphatidylglycerol lysinylation domain. Six isolates (MSa1, MSa4, MSa5, MSa6, MSa7 and MSa8) included in this wide group of mutations showed some amino acid changes in positions previously associated with DAP non-susceptibility (S295, P314, T345) [7,11,16], but other isolates (MSa2, MSa3 and MSa9) showed mutations previously not reported in association with DAP non-susceptibility. Moreover, they showed no mutations in the area comprising between S295 and I420, where most DAP non-susceptibility-associated mutations reported are located, though they showed more than 30 amino acid changes throughout the gene. This study reflects a large genetic variability between our clinical isolates and the strain used as a reference strain (S. aureus ATCC 25923), especially in some of the genes studied. Some of the mutations observed in the different genes have been previously described to be associated with daptomycin resistance in previous studies [7,8,11,16,17], but others have not. As already mentioned in the results section, the absence of MLST forced us to consider the possibility that at least some of these previously undescribed changes are not associated with daptomycin resistance but with the genetic variability existing between the different MLST types.
All the isolates showed rpoB mutations. The rpoB and rpoC genes encode for the bacterial RNA polymerase β and β’ subunits [7,8]. The mutations described in rpoB and rpoC, associated with DAP non-susceptibility, are different from those affected by other antibiotics, such as rifampicin [25]. Unlike mprF mutations that, at least in vitro, usually emerge at relatively early times during the selection process, rpoB and rpoC mutations emerge later [7]. Some studies have reported rpoB changes in mutants selected in vitro but not in clinical isolates [7], while other studies [11] did not detect rpoB changes in strains selected in vitro and detected them only in clinical isolates. Some authors [25,26] have shown that single-point mutations in rpoB, such as A477D or A621E, can reduce susceptibility to both daptomycin and vancomycin. Such mutations have been shown to cause cell wall thickening and reduction of the negative charge of the outer layer in S. aureus. All the isolates tested in this study showed a rpoB mutation (F737Y) that had not been previously described in non-susceptible isolates, whether clinical or obtained in vitro. Changes in the rpoC gene were also detected in all isolates, excepting MSa5. Single rpoC mutations (N341D) have also been associated with treatment failures in staphylococcal infections [27]. Mutations in the walkR sequence, a regulator of cell wall metabolism and virulence in S. aureus, B. subtilis and S. pneumoniae [28], have been associated with vancomycin resistance [29] and also with reduced DAP susceptibility, in association or not with mprF and agrA mutations [10,29].
fakA has only been associated with DAP non-susceptibility in one study, and only in mutants selected in vitro [10]. In that study, the authors found a L133H mutation associated with DAP non-susceptibility. In our study, this protein showed different changes in eight out of nine clinical isolates. Since the function of this protein is unknown, it is not possible to know its role in DAP susceptibility or even if there is really a cause–effect relationship.
cls genes encode for a cardiolipin synthase, and mutations in these genes have been associated with low DAP susceptibility in staphylococci [11]. Cls proteins are membrane-bound enzymes that synthesize cardiolipin, an important anionic membrane phospholipid, from the phosphatidyl moiety of two PG molecules [30]. While in logarithmic growth, PG is the main membrane phospholipid, in stationary growth conditions and under conditions of stress, such as unfavorable growth conditions or cell wall acting antibiotics, cardiolipin can accumulate up to 25–30% of the membrane phospholipid. cls2 has been the most frequently reported cls gene in this aspect. In this study, all isolates excepting MSa5 showed mutations in this gene. Most isolates shared two mutations (V135I, H205R), and two of them added a third mutation (A459L, A471E). None of these mutations had been described previously. The mutations previously reported appeared between amino acids 20 and 60 (A23V, T33N, L52F, F60S) [11] in the two transmembrane domains described in this protein, while our mutations appeared outside these domains and the cardiolipin synthase active domains.
Mutations in cls1 have been described with a much lower frequency in association to reduced DAP susceptibility. Nevertheless, we found mutations in this gene in the same isolates in which we found mutations in cls2, and the number of mutations in this gene/isolate ranged from six to sixteen. Most mutations found in the isolates tested in this study appeared outside the transmembrane and cardiolipin synthase active domains, but V18A (in cls1), which appeared in all isolates excepting MSa5 and G20A and appeared in MSa6 and MSa9 (Table 3), was in the first transmembrane domain, and I238V, which also appeared in all isolates excepting MSa5, and I421M (isolates MSa1, MSa4 and MSa9) appeared in the cardiolipin synthase domains. Results concerning cls1 and cls2 suggest that, though DAP non-susceptibility-associated mutations are more frequently described in cls2, in the isolates tested in this study, it seems more likely that the most transcendent mutations are those observed in cls1, since, unlike cls2, in almost all isolates, we found mutations that specifically affect the transmembrane and cardiolipin synthase functional domains. Mutations in these areas may have a summation effect with those found in mprF, since the negative charge provided by cardiolipin would be reduced or even suppressed, reducing the attraction between DAP and the bacterial membrane [31].

5. Conclusions

The present study confirms that the acquisition of daptomycin resistance may be associated with different mutational profiles. Therefore, various combinations of mutations in different genes may end up leading to the same result of low susceptibility to daptomycin. However, resistance is mainly associated with mutations in the mprF gene involved in the amount of and situation on the membrane of lysyl phosphatidyl glycerol molecules; rpoB, which encodes an RNA polymerase; and the cls genes, which encode a cardiolipin synthase involved in the metabolism of membrane phospholipids. In the isolates analyzed in this study, mprF, cls1 and cls2 showed much higher numbers of mutations than reported in other studies, many of which had not been described so far. There is no relationship between the number of changes in the genes studied and the degree of sensitivity (MICs). That is, we could find strains with the same MICs and different genetic profiles. Future studies should clarify the real role of each of these mutations.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/pathogens13030206/s1, Table S1: Oligonucleotide specific primers.

Author Contributions

Conceptualization, J.L.M.-B. and N.G.-C. Study direction: J.L.M.-B. Study development: N.G.-C., M.N.G.-Z. and A.M.B.d.C. Conclusions and writing: N.G.-C. and J.L.M.-B. All authors have read and agreed to the published version of the manuscript.

Funding

This study was partially financed by Grant GRS 1831/A/18, Gerencia Regional de Salud, Consejería de Sanidad, Junta de Castilla y León.

Institutional Review Board Statement

This study was conducted in accordance with the Declaration of Helsinki. Since the study was conducted exclusively with microorganisms, and no human subjects or human tissues were involved, the Hospital Ethics Committee considered that his approval was not necessary.

Informed Consent Statement

Not applicable.

Data Availability Statement

The authors declare that data related to this research are available from the authors upon reasonable request.

Acknowledgments

We thank Miguel Fajardo (Hospital General Universitario de Badajoz) for providing us with part of the isolates studied.

Conflicts of Interest

The authors declare they have no conflicts of interest.

References

  1. Eisenstein, B.I.; Oleson, F.B., Jr.; Baltz, R.H. Daptomycin: From the mountain to the clinic, with essential help from Francis Tally, MD. Clin. Infect. Dis. 2010, 50 (Suppl. 1), S10–S15. [Google Scholar] [CrossRef]
  2. Steenbergen, J.N.; Alder, J.; Thorne, G.M.; Tally, F.P. Daptomycin: A lipopeptide antibiotic for the treatment of serious Gram-positive infections. J. Antimicrob. Chemother. 2005, 55, 283–288. [Google Scholar] [CrossRef]
  3. Anastasiou, D.M.; Thorne, G.M.; Luperechio, S.A.; Alder, J.D. In vitro activity of daptomycin against clinical isolates with reduced susceptibilities to linezolid and quinupristin/ dalfopristin. Int. J. Antimicrob. Agents 2006, 28, 385–388. [Google Scholar] [CrossRef]
  4. Cotroneo, N.; Harris, R.; Perlmutter, N.; Beveridge, T.; Silverman, J.A. Daptomycin exerts bactericidal activity without lysis of Staphylococcus aureus. Antimicrob. Agents Chemother. 2008, 52, 2223–2225. [Google Scholar] [CrossRef] [PubMed]
  5. Bhullar, K.; Waglechner, N.; Pawlowski, A.; Koteva, K.; Banks, E.D.; Johnston, M.D.; Barton, H.A.; Wright, G.D. Antibiotic resistance is prevalent in an isolated cave microbiome. PLoS ONE 2012, 7, e34953. [Google Scholar] [CrossRef] [PubMed]
  6. Bayer, A.S.; Schneider, T.; Sahl, H.G. Mechanisms of daptomycin resistance in Staphylococcus aureus: Role of the cell membrane and cell wall. Ann. N. Y. Acad. Sci. 2013, 1277, 139–158. [Google Scholar] [CrossRef] [PubMed]
  7. Friedman, L.; Alder, J.D.; Silverman, J.A. Genetic changes that correlate with reduced susceptibility to daptomycin in Staphylococcus aureus. Antimicrob. Agents Chemother. 2006, 50, 2137–2145. [Google Scholar] [CrossRef]
  8. Mishra, N.N.; Rubio, A.; Nast, C.C.; Bayer, A.S. Differential Adaptations of Methicillin-Resistant Staphylococcus aureus to Serial In Vitro Passage in Daptomycin: Evolution of Daptomycin Resistance and Role of Membrane Carotenoid Content and Fluidity. Int. J. Microbiol. 2012, 2012, 683450. [Google Scholar] [CrossRef] [PubMed]
  9. Pillai, S.K.; Gold, H.S.; Sakoulas, G.; Wennersten, C.; Moellering, R.C., Jr.; Eliopoulos, G.M. Daptomycin nonsusceptibility in Staphylococcus aureus with reduced vancomycin susceptibility is independent of alterations in MprF. Antimicrob. Agents Chemother. 2007, 51, 2223–2225. [Google Scholar] [CrossRef] [PubMed]
  10. Song, Y.; Rubio, A.; Jayaswal, R.K.; Silverman, J.A.; Wilkinson, B.J. Additional routes to Staphylococcus aureus daptomycin resistance as revealed by comparative genome sequencing, transcriptional profiling, and phenotypic studies. PLoS ONE 2013, 8, e58469. [Google Scholar] [CrossRef] [PubMed]
  11. Peleg, A.Y.; Miyakis, S.; Ward, D.V.; Earl, A.M.; Rubio, A.; Cameron, D.R.; Pillai, S.; Moellering, R.C.; Eliopoulos, G.M., Jr. Whole genome characterization of the mechanisms of daptomycin resistance in clinical and laboratory derived isolates of Staphylococcus aureus. PLoS ONE 2012, 7, e28316. [Google Scholar] [CrossRef] [PubMed]
  12. Mehta, S.; Cuirolo, A.X.; Plata, K.B.; Riosa, S.; Silverman, J.A.; Rubio, A.; Rosato, R.R.; Rosato, A.E. VraSR two-component regulatory system contributes to mprF-mediated decreased susceptibility to daptomycin in in vivo-selected clinical strains of methicillin-resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 2012, 56, 92–102. [Google Scholar] [CrossRef]
  13. CLSI. Performance Standards for Antimicrobial Susceptibility Testing, 27th ed.; CLSI Supplement M100S; Clinical Laboratory Standards Institute: Wayne, PA, USA, 2017. [Google Scholar]
  14. Fujimura, S.; Nakano, Y.; Watanabe, A. A correlation between reduced susceptibilities to vancomycin and daptomycin among the MRSA isolates selected in mutant selection window of both vancomycin and daptomycin. J. Infect. Chemother. 2014, 20, 752–756. [Google Scholar] [CrossRef] [PubMed]
  15. Humphries, R.M.; Pollett, S.; Sakoulas, G. A current perspective on daptomycin for the clinical microbiologist. Clin. Microbiol. Rev. 2013, 26, 759–780. [Google Scholar] [CrossRef] [PubMed]
  16. Cameron, D.R.; Mortin, L.I.; Rubio, A.; Mylonakis, E.; Moellering, R.C., Jr.; Eliopoulos, G.M.; Peleg, A.Y. Impact of daptomycin resistance on Staphylococcus aureus virulence. Virulence 2015, 6, 127–131. [Google Scholar] [CrossRef]
  17. Murthy, M.H.; Olson, M.E.; Wickert, R.W.; Fey, P.D.; Jalali, Z. Daptomycin non-susceptible methicillin-resistant Staphylococcus aureus USA 300 isolate. J. Med. Microbiol. 2008, 57, 1036–1038. [Google Scholar] [CrossRef] [PubMed]
  18. Gómez-Casanova, N.; Siller Ruiz, M.; Muñoz Bellido, J.L. Mechanisms of resistance to daptomycin in Staphylococcus aureus. Rev. Esp. Quimioter. 2017, 30, 391–396. [Google Scholar] [PubMed]
  19. Ernst, C.M.; Peschel, A. Broad spectrum antimicrobial peptide resistance by MprF-mediated aminoacylation and flipping of phospholipids. Mol. Microbiol. 2011, 80, 290–299. [Google Scholar] [CrossRef]
  20. Ernst, C.M.; Staubitz, P.; Mishra, N.; Yang, S.J.; Hornig, G.; Kalbacher, H.; Bayer, A.S.; Kraus, D.; Peschel, A. The bacterial defensin resistance protein MprF consists of separable domains for lipid lysinilation and antimicrobial peptide repulsion. PLoS Pathog. 2009, 5, e1000660. [Google Scholar] [CrossRef]
  21. Kristian, S.A.; Dürr, M.; Van Strijp, J.A.; Neumeister, B.; Peschel, A. MprF-mediated lysinylation of phospholipids in Staphylococcus aureus leads to protection against oxygen-independent neutrophil killing. Infect. Immun. 2003, 71, 546–549. [Google Scholar] [CrossRef]
  22. Yang, S.J.; Nast, C.C.; Mishra, N.N.; Yeaman, M.R.; Fey, P.D.; Bayer, A.S. Cell wall thickening is not a universal accompaniment of the daptomycin nonsusceptibility phenotype in Staphylococcus aureus: Evidence for multiple resistance mechanisms. Antimicrob. Agents Chemother. 2010, 54, 3079–3085. [Google Scholar] [CrossRef]
  23. Bayer, A.S.; Mishra, N.N.; Sakoulas, G.; Nonejuie, P.; Nast, C.C.; Pogliano, J.; Chen, K.T.; Ellison, S.N.; Yeaman, M.R.; Yang, S.J. Heterogeneity of mprF sequences in methicillin-resistant Staphylococcus aureus clinical isolates: Role in cross-resistance between daptomycin and host defense antimicrobial peptides. Antimicrob. Agents Chemother. 2014, 58, 7462–7467. [Google Scholar] [CrossRef]
  24. Sabat, A.J.; Tinelli, M.; Grundmann, H.; Akkerboom, V.; Monaco, M.; del Grosso, M.; Errico, G.; Pantosti, A.; Friedrich, A.W. Daptomycin resistant Staphylococcus aureus clinical strain with novel non-synonymous mutations in the mprF and vraS genes: A new insight into daptomycin resistance. Front. Microbiol. 2018, 9, 2705. [Google Scholar] [CrossRef]
  25. Cui, L.; Isii, T.; Fukuda, M.; Ochiai, T.; Neoh, H.; Da Camargo, I.L.B.C.; Watanabe, Y.; Shoji, M.; Hiramatsu, K. An rpoB mutation confers dual heteroresistance to daptomycin and vancomycin in S. aureus. Antimicrob. Agents Chemother. 2010, 54, 5222–5233. [Google Scholar] [CrossRef] [PubMed]
  26. Bæk, K.T.; Thøgersen, L.; Mogenssen, R.G.; Mellergaard, M.; Thomsen, L.E.; Petersen, A.; Skov, S.; Cameron, D.R.; Peleg, A.Y.; Frees, D. Stepwise decrease in daptomycin susceptibility in clinical S. aureus isolates associated with an initial mutation in rpoB and a compensatory inactivation of the clpX gene. Antimicrob. Agents Chemother. 2015, 59, 6983–6991. [Google Scholar] [CrossRef]
  27. Hagiya, H.; Sugawara, Y.; Kimura, K.; Hamaguchi, S.; Nishi, I.; Hayashi, M.; Akeda, Y.; Tomono, K. Emergence of daptomycin non-susceptible coagulase-negative Staphylococci in patients with cardiovascular device infections: Two cases report investigated by whole genome analysis. Medicine 2018, 97, e13487. [Google Scholar] [CrossRef]
  28. Dubrac, S.; Bisicchia, P.; Devine, K.M.; Msadek, T. A matter of life and death: Cell wall homeostasis and the WalKR (YycGF) essential signal transduction pathway. Mol. Microbiol. 2008, 7, 1307–1322. [Google Scholar] [CrossRef] [PubMed]
  29. Howden, B.P.; McEvoy, C.R.; Allen, D.L.; Chua, K.; Gao, W.; Harrison, P.F.; Bell, J.; Coombs, G.; Bennett-Wood, V.; Porter, J.L.; et al. Evolution of multidrug resistance during Staphylococcus aureus infection involves mutation of the essential two component regulator WalKR. PLoS Pathog. 2011, 7, e1002359. [Google Scholar] [CrossRef]
  30. Koprivnjak, T.; Zhang, D.; Ernst, C.M.; Peschel, A.; Nauseef, W.M.; Weiss, J.P. Characterization of Staphylococcus aureus cardiolipin synthases 1 and 2 and their contribution to accumulation of cardiolipin in stationary phase and within phagocytes. J. Bacteriol. 2011, 193, 4134–4142. [Google Scholar] [CrossRef]
  31. Camargo, I.L.; Neoh, H.M.; Cui, L.; Hiramatsu, K. Serial daptomycin selection generates daptomycin-nonsusceptible Staphylococcus aureus strains with a heterogeneous vancomycin-intermediate phenotype. Antimicrob. Agents Chemother. 2008, 52, 4289–4299. [Google Scholar] [CrossRef] [PubMed]
Pathogens 13 00206 g001
Figure 1. Number of mutations of each clinical isolate for each gene studied.
Figure 1. Number of mutations of each clinical isolate for each gene studied.
Pathogens 13 00206 g001
Table 1. Antibiotic susceptibility of the isolates studied (MIC, mg/L). DAP: daptomycin; VAN: vancomycin; OXA: oxacillin; LZD: linezolid; DLV: dalbavancin; TLV: telavancin. S: susceptible; R: resistant.
Table 1. Antibiotic susceptibility of the isolates studied (MIC, mg/L). DAP: daptomycin; VAN: vancomycin; OXA: oxacillin; LZD: linezolid; DLV: dalbavancin; TLV: telavancin. S: susceptible; R: resistant.
StrainsDAPVANOXALZDDLVTLV
MSa14.0 (R)1.0 (S)16 (R)2.0 (S)0.064 (S)0.125 (S)
MSa21.0 (S)1.0 (S)256 (R)2.0 (S)0.047 (S)0.047 (S)
MSa34.0 (R)2.0 (S)0.125 (S)2.0 (S)0.100 (S)0.125 (S)
MSa4 *2.0 (R)2.0 (S)2.0 (S)2.0 (S)0.064 (S)0.125 (S)
MSa54.0 (R)1.0 (S)0.064 (S)2.0 (S)0.047 (S)0.125 (S)
MSa64.0 (R)1.0 (S)4.0 (R)2.0 (S)0.032 (S)0.125 (S)
MSa7 *2.0 (R)1.0 (S)2.0 (S)2.0 (S)0.064 (S)0.094 (S)
MSa84.0 (R)1.0 (S)0.125(S)2.0 (S)0.047 (S)0.125 (S)
MSa92.0 (R)1.0 (S)0.5 (S)2.0 (S)0.032 (S)0.094 (S)
* MRSA (mecA+).
Table 2. Mutations in mprF gene.
Table 2. Mutations in mprF gene.
N° of Isolate (MSax)Amino Acid Mutations 1Base Changes *
1, 2, 3, 4, 6, 7, 8, 9V26A; N160D; A171V;
L174F; Y194F; V223A;
L371I; Y400F; I406L;
I409T;
L413F; V426A; A430V;
I446V; L451I;
I459L; I464V; F473L;
V478I; K489R;
I494L; V503I;
A505M;
N522K; E525D; D531N;
D554N; N556T;
I575L; T696E;
N710E		T1353395C; A1353696G; C1353730T-T1353731A; G1353740T; A1353799T; T1353886C-C1353887T; C1354329A; A1354417T; A1354434T-C1354436A; T1354444C-T1354445A; A1354457C; T1354495C; C1354507T; A1354554G-T1354556A; C1354569A; A1354593T; A1354608G-C1354610T; T1354637A; G1354650A; A1354683C-A1354684G-A1354685C; A1354698C; G1354725A-T1354727A; G1354731A-C1354732T-A1354733G; T1354784A; G1354793T; G1354809A; G1354878A; A1354885C-T1354886A; A1354941T- C1354943A; A1355305G-C1355304A; A1355346G-T1355348A
1, 4I375M; I461TA1354343G; T1354600C-A1354601T
1, 5P314TC1354158A-G1354160T
6I9V; G105A;
P314L 2; P721T		A1353243G; G1353532C;
C1354159T-G1354160T; C1355379
3L291IT1354089A
4, 7T345I 2,4,5, C1354252T
8V287I; S295P 3;
A500S		G1354077A-G1354079A; T1354101C; G1354716T- A1354718T
1 Amino acid numbering corresponds to their positions within the mprF gene. * Base numbering corresponds to the complete genome of S. aureus subsp. aureus strain ATCC 25923 (GenBank: CP009361.1). 2 Friedman [7]; 3 Friedman [7] and Peleg [11] S295L; 4 Mishra [8]; 5 Friedman [7], Mishra [8], Murthy [17] and Cameron [16]: T345A.
Table 3. Mutations in cardiolipin synthase genes (cls1 and cls2).
Table 3. Mutations in cardiolipin synthase genes (cls1 and cls2).
GenesN° of Strain (MSax)Amino Acid Mutations 1 Base Changes *
cls11, 4, 6, 8, 9K147Q; K170Q;
H174K; N197K		A1306452C; A1306521C; C1306533A-T1306535A; T1306604A
1, 4, 9F389Y; I421M; N448KT1307179A; C1307276G; T1307357G
1, 2, 3, 4, 6, 7, 8, 9I238VA1306725G-T1306727A
1, 2, 3, 4, 7, 8E469KG1307418A
1, 2, 3, 4, 7, 8Q2R; F3Y; S4TA1306018G; T1306021A; T1306023A
1, 2, 3, 4, 7, 8V18AT1306166C
6, 9V18A; G87A;
V132I;
A175V		T1306166C-C1306167A; G1306273C-A1306274G; G1306407A; C1306537T-T1306538G
9S43A; T44KT1306140G; C1306144A-T1306145A
6, 9G20AG1306072C-A1306073C
6V300E;
G308K;
P309S; L310F;
S313A; V455A		T1306912A-T1306913A; G1306935A-G1306936A; C1306938T; G1306943C; T1306950G-A1306952G; T1307377C
5NCNC
cls21, 2, 3, 4, 6, 7, 8, 9V135I; H205RG2125012A; A2125223G
6A471EC2126021A
9I459LA2125984T
5NCNC
1 Amino acid numbering corresponds to their positions within the cls1 and cls2 genes. * Base numbering corresponds to the complete genome of S. aureus subsp. aureus strain ATCC 25923 (GenBank: CP009361.1). NC: no change.
Table 4. Mutations in rpoB, rpoC and fakA genes.
Table 4. Mutations in rpoB, rpoC and fakA genes.
GenesN° of Strain (MSax)Amino Acid Mutations 1Base Changes *
rpoB1, 2, 3, 4, 5, 6, 7, 8, 9F737YT545943A
3M513IG545272A
rpoC1, 2, 3, 4, 6, 7, 8, 9V864IG550011A-T550013A
3P100LC547720T
5NCNC
fakA1, 2, 3, 4, 6, 7, 8, 9L214I; D497EC1191817A; T1192668A
9E431DA1192470C
6I144V; E277K;
Y287H; A513E		A1191607G; G1192006A; T1192036C; C1192715A
5NCNC
1 Amino acid numbering corresponds to their positions within the rpoB, rpoC and fakA (mw1109) genes. * Base numbering corresponds to the complete genome of S. aureus subsp. aureus strain ATCC 25923 (GenBank: CP009361.1). NC: no change.
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Gómez-Casanova, N.; Gutiérrez-Zufiaurre, M.N.; Blázquez de Castro, A.M.; Muñoz-Bellido, J.L. Genomic Insights into Staphylococcus aureus Isolates Exhibiting Diminished Daptomycin Susceptibility. Pathogens 2024, 13, 206. https://doi.org/10.3390/pathogens13030206

AMA Style

Gómez-Casanova N, Gutiérrez-Zufiaurre MN, Blázquez de Castro AM, Muñoz-Bellido JL. Genomic Insights into Staphylococcus aureus Isolates Exhibiting Diminished Daptomycin Susceptibility. Pathogens. 2024; 13(3):206. https://doi.org/10.3390/pathogens13030206

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Gómez-Casanova, Natalia, Mª Nieves Gutiérrez-Zufiaurre, Ana Mª Blázquez de Castro, and Juan Luis Muñoz-Bellido. 2024. "Genomic Insights into Staphylococcus aureus Isolates Exhibiting Diminished Daptomycin Susceptibility" Pathogens 13, no. 3: 206. https://doi.org/10.3390/pathogens13030206

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