Abstract
This study aimed at determining the mechanisms of linezolid resistance and the molecular characteristics of clinical Staphylococcus aureus (n = 2) and coagulase-negative staphylococci (n = 15) isolates obtained from four Spanish hospitals. The detection of linezolid resistance mechanisms (mutations and acquisition of resistance genes) was performed by PCR/sequencing. The antimicrobial resistance and virulence profile was determined, and the isolates were typed by different molecular techniques. Moreover, the genetic environment of the cfr gene was determined by whole-genome sequencing. The cfr gene was detected in one methicillin-resistant S. aureus (MRSA) that also displayed the amino acid change Val118Ala in the ribosomal protein L4. The second S. aureus isolate was methicillin susceptible and showed different alterations in the ribosomal protein L4. All remaining linezolid-resistant Staphylococcus epidermidis (n = 14) and Staphylococcus hominis isolates (n = 1) showed the mutation G2576T (n = 14) or C2534T (n = 1) in the 23S rRNA. Moreover, different amino acid changes were detected in the ribosomal proteins L3 and L4 in S. epidermidis isolates. All S. epidermidis isolates belonged to the multilocus sequence type ST2. Linezolid-resistant staphylococci (LRS) showed a multiresistance phenotype, including methicillin resistance that was detected in all isolates but one, and was mediated by the mecA gene. The cfr gene in the MRSA isolate was located together with the fexA gene on a conjugative 38,864 bp plasmid. Linezolid- and methicillin-resistant S. epidermidis ST2 showing mutations in the 23S rRNA and in the ribosomal proteins L3 and L4 are spread among Spanish hospitals, whereas LRS carrying acquired linezolid resistance genes are rarely detected.
Introduction
Methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant coagulase-negative staphylococci (MRCoNS) are important pathogens involved in community- and hospital-associated infections. Frequently, these bacteria are not only resistant to β-lactam antibiotics, but also to several other classes of antimicrobial agents.1,2 This fact, in addition to the capacity of certain staphylococcal species, such as Staphylococcus epidermidis, to produce biofilms, compromise the therapeutic success. 2
In this context, linezolid is the first member of the oxazolidinone class of antimicrobial agents, which has demonstrated good efficacy against multiresistant Gram-positive pathogens, including MRSA and MRCoNS.1,3,4 Nearly two decades after its introduction into clinical use, linezolid remains active against ∼99% of Gram-positive bacteria. 5
In Staphylococcus spp., the main mechanism of linezolid resistance involves point mutations in the central loop of domain V of the 23S rRNA. Moreover, decreased susceptibility to linezolid has also been related to amino acid changes and alterations in the ribosomal proteins L3 (rplC), L4 (rplD), and L22 (rplV).1,4–6 However, linezolid resistance mediated by acquired resistance genes is concerning because of its great capacity of dissemination. Three transferable linezolid resistance genes have been detected in staphylococci so far. The cfr gene mediates resistance to five classes of antimicrobial agents (phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A) leading to a multiresistance phenotype. Since its first detection in Staphylococcus sciuri,7,8 cfr has been reported in several Gram-positive and Gram-negative bacteria of diverse origins. 9 More recently, the optrA and poxtA genes were described in Enterococcus spp. and S. aureus, respectively. Both genes confer reduced susceptibility to oxazolidinones as well as to phenicols. Additionally, the poxtA gene decreases the tetracycline susceptibility.10,11
Given the importance of linezolid as a last-resort antimicrobial agent in human medicine, it is critical to assess the current molecular mechanisms of resistance and especially to evaluate the presence of the abovementioned linezolid resistance genes in the clinical setting. Therefore, the objective of the present study was to identify the mechanisms of linezolid resistance, and to study the molecular characteristics of linezolid-resistant staphylococci (LRS) recovered from four Spanish hospitals located in different regions.
Materials and Methods
Bacterial collection
During the period 2017–2019, LRS exhibiting a minimum inhibitory concentration (MIC) of >4 mg/L to linezolid, 12 as confirmed by E-test (bioMérieux, Durham), were recovered from four Spanish hospitals located in four different geographic regions. The hospitals that took part in this study are the following: Hospital San Pedro (Logroño), Hospital Royo-Villanova (Zaragoza), Hospital Verge de la Cinta (Tortosa), and Hospital Arnau Vilanova (Lleida).
Isolates of each patient that belonged to different staphylococcal species and/or showed different antimicrobial resistance phenotypes were characterized. These included 14 S. epidermidis, two S. aureus, and one Staphylococcus hominis, recovered from 15 different patients. Three isolates were obtained from the same patient: S. epidermidis X316 and S. hominis X315 were recovered at the same time point, and S. epidermidis X507 one week later. LRS were recovered from blood (n = 7), nasal/pharyngeal samples (n = 3), and other sources (n = 7) (Table 1).
Characterization of the 17 Linezolid-Resistant Staphylococcus spp. Recovered from Four Spanish Hospitals
In brackets the mutations, amino acid changes, deletions (del), or insertions (in) detected.
Ser84Leu in GyrA protein.
Ala457Thr in GyrA protein.
Ser80Phe in GrlA protein.
Tyr410Phe in GrlA protein.
IEC type E contains scn and sak genes.
Ser84Tyr in GyrA protein.
Glu88Lys in GyrA protein.
Asp84Tyr in GrlA protein.
Ser84Phe in GyrA protein.
Gly84Asp in GrlA protein.
Intermediate resistance to vancomycin.
OXA, oxacillin; PEN, penicillin; AMP, ampicillin; ERY, erythromycin; CLI, clindamycin; GEN, gentamicin; TOB, tobramycin; STR, streptomycin; TET, tetracycline; CIP, ciprofloxacin; LEV, levofloxacin; VAN, vancomycin; CHL, chloramphenicol; FFN, florfenicol; LZD, linezolid; TEI, teicoplanin; FOS, fosfomycin; MUP, mupirocin; FUS, fusidic acid; SXT, trimethoprim/sulfamethoxazole; HAV, Hospital Arnau Villanova; HRV, Hospital Royo Villanova; HSP, Hospital San Pedro; HVC, Hospital Verge de la Cinta; IEC, immune evasion cluster; MIC, minimum inhibitory concentration; MLST, multilocus sequence typing.
Antimicrobial resistance pheno- and genotypes
The susceptibility to penicillin, oxacillin, ampicillin, erythromycin, clindamycin, gentamicin, tobramycin, streptomycin, tetracycline, ciprofloxacin, levofloxacin, vancomycin, teicoplanin, daptomycin, fosfomycin, mupirocin, fusidic acid, and trimethoprim/sulfamethoxazole was studied using the MicroScan® WalkAway (Beckman Coulter, Brea). The MIC to rifampicin, chloramphenicol, and florfenicol was measured by broth macrodilution for the cfr-positive isolate, using S. aureus ATCC 29213 as quality control. The CLSI standard M10012 was used to evaluate the MIC results of all antimicrobial agents, except fosfomycin, mupirocin, fusidic acid, 13 and streptomycin, 14 for which the methods and breakpoints recommended by the Committée de l'Antibiogramme de la Société Française de Microbiologie were employed.
Isolates were PCR screened for the presence of the linezolid resistance genes cfr, optrA, and poxtA (Supplementary Table S1). Moreover, mutations in 23S rRNA were investigated by PCR and amplicon sequencing, and by digestion with the NheI restriction enzyme. 15 The presence of amino acid changes in the genes encoding the ribosomal proteins L3 (rplC), L4 (rplD), and L22 (rplV) were determined in all isolates by PCR and sequencing (Supplementary Table S1). The obtained sequences were compared with those of linezolid-susceptible S. aureus NCTC 8325 (GenBank accession number CP000253) and S. epidermidis ATCC 12228 (GenBank accession number CP022247) using the EMBOSS Needle software for nucleotide or amino acid (BLOSUM 62 cost matrix) alignments. The sequence chromatogram of the 23S rRNA, rplC, rplD, and rplV were carefully checked to avoid false-positive observations.
According to the antimicrobial resistance phenotype, the presence of the antimicrobial resistance genes blaZ, mecA, mecB, mecC, erm(A), erm(B), erm(C), msr(A), mph(C), lnu(A), lnu(B), lsa(B), vga(A), aac(6′)-Ie-aph(2″)-Ia, ant(4′)-Ia, str, ant(6)-Ia, tet(L), tet(M), tet(K), vanA, vanB, mupA, fusB, fusC, dfrA, dfrD, dfrG, and dfrK was studied by PCR (Supplementary Table S1). Positive controls from the collection of the University of La Rioja were included in all PCR assays.
In the isolates that showed resistance to fluoroquinolones, amino acid changes in the deduced sequences of GyrA and GrlA proteins were investigated by PCR and sequencing, and compared with the wild-type reference strains S. aureus NCTC 8325 (GenBank accession number CP000253) and S. epidermidis ATCC 12228 (GenBank accession number CP022247) (Supplementary Table S1).
Molecular typing
Characterization by spa typing was performed in S. aureus isolates by PCR and sequencing, and the obtained sequences were analyzed using Ridom Staph-Type© software (Ridom GmbH, Münster, Germany). S. aureus and S. epidermidis isolates were subjected to MLST (multilocus sequence typing). In addition, methicillin-resistant staphylococci were characterized by SCCmec (Staphylococcal Cassette Chromosome mec) typing (Supplementary Table S1).
Virulence gene content
The presence of the genes encoding the virulence factors Panton-Valentine leucocidin (PVL) (lukF/S-PV), toxic shock syndrome toxin (tst), and exfoliative toxins (eta, etb, and etd) was investigated in all staphylococcal isolates by PCR. The five genes (scn, chp, sak, sea, and sep) that comprise the IEC (immune evasion cluster) system were investigated in S. aureus isolates (Supplementary Table S1).
Whole-genome sequencing and genetic environment of the cfr gene
The genetic context of the cfr gene was determined by whole-genome sequencing (WGS). The DNA extraction was performed using the QIAamp® DNA Mini Kit (QIAGEN, Hilden, Germany) with modifications. Before starting the protocol, the cells were mixed with 25 μL lysostaphin solution (0.1 mg/mL) and incubated for 25 min at 37°C. After that, 75 μL TE buffer and 25 μL proteinase K (0.1 mg/L) were added and incubated for 25 min at 37°C. Then, 75 μL phosphate-buffered saline and 2 μL RNAse (2 μg/μL) were added and slightly mixed. After this, the protocol for the kit was followed starting with the addition of AL buffer. The libraries for WGS were prepared using the Nextera XT Library Preparation Kit (Illumina, Inc., San Diego) according to the manufacturer's instructions. The 2 × 300 bp paired-end sequencing in 40-fold multiplexes was performed on the Illumina MiSeq platform (Illumina, Inc.). Genome sequences were de novo assembled using the software MIRA 4.0 (Biomatters, Auckland, New Zealand) and annotated using RAST. 16 The nucleotide sequences were analyzed using Geneious v 2019.0.4 (Biomatters, Auckland, New Zealand), and with the online tools ResFinder 17 and VirulenceFinder 18 of the Center for Genomic Epidemiology website. Nucleotide alignments were performed using Geneious alignment with default settings and amino acid alignments with the BLOSUM62 cost matrix.
A set of primers (F-CCTTGTAAGTTGTGAAACAAACACA and R-AGTCTAAATGGCTTTCATCTGCTTT) was designed to complete the cfr-carrying plasmid based on the sequence of S. epidermidis strain 12-02300 plasmid p12-02300 (GenBank accession number KM521837).
Conjugation experiments
Conjugation experiments were performed to evaluate the transfer of the cfr gene by the filter-mating method 19 using a rifampicin-resistant mutant of the S. aureus ATCC 29213 as recipient strain. The selection of the transconjugants was performed using three different strategies: (1) Selection on brain heart infusion (BHI) agar containing 50 mg/L rifampicin and 10 mg/L chloramphenicol, (2) Selection on BHI agar containing 50 mg/L rifampicin and 10 mg/L florfenicol, and (3) Selection on BHI agar containing 50 mg/L rifampicin and 4 mg/L linezolid. The identification of the transconjugants was performed by spa typing (Supplementary Table S1). MICs of the transconjugants to clindamycin, chloramphenicol, florfenicol, and linezolid 12 were determined by broth macrodilution and their respective resistance genotype was determined by PCR (Supplementary Table S1).
Results
Mechanisms of linezolid resistance
The detailed characteristics of the LRS investigated in this study are shown in Table 1. The isolates showed different combinations of linezolid resistance mechanisms. The single MRSA isolate (linezolid MIC 16 mg/L) harbored the multiresistance gene cfr and the amino acid change Val118Ala in the ribosomal protein L4. The methicillin-susceptible S. aureus (MSSA) isolate showed a two amino acid deletion at positions 66 and 67 and the amino acid change Val118Ala in the ribosomal protein L4 with respect to the wild-type sequence. All linezolid-resistant S. epidermidis (LRSE, n = 14) displayed the highest linezolid MICs identified in this study (MICs >256 mg/L). Mutations within the domain V of the 23S rRNA gene were detected in all cases: G2576T (n = 13) and C2534T (n = 1). Different amino acid changes and insertions were found in the deduced sequences of the ribosomal proteins L3 and/or L4 among S. epidermidis isolates, while all the isolates were wild type for L22 (Table 1). The S. hominis isolate, which showed a linezolid MIC of 32 mg/L, also showed the mutation G2576T in the 23S rRNA nucleotide sequence. Neither optrA nor poxtA genes were detected among the LRS.
Molecular typing, resistance to other antimicrobial agents and virulence gene content
The MRSA and the MSSA isolates were typed as t2220-ST125 and t1688-ST123, respectively, and all LRSE were assigned to the sequence type ST2 (Table 1). All LRS showed a multiresistance phenotype (resistance to three or more classes of antimicrobial agents). All LRS, except one S. aureus, were methicillin resistant and carried the mecA gene, whereas mecB and mecC genes were not detected. The SCCmec type IV was identified in the MRSA isolate, whereas all methicillin-resistant S. epidermidis carried the SCCmec type III. The S. hominis isolate carried a non-typeable SCCmec element. Thirteen LRS isolates displayed macrolide and/or lincosamide resistance, which was mediated by erm(A), erm(C), msr(A), lnu(A), and/or vga(A) genes. Resistance to at least one aminoglycoside was detected in all LRS with the exception of the MSSA isolate. The aminoglycoside resistance genes, aac(6′)-Ie-aph(2″)-Ia, ant(4′)-Ia, aph(3′)-III, str, and/or ant(6)-Ia, were detected (Table 1). The MSSA and one S. epidermidis isolate displayed resistance to tetracycline and the tet(L) and tet(K) genes were detected, respectively. Fluoroquinolone resistance, seen in all MRCoNS, and in the MRSA isolate was mediated by amino acid changes in the deduced sequences of the GyrA (Ser84Leu, Ser84Tyr, Ser84Phe, Glu88Lys, and/or Ala457Thr) and the GrlA (Ser80Phe, Asp84Tyr, Gly84Asp, and/or Tyr410Phe) proteins. The fexA gene was found in the MRSA isolate which displayed MICs to chloramphenicol and florfenicol of 128 and 512 mg/L, respectively. The mupA gene was detected in four out of the seven mupirocin-resistant isolates. All S. epidermidis isolates showed resistance to fusidic acid, which was mediated by the fusB gene. The dfrA (n = 14) and dfrG (n = 1) genes were responsible for the resistance to trimethoprim detected in all MRCoNS. One S. epidermidis isolate was classified as intermediate to vancomycin, but neither vanA nor vanB resistance genes were detected.
None of the virulence associated genes tested (lukS/F-PV, tst, eta, etb, and etd) was detected among our isolates. The MSSA isolate carried the scn and sak genes, and therefore was ascribed to IEC type E. The WGS allowed us to identify the presence of different virulence genes in the MRSA isolate, including hemolysins (hlgA, hlgB, hlgC), leukotoxins (lukD, lukE), aureolysin (aur), enterotoxins (seg), and proteases (splA, splB).
Genetic environment of the cfr gene and conjugation assays
The analysis of the whole-genome sequence of isolate S. aureus C9026 identified the cfr gene as located in combination with the fexA gene on a 38,864 bp plasmid. A very similar plasmid as the one detected in this study, was previously described in the S. epidermidis strain 12-02300 (GenBank accession number KM521837). 20 The only difference between the plasmid described in the present study and the p12-02300 was the amino acid change Asp184Tyr in one hypothetical protein. The plasmid carrying the cfr and fexA genes was successfully transferred into S. aureus ATCC 29213 using linezolid for selection of transconjugants. The transconjugants displayed resistance to clindamycin (32 mg/L), chloramphenicol (128 mg/L), florfenicol (256 mg/L), and linezolid (8 mg/L) (Table 2). Apart from cfr and fexA, no other resistance genes were detected in the transconjugants.
Minimum Inhibitory Concentrations of the Recipient Staphylococcus aureus ATCC 29213, the Donor S. aureus C9026, and the Transconjugant C9026-TC Isolate
Discussion
When linezolid was introduced in 2000, it was thought that, due to its unique mechanism of action and not being structurally related to other known family of antimicrobial agents, it would be difficult for bacteria to develop resistance. 3 However, 1 year after its introduction, the first linezolid-resistant S. aureus isolate showing the G2576T mutation in the 23S rRNA was reported. 21 Since then, several studies have described the emergence of linezolid resistance among Staphylococcus spp. worldwide.1,6,20,22–28
Previous studies have reported the presence of the poxtA gene in staphylococci recovered from humans,11,29 whereas, to the best of our knowledge, the optrA gene has not been described in clinical LRS. In this study, we have only detected the presence of the cfr gene in one MRSA isolate. This gene has been reported in clinical LRS in different countries,5,20,22,25,27 and, as in our study, it has been previously described to co-occur with other mechanisms of linezolid resistance.20,25
As also observed by other authors,5,25,27 changes in different target sites associated with oxazolidinone resistance have become common among LRS. Several studies have demonstrated that the main mechanism of linezolid resistance among Staphylococcus spp. is attributable to point mutations in the 23S rRNA and, as in our study, the change G2576T is the most commonly detected worldwide.5,6,20,24,25,27 The mutation C2534T that displayed one S. epidermidis isolate of this study has been previously detected among clinical isolates of this species. 30 Several of the nonsynonymous mutations in the gene encoding the ribosomal protein L3 detected in this study (e.g., Val154Leu, Ala157Arg, Met156Thr, Leu101Val) have been previously reported among clinical linezolid-resistant S. epidermidis isolates.5,6,25,27,30 However, no data exist regarding other amino acid changes, such as Gly137Val. As it was observed in several studies, the gene encoding the ribosomal protein L4 seems to have more probable insertions and deletions,5,6,30 and the insertion of a glycine at position 71 detected in seven S. epidermidis isolates in this study, has been previously described.5,6 Conversely, amino acid changes in the ribosomal protein L22 remain uncommon among LRS.5,6,25,27 The association between some of these amino acid changes in the ribosomal proteins L3 and L4, and the decreased susceptibility to linezolid has not yet been found, since other works have reported changes (e.g., Leu101Val), which do not seem to be involved in linezolid susceptibility.22,24,28
Among CoNS, S. epidermidis is the most clinically relevant species, primarily associated with foreign body-related infections. 2 Linezolid resistance has been previously detected in S. epidermidis isolates of different STs (e.g., ST22, ST5, ST23, ST24, ST185, ST186),30,31 but the most prevalent one is ST2. S. epidermidis belonging to ST2 (clonal complex CC5) is the most important genetic lineage related to hospital-associated infections and involved in linezolid resistance worldwide.2,5,22,24 Becker et al. 2 attributed the spread of this clonal type in clinical settings to its capacity to produce biofilm and the presence of numerous resistance genes. In the case of Spain, linezolid-resistant S. epidermidis of ST2 have been previously described in hospitals of the same and different regions where the hospitals that took part in this work are located.23,26,27
In this study, linezolid resistance was associated with a multiresistance phenotype in all cases, and with methicillin resistance in all but one isolate, which is in accordance with previous works.2,6,25 As it was observed by other authors, resistance to vancomycin, teicoplanin, and daptomycin is uncommon among both linezolid-resistant CoNS and S. aureus. However, LRS frequently display resistance to macrolides and lincosamides, gentamicin, and fluoroquinolones.6,25
Regarding virulence-associated genes, none of the CoNS isolates carried any of the ones studied. The virulence genes investigated are strongly associated with S. aureus but, although few studies do exist about their presence among CoNS, they have been previously detected in different species of diverse origins.32–35 The MSSA isolate harbored the staphylococcal complement inhibitor (scn) and the staphylokinase (sak) genes, and so, it was ascribed to the IEC type E. The IEC facilitates human colonization and invasion and, therefore, its detection is common in lineages adapted to humans. 36 The MRSA isolate harbored several genes, which are major contributors to S. aureus virulence. Some of these virulence-associated genes, such as lukDE, aur, and the hemolysin genes are frequently detected in S. aureus isolates of diverse origins.37,38
In this study, we have detected a cfr- and fexA-carrying plasmid in one MRSA isolate. The genetic structure containing the cfr and fexA genes, including the transposases tnpA, tnpB, and tnpC, is similar to the Tn558 variant described in the plasmid pSCFS7 of a clinical MRSA strain (GenBank accession number FR675942). 39 The pSCFS7-like plasmids have been reported many times in different staphylococcal species in European countries, including Spain.40,41 Moreover, the first cfr-carrying S. epidermidis isolate in Spain belonged to the lineage ST22 and also harbored the cfr and fexA genes in a similar structure to pSCFS7. 31 Although, the whole-genome sequence analysis did not reveal the presence of known conjugation-associated machinery in the cfr-MRSA isolate detected in our study, this plasmid was transferred by conjugation. However, the horizontal gene transmission of pSCFS7-like plasmids by conjugation, even in the absence of conjugation machinery, has been previously reported. 42
In conclusion, linezolid-resistant S. epidermidis ST2 with mutations in different oxazolidinone target sites are present in Spanish hospitals. LRS, carrying acquired linezolid resistance genes, are uncommon in isolates recovered from humans. LRS showed a multiresistance phenotype, but remained susceptible to some last-resort antimicrobial agents, such as daptomycin. These results highlight the need for continued epidemiological surveillance to better understand the characteristics of LRS.
Acknowledgment
L.R. has a predoctoral fellowship from the Universidad de La Rioja (Spain).
Footnotes
Disclosure Statement
No competing financial interests exist.
Funding Information
This work was partially supported by project SAF2016-76571-R from the Agencia Estatal de Investigación (AEI) of Spain and the Fondo Europeo de Desarrollo Regional (FEDER) of EU and the Federal Ministry of Education and Research (BMBF) under project number 01KI1727D as part of the Research Network Zoonotic Infectious Diseases.
References
Supplementary Material
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