Emergence of optrA-Mediated Linezolid Resistance in Enterococcus faecium: A Molecular Investigation in a Tertiary Hospital of Southwest China from 2014–2018

Introduction

Enterococcus is a Gram-positive opportunistic pathogen, normally residing in the gastrointestinal tracts of humans, which is regarded as the most common cause of nosocomial infections, such as meningitis, bacteremia, pneumonia, surgical wound infection, and urinary tract infection.¹ Its strong intrinsic and acquired resistance leads to resistance to a major group of antibiotics such as vancomycin. Hence, a better understanding of the mechanism of resistance and transmission could support better strategies to monitor and control drug resistance.

The oxazolidinone linezolid targeted at the large (50S) subunit of bacterial ribosomes is considered as the last resort to methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), and other multi-drug Gram-positive bacteria.² However, linezolid-resistant isolates have been increasingly monitored since their clinical use in 2000.^3,4 Linezolid-resistant Enterococcus (LRE) represents a significant threat to clinical treatment. The most common resistance mechanism is mutations in the central loop of the domain V region of the 23S rRNA gene, especially G2576T and varying copy numbers.⁵ Moreover, mutation or deletion of genes that encode the 50S ribosomal subunit proteins L3, L4, L22 and acquisition of resistance genes such as optrA, poxtA also lead to increased linezolid MIC.^6–8 Oxazolidinone and phenicol transferable gene optrA was first detected in a clinical Enterococcus faecalis from China in 2015.⁷ Following its first report, optrA has also been discovered in many countries such as Colombia, Tunisia, Poland.^9–11 It is mostly reported in Enterococcus but also detected in Staphylococcus sciuri, Streptococcus suis, and other Gram-positive or Gram-negative strains.¹² OptrA is often located on chromosomes or plasmids and can be transmitted by mobile genetic elements such as transposons and insertion sequences.^13–15 The most recently reported poxtA gene, mediating resistance to oxazolidinones, tetracyclines, and phenicols, was first described in an MRSA isolate from respiratory secretion of an Italian patient.⁸ Since its first report, it has also been detected in isolates from animals and humans in many countries, including Italy, Greece, and China.^8,16,17 Otherwise, poxtA is found more in the environment or food-producing animals than human samples and Enterococcus faecium has higher prevalence than Enterococcus faecalis.

Our previous transcriptomics and proteomics studies showed that optrA played an important role in linezolid-resistance E. faecalis at the First Affiliated Hospital of Chongqing Medical University and that sexual pheromones could promote optrA transmission.^18–21 However, the mechanism of linezolid in E. faecium is not yet clear. Therefore, the purpose of this study is to investigate the mechanism of linezolid in E. faecium and reveal its transmission by whole-genome sequencing. To the best of our knowledge, we monitored the emergence of optrA-mediated linezolid resistance in E. faecium. Besides, this is the first report of optrA variants, P and EYDNDM identified in E. faecium, and optrA and poxtA co-exist in the same strain in southwest China.

Materials and Methods

Bacterial Strains and Antimicrobial Susceptibility Tests

A total of 1891 E. faecium strains were obtained at the First Affiliated Hospital of Chongqing Medical University from January 2014 to December 2018. Excluding duplicate strains, 15 LNSEFMs were collected from six types of samples, including urine, blood, secretion, seroperitoneum, drainage, and bile. Then, they were stocked at −80°C with glycerol. Antimicrobial susceptibility tests were initially confirmed by AST-GP67 cards (BioMérieux) on the VITEK-2 Compact system (bioMérieux, Lyon, France), including linezolid (LZD), clindamycin (CLI), dalfopristin (DAF), tetracycline (TET), erythromycin (ERY), ciprofloxacin (CIP), moxifloxacin (MOX), levofloxacin (LEV), vancomycin (VAN), ampicillin (AMP), penicillin (PEN), tigecycline (TIG), streptomycin (STR), and gentamicin (GEN), and then linezolid MIC is manually reconfirmed by the broth microdilution method. All results were determined according to the CLSI guidelines,²² and the E. faecalis ATCC29212 was used as a reference strain.

DNA Extraction and Molecular Detection of Mutation and Resistance Genes

Following the manufacturer’s protocol, genomic DNA was extracted from bacteria cultured in the logarithmic growth phase using the HiPure Bacterial DNA Kit (Magen, Guangzhou, China). To investigate the mechanism of linezolid resistance, the mutation of 23s rRNA ribosomal proteins L3 (rplC), L4 (RplD), L22 (rplV) and the presence of cfr, optrA, and poxtA were identified using a previously described method.¹⁸ Primers and reaction conditions are shown in Supplementary Table S2. All positive PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for bidirectional sequencing and blasted against the NCBI nucleotide database. Nucleotides of 23S rRNA and amino acid sequences of L3, L4, and L22 were compared with the reference E. faecium Aus0004 (GenBank Accession No. CP003351) using DNASTAR package MegAlign (Version 7.1.0). The optrA sequence was compared with plasmid pE349 (GenBank Accession No. NG_048023.1).

Multi-Locus Sequence Typing (MLST)

According to the E. faecium MLST scheme, 7 housekeeping genes (adk, pstS, gyd, purK, gdh, ddl, atpA) were amplified using multi-locus sequence typing (MLST) for an analysis of sequences on the Enterococcus faecium database (https://pubmlst.org/mlst). Primers are listed in Supplementary Table S2.

Whole Genome Sequencing (WGS) and Bioinformatic Analysis

Whole-genome sequencing and bioinformatic analysis on optrA-and-poxtA co-harboring isolate EFM-7150 were conducted by Shanghai Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China). Following the manufacturer’s protocol, Wizard^® Genomic DNA Purification Kit (Promega) was used to extract genomic DNA. Genomic DNA was sequenced using a combination of Illumina and Nanopore sequencing platforms. For Illumina sequencing, 5 μg of genomic DNA was used for library construction. An Illumina HiSeq X Ten with 2 × 150 bp paired-end reads (Illumina) was used to sequence those libraries. For Nanopore sequencing, Covaris G-TUBE (Covaris, MA) was used for spinning 15 μg of genomic DNA to cut the genomic DNA into ∼10 kb fragments, followed by magnetic bead purification and sequencing adapter connection to both ends. After removing low-quality reads, the following reads were assembled into a contig using HGAP and canu.²³ In the end, Pilon (1.23) was applied for error correction of Nanopore assembly results. Gene prediction was performed using bioinformatics software Glimmer (3.0) and GeneMarkS.²⁴ Each set of query nucleotide sequences was aligned with NR, Swiss-Prot and Pfam databases. Circular representation of complete plasmids sequences was visualized using the GView server.²⁵ And Plasmid replicons were identified using PlasmidFinder (2.1) (https://cge.cbs.dtu.dk/services/PlasmidFinder/).

Nucleotide Sequence Accession Number

Nucleotide sequences of EFM_7150 complete chromosome and two key plasmids pEF7150-3, pEF7150-5 were collected in GenBank under accession numbers CP079927, CP079928, CP079929, respectively.

Clinical Data

Patient demographics and clinical data including patient age, sex, date, sample, wards, and antibiotic usage were collected from the hospital information system (HIS) and laboratory information system (LIS).

Ethical Approval Statements

This retrospective study was approved by the Evaluation Committee and the Biomedical Ethics Committee of Chongqing Medical University (2021–515). In light of the retrospective and anonymous nature of the study, the Ethics Committee did not require written informed consent provided by participants.

Results

Clinical Information and Antimicrobial Susceptibility Testing

A total of 15 nonduplicated LNSEFM isolates were recovered from 1891 Enterococcus according to VITEK-2 Compact by the BMD method (Supplementary Table S1). Table 1 shows that 15 isolates were obtained from eleven different wards, the most common is gastrointestinal surgery (n = 3) and urology surgery (n = 3), and there is only one isolate in other wards. Urine is the most common source, followed by secretion. According to clinical data, 10 patients were discharged, and 5 patients were transferred. Penicillin, aminoglycosides, carbapenems and cephalosporins were used during the treatment period.

Table 1 Clinical Information and Screening of Linezolid-Resistant Mechanism

The 15 strains of LNSEFM linezolid MIC spread from 4 to 16 mg/L, of which 5 strains were intermediary, and 10 strains were resistant to linezolid. In addition, the highest drug resistance rate is penicillin (93.3%), erythromycin (93.3%), and ampicillin (93.3%), followed by levofloxacin (86.7%), moxifloxacin (86.7%), and ciprofloxacin (86.7%). All strains are sensitive to vancomycin and tigecycline (Supplementary Table S1).

Screening of Linezolid-Resistant Mechanism and Sequence Type (STs)

The most common mechanism of linezolid resistance in Enterococcus faecium is 23S rRNA gene mutation, but the presence of resistance genes was found to be the main cause in this study.²⁶ The molecular mechanism of linezolid resistance in 15 LNSEFM is detected in Table 1, showing that the positive rate of optrA gene is 46.7%, while the detection rate of poxtA is 13.3%. Of these, one strain carried both poxtA and optrA. The majority of LR E. faecium (70%, 7/10) had optrA. Compared with pE394, two of optrA proteins are wild-type. Four strains had a single point mutation T481P (P variant), and EFM_7150 had K3E, N12Y, Y176D, D247N, G393D, I622M (EYDNDM variant). One LR E. faecium had a L22 protein alteration. No isolates contained cfr gene and L3/L4 alteration. No genetic mechanism was identified in 6 isolates (40%), among which 5 were LI E. faecium. As shown in Table 1, a total of 9 sequence types (ST) were identified among 15 isolates, of which 3 isolates were new ST with 1 to 2 alle mutation. Besides, 46.7% isolates belonged to CC17 clone complex.

Characteristics of Plasmids and the Genetic Environment of optrA and poxtA

The complete genome of Enterococcus faecium_7150 was constructed using the data from the Illumina HiSeq and Oxford Nanopore MinION. The complete genome has 3,083,859 bp nucleotide and 307,877 reads with a GC content of 37.93%. The plasmids carrying optrA or poxtA are shown in Figure 1. The majority of CDSs code gene in the forward orientation are shown in Supplementary Tables S3 and S4. pEF7150-3 with the GC content of 36.91% has a size of 72,048 bp and 85 Coding sequences were identified. While pEF7150-3 is not completely identical to any other plasmid in the Gene Bank at the moment, as shown from the result of blast analysis, the region around 16,790 bp containing the optrA and fexA genes demonstrated 99.9% similarity and a query coverage of 22% to Enterococcus avium C674 (GenBank accession no. MH018573.1), Staphylococcus sciuri S49-1 optrA gene cluster (GenBank accession no. KX447572.1). Tn558 mediating fexA gene transfer was identified in pEF7150-3 and chromosome. Tn558 and optrA were inserted downstream of the radC gene (encoding a DNA repair protein). Moreover, the transcriptional regulator gene araC was located upstream of optrA.

Figure 1 Structure of two resistant gene plasmids in E. faecium_7150. (A) Structure of the optrA-carrying plasmid pEF7150-3. (B) Structure of the poxtA-carrying plasmid pEF7150-5. The peripheral circle represents CDS, and arrows indicate the CDSs and their transcription directions. The second and third circle shows GC skew and GC content respectively. The purple square refers to other function gene. The yellow square refers to other resistant gene. The red square refers to linezolid resistant gene. The grey square refers to mobile genetic elements.

pEF7150-5 with GC content of 36.11% is 21,754 bp in size and has 22 CDSs. It belongs to rep2 family and the incompatibility (Inc) 18 group plasmids.^27,28 pEF7150-5 shared 100% identity with a query coverage of 100% to pM16/0594 (GenBank accession no. MN831411.1), pC25-1 (GenBank accession no. MH784601.1), pC27-2 (GenBank accession no. MH784602.1), pHN11 (GenBank accession no. CP038176.1). Moreover, on plasmid pEF7150-5, the tetracycline resistance gene tet (L) and tet (M) were also identified. The poxtA gene was flanked by two IS1216E elements in the same orientation, which is responsible for horizontal gene transfer of poxtA, as previously described in S. aureus AOUC-0915, E. faecium Efa-955.^8,29

Discussion

Linezolid is an effective drug for treating multidrug-resistant Gram-positive bacterial infection. However, the rate of linezolid-resistant has steadily risen in recent years, posing a threat to public health. Zyvox^® Annual Appraisal of Potency and Spectrum (ZAAPS), Linezolid Experience and Accurate Determination of Resistance (LEADER), which monitor global pathogens and the changes in resistance to linezolid over time, suggest that the rates of linezolid-non-sensitive were 0.70% and 0.74%, respectively.^30,31 In our study, the 5-year prevalence rate of linezolid-resistant E. faecium in our hospital was 1.9%, which is higher than that of China (1.0%).³² Most strains (93.3%) display a multidrug-resistant phenotype (resistant to at least three antimicrobial categories), but all strains are susceptible to vancomycin. It should be noted that nearly half of the isolates belong to CC17 clone that has posed a potential threat to public health, resulting in restricted treatment options worldwide. On antibiotics usage, infected patients had no history of linezolid use during hospitalization, indicating that LNSEFM infection was not associated with linezolid use.

It is well known that mutations in the domain V of the 23S rRNA are the most common mechanism of linezolid resistance among E. faecium, and optrA appears to be almost ubiquitous among the linezolid-non-susceptible E. faecalis.²⁶ Surprisingly, we did not find 23S rRNA mutations but a high prevalence of optrA, which is at odds with the Sentry data.³³ This discrepancy may be explained by geographical variation. Almost all resistant isolates, except intermediates, have resistance genes. Nonetheless, we fail to find any previously described mediated genes in linezolid resistance in one isolate (MIC 8mg/L). We speculate that the observed phenotype could be related to cell wall thickness or biofilm formation.³⁴ Although it has been shown that L22 protein mutation could reduce linezolid sensitivity by interfering with binding sites, this mutation is uncommon in Enterococcus.⁶ Ser77Thr was detected in one LR E. faecium in our study, but the relationship between L22 protein mutation and linezolid MIC remains unclear. Increased copy number of 23S rRNA gene resulted in increased resistance expression in the previous study,³⁵ but there is no evident correlation between optrA variants and oxazolidinone mic in Enterococcus.³⁶ Cai et al revealed that different optrA variants and their genetic context have the potential to regulate linezolid MIC at a variety of levels.³⁷ It is still important to investigate other optrA variants and oxazolidinone resistance level. We identified new types of optrA variants, P and EYDNDM in E. faecium. P variant was distributed in Clostridium difficile and Campylobacter jejuni and EYDNDM variants were found in Enterococcus faecalis, Staphylococcus aureus, Staphylococcus sciuri, Enterococcus avium.¹² Given that the relationship between optrA variants and linezolid MIC remains unclear, novel optrA variants in E. faecium is of concern.

To the best of our knowledge, mobile genetic elements contribute significantly to the transmission of resistant genes.³⁸ Tn558 carrying fexA gene is located on both the chromosome and the plasmids in EFM_7150. It integrates optrA through the radC gene in pEF7150-3, resulting in a similar genetic environment to many isolates, such as E. avium C674 isolated from the stool samples of healthy populations in Hangzhou and S. sciuri isolated from Sichuan province and Guangdong province.^37,39,40 Notably, Fan et al revealed that optrA can exist in methicillin-resistant coagulase-negative staphylococci including S. sciuri S49-1.³⁹ Clinicians need to be alert to the prevalence of optrA in other superbugs such as VRE.

The recently discovered poxtA, a member of the ABC-F proteins family, shares the homology of 32% with optrA. PoxtA was detected more often in the natural environment than in the clinical setting. The IS1216E-PoxtA-IS1216E segment in our study is similar to S. aureus AOUC-0915, and clinical E. faecium from Italy, Spain, indicating that the genetic background of poxtA is relatively single.^8,16,41 In addition to the conjugative plasmid pE035 detected in China, Enterococcus harboring both the optrA and poxtA genes were also found in the environment and human samples from Pakistan, Ireland, Spain, France^17,42–45 Unexpectedly, there does not seem to be any obvious synergistic effect when optrA and poxtA coexist. Because the MIC of EFM_7150 is not only similar to optrA or poxtA alone isolates in our study but also to E. faecium C10004 isolated from Spain (8mg/L).⁴⁴ Furthermore, it is higher than strain isolated from Shanghai (0.5mg/L).⁴⁶ Despite the emergence of co-existence of optrA and poxtA worldwide, limited genetic context was identified and we hope to obtain more genomic data to explain whether there are other mechanisms or negative regulation.

Last but not the least, these results must be taken cautiously and with some limitations in mind. First, since it included just one center and the relative small number LNSEFM, the results of this study may deviate from other sets of studies. Second, the evidence that only one isolate co-harboring optrA and poxtA is not sufficient. Additional sequencing results that reveal the genetic environment will be more convincing. Nonetheless, our findings indicate the emergence of optrA in Enterococcus faecium. Furthermore, optrA and poxtA can coexist in clinical settings and may be transmitted through MGEs. This is real-world clinical experience and providing a useful information for enterococcal infection.

Conclusion

In summary, this research revealed the emergence of optrA in E. faecium, and the prevalence of optrA gene is higher than that of poxtA in Enterococcus faecium from 2014–2018 in our hospital. MGEs, particularly Transposons and insertion sequences may contribute significantly to the dissemination of optrA and poxtA, respectively. Although linezolid is currently effective in the treatment of enterococcal infections, advanced monitoring of changes in the resistance mechanism of linezolid is needed in the future.