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Linezolid-resistant Enterococcus faecium clinical isolates from Pakistan: a genomic analysis

BMC Microbiology volume 24, Article number: 347 (2024) Cite this article

Abstract

Background

Linezolid-resistant Enterococcus faecium (LRE) is a global priority pathogen. Thirteen LRE were reported from clinical specimens between November 2021 and April 2023 at two laboratories in Karachi, Pakistan. We aimed to investigate the strain types and genes associated with linezolid resistance among these isolates. Whole genome sequencing (WGS) was performed and analyzed by multilocus sequence typing (MLST). The presence of linezolid resistance genes was identified using ResFinder v4.1.11 and the LRE-finder tool.

Results

Twelve isolates belonged to clonal complex 17 (CC17); ST80 (n = 10), ST612 (n = 1) and ST1380 (n = 1). Six isolates showed the presence of optrA gene and G2576T mutations in the 23S rRNA gene, while six showed poxtA and cfr(D) genes. One isolate showed the combination of optrA, cfr(D) and poxtA genes.

Conclusion

Our findings show the circulation of CC17 sequence types with a known outbreak potential and we identified molecular mechanisms of resistance that were not previously reported from Pakistan.

Peer Review reports

Background

Enterococcus, a genus of Gram-positive, facultatively anaerobic bacteria, has obtained increasing attention in microbiology and clinical research. Certain strains within the Enterococcus species have emerged as significant human, animal and plant pathogens [1]. In humans, Enterococcus faecium and Enterococcus faecalis are the main species most associated with urinary tract infections, peritonitis, and endocarditis [2]. Over the last two decades, E. faecalis has been replaced with E. faecium as the more frequent causative agent of infections in humans. Using population genetics E. faecium can be categorized into two major subpopulations; commensals of the GI tract (usually not involved in clinical infections), and a hospital-associated (HA) lineage [3, 4]. Clonal complex 17 (CC17) of Enterococcus faecium is associated with the colonization of hospital surfaces and is responsible for outbreaks across the globe. The presence of antimicrobial resistance and virulence genes in the HA lineage enhances biofilm formation and colonization which further facilitates infections [5,6,7].

The emergence of vancomycin resistance in strains isolated from human clinical specimens has led Enterococci to be recognized as organisms of global concern and prioritized as high-priority pathogens [8]. Linezolid is a common treatment option against these resistant strains. Reports of linezolid resistance in Staphylococcus and Enterococcus species have started to emerge globally [9, 10]. Thus far, the two major reported mechanisms for oxazolidinone resistance in Enterococci are (i) chromosomal mutations at positions G2447U, G2504A and G2576U in the V domain of 23S rRNA and gene encoding ribosomal protein L3, L4 and L22 and (ii) the acquisition of plasmids carrying genes cfr, poxtA and optrA [11].

The presence of the cfr, optrA and poxtA genes in Enterococcus species has been reported in isolates from foods of animal origin, including pigs and chickens [12,13,14]. Studies have identified resistance genes in other environmental samples such as lakes and rivers [15, 16]. In Pakistan, poxtA and cfr genes in linezolid-susceptible and linezolid-intermediate E. faecium have been detected in environmental specimens [17]. A case of linezolid-resistant, vancomycin-resistant E. faecium was also reported in VP shunt-associated meningitis [18]. From our geographical region, India and Iran have reported linezolid-resistant Enterococcus (LRE) cases in clinical specimens [19,20,21].

In Pakistan, the use of linezolid is unavoidable in certain clinical conditions. Due to nonavailability of daptomycin, it is the only available option for vancomycin-resistant Enterococcus bacteremia. Linezolid is frequently prescribed as empiric treatment for skin and soft tissue infections in outpatient settings and is among the few widely available second-line anti-tuberculosis drugs in Pakistan which has one of the highest prevalence of tuberculosis in the world. Furthermore, the injudicious use of linezolid and other antibiotics without a physician’s prescription is also alarming [22, 23].

Considering LREs as a potential future threat, this study aimed to determine the mechanisms of linezolid resistance and study the molecular characteristics of linezolid-resistant E. faecium recovered from clinical specimens in a tertiary care hospital laboratory in Karachi, Pakistan.

Methods

Isolates selection, identification, and susceptibility testing methods

Between November 2021 and April 2023, 13 clinical isolates of linezolid-resistant Enterococcus faecium (LRE) from blood, urine, and central venous catheter (CVC) cultures were identified from two large hospitals in Karachi, Pakistan. Of these, 12 were reported from the Aga Khan University Hospital, and 1 from the National Institute of Cardiovascular Diseases.

Bile-esculin and growth in 6.5% NaCl were used as initial biochemical tests. The identification of linezolid-resistant isolates was confirmed by API 20 Strep (bioMérieux, France). Susceptibility testing was done by the Kirby-Bauer disc diffusion method on Muller-Hinton agar (MHA) and results were confirmed by minimum inhibitory concentration (MIC) on VITEK 2 (bioMérieux). Susceptibilities were interpreted according to CLSI M100, 31st edition, and EUCAST breakpoints in the case of tigecycline [24, 25].

Isolate archiving and retrieval for DNA extraction

Pure growths of the isolates were stored in 1.5 mL Eppendorf vials with sterile glycerol phosphate broth (GPB) and archived in ultralow freezers with unique identification numbers. Enterococcus faecium Pakistan EFPK_1–12 isolates and EFPK_ 14 were LREs, and EFPK_13 and 15 were linezolid susceptible isolates. Data was maintained in the laboratory software system. Strains were revived by subculturing on sheep blood agar (SBA) and incubated at 37◦C in ambient air for 24–48 h. Pure bacterial overnight growth was used to prepare a homogenous suspension in sterile water for extraction of nucleic acid.

QIAamp DNA kit was used for DNA extraction with slight modification. Briefly, after centrifugation at 7500 rpm for 10 min, bacterial pellets were suspended in 180 µL of 20 mg/mL lysozyme solution mix and incubated at 37◦C for 30 minutes followed by the manufacturer’s instructions. Extracted DNA was saved at -20◦C until processed for whole genome sequencing libraries.

Whole genome sequencing

The genomic DNA of 13 linezolid-resistant and two linezolid-susceptible E. faecium isolates were sequenced. Briefly, DNA libraries for Illumina sequencing were prepared using the Nextera XT DNA library preparation kit (Illumina, USA) [26]. Equimolar libraries were pooled and sequenced on the MiniSeq platform (Illumina) to generate paired-end 153 bp reads. The quality of raw reads was determined by FastQC v0.11.9 (https://github.com/s-andrews/FastQC) and the Nextera XT adaptor sequence was removed using Trimmomatic v0.39 (https://github.com/usadellab/Trimmomatic). Reads were assembled into a draft genome using SPAdes de-novo assembler v3.15.5 [27]. Draft genome assemblies from this study and the public database were annotated with Prokka v1.14.6 [28]. MLST sequence types (STs) were determined using the MLST tool (https://github.com/tseemann/mlst) and the PubMLST database of Enterococcus faecium (https://pubmlst.org/organisms/enterococcus-faecium). Core genome alignment was performed using Roary v3.13.0 and the maximum likelihood tree was constructed from the core genome alignment file using Fasttree v.2.1.11 [29, 30].

The Newick tree file was visualized and annotated in iTOL (https://itol.embl.de/). Genomic sequences from this study (EFPK_01-EFPK_15) were submitted to NCBI with accession numbers (PRJNA1013219). For comparative genomic analysis, we have downloaded eight publicly available genome sequences of E. faecium clinical isolates from Pakistan (GCA_004152605.1, GCA_004152365.1, GCA_004151675.1, GCA_004151685.1, GCA_004152375.1, GCA_004151665.1, GCA_004151705.1, GCA_004152325.1, GCA_004151695.1) and other global sequences of linezolid-resistant E. faecium isolates GCA_004152365.1, NZ_CP040236.1, NZ_CP040368.1 and CP079927.1 for phylogenetic analysis. Genome comparison was carried out using BRIG [31].

ResFinder v4.1.11 (https://bitbucket.org/genomicepidemiology/resfinder/) was used to identify the antimicrobial resistance genes (ARGs) for multiple antibiotics present in the isolates. To identify the G2576T and G2505A mutation (Escherichia coli numbering) in domain V of 23S ribosomal RNA, responsible for linezolid resistance, LRE-Finder was used (https://bitbucket.org/genomicepidemiology/lre-finder/) on FASTQ read files [32].

Results

Clinical and microbiological profiles of the patients

A total of 13 isolates were included (eight from blood, four from urine and one from central venous catheter tip). The mean age of patients was 51 ± 22 years. Linezolid was used in only two patients (EFPK_8 = 4 days and EFPK_ 4 = 31 days) before the isolation of LRE in clinical specimens. Six patients who died in this cohort were critically ill at the time of admission and had prior comorbidities: chronic renal failure secondary to diabetes mellitus and hypertension, renal and liver transplant recipients, COVID-19 and congestive heart failure. All six were treated with broad-spectrum antibiotics. One patient (EFPK_14) had also received targeted therapy with tigecycline.

In all isolates, linezolid MICs (VITEK) were ≥ 8 µg/mL and all isolates were susceptible to tigecycline (MICs range: < 0.12–0.25 µg/mL). One isolate was also susceptible to vancomycin (Table 1).

Table 1 Antimicrobial susceptibility and associated genomic profile of linezolid-resistant Enterococcus faecium
Full size table

Whole genome sequence analysis

The genome sequenced in this study was assembled using SPAdes de-novo assembler, and the genome with more than 20X depth was further processed for the phylogenetics analysis; details of assembly size and read coverage are provided in the supplementary table.

Isolates from this study were largely phylogenetically clustered based on either carriage of the resistance genes poxtA and cfr(D) gene or optrA gene carriage and mutation in the 23S rRNA gene at position G2576T. Six isolates were found to be phylogenetically similar as they clustered together and mainly belonged to the MLST sequence type 80. In EFPK_06, one of the alleles was partially matched using a contigs file but reanalysis with the scaffold.fasta assigned its MLST as ST80, whereas ST612 and ST1380 was identified in EFPK_01 and EFPK_12 respectively. EFPK_04 was truly non-typable as all 7 alleles matched perfectly but the pattern was not found in the database. (Table 1; Fig. 1).

Fig. 1
figure 1

Relatedness of the fifteen Enterococcus faecium strains from Pakistan with global isolates. The phylogenetic tree of fifteen isolates from Pakistan (EFPK_1 to EFPK_15) generated with strains from three countries: India, China and USA were visualized and annotated in iTOL (https://itol.embl.de/upload.cgi). The isolates were annotated based on country, MLST, source of specimen (blood, urine and central venous catheter tip), Vancomycin resistance (resistant = purple), Linezolid resistance (resistant = blue) and presence of genes vanHAX, optrA, poxtA, cfr(D), 23S G2576T (presence is labelled as black)

Full size image

The plasmid-borne genes (optrA, poxtA and cfr(D)) were found in different combinations. The combination of poxtA and cfr(D) genes was identified in six isolates, and a point mutation (amino acid change G2576T) in the central loop of domain V of the 23rDNA, was identified in combination with the optrA gene in the other six strains. EFPK_08 is phylogenetically unique due to its distinct pattern carrying all three plasmid-borne genes (optrA, poxtA and cfr(D)). (Table 1; Fig. 1).

The purpose of selecting non-study isolates from India, Iran and China was to assess the phylogenetic similarities and resistance mechanisms in countries sharing geographical boundaries with Pakistan.

WGS-based analysis of the strains revealed the presence of genes associated with resistance to multiple other antibiotics including glycopeptides (vanHAX gene), aminoglycosides (aac(6’)-li, aph(2″), aph(3’)-IIIa, ant [6]-I), macrolides (erm(A), erm(B), msr(C)), tetracyclines (tet(L) gene), and trimethoprim (dfrG gene). (Table 2)

Table 2 Antimicrobial-resistant genes (ARGs) identified in the genome of E. faecium strains using ResFinder tool
Full size table

The BRIG plot of four of our representative clinical isolates, having different combinations of resistance mechanisms, compared with the reference strain from India found 90 similarities in nuclear contents. Genome sequences of E. faecium clinical isolates harboring variable genes and mutations conferring resistance to linezolid were compared with the VB3025 genome sequence from India (NZ_CP040236), a sequence reported to be positive with G2576T mutation and optrA gene (Fig. 2).

Fig. 2
figure 2

BRIG plot of Enterococcus faecium strains. The coverage map of four Enterococcus faecium sequences (EFPK_01, EFPK_08, EFPK_10 and EFPK_13) are labelled as blue, red, green, and magenta, and are compared with the reference strain from India (VB3025, grey). All four sequences show more than 90% similarity with the reference strain. Additional GC content and GC Skew features are displayed. Antimicrobial resistance genes were annotated correspond to the reference (VB3025). The tool used to draw figure is BRIG tool (https://brig.sourceforge.net/)

Full size image

Discussion

Our study provides insight into the genotypic patterns of emerging linezolid resistance in E. faecium isolated from clinical specimens in Pakistan. A previous study by Wardenburg et al. identified linezolid non-susceptible Enterococcus strains in clinical as well as environmental isolates from Pakistan [17]. This study identified the efflux pump genes (optrA and poxtA) and the cfr gene in only environmental isolates. Our study adds to this earlier work by documenting resistance mechanisms in clinical LRE isolates. Sequencing showed efflux pump genes in all 13 LRE isolates, and 6 isolates were found to have additional G2576T mutations in the 23S rRNA gene. The presence of plasmid genes, involved in transferable resistance to other bacteria including S. aureus is an area of treatment concern given the added potential for causing outbreaks in hospitals [33, 34].

A recent study from India documented the first optrA-carrying E. faecium [35] which we also observed in six of our LRE isolates. A recent study from Iran also identified through PCR the combination of all three efflux pump genes (optrA, poxtA, cfr(D)) in clinical isolates as identified in our study isolate EFPK_08 [21]. A similar pattern has also been reported in China [36, 37]. In contrast, 23S rRNA mutations are the most common mechanisms of linezolid resistance in LRE isolates from the USA and Europe [17, 38,39,40]. Emerging patterns of linezolid resistance mechanisms among LRE require further exploration as they may be representative of geographical spread.

Studies from China have identified a considerable presence of linezolid resistance genes in poultry sources and discussed their potential for outbreaks [41,42,43]. Similar patterns of genes in livestock sources have been identified in other countries [44]. Unfortunately, surveillance of such resistance patterns in environmental and livestock sources from Pakistan is lacking and our study highlights the need to bridge this gap. All sequence types identified in our study (ST80, ST1380 and ST612), however, belong to the CC17 lineage, known for its potential as a hospital environment colonizer [38].

Interestingly, our study suggests that while the source of acquisition of LRE may be exogenous (healthcare setting), antibiotic pressure is also likely to have an impact. EFPK_08 is the only isolate where sequencing revealed all three transferable resistant genes. This patient had a history of prior linezolid use for 4 days. This indicates that linezolid exposure may lead to the acquisition of additional resistance genes, since none of the other isolates showed this pattern. However, a larger sample size with more clinical information is important to confirm this finding in future studies.

Prior linezolid use was not identified as a risk factor for the development of LRE in other cases in our study. Evidence from the literature reports high VRE colonization in hospitalized patients posing a potential threat for hospital-acquired infections, highlighting the importance of strengthening infection prevention and control teams to reduce the chance of HAI from these drug-resistant strains [45, 46].

The resistance genes identified optrA, poxtA and cfr also confer reduced susceptibility to other groups of antibiotics (e.g., tetracyclines and phenicols) [47]. Therefore the implication of linezolid therapy predicts not only potential resistance to oxazolidinones but also to other antibiotics, further limiting treatment options.

Our study has several limitations. The use of VITEK 2 to ascertain linezolid resistance meant that true MICs could not be determined to be correlated with mechanisms of resistance and clinical patterns (e.g., prior linezolid use), as all isolates had MICs higher than the VITEK 2 test limit (≥ 8 µg/mL). The clinical histories were incomplete or not fully available in some cases regarding previous linezolid exposure or healthcare exposure in other hospitals.

In conclusion, our study provides valuable insight into genotypic patterns of LRE and the co-existence of multiple mechanisms of resistance in E. faecium isolates in clinical specimens highlights the importance of this emerging challenge. To avoid the expansion of LRE in Pakistan there is a need for a national consensus document that should emphasize and guide the physicians for judicious linezolid use only in recommended clinical conditions and strengthening infection control practices that could help in reducing healthcare-associated infections.

Data availability

The datasets generated and analyzed during the current study are available in the NCBI repository, BioProject ID: PRJNA1013219. (Availability of the datasets will be made public after December 2024 or the publication of this study, whichever comes first)

References

  1. Krawczyk B, Wityk P, Gałęcka M, Michalik M. The many faces of Enterococcus spp.—commensal, probiotic and opportunistic pathogen. Microorganisms. 2021;9(9):1900.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. García-Solache M, Rice LB. The Enterococcus: a model of adaptability to its environment. Clin Microbiol Rev. 2019;32(2). https://doi.org/10.1128/cmr. 00058 – 18.

  3. Horner C, Mushtaq S, Allen M, Hope R, Gerver S, Longshaw C, et al. Replacement of Enterococcus faecalis by Enterococcus faecium as the predominant enterococcus in UK bacteraemias. JAC-Antimicrobial Resist. 2021;3(4):dlab185.

    Article  Google Scholar 

  4. Top J, Willems R, Blok H, De Regt M, Jalink K, Troelstra A, et al. Ecological replacement of Enterococcus faecalis by multiresistant clonal complex 17 Enterococcus faecium. Clin Microbiol Infect. 2007;13(3):316–9.

    Article  CAS  PubMed  Google Scholar 

  5. Zhou X, Willems RJ, Friedrich AW, Rossen JW, Bathoorn E. Enterococcus faecium: from microbiological insights to practical recommendations for infection control and diagnostics. Antimicrob Resist Infect Control. 2020;9:1–13.

    Article  CAS  Google Scholar 

  6. Echeverria-Esnal D, Sorli L, Navarrete-Rouco ME, Prim N, Barcelo-Vidal J, Conde-Estévez D et al. Ampicillin-resistant and Vancomycin-susceptible Enterococcus faecium bacteremia: a clinical narrative review. Expert Rev Anti-infective Therapy. 2023(just-accepted).

  7. Peng Z, Yan L, Yang S, Yang D. Antimicrobial-resistant evolution and global spread of Enterococcus faecium Clonal Complex (CC) 17: Progressive Change from Gut colonization to Hospital-adapted Pathogen. China CDC Wkly. 2022;4(2):17.

    Article  PubMed  PubMed Central  Google Scholar 

  8. World Health Organization (WHO.) Global priority list of antibiotic-resistant bacteria to guide research, discovery, and development of new antibiotics WHO, Geneva, Switzerland [Internet]. 2017.

  9. Bi R, Qin T, Fan W, Ma P, Gu B. The emerging problem of linezolid-resistant enterococci. J Global Antimicrob Resist. 2018;13:11–9.

    Article  Google Scholar 

  10. Stefani S, Bongiorno D, Mongelli G, Campanile F. Linezolid resistance in staphylococci. Pharmaceuticals. 2010;3(7):1988–2006.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Rogers R, Rice LB. State-of-the-art review: persistent enterococcal bacteremia. Clin Infect Dis. 2023:ciad612.

  12. Zarzecka U, Zakrzewski AJ, Chajęcka-Wierzchowska W, Zadernowska A. Linezolid-resistant Enterococcus spp. Isolates from Foods of Animal Origin—the genetic basis of Acquired Resistance. Foods. 2022;11(7):975.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Ni J, Long X, Wang M, Ma J, Sun Y, Wang W, et al. Transmission of linezolid-resistant Enterococcus isolates carrying optrA and poxtA genes in slaughterhouses. Front Sustainable Food Syst. 2023;7:1179078.

    Article  Google Scholar 

  14. Yoon S, Kim YB, Seo KW, Ha JS, Noh EB, Lee YJ. Characteristics of linezolid-resistant Enterococcus faecalis isolates from broiler breeder farms. Poult Sci. 2020;99(11):6055–61.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Fioriti S, Coccitto SN, Cedraro N, Simoni S, Morroni G, Brenciani A, et al. Linezolid resistance genes in enterococci isolated from sediment and zooplankton in two Italian coastal areas. Appl Environ Microbiol. 2021;87(9):e02958–20.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Nüesch-Inderbinen M, Raschle S, Stevens MJ, Schmitt K, Stephan R. Linezolid-resistant Enterococcus faecalis ST16 harbouring optrA on a Tn6674-like element isolated from surface water. J Global Antimicrob Resist. 2021;25:89–92.

    Article  Google Scholar 

  17. Wardenburg KE, Potter RF, D’Souza AW, Hussain T, Wallace MA, Andleeb S, et al. Phenotypic and genotypic characterization of linezolid-resistant Enterococcus faecium from the USA and Pakistan. J Antimicrob Chemother. 2019;74(12):3445–52.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Irfan S, Qamar S. A case report on isolation of linezolid-and Vancomycin-resistant Enterococcus faecium species from cerebrospinal fluid of a patient suffering from ventriculoperitoneal shunt-associated meningitis. Cureus. 2019;11(9).

  19. Kumar S, Bandyoapdhyay M, Chatterjee M, Mukhopadhyay P, Poddar S, Banerjee P. The first linezolid-resistant Enterococcus faecium in India: high level resistance in a patient with no previous antibiotic exposure. Avicenna J Med. 2014;4(01):13–6.

    Article  PubMed  PubMed Central  Google Scholar 

  20. Yasliani S, Mobarez A, Doust R, Satari M, Teymornejad O. Linezolid Vancomycin resistant Enterococcus isolated from clinical samples in Tehran hospitals. Indian J Med Sci. 2009;63(7):297.

    Article  CAS  PubMed  Google Scholar 

  21. Torabi M, Faghri J, Poursina F. Detection of genes related to Linezolid Resistance (poxtA, cfr, and optrA) in clinical isolates of Enterococcus spp. from humans: a First Report from Iran. Adv Biomedical Res. 2023;12(1):205.

    Article  CAS  Google Scholar 

  22. Saleem Z, Hassali MA, Godman B, Fatima M, Ahmad Z, Sajid A, et al. Sale of WHO AWaRe groups antibiotics without a prescription in Pakistan: a simulated client study. J Pharm Policy Pract. 2020;13:1–8.

    Article  Google Scholar 

  23. Alizai Q, Haseeb A, Hamayun S, Khan S, Ali F, Roghani M et al. Community-acquired skin and soft tissue infections: epidemiology and management in patients presenting to the Emergency Department of a Tertiary Care Hospital. Cureus. 2023;15(1).

  24. Testing ECoAS. Breakpoint tables for interpretation of MICs and zone diameters. version; 2019.

  25. Clinical, Institute LS. Performance standards for antimicrobial susceptibility testing. Clinical and Laboratory Standards Institute Wayne, PA; 2021. pp. 106 – 12.

  26. Baym M, Kryazhimskiy S, Lieberman TD, Chung H, Desai MM, Kishony R. Inexpensive multiplexed library preparation for megabase-sized genomes. PLoS ONE. 2015;10(5):e0128036.

    Article  PubMed  PubMed Central  Google Scholar 

  27. Prjibelski A, Antipov D, Meleshko D, Lapidus A, Korobeynikov A. Using SPAdes De Novo Assembler. Curr Protoc Bioinf. 2020;70(1):e102.

    Article  CAS  Google Scholar 

  28. Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics. 2014;30(14):2068–9.

    Article  CAS  PubMed  Google Scholar 

  29. Page AJ, Cummins CA, Hunt M, Wong VK, Reuter S, Holden MT, et al. Roary: rapid large-scale prokaryote pan genome analysis. Bioinformatics. 2015;31(22):3691–3.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Price MN, Dehal PS, Arkin AP. FastTree 2–approximately maximum-likelihood trees for large alignments. PLoS ONE. 2010;5(3):e9490.

    Article  PubMed  PubMed Central  Google Scholar 

  31. Alikhan NF, Petty NK, Ben Zakour NL, Beatson SA. BLAST Ring Image Generator (BRIG): simple prokaryote genome comparisons. BMC Genomics. 2011;12:402.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Beukers AG, Hasman H, Hegstad K, van Hal SJ. Recommendations to address the difficulties encountered when determining Linezolid Resistance from whole-genome sequencing data. Antimicrob Agents Chemother. 2018;62(8).

  33. Wang Y, Lv Y, Cai J, Schwarz S, Cui L, Hu Z, et al. A novel gene, optrA, that confers transferable resistance to oxazolidinones and phenicols and its presence in Enterococcus faecalis and Enterococcus faecium of human and animal origin. J Antimicrob Chemother. 2015;70(8):2182–90.

    Article  CAS  PubMed  Google Scholar 

  34. Antonelli A, D’Andrea MM, Brenciani A, Galeotti CL, Morroni G, Pollini S, et al. Characterization of poxtA, a novel phenicol–oxazolidinone–tetracycline resistance gene from an MRSA of clinical origin. J Antimicrob Chemother. 2018;73(7):1763–9.

    Article  CAS  PubMed  Google Scholar 

  35. Bakthavatchalam YD, Vasudevan K, Babu P, Neeravi AR, Narasiman V, Veeraraghavan B. Genomic insights of optra-carrying linezolid-resistant Enterococcus faecium using hybrid assembly: first report from India. J Global Antimicrob Resist. 2021;25:331–6.

    Article  CAS  Google Scholar 

  36. Zhang Y, Dong G, Li J, Chen L, Liu H, Bi W, et al. A high incidence and coexistence of multiresistance genes cfr and optrA among linezolid-resistant enterococci isolated from a teaching hospital in Wenzhou, China. Eur J Clin Microbiol Infect Dis. 2018;37:1441–8.

    Article  CAS  PubMed  Google Scholar 

  37. Tang B, Zou C, Schwarz S, Xu C, Hao W, Yan XM, et al. Linezolid-resistant Enterococcus faecalis of Chicken Origin harbored chromosome-borne optrA and plasmid-borne cfr, cfr(D), and poxtA2 genes. Microbiol Spectr. 2023;11(3):e0274122.

    Article  PubMed  Google Scholar 

  38. Mališová L, Jakubů V, Pomorská K, Musílek M, Žemličková H. Spread of linezolid-resistant Enterococcus spp. in human clinical isolates in the Czech Republic. Antibiotics. 2021;10(2):219.

    Article  PubMed  PubMed Central  Google Scholar 

  39. Spiliopoulou I, Damani A, Chini V, Zerva L, Kolonitsiou F, Anastassiou ED, et al. Linezolid-resistant enterococci in Greece: epidemiological characteristics. Chemotherapy. 2011;57(3):181–5.

    Article  CAS  PubMed  Google Scholar 

  40. Krawczyk B, Wysocka M, Kotłowski R, Bronk M, Michalik M, Samet A. Linezolid-resistant Enterococcus faecium strains isolated from one hospital in Poland–commensals or hospital-adapted pathogens? PLoS ONE. 2020;15(5):e0233504.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Wang L, He Y, Xia Y, Wang H, Liang S. Investigation of mechanism and molecular epidemiology of linezolid-resistant Enterococcus faecalis in China. Infect Genet Evol. 2014;26:14–9.

    Article  PubMed  Google Scholar 

  42. Sun W, Liu H, Liu J, Jiang Q, Pan Y, Yang Y, et al. Detection of optrA and poxtA genes in linezolid-resistant Enterococcus isolates from fur animals in China. Lett Appl Microbiol. 2022;75(6):1590–5.

    Article  CAS  PubMed  Google Scholar 

  43. Tang B, Wang Y, Luo Y, Zheng X, Qin X, Yang H et al. Coexistence of optrA and fexA in Campylobacter. mSphere. 2021;6(3).

  44. Cinthi M, Coccitto SN, Fioriti S, Morroni G, Simoni S, Vignaroli C, et al. Occurrence of a plasmid co-carrying cfr (D) and poxtA2 linezolid resistance genes in Enterococcus faecalis and Enterococcus casseliflavus from porcine manure, Italy. J Antimicrob Chemother. 2022;77(3):598–603.

    Article  CAS  PubMed  Google Scholar 

  45. Davis E, Hicks L, Ali I, Salzman E, Wang J, Snitkin E, et al. editors. Epidemiology of vancomycin-resistant Enterococcus faecium and Enterococcus faecalis colonization in nursing facilities. Open Forum Infectious Diseases; 2020: Oxford University Press US.

  46. Chen P-Y, Chuang Y-C, Wang J-T, Sheng W-H, Chen Y-C, Chang S-C. Predictors for Vancomycin resistant Enterococcus faecium transforming from colonization to infection: a case control study. Antimicrob Resist Infect Control. 2019;8(1):1–11.

    Article  PubMed  PubMed Central  Google Scholar 

  47. Schwarz S, Zhang W, Du X-D, Krüger H, Feßler AT, Ma S, et al. Mobile oxazolidinone resistance genes in Gram-positive and Gram-negative bacteria. Clin Microbiol Rev. 2021;34(3). https://doi.org/10.1128/cmr. 00188 – 20.

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Acknowledgements

Safia Moin, Fizza Farooqui and Farheen Ali.

Funding

This work was supported in part by an award from the Centers for Disease Control and Prevention; PTE Federal Award No 6 NU3HCK000007-01-01, Subaward No 60060796 AKU.

Author information

Authors and Affiliations

  1. Aga Khan University Hospital, Karachi, Sindh, Pakistan

    Syed Ali Raza Nasir, Mohammad Zeeshan, Najia Ghanchi, Noureen Saeed, Hassan Ghayas, Sadaf Zaka, Javaria Ashraf, Kauser Jabeen, Joveria Farooqi, Zahra Hasan, Syed Faisal Mahmood, Mehreen Arshad, Erum Khan & Rumina Hasan

  2. National Institute of Cardiovascular Diseases, Karachi, Sindh, Pakistan

    Najia Ghanchi, Tazeen Fatima & Faiza Rezwan

  3. Northwestern University Feinberg School of Medicine, Chicago, IL, USA

    Egon A. Ozer

  4. Microbiology lab, Sopariwala building, Aga Khan University Hospital, Karachi, Pakistan

    Rumina Hasan

Authors
  1. Syed Ali Raza Nasir
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  2. Mohammad Zeeshan
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  3. Najia Ghanchi
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  4. Noureen Saeed
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  5. Hassan Ghayas
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  6. Sadaf Zaka
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  7. Javaria Ashraf
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  8. Kauser Jabeen
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  9. Joveria Farooqi
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  10. Zahra Hasan
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  11. Tazeen Fatima
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  12. Faiza Rezwan
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  13. Syed Faisal Mahmood
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  14. Mehreen Arshad
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  15. Erum Khan
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  16. Egon A. Ozer
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  17. Rumina Hasan
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Contributions

SAR, MZ, EK and RH conceptualized the study. NS, SZ, TF and FR carried out isolate identification and isolation. NG, HG, JA and EO were responsible for the genomic and bioinformatic analysis. SAR, MZ, JF, KJ, ZH, JA, SFM, MA, EO and RH carried out writing of and validation of the different sections of the manuscript. All authors reviewed the manuscript.

Corresponding author

Correspondence to Rumina Hasan.

Ethics declarations

Ethics approval and consent to participate

Since the study was centered around genomic analysis of microbiological isolates and did not involve human participants, exemption from ethical approval was obtained from the Ethics Review Committee, Faculty of Health Sciences at Aga Khan University Hospital (Review reference: 2022-7156-23110), as the patients admitted at Aga Khan University Hospital already provide consent at the time of admission for their medical record to be used for research purposes (Policy: AKUH/PBSD/008). For outpatients, informed consent was taken after approval from the same committee (Review reference: 2023-6798-27300).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

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Nasir, S.A.R., Zeeshan, M., Ghanchi, N. et al. Linezolid-resistant Enterococcus faecium clinical isolates from Pakistan: a genomic analysis. BMC Microbiol 24, 347 (2024). https://doi.org/10.1186/s12866-024-03491-2

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  • DOI: https://doi.org/10.1186/s12866-024-03491-2

Keywords

BMC Microbiology

ISSN: 1471-2180

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