
==== Front
BMC Microbiol
BMC Microbiol
BMC Microbiology
1471-2180
BioMed Central London

3491
10.1186/s12866-024-03491-2
Research
Linezolid-resistant Enterococcus faecium clinical isolates from Pakistan: a genomic analysis
Nasir Syed Ali Raza 1
Zeeshan Mohammad 1
Ghanchi Najia 12
Saeed Noureen 1
Ghayas Hassan 1
Zaka Sadaf 1
Ashraf Javaria 1
Jabeen Kauser 1
Farooqi Joveria 1
Hasan Zahra 1
Fatima Tazeen 2
Rezwan Faiza 2
Mahmood Syed Faisal 1
Arshad Mehreen 1
Khan Erum 1
Ozer Egon A. 3
Hasan Rumina rumina.hasan@aku.edu

14
1 https://ror.org/05xcx0k58 grid.411190.c 0000 0004 0606 972X Aga Khan University Hospital, Karachi, Sindh Pakistan
2 https://ror.org/0425pdj49 grid.419561.e 0000 0004 0397 154X National Institute of Cardiovascular Diseases, Karachi, Sindh Pakistan
3 grid.16753.36 0000 0001 2299 3507 Northwestern University Feinberg School of Medicine, Chicago, IL USA
4 https://ror.org/05xcx0k58 grid.411190.c 0000 0004 0606 972X Microbiology lab, Sopariwala building, Aga Khan University Hospital, Karachi, Pakistan
14 9 2024
14 9 2024
2024
24 34718 3 2024
2 9 2024
© The Author(s) 2024
2024
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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.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12866-024-03491-2.

Keywords

Linezolid resistant Enterococcus
Pakistan
Mechanism of resistance
Whole genome sequencing
issue-copyright-statement© BioMed Central Ltd., part of Springer Nature 2024
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pmcBackground

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 [5–7].

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 [12–14]. 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 [19–21].

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

SeqID	Specimen type	MLST	23S:
G2576T mutation	Acquired resistance genes	LNZ	VAN	AMP	TE	TGC	CN	F	FOT	CIP	LEV	C	E	
optrA	poxtA	cfr	
EFPK_01	Blood	612	+	+	-	-	R	S	R	S	S	S	S	NT	NT	NT	R	R	
EFPK_02	Urine	80	+	+	-	-	R	R	R	S	S	NT	I	R	R	R	NT	R	
EFPK_03	CVP tip	80	+	+	-	-	R	R	R	S	S	NT	S	NT	NT	NT	R	R	
EFPK_04	Blood	non-typeable	+	+	-	-	R	R	R	S	S	R	I	NT	NT	NT	R	R	
EFPK_05	Urine	80	-	-	+	+	R	R	R	S	S	NT	I	R	R	R	NT	R	
EFPK_06	Urine	80	-	-	+	+	R	R	R	S	S	NT	R	R	R	R	NT	S	
EFPK_07	Blood	80	+	+	-	-	R	R	R	S	S	R	I	NT	NT	R	R	R	
EFPK_08	Urine	80	-	+	+	+	R	R	R	S	NT	NT	R	R	R	NT	NT	R	
EFPK_09	Blood	80	-	-	+	+	R	R	R	R	S	R	I	NT	NT	R	R	R	
EFPK_10	Blood	80	-	-	+	+	R	R	R	S	S	R	R	NT	NT	R	I	R	
EFPK_11	Blood	80	-	-	+	+	R	R	R	S	S	NT	NT	NT	NT	NT	NT	NT	
EFPK_12	Blood	1380	+	+	-	-	R	R	R	R	S	R	I	NT	NT	R	R	R	
EFPK_14	Blood	80	-	-	+	+	R	R	R	R	S	R	R	NT	NT	NT	S	S	
EFPK_13	Urine	80	+	-	-	-	S	R	R	R	NT	NT	S	S	R	NT	NT	NT	
EFPK_15	Urine	80	-	-	-	-	S	S	S	R	NT	NT	S	R	R	NT	NT	NT	
(+) detected, (-) not-detected, R = resistant, S = susceptible, I = intermediate, AMP = ampicillin, C = chloramphenicol, CN = gentamicin, CIP = ciprofloxacin E = erythromycin, F = nitrofurantoin, FOT = fosfomycin, LEV = levofloxacin, LNZ = linezolid, TE = tetracycline, TGC = tigecycline, VAN = vancomycin, MLST = multilocus sequence type, NT = not tested

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 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)

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

Antibiotics	ARGs	Mechanisms of resistance	EFPK_1	EFPK_2	EFPK_3	EFPK_4	EFPK_5	EFPK_6	EFPK_7	EFPK_8	EFPK_9	EFPK_10	EFPK_11	EFPK_12	EFPK_13	EFPK_14	EFPK_15	
Vancomycin, Teicoplanin	vanHAX	Coding for structural enzymes - for cell wall modelling	○	●	●	●	●	●	●	●	●	●	●	●	●	●	○	
Amikacin,

Tobramycin

	aac(6)-Ii	Aminoglycoside-acetyltransferase	●	●	●	●	●	●	●	●	●	●	●	●	●	●	●	
aph(2)	Aminoglycoside phosphotransferase (HLGR)	○	●	○	●	●	○	●	●	●	●	●	●	●	●	○	
aph(3)-III	Aminoglycoside phosphotransferase (HLKR)	○	○	○	○	●	○	○	●	○	●	●	●	●	○	●	
Streptomycin	ant(6)-Ia	Aminoglycoside nucleotidyltransferase (HLAR)	○	○	○	○	●	○	○	●	●	●	●	●	●	○	●	
Erythromycin	erm(A)	Erythromycin ribosome methylase – methylate nucleotide in peptidyl transferase − 23S rRNA	●	●	●	●	○	○	●	●	●	○	○	●	○	○	○	
erm(B)	○	●	●	○	●	○	●	●	○	●	●	○	●	○	●	
msr(C)	ABC-F subfamily-active efflux of macrolide	●	●	●	●	●	●	●	●	●	●	●	●	●	●	●	
Linezolid	optrA	ABC transport – active efflux	●	●	●	●	○	○	●	●	○	○	○	●	○	○	○	
poxtA	ABC transport – active efflux	○	○	○	○	●	●	○	●	●	●	●	○	○	●	○	
cfr(D)	rRNA methyltransferase – methylate nucleotide A2503	○	○	○	○	●	●	○	●	●	●	●	○	○	●	○	
Tetracycline, Doxycycline.	tet(L)	Tetracycline efflux protein	○	○	○	●	○	○	○	○	●	○	○	●	●	●	○	
Trimethoprim	dfrG	Mutation of dihydrofolate reductase	○	○	○	○	○	○	○	●	○	○	○	○	●	○	○	
●=gene detected, ○=gene not detected, ABC = ATP binding cassette, ARG = antibiotic resistance gene, HLAR = high-level aminoglycoside resistance, HLGR = high-level gentamicin resistance, HLKR = high level kanamycin resistance

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 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/)

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, 38–40]. 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 [41–43]. 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.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1

Acknowledgements

Safia Moin, Fizza Farooqui and Farheen Ali.

Author 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.

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.

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)

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.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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