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Detection of Genes Related to Linezolid Resistance (poxtA, cfr, and optrA) in Clinical Isolates of Enterococcus spp. from Humans: A First Report from Iran
Majid Torabi, Jamshid Faghri, Farkhondeh Poursina
Department of Bacteriology and Virology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
| Date of Submission | 25-Feb-2023 |
| Date of Decision | 02-May-2023 |
| Date of Acceptance | 10-May-2023 |
| Date of Web Publication | 31-Jul-2023 |
Correspondence Address:
Farkhondeh Poursina
Department of Bacteriology and Virology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Postal Code: 8174673461
Iran
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/abr.abr_74_23
Background: Enterococci may develop resistance to linezolid through chromosomal mutations that involve specific linezolid resistance genes, such as cfr, optrA, and poxtA. The objective of this study was to evaluate the antibiotic susceptibility of enterococcal isolates and identify cfr, optrA, and poxtA genes in MDR isolates.
Materials and Methods: Enterococcal isolates were collected from various clinical specimens at Al-Zahra, Amin, and Khorshid Hospitals in Isfahan. The Enterococcus isolates were identified as belonging to the E. faecalis and E. faecium species by using specific gene (D alanine D alanine ligase ddl) sets in PCR. To detect cfr, optrA, and poxtA genes among the species, a multiplex-PCR assay was performed.
Results: Out of 175 isolates, E. faecalis predominated 129/175 (73.7%). Furthermore, the prevalence of vancomycin-resistant Enterococci (VRE) and linezolid-resistant Enterococci (LRE) was 29.7% and 4%, respectively. The overall prevalence of MDR was 91.1%, 68.9%, and 66.6% of E. faecium, E. faecalis, and other Enterococcus spp., respectively. Interestingly, the frequency of optrA (71.4%) in E. faecium and poxtA and crf (42.8%) in E. faecalis were detected among LRE species. A statistically significant relationship (P < 0.05) was found between the presence of the three genes and the occurrence of LRE.
Conclusion: This is the first study to report the detection of linezolid resistance genes (cfr, optrA, and poxtA) in clinical Enterococcus spp. isolates from Iran, conducted at Isfahan University of Medical Sciences hospitals. The emergence of enterococcal strains that resist linezolid is concerning as it can lead to the spread of resistant strains among patients, resulting in treatment failure.
Keywords: Enterococcus, drug resistance, humans, linezolid
How to cite this article:
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 Biomed Res 2023;12:205 |
How to cite this URL:
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 Biomed Res [serial online] 2023 [cited 2023 Sep 28];12:205. Available from: https://www.advbiores.net/text.asp?2023/12/1/205/382555 |
| Introduction | |  |
Linezolid is commonly reserved as a final option for treating severe infections caused by multidrug-resistant (MDR) gram-positive organisms, including vancomycin-resistant Enterococcus spp. (VRE), methicillin-resistant Staphylococcus spp., and Streptococcus pneumoniae.[1],[2],[3] While the majority of gram-positive cocci remain susceptible to linezolid, resistant Enterococci isolates have been reported across the globe.[3],[4] Bacteria can develop resistance to linezolid through either chromosomal mutations or the uptake of mobile genetic elements that harbor certain linezolid-resistance genes, such as cfr, optrA, and poxtA.[5],[6],[7],[8]
The cfr gene, which codes for an rRNA methyltransferase, was initially identified on the plasmid pSCFS1 of Staphylococcus sciuri and has since been detected in a range of gram-positive and gram-negative bacteria from different sources.[9],[10] The cfr gene confers resistance to a group of antibiotics known as PhLOPSA, which includes phenicols, lincosamides, pleuromutilins, streptogramin A, and oxazolidinones. In addition, clinical enterococcal isolates have been shown to exhibit cfr gene variations [cfr(B) and cfr(D)].[11],[12],[13] However, the role of cfr-like genes in reducing linezolid susceptibility in Enterococci is still debated. OptrA is an ATP-binding cassette F (ABC-F) protein that gives resistance to phenicols and oxazolidinones. The optrA gene was initially identified among Enterococcus faecalis (Efa) and Enterococcus faecium (Efm) isolates from both animals and humans in China.[14],[15],[16] poxtA is a recently discovered gene that provides resistance to oxazolidinones, phenicols, and tetracycline. It was initially identified in a clinical strain of methicillin-resistant Staphylococcus aureus. This gene, like optrA, encodes antibiotic resistance (ARE) ABC-F protein; however, it only shares 32 percent of optrA's amino acid sequence. poxtA is also found in efa and efm, and it is frequently found in a composite transposon.[16],[17]
Due to this emerging problem, an accurate and thorough assessment of the rate and magnitude of linezolid resistance between clinical isolates is required.[18],[19],[20],[21] The objective of this study was to evaluate the antibiotic susceptibility patterns of clinical MDR Enterococcus isolates in several teaching hospitals affiliated with Isfahan University of Medical Sciences and to investigate the emergence of linezolid resistance as well as the prevalence of cfr, optrA, and poxtA genes in these isolates.
| Materials and Methods | | |
Clinical samples
Between May 2020 and December 2021, a total of 175 Enterococcal isolates were collected from various clinical specimens, including urine, blood, body fluids, wounds, and catheters, at three hospitals located in Isfahan, a central province in Iran. The hospitals included Al-Zahra, Amin, and Khorshid. The isolates were confirmed as non-duplicate strains to ensure that each isolate was unique and representative of a different infection episode.
Enterococcus species identification
For bacterial identification, standard biochemical tests were utilized, including assessments of catalase activity, growth in 6.5% sodium chloride, bile esculin hydrolysis, and pyrrolidinyl aminopeptidase activity.[22] The ddl gene was detected using polymerase chain reaction (PCR) to confirm the Enterococcus species.[23]
Antibiotic susceptibility testing
As described in the Clinical and Laboratory Standards Institute Guidelines (CLSI-2022), all isolates of enterococci were tested for antimicrobial agents (PadtanTEB Co., IR) in Muller-Hinton agar (IBRESCO Co., IR) using Kirby-Bauer disk diffusion methods: ampicillin (AMP, 10 μg), vancomycin (VAN, 30 μg), teicoplanin (TEC, 30 μg), ciprofloxacin (CIP, 5 μg), nitrofurantoin (NIT, 300 μg), chloramphenicol (CHL, 30 μg), gentamicin (GEN, 120 μg), rifampin (RIF, 5 μg), erythromycin (ERY, 15 μg), teicoplanin (TEC, 30 μg), linezolid (LZD, 30 μg) and fosfomycin (FOS, 200 μg). E. faecalis ATCC 29,212 was used as the control strain. A brain-heart infusion (BHI) screening agar with vancomycin (6 mg/mL) was used to retest bacteria that showed intermediate or resistant responses to vancomycin disk in Kirby-Bauer.[22] After that, WHONET 2021 software (https://whonet.org/software.html) was utilized to determine the MDR of each strain.[24]
PCR detection
To confirm the two species E. faecalis and E. faecium, amplification of ddl gene was performed.[23] For the detection of cfr, optrA, and poxtA genes, a multiplex-PCR method as described by K. Bender et al.[25] was employed in this study. The expected fragment sizes for optrA, poxtA, and cfr were 422 bp, 533 bp, and 746 bp, respectively.
Sequencing of PCR products
The PCR products of samples that showed bands for the mentioned ranges (for optrA, the expected fragment size was 422 bp, for poxtA it was 533 bp, and for cfr it was 746 bp) was sequenced and checked with a database PubMLST (https://pubmlst.org/), and then these positive genes were used as control of gene amplification.
Statistical analysis
The statistical analyses for this study were conducted using SPSS v26 software. A P value less than 0.05 was considered significant. The data were analyzed using both Chi-square and Fisher's exact tests to determine statistical significance.
| Results | | |
Identification of Enterococcal isolates
To identify the Enterococcal isolates, both phenotypic and PCR methods were employed for E. faecalis and E. faecium with a high level of agreement. Out of 175 clinical Enterococcal isolates, the majority (73.7%) were identified as E. faecalis, while 19.4% were identified as E. faecium. The remaining 6.8% of the isolates were classified as other species of Enterococcus.
Antimicrobial susceptibility results
A summary of the ARE profiles found among all isolates using the disk diffusion method can be found in [Table 1].
According to our data, the Enterococcal isolates exhibited varying levels of ARE. Erythromycin had the highest rate of resistance at 84.6%, while Fosfomycin had the lowest rate at 0.6%. The prevalence of vancomycin-resistant Enterococci was 29.7%, and 52/175 (29.7%) and 7/175 (4%)., Further details regarding the resistance patterns of different Enterococcus species are provided in [Table 1]. MDR was observed in a significant number of Enterococcal isolates. Specifically, the prevalence of MDR was found to be 91.1% (31/34) in E. faecium, 68.9% (89/129) in E. faecalis, and 66.6% (8/12) in other Enterococcus species, respectively. This indicates that these bacteria have developed resistance to multiple antibiotics.
Prevalence of optrA, cfr, and poxtA genes among Enterococcus strains
The prevalence of optrA, cfr, and poxtA genes among the total MDR isolates was 5.4% (7/128), 3.1% (4/128), and 2.3% (3/128), respectively. There was no significantly correlation between the prevalence of resistance genes (optrA, cfr, and poxtA) and MDR isolates (P > 0.05) [Table 2].
Among the Enterococcal isolates that we tested, we identified several strains that contained multiple ARE genes. Specifically, two isolates (1 E. faecalis and 1 E. faecium) were found to harbor all three resistance genes (optrA, cfr, and poxtA), while two other isolates (1 E. faecalis and 1 E. faecium) carried two resistance genes (optrA and poxtA). Additionally, three strains (2 E. faecalis and 1 E. faecium) carried a single resistance gene (optrA), and one strain (other species of Enterococcus) was found to harbor a single resistance gene (cfr).
| Discussion | | |
Linezolid, which is one of the final options for treating MDR gram-positive pathogens, stands out due to its distinctive mechanism of action and relatively low global resistance rates.[9] Additionally, it boasts a near-perfect bioavailability when taken orally and has an excellent safety record. Unfortunately, the combination of these factors has led to excessive and widespread use of linezolid, ultimately resulting in the development of resistance to this antibiotic.[9],[26]
Our study's findings corroborate previous Iranian research that identified E. faecalis as the predominant clinical Enterococcus species. Additionally, our results support the notion that E. faecium exhibits greater multidrug ARE compared to other Enterococcus species.[22],[23],[27],[28],[29],[30]
Despite the lack of a reliable report on the prevalence of LRE in Iran, our findings indicate that MDR strains had a 4.5% rate of LRE, while intermediate strains had a rate of 3.9%. In contrast, Jahansepas et al.[22] reported 8.7% prevalence of LRE in their study conducted in northern Iran. Contrary to our study, no LRE strain was shown in published studies from south-western Iran in 2017.[31],[32] It seems that the differences in the prevalence of resistance is related to the geographical and the treatment strategy in different areas.
As far as we know, this is the initial report on the occurrence of cfr, optrA, and poxtA linezolid resistance genes in human clinical Enterococcus spp. isolates in Iran. Our study revealed a high prevalence of the optrA and poxtA genes in LREs. We observed a significant association (P < 0.05) between the presence of these genes and resistance to linezolid. These results suggest that optrA and poxtA may play a key role in the development of linezolid resistance in Enterococci and highlight the need for continued surveillance and investigation into the mechanisms underlying ARE in these bacteria. Another study conducted in Pakistan and the USA also reported high prevalence rates of poxtA and optrA among LREs.[33] Similarly, Zaira Moure et al.[34] from Spain reported an optrA frequency of 85.2% among LREs. In various studies, the relationship of these genes with LRE strains has been reported.[5],[6],[7],[8]
According to our research, a significant proportion of LREs had multiple ARE genes. Specifically, in some LREs (28.5%) was detected all three genes (optrA, cfr, and poxtA) at the same time, while others (28.5%) had two genes (optrA and poxtA) simultaneously. Additionally, in a large portion of LREs (42.8%), there was only optrA gene. Interestingly, these results are very similar to those found in a study conducted by Zaira Moure et al.[34] in Spain.
While the prevalence of LRE strains may be relatively low, the identification of even a single isolate carrying the optrA, poxtA, or cfr resistance genes in Enterococcus spp. is a cause for concern. Such findings underscore the potential for these genes to spread across clinical and nonclinical settings, as well as among different species. Therefore, continued surveillance and implementation of effective infection control measures are crucial in preventing the dissemination of antibiotic-resistant Enterococci.[35],[36],[37],[38]
As a conclusion, the prevalence of antibiotic resistance is a growing concern worldwide, and Enterococci strains among Iranian patients are no exception. These strains have been found to carry and spread resistance genes, such as optrA, poxtA, and cfr, leading to the emergence of last-resort ARE strains. To address this issue, active surveillance is recommended. This includes identifying the different epidemiological contexts in which linezolid resistance develops, evaluating ARE in Enterococcus spp., and implementing antibiotic stewardship of linezolid to prevent the spread of resistance. Additionally, it is important to study the prevalence of these resistance genes in other bacterial strains and environments to better understand the scope of this issue. Implementation of effective infection control measures is essential in combating the spread of ARE and preserving the efficacy of our current treatments. By taking such measures, we can work toward mitigating the impact of ARE and ensuring that effective treatments remain available for patients in need.
Acknowledgments
Medical University of Isfahan, Iran.
Financial support and sponsorship
The study was granted by the Medical University of Isfahan, Iran (Council of Medical Science, grant No. 399097).
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2]
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