Characterization of linezolid- and methicillin-resistant coagulase-negative staphylococci in a tertiary hospital in China

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It is very important to understand the resistance mechanisms, molecular epidemiology and probable transmission of linezolid-resistant CoNS in the hospital. Methods The antimicrobial susceptibilities of all the isolates were determined by the microdilution method. The resistance mechanisms and molecular characteristics of the strains were detected using whole-genome sequencing and PCR. Results All the strains were resistant to oxacillin and carried the mecA gene; 13 patients (36.1%) had received prior linezolid exposure. The majority of the S. epidermidis and S. hominis isolates were ST22 and ST1, respectively. MLST typing and evolutionary analysis indicated that the majority of linezolid-resistant CoNS were genetically related. This study revealed that distinct CoNS strains have different linezolid resistance mechanisms. Among ST22-type S. epidermidis , the acquisition of the T2504A and C2534T mutations in the V domain of the 23S rRNA gene as well as the mutations in the ribosomal proteins L3 (L101V, G152D, and D159Y) and L4 (N158S) were linked to the development of linezolid resistance. In the S. cohnii isolates, the cfr , S158Y and D159Y mutations in the ribosomal protein L3 were found. Additionally, the emergence of the G2576T mutation and the cfr gene were major causes of linezolid resistance in S. hominis isolates. The cfr gene, the G2576T and C2104T mutations, the M156T change in L3, and the I188S change in the L4 protein were found in S. capiti s isolates. Conclusion The emergence of linezolid-resistant CoNS in our environment is concerning because it involves clonal dissemination and frequently coexists with various drug resistance mechanisms. coagulase-negative staphylococci linezolid-resistant G2576T mutation cfr gene Figures Figure 1 Background Coagulase-negative staphylococci (CoNS) have become important pathogens in patients with healthcare-associated infections caused by indwelling medical devices or immunocompromised patients [ 1 , 2 ]. According to the monitoring data of the China Antimicrobial Resistance Surveillance System (CARSS, www.carss.cn/ ), the frequency of methicillin-resistant coagulase-negative staphylococci (MRCoNS) was extremely high, and the prevalence of MRCoNS was 74.5% in 2021. Linezolid is an oxazolidinone antibiotic that is a last-resort antibiotic for the treatment of serious infections caused by gram-positive bacteria, including resistant organisms, such as methicillin-resistant staphylococci and vancomycin-resistant enterococci [ 3 , 4 ]. The first clinical isolates of linezolid-resistant staphylococci and enterococci were reported in 2001 [ 5 , 6 ]. Since then, linezolid-resistant organisms have been sporadically reported worldwide [ 7 – 10 ]. However, the detection rate of linezolid-resistant coagulase-negative staphylococci has progressively increased in recent years in China. Point mutation in the domain V region of the 23S rRNA gene was the most common mechanism of linezolid resistance, and the most frequent mutation was G2576T. In addition, T2500A, T2604C, C2532T, C2551T, G2603T, G2614T, C2190T, and G2447T have been reported [ 11 – 13 ]. Furthermore, other mechanisms involved in linezolid resistance in staphylococci have also been reported, such as the presence of the cfr gene, which encodes an rRNA methyltransferase, and mutations in the ribosomal proteins L3, L4 and L22 [ 14 – 16 ]. In China, the prevalence of linezolid-resistant staphylococci has gradually increased in recent years; however, very few studies have investigated linezolid resistance. In this study, the clinical characteristics, antibiotic susceptibility, and resistance mechanisms of linezolid-resistant MRCoNS isolates that were recovered from 2019 to 2023 in a Chinese tertiary hospital were examined. Materials and Methods Bacterial isolates Thirty-seven linezolid-resistant MRCoNS isolates, including thirteen Staphylococcus capitis , nine Staphylococcus hominis , eight Staphylococcus epidermidis , six Staphylococcus cohnii and one Staphylococcus haemolyticus , were collected from December 2019 to March 2023 in a large tertiary teaching hospital (The First Affiliated Hospital of Zhengzhou University, Zhengzhou, with 7,000 beds located in east-central China). Two strains were isolated from pleural fluid, two from catheter tips, one from abdominal dropsy fluid, and one from cerebrospinal fluid; the remaining strains were recovered from blood cultures. Thirty-seven linezolid-resistant MRCoNS isolates were isolated from 36 patients, 25 of whom were male and 11 of whom were female. Linezolid-resistant S. epidermidis -5 and S. capitis -33 were isolated from the same patient, a 76-year-old man who was admitted to the respiratory intensive care unit. In addition, all patients were administered antibiotics, and 13 patients (36.1%) had received prior linezolid treatment. The organism characteristics and associated clinical data are listed in Table 1. Identification of the organisms was carried out using a VITEK®2 Compact system and VITEK® MS (bioMérieux, Marcy-l’Étoile, France). Antimicrobial susceptibility testing The antimicrobial susceptibilities of all the isolates were determined using the microdilution method. S. aureus ATCC 29213 was used for quality control for susceptibility testing. The breakpoint for tigecycline treatment was interpreted according to the European Committee on Antimicrobial Susceptibility Testing Guidelines ( http://www.eucast.org/clinical_breakpoints/ ). The results of susceptibility testing for the other antimicrobial agents were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) M100-S33 breakpoints. Genomic DNA extraction and whole-genome sequencing Genomic DNA was extracted using the Puregene Yeast/Bacteria Kit (QIAGEN) according to the manufacturer’s instructions for gram-positive bacteria. Whole-genome sequencing (WGS) was performed using the Illumina HiSeq PE150 platform (Novogene Bioinformatics Technology Co., Ltd., Beijing, China). Acquired antibiotic resistance genes (ARGs) carried by the isolates were analyzed using KmerResistance v 2.2[ 17 ] with raw reads. The results are reported for ≥ 90% nucleotide identity, ≥ 90% coverage of the query, and a sequence depth of ≥ 10×. The raw reads were assembled into scaffolds using SPAdes v 3.13.1[ 18 ]. The multilocus sequence typing (MLST) of the isolates was determined using MLST 2.0 ( https://cge.food.dtu.dk/services/MLST/ ) and further validated with PubMLST ( https://pubmlst.org ) for S. epidermidis , S. hominis , and S. hemolyticus . Additionally, the phylogenetic tree of 37 linezolid-resistant MRCoNS isolates was analyzed at http://bacdb.cn/BacWGSTdb/analysis_multiple.php . Molecular detection of resistance genes and mutations Domain V of the 23S rRNA gene and cfr gene was amplified and sequenced as previously described (19, 20), and the rplC , rplD , and rplV genes, which encode the ribosomal proteins L3, L4 and L22, were tested using previously described conditions and primers (10, 21). Results Antimicrobial susceptibility All 37 isolates displayed varying degrees of resistance to penicillin, oxacillin, and linezolid, and the mecA gene was detected in all 37 isolates. These iaolates were all resistant to chloramphenicol, levofloxacin, ciprofloxacin, clindamycin, erythromycin, and gentamicin, with resistance rates of 94.6% (35/37), 89.2% (33/37), 89.2% (33/37), 78.4% (29/37), 67.6% (25/37), and 54.1% (20/37), respectively. Interestingly, only 1 of the 8 S. epidermidis isolates was resistant to erythromycin; however, all thirteen S. capitis strains were resistant to erythromycin. The resistance rates of the linezolid-resistant MRCoNS strains to trimethoprim/sulfamethoxazole, tetracycline and rifampicin were 27.0% (10/37), 16.2% (6/37), and 10.8% (4/37), respectively; however, all six S. cohnii isolates were sensitive to trimethoprim/sulfamethoxazole. No resistance to tigecycline, vancomycin or teicoplanin was observed (Table 2). Molecular characteristics and the mechanisms of linezolid resistance Seven of the eight S. epidermidis isolates belonged to the same clone, ST22. Two point mutations (T2504A and C2534T) in domain V of the 23S rRNA gene were discovered, and seven additional isolates also exhibited L101V, G152D, and D159Y changes in their deduced amino acid sequences of the L3 protein and N158S changes in the L4 protein. No cfr genes were detected in any of the isolates. Another S. epidermidis isolate was of the ST2 type, with positive carriage of the cfr gene and no point mutations in domain V of the 23S rRNA gene (Table 3). Six S. cohnii isolates carried the cfr gene, and five of these isolates were found to have S158Y and D159Y changes in the L3 protein. No mutation in domain V of the 23S rRNA gene was detected in any of the S. cohnii isolates. The nine S. hominis isolates were divided into 3 distinct clones: ST1 (n = 5), ST2 (n = 2) and ST85 (n = 2). The novel ST type (ST85) was found for the first time in this study. The cfr gene was identified among the six S. hominis isolates, and the other three isolates had the 23S rRNA G2576T mutation. In the present study, all the S. capitis isolates were found to belong to the same clone by phylogenetic analysis (Fig. 1 ), and the G2576T and C2104T 23S rRNA mutations were identified. Additionally, 8 isolates (61.5%) harbored the cfr gene. Furthermore, M156T, V154L, and I188S changes were identified in the L3 and L4 proteins. S. haemolyticus -37 harbored the cfr gene and had an additional R138V change in L3. A mutation in the L22 protein was not found in any of the linezolid-resistant MRCoNS isolates in this study. In addition to linezolid-related resistance genes, other resistance genes were also detected in our study, and the mecA gene was detected in all linezolid-resistant MRCoNS (Table 3). Discussion In the present study, 37 linezolid-resistant MRCoNS isolates were obtained from a large tertiary teaching hospital from December 2019 to March 2023. In our setting, linezolid-resistant MRCoNS were first found in 2016; however, the detection rate of linezolid-resistant MRCoNS has steadily increased in recent years. Linezolid is an important alternative for the management of MRCoNS infections. The rapid emergence of linezolid-resistant MRCoNS is alarming and requires ongoing surveillance. Previous studies have indicated that linezolid administration is a significant risk factor for linezolid-resistant gram-positive cocci during hospital outbreaks [ 22 , 23 ]. Our data showed that 13 patients (36.1%) had received prior linezolid therapy; thus, we speculated that increasing selective pressure most likely contributed to drug resistance. All 37 isolates were resistant to multiple antibiotics, and various resistance genes were detected, which indicated that the treatment options were limited. Multilocus sequence typing indicated that ST22 was the dominant clone among the S. epidermidis isolates in the present study. Our research result was consistent with the S. epidermidis lineage in Greece and Spain [ 24 , 25 ] and different from the findings of German and France (ST2) [ 26 , 27 ]. ST2, ST5, and ST22 are clustered into the CC5 clone, which is the most prevalent clonal complex among the nosocomial S. epidermidis population according to the literature[ 28 ]. Although linezolid-resistant CoNS have been reported sporadically worldwide, S. hominis infections caused by linezolid are uncommon. Nine S. hominis isolates were isolated in the present study; these strains belonged to 3 distinct clones: ST1 was the predominant clone, and ST85, a novel ST type, was found for the first time in our study. MLST typing indicated that the bulk of linezolid-resistant CoNS were genetically related, which suggested the transmission of resistant clones from patient to patient and a clonal spread within the intensive care unit in our hospital. The primary causes of linezolid resistance among staphylococci include modification of the target site of 23S rRNA, acquisition of the cfr gene, and mutation of the ribosomal proteins L3 and L4 [ 11 – 16 ]. The current study revealed that linezolid resistance was associated with the acquisition of the T2504A and C2534T mutations as well as with the acquisition of the ribosomal proteins L3 and L4 among S. epidermidis isolates. The cfr gene was not detected among ST22-type S. epidermidis. Interestingly, the cfr gene was found in ST2-type S. epidermidis , and the research findings indicated that distinct clone strains had diverse mechanisms for linezolid resistance. Additionally, this study revealed L101V, G152D, and D159Y changes in the L3 protein and N158S changes in the L4 protein for the first time in S. epidermidis. G2576T was the most frequently detected mutation in domain V of the 23S rRNA gene. In addition to the G2576T mutation, the C2104T mutation was also detected in thirteen S. capitis isolates, and similar results have also been reported in previous studies [ 29 , 30 ]. Notably, the M156T mutation in the L3 protein and the I188S mutation in the L4 protein were also discovered for the first time. Furthermore, the G2576T mutation was also present among the three ST1-type S. hominis isolates, and no mutation in domain V of the 23S rRNA gene was detected in S. cohnii or S. haemolyticus . The cfr gene was detected in one S. epidermidis , one S. haemolyticus , five S. cohnii , six S. hominis and eight S. capitis strains . In the present study, the main mechanisms of linezolid resistance among S. cohnii isolates were the presence of the cfr gene and the acquisition of the S158Y and D159Y mutations in the ribosomal protein L3. Interestingly, our data demonstrated that the mechanism of linezolid resistance in S. epidermidis and S. capitis was complex, and the linezolid MIC was greater than that in S. cohnii and S. hominis . In conclusion, there has been an increase in the prevalence of linezolid resistance among CoNS in our hospital's intensive care units in recent years. Additionally, many of the isolates were clonally related, suggesting the intrahospital dissemination of resistant clones. Resistance was related to the presence of the cfr gene, a point mutation in the V domain of the 23S rRNA gene and/or a mutation in the ribosomal L3 and L4 proteins, and multiple drug resistance mechanisms often coexisted. Notably, distinct CoNS have different linezolid resistance mechanisms. Taken together, the findings on the spread of linezolid-resistant CoNS in our setting highlight the importance of monitoring linezolid resistance in MRCoNS. Strict control measures should be taken to prevent further dissemination, and the relevant use of antibiotics needs to be further emphasized. Abbreviations CoNS coagulase-negative staphylococci MRCoNS methicillin-resistant coagulase-negative Staphylococci MLST Multilocus sequence typing CARSS China Antimicrobial Resistance Surveillance System CLSI Clinical and Laboratory Standards Institute WGS Whole genome sequencing Declarations Acknowledgments None. Authors’ contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Cailin Liu, Jing Yu, Chunguang Chen, Xiaogai Li, Yafei Ye, Yani Dong, Xinxin Ying, Haijun Li,Wanhai Wang. The first draft of the manuscript was written by Cailin Liu, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscrip. Funding This work was supported by grants from the National Natural Science Foundation of China (Project Number: 82102438). Availability of data and materials The sequence data generated in this study have been submitted to the GenBank nucleotide sequence database (https://www.ncbi.nlm.nih.gov/) under accession numbers SUB14257732. Ethical approval The study protocol was approved by the First Affiliated Hospital of Zhengzhou University Ethics Committee for Research in Health. The First Affiliated Hospital of Zhengzhou University Ethics Committee also approved the waiver of informed consent to participate in this study. All patient data were anonymised prior to analysis(2020-KY-173). Consent for publication Not applicable Conflict of interest The authors declare no conflicts of interest. Author details 1 Department of Clinical Laboratory, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China 2 Key Clinical Laboratory of Henan Province, Zhengzhou, China 3 Department of Clinical Laboratory, The Third Affiliated Hospital of Zhengzhou University, Zhengzhou, China 4 Department of Clinical Laboratory, The Sixth People’s Hospital of Zhengzhou City, Zhengzhou, China 5 Department of Clinical Laboratory, Yichuan People's Hospital, Zhengzhou, China 6 Department of Clinical Laboratory, Luohe Sixth People's Hospital, Zhengzhou, China References Decousser, J.W.; Desroches, M.; Bourgeois-Nicolaos, N.; Potier, J.; Jehl, F.; Lina, G., et al . Susceptibility trends including emergence of linezolid resistance among coagulase-negative staphylococci and meticillin-resistant Staphylococcus aureus from invasive infections. Int. J. Antimicrob. Agents 2015, 46(6), 622-630. http://dx.doi.org/10.1016/j.ijantimicag.2015.07.022. Angela França. 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Tables Tables 1 to 3 are available in the Supplementary Files section Additional Declarations No competing interests reported. Supplementary Files Table1.xlsx Table 1Clinical characteristics of patients with linezolid-resistant MRCoNS Table2.xlsx Table 2 Antimicrobial susceptibility (μg/ml) of the linezolid-resistant MRCoNS Table3.xlsx Table 3Characteristics and drug resistance mechanisms of linezolid-resistant MRCoNS Cite Share Download PDF Status: Published Journal Publication published 10 May, 2024 Read the published version in BMC Infectious Diseases → Version 1 posted Editorial decision: Revision requested 26 Mar, 2024 Reviews received at journal 06 Mar, 2024 Reviews received at journal 25 Feb, 2024 Reviewers agreed at journal 20 Feb, 2024 Reviewers agreed at journal 20 Feb, 2024 Reviewers agreed at journal 20 Feb, 2024 Reviewers invited by journal 20 Feb, 2024 Editor assigned by journal 20 Feb, 2024 Editor invited by journal 20 Feb, 2024 Submission checks completed at journal 20 Feb, 2024 First submitted to journal 04 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAz0lEQVRIiWNgGAWjYFACxsYDCQz/5NiYmQ8c+PCDOC0NQC0HjPnZ2xIPzuwh0p4DQJQ4s+eM8WEONiKU87cfbjjwcMcdxg03cj4cZuBhkOcXO4Bfi8SZxIYDiWeeMRvcyN1wuMCCwXDm7AT8WgwYQFramNnAWmbwMCQY3Cakhf8hWAuPwY2cB4d52IjRIgG25bCEZM8ZBuK0SNwA25JmAAxkA2AgSxD2C39/+sOHP9ts6tuYmR9/+PDDRp5fmoAWDFtJUz4KRsEoGAWjADsAAJ3tT7Z7HfQQAAAAAElFTkSuQmCC","orcid":"","institution":"The First Affiliated Hospital of Zhengzhou University","correspondingAuthor":true,"prefix":"","firstName":"Cailin","middleName":"","lastName":"Liu","suffix":""},{"id":274021041,"identity":"f3d240bb-d9fd-4b34-bbf6-98d88db26a58","order_by":1,"name":"Jing Yu","email":"","orcid":"","institution":"The Third Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Yu","suffix":""},{"id":274021042,"identity":"49d7a957-7729-4f13-a047-82f5120ff473","order_by":2,"name":"Chunguang Chen","email":"","orcid":"","institution":"The Sixth People’s Hospital of Zhengzhou City","correspondingAuthor":false,"prefix":"","firstName":"Chunguang","middleName":"","lastName":"Chen","suffix":""},{"id":274021043,"identity":"b5caab80-df67-4c23-8299-e1b191f8866c","order_by":3,"name":"Xiaogai Li","email":"","orcid":"","institution":"The First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Xiaogai","middleName":"","lastName":"Li","suffix":""},{"id":274021044,"identity":"69651457-7e97-4d30-8431-3d0ca6494378","order_by":4,"name":"Yafei Ye","email":"","orcid":"","institution":"The First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Yafei","middleName":"","lastName":"Ye","suffix":""},{"id":274021045,"identity":"99cdd338-4eeb-4316-8612-f14e262152df","order_by":5,"name":"Yani Dong","email":"","orcid":"","institution":"Yichuan People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yani","middleName":"","lastName":"Dong","suffix":""},{"id":274021046,"identity":"82c01162-5c00-4ad7-950f-dc633c0071f9","order_by":6,"name":"Xinxin Ying","email":"","orcid":"","institution":"Luohe Sixth People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xinxin","middleName":"","lastName":"Ying","suffix":""},{"id":274021047,"identity":"bb4eaabf-1605-4642-908b-a72e32e9796d","order_by":7,"name":"Haijun Li","email":"","orcid":"","institution":"The First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Haijun","middleName":"","lastName":"Li","suffix":""},{"id":274021048,"identity":"c6f08975-630b-4daf-a85b-115bed0a1550","order_by":8,"name":"Wanhai Wang","email":"","orcid":"","institution":"The First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Wanhai","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2024-02-04 14:59:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3927977/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3927977/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12879-024-09376-z","type":"published","date":"2024-05-10T21:17:44+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":51507656,"identity":"fcbb7650-8456-495c-907f-1171dba28382","added_by":"auto","created_at":"2024-02-22 19:41:28","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":277761,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree (based on the SNP strategy) analysis of the linezolid-resistant MRCoNS\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3927977/v1/ba8acbbafe9e3521efa6d386.jpg"},{"id":56488110,"identity":"54fdadab-d314-4dd3-b87e-d8da38fa5d29","added_by":"auto","created_at":"2024-05-14 21:29:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":544766,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3927977/v1/2f98d042-a5d0-4997-9887-7022933640d3.pdf"},{"id":51507654,"identity":"e6ae7613-1f7a-4ba4-81b0-4f1c9d1624b2","added_by":"auto","created_at":"2024-02-22 19:41:27","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":21669,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003eClinical characteristics of patients with linezolid-resistant MRCoNS\u003c/p\u003e","description":"","filename":"Table1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3927977/v1/b972a16fe162d86ba20f0f2f.xlsx"},{"id":51507657,"identity":"4b517808-411f-4c05-9fbc-af7f33a41a9e","added_by":"auto","created_at":"2024-02-22 19:41:28","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":27288,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e Antimicrobial susceptibility (μg/ml) of the linezolid-resistant MRCoNS\u003c/p\u003e","description":"","filename":"Table2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3927977/v1/a611a1054cf18167406d6d1c.xlsx"},{"id":51507835,"identity":"1cc8f96a-2071-440c-a400-e417eaa942f9","added_by":"auto","created_at":"2024-02-22 19:49:28","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":19277,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 3\u003c/strong\u003eCharacteristics and drug resistance mechanisms of linezolid-resistant MRCoNS\u003c/p\u003e","description":"","filename":"Table3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3927977/v1/9a7231741fa7f8540f1a5393.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Characterization of linezolid- and methicillin-resistant coagulase-negative staphylococci in a tertiary hospital in China","fulltext":[{"header":"Background","content":"\u003cp\u003eCoagulase-negative \u003cem\u003estaphylococci\u003c/em\u003e (CoNS) have become important pathogens in patients with healthcare-associated infections caused by indwelling medical devices or immunocompromised patients [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. According to the monitoring data of the China Antimicrobial Resistance Surveillance System (CARSS, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://www.carss.cn/\" target=\"_blank\"\u003ewww.carss.cn/\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.carss.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), the frequency of methicillin-resistant coagulase-negative \u003cem\u003estaphylococci\u003c/em\u003e (MRCoNS) was extremely high, and the prevalence of MRCoNS was 74.5% in 2021.\u003c/p\u003e \u003cp\u003eLinezolid is an oxazolidinone antibiotic that is a last-resort antibiotic for the treatment of serious infections caused by gram-positive bacteria, including resistant organisms, such as methicillin-resistant \u003cem\u003estaphylococci\u003c/em\u003e and vancomycin-resistant \u003cem\u003eenterococci\u003c/em\u003e [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The first clinical isolates of linezolid-resistant \u003cem\u003estaphylococci\u003c/em\u003e and \u003cem\u003eenterococci\u003c/em\u003e were reported in 2001 [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Since then, linezolid-resistant organisms have been sporadically reported worldwide [\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, the detection rate of linezolid-resistant coagulase-negative \u003cem\u003estaphylococci\u003c/em\u003e has progressively increased in recent years in China.\u003c/p\u003e \u003cp\u003ePoint mutation in the domain V region of the 23S rRNA gene was the most common mechanism of linezolid resistance, and the most frequent mutation was G2576T. In addition, T2500A, T2604C, C2532T, C2551T, G2603T, G2614T, C2190T, and G2447T have been reported [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Furthermore, other mechanisms involved in linezolid resistance in \u003cem\u003estaphylococci\u003c/em\u003e have also been reported, such as the presence of the \u003cem\u003ecfr\u003c/em\u003e gene, which encodes an rRNA methyltransferase, and mutations in the ribosomal proteins L3, L4 and L22 [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn China, the prevalence of linezolid-resistant \u003cem\u003estaphylococci\u003c/em\u003e has gradually increased in recent years; however, very few studies have investigated linezolid resistance. In this study, the clinical characteristics, antibiotic susceptibility, and resistance mechanisms of linezolid-resistant MRCoNS isolates that were recovered from 2019 to 2023 in a Chinese tertiary hospital were examined.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBacterial isolates\u003c/h2\u003e \u003cp\u003eThirty-seven linezolid-resistant MRCoNS isolates, including thirteen \u003cem\u003eStaphylococcus capitis\u003c/em\u003e, nine \u003cem\u003eStaphylococcus hominis\u003c/em\u003e, eight \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e, six \u003cem\u003eStaphylococcus cohnii\u003c/em\u003e and one \u003cem\u003eStaphylococcus haemolyticus\u003c/em\u003e, were collected from December 2019 to March 2023 in a large tertiary teaching hospital (The First Affiliated Hospital of Zhengzhou University, Zhengzhou, with 7,000 beds located in east-central China). Two strains were isolated from pleural fluid, two from catheter tips, one from abdominal dropsy fluid, and one from cerebrospinal fluid; the remaining strains were recovered from blood cultures. Thirty-seven linezolid-resistant MRCoNS isolates were isolated from 36 patients, 25 of whom were male and 11 of whom were female. Linezolid-resistant \u003cem\u003eS. epidermidis\u003c/em\u003e-5 and \u003cem\u003eS. capitis\u003c/em\u003e-33 were isolated from the same patient, a 76-year-old man who was admitted to the respiratory intensive care unit. In addition, all patients were administered antibiotics, and 13 patients (36.1%) had received prior linezolid treatment. The organism characteristics and associated clinical data are listed in Table\u0026nbsp;1. Identification of the organisms was carried out using a VITEK\u0026reg;2 Compact system and VITEK\u0026reg; MS (bioM\u0026eacute;rieux, Marcy-l\u0026rsquo;\u0026Eacute;toile, France).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eAntimicrobial susceptibility testing\u003c/h2\u003e \u003cp\u003eThe antimicrobial susceptibilities of all the isolates were determined using the microdilution method. \u003cem\u003eS. aureus\u003c/em\u003e ATCC 29213 was used for quality control for susceptibility testing. The breakpoint for tigecycline treatment was interpreted according to the European Committee on Antimicrobial Susceptibility Testing Guidelines (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.eucast.org/clinical_breakpoints/\u003c/span\u003e\u003cspan address=\"http://www.eucast.org/clinical_breakpoints/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The results of susceptibility testing for the other antimicrobial agents were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) M100-S33 breakpoints.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eGenomic DNA extraction and whole-genome sequencing\u003c/h2\u003e \u003cp\u003eGenomic DNA was extracted using the Puregene Yeast/Bacteria Kit (QIAGEN) according to the manufacturer\u0026rsquo;s instructions for gram-positive bacteria. Whole-genome sequencing (WGS) was performed using the Illumina HiSeq PE150 platform (Novogene Bioinformatics Technology Co., Ltd., Beijing, China). Acquired antibiotic resistance genes (ARGs) carried by the isolates were analyzed using KmerResistance v 2.2[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] with raw reads. The results are reported for \u0026ge;\u0026thinsp;90% nucleotide identity, \u0026ge;\u0026thinsp;90% coverage of the query, and a sequence depth of \u0026ge;\u0026thinsp;10\u0026times;. The raw reads were assembled into scaffolds using SPAdes v 3.13.1[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The multilocus sequence typing (MLST) of the isolates was determined using MLST 2.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cge.food.dtu.dk/services/MLST/\u003c/span\u003e\u003cspan address=\"https://cge.food.dtu.dk/services/MLST/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and further validated with PubMLST (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubmlst.org\u003c/span\u003e\u003cspan address=\"https://pubmlst.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for \u003cem\u003eS. epidermidis\u003c/em\u003e, \u003cem\u003eS. hominis\u003c/em\u003e, and \u003cem\u003eS. hemolyticus\u003c/em\u003e. Additionally, the phylogenetic tree of 37 linezolid-resistant MRCoNS isolates was analyzed at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bacdb.cn/BacWGSTdb/analysis_multiple.php\u003c/span\u003e\u003cspan address=\"http://bacdb.cn/BacWGSTdb/analysis_multiple.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eMolecular detection of resistance genes and mutations\u003c/h2\u003e \u003cp\u003eDomain V of the 23S rRNA gene and \u003cem\u003ecfr\u003c/em\u003e gene was amplified and sequenced as previously described (19, 20), and the \u003cem\u003erplC\u003c/em\u003e, \u003cem\u003erplD\u003c/em\u003e, and \u003cem\u003erplV\u003c/em\u003e genes, which encode the ribosomal proteins L3, L4 and L22, were tested using previously described conditions and primers (10, 21).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAntimicrobial susceptibility\u003c/h2\u003e \u003cp\u003eAll 37 isolates displayed varying degrees of resistance to penicillin, oxacillin, and linezolid, and the \u003cem\u003emecA\u003c/em\u003e gene was detected in all 37 isolates. These iaolates were all resistant to chloramphenicol, levofloxacin, ciprofloxacin, clindamycin, erythromycin, and gentamicin, with resistance rates of 94.6% (35/37), 89.2% (33/37), 89.2% (33/37), 78.4% (29/37), 67.6% (25/37), and 54.1% (20/37), respectively. Interestingly, only 1 of the 8 \u003cem\u003eS. epidermidis\u003c/em\u003e isolates was resistant to erythromycin; however, all thirteen \u003cem\u003eS. capitis\u003c/em\u003e strains were resistant to erythromycin. The resistance rates of the linezolid-resistant MRCoNS strains to trimethoprim/sulfamethoxazole, tetracycline and rifampicin were 27.0% (10/37), 16.2% (6/37), and 10.8% (4/37), respectively; however, all six \u003cem\u003eS. cohnii\u003c/em\u003e isolates were sensitive to trimethoprim/sulfamethoxazole. No resistance to tigecycline, vancomycin or teicoplanin was observed (Table\u0026nbsp;2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eMolecular characteristics and the mechanisms of linezolid resistance\u003c/h2\u003e \u003cp\u003eSeven of the eight \u003cem\u003eS. epidermidis\u003c/em\u003e isolates belonged to the same clone, ST22. Two point mutations (T2504A and C2534T) in domain V of the 23S rRNA gene were discovered, and seven additional isolates also exhibited L101V, G152D, and D159Y changes in their deduced amino acid sequences of the L3 protein and N158S changes in the L4 protein. No \u003cem\u003ecfr\u003c/em\u003e genes were detected in any of the isolates. Another \u003cem\u003eS. epidermidis\u003c/em\u003e isolate was of the ST2 type, with positive carriage of the \u003cem\u003ecfr\u003c/em\u003e gene and no point mutations in domain V of the 23S rRNA gene (Table\u0026nbsp;3).\u003c/p\u003e \u003cp\u003eSix \u003cem\u003eS. cohnii\u003c/em\u003e isolates carried the \u003cem\u003ecfr\u003c/em\u003e gene, and five of these isolates were found to have S158Y and D159Y changes in the L3 protein. No mutation in domain V of the 23S rRNA gene was detected in any of the \u003cem\u003eS. cohnii\u003c/em\u003e isolates.\u003c/p\u003e \u003cp\u003eThe nine \u003cem\u003eS. hominis\u003c/em\u003e isolates were divided into 3 distinct clones: ST1 (n\u0026thinsp;=\u0026thinsp;5), ST2 (n\u0026thinsp;=\u0026thinsp;2) and ST85 (n\u0026thinsp;=\u0026thinsp;2). The novel ST type (ST85) was found for the first time in this study. The \u003cem\u003ecfr\u003c/em\u003e gene was identified among the six \u003cem\u003eS. hominis\u003c/em\u003e isolates, and the other three isolates had the 23S rRNA G2576T mutation.\u003c/p\u003e \u003cp\u003eIn the present study, all the \u003cem\u003eS. capitis\u003c/em\u003e isolates were found to belong to the same clone by phylogenetic analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), and the G2576T and C2104T 23S rRNA mutations were identified. Additionally, 8 isolates (61.5%) harbored the \u003cem\u003ecfr\u003c/em\u003e gene. Furthermore, M156T, V154L, and I188S changes were identified in the L3 and L4 proteins. \u003cem\u003eS. haemolyticus\u003c/em\u003e-37 harbored the \u003cem\u003ecfr\u003c/em\u003e gene and had an additional R138V change in L3.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA mutation in the L22 protein was not found in any of the linezolid-resistant MRCoNS isolates in this study.\u003c/p\u003e \u003cp\u003eIn addition to linezolid-related resistance genes, other resistance genes were also detected in our study, and the \u003cem\u003emecA\u003c/em\u003e gene was detected in all linezolid-resistant MRCoNS (Table\u0026nbsp;3).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study, 37 linezolid-resistant MRCoNS isolates were obtained from a large tertiary teaching hospital from December 2019 to March 2023. In our setting, linezolid-resistant MRCoNS were first found in 2016; however, the detection rate of linezolid-resistant MRCoNS has steadily increased in recent years. Linezolid is an important alternative for the management of MRCoNS infections. The rapid emergence of linezolid-resistant MRCoNS is alarming and requires ongoing surveillance. Previous studies have indicated that linezolid administration is a significant risk factor for linezolid-resistant gram-positive cocci during hospital outbreaks [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Our data showed that 13 patients (36.1%) had received prior linezolid therapy; thus, we speculated that increasing selective pressure most likely contributed to drug resistance. All 37 isolates were resistant to multiple antibiotics, and various resistance genes were detected, which indicated that the treatment options were limited.\u003c/p\u003e \u003cp\u003eMultilocus sequence typing indicated that ST22 was the dominant clone among the \u003cem\u003eS. epidermidis\u003c/em\u003e isolates in the present study. Our research result was consistent with the \u003cem\u003eS. epidermidis\u003c/em\u003e lineage in Greece and Spain [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] and different from the findings of German and France (ST2) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. ST2, ST5, and ST22 are clustered into the CC5 clone, which is the most prevalent clonal complex among the nosocomial \u003cem\u003eS. epidermidis\u003c/em\u003e population according to the literature[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Although linezolid-resistant CoNS have been reported sporadically worldwide, \u003cem\u003eS. hominis\u003c/em\u003e infections caused by linezolid are uncommon. Nine \u003cem\u003eS. hominis\u003c/em\u003e isolates were isolated in the present study; these strains belonged to 3 distinct clones: ST1 was the predominant clone, and ST85, a novel ST type, was found for the first time in our study.\u003c/p\u003e \u003cp\u003eMLST typing indicated that the bulk of linezolid-resistant CoNS were genetically related, which suggested the transmission of resistant clones from patient to patient and a clonal spread within the intensive care unit in our hospital.\u003c/p\u003e \u003cp\u003eThe primary causes of linezolid resistance among \u003cem\u003estaphylococci\u003c/em\u003e include modification of the target site of 23S rRNA, acquisition of the \u003cem\u003ecfr\u003c/em\u003e gene, and mutation of the ribosomal proteins L3 and L4 [\u003cspan additionalcitationids=\"CR12 CR13 CR14 CR15\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The current study revealed that linezolid resistance was associated with the acquisition of the T2504A and C2534T mutations as well as with the acquisition of the ribosomal proteins L3 and L4 among \u003cem\u003eS. epidermidis\u003c/em\u003e isolates. The \u003cem\u003ecfr\u003c/em\u003e gene was not detected among ST22-type \u003cem\u003eS. epidermidis.\u003c/em\u003e Interestingly, the \u003cem\u003ecfr\u003c/em\u003e gene was found in ST2-type \u003cem\u003eS. epidermidis\u003c/em\u003e, and the research findings indicated that distinct clone strains had diverse mechanisms for linezolid resistance. Additionally, this study revealed L101V, G152D, and D159Y changes in the L3 protein and N158S changes in the L4 protein for the first time in \u003cem\u003eS. epidermidis.\u003c/em\u003e\u003c/p\u003e \u003cp\u003eG2576T was the most frequently detected mutation in domain V of the 23S rRNA gene. In addition to the G2576T mutation, the C2104T mutation was also detected in thirteen \u003cem\u003eS. capitis\u003c/em\u003e isolates, and similar results have also been reported in previous studies [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Notably, the M156T mutation in the L3 protein and the I188S mutation in the L4 protein were also discovered for the first time. Furthermore, the G2576T mutation was also present among the three ST1-type \u003cem\u003eS. hominis\u003c/em\u003e isolates, and no mutation in domain V of the 23S rRNA gene was detected in \u003cem\u003eS. cohnii\u003c/em\u003e or \u003cem\u003eS. haemolyticus\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003ecfr\u003c/em\u003e gene was detected in one \u003cem\u003eS. epidermidis\u003c/em\u003e, one \u003cem\u003eS. haemolyticus\u003c/em\u003e, five \u003cem\u003eS. cohnii\u003c/em\u003e, six \u003cem\u003eS. hominis\u003c/em\u003e and eight \u003cem\u003eS. capitis strains\u003c/em\u003e. In the present study, the main mechanisms of linezolid resistance among \u003cem\u003eS. cohnii\u003c/em\u003e isolates were the presence of the \u003cem\u003ecfr\u003c/em\u003e gene and the acquisition of the S158Y and D159Y mutations in the ribosomal protein L3. Interestingly, our data demonstrated that the mechanism of linezolid resistance in \u003cem\u003eS. epidermidis\u003c/em\u003e and \u003cem\u003eS. capitis\u003c/em\u003e was complex, and the linezolid MIC was greater than that in \u003cem\u003eS. cohnii\u003c/em\u003e and \u003cem\u003eS. hominis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIn conclusion, there has been an increase in the prevalence of linezolid resistance among CoNS in our hospital's intensive care units in recent years. Additionally, many of the isolates were clonally related, suggesting the intrahospital dissemination of resistant clones. Resistance was related to the presence of the \u003cem\u003ecfr\u003c/em\u003e gene, a point mutation in the V domain of the 23S rRNA gene and/or a mutation in the ribosomal L3 and L4 proteins, and multiple drug resistance mechanisms often coexisted. Notably, distinct CoNS have different linezolid resistance mechanisms. Taken together, the findings on the spread of linezolid-resistant CoNS in our setting highlight the importance of monitoring linezolid resistance in MRCoNS. Strict control measures should be taken to prevent further dissemination, and the relevant use of antibiotics needs to be further emphasized.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCoNS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecoagulase-negative \u003cem\u003estaphylococci\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMRCoNS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emethicillin-resistant coagulase-negative \u003cem\u003eStaphylococci\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMLST\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMultilocus sequence typing\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCARSS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eChina Antimicrobial Resistance Surveillance System\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCLSI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eClinical and Laboratory Standards Institute\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWGS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWhole genome sequencing\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Cailin Liu, Jing Yu, Chunguang Chen, Xiaogai Li, Yafei Ye, Yani Dong, Xinxin Ying, Haijun Li,Wanhai Wang. The first draft of the manuscript was written by Cailin Liu, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscrip.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the National Natural Science Foundation of China (Project Number: 82102438).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sequence data generated in this study have been submitted to the GenBank\u0026nbsp;\u003c/p\u003e\n\u003cp\u003enucleotide sequence database (https://www.ncbi.nlm.nih.gov/) under accession numbers SUB14257732.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study protocol was approved by the First Affiliated Hospital of Zhengzhou University Ethics Committee for Research in Health. The First Affiliated Hospital of Zhengzhou University Ethics Committee also approved the waiver of informed consent to participate in this study. All patient data were anonymised prior to analysis(2020-KY-173).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eDepartment of Clinical Laboratory, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u003c/sup\u003eKey Clinical Laboratory of Henan Province, Zhengzhou, China\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e3\u003c/sup\u003eDepartment of Clinical Laboratory, The Third Affiliated Hospital of Zhengzhou University, Zhengzhou, China\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e4\u003c/sup\u003eDepartment of Clinical Laboratory, The Sixth People\u0026rsquo;s Hospital of Zhengzhou City, Zhengzhou, China\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e5\u003c/sup\u003eDepartment of Clinical Laboratory, Yichuan People\u0026apos;s Hospital, Zhengzhou, China\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e6\u003c/sup\u003eDepartment of Clinical Laboratory, Luohe Sixth People\u0026apos;s Hospital, Zhengzhou, China\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDecousser, J.W.; Desroches, M.; Bourgeois-Nicolaos, N.; Potier, J.; Jehl, F.; Lina, G., \u003cem\u003eet al\u003c/em\u003e. Susceptibility trends including emergence of linezolid resistance among coagulase-negative \u003cem\u003estaphylococci\u003c/em\u003e and meticillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e from invasive infections. Int. J. Antimicrob. Agents 2015, 46(6), 622-630. http://dx.doi.org/10.1016/j.ijantimicag.2015.07.022.\u003c/li\u003e\n\u003cli\u003eAngela Fran\u0026ccedil;a. The Role of Coagulase-Negative \u003cem\u003eStaphylococci \u003c/em\u003eBiofilms on Late-Onset Sepsis: Current Challenges and Emerging Diagnostics and Therapies. Antibiotics (Basel)2023, 10;12(3):554. . https://doi.org/10.3390/antibiotics12030554\u003c/li\u003e\n\u003cli\u003eHashemian SMR, Farhadi T, Ganjparvar M. Linezolid: a review of its properties, function, and use in critical care. Drug Des Devel Ther2018, 18;12:1759-1767. https://www.dovepress.com/by 129.130.89.83.\u003c/li\u003e\n\u003cli\u003eMeka, V.G.; Gold, H.S. Antimicrobial resistance to linezolid. Clin. Infect. Dis 2004, 39(7), 1010-1015. http://dx.doi.org/10.1086/423841.\u003c/li\u003e\n\u003cli\u003eGonzales, R. D., Schreckenberger, P. C., Graham, M. B., Kelkar, S., DenBesten, K. \u0026amp; Quinn, J. P. Infections due to vancomycin resistant \u003cem\u003eEnterococcus faecium \u003c/em\u003eresistant to linezolid. Lancet 2001, 357(9263):1179.\u003c/li\u003e\n\u003cli\u003eTsiodras, S., Gold, H. S., Sakoulas, G., Eliopoulos, G. M., Wennersten, C., Venkataraman, L., \u003cem\u003eet al\u003c/em\u003e. Linezolid resistance in a clinical isolate of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e. Lancet 2001, 358, 207\u0026ndash;208.\u003c/li\u003e\n\u003cli\u003eBrescini L, Fioriti S, Coccitto SN, Cinthi M, Mingoia M, Cirioni O, \u003cem\u003eet al\u003c/em\u003e. Genomic Analysis of a Linezolid-Resistant \u003cem\u003eStaphylococcus capitis\u003c/em\u003e Causing Bacteremia: Report from a University Hospital in Central Italy. Microb Drug Resist 2023. doi: 10.1089/mdr.2022.0330.\u003c/li\u003e\n\u003cli\u003eDadashi M, Sharifian P, Bostanshirin N, Hajikhani B, Bostanghadiri N, Khosravi-Dehaghi N, \u003cem\u003eet al\u003c/em\u003e. The Global Prevalence of Daptomycin, Tigecycline, and Linezolid-Resistant \u003cem\u003eEnterococcus faecalis \u003c/em\u003eand \u003cem\u003eEnterococcus faecium\u003c/em\u003e Strains From Human Clinical Samples: A Systematic Review and Meta-Analysis. Front Med (Lausanne) 2021. doi: 10.3389/fmed.2021.720647.\u003c/li\u003e\n\u003cli\u003eKevin Bouiller, Dejan Ilic, Paul Henry Wicky, Pascal Cholley, Catherine Chirouze, Xavier Bertrand. Spread of clonal linezolid-resistant \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e in an intensive care unit associated with linezolid exposure. 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Linezolid resistance in \u003cem\u003eStaphylococcus epidermidis \u003c/em\u003eassociated with a G2603T mutation in the 23S rRNA gene. Int J Antimicrob Agents 2009, 34(3):281\u0026ndash;282. https://doi.org/10.1016/j. ijantimicag.2009.02.023.\u003c/li\u003e\n\u003cli\u003eGajanand Mittal,Vasundhra Bhandari,Rajni Gaind,Vandana Rani,Shimpi Chopra,Reetika Dawar, \u003cem\u003eet al\u003c/em\u003e. Linezolid resistant coagulase negative \u003cem\u003estaphylococci \u003c/em\u003e(LRCoNS) with novel mutations causing blood stream infections (BSI) in India. BMC Infect Dis . 2019,19(1):717.\u003c/li\u003e\n\u003cli\u003eMendes RE, Deshpande LM, Jones RN. Linezolid update: stable in vitro activity following more than a decade of clinical use and summary of associated resistance mechanisms. Drug Resist Updat 2014, 17:1\u0026ndash;12.\u003c/li\u003e\n\u003cli\u003eAnders Michael Bernth Giessing, S\u0026oslash;ren Skov Jensen, Anette Rasmussen, Lykke Haastrup Hansen, Andrzej Gondela, Katherine Long, \u003cem\u003eet al\u003c/em\u003e. Identification of 8-methyladenosine as the modification catalyzed by the radical SAM methyltransferase Cfr that confers antibiotic resistance in bacteria. RNA 2009, 15:327\u0026ndash;36.\u003c/li\u003e\n\u003cli\u003eLong KS, Vester B. Resistance to linezolid caused by modifications at its binding site on the ribosome. Antimicrob Agents Chemother 2012, 56:603\u0026ndash;612. https://doi.org/10.1128/AAC.05702-11.\u003c/li\u003e\n\u003cli\u003eClausen PT, Zankari E, Aarestrup FM, Lund O. Benchmarking of methods for identification of antimicrobial resistance genes in bacterial whole genome data. J Antimicrob Chemother 2016, 71(9):2484-8.\u003c/li\u003e\n\u003cli\u003eNurk S, Bankevich A, Antipov D, Gurevich AA, Korobeynikov A, Lapidus A, \u003cem\u003eet al\u003c/em\u003e. Assembling single-cell genomes and mini-metagenomes from chimeric MDA products. J Comput Biol 2013, 20(10):714-37.\u003c/li\u003e\n\u003cli\u003eLi Ding,Pei Li,Yang Yang,Dongfang Lin,Xiaogang Xu. The epidemiology and molecular characteristics of linezolid-resistant \u003cem\u003eStaphylococcus capitis\u003c/em\u003e in Huashan Hospital, Shanghai. J Med Microbiol 2020, 69(8):1079-1088.\u003c/li\u003e\n\u003cli\u003eKehrenberg, C. Schwarz, S. Distribution of florfenicol resistance genes fexA and cfr among chloramphenicol-resistant \u003cem\u003eStaphylococcus isolates\u003c/em\u003e. Antimicrob Agents Chemother 2006, 50, 1156\u0026ndash;1163.\u003c/li\u003e\n\u003cli\u003eMendes RE, Deshpande LM, Farrell DJ, Spanu T, Fadda G, Jones RN. Assessment of linezolid resistance mechanisms among \u003cem\u003eStaphylococcus epidermidis \u003c/em\u003ecausing bacteraemia in Rome, Italy. J Antimicrob Chemother 2010, 65:2329\u0026ndash;35.\u003c/li\u003e\n\u003cli\u003eKevin Bouiller, Dejan Ilic, Paul Henry Wicky, Pascal Cholley, Catherine Chirouze, Xavier Bertrand. Spread of clonal linezolid-resistant \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e in an intensive care unit associated with linezolid exposure. Eur J Clin Microbiol Infect Dis 2019, 39(7):1271-1277. https://doi.org/10.1007/s10096-020-03842-7.\u003c/li\u003e\n\u003cli\u003eK\u0026eacute;vin Bouiller, Julien Bador, R\u0026eacute;mi Bruy\u0026egrave;re, Amaury Toitot, S\u0026eacute;bastien Prin, Jean-Pierre Quenot, Pierre-Emmanuel Charles. Recent exposure to linezolid is strongly associated with the isolation of linezolid-resistant coagulase-negative \u003cem\u003estaphylococcus\u003c/em\u003e species in patients with related infection or colonization: a case‒control study in an intensive care unit. Int J Antimicrob Agents 2017, 50(5):693-694. http://dx.doi.org/doi: 10.1016/j.ijantimicag.2017.08.026.\u003c/li\u003e\n\u003cli\u003eKaravasilis V, Zarkotou O, Panopoulou M, Kachrimanidou M, Themeli-Digalaki K, Stylianakis A, \u003cem\u003eet al\u003c/em\u003e. On behalf of the Greek Study Group on \u003cem\u003eStaphylococcal \u003c/em\u003eLinezolid Resistance Wide dissemination of linezolid-resistant \u003cem\u003eStaphylococcus epidermidis \u003c/em\u003ein Greece is associated with a linezolid-dependent ST22 clone. J Antimicrob Chemother 2015, 70(6):1625\u0026ndash;1629. https://doi.org/10.1093/jac/dkv028.\u003c/li\u003e\n\u003cli\u003eLozano C, Ruiz-Garc\u0026iacute;a M, G\u0026oacute;mez-Sanz E, L\u0026oacute;pez-Garc\u0026iacute;a P, Royo Garc\u0026iacute;a G, Zarazaga M, Torres C. Characterization of a cfr positive methicillin-resistant \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e strain of the lineage ST22 implicated in a life-threatening human infection. Diagn Microbiol Infect Dis 2012, 73(4):380\u0026ndash;382. https://doi.org/10.1016/j.diagmicrobio.2012.04.013.\u003c/li\u003e\n\u003cli\u003eDortet L, Glaser P, Kassis-Chikhani N, Girlich D, Ichai P, Boudon M, \u003cem\u003eet al\u003c/em\u003e. Long-lasting successful dissemination of resistance to oxazolidinones in MDR \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e clinical isolates in a tertiary care hospital in France. J Antimicrob Chemother 2018, 73(1):41\u0026ndash;51. https://doi.org/10.1093/jac/dkx370.\u003c/li\u003e\n\u003cli\u003eBender J, Strommenger B, Steglich M, Zimmermann O, Fenner I, Lensing C, \u003cem\u003eet al\u003c/em\u003e. Linezolid resistance in clinical isolates of \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e from German hospitals and characterization of two cfr carrying plasmids. J Antimicrob Chemother 2015, 70(6):1630\u0026ndash;1638. https://doi.org/10.1093/jac/dkv025\u003c/li\u003e\n\u003cli\u003eThomas JC, Vargas MR, Miragaia M, Peacock SJ, Archer GL, Enright MC. Improved multilocus sequence typing scheme for \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e. J Clin Microbiol 2017, 45:616\u0026ndash;619. https://doi.org/10.1128/JCM.01934-06.\u003c/li\u003e\n\u003cli\u003eJia Chang Cai,Yan Yan Hu, Rong Zhang, Hong Wei Zhou, Gong-Xiang Chen. Linezolid-resistant clinical isolates of meticillin-resistant coagulase-negative \u003cem\u003estaphylococci\u003c/em\u003e and \u003cem\u003eEnterococcus faecium \u003c/em\u003efrom China. J Med Microbiol 2012, 61(Pt 11):1568-1573.\u003c/li\u003e\n\u003cli\u003eDing L, Li P, Yang Y, Lin D, Xu X. The epidemiology and molecular characteristics of linezolid-resistant \u003cem\u003eStaphylococcus capitis\u003c/em\u003e in Huashan Hospital, Shanghai. Med Microbiol 2020, 69(8):1079-1088.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 3 are available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"infd","sideBox":"Learn more about [BMC Infectious Diseases](http://bmcinfectdis.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/infd","title":"BMC Infectious Diseases","twitterHandle":"#bmcinfectdis","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"coagulase-negative staphylococci, linezolid-resistant, G2576T mutation, cfr gene","lastPublishedDoi":"10.21203/rs.3.rs-3927977/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3927977/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground \u003c/strong\u003eRecently, linezolid-resistant staphylococci have become an emerging problem worldwide. It is very important to understand the resistance mechanisms, molecular epidemiology and probable transmission of linezolid-resistant CoNS in the hospital.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods \u003c/strong\u003eThe antimicrobial susceptibilities of all the isolates were determined by the microdilution method. The resistance mechanisms and molecular characteristics of the strains were detected using whole-genome sequencing and PCR.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e All the strains were resistant to oxacillin and carried the \u003cem\u003emecA\u003c/em\u003e gene; 13 patients (36.1%) had received prior linezolid exposure. The majority of the \u003cem\u003eS. epidermidis\u003c/em\u003e and \u003cem\u003eS. hominis \u003c/em\u003eisolates were ST22 and ST1, respectively. MLST typing and evolutionary analysis indicated that the majority of linezolid-resistant CoNS were genetically related. This study revealed that distinct CoNS strains have different linezolid resistance mechanisms. Among ST22-type \u003cem\u003eS. epidermidis\u003c/em\u003e, the acquisition of the T2504A and C2534T mutations in the V domain of the 23S rRNA gene as well as the mutations in the ribosomal proteins L3 (L101V, G152D, and D159Y) and L4 (N158S) were linked to the development of linezolid resistance. In the \u003cem\u003eS. cohnii \u003c/em\u003eisolates, the \u003cem\u003ecfr\u003c/em\u003e, S158Y and D159Y mutations in the ribosomal protein L3 were found. Additionally, the emergence of the G2576T mutation and the \u003cem\u003ecfr \u003c/em\u003egene were major causes of linezolid resistance in \u003cem\u003eS. hominis \u003c/em\u003eisolates.\u003cem\u003eThe cfr\u003c/em\u003e gene, the G2576T and C2104T mutations, the M156T change in L3, and the I188S change in the L4 protein were found in \u003cem\u003eS. capiti\u003c/em\u003es\u003cem\u003e \u003c/em\u003eisolates.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e The emergence of linezolid-resistant CoNS in our environment is concerning because it involves clonal dissemination and frequently coexists with various drug resistance mechanisms.\u003c/p\u003e","manuscriptTitle":"Characterization of linezolid- and methicillin-resistant coagulase-negative staphylococci in a tertiary hospital in China","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-22 19:41:23","doi":"10.21203/rs.3.rs-3927977/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-03-26T06:05:53+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-03-06T12:55:17+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-02-25T05:15:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"8729c49d-ceac-4232-b556-c78a73a9c308","date":"2024-02-20T11:33:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"35d87af8-3639-477b-9b69-87e47a6d32a0","date":"2024-02-20T11:25:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"8f7d31e6-e686-4ef0-9d03-1a9af3f23733","date":"2024-02-20T10:05:14+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-02-20T08:58:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-20T08:53:02+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-02-20T08:46:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-02-20T08:27:03+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Infectious Diseases","date":"2024-02-04T14:50:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"infd","sideBox":"Learn more about [BMC Infectious Diseases](http://bmcinfectdis.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/infd","title":"BMC Infectious Diseases","twitterHandle":"#bmcinfectdis","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d9f64363-5f93-4c37-832b-6b30468b5138","owner":[],"postedDate":"February 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-05-14T21:21:32+00:00","versionOfRecord":{"articleIdentity":"rs-3927977","link":"https://doi.org/10.1186/s12879-024-09376-z","journal":{"identity":"bmc-infectious-diseases","isVorOnly":false,"title":"BMC Infectious Diseases"},"publishedOn":"2024-05-10 21:17:44","publishedOnDateReadable":"May 10th, 2024"},"versionCreatedAt":"2024-02-22 19:41:23","video":"","vorDoi":"10.1186/s12879-024-09376-z","vorDoiUrl":"https://doi.org/10.1186/s12879-024-09376-z","workflowStages":[]},"version":"v1","identity":"rs-3927977","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3927977","identity":"rs-3927977","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}