Colocalization of Linezolid Resistance (cfr) and Virulence Factors Cytolysin and Hemolysin (cln and hln) on a Plasmid in Enterococcus faecalis.
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ABSTRACT:
SUBMITTER: Singh NV
PROVIDER: S-EPMC10269126 | biostudies-literature | 2023 Jun
REPOSITORIES: biostudies-literature
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Colocalization of Linezolid Resistance (<i>cfr</i>) and Virulence Factors Cytolysin and Hemolysin (<i>cln</i> and <i>hln</i>) on a Plasmid in Enterococcus faecalis.
Singh Nikita V NV Singh Kavindra V KV Dinh An Q AQ Arias Cesar A CA Shropshire William C WC Hanson Blake M BM Murray Barbara E BE
Antimicrobial agents and chemotherapy 20230510 6
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Project description:Nonmutational resistance to linezolid is due to the presence of cfr, which encodes a methyltransferase responsible for methylation of A2503 in the 23S rRNA. The cfr gene was first described in animal isolates of staphylococci, and more recently, it has been identified in Staphylococcus aureus from human clinical infections, including in an outbreak of methicillin-resistant S. aureus. In enterococci, cfr has been described in an animal isolate of Enterococcus faecalis from China. Here, we report an isolate of linezolid-resistant E. faecalis (603-50427X) recovered from a patient in Thailand who received prolonged therapy with the antibiotic for the treatment of atypical mycobacterial disease. The isolate lacked mutations in the genes coding for 23S rRNA and L3 and L4 ribosomal proteins and belonged to the multilocus sequence type (MLST) 16 (ST16), which is commonly found in enterococcal isolates from animal sources. Resistance to linezolid was associated with the presence of cfr on an ~97-kb transferable plasmid. The cfr gene environment exhibited DNA sequences similar to those of other cfr-carrying plasmids previously identified in staphylococci (nucleotide identity, 99 to 100%). The cfr-carrying plasmid was transferable by conjugation to a laboratory strain of E. faecalis (OG1RF) but not to Enterococcus faecium or S. aureus. The cfr gene was flanked by IS256-like sequences both upstream and downstream. This is the first characterization of the potential horizontal transferability of the cfr gene from a human linezolid-resistant isolate of E. faecalis.
| S-EPMC3393385 | biostudies-literature
Project description:The aim of this study was to investigate the transferability of acquired linezolid resistance genes and associated mobile genetic elements in an Enterococcus faecalis isolate QZ076, cocarrying optrA, cfr, cfr(D), and poxtA2 genes. MICs were determined by broth microdilution. Whole-genome sequencing (WGS) was performed using the Illumina and Nanopore platforms. The transfer of linezolid resistance genes was investigated by conjugation, using E. faecalis JH2-2 and clinical methicillin-resistant Staphylococcus aureus (MRSA) 109 as recipients. E. faecalis QZ076 harbors four plasmids, designated pQZ076-1 to pQZ076-4, with optrA located in the chromosomal DNA. The gene cfr was located on a novel pseudocompound transposon, designated Tn7515, integrated into the 65,961-bp pCF10-like pheromone-responsive conjugative plasmid pQZ076-1. Tn7515 generated 8-bp direct target duplications (5'-GATACGTA-3'). The genes cfr(D) and poxtA2 were colocated on the 16,397-bp mobilizable broad-host-range Inc18 plasmid pQZ076-4. The cfr-carrying plasmid pQZ076-1 could transfer from E. faecalis QZ076 to E. faecalis JH2-2, along with the cfr(D)- and poxtA2-cocarrying plasmid pQZ076-4, conferring the corresponding resistant phenotype to the recipient. Moreover, pQZ076-4 could also transfer to MRSA 109. To the best of our knowledge, this study presented the first report of four acquired linezolid resistance genes [optrA, cfr, cfr(D), and poxtA2] being simultaneously present in the same E. faecalis isolate. The location of the cfr gene on a pseudocompound transposon in a pheromone-responsive conjugative plasmid will accelerate its rapid dissemination. In addition, the cfr-carrying pheromone-responsive conjugative plasmid in E. faecalis was also able to mobilize the interspecies transfer of the cfr(D)- and poxtA2-cocarrying plasmid between enterococci and staphylococci. IMPORTANCE In this study, the simultaneous occurrence of four acquired oxazolidinone resistance genes [optrA, cfr, cfr(D), and poxtA2] was identified in an E. faecalis isolate of chicken origin. The association of the cfr gene with a novel pseudocompound transposon Tn7515 integrated into a pCF10-like pheromone-responsive conjugative plasmid will accelerate its dissemination. Moreover, the location of the resistance genes cfr(D) and poxtA2 on a mobilizable broad-host-range Inc18 family plasmid represents the basis for their intra- and interspecies dissemination with the aid of a conjugative plasmid and further accelerates the spreading of acquired oxazolidinone resistance genes, such as cfr, cfr(D), and poxtA2, among Gram-positive pathogens.
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Project description:Despite recent recognition of the ATP-binding cassette protein OptrA as an important mediator of linezolid resistance in Enterococcus faecalis worldwide, the mechanisms of optrA gene acquisition and transfer remain poorly understood. In this study, we performed comprehensive molecular and phenotypic profiling of 44 optrA-carrying E. faecalis clinical isolates with linezolid resistance. Pulse-field gel electrophoresis and DNA hybridization revealed the presence of optrA in the plasmid in 26 (59%) isolates and in the chromosome in 18 (41%) isolates. Conjugation experiments showed a successful transfer of optrA in 88.5% (23/26) of isolates carrying optrA in plasmids while no transfer occurred in any isolates carrying optrA in the chromosome (0/18). All 23 transconjugants exhibited in vitro resistance to linezolid and several other antibiotics and were confirmed to contain optrA and other resistance genes. Plasmid typing demonstrated a predominance (18/23,78%) of rep 9-type plasmids (pCF10 prototype) known to be the best studied sex pheromone responsive plasmids. Full plasmid genome sequencing of one isolate revealed the presence of drug resistance genes (optrA and fexA) and multiple sex pheromone response genes in the same plasmid, which represents the first sex pheromone responsive plasmid carrying optrA from a clinical isolate. PCR-based genotyping revealed the presence of three key sex pheromone response genes (prgA, prgB, and prgC) in 23 optrA-carrying isolates. Finally, functional studies of these isolates by clumping induction assay detected different degrees of clumping in 17 isolates. Our analysis suggests that optrA-mediated linezolid resistance can be widely disseminated through sex pheromone plasmid transfer.
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Project description:Linezolid (LZD) has become one of the most important antimicrobial agents for infections caused by gram-positive bacteria, including those caused by Enterococcus species. LZD-resistant (LR) genetic features include mutations in 23S rRNA/ribosomal proteins, a plasmid-borne 23S rRNA methyltransferase gene cfr, and ribosomal protection genes (optrA and poxtA). Recently, a cfr gene variant, cfr(B), was identified in a Tn6218-like transposon (Tn) in a Clostridioides difficile isolate. Here, we isolated an LR Enterococcus faecalis clinical isolate, KUB3006, from a urine specimen of a patient with urinary tract infection during hospitalization in 2017. Comparative and whole-genome analyses were performed to characterize the genetic features and overall antimicrobial resistance genes in E. faecalis isolate KUB3006. Complete genome sequencing of KUB3006 revealed that it carried cfr(B) on a chromosomal Tn6218-like element. Surprisingly, this Tn6218-like element was almost (99%) identical to that of C. difficile Ox3196, which was isolated from a human in the UK in 2012, and to that of Enterococcus faecium 5_Efcm_HA-NL, which was isolated from a human in the Netherlands in 2012. An additional oxazolidinone and phenicol resistance gene, optrA, was also identified on a plasmid. KUB3006 is sequence type (ST) 729, suggesting that it is a minor ST that has not been reported previously and is unlikely to be a high-risk E. faecalis lineage. In summary, LR E. faecalis KUB3006 possesses a notable Tn6218-like-borne cfr(B) and a plasmid-borne optrA. This finding raises further concerns regarding the potential declining effectiveness of LZD treatment in the future.
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