Bile Carriage of optrA-Positive Enterococcus faecium in a Patient with Choledocholith

Oxazolidinone are effective antimicrobial agents for the treatment of infections caused by multidrug-resistant Gram-positive bacteria, including vancomycin-resistant enterococci. The emergence and global spread of transferable oxazolidinone resistance genes such as optrA is worrisome. Enterococcus spp. can become causes of hospital-associated infections and are also widely distributed in the gastrointestinal tracts of animals and the natural environment. ABSTRACT We isolated one Enterococcus faecium isolate SZ21B15 from a bile sample of a patient with choledocholith in Shenzhen, China in 2021. It was positive for oxazolidinone resistance gene optrA and was intermediate to linezolid. The whole genome of E. faecium SZ21B15 was sequenced by Illumina Hiseq. It belonged to ST533 within the clonal complex 17. The optrA gene and additional two resistance genes fexA and erm(A) were located within a 25,777-bp multiresistance region, which was inserted into the chromosomal radC gene, being chromosomal intrinsic resistance genes. The chromosomal optrA gene cluster found in E. faecium SZ21B15 was closely related to the corresponding regions of multiple optrA-carrying plasmids or chromosomes from Enterococcus, Listeria, Staphylococcus, and Lactococcus strains. It further highlights the ability of the optrA cluster that transfers between plasmids and chromosomes and evolves by a series of molecular recombination events. IMPORTANCE Oxazolidinone are effective antimicrobial agents for the treatment of infections caused by multidrug-resistant Gram-positive bacteria, including vancomycin-resistant enterococci. The emergence and global spread of transferable oxazolidinone resistance genes such as optrA is worrisome. Enterococcus spp. can become causes of hospital-associated infections and are also widely distributed in the gastrointestinal tracts of animals and the natural environment. In this study, one E. faecium isolate from bile sample carried chromosomal optrA, being intrinsic resistance gene. optrA-positive E. faecium in bile not only makes the treatment of gallstones difficult, but also may become a reservoir of resistance genes in the body.

IMPORTANCE Oxazolidinone are effective antimicrobial agents for the treatment of infections caused by multidrug-resistant Gram-positive bacteria, including vancomycin-resistant enterococci. The emergence and global spread of transferable oxazolidinone resistance genes such as optrA is worrisome. Enterococcus spp. can become causes of hospital-associated infections and are also widely distributed in the gastrointestinal tracts of animals and the natural environment. In this study, one E. faecium isolate from bile sample carried chromosomal optrA, being intrinsic resistance gene. optrA-positive E. faecium in bile not only makes the treatment of gallstones difficult, but also may become a reservoir of resistance genes in the body. KEYWORDS bile, E. faecium, optrA L inezolid, the first commercially available oxazolidinone, is a crucial antimicrobial agent against infections caused by multidrug-resistant Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci, and penicillin-resistant pneumococci (1). The emergence and wide spread of linezolid-resistant bacteria is worrying. The multiresistance gene cfr, as the first transferable oxazolidinone resistance gene, has been globally reported among Grampositive and Gram-negative bacteria from different origins, particularly in China (1). In 2015, a novel transferable oxazolidinone resistance gene optrA, located on a conjugative plasmid pE349, was identified in Enterococcus faecalis of human origin in China, and further detected in E. faecalis and E. faecium from food-producing animals and humans in China (2). Recently, poxtA, as a novel phenicol-oxazolidinone-tetracycline resistance gene, was found in a clinical MRSA in Italy (3). It has been also identified in Enterococcus and Staphylococcus isolates from human, food-producing animals and food products (1). Here, we reported the detection of optrA in one E. faecium strain from bile of patient with choledocholith.
E. faecium isolate SZ21B15 was obtained from a bile sample collected from a 71-yearold male patient with choledocholith in a tertiary hospital in Shenzhen, China in October 2021. As part of routine surveillance, the oxazolidinone resistance genes (cfr, optrA, and poxtA) were detected as previously described (2,4,5) and optrA was identified in SZ21B15. MICs of 13 antimicrobial agents were tested by using the broth microdilution method. The results were interpreted by Clinical and Laboratory Standards Institute (CLSI) M100, 30 th edition. Gentamicin ($64 mg/L) was interpreted according to the epidemiological cutoff value for E. faecium set by European Committee on Antimicrobial Susceptibility Testing (EUCAST; https://www.eucast.org/). The isolate SZ21B15 exhibited MIC of 4 mg/L to linezolid, classified as intermediate to linezolid. Similar results were previously observed in optrA-positive enterococci (6,7). It was resistant to erythromycin, chloramphenicol, and ciprofloxacin, and intermediate to nitrofurantoxin, but was susceptible to penicillin, ampicillin, gentamicin, daptomycin, teicoplanin, vancomycin, fosfomycin, and high-level mupirocin (Table S1).
To characterize this optrA-positive isolate, we used Illumina Hiseq platform to sequence the whole genome of E. faecium SZ21B15. Genomic DNA extraction, library preparation, and sequencing were performed as previously described (8,9). A total of 224 contigs (.200 bp) (GenBank accession on. PRJNA861704) were obtained by using SPAdes version 3.15.4 to assemble the raw data. We used the Center for Genomic Epidemiology (CGE) pipeline (https://cge.cbs.dtu.dk/) to analyze the sequence type (ST) and antimicrobial resistance genes. The optrA-positive E. faecium strain SZ21B15 belonged to ST533 within the commonly observed clonal complex 17. Thus far, only three E. faecium ST533 isolates were retrieved from PubMLST Enterococcus faecium database (https://pubmlst.org/organisms/enterococcus-faecium) originating from patients (n = 2) and the environment (n = 1) in Europe.
E. faecium SZ21B15 harbored five resistance genes, including the linezolid resistance gene optrA, the phenicol exporter gene fexA, the aminoglycoside resistance gene aac(6')-Ii, and the macrolide resistance genes erm(A) and msr(C). The optrA gene and additional two resistance genes [fexA and erm(A)] was carried by the largest contig (294, 884bp) corresponding to the chromosome of E. faecium. As shown in Fig. 1, chromosomal optrA gene cluster found in E. faecium SZ21B15 was closely related to the corresponding regions of multiple optrA-carrying plasmids or chromosomes from Enterococcus, Listeria, Staphylococcus, and Lactococcus strains. The 25,777-bp multiresistance region (MRR) was inserted into the radC gene, encoding a DNA repair protein (Fig. 1). Similar insertion of the fexA-optrA-erm(A) region into radC was also found in E. faecium strains from pig and river water and Listeria innocua isolate LI47 from food in China (Fig. 1). The chromosomal radC gene is a common integration hot spot for Tn554 family transposons, such as Tn558 and optrA-positive Tn6674 (10).
The optrA gene was first identified in E. faecalis from a human in China and was more frequently in E. faecalis from food-producing animals than from humans (2). Since the discovery of optrA in enterococci, it has been successively and globally identified in Gram-positive bacteria such as Staphylococcus, Streptococcus, Clostridium, Lactococcus, Listeria, and Gram-negative pathogen Campylobacter from different sources (1,11). Various plasmids, transposons, integrative and conjugative elements, prophages, and insertion sequences are responsible for horizontal transmission of optrA (1). The chromosomal integration of optrA together with additional resistance genes such as erm(A) and/or fexA has been previously described in E. faecalis and E. faecium from pigs and humans, being chromosomal intrinsic resistance genes (6,7). Diverse but related optrA genetic structures are observed in plasmids or chromosomes from different species, suggesting that the optrA cluster may jump between plasmids and chromosomes via mobile elements and evolve by capturing, losing and rearranging molecular modules. Furthermore, Enterococcus spp. has become the common bacteria in bile culture with biliary diseases (12). This study is the first report of optrA in E. faecium from bile. optrA-positive E. faecium in bile not only makes the treatment of gallstones difficult, but also may become the resistance gene pool of other pathogens in the body. It has always been said that the intestine is a reservoir of drug-resistant genes. Our study found that drug-resistant bacteria can also exist in the gallbladder upstream of the intestine. It is worth paying attention to whether drug-resistant bacteria can colonize in the intestine for a long time or transmit resistance genes to other bacteria in the intestine. Ethical approval. This research involving clinical isolates were reviewed and approved by the Ethical Committee of the Shenzhen University Health Science Center (PN202200030).

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