Characterization of multiresistance gene optrA in Bacillus subtillis from China

Sir,

Antibiotic resistances have significantly reduced the effectiveness of antibiotic treatment, posing a grave threat to human and animal health. Moreover, antibiotic resistance genes (ARGs) are one of the important factors contributing to bacterial resistance and are now ubiquitous in non-clinical environments. Specifically, water environments are considered major reservoirs of ARGs and cause them to spread among different bacterial species. The resistant bacteria carrying ARGs in water environments can be disseminated between human and animal.

Linezolid, the first member of the oxazolidinones family, is an effective antimicrobial agent for treating the human infection of multidrug-resistant Gram-positive bacteria.¹ However, due to the abuse and inappropriate use of linezolid, acquired resistance has arisen in various bacteria. So far, the resistance to linezolid should be attributed to three resistance genes: the optrA gene that encodes an ABC-F protein resulting in resistance to oxazolidinones and phenicols^2,3; the cfr gene that encodes an rRNA methyltransferase that adds a methyl group at the C-8 position of 23S rRNA nucleotide A2503^3,4 and the poxtA gene that encodes a ribosomal protection protein of the ARE ABC-F family.^3,5 Also, the optrA gene has been reported in some bacteria,⁶ including Enterococcus sp., Staphylococcus sp., Streptococcus sp., Lactococcus sp., Campylobacter sp., Clostridium perfringens and Vagococcus lutrae. Thus, analysis of the prevalence and distribution of optrA is important for evaluating the risk to public health and for development of control measures.

A routine surveillance was performed with the aim of detecting the optrA gene in bacteria of drinking water origin from pig farms. These bacterial strains were restored to an inoculate in Tryptic Soy Agar supplemented with 5% calf serum and were tested by PCR for the presence of optrA (Table S1, available as Supplementary data at JAC Online). Interestingly, 16S rRNA sequence analysis showed that one optrA-positive isolate (called FJYA24) was Bacillus subtilis. Susceptibility testing, carried out by reference broth microdilution (CLSI) and interpreted according to the CLSI clinical breakpoints (CLSI, version 2018), revealed that MICs of linezolid, tedizolid, florfenicol and chloramphenicol for B. subtilis strain FJYA24 were 4, 2, 128 and >256 mg/L, respectively (Table S2).

To characterize the optrA genetic element, whole-genome sequencing of B. subtilis strain FJYA24 was carried out using the Illumina Hiseq and Oxford Nanopore MinIon platforms and complete sequences were obtained by hybrid assembly using Unicycler version 0.5.0 (accession no. CP173415).⁷ The functional element prediction analysis was analysed using Prokka.⁸ The sequences were annotated by comparing the predicted gene sequences with functional databases such as CARD⁹ by BLAST+. MLST analysis indicated that the strain was ST191 (http://mlst.zoo.ox.ac.uk). Bioinformatics analysis revealed that the optrA gene was located on a new 4635 bp plasmid, named pFJYA1 (accession no. PQ582411). The map of the plasmid is shown in Figure S1. Three relevant areas were detectable in pFJYA1. (i) An antibiotic resistance region containing optrA gene (responsible for phenicol and oxazolidinone resistance). The deduced amino acid (aa) sequence of optrA gene was 99% identical to the aa reference sequence (GenBank accession no. CP082206.1). Compared with optrA from pE394, the optrA gene variant is a DKDMD variant.³ The Genetic evolutionary tree of the aa sequence of optrA gene showed that the optrA gene from B. subtilis had a close relationship to one from other all bacterial species (Figure 1a). Inverse PCR experiments, using outward-directed primer pairs targeting the optrA gene, showed that the plasmid was circular. (ii) A segment harbouring the rep gene (responsible for plasmid replication). The rep gene was classified as rep1 by PlasmidFinder (https://cge.food.dtu.dk/services/PlasmidFinder/). Sequence comparison analysis showed that the plasmid was significantly similar to some plasmids from the Staphylococcus genus (Figure 1b). These plasmids were all small plasmids, from 2168 to 5079 bp in size, which carried the rep gene, mob gene and different functioning genes or resistant genes. The result revealed that the plasmid pFJYA1 from B. subtilis might derive from Staphylococcus genus. (iii) A mobilization region containing the mob gene. Using E. faecalis JH2-2 as recipient, a conjugation transfer and transformation test for the pFJYA1 were performed. However, the transconjugant could not be obtained and the plasmid successfully transformed into E. faecalis JH2-2.

Figure 1.

Evolutionary analysis of OptrA protein and sequence comparison analysis of seven small plasmids. (a) Evolutionary analysis of deduced aa sequences of optrA gene was conducted with MEGA v.11.0, and the analysis involved 15 optrA genes from various Gram-positive bacterial strains. Initial trees for the heuristic search were obtained automatically by applying neighbour-joining methods and BioNJ algorithms. The interval size of the evolutionary tree is 0.0010. (b) Sequence comparison analysis of seven small plasmids from six Staphylococcus sp. and one Bacillus subtilis was performed using Easyfig v.2.2.5, and one map was generated using Adobe Illustrator software. The extents and directions of resistance genes, mob genes and rep genes are shown by arrows labelled with gene names. The resistance genes are shown in green, mob genes are shown in light blue, rep genes are shown in blue and levels of homology are shown in grey.

B. subtilis, a species of the Bacillus genus, is widely distributed in soil and water, and is used extensively in some fields such as medicine, health preservation and food.¹⁰ To the best of our knowledge, this is the first report of the optrA gene in a Bacillus species carried by a small plasmid. Because B. subtilis has been frequently used in food and feed, this will accelerate the optrA gene spread. Therefore, to prevent optrA gene transfer and spread it is important to monitor and detect this gene in farm feeds.

Funding

This study was supported by Science and Technology Plan Project from Fujian Academy of Agricultural Sciences (no. YDXM2023007), Central Guidance for Local Science and Technology Development Funds Project for Fujian Province (2022L3033), basic scientific research projects of provincial public welfare scientific research institutions (Fujian Province), China (No. 2022R10260013) and the key project of Fujian 5511 collaborative innovation project, China (No. XTCXGC2021008).

Transparency declarations

None to declare.

Supplementary data

Figure S1 and Tables S1 and S2 are available as Supplementary data at JAC Online.