Complete sequence of carbapenem-resistant Ralstonia mannitolilytica clinical isolate co-producing novel class D β-lactamase OXA-1176 and OXA-1177 in Japan

ABSTRACT In 2020, the Ralstonia mannitolilytica strain JARB-RN-0044 was isolated from a midstream urine sample of an elderly hospitalized patient in Japan and was highly resistant to carbapenem (i.e., imipenem, meropenem, and doripenem). Whole-genome sequencing revealed that the complete genome consists of two replicons, a 3.5-Mb chromosome and a 1.5-Mb large non-chromosomal replicon which has not been reported in R. mannitolilytica, and referred to as the “megaplasmid” in this study based on Cluster of Orthologous Group of proteins functional analysis. The strain JARB-RN-0044 harbored two novel OXA-60 and OXA-22 family class D β-lactamase genes blaOXA-1176 and blaOXA-1177 on the megaplasmid. Cloning experiments indicated that Escherichia coli recombinant clone expressing blaOXA-1176 gene showed increased minimum inhibitory concentrations (MICs) of imipenem, meropenem, and doripenem, indicating that blaOXA-1176 gene encodes carbapenemase. In contrast, E. coli recombinant clone expressing blaOXA-1177 gene showed increased MICs of piperacillin and cefazolin, but not of carbapenem. Interestingly, the 44.6 kb putative prophage region containing genes encoding phage integrase, terminase, head and tail protein was identified in the downstream region of blaOXA-1176 gene, and comparative analysis with some previously reported R. mannitolilytica isolates revealed that the prophage region was unique to strain JARB-RN-0044. The existence of a highly carbapenem-resistant R. mannitolilytica isolate may raise human health concerns in Japan, where the population is rapidly aging. IMPORTANCE Ralstonia mannitolilytica is an aerobic non-fermenting Gram-negative rod commonly found in aquatic environments and soil. The bacteria can occasionally cause severe hospital-acquired bloodstream infections in immunocompromised patients and it has been recently recognized as an emerging opportunistic human pathogen. Furthermore, some R. mannitolilytica isolates are resistant to various antimicrobial agents, including β-lactams and aminoglycosides, making antimicrobial therapy challenging and clinically problematic. However, clinical awareness of this pathogen is limited. To our knowledge, in Japan, there has been only one report of a carbapenem-resistant R. mannitolilytica clinical isolate from urine by Suzuki et al. in 2015. In this study, whole-genome sequencing analysis revealed the presence and genetic context of novel blaOXA-1176 and blaOXA-1177 genes on the 1.5 Mb megaplasmid from highly carbapenem-resistant R. mannitolilytica isolate and characterized the overall distribution of functional genes in the chromosome and megaplasmid. Our findings highlight the importance of further attention to R. mannitolilytica isolate in clinical settings.

R alstonia mannitolilytica is an aerobic non-fermenting Gram-negative rod commonly found in aquatic environments and soil (1).The bacteria can occasionally cause severe hospital-acquired bloodstream infections in immunocompromised patients (2)(3)(4)(5).The disease caused by R. mannitolilytica can progress rapidly and sometimes lead to septic shock symptoms and multiple organ dysfunction syndromes (1,6).However, clinical awareness of this rare pathogen is limited.In addition, some R. mannitolilytica isolates are resistant to various antimicrobial agents, including β-lactams and amino glycosides, making antimicrobial therapy challenging and clinically problematic (4,5).Recently, healthcare-associated infections due to R. mannitolilytica related to contamina ted water and medical devices have been reported worldwide (7)(8)(9), and it is increasingly recognized as an emerging opportunistic human pathogen.
In 2020, the R. mannitolilytica strain JARB-RN-0044 was isolated from a midstream urine sample of a female patient in her 70s in a general hospital in Mie Prefecture, southwest of the Chubu region, Japan.Antimicrobial susceptibility testing was per formed with Neg MIC EN 2J (Beckman Coulter, Brea, CA, USA) and NQJ2 (Eiken Chemical Co., Tokyo, Japan) panels and interpreted according to minimum inhibitory concen tration (MIC) breakpoints for Pseudomonas aeruginosa from Clinical and Laboratory Standards Institute documents M100-ED30 guidelines (10).The isolate exhibited high MICs against piperacillin, imipenem, meropenem, and doripenem; however, it remained susceptible to levofloxacin and ciprofloxacin (Table S1).The isolate was negative for screenings of carbapenemase producers using modified carbapenem inactivation methods, mCIM and CIMTris (11,12).
The R. mannitolilytica strain JARB-RN-0044 was identified by using matrix-assisted laser desorption ionization-time of flight mass spectrometry (Bruker Daltonics Japan, Yokohama, Japan) with a score of 2.38.Pair-wise average nucleotide identity based on MUMmer calculation (ANIm) analysis using JSpeciesWS online tool (18) revealed that strain had the highest ANIm value of 99.4% with the R. mannitolilytica strain WCHRM065837 (GenBank assembly accession number GCA_002939145).The hybrid assembly yielded a large circular replicon with a size of 1.5 Mb in addition to a chro mosome with a size of 3.5 Mb with a mean G + C content of 66.1% (Fig. S1).To date, the functional role of large non-chromosomal replicon as the "megaplasmid" in other Ralstonia spp.has been clarified, while it has been completely unknown in R. mannitolily tica.The 3.5 Mb chromosome harbored by strain JARB-RN-0044 carries several localized housekeeping genes (rpoB, rnpA, rpmH, dnaA, dnaN, and gyrB) and two copies each of the 16S, 23S, and 5S rRNA genes; however, no rRNA genes were found on the 1.5 Mb replicon.The 1.5 Mb replicon carried the repA-parA-parB gene cluster involved in plasmid replication and partitioning systems and 12 conserved iteron repeats (G/CCGTACCCG/ ATTTCTGCG) essential for RepA binding previously identified in Ralstonia solanacearum isolate (18).Overall, the 3.5 Mb chromosome and 1.5 Mb replicon consisted of 3,336 and 1,390 protein-coding genes, respectively, and the COG were mainly classified into 19 categories, more than half of which had different distribution characteristics between the two replicons (Fig. 1).Particularly, gene families involved in replication, recombination, repair, and metabolism of nucleotides and coenzymes were distributed primarily on the chromosome compared with the 1.5 Mb replicon (Fisher's exact test, P < 0.001); however, those involved in transcription and cell motility were significantly abundant on the 1.5 Mb replicon (P < 0.001).As this was similar to a previously described functional distribution found in megaplasmids possessed by R. solanacearum strains (19)(20)(21)(22), we refer to the 1.5 Mb replicon as a presumptive "megaplasmid" in this study.
The R. mannitolilytica strain JARB-RN-0044 harbored two novel OXA-60 and OXA-22 family class D β-lactamase genes encoding carbapenemase and narrow spectrum oxacillinase on the megaplasmid, which were newly assigned as bla OXA-1176 (RefSeq accession number NG_148663) and bla OXA-1177 (RefSeq accession number NG_148664) genes, respectively.OXA-1176 and OXA-1177 had two amino acid substitutions compared with OXA-571 (GenBank accession number MG736318) and OXA-572 (GenBank accession number MG736319) possessed by the R. mannitolilytica isolate WCHRM065837 in China, respectively, and both shared 99.3% amino acid identity each.The amplified products of bla OXA-1176 and bla OXA-1177 genes were subcloned into the pTAKN-2 vector (BioDynamics Laboratory Inc., Tokyo, Japan) carrying kanamycin resistance gene and chemically transformed into ECOS X Competent Escherichia coli DH5α (Nippon Gene, Tokyo, Japan).The cloning and expression of bla OXA-1176 gene clearly indicated that the E. coli DH5α recombinant clone harboring pTAKN-2/OXA-1176 showed more than fourfold increases in the MICs of imipenem, meropenem, and doripenem, indicating that OXA-1176 is presumed to have hydrolytic activity against carbapenem, consistent with previous report (23) (Table S1).In contrast, the E. coli DH5α recombinant clone harboring pTAKN-2/OXA-1177 was susceptible to all carbapenems tested; however, it showed a 16-fold increase in the MICs of piperacillin and cefazolin, but not of carbapenem.
Notably, the ca.44.6 kb putative prophage region containing genes encoding phage integrase, terminase, head and tail protein was identified in the downstream region of bla OXA-1176 gene (Fig. 2).Attachment sites attL and attR were not detec ted at the ends of this prophage region.Comparative analysis with previously repor ted R. mannitolilytica isolates WCHRM065837 and MRY14-0246 (GenBank assembly accession number GCA_000953875.1) harboring OXA-60 family class D β-lactamase genes bla OXA-571 and bla OXA-444 , respectively, revealed that the upstream regions of these genes were conserved, while the downstream regions were relatively variable, and the prophage region was unique to strain JARB-RN-0044, even though the disrupted int gene was found in strain WCHRM065837.BLAST searches did not show any homol ogous sequences to this prophage region; thus, its origin could not be inferred, and future studies to elucidate the function of this prophage and its association with the bla OXA-1176 gene are warranted.Meanwhile, bla OXA-1177 gene was flanked upstream by tRNA Leu gene and downstream by the genes encoding the ArsR family transcriptional regulator and major facilitator superfamily (MFS) transporter, both of which were also found in strain WCHRM065837 harboring bla OXA-572 gene, and not in strain MRY14-0246 harboring bla OXA-443 gene (Fig. S2).
In conclusion, this study confirmed the presence and genetic context of novel bla OXA-1176 and bla OXA-1177 genes on the 1.5 Mb megaplasmid and the overall distribution of functional genes.However, there were several essential genes on the megaplasmid that may be crucial for bacterial survival, such as amino acid and nucleotide metabolic systems, as well as on the chromosome, and further studies are required to better understand the role of this large replicon.To the best of our knowl edge, in Japan, there has been only one report of a carbapenem-resistant R. mannito lilytica strain from urine in 2015, as described above (24).The current trends of this opportunistic pathogen in Japan, which has limited treatment options, remain elusive.A recent study has also reported bloodstream infection caused by R. mannitolilytica isolate in a hospitalized patient with coronavirus disease 2019 (COVID-19) and the higher abundance of Ralstonia spp. in the gut microbiota of patients with severe COVID-19 (25,26).The existence of a highly carbapenem-resistant R. mannitolilytica isolate may raise human health concerns in Japan, where the population is rapidly aging and requires further attention to this rare pathogen in clinical settings.S1 (Spectrum03919-23-s0003.pdf).MICs of antimicrobial agents for the R. mannitolilytica strain JARB-RN-0044 and E. coli DH5α transformants expressing OXA-1176 and OXA-1177.