Emergence of fosfomycin resistance gene fosZ in carbapenem and fosfomycin-resistant Pseudomonas juntendi isolates in China

With the emergence and spread of plasmid-mediated fosfomycin resistance genes[1-3], fosfomycin has been moved up to the highest priority critically important antimicrobials in the 2024 World Health Organization list of medically important antimicrobials[4,5]. Due to the reduction in treatment options for multidrug resistant bacterial infections, fosfomycin has been reintroduced to treat infections caused by multidrug-resistant Pseudomonas (P.) aeruginosa and carbapenem-resistant Enterobacteriaceae. Carbapenem-resistant-P. aeruginosa have been listed in the high-priority pathogens in the 2024 World Health Organization Bacterial Priority Pathogens List. The increasing prevalence of fosfomycin resistance genes has become a serious challenge in both animals and clinical environments[1-3,6,7]. Recently, a novel plasmid-mediated fosfomycin resistance mechanism, fosZ, has been described in a conjugative IncP-2 plasmid recovered from a carbapenem-resistant P. aeruginosa strain in China[8]. It shows less than 70% amino acid identity with known fosA proteins and displays a fosfomycin minimum inhibitory concentration (MIC) of 128 mg/L. Although the fosZ gene has been identified in six species, including P. aeruginosa, P. putida, P. fulva, P. asiatica, P. oleovorans, and P. defluvii, there are gaps in our understanding of the host range of fosZ. Here, we report the first identification of fosZ from a P. juntendi isolate of human origin in China. A Pseudomonas spp. strain DS24IMWYN200_XXB469 was isolated from the urine sample of a 79-year-old male in a nursing home in Qingpu District, Shanghai, on July 2, 2024, using a Columbia blood agar plate. The species was identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and whole- genome sequencing. Antimicrobial susceptibility testing was performed using broth microdilution and interpreted according to the Clinical and Laboratory Standards Institute 2023 standards and the European Committee on Antimicrobial Susceptibility Testing. DS24IMWYN200_XXB469 was resistant to ampicillin, ceftazidime, cephalexin, cefoxitin, cefazolin, cefotaxime, cefuroxime, ceftazidime/avibactam, ampicillin/sulbactam, amoxicillin/ clavulanate, ertapenem, meropenem, imipenem, chloramphenicol, florfenicol, nalidixic acid, ciprofloxacin, fosfomycin, gentamicin, streptomycin, trimethoprim/sulfamethoxazole, tetracycline, and tigecycline, while non- susceptible to polymyxin, colistin, and cefepime, but susceptible to amikacin, and azithromycin (Figure 1A and Supplementary Table 1). The exact MIC values and corresponding CLSI 2023 and EUCAST interpretive criteria were determined for imipenem, meropenem, and tigecycline (Supplementary Table 1).Figure 1: Comparative analysis of fosZ-harbor P. juntendi genomes and comparison of the genetic context of fosZ. A: Susceptibility of the fosZ-positive strain of P. juntendi to commonly used antibiotics. R: resistant; I: intermediate; S: sensitive. B: Phylogenetic analysis of fosZ-harbor P. juntendi and 69 publicly available genomes. From the inner to outer circles are year, source, province (only isolates in China), country, continent, bla NDM, bla KPC, bla VIM-2, and fosZ. The P. juntendi PP_2463 (GCA_021560075.1) was used as the reference genome. C: Genetic environment of the fosZ gene in P. juntendi and P. aeruginosa HS17-127 (OR827686.1). Different genes are shown in different colors. Arrows indicate: insertion sequence (light blue), transposase genes tnpA/tnpB (dark blue), resistance gene fosZ (yellow). Regions of >84% identity are indicated by dark grey shading. D: Genetic environment of the bla vim -2 gene in P. juntendi genome. P. putida NY5709 (MN961670.1) and P. putida ZXPA-20 (CP061724.1). Different genes are shown in different colors. Arrows indicate: bla VIM-2 (green), Tn3 transposon (purple), and other genes (orange). E: Genetic context of tmexC3-tmexD3-topr J3 in the fosZ-positive P. juntendi genome. P. fulva ZDHY316 (CP064945.1), and P. fulva ZDHY414 (CP064948.1). Different genes are shown in different colors. Arrows indicate: tmexC3-tmexD3-toprJ3 (pink), and other genes (orange).The genomic DNA of DS24IMWYN200_XXB469 was extracted and sequenced by the DNBSEQ-T7 platform and assembled by Unicycler v0.5.0 (https://github.com/rrwick/Unicycler), and then generated a total length of 6070833 bp with an N50 of 59046 bp and GC content of 62%. The species was identified by both gtdb-Tk v2.6.1 (https://github.com/Ecogenomics/GTDBTk) and MetaPhlAn v4.2.4 (https://github.com/biobakery/MetaPhlAn), indicating that DS24IMWYN200_XXB469 belongs to P. juntend. DS24IMWYN200_XXB469 harbored one chromosome and one plasmid using MOB-suite v3.1.9 (https://github.com/phac-nml/mob-suite). The completeness and contamination of the genome was 99.94% and 0.55%, respectively, according to CheckM2 (https://github.com/chklovski/CheckM2). A total of 5719 predicted coding sequences were identified. BLAST analysis showed that DS24IMWYN200_XXB469 harbored multiple antibiotic resistance genes (ARGs), including fosZ, blaVIM.2, aph(3'')-Ib, aac(6')-Ib3, aac(6')-IIa, aph(6)-Id, CmlA9, qnrVC6, tet(G), sull, dfrBl, and transferable resistance-nodulation-division pump gene cluster tmexCD3-toprJ3, using ResFinder (https://github.com/genomicepidemiology/resfinder) and the Comprehensive Antibiotic Resistance Database (https://card.mcmaster.ca/). Notably, aac(6')-IIa, CmlA9, and tet(G) were predicted in the plasmid; other ARGs were predicted in the chromosome. Phylogenetic analysis indicated that the fosZ-positive strain was clustered together with a genome from human urine in Jinhua, Zhejiang, in 2024, and a genome from a fish intestinal isolate in Wenzhou, Zhejiang, in 2022 (Figure 1B and Supplementary Table 2). The strains from different regions of China have significant genetic differences and are distributed on different branches, clustering separately from other regions (for example, Europe and Australia). Among these 70 P. juntendi isolates used in this work, only one fosfomycin resistance gene, fosZ, was identified in DS24IMWYN200_XXB469. To the best of our knowledge, this is the first report of the identification of new discovered fosfomycin resistance gene, fosZ, in a carbapenem- and fosfomycin-resistant P. juntendi isolate. In addition, a total of 35 carbapenem resistance genes, including blaVIM-2 (n=13), blaIMP-1 (n=4), blaIMP-15 (n=3), blaIMP-16 (n= 3) blaIMP-8 (n=1) blaNDM-1 (n= 7) blaNDM-14 (n=1) blaKPC-2 (n=2), and blaBKC-1 (n=1) were identified in P. juntendi genomes (Supplementary Table 2). Further analysis showed that the fosZ- positive strain harbored multiple virulence factors (Supplementary Table 3). Genetic environment analysis revealed that fosZ is located next to an insertion sequence (IS) of the IS66 family. The genetic context of fosZ in carbapenem- and fosfomycin- resistant P. juntendi isolate is TnpA-TnpB-ISPa75-fosZ (Figure 1C), which is similar to those reported previously[8]. The genetic context of blaVIM-2 located on the chromosome of P. juntendi was dfrB1-aac(6')-Ib3-blaVIM-2-TniR-TniQ-TniB-TniA (Figure 1D), which was similar to the previous report that blaVIM-2 was in a P. putida plasmid of migratory bird origin[9]. The genetic context of the RND-type efflux pump gene cluster tmexC3-tmexD3-toprJ3 is similar to clinical P. fulva strains isolated from the bile specimen of a liver transplant patient in Zhengzhou, Henan Province, on May 4, 2019[10], that carry plasmid-mediated blaVIM.24 and tmexCD3-toprJ3. No other mobile genetic elements were observed around tmexC3- tmexD3-toprJ3 (Figure 1E). In summary, to our knowledge, this is the first report of the convergence of fosZ, blaVIM-2, and tmexC3-tmexD3-toprJ3 in P. juntendi isolated from a human, which expanded our knowledge of the host reservoir of fosZ. To better protect human, animal, and environmental health worldwide, a continuous surveillance of carbapenem, tigecycline, and fosfomycin resistance in different environments is urgently needed to fight the emergence and dissemination of these resistant pathogens. Overall, our study highlights the emergence of blaVIM-2, fosZ, and tmexC3-tmexD3- toprJ3, which are predicted to be associated with resistance to the last-line antibiotics, carbapenem, fosfomycin, and tigecycline, and may pose a huge challenge to clinical therapy in the future. Ethical approval The participant provided written informed consent and allowed analysis of strains and data for medical case report. Conflict of interest statement The authors declare that there is no conflict of interest. Funding This study was supported by the Project for the Young Scientist of the Joint Funds of Science and Technology Research and Development Plan of Henan Province, China (235200810058), the National Key Research and Development Program of China (2023YFC2307101), Natural Science Foundation of Henan (262300421124), and the Young TopNotch Talents Foundation of Henan Agricultural University (30501278). Data availability statement The data supporting the findings of this study are available from the corresponding author upon request. Authors’ contributions Wang YN and Xu XB conceived and designed the project. Xu XB collected samples, strains, and epidemiological data. Yang ZR performed bioinformatics analysis, created the figures and tables. Wang YN, Yang ZR, and Xu XB drafted and revised the manuscript. Publisher’s note The Publisher of the Journal remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.