Co-occurrence of fosA5, blaSHV-145 and blaOXA-48 among a Klebsiella pneumoniae high-risk ST16 from a tertiary hospital in China: focusing on the phylogeny of OXA-48 genes from global Klebsiella pneumoniae isolates

Since the OXA-48-type carbapenem-hydrolyzing class D β-lactamase was reported in a Klebsiella pneumoniae isolate from Istanbul (Turkey) in 2001 [1], this carbapenemase has been widely distributed among Enterobacterales, with significant geographical differences [2, 3]. Albeit outbreak of nosocomial infections caused by OXA-48-producing K. pneumoniae has been frequently reported in the Mediterranean area and European countries [4–6]. The situation is less common in China, until the nosocomial outbreak of OXA-48-producing K. pneumoniae ST147 and ST383 was reported in a Chinese Hospital [7]. Subsequently, clonal dissemination of KPC-2and OXA-48-coproducing K. pneumoniae sequence type 11 and the nosocomial outbreak of OXA48-producing K. pneumoniae caused by clonal expansion of ST11-KL64 sublineages were found in Taiwan [8, 9]. In this study, we provided data on the genomic characterization of an imipenem intermediate K. pneumoniae strain isolated from the blood culture of a 56-year-old male patient who suffered from bacteremia and septic shock in May 2017. Antimicrobial susceptibility testing toward imipenem, meropenem, piperacillin, ticarcillin, cefoperazone, cefuroxime, cefazolin, cefoxitin, ampicillin, cefepime, ceftriaxone ceftazidime, amikacin, gentamicin, chloramphenicol, levofloxacin, ciprofloxacin, and trimethoprim/sulfamethoxazole was performed by broth microdilution and that of fosfomycin was determined by the agar dilution method using Mueller Hinton medium supplemented with 25 μg/mL glucose-6-phosphate, and the results were interpreted according to the CLSI 2020 (M100-30ED) guideline [10]. To characterize this strain in detail, whole genome sequencing was further performed using an Illumina MiniSeq plus Pacbio Sequencing. Based on the whole genome sequence, subsequent multi-locus sequence typing (MLST) was determined by uploading the genome to the webtool MLST v2.0 (https:// cge. cbs. dtu. dk/ servi ces/ MLST/), and antimicrobial resistance genes and plasmid replicons were identified by ResFinder 3.2 and PlasmidFinder 2.1 (https:// cge. cbs. dtu. dk/ servi ces/). The plasmid carrying blaOXA-48 was detailly characterized by circular plasmid map and comparison of plasmid structures. Additionally, the phylogenomic tree of blaOXA-48 from global K. pneumoniae isolates was further constructed to observe the evolutionary relationship of blaOXA-48. Briefly, the nucleotide sequences of blaOXA-48 gene of 576 strains were compared by muscle, and then single nucleotide polymorphism (SNP) sites were used to extract SNPs from multiple alignment species. Finally, the maximum likelihood tree was constructed using RAxML software [11]. Yanmei Sun and Wei Chen equally contributed to this work

Since the OXA-48-type carbapenem-hydrolyzing class D β-lactamase was reported in a Klebsiella pneumoniae isolate from Istanbul (Turkey) in 2001 [1], this carbapenemase has been widely distributed among Enterobacterales, with significant geographical differences [2,3]. Albeit outbreak of nosocomial infections caused by OXA-48-producing K. pneumoniae has been frequently reported in the Mediterranean area and European countries [4][5][6]. The situation is less common in China, until the nosocomial outbreak of OXA-48-producing K. pneumoniae ST147 and ST383 was reported in a Chinese Hospital [7]. Subsequently, clonal dissemination of KPC-2-and OXA-48-coproducing K. pneumoniae sequence type 11 and the nosocomial outbreak of OXA-48-producing K. pneumoniae caused by clonal expansion of ST11-KL64 sublineages were found in Taiwan [8,9].
In this study, we provided data on the genomic characterization of an imipenem intermediate K. pneumoniae strain isolated from the blood culture of a 56-year-old male patient who suffered from bacteremia and septic shock in May 2017. Antimicrobial susceptibility testing toward imipenem, meropenem, piperacillin, ticarcillin, cefoperazone, cefuroxime, cefazolin, cefoxitin, ampicillin, cefepime, ceftriaxone ceftazidime, amikacin, gentamicin, chloramphenicol, levofloxacin, ciprofloxacin, and trimethoprim/sulfamethoxazole was performed by broth microdilution and that of fosfomycin was determined by the agar dilution method using Mueller Hinton medium supplemented with 25 µg/mL glucose-6-phosphate, and the results were interpreted according to the CLSI 2020 (M100-30ED) guideline [10]. To characterize this strain in detail, whole genome sequencing was further performed using an Illumina MiniSeq plus Pacbio Sequencing. Based on the whole genome sequence, subsequent multi-locus sequence typing (MLST) was determined by uploading the genome to the webtool MLST v2.0 (https:// cge. cbs. dtu. dk/ servi ces/ MLST/), and antimicrobial resistance genes and plasmid replicons were identified by ResFinder 3.2 and PlasmidFinder 2.1 (https:// cge. cbs. dtu. dk/ servi ces/). The plasmid carrying bla OXA-48 was detailly characterized by circular plasmid map and comparison of plasmid structures. Additionally, the phylogenomic tree of bla OXA-48 from global K. pneumoniae isolates was further constructed to observe the evolutionary relationship of bla OXA-48 . Briefly, the nucleotide sequences of bla OXA-48 gene of 576 strains were compared by muscle, and then single nucleotide polymorphism (SNP) sites were used to extract SNPs from multiple alignment species. Finally, the maximum likelihood tree was constructed using RAxML software [11].
Whole-genome sequencing dat a by Illumina MiSeq plus Pacbio found that the K. pneumoniae isolate contained a 5.68-Mb genome, including a 5.31-Mb chromosome and seven different plasmids ( Table 2). Resistant determinants including fosA5, oqxAB, and bla SHV-145 were found in chromosome, whereas, bla OXA-48 was found in a conjugative IncL/M plasmid (Fig. 1). The size of this plasmid is 66,076 bp, with GC content being 55.23% (Fig. 2). The circular structure of plasmid showed that this plasmid is likely to be a conjugative mobile plasmid because of the frequent binding mobile element tra-associated operons region within this plasmid [13] (Fig. 1). As known, IncL/M plasmid has been reported to be the vector for bla OXA-48 [14]. Analysis of f lanking elements of bla OXA-48 gene displayed mobile elements including inser tion sequence 4 (IS4), transposase, and IS kra 4. We found that IS4 was distributed among both of the downstream and upstream of bla OXA-48 gene (Fig. 2). So far, IS4 family element has been repor ted to be involved in mobilization and expression of ß-lactam resistance genes including bla VEB-1 and bla OXA-48 [15]. Transposase has been found to mediate chromosomal integration of exogenous genes in Acidithiobacillus ferrooxidans [16]. Altogether, our study suggested that the bla OXA-48 gene might be rapidly spread by a broad host-range conjugative plasmid. Noteworthily, this is the first time that we identified SHV-145 in a clinical K. pneumoniae isolate in China. SHV-145 is an extended-spectrum beta-lactamase that has been previously found in a K. pneumonia clinical isolate recovered from a hospitalized patient in Portugal (unpublished data: accession number AFN88952.1). To date, SHV-145 has been predicted by the protein homolog model (AMR detection model) as a beta-lactamase which could lead to the inactivation of penam, cephalosporin and carbapenem (https:// card. mcmas ter. ca/ ontol ogy/ 37565# resis tomes-table). Furthermore, SHV-ESBLs are usually encoded by selftransmissible plasmids. However, in our study, bla SHV-145 was identified in Chromosome, which may be mobilized by mobile elements.
The phylogenetic tree based on the bla OXA-48 gene from global 576 K. pneumoniae isolates displayed 2 clades (Fig. 3). The simple evolutionary relationship suggests that this gene is relatively conservative. Concurrently, this may also indicate that bla OXA-48 has a strong transmission ability among K. pneumoniae isolates.
To the best of our knowledge, this is the first report on the co-occurrence of fosA5, bla SHV-145 and bla OXA-48 among the K. pneumoniae ST16 in China and for the first time, we prescribed the bla OXA-48 evolutionary phylogenomic of global-producing K. pneumoniae.   The bla OXA-48 phylogenetic tree based on the global 576 OXA-48-producing Klebsiella pneumoniae isolates. The nucleotide sequences of blaOXA-48 gene of 576 strains were compared by muscle, and then single nucleotide polymorphism (SNP) sites were used to extract SNPs from mul-tiple alignment species. The maximum likelihood tree was constructed using RAxML. Red, the OXA-48-producing K. pneumoniae isolate in our study