Increased clonal dissemination of OXA-232-producing ST15 Klebsiella pneumoniae in Zhejiang, China from 2018 to 2021

Background OXA-232-producing Klebsiella pneumoniae was first identified in China in 2016, and its clonal transmission was reported in 2019. However, there are no prevalence and genotypic surveillance data available for OXA-232 in China. Therefore, we investigated the trends and characteristics of OXA-232 type carbapenemase in Zhejiang Province, China from 2018 to 2021. Methods A total of 3278 samples from 1666 patients in the intensive care units were collected from hospitals in Zhejiang Province from 2018 to 2021. Carbapenem-resistant isolates were initially selected by China Blue agar plates supplemented with 0.3 μg/ml meropenem, and further analyzed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry identification, immune colloidal gold technique, conjugation experiment, antimicrobial susceptibility testing and whole genome sequencing. Results A total of 79 OXA-producing strains were recovered, with the prevalence increased from 1.8% [95% confidence interval (CI): 0.7–3.7%] in 2018 to 6.0% (95% CI: 4.4–7.9%) in 2021. Seventy-eight strains produced OXA-232 and one produced OXA-181. The blaOXA-232 gene in all strains was located in a 6141-bp ColKP3-type non-conjugative plasmid and the blaOXA-181 gene was located in a 51,391-bp ColKP3/IncX3-type non-conjugative plasmid. The blaOXA-232-producing K. pneumoniae was dominated (75/76) by isolates of sequence type 15 (ST15) that differed by less than 80 SNPs. All OXA-producing strains (100%, 95% CI: 95.4–100.0%) were multidrug-resistant. Conclusions From 2018 to 2021, OXA-232 is the most prevalent OXA-48-like derivative in Zhejiang Province, and ST15 K. pneumoniae isolates belonging to the same clone are the major carriers. The transmission of ColKP3-type plasmid to E. coli highlighted that understanding the transmission mechanism is of great importance to delay or arrest the propagation of OXA-232 to other species. Supplementary Information The online version contains supplementary material available at 10.1186/s40249-023-01051-w.


Background
Antibiotic resistance has become one of the major stumbling blocks on the road to human health. It is worth noting that carbapenem-resistant Enterobacterales (CRE) played a significant role in this challenge. The oxacillinase OXA-48 was first identified in 2004 from a clinically collected Klebsiella pneumoniae isolate in Turkey [1]. Since then, OXA-48-like enzymes have emerged and spread rapidly around the world and remain endemic in the Mediterranean Region, such as Turkey, Lebanon, and Egypt [29]. OXA-181, which contains four amino acid substitutions compared to OXA-48, was first discovered in India and is prevalent in the Indian subcontinent [3][4][5][6]. OXA-232 was first reported in 2013 from K. pneumoniae and Escherichia coli obtained from three French patients with a travel history to India. Such carbapenemases have also caused nosocomial outbreaks in different countries [7][8][9][10][11]. OXA-181 and OXA-232 are the most common OXA-48 derivatives, differing by only one amino acid substitution [7,12].
Bacterial strains carrying the bla OXA-232 gene were first reported in China in 2016. The gene is located on a ColKP3-type nonconjugative plasmid (also known as ColE-type) from sequence type (ST) 15 K. pneumoniae, and the subsequent clonal dissemination was reported in 2018 [8,9]. The ColKP3-type plasmids have been identified in many countries, such as ST14 K. pneumoniae in South Korea, ST14, ST231, ST395 K. pneumoniae and ST167 E. coli in Canada, ST16 K. pneumoniae in Italy [10,13,14]. OXA-181-producing E. coli and K. pneumoniae isolates had been reported sporadically in China with all bla OXA-181 genes located on the IncX3-type plasmid [15][16][17][18]. Moreover, bacteria co-producing OXA-48 family and other carbapenemases such as K. pneumoniae carbapenemase (KPC) and the New Delhi metallo-βlactamase (NDM) were intermittently reported worldwide [19][20][21]. In 2017, OXA-232 associating with ST147 K. pneumoniae was reported in Tunisia with the coproduction of extended-spectrum β-lactamase (ESBL) CTX-M-15 [22]. The co-production of OXA-232 with various key enzymes could increase the MICs to carbapenems and cause resistance to other common antibiotics such as extended-spectrum cephalosporins, ceftazidime/avibactam, and therefore pose a looming threat to human health. So far, only a limited number of nosocomial OXA-48-like carbapenemases reports were published in Zhejiang Province, long-term surveillance focusing on its prevalence and molecular characteristics is therefore urgently needed to provide a reference to making future surveillance strategy [8,18]. Herein, we designed this study to identify the prevalence and molecular characteristics of bla OXA -producing CRE in Zhejiang Province from 2018 to 2021.

Sample collection
This experiment was designed to investigate the prevalent trends and characteristics of OXA type carbapenemase in Zhejiang, China, from 2018 to 2021. A total of 2512 respiratory and rectal swabs from 1283 ICU patients were collected in six regions of Zhejiang Province, China, from March 2020 to June 2021. 1576 respiratory and rectal swabs from 788 ICU patients were collected from six cities in Zhejiang Province, including Hangzhou, Taizhou, Jinhua, Wenzhou, Lishui, and Quzhou in 2021. 936 respiratory and rectal swabs of 495 patients were collected from eight cities in Zhejiang province, including Hangzhou, Taizhou, Jiaxing, Huzhou, Shaoxing, Zhoushan, Ningbo, and Wenzhou in 2020 (Additional file 2: Table S1). In addition, ten OXA-producing Enterobacterales were recovered from 766 respiratory and rectal swabs of 383 ICU patients from six cities in Zhejiang Province in 2018 were also included in this study [8]. All samples were collected from patients who signed informed consent form when they were completing the admission procedures.

Bacterial cultivation and resistance genes primary screening
Firstly, the respiratory and rectal swabs were incubated overnight at 37 °C in Luria-Bertani (LB) broth (Oxoid, UK) for enrichment. The broth suspensions were then inoculated onto China Blue agar plates containing 0.3 μg/ml meropenem and incubated overnight in 37 °C. All colonies were subjected to identification using the matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometry (MALDI-TOF MS) (Fosun Diagnostics Co., Ltd, Shanghai, China). The presences of carbapenemase genes were screened by NG-Test ® CARBA 5 (Zhongshengzhongjie Bio-Technology Co., Ltd., Changsha, China).

Conjugation
The conjugation experiment was performed by the filtermating method using rifampin-resistant E. coli EC600 as recipient as previously reported [25]. In short, the donor strain and recipient strain E. coli EC600 were cultivated at 37 °C in LB broth for four hours, respectively, and then co-incubated overnight on a membrane placed on Columbia Blood Agar plate. Membranes were swirled in Luria-Bertani (LB) broth, the suspension cultured overnight at 37 °C on Mueller-Hinton (MH) agar plates containing 1 μg/ml meropenem and 600 μg/ml rifampin. Finally, the transconjugants were verified by THE MALDI-TOF MS and Polymerase Chain Reaction [26,27].

Whole genome sequencing and bioinformatics analysis
Genomic DNA of all the OXA-producing isolates were extracted using the PureLink Genomic DNA Mini Kit (Invitrogen, Carlsbad, CA, USA) following manufacturer's instructions and submitted for whole genome sequencing using the Illumina HiSeq X Ten platform (Novogene, Beijing, China). The Illumina reads were de novo assembled using SPAdes Genome Assembler version 3.15.1 [28]. Assembled draft genome sequences were annotated with Prokka version 1.14.5 [29]. Single nucleotide polymorphisms (SNPs) were identified via mapping of Illumina raw reads to genome of strain K210003 as reference. An alignment of core SNPs was produced using Snippy and used to build a high-resolution phylogeny [30]. Lineages were defined based on patristic distances in the maximum-likelihood (ML) tree using IQ-Tree [31]. The output tree was then merged to attain a dated tree with the online TreeAnnotator software iTOL [32]. Bioinformatics analysis including species identity, multilocus sequence typing (MLST) and identification of antimicrobial resistance genes (ARGs) of K. pneumoniae was conducted with Kleborate [33]. Capsular typing on the assembled sequences was performed using Kaptiveg [34]. Multilocus sequence typing of E. coli were confirmed on the center for genomic epidemiology platform [35]. Plasmid replicons were identified by PlasmidFinder [36].
To obtain the complete genome of strains K210049 and K210065, genomic DNA of these two strains were also subjected to the long-read Oxford Nanopore Technologies MinION platform (Oxford Nanopore Technologies, Abingdon, United Kingdom) after treated with supplementary sequencing kit (Oxford Nanopore Technologies, Abingdon, United Kingdom). Both short and long reads were de novo hybrid assembled using Unicycler version 0.4.8 [37]. Alignment of plasmids with similar structures were generated by Easyfig_win_2.1 and BLAST Ring Image Generator (BRIG) version 0.95.22 [38,39].

Data analysis
Data was organized and analyzed by Statistical Products and Services Solutions (SPSS) software (IBM, USA), and 95% confidence intervals (CIs) were calculated by the exact binomial method.

Genomic characteristics of OXA-producing strains
All 79 OXA-producing isolates were subjected to WGS, and 77 were identified to be K. pneumoniae and two were E. coli ( Table 1). The bla OXA genes were found to be bla OXA-232 (78/79) and bla OXA-181 (1/79). Among the 77 OXA-producing K. pneumoniae strains, 75 belonged to ST15/KL112, one belonged to ST37/KL118 and one belonged to ST101/KL106. The ST101 strain K210184, which was recovered from a patient (P16) when he was hospitalized in Jinhua city, also harbored the bla KPC-2 gene except for the bla OXA-232 gene (Fig. 1). In addition, ST15 strain K210005 which harbored the bla OXA-232 gene and ST11 strain K210004 which harbored the bla KPC-2 gene (not included in this study) were also separated from the same patient during his earlier hospitalization in Hangzhou city. The OXA-232 positive strain K210284 recovered from patient P45 also carried the bla NDM-1 gene. While the ST37 strain K210065 collected in 2021 harbored the bla OXA-181 gene other than the bla OXA-232 gene (Fig. 1). The two OXA-producing E. coli strains belonged to ST38 and ST39, respectively, and were collected from patients P1 and P2 in the same ward. Notably, the OXA-232-producing K. pneumoniae strains K210037 and K210022 were isolated from patients P1 and P2, respectively, indicating potential transmission of the bla OXA-232 -carrying determinants from K. pneumoniae to E. coli (Fig. 1).
As the ten K. pneumoniae stains isolated in 2018 exhibited only a few SNPs and belonged to a single clone, we performed pairwise SNP analysis of all the ST15 K. pneumoniae stains. The strains isolated in 2020 from Jiaxing and Taizhou differed from the isolates in 2018 with < 50 SNPs (Additional file 3: Table S2). The nine isolates from Taizhou in 2021 together with three isolates from Hangzhou exhibited SNPs < 8. 31 strains isolated from Hangzhou in 2021 exhibited SNPs < 10. Another nine strains isolated in this region exhibited SNPs < 4. These two clones differed with < 10 SNPs. The ST15 OXA-producing K. pneumoniae stains have circulated in Zhejiang Province in the past few years as all the ST15 K. pneumoniae stains differed with SNPs < 80. The ST15 remained the most prevalent OXA-232 K.

Discussion
Antimicrobial resistance has become one of the major global concerns, and carbapenems have become antibiotics of last resort. OXA-48-like enzymes possessed a stronger ability to hydrolyze oxacillin but had low activity against carbapenems and is therefore known as oxacillinases [40]. In the current study, numerous OXA-positive strains possess a similar carbapenem resistance profile, only resistant to ertapenem but susceptible to meropenem and imipenem, known as 'the phantom menace' in the literature [41]. Previous studies of OXA-232 only described its genetic characteristics but did not describe its prevalent situation in Zhejiang hospitals [8]. Our study collected samples from 2018 to 2021 and described both prevalence and genetic characteristics of OXA in hospitals in Zhejiang Province.
Two clones from Hangzhou in 2021 differed with < 10 SNPs, suggesting that they might originate from a single clone. The dominant clone transmission and the increased prevalence of OXA suggest that close monitoring of OXA is needed to curtail CRE spread and thus reduce the incidence of disease. Moreover, our study has found that OXA-232 was reported in more cities' hospitals in Zhejiang in recent years than before, which  indicated its wide transmission trend. So continuous surveillance was strongly recommended to minimize the problems associated with oxacillinases. The ColKP3-type nonconjugative plasmid is approximately 6 kb in length harbored by all strains collected in our study. To date, only ST15 K. pneumoniae had been reported to contain the bla OXA-232 gene situated within the OXA-232 plasmid in China [8,9,18], ST101 K. pneumoniae and two E. coli strains were shown to produce OXA-232 in our study. Additionally, the presence of OXA-232-positive E. coli and K. pneumoniae in the same patient reflects the horizontal transfer of ColKP3type plasmid. However, this small plasmid does not contain genes sufficient for self-transfer. We speculate that it could transmit with the help of other plasmids such as pKP3-A. Further research is needed to investigate the transmissible mechanism of ColKP3 type plasmid to prevent the further transmission to other species.
One of the limitations of our study was that we cannot apply random sampling given the importance of consent. The hospitals that we selected were only the ones agreed to join this study. Samples from different Provinces are highly recommended to be collected in further studies to reflect the prevalence of OXA in China.
Our study highlighted the importance of the combined antimicrobic susceptibility to avoid potential threats to patients, especially to those in ICU, and to reduce the spreading due to under detection.

Conclusions
From 2018 to 2021, OXA-232 is the most prevalent OXA-48-like derivative in Zhejiang Province, and ST15 K. pneumoniae isolates belonging to the same clone are the major carriers. The transmission of ColKP3-type plasmid to E. coli highlighted that understanding the transmission mechanism is of great importance to delay or arrest the propagation of OXA-232 to other species.