Carriage of distinct blaKPC-2 and blaOXA-48 plasmids in a single ST11 hypervirulent Klebsiella pneumoniae isolate in Egypt

Background Carbapenem-resistant hypervirulent K. pneumoniae (CR-hvKP) causes serious infections with significant morbidity and mortality. However, the epidemiology and transmission mechanisms of CR-hvKP and the corresponding carbapenem-resistant plasmids require further investigation. Herein, we have characterized an ST11 K. pneumoniae strain EBSI041 from the blood sample encoding both hypervirulence and carbapenem resistance phenotypes from a patient in Egypt. Results K. pneumoniae strain EBSI041 showed multidrug-resistance phenotypes, where it was highly resistant to almost all tested antibiotics including carbapenems. And hypervirulence phenotypes of EBSI041 was confirmed by the model of Galleria mellonella infection. Whole-genome sequencing analysis showed that the hybrid plasmid pEBSI041-1 carried a set of virulence factors rmpA, rmpA2, iucABCD and iutA, and six resistance genes aph(3′)-VI, armA, msr(E), mph(E), qnrS, and sul2. Besides, blaOXA-48 and blaSHV-12 were harboured in a novel conjugative IncL-type plasmid pEBSI041-2. The blaKPC-2-carrying plasmid pEBSI041-3, a non-conjugative plasmid lacking the conjugative transfer genes, could be transferred with the help of pEBSI041-2, and the two plasmids could fuse into a new plasmid during co-transfer. Moreover, the emergence of the p16HN-263_KPC-like plasmids is likely due to the integration of pEBSI041-3 and pEBSI041-4 via IS26-mediated rearrangement. Conclusion To the best of our knowledge, this is the first report on the complete genome sequence of KPC-2- and OXA-48-coproducing hypervirulent K. pneumoniae from Egypt. These results give new insights into the adaptation and evolution of K. pneumoniae during nosocomial infections. Supplementary Information The online version contains supplementary material available at 10.1186/s12864-021-08214-9.

them, the IncF and IncA/C type plasmids are predominantly responsible for the transfer of bla KPC and bla NDM , respectively [4,5]. Besides, the bla KPC gene is strongly associated with Tn4401 flanking by ISKpn6 and ISKpn7, and the bla NDM gene is closely related to Tn125 structure with two ISAba125 elements [6,7]. Unlike KPCs and NDMs, IncL group plasmid has been shown to be the major genetic carrier for bla OXA in K. pneumoniae and the composite transposon Tn1999 is mainly responsible for integration of the bla OXA gene [4]. Furthermore, dissemination of KPC-producing K. pneumoniae in worldwide is largely caused by expansion of the dominant ST258 clones [8]. Differently, the bla OXA and bla NDM genes are detected in various K. pneumoniae clones, in which ST11 is a major high-risk sequence type in many countries, such as China [9,10], Australia [11], Poland [12], Spain [13] and Turkey [14]. Co-carriage of different carbapenems resistance genes is not common but renders clinical K. pneumoniae strains extremely highly resistant to different carbapenems, which leads to more difficult infection treatment [15,16].
Seriously, carbapenem-resistant hypervirulent K. pneumoniae (CR-hvKP) has been increasingly reported in nosocomial infection and can cause higher mortality [17,18]. The plasmid-mediated genetic factors conferring the hypervirulent phenotype including rmpA and rmpA2 (regulators that increase capsule production), and several siderophore gene clusters [19]. Notably, a multinational prospective cohort study warns of the severity of carbapenem resistance in low-income and middleincome countries, including Egypt [3]. In the previous studies, the high prevalence of carbapenemase-mediated resistance in K. pneumoniae isolates in the clinical setting from Egypt was reported [20][21][22][23], but few studies have analyzed the genome characteristic of carbapenemresistant K. pneumoniae by whole-genome sequencing. In this study, we report the in-depth characterization of co-producing bla KPC-2 and bla OXA-48 hypervirulent ST11 K. pneumoniae strain from Egypt. The dissemination of two plasmids carrying bla OXA-48 and bla KPC-2 and novel plasmid structures were identified.
In vitro conjugation demonstrated that carbapenem resistance genetic factors in EBSI041 can be transferred to the recipient strains E. coli J53 and EC600. Further S1-PFGE and PCR assay revealed OXA-48-carrying plasmid (~ 90 kb) can be self-transferred and KPC-2-producing plasmid has no transferability. However, KPC-2-producing plasmid can be transferred to the recipient strains with the help of OXA-48-carrying plasmid, and the two plasmids may be fused a larger plasmid (~ 180 kb) carring both bla OXA-48 and bla KPC-2 according to results of S1-PFGE (Fig. S2).
For source tracking bacterial pathogens, EBSI041 was found to be similar to a ST11 K. pneumoniae strain (WJTB01) isolated from bronchoalveolar lavage of patient in China, with 317 SNPs, based on SNP (sequence-based) strategy using BacWGSTdb 2.0 [24]. The core-genome-based MLST (cgMLST) analysis showed that 67 strains were less than or equal to 50 alleles different from EBSI041. All strains were isolated from China, except one from USA. EBSI041 and HA_74 (PJOU01) were on the same branch of the phylogenetic tree (Fig. S3). ST11 K. pneumoniae strain HA_74 was isolated from China in 2015. However, because of limited sources from Egypt or Africa, we were not able to clarify the domestic transmission route.

The MDR pEBSI041-1 co-harbouring virulence genes via recombination
The plasmid pEBSI041-1 is 299,522 bp in size and belongs to an IncFIB:IncHI1B type hybrid plasmid. A 27,568-bp multidrug-resistance (MDR) module in pEBSI041-1 harboured six resistance genes armA, msr(E), mph(E), qnrS, aph(3′)-VI and sul2. The armA gene was mediated by an intact IS26 (IS6 family, 820 bp) element upstream. With a 2300-bp space to armA downstream, a locus of msr(E) and mph(E) was flanked by an intact ISEc29 (IS4 family, 1325 bp) (Fig. 1). For the rest of ARGs, each of them was flanked independently by different IS elements. This module was almost identical to the sequences in plasmid p51015_NDM_1 (CP050380) which was identified in a human K. pneumoniae isolate in the Czech Republic. The only difference between them was an inversion of a 7710-bp sequence bounded by two IS26 occurred in p51015_NDM_1. Besides this MDR module, pEBSI041-1 harbours the plasmid-located virulence factors, including regulator of the mucoid phenotype (rmpA), the regulator of mucoid phenotype 2 (rmpA2), aerobactin (iucABCD, iutA) ( Fig. 1). A BLASTn search showed that a 36,929-bp sequence containing the virulence genes in pEBSI041-1 was identical to the sequences in pF16KP0084-1 (CP052159.1; South Korea). Furthermore, close to this sequence, a 37,030-bp sequence was also found to be identical to the sequence in pF16KP0084-1 with a reversion order, while the sequence harbours the virulence gene cluster iroBCD and iroN in pF16KP0084-1 was lacking in our plasmid (Fig. 1). Also, these two sequence fragments in pEBSI041-1 caused the main difference to plasmid pKpvST101_5 (CP031372.2; United Kingdom), indicating that the emergence of the MDR-virulent pEBSI041-1 was due to the transfer of virulence determinants into a pKpvST101_5-like MDR plasmid. Sequence alignments showed that pEBSI041-1 shared > 99% identity with plasmid pKpvST147B_virulence (CP040726.1; United Kingdom), pKpvST383L (CP034201.2; United Kingdom), and p51015_NDM_1 (CP050380.1) with query coverages of 96-99.5%, all of which are MDR-virulent hybrid plasmids (Fig. 1).  2). pEBSI041-2 exhibited a high similarity (80% coverage, 99.58% identity) with plasmid pOXA-48_1639 (CP025105.1), which also encodes OXA-48 and identified from E. coli. Besides, pEBSI041-2 was also similar to plasmids pOXA-48_920 (LR025095.1) and pUR17313-1 (KP061858.1), which are from K. pneumoniae and Enterobacter cloacae respectively. This indicates that pOXA-48_1639-like plasmids have been widely spread among bacteria of different species. However, compared with pOXA-48_1639, an 18,779-bp sequence harbouring bla SHV-12 was missing in pEBSI036-2. The bla SHV-12 gene was surrounded by two IS26 (IS6 family, 820 bp) elements, and this 6100-bp fragment was  (Fig. 2). The mobilization of bla SHV-12 can be attributed to the gene exchange by recombination, while the acquisition of exogenous chromosome sequence needs to be further considered. Indeed, the conjugation experiment demonstrated that pEBSI041-2 transferred from the donor strain K. pneumoniae EBSI041 to the recipients E. coli J53 and EC600.

Discussion
ST11 is the dominant clone of carbapenemases-producing K. pneumoniae in Asia, especially in China, and ST11 clone was reported to account for up to 60% of carbapenem-resistant K. pneumoniae [9,29,30]. Recently, ST11 has been reported in several clinical infection cases from African countries, such as Egypt [31,32] and Tunisian [33]. And in ST11 carbapenem-resistant K. pneumoniae isolates, the most predominant carbapenemase genes are bla KPC-2 , bla NDM-1 and bla OXA-48 [10,34]. The emerging threat of carbapenem resistance of K. pneumoniae in Egyptian hospitals has been highlighted over recent years [3,35]. To the best of our knowledge, this is the first report on the complete genome sequence of KPC-2and OXA-48-coproducing virulent K. pneumoniae from Egypt.
The uncommon co-carriage of genes encoding different classes of carbapenemases endowed EBSI041 with high carbapenems resistance. Not restricted in this study, the co-harboring bla KPC-2 and bla OXA-48 in K. pneumoniae isolates were also found from other clinical setting [15]. The presence of carbapenemase genes on mobile elements greatly promotes the spread and stacking of carbapenems resistance. The bla OXA-48 -carrying pEBSI041-2 was identified as an IncL/M-type plasmids, which are commonly self-conjugative among Enterobacteriaceae according to the previous studies [36]. It's worth noting that non-conjugative pEBSI041-3 carried bla KPC-2 gene, was successfully transferred with the help of pEBSI041-2. The results warn that the mechanisms of bla OXA-48 or bla KPC-2 -carrying plasmid transfer need to be further studied to better control the spread of carbapenemaseproducing K. pneumoniae.
The genetic structure of bla OXA-48 or bla KPC-2 -carrying plasmid in this study is different from that of reported plasmids due to the presence of multiple transposons and insertion sequences. The bla OXA-48 -carrying pEBSI041-2 was similar to other IncL/M plasmids previously sequenced, the majority of which only carry bla OXA-48 [15]. However, pEBSI041-2 harboured an exogenetic chromosome-located fragment and acquired additional resistance gene bla SHV-12 due to the recombination of the IS26-like elements. In pEBSI041-3, the bla KPC-2 gene was located on a transposon unit with the core structure of ΔISKpn6-bla KPC-2 -ISKpn27 as that reported [15,25]. Further, pEBSI041-3 and MDR plasmid pEBSI041-4, which carried eight IS26 elements, almost constitutes another KPC-2-producing plasmids [27,28]. Therefore, the novel genetic structure of these plasmids are likely to be created by IS-mediated recombination.

Conclusion
This study reported the co-carriage of distinct bla KPC-2 and bla OXA-48 plasmids in a single ST11 hypervirulent Klebsiella pneumoniae isolate in Egypt. The recombination and rearrangement of MDR plasmids and virulent plasmids have occurred during evolution. These results give new insights into the adaptation and evolution of K. pneumoniae plasmids during nosocomial infections in Egypt.

Bacterial strain
K. pneumoniae EBSI041 was collected from the blood sample of a patient in Egypt. The clinical strain was initially isolated on MacConkey agar (Oxoid, UK). Species identification was determined primarily with an automated VITEK ® 2 AST-16 Gram-negative susceptibility card (bioMérieux, Marcy-l'Étoile, France) and confirmed by matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS). Investigation of carbapenemase production in the routine hospital laboratory procedure using the Modified Hodge Test (MHT) [37] and Carba NP test [38] showed that EBSI041 was resistant to carbapenems. We tested EBSI041 for carbapenems-resistant genes and confirmed that it carried bla OXA-48 and bla KPC-2 . Then, EBSI041 was selected for whole-genome sequencing for further identification. Ethical approval for this study was given by Zhongshan School of Medicine of Sun Yat-sen University under approval number 068. All methods involved in this study were carried out in accordance with relevant guidelines and regulations.

In vitro conjugation and S1-PFGE
The horizontal transfer of plasmids was examined by in vitro conjugation using K. pneumoniae EBSI041 as a donor and E. coli strain EC600 and J53 as a recipient, respectively. Briefly, the EBSI041, EC600 and J53 strains were cultured to OD 600 0.4-0.6, mixed in a 1: 1 donorto-recipient ratio, platted onto Luria-Bertani (LB) agar plates and incubated at 37 °C overnight. One ml of sterile saline was used to remove the conjugation mix from the LB agar plates. Transconjugants were then selected by plating LB agar plates containing rifampicin (Rif; 500 μg/ ml) and imipenem (Imp; 2 μg/ml) for EC600, and sodium azide (NaN3; 100 μg/ml) and imipenem (Imp; 2 μg/ml) for J53. The transfer of the plasmid was checked by PCR analysis and MICs. The bla KPC-2 and bla OXA-48 genes were confirmed by PCR and sequencing with primers KPC-A (TGT AAG TTA CCG CGC TGA GG), KPC-B (CCA GAC GAC GGC ATA GTC ATF) [40], and OXA-A (TTG GTG GCA TCG ATT ATC GG), OXA-B (GAG CAC TTC TTT TGT GAT GGC) [41]. And S1-PFGE was used to determine the sizes and numbers of plasmids harboured by the isolate EBSI041 and transconjugants [42].

Galleria mellonella virulence assay
The virulence of strain EBSI041 was tested using the wax moth (Galleria mellonella) larvae model. Briefly, 30 larvae weighing about 300 mg were randomly selected for each isolate and maintained on woodchips in the dark at 15 °C until being used. Overnight cultures of K. pneumoniae strains were washed with phosphate-buffered saline (PBS) and further adjusted with PBS to concentrations of 1×10 6 CFU/mL (10 ul for injection). Colony counts were conducted by serial dilution with final plating on LB agar. The G. mellonella were infected with the tested bacteria, as previously described [43]. PBS injection controls and the negative controls (receiving no injection) were used to evaluate trauma and attrition, respectively. EC600 strain was used as non-virulent control, while, HvKP4 strain was used as the hypervirulent control [18]. HvKP4, a ST11 carbapenem-resistant hypervirulent K. pneumoniae outbreak strain, was isolated from China. The larvae were incubated at 37 °C in the dark and observed every 12 h for 7 days. We recorded the survival rate of the G. mellonella over 48 h post-infection. Results were not included if greater than or equal to two larvae died in either of the control groups. All experiments were done in triplicate.