Emergence of Hypervirulent Carbapenem-Resistant Klebsiella pneumoniae Coharboring a blaNDM-1-Carrying Virulent Plasmid and a blaKPC-2-Carrying Plasmid in an Egyptian Hospital

ABSTRACT The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates in Egyptian hospitals has been reported. However, the genetic basis and analysis of the plasmids associated with carbapenem-resistant hypervirulent K. pneumoniae (CR-HvKP) in Egypt have not been presented. Therefore, we attempted to decipher the plasmid sequences that are responsible for transferring the determinants of carbapenem resistance, particularly blaNDM-1 and blaKPC-2. Out of 34 K. pneumoniae isolates collected from two tertiary hospitals in Egypt, 31 were CRKP. Whole-genome sequencing revealed that our isolates were related to 13 different sequence types (STs). The most prevalent ST was ST101, followed by ST383 and ST11. Among the CRKP isolates, one isolate named EBSI036 has been reassessed by Nanopore sequencing. Genetic environment analysis showed that EBSI036 carried 20 antibiotic resistance genes and was identified as a CR-HvKP strain: it harbored four plasmids, namely, pEBSI036-1-NDM-VIR, pEBSI036-2-KPC, pEBSI036-3, and pEBSI036-4. The two carbapenemase genes blaNDM-1 and blaKPC-2 were located on plasmids pEBSI036-1-NDM-VIR and pEBSI036-2-KPC, respectively. The IncFIB:IncHI1B hybrid plasmid pEBSI036-1-NDM-VIR also carried some virulence factors, including the regulator of the mucoid phenotype (rmpA), the regulator of mucoid phenotype 2 (rmpA2), and aerobactin (iucABCD and iutA). Thus, we set out in this study to analyze in depth the genetic basis of the pEBSI036-1-NDM-VIR and pEBSI036-2-KPC plasmids. We report a high-risk clone ST11 KL47 serotype of a CR-HvKP strain isolated from the blood of a 60-year-old hospitalized female patient from the intensive care unit (ICU) in a tertiary care hospital in Egypt, which showed the cohabitation of a novel hybrid plasmid coharboring the blaNDM-1 and virulence genes and a blaKPC-2-carrying plasmid. IMPORTANCE CRKP has been registered in the critical priority tier by the World Health Organization and has become a significant menace to public health. The emergence of CR-HvKP is of great concern in terms of both disease and treatment. In-depth analysis of the carbapenemase-encoding and virulence plasmids may provide insight into ongoing recombination and evolution of virulence and multidrug resistance in K. pneumoniae. Thus, this study serves to alert contagious disease clinicians to the presence of hypervirulence in CRKP isolates in Egyptian hospitals.

ABSTRACT The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates in Egyptian hospitals has been reported. However, the genetic basis and analysis of the plasmids associated with carbapenem-resistant hypervirulent K. pneumoniae (CR-HvKP) in Egypt have not been presented. Therefore, we attempted to decipher the plasmid sequences that are responsible for transferring the determinants of carbapenem resistance, particularly bla NDM-1 and bla KPC-2 . Out of 34 K. pneumoniae isolates collected from two tertiary hospitals in Egypt, 31 were CRKP. Whole-genome sequencing revealed that our isolates were related to 13 different sequence types (STs). The most prevalent ST was ST101, followed by ST383 and ST11. Among the CRKP isolates, one isolate named EBSI036 has been reassessed by Nanopore sequencing. Genetic environment analysis showed that EBSI036 carried 20 antibiotic resistance genes and was identified as a CR-HvKP strain: it harbored four plasmids, namely, pEBSI036-1-NDM-VIR, pEBSI036-2-KPC, pEBSI036-3, and pEBSI036-4. The two carbapenemase genes bla NDM-1 and bla KPC-2 were located on plasmids pEBSI036-1-NDM-VIR and pEBSI036-2-KPC, respectively. The IncFIB:IncHI1B hybrid plasmid pEBSI036-1-NDM-VIR also carried some virulence factors, including the regulator of the mucoid phenotype (rmpA), the regulator of mucoid phenotype 2 (rmpA2), and aerobactin (iucABCD and iutA). Thus, we set out in this study to analyze in depth the genetic basis of the pEBSI036-1-NDM-VIR and pEBSI036-2-KPC plasmids. We report a high-risk clone ST11 KL47 serotype of a CR-HvKP strain isolated from the blood of a 60-year-old hospitalized female patient from the intensive care unit (ICU) in a tertiary care hospital in Egypt, which showed the Citation Ahmed MAE-GE-S, Yang Y, Yang Y, Yan B, Chen G, Hassan RM, Zhong L-L, Chen Y, Roberts AP, Wu Y, He R, Liang X, Qin M, Dai M, Zhang L, Li H, Yang F, Xu L, Tian G-B. 2021. Emergence of hypervirulent carbapenemresistant Klebsiella pneumoniae coharboring a bla NDM-1 -carrying virulent plasmid and a bla KPC-IMPORTANCE CRKP has been registered in the critical priority tier by the World Health Organization and has become a significant menace to public health. The emergence of CR-HvKP is of great concern in terms of both disease and treatment. In-depth analysis of the carbapenemase-encoding and virulence plasmids may provide insight into ongoing recombination and evolution of virulence and multidrug resistance in K. pneumoniae. Thus, this study serves to alert contagious disease clinicians to the presence of hypervirulence in CRKP isolates in Egyptian hospitals.
KEYWORDS Klebsiella pneumoniae, NDM-1, KPC-2, hybrid plasmid, virulent plasmid, Egypt S everal studies have reported the emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates in Egyptian hospitals (1)(2)(3)(4); however, to the best of our knowledge, the genetic basis and analysis of the plasmids associated with CR-hypervirulent K. pneumoniae (CR-HvKP) in Egypt have not been presented. Furthermore, carbapenem resistance has been reported to be associated with increased length of hospital stay and mortality of bloodstream infection (BSI) patients in low-and middle-income countries (5). Therefore, we sought to analyze in depth the genetic basis of pEBSI036-1-NDM-VIR (a novel hybrid plasmid harboring bla NDM-1 and virulence genes) and pEBSI036-2-KPC (a bla KPC-2 -carrying plasmid), which have been identified in a clinical K. pneumoniae strain from a blood sample of a patient in Egypt.
A total of 34 nonduplicate K. pneumoniae isolates were recovered from the blood of hospitalized patients in two tertiary care hospitals, namely, El-Demerdash Hospital (Cairo, Egypt) and the National Cancer Institute (Cairo, Egypt), in the period between June 2017 and March 2018 as a part of a study for the monitoring of antimicrobial resistance. Our isolates were selected based on their clinical characteristics, where all of them were primarily identified by Vitek 2 and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) as K. pneumoniae causing bloodstream infections (BSIs), among which 31 were confirmed phenotypically and genotypically as CRKP isolates. Overall, the 34 isolates were isolated from the blood of 55.9% (19/34) female and 44.1% (15/34) male hospitalized patients from 9 days to 75 years of age. MICs of all 34 isolates were determined for 17 antibiotics using the agar microdilution method according to CLSI (6), except for tigecycline and colistin, for which MICs were obtained by the broth microdilution method according to EUCAST (7). Out of 34 isolates, 91.2% (31/34) were resistant to ertapenem, while 73.5% (25/34) and 61.8% (21/34) were resistant to imipenem and meropenem, respectively. However, all isolates were susceptible to colistin.
All isolates were assessed by whole-genome sequencing (WGS) using an Illumina HiSeq 2000 platform. In silico multilocus sequence typing showed that our isolates belong to 13 different sequence types (STs). The most prevalent ST was ST101 (13/34 [38.2%]), followed by ST383 (5/34 [14.7%]). One isolate, EBSI036, belongs to ST11: ST11 is the dominant ST clone responsible for the prevalence of CRKP worldwide and is considered an emerging high-risk clone (1,(8)(9)(10). Among 31 CRKP isolates, the prevalence of carbapenemase genes bla NDM-1 and bla OXA-48 was 45.2% (14/31)-in addition, four strains carried both genes. Moreover, strain EBSI036 coharbors bla NDM-1 and bla KPC-2 . According to the clinical data, the K. pneumoniae strain EBSI036 was isolated from the blood of a 60-year-old female patient 2 days after admission to the Gastroenterology Department of El-Demerdash Hospital with symptoms of pneumonia, diarrhea, and fever. The patient's symptoms improved following the administration of intravenous ceftriaxone and colistin, and she was discharged from the hospital 8 days posthospitalization. With the further genome analysis of EBSI036, the plasmid-associated virulence determinants rmpA/rmpA2, iucABCD, and iutA in this strain were predicted using the Virulence Factor Database (VFDB [http://www.mgc.ac.cn/VFs/main.htm]). EBSI036 was determined as a KL47 capsular serotype by using Kaptive software (https://github .com/katholt/Kaptive). The serotype KL47 was the most reported type among CRKP infections in Asia (11)(12)(13). The virulence level of EBSI036 was confirmed using the Galleria mellonella larva model as previously described (11,14) (see Fig. S1 in the supplemental material). These results revealed that EBSI036 is a CR-HvKP strain.
An , and IS26 elements. This fragment with 15,448 bp containing the resistance genes mentioned above was similar to plasmid pKpvST383L (Fig. 1). The aph(39)-Ia gene was flanked by IS26 elements; a similar structure was also found downstream of the resistance region in pKpvST383L. The segment IS26-armA-IS26-msr(E)-mph(E)-ORF-ORF-IS26-DTn2 in pEBSI036-1-NDM-VIR was also found to be identical to an inverted sequence in pKpvST383L. In addition, the bla NDM-1 and qnrS genes were on either side of this fragment, while they were absent in pKpvST383L. By comparing the complete sequences of pEBSI036-1-NDM-VIR and pKpvST383L, it was found that pKpvST383L had another resistance region (26,683 bp) carrying bla NDM-5 and bla OXA-9 . Compared with plasmid p51015_NDM_1, the resistance region of plasmid pEBSI036-1-NDM-VIR lacked the aph(39)-VI and sul2 genes (Fig. 1). In pEBSI036-1-NDM-VIR, the MDR region contained six IS26 elements and other transposon elements. Some studies demonstrated that the resistance loci containing IS26 can be hot spots for the capture of further resistance genes to constitute a novel MDR region (18).
In conclusion, we have reported a high-risk clone of ST11 KL47 of a CR-HvKP strain isolated from the blood of a patient from an ICU in Egypt, which coharbors two plasmids: one is a novel hybrid plasmid harboring the carbapenemase gene bla NDM-1 and virulence genes, and the other carries bla KPC-2 . Further countrywide surveillance studies are needed to elucidate the rate of prevalence of this high-risk clone in Egypt and its burden on hospital-acquired infections.
Accession numbers. The sequences of the plasmids pEBSI036-1-NDM-VIR and pEBSI036-2-KPC have been deposited in GenBank under accession no. MT648512 and MT648513.

SUPPLEMENTAL MATERIAL
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