Klebsiella pneumoniae ST147 harboring blaNDM-1, multidrug resistance and hypervirulence plasmids

ABSTRACT The spread of hypervirulent (hv) and carbapenem-/multidrug-resistant Klebsiella pneumoniae is an emerging problem in healthcare settings. The New Delhi metallo-β-lactamase-1 (blaNDM-1) is found in Enterobacteriaceae including K. pneumoniae. The blaNDM-1 is capable of hydrolyzing β-lactam antibiotics which are used for treatment of severe infections caused by multidrug-resistant Gram-negative bacteria. This is associated with the unacceptably high mortality rate in immunocompromised burn injury patients. This study reports on the characterization of blaNDM-1 gene and virulence factors in hv carbapenem-/multidrug-resistant K. pneumoniae ST147 in the burns unit of a tertiary teaching hospital during routine surveillance. Two K. pneumoniae strains were obtained from wounds of burn-infected patients from May 2020 to July 2021. The hypervirulence genes and genetic context of the blaNDM-1 gene and mobile genetic elements potentially involved in the transposition of the gene were analyzed. We identified a conserved genetic background and an IS26 and open reading frame flanking the blaNDM-1 gene that could suggest its involvement in the mobilization of the gene. The plasmid harbored additional antibiotic resistance predicted regions that were responsible for resistance to almost all the routinely used antibiotics. To ensure the identification of potential outbreak strains during routine surveillance, investigations on resistance genes and their environment in relation to evolution are necessary for molecular epidemiology. IMPORTANCE Data obtained from this study will aid in the prompt identification of disease outbreaks including evolving resistance and virulence of the outbreak bacteria. This will help establish and implement antimicrobial stewardship programs and infection prevention protocols in fragile health systems in countries with limited resources. Integration of molecular surveillance and translation of whole-genome sequencing in routine diagnosis will provide valuable data for control of infection. This study reports for the first time a high-risk clone K. pneumoniae ST147 with hypervirulence and multidrug-resistance features in Ghana.

β-lactamase, after its first detection in Sweden in 2008 from an Indian patient, is a major public health concern (7)(8)(9).First discovered in K. pneumoniae and Escherichia coli, New Delhi metallo-β-lactamase-1 (bla NDM-1 ) has been identified in other Enterobac teriaceae including Citrobacter freundii, Enterobacter cloacae, Morganella morganii, and Proteus vulgaris (10,11).The bla NDM-1 -positive Enterobacteriaceae has been found in both hospital and community-acquired infections, food, and water sources (12).This has led to the emergence of hypervirulent (hv) and carbapenem and/or multidrug-resist ant K. pneumoniae spp.that cause invasive infections including endophthalmitis and meningitis in immunocompromised patients (13).Hypervirulent K. pneumoniae (hv-KP) is associated with a variety of factors including capsular serotypes, hypermucoviscosity, sequence types (ST11 and ST23), pathogenicity island, virulence plasmid, and virulence factors such as lipopolysaccharide, type VI secretion system, siderophore production and allantoin metabolism (14).Hv-KP pathogens are responsible for pyogenic liver abscesses, osteomyelitis and endophthalmitis in younger populations in the community (15).Over 78 capsular serotypes of K. pneumoniae have been reported; K1 and K2 serotypes are the most prevalent in hv-KP (16)(17)(18)(19).Most hv-KP strains are susceptible to the commonly prescribed antibiotics, including carbapenems (20).However, these strains can acquire carbapenem or multidrug resistance that can lead to the emergence of isolates that have combined resistance and virulence.Alternatively, multidrug-resistant K. pneumo niae strain can acquire virulence plasmids (pLVPK/pVir-CR-HVKP4) (13,21,22).This has compromised the options for the treatment of life-threatening infections caused by K. pneumoniae with combined multidrug/carbapenem and hypervirulence factors (23).
Bacteria that cause invasive wound infection can lead to systemic sepsis.It is important to report on the pathogenic potential of bacteria that cause invasive wound infection in burn patients.This study characterized the genetic environment of the plasmid-borne bla NDM-1 and hypervirulence genes in K. pneumoniae ST147 obtained from the burns unit of a tertiary care hospital in Ghana.Data analyses revealed that other mechanisms may initiate transposition of the bla NDM-1 than cointegration of IS26 into the adjacent IS26 region.The information obtained together with the phenotypic data will be valuable in routine surveillance and will guide infection prevention and control in our health institutions to prevent possible outbreaks.

K. pneumoniae ST147 phylogeny and genetic environment of bla NDM-1 and bla oxa-1 genes
Isolates 016W_16082020 and 017WC1_20082020 were assigned to ST147.A phylo genetic tree was constructed using the whole-genome single nucleotide polymor phism (SNP) phylogeny approach to compare the genetic relatedness of our isolates with other global ST147 K. pneumoniae strains (Fig. 1).Isolates 016W_16082020 and  017WC1_20082020 clustered with a Nigerian strain ERS2604946.The isolates differed by 58 and 72 SNPs, respectively.Both isolates harbored similar plasmids: IncQ and IncF and an additional plasmid IncH1 for isolate 017WC1_20082020.We compared their genetic environment to assess whether the bla NDM-1 gene from both isolates originated from a similar source and were genetically related.The bla NDM-1 gene was located on untyped plasmid pKP016W_16082020 and pKP017WC1_20082020 with plasmid sizes of 84,272 and 91,465 bp, respectively (Fig. 2).The two isolates shared a highly conserved backbone structure (Fig. 3A); however, the bla NDM-1 gene of isolate 017WC1_20082020 was oriented in the opposite direction.Both isolates were flanked upstream by an open reading frame (ORF) and downstream by the putative ancestral genes: ble MBL -iso-DsbD-cutA (Fig. 3A).These genes code for bleomycin resistance, phosphoribosylanthranilate isomerase, protein disulfide reductase, and heavy metal resistance.This segment was followed by an insertion sequence IS26.However, isolate 016W_16082020 had two copies of IS26 and ble MBL compared to 017WC1_20082020.The two isolates lacked the ISAba125, groEL, groES, tat, and IS30tnp genes when compared with KP3771 reference strain (Fig. 3A).The genetic environment surrounding the bla oxa-1 gene of both isolates shared a similar structure but in the opposite orientation.The bla oxa-1 gene was located with other resistance genes, bla oxa-1 -catB-Arr-SMR-sul2-IS26 downstream for isolate 016W_16082020.Upstream contained class 1 integron with the structure of intl1-aac(6′)-Ib-cr-bla oxa-1 (Fig. 3B), while the bla oxa-1 gene of the KP3771 reference strain was flanked by the composite IS6 insertion sequence.For the class A beta-lactamase gene bla CTX-M15 , it was bound upstream by the IS2 and downstream by aminoglycoside aac(6′)-Ib/aac(6′)-II and bla TEM (data not shown).The pKP016W_16082020 (Fig. 2, top) and pKP017WC1_20082020 (Fig. 2, bottom) plasmids harbored predicted antibiotic resistance regions that have been shown to confer resistance to amikacin, aztreonam, cefepime, ciprofloxacin, ertapenem, fosfomycin, gentamicin, imipenem, levofloxacin, meropenem, piperacillin-tazobactam, tetracycline, tobramycin, and trimethoprim-sulfamethoxazole.Both plasmids did not contain the conjugal transfer gene.

Detection of antibiotic and virulence genes
The antibiotic resistance genes identified for both isolates coded for aminoglycosides, beta-lactams, carbapenems, cephalosporins, fluoroquinolones, phenicol, and sulfona mides (Table 3).The antibiotic resistance genes were located on untyped plasmids pKP016W_16082020 and pKP017WC1_20082020 and lacked the conjugal transfer gene (Fig. 2).A variety of hypervirulence-associated factors were identified in both K. pneumoniae strains.This includes capsule-associated genes (K64), lipopolysaccharides, type VI secretion system, fimbrial adhesin, virulence-associated regulators, siderophores (aerobactin, enterobactin, and yersiniabactin), allantoin, and efflux pumps (Table 4).Isolate 017WC1_20082020 had additional genes coding for lipopolysaccharide core biosynthesis and transport system: rfbD (o-antigen) and wzzE.These isolates tested negative for hypermucoviscosity according to the string test.The hypervirulence genes of both isolates were located on the IncQ plasmid, which harbored the conjugal transfer gene traG.

DISCUSSION
Routine surveillance of antimicrobial-resistant (AMR) pathogens is being widely explored in low-and middle-income countries (LMICs) to inform treatment outcomes.How ever, genomic surveillance to monitor bacterial evolution, antimicrobial resistance, and virulence determinants over time is relatively few in LMICs (21,(41)(42)(43).According to the World Health Organization's list of AMR bacteria, K. pneumoniae has been reported as a high-priority antibiotic-resistant pathogen responsible for global nosocomial infections (44).Here, we report on the genetic environment of carbapenem-resistant bla NDM-1 gene and hypervirulence of K. pneumoniae obtained during routine surveillance in the burns unit of a tertiary teaching hospital in Ghana to gain insights into emerging high-risk clone ST147.
Extended-spectrum β-lactamase-producing K. pneumoniae ST13, ST15, ST22, ST25, ST36, ST70, ST110, ST147, ST334, ST405, ST414, ST502, and ST530 have been reported in bloodstream infections, poultry, and wounds of patients (45)(46)(47).Of these, ST147 has emerged as a high-risk clone reported in hospital outbreaks globally (48).Hospi tal outbreaks of K. pneumoniae ST147 have been reported in China (49), Greece (50), Slovenia (51), and Tunisia (52,53).In Africa, cases have been reported from wound infections and community samples from Algeria (32,54), Burkina Faso (55), Egypt (56), Kenya (57,58), Libya (59), Nigeria (60), and Tunisia (61)(62)(63)(64).Other sources such as animals from Senegal (65) and poultry products from Ghana (46) have been shown to harbor K. pneumoniae ST147.In a decade, there has been a global dissemination of ST147 high-risk clone.The close relatedness of our strains with a strain from Nigeria may suggest recent travel history of patients or contact with colonized persons in Ghana.The detection of ST147 underscores the importance of molecular surveillance in healthcare settings to prevent nosocomial outbreaks.Carbapenem-/multidrug-resistant K. pneumoniae isolates were resistant to the prescribed antibiotics.The strains were also resistant to other antibiotics prescribed in the burns unit: ceftazidime, ciprofloxacin, imipenem, and meropenem except for gentamicin, tigecycline, and trimethoprim/sulfamethoxazole.Combination therapy for the treatment of carbapenem-resistant pathogens, including ceftazidime-avibactam, and azetreonam-avibactam, or monotherapy using cefiderocol is not the current protocol for the treatment of patients in the burns unit; hence, antimicrobial susceptibility test for these antibiotics was not tested.Due to the intermediate resistance observed, the lastresort antimicrobial, colistin, was not administered to the patients.Gentamicin and trimethoprim/sulfamethoxazole predicted resistance regions were detected; however, the strains were phenotypically susceptible at concentrations of ≤4 µg/mL and ≤2/38 µg/mL, respectively.The reasons for this observation were not explored in this study.Multidrug resistance leads to serious illness, prolonged hospital stays, and poor patient outcomes due to treatment failure.There is an urgent need to promote the appropriate use of antimicrobials to reduce microbial resistance.Hv-KP strains are highly pathogenic and known to cause invasive infections, specifically pyogenic liver abscess, meningitis, and endophthalmitis, in immunocompromised and healthy individuals (15,66).Capsular serotypes including K5, K16, K20, K28, K54, K57, K63, K64 (identified in this study), and KN1 have been detected in hv-KP and associated with high pathogenicity and transmissibility (67,68).The hv-KP acts by evading the host immune system, leading to severe invasive infections (19).Given the repertoire of virulence factors identified in the two carbapenem-resistant K. pneumoniae isolates, we envisage that the virulence factors could cause wound abscess, severe infection leading to sepsis, and multiple organ dysfunction syndrome, the primary cause of death in burn patients (69).
K. pneumoniae ST147 strains have acquired resistance genes on plasmids, including oxacillinases: bla oxa-48 on IncL (70), bla oxa-181 on the chromosome (71,72), bla oxa-204 on IncA/C (73), metallo-beta-lactamase bla NDM-1 on IncFIIA (74), IncA/C (75), and IncX3 ( 76), and bla KPCs on pKpQIL (77) and IncN (78).Several mobile genetic elements (MGEs) such as plasmids, integrons, and transposons are associated with acquiring and transmitting carbapenem resistance genes and other AMR determinants between bacteria (79,80).Insertion sequences (ISs) and transposons including ISAba125, IS3000, IS26, IS5, ISCR1, ISCR27, Tn3, Tn125, and Tn3000 have played vital roles in the dissemination of bla NDM genes.There has been a temporal role of plasmids, ISs, and transposons in the mobili zation of bla NDM genes.First Tn125 and then Tn3000 and IS26 were involved in the transposition of the carbapenem gene (81,82).In this study, the ST147 strains harbored the bla NDM-1 genes on plasmids pKP016W_16082020 and pKP017WC1_20082020.The IS26 was found in two positions on both plasmids about 36-52 kb apart; the second IS26 flanked bla oxa-1 in the opposite orientation, which suggests involvement in two independent activities of mobilization of antibiotic-resistant genes.Given that transposi tion of IS26 with the bla NDM-1 gene occurs via cointegration into the adjacent IS26 with the same orientation (83), it is unlikely that the transposition will occur successfully in our strains for the following reasons: (i) the bla NDM-1 gene was not flanked immediately on both sides by IS26 to facilitate transposition of the bla NDM-1 gene, and (ii) the orientation of the second IS26 is in the opposite direction compared to experiments shown to demonstrate mobilization of the bla NDM-1 gene.Since the bla NDM-1 gene is carried on non-conjugative plasmids, additional processes including recombination events may be involved in the transposition of the bla NDM-1 gene.Further studies are needed to improve our understanding of how IS26 pseudo-composite transposon observed in isolate 016W_16082020 will mobilize the bla NDM-1 gene to adjacent IS26 that is oriented in the opposite direction.Considering that the mobility of the bla NDM-1 gene is restricted due to the absence of the conjugal transfer gene and orientation of the second IS26, we suggest a combined role of the flanking IS26 and the ORF gene in the dissemination of the bla NDM-1 gene.Alternatively, other means of transfer, including mobilization by the virulence conjugative plasmid, IncQ (helper plasmid), or transduction by a bacteriophage, could be used to transfer the carbapenem resistance genes to other bacteria.With the high number of predicted regions for AMR, further studies are needed to demonstrate the dissemination of these genes across different bacterial genera.
Our study characterized the genetic environment and virulence factors of hypervir ulent carbapenem-/multidrug-resistant K. pneumoniae strains harboring bla NDM-1 on plasmids pKP016W_16082020 and pKP017WC1_20082020 and the MGEs involved in its complex evolutionary dissemination.This highlights the virulence potential and limited options for antimicrobial therapy of patients whose wounds are infected with hv carbapenem-/multidrug-resistant K. pneumoniae strains.It is important to imple ment genomic surveillance in low-resource settings to promptly identify and control the spread of carbapenem resistance and monitor evolving resistant bacteria in our healthcare settings.

Cultivation of hv multidrug-resistant K. pneumoniae and antimicrobial susceptibility
Two K. pneumoniae strains, 016W_16082020 and 017WC1_20082020, were obtained from patients in the burns unit between May 2020 and July 2021.Clinical isolates were selected from these patients because their burn injuries were infected with hv carbapenem-/multidrug-resistant K. pneumoniae.The clinical samples were cultured on MacConkey agar (Oxoid Ltd, Basingstoke, UK) and incubated at 37°C for 24 hours with the reference strain K. pneumoniae ATCC 700603.Subcultures were made on nutrient agar (Oxoid Ltd) plates supplemented with 100-μg/mL imipenem-meropenem antibiotics.Identification was done using matrix-assisted laser desorption ionizationtime of flight mass spectrometry with a microflex LT Biotyper v.3.0 (Bruker; Daltonics, Bremen, Germany) according to the manufacturer's instructions.

FIG 3
FIG 3 Genetic environment of beta-lactamase genes in K. pneumoniae strains.(A) Genetic structure of bla NDM-1 gene in the genomes of 016W_16082020 and 017WC1_20082020 strains in comparison with KP3771 reference strain.(B) Genetic structure of the bla oxa-1 gene in the genomes of 016W_16082020 and 017WC1_20082020 strains in comparison with KP3771 reference strain.The colored arrows denote the following: gray, mobile genetic elements including ISs and class 1 integron; green, bla NDM-1 gene; blue, antibiotic resistance genes and hypothetical genes.Abbreviations: arr, rifampin ADP ribosyl transferase; hypo, hypothetical gene; SMR, small multidrug-resistance efflux transpoter.

TABLE 2
Antimicrobial susceptibility test of hv carbapenem-/multidrug-resistant K. pneumoniae isolates a