The making of hypervirulent Klebsiella pneumoniae

Abstract

Klebsiella pneumoniae is a notorious bacterium in clinical practice. Virulence, carbapenem‐resistance and their convergence among K. pneumoniae are extensively discussed in this article. Hypervirulent K. pneumoniae (HvKP) has spread from the Asian Pacific Rim to the world, inducing various invasive infections, such as pyogenic liver abscess, endophthalmitis, and meningitis. Furthermore, HvKP has acquired more and more drug resistance. Among multidrug‐resistant HvKP, hypervirulent carbapenem‐resistant K. pneumoniae (Hv‐CRKP), and carbapenem‐resistant hypervirulent K. pneumoniae (CR‐HvKP) are both devastating for their extreme drug resistance and virulence. The hypervirulence of HvKP is primarily attributed to hypercapsule, macromolecular exopolysaccharides, or excessive siderophores, although it has many other factors, for example, lipopolysaccharides, fimbriae, and porins. In contrast with classical determination of HvKP, that is, animal lethality test, molecular determination could be an optional and practical method after improvement. HvKP, including Hv‐CRKP and CR‐HvKP, has been progressing. R‐M and CRISPR‐Cas systems may play pivotal roles in such evolutions. Hv‐CRKP and CR‐HvKP, in particular the former, should be of severe concern due to their being more and more prevalent.

Factors involved in HvKP.

1. INTRODUCTION

Klebsiella pneumoniae (K. pneumoniae) is a gram‐negative and non‐motile bacterium and belongs to Enterobacteriaceae, which was first described by Carl Friedlander in 1882 as a bacterium and isolated from the lungs of patients who had died of pneumonia. K. pneumoniae is ubiquitously found in water, soil, humans, and animals and can colonize healthcare‐related sites. ¹ , ² K. pneumoniae could colonize a variety of sites in human body, for example, axilla, intestinal tract, and nasopharynx. ³ , ⁴ , ⁵ In the past decades, K. pneumoniae was of greater and greater concern worldwide, mainly due to its enhanced resistance and recently focused hypervirulence. ⁶ , ⁷ Despite SHV‐1 β‐lactamase, encoded on the chromosome and being intrinsic resistance, K. pneumoniae could antagonize antimicrobials though various pathways, for example, hydrolyzing enzymes, missing porins, efflux overexpression, topoisomerase, and lipopolysaccharide (LPS) modification. ⁸ Alarmingly, carbapenem‐resistant K. pneumoniae (CR‐KP) has reached a rate of over 30.0% among K. pneumoniae strains and brought formidable challenges in clinical practice. ⁹ In 2016, The World Health Organization (WHO) made a list of the most “critical” bacteria at a global level with an urgent need for new treatments, among which carbapenem‐resistant Enterobacteriaceae (CRE) was classified as a critical priority organism.

CR‐KP could come into existence via multiple mechanisms, that is, (i) production of carbapenemase; (ii) decreased expression or loss of outer membrane proteins with overexpression of extended‐spectrum β‐lactamases and AmpC cephalosporinases; and (iii) activation of efflux pumps. ¹⁰ Carbapenemases include K. pneumoniae carbapenemases, New Delhi metallo‐β‐lactamase, Verona integron‐encoded metallo‐β‐lactamase, imipenemase, and oxacillinase. ¹¹ Disruption or deficiency of OmpK35 and OmpK36 could result in CR‐KP. ¹² Overexpression of efflux pumps also confers carbapenem resistance. ¹³ While the reasons of CR‐KP differ remarkably worldwide, it is dominantly conferred by the mobile genetic elements harboring a variety of antibiotic‐resistance genes, for example, beta lactamase K. pneumoniae carbapenemases gene (bla _KPC), New Delhi metallo‐β‐lactamase gene (bla _NDM), and oxacillinase‐48 gene (bla _OXA‐48). ¹⁴ , ¹⁵

Hypervirulent K. pneumoniae (HvKP), first recognized as a unique clinical pathogen in the 1980s in Taiwan, ¹⁶ is an entity of being more virulent than classical K. pneumoniae (cKP), which is determined by animal (mice, wax moth larvae) lethality tests, neutrophil assay, and so on. ¹⁷ , ¹⁸ , ¹⁹ cKP is often associated with nosocomial infections, for example, pneumonia, urinary tract infection (UTI), and bacteremia, among those at extremes of age or with underlying immunodeficiencies while HvKP often induces infections, such as pyogenic liver abscess (PLA), lung abscess, endophthalmitis, in otherwise healthy individuals (Table 1). The prevalence of HvKP varies in different regions, ranging from 12% to 45% in HvKP‐endemic areas. ²⁰ , ²¹ , ²² , ²³ , ²⁴ Recent studies unveiled a convergent trend of CR‐KP and HvKP, ²⁵ which needs our deeper insights. Here, we try to focus on the determinants of HvKP, detection, and its evolution.

TABLE 1.

Characteristics of cKP and HvKP

Parameters	cKP	HvKP	Hv‐CRKP or CR‐HvKP	References
Typical infections	Pneumonia, UTI, bacteremia	PLA; lung, neck and kidney abscesses; endophthalmitis; necrotizing fasciitis; meningitis; pneumonia; cellulitis; myositis; septic arthritis; osteomyelitis	Combined infections by cKP and HvKP	19, 26, 27, 28, 29, 30, 31, 32, 33
Susceptible populations	Immunocompromised (diabetics, patients with malignancies or transplant, bedridden individuals)	Diabetics, otherwise healthy individuals	Combined populations	19, 20, 28, 29, 34, 35, 36, 37
Serotypes	K1‐K79	Mostly K1 and K2, seldom K5 and K57	K64, K47, K20, K2 and K20	1, 28, 29, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50
Siderophores (positive rates, %)	Enterobactin (100), yersiniabactin (17–46), salmochelin (2–4), aerobactin (6)	Enterobactin (100), yersiniabactin (90), salmochelin (>90), aerobactin (>93)	Enterobactin (100), yersiniabactin (90), salmochelin (40), aerobactin (>93)	40, 51, 52, 53, 54, 55, 56
Geographical prevalence	Worldwide	Mostly the Asian Pacific Rim, the trend to the world	Mainly Asia, the trend to the world	29, 30, 32, 38, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76
Commonly acquired infection type	Primarily nosocomial	Community acquired	Often nosocomial and seldom community‐acquired	29, 59, 77, 78, 79, 80
Drug‐resistance	Frequent (for example ESBLs and carbapenemase‐producing)	Rare except penicillin‐resistance	Carbapenemase‐producing	9, 19, 28, 29, 81, 82

Abbreviations: cKP, classical Klebsiella pneumoniae; CR‐HvKP, carbapenem‐resistant hypervirulent Klebsiella pneumoniae; ESBLs, extended‐spectrum β‐lactamases; HvKP, hypervirulent Klebsiella pneumoniae; Hv‐CRKP, hypervirulent carbapenem‐resistant Klebsiella pneumoniae; PLA, pyogenic liver abscess; UTI, urinary tract infection ; PLA, pyogenic liver abscess.

2. CONCEPTION AND CLASSIFICATION

cKP is a group of K. pneumoniae that lacks hypercapsule, macromolecular exopolysaccharide, or excessive siderophores, which incurs a high median lethality dose (LD₅₀) and rarely induces diseases in otherwise healthy individuals (except UTI) regardless of its being multidrug‐resistant (MDR). cKP often causes infections, for example, pneumonia, UTI, bacteremia, or meningitis in immunocompromised individuals. ²⁶ , ²⁷ , ²⁸ cKP brings a LD₅₀ of >10⁷ colony forming unit (CFU) in a BALB/c mouse pneumonia model. ²⁹ By contrast, HvKP is another type of K. pneumoniae that harbors hypercapsule, macromolecular exopolysaccharide, or excessive siderophores, which incurs a lower LD₅₀ and induces infections in both immunocompromised and otherwise healthy individuals. HvKP yields a LD₅₀ of <10² CFU in a BALB/c mouse pneumonia model. ²⁹ , ³⁰ HvKP could be divided into two categories: drug‐sensitive and MDR. Among MDR‐HvKP, hypervirulent carbapenem‐resistant K. pneumoniae (Hv‐CRKP) and carbapenem‐resistant hypervirulent K. pneumoniae (CR‐HvKP) are both notorious for their super drug‐resistance and hypervirulence, the former being much more frequent than the latter. The characteristics of cKP and HvKP are listed in Table 1. ³¹

The basis of Hv‐CRKP is classical CR‐KP while CR‐HvKP evolves from HvKP (serotypes K1, K2, K5, K10, K20, K25, K27, and K57) acquiring carbapenem‐resistant plasmids. ⁶⁹ , ⁸³ As reported in the document, ⁶² 521 K. pneumoniae strains were collected from GenBank as of May 13, 2020; Molecular combinations predicted 29 strains to be bla _KPC‐positive and hypervirulent, of which 7 were CR‐HvKP and 22 were Hv‐CRKP; 94 and 165 strains were HvKP and bla _KPC‐positive K. pneumoniae, respectively. As of May 31, 2021, 890 K. pneumoniae genomes from GenBank were analyzed, and 53 Hv‐CRKP and 17 CR‐HvKP strains were designated ⁸⁴ ; 478 and 168 strains were CR‐KP and HvKP, respectively. Among another 530 clinical K. pneumoniae strains collected in Mainland China from January 2017 to February 2018, 28 and 6 were Hv‐CRKP and CR‐HvKP respectively ⁸⁴ ; 227 and 171 were CR‐KP and HvKP, respectively, showing constituent ratios of 12.3% for Hv‐CRKP among CRKP and 3.5% for CR‐HvKP among HvKP. Such surveillance results suggest Hv‐CRKP is far more prevalent than CR‐HvKP.

3. DETERMINANTS

3.1. Hypercapsule

Capsule (Figure 1) is an essential layer of polysaccharide bound on the surface protein Wzi of K. pneumoniae, ¹ , ⁵³ , ⁸⁵ , ⁸⁶ the loss of which would render K. pneumoniae remarkably less virulent or nonvirulent. ³⁰ , ⁸⁵ , ⁸⁷ , ⁸⁸ To date, there are in total 79 serotypes for K. pneumoniae. ⁵² In contrast with cKP, HvKP could produce a hypercapsule, which contributes to hypervirulence. ⁵² , ⁶⁵ A basic production and the serotype of capsule is controlled by a chrosomal operon, cps, which harbors a couple of genes, that is, wzi, wza, wzb, wzc, gnd, wca, cpsB, cpsG, and galF ⁸⁹ , ⁹⁰ (Table 2). Sequencing of wzi and wzc could be a shortcut to determine serotypes of K. pneumoniae instead of a traditional serological method. ¹⁸ , ⁵⁵ , ⁹¹ More importantly, hypercapsule could be regulated by several specific virulence genes, that is, c‐rmpA, c‐rmpA2, p‐rmpA, p‐rmpA2, and wzy‐K1. Genes c‐rmpA, c‐rmpA2, and wzy‐K1 are both in chromosome while p‐rmpA and p‐rmpA2 are plasmid‐borne. ⁹² , ⁹³ , ⁹⁴ Each of the 5 virulence genes could result in hypercapsule. However, their different combinations may yield different production of capsule. c‐rmpA and c‐rmpA2 in the ICEKp genomic island exist only in serotype K1 K. pneumoniae with a positive rate of <50.0%. ⁹⁵ HvKP shows positive rates of 55–100% for rmpA or rmpA2 while cKP rarely harbors. ²¹ , ⁹⁵ RmpA, described in 1989, is a regulator of mucoid phenotype in pK100, ⁹⁶ along with RmpB, another virulence plasmid‐encoded regulator. ⁹⁷

FIGURE 1.

The schematic diagram of Klebsiella pneumoniae and the difference between cKP and HvKP

TABLE 2.

Locations of virulence‐related genes in this study

Genes	Related factor	Plasmid	Chromosome
wzy‐K1, wzx, wzc, wza, wzb, wzi, gnd, wca, cpsA, cpsB, cpsG, galF	Capsule/exopolysaccharide		√
allS	Allantoin		√
rmpA, rmpB, rmpA2	Capsule	√	√
kvrA, kvrB; rcsA, rcsB	Capsule		√
p‐rmpA, p‐rmpA2	Capsule	√
c‐rmpA, c‐rmpA2	Capsule		√
entABCDEF	Enterobactin	entC/E/F	√
fepABCDG	Enterobactin	√	√
irp, ybt, fyu	Yersiniabactin	√	√
iroABCDE	Salmochelin	√	√
iucABCD, iutC	Aerobactin	√	√
fim	Type 1 fimbria	fimC/D	√
mrkABCD	Type 3 fimbria		√
wb, waa, lpx, uge, wabG	LPS		√
pks, clbA‐S	Colibactin	√	√
terZA‐Z, terWXY	Tellurite resistance	√
silS	Silver resistance	√
peg‐344	Metabolite transporter	√

Abbreviations: LPS, lipopolysaccharide.

The expression of rmpA depends on RcsB, KvrA, and KvrB. ⁹⁸ KvrA, KvrB, and RcsB contribute to capsule regulation through the control of the rmpA promoter and through additional mechanisms. K. pneumoniae strains with deletions of kvrA and kvrB are less virulent than wild type. ⁹⁹ Genes c‐rmpA, c‐rmpA2, p‐rmpA, and p‐rmpA2 positively regulate the cps locus at the transcriptional level. ⁹⁴ , ⁹⁷ , ¹⁰⁰ , ¹⁰¹ Gene wzy‐K1 was firstly termed as magA, which was discovered in 2004 ¹⁰² and specific to serotype K1 K. pneumoniae. ¹⁰³ , ¹⁰⁴ , ¹⁰⁵ Wzy is a polymerase present in the inner membrane, ¹⁰⁶ which combines Wzx into “Wzx/Wzy pathway” ¹⁰⁷ or “Wzx/Wzy secretion system” ¹⁰⁸ and is imperative for polymerization of undecaprenyl diphosphate‐linked K‐antigen sugar moiety in the periplasmic region. Wzy polymerizes the repeating oligosaccharides following their being flipped by Wzx in a blockwise manner. ¹⁰⁹ Wzy then releases the oligosaccharide moiety from its lipid carrier to the nascent polymer. ¹¹⁰ In addition, the regulation of capsule A (cpsA) and B (cpsB) genes can also result in hypercapsule. Glucose could also be a signal to increase capsule production. ⁹⁴ , ¹¹¹

Capsule could protect K. pneumoniae through the following strategies: inhibiting phagocytosis by hosts' immune cells, preventing activation of the early immune response, and hampering lysis by complement and antimicrobial peptides. ³¹ Capsule confers resistance to opsonophagocytosis in K. pneumoniae regardless of opsonins. ¹¹² Capsule could also contribute to biofilm formation, ¹¹³ , ¹¹⁴ which is contrary to another report. ¹¹⁵ Capsule induces a defective immunological host response, for example, maturation of dendritic cells (DCs) and pro‐Th1 cytokine production by hampering bacterial binding and internalization. ¹¹⁶ The rate of phagocytosis by immune cells is inversely proportional to the amount of capsule on the bacterial cell surface. Capsule induces DCs maturation with upregulation of CD83, CD86, and toll‐like receptor (TL) and downregulation of CD14 and DC‐SIGN. Capsule also suppresses the host immunological responses by inducing lower cytokines TNF‐α, IFN‐γ, and IL‐6 in the early stages of lung infection, reverse in the late stage. ⁸⁸ , ¹¹⁷ In contrast, capsule also induces persistently higher IL‐10 level, which down‐regulates the expression of pro‐inflammatory cytokines. ¹¹⁷ Klebsiella inhibits Rac1 activation; and inhibition of Rac1 activity triggers a NOD1‐mediated CYLD and MKP‐1 expression, which in turn attenuates IL‐1β‐induced IL‐8 secretion. Purified capsular polysaccharide (CPS) neither reduces IL‐1β‐induced IL‐8 secretion nor induces the expression of CYLD and MKP‐1, thereby indicating that CPS is necessary but not sufficient to attenuate inflammation. ¹¹⁸ More immune cells were recruited to lungs infected with acapsular than capsular K. pneumoniae strains. ⁸⁸ Capsule could protect K. pneumoniae against human defensin‐mediated bactericidal activity, ¹¹⁹ attenuate the production of human defensins in vitro, ¹¹⁹ and enhance pneumonia in mouse models. ¹²⁰ , ¹²¹ , ¹²² The hypercapsule would render HvKP more prominent defense in comparison with cKP.

CPS, but not LPS O side chain is a major complement resistance factor in K. pneumoniae isolates due to its modulating the deposition of C3 and protecting the microorganisms against human alveolar macrophage phagocytosis. ⁸⁷ , ¹²³ Anionic capsule, but not cationic or uncharged, blocked the bactericidal activity of antimicrobial peptides or proteins, for example, human neutrophil defensin 1, beta‐defensin 1, lactoferrin, and protamine sulfate, by binding them, thereby reducing the amount of peptides reaching K. pneumoniae surface. ¹²⁴ , ¹²⁵ Capsule both protects K. pneumoniae from the bactericidal action of defensins and impedes their expression via the expression of CYLD and MKP‐1. ¹¹⁹

3.2. Macromolecular exopolysaccharide

Despite of polysaccharides bound on Wzi, namely CPS, Enterobacteriaceae including K. pneumoniae could also synthesize and secret a variety of extracellular saccharides with low or high molecular weight, included in which are alginate, cellulose, colonic acid, curdlan, dextran, diutan, gellan, hyaluronic acid, levan, succinoglycan, welan, and xanthan. ¹²⁶ Among such exopolysaccharides, colonic acid is closely related to HvKP for its macromolecular weight and ability to form biofilm, which is produced via “Wzx/Wzy pathway”. ¹²⁷ , ¹²⁸ , ¹²⁹

Exopolysaccharides could be produced via 4 pathways: (1) the so‐called Wzx/Wzy‐dependent pathway; (2) the ATP‐binding cassette (ABC) transporter‐dependent pathway; (3) the synthase‐dependent pathway and (4) the extracellular synthesis by use of a single sucrase protein. ¹²⁶ “Wzx/Wzy pathway” is closely related with HvKP, which per se includes a couple of virulence proteins, that is, Wzx, Wzy, Wzc, Wza and Wzb. Hypervirulence via “Wzx/Wzy pathway” is limited to serotype K1 K. pneumoniae strains. Wzy‐K1, wzx, and wzc but not wza and wzb are always positive simultaneously, suggesting they are one integrity to form hypervirulence; The following knockout of each of them resulted in non‐mucoviscous colonies by “string test” ⁴⁴ and disappeared macromolecular exopolysaccharides by Periodic Acid Schiff stain. ¹⁰²

Wzx is a flippase ¹⁰⁹ and Wzc is an inner membrane tyrosine autokinase. Following the action of Wzx and Wzy, nascent K antigen is translocated onto the bacterial surface by synergetic action of Wza, Wzc, and Wzb. ¹³⁰ Wzc and Wzb control the length and amount of K antigen. ¹³¹ , ¹³² Wzc and Wzb are cognate low molecular weight phosphotyrosine phosphatase. ¹³³ Wzc belongs to polysaccharide copolymerse 2a subfamily, which is essential for the assembly of Group 1 capsule 1. ¹³⁴ Wza is an outer membrane translocon that translocates the nascent capsular polysaccharides on the bacterial surface. ¹³⁵ , ¹³⁶ Wza octamerizes across the periplasmic and outer membrane regions. ¹³⁷ , ¹³⁸ Wza is widely distributed in various K. pneumoniae including HvKP and cKP, loss of which induces ineffective exporting CPS. ¹³⁹ , ¹⁴⁰ To date, the structures and exact functions of Wzx, Wzy, Wzc, Wza, and Wzb were primarily elucidated in Escherichia coli, which is a close member with K. pneumoniae in Enterobacteriaceae.

3.3. Excessive siderophores

Iron is an essential element for bacteria to strive during infection, which is restricted by the host, ¹⁴¹ a process called nutritional immune. ¹⁴² Iron supply could affect the proliferation of K. pneumoniae and consequent bacterial count, which is also a vital factor for virulence. The majority of iron is deposited in a bound manner in the host, for example, transferrin and ferritin, little being free. Therefore, to capture iron is a prerequisite for K. pneumoniae to survive and propagate. ¹⁴³ K. pneumoniae could harbor 4 kinds of siderophores, that is, enterobactin, yersiniabactin, salmochelin, and aerobactin, which possess higher affinity than host transport proteins and can thus steal iron successfully from hosts' iron‐chelating proteins. ¹⁴³ At least one siderophore is harbored by K. pneumoniae with the positive rate of enterobactin being 100.0%. The affinity of the 4 siderophores is not equal, ranging from aerobactin with the lowest to enterobactin with the highest. ¹⁴⁴ , ¹⁴⁵

Enterobactin is the basic iron uptake system in K. pneumoniae, ⁵⁷ , ¹⁴⁶ , ¹⁴⁷ , ¹⁴⁸ with its biosynthesis and transport being encoded by the chromosomal gene cluster entABCDEF and fepABCDG. ⁵⁸ , ¹⁴⁹ Enterobactin could be neutralized by the host‐secreted lipocalin‐2, ⁵⁹ , ¹⁵⁰ , ¹⁵¹ which has several antimicrobial capabilities and is secreted by many cell lineages, for example, neutrophils. Lipocalin‐2 is upregulated by the host in the respiratory tract infection of K. pneumoniae. ¹⁵² , ¹⁵³ , ¹⁵⁴ In addition, lipocalin‐2 also has proinflammatory effects, leading to neutrophil recruitment to the site of infection via IL‐8. ¹⁵⁵ , ¹⁵⁶ The presence of lipocalin‐2 aids in the clearance of K. pneumoniae with only one siderophore: enterobactin. ⁶⁰

Yersiniabactin is another “basic” siderophore in K. pneumoniae with a positive rate of over 75.0%, ⁶² which originated from Yersinia and is also encoded by chromosome. Yersiniabactin is positive in 18% of cKP strains and 90% in HvKP. ⁵⁸ , ⁶⁰ irp gene cluster encodes proteins for yersiniabactin synthesis, ybt and fyu encode transporters for the secretion of enterobactin and ybtO encodes the uptake receptor of enterobactin. ⁵⁸ , ⁵⁹ , ¹⁵⁷ During lung infection, yersiniabactin together with enterobactin is highly expressed and it is not inhibited by lipocalin‐2 in vivo. ⁵⁹ , ⁶⁰ , ¹⁴³ Yersiniabactin alone is not capable of acquiring the iron for K. pneumoniae and lack of the other 3 siderophores would render K. pneumoniae not capable of disseminating from the lungs, ⁶⁰ which may be the reason why the positive rate of enterobactin is 100.0%. In addition to lung infection, yersiniabactin may be important for K. pneumoniae to induce PLA. ⁵⁸ , ¹⁵⁸

Salmochelin is an additional siderophore in K. pneumoniae, which is per se a c‐glucosylated form of enterobactin. ¹⁵⁹ , ¹⁶⁰ The c‐glucosylation is carried out by iro gene cluster, that is, iroABCDE on either the chromosome or a plasmid. ⁵⁸ IroN contributes to the transport of salmochelin carrying iron. ¹⁴⁹ , ¹⁶¹ Salmochelin is not neutralized by lipocalin‐2 ¹⁵⁹ and induces K. pneumoniae colonization of the nasopharynx ¹⁵⁵ and consequent pneumonia. Salmochelin is seldom present in cKP with a rate of 2–4% but usual in HvKP with a rate of > 90%. ⁵⁷ , ⁵⁸ , ⁶⁰

Aerobactin is a citrate‐hydroxamate siderophore, which is rarely present in cKP with a rate of ~ 6% but common in HvKP rating over 90.0%. ⁴⁶ , ⁵⁷ , ⁶¹ , ¹⁴⁶ Aerobactin is usually associated with a hypercapsule while K. pneumoniae with hypercapsule does not inevitably harbor aerobactin. ⁴⁶ , ⁶¹ , ¹⁴³ Aerobactin is controlled by the gene cluster iucABCD and its transport is determined by iutA, both of which are often present on the same pLVPK‐like plasmids carrying p‐rmpA. ⁵⁸ , ⁹³ , ⁹⁷ , ¹⁶² , ¹⁶³ Aerobactin is not neutralized by lipocalin‐2. Aerobactin is crucial for some HvKP causing lung infection ¹⁵⁸ and it accounts for the majority of the total siderophores in HvKP. ¹⁶⁴ Aerobactin, but not enterobactin, yersiniabactin, or salmochelin, is essential for successful infection by HvKP in pneumonic and subcutaneous mouse infection models. ¹⁵⁸ , ¹⁶⁴ However, in a mouse i.p. HvKP infection model, aerobactin along with yersiniabactin and salmochelin is seemingly redundant unless the 3 are all deleted. ⁵⁸

In addition to the aforementioned 4 siderophores, other iron‐stealing systems may also support the pathogenesis of HvKP. K. pneumoniae NTUH‐K2044 also encodes ferrichrome, ferric, ferrous, and haem‐iron uptake systems. ⁵⁸ Intriguingly, another study reported a CR‐HvKP strain with a positive string test and K2 ST14 types, ¹⁶⁵ which lacked rmpA, rmpA2, and yersiniabactin, but harbored receptors (not biosynthetic genes) for aerobactin and salmochelin. It suggests curious capability of “stealing” siderophores from other species with their coexistence and the following transformation into being hypervirulent.

3.4. Other virulence factors

Apart from hypercapsule, exopolysaccharide, and excessive siderophores, which are all vital for HvKP, HvKP could also possess a couple of other virulence factors, such as fimbriae, LPS, colibactin, tellurite and silver resistance and allantoin metabolism.

Fimbriae in KP are classified as type 1 and 3 with type 1 being thin, thread‐like protrusions and type 3 being helix‐like filaments. ³¹ Type 1 fimbriae, encoded by fim gene cluster, ³¹ are widely distributed in K. pneumoniae with a positive rate of over 90.0%, ¹⁶⁶ , ¹⁶⁷ which was over 95.0% in our study. ⁶² Type 3 fimbriae, encoded by mrkABCD gene cluster, ¹⁶⁸ are “mannose insensitive” and therefore do not bind mannose. Type 1 fimbriae contribute to UTI ¹⁶⁹ and biofilm formation, including on urinary catheters. ¹⁷⁰ Type 3 fimbriae have been shown to bind extracellular matrix proteins such as type IV and V collagens ¹⁷¹ and contribute to biofilm formation. ¹⁷⁰ , ¹⁷² Type 3 fimbriae are also over 95.0% positive in K. pneumoniae strains. ⁶²

LPS, also termed as endotoxin, typically consists of an O antigen, a core oligosaccharide and lipid A, which are ubiquitous and encoded by wb, waa, and lpx gene clusters respectively. ¹⁷³ , ¹⁷⁴ , ¹⁷⁵ , ¹⁷⁶ LPS protects K. pneumoniae against humoral defenses and also serves as a strong immune activator. Lipid A is a potent ligand for TLR4, which leads to the production of cytokines and chemokines, followed by the recruiting and activating of neutrophils and macrophages. Lipid A protects K. pneumoniae against some cationic antimicrobial peptides. ¹⁷⁷ , ¹⁷⁸ K. pneumoniae strains with K1, K10, and K16 serotypes could mask their LPS, ¹⁷⁹ which was also found in HvKP. ¹⁸⁰ K. pneumoniae could also modify its LPS as unrecognizable by certain immune receptors. ¹⁷⁷ For K. pneumoniae, LPS is the primary means of protection against complement, even in the presence of capsule. ¹⁷⁹ The O antigen of LPS protects against C3 by binding C3b and abrogates pore formation. ¹⁷⁹ , ¹⁸¹ , ¹⁸² , ¹⁸³ uge, which encodes a UDP galacturonate 4‐epimerase, and wabG, which encodes a GalA transferase, are also involved in the production of LPS. ¹⁸⁴ , ¹⁸⁵

Colibactin, also termed as “genotoxin,” is a natural and genotoxic chemical compound, which is encoded by pks genomic island with a length of 54 Kb. ¹⁸⁶ The pks island represents a total of 19 genes, that is, clbA to clbS. ¹⁸⁶ The pks locus is usually present in a chromosomal integrative and conjugative element (ICE). ⁴⁷ , ¹⁸⁷ Colibactin could induce DNA double‐strand breaking, chromosome aberrations, and cell cycle arrest in the G2/M phase. ¹⁸⁶ , ¹⁸⁸ In addition, colibactin contributes to colonization and survival of K. pneumoniae ¹⁸⁹ and the global spread of clonal group (CG) 23. ¹⁹⁰ The pks‐related island could also encode numerous compounds ¹⁹¹ and contribute to the anti‐inflammatory, ¹⁹² antibiotic, ¹⁹³ , ¹⁹⁴ and analgesic effects ¹⁹⁵ for colibactin‐producing K. pneumoniae. Prevalence of pks differs in different regions, ranging from 3.5% (5/141) in Europe to 25.6% (53/207) in Taiwan. ¹⁸⁷ , ¹⁹⁶ In particular, pks is highly prevalent in serotype K1 K. pneumoniae rating from 71.4% (35/41) and 78.8% (26/33). ¹⁸⁷ , ¹⁹⁷ Carriage of rmpA, iutC, and ybtA was significantly higher in the pks‐positive isolates than the pks‐negative isolates (95.5% vs. 13.2%, p < 0.001), which indicates the emerging pks genotoxic trait is associated with increasing HvKP strains. ¹⁹⁷

Tellurite and silver resistance is encoded by terZA to Z, terWXY and silS, respectively, which may be important for systemic infections. ¹⁶³ , ¹⁹⁸ Such genes are in the virulence plasmid and not HvKP specific, ⁹² , ¹⁹⁹ loss of which decreases tellurite and silver resistance in K. pneumoniae but did not affect virulence in a mouse pneumonia model. ¹⁹⁹

Allantoin metabolism is one pathway for K. pneumoniae to obtain carbon and nitrogen from allantoin, ²⁰⁰ a degradation product from nucleic acids that some microbials can use as a source of nitrogen. ²⁰¹ Allantoin metabolism is under control of all gene cluster ²⁰² ; all gene cluster is enriched in strains associated with PLA versus commensal strains. ²⁰³ , ²⁰⁴ allS is present in K1 but no other serotype HvKP causing PLA with a rate of 100.0%, ⁴⁶ which suggests an association with K1 serotype. However, if specimen types are of consideration, allS is not inevitably in line with K1 serotype, vice versa. The absence of allS does not reduce virulence. ⁴⁶

Peg‐344 is a metabolite transporter encoded by HvKP virulence plasmid. ²⁰⁵ The exact function of peg‐344 needs to be elucidated. Peg‐344 could increase RNA abundance when K. pneumoniae is grown in human ascites, which suggests Peg‐344 may transport an unidentified growth factor present in ascites. ²⁰⁵ It may be per se a coincidence that peg‐344 could be a rather good indicator of HvKP because of its being in virulence plasmid.

4. ROLE OF RACE FACTOR

PLA is a typical infection caused by HvKP. PLA is endemic in East Asia, ⁶⁹ , ⁷⁵ , ⁸³ , ²⁰⁶ which indicates a close relationship between certain races and HvKP. In North America, 78.3% of patients were of Asian origin, ⁶³ who were with K. pneumoniae‐induced PLA. A surveillance showed high carriage rate of K. pneumoniae (21.1%) in Korean intestinal tract ²⁰⁷ ; among the strains, 23.0% were K1 serotype, predominant in PLA‐derived K. pneumoniae strains. ²⁰⁸ Chinese ethnicity is also a major factor predisposing to intestinal colonization by serotype K1/K2 K. pneumoniae isolates. ³ The colonization of HvKP in intestinal tract is a vital step for the formation of PLA. As carbapenem resistance is taken into consideration, CR‐HvKP and Hv‐CRKP are mainly distributed in healthcare settings ²⁰⁹ and presents no significant correlation with race.

On the contrary, the role of race factor is some limited. For instance, immigration to western countries decreased the carriage of K. pneumoniae (5.6% vs 24.1%, p = 0.024) in Korean intestinal tract, ²⁰⁷ which indicates the important role of environmental factors. ²¹⁰

5. DETERMINATION

To determine a K. pneumoniae strain as HvKP, the golden standard should be animal tests, such as mouse lethality test, ¹⁵ Galleria mellonella lethality test. ¹⁸ , ¹⁹ , ²⁵ Besides, serum killing assay is also an alternative method. ²¹¹ However, the aforementioned tests are cumbersome, sometimes confusing ²¹² and not ready to use for clinical laboratories. It is essential to find novel methods of determining HvKP for clinical practice.

“String test” was once used for HvKP. The formation of a viscous string >5 mm is considered positive when the colony is stretched out using a loop from a blood‐agar plate. ¹⁰² “String test” showed a sensitivity of 89% and a specificity of 91%, ⁹² which are inconsistent with another report. ²¹³ Sequence types (ST) and serotypes are both not so specific for HvKP, but the virulence gene repertoire may be one optimal clue to choose markers. ⁵⁴ , ⁶⁵ , ⁸¹ , ²¹⁴ Virulence genes in the virulence plasmids (for example, pK2044 and pLVPK) ⁴⁷ , ⁹³ , ²¹⁵ are more accurate for defining HvKP than those in integrative and conjugative elements (ICE), ¹⁸⁷ , ¹⁹⁰ , ¹⁹⁷ , ²¹⁶ among which are genes encoding yersiniabactin and colibactin, not vital for hypervirulence. Nevertheless, iroB, iucA, peg‐344, rmpA, and rmpA2 in the virulence plasmids are all optimal biomarkers for defining HvKP, ⁹² among which iuc and/or either rmpA or rmpA2 would be the best combined markers. In the recent report of 5 HvKP strains, ²⁵ iro, peg‐344, and rmpA were all negative in the relevant plasmid. With the recognition on molecular biomarkers progressing for defining HvKP, novel single or combined markers are likely to be designated in the near future.

On the other hand, hypervirulence of K. pneumoniae is restricted by series of virulence genes (Table 2), which means that the polymorphism and deletion of any of such genes may affect the eventual virulence. The lack of hypervirulent phenotype in virulence plasmid‐bearing CR‐KP strains was found to be due to the mutation's presence on rmpA and rmpA2 genes, which rendered them non‐functional, while some strains carrying wild type rmpA did not exhibit hypervirulent phenotype either suggesting that other factors might also contribute to the hypervirulence of CR‐KP. ²¹⁷ A large proportion (58%) of CR‐KP strains in China mainland during 2014–2017 were found to harbor a virulence plasmid, while only 13% of such strains exhibited a hypervirulent phenotype by string test and neutrophil assay. ²¹⁷ Therefore, the complexity of virulence in K. pneumoniae indicates the molecular combinations for hypervirulence need further investigations and optimizations.

6. EVOLUTION

Clonal group (CG) 23 is associated with K1 capsule and HvKP, which accounts for over 30% of (ST) for HvKP‐inducing PLA. ²¹⁴ , ²¹⁸ , ²¹⁹ , ²²⁰ Another study showed over 80% of CG23 K. pneumoniae strains inducing PLA belong to CG23‐I, which emerged in ~1928 following acquisition of ICEKp10, and then disseminated globally ¹⁹⁰ ; Ninety‐four of the 97 strains possessed plasmid‐borne iro, iuc, rmpA, and rmpA2. ¹⁹⁰ The possible dates for the most recent common ancestors for the entire CG23 population, the CG23‐I sublineage, and the equine strains could be 1878, 1928, and 1972, respectively.

Plasmids pK2044 (224,152 bp) and pLVPK (219,385 bp) ⁹³ , ²¹⁵ are the 2 classical ones conferring hypervirulence in K. pneumoniae, ⁹⁷ , ¹⁶² , ¹⁶³ which is mediated through iuc, rmpA, and rmpA2 genes. pK2044 and pLVPK are highly similar. ²²⁰ , ²²¹ Their descendants varied in the length, for example, 121 Kb, 90 Kb, 200 Kb, and 178 Kb. ²⁵ , ⁴⁷ , ²²⁰

The first KPC‐producing K. pneumoniae was found in a patient in a North Carolina Hospital in 1996, ²²² while the first KPC‐2‐producing K. pneumoniae in China was found in Zhejiang Province in 2007. ²²³ Furthermore, the first Hv‐CRKP, showing K2 type and ST65 emerged in China in 2013. ¹⁵ Another recent study unveiled an outbreak of Hv‐CRKP with ST11 and a pLVPK‐like virulence plasmid pVir‐CR‐hvKP4 (178,154 bp). ²⁵ Compared with pLVPK, pVir‐CR‐hvKP4 had a deletion of 41,231 bp fragment, which included the virulence genes rmpA and iro. Another report from Taiwan investigated a strain TVGHCRE225 with ST11 and a pVir plasmid (297,984 bp), a hybrid HvKp virulence plasmid. ²²⁴ 38% of pVir shared 49% and 47% of identities with pK2044 and pLVPK, respectively, the remaining portion possessing 61% coverage with pPMK‐NDM, a resistance plasmid, at 99% identity.

MDR and extremely drug‐resistant (XDR) HvKP are concerning pathogens, which accounts for 7.4–15.0% among CR‐KP ²²⁵ albeit 57% (20/35) HvKP strains inducing bacteremia were concurrently CR‐KP in a 2016 investigation from China. ²²⁶ Hv‐CRKP and CR‐HvKP, the most notorious ones among MDR and XDR HvKP, are now seemingly emerging worldwide. ²⁵ , ²²⁷ Such evolution may occur through 2 mechanisms. The first pathway is via HvKP acquiring a plasmid carrying drug resistance determinants ²²⁸ , ²²⁹ or by the insertion of resistance genes into virulence plasmid or chromosome harbored by HvKP. ²³ , ²³⁰ The second pathway is via MDR/XDR cKP acquiring a pK2044‐ or pLVPK‐like virulence plasmid or integrated virulence genes into drug resistance plasmid. ²⁵ The virulence plasmids themselves are usually non‐conjugative and therefore non‐self‐mobilizable. However, they could be mobilized or co‐transferred with the help of the self‐transferable incompatibility group F plasmids. ⁸⁴ , ²³¹ Due to the hypercapsule of HvKP itself, which could mask the fimbriae and hamper the conjugation, the second pathway may be more convenient. Hv‐CRKP is far more prevalent than CR‐HvKP. ⁸⁴ ST11 accounted for over 70.0% among CR‐HvKP/Hv‐CRKP strains. ⁶² However, such overall assessment is lacking (Table 3). ⁷ Hv‐CRKP and CR‐HvKP are both still progressing, obtaining other resistance, for example, to colistin. ²³²

TABLE 3.

Characteristics of Hv‐CRKP and CR‐HvKP

Classification	Location	ST	Serotype	Clinical context	Note	References
Hv‐CRKP	China	ST11	K47	Ventilator‐associated pneumonia	Few cases: 5 isolates; XDR; 178 Kb pLVPK‐like plasmid; One clone	25
Hv‐CRKP	China	ST11	K47	Retrospective study	One case; pLVPK‐like plasmid and 2 drug resistance plasmid; unique feature of 5 tandem copies of bla KPC‐2	233
CR‐HvKP	USA	ST23	K1	UTI	One case; bla SHV‐36, fosA, oqxAB on chromosome; drug resistance plasmid with bla KPC‐2, bla TEM‐1A and truncated bla OXA‐9	227
CR‐HvKP	China	ST23	K1	Sepsis	One case; pLVPK‐like plasmid with insertion of bla KPC‐2 and dfrA14	23, 234
CR‐HvKP	China	ST36	K62	Bloodstream, burn wounds	One case; pLVPK‐like plasmid and drug resistance plasmid with bla KPC‐2, fosA, oqxAB	229
CR‐HvKP	China	ST86	K2	Burn wound	One case; bla KPC‐2 and bla NDM‐1; 215 Kb virulence plasmid	235
CR‐HvKP	China	ST65	K2	Septicemia	One case; Encodes enterobactin and aerobactin but not yersiniabactin or kfu; bla SHV‐11, bla TEM‐53, ompK35/36 decreased expression	15
CR‐HvKP	Canada	ST86	KL2	UTI	One case; Plasmid with bla KPC‐2 as well as bla SHV‐1 and fosA	236

Abbreviations: CR‐HvKP, carbapenem‐resistant hypervirulent Klebsiella pneumoniae; Hv‐CRKP, hypervirulent carbapenem‐resistant Klebsiella pneumoniae; ST, sequence type; XDR, extreme drug resistance; UTI, urinary tract infection.

Integration and conjugation (Figure 2) are two primary means of acquiring foreign nucleic acids. tRNA sites are often targets for integration and 73% of K. pneumoniae strains had an ICE inserted into one or more of the four asparagine tRNA genes. ²¹⁶ ICEKp1, 76 Kb long in NTUH‐2044, was more prevalent in HvKP than in cKP: 38/42 vs 5/32. ²³⁷ Later, a more widely conserved ICEKp (KPHPI208‐GM1 also designated ICEKp10) was described to possess 8 genomic modules, ¹⁸⁷ which is ubiquitous in CG23 HvKP strains. ⁴⁷ ICEKp10, the most common ICE, was found in 77% of ST23, 40% of ST258, and in 25 other STs strains. ²³⁸ However, ICEKp10 is poorly conserved in non‐CG23 HvKP. The comparative analysis of 97 CG23 genomes showed the 81 members of sublineage CG23‐I had acquired ICEKp10 containing genes that encode yersiniabactin and colibactin. ¹⁹⁰ , ²³⁸ ICEKp is widely distributed in both cKP and HvKP, which bore 14 variants. ²³⁸ ICE could also carry other virulence genes apart from those encoding yersiniabactin and colibactin. ICEKp1 encodes RmpA and salmochelin. ²³⁷ In addition, the integration of other ICE or genomic islands is also a commonplace. ²¹⁶ , ²³⁹ The yersiniabactin carried by ICE is more beneficial in cKP than in HvKP. ⁶⁰ , ¹⁵⁸ Colibactin contributes to colonization, mucosal invasion, and/or dissemination of K. pneumoniae. ¹⁸⁹ Furthermore, the other factors carried by ICEKp or other genomic islands may prove to play critical roles in various settings.

FIGURE 2.

The formation and transfer of drug resistance or virulence elements. (A) the formation of drug resistance or virulence elements. (B) the transfer of drug resistance or virulence elements

The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated proteins (Cas), restriction and modification (R‐M) are the two primary immune systems in bacteria, which are both responsible for surveillance of nucleic acids and may play salient roles in horizontal gene transfer (HGT) of drug resistance or virulence genes. ²⁴⁰ CRISPR‐Cas system could cut exogenous DNA by targeting proto‐spacers. ²⁴¹ , ²⁴² , ²⁴³ Recent studies ²⁴⁴ , ²⁴⁵ confirmed that CG258 K. pneumoniae strains lack type I‐E CRISPR‐Cas system and another investigation suggested such absence is associated with the dissemination of IncF epidemic drug resistance plasmids in CG258. ²⁴⁶ Furthermore, CG258 K. pneumoniae strains also has an impaired R‐M system. ²⁴⁷ Type I R‐M system consists of HsDR (slicing), HsdM (methylating) and HsdS (targeting), ²⁴⁰ , ²⁴⁸ which could hamper invasion of foreign DNA via methyltransferase and restriction endonuclease. ²⁴⁸ In addition, CRISPR‐Cas and R‐M systems could cooperate to combat exogenous nucleic acids. ²⁴⁹ , ²⁵⁰ Therefore, lack of CRISPR‐Cas and R‐M renders CG258 more propense to acquire outer plasmids carrying drug resistance or virulence genes, which is called HGT. Armed with extreme drug resistance and virulence, CR‐HvKP and Hv‐CRKP strains can then survive better in both community and healthcare settings and cause consequent both vertical transfer and HGT, forming a vicious cycle. KPC(+) ST11 CR‐KP accounted for 11 mobile genetic element clusters with type A and F sharing 20.83% and 54.76%, respectively, ²⁵¹ showing an evident combination of HGT and vertical transfer which is likely present in Hv‐CRKP.

7. CONCLUSIONS

Klebsiella pneumoniae was first described in the late 19th century, which was more and more documented for its hypervirulence and drug resistance. Hypervirulence of K. pneumoniae is primarily dependent on some factors, that is, hypercapsule, macromolecular exopolysaccharide, or excessive siderophores albeit K. pneumoniae harbors numerous virulence genes. Molecular determination of HvKP is a practical pathway in contrast with the traditional laborious lethality tests while further improvement is needed. HvKP, including Hv‐CRKP and CR‐HvKP, could bring great challenges worldwide in clinical practice in the future due to their extreme virulence and drug resistance. The impaired immune systems (CRISPR‐Cas and R‐M) could enhance such trend.

As confirmed now and speculated in the future, Hv‐CRKP is far more prevalent than CR‐HvKP. They would not bring more and more novel virulence, but present more and more drug resistance, for example, polymyxin resistance. Their targeted options are rather limited. More researches are needed to be done to fight against it.

AUTHOR CONTRIBUTIONS

Piaopiao Dai and Dakang Hu conceived of and wrote the review, which was revised by Dakang Hu.

FUNDING INFORMATION

The study is supported by Zhejiang Medical and Health Research Program (2023KY1326).

CONFLICT OF INTEREST

None declared.

Dai P, Hu D. The making of hypervirulent Klebsiella pneumoniae . J Clin Lab Anal. 2022;36:e24743. doi: 10.1002/jcla.24743

DATA AVAILABILITY STATEMENT

Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.