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Introduction
The Gram-negative bacilli Klebsiella pneumoniae are an opportunistic pathogen with high pathogenic and epidemic potential, contributing to infection outbreaks worldwide. K. pneumoniae are the etiological factors of respiratory tract infections, mainly pneumonia, meningitis, septic infections and difficult-to-treat urinary tract infections. Increasing drug resistance, high mortality among patients infected with this pathogen and difficulties in treating the infection resulted in the World Health Organization (WHO) including these bacteria on the list of one of the most dangerous pathogens in the world (WHO 2024).
General characteristics of Klebsiella genus
Nomenclature and taxonomy
The generic name Klebsiella comes from the surname of the German microbiologist Edwin Klebs (1834–1913). Bacteria was first isolated in 1882 by Carl Friedländer from a patient who died of pneumonia (Grimont and Grimont 2015). The first species belonging to the genus Klebsiella described by Karl von Frisch was the bacterium Klebsiella rhinoscleromatis isolated from a patient with scleroma (Grimont and Grimont 2015).
In routine microbiological practice, exploitation of the 16S rRNA gene as a molecular marker led to the correction of previous findings regarding the taxonomy of bacilli belonging to the genus Klebsiella (Ma et al. 2021). This gene contains conserved regions (regions common to many bacteria) and species-specific regions, which allows precise identification of the genus or species of an isolated bacterial strain based on comparing determined sequences with sequences available in public databases (Srinivasan et al. 2015). According to the current state of scientific knowledge acquired in the course of many molecular studies, it has been shown that other genes with evolutionarily conserved sequences, for example, selected housekeeping genes, including the rpo B gene encoding the β subunit of RNA polymerase, are also characterized by a high potential differentiating (He et al. 2016). Currently, the identification of various groups of bacteria is carried out based on phenotypic and genotypic features based on rRNA coding sequences or sequenced genomes, additionally supplemented with information obtained from the amino acid structure of proteins performing critical functions in cells (Kim et al. 2021). On this basis, changes in the taxonomy of gamma-proteobacteria were proposed in 2016, the name of the Enterobacteriales order was modified, and a new division of families separated from the Enterobacteriaceae family, which included genus Klebsiella was introduced within the Enterobacterales order novum (Adeolu et al. 2016). Based on the findings, the monotypic order Enterobacteriales, containing one family of Enterobacteriaceae, was transformed into the polytypic order Enterobacterales order novum, consisting of seven new families, including Enterobac-teriaceae, Erwiniaceae, Pectobacteriaceae, Yersiniaceae, Hafniaceae, Morganellaceae and Budviciaceae (Adeolu et al. 2016). It was also agreed that bacteria previously classified to the Enterobacteriaceae family, including species of the Klebsiella genus, will now be classified as a taxon in the order Enterobacterales (Fig. 1) (Adeolu et al. 2016; Schoch and Karsch-Mizrachi 2020).
Fig. 1.
The current taxonomic position of the species K. pneumoniae.
In the course of the conducted phylogenetic studies, considering the latest divisions of microorganisms within the Klebsiella genus, a new species, Klebsiella aerogenes, appeared, referred to as a nomenclature (homotypic) synonym as Klebsiella mobilis (Szewczyk 2019). These bacteria were first described in 1885 by Theodor Escherich as “Bacterium lactis aerogenes”, then renamed “Bacillus aerogenes” in 1896 by Walther Kruse, then “Aerobacter aerogenes” and finally named in 1960 by Estenio Hormaeche and Peter Geoffrey Edwards as Enterobacter aerogenes (Tindall et al. 2017). Klebsiella mobilis is an opportunistic pathogen responsible for nosocomial infections (Szewczyk 2019).
The genus Klebsiella also contains species previously counted in other taxonomic groups, including Klebsiella oxytoca and Klebsiella ozaenae (Tachibana et al. 2022; Yang et al. 2022). The first was isolated from sour milk and first described in 1886 by Carl Flügge as “Bacillus oxytocus perniciosus”, then renamed in 1923 by David Hendricks Bergey as “Aerobacter oxytocum” and finally named K. oxytoca by Hans Lautrop in 1956 (Yang et al. 2022). The species name K. oxytoca comes from the Greek language and consists of the two elements “oxus”, meaning “sour”, and “tokos”, meaning “production” (Yang et al. 2022). The other species included in the genus Klebsiella was K. ozaenae. These bacteria were observed in 1893 by Rudolf Abel in the nasal discharge of patients with ozena, or chronic atrophic malodorous rhinitis. Initially, these capsulated bacteria were known as “Bacillus mucosus ozaenae” and finally changed its name to K. ozenae (Tachibana et al. 2022). Another species included in the Klebsiella genus was Klebsiella granulomatis with the former name “Donovania granulomatis”, otherwise called “Calymmatobacterium granulomatis” - the etiological agent of inguinal granuloma (donovanosis), i.e. an infectious granulomatous disease affecting the genitals and groin (Belda Junior 2020). The genus Klebsiella has also been enriched with a new species, Klebsiella variicola, isolated mainly from elements of edible plants such as roots, leaves and banana stem (Latin Musa spp.), corn shoots (Latin Zea mays L.), rice roots (Latin Oryza sativa L.) (Ma et al. 2021). In 2001, three other species of Klebsiella, namely Klebsiella ornitynolytica, Klebsiella planticola, and Klebsiella terrigena isolated from the environment previously classified as “Klebsiella-like organisms” were transferred to the newly created genus Raoultella (Kimura et al. 2014; Ma et al. 2021).
There are several taxonomic classification systems of rods belonging to the genus Klebsiella in use in the world, including the Cowan classification system introduced in 1960, the Bascomb classification system introduced in 1971, and the Ørskov classification system introduced in 1984 (Grimont and Grimont 2015). Most scientific teams rely on the classification developed by Ørskov (Grimont and Grimont 2015). Currently, the genus Klebsiella includes 22 species (Table I).
Clinical significance of selected Klebsiella species presented in alphabetical order
Species
Special features
References
1. K. aerogenes
The opportunistic pathogen, an etiological agent of nosocomial infections, present in various sewage wastes, chemicals and soil. Commercially important bacterium, „preeminent producer of hydrogen” produced by anaerobic fermentation, used as a substrate in molasses experiments, and a common cause of spoilage in maple sap and syrup.
The etiological agent of inguinal granuloma (donovanosis), an infectious disease occurring in tropical and subtropical regions of Southeast Asia, India, Africa and Central America. The diagnosis of donovanosis is based on the history taking, the characteristic clinical picture (no changes in the lymph nodes) and the detection of the presence of vacuole in the tissue smear, the so-called Donovan bodies surrounding bacteria.
The opportunistic pathogen. First detected in Michigan. The bacterium was first isolated in Europe from blood and rectal swabs from an immunosuppressed patient.
The second important species pathogenic for humans after K. pneumoniae. Isolated from pneumonia, and UTIs. Common cause of nosocomial infections in neonatal wards.
The most frequently isolated in about 95% of all Klebsiella strains. An opportunistic pathogen. Isolated from: sepsis, endotoxic shock, pneumonia, lung abscesses, infections of the urinary, digestive and biliary tracts. In addition, it causes inflammation of the sinuses, middle ear, inflammation of soft tissues, osteomyelitis, and meningitis in newborns.
Frisch’s bacillus, the etiological agent of heart disease („rhinoscleroma”) known as „Slavic leprosy”, a chronic infectious granulomatous disease of the respiratory tract covering mainly the nasal cavity, as well as the oral cavity, pharynx, larynx, trachea, and bronchi; is now very rare in Poland.
The name derives from „quasipneumoniae” which means almost like „pneumoniae”. The opportunistic pathogen. Pathogenicity as in K. pneumoniae, mainly the etiological agent of pneumonia.
Name derived from „similis” which means similar to „pneumoniae”. The opportunistic pathogen. Pathogenicity as in K. pneumoniae, mainly the etiological agent of pneumonia.
These rods account for less than 10% of Klebsiella clinical isolates previously classified as K. pneumoniae. Hypervirulent isolates have been identified, and colistin-resistant isolates of this species are also reported. Abundant in the environment (mainly rivers), edible plants, e.g. root, leaves, banana stem, sugar cane stem, corn shoots, rice roots. The etiological agent of mastitis in cattle.
Bacteria of the genus Klebsiella are microorganisms widely distributed in the natural environment (Navon-Venezia et al. 2017; Khan et al. 2019; Huang et al. 2020). Moreover, Klebsiella is part of the microbiota in humans and various animals (dogs, cats, horses and pigs) (Navon-Venezia et al. 2017). Klebsiella, excreted in human and animal feces, is commonly found in soil, groundwater, surface and seawater, and on various plants such as banana, corn, rice and sorghum (Khan et al. 2017; Huang et al. 2020). These bacteria are also a component of industrial sewage (Navon-Venezia et al. 2017). The high adaptability of many species of Klebsiella bacilli, mainly K. pneumoniae, enables them to colonize hospital environments where multidrugresistant hospital strains are selected. Recent studies show these strains present in the hospital environment, primarily in anesthesiology, intensive care, cardiology, neurosurgery, and neonatal departments. In patients and medical staff, these bacteria are part of the physiological permanent or transient microbiota (Ali et al. 2022). Outside the hospital, premature infants, newborns, older adults, as well as immunocompromised patients and alcoholics, are most at risk for infections caused by Klebsiella bacilli, in particular K. pneumoniae (Chang et al. 2021).
Characteristics of the Klebsiella pneumoniae species
Morphology, growth conditions, culture and biochemical profile
Klebsiella pneumoniae (formerly called Friedländer’s bacilli) is a cylindrical, capsulated, ciliated, non-sporeforming bacterium measuring 0.3 to 1 μm in width and 0.6 to 6 μm in length (Fig. 2) (Ali et al. 2022). Some clinical strains of K. pneumoniae may be equipped with a single flagellum, which determines motility. The presence of these flagella is considered a virulence factor (Carabarin-Lima et al. 2016). K. pneumoniae in culture microscope preparations are arranged in pairs or short chains, the cells generally joining poles (Szewczyk 2019).
Fig. 2.
Schematic representation of the differences in cell morphology of classical (cKp) and hypervirulent (hvKp) K. pneumoniae, taking into account virulence factors.
Schematic representation of the subsequent stages of bacterial biofilm formation and its extracellular polymeric substance (EPS).
Own graphic design according to (Zhao et al. 2023).
K. pneumoniae are facultative anaerobes that grow at an optimal temperature of 37°C. These bacilli survive in the inanimate environment in a wide temperature range (12°C–42°C). ‘The ability to break down glucose, especially at high temperatures (up to 44.5°C), gives bacteria an advantage in non-living environments by providing energy for key life processes, supporting biofilm production and enabling adaptation. As a result, bacteria can survive longer in harsh environments, enhancing their ability to infect new hosts (Mason and Ztirich 1987; Centeleghe et al. 2023; Horng et al. 2023).
These bacteria are catalase-positive and indole-negative, produce urease, ferment lactose, produce lysine decarboxylase, do not produce ornithine decarboxylase, reduce nitrates to nitrites, do not produce deoxyribonucleases (DNases), use malonic acid and citrate as a carbon source, are oxidase-negative, do not cause deamination of phenylalanine (Brisse et al. 2014; Mączyńska 2015; Szewczyk 2019). Broth cultures of K. pneumoniae are uniformly turbid with a ring or a characteristic film located on the surface of the culture. Like other species of Enterobacterales, K. pneumoniae grow well and abundantly on solid substrates, forming characteristic mucous, shiny, convex, smooth, grey-white colonies (Murray et al. 2022). K. pneumoniae bacilli cultures can be performed on non-selective media such as tryptic soy agar (TSA) and blood agar (BA), as well as on selective-differentiating (selective) media such as: (1) MacConkey agar containing selective factors (crystal violet and sodium deoxycholate) and a differentiating factor (lactose) – K. pneumoniae form pink colonies, (2) eosin-methylene blue agar (EMB), medium containing selective factors (eosin and methylene blue) and differentiation factors (glucose and/or sucrose) – K. pneumoniae form blue-black colonies, (3) as well as Drigalski medium containing selective factors (crystal violet and sodium deoxycholate) (4) and bromothymol blue agar (BTB), differentiation medium on which the distinguishing of bacilli from the family Enterobacteriaceae is based on the ability to ferment the lactose in the presence of bromothymol blue – K. pneumoniae form yellow colonies (Brisse et al. 2014; Szewczyk 2019).
Species identification
Various microbiological methods are used to identify the species of K. pneumoniae bacilli, from microscopic techniques through traditional phenotypic methods to advanced molecular analyses (Grimont and Grimont 2015; Cheng et al. 2018; Froböse et al. 2020). During the cultivation of bacilli on solid media containing carbohydrates, the isolation and initial classification of bacteria into the Klebsiella genus is facilitated by the visible mucous appearance of bacterial colonies as a result of the production of a multi-sugar bacterial coating capsular polysaccharide (CPS) by K. pneumoniae strains (Szewczyk 2019; Murray et al. 2022). The bacterial capsule of K. pneumoniae can be visualized using various staining techniques, including the negative-positive method (Burri-Gins) using Chinese ink and alkaline fuchsin. Identification of K. pneumoniae strains characterized by the ability to create a hypermucoviscosity (HM) phenotype typical of highly pathogenic isolates is carried out using the string test (Eisenmenger et al. 2021). Identification of K. pneumoniae is based on traditional bacteriological methods involving the analysis of biochemical features using manual methods or standardized sets, e.g. API® 20E strips, or automatic methods using compact systems for identifying bacteria with the simultaneous determination of antimicrobial susceptibility of microorganisms, for example using the VITEK® 2 Compact system (Master et al. 2013). According to Monnet et al. (1991), for conventional methods, incorrect identification was made in 13% of the K. pneumoniae strains tested (Monnet et al. 1991). Precise identification to the species level can be performed using highly specialized techniques using matrix-assisted laser description/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) (Váradi et al. 2017).
Among the molecular methods used for species identification of K. pneumoniae, the polymerase chain reaction (PCR) method is commonly used (Järvinen et al. 2009). For the molecular identification of K. pneumoniae, as well as typing of the most pathogenic strains, numerous genetic methods based on the multiplex-PCR technique have been developed, aiming to detect genes encoding pathogenicity factors, as well as to determine the serotype of the envelope (Chen et al. 2014; Fonseca et al. 2017). Multiplex-PCR has a high sensitivity and test specificity of over 90% (Dessajan and Timsit 2024). PCR also forms the basis of other techniques used in K. pneumoniae identification/differentiation based on regions of the rrn operon. These techniques use a variety of methods, including amplification of a variable region within the gene encoding 16S or 23S rRNA, amplification of polymorphic sequences located between the genes encoding 16S and 23S rRNA (Internal Transcribed Spacer-PCR, or ITS-PCR) (Liu et al. 2008) and Real-Time PCR for detecting K. pneumoniae with rmpA or magA genes associated with the hypermucoviscosity phenotype (Hartman et al. 2009). The Real-Time PCR technique has high sensitivity and specificity (Hartman et al. 2009). Droplet digiatal (ddPCR) is used to detect K. pneumoniae in stool samples (Feng et al. 2024).
Another innovation is the Loop-Mediated Isothermal Amplification (LAMP) method (Poirier et al. 2021). LAMP (like the method with PCR) uses technology based on amplification and detecting specific DNA sequences. It relies on the isothermal amplification reaction of nucleic acids. The LAMP method is highly specific, as six primers (3 pairs) are used in the reaction, and amplification of genetic material occurs only if the primers recognize 6 to 8 specific DNA sequences of the pathogen under study (Poirier et al. 2021). The LAMP technique found particular application in a study by Poirier et al. (2021), who identified three target genes (yhaI, epsL and xcpW) common to K. pneumoniae isolates from both China and Europe and designed LAMP assays for detecting K. pneumoniae in clinical samples (Poirier et al. 2021). In turn, Dong et al. (2015) described the LAMP method for rapid detection of the synthesis of the envelope polysaccharide regulating the rcsA gene from K. pneumoniae (Dong et al. 2015). The LAMP method is also being used to detect carbapenem resistance genes (blaKPC, blaNDM-1, blaOXA-48-like, blaIMP-1 group, and blaVIM) in K. pneumoniae (Poirier et al. 2021; Kgim et al. 2022).
In addition to PCR-based methods, methods on Sanger sequencing, next-generation sequencing (NGS) and whole-genome sequencing (WGS) are used for proper molecular identification of K. pneumoniae species (Nafea et al. 2024). The main advantage of NGS over the conventional method is the simultaneous use of many genetic markers with high-resolution genetic data (Nafea et al. 2024). In turn, the high resolution of WGS analyses can provide information on the origin of bacteria, their routes of transmission, and biological traits (e.g., serotype). WGS also enables the identification of virulence genes and antibiotic-resistance genes. WGS analyses are suitable for introduction into routine laboratory testing. With comparative analysis of the entire genome, WGS could become the primary typing method used for early detection of epidemic outbreaks and monitoring the dynamics of the spread of a given pathogen (Nafea et al. 2024).
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-Cas) is increasingly used in microbiology. One of the first applications of this method was the typing of bacterial strains. CRISSP-Cas can also help develop new antimicrobial strategies (Barrangou et al. 2016; Ding et al. 2020). These techniques may find application in advanced studies of K. pneumoniae.
Molecular methods are increasingly being used in the identification of K. pneumoniae over classical phenotypic methods because they are independent of culture conditions, are more reproducibly sensitive and allow for shorter waiting times for results. The versatility of these methods is due to their applicability to the examination of virtually any biological material with minor adjustments to laboratory procedures. The limitations of these methods are limited availability and the inability to distinguish whether specific genetic material comes from live or dead bacteria.
Pathogenicity
Klebsiella spp. constitute a heterogeneous group of closely related enteric bacilli. Klebsiella rods are commonly found in hospital and non-hospital environments. They are part of the KESC subgroup (Klebsiella, Enterobacter, Serratia and Citrobacter subgroup), which collects genera with the closest relatedness and a similar biochemical profile (Szewczyk 2019). Microorganisms classified as KESC are characterized by multidrug resistance (MDR), and the presence of factors that enable them to survive freely in the hospital environment makes them a frequent cause of nosocomial infections (Szewczyk 2019). The species of the Klebsiella genus that most often causes infections in humans is K. pneumoniae. The second most frequently isolated species from clinical materials is K. oxytoca. The remaining Klebsiella species, much less frequently, may also be the etiological factor of infections (Chang et al. 2021).
K. pneumoniae is responsible for most (about 95%) severe human infections (Murray et al. 2022). Risk fac-tors predisposing to K. pneumoniae infections include age (premature infants, newborns, the elderly), reduced immunity, debilitating diseases (cancer), frequent or prolonged hospitalization, assisted breathing, surgical interventions in the abdominal cavity, use of catheters (vascular, urological) drains and other implants, alcoholism, smoking, residence in nursing homes, colonization of the gastrointestinal tract by hospital strains (Fig. 4) (Mączyńska 2015).
Fig. 4.
Schematic representation of the risk factors that increase predisposition to K. pneumoniaepremature babies, newborns, elderly people), b – lowered immunity, c – debilitating diseases (cancer), d – concomitant diseases (e.g. diabetes), e – smoking, f – alcoholism, g – frequent or long-term hospitalization, h – use of vascular and urological catheters, drains and other implants, i – assisted breathing, j – stay in nursing homes, k – surgical interventions in the abdominal cavity, l – colonization of the gastrointestinal tract by hospital strains.
Klebsiella can cause both healthcare-acquired infections (HAIs) and community-acquired infections (CAIs) (Chang et al. 2021). HAIs usually affect premature and frail neonates, older adults, and immunocompromised patients and include pneumonia, urinary tract infections (UTI), septic infections, endocarditis, central nervous system infections, purulent infections, wound infections, gastrointestinal infections associated with toxin production (Chang et al. 2021). CAIs include pneumonia, primary liver abscesses (PLA) combined with a characteristic invasive syndrome characterized by blood-borne infections spreading to other organs (bones and joints, eye, brain, lung, prostate, spleen) and rare infections occurring endemically (ozena, scleroderma and donovanosis) (Table I) (Fig. 5) (Fusconi et al. 2018; Belda Junior 2020; Tachibana et al. 2022).
Fig. 5.
Schematic representation of the pathogenicity of K. pneumoniae: a – pneumonia, b – central nervous system infections, c – primary liver abscess, d – cholecystitis, e – septic infections, f – gastrointestinal infections associated with toxin production, g – urinary tract infections, h – bone and joint infection, i – soft tissue infections, j – purulent and wound infections.
K. pneumoniae is crucial in primary hospital-acquired pneumonia (HAPs) and community-acquired pneumonia (CAPs). In hospitalized patients, K. pneumoniae strains cause 7–14% of HAPs. The frequency of HAPs depends on the ward and condition of the patients and the intensity of invasive medical procedures (intubation, tracheotomy) associated with respiratory support (Mączyńska 2015). These factors increase the risk of pulmonary infections, including those associated with ventilator-acquired pneumonia (VAP), for which K. pneumoniae is also a significant etiologic agent. K. pneumoniae is generally the only Enterobacteriaceae causing 4–5% of out-of-hospital respiratory tract infections occurring most often in patients over 60 years of age, in poor health, often accompanying another severe underlying disease (e.g., diabetes, cardiovascular disease) but also much more common in smokers and alcohol abusers. These infections are characterized by a sudden onset, a severe course and a relatively high mortality rate. The characteristic symptom of the disease is the expectoration of a large amount of purulent, thick secretion, often colored by blood (Mączyńska 2015; Murray et al. 2022). The most important virulence factor of K. pneumoniae causing pneumonias is the polysaccharide envelope, which protects the bacteria from phagocytosis, and lysis by the complement system. It plays a crucial role in pathogenesis. Adhesins (especially fimbriae types 1 and 3) facilitate colonization of the airway epithelium and the production of mucus (exopolysaccharide) that is part of the biofilm structure that K. pneumoniae can form, for example, in the patient’s lungs and endotracheal tubes or tracheostomy tubes, are also important in pneumonia (Alcántar-Curiel et al. 2013; Mączyńska 2015; Cader et al. 2020; Ochońska et al. 2021; Murray et al. 2022).
In addition to pneumonia, K. pneumoniae is often responsible for causing urinary tract infections – UTIs. K. pneumoniae causes 6–17% of hospital-acquired UTIs (Campana et al. 2017; Murray et al. 2022). UTIs can progress as pyelonephritis, typical bladder infections, but can also be recurrent and lead to permanent kidney changes. Among the most critical pathogenicity factors of uropathogenic K. pneumoniae strains are fimbriae, responsible for bacterial adhesion to urinary tract epithelial cells, which prevents the washout of microorganisms during micturition. The ability to produce urease and form biofilm structures on urinary catheters are also important virulence factors (Campana et al. 2017).
Septic infections caused by K. pneumoniae can manifest as asymptomatic bacteremia; these bacilli can also cause sepsis. The most common causes of sepsis are untreated urinary tract infections, respiratory tract infections and inflammation and obstruction of the intestines in immunocompromised patients (Carabarin-Lima et al. 2016).
A primary liver abscess (PLA) and the characteristic invasive syndrome are mainly caused by hvKp strains. The ability of hvKp strains to cause PLA with a tendency to spread to multiple tissues and organs (central nervous system, kidney, bones, eyes, lungs, prostate, skin and subcutaneous tissues, pancreas) is significant. The most common route of infection is bacterial translocation from the intestine. PLA caused by K. pneumoniae is localized in the right lobe of the liver and is limited in nature. Multiple abscesses may occur at different locations with greater or lesser frequency, commonly as liver abscesses, lung abscesses, biliary tract abscesses, skin and soft tissue abscesses, pleural abscesses, peritonitis, and inflammation of the external coating of the eye (endophtalmitis) (Ali et al. 2022). The rapid course of infection, having a poor prognosis and a tendency to develop generalized infection, is referred to as “characteristic invasive syndrome” (DIS) (Mączyńska 2015).
Among the isolates of K. pneumoniae are distinguished between classical K. pneumoniae (cKp) strains and hypervirulent K. pneumoniae (hvKp) strains (Fig. 2) (Russo et al. 2024). The cKp strains commonly cause infections in immunocompromised individuals. These are community-acquired pneumonia, UTIs, bacteremia or meningitis (Dai and Hu 2022). In addition, cKps strains are isolated from elderly patients and those with risk factors such as alcohol abuse and smoking (Russo et al. 2024). cKp is a group of K. pneumoniae that lacks hypercapsule, macromolecular exopolysaccharide or excessive siderophores and rarely causes disease in healthy individuals (except for UTIs), although it is MDR (Dai and Hu 2022). In contrast, hvKp are highly virulent strains responsible for community-acquired pneumonia characterized by a severe course and a high mortality rate reaching up to 40% in some regions of the world (Russo et al. 2024). As a result of the translocation process from the gut, strains of hvKP can spread to other organs and tissues (central nervous system, lungs, bones, prostate, skin and subcutaneous tissue) and also contribute to the formation of PLAs (Russo et al. 2024). hvKp is another type of K. pneumoniae that harbors hypercapsule, macromolecular exopolysaccharide, or highly active siderophores and induces infections in both immunocompromised and otherwise healthy individuals (Dai and Hu 2022).
Virulence factors
The pathogenicity of K. pneumoniae is determined by many virulence factors (Compain et al. 2014; de Souza et al. 2024). Their presence can lead to infection and antibiotic resistance. The major virulence factors playing an essential role in the pathogenesis of infections caused by K. pneumoniae are capsule polysaccharides (CPS, K-antigen) and lipopolysaccharides (LPS, O-antigen). These critical virulence factors help to enter the bloodstream and cause septic shock in the host. Fimbrial and non-fimbrial adhesins, siderophores (aerobactin, enterobactin, salmochelin and yersiniabactin), heat-stable and heat-labile enterotoxins, cytolysins and the ability to form a biofilm are also important (Fig. 2) (Ali et al. 2022). The genes encoding many virulence factors are located in the large mosaic virulence plasmid (pLVPK) in K. pneumoniae isolates (Mączyńska et al. 2015; Clegg and Murphy 2016).
Capsule polysaccharide (CPS)
The capsule polysaccharide (CPS) is the essential, well-known virulence factor of K. pneumoniae, forming a layer hermetically surrounding the bacterial cell wall (Ali et al. 2022). It is responsible for the initial interaction between bacteria and host. Moreover, the CPS is vital for the survival of K. pneumoniae in the host tissue, allowing the pathogen to escape phagocytosis (Ali et al. 2022). Structurally, K. pneumoniae CPS is a heteropolymer comprised of repeated sugar moieties of hexoses (D-glucose, D-galactose and D-mannose), deoxyhexoses (D-fucose and D-rhamnose) and glucuronic and galacturonic acids. The capsule may also include non-sugar components such as O-acyl, succinate, formate and pyruvate residues (Ali et al. 2022). The CPS of K. pneumoniae is characterized by the diversity of the structure of polysaccharides that build them and high serological variability. Genes involved in capsule production are located on the chromosome’s capsular polysaccharide synthesis (cps) region. The region of the cps cluster (from galF to ugd) harbors over 20 genes, mainly driven by three promotors located upstream of genes, galF, wzi, and manC, respectively (Fig. 6) (Zhu et al. 2020). A group of six genes mainly carries out the synthesis of the capsule at the 5’ end of the cps operon (galF, cpsACP, wzi, wza, wzb, wzc) and the ugd gene located at the 3’ end (Shu et al. 2009; Wyres et al. 2016). The diversity of serotypes is the result of the action of various glycosyl-transferases (GTs), whose genes wbaP, wbaZ, wcaN, wcaJ and wcaO, are located in the middle part of the cps operon (Shu et al. 2009). The protein encoded by wbaP mediated the first step in capsule biosynthesis. Glycosylation of the repeating unit is initiated by WbaP (when the initializing sugar linked to the undecaprenol-pyrophosphate – Und-PP – is galactose) or by WcaJ (when the initializing sugar linked to Und-PP is glucose). Current research has shown that some wbaP mutations increased pathogenicity by increasing biofilm formation and invasion of bladder epithelial cells in urinary tract infections (UTIs) (Zhu et al. 2020). Several genes that regulate the biosynthesis of CPS sugar, rmlA, rmlB, rmlC, rmlD, manB and manC, are also located towards the end of the cps locus (Ali et al. 2022). Differences in K. pneumoniae capsule types result from changes in the nucleotide sequences of the cps locus and genes involved in CPS biosynthesis, assembly and translocation (Ali et al. 2022).
Fig. 6.
(A) Schematic representation of the capsule (CPS) biosynthetic pathway in K. pneumoniae. Own graphic design according to (Patro and Rathinavelan 2019, Rendueles 2020, Patro et al. 2020), (B) Scheme of the representative locus cps system of K. pneumoniae using the K1 serotype as an example. Own graphic design according to (Rendueles 2020, Patro et al. 2020).
Enhanced capsule production in K. pneumoniae may result from the activity of other genes in addition to those located in the cps cluster: the capsular synthesis gene B (rcsB), the mucoid phenotype regulators A and A2 (rmpA and rmpA2) and Klebsiella virulence regulators (kvrA and kvrB) and wzy-K1 (Zhu et al. 2020). Different combinations of these genes can result in the production of capsules with different structures. The rmpA or rmpA2 genes are found in 55–100% hvKp strains, while they are less frequently found in cKp strains. RmpA regulates mucoid phenotype in pK100 and RmpB (Dai and Hu 2022). The expression of rmpA depends on RcsB, KvrA, and KvrB. The newly described regulators, kvrA and kvrB, affect the virulence of K1/K2 hvKp strains due to the activation of capsule gene expression, which is not present in cKp strains. Different from rcsB found in chromosome, both rmpA and rmpA2 could be located in plasmid or chromosome. Chromosomal rmpA (c-rmpA and c-rmpA2) are located in an integrative and conjugative element (ICEKp1) and are only found in < 50% K. pneumoniae strains of serotype K1 (Zhu et al. 2020; Dai and Hu 2022). Plasmidic rmpA (p-rmpA and p-rmpA2) are more prevalent. The wzy-K1 gene is specific to the K1 serotype of K. pneumoniae. The function of a wzx gene product is to transport the polymer from the cytoplasm to the periplasm. The Wzy protein is involved in the polymerization via a catch-and-release mechanism (Zhu et al. 2020). Proteins encoded by wza and wzc genes form a translocation complex responsible for assembling capsular polysaccharides and transporting them from the periplasm to the surface of the bacteria (Pan et al. 2013). The Wzb protein, as the cognate phosphatase of Wzc, combines with the catalytic domain on Wzc and, in turn, dephosphorylates Wzc (Zhu et al. 2020).
Hypercapsules can be regulated by the capsule A (cpsA) and B (cpsB) genes (Dai and Hu 2022). 70% of hvKp strains produce a hypercapsule composed of types K1 and K2. This kind is more stable than the typical capsule found in cKp strains, contributing to their increased virulence in hvKp strains. Other capsule types occur in cKp strains (Marr and Russo 2019). In K. pneumoniae, the capsule binds to the surface protein Wzi. Loss of this protein can reduce or lose virulence (Ali et al. 2022). There are 79 serotypes of capsulated K. pneumoniae strains (K1 to K79). The eight most common types have been described in hvKp strains: K1, K2, K5, K16, K20, K54, K57, and KN1 (Zhu et al. 2020). Recently, a classification scheme has been proposed based on the sequence of conserved wzi and/or wzc genes in the cps locus (Ali et al. 2022).
In K. pneumoniae, the WGS method is specifically used to identify cps locus variants (Wyres et al. 2016). In a study conducted by Wyres et al. (2016), among 2503 K. pneumoniae genomes, the diversity of capsid fusion loci (K-loci) was examined. The study included analysis of full-length K-locus nucleotide sequences and clustering protein-coding sequences to identify, annotate and compare K-locus structures. A total of 134 distinct K-locuses were identified, including 31 new types. Comparative analyses revealed 508 unique clusters of protein-coding genes that appear to reassort through homologous recombination. In addition, a high diversity of intra- and inter-locus nucleotides was detected among wzi and wzc genes. Based on the results, a standardized nomenclature for K loci was proposed, a reference database was presented, and a new software tool – Kaptive, was developed to automate the process of identifying K loci based on complete locus information extracted from the whole-genome sequence (https://github.com/katholt/Kaptive) (Wyres et al. 2016).
Assessment of the prevalence of specific serotypes in the K. pneumoniae population is valuable for epidemiological investigations. The prevalence of individual serogroups/serotypes depends on the geographical location, patient age, and changes over the years. Strains of different serotypes differ in their resistance to phagocytosis in vitro and their ability to activate the humoral response. Some serotypes are more frequently associated with human diseases and epidemics. Hypervirulent strains of K. pneumoniae (hvKp) with high virulence usually have the K1 or K2 envelope antigen (Ali et al. 2022). K. pneumoniae strains represent the same epidemic clone and have the same capsule type (Choi et al. 2020). Serotyping is not sufficient for epidemiological purposes due to its poor resolution. However, the synthesis of the bacterial capsule is determined by a set of cps genes located in the chromosome and plasmid. Thanks to knowledge of the allele sequence in the cps locus, the PCR method is now more widely used for their detection (Walker and Miller 2020; Ali et al. 2022).
Lipopolysaccharide (LPS)
Lipopolysaccharide (LPS), also called endotoxin, is an integral and essential component of the cell membrane of Gram-negative bacteria (Ali et al. 2022). LPS has strong cytotoxic, immunomodulatory and pro-inflammatory properties. LPS remains one of the most important pathogenic factors and the main antigen of the K. pneumoniae cell wall. It plays a role in the pathomechanism of infection, especially in endotoxic shock accompanying central nervous system infections, blood infections and pneumonia (Choi et al. 2020). It is connected with a massive release of LPS after bacterial cells lysis. Increased release of LPS can occur under the influence of various groups of antibiotics that cause lysis of bacterial cells or interfere with their function, including β-lactams (Eng et al. 1993; Kirikae et al. 1997; Holzheimer 2001).
LPS is a substance with a conservative structure consisting of three fundamental components, i.e. lipid A, the core oligosaccharide and a polysaccharide that determines antigenic specificity (chain O, somatic antigen O). Serological typing of K. pneumoniae is based on two main groups of antigens, i.e. the somatic polysaccharide O and the typical-specific capsular antigen K (Choi et al. 2020).
Lipid A is a crucial virulence factor responsible for the endotoxic effects of LPS (Navon-Venezia et al. 2017). It is recognized as the most structurally conserved region. Several enzymes encoded by the lpx gene cluster are involved in the synthesis of lipid A components. The host immune cell receptor, Toll-like receptor 4 (TLR4), recognizes and binds lipid A of LPS, which initiates a cascade of host immune reactions. Although modifications of the lipid A component help the pathogen escape recognition by the host immune cells by favoring the pathogen to establish the infection successfully (Ali et al. 2022). Lipid A is the hydrophobic part of endotoxin, responsible for anchoring the heteropolymer in the outer membrane of the host, thanks to which it creates a specific barrier that inhibits the penetration of substances, including antibiotics and detergents, into the microorganism while generating resistance to these compounds. In K. pneumoniae, the ineffective antimicrobial effect of colistin can occur through plasmid-mediated transfer of mcr-1, a resistance gene, causing modification of LPS lipid A and disruption of the interaction between polymyxins and lipid A (MacDermott-Opeskin et al. 2022). Acylation of lipopolysaccharides plays a key role in providing Gramnegative bacteria with some resistance to structural and intrinsic defense mechanisms, particularly the antibacterial properties of detergents (e.g., bile) and cationic defensins (Clements and Strugnell et al. 2022).
The core oligosaccharide component of the LPS connects lipid A to the terminal side chains called the O antigen. The genes encoding core oligosaccharides are located in the waa locus, and the ligase enzyme WaaL, which links the core structure to the antigen O chain, is encoded by waaL. The outer part of the LPS structure, O antigen, comprises multiple repeating units of oligosaccharides: glucose, galactose, mannose and ribose residues. Epidemiological investigations within a species are based on their structure. The wb gene cluster regulates the O antigen’s synthesis, assembly and translocation. The variation in the oligosaccharide repeats underlies the LPS diversification structurally and functionally. So far, up to nine O K. pneumoniae antigens have been identified based on the composition of the sugar molecules (Ali et al. 2022).
Fimbrial and non-fimbrial adhesins
The adhesive properties of K. pneumoniae are also due to the possession of fimbrial and non-fimbrial adhesins. The fimbrial adhesins include type 1 mannose-sensitive (MS) fimbriae, type 3 mannose-resistant (MR) fimbriae, type 6 fimbriae, KPF-28 fimbriae. Klebsiella pneumoniae fimbriae with a fimbrin molecular mass of 28 kDa) and Kp (a-g) fimbriae (Klemm et al. 2000; Struve et al. 2009; Chen et al. 2011; Alcantal-Curiel et al. 2013; Mączyńska 2015; Alcantal-Curiel et al. 2018; Khonsari et al. 2021). Non-fibrillar adhesins include the non-fibrillar P-type adhesion factor and CF29K adhesion protein (CF29K adhesion factor) (Staniszewska et al. 2000; Chan et al. 2012; Hennequin et al. 2016).
Type 1 fimbriae are among the best characterized. They are expressed in about 90% of K. pneumoniae strains (Mączyńska 2015). They are mannose-sensitive hemagglutinins (MSHAs), forming long, thick, stiff filaments 1 to 2 μm long and about 7 nm in diameter (Chen et al. 2011). Type 1 fimbriae are protein heterocomplexes of the major fimbriae subunit (FimA) that form the protuberance’s structure. Smaller subunits (FimB, FimC, FimD, FimE, FimF, FimG, FimH, FimK, FimS and FimX), in addition to adhesion functions, are responsible for protuberance elongation and stability (Alcantal-Curiel et al. 2013; Mączyńska 2015). Receptors for FimH are mannosides. The protein determining adhesion properties can be located at the top of the fimbriae and distributed along the spear’s entire length (Alcantal-Curiel et al. 2013, Mączyńska 2015). The individual subunits of the fimbriae are linked by hydrophobic bonds and form a right-handed helix stabilized by hydrogen bonds. The structural and functional integrity of the elements formed is called the “fimbriae-adhesin complex” (Mączyńska 2015). A set of fim genes located in a chromosome or plasmid is responsible for the expression of type 1 fimbriae. Synthesis of type 1 fimbriae follows the “all-or-nothing” principle (Mączyńska 2015). Recent evidence shows that the expression of fimbriae’s subunit genes responsible for turning on or off fimbriae synthesis can be directly influenced by oxygen availability, elevated temperature but also by the presence of sub-minimal inhibitory concentrations (sub-MICs) of an antibiotic (e.g. streptomycin), which can affect the production of longer fimbriae lacking the ability to bind mannose (Shibl et al. 1985; Klemm et al. 2000; Struve et al. 2009; Mączyńska 2015). Streptomycin induces fimbriae formation that is both functionally and morphologically abnormal. This may have resulted from amino acid substitutions in fimbrial proteins due to the misreading of mRNA by ribosomes (Shibl et al. 1985).
Type 3 fimbriae are expressed on the surface in more than 80% of K. pneumoniae strains (Murphy and Clegg 2012; Khonsari et al. 2021). These are protein heterocomplexes and are mannose-resistant hemagglutinins (MRHA). They form short and thin filaments about 2–4 nm wide and 0.5–2 μm long (Murphy and Clegg 2012). At least nine genes from the mrk cluster are required to express type 3 fimbriae. The mrk gene cluster can be located in chromosomal or plasmid DNA. The mrkA gene encodes the main structural subunit of the fimbriae, while the mrkD gene encodes the actual adhesin. This protein determines the specific interaction of the protuberance with the receptor. Smaller fimbriae subunits MrkB, MrkC, and MrkD form the characteristic structure, and their genes mrkB, mrkC and mrkD regulate the spears’ expression. The product of the mrkF gene stabilizes the fimbriae structure on the bacterial cell surface (Murphy and Clegg 2012; Alcantal-Curiel et al. 2013; Mączyńska 2015).
Type 6 fimbriae are the longest, thick spears present in small numbers on the bacterial surface. Type 6 fimbriae have only been confirmed in the species K. pneumoniae subsp. ozenae and their role in pathogenicity is little understood (Darfeuille-Michaud et al. 1992; Mączyńska 2015).
KPF28 fimbriae (Klebsiella pneumoniae fimbriae with a fimbrin molecular mass of 28 kDa) are a long, thin, and flexible, about 4 to 5 nm in diameter and 0.5 to 2 mm long 6). The N-terminal amino acid sequence of the KPF-28 major fimbrial subunit showed no homology with type 1 and type 3 pili of K. pneumoniae. Still, it showed 61.7% identity with residues 6 to 19 of the N-terminal amino acid sequence of PapA, the Pap major pilus subunit expressed by uropathogenic Escherichia coli strains (UPEC) (Di Martino et al. 1996). In a study of K. pneumoniae responsible for nosocomial infections, KPF-28 was shown to be present in strains producing the extended-spectrum β-lactamase CAZ-5/SHV-4 (current name SHV-4) (Di Martino et al. 1996). KPF-28 fimbriae are plasmid-encoded, specifically in plasmid R, which contains blaSHV-4 gene (Di Martino et al. 1996). A study by Di Martino et al. (1996) involving K. pneumoniae strain CF914-1 isolated from urine from a patient in ICU and 78 other K. pneumoniae isolates involved in nosocomial infections showed that fimbriae KPF-28 were present in K. pneumoniae strain CF914-1, as well as in vast majority (83%) of clinical K. pneumoniae strains producing SHV-4 extended-spectrum β-lactamase (DiMartino et al. 1996). Further studies on the occurrence of KPF28-type fimbriae in K. pneumoniae strains causing UTIs are needed.
Kp-type fimbriae (Kpa, Kpb, Kpc, Kpd, Kpe, Kpf and Kpg) are another seven types of fimbriae detected in K. pneumoniae (Wu et al. 2010). A study by researchers in Taiwan showed that Kp-type fimbriae are only found in K. pneumoniae strains with the K1 capsule antigen and increase the ability of strains to form a biofilm (Wu et al. 2010).
A non-fimbrial P-type adhesion factor is a protein heteropolymer that exhibits hemagglutination properties similar to the analogous properties of P fimbriae in E. coli. It has no fimbrial filament-forming subunits. The receptor for the non-fimbrial P-type adhesion factor is the globoside receptor α – D – Galp – (1 4) – D – Galp. The P-type factor involves bacterial adhesion to the epithelium of the urinary, gastrointestinal and respiratory tract (Staniszewska et al. 2000).
CF29K (nonfimbrial protein of 29 kDa) (CF29K adhesion factor) – nonfimbrial adhesion protein was found in K. pneumoniae strains characterized by high adhesion capacity to intestinal cell lines. It is encoded by the cf29A gene located on a plasmid that also contains the gene encoding TEM-5 β-lactamase. It shows high homology to the CS31A-L protein encoded by the clpG gene and produced by enterotoxigenic E. coli (ETEC) strains (Hennequin et al. 2016).
Siderophores
Siderophores such as aeroactin, enterobactin, salmochelin and yersiniabactin are virulence factors synthesized by K. pneumoniae (Farzand et al. 2021). Bacterial siderophores, called iron carriers, are low-molecular, organic chemical compounds of a nonprotein and non-porphyrin nature, chelating iron ions and secreted extracellularly by some microorganisms to capture this element (Farzand et al. 2021). In bacterial cells, iron is an element necessary for the synthesis of cytochromes and ribonucleotide reductase, which are involved in the DNA synthesis process, as well as other enzymes. The survival of K. pneumoniae in the environment depends on siderophores to meet the demand for iron. They compete with the host for the available iron pool (Chhabra et al. 2020).
Enterobactin is the primary iron uptake system in K. pneumoniae and is the most commonly but not only siderophore synthesized in this bacterial species. Studies show that hypervirulent hvKp strains quantitatively produce more siderophores than cKp strains (Dai and Hu 2022). Enterobactin is catecholate. The chromosomal gene cluster entABCDEF and fepABCDG encode its biosynthesis and transport. ybt and fyu genes encode transporters for the secretion of enterobactin, and ybtO encodes the uptake receptor of enterobactin. Lipocalin-2 (LCN2), known as neutrophil gelatinase-associated lipocalin (NGAL), is extremely important in immune processes and can bind and neutralize enterobactin. LCN2 participate in the regulation of cell aging, cell differentiation and modeling of the immune response (Xiao et al. 2017). In K. pneumoniae respiratory tract infection, LCN2 is up-regulated by the host. This lipocalin also has pro-inflammatory effects, leading to IL-8-mediated recruitment of neutrophils to the site of infection (Dai and Hu 2022).
Aerobactin is a citrate-hydroxamate siderophore found mainly in more than 90% hvKp strains, while it is less common (6%) in (cKp) strains. Aerobactin was present in hvKp-caused lung infections and is the dominant siderophore in hvKp strains. Aerobactin production is usually associated with hypercapsule, while K. pneumoniae with hypercapsule does not always contain aerobactin. The iucABCD gene cluster controls aerobactin synthesis, while its transport is determined by iutA. They are often present in the same pLVPK-like plasmids carrying p-rmpA (Dai and Hu 2022). LCN2 does not neutralize aerobactin (Xiao et al. 2017).
Salmochelin is a c-glucosylated form of enterobactin and another siderophore in K. pneumoniae. Glucosylation is carried out by the iro gene cluster, iroABCDE, which can be localized in a chromosome or plasmid. IroN contributes to the transport of iron-carrying salmochelin. Salmochelin is not neutralyzed by LCN2. This siderophore induces colonization of the nasopharyngeal cavity by K. pneumoniae, leading to pneumonia. Salmochelin, like aerobactin, is usually found in hvKp strains with a frequency of over 90% and only 2–4% in cKp strains (Dai and Hu 2022).
Yersiniabactin is another siderophore in K. pneumoniae whose production is likely due to horizontal gene transfer (HGT) genes from Yersinia. Yersiniabactin was found in 18% of cKp strains and 90% hvKp. Located in chromosome irp gene cluster encodes proteins for yers-iniabactin synthesis. Yersiniabactin and enterobactin are highly expressed during lung infection, and LCN2 does not inhibit it in vivo. However, yersiniabactin alone cannot acquire the iron for K. pneumoniae, and the lack of the other three siderophores would prevent K. pneumoniae from colonizing the lungs (Holden and Bachman 2015; Dai and Hu 2022).
Heat-stable and heat-labile enterotoxins
Important extracellular pathogenicity factors of K. pneumoniae are also enterotoxins – protein toxins similar to enteroaggregative E. coli heat-stable enterotoxin 1 (EAST 1), heat-stabile (ST) and heat-labile (LT) enterotoxins (O’Ryan 2011). Plasmids carrying enterotoxin-coding genes acquired by multidrug-resistant strains may contribute to the emergence of epidemics. Bacterial diarrhea occurring in the hospital environment can cause the spread of bacteria, and strain ability to produce enterotoxins can be a factor that predisposes to cause an epidemic outbreak (O’Ryan 2011).
Hemolysins
Until recently, Klebsiella spp. were considered nonhemolytic, and only single papers of one research group from the 1980s described the hemolysin produced by K. pneumoniae and K. oxytoca (Barberis et al. 1986). According to them, these hemolysins are thiol-activated cytolysins and are supposed to belong to the TACY group. The cytolytic activity of the TACY group toxins is observed upon the addition of thiol compounds, for example, 2-mercaptoethanol or dithiothreitol (DTT). The hemolysin produced by these bacteria was named klebolysin, and it was established that it is inactivated in the presence of cholesterol and crossreacts with antibodies directed against streptolysin O (Szramka 2001).
K. pneumoniae biofilm
Over the past few years, there has been a growing problem of infections caused by K. pneumoniae due to the bacteria’s ability to form a biofilm (Dsouza et al. 2019). K. pneumoniae exhibits many pathogenic properties that facilitate their survival, spread in the hospital environment, and adhesion to biotic or abiotic surfaces (Piperaki et al. 2017). K. pneumoniae is characterized by a high ability to adhere and form biofilm structures, which plays an essential role in the colonization and persistence of these microorganisms on mucous membranes of the body and artificial surfaces of catheters, implants and others. Important pathogenic features of K. pneumoniae involved in biofilm formation include overproduction of mucus (hvKp strains), selection of strains with a specific type of envelope and transfer of adhesin genes in plasmids (Guerra et al. 2022).
Biofilm is defined as a complex organized multicellular, single- or multi-species structure in which bacterial cells are embedded in a matrix made of extracellular polymeric slime (EPS), where they adhere to each other and/or show adhesion to various surfaces (Piperaki et al. 2017). The phenomenon of biofilm formation is a process that occurs in several stages: reversible adhesion, irreversible adhesion, maturation and dispersion (Fig. 3) (Piperaki et al. 2017). Then, as a result of the movement of bacterial cells along with blood and other body fluids, the colonisation process of new niches begins, giving rise to a new biofilm. Mature biofilm structures are characterized by bacterial persister cells (PCs), which enable the renewal of the biofilm population (She et al. 2022). The biofilm formation process is controlled by “quorum sensing”, a unique intercellular communication system regulated by chemicals called signaling molecules (Piperaki et al. 2017).
Bacteria in biofilms, including pathogens such as K. pneumoniae, display highly developed adaptive capabilities that enable them to survive under challenging conditions, colonize new environments, and evade the host immune system (Piperaki et al. 2017; Dsouza et al. 2019; Thoraninsdottir et al. 2020; Guerra et al. 2022; Centeleghe et al. 2023). Mature biofilm provides a protective barrier against the effects of antibiotics and disinfectants. One of the possible mechanisms of antibiotic resistance is limited penetration of antibiotics into the bacterial cell and reduced metabolism of cells located inside the biofilm. Even if antibiotics reach the bacteria, the cells inside the biofilm are less metabolically active, making antibiotics that target rapidly dividing cells (e.g., β-lactams) less effective. Biofilm constitutes a physical barrier for immune cells such as neutrophils and macrophages, hindering their access to the bacteria inside (Piperaki et al. 2017; Dsouza et al. 2019; Thoraninsdottir et al. 2020; Guerra et al. 2022; Centeleghe et al. 2023). The biofilm matrix can also bind components of the complement system, which hinders its activation and limits the effectiveness of the immune response. Bacteria in a biofilm can survive in conditions lethal to planktonic (free-swimming) bacteria. Low nutrient or oxygen availability is compensated by bacteria differentiating into different metabolic states. Currently, the phenomenon of biofilm formation is related mainly to the rapid development of biomaterials engineering (biomedical materials) and their extensive use in various fields of modern medicine (Chung et al. 2016; Piperaki et al. 2017; Zheng et al. 2018; Dsouza et al. 2019; Thoraninsdottir et al. 2020; Ochońska et al. 2021).
Biomaterial surfaces with biofilm formation are an essential reservoir of etiological agents of biomaterials associated infections (BAIs) (Chung et al. 2016; Piperaki et al. 2017; Thoraninsdottir et al. 2020). Targeted at improving patient comfort and function, the comprehensive use of biomaterials contributes to an increase in the frequency of the risk of developing BAIs. It is currently estimated that BAIs are responsible for approximately 65–80% of all infections occurring in humans and animals (Garcia and Percival 2011; Guerra et al. 2022). BAIs include device-related and non-device-related infections due to streptococci, staphylococci, Gram-negative bacteria and/or fungal infections (Jamal et al. 2018). A study of clinical strains of K. pneumoniae reported that 72.7% of tested isolates detected on medical devices were biofilm producers. However, they remained susceptible to different classes of antibiotics (Folliero et al. 2021). Another study confirmed the survival of K. pneumoniae on dry surfaces in biofilm (Centeleghe et al. 2023). The presence of viable but non-culturable (VBNC) bacteria indicated that K. pneumoniae could survive on surfaces for up to 4 weeks. It was possible to remove these bacteria from surfaces by mechanical wiping. The study proved the need for robust cleaning regimens in the hospital (Centeleghe et al. 2023). Another research showed that 44.4% of the tested clinical strains of K. pneumoniae could form biofilm on the surfaces of tracheostomy tubes made of polyethylene and polyvinyl chloride. The biofilms formed on the inner part of these surfaces were observed using scanning electron microscopy (SEM) after only 48 hours of exposure to a bacterial suspension at a concentration of 106 CFU/ml (Ochońska et al. 2021). A varied degree of biofilm formation by clinical K. pneumoniae strains was also found on venous catheters made of polyurethane and urinary catheters made of latex, polyvinyl chloride and silicone. In a study of the penetration of antibiotics (β-lactams, quinolones, aminoglycosides and trimethoprim) and disinfectants (chlorhexidine, ethacridine lactate, hydrogen peroxide, polyhexanidine, povidone-iodine and octenidine) into the biofilm formed by K. pneumoniae, the minimum inhibitory concentration (MIC) for bacteria in the biofilm was higher than in the planktonic form (Bartosze-wicz et al. 2011). Preliminary analyses of the effect of erythromycin on the biofilm formed by K. pneumoniae strains showed that macrolides affect the synthesis of the AI-2 autoinducer system, for example, by reducing the expression level of luxS genes, blocking the autoinducer synthase enzyme or the signal molecule itself (Martínez and Baquero 2002). The search for agents that would prevent the formation of K. pneumoniae biofilm or cause its breakdown is still ongoing, e.g., studies involving attempts to coat catheters with various substances, e.g., silver (Mousavi et al. 2023) or to use specific antimicrobial preparations such as octenidine hydrochloride or sodium hypochlorite (Stoffel et al. 2020; Huang et al. 2022). Attempts are also being made to use some enzymes that eliminate slime or block the metabolic pathways of bacteria, leading to their multiplication and biofilm formation (e.g. DNA-ze, oxindole-L-alanine, a tryptophanase inhibitor regulating the disintegration of tryptophan to indole) (Mączyńska et al. 2015). In addition, research is being conducted on regulating K. pneumoniae biofilm production. New genes related to this process are being discovered, such as luxS – synthesis of autoinducer, luxR – coordination of biofilm formation steps, e.g. regulation of synthesis of various virulence factors (including “quorum sensing”), fimA – regulation of specific adhesion, magA, rmpA, rmpA2 – regulation of mucus production in hvKp strains (Widmer et al. 2007; Mączyńska 2015).
Fimbrial and non-fimbrial adhesins also play a vital role in BAIs by K. pneumoniae (Alcántar-Curiel et al. 2013). These bacterial structures actively participate in adhesion to epithelial cells, which facilitates colonization, which is the first stage of infection. A thorough understanding of the structures involved in bacterial adhesion and invasion may contribute to the discovery of effective inhibitors of these processes, which will allow for effective treatment at the beginning of the disease, which will enable effective suppression of the disease development (Davies et al. 2009; Kalia 2013; Gopu et al. 2015; Ribeiro et al. 2015; Wang et al. 2022).
The main approaches to reduce biofilm development involve modifying the surface of materials to reduce microbial adhesion. In recent years, many research teams have focused on low-molecular-weight compounds (small molecules) capable of inhibiting biofilm development. In a study by Davies and Marques (2009), it was shown that cis-2-decylenic acid, produced by a strain of Pseudomonas aeruginosa, can eradicate the mature biofilm of various bacterial species including K. pneumoniae (Davies et al. 2009). In another study, new chemical entities (NCEs) with activity against K. pneumoniae and Acinetobacter baumannii could be used in new therapies for drug-resistant infections (Blasco and Piddock 2024). A promising strategy to combat BAIs is application coatings that exhibit bacteriostatic or bactericidal properties (Siddique and Muzammil 2020). Siddique and Muzammil (2020) demonstrated the efficacy of silver nanoparticles (AgNPs) as safe antimicrobial and antibiofilm compounds against MDR K. pneumoniae (Siddique and Muzammil 2020). Among the intensively researched biological methods of K. pneumoniae biofilm eradication, phagotherapy appears promising (Zurabov et al. 2023). Another strategy for biological control of K. pneumoniae biofilm is using enzymes targeting the polysaccharide matrix (matrix-targeting enzymes) (Ribeiro et al. 2015). Interference with the structure or degradation of the extracellular polymeric matrix of the biofilm can effectively weaken it or lead to its dispersal (Ribeiro et al. 2015).
Inhibition of quorum sensing (QS) systems, called Quorum Quenching (QQ), is now considered another promising strategy to combat biofilm-forming bacteria (Kalia 2013). Many substances of natural and synthetic origin with the function of quorum sensing inhibitors (QSIs) have become known and may have potential therapeutic applications. Plant compounds are considered one of the most important groups of QSIs due to their chemical structure similarities to acylated homoserine lactone (AHL) and their ability to degrade protein transcriptional regulators (LuxR/LasR) (Kalia 2013). A study by Gopu et al. (2015) showed the quorum quenching activity of anthocyanin malvidin from Syzygium cumini (L.) Skeels against K. pneumoniae (Gopu et al. 2015). Promising results were obtained for two molecules (3-methyl-2(5H)-furanone and 2-hydroxycinnamic acid) that can be developed as a complement to antibiotics (Cadavid and Echeverri 2019). A study by Ahmad et al. (2020) attempted to identify new inhibitors of SdiA (a homolog of the transcriptional regulator LuxR) of K. pneumoniae using various computational techniques (Ahmad et al. 2020). A study by Liu et al. (2020) showed that tea polyphenols can act as an effective QS inhibitor, enhance resistance to K. pneumoniae infection in a Caenorhabditis elegans model, and may serve as a novel antiviral agent to combat bacterial pathogens (Liu et al. 2020). A study by Wang et al. (2022) showed that chlorogenic acid (CA) may be an effective antimicrobial and antiviral compound that can target QS in hvKp infections, thus providing a new therapeutic direction for treating bacterial infections (Wang et al. 2022).
Conclusion
K. pneumoniae is an example of a microorganism that has evolved from a common opportunistic microorganism to one of the most dangerous pathogens causing serious healthcare-acquired infections – HAIs. Infections caused by this bacterium are characterized by a severe and progressive course, requiring prolonged hospitalization of patients. They are often challenging to treat due to the ease of acquiring new virulence and antibiotic-resistance traits by K. pneumoniae. The accumulation of virulence factors in bacterial strains of K. pneumoniae significantly impacts their ability to cause disease and survive in the host. Through mechanisms of horizontal gene transfer, regulation of gene expression, biofilm formation and increased envelope production, these bacteria can effectively evade the host immune system. In addition, the spread of virulence mechanisms is facilitated by the development of civilization and the faster and unrestricted movement of highly virulent K. pneumoniae strains. In the face of these threats, knowledge about K. pneumoniae should be continuously updated to capture the changing pathogenicity characteristics to prevent future infections.
Orcid profile
