The process of biofilm formation includes several well-orchestrated events, starting with attachment to the colonizing surface, and continuing with production of microcolonies, biofilm maturation and further organization and finally, detachment of planktonic cells that can spread and colonize or infect sites nearby or at distant sites (Chao et al., 2014). In the attachment process, the microbial cells adhere to the surface through structures such as surface adhesins, as well as fimbriae and flagella (Marks et al., 2014; Piperaki et al., 2017). After adhesion, the bacteria multiply, generating microcolonies surrounded by self-produced extracellular polymeric substance (EPS), including polysaccharides, proteins, nucleic acids and lipids, which continues to be secreted throughout the maturation stage, providing the three-dimensional characteristic of the biofilm (Hall-Stoodley et al., 2004; Flemming and Wingender, 2010). In most cases, colonization of host surfaces likely results in incorporation also of host factors and molecules from other microbes in the resident microflora, including proteins and extracellular DNA that are used as a scaffold and further contributes to the biofilm three-dimensional structure (Okshevsky et al., 2015; Karygianni et al., 2020; Mirzaei and Ranjbar, 2022). The voids are mainly filled with water, functioning as a drainage system of waste and also acting as channels for nutrient acquisition and distribution between the micro communities (Flemming and Wingender, 2010; Jamal et al., 2018).

The dispersal step involves phenotypic changes in a fraction of the bacterial community, which detach from the structure and become planktonic. The microbial communities positively regulate the expression of proteins related to motility structures such as flagella and produce different sacrolytic enzymes that contribute to surface detachment, in response to signals from the environment, such as mechanical stress, nutrient availability, temperature variations, and the presence of extracellular ATP and other damage associated molecular patterns (DAMPs) (Baselga et al., 1994; Chao et al., 2014). Biofilm bacteria are less affected and killed than planktonic bacteria by antimicrobials and host defense systems (Guilhen et al., 2016); therefore, biofilms are regarded as a reservoir of bacteria during host infection.

It has become more and more evident that bacteria on medical devices or in host niches co-exist and synergize in polymicrobial biofilm communities (Elias and Banin, 2012; Flemming et al., 2016; Roder et al., 2016). These interactions often lead to an increased resistance against host and antimicrobial agents for all species involved (Fux et al., 2005; Attinger and Wolcott, 2012; Kong et al., 2016; Wang et al., 2022). This occurs either through increased tolerance or adaptive resistance resulting from inter-species synergy or by antibiotic-resistant species in a polymicrobial biofilm protecting other species in the biofilm against antibiotic treatment (Chatterjee et al., 2014; Puca et al., 2021). Species in polymicrobial biofilms often show increased virulence (Pastar et al., 2013), an increased ability to degrade and utilize organic compounds in their environment (Yoshida et al., 2009; Elias and Banin, 2012), and provide an environment for intra- and interspecies spread of adaptive traits and antimicrobial resistance genes (Roberts and Kreth, 2014).

The complex oral microflora may be considered one of the best studied polymicrobial biofilm environments, with well-established model systems based on physiological media, advanced imaging, and species identification by 16S rRNA gene sequencing (Bowen et al., 2018; Luo et al., 2022). Here, species composition and extracellular matrix components influence and specify the function of the biofilm by affecting cell-cell interactions and the microenvironment to modulate virulence of specific organisms (Flemming et al., 2016; Koo and Yamada, 2016). Greater diversity in the microbial community is generally associated with health (Bowen et al., 2018). Inter-kingdom interactions between bacterial and fungal species, including Candida albicans, are often observed in this niche and have also been well studied (Lohse et al., 2018; Ponde et al., 2021). Fungal-bacterial interactions are also important during skin and lung infections and in the gastrointestinal system, where Candida often helps to protect anaerobic species (Ponde et al., 2021).

Dual and polymicrobial interactions are also observed during wound infections (Goodwine et al., 2019; Warrier et al., 2021) and have been associated with urinary tract infections in patients originating from biofilms detected on urologic devices (Chatterjee et al., 2014). In the latter study, data from all culture positive catheters showed that the majority were positive for two or more bacterial species. Pseudomonas aeruginosa, Escherichia coli and K. pneumoniae were the most commonly isolated agents and found to co-exist (Chatterjee et al., 2014).

Besides studies investigating single-species biofilms with K. pneumoniae, described in detail below, several investigations have been conducted to understand the interaction with other relevant organisms. K. pneumoniae and P. aeruginosa, two organisms that often co-exist in the environment and potentially also in the gastrointestinal tract, synergize to form biofilms with distinct structures from their respective mono-biofilms (Lee et al., 2014). Mixed biofilms of these two organisms also display increased resistance to antibiotics, such as tobramycin, as well as resistance to detergent treatment (Lee et al., 2014). These interactions were not affected by the P. aeruginosa quorum sensing systems Las and Rhl (Subramoni et al., 2021) but optimal biofilm formation required the Pseudomonas type IV pilus, involved in motility as well as the ability of K. pneumoniae to produce extracellular matrix as a non-mucoid variant of K. pneumoniae grew poorly in the presence of P. aeruginosa (Booth and Rice, 2020). In another study, carbapenemase-resistant K. pneumoniae were examined in the context of environmental biofilms collected from sinks in patient rooms at hospital wards (Santiago et al., 2020). Using phage therapy, the authors were able to show that a phage cocktail was able to kill K. pneumoniae selectively without affecting the environmental bacteria.

The polysaccharide capsule is an important protection mechanism for the bacterium that inhibits complement deposition and prevents bacterial opsonization and phagocytosis (Piperaki et al., 2017). It is also a highly variable structure. To date, 134 distinct capsule synthesis loci (K-loci) have been identified K. pneumoniae isolates through genome sequencing and comparative genomics (Wyres et al., 2016).

The polysaccharide capsule has been shown to influence different stages of biofilm formation in K. pneumoniae, including initial surface adhesion and maturation (Balestrino et al., 2008). Mutant strains with defects in capsule production displayed impaired biofilm formation (Zheng et al., 2018). The contribution of capsular polysaccharides to biofilm formation has been confirmed in further studies. A study evaluating biofilm formation in K. pneumoniae bacteremia strains found a positive association with the expression level of the virulence gene wcaG, involved in capsule biosynthesis (Zheng et al., 2018). Moreover, wcaG silencing led to a reduction in biofilm formation in these bacteria. Interestingly, the hypermucoviscosity phenotype – a common trait in bacteremic K. pneumoniae – was not associated with increased biofilm formation in these strains (Zheng et al., 2018).

Two other genes associated with polysaccharide production, treC (an enzyme that splits trehalose-6-phosphate into glucose and glucose-6-phosphate) and sugE (which is predicted to encode an inner-membrane protein with a very short tail facing the cytoplasm), influence biofilm formation by K. pneumoniae isolated by pyogenic liver abscess (Wu et al., 2011). The same study described a role for treC during colonization of the gastrointestinal tract, suggesting that biofilm formation is important for successful colonization and further infection (Wu et al., 2011).

In addition to their direct contribution to biofilm formation, capsular polysaccharides from K. pneumoniae display anti-biofilm properties against other bacteria, providing a competitive advantage in mixed bacteria environments (Goncalves Mdos et al., 2014). Furthermore, the anti-biofilm activity of the capsular polysaccharides towards other bacteria was ubiquitous and not dependent on the K. pneumoniae serotype.

Studies on the capsule interference in fimbriae-mediated biofilm formation show that the adhesive properties of fimbriae are influenced by capsule expression, as the presence of the capsule masks the fimbriae, reduces adhesion and consequently impairs biofilm formation (Schembri, Dalsgaard, and Klemm 2004; Schembri et al., 2005).

In a study that investigate the contribution of the capsule in K. pneumoniae fitness under different growth conditions, Buffet et al, showed that in a poor nutrient environment, the encapsulated strains had a fitness advantage while in a nutrient rich environment the presence of the capsule represented a disadvantage. To further highlight the correlation between capsule and biofilm formation, this study also claimed that non-encapsulated strains showed more adherence in all environments, while the capsule production of the encapsulated strain masked the fimbriae and reduced biofilm formation. However, they suggest that the ability to form biofilm is not affected by the presence of the capsule, but that amount of capsule production impairs biofilm formation (Buffet, Rocha, and Rendueles 2021).

Lipopolysaccharide (LPS) is an important component of the external membrane in Gram-negative bacteria, including K. pneumoniae. The LPS also contributes to K. pneumoniae biofilm formation. Balestrino et al. demonstrated that LPS promotes the initial attachment of K. pneumoniae to abiotic surfaces and, therefore, is a critical factor in the early phases of biofilm formation (Balestrino et al., 2008). The authors showed that K. pneumoniae mutant strains lacking genes associated with LPS biosynthesis (wbbM gene) or transport (wzm gene) present a delay in biofilm formation. Their hypothesis is that LPS charge is necessary for the correct folding of Type 1 pilli which may explain their observation. More recently, Vuotto et al. emphasized the involvement of the wbbM and wzm genes in K. pneumoniae biofilm production by showing an upregulation of both genes in K. pneumoniae grown as biofilms in comparison with the expression in planktonic K. pneumoniae cells during the exponential phase (Vuotto et al., 2017).

K. pneumoniae expresses two main classes of fimbriae, named type I and type III. Fimbriae function as adhesins, promoting binding to biological surfaces (with subsequent tissue invasion) but also to abiotic surfaces, including medical devices, where the bacteria form biofilm (Piperaki et al., 2017). The genome of K. pneumoniae harbors at least 10 clusters of genes encoding chaperones, ushers, and adhesin proteins for the assembly of fimbriae, including fim, mrk, ecp and kpa to kpg gene clusters (Wu et al., 2010; Alcantar-Curiel et al., 2013; Khater et al., 2015). Among these, the most experimentally well-characterized are fim, mrk, ecp and kpf, which encode type I and type III fimbriae (Paczosa and Mecsas, 2016), common pilus (Alcantar-Curiel et al., 2013), and the type I-like fimbriae (Gomes et al., 2020).

The two main classes of fimbriae expressed by K. pneumoniae are type I and type III fimbriae. Type I fimbriae bind to receptors containing mannose, found in various tissues in the human host (Klemm and Schembri, 2000). Type III fimbriae have been shown to bind to different cell types in vitro, including kidney, lung and bladder epithelial cells. Although MrkD – the adhesin in type III fimbriae – has been shown to interact with collagen structures (Jagnow and Clegg, 2003), the specific cellular receptor for this molecule has not been identified.

In terms of biofilm formation, the type III fimbriae appear to be consistently associated with increased biofilm formation, whereas the role of type I fimbriae appears to be more complex and varies depending on experimental conditions and host niche. A study evaluating 33 strains of K. pneumoniae found a positive correlation between type III fimbriae expression and biofilm formation on abiotic surfaces, while a strain expressing only type I fimbriae did not form biofilms (Di Martino et al., 2003). This data was corroborated by Schroll et al. who found that type III, but not type I fimbriae, increase biofilm formation in a flow-cell system (Schroll et al., 2010). They also found that a mutant strain lacking type I fimbriae was not affected in its ability to form biofilms. Furthermore, in biofilms formed with bacteria lacking type III fimbriae, the fim operator was OFF, suggesting repression of type I fimbriae expression under biofilm conditions (Schroll et al., 2010). Also, the role of type III fimbriae in biofilm formation is supported by the observation that mutant K. pneumoniae strains lacking this gene cluster produced significantly less biofilm in the presence of lung surfactant, with cholesterol and phosphatidylcholine identified as surfactant components promoting type III fimbriae expression (Willsey et al., 2018). This study investigated factors of importance for survival in the lung and did not identify a role for Type I fimbriae. These studies combined would indicate a limited role of type I fimbriae in biofilm formation.

However, in contrast, a later study found that both type I and type III fimbriae promote biofilm formation in a model of catheter associated bladder infection using human urine as a culture medium (Stahlhut et al., 2012). In this study, only the double-negative mutant was significantly attenuated in its ability to form biofilms. Also, high expression of both fimbrial types was detected in mature 70 h biofilms, indicating that these structures are important also during later stages of biofilm development or maintenance (Stahlhut et al., 2012). Interestingly, immunostaining of biofilm bacteria using specific antibodies revealed that type I and type III fimbriae are not expressed simultaneously, suggesting overlapping roles for bacterial virulence. Finally, transformation of non-fimbriated E. coli with plasmids carrying either type I or type III fimbriae promoted an increase in biofilm formation on catheters, reinforcing the individual contribution of these structures to biofilm formation.

As the production of type I and III fimbriae has been shown to impact biofilm formation, how these fimbriae are regulated has important implications for the early stages of bacterial adhesion that lead to biofilm development. Regulation of fimbriae expression is an intricate process influenced by factors such as iron availability, oxidative stress and DNA binding regulators (Wu et al., 2012; Ares et al., 2016). Regarding the regulation of type III fimbrial genes and biofilm formation, Johnson et al. showed that the MrkH and MrkI regulators positively control the expression of type III fimbriae in K. pneumoniae, while mutant strains lacking both these regulators have a significantly decreased ability to form biofilms (Johnson et al., 2011). The mrkH and mrkI genes encode for proteins with a c-di-GMP-binding PilZ domain and a LuxR-type transcriptional regulator, and their regulatory effect is mediated by c-di-GMP. In the presence of this effector molecule, MrkH activates transcription of the MrkA promoter, leading to type III fimbriae expression and increased biofilm production (Wilksch et al., 2011). Also, Lin et al. showed that the global regulator IscR represses the expression of type III fimbriae in K. pneumoniae and strains depleted of IscR show an increased ability to form biofilms (Lin et al., 2017). According to those authors, IscR represses type III fimbriae by directly repressing the synthesis of MrkH and MrkI regulators (Johnson et al., 2011; Lin et al., 2017).

Type I fimbriae expression is controlled at the transcriptional level by phase variation and by the action of transcriptional regulators. Phase variation is mediated by an invertible DNA segment, named the fimS element, which contains the promoter of the fim operon and whose orientation determines the fimbriated or the nonfimbriated phenotypes (Abraham et al., 1985). The ON-orientation, corresponding to activation of fimbriae expression, was found in K. pneumoniae infecting the bladder, while the OFF-orientation was prevalent during gut and lung colonization with the same strain (Struve et al., 2008). This result suggests that type I fimbriae expression is influenced by the colonizing site and environment during infection. In addition to the phase variation, two type I fimbriae repressors, FimK and KpfR, represent inhibitory factors for biofilm formation by K. pneumoniae, since loss of fimK or kpfR genes renders mutant strains with a hyperfimbriated phenotype and an enhanced ability to form biofilms (Rosen et al., 2008; Gomes et al., 2020). FimK harbors an EAL domain in its carboxy-terminal region and it is involved in cleavage of c-di-GMP (Rosen et al., 2008). The presence of FimK was also associated with impaired adherence to the bladder of K. pneumoniae in comparison with UPEC in the early stages of infection, due to the inhibition of fimbriae production. However, the mechanism of fimbriae inhibition by FimK, as well as the factors regulating its action, are unknown. The kpfR gene encodes a transcriptional regulator of the kpf gene cluster, which encodes for a type I-like fimbriae. Our group has shown that KpfR expression is regulated by Fur (ferric uptake regulator) through an iron-dependent mechanism (Gomes et al., 2020).

Besides FimK and KpfR, our group has recently described a quorum-sensing regulator, SdiA, which controls the expression of virulence factors, including type I fimbriae, and affects biofilm formation (Pacheco et al., 2021). Mutant strains lacking SdiA showed increased biofilm formation in vitro, which correlates with an up-regulation of type 1 fimbriae, suggesting that SdiA acts as a repressor for fimbriae production and biofilm formation.

In addition to type I and III fimbriae, the two other fimbrial gene clusters experimentally characterized in terms of biofilm formation and cell adherence were the ecp and kpf gene clusters. The ecp operon is composed of the ecpR-A-B-C-D-E genes and encodes ECP, a pilus homologous to the Escherichia coli common pilus that is required for biofilm formation (Alcantar-Curiel et al., 2013), The kpf gene cluster comprises the kpfR-A-B-C-D genes and encodes type I-like fimbriae (Wu et al., 2010; Gomes et al., 2020). K. pneumoniae cells depleted of kpfR gene, which encodes a transcriptional repressor of fimbrial expression, present up-regulation of both type I and type I-like fimbriae, resulting in enhanced biofilm formation and greater adhesion to epithelial host cells (Gomes et al., 2020)

In an attempt to better understand the systems of chaperones, ushers, and adhesins encoded by K. pneumoniae for fimbriae assembly, Khater et. al, managed to construct and characterize mutant K. pneumoniae cells for the usher-encoding genes from each of the kpa to kpg fimbrial loci (Khater et al., 2015). According to the authors, only the deletion of the usher-encoding genes from kpa and kpg loci results in a significant reduction in biofilm formation compared to the wild-type strain. Moreover, only the strain depleted of the usher-encoding gene from the kpg adheres less to human cells than the wild-type strain (Khater et al., 2015). The broad number of chaperone, usher, and adhesin encoding genes on the K. pneumoniae genome highlights the impressive adhesion capacity of this bacterium and emphasizes the need to deepen the understanding of the colonization ability of K. pneumoniae.

In summary, K. pneumoniae fimbriae play important roles during biofilm formation, which vary according to the infection site (or abiotic surface) and are not limited to initial attachment of bacteria. Regulation of fimbriae expression is a complex process controlled by several regulators, which respond to diverse environmental signals in different host niches, while the absence of co-expression of type I and type III fimbriae suggests a co-regulation system with mutual inhibitory effects.

In pathogenic bacteria, one signal that triggers infection and colonization is the iron deprivation that the pathogen faces when in contact with the host. In fact, iron plays a crucial role in the regulation of numerous virulence factors, as well as biofilm formation (Gomes et al., 2020). Pathogens obtain iron through the activation of iron uptake systems. One of the most effective strategies to acquire iron from mammalian hosts is through the production of iron chelator molecules, named siderophores, which have a high affinity for iron. K. pneumoniae has four siderophore-mediated iron uptake systems, mediated by the enterobactin, yersiniabactin, salmochelin, and aerobactin siderophores (Fang et al., 2000; Dao et al., 2014; Paczosa and Mecsas, 2016). Enterobactin has the highest affinity for iron and it is widely spread among K. pneumoniae strains. On the other hand, aerobactin and salmochelin are typically found in hypervirulent K. pneumoniae strains and represent critical virulence factors in these strains ( (Russo et al., 2014; Paczosa and Mecsas, 2016).

Guilhen at al. found that the expression of all siderophore-encoding genes are greatly down-regulated in K. pneumoniae biofilm-dispersed and sessile cells compared to planktonic cells (Guilhen et al., 2016). In a separate study, Guilhen at al. reported that biofilm-dispersed and sessile cells elicit a lower innate immune response than planktonic cells. Taking both results together, the authors suggest a possible immune evasion strategy of biofilm-dispersed cells by repressing siderophore genes. Since siderophores are known to induce strong pro-inflammatory responses, by producing fewer siderophores the biofilm-dispersed cells would hide from the immune system by avoiding overstimulation of the immune response (Guilhen et al., 2019).

The influence of iron levels on biofilm development is also evidenced by studies conducted using iron chelators and antagonists. For instance, Hancock et al. demonstrated that biofilm formation by uropathogenic E. coli strains is impaired in the presence of divalent metal ions such as Zn(II) and Co(II) (Hancock et al., 2010). Similarly, Chhibber et al. (2013) investigated the ability of K. pneumoniae to form biofilm in the presence of divalent Co[II] ions (an iron antagonizing ion), in combination with a bacteriophage encoding depolymerase enzyme that degrades exopolysaccharides on the biofilm structure. The authors observed a significant reduction in biofilm formation by K. pneumoniae when both elements were present (Chhibber et al., 2013). According to Chhibber and collegues, the combination of Co[II] and depolymerase producing phage caused an inhibition of biofilm formation as well as a disruption of mature biofilms, suggesting a possible adjuvant effect in treatment of persistent, biofilm forming K. pneumoniae infections. Finally, Chen et al. (2020) demonstrated that liver abscess-causing K. pneumoniae strains grown in an iron-supplemented medium had a strong biofilm formation, whereas the addition of the iron chelator 2,2’-Dipyridyl resulted in decreased growth and inhibited biofilm formation.

In K. pneumoniae, as in most bacteria, the iron homeostasis is controlled by the transcriptional regulator Fur (Gomes et al., 2020). Fur exerts transcriptional activation or repression of target genes by either forming a complex with its cofactor, iron, or remaining in its apo-form (no cofactor bound) (Escolar et al., 1999; Gomes et al., 2020). Considered a global transcriptional regulator, Fur modulates the expression not only of genes related to iron metabolism, but also numerous genes associated with virulence factors, including some involved on biofilm development (Troxell and Hassan, 2013). For instance, Fur plays a role, in the presence of iron, in biofilm formation by regulating the expression of type I (Gomes et al., 2020; Pacheco et al., 2021) and type III (Wu et al., 2012) fimbrial genes, and also the expression of transcriptional regulators of fimbriae (Wu et al., 2012; Gomes et al., 2020).

All the steps of biofilm formation are regulated by signaling molecules, which are part of the quorum sensing system (Guilhen et al., 2019; Saxena et al., 2019). Due to varying environmental stimuli, the bacterium inside the biofilm needs to be able to quickly adapt and change gene expression, therefore the transcriptional regulation is also controlled by quorum sensing (Wang et al., 2020).

Quorum sensing (QS) is a sophisticated mechanism that allows communication between bacteria of the same species or between different species within the same community. Communication is based on the production, secretion, and detection of small molecules called AutoInducers (AI) (Balestrino et al., 2005). When the concentration of these molecules in the extracellular matrix reaches a threshold, the signal is detected by the bacteria and induces a change in the expression of certain genes, modifying the bacterial phenotype, expression of virulence factors, acid tolerance and biofilm formation (Balestrino et al., 2005; Saxena et al., 2019). Besides its role in coordinating biofilm formation, quorum sensing is also important for the maintenance of mature biofilms (Abraham, 2016).

Two main types of QS communication systems have been described on Gram-negative bacteria (Miller and Bassler, 2001). The type 1 QS is primarily used for intra-species communication and uses acyl-homoserine lactones (AHL) as autoinducers of type 1 (AI-1). AHL binds to its cognate receptor, the transcriptional regulator LuxR, to regulate the transcription of target genes. K. pneumoniae does not produce AI-1 molecules (Balestrino et al., 2005), but encodes SdiA, a LuxR-type receptor that, interestingly, responds to AHL molecules synthesized by other bacterial species (Pacheco et al., 2021). The type 2 QS allows intra- and inter-species communication and involves cyclic furanone compounds as autoinducers of type 2 (AI-2). LuxS synthase is the key enzyme in AI-2 molecules production, and a homolog of the luxS gene has been identified in the K. pneumoniae genome (Balestrino et al., 2005).

Previous studies have demonstrated the regulatory effect of both type 1 and type 2 QS in biofilm formation by K. pneumoniae. Firstly, Balestrino et al. revealed that a luxS-deficient mutant strain of K. pneumoniae shows a reduced capacity to develop microcolonies during biofilm development, although the strain is still able to form a mature biofilm (Balestrino et al., 2005). These data suggest that AI-2 molecules play a role in the early steps of biofilm formation.QS also regulates K. pneumoniae biofilm formation by modulating the expression of genes that play an import ant role in biofilm development, such as genes encoding bacterial surface structures (including fimbriae) and exopolysaccharides (such as LPS). According to De Araujo et al., K. pneumoniae strains deficient in both AI-2 synthesis and transport show increased expression of LPS related genes, leading the mutant strains to form a biofilm with greater biomass, although with altered architecture. The authors hypothesized a role of AI-2 molecules in regulating biofilm formation and LPS biosynthesis in sessile K. pneumoniae cells (De Araujo et al., 2010).

Quorum sensing and intra- and interspecies communication is crucial for coordinated behaviors of bacteria in biofilm communities at both those consisting of single species and polymicrobial biofilms (Thompson J. A. et al., 2015; Mukherjee and Bassler, 2019; Warrier et al., 2021). Quorum sensing systems are responsible for multiple coordinated events, including virulence gene expression (Bronesky et al., 2016), metabolite production and use (Barnard et al., 2007), as well as competence and DNA uptake and integration and spread of fitness traits and resistance genes (Marks et al., 2014).