Figure - available from: Scientific Reports
This content is subject to copyright. Terms and conditions apply.
Schematic illustration of (top) the Wzx/Wzy dependent pathway involved in the Klebsiella spp CPS transportation and surface expression, and (bottom) the representative cps locus arrangement.
Source publication
A computational method has been developed to distinguish the Klebsiella species serotypes to aid in outbreak surveillance. A reliability score (estimated based on the accuracy of a specific K-type prediction against the dataset of 141 distinct K-types) average (ARS) that reflects the specificity between the Klebsiella species capsular polysaccharid...
Citations
... Interestingly, the NCBI blast result indicates that both mutations are located in the wzc of the cps gene cluster that is responsible for putative tyrosine-protein kinase. Some recent studies reported that, among the capsular type defined gene in the cps biosynthesis gene cluster, wzc genes are involved in tyrosine-protein kinase (Patro et al., 2020;Yang et al., 2021). Moreover, a previous study reported that rmpA and rmpA2 regulate the cps gene and enhance capsular polysaccharide synthesis (Choby et al., 2020). ...
Klebsiella pneumoniae is an opportunistic pathogen that mainly causes nosocomial infections and hospital-associated pneumonia in elderly and immunocompromised people. However, multidrug-resistant hypervirulent K. pneumoniae (MDR-hvKp) has emerged recently as a serious threat to global health that can infect both immunocompromised and healthy individuals. It is scientifically established that plasmid-mediated regulator of mucoid phenotype genes ( rmpA and rmpA2 ) and other virulence factors (aerobactin and salmochelin) are mainly responsible for this phenotype. In this study, we collected 23 MDR-hvKp isolates and performed molecular typing, whole genome sequencing, comparative genomic analysis, and phenotypic experiments, including the Galleria mellonella infection model, to reveal its genetic and phenotypic features. Meanwhile, we discovered two MDR-hvKp isolates (22122315 and 22091569) that showed a wide range of hypervirulence and hypermucoviscosity without rmpA and rmpA2 and any virulence factors. In phenotypic experiments, isolate 22122315 showed the highest hypervirulence (infection model) with significant mucoviscosity, and conversely, isolate 22091569 exhibited the highest mucoviscosity (string test) with higher virulence compared to control. These two isolates carried carbapenemase ( bla KPC − 2 ), β-lactamase ( bla OXA − 1 , bla TEM − 1B ), extended-spectrum β-lactamase (ESBL) genes ( bla CTX − M − 15 , bla SHV − 106 ), outer membrane protein-coding genes ( ompA ), fimbriae encoding genes ( ecpABCDER ), and enterobactin coding genes ( entAB , fepC ). In addition, single nucleotide polymorphism analysis indicated that both isolates, 22122315 and 22091569, were found to have novel mutations in loci FEBNDAKP_03184 (c. 2084A > C, p. Asn695Thr), and EOFMAFIB_02276 (c. 1930C > A, p. Pro644Thr), respectively. Finally, NCBI blast analysis suggested these mutations are located in the wzc of the capsule polysaccharide ( cps ) region and are responsible for putative tyrosine kinase. This study would be a strong reference for enhancing the current understanding of identifying the MDR-hvKp isolates that lacked both mucoid regulators and virulence factors.
... To aid in the identification process, we utilized a reference database of 81 Klebsiella K-serotypes with known polysaccharide structures and cps genotypes (Table S1). Additionally, two open-source tools, namely K-Pam (Patro et al., 2020) and Kaptive (Wick et al., 2018), were used to verify the serotype prediction. ...
... K-typing of KV-3 was also performed using K-Pam (Patro et al., 2020) and Kaptive (Wick et al., 2018), both of which yielded results consistent with the findings obtained from the MultiGeneBlast analysis. These combined results provided strong evidence that K. pneumoniae KV-3 belongs to the capsular type K54. ...
Klebsiella pneumoniae poses a major global challenge due to its virulence, multidrug resistance, and nosocomial nature. Thus, bacteriophage-derived proteins are extensively being investigated as a means to combat this bacterium. In this study, we explored the enzymatic specificity of depolymerase gp531, encoded by the jumbo bacteriophage vB_KleM_RaK2 (RaK2). We used two different methods to modify the reducing end of the oligosaccharides released during capsule hydrolysis with gp531. Subsequent acidic cleavage with TFA, followed by TLC and HPLC-MS analyses, revealed that RaK2 gp531 is a β-(1→4)-endoglucosidase. The enzyme specifically recognizes and cleaves the capsular polysaccharide (CPS) of the Klebsiella pneumoniae K54 serotype, releasing K-unit monomers (the main product), dimers, and trimers. Depolymerase gp531 remains active from 10 to 50 °C and in the pH 3–8 range, indicating its stability and versatility. Additionally, we demonstrated that gp531′s activity is not affected by CPS acetylation, which is influenced by the growth conditions of the bacterial culture. Overall, our findings provide valuable insights into the enzymatic activity of the first characterized depolymerase targeting the capsule of the clinically relevant K54 serotype of K. pneumoniae.
... The surface CPS/LPS are enormously variable among bacteria and can be changed or modified by gene mutations, deletions or insertion in the carbohydrate biosynthesis loci 32 . The diversities in the sugar composition and structure of O-or K-antigen distinguish immunologically distinct strains by the corresponding O-or K-serotyping schemes 40 . Correlatively, depolymerases with a high serotype specificity also exhibit a high diversity to target different O-or K-types of bacteria. ...
Predatory bacteriophages have evolved a vast array of depolymerases for bacteria capture and
deprotection. These depolymerases are enzymes responsible for degrading diverse bacterial surface carbohydrates.
They are exploited as antibiofilm agents and antimicrobial adjuvants while rarely inducing
bacterial resistance, making them an invaluable asset in the era of antibiotic resistance. Numerous depolymerases
have been investigated preclinically, with evidence indicating that depolymerases with appropriate
dose regimens can safely and effectively combat different multidrug-resistant pathogens in animal
infection models. Additionally, some formulation approaches have been developed for improved stability
and activity of depolymerases. However, depolymerase formulation is limited to liquid dosage form and
remains in its infancy, posing a significant hurdle to their clinical translation, compounded by challenges
in their applicability and manufacturing. Future development must address these obstacles for clinical
utility. Here, after unravelling the history, diversity, and therapeutic use of depolymerases, we summarized
the preclinical efficacy and existing formulation findings of recombinant depolymerases. Finally,
the challenges and perspectives of depolymerases as therapeutics for humans were assessed to provide
insights for their further development.
... Moreover, the majority of patients exhibit signs of inflammation and fever, and our findings showed that the ratios of WBC/PCT and CRP/PCT were potential clinical markers for identifying suppurative infection caused by hvKp and cKp, which is consistent with previous reports showing that CRP and PCT are reliable indicators to differentiate between hvKp and cKp (29,30). Based on the presence of specific marker genes (9,11), the majority (33 of the 54 cases) of pyogenic infections were caused by hvKp, which is consistent with its characteristics of being more virulent and aggressive (2,3). The results of this study indicated that K1-ST23 types were the most prevalent hvKp clones and that these were primarily observed in liver abscesses, in agreement with reports from China and other Asian countries (31,32). ...
... from conventional Klebsiella spp. using the marker genes iroB, iucA, rmpA, rmpA2, and peg-344 (11). The server classifies a strain as hvKp if four of the five genes are present (10,11,51). ...
... using the marker genes iroB, iucA, rmpA, rmpA2, and peg-344 (11). The server classifies a strain as hvKp if four of the five genes are present (10,11,51). ...
Treatment of Klebsiella pneumoniae causing pyogenic infections is challenging. The clinical and molecular characteristics of Klebsiella pneumoniae causing pyogenic infections are poorly understood, and antibacterial treatment strategies are limited. We analyzed the clinical and molecular characteristics of K. pneumoniae from patients with pyogenic infections and used time-kill assays to reveal the bactericidal kinetics of antimicrobial agents against hypervirulent K. pneumoniae (hvKp). A total of 54 K. pneumoniae isolates were included, comprising 33 hvKp and 21 classic K. pneumoniae (cKp) isolates, and the hvKp and cKp isolates were identified using five genes (iroB, iucA, rmpA, rmpA2, and peg-344) that have been applied as hvKp strain markers. The median age of all cases was 54 years (25th and 75th percentiles, 50.5 to 70), 62.96% of individuals had diabetes, and 22.22% of isolates were sourced from individuals without underlying disease. The ratios of white blood cells/procalcitonin and C-reactive protein/procalcitonin were potential clinical markers for the identification of suppurative infection caused by hvKp and cKp. The 54 K. pneumoniae isolates were classified into 8 sequence type 11 (ST11) and 46 non-ST11 strains. ST11 strains carrying multiple drug resistance genes have a multidrug resistance phenotype, while non-ST11 strains carrying only intrinsic resistance genes are generally susceptible to antibiotics. Bactericidal kinetics revealed that hvKp isolates were not easily killed by antimicrobials at susceptible breakpoint concentrations compared with cKp. Given the varied clinical and molecular features and the catastrophic pathogenicity of K. pneumoniae, it is critical to determine the characteristics of such isolates for optimal management and effective treatment of K. pneumoniae causing pyogenic infections. IMPORTANCE Klebsiella pneumoniae may cause pyogenic infections, which are potentially life-threatening and bring great challenges for clinical management. However, the clinical and molecular characteristics of K. pneumoniae are poorly understood, and effective antibacterial treatment strategies are limited. We analyzed the clinical and molecular features of 54 isolates from patients with various pyogenic infections. We found that most patients with pyogenic infections had underlying diseases, such as diabetes. The ratio of white blood cells to procalcitonin and the ratio of C-reactive protein to procalcitonin were potential clinical markers for differentiating hypervirulent K. pneumoniae strains from classical K. pneumoniae strains that cause pyogenic infections. K. pneumoniae isolates of ST11 were generally more resistant to antibiotics than non-ST11 isolates. Most importantly, hypervirulent K. pneumoniae strains were more tolerant to antibiotics than classic K. pneumoniae isolates.
... The diversity of the 12 protein sequences (notably ItrB4 was not considered as it is present only in KL75) corresponding to 237 serotypes of A. baumannii was investigated to effectively utilize them to distinguish different serotypes, as described earlier (Patro et al., 2020). For this, multiple sequence alignment (MSA) and percentage identity matrix (PIM) of all the 12 protein sequences were constructed using Clustal Omega (Madeira et al., 2022). ...
... The reliability score (RS) and average reliability score (ARS) for each protein were calculated individually for all 237 serotypes based on the protein's ability to predict a unique serotype when searched against the reference dataset, as described previously (Patro et al., 2020). The reliability score used for the K-typing provides the uniqueness of proteins in the Wzx/Wzy biosynthesis pathway across different serotypes. ...
... The structural information presented in Table 1 was utilized to model and create the 3D structural repository of A. baumannii K-antigen, similar to the K-and O-antigen structural repositories of E. coli (Rojas-Macias et al., 2015;Kunduru et al., 2016) and Klebsiella spp. (Patro et al., 2020). Here, 64 known K-antigen threedimensional structures were modeled, and their repository was created in a Linux-based server (Figure 3). ...
Acinetobacter baumannii is an emerging opportunistic pathogen. It exhibits multi-, extreme-, and pan-drug resistance against several classes of antibiotics. Capsular polysaccharide (CPS or K-antigen) is one of the major virulence factors which aids A. baumannii in evading the host immune system. K-antigens of A. baumannii exploit the Wzx/Wzy-dependent pathway that involves 13 different proteins for its assembly and transport onto the outer membrane. A total of 64 (out of 237 K-locus(KL) types) known K-antigen sugar repeating structures are discussed here and are classified into seven groups based on their initial sugars, QuiNAc4NAc, GalNAc, GlcNAc, Gal, QuiNAc/FucNAc, FucNAc, and GlcNAc along with Leg5Ac7Ac/Leg5Ac7R. Thus, the corresponding seven initializing glycosyltransferases (ItrA1, ItrA2, ItrA3, ItrA4, ItrB1, ItrB3, and ItrA3 along with ItrB2) exhibit serotype specificity. The modeled 3D-structural repository of the 64 K-antigens can be accessed at https://project.iith.ac.in/ABSD/k_antigen.html . The topology of K-antigens further reveals the presence of 2-6 and 0-4 sugar monomers in the main and side chains, respectively. The presence of negatively (predominant) or neutrally charged K-antigens is observed in A. baumannii . Such diversity in the K-antigen sugar composition provides the K-typing specificity ( viz ., 18–69% in terms of reliability) for Wza, Wzb, Wzc, Wzx, and Wzy proteins involved in the Wzx/Wzy-dependent pathway. Interestingly, the degree of uniqueness of these proteins among different K-types is estimated to be 76.79%, considering the 237 reference sequences. This article summarizes the A. baumannii K-antigen structural diversity and creation of a K-antigen digital repository and provides a systematic analysis of the K-antigen assembly and transportation marker proteins.
... Conserved genes involved in assembly and export of the capsule (wza, wzb, wzc, wzi) and initiating glycosyl-transferase (wcaJ, wbaP) are labelled. The chemical composition of the capsule (monomers and their organisation), is displayed on the right of each locus (predicted by K-PAM [69]). B. Matrix of phage infection. ...
Bacterial evolution is affected by mobile genetic elements such as phages and conjugative plasmids, which may provide novel adaptive traits but also incur in fitness costs. Infection by these elements is affected by the bacterial capsule. Yet, its importance has been difficult to quantify and characterise because of the high diversity of bacterial genomes regarding confounding mechanisms such as anti-viral systems. We swapped capsule loci between Klebsiella pneumoniae strains to quantify their effect on transfer of conjugative plasmids and phages independently of the genetic background. Capsule swaps systematically invert phage susceptibility, demonstrating that serotypes are key determinants of phage infection. Capsule types also affect conjugation efficiency in both donor and recipient cells depending on the serotype, a mechanism shaped by the capsule volume and depending on the structure of the conjugative pilus. Comparative genomics confirmed that more permissive serotypes in the lab correspond to the strains acquiring more conjugative plasmids in nature. The pili least sensitive to capsules (F-like) are also the most frequent in the species’ plasmids, and are the only ones associated with both antibiotic resistance and virulence factors, driving the convergence between virulence and antibiotics resistance in the population. These results show how the traits of cellular envelopes define slow and fast lanes of infection by mobile genetic elements, with implications for population dynamics and horizontal gene transfer.
Graphical abstract
... 11 Since conventional serotyping is a timeconsuming process, in silico serotyping has become an alternative. Such serotyping relies on the genotype-serotype specificity, which has successfully been utilized in the serotyping of E. coli, 12 Klebsiella spp., 13 Acinetobacter baumannii 14 and even Salmonella spp. 3,[15][16][17] In this investigation, the protein/gene sequences involved in O-antigen biosynthesis and H-antigen formation are utilized in serotyping. ...
... 19,39 Thus, to speed up the O-typing, Wzx and Wzy were alone used for the majority of O-types, unlike in the case of Klebsiella spp., where eight genes were required for the accurate prediction of serotype. 13 The analysis further indicated that 150 additional glycosyl transferases were unique among different O-types (Supplementary Table S5) which can be used for serotyping in the absence of Wzx and/or Wzy. Notably, the initializing glycosyl transferases WbaP, WecA and GNE were among them, which led to three different classifications of Salmonella species based on the initializing sugars: Gal (WbaP), GlcNAc (WecA) and GalNAc (WecA and GNE) (Supplementary Figure S3). ...
Over 2500 Salmonella species (alternatively, serovars) encompassing different combinations of O-, H1- and H2-antigens are present in nature and cause millions of deaths worldwide every year. Since conventional serotyping is time-consuming, a user-friendly Salmonella species serotyping (SSP) web tool
(https://project.iith.ac.in/SSP/) is developed here to predict the serotypes using Salmonella protein(s) or whole proteome sequences. Prior to SSP implementation, a detailed analysis of protein sequences involved in O-antigen biosynthesis and H-antigen formation is carried out to assess their serotype specificity. Intriguingly, the results indicate that the initializing transferases WbaP, WecA and GNE can efficiently distinguish the O-antigens, which have Gal, GlcNAc and GalNAc as initial sugars respectively. Rigorous analysis shows that Wzx and Wzy are sufficient to distinguish the O-types. Exceptionally, some situations warrant additional proteins. Thus, 150 additional transferases, RfbE for O2, O9 and O9,46 types, Orf17.4 for O3,10 and O1,3,19 types, WecB, WbbE and WbbF for O54 and, Wzm and Wzt for O67 are utilized in serotyping. An in-depth analysis of 302 reference datasets representing 56 H1- and 20 H2-types leads to the identification and utilization of 61 unique sequence patterns of FliC and FljB in H-typing. A test dataset of 2136 whole proteome sequences covering 740 Salmonella serovars, including 13 new species are successfully predicted with 99.72% accuracy. Prior to this, all the O-, H1- and H2-antigens are predicted accurately when tested independently. Indeed, SSP also identifies wrongly annotated Salmonella species; hence, it can easily identify new species that emerge with any combination of O-, H1- and H2-antigens. Thus, SSP can act as a valuable tool in the surveillance of Salmonella species.
... Hypervirulence factors of hypermucoviscous strains were detected by Polymerase Chain Reaction (PCR) using primers and thermal profiles reported earlier [12]. The marker genes used to distinguish hvKp from cKp are: rmpA/rmpA2 encoding regulator of mucoid phenotype (hypermucoid); iucA gene in the locus of aerobactin siderophore; iroB gene in the locus of salmochelin siderophore; and peg-344, which is a putative transporter. ...
... Through molecular characterization, it was found that seven strains of the study displayed at least 4 hypervirulence genes. According to what is reported in the literature, a K. pneumoniae strain is defined as hypervirulent if it has at least four of the aforementioned genes [12]. However, it should also be taken into account that the strains MDR 1682 and MDR 3605 carry the rmpA2 gene alone, which according to the ECDC risk assessment [1] is one of the major factors of hypervirulence. ...
This study focused on the characterization of 19 hypermucoviscous Klebsiella pneumoniae strains, that were identified from 26 hypermucosal strains. In order to identify hypermucoviscous strains of K. pneumoniae, the string test was applied. This phenotype is known in the literature as one of the virulence factors of this species together with the production of biofilm and other hypervirulence factor genes such as: rmpA, rmpA2, iucA, iroB, peg-344. We also investigated presence of magA gene that correlates with the hyper-production of capsule of K1 serotype. Of the strains under study, 13 out of 19 harboured at least one virulence factor.
Sequence type (ST) was determined in order to identify known high-risk clones or new emerging high-risk clones and their variability in a single clinical setting. Important STs found among these strains were ST65 and ST29. Carbapenem resistance was also investigated and 4 out of 19 strains harboured at least a carbapenemase: one strain harboured a KPC enzyme alone, one strain carried a KPC and an OXA-48 like, one strain produced OXA-48-like alone, and the last strain harboured two metallo-β-lactamases (VIM-1 and NDM-5) plus OXA-48-like. In particular, this latter strain belongs to ST383, which was recently reported in Northern Italy as a hypervirulent and XDR strain.
The global spread of hypervirulent K. pneumoniae is an important epidemiological issue that should be considered in diagnostic and therapeutic managements of patients with K. pneumoniae infections.
... Although all K. pneumoniae isolates carried enterobactin genes and the iroE gene, which is part of the locus that encodes for salmochelin, in order to designate isolates as hvKp, at least four of the following genes need to be present in a K. pneumoniae isolate: rmpA, rmpA2, iucA, iroB and peg-344 [47,48]. Moreover, other authors differentiated commensal K. pneumoniae from hvKp due to the presence in hvKp of salmochelin and aerobactin [49], as well as the presence of specific capsule serotypes for hvKp [50]. ...
Antimicrobial resistance (AMR) is a public health issue attributed to the misuse of antibiotics in human and veterinary medicine. Since AMR surveillance requires a One Health approach, we sampled nine interconnected compartments at a hydrological open-air lab (HOAL) in Austria to obtain six bacterial species included in the WHO priority list of antibiotic-resistant bacteria (ARB). Whole genome sequencing-based typing included core genome multilocus sequence typing (cgMLST). Genetic and phenotypic characterization of AMR was performed for all isolates. Eighty-nine clinically-relevant bacteria were obtained from eight compartments including 49 E. coli, 27 E. faecalis, 7 K. pneumoniae and 6 E. faecium. Clusters of isolates from the same species obtained in different sample collection dates were detected. Of the isolates, 29.2% were resistant to at least one antimicrobial. E. coli and E. faecalis isolates from different compartments had acquired antimicrobial resistance genes (ARGs) associated with veterinary drugs such as aminoglycosides and tetracyclines, some of which were carried in conjugative and mobilizable plasmids. Three multidrug resistant (MDR) E. coli isolates were found in samples from field drainage and wastewater. Early detection of ARGs and ARB in natural and farm-related environments can identify hotspots of AMR and help prevent its emergence and dissemination along the food/feed chain.
... The cpxR/cpxA genes encode the two-component system and are associated with virulence features and biofilm formation [30]. The wzi gene is associated with the K-antigen synthesis [31]. The proximity of O-antigen-and K-antigen-related genes on the chromosome possibly indicates a tight connection between biosynthesis pathways of O-specific and capsular polysaccharides. ...
Providencia is a genus of Gram-negative bacteria belonging to the Morganellaceae family. This genus includes nine species (P. stuartii, P. sneebia, P. rettgeri, P. rustigianii, P. heimbachae, P. burhodogranariea, P. alcalifaciens, P. huaxiensis , and P. vermicola ) with varying degrees of virulence, capable of infecting humans and insects [1, 2].
For Gram-negative bacteria, the somatic antigen (O-antigen) has become one of the key virulence factors. It is the highly immunogenic part of lipopolysaccharides due to the distal location. O-antigens are characterized by structural heterogeneity, providing varying degrees of inter- and intraspecific virulence. At the genetic level, somatic antigens have an operon structure. Operon genes responsible for the synthesis and transformation of O-polysaccharide are transcribed together. Analysis of O-antigen operon organization determines genes specific for each O-serogroup. It is beneficial for molecular typing of strains and for studies of bacterial evolution.
This study focuses on identifying and comparing candidates for O-antigen operons in Providencia species with different levels of virulence. The hypothesis is the presence of an association between the O-antigen operon composition and the bacteria lifestyle. Data processing and analysis are carried out by a pipeline developed by the authors. Pipeline combines five steps of the genome analysis: genome quality evaluation, assembly annotation, operon identification with verification of operon boundaries, and visualization of O-antigen operons. The results reveal previously undescribed O-antigen genes and the changes in the O-antigen operons structure. Among the changes are a transposon insertion leading to tetracycline resistance and the presence of IS elements.
