Hybrid Materials Obtained by Immobilization of Biosynthesized Ag Nanoparticles with Antioxidant and Antimicrobial Activity.

Ag nanoparticles (AgNPs) were biosynthesized using sage (Salvia officinalis L.) extract. The obtained nanoparticles were supported on SBA-15 mesoporous silica (S), before and after immobilization of 10% TiO2 (Degussa-P25, STp; commercial rutile, STr; and silica synthesized from Ti butoxide, STb). The formation of AgNPs was confirmed by X-ray diffraction. The plasmon resonance effect, evidenced by UV-Vis spectra, was preserved after immobilization only for the sample supported on STb. The immobilization and dispersion properties of AgNPs on supports were evidenced by TEM microscopy, energy-dispersive X-rays, dynamic light scattering, photoluminescence and FT-IR spectroscopy. The antioxidant activity of the supported samples significantly exceeded that of the sage extract or AgNPs. Antimicrobial tests were carried out, in conditions of darkness and white light, on Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Candida albicans. Higher antimicrobial activity was evident for SAg and STbAg samples. White light increased antibacterial activity in the case of Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa). In the first case, antibacterial activity increased for both supported and unsupported AgNPs, while in the second one, the activity increased only for SAg and STbAg samples. The proposed antibacterial mechanism shows the effect of AgNPs and Ag+ ions on bacteria in dark and light conditions.

O-Antigen decorations in Salmonella enterica play a key role in eliciting functional immune responses against heterologous serovars in animal models.

Introduction: Different serovars of Salmonella enterica cause systemic diseases in humans including enteric fever, caused by S. Typhi and S. Paratyphi A, and invasive nontyphoidal salmonellosis (iNTS), caused mainly by S. Typhimurium and S. Enteritidis. No vaccines are yet available against paratyphoid fever and iNTS but different strategies, based on the immunodominant O-Antigen component of the lipopolysaccharide, are currently being tested. The O-Antigens of S. enterica serovars share structural features including the backbone comprising mannose, rhamnose and galactose as well as further modifications such as O-acetylation and glucosylation. The importance of these O-Antigen decorations for the induced immunogenicity and cross-reactivity has been poorly characterized. Methods: These immunological aspects were investigated in this study using Generalized Modules for Membrane Antigens (GMMA) as delivery systems for the different O-Antigen variants. This platform allowed the rapid generation and in vivo testing of defined and controlled polysaccharide structures through genetic manipulation of the O-Antigen biosynthetic genes. Results: Results from mice and rabbit immunization experiments highlighted the important role played by secondary O-Antigen decorations in the induced immunogenicity. Moreover, molecular modeling of O-Antigen conformations corroborated the likelihood of cross-protection between S. enterica serovars. Discussion: Such results, if confirmed in humans, could have a great impact on the design of a simplified vaccine composition able to maximize functional immune responses against clinically relevant Salmonella enterica serovars.

Characterization of Actinobacillus pleuropneumoniae biovar 2 isolates reportedly reacted with the serovar 4 antiserum, and development of a multiplex PCR for O-antigen typing.

We have analyzed the capsule (CPS) and the lipopolysaccharide O-Antigen (O-Ag) biosynthesis loci of twelve Spanish field isolates of Actinobacillus pleuropneumoniae biovar 2, eleven of them previously typed serologically as serovar 4 and one non-typable (NT) (Maldonado et al., 2009, 2011). These isolates have the common core genes of the type I CPS locus, sharing >98% identity with those of serovar 2. However, the former possesses the O-Ag locus as serovar 4, and the latter possesses the O-Ag locus as serovar 7. The main difference found between the CPS loci of the 11 isolates and that of serovar 2 reference strain S1536 are two deletions, one of an 8 bp sequence upstream of the coding sequence and one of 111 bp sequence at the 5' end of the cps2G gene. The deletion mutations mentioned lead to a defect in the production of CPS in these isolates, which contributed to their previous mis-identification. In order to complement the serotyping of A. pleuropneumoniae in diagnostics and epidemiology, we have developed a multiplex PCR for the comprehensive O-Ag typing of all A. pleuropneumoniae isolates.

Chemical synthesis of 6-deoxy-D-talose containing a tetrasaccharide repeating unit of the O-specific polysaccharide from Enterobacter cloacae G3422 in the form of its 2-aminoethyl glycoside.

Chemical synthesis of the tetrasaccharide repeating unit of the O-specific polysaccharide from Enterobacter cloacae G3422 is reported. The synthesis of the target tetrasaccharide is achieved through a convergent [2 + 2]-block strategy. The conjugation ready target oligosaccharide is attractive for further glycoconjugate formation with a suitable aglycon. Synthesis of the challenging 6-deoxy-L-talose moiety is reported using two different approaches and the obvious difficulties are discussed.

Mapping the architecture of the initiating phosphoglycosyl transferase from S. enterica O-antigen biosynthesis in a liponanoparticle.

Bacterial cell surface glycoconjugates are critical for cell survival and for interactions between bacteria and their hosts. Consequently, the pathways responsible for their biosynthesis have untapped potential as therapeutic targets. The localization of many glycoconjugate biosynthesis enzymes to the membrane represents a significant challenge for expressing, purifying, and characterizing these enzymes. Here, we leverage cutting-edge detergent-free methods to stabilize, purify, and structurally characterize WbaP, a phosphoglycosyl transferase (PGT) from the Salmonella enterica (LT2) O-antigen biosynthesis. From a functional perspective, these studies establish WbaP as a homodimer, reveal the structural elements responsible for dimerization, shed light on the regulatory role of a domain of unknown function embedded within WbaP, and identify conserved structural motifs between PGTs and functionally unrelated UDP-sugar dehydratases. From a technological perspective, the strategy developed here is generalizable and provides a toolkit for studying other classes of small membrane proteins embedded in liponanoparticles beyond PGTs.

Mutation of wbtJ, a N-formyltransferase involved in O-antigen synthesis, results in biofilm formation, phase variation and attenuation in Francisella tularensis.

Two clinically important subspecies, Francisella tularensis subsp. tularensis (type A) and F. tularensis subsp. holarctica (type B) are responsible for most tularaemia cases, but these isolates typically form a weak biofilm under in vitro conditions. Phase variation of the F. tularensis lipopolysaccharide (LPS) has been reported in these subspecies, but the role of variation is unclear as LPS is crucial for virulence. We previously demonstrated that a subpopulation of LPS variants can constitutively form a robust biofilm in vitro, but it is unclear whether virulence was affected. In this study, we show that biofilm-forming variants of both fully virulent F. tularensis subspecies were highly attenuated in the murine tularaemia model by multiple challenge routes. Genomic sequencing was performed on these strains, which revealed that all biofilm-forming variants contained a lesion within the wbtJ gene, a formyltransferase involved in O-antigen synthesis. A ΔwbtJ deletion mutant recapitulated the biofilm, O-antigen and virulence phenotypes observed in natural variants and could be rescued through complementation with a functional wbtJ gene. Since the spontaneously derived biofilm-forming isolates in this study were a subpopulation of natural variants, reversion events to the wbtJ gene were detected that eliminated the phenotypes associated with biofilm variants and restored virulence. These results demonstrate a role for WbtJ in biofilm formation, LPS variation and virulence of F. tularensis.

Deciphering the Chemical Language of the Immunomodulatory Properties of Veillonella parvula Lipopolysaccharide.

Veillonella parvula, prototypical member of the oral and gut microbiota, is at times commensal yet also potentially pathogenic. The definition of the molecular basis tailoring this contrasting behavior is key for broadening our understanding of the microbiota-driven pathogenic and/or tolerogenic mechanisms that take place within our body. In this study, we focused on the chemistry of the main constituent of the outer membrane of V. parvula, the lipopolysaccharide (LPS). LPS molecules indeed elicit pro-inflammatory and immunomodulatory responses depending on their chemical structures. Herein we report the structural elucidation of the LPS from two strains of V. parvula and show important and unprecedented differences in both the lipid and carbohydrate moieties, including the identification of a novel galactofuranose and mannitol-containing O-antigen repeating unit for one of the two strains. Furthermore, by harnessing computational studies, in vitro human cell models, as well as lectin binding solid-phase assays, we discovered that the two chemically diverse LPS immunologically behave differently and have attempted to identify the molecular determinant(s) governing this phenomenon. Whereas pro-inflammatory potential has been evidenced for the lipid A moiety, by contrast a plausible "immune modulating" action has been proposed for the peculiar O-antigen portion.

Cholera toxin and O-specific polysaccharide immune responses after oral cholera vaccination with Dukoral in different age groups of Bangladeshi participants.

Vaccination is important to prevent cholera. There are limited data comparing anti-O-specific polysaccharide (OSP) and anti-cholera toxin-specific immune responses following oral whole-cell with cholera toxin B-subunit (WC-rBS) vaccine (Dukoral, Valneva) administration in different age groups. An understanding of the differences is relevant because young children are less well protected by oral cholera vaccines than older children and adults. We compared responses in 50 adults and 49 children (ages 2 to <18) who were administered two doses of WC-rBS at a standard 14-day interval. All age groups had significant IgA and IgG plasma-blast responses to the OSP and cholera toxin B-subunit (CtxB) antigens that peaked 7 days after vaccination. However, in adults and older children (ages 5 to <18), antibody responses directed at the OSP antigen were largely IgA and IgG, with a minimal IgM response, while younger children (ages 2 to <5) mounted significant increases in IgM with minimal increases in IgA and IgG antibody responses 30 days after vaccination. In adults, anti-OSP and CtxB memory B-cell responses were detected after completion of the vaccination series, while children only mounted CtxB-specific IgG memory B-cell responses and no OSP-memory B-cell responses. In summary, children and adults living in a cholera endemic area mounted different responses to the WC-rBS vaccine, which may be a result of more prior exposure to Vibrio cholerae in older participants. The absence of class-switched antibody responses and memory B-cell responses to OSP may explain why protection wanes more rapidly after vaccination in young children compared to older vaccinees.IMPORTANCEVaccination is an important strategy to prevent cholera. Though immune responses targeting the OSP of V. cholerae are believed to mediate protection against cholera, there are limited data on anti-OSP responses after vaccination in different age groups, which is important as young children are not well protected by current oral cholera vaccines. In this study, we found that adults mounted memory B-cell responses to OSP, which were not seen in children. Adults and older children mounted class-switched (IgG and IgA) serum antibody responses to OSP, which were not seen in young children who had only IgM responses to OSP. The lack of class-switched antibody responses and memory B-cell responses to OSP in younger participants may be due to lack of prior exposure to V. cholerae and could explain why protection wanes more rapidly after vaccination in young children.

Specific labeling of newly synthesized lipopolysaccharide via metabolic incorporation of azido-galactose.

Gram-negative bacteria possess an asymmetric outer membrane (OM) primarily composed of lipopolysaccharides (LPS) on the outer leaflet and phospholipids on the inner leaflet. The outer membrane functions as an effective permeability barrier to compounds such as antibiotics. Studying LPS biosynthesis is therefore helpful to explore novel strategies for new antibiotic development. Metabolic glycan labeling of the bacterial surface has emerged as a powerful method to investigate LPS biosynthesis. However, the previously reported methods of labeling LPS are based on radioactivity or difficult-to-produce analogs of bacterial sugars. In this study, we report on the incorporation of azido galactose into the LPS of the Gram-negative bacteria Escherichia coli and Salmonella typhi via metabolic labeling. As a common sugar analog, azido galactose successfully labeled both O-antigen and core of Salmonella LPS, but not E. coli LPS. This labeling of Salmonella LPS, as shown by SDS-PAGE analysis and fluorescence microscopy, differs from the previously reported labeling of either O-antigen or core of LPS. Our findings are useful for studying LPS biogenesis pathways in Gram-negative bacteria like Salmonella. In addition, our approach is helpful for screening for agents that target LPS biosynthesis as it allows for the detection of newly synthesized LPS that appears in the OM. Furthermore, this approach may also aid in isolating chemically modified LPS for vaccine development or immunotherapy.

Structure, Physicochemical Properties and Biological Activity of Lipopolysaccharide from the Rhizospheric Bacterium Ochrobactrum quorumnocens T1Kr02, Containing d-Fucose Residues.

Lipopolysaccharides (LPSs) are major components of the outer membranes of Gram-negative bacteria. In this work, the structure of the O-polysaccharide of Ochrobactrum quorumnocens T1Kr02 was identified by nuclear magnetic resonance (NMR), and the physical-chemical properties and biological activity of LPS were also investigated. The NMR analysis showed that the O-polysaccharide has the following structure: →2)-ß-d-Fucf-(1→3)-ß-d-Fucp-(1→. The structure of the periplasmic glucan coextracted with LPS was established by NMR spectroscopy and chemical methods: →2)-ß-d-Glcp-(1→. Non-stoichiometric modifications were identified in both polysaccharides: 50% of d-fucofuranose residues at position 3 were O-acetylated, and 15% of d-Glcp residues at position 6 were linked with succinate. This is the first report of a polysaccharide containing both d-fucopyranose and d-fucofuranose residues. The fatty acid analysis of the LPS showed the prevalence of 3-hydroxytetradecanoic, hexadecenoic, octadecenoic, lactobacillic, and 27-hydroxyoctacosanoic acids. The dynamic light scattering demonstrated that LPS (in an aqueous solution) formed supramolecular particles with a size of 72.2 nm and a zeta-potential of -21.5 mV. The LPS solution (10 mkg/mL) promoted the growth of potato microplants under in vitro conditions. Thus, LPS of O. quorumnocens T1Kr02 can be recommended as a promoter for plants and as a source of biotechnological production of d-fucose.

Genomic surveillance of STEC/EHEC infections in Germany 2020 to 2022 permits insight into virulence gene profiles and novel O-antigen gene clusters.

Shiga toxin-producing E. coli (STEC), including the subgroup of enterohemorrhagic E. coli (EHEC), are important bacterial pathogens which cause diarrhea and the severe clinical manifestation hemolytic uremic syndrome (HUS). Genomic surveillance of STEC/EHEC is a state-of-the-art tool to identify infection clusters and to extract markers of circulating clinical strains, such as their virulence and resistance profile for risk assessment and implementation of infection prevention measures. The aim of the study was characterization of the clinical STEC population in Germany for establishment of a reference data set. To that end, from 2020 to 2022 1257 STEC isolates, including 39 of known HUS association, were analyzed and lead to a classification of 30.4 % into 129 infection clusters. Major serogroups in all clinical STEC analyzed were O26, O146, O91, O157, O103, and O145; and in HUS-associated strains were O26, O145, O157, O111, and O80. stx1 was less frequently and stx2 or a combination of stx, eaeA and ehxA were more frequently found in HUS-associated strains. Predominant stx gene subtypes in all STEC strains were stx1a (24 %) and stx2a (21 %) and in HUS-associated strains were mainly stx2a (69 %) and the combination of stx1a and stx2a (12.8 %). Furthermore, two novel O-antigen gene clusters (RKI6 and RKI7) and strains of serovars O45:H2 and O80:H2 showing multidrug resistance were detected. In conclusion, the implemented surveillance tools now allow to comprehensively define the population of clinical STEC strains including those associated with the severe disease manifestation HUS reaching a new surveillance level in Germany.

Characterization of Acinetobacter baumannii core oligosaccharide synthesis reveals novel aspects of lipooligosaccharide assembly.

A fundamental feature of Gram-negative bacteria is their outer membrane that protects the cell against environmental stressors. This defense is predominantly due to its asymmetry, with glycerophospholipids located in the inner leaflet and lipopolysaccharide (LPS) or lipooligosaccharide (LOS) confined to the outer leaflet. LPS consists of a lipid A anchor, a core oligosaccharide, and a distal O-antigen while LOS lacks O-antigen. While LPS/LOS is typically essential for growth, this is not the case for Acinetobacter baumannii. Despite this unique property, the synthesis of the core oligosaccharide of A. baumannii LOS is not well-described. Here, we characterized the LOS chemotypes of A. baumannii strains with mutations in a predicted core oligosaccharide locus via tandem mass spectrometry. This allowed for an extensive identification of genes required for core assembly that can be exploited to generate precise structural LOS modifications in many A. baumannii strains. We further investigated two chemotypically identical yet phenotypically distinct mutants, ∆2903 and ∆lpsB, that exposed a possible link between LOS and the peptidoglycan cell wall-two cell envelope components whose coordination has not yet been described in A. baumannii. Selective reconstruction of the core oligosaccharide via expression of 2903 and LpsB revealed that these proteins rely on each other for the unusual tandem transfer of two residues, KdoIII and N-acetylglucosaminuronic acid. The data presented not only allow for better usage of A. baumannii as a tool to study outer membrane integrity but also provide further evidence for a novel mechanism of core oligosaccharide assembly. IMPORTANCE: Acinetobacter baumannii is a multidrug-resistant pathogen that produces lipooligosaccharide (LOS), a glycolipid that confers protective asymmetry to the bacterial outer membrane. The core oligosaccharide is a ubiquitous component of LOS that typically follows a well-established model of synthesis. In addition to providing an extensive analysis of the genes involved in the synthesis of the core region, we demonstrate that this organism has evidently diverged from the long-held archetype of core synthesis. Moreover, our data suggest that A. baumannii LOS assembly is important for cell division and likely intersects with the synthesis of the peptidoglycan cell wall, another essential component of the Gram-negative cell envelope. This connection between LOS and cell wall synthesis provides an intriguing foundation for a unique method of outer membrane biogenesis and cell envelope coordination.

PEtN-Modified O-Antigen Enhances Shigella Pathogenesis by Promoting Epithelial Cell Invasion and Inhibiting Complement Binding.

Shigellosis poses an ongoing global public health threat. The presence and length of the O-antigen in lipopolysaccharide play critical roles in Shigella pathogenesis. The plasmid-mediated opt gene encodes a phosphoethanolamine (PEtN) transferase that catalyzes the addition of PEtN to the O-antigen of Shigella flexneri serotype X and Y strains, converting them into serotype Xv and Yv strains, respectively. Since 2002, these modified strains have become prevalent in China. Here we demonstrate that PEtN-mediated O-antigen modification in S. flexneri increase the severity of corneal infection in guinea pigs without any adaptive cost. This heightened virulence is associated with epithelial cell adhesion and invasion, as well as an enhanced inflammatory response of macrophage. Notably, PEtN addition allow S. flexneri to attenuate the binding of complement C3 and better resist phagocytosis, potentially contributing to the retention of S. flexneri in the host environment.

Recent chemical synthesis and immunological evaluation of glycans related to bacterial lipopolysaccharides.

O-Antigens and core oligosaccharides from bacterial lipopolysaccharides (LPS) are often structurally unique and immunologically active, have become attractive targets in the development of antibacterial vaccines. Structurally well-defined and pure oligosaccharides can be used in identifying protective epitopes of the carbohydrate antigens, which is important for the design of an effective vaccine. Here, the recent progress on chemical synthesis and immunological evaluation of glycans related to O-antigens and core oligosaccharides from bacterial LPS are summarized.

Total Synthesis of a Conjugation-Ready Tetrasaccharide Repeating Unit of Vibrio cholerae O:3 O-antigen Polysaccharide.

Herein, we report the first total synthesis of the tetrasaccharide repeating unit of Vibrio cholerae O:3 O-antigen polysaccharide. The highly complex tetrasaccharide contains rare amino sugars such as d-bacillosamine and l-fucosamine, highly labile sugar ascarylose, and higher carbon sugar d-d-heptose. Stereoselective glycosylation of the notoriously reactive ascarylose with d-d-heptose, poor nucleophilicity of the axial C4-OH of l-fucosamine, and amide coupling are the key challenges encountered in the total synthesis, which was completed via a longest linear sequence of 23 steps in 4.2% overall yield.

Structure of the O-polysaccharide from the moderately halophilic bacterium Halomonas fontilapidosi KR26.

Lipopolysaccharide was obtained from the aerobic moderately halophilic bacterium Halomonas fontilapidosi KR26. The O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide and was examined by chemical methods and by 1H and 13C NMR spectroscopy, including 1H,1H COSY, TOCSY, ROESY, and 1H,13C HSQC, and HMBC experiments. The following structure of the linear tetrasaccharide repeating unit was deduced. →2)-α-l-Rhap-(1→2)-α-l-Rhap-(1→3)-α-l-Rhap-(1→3)-ß-d-Galp-(1→.

Lipopolysaccharides of Herbaspirillum species and their relevance for bacterium-host interactions.

The lipopolysaccharides of Herbaspirillum lusitanum P6-12T (HlP6-12T) and H. frisingense GSF30T (HfGSF30T) was isolated by phenol-water extraction from bacterial cells and was characterized using chemical analysis and SDS-PAGE. It was shown that these bacteria produce LPSs that differ in their physicochemical properties and macromolecular organization. In this paper, the lipid A structure of the HlP6-12T LPS, was characterized through chemical analyses and matrix-assisted laser desorption ionization (MALDI) mass spectrometry. To prove the effect of the size of micelles on their bioavailability, we examined the activity of both LPSs toward the morphology of wheat seedlings. Analysis of the HlP6-12T and HfGSF30T genomes showed no significant differences between the operons that encode proteins involved in the biosynthesis of the lipids A and core oligosaccharides. The difference may be due to the composition of the O-antigen operon. HfGSF30T has two copies of the rfb operon, with the main one divided into two fragments. In contrast, the HlP6-12T genome contains only a single rfb-containing operon, and the other O-antigen operons are not comparable at all. The integrity of O-antigen-related genes may also affect LPS variability of. Specifically, we have observed a hairpin structure in the middle of the O-antigen glycosyltransferase gene, which led to the division of the gene into two fragments, resulting in incorrect protein synthesis and potential abnormalities in O-antigen production.

Combined usage of serodiagnosis and O antigen typing to isolate Shiga toxin-producing Escherichia coli O76:H7 from a hemolytic uremic syndrome case and genomic insights from the isolate.

IMPORTANCE: Hemolytic uremic syndrome (HUS) is a life-threatening disease caused by Shiga toxin-producing Escherichia coli (STEC) infection. The treatment approaches for STEC-mediated typical HUS and atypical HUS differ, underscoring the importance of rapid and accurate diagnosis. However, specific detection methods for STECs other than major serogroups, such as O157, O26, and O111, are limited. This study focuses on the utility of PCR-based O-serotyping, serum agglutination tests utilizing antibodies against the identified Og type, and isolation techniques employing antibody-conjugated immunomagnetic beads for STEC isolation. By employing these methods, we successfully isolated a STEC strain of a minor serotype, O76:H7, from a HUS patient.

Effect of O antigen glycosyl isomerase gene mutation on biological property and pathogenicity of Burkholderia pseudomallei strain BPC006.

Burkholderia pseudomallei, an intracellular pathogen, is responsible for melioidosis, a zoonotic disease. Its pathogenesis involves several virulence factors, among which lipopolysaccharide (LPS) plays a crucial role. Our research reveals that the O antigen present within the LPS significantly regulates the host immune response. In a previous study, we obtained a B. pseudomallei mutant strain ΔwbiI. Here, the purification of LPS from ΔwbiI and a gas chromatography-mass spectrometry (GC-MS) analysis were conducted. The results confirmed the absence of specific sugar 6-deoxy-Talp, which is a typical component of the O antigen in the wild type B. pseudomallei. Our findings underscore the potent impact the O antigen exerts on the virulence of B. pseudomallei. The ΔwbiI strain displayed significantly increased invasiveness and cytotoxicity in vitro. This enhanced cytotoxicity seems to be related to the exposure of lipid A and an increased cell membrane hydrophobicity resulting from the deletion of the O antigen. Additionally, in mouse models, the ΔwbiI strain resulted in a heightened host lethality and an excessive inflammatory response in mice. These findings indicate that the O-antigenic polysaccharide moiety of B. pseudomallei plays a role in its pathogenicity in vitro and in vivo.

The OL101 O antigen locus specifies a novel Klebsiella pneumoniae serotype O13 structure.

Klebsiella pneumoniae is one of the priority objects for the development of new therapies against infections. The species has been perceived as of limited variety of O antigens (11 O serotypes identified to date). That trait makes lipopolysaccharide an attractive target for protective antibodies. Nowadays, K. pneumoniae O antigens encoding genes are often analysed by bioinformatic tools, such as Kaptive, indicating higher actual diversity of the O antigen loci. One of the novel K. pneumoniae O loci for which the antigen structure has not been elucidated so far is OL101. In this study, four clinical isolates predicted as OL101 were characterized and found to have the O antigen structure composed of ß-Kdop-[→3)-α-l-Rhap-(1→4)-α-d-Glcp-(1→]n, representing a novel serotype O13. Identification of the ß-Kdop terminus was based on the analysis of the complete LPS molecule by the HR-MAS NMR spectroscopy. The bioinformatic analysis of 71,377 K. pneumoniae genomes from public databases (July 2023) revealed a notable OL101 prevalence of 6.55 %.