Mucoid Phenotype Research Articles

A strategy for controlling Hypervirulent Klebsiella pneumoniae: inhibition of ClpV expression

The emergence and prevalence of hypervirulent Klebsiella pneumoniae (hvKP) have proposed a great challenge to control this infection. Therefore, exploring some new drugs or strategies for treating hvKP infection is an urgent issue for scientific researchers. In the present study, the clpV gene deletion strain of hvKP (ΔclpV-hvKP) was constructed using CRISPR-Cas9 technology, and the biological characteristics of ΔclpV-hvKP were investigated to explore the new targets for controlling this pathogen. The results showed that clpV gene deletion did not affect the growth ability of hvKP. However, knocking out the clpV gene markedly decreased the mucoid phenotype and the biofilm formation ability of hvKP. It reduced the interspecific competition of hvKP with Escherichia coli, Salmonella, Pseudomonas aeruginosa, and Staphylococcus aureus. The clpV deletion significantly changed the transcriptome profile of hvKP, inhibited the expression of virulence factors, and decreased the lethality of hvKP against Galleria mellonella larvae. In vitro experiments showed that lithocholic acid could inhibit the expression of the clpV gene and reduce the virulence of hvKP. Our data suggested that the clpV gene may be a potential target for decreasing hvKP infection risk.

Molecular Epidemiology and In-Depth Characterization of Klebsiella pneumoniae Clinical Isolates from Armenia.

The global dissemination of Klebsiella pneumoniae pathotypes with multidrug-resistant (MDR) and hypervirulent traits poses a threat to public health. The situation in Armenia is unclear, and we performed a comprehensive characterisation of 48 clinical isolates of K. pneumoniae, collected from 2018 to 2024. The majority of the isolates (64.58%) were extensively drug-resistant (XDR) and MDR. Genomic analysis of 21 isolates revealed the presence of international high-risk MDR clones (ST395, ST15, and ST307). The ST395 strains were isolated from children and resisted the first-line drugs such as beta-lactams. These isolates harboured a range of virulence determinants, from capsule polysaccharides to siderophores to regulators of the mucoid phenotype. The ST395 strains are enriched by ICEs, plasmids, and prophages, on which antimicrobial resistance (AMR) and virulence genes are located and which may lead to the convergence of MDR and hypervirulent traits. There is a widespread non-specific AMR mechanism among our K. pneumoniae strains. These are mutations in the porin genes, which reduce permeability to antimicrobials, and mutations in the regulators of efflux pumps, which lead to overexpression of drug efflux pumps such as AcrAB. These mechanisms may contribute to the elevated MICs and confer AMR to strains with no specific AMR genes.

Diverse polysaccharide production and biofilm formation abilities of clinical Klebsiella pneumoniae

Klebsiella pneumoniae infections have become a growing threat for human health. The lack of understanding of the relationship between antibiotic resistance, mucoviscosity, and biofilm formation impedes our abilities to effectively predict K. pneumoniae infection outcomes. The Multidrug-Resistant Organism Repository and Surveillance Network offers a unique opportunity into the genetic and phenotypic variabilities in the K. pneumoniae isolates. To this end, we compared the genetic profiles of these isolates with the phenotypic biofilm formation, percent mucoviscosity, and growth rates. There was a significant phenotype-genotype correlation with decreased biofilm formation and an insertion sequence in the transcriptional activator of the type III fimbrial system. Interestingly, the most mucoid strains in the populations were lacking the genetic element regulating the mucoid phenotype and three of these isolates were able to form robust biofilms. The combination of phenotypic, genomic, and image analyses revealed an intricate relation between growth, mucoviscosity and specific virulence-associated genetic determinants.

Ceftolozane/tazobactam disrupts Pseudomonas aeruginosa biofilms under static and dynamic conditions.

Pseudomonas aeruginosa biofilms limit the efficacy of currently available antibacterial therapies and pose significant clinical challenges. Pseudomonal biofilms are complicated further when other markers of persistence such as mucoid and hypermutable phenotypes are present. There is currently a paucity of data regarding the activity of the newer β-lactam/β-lactamase inhibitor combination ceftolozane/tazobactam against P. aeruginosa biofilms. We evaluated the efficacy of ceftolozane/tazobactam against clinical P. aeruginosa isolates, the laboratory isolate PAO1 and its isogenic mutS-deficient hypermutator derivative (PAOMS) grown under static and dynamic biofilm conditions. The clinical isolate collection included strains with mucoid and hypermutable phenotypes. Ceftolozane/tazobactam exposure led to a bactericidal (≥3 log cfu/cm2) biofilm reduction in 15/18 (83%) clinical isolates grown under static conditions, irrespective of carbapenem susceptibility or mucoid phenotype, with greater activity compared with colistin (P < 0.05). Dynamically grown biofilms were less susceptible to ceftolozane/tazobactam with active biofilm reduction (≥1 log cfu/cm2) observed in 2/3 isolates. Hypermutability did not affect the antibiofilm efficacy of ceftolozane/tazobactam in either static or dynamic conditions when comparing PAO1 and PAOMS. Consistent with the activity of ceftolozane/tazobactam as a potent inhibitor of PBP3, dramatic impacts on P. aeruginosa morphology were observed. Our data demonstrate that ceftolozane/tazobactam has encouraging properties in the treatment of P. aeruginosa biofilm infections, and its activity is not diminished against mucoid or hypermutable variants at the timepoints examined.

Arginine Regulates the Mucoid Phenotype of Hypervirulent Klebsiella pneumoniae.

Hypervirulent Klebsiella pneumoniae is associated with severe community-acquired infections. Hypervirulent K. pneumoniae colonies typically exhibit a mucoid phenotype. K. pneumoniae mucoidy is influenced by a complex combination of environmental factors and genetic mechanisms. Mucoidy results from altered capsular polysaccharide chain length, yet the specific environmental cues regulating this phenotype and their impact on pathogenesis remain unclear. This study demonstrates that casamino acids enhance the mucoidy phenotype but do not affect total capsular polysaccharide levels. Through targeted screening of each amino acid present in casamino acids, we identified that arginine is necessary and sufficient to stimulate the mucoid phenotype without altering capsule abundance. Furthermore, arginine activates the rmpADC promoter, increasing rmpD transcript levels, which in turn modulates capsular polysaccharide chain length and diversity. The arginine regulator, ArgR, plays a pivotal role in this regulatory cascade since deleting argR decreases mucoidy and increases capsular polysaccharide chain length diversity. Additionally, the ∆argR mutant displays increased macrophage association and has a substantial competitive defect in the lungs of mice, suggesting a link between arginine-dependent gene regulation, immune evasion and in vivo fitness. We discovered that arginine-dependent regulation of mucoidy is conserved in four additional hypervirulent K. pneumoniae isolates likely via a conserved ARG binding box present in rmp promoters. Our findings support a model in which arginine activates ArgR and increases mucoidy in hypervirulent K. pneumoniae. As a result, it is possible that arginine-dependent regulation of mucoidy allows hypervirulent K. pneumoniae to adapt the cell surface across different niches. This study underscores the significance of arginine as a regulatory signal in bacterial virulence.

At what cost? The impactof bacteriophage resistance on the growth kinetics and protein synthesis of Escherichia coli.

Cost of bacteriophage resistance (COR) is important in explaining processes of diversification and coexistence in microbial communities. COR can be expressed in different traits, and the lack of universally applicable methods to measure fitness trade-offs makes COR challenging to study. Due to its fundamental role in growth, we explored protein synthesis as a target for quantifying COR. In this study, the growth kinetics of three genome-sequenced strains of phage-resistant Escherichia coli, along with the phage-susceptible wild-type, were characterized over a range of glucose concentrations. Bioorthogonal non-canonical amino acid tagging (BONCAT) was used to track differences in protein synthetic activity between the wild-type and phage-resistant E. coli. Two of the resistant strains, with different levels of phage susceptibility, showed mucoid phenotypes corresponding with mutations in genes associated with the Rcs phosphorelay. These mucoid isolates, however, had reduced growth rates and potentially lower protein synthetic activity. Another resistant isolate with a different mutational profile maintained the same growth rate as the wild-type and showed increased BONCAT fluorescence, but its yield was lower. Together, these findings present different patterns of trade-offs resulting from the phage-induced mutations and demonstrate the potential applicability of BONCAT as a tool for measuring COR.

Complete genome sequence of Kozakia baliensis LTH 7215, a newly isolated strain from grape must.

We report the complete genome sequence of the novel Kozakia baliensis strain LTH 7215, isolated from grape must. This strain exhibits a mucoid phenotype on solid medium, associated with exopolysaccharide production. The high-quality and full-length genome sequence was obtained using PacBio Sequencing technology, refined with Illumina sequencing.

Genome sequence of the hypervirulent ST65 Klebsiella pneumoniae isolate carrying mcr-8 from China

ObjectiveIn this study we report the complete genome sequence of a hypervirulent ST65 Klebsiella pneumoniae isolate harbouring mcr-8 from China. The aim was to investigate its molecular characteristics and resistance mechanisms. MethodsColistin-resistant hypervirulent K. pneumonia was isolated from an in-patient in China. The entire genome was sequenced on the Illumina NovaSeq 6000 System and long-read ONT Platform. De novo assembly was conducted using SPAdes and Unicycler. S1 nuclease pulsed-field gel electrophoresis, Southern blot, and antimicrobial susceptibility testing were performed. Sequence type, antimicrobial resistance, and virulence-related genes were predicted from the sequence. Circular maps of multiple plasmid comparisons were drawn by the BLAST Ring Image. ResultsThe complete genome sequence of K. pneumoniae ACESH00926 consists of one chromosome and two plasmids. ACESH00926 belongs to K2 ST65 according to multilocus sequence typing. ACESH00926 also showed high resistance to colistin (MIC >8 µg/mL). Several ARGs were identified, including the mobile colistin-resistant gene, mcr-8, which was located in an IncFIl(K)/IncFIA(HI1)-type plasmid. The larger plasmid was a pK244-like virulence plasmid that carries a series of virulence genes, such as regulators of the mucoid phenotype (rmpA and rmpA2), salmochelin siderophore biosynthesis (iroB), ABC transporter (iroC), ferric aerobactin receptor (iutA), aerobactin siderophore biosynthesis protein (iucC), and aerobactin synthetase (iucA)-encoded genes, in addition to another plasmid carrying mcr-8 with a conserved genetic context (dgkA-sasA-copR-mcr-8-ccdA-ccdB-xerD-repE-parM-umuC-lexA-klcA). ConclusionsThe necessity of monitoring a combination of colistin-resistant and hypervirulent K. pneumoniae strains in the future is emphasised in this article.

First Report of Virulence Factors in Carbapenem-resistant Klebsiella pneumoniae from Maharashtra, India

Introduction: Klebsiella pneumoniae (KPN) is a major multidrug-resistant (MDR) organism all over the world which can cause various infections. These variations in the infections are due to the presence or expression of virulence factors along with the presence of antimicrobial resistance. These virulence factors play an important role in adherence, invasion, spread, and in turn the range of infections. Various virulence factors such as capsules, lipopolysaccharides, siderophores, and fimbriae (FimH) are reported. Objective: Molecular screening of the multidrug-resistant KPN (MDR KPN) for the most common multidrug resistant gene to characterize the virulence factors in the clinical isolates and find out their prevalence in the Maharashtra region of India. Materials and Methods: The study was conducted for 2 years at a tertiary care teaching hospital in western Maharashtra. A total of 931 extended-spectrum beta-lactamase (ESBL)-producing MDR KPN were isolated. Polymerase chain reaction (PCR) was performed for 45 purified isolates to check the presence of various multidrug-resistant (MDR) genes and to detect virulence factors. Results: Multiplex PCR was performed on 45 isolates, and the presence of ESBLs genes was detected in majority of the isolates. Among these, 97.8% harbored blaTEM and blaSHV genes, and blaCTX-M gene was found in 88.9% of isolates, whereas enterobactin (entB) and FimH genes contributed to 93.33% (14 of 15 isolates). However, all these isolates did not harbor mucoid phenotype regulator, K2, and Aerobactin genes screened for. The majority of the isolates expressed both entB and FimH simultaneously. Conclusion: The current study observed that the MDR isolates of KPN-producing β-lactamases harbor multiple types of virulence genes and virulence factors. Prevalence of entB and FimH virulence factors was found while other virulence factors may be absent in the Maharashtra region for MDR KPN.

Clinical, phenotypic characterization and genomic analysis of the mucoid Acinetobacter baumannii from a teaching hospital

BackgroundAcinetobacter baumannii (A. baumannii) has become a significant nosocomial pathogen globally over the past decade due to the increasing prevalence of antibiotic-resistant isolates. The formation of the mucoid phenotype is a crucial adaptive defense response to external pressure, but the clinical, phenotypic and genotypic characteristics and their relationship with sequence types (ST) and K locus (KL) types remain unclear. MethodsIn this study, we screened a total of 736 A. baumannii isolates, from which we identified and characterized 13 mucoid isolates. The study explored the clinical characteristics of patients with mucoid isolates, investigated the mucoid phenotype, performed capsule observation, quantified capsule production, and assessed antimicrobial susceptibility. Subsequently, whole-genome sequencing (WGS) was used to analyze the sequence types (ST), loci for capsular polysaccharide (KL), antibiotic resistance genes, virulence genes, and core-genome single-nucleotide polymorphisms (SNPs). Additionally, the virulence of all mucoid strains was evaluated through serum resistance assay, biofilm-forming assay, and Galleria mellonella survival assay. ResultsAll mucoid A. baumannii isolates were found to be encapsulated and extremely drug-resistant. Among patients infected with these isolates, 92.3 % had pulmonary infections, and the 30-day mortality rate was 61.5 %. The analysis revealed that not all strains are highly virulent. Whole-genome sequencing (WGS) identified the sequence types as ST136, ST208, ST381, ST195, and ST281, and the capsular types as KL77, KL7, KL33, KL2, and KL3. The ST208 and KL7 isolates exhibited higher virulence and greater biofilm formation, with KL7 isolates also showing higher capsule production. Despite these differences, no significant variations in virulence genes were observed among the mucoid isolates, except for biofilm-associated and quorum-sensing genes. The highly virulent ST208/KL7 strains (AB276, AB313, and AB552) lacked biofilm-associated genes (csuA/BABCDE), indicating these genes do not directly cause differences in biofilm formation. ConclusionThe mucoid A. baumannii isolates were extensively drug-resistant, and infections caused by these isolates could lead to higher mortality. However, not all strains had high virulence, with variations likely related to specific sequence types (ST) and K locus (KL) types.

Adaptive evolution of carbapenem-resistant hypervirulent Klebsiella pneumoniae in the urinary tract of a single patient

The emergence of carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKp) is a growing concern due to its high mortality and limited treatment options. Although hypermucoviscosity is crucial for CR-hvKp infection, the role of changes in bacterial mucoviscosity in the host colonization and persistence of CR-hvKp is not clearly defined. Herein, we observed a phenotypic switch of CR-hvKp from a hypermucoviscous to a hypomucoviscous state in a patient with scrotal abscess and urinary tract infection (UTI). This switch was attributed to decreased expression of rmpADC, the regulator of mucoid phenotype, caused by deletion of the upstream insertion sequence ISKpn26. Postswitching, the hypomucoid variant showed a 9.0-fold decrease in mice sepsis mortality, a >170.0-fold reduction in the ability to evade macrophage phagocytosis in vitro, and an 11.2- to 40.9-fold drop in growth rate in normal mouse serum. Conversely, it exhibited an increased residence time in the mouse urinary tract (21 vs. 6 d), as well as a 216.4-fold boost in adhesion to bladder epithelial cells and a 48.7% enhancement in biofilm production. Notably, the CR-hvKp mucoid switch was reproduced in an antibiotic-free mouse UTI model. The in vivo generation of hypomucoid variants was primarily associated with defective or low expression of rmpADC or capsule synthesis gene wcaJ, mediated by ISKpn26 insertion/deletion or base-pair insertion. The spontaneous hypomucoid variants also outcompeted hypermucoid bacteria in the mouse urinary tract. Collectively, the ISKpn26-associated mucoid switch in CR-hvKp signifies the antibiotic-independent host adaptive evolution, providing insights into the role of mucoid switch in the persistence of CR-hvKp.

Diverse polysaccharide production and biofilm formation abilities of clinical Klebsiella pneumoniae.

Klebsiella pneumoniae infections have become a growing threat for human health. The lack of understanding of the relationship between antibiotic resistance, mucoviscosity, and biofilm formation in clinical isolates impedes our abilities to effectively predict K. pneumoniae infection outcomes. These traits are also associated with fitness in natural populations and more specifically within a host. The Multidrug-Resistant Organism Repository and Surveillance Network offers a unique opportunity into the genetic and phenotypic variabilities in the K. pneumoniae isolates encountered in the clinics today. To this end, we compared the genetic profiles of these isolates with the phenotypic biofilm formation abilities, percent mucoviscosity, and growth rates. We found most isolates formed limited biofilm, although a select group of isolates could form extremely robust biofilms. Variation in biofilm formation could not be explained by difference in growth rate, suggesting specific genetic and physical determinants. Interestingly, the most mucoid strains in the populations were lacking the genetic element regulating the mucoid phenotype and three of these isolates were able to form robust biofilms. There was a significant phenotype-genotype correlation with decreased biofilm formation and an insertion sequence in the transcriptional activator of the type III fimbrial system. Finally, confocal microscopy highlighted the structural and spatial heterogeneity of biofilm among the most robust biofilm formers not detected by traditional methods. The combination of phenotypic, genomic and image analyses allowed us to reveal an unexpected phenotypic diversity and an intricate relation between growth, mucoviscosity and specific virulence-associated genetic determinants.

Genomic Comparative of Pseudomonas aeruginosa Small Colony Variant, Mucoid and Non-mucoid Phenotypes Obtained from a Patient with Cystic Fibrosis During Respiratory Exacerbations.

Pseudomonas aeruginosa, the most prevalent opportunistic pathogen in chronic obstructive pulmonary disease, associated with high morbidity and mortality in patients with cystic fibrosis (CF), is practically impossible to be eradicated from the airways in chronicity. Its extraordinary genomic plasticity is possibly associated with high antimicrobial resistance, virulence factors, and its phenotypic diversity. The occurrence of P. aeruginosa isolates promoting airway infection, showing mucoid, non-mucoid, and small colony variant (SCV) phenotypes, was observed simultaneously, in the present study, in sputum cultures obtained from a male CF young patient with chronic pulmonary infection for over a decade. The isolates belonged to a new ST (2744) were obtained in two moments of exacerbation of the respiratory disease, in which he was hospitalized. Genetic background and phenotypic analysis indicated that the isolates exhibited multi- and pan-antimicrobial resistant profiles, as well as non-susceptible to polymyxin and predominantly hypermutable (HPM) phenotypes. Whole genome sequencing showed variations in genome sizes, coding sequences and their determinants of resistance and virulence. The annotated genomes were compared for antimicrobial resistance, hypermutability, and SCV characteristics. We highlight the lack of reported genetic determinants of SCV emergence and HPM phenotypes, which can be explained in part due to the very short time between collections of isolates. To the best of our knowledge, this is the first report of genome sequencing of P. aeruginosa SCV from a CF patient in Brazil.

Adaptive attenuation of virulence in hypervirulent carbapenem-resistant Klebsiella pneumoniae.

The emergence of nosocomial infections caused by hypervirulent and carbapenem-resistant K. pneumoniae (hv-CRKP) has become a significant public health challenge. The genetic traits of virulence and resistance plasmids in hv-CRKP have been extensively studied; however, research on the adaptive evolution strategies of clinical strains inside the host was scarce. This study aimed to understand the effects of antibiotic treatment on the phenotype and genotype characteristics of hv-CRKP. We investigated the evolution of hv-CRKP strains isolated from the same patient to elucidate the transition between hospital invasion and colonization. A comparative genomics analysis was performed to identify single nucleotide polymorphisms in the rmpA promoter. Subsequent validation through RNA-seq and gene deletion confirmed that distinct rmpA promoter sequences exert control over the mucoid phenotype. Additionally, biofilm experiments, cell adhesion assays, and animal infection models were conducted to illuminate the influence of rmpA promoter diversity on virulence changes. We demonstrated that the P12T and P11T promoters of rmpA possess strong activity, which leads to the evolution of CRKP into infectious and virulent strains. Meanwhile, the specific sequence of polyT motifs in the rmpA promoter led to a decrease in the lethality of hv-CRKP and enhanced cell adhesion and colonization. To summarize, the rmpA promoter of hv-CRKP is utilized to control capsule production, thereby modifying pathogenicity to better suit the host's ecological environment.IMPORTANCEThe prevalence of hospital-acquired illness caused by hypervirulent carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) is significant, leading to prolonged antibiotic treatment. However, there are few reports on the phenotypic changes of hv-CRKP in patients undergoing antibiotic treatment. We performed a comprehensive examination of the genetic evolutionary traits of hv-CRKP obtained from the same patient and observed variations in the promoter sequences of the virulence factor rmpA. The strong activity of the promoter sequences P11T and P12T enhances the consistent production of capsule polysaccharides, resulting in an invasive strain. Conversely, weak promoter activity of P9T and P10T is advantageous for exposing pili, hence improving bacterial cell attachment ability and facilitating bacterial colonization. This finding also explains the confusion of some clinical strains carrying wild-type rmpA but exhibiting a low mucoid phenotype. This adaptive alteration facilitates the dissemination of K. pneumoniae within the hospital setting.

The Increased Prevalence of rmpA Gene in Klebsiella pneumoniae Isolates Coharboring blaNDM and blaOXA-48-like Genes.

The emergence of carbapenemase-producing Klebsiella pneumoniae poses a substantial risk to public health. It is essential to comprehend the influence of carbapenemase on the virulence characteristics of K. pneumoniae in order to devise successful strategies for combating these infections. In this study, we explored the distribution disparity of virulence determinants between carbapenemase-producing (CP-Kp, n = 52) and carbapenemase-nonproducing (CN-Kp, n = 43) isolates. The presence of carbapenemases was detected via the modified carbapenem inactivation method and confirmed by PCR. The New Delhi metallo-β-lactamase (blaNDM) and Oxacillinase-48-like (blaOXA-48-like) genes were the most prevalent (94.23% and 76.92%, respectively) in CP-Kp isolates. Coexistence of blaNDM and blaOXA-48-like was observed in 71.15% of isolates, whereas 5.77% coharbored blaNDM and blaKPC. PCR analysis revealed the presence of several virulence genes, including adhesins (fimH, 92.63%, mrkD, 97.89%), capsule-associated virulence (uge, 90.53%), the K2 capsule serotype (k2, 6.32%), the iron acquisition system (kfu, 23.16%), and the regulator of mucoid phenotype (rmpA, 28.42%). A significantly higher prevalence of rmpA was detected in the CP-Kp compared with the CN-Kp (24/52 vs. 3/43, p < 0.0001), indicating a potential association between rmpA and carbapenemase acquisition. In addition, the majority of rmpA (22/24) positive isolates in the CP-Kp isolates coharbored blaNDM and either blaOXA-48-like or blaKPC.

Genomic analysis of extensively drug resistant (XDR) Klebsiella pneumoniae high-risk clone ST14 co-harboring blaNDM and blaOXA-48 recovered from Saudi Arabia

BackgroundThis study presents a comprehensive genomic analysis of NDM and OXA-48-producing Klebsiella pneumoniae in the Western region of Saudi Arabia, traversed by tens of millions of Muslims from various countries annually. This significant influx of visitors invariably leads to the spread and diversity of MDR bacteria. MethodsGenome sequencing was performed using MiSeq system of 29 CPKP isolates that were NDM and OXA-48-positive isolated from nosocomial infections and demonstrated resistance to most antibiotics, including carbapenems. ResultsWGS analysis showed that 12 (41.3%) isolates co-harbored blaOXA-48,blaCTX-M-15 and blaNDM genes. Notably, 16 (55.1%) isolates were identified as high-risk clone ST14, with 50% of these isolates co-harbored blaOXA-48, blaNDM and blaCTX-M-15 genes. All ST14 isolates were identified as capsular genotype KL2 and O1/O2v1 antigen with yersiniabactin locus ypt 14 carried by ICEKp5. The two isolates were identified as ST2096/KL64 hypervirulent K. pneumoniae (hvKp) clone harboring several virulence factors, including the regulator of the mucoid phenotype rmpA2 and aerobactin (iuc-1). Interestingly, two of the hvKp ST383/KL30 isolates were resistant to all tested antimicrobials except colistin and tigecycline, and simultaneously carried numerous ESBLs and carbapenemase genes. These isolates also harbor several virulence factors such as rmpA1, rmpA2, carried on KpVP-1, and aerobactin (iuc-1). Conclusionthis study provides insights into the spread and prevalence of high-risk clones of CPKP in the Western region of Saudi Arabia. The ST14 high-risk clone appears to be the predominant CPKP clone in this region, posing a significant threat to public health. This study also reports the presence of two globally disseminated hypervirulent K. pneumoniae (hvKp) clones, namely ST2096 and ST383. Therefore, it is essential to improve surveillance and implement strict infection control measures in this region, which receives a substantial number of visitors to effectively monitor and reduce the spread of high-risk clones of antimicrobial-resistant bacteria, including CPKP.

Impact of LuxS on virulence and pathogenicity in Klebsiella pneumoniae exhibiting varied mucoid phenotypes.

How the LuxS/AI-2 quorum sensing (QS) system influences the pathogenicity of K. pneumoniae is complicated by the heterogeneity of the bacterial mucoid phenotypes. This study aims to explore the LuxS-mediated regulation of the pathogenicity of K. pneumoniae with diverse mucoid phenotypes, including hypermucoid, regular-mucoid, and nonmucoid. The wild-type, luxS knockout, and complemented strains of three K. pneumoniae clinical isolates with distinct mucoid phenotypes were constructed. The results revealed the downregulation of virulence genes of regular-mucoid, and nonmucoid but not hypermucoid strains. The deletion of luxS reduced the pathogenicity of the regular-mucoid, and nonmucoid strains in mice; while in hypermucoid strain, luxS knockout reduced virulence in late growth but enhanced virulence in the early growth phase. Furthermore, the absence of luxS led the regular-mucoid and nonmucoid strains to be more sensitive to the host cell defense, and less biofilm-productive than the wild-type at both the low and high-density growth state. Nevertheless, luxS knockout enhanced the resistances to adhesion and phagocytosis by macrophage as well as serum-killing, of hypermucoid K. pneumoniae at its early low-density growth state, while it was opposite to those in its late high-density growth phase. Collectively, our results suggested that LuxS plays a crucial role in the pathogenicity of K. pneumoniae, and it is highly relevant to the mucoid phenotypes and growth phases of the strains. LuxS probably depresses the capsule in the early low-density phase and promotes the capsule, biofilm, and pathogenicity during the late high-density phase, but inhibits lipopolysaccharide throughout the growth phase, in K. pneumoniae.IMPORTANCECharacterizing the regulation of physiological functions by the LuxS/AI-2 quorum sensing (QS) system in Klebsiella pneumoniae strains will improve our understanding of this important pathogen. The genetic heterogeneity of K. pneumoniae isolates complicates our understanding of its pathogenicity, and the association of LuxS with bacterial pathogenicity has remained poorly addressed in K. pneumoniae. Our results demonstrated strain and growth phase-dependent variation in the contributions of LuxS to the virulence and pathogenicity of K. pneumoniae. Our findings provide new insights into the important contribution of the LuxS/AI-2 QS system to the networks that regulate the pathogenicity of K. pneumoniae. Our study will facilitate our understanding of the regulatory mechanisms of LuxS/AI-2 QS on the pathogenicity of K. pneumoniae under the background of their genetic heterogeneity and help develop new strategies for diminished bacterial virulence within the clinical K. pneumoniae population.

Characterisation of highly virulent and colistin-resistant ST367-KL1 Klebsiella quasipneumoniae subsp. similipneumoniae Strain

ObjectivesTo elucidate the characteristics of a colistin-resistant and hypervirulent Klebsiella quasipneumoniae subsp. similipneumoniae strain (KP8) using whole genome sequencing and various phenotypic assays. MethodsAntimicrobial susceptibility testing was performed using broth microdilution. Whole genome sequencing and comparative genomics were utilised to elucidate genomic characteristics. Phenotypic assays to evaluate virulence factors included measurements of mucosal viscosity, biofilm production, siderophore production, infection of A549 cells, serum-killing assays, and Galleria mellonella infection models. ResultsWhole-genome sequencing revealed that the strain (KP8) belongs to sequence type 367 (ST367) and capsular type 1 (KL1), and it harbours several virulence genes, including regulator of mucoid phenotype (rmpA/A2), salmochelin (iroBCDN) and aerobactin (iucABCDiutA). Antibiotic susceptibility tests showed that KP8 was resistant to colistin. Genome analysis showed that the colistin resistance of KP8 might be related to amino acid insertions in pmrB (L215_D217, insL) and pagP (M1_S3, insV). Importantly, KP8 demonstrated comparable mucosal viscosity, biofilm production capacity, siderophore production levels to hvKP. Serum-killing experiments, A549 cell infection models, and G. mellonella infection models further indicated that KP8 displayed high virulence, akin to the hypervirulent strain NUTH-K2044. Notably, global genome analysis of the K. quasipneumoniae subsp. similipneumoniae strains highlighted that the ST367 lineage has a higher tendency to carry virulence-associated genes compared to other sequence types. The prevalence of virulence-associated factors concentrated within Chinese ST367 isolates reinforces this observation. ConclusionThese findings further enhance our understanding of the resistance and pathogenicity of ST367 K. quasipneumoniae subsp. similipneumoniae strain and also providing a broader perspective on the global epidemiological landscape.

Pseudomonas aeruginosa mucinous phenotypes and algUmucABD operon mutant characteristics obtained from inpatients with bronchiectasis and their correlation with acute aggravation.

Although the mechanism is unclear, Pseudomonas aeruginosa (PA) infection directly affects the frequency of acute exacerbations in patients with bronchiectasis. The aims of this article are to analyze the genetic mutation characteristics of the algUmucABD operon in PA, isolated from hospitalized patients with bronchiectasis, and to explore independent risk factors for frequent acute exacerbations of bronchiectasis. Based on the number of acute exacerbations that occurred in the past year, these patients with bronchiectasis were divided into those with frequent acute exacerbations (Group A) and those with non-frequent acute exacerbations (Group B). We identified the distribution of mucoid phenotypes (MPs) and alginate morphotypes (AMs) in PA, and classified them into I-IV categories based on their different AMs; otherwise, the gene mutation types (GMTs) of the algUmucABD operon were tested. Subsequently, the relationship between GMT, MP, and AM and the independent risk factors for frequent acute exacerbations in patients with bronchiectasis were explored. A total of 93 patients and 75 PA strains, from January 2019 to August 2023, were included in this study. The MP and AM distributions of PA were as follows: 64 strains (85.33%) of mucoid (the AMs were 38 strains of type I, 3 strains of type II, and 23 strains of type IV) and 11 strains of non-mucoid (the AM was type III only). Mucoid PA with algU, mucA, mucB, and mucD mutations accounted for 19.61%, 74.51%, 31.37%, and 50.98%, respectively. GMT was divided into the following: mucA mutations only, mucA combined with other gene mutations, other gene mutations without mucA mutations, and without gene mutations. In 91.7% of PA with type I of AM, only mucA mutations occurred, and in both separate MP and AM, the GMT differences were statistically significant. Lastly, the number of lung lobes with bronchiectasis and the number of PA with mucA mutations only were the independent risk factors for frequent acute exacerbations. The mucA mutation was primarily responsible for the mucoid of MP and type I of AM in PA, and it was also an independent risk factor for frequent exacerbations of bronchiectasis.

Clinical and Molecular Characterisation of Multi Drug Resistant Hypervirulent Klebsiella pneumoniae (MDR-HvKp) Isolated from Patient Attending ATBU Teaching Hospital Bauchi

Background and Aim: K. pneumoniae exhibits increasing multidrug resistance and a global rise in hypervirulent strains, particularly prevalent in various African countries, posing significant challenges in managing infections ranging from pneumonia to neonatal septicemia and urinary tract infections. The study aimed to identify and detect virulence characteristics of MDR-HvKp from clinical samples. Method: Two hundred and fourteen (214) clinical samples were identified and drug resistance was measured using the Vitek 2 system, the isolates were phenotypically characterized for mucoviscosity and hypermucoviscosity using a string test and mucoid sedimentation test. The isolated K. pneumoniae were further assessed for the presence of rmpA, magA, BlaCTX-M, and BlaKPC-2 genes using PCR, Gel electrophoresis, and Sequencing, the rmpA gene sequence peculiar to K. pneumoniae was used for phylogenetic analysis using NCBI BLAST for nucleotide suite. Result: Twenty-four (24) K. pneumoniae were identified, phenotypically and molecularly characterized for drug resistance and virulence genes. The prevalence of K. pneumoniae was recorded at 24(17.3%), with higher rates observed among females 15(10.79%) compared to male patients with 9(6.47%), while the difference in prevalence between genders was not statistically significant, p = 0.06 (χ2 = 2.59), this is also similar to marital status with p = 0.025 (χ2 = 5.37). A total of 11(45.83%) K. pneumoniae were isolated from urine 59(42.45%), which is the highest followed by sputum 29(20.86%), blood 26(18.70%), and swab 25(17.99%); HvKp strain Kp128 demonstrated mucoviscosity and carried genes associated with mucoid phenotype and resistance, Antibiotic susceptibility testing revealed resistance to several antibiotics, particularly beta-lactams and carbapenems. Conclusion: The study reveals a rising prevalence of MDR-HvKp strains in Nigerian and Bauchi state in particular, emphasizing the critical need for effective management strategies against infections across diverse clinical settings.