Flow Cytometry and Automated Microscopy Integration for Phagocytic Analysis of Hypervirulent Klebsiella pneumoniae in Dictyostelium discoideum.

This chapter introduces protocols for culturing and maintaining Dictyostelium discoideum and methods for conducting virulence assays in this organism to study bacterial pathogenicity. It outlines advanced techniques, such as automated microscopy and flow cytometry, for detailed cellular analysis and traditional microbiological approaches. These comprehensive protocols will enable researchers to probe the virulence factors of pathogens like Klebsiella pneumoniae and to elucidate the details of host-pathogen interactions within a cost-effective and adaptable laboratory framework.

Establishment of a Rapid Detection Method for Highly Virulent Klebsiella Pneumoniae Based on Multienzyme Isothermal Rapid Amplification Technology.

BACKGROUND: This study aimed to establish a method for the rapid detection of highly virulent Klebsiella pneumoniae (hvKP) by using multienzyme isothermal rapid amplification (MIRA) technology. The laboratory can quickly, accurately, and conveniently diagnose highly virulent Klebsiella pneumoniae infection. METHODS: For this study, 7 laboratory standard strains and 184 clinical isolates (including 70 strains of Klebsiella pneumoniae) were collected and screened for highly virulent Klebsiella pneumoniae based on its colony morphology, wire drawing test, and next-generation sequencing (NGS) results. Based on the nucleic acid sequence of the peg344 gene of highly virulent Klebsiella pneumoniae on GenBank (no. AP006726.1), specific conserved regions were selected to design MIRA and real-time fluorescence quantitative PCR (qPCR) specific primers and probes. The MIRA and qPCR methods were used to detect the tested strain, and the specificity, sensitivity, and clinical performance of the MIRA method for detecting hvKP were evaluated. RESULTS: In total, 21 cases of hvKP were screened from clinical isolates. The MIRA detection method utilizes specific primers and probes to transmit significant fluorescence signals at 39°C, and the detection process takes 30 minutes. The specificity test results showed that only hvKP had a specific amplification curve, while the rest of non-highly virulent Klebsiella pneumoniae (non-hvKP) had no specific amplification curve. The sensitivity test results showed that the sensitivity of MIRA for detecting hvKP is 7 × 102 CFU/mL, which is consistent with the sensitivity of the real-time fluorescence qPCR method. A simultaneous detection of 184 clinical isolates was accomplished by using MIRA and qPCR methods. Twenty-one strains of hvKP have specific amplification curves, while the remaining 163 strains of non-hvKP have no specific amplification curves. The accuracy of both methods for detecting hvKP is 100%. CONCLUSIONS: The established multienzyme isothermal rapid amplification (MIRA) has the following characteristics: a short detection time, high sensitivity, and a strong specificity, and it can be used as a powerful tool for an early diagnosis and epidemiological monitoring of hvKp.

Evaluation of Klebsiella pneumoniae pathogenicity through holistic gene content analysis.

Klebsiella pneumoniae is a Gram-negative bacterium that causes both community- and healthcare-associated infections. Although various virulence factors and highly pathogenic phenotypes have been reported, the pathogenicity of K. pneumoniae is still not fully understood. In this study, we utilized whole-genome sequencing data of 168 clinical K. pneumoniae strains to assess pathogenicity. This work was based on the concept that the genetic composition of individual genomes (referred to as holistic gene content) of the strains may contribute to their pathogenicity. Holistic gene content analysis revealed two distinct groups of K. pneumoniae strains ('major group' and 'minor group'). The minor group included strains with known highly pathogenic clones (ST23, ST375, ST65 and ST86). The minor group had higher rates of capsular genotype K1 and presence of nine specific virulence genes (rmpA, iucA, iutA, irp2, fyuA, ybtS, iroN, allS and clbA) compared to the major group. Pathogenicity was assessed using Galleria mellonella larvae. Infection experiments revealed lower survival rates of larvae infected with strains from the minor group, indicating higher virulence. In addition, the minor group had a higher string test positivity rate than the major group. Holistic gene content analysis predicted possession of virulence genes, string test positivity and pathogenicity as observed in the G. mellonella infection model. Moreover, the findings suggested the presence of as yet unrecognized genomic elements that are either involved in the acquisition of virulence genes or associated with pathogenicity.

The Response of Murine Gut Microbiome in the Presence of Altered rpoS Gene of Klebsiella pneumoniae.

The murine model is invaluable for studying intricate interactions among gut microbes; hosts; and diseases. However; the impact of genetic variations in the murine microbiome; especially in disease contexts such as Klebsiella pneumoniae (Kp) infection; still needs to be explored. Kp; an opportunistic global pathogen; is becoming increasingly prevalent in regions like Asia; especially China. This study explored the role of the gut microbiota during Kp infection using mouse model; including wild-type and rpoS mutants of Kp138; KpC4; and KpE4 from human; maize; and ditch water; respectively. Under stress conditions; RpoS reconfigures global gene expression in bacteria; shifting the cells from active growth to survival mode. Our study examined notable differences in microbiome composition; finding that Lactobacillus and Klebsiella (particularly in WKp138) were the most abundant genera in mice guts at the genus level in all wild-type treated mice. In contrast; Firmicutes were predominant in the healthy control mice. Furthermore; Clostridium was the dominant genus in all mutants; mainly in ∆KpC4; and was absent in wild-type treated mice. Differential abundance analysis identified that these candidate taxa potentially influence disease progression and pathogen virulence. Functional prediction analysis showed that most bacterial groups were functionally involved in biosynthesis; precursor metabolites; degradation; energy generation; and metabolic cluster formation. These findings challenge the conventional understanding and highlight the need for nuanced interpretations in murine studies. Additionally; this study sheds light on microbiome-immune interactions in K. pneumoniae infection and proposes new potential therapeutic strategies.

An expanded database and analytical toolkit for identifying bacterial virulence factors and their associations with chronic diseases.

Virulence factor genes (VFGs) play pivotal roles in bacterial infections and have been identified within the human gut microbiota. However, their involvement in chronic diseases remains poorly understood. Here, we establish an expanded VFG database (VFDB 2.0) consisting of 62,332 nonredundant orthologues and alleles of VFGs using species-specific average nucleotide identity ( https://github.com/Wanting-Dong/MetaVF_toolkit/tree/main/databases ). We further develop the MetaVF toolkit, facilitating the precise identification of pathobiont-carried VFGs at the species level. A thorough characterization of VFGs for 5452 commensal isolates from healthy individuals reveals that only 11 of 301 species harbour these factors. Further analyses of VFGs within the gut microbiomes of nine chronic diseases reveal both common and disease-specific VFG features. Notably, in type 2 diabetes patients, long HiFi sequencing confirms that shared VF features are carried by pathobiont strains of Escherichia coli and Klebsiella pneumoniae. These findings underscore the critical importance of identifying and understanding VFGs in microbiome-associated diseases.

A new protocol for multispecies bacterial infections in zebrafish and their monitoring through automated image analysis.

The zebrafish Danio rerio has become a popular model host to explore disease pathology caused by infectious agents. A main advantage is its transparency at an early age, which enables live imaging of infection dynamics. While multispecies infections are common in patients, the zebrafish model is rarely used to study them, although the model would be ideal for investigating pathogen-pathogen and pathogen-host interactions. This may be due to the absence of an established multispecies infection protocol for a defined organ and the lack of suitable image analysis pipelines for automated image processing. To address these issues, we developed a protocol for establishing and tracking single and multispecies bacterial infections in the inner ear structure (otic vesicle) of the zebrafish by imaging. Subsequently, we generated an image analysis pipeline that involved deep learning for the automated segmentation of the otic vesicle, and scripts for quantifying pathogen frequencies through fluorescence intensity measures. We used Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae, three of the difficult-to-treat ESKAPE pathogens, to show that our infection protocol and image analysis pipeline work both for single pathogens and pairwise pathogen combinations. Thus, our protocols provide a comprehensive toolbox for studying single and multispecies infections in real-time in zebrafish.

A unique combination of natural fatty acids from Hermetia illucens fly larvae fat effectively combats virulence factors and biofilms of MDR hypervirulent mucoviscus Klebsiella pneumoniae strains by increasing Lewis acid-base/van der Waals interactions in bacterial wall membranes.

Introduction: Hypervirulent Klebsiella pneumoniae (hvKp) and carbapenem-resistant K. pneumoniae (CR-Kp) are rapidly emerging as opportunistic pathogens that have a global impact leading to a significant increase in mortality rates among clinical patients. Anti-virulence strategies that target bacterial behavior, such as adhesion and biofilm formation, have been proposed as alternatives to biocidal antibiotic treatments to reduce the rapid emergence of bacterial resistance. The main objective of this study was to examine the efficacy of fatty acid-enriched extract (AWME3) derived from the fat of Black Soldier Fly larvae (Hermetia illucens) in fighting against biofilms of multi-drug resistant (MDR) and highly virulent Klebsiella pneumoniae (hvKp) pathogens. Additionally, the study also aimed to investigate the potential mechanisms underlying this effect. Methods: Crystal violet (CV) and ethidium bromide (EtBr) assays show how AWME3 affects the formation of mixed and mature biofilms by the KP ATCC BAA-2473, KPi1627, and KPM9 strains. AWME3 has shown exceptional efficacy in combating the hypermucoviscosity (HMV) virulent factors of KPi1627 and KPM9 strains when tested using the string assay. The rudimentary motility of MDR KPM9 and KP ATCC BAA-2473 strains was detected through swimming, swarming, and twitching assays. The cell wall membrane disturbances induced by AWME3 were detected by light and scanning electron microscopy and further validated by an increase in the bacterial cell wall permeability and Lewis acid-base/van der Waals characteristics of K. pneumoniae strains tested by MATS (microbial adhesion to solvents) method. Results: After being exposed to 0.5 MIC (0.125 mg/ml) of AWME3, a significant reduction in the rudimentary motility of MDR KPM9 and KP ATCC BAA-2473 strains, whereas the treated bacterial strains exhibited motility between 4.23 ± 0.25 and 4.47 ± 0.25 mm, while the non-treated control groups showed significantly higher motility ranging from 8.5 ± 0.5 to 10.5 ± 0.5 mm. Conclusion: In conclusion, this study demonstrates the exceptional capability of the natural AWME3 extract enriched with a unique combination of fatty acids to effectively eliminate the biofilms formed by the highly drug-resistant and highly virulent K. pneumoniae (hvKp) pathogens. Our results highlight the opportunity to control and minimize the rapid emergence of bacterial resistance through the treatment using AWME3 of biofilm-associated infections caused by hvKp and CRKp pathogens.

RNA interactome of hypervirulent Klebsiella pneumoniae reveals a small RNA inhibitor of capsular mucoviscosity and virulence.

Hypervirulent Klebsiella pneumoniae (HvKP) is an emerging bacterial pathogen causing invasive infection in immune-competent humans. The hypervirulence is strongly linked to the overproduction of hypermucoviscous capsule, but the underlying regulatory mechanisms of hypermucoviscosity (HMV) have been elusive, especially at the post-transcriptional level mediated by small noncoding RNAs (sRNAs). Using a recently developed RNA interactome profiling approach iRIL-seq, we interrogate the Hfq-associated sRNA regulatory network and establish an intracellular RNA-RNA interactome in HvKP. Our data reveal numerous interactions between sRNAs and HMV-related mRNAs, and identify a plethora of sRNAs that repress or promote HMV. One of the strongest HMV repressors is ArcZ, which is activated by the catabolite regulator CRP and targets many HMV-related genes including mlaA and fbp. We discover that MlaA and its function in phospholipid transport is crucial for capsule retention and HMV, inactivation of which abolishes Klebsiella virulence in mice. ArcZ overexpression drastically reduces bacterial burden in mice and reduces HMV in multiple hypervirulent and carbapenem-resistant clinical isolates, indicating ArcZ is a potent RNA inhibitor of bacterial pneumonia with therapeutic potential. Our work unravels a novel CRP-ArcZ-MlaA regulatory circuit of HMV and provides mechanistic insights into the posttranscriptional virulence control in a superbug of global concern.

Clonal background and routes of plasmid transmission underlie antimicrobial resistance features of bloodstream Klebsiella pneumoniae.

Bloodstream infections caused by the opportunistic pathogen Klebsiella pneumoniae are associated with adverse health complications and high mortality rates. Antimicrobial resistance (AMR) limits available treatment options, thus exacerbating its public health and clinical burden. Here, we aim to elucidate the population structure of K. pneumoniae in bloodstream infections from a single medical center and the drivers that facilitate the dissemination of AMR. Analysis of 136 short-read genome sequences complemented with 12 long-read sequences shows the population consisting of 94 sequence types (STs) and 99 clonal groups, including globally distributed multidrug resistant and hypervirulent clones. In vitro antimicrobial susceptibility testing and in silico identification of AMR determinants reveal high concordance (90.44-100%) for aminoglycosides, beta-lactams, carbapenems, cephalosporins, quinolones, and sulfonamides. IncF plasmids mediate the clonal (within the same lineage) and horizontal (between lineages) transmission of the extended-spectrum beta-lactamase gene blaCTX-M-15. Nearly identical plasmids are recovered from isolates over a span of two years indicating long-term persistence. The genetic determinants for hypervirulence are carried on plasmids exhibiting genomic rearrangement, loss, and/or truncation. Our findings highlight the importance of considering both the genetic background of host strains and the routes of plasmid transmission in understanding the spread of AMR in bloodstream infections.

Pulmonary abscess secondary to epididymitis caused by extended spectrum ß-lactamase-producing hypervirulent Klebsiella pneumoniae: a case report.

BACKGROUND: Pulmonary abscesses resulting from epididymitis caused by extended spectrum ß-lactamase-producing hypervirulent Klebsiella pneumoniae (ESBL-hvKp) in a nondiabetic patient are extremely uncommon. The infection caused by this disseminated drug-resistant bacteria, which is generally considered an intractable case, poses a potential challenge in clinical practice. CASE PRESENTATION: In this case report, we present the clinical course of a 71-year-old male patient with epididymitis, who subsequently developed cough and dyspnea following anti-infection treatment. Imaging examinations revealed severe pneumonia and pulmonary abscess. The infection of ESBL-hvKp in the epididymis led to bacteremia and subsequent lung lesions. Due to poor response to anti-infection therapy, the patient required an extended duration of anti-infection treatment and ultimately chosed to discontinue treatment. CONCLUSIONS: Acute epididymitis caused by ESBL-hvKP infection can result in the spread of the infection through the bloodstream, leading to severe pneumonia and lung abscess. Given the critical condition of the patient, even with active anti-infection treatment, there is a risk of treatment failure or potentially fatal outcomes.

Hypervirulent and carbapenem-resistant Klebsiella pneumoniae: A global public health threat.

The evolution of hypervirulent and carbapenem-resistant Klebsiella pneumoniae can be categorized into three main patterns: the evolution of KL1/KL2-hvKp strains into CR-hvKp, the evolution of carbapenem-resistant K. pneumoniae (CRKp) strains into hv-CRKp, and the acquisition of hybrid plasmids carrying carbapenem resistance and virulence genes by classical K. pneumoniae (cKp). These strains are characterized by multi-drug resistance, high virulence, and high infectivity. Currently, there are no effective methods for treating and surveillance this pathogen. In addition, the continuous horizontal transfer and clonal spread of these bacteria under the pressure of hospital antibiotics have led to the emergence of more drug-resistant strains. This review discusses the evolution and distribution characteristics of hypervirulent and carbapenem-resistant K. pneumoniae, the mechanisms of carbapenem resistance and hypervirulence, risk factors for susceptibility, infection syndromes, treatment regimens, real-time surveillance and preventive control measures. It also outlines the resistance mechanisms of antimicrobial drugs used to treat this pathogen, providing insights for developing new drugs, combination therapies, and a "One Health" approach. Narrowing the scope of surveillance but intensifying implementation efforts is a viable solution. Monitoring of strains can be focused primarily on hospitals and urban wastewater treatment plants.

VacSol-ML(ESKAPE): Machine learning empowering vaccine antigen prediction for ESKAPE pathogens.

The ESKAPE family, comprising Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp., poses a significant global threat due to their heightened virulence and extensive antibiotic resistance. These pathogens contribute largely to the prevalence of nosocomial or hospital-acquired infections, resulting in high morbidity and mortality rates. To tackle this healthcare problem urgent measures are needed, including development of innovative vaccines and therapeutic strategies. Designing vaccines involves a complex and resource-intensive process of identifying protective antigens and potential vaccine candidates (PVCs) from pathogens. Reverse vaccinology (RV), an approach based on genomics, made this process more efficient by leveraging bioinformatics tools to identify potential vaccine candidates. In recent years, artificial intelligence and machine learning (ML) techniques has shown promise in enhancing the accuracy and efficiency of reverse vaccinology. This study introduces a supervised ML classification framework, to predict potential vaccine candidates specifically against ESKAPE pathogens. The model's training utilized biological and physicochemical properties from a dataset containing protective antigens and non-protective proteins of ESKAPE pathogens. Conventional autoencoders based strategy was employed for feature encoding and selection. During the training process, seven machine learning algorithms were trained and subjected to Stratified 5-fold Cross Validation. Random Forest and Logistic Regression exhibited best performance in various metrics including accuracy, precision, recall, WF1 score, and Area under the curve. An ensemble model was developed, to take collective strengths of both the algorithms. To assess efficacy of our final ensemble model, a high-quality benchmark dataset was employed. VacSol-ML(ESKAPE) demonstrated outstanding discrimination between protective vaccine candidates (PVCs) and non-protective antigens. VacSol-ML(ESKAPE), proves to be an invaluable tool in expediting vaccine development for these pathogens. Accessible to the public through both a web server and standalone version, it encourages collaborative research. The web-based and standalone tools are available at http://vacsolml.mgbio.tech/.

Molecular characteristics and pathogenic mechanisms of KPC-3 producing hypervirulent carbapenem-resistant Klebsiella pneumoniae (ST23-K1).

Objective: This study aimed to comprehensively investigate hypervirulent carbapenem-resistant Klebsiella pneumoniae (CR-hvKP) in the Ningbo region. Importantly, we sought to elucidate its molecular characteristics and pathogenic mechanisms. This information will provide evidence-based insights for preventing and controlling nosocomial infections and facilitate improved clinical diagnosis and treatment in this region. Methods: 96 carbapenem-resistant Klebsiella pneumoniae strains were collected from the Ningbo region between January 2021 and December 2022. Whole genome sequencing and bioinformatic methods were employed to identify and characterize CR-hvKP strains at the molecular level. The minimum inhibitory concentrations (MICs) of common clinical antibiotics were determined using the VITEK-2 Compact automatic microbiological analyzer. Plasmid conjugation experiments evaluated the transferability of resistance plasmids. Finally, mouse virulence assays were conducted to explore the pathogenic mechanisms. Results: Among the 96 strains, a single CR-hvKP strain, designated CR-hvKP57, was identified, with an isolation frequency of 1.04%. Whole-genome sequencing revealed the strain to be ST23 serotype with a K1 capsule. This strain harbored three plasmids. Plasmid 1, a pLVPK-like virulence plasmid, carried multiple virulence genes, including rmpA, rmpA2, iroB, iucA, and terB. Plasmid 2 contained transposable element sequences such as IS15 and IS26. Plasmid 3, classified as a resistance plasmid, harbored the bla KPC-3 carbapenem resistance gene. Mouse virulence assays demonstrated a high mortality rate associated with CR-hvKP57 infection. Additionally, there was a significant increase in IL-1ß, IL-6, and TNF-α levels in response to CR-hvKP57 infection, indicating varying degrees of inflammatory response. Western blot experiments further suggested that the pathogenic mechanism involves activation of the NF-κB signaling pathway. Conclusion: This study confirms the emergence of hypervirulent CR-hvKP in the Ningbo region, which likely resulted from the acquisition of a pLVPK-like virulence plasmid and a bla KPC-3 resistance plasmid by the ST23-K1 type Klebsiella pneumoniae. Our findings highlight the urgent need for more judicious use of antibiotics to limit the emergence of resistance. Additionally, strengthening infection prevention and control measures is crucial to minimize the spread of virulence and resistance plasmids.

Deciphering the relative importance of genetic elements in hypervirulent Klebsiella pneumoniae to guide countermeasure development.

BACKGROUND: Quantitating the contribution of phenotype-responsible elements in hypervirulent Klebsiella pneumoniae is needed. METHODS: Isogenic mutants of four hypervirulent clinical isolates that produced K1 (ST23), K2 (ST86), K20 (ST1544), or K54 (ST29) capsules (mean 2.2 log10 LD50 (range 1.5-2.9)) were created to measure the effects on LD50 in a murine model of the hypervirulence-associated plasmid (pVir), iucA, prmpA, prmpA2 (truncated), irp2, and clbBC. FINDINGS: Curing pVir had the greatest increase in survival (mean LD50 to 7.6 (range 7.0-9.0, p ≤ 0.0001), a dosage comparable to classical K. pneumoniae. Results also showed increased mean LD50s for ΔprmpA (5.9, p ≤ 0.0001), ΔiucA (3.6, p ≤ 0.0001), Δirp2 (3.4), ΔrmpAΔiucA (6.3, p ≤ 0.0001), and ΔpVirΔirp2 (8.7, p ≤ 0.0001). Notably ΔpVir had an additional mean LD50 increase of 1.3 compared to the pVir-encoded ΔprmpAΔiucA (p ≤ 0.01), suggesting presence of additional pVir-virulence genes. Truncated pRmpA2 did not contribute to virulence. Odd ratios in the absence of pVir/yersiniabactin, pVir, pRmpA/aerobactin, pRmpA, aerobactin, yersiniabactin, and colibactin demonstrated a 250-fold, 67-fold, 20-fold, 16.7-fold, 9.6-fold, and 1.7-fold decrease in lethality respectively. INTERPRETATION: These data can guide countermeasure development. FUNDING: This work was supported by NIH R21 AI123558-01 and 1R21AI141826-01A1 (Dr. Russo) and the Department of Veterans Affairs VA Merit Review (I01 BX004677-01) (Dr. Russo). This study was also partially funded by the U.S. Defense Health Program (DHP) Operations and Maintenance.

Relationship between virulence and carbapenem resistance phenotype of Klebsiella pneumoniae from blood infection: identification of a carbapenem-resistant and hypervirulent strain. / è¡€æ¶²æ„ŸæŸ“æ¥æºè‚ºç‚Žå ‹é›·ä¼¯èŒæ¯’åŠ›ä¸Žç¢³é’éœ‰çƒ¯ç±»è€è¯è¡¨åž‹çš„å ³ç³»åŠç¢³é’éœ‰çƒ¯ç±»è€è¯é«˜æ¯’åŠ›èŒæ ªçš„é‰´å®š.

OBJECTIVES: To investigate the relationship between the virulence and the carbapenem resistance phenotype of Klebsiella pneumoniae from blood infection, and to identify carbapenem-resistant and hypervirulent Klebsiella pneumoniae (CR-HVKP)strains. METHODS: A total of 192 Klebsiella pneumoniae strains were isolated from blood culture of patients with bloodstream infections from 2016 to 2019, of which 96 isolates were carbapenem-resistant Klebsiella pneumoniae (CRKP) and 96 were carbapenem-sensitive Klebsiella pneumoniae (CSKP). The drug susceptibility was detected by VITEK-2 automatic microbial analyzer; carbapenemase genes, virulence genes and capsule typing were detected by polymerase chain reaction; the high viscosity phenotype of strains was detected by string test, and the genome characteristics of CR-HVKP were detected by whole genome sequencing. Serum killing and biofilm formation test were used to further verify the virulence of CR-HVKP. RESULTS: There were significant differences in drug resistance to common antibiotics, except for minocycline between CSKP and CRKP isolates (all P<0.05). 92 out of 96 CRKP isolates carried carbapenemase genes, mainly blaKPC-2. The string tests were positive in 4 isolates of CRKP and 36 isolates of CSKP (P<0.05). The detection rates of virulence genes Kfu, aerobictin, iutA, ybtS, rmpA, magA, allS, and capsule antigen K1 and K2 in CSKP group were significantly higher than those in CRKP group (all P<0.05). One HVKP strain was detected in the CRKP group (CR-HVKP) and 36 HVKP was detected in the CSKP group (P<0.05). The CR-HVKP strain belonged to the MLST412, serotype K57, expressed iutA, entB, mrkD, fimH, and rmpA virulence genes, and showed strong biofilm formation and significantly increased serum resistance. Whole genome sequencing results showed that this CR-HVKP isolate carried blaSHV-145, blaTEM-1, blaCTX-M-3, fosA6, oqxA5, oqxB26, and aac(3)-IId resistance genes, accompanied by abnormalities in outer membrane protein K (OmpK) 35 and OmpK36. CONCLUSIONS: The drug resistance of CRKP is significantly higher than that of CSKP, while CRKP carrying fewer virulence genes in both number and types compared to CSKP. A new MLST type of carbapenem-resistant and hypervirulent Klebsiella pneumoniae strain has been detected, which requires clinical awareness and epidemiological monitoring.

Molecular characterization of NDM and OXA-48-like-producing Klebsiella pneumoniae ST16 and hypervirulent ST337 clone among two patients; a case report.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) infections are a major public health problem, requiring the use of last-resort antibiotics such as colistin. However, there is concern regarding the emergence of isolates resistant to this agent. The report describes two patients with urinary tract infection (UTI) and ventilator-associated pneumonia (VAP) infection caused by CRKP strains. The first case was a 23-year-old male with UTI caused by a strain of ST16 co-harboring blaCTX-M, blaTEM, blaSHV, blaNDM, blaOXA-48-like genes. The second case was a 39-year-old woman with VAP due to hypervirulent ST337-K2 co-harboring blaSHV, blaNDM, blaOXA-48-like, iucA, rmpA2 and rmpA. The patients' general condition improved after combination therapy with colistin (plus meropenem and rifampin, respectively) and both of them recovered and were discharged from the hospital. This study highlights the necessary prevention and control steps to prevent the further spread of CRKP strains should be a priority in our hospital.

Virulence gene distribution and molecular epidemiological characteristics of carbapenem-resistant Klebsiella pneumoniae in the ICU. / ICUç¢³é’éœ‰çƒ¯è€è¯è‚ºç‚Žå ‹é›·ä¼¯èŒçš„æ¯’åŠ›åŸºå› åˆ†å¸ƒåŠåˆ†å­æµè¡Œç— å­¦ç‰¹å¾.

OBJECTIVES: The drug-resistant genes carried by carbapenem-resistant Klebsiella pneumoniae (CRKP) limit clinical treatment options, and its virulence genes severely affect patient prognosis. This study aims to investigate the distribution of virulence genes, capsular serotypes, and molecular epidemiological characteristics of CRKP in ICU, to understand the characteristics of CRKP infections in ICU, and to provide a scientific basis for effective monitoring and control of CRKP infections in ICU. METHODS: A total of 40 non-duplicate strains of CRKP isolated from the ICU of Guangdong Provincial People's Hospital between January 2021 and December 2022 were collected and analyzed. Whole-genome sequencing was used to analyze the distribution of resistance genes, virulence genes, and capsular serotypes of the strains. The sequences of 7 housekeeping genes of CRKP genome were uploaded to the Klebsiella pneumoniae (KPN)multilocus sequence typing (MLST) database to determine the sequence types (STs) of the strains. RESULTS: The age of the 40 ICU CRKP-infected patients was (69.03±17.82) years old, with various underlying diseases, and there were 20 patients with improved clinical outcome and 20 patients with death. The isolated strains primarily originated from mid-stream urine and bronchoalveolar lavage fluid. Whole-genome sequencing results revealed that the strains predominantly carried blaKPC-1 (29 strains, 72.5%) and blaNDM-1 (6 strains, 15.0%), with 5 strains carrying both blaKPC-1 and blaNDM-1. Various virulence genes were detected, among which the carriage rates of genes such as entA, entB, entE, entS, fepA, fepC, fepG, yag/ecp, and ompA reached 100%, while the carriage rates of genes such as entD, fimB, iroB, iroD, fes,and pla were low. The CRKP strains isolated from ICU were predominantly ST11 (27 cases, 67.5%), with KL64 being the main capsular serotype (29 cases, 72.5%). A total of 23 ST11-KL64 CRKP strains were detected, accounting for 57.5%. CONCLUSIONS: The main type of ICU CRKP is ST11-KL64, carrying various virulence genes, primarily those related to iron absorption. Furthermore, blaKPC has shifted from blaKPC-2 to blaKPC-1. Therefore, close monitoring of the molecular epidemiological changes of CRKP is necessary, and strict control measures should be implemented to effectively curb the occurrence of CRKP infections.

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.

A Case of Klebsiella pneumoniae infection.

INTRODUCTION: In recent years, hypervirulent Klebsiella pneumoniae (hvKp) has attracted increasing attention. It usually causes liver abscesses, which spread through the bloodstream to other parts such as the eyes, brain, lungs. 5.5% of all paroxysmal sympathetic hyperactivity syndrome are associated with infection, hydrocephalus, brain tumors, and some unknown causes. Younger patients with focal lesions of the brain parenchyma are at higher risk of paroxysmal sympathetic hyperactivity (PSH). CASE PRESENTATION: This case report details the clinical features of Klebsiella pneumoniae diagnosed in a healthy individual. In addition to liver abscesses, bacteremia, and hyperglycemia, there are also brain abscesses, hernias, and postoperative paroxysmal sympathetic hyperactivity, an unexpected association between diseases or symptoms. The patient stabilized after comprehensive treatment, including early drainage of abscesses, rapid pathogen diagnosis, and timely and appropriate antibiotics. At a two-month follow-up, no signs of infection recurrence were noted, and the patient regained neurological function and could participate in regular physical activity. DISCUSSION: Symptoms of Klebsiella pneumoniae infection usually appear gradually, and misdiagnosis is common. When young patients suddenly develop high fever and abscess at a particular site, Klebsiella pneumoniae infection should be considered routine. Paroxysmal sympathetic hyperactivity syndrome caused by infection is rare, but a clinical score (PSH assessment measure, PSH-AM score) should be performed when clinical features appear. Early diagnosis and treatment can improve the prognosis.

Clinical and laboratory insights into the threat of hypervirulent Klebsiella pneumoniae.

Hypervirulent Klebsiella pneumoniae (hvKP) typically causes severe invasive infections affecting multiple sites in healthy individuals. In the past, hvKP was characterized by a hypermucoviscosity phenotype, susceptibility to antimicrobial agents, and its tendency to cause invasive infections in healthy individuals within the community. However, there has been an alarming increase in reports of multidrug-resistant hvKP, particularly carbapenem-resistant strains, causing nosocomial infections in critically ill or immunocompromised patients. This presents a significant challenge for clinical treatment. Early identification of hvKP is crucial for timely infection control. Notably, identifying hvKP has become confusing due to its prevalence in nosocomial settings and the limited predictive specificity of the hypermucoviscosity phenotype. Novel virulence predictors for hvKP have been discovered through animal models or machine learning algorithms, while standardization of identification criteria is still necessary. Timely source control and antibiotic therapy have been widely employed for the treatment of hvKP infections. Additionally, phage therapy is a promising alternative approach due to escalating antibiotic resistance. In summary, this narrative review highlights the latest research progress in the development, virulence factors, identification, epidemiology of hvKP, and treatment options available for hvKP infection.