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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.

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Dictyostelium , Citometría de Flujo , Klebsiella pneumoniae , Dictyostelium/microbiología , Citometría de Flujo/métodos , Klebsiella pneumoniae/patogenicidad , Fagocitosis , Virulencia , Interacciones Huésped-Patógeno , Microscopía/métodos

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Studying host-pathogen interactions is essential for understanding infectious diseases and developing possible treatments, especially for priority pathogens with increased virulence and antibiotic resistance, such as Klebsiella pneumoniae. Over time, this subject has been approached from different perspectives, often using mammal host models and invasive endpoint measurements (e.g., sacrifice and organ extraction). However, taking advantage of technological advances, it is now possible to follow the infective process by noninvasive visualization in real time, using optically amenable surrogate hosts. In this line, this chapter describes a live-cell imaging approach to monitor the interaction of K. pneumoniae and potentially other bacterial pathogens with zebrafish larvae in vivo. This methodology is based on the microinjection of fluorescent bacteria into the otic vesicle, followed by time-lapse observation by automated fluorescence microscopy with environmental control, monitoring the dynamics of immune cell recruitment, bacterial load, and larvae survival.

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Interacciones Huésped-Patógeno , Infecciones por Klebsiella , Klebsiella pneumoniae , Larva , Microinyecciones , Microscopía Fluorescente , Pez Cebra , Animales , Pez Cebra/microbiología , Klebsiella pneumoniae/inmunología , Microinyecciones/métodos , Larva/microbiología , Larva/inmunología , Microscopía Fluorescente/métodos , Interacciones Huésped-Patógeno/inmunología , Infecciones por Klebsiella/microbiología , Infecciones por Klebsiella/inmunología , Modelos Animales de Enfermedad

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Background Antibiotic resistance is a global health concern, yet research on the identities and proportions of antibiotic-resistant bacteria in the oral microflora of healthy children remains limited. These resistant bacteria could play a role in various conditions, such as dental infections, periodontitis, or systemic infections following dental procedures, particularly in immunocompromised individuals. This study aimed to assess the prevalence, proportions, and identities of antibiotic-resistant bacteria in the oral microflora of healthy children. Methodology This cross-sectional study, conducted from January to December 2023 across three tertiary care hospitals in Peshawar, Pakistan, involved 364 healthy children aged 7-13 years. Data on antibiotic use were collected via structured interviews and medical records, detailing specific antibiotics (e.g., amoxicillin, ceftriaxone, azithromycin), including dosage, duration, and reason for use. Oral swabs were taken from various sites in the oral cavity using sterile techniques and analyzed using microbiological culture methods and polymerase chain reaction to identify antibiotic-resistant bacteria. Statistical analysis was performed with SPSS version 27.0, utilizing chi-square tests to explore associations between demographic factors and resistance patterns, with significance set at p < 0.05. Results Significant proportions of antibiotic use were found among the participants: 172 (47.25%) received penicillins, 91 (25.00%) cephalosporins, and 101 (27.75%) macrolides, with associated side effects including gastrointestinal disorders and allergic responses. Age and gender differences were observed in antibiotic resistance proportions: among 7-9-year-olds, resistance to penicillins was 44 (18.33%), to cephalosporins 29 (12.08%), and to macrolides 33 (13.75%). In contrast, among 10-13-year-olds, these proportions increased to 55 (22.92%), 36 (15.00%), and 43 (17.92%), respectively. Male participants exhibited resistance to cephalosporins at a proportion of 24 (10.00%), to macrolides 45 (18.75%), and to penicillin 46 (19.17%), while female participants showed proportions of 53 (22.08%), 41 (17.08%), and 31 (12.92%), respectively. Among oral swab locations, the buccal mucosa had the highest resistance proportions: 35 (14.58%) to penicillins, 27 (11.25%) to cephalosporins, and 33 (13.92%) to macrolides. Specific bacterial species showed distinct resistance patterns, with notable proportions observed in Staphylococcus aureus (n=18; 50.00%, n=12; 33.33%, and n=6; 16.67%, respectively), Escherichia coli (n=16; 40.00%, n=10; 25.00%, and n=14; 35.00%, respectively), Enterococcus faecalis (n=29; 45.31%, n=14; 21.88%, and n=21; 32.81%, respectively), and Klebsiella pneumoniae (n=19; 36.54%, n=13; 25.00%, and n=20; 38.46%, respectively). Conclusions This study focused on healthy children aged 7-13 years in Peshawar, Pakistan, to assess the prevalence and types of antibiotic-resistant bacteria in their oral microbiota. The findings highlight resistance patterns by age, gender, and bacterial species. However, the regional context may limit the generalizability of these results. Differences in local antibiotic use, healthcare practices, and environmental factors could influence resistance patterns in other regions. Future research should expand to include diverse geographic locations to evaluate the broader applicability of these findings and identify region-specific factors affecting antibiotic resistance.

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Background: Multidrug-resistant Klebsiella pneumoniae (MDR-KP) poses a significant global health threat due to its involvement in severe infections and high mortality rates. The emergence of MDR strains necessitates the exploration of alternative therapeutic strategies. Methods: K. pneumoniae isolates were obtained from human and animal sources. Antibacterial susceptibility testing was performed, followed by the evaluation of essential oil activity through inhibition zone, MIC, and MBC determinations. Checkerboard assays were conducted to assess synergistic effects with amikacin. Gene expression analysis and transmission electron microscopy were employed to elucidate the mechanisms of action. Molecular docking studies were performed to identify potential binding targets of bioactive compounds. Results: Klebsiella pneumoniae was isolated from 25 of the100 samples examined, representing a prevalence rate of 25%. All isolates were found to be multidrug-resistant. Tea tree and thyme essential oils exhibited potent antibacterial activity and synergistic effects with amikacin. Notably, these combinations significantly downregulated the expression of key capsule virulence genes (wcaG, rmpA, magA, uge, and wabG), suggesting a novel mechanism for enhancing amikacin efficacy. Transmission electron microscopy revealed disrupted cell integrity in MDR-KP cells treated with the combinations. Molecular docking analysis identified Terpinen-4-ol, Farnesol, 1,4-Dihydroxy-p-menth-2-ene, and 7-Oxabicyclo [4.1.0] heptane as potential bioactive compounds responsible for the observed effects. Conclusion: By effectively combating MDR-KP, this research holds promise for reducing antibiotic resistance, improving treatment outcomes, and ultimately enhancing potential care.

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Objectives: Our objective was to establish a rapid and precise method for detecting hypervirulent Klebsiella pneumoniae (hvKP) by utilizing a duplex real-time multienzyme isothermal rapid amplification (real-time MIRA) and to evaluate its performance in clinical spiked blood specimens. Methods: The research comprised two phases: an initial pilot study to establish the methodology and a clinical validation study to assess its effectiveness. In the pilot phase, we designed specific primers and probes targeting the hvKP pg344 and incA genes and subsequently developed a duplex real-time MIRA assay to evaluate its detection limits, specificity, and efficiency. In the clinical validation phase, we analyzed thirty-three spiked blood specimens using the duplex real-time MIRA assay. Results: The duplex real-time MIRA assay demonstrated no cross-reactivity with other strains. Sensitivity experiments confirmed that the assay had a detection limit as low as 8 × 102 CFU per reaction for hvKP. The analysis of clinical spiked blood specimens indicated that the sensitivity and specificity of the duplex real-time MIRA assay were on par with those of duplex real-time PCR. Conclusions: These findings confirm that the duplex real-time MIRA assay is a fast, straightforward, and dependable method for detecting hvKP.

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Objective: Antimicrobial resistance is an emerging problem and multi-drug resistant (MDR) Klebsiella pneumoniae (K. pneumoniae) represents an enormous risk of failing therapy in hospital-acquired pneumonia. The current study aimed to determine the immunomodulatory effect of topical flagellin in addition to antibiotic treatment during respiratory infection evoked by hypervirulent antibiotic-susceptible and antibiotic-resistant K. pneumoniae in mice. Methods: C57BL6 mice were inoculated intranasally with hypervirulent K. pneumoniae (K2:O1) which was either antibiotic-susceptible or multi-drug resistant. Six hours after infection, mice were treated with antibiotics intraperitoneally and flagellin or vehicle intranasally. Mice were sacrificed 24 hours after infection. Samples were analyzed for bacterial loads and for inflammatory and coagulation markers. Results: Flagellin therapy induced neutrophil influx in the lung during antibiotic-treated pneumonia evoked by either antibiotic-susceptible or -resistant K. pneumoniae. The pulmonary neutrophil response was matched by elevated levels of neutrophil-attracting chemokines, neutrophil degranulation products, and local coagulation activation. The combined therapy of effective antibiotics and flagellin did not impact K. pneumoniae outgrowth in the lung, but decreased bacterial counts in distant organs. Neutrophil depletion abrogated the flagellin-mediated effect on bacterial dissemination and local coagulation responses. Conclusion: Topical flagellin administration as an adjunctive to antibiotic treatment augments neutrophil responses during pneumonia evoked by MDR-K. pneumoniae, thereby reducing bacterial dissemination to distant organs.

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Farmacorresistencia Bacteriana Múltiple , Flagelina , Infecciones por Klebsiella , Klebsiella pneumoniae , Ratones Endogámicos C57BL , Neutrófilos , Animales , Flagelina/inmunología , Flagelina/administración & dosificación , Neutrófilos/inmunología , Neutrófilos/efectos de los fármacos , Infecciones por Klebsiella/inmunología , Infecciones por Klebsiella/tratamiento farmacológico , Infecciones por Klebsiella/microbiología , Ratones , Pulmón/inmunología , Pulmón/microbiología , Antibacterianos/administración & dosificación , Antibacterianos/farmacología , Administración Tópica , Modelos Animales de Enfermedad , Infiltración Neutrófila/efectos de los fármacos , Carga Bacteriana/efectos de los fármacos

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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.

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Infecciones por Klebsiella , Klebsiella pneumoniae , Factores de Virulencia , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/patogenicidad , Factores de Virulencia/genética , Virulencia/genética , Animales , Infecciones por Klebsiella/microbiología , Humanos , Secuenciación Completa del Genoma/métodos , Genoma Bacteriano , Mariposas Nocturnas/microbiología , Larva/microbiología , Proteínas Bacterianas/genética

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There have been increasing reports of Klebsiella pneumoniae resistant to ß-lactam antibiotics. This study aimed to determine the prevalence of some selected carbapenemase genes among clinical isolates of Klebsiella pneumoniae recovered from patients attending a private tertiary hospital in Southwestern Nigeria. The study was conducted over two months (February-March 2024). A total of 50 clinical isolates of Klebsiella pneumoniae from different clinical specimens were obtained from the Medical Microbiology Department, Babcock University Teaching Hospital (BUTH). The clinical isolates were then characterized using standard microbiological procedures and were tested for susceptibility to meropenem and other classes of antibiotics according to Clinical and Laboratory Standards Institute (CLSI) guidelines. Polymerase Chain Reaction (PCR) detection for OXA-48 and NDM-1 carbapenemase genes was performed on the 50 clinical isolates. PCR analysis showed that 9 (18%) clinical isolates were positive for the OXA-48 gene, 22 (44%) were positive for the NDM-1 gene, 4 (8%) possessed both the OXA-48 and NDM-1 genes, and 23 (46%) possessed neither the OXA-48 nor NDM-1 genes. Antibiotic Susceptibility Testing (AST) revealed that all the clinical isolates were resistant to meropenem. In conclusion, this study demonstrates the presence of OXA-48 and NDM-1 genes in clinical isolates of Klebsiella pneumoniae recovered from patients attending a private tertiary hospital in Southwestern Nigeria, highlighting the role of ESBL (extended-spectrum beta-lactamase) as a major resistance mechanism alongside other mechanisms. Population-based surveillance programs should be implemented to monitor the prevalence and epidemiology of Klebsiella pneumoniae infections at the community level, facilitating early detection of outbreaks and identification of emerging antimicrobial resistance patterns. CORE TIP: This study highlights the significant prevalence of NDM-1 and OXA-48 carbapenemase genes among Klebsiella pneumoniae clinical isolates in a private tertiary hospital in Southwestern Nigeria, with 44% and 18% of isolates harboring these genes, respectively. Notably, 46% of isolates were resistant to carbapenems despite lacking these genes, suggesting alternative resistance mechanisms. The findings underscore the urgent need for enhanced surveillance, infection control measures, and antibiotic stewardship programs to combat the spread of multidrug-resistant Klebsiella pneumoniae in healthcare settings.

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Antibacterianos , Proteínas Bacterianas , Infecciones por Klebsiella , Klebsiella pneumoniae , Pruebas de Sensibilidad Microbiana , Centros de Atención Terciaria , beta-Lactamasas , beta-Lactamasas/genética , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/aislamiento & purificación , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/enzimología , Humanos , Centros de Atención Terciaria/estadística & datos numéricos , Infecciones por Klebsiella/microbiología , Infecciones por Klebsiella/epidemiología , Nigeria/epidemiología , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Masculino , Femenino , Persona de Mediana Edad , Adulto , Anciano , Adulto Joven

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BACKGROUND: Extended-spectrum ß-lactamase -producing Enterobacterales (ESBL-E) are important zoonotic pathogens that can cause serious clinical infections, also in horses. Preventing the spread of ESBL-E, especially in the equine hospital environment, is key to reducing the number of difficult-to-treat infections. Estimating the local prevalence of ESBL-E in horses is crucial to establish targeted infection control programs at equine hospitals. We conducted a prevalence and risk factor study in equine patients on admission to an equine teaching hospital in Finland through a rectal ESBL-E screening specimen of the horse and a questionnaire. RESULTS: The prevalence of ESBL-E in admitted horses was 3% (5/161, 95% CI 1-7%); none of the tested factors remained statistically significant in multivariate analysis, although antimicrobial treatment within three months was borderline significant (p = 0.052). Extended-spectrum ß-lactamase -producing Klebsiella pneumoniae ST6179:CTX-M-15 was detected in three horses using whole-genome sequencing, which in combination with patient records suggested nosocomial transmission. Escherichia coli isolates were ST1250:CTX-M-1 (n = 1), ST1079:CTX-M-1 (n = 1), and ST1245:CTX-M-14 (n = 1). Multiple virulence genes were detected in the ESBL-E isolates. In the ESBL-E positive horses enrolled in a one-year follow-up study, ESBL-E were unlikely to be isolated in rectal screening specimens after the initial positive specimen. CONCLUSIONS: The prevalence of ESBL-E in horses visiting a veterinary teaching hospital in Finland is low, indicating an overall low prevalence estimate in the country's equine population. No statistically significant risk factors were identified, likely due to the low number of cases. The duration of ESBL-E carriage is likely to be very short in horses.

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Infecciones por Enterobacteriaceae , Enfermedades de los Caballos , Hospitales Veterinarios , beta-Lactamasas , Animales , Caballos , Enfermedades de los Caballos/microbiología , Enfermedades de los Caballos/epidemiología , beta-Lactamasas/metabolismo , beta-Lactamasas/genética , Prevalencia , Factores de Riesgo , Finlandia/epidemiología , Infecciones por Enterobacteriaceae/veterinaria , Infecciones por Enterobacteriaceae/epidemiología , Infecciones por Enterobacteriaceae/microbiología , Masculino , Femenino , Klebsiella pneumoniae/enzimología , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/aislamiento & purificación , Infección Hospitalaria/epidemiología , Infección Hospitalaria/veterinaria , Infección Hospitalaria/microbiología , Escherichia coli/enzimología , Escherichia coli/aislamiento & purificación , Enterobacteriaceae/enzimología , Enterobacteriaceae/aislamiento & purificación , Enterobacteriaceae/efectos de los fármacos , Antibacterianos/farmacología

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Klebsiella pneumoniae (Kp) is increasingly recognized as a reservoir for a range of antibiotic resistance genes and a pathogen that frequently causes severe infections in both hospital and community settings. In this study, we have identified a novel mechanism of conjugative transfer of a non-conjugative virulence plasmid through the formation of a fusion plasmid between the virulence plasmid and a novel 59,162 bp IncN- plasmid. This plasmid was found to be a multidrug-resistance (MDR) plasmid and carried a T4SS cluster, which greatly facilitated the efficient horizontal transfer of the fusion plasmid between Kp strains. The fused virulence plasmid conferred the resistance of serum killing and macrophage phagocytosis to the transconjugants. Importantly, this plasmid was shown to be essential for Kp virulence in a mouse model. Mechanistic analysis revealed that the virulence factors encoded by this virulence plasmid contributed to resistance to in vivo clearance and induced a high level of proinflammatory cytokine IL-1ß, which acts as an inducer for more neutrophil recruitment. The transmission of the fusion plasmid in Kp has the potential to convert it into both MDR and hypervirulent Kp, accelerating its evolution, and posing a serious threat to human health. The findings of this study provide new insights into the rapid evolution of MDR and hypervirulent Kp in recent years.

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Antibiotic resistance poses a considerable worldwide concern, particularly in clinical environments where drug-resistant Gram-negative bacteria like Klebsiella pneumoniae present a major challenge. The objective of this research was to investigate the mechanisms by which isobavachalcone (IBC) restores the sensitivity of K. pneumoniae to colistin in vitro and to validate the synergistic therapeutic effect in vivo. The results indicate that the combined administration of colistin and IBC exhibits a potent antibacterial effect both in vitro and in vivo. The in vitro concurrent administration of colistin and IBC resulted in increased membrane permeability, compromised cell integrity, diminished membrane fluidity, and disrupted membrane homeostasis. Additionally, this combination reduced biofilm production, inhibited the synthesis of the AI-2 factor, altered membrane potential, and affected levels of reactive oxygen species and adenosine triphosphate synthesis, ultimately leading to bacterial death. In vivo experiments on Galleria mellonella and mice demonstrated that the co-administration of colistin and IBC increased the survival rate and significantly reduced pathological damage compared to colistin alone. These results suggested that IBC effectively restores the sensitivity of colistin by inducing physical disruption of bacterial membranes and oxidative stress. The combination therapy of colistin and IBC presents a viable and safe strategy to combat drug-resistant K. pneumoniae-associated infections.

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Background: The emergence of coronavirus disease in 2019 (COVID-19) appears to be having an impact on antibiotic resistance patterns. Specific circumstances during the COVID-19 era may have played a role in the spread of antimicrobial resistance (AMR). This study aimed to look at the changes in AMR patterns of Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii at Al-Zahra Hospital. Materials and Methods: From March 2021 to January 2023, 3651 clinical samples were collected from patients hospitalized at Isfahan's Al-Zahra Hospital. The Clinical and Laboratory Standards Institute recommended procedures for detecting gram-negative bacteria and assessing antibiotic susceptibility were used. We divided the information into three years. Results: Highest resistance rates were seen in A. baumannii to Ciprofloxacin (98.0%) and Ampicillin-Sulbactam (97.0%). For P. aeruginosa the resistance rate for Ceftazidime (36.1), Levofloxacin (37.8), and Meropenem (47.1) dropped seriously in 2022. Conclusion: During the second year of the pandemic in central Iran, all three species studied showed rising rates of AMR. This can be attributable to two peaks within Iran on May 6, 2021 and August 27, 2021. The results of this study show that P. aeruginosa, K. pneumoniae, and A. baumannii bacteria in central Iran have a higher level of antibiotic resistance than previously studied strains before the pandemic.

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Phage-antibiotic synergy (PAS) represents a superior treatment strategy for pathogen infections with less probability of resistance development. Here, we aim to understand the molecular mechanism by which PAS suppresses resistance in terms of population evolution. A novel hypervirulent Klebsiella pneumoniae (KP) phage H5 was genetically and structurally characterized. The combination of H5 and ceftazidime (CAZ) showed a robust synergistic effect in suppressing resistance emergence. Single-cell Raman analysis showed that the phage-CAZ combination suppressed bacterial metabolic activities, contrasting with the upregulation observed with phage alone. The altered population evolutionary trajectory was found to be responsible for the contrasting metabolic activities under different selective pressures, resulting in pleiotropic effects. A pre-existing wcaJ point mutation (wcaJG949A) was exclusively selected by H5, conferring a fitness advantage and up-regulated activity of carbohydrate metabolism, but also causing a trade-off between phage resistance and collateral sensitivity to CAZ. The wcaJ point mutation was counter-selected by H5-CAZ, inducing various mutations in galU that imposed evolutionary disadvantages with higher fitness costs, and suppressed carbohydrate metabolic activity. H5 and H5-CAZ treatments resulted in opposite effects on the transcriptional activity of the phosphotransferase system and the ascorbate and aldarate metabolism pathway, suggesting potential targets for phage resistance suppression. Our study reveals a novel mechanism of resistance suppression by PAS, highlighting how the complexity of bacterial adaptation to selective pressures drives treatment outcomes. IMPORTANCE: Phage-antibiotic synergy (PAS) has been recently proposed as a superior strategy for the treatment of multidrug-resistant pathogens to effectively reduce bacterial load and slow down both phage and antibiotic resistance. However, the underlying mechanisms of resistance suppression by PAS have been poorly and rarely been studied. In this study, we tried to understand how PAS suppresses the emergence of resistance using a hypervirulent Klebsiella pneumoniae (KP) strain and a novel phage H5 in combination with ceftazidime (CAZ) as a model. Our study reveals a novel mechanism by which PAS drives altered evolutionary trajectory of bacterial populations, leading to suppressed emergence of resistance. The findings advance our understanding of how PAS suppresses the emergence of resistance, and are imperative for optimizing the efficacy of phage-antibiotic therapy to further improve clinical outcomes.

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The successful evolution of KPC-2 in bacteria has limited the clinical practice of carbapenems. This dilemma deteriorated the prognosis of associated infections and hence attracted increasing attention from researchers to explore alternative therapeutic options. Here, the enzyme inhibition assay was first performed to screen for a potent KPC-2 inhibitor. The synergistic effect of the candidate with carbapenems was further confirmed by checkboard minimum inhibitory concentration (MIC) assay, time-killing assay, disk diffusion method, and live/dead bacteria staining analysis. The mechanisms by which the candidate acts were subsequently explored through molecular dynamics (MD) simulations, etc. Our study found that Ginkgolic Acid (C13:0) (GA) exhibited effective KPC-2 inhibitory activity in both laboratory strain and clinical strain containing KPC-2. It could potentiate the killing effect of carbapenems on KPC-2-positive Klebsiella pnenmoniae (K. pnenmoniae). Further explorations revealed that GA could competitively bind to the active pocket of KPC-2 with meropenem (MEM) via residues Trp104, Gly235, and Leu166. The secondary structure and functional groups of KPC-2 were subsequently altered, which may be the main mechanism by which GA exerted its KPC-2 inhibitory effect. In addition, GA was also found to synergize with MEM to disrupt membrane integrity and increase membrane permeability, which may be another mechanism by which GA reinforced the bactericidal ability of carbapenems. Our study indicated that GA was a significant KPC-2 inhibitor that could prolong the lifespan of carbapenems and improve the prognosis of patients.

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This study investigated the molecular characteristics of urinary carbapenemase-producing Klebsiella pneumoniae isolates (n = 194) in Gauteng, South Africa, using simple, cost-effective PCR methodologies. Extensively drug resistant (XDR) ST307 with blaOXA-181 on IncX3 plasmids was endemic in Gauteng community hospitals leaving limited options for treating in- and outpatient urinary tract infections. High-level ceftazidime/avibactam resistance was detected among isolates harbouring blaOXA-48-like including blaOXA-181. These findings highlighted the need for genomic methodologies suitable for lower- and middle-income countries to track XDR clones and plasmids in community hospitals. Such results will aid with treatment and stewardship strategies.

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BACKGROUND: Klebsiella pneumoniae is infamous for hospital-acquired infections and sepsis, which have also been linked to Alzheimer disease (AD)-related neuroinflammatory and neurodegenerative impairment. However, its causative and mechanistic role in AD pathology remains unstudied. METHODS: A preclinical model of K. pneumoniae enteric infection and colonization is developed in an AD model (3xTg-AD mice) to investigate whether and how K. pneumoniae pathogenesis exacerbates neuropathogenesis via the gut-blood-brain axis. RESULTS: K. pneumoniae, particularly under antibiotic-induced dysbiosis, was able to translocate from the gut to the bloodstream by penetrating the gut epithelial barrier. Subsequently, K. pneumoniae infiltrated the brain by breaching the blood-brain barrier. Significant neuroinflammatory phenotype was observed in mice with K. pneumoniae brain infection. K. pneumoniae-infected mice also exhibited impaired neurobehavioral function and elevated total tau levels in the brain. Metagenomic analyses revealed an inverse correlation of K. pneumoniae with gut biome diversity and commensal bacteria, highlighting how antibiotic-induced dysbiosis triggers an enteroseptic "pathobiome" signature implicated in gut-brain perturbations. CONCLUSIONS: The findings demonstrate how infectious agents following hospital-acquired infections and consequent antibiotic regimen may induce gut dysbiosis and pathobiome and increase the risk of sepsis, thereby increasing the predisposition to neuroinflammatory and neurobehavioral impairments via breaching the gut-blood-brain barrier.

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Enfermedad de Alzheimer , Barrera Hematoencefálica , Modelos Animales de Enfermedad , Disbiosis , Microbioma Gastrointestinal , Infecciones por Klebsiella , Klebsiella pneumoniae , Ratones Transgénicos , Enfermedades Neuroinflamatorias , Animales , Ratones , Disbiosis/microbiología , Disbiosis/inducido químicamente , Enfermedad de Alzheimer/microbiología , Enfermedades Neuroinflamatorias/microbiología , Microbioma Gastrointestinal/efectos de los fármacos , Infecciones por Klebsiella/microbiología , Barrera Hematoencefálica/microbiología , Encéfalo/patología , Encéfalo/microbiología , Antibacterianos/farmacología , Eje Cerebro-Intestino , Masculino , Humanos

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INTRODUCTION: Chronic obstructive pulmonary disease is a lung condition characterized by chronic respiratory symptoms (breathlessness, cough, and expectoration). In the advanced stages, patients often report to the Accident & Emergency department due to worsening of symptoms. Because of the repeated exposure to corticosteroids during the management of exacerbations, these patients are susceptible to super additional infections. Pulmonary aspergillosis can be divided into three main categories: invasive pulmonary aspergillosis, allergic bronchopulmonary aspergillosis and chronic pulmonary aspergillosis. Aspergillus overlap syndrome is defined as the presence of more than one form of Aspergillus in a single patient. However, coinfection with Klebsiella and pulmonary aspergillosis overlap syndrome is rare and poses a treatment challenge. As per a pub med search, no such case report has been reported in a case of chronic obstructive pulmonary disease. CASE REPORT: We report the case of a 66-year-old male, Punjabi Hindu by ethnicity, who was a reformed smoker with a known case of COPD. He presented with a history of breathlessness (mMRC grade 4) associated with cough with expectoration and wheezing for 15 days and intermittent episodes of hemoptysis for more than 6 months. The examination revealed tachypnea and wheezing throughout the lung fields. He was initially managed with parenteral steroids and frequent nebulization with bronchodilators. On day 5 of hospitalization, the patient experienced worsening of symptoms and cardiac arrest; he was intubated and return of spontaneous circulation was achieved within 5 minutes of cardio pulmonary resuscitation. Tracheal aspirate and culture revealed Aspergillus fumigatus and Klebsiella pneumoniae respectively. He underwent chest CT, which showed features suggestive of allergic bronchopulmonary aspergillosis and invasive pulmonary aspergillosis. He was found to have elevated ß-D-glucan, galactomannan, and aspergillus IgE and IgG. Severe pneumonia and pulmonary Aspergillus overlap syndrome were managed with antibiotics, steroids, and antifungals. Over the next 15-20 days, his general condition improved. He was discharged after 45 days of hospitalization and continued on oral corticosteroids, antifungals, and inhaled bronchodilators. CONCLUSION: Coinfection with bacteria and fungi worsens the outcome. Clinicians should be aware of the polymicrobial manifestations and various drug interactions involved. Timely diagnosis aids in better management strategies and improved patient outcomes.

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Antifúngicos , Coinfección , Infecciones por Klebsiella , Klebsiella pneumoniae , Enfermedad Pulmonar Obstructiva Crónica , Humanos , Masculino , Enfermedad Pulmonar Obstructiva Crónica/complicaciones , Anciano , Infecciones por Klebsiella/complicaciones , Infecciones por Klebsiella/tratamiento farmacológico , Infecciones por Klebsiella/diagnóstico , Klebsiella pneumoniae/aislamiento & purificación , Antifúngicos/uso terapéutico , Paro Cardíaco/etiología , Aspergilosis Pulmonar/complicaciones , Aspergilosis Pulmonar/tratamiento farmacológico , Aspergilosis Pulmonar/diagnóstico , Antibacterianos/uso terapéutico

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Spread of hypervirulent and multi-drug resistant Klebsiella pneumoniae in raw milk is public health concern due to its potential impact on food safety and public health. Therefore, this study investigated antibiotic susceptibility test (AST), antibiotic resistance genes (ARGs), mutations conferring ARGs, virulence factor and plasmid replicons to check prevalence of fosfomycin resistant MDR K. pneumoniae isolated from raw milk samples collected from Saurashtra region of Gujarat, India. K. pneumoniae isolated from raw milk and subjected to disk diffusion assay. From that, MDR along with fosfomycin resistant isolates were analysed for multi locus sequence typing, presence of ARGs, mutations conferring resistance, virulence factors and plasmid replicon types by using its whole genome sequence. Results shows that, among 32 K. pneumoniae, 8 were phenotypically resistant to fosfomycin. As per WGS analysis, 8 MDR isolates were assigned into different sequence types such as ST3321, ST37, ST2715, ST1087, ST3157, ST299 and ST29. Among that, ST37 is well recognized MDR high risk clone reported worldwide and first time reported from raw milk of Saurashtra region of Gujarat, India. ARGs responsible for resistance to fosfomycin (fosA) were found in all 8 isolates. Other ARGs such as blaSHV, kdeA, OqxA, OqxB, dfrA1, sul1, qnrB4, aadA2 and ere(A) were also detected. High diversity of virulence factors was also identified by detection of genes encoding virulence factors related to iron uptake such as entE, fepD, entA, entB, Irp2, fepG, ybtU, ybtP, fepC, ybtA, ybtE, fepB, ybtS, fyuA, ybtQ, ybtT, ybtX, Irp1, adherence such as yagZ/ecpA, yagV/ecpE, yagX/ecpC, yagV/ecpE, ykgK/ecpR and invasion such as fimA, pla, fimC, fimH, fimB, fimE were detected in eight genomes. Mutations in murA, uhpT and glpT conferring a fosfomycin resistance were also present in genomes of 8 K. pneumoniae. IncF was the most common plasmid replicon type detected in all 8 genomes. The study reports high diversity of virulent and multidrug resistant K. pneumoniae in raw milk. Hence, genomic surveillance plans are urgently required for food borne pathogens.

RESUMEN

The rise of carbapenem-resistant Klebsiella pneumoniae (CRKP) presents a significant global challenge in clinical and healthcare settings, severely limiting treatment options. This study aimed to utilize a bacteriophage as an alternative therapy against carbapenem-resistant K. pneumoniae. A novel lytic N4-like Klebsiella phage, vB_kpnP_KPYAP-1 (KPYAP-1), was isolated from sewage. It demonstrated efficacy against the K62 serotype polysaccharide capsule of blaOXA-48-producing K. pneumoniae. KPYAP-1 forms small, clear plaques, has a latent period of 20 min, and reaches a growth plateau at 35 min, with a burst size of 473 plaque-forming units (PFUs) per infected cell. Phylogenetic analysis places KPYAP-1 in the Schitoviridae family, Enquatrovirinae subfamily, and Kaypoctavirus genus. KPYAP-1 employs an N4-like direct terminal repeat mechanism for genome packaging and encodes a large virion-encapsulated RNA polymerase. It lacks integrase or repressor genes, antibiotic resistance genes, bacterial virulence factors, and toxins, ensuring its safety for therapeutic use. Comparative genome analysis revealed that the KPYAP-1 genome is most similar to the KP8 genome, yet differs in tail fiber protein, indicating variations in host recognition. In a zebrafish infection model, KPYAP-1 significantly improved the survival rate of infected fish by 92% at a multiplicity of infection (MOI) of 10, demonstrating its potential for in vivo treatment. These results highlight KPYAP-1 as a promising candidate for developing phage-based therapies targeting carbapenemase-producing K. pneumoniae.

Asunto(s)

Bacteriófagos , Infecciones por Klebsiella , Klebsiella pneumoniae , Pez Cebra , Klebsiella pneumoniae/virología , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/genética , Animales , Bacteriófagos/genética , Bacteriófagos/fisiología , Bacteriófagos/aislamiento & purificación , Infecciones por Klebsiella/terapia , Infecciones por Klebsiella/microbiología , Filogenia , Genoma Viral , Carbapenémicos/farmacología , Enterobacteriaceae Resistentes a los Carbapenémicos/genética , Enterobacteriaceae Resistentes a los Carbapenémicos/efectos de los fármacos , Antibacterianos/farmacología , Terapia de Fagos

RESUMEN

Sepsis is an uncontrolled reaction of the body to an infection, and if not effectively treated, it can progress to septic shock, multiple organ failure, and ultimately, death. OBJECTIVE: To determine the resistance profile of Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) strains isolated in sepsis cases diagnosed at the Infectious Diseases Clinic in Craiova, Romania. METHODS: The bacteria responsible for sepsis cases were identified using the Vitek 2 Systems version 06.01, which was then employed to assess their antimicrobial susceptibility (Global CLSI and Phenotypic 2017). RESULTS: We have identified 989 patients diagnosed with bacterial sepsis. Among these, 953 cases were caused by Gram-negative rods, with 415 attributed to E. coli and 278 to K. pneumoniae. High levels of resistance to ampicillin were recorded for E. coli strains isolated in sepsis cases (64.6%); adding sulbactam lowers the level of resistance to 41.8%. Resistance to 3rd generation cephalosporins varied between 7.47 and 14.6% and another 3.41 to 11.1% are dose-dependent susceptibility strains. Resistance to carbapenems (i.e., ertapenem, meropenem) is low-2.18-2.42%. More than 95% of the tested K. pneumoniae strains were resistant to ampicillin and adding sulbactam as a ß-lactamase inhibitor only halves that level. Resistance to 3rd generation cephalosporins varied between 20.7% and 22.5%; resistance levels for K. pneumoniae were notably higher than those for E. coli. Over 95% of K. pneumoniae strains showed resistance to ampicillin, and resistance to 3rd generation cephalosporins varied between 20.7% and 22.5%. Additionally, K. pneumoniae exhibited higher resistance to carbapenems (13.7-19.5%) compared to E. coli (2.18-2.42%). CONCLUSIONS: Antimicrobial resistance levels are generally lower than continental and national data, except for ampicillin and carbapenems (meropenem and ertapenem). K. pneumoniae strains are significantly more resistant than E. coli strains.