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Novel bacteriophages effectively target multidrug-resistant clinical isolates of Klebsiella pneumoniae
Authors: Malavika Binitha Hari
Abstract
Purpose: The global rise in multidrug-resistant (MDR) Klebsiella pneumoniae, a critical ESKAPE pathogen, has outpaced the development of effective antibiotics. Bacteriophage therapy offers a promising alternative, but therapeutic candidates must be carefully selected for broad activity, genetic safety, synergistic cocktail performance, and clinical stability.
Methods: We isolated and characterized six novel lytic phages (vB_Kpn_AM.K1 to vB_Kpn_AM.K6) targeting K. pneumoniae by assessing morphology, host range, growth kinetics, physicochemical stability, and resistance frequency. Genomes were sequenced to confirm absence of lysogeny and virulence genes. Infection dynamics was visualized via fluorescence microscopy. Phage activity was tested across 60 different MDR K. pneumoniae clinical isolates, obtained from diverse sources such as blood, sputum, occult feces, urine etc.
Results: All six isolated phages were identified as novel dsDNA phages belonging to Caudoviricetes, with genome sizes ranging from 111 to 169 Kbp, devoid of virulence and AMR genes and demonstrating strong bacteriolytic activity. Growth kinetics indicated burst sizes varying from 12-148 PFU/infected cell. The phages displayed stability between 4-50°C, pH 4 -10 and sustained complete activity after lyophilization. More significantly, the phages and their cocktail combinations could effectively kill 93% of MDR K. pneumoniae clinical isolates.
Conclusion: These findings establish a panel of genetically safe, phenotypically diverse phages with broad and synergistic activity against MDR K. pneumoniae. The unique replication phenotypes and formulation stability highlight their potential for therapeutic development and deployment in clinical or resource-limited settings.”
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Evaluation of VITEK 2 and three protocols for direct susceptibility testing on Gram-negative bacilli from positive blood culture bottles. Eur J Clin Microbiol Infect Dis (2025).
Authors: Özlem Aydemir
Abstract
Background : Bloodstream infections are associated with high mortality, while early and appropriate antibiotic therapy can significantly reduce fatal outcomes. Direct antibiotic susceptibility testing (AST) from positive blood cultures has the potential to enable earlier administration of antimicrobials compared with routine AST. The aim of the study was to evaluate the performance of three direct AST protocols for Gram-negative bacilli using positive blood culture fluids.
Methods: AST performed on subcultured isolates with VITEK 2 was defined as protocol 1 and used as the reference method. From positive blood culture bottles signaling Gram-negative bacilli, VITEK 2 (protocol 2), modified disk diffusion (protocol 3) and rapid disk diffusion (protocol 4) recommended by EUCAST. The results of protocols 2, 3 and 4 were compared with protocol 1.
Results: The categorical agreements (CA) of protocols 2, 3, and 4 compared with protocol 1 for all strains were 95.9%, 80.7%, and 80.6%, respectively. Very major discrepancy (VMD) rates were 0.5%, 9%, and 1%, major discrepancy (MD) rates were 2.5%, 2.9%, and 6.9%, while minor discrepancy rates were 2.4%, 3.7%, and 3.5%, respectively.
Conclusion: VITEK 2 AST performed directly from blood culture bottles demonstrated the highest categorical agreement and the lowest discrepancy rates across all isolates and antibiotics. This approach is practical in laboratories equipped with mass spectrometry for direct identification and offers a cost-effective option for widespread use in clinical microbiology settings.
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Multicenter evaluation of the QuickMIC® rapid AST system in clinical practice: impact on turnaround time compared to routine AST systems.
Authors: Anna Olsson
Abstract
Increased antibiotic resistance highlights the need for new, rapid antibiotic susceptibility tests to guide therapy, especially in critical disease such as bloodstream infections and sepsis. This study investigates a new ultra-rapid AST system in multiple clinical laboratories, with respect to accuracy, speed and turnaround time in comparison to commonly used AST systems. The QuickMIC system is compared to the commonly used automated AST systems BD Phoenix™ (BD, USA), MicroScan WalkAway plus (Beckman Coulter, USA) and VITEK® 2 (bioMérieux, France) by concurrent testing of incoming positive blood-cultures with Gram-negative bacteria in four clinical laboratories located in the EU and USA, on the basis of agreement of results, time-to-result (TTR, analysis time) and turnaround time (TAT, time from blood culture positivity or blood culture processing to actionable result). A total of 155 patient samples were included, totaling 10 species of Gram-negative bacteria. The overall EA and CA between QuickMIC® GN and each routine AST system was > 95%, while overall bias was within the acceptable range of ± 30%. QuickMIC time-to-result was on average 3 h and 4 min, compared to 9–19 h for the routine systems. The average QuickMIC system turnaround-time ranged from 10 to 11 h 30 min, compared to 22–45 h for the routine systems. The QuickMIC system represents a promising rapid AST technology with potential to reduce time-to-result and overall turnaround-time of clinically actionable AST results compared with the most common routinely used automated AST methods, while maintaining good accuracy and quality of results.
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Artificial intelligence in clinical microbiology: results from the first National survey by the Italian association of clinical microbiologists.
Authors: Alberto Rizzo
Abstract
Methods: A structured 63-question survey was developed by the AI/ML in Microbiology Study Group and distributed to all members of the Italian Association of Clinical Microbiologists between December 2024 and March 2025. Responses were collected anonymously and analyzed using descriptive statistics analysis.
Results: A total of 163 professionals completed the survey. While 25.4% reported current AI/ML usage—primarily in bacteriology and virology—the majority had limited experience with AI technologies. Only 13.6% of respondents had a good understanding of AI/ML concepts, and 2.5% reported having trained data scientists on staff. Major barriers included lack of trained personnel and insufficient infrastructure. Most participants (99.0%) expressed interest in targeted AI training, and 57.5% showed willingness to collaborate on AI-related initiatives. Large language models (LLMs) were seen as promising, especially for data interpretation, despite low adoption rates.
Conclusion: The survey might provide valuable insights to identify priority areas for intervention, guide future training initiatives and develop targeted strategies to promote the adoption of these technologies through a fruitful dialogue with companies and IT professionals.”
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Enhancing Bloodstream Infection Management: A Systematic Review of Rapid Diagnostic Tests and Their Integration Into Antimicrobial Stewardship Programs.
DOI: 10.7759/cureus.92866
Authors: Singla S et al
Abstract
This systematic review evaluates the effect of rapid diagnostic tests (RDTs) on clinical outcomes in patients with bloodstream infections (BSIs), with particular emphasis on their integration into antimicrobial stewardship programs (ASPs). A comprehensive literature search across PubMed, Scopus, Embase, and the Cochrane Central Register of Controlled Trials identified randomized controlled trials (RCTs) and post-hoc analyses that compared RDTs, including multiplex polymerase chain reaction (PCR), matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS), and rapid phenotypic antimicrobial susceptibility testing (AST), with conventional microbiological workflows. Studies were assessed for methodological quality using the Cochrane risk of bias tool. Outcomes of interest included time to effective therapy, antimicrobial optimization, mortality, and hospital length of stay. The included RCTs consistently demonstrated that RDTs, particularly when paired with ASP interventions, accelerated therapeutic decisions and improved antibiotic targeting compared with conventional methods. Importantly, this review is novel in restricting inclusion to RCT evidence and in examining individual diagnostic modalities, offering more granular insights than prior reviews that pooled heterogeneous study types. However, variability in study design, population characteristics, and outcome definitions highlights the need for standardized research approaches and consistent integration with stewardship frameworks. Overall, the findings support the clinical value of RDTs in enhancing BSI management and optimizing antimicrobial therapy, while underscoring that their impact is maximized when embedded within structured ASPs. These results carry important implications for both high-income and resource-limited healthcare settings, reinforcing the role of diagnostic stewardship in translating rapid results into improved patient outcomes.
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Invasive candidiasis in intensive care medicine: shaping the future of diagnosis and therapy.
Authors: Martin Loeches I, et al
Abstract
Background: Invasive candidiasis (IC) remains one of the most challenging infections in critical care, contributing significantly to morbidity, prolonged organ support, and mortality among intensive care unit (ICU) patients. The clinical landscape of IC is evolving, with increasing recognition of Candida non-albicans species and other yeasts formerly classified as Candida spp., alongside emerging multidrug resistance and growing complexity in host immune profiles.
Objectives: This contemporary, multidisciplinary narrative review-authored by intensivists, infectious diseases specialists, clinical microbiologists, and pharmacologists-aims to provide ICU clinicians with practical, up-to-date insights into the diagnosis and management of IC. To maintain clinical focus, the review excludes non-IC fungal infections and non-ICU patient populations. Methods: Relevant literature and expert consensus were critically reviewed to summarize current diagnostic and therapeutic approaches for IC in critically ill patients. Emphasis was placed on pragmatic clinical application, diagnostic limitations, antifungal stewardship, and personalized therapeutic decision-making.
Results: Despite the availability of novel antifungal agents with improved pharmacokinetic properties, treatment success in IC depends equally on timely and accurate diagnosis and individualized, context-aware therapy. Blood cultures continue to demonstrate limited sensitivity (~40%). Non-culture assays, including β-D-glucan and molecular diagnostics, provide faster detection and high negative predictive value but suffer from low positive predictive value and inconsistent adoption in clinical practice. The absence of validated host-derived biomarkers further limits risk stratification, antifungal discontinuation decisions, and personalized care.
Conclusions: Emerging antifungal agents, stewardship strategies, and multidisciplinary care models are essential to improve clinical outcomes and reduce antifungal resistance. This review underscores the need for integrated, team-based diagnostic and therapeutic approaches to close persistent gaps in IC management, ultimately promoting more effective, timely, and individualized care for critically ill patients with Candida spp.
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The potential of the microbiome as a target for prevention and treatment of carbapenem-resistant Enterobacteriaceae infections.
Authors: Zhang L et al
Abstract
Carbapenem-resistant Enterobacteriaceae (CRE) present an escalating threat to global health due to their high transmissibility, limited treatment options, and high mortality rates. The gastrointestinal tract serves as both a major reservoir and a transmission hub for CRE, especially under conditions of antibiotic-induced dysbiosis. This review highlights the growing interest in the gut microbiome as a potential target for preventing and managing CRE infections. Building upon the understanding of CRE pathogenesis, it has been examined how commensal microbiota contribute to colonization resistance through mechanisms such as nutrient competition, spatial niche exclusion, immune modulation, and the production of antimicrobial metabolites. The authors further discuss microbiome-based therapeutic strategies, including probiotic administration, fecal microbiota transplantation (FMT), and supplementation with short-chain fatty acids (SCFAs), that have shown encouraging results in reducing intestinal CRE colonization. In addition, emerging microbiome engineering approaches, particularly CRISPR-Cas9-mediated systems have been explored, which enable the selective elimination of resistant strains while maintaining microbial homeostasis. Current microbiome-based approaches have shown promise in the treatment and prevention of CRE infections, but further research is still needed to clarify their mechanisms, evaluate long-term safety, and determine their effectiveness in different clinical settings. With continued studies and thoughtful integration into existing infection control and antibiotic stewardship practices, these strategies may gradually contribute to a more practical and sustainable way to manage CRE.
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Understanding the impact of antibiotic treatment on the diversity of gut microbiota species.
Authors: Charitos IA, et al
Abstract
Introduction: The community of microorganisms that colonize the intestine plays a vital role in regulating human metabolism and immune system function. According to translational medicine studies, administering antibiotics disrupts the balance of microorganisms in the gastrointestinal system, increasing the risk of multiple diseases. Dysregulation and reduction of the biodiversity of intestinal microbiota increase the risk of developing unhealthy conditions for the host. In this review, the aouthors have tried to offer a current vision regarding the use of antibiotics and the modification of the intestinal microbiota. Areas covered: Research has also shown that, in addition to antibiotics, the composition and balance of the intestinal microbiota depend on diet from the first days of life (breastfeeding or formula) and the mode of delivery (vaginal birth or cesarean section). Having this in mind, an extensive literature search was performed in PubMed, Scopus, Embase, and Web of Science on the relationships between human microbiota and antibiotics. Expert opinion: Effective antimicrobial stewardship programs are urgently needed to reduce the misuse of antibiotics to avoid bacteria becoming more resistant, resulting in the ineffectiveness of antibiotics. Alternatives to antibiotics, e.g., targeted probiotics or bacteriophages, are increasingly considered as a strategy to preserve the intestinal microbiota diversity and maintain a good health status.
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Langya henipavirus (LayV) as an emerging zoonotic disease: a mini-review.
Authors: Shafaati M, et al
Abstract
Henipavirus is one of the genera in the Orthoparamyxovirinae subfamily, which includes several emerging viruses that pose a major public health threat. The predominant members of the virus genus, Hendra and Nipah viruses, are extremely virulent zoonotic viruses that cause neurological and respiratory infections and outbreaks in humans. The recently discovered Langya henipavirus, a new henipavirus phylogenetically related to Mojiang henipavirus (MojV), has been associated with febrile illness in patients from China who are mainly agricultural workers. Active surveillance must be conducted worldwide in an open and collaborative manner to reduce the likelihood of this new virus causing a health crisis. More research is needed to address the remaining difficulties.
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Antimicrobial resistance among Gram-negative agents of bacteraemia in the UK and Ireland: trends from 2001 to 2019.
DOI: 10.1093/jac/dkaf250
Authors: Reynolds R, et al
Abstract
Objectives: The BSAC Bacteraemia Resistance Surveillance Programme collected isolates from UK and Irish hospitals for central testing. Concurrent UKHSA surveillance collected English hospitals’ own susceptibility data. Results were reviewed and compared.
Methods: The BSAC surveillance collected fixed quotas of isolates per site annually from 2001 to 2019. MIC testing was by BSAC agar dilution. Resistance mechanisms were investigated by synergy tests, interpretive reading and PCR. The UKHSA seeks data on all bacteraemia isolates in England.
Results: For Escherichia coli, which now causes >30% of all bacteraemias, there were marked early (2002-06) rises in resistance to cephalosporins, fluoroquinolones and gentamicin, followed by small falls, stabilization, then from around 2015, very slow rises, with similar patterns seen for Klebsiella pneumoniae. Most cephalosporin resistance in these two species involved ESBLs, principally CTX-M types. Both species had frequent co-amoxiclav resistance. Cephalosporin resistance-mostly AmpC-mediated-declined in Enterobacter and Serratia spp., as did fluoroquinolone resistance, likely reflecting reduced use and selection pressure. Proteeae showed few changes; increasing dominance of Proteus mirabilis in the BSAC collection was not confirmed by the UKHSA dataset. Resistance in Pseudomonas aeruginosa was uncommon and showed little temporal change in either dataset. Carbapenemases remained extremely rare in all species. Newer and developmental agents covered many resistance types, but none covered all types.
Conclusions: Except for early rises of cephalosporin, fluoroquinolone and gentamicin resistance in E. coli and K. pneumoniae, there was little evidence for rising resistance and some evidence of declining resistance, notably in species where it predominantly involves AmpC derepression.