Abi Manesh

Introduction. Klebsiella pneumoniae (Kp) is a major cause of nosocomial infections, with its evolving pathotypes including multidrug-resistant, hypervirulent (hvKp) and convergent strains posing significant diagnostic and treatment challenges due to combined antimicrobial resistance and virulence. Gap Statement. While there is a pressing requirement for thorough detection of Kp pathotypes, current assays in resource-limited environments are unable to effectively focus on essential carbapenemase and hypervirulence genes with the necessary reliability and precision. Aim. To develop and validate a multiplex PCR (m-PCR) assay capable of simultaneously detecting Kp isolates including those carrying partial or full virulence markers, alongside antimicrobial resistance. Methodology. In this study, an m-PCR assay was designed and optimized for the simultaneous detection of key biomarkers associated with hypervirulent ( rmpA , rmpA2 , iucA , peg344 and iroB ), carbapenem-resistant ( bla _NDM , bla _OXA-48-like and bla _KPC ) and convergent Kp pathotypes in clinical isolates. The assay was evaluated on clinical isolates and validated against whole-genome sequencing (WGS) data for accuracy, specificity and sensitivity. Results. The developed m-PCR assay exhibited 100% specificity when compared to WGS data, successfully detecting all target genes without cross-amplification in ATCC control strains. The assay demonstrated high sensitivity, efficiently amplifying bacterial genomes from minimal DNA input as low as 1 ng µl ⁻¹ . Additionally, validation through sequencing confirmed the accuracy of detected amplicons. Conclusion. This m-PCR assay offers a rapid, sensitive and specific diagnostic tool for differentiating Kp pathotypes in clinical settings, aiding in timely intervention and improved infection control measures.

New Delhi metallo-β-lactamase (NDM)-producing enterobacterales infections pose significant challenges due to limited treatment options. Recently, a triple combination of ceftazidime/avibactam and aztreonam has been utilized to combat these infections. However, a four-amino-acid insertion in PBP3 and/or the ampC β-lactamase has been linked to the development of resistance to this triple combination in several recent investigations. To evaluate the effectiveness of cefiderocol and cefepime/tazobactam using both in-vitro and in-silico methods, this study examines current non-duplicate isolates of carbapenem-resistant E. coli (n = 40) and carbapenem-resistant K. pneumoniae (n = 40) derived from blood cultures. The MIC of cefepime/taniborbactam and cefiderocol was ascertained using the micro-broth dilution technique. Additionally, computational analysis was performed using molecular docking with AutoDock tools and dynamic simulation with GROMACS, followed by MM-PBSA analysis for a duration of 50 ns. In MIC assays, cefiderocol and cefepime/taniborbactam showed minimal effectiveness against NDM-producing K. pneumoniae and E. coli that possessed OXA-48-like traits. However, both antibiotics showed strong affinity against OXA-48-like producers. Docking studies varied from −4.3 to −8.1 kcal/mol, indicating a higher binding affinity and stronger hydrogen bond formation in OXA-48-like compared to NDM producers. All complexes exhibited stable conformational states, as indicated by analyses of the RMSD, RMSF, Rg, SASA, and hydrogen bond trajectories. Further evidence supports stable BL/BLI interactions within binding sites, mediated by PCA, FEL, and DCCM, as indicated by the binding free energy. Computational studies confirmed better binding affinity for both OXA-48-like and NDM. However, in-vitro studies demonstrated that cefepime/taniborbactam and cefiderocol represent viable alternative options, specifically for OXA-48-like producers, as their effectiveness is significantly compromised in the presence of NDM.

Introduction: Klebsiella pneumoniae is a major cause of nosocomial infections, with its evolving pathotypes—including multidrug-resistant (MDR), hypervirulent (hvKp), and convergent strains—posing significant diagnostic and treatment challenges. The convergence of antimicrobial resistance and hypervirulence in clinical settings complicates timely identification, leading to difficult-to-treat infections with limited therapeutic options. Materials and Methods: In this study, we designed and optimized a multiplex polymerase chain reaction (m-PCR) assay for the simultaneous detection of key biomarkers/genes associated with hypervirulent ( rmpA, rmpA2, iucA, peg344, iroB ), carbapenem-resistant ( bla- _NDM , bla- _OXA-48-like ), and convergent K. pneumoniae (hv-CRKp) pathotypes in clinical isolates. Specific primers were designed for these targets in a five-pair combination, with rigorous optimization of reaction conditions, including annealing temperatures, specificity, and sensitivity. Results and Conclusion: The developed m-PCR assay exhibited 100% specificity when compared to whole genome sequencing data, successfully detecting all target genes without cross-amplification in ATCC control strains. The assay demonstrated high sensitivity, efficiently amplifying bacterial genomes from minimal DNA input as low as 1 ng/µL. Additionally, validation through sequencing confirmed the accuracy of detected amplicons. This m-PCR assay offers a rapid, sensitive, and specific diagnostic tool for differentiating K. pneumoniae pathotypes in clinical settings, aiding in timely intervention and improved infection control measures. Impact Statement: This study presents a multiplex PCR assay for the rapid and accurate detection of Klebsiella pneumoniae pathotypes, addressing the challenge of defining hypervirulence due to the diverse and evolving set of markers. By analyzing genomic data from public databases, the most well-established virulence and resistance markers were carefully selected to enable comprehensive strain differentiation. The assay simultaneously detects hypervirulent, carbapenem-resistant, and classical strains, offering a faster and more targeted alternative to conventional methods. Additionally, its adaptable design allows for modifications based on regional strain variations, making it a valuable tool for both clinical and epidemiological applications. This approach enhances infection control efforts and antimicrobial stewardship by enabling timely and precise pathogen identification, particularly in resource-limited settings. By bridging genomic insights with practical diagnostics, this assay provides a cost-effective, scalable solution to monitor and manage high-risk K. pneumoniae infections more effectively.

In the ATTACK Phase 3 trial examining the efficacy of sulbactam (SUL)/durlobactam (DUR) to treat primarily Acinetobacter baumannii complex (ABC) infections, imipenem (IPM)/cilastatin was added as a common therapy to both the SUL/DUR and the comparator colistin arms. This raised the question of whether the use of IPM in the SUL/DUR arm of the study influenced the efficacy of SUL/DUR. To investigate this issue on a microbiological and molecular level, we performed static concentration time-kill studies and molecular modeling of binding of SUL to PBP1a and PBP3, IPM to PBP1a, PBP2, and PBP3, and DUR to OXA-23 and OXA-51. The time-kill studies performed using carbapenemase- and non-carbapenemase-producing isolates demonstrated synergy between SUL and IPM in the presence of DUR, supporting the notion that the efficacy of the SUL/DUR arm against Acinetobacter spp. in the ATTACK trial was enhanced by the addition of IPM. We also hypothesize that the protection of SUL and IPM from OXA carbapenemases by DUR enabled IPM and SUL to synergistically deactivate multiple PBPs (“target redundancy”). Docking simulations supported the favorable binding of SUL to PBP1a and PBP3, resulting in the formation of acyl–enzyme complexes. Molecular docking analysis of OXA carbapenemase enzymes with DUR also revealed favorable interactions. Although clinical trials are warranted, these analyses provide mechanistic support for the addition of IPM to SUL/DUR.

Introduction International guidelines recommend definitive combination antibiotic therapy for the management of serious infections involving carbapenem-resistant Acinetobacter (CRAB) species. The commonly available combination options include high-dose sulbactam, polymyxins, tetracyclines, and cefiderocol. Scanty prospective data exist to support this approach. Methods Patients with CRAB bacteraemia, ventilator-associated pneumonia (VAP), or both were categorized based on whether they received combination therapy or monotherapy. The 30-day mortality was compared between the two groups. Inverse probability treatment weighting (IPTW) was done using propensity score (PS) for a balanced comparison between groups. Results Between January 2021 and May 2023, of the 161 patients with CRAB bacteraemia (n = 55, 34.2%), VAP (n = 46, 28.6%), or both (n = 60, 37.3%) who received appropriate intravenous antibiotic therapy, 70% (112/161) received monotherapy, and the rest received combination therapy. The overall 30-day mortality was 62% (99/161) and not different (p = 0.76) between the combination therapy (31/49, 63.3%) and monotherapy (68/112, 60.7%) groups. The propensity score matching using IPTW did not show a statistical difference (p = 0.47) in 30-day mortality for receiving combination therapy with an adjusted odds ratio (OR) P of 1.29 (0.64, 2.58). Conclusion Combination therapy for CRAB infections needs further study in a randomised controlled trial, as this observational study showed no difference in 30-day mortality between monotherapy and combination therapy.

Tuberculosis (TB) is an important cause of morbidity and mortality globally. About 3-4% of hospitalized TB patients require admission to the intensive care unit (ICU); the mortality in these patients is around 50-60%. There is limited literature on the evaluation and management of patients with TB who required ICU admission. The Indian Society of Critical Care Medicine (ISCCM) constituted a working group to develop a position paper that provides recommendations on the various aspects of TB in the ICU setting based on available evidence. Seven domains were identified including the categorization of TB in the critically ill, diagnostic workup, drug therapy, TB in the immunocompromised host, organ support, infection control, and post-TB sequelae. Forty-one questions pertaining to these domains were identified and evidence-based position statements were generated, where available, keeping in focus the critical care aspects. Where evidence was not available, the recommendations were based on consensus. This position paper guides the approach to and management of critically ill patients with TB.