Eswarappa Pradeep Bulagonda

Airborne microorganisms significantly contribute to hospital-associated infections (HAIs), particularly causing respiratory tract infections (RTIs). The spread of Multi-Drug Resistant (MDR) airborne bacteria further complicates disease prevention, challenging existing infection control strategies. Current air decontamination technologies are found to have limitations, necessitating novel approaches. In this study, we demonstrated the utility of zeta potential, a natural physicochemical electro-kinetic property of bacteria as a key target, which can be exploited to trap and eliminate MDR airborne bacteria. Multiple respiratory pathogens were included in the study, harbouring various resistance phenotypes. Zeta potentials of these clinical isolates were measured and compared against corresponding ATCC strains. Clinical isolates were aerosolized in a certified BSL-2 setting containing a ZeBox-powered air sterilization device. Viable bacteria were enumerated at various time points, before and after exposure to the air decontamination device. Our analyses revealed that Zeta potential is relatively independent of the origin and antibiotic susceptibility of the tested isolates. Exposure to ZeBox powered device for 5 min resulted in a minimum of 5 log reduction (99.999%) among majority of the isolates, irrespective of their genus and origin. Zeta potential measurements correlated to the kill kinetics of ZeBox technology. The current study underscores the reliability of zeta potential based air decontamination technologies such as ZeBox for potential elimination of diverse, airborne respiratory pathogens in healthcare and domestic settings, offering a promising strategy to combat HAIs in post antibiotic and post pandemic era.

Urinary tract infections (UTIs) caused by Uropathogenic Escherichia coli (UPEC) belonging to global strains such as ST-131 pose a significant health challenge. To understand the evolutionary landscape and molecular mechanisms defining ST-131 UPEC, the complete genome of E. coli NS30 was generated and analyzed. The complete genome assembly of E. coli NS30, belonging to high-risk ST-131, C2 subclade, revealed a chromosome and two plasmids. A large conjugative plasmid, pNS30-1, harboured a multi-drug resistance (MDR) cassette within a Tn402-like class 1 integron, which was functionally demonstrated to be transferable. Comparative genomic analysis identified four distinct genomic islands (GIs) that are absent in its closest ST-131 neighbour. Two of these, including a novel pathogenicity island (PAI), were acquired from other E. coli lineages, harbouring Virulence factors (VFs) and efflux pump genes. The remaining two GIs are phage-like elements contributing to genome plasticity. E. coli NS30 is distinct from the other ST-131 UPEC genomes by the acquisition of novel GIs. The presence of GIs, virulence factors and AMR genes in a conjugative MDR plasmid has driven its evolution into a formidable uropathogen with a high potential to spread resistance and virulence traits.

Background: Airborne microorganisms contribute significantly to hospital-associated infections (HAIs), particularly causing respiratory tract infections (RTIs). Multi-drug resistant (MDR) airborne pathogens pose a global clinical threat, challenging existing air decontamination technologies. While current methods effectively trap microbes, they often lack microbicidal properties. Exploitation of Zeta potential of microbes which are naturally charged could be a potential strategy for air decontamination, independent of their antimicrobial resistance. In this study, we tested the efficacy of ZeBox technology which relies on zeta potential to trap and eliminate airborne pathogens such as clinically isolated, MDR Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli of respiratory origin and a non RTI isolate Staphylococcus aureus . Methods: Five MDR bacterial pathogens which include K. pneumoniae (2 nos), A. baumannii, P. aeruginosa , and E. coli from patients with RTI and one pus isolate of S. aureus were included in the study. These isolates were aerosolized in a certified BSL-2 setting containing a ZeBox-powered Air Sterilization device. Viable bacteria were enumerated before and after exposure to the Zebox powered device. Clinical and laboratory isolated bacterial strains were assessed for zeta potential variations to understand its association with antibiotic resistance and pathogenicity. Results: Our analyses revealed that Zeta potential is species specific, but independent of the pathogenicity and antibiotic susceptibility of the tested bacteria. Exposure to Zebox powered device for 5 mins resulted in a remarkable decline of microbial load with a minimum 5 log reduction (99.999%) of all the tested isolates, irrespective of their species. Zeta potential measurements further indicated a consistent killing mechanism of MDR pathogens by Zebox technology. Conclusions: The current study underscores the reliability of zeta potential based air decontamination strategies for potential elimination of diverse, air borne MDR bacteria in healthcare settings. Highlights: Zeta potential value of bacteria is not influenced by AST or pathogenicity of bacteria. Zeta potential driven device traps and kills airborne MDR bacteria with 99.999% efficiency. The efficacy of ZeBox technology is consistent across pathogens.

The rapid and early detection of infections and antibiotic resistance markers is a critical challenge in healthcare. Currently, most commercial diagnostic tools for analyzing antimicrobial resistance patterns of pathogens require elaborate culture-based testing. Our study aims to develop a rapid, accurate molecular detection system that can be used directly from culture, thereby introducing molecular testing in conjunction with culture tests to reduce turnaround time and guide therapy. PathCrisp assay, a combination of loop-mediated isothermal amplification and CRISPR-based detection, maintained at a single temperature, was designed and tested on clinical isolates. The specificity and sensitivity of the assay was analyzed, post which the assay was compared with the polymerase chain reaction (PCR) method to detect the New Delhi metallo-beta-lactamase (NDM) gene in carbapenem-resistant enterobacteriaceae clinical samples. Our PathCrisp assay demonstrated the ability to detect as few as 700 copies of the NDM gene from clinical isolates. Our assay demonstrated 100% concordance with the PCR-Sanger sequencing method, more commonly used. Additionally, the lack of the need for a kit-based DNA purification step, rather a crude extraction via heating, enables the direct use of culture samples. The PathCrisp assay is precise, specific and rapid, providing results in approximately 2 h, and operates at a constant temperature, reducing the need for complex equipment handling. In the near future, we hope that this assay can be further optimized and designed as a point-of-care test kit, facilitating its use in various healthcare settings and aiding clinicians in the choice of antibiotics for therapy.

Antimicrobial resistance (AMR) is a global health crisis that has already claimed millions of lives and is projected to affect millions more unless urgent action is taken. Effective control of AMR requires the correct choice and dosage of antibiotics, as well as robust surveillance and research. Understanding the mechanisms of antibiotic action and the emergence of resistance phenotypes along with their genotypes is essential. This knowledge, combined with insights into resistance prevalence and spread, empowers clinicians to propose alternative therapies. Nitrofurantoin, a 70-year-old antibiotic, remains effective for the treatment of uncomplicated lower UTIs. Preventing emergence and spread of nitrofurantoin-resistant superbugs would preserve the efficacy of this antibiotic which is crucial for ongoing and future AMR efforts. Nitrofurantoin resistance evolves slowly, leading to low prevalence compared to other antibiotics. However, it is often linked with extensive drug resistance, complicating treatment outcomes. Even a minor percentage of nitrofurantoin-resistant bacteria can cause significant clinical challenges due to irreversible evolution. While detailed study of these mechanisms can guide the development of strategies to combat nitrofurantoin resistance, early detection of resistant infections is critical for saving lives. The current review aimed to provide a comprehensive analysis of nitrofurantoin’s mechanisms of action, resistance evolution, prevalence, and resistance prediction. Our goal is to offer valuable insights for researchers and clinicians to enhance nitrofurantoin use and address the challenges posed by AMR.

Objective: The rapid and early detection of infections and antibiotic resistance markers is a critical challenge in healthcare. Currently, most commercial diagnostic tools for analyzing antimicrobial resistance patterns of pathogens require elaborate culture-based testing. Our study aims to develop a rapid, accurate molecular detection system that can be used directly from culture, thereby introducing molecular testing in conjunction with culture tests to reduce turnaround time (TAT) and guide therapy. Methods: PathCrisp assay, a combination of Loop-mediated Isothermal Amplification (LAMP) and CRISPR-based detection, maintained at a single temperature, was designed and tested on clinical isolates. The specificity and sensitivity of the assay was analyzed, post which the assay was compared with the Polymerase Chain Reaction (PCR) method to detect the New Delhi metallo-beta-lactamase (NDM) gene in carbapenem-resistant Enterobacteriaceae (CRE) clinical samples. Results: Our PathCrisp assay demonstrated the ability to detect as few as 700 copies of the NDM gene from clinical isolates. Our assay demonstrated 100% concordance with the PCR-Sanger sequencing method, more commonly used. Additionally, the lack of the need for a kit-based DNA purification step, rather a crude extraction via heating, enables the direct use of culture samples. Conclusions: The PathCrisp assay is precise, specific and rapid, providing results in approximately 2 hours, and operates at a constant temperature, reducing the need for complex equipment handling. In the near future, we hope that this assay can be further optimized and designed as a point-of-care test kit, facilitating its use in various healthcare settings and aiding clinicians in the choice of antibiotics for therapy. Plain language summary: Resistance to Carbapenem, a last-line antibiotic for treatment, is a global threat. Timely diagnosis is critical for a clinician to decide upon the treatment. However, present available methods to detect resistance are either expensive or have longer turnaround time. Here, in this study, we aim to tackle both limitations by developing a rapid, instrument-light, point-of-care assay called PathCrisp . Our PathCrisp assay is a combination of isothermal amplification (a single temperature) and the CRISPR/Cas system used for diagnosis. This provides results within 2 hours and operates at a constant temperature. Our study validated the assay using Carbapenem-resistant Enterobacteriaceae clinical samples to detect the NDM gene, compared to the PCR and sequencing technique previously used. Furthermore, the PathCrisp assay can detect as few as 700 copies of target DNA when tested upon serial dilution, works on crude samples (does not require pure isolated DNA), and can detect NDM-positive samples directly from the culture.

Introduction. Pseudomonas aeruginosa is now considered as a major bacterial pathogen associated with hospital infections. Frequently, multidrug-resistant (MDR) and extensively drug-resistant (XDR) P. aeruginosa are being encountered. Unusual increase in the P. aeruginosa infections led to the suspicion of outbreaks in the urology ward and cardiothoracic and vascular surgery intensive care unit (CTVS-ICU). Hypothesis. We hypothesize that the localized outbreaks may have originated from environmental sources within the hospital premises. An alternative possibility is the transmission from a previously infected patient or hospital attendant. Understanding the drug-resistance profile and genome characteristics of these clinical samples would determine the likely source of infection and spread. Aim. To perform epidemiological and molecular investigations on the suspected outbreaks of P. aeruginosa in the study centre and identify potential sources of infection. Methodology. Fourteen drug-resistant P. aeruginosa isolated from patients of the urology ward, CTVS-ICU and tap waters collected during the suspected outbreaks were subjected to microbiological and genomic analysis. Comparative genome (CG) analysis of these 14 study genomes with 284 complete P. aeruginosa genomes was performed. Results. Multilocus sequence typing analysis revealed that the isolates belonged to five different sequence types (ST235, ST357, ST639, ST654 and ST1203) and clustered into three distinct groups while two CTVS-ICU isolates remained as singletons. Genome analysis distinguished that the outbreaks in the urology ward and CTVS-ICU are independent, epidemiologically unrelated to each other and with the tap-water isolates. Conclusion. This study highlights the presence of distinct, clonally unrelated, drug-resistant P. aeruginosa within a hospital setting. The genome analysis of the two localized outbreaks revealed their distinct genetic background and phylogenetically unrelated origin. Vigilant screening and effective implementation of infection control measures led to the successful containment of potential environmental reservoirs of P. aeruginosa within the premises.

Dear Sir/Madam,Mithlesh Kumari et al. described the emergence of blaNDM-1 and blaVIM among ventilator associated pneumonia causing gram negative bacterial pathogens. It's an excellent study; the tables, figures, methods, and results are very well pre...