A Comprehensive Review of Innovative Paradigms in Microbial Detection and Antimicrobial Resistance: Beyond Traditional Cultural Methods.

Microbial detection and antimicrobial resistance (AMR) surveillance are critical components of public health efforts to combat infectious diseases and preserve the efficacy of antimicrobial agents. While foundational in microbial identification, traditional cultural methods are often laborious, time-consuming, and limited in their ability to detect AMR markers. In response to these challenges, innovative paradigms have emerged, leveraging advances in molecular biology, genomics, proteomics, nanotechnology, and bioinformatics. This comprehensive review provides an overview of innovative approaches beyond traditional cultural methods for microbial detection and AMR surveillance. Molecular-based techniques such as polymerase chain reaction (PCR) and next-generation sequencing (NGS) offer enhanced sensitivity and specificity, enabling the rapid identification of microbial pathogens and AMR determinants. Mass spectrometry-based methods provide rapid and accurate detection of microbial biomarkers, including matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and biosensor technologies. Nanotechnology approaches, such as nanoparticle-based assays and nanopore sequencing, offer novel platforms for sensitive and label-free detection of pathogens and AMR markers. Embracing these innovative paradigms holds immense promise for improving disease diagnosis, antibiotic stewardship, and AMR containment efforts. However, challenges such as cost, standardization, and integration with existing healthcare systems must be addressed to realize the full potential of these technologies. By fostering interdisciplinary collaboration and innovation, we can strengthen our ability to detect, monitor, and combat AMR, safeguarding public health for generations.

Identifying antibiotic-resistant strains via cell sorting and elastic-light-scatter phenotyping.

The proliferation and dissemination of antimicrobial-resistant bacteria is an increasingly global challenge and is attributed mainly to the excessive or improper use of antibiotics. Currently, the gold-standard phenotypic methodology for detecting resistant strains is agar plating, which is a time-consuming process that involves multiple subculturing steps. Genotypic analysis techniques are fast, but they require pure starting samples and cannot differentiate between viable and non-viable organisms. Thus, there is a need to develop a better method to identify and prevent the spread of antimicrobial resistance. This work presents a novel method for detecting and identifying antibiotic-resistant strains by combining a cell sorter for bacterial detection and an elastic-light-scattering method for bacterial classification. The cell sorter was equipped with safety mechanisms for handling pathogenic organisms and enabled precise placement of individual bacteria onto an agar plate. The patterning was performed on an antibiotic-gradient plate, where the growth of colonies in sections with high antibiotic concentrations confirmed the presence of a resistant strain. The antibiotic-gradient plate was also tested with an elastic-light-scattering device where each colony's unique colony scatter pattern was recorded and classified using machine learning for rapid identification of bacteria. Sorting and patterning bacteria on an antibiotic-gradient plate using a cell sorter reduced the number of subculturing steps and allowed direct qualitative binary detection of resistant strains. Elastic-light-scattering technology is a rapid, label-free, and non-destructive method that permits instantaneous classification of pathogenic strains based on the unique bacterial colony scatter pattern. KEY POINTS: • Individual bacteria cells are placed on gradient agar plates by a cell sorter • Laser-light scatter patterns are used to recognize antibiotic-resistant organisms • Scatter patterns formed by colonies correspond to AMR-associated phenotypes.

Impact of the COVID-19 pandemic on extended-spectrum ß-lactamase producing Escherichia coli in urinary tract and blood stream infections: results from a nationwide surveillance network, Finland, 2018 to 2022.

BACKGROUND: Before the COVID-19 pandemic there has been a constant increase in antimicrobial resistance (AMR) of Escherichia coli, the most common cause of urinary tract infections and bloodstream infections. The aim of this study was to investigate the impact of the COVID-19 pandemic on extended-spectrum ß-lactamase (ESBL) production in urine and blood E. coli isolates in Finland to improve our understanding on the source attribution of this major multidrug-resistant pathogen. METHODS: Susceptibility test results of 564,233 urine (88.3% from females) and 23,860 blood E. coli isolates (58.8% from females) were obtained from the nationwide surveillance database of Finnish clinical microbiology laboratories. Susceptibility testing was performed according to EUCAST guidelines. We compared ESBL-producing E. coli proportions and incidence before (2018-2019), during (2020-2021), and after (2022) the pandemic and stratified these by age groups and sex. RESULTS: The annual number of urine E. coli isolates tested for antimicrobial susceptibility decreased 23.3% during 2018-2022 whereas the number of blood E. coli isolates increased 1.1%. The annual proportion of ESBL-producing E. coli in urine E. coli isolates decreased 28.7% among males, from 6.9% (average during 2018-2019) to 4.9% in 2022, and 28.7% among females, from 3.0 to 2.1%. In blood E. coli isolates, the proportion decreased 32.9% among males, from 9.3 to 6.2%, and 26.6% among females, from 6.2 to 4.6%. A significant decreasing trend was also observed in most age groups, but risk remained highest among persons aged ≥ 60 years. CONCLUSIONS: The reduction in the proportions of ESBL-producing E. coli was comprehensive, covering both specimen types, both sexes, and all age groups, showing that the continuously increasing trends could be reversed. Decrease in international travel and antimicrobial use were likely behind this reduction, suggesting that informing travellers about the risk of multidrug-resistant bacteria, hygiene measures, and appropriate antimicrobial use is crucial in prevention. Evaluation of infection control measures in healthcare settings could be beneficial, especially in long-term care.

Pseudomonas aeruginosa infection correlates with high MFI donor-specific antibody development following lung transplantation with consequential graft loss and shortened CLAD-free survival.

BACKGROUND: Donor-specific antibodies (DSAs) are common following lung transplantation (LuTx), yet their role in graft damage is inconclusive. Mean fluorescent intensity (MFI) is the main read-out of DSA diagnostics; however its value is often disregarded when analyzing unwanted post-transplant outcomes such as graft loss or chronic lung allograft dysfunction (CLAD). Here we aim to evaluate an MFI stratification method in these outcomes. METHODS: A cohort of 87 LuTx recipients has been analyzed, in which a cutoff of 8000 MFI has been determined for high MFI based on clinically relevant data. Accordingly, recipients were divided into DSA-negative, DSA-low and DSA-high subgroups. Both graft survival and CLAD-free survival were evaluated. Among factors that may contribute to DSA development we analyzed Pseudomonas aeruginosa (P. aeruginosa) infection in bronchoalveolar lavage (BAL) specimens. RESULTS: High MFI DSAs contributed to clinical antibody-mediated rejection (AMR) and were associated with significantly worse graft (HR: 5.77, p < 0.0001) and CLAD-free survival (HR: 6.47, p = 0.019) compared to low or negative MFI DSA levels. Analysis of BAL specimens revealed a strong correlation between DSA status, P. aeruginosa infection and BAL neutrophilia. DSA-high status and clinical AMR were both independent prognosticators for decreased graft and CLAD-free survival in our multivariate Cox-regression models, whereas BAL neutrophilia was associated with worse graft survival. CONCLUSIONS: P. aeruginosa infection rates are elevated in recipients with a strong DSA response. Our results indicate that the simultaneous interpretation of MFI values and BAL neutrophilia is a feasible approach for risk evaluation and may help clinicians when to initiate DSA desensitization therapy, as early intervention could improve prognosis.

Genomic characterization of Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae (KPC-Kp) strains circulating in three university hospitals in Northern Italy over three years.

OBJECTIVES: Genomic surveillance of Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae (KPC-Kp) is crucial for virulence, drug-resistance monitoring, and outbreak containment. METHODS: Genomic analysis on 87 KPC-Kp strains isolated from 3 Northern Italy hospitals in 2019-2021 was performed by whole genome sequencing (WGS), to characterize resistome, virulome, and mobilome, and to assess potential associations with phenotype resistance and clinical presentation. Maximum Likelihood and Minimum Spanning Trees were used to determine strain correlations and identify potential transmission clusters. RESULTS: Overall, 15 different STs were found; the predominant ones included ST307 (35, 40.2%), ST512/1519 (15, 17.2%), ST20 (12, 13.8%), and ST101 (7, 8.1%). 33 (37.9%) KPC-Kp strains were noticed to be in five transmission clusters (median number of isolates in each cluster: 5 [3-10]), four of them characterized by intra-hospital transmission. All 87 strains harbored Tn4401a transposon, carrying blaKPC-3 (48, 55.2%), blaKPC-2 (38, 43.7%), and in one case (1.2%) blaKPC-33, the latter gene conferred resistance to ceftazidime/avibactam (CZA). Thirty strains (34.5%) harbored porin mutations; of them, 7 (8.1%) carried multiple Tn4401a copies. These strains were characterized by significantly higher CZA minimum inhibitory concentration compared with strains with no porin mutations or single Tn4401a copy, respectively, even if they did not overcome the resistance breakpoint of 8 ug/mL. Median 2 (IQR:1-2) virulence factors per strain were detected. The lowest number was observed in ST20 compared to the other STs (p<0.001). While ST307 was associated with infection events, a trend associated with colonization events could be observed for ST20. CONCLUSIONS: Integration of genomic, resistance score, and clinical data allowed us to define a relative diversification of KPC-Kp in Northern Italy between 2019 and 2021, characterized by few large transmission chains and rare inter-hospital transmission. Our results also provided initial evidence of correlation between KPC-Kp genomic signatures and higher MIC levels to some antimicrobial agents or colonization/infection status, once again underlining WGS's importance in bacterial surveillance.

Antibiotic usage patterns in COVID-19 patients in five tertiary hospitals from Bangladesh: A countrywide picture.

Objectives: Irrational and injudicious use of antibiotics in COVID-19 patients could be detrimental in a tropical country with a weak antibiotic stewardship policy such as Bangladesh. This study aimed to focus on the antibiotic usage patterns in COVID-19 patients in Bangladesh. Methods: This prospective observational study was performed from July 2020 to June 2021 in five tertiary hospitals in Bangladesh. Data on demographic profile, disease severity, and antibiotic usage were collected directly from the patients' hospital documents. Results: A total of 3486 (94.4%) patients were treated with at least one antibiotic; 3261 (93.6%) patients received a single antibiotic, and 225 (6.5%) received multiple antibiotics. The most used antibiotics were ceftriaxone (37.3%), co-amoxiclav (26.3%), azithromycin (10.6%), and meropenem (10.3%). According to the World Health Organization AWaRe categorization, most (2260; 69.6%) of the antibiotics prescribed in this study belonged to the "Watch" group. Culture and sensitivity reports were available in 111 cases from one center. Only 18.9% of the patients were found to be co-infected with multi-drug-resistant bacteria (52.4% yield from sputum, 28.6% from urine, and 14.3% from blood). Conclusions: Strict antibiotic prescribing policy and antibiotic stewardship should be implemented immediately to limit the future threat of antimicrobial resistance in countries such as Bangladesh.

Modified HT-SPOTi: An Antimicrobial Susceptibility Testing to Evaluate Formulated Therapeutic Combinations Against Bacterial Growth and Viability.

Antimicrobial resistance (AMR) poses a serious threat to global health, potentially causing 10 million deaths per year globally by 2050. To tackle AMR, researchers from all around the world have generated a selection of various formulated (viz. nanoparticulate, liposomal) therapeutic combinations to be evaluated for new antimicrobial drug discovery. To meet the urgent need for accelerating new antibacterial drug development, we need rapid but reliable whole-cell assay methods and models to test formulated therapeutic combinations against several pathogens in different in vitro conditions as models of actual infections.Over the past two decades, high-throughput spot-culture growth inhibition assay (HT-SPOTi) has been demonstrated to be a gold-standard drug susceptibility method for evaluating novel chemotherapeutic entities and existing drugs against various microbes of global concern. Our modified HT-SPOTi method serves the purpose of evaluating drug combinations against Gram-positive/negative microorganisms as well as acid-fast bacilli. The newly developed and modified HT-SPOTi assay builds upon the limitations of our previously published method to incorporate antimicrobial susceptibility testing with formulated therapeutic combinations. The modified HT-SPOTi is compared with a range of other antimicrobial susceptibility testing methods and validated using a library of existing antibiotics as well as formulated therapeutic combinations. The modified HT-SPOTi assay can serve as an efficient and reliable high-throughput drug screening platform to discover new potential antimicrobial molecules, including as part of therapeutic formulations.This chapter describes the generation of drug susceptibility profile for formulated therapeutic combinations using modified HT-SPOTi in a semi-automated system.

Analysis of Whole Genome Sequencing Data for Detection of Antimicrobial Resistance Determinants.

Genomic sequencing has revolutionized microbial typing methods and transformed high-throughput methods in reference, clinical, and research laboratories. The detection of antimicrobial-resistant (AMR) determinants using genomic methods can provide valuable information on the emergence of resistance. Here we describe an approach to detecting AMR determinants using an open access and freely available platform which does not require bioinformatic expertise.

Slaughtering processes impact microbial communities and antimicrobial resistance genes of pig carcasses.

Several steps in the abattoir can influence the presence of microbes and associated resistance genes (ARGs) on the animal carcasses used for further meat processing. We investigated how these processes influence the resistome-microbiome of groups of pigs with different on-farm antimicrobial exposure status, from the moment they entered the abattoir until the end of carcass processing. Using a targeted enrichment metagenomic approach, we identified 672 unique ARGs conferring resistance to 43 distinct AMR classes from pooled skin (N = 42) and carcass swabs (N = 63) collected sequentially before, during, and after the slaughter process and food safety interventions. We observed significant variations in the resistome and microbial profiles of pigs before and after slaughter, as well as a significant decline in ARG counts, diversity, and microbial DNA load during slaughter and carcass processing, irrespective of prior antimicrobial treatments on the farm. These results suggest that existing interventions in the abattoir are effective in reducing not only the pathogen load but also the overall bacterial burden, including ARGs on pork carcasses. Concomitant with reductions in microbial and ARG counts, we observed an increase in the relative abundance of non-drug-specific ARGs, such as those conferring resistance to metals and biocides, and in particular mercury. Using a strict colocalization procedure, we found that most mercury ARGs were associated with genomes from the Pseudomonadaceae and Enterobacteriaceae families. Collectively, these findings demonstrate that slaughter and processing practices within the abattoir can shape the microbial and ARG profiles of pork carcasses during the transition from living muscle to meat.

Embedding community and public voices in co-created solutions to mitigate antimicrobial resistance (AMR) in Thailand using the 'Responsive Dialogues' public engagement framework.

The use of antimicrobials in Thailand has been reported as one of the highest in the world in human and animal sectors. Our engagement project aimed to improve our understanding of the issue of antimicrobial use and antimicrobial resistance (AMR) among adult Thai communities, and co-create locally relevant solutions to AMR, especially those focusing on raising awareness to improve related policies in Thailand.We conducted a series of online and in-person 'conversations' according to Wellcome's 'Responsive Dialogues' engagement approach, designed to bring together different voices to understand complex AMR problems and find potential solutions. This approach enabled key AMR stakeholders and policy makers to hear directly from communities and members of the public, and vice versa. Conversations events took place between 25 November 2020 and 8 July 2022, and we engaged 179 AMR key stakeholders and members of the public across Thailand.The issues found were: there were quite a lot of misunderstandings around antimicrobials and AMR; participants felt that communications and engagement around antimicrobial resistance had limited reach and impact; asking for and taking antibiotics for self-limiting ailments is a social norm in Thailand; and there appeared to be a wide availability of cheap antimicrobials. To mitigate the spread of AMR, participants suggested that the messages around AMR should be tailored to the target audience, there should be more initiatives to increase general health literacy, there should be increased availability of AMR related information at the local level and there should be increased local leadership of AMR mitigation efforts.Trial registration Thaiclinicaltrials.org registration: TCTR20210528003 (28/05/2021).

An overview of the occurrence, impact of process parameters, and the fate of antibiotic resistance genes during anaerobic digestion processes.

Antibiotic resistance genes (ARGs) have emerged as a significant global health threat, contributing to fatalities worldwide. Wastewater treatment plants (WWTPs) and livestock farms serve as primary reservoirs for these genes due to the limited efficacy of existing treatment methods and microbial adaptation to environmental stressors. Anaerobic digestion (AD) stands as a prevalent biological treatment for managing sewage sludge and manure in these settings. Given the agricultural utility of AD digestate as biofertilizers, understanding ARGs' fate within AD processes is essential to devise effective mitigation strategies. However, understanding the impact of various factors on ARGs occurrence, dissemination, and fate remains limited. This review article explores various AD treatment parameters and correlates to various resistance mechanisms and hotspots of ARGs in the environment. It further evaluates the dissemination and occurrence of ARGs in AD feedstocks and provides a comprehensive understanding of the fate of ARGs in AD systems. This review explores the influence of key AD parameters such as feedstock properties, pretreatments, additives, and operational strategies on ARGs. Results show that properties such as high solid content and optimum co-digestion ratios can enhance ARG removal, while the presence of heavy metals, microplastics, and antibiotics could elevate ARG abundance. Also, operational enhancements, such as employing two-stage digestion, have shown promise in improving ARG removal. However, certain pretreatment methods, like thermal hydrolysis, may exhibit a rebounding effect on ARG levels. Overall, this review systematically addresses current challenges and offers future perspectives associated with the fate of ARGs in AD systems.

The significance of environmentally bioavailable antimicrobials in driving antimicrobial resistance in soils.

Antimicrobial resistance (AMR) stands as an escalating public health crisis fueled by antimicrobial residues in the environment, particularly in soil, which acts as a reservoir for antimicrobial resistance genes (ARGs). Merely quantifying the total extractable concentration of antimicrobials, instead of bioavailable fractions, may substantially underestimate their minimal selection concentration for propagating ARGs. To shed light on the role of bioavailability in ARG abundance within soil, a systematic bioavailability assessment method was established for accurately quantifying the partitioning of multi-class antimicrobials in representative Chinese soils. Microcosm studies unveiled that antimicrobials persisting in the bioavailable fraction could potentially prolong their selection pressure duration to trigger AMR. Notably, the co-occurrence of pesticide or steroid hormone influenced the development trends of ARG subtypes, with fluoroquinolone resistance genes (RGs) being particularly susceptible. Partial least squares path model (PLS-PM) analysis uncovered potentially distinct induction mechanisms of antimicrobials: observable results suggested that extractable residual concentration may exert a direct selection pressure on the development of ARGs, while bioavailable concentration could potentially play a stepwise role in affecting the abundance of mobile genetic elements and initiating ARG dissemination. Such unprecedented scrutinization of the interplay between bioavailable antimicrobials in soils and ARG abundance provides valuable insights into strategizing regulatory policy or guidelines for soil remediation.

Bioactive Phyto-Compounds with Antimicrobial Effects and AI: Results of a Desk Research Study.

Resistance of microorganisms to antibiotics represents a formidable global challenge, manifesting in intricate public health ramifications including escalated mortality rates and augmented healthcare costs. The current efforts to manage antimicrobial resistance (AMR) are limited mainly to the standard therapeutic approaches. The aim of this study is to present and analyze the role of artificial intelligence (AI) in the search for new phyto-compounds and novel interactions with antimicrobial effects. The ambition of the current research study is to support researchers by providing summarized information and ideas for future research in the battle with AMR. Inevitably, the AI role in healthcare is growing exponentially. The reviewed AI models reveal new data on essential oils (EOs) as potential therapeutic agents. In terms of antibacterial activity, EOs show activity against MDR bacteria, reduce resistance by sensitizing bacteria to the action of antibiotics, and improve therapeutic efficiency when combined with antibiotics. AI models can also serve for the detailed study of other therapeutic applications of EOs such as respiratory diseases, immune diseases, neurodegenerative diseases, and oncological diseases. The last 5 years have seen an increasing application of AI in the search for potential plant sources to control AMR. For the time being, the application of machine-learning (ML) models is greater in the studies of EOs. Future attention of research teams may also be directed toward a more efficient search for plant antimicrobial peptides (PAMPs). Of course, investments in this direction are a necessary preface, but the excitement of new possibilities should not override the role of human intelligence in directing research processes. In this report, tradition meets innovation to address the "silent pandemic" of AMR.

Displacement of Hospital-Acquired, Methicillin-Resistant Staphylococcus aureus Clones by Heterogeneous Community Strains in Kenya over a 13-Year Period.

We determined antibiotic susceptibility and employed Oxford Nanopore whole-genome sequencing to explore strain diversity, resistance, and virulence gene carriage among methicillin-resistant Staphylococcus aureus (MRSA) strains from different infection sites and timepoints in a tertiary Kenyan hospital. Ninety-six nonduplicate clinical isolates recovered between 2010 and 2023, identified and tested for antibiotic susceptibility on the VITEK ID/AST platform, were sequenced. Molecular typing, antibiotic resistance, and virulence determinant screening were performed using the relevant bioinformatics tools. The strains, alongside those from previous studies, were stratified into two periods covering 2010-2017 and 2018-2023 and comparisons were made. Mirroring phenotypic profiles, aac(6')-aph(2″) [aminoglycosides]; gyrA (S84L) and grlA (S80Y) [fluoroquinolones]; dfrG [anti-folates]; and tet(K) [tetracycline] resistance determinants dominated the collection. While the proportion of ST239/241-t037-SCCmec III among MRSA reduced from 37.7% to 0% over the investigated period, ST4803-t1476-SCCmec IV and ST152-t355-SCCmec IV were pre-eminent. The prevalence of Panton-Valentine leucocidin (PVL) and arginine catabolic mobile element (ACME) genes was 38% (33/87) and 6.8% (6/87), respectively. We observed the displacement of HA-MRSA ST239/241-t037-SCCmec III with the emergence of ST152-t355-SCCmec IV and a greater clonal heterogeneity. The occurrence of PVL+/ACME+ CA-MRSA in recent years warrants further investigations into their role in the CA-MRSA virulence landscape, in a setting of high PVL prevalence.

EU surveys insights: analytical tools, future directions, and the essential requirement for reference materials in wastewater monitoring of SARS-CoV-2, antimicrobial resistance and beyond.

BACKGROUND: Wastewater surveillance (WWS) acts as a vigilant sentinel system for communities, analysing sewage to protect public health by detecting outbreaks and monitoring trends in pathogens and contaminants. To achieve a thorough comprehension of present and upcoming practices and to identify challenges and opportunities for standardisation and improvement in WWS methodologies, two EU surveys were conducted targeting over 750 WWS laboratories across Europe and other regions. The first survey explored a diverse range of activities currently undertaken or planned by laboratories. The second survey specifically targeted methods and quality controls utilised for SARS-CoV-2 surveillance. RESULTS: The findings of the two surveys provide a comprehensive insight into the procedures and methodologies applied in WWS. In Europe, WWS primarily focuses on SARS-CoV-2 with 99% of the survey participants dedicated to this virus. However, the responses highlighted a lack of standardisation in the methodologies employed for monitoring SARS-CoV-2. The surveillance of other pathogens, including antimicrobial resistance, is currently fragmented and conducted by only a limited number of laboratories. Notably, these activities are anticipated to expand in the future. Survey replies emphasise the collective recognition of the need to enhance the accuracy of results in WWS practices, reflecting a shared commitment to advancing precision and effectiveness in WWS methodologies. CONCLUSIONS: These surveys identified a lack of standardised common procedures in WWS practices and the need for quality standards and reference materials to enhance the accuracy and reliability of WWS methods in the future. In addition, it is important to broaden surveillance efforts beyond SARS-CoV-2 to include other emerging pathogens and antimicrobial resistance to ensure a comprehensive approach to protecting public health.

Impact of COVID-19 pandemic on the utilization and quality of antibiotic use in the primary care setting in England, March 2019-March 2023: a segmented interrupted time series analysis of over 53 million individuals.

BACKGROUND: Amid the COVID-19 pandemic, we evaluated the short-term impact of COVID-19 on antibiotic use in primary care in England, focusing on both antibiotic quantity (overuse) and quality (misuse) of use. RESEARCH DESIGN AND METHODS: A population-based segmented interrupted analysis was applied on monthly dispensed antibiotics prescriptions using the Prescription Cost Analysis dataset (March/2019-March/2023). The quantity was assessed using number of items dispensed per 1000 inhabitants (NTI) and defined daily doses per 1000 inhabitants per day (DID), while quality was evaluated using WHO's Access Watch Reserve (AWaRe) classification, the proportion of '4C' antibiotics and the percentage of broad- to narrow-spectrum antibiotics. RESULTS: Findings indicate 8.6 (17.2%) and 0.4 (2.6%) increase in the NTI and DID, respectively, with a statistically significant uptick in trend noted after the second lockdown (ß5) for 'total antibiotics' for NTI only (ß5 = 1.6; 95% CI:0.17, 3.1). Quality assessment showed an increase in 'Access' antibiotics from 77% in March/2019 to 86% in March/2023; however, COVID-19 had no significant impact on WHO AWaRe classes. CONCLUSION: COVID-19's impact on antibiotic use quality and quantity appeared to be minimal, though an increase in utilization post-second lockdown coincided with healthcare system recovery. This suggests a nuanced impact of the pandemic, highlighting the importance of continued antimicrobial stewardship.

Salmonella Typhi Haplotype 58 (H58) Biofilm Formation and Genetic Variation in Typhoid Fever Patients with Gallstones in an Endemic Setting in Kenya.

The causative agent of typhoid fever, Salmonella enterica serovar Typhi, is a human restricted pathogen. Human carriers, 90% of whom have gallstones in their gallbladder, continue to shed the pathogen after treatment. The genetic mechanisms involved in establishing the carrier state are poorly understood, but S. Typhi is thought to undergo specific genetic changes within the gallbladder as an adaptive mechanism. In the current study, we aimed to identify biofilm forming ability and the genetic differences in longitudinal clinical S. Typhi isolates from asymptomatic carriers with gallstones in Nairobi, Kenya. Whole genome sequences were analyzed from 22 S. Typhi isolates, 20 from stool and 2 from blood samples, all genotype 4.3.1 (H58). Nineteen strains were from four patients also diagnosed with gallstones, of whom, three had typhoid symptoms and continued to shed S. Typhi after treatment. All isolates had point mutations in the quinolone resistance determining region (QRDR) and only sub-lineage 4.3.1.2EA3 encoded multidrug resistance genes. There was no variation in antimicrobial resistance patterns among strains from the same patient/household. Non-multidrug resistant (MDR), isolates formed significantly stronger biofilms in vitro than the MDR isolates, p<0.001. A point mutation within the treB gene (treB A383T) was observed in strains isolated after clinical resolution from patients living in 75% of the households. Missense mutations in Vi capsular polysaccharide genes, tviE P263S was also observed in 18% of the isolates. This study provides insights into the role of typhoid carriage, biofilm formation, AMR genes and genetic variations in S. Typhi from asymptomatic carriers.

Unveiling the Druggable Landscape of Bacterial Peptidyl tRNA Hydrolase: Insights into Structure, Function, and Therapeutic Potential.

Bacterial peptidyl tRNA hydrolase (Pth) or Pth1 emerges as a pivotal enzyme involved in the maintenance of cellular homeostasis by catalyzing the release of peptidyl moieties from peptidyl-tRNA molecules and the maintenance of a free pool of specific tRNAs. This enzyme is vital for bacterial cells and an emerging drug target for various bacterial infections. Understanding the enzymatic mechanisms and structural intricacies of bacterial Pth is pivotal in designing novel therapeutics to combat antibiotic resistance. This review provides a comprehensive analysis of the multifaceted roles of Pth in bacterial physiology, shedding light on its significance as a potential drug target. This article delves into the diverse functions of Pth, encompassing its involvement in ribosome rescue, the maintenance of a free tRNA pool in bacterial systems, the regulation of translation fidelity, and stress response pathways within bacterial systems. Moreover, it also explores the druggability of bacterial Pth, emphasizing its promise as a target for antibacterial agents and highlighting the challenges associated with developing specific inhibitors against this enzyme. Structural elucidation represents a cornerstone in unraveling the catalytic mechanisms and substrate recognition of Pth. This review encapsulates the current structural insights of Pth garnered through various biophysical techniques, such as X-ray crystallography and NMR spectroscopy, providing a detailed understanding of the enzyme's architecture and conformational dynamics. Additionally, biophysical aspects, including its interaction with ligands, inhibitors, and substrates, are discussed, elucidating the molecular basis of bacterial Pth's function and its potential use in drug design strategies. Through this review article, we aim to put together all the available information on bacterial Pth and emphasize its potential in advancing innovative therapeutic interventions and combating bacterial infections.