OBJECTIVE: 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.

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.

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.

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.

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.

Repurposing pharmaceuticals is a technique used to find new, alternate clinical applications for approved drug molecules. It may include altering the drug formulation, route of administration, dose or the dosage regimen. The process of repurposing medicines starts with screening libraries of previously approved drugs for the targeted disease condition. If after an the initial in silico, in vitro or in vivo experimentation, the molecule has been found to be active against a particular target, the molecule is considered as a good candidate for clinical trials. As the safety profile of such molecules is available from the previous data, significant time and resources are saved. These advantages of drug repurposing approach make it especially helpful for finding treatments for rapidly evolving conditions including bacterial infections. An ever-increasing incidence of antimicrobial resistance, owing to the mutations in bacterial genome, leads to therapeutic failure of many approved antibiotics. Repurposing the approved drug molecules for use as antibiotics can provide an effective means for the combating life-threatening bacterial diseases. A number of drugs have been considered for drug repurposing against bacterial infections. These include, but are not limited to, Auranofin, Closantel, and Toremifene that have been repurposed for various infections. In addition, the reallocation of route of administration, redefining dosage regimen and reformulation of dosage forms have also been carried out for repurposing purpose. The current chapter addresses the drug discovery and development process with relevance to repurposing against bacterial infections.

UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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 (β5) for \'total antibiotics\' for NTI only (β5 = 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.
UNASSIGNED: 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.

The aim of this paper is to explore and assess various strategies for monitoring antimicrobial consumption (AMC) in animals, within the context of the One Health approach. Recent studies have shed light on the limited surveillance and data collection for AMC in animals. Using the United States Center for Disease Control and Prevention Policy Analytical Framework, we assess global, national, and farm-level surveillance strategies on public health impact and feasibility using evidence from primary, secondary, and grey literature. From this, we identify key policy mechanisms that support the adoption of surveillance while providing specific recommendations. We find that a global strategy, though valuable for benchmarking and policy guidance, faces participation and data visibility challenges. National-level surveillance offers direct inputs into national action plans but struggles with data uniformity and comparability. Farm-level surveillance, while resource-intensive, provides the most granular data for informing specific interventions. We advocate for a multi-faceted approach to AMC surveillance, emphasizing that legal mandates and financial incentives are crucial for encouraging surveillance participation, along with international cooperation for enhancing participation and data quality. Drawing parallels with public reporting challenges in other sectors can provide valuable lessons on how to address data collection, analysis, and reporting barriers.

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.

Antimicrobial resistance (AMR) is a global public health threat, and the environment has been identified as an important reservoir for resistant microorganisms and genes. Storm overflows (SOs) discharge wastewater and stormwater, and are found throughout many wastewater networks. While there are no data currently showing the impact of SOs on the environment with respect to AMR in the UK, there is a small but growing body of evidence globally highlighting the potential role of SOs on environmental AMR. This review aims to provide an overview of the current state of SOs, describe global data investigating the impact of SOs on environmental AMR, and discuss the implications of SOs regarding AMR and human health. In addition, the complexities of studying the effects of SOs are discussed and a set of priority research questions and policy interventions to tackle a potentially emerging threat to public health are presented.