Human Clinical Samples Research Articles

Genomic characteristics of cfr and fexA carrying Staphylococcus aureus isolated from pig carcasses in Korea

The emergence of transferable linezolid resistance genes poses significant challenges to public health, as it does not only confer linezolid resistance but also reduces susceptibility to florfenicol, which is widely used in the veterinary field. This study evaluated the genetic characteristics of linezolid-resistant Staphylococcus aureus strains isolated from pig carcasses and further clarified potential resistance and virulence mechanisms in a newly identified sequence type. Of more than 2500 strains isolated in a prior study, 15 isolated from pig carcasses exhibited linezolid resistance (minimum inhibitory concentration ≥ 8 mg/L). The strains were characterized in detail by genomic analysis. Linezolid-resistant S. aureus strains exhibited a high degree of genetic lineage diversity, with one strain (LNZ_R_SAU_64) belonging to ST8004, which has not been reported previously. The 15 strains carried a total of 21 antibiotic resistance genes, and five carried mecA associated with methicillin resistance. All strains harbored cfr and fexA, which mediate resistance to linezolid, phenicol, and other antibiotics. Moreover, the strains carried enterotoxin gene clusters, including the hemolysin, leukotoxin, and protease genes, which are associated with humans or livestock. Some genes were predicted to be carried in plasmids or flanked by ISSau9 and the transposon Tn554, thus being transmittable between staphylococci. Strains carrying the plasmid replicon repUS5 displayed high sequence similarity (99%) to the previously reported strain pSA737 in human clinical samples in the United States. The results illustrate the need for continuous monitoring of the prevalence and transmission of linezolid-resistant S. aureus isolated from animals and their products.

Evaluating Mass Spectrometry-Based Hydroxyl Radical Protein Footprinting of a Benchtop Flash Oxidation System against a Synchrotron X-ray Beamline.

Hydroxyl radical protein footprinting (HRPF) using synchrotron X-ray radiation (XFP) and mass spectrometry is a well-validated structural biology method that provides critical insights into macromolecular structural dynamics, such as determining binding sites, measuring affinity, and mapping epitopes. Numerous alternative sources for generating the hydroxyl radicals (•OH) needed for HRPF, such as laser photolysis and plasma irradiation, complement synchrotron-based HRPF, and a recently developed commercially available instrument based on flash lamp photolysis, the FOX system, enables access to laboratory benchtop HRPF. Here, we evaluate performing HRPF experiments in-house with a benchtop FOX instrument compared to synchrotron-based X-ray footprinting at the NSLS-II XFP beamline. Using lactate oxidase (LOx) as a model system, we carried out •OH labeling experiments using both instruments, followed by nanoLC-MS/MS bottom-up peptide mass mapping. Experiments were performed under high glucose concentrations to mimic the highly scavenging conditions present in biological buffers and human clinical samples, where less •OH are available for reaction with the biomolecule(s) of interest. The performance of the FOX and XFP HRPF methods was compared, and we found that tuning the •OH dosage enabled optimal labeling coverage for both setups under physiologically relevant highly scavenging conditions. Our study demonstrates the complementarity of FOX and XFP labeling approaches, demonstrating that benchtop instruments such as the FOX photolysis system can increase both the throughput and the accessibility of the HRPF technique.

CircLZIC regulates ox-LDL-induced HUVEC cell proliferation and apoptosis via Micro-330-5p/NOTCH2 axis in atherosclerosis.

Atherosclerosis (AS) is a major chronic non-communicable disease and a primary cause of cardiovascular disease. Recent studies have shown that circRNAs are potential epigenetic factors that regulate vascular endothelial inflammatory responses and AS progression. Therefore, identification of the circRNAs that regulate ox-LDL levels is a critical step to understanding the pathology of AS. Our study is aim to investigate how circLZIC regulates atherosclerosis (AS) via the Micro-330-5p/NOTCH2 regulatory axis. The results showed that CircLZIC and NOTCH2 are highly expressed in human AS clinical samples, while Micro-330-5p is expressed locally. The CCK-8 experiment results showed that circLZIC promotes the proliferation of HUVECS cells. Flow cytometry analysis showed that circLZIC act as an inhibitor of HUVEC cell apoptosis. The expression level of Micro-330-5p can be up-regulated by transfection of small interfering RNA against circLZIC. Further, Starbase predicted that Micro-330-5p could target and regulate NOTCH2. Next, we confirmed that overexpression of Micro-330-5p could significantly reduce the expression of fluorescein using the double Luciferase reporter assay. RIP-qRT-PCR experiment showed that Micro-330-5p and NOTCH2 mRNAs are effectively enriched by ago2 protein. Further, we found that knocking down circLZIC increases the expression of Micro-330-5p and promotes cell apoptosis, while inhibiting the expression of NOTCH2 and cell activity. On the other hand, co-transfection of Micro-330-5p inhibitor decreases Micro-330-5p expression and inhibit cell apoptosis, while increasing NOTCH2 expression and cell activity. In conclusion, CircLZIC regulates HUVEC cell activity by the Micro-330-5p/NOTCH2 signaling pathway, suggesting that circLZIC plays a key role in atherosclerosis development.

A MICROPHYSIOLOGICAL SYSTEM OF INFLAMMATION AND FIBROSIS IN TENDON FIBROVASCULAR SCAR

Vascular inflammation and activation of myofibroblasts are significant contributors to the progression of fibrosis, which can severely impair tissue function. In various tissues, including tendons, Transforming growth factor beta 1 (TGF-β1) has been identified as a critical driver of adhesion and scar formation. Nevertheless, the mechanisms that underlie fibrotic peritendinous adhesions are still not well comprehended, and human microphysiological systems to help identify effective therapies remain scarce. To address this issue, we developed a novel human Tendon-on-a-Chip (hToC), comprised of an endothelialized vascular compartment harboring circulating monocytes and separated by a 5 μm/100 nm dual-scale ultrathin porous membrane from a type I/III collagen hydrogel with primary tendon fibroblasts and tissue-resident macrophages, all under defined serum-free conditions. The hToC models the crosstalk of the various cells in the system leading to the induction of inflammatory and fibrotic pathways including the activation of mTOR signaling. Consistent with phenotypes observed in vivo in mouse models and clinical human samples, we observed myofibroblast differentiation and senescence, tissue contraction, excessive extracellular matrix deposition, and monocytes’ transmigration and macrophages’ secretion of inflammatory cytokines, which were dependent on the presence of the endothelial barrier. This model offers novel insights on the role of vasculature in the pathophysiology of adhesions, which were previously underappreciated. Moreover, in testing whether the hToC could be used to evaluate efficacy of therapeutics, we were able to capture donor-specific variability in the response to Rapamycin treatment, which reduced myofibroblast activation regardless. Thus, our findings demonstrate the value of the hToC as a human microphysiological system for investigating the pathophysiology of fibrotic conditions in the context of peritendinous injury and similar fibrotic conditions, providing an alternative to animal testing.

Identification and antifungal susceptibility testing of Candida haemulonii complex isolated from clinical samples.

Candida haemulonii complex (Candida haemulonii [I], Candida duobushaemulonii [II], and Candida haemulonii var. vulnera [III]) has become relevant in recent times, not so much because of a high incidence in human clinical sample cultures but because of its remarkable antifungal resistance. The objective of this study was to evaluate several methods for the identification of this uncommon species of Candida. Ten isolates of C. haemulonii were identified by biochemical and proteomic methods, and their antifungal susceptibility testing was performed by both commercial and reference methods. MALDI-TOF MS (Vitek MS and Vitek MS PRIME) and Vitek2 correctly identified these genera but API method did not. There was a good correlation between the commercial methods and the reference methods for the AST. In conclusion Vitek MS, Vitek MS PRIME, and Vitek2 systems, but not API32C, are reliable for identification of C. haemulonii complex. Furthermore, MALDI-TOF MS systems could identify to the subspecies level. Commercial methods for antifungal susceptibility testing are valid for the study of this species and confirm amphotericin B and to azole resistance.

Circulating small extracellular vesicles microRNAs plus CA-125 for treatment stratification in advanced ovarian cancer

BackgroundNo residual disease (R0 resection) after debulking surgery is the most critical independent prognostic factor for advanced ovarian cancer (AOC). There is an unmet clinical need for selecting primary or interval debulking surgery in AOC patients using existing prediction models.MethodsRNA sequencing of circulating small extracellular vesicles (sEVs) was used to discover the differential expression microRNAs (DEMs) profile between any residual disease (R0, n = 17) and no residual disease (non-R0, n = 20) in AOC patients. We further analyzed plasma samples of AOC patients collected before surgery or neoadjuvant chemotherapy via TaqMan qRT-PCR. The combined risk model of residual disease was developed by logistic regression analysis based on the discovery-validation sets.ResultsUsing a comprehensive plasma small extracellular vesicles (sEVs) microRNAs (miRNAs) profile in AOC, we identified and optimized a risk prediction model consisting of plasma sEVs-derived 4-miRNA and CA-125 with better performance in predicting R0 resection. Based on 360 clinical human samples, this model was constructed using least absolute shrinkage and selection operator (LASSO) and logistic regression analysis, and it has favorable calibration and discrimination ability (AUC:0.903; sensitivity:0.897; specificity:0.910; PPV:0.926; NPV:0.871). The quantitative evaluation of Net Reclassification Improvement (NRI) and Integrated Discrimination Improvement (IDI) suggested that the additional predictive power of the combined model was significantly improved contrasted with CA-125 or 4-miRNA alone (NRI = 0.471, IDI = 0.538, p < 0.001; NRI = 0.122, IDI = 0.185, p < 0.01).ConclusionOverall, we established a reliable, non-invasive, and objective detection method composed of circulating tumor-derived sEVs 4-miRNA plus CA-125 to preoperatively anticipate the high-risk AOC patients of residual disease to optimize clinical therapy.

Abstract A162: A comprehensive platform for unraveling the molecular mechanisms and vulnerabilities of colorectal cancer: A step forward in target discovery

Abstract Colorectal cancer (CRC) is a heterogeneous malignancy that originates from the colonic and rectal epithelium. It ranks among the most prevalent and deadly cancers globally, representing about 10% of cancer incidence and 9% of cancer mortality in 2018. The oncogenesis and evolution of CRC are driven by the accumulation of genetic and epigenetic alterations that dysregulate key signaling pathways, such as Wnt, RAS, TGF-β, and DNA mismatch repair. However, the molecular mechanisms and vulnerabilities of CRC remain poorly understood, and many patients exhibit poor response or resistance to the current therapies. Therefore, there is an urgent need for novel and effective therapies that can target the specific features and dependencies of different CRC subtypes. To better understand the molecular mechanisms and vulnerabilities of CRC, we established a comprehensive platform based on the CRC model cells derived from human intestinal stem cells (hISCs), patient-derived xenografts (PDXs), and clinical samples, on which we conducted integrative genomic and functional analyses. We employed CRISPR/Cas9 to introduce common CRC mutations, such as APC truncation, KRAS G12D, TP53 and SMAD4 knockouts into hISCs and generated isogenic CRC model cells with different mutational backgrounds. We also developed PDXs from CRC patients and collected biopsy samples from primary and metastatic tumors. We characterized the transcriptomic and genomic profiles of all the samples using RNA-seq and whole-exome sequencing (WES). Then, we conducted high-throughput drug screening (drug HTS) with a collection of standard-of-care drugs and CRISPR/Cas9 dropout screening with a genome-wide library on the CRC model cells. RNA-seq and WES analyses identified distinct molecular signatures and pathways associated with different mutational variants. We validated the presence of driver mutations in the patient-derived cultures, such as APC, KRAS, and TP53, and compared them with the model cells. Our primary cancer cell culture platform was proven suitable for high-throughput identification of new molecular targets through CRISPR/Cas9 dropout screening with a genome-wide library performed on CRC model cells. Additionally, our in-house drug HTS, utilizing a collection of standard-of-care drugs, met industrial standards of QC values such as signal-to-noise ratio, Z-factor, and robustness.. The QC values, such as read depth, library complexity, and alignment rate met the established criteria and we were able to identify essential and tumor suppressor gene sets. Moreover, by utilizing normal hIPCs, we were able to identify among the essential genes those that are critical only for the transformed cells. This innovative platform provides a powerful tool for target discovery and validation in CRC, with potential applicability to other cancer types and personalized medicine approaches. Our findings underscore the importance of using primary material-derived cells in the process of understanding the molecular intricacies of CRC and pave the way for the development of novel, effective therapies. Citation Format: Marcin Duleba, Joanna Szuszkiewicz, Marcin Serocki, Joanna Szklarczyk, Olga Dracz, Anna Ledwon, Michał Combik, Oleksii Bryzghalov, Agata Stachowicz, Michal Mikula, Krzysztof Brzozka, Rafal Dziadziuszko, Tomasz Rzymski, Eliza Zimolag, Andrzej Mazan. A comprehensive platform for unraveling the molecular mechanisms and vulnerabilities of colorectal cancer: A step forward in target discovery [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A162.

A Protein Suspension-Trapping Sample Preparation for Tear Proteomics by Liquid Chromatography-Tandem Mass Spectrometry.

Tear fluid is one of the easily accessible biofluids that can be collected non-invasively. Tear proteomics has the potential to discover biomarkers for several ocular diseases and conditions. The suspension trapping column has been reported to be an efficient and user-friendly sample preparation workflow for the broad application of downstream proteomic analysis. Yet, this strategy has not been well-studied in the analysis of human tear proteome. The present protocol describes an integrated workflow from clinical human tear samples to purified peptides for non-invasive tear protein biomarker research using mass spectrometry, which provides insights into disease biomarkers and monitoring when combined with bioinformatics analysis. A protein suspension trapping sample preparation was applied and demonstrated the discovery of tear proteome with fast, reproducible, and user-friendly procedures, as a universal, optimized sample preparation for human tear fluid analysis. In particular, the suspension trapping procedure outperformed in-solution sample preparation in terms of peptide recovery, protein identification, and shorter sample preparation time.

Redeveloping antigen detection kits for the diagnosis of rat hepatitis E virus.

The emergence of Rocahepevirus ratti [species HEV ratti (r HEV)] as a causative agent of hepatitis E in humans presents a new potential threat to global public health. The R. ratti genotype 1 (r-1 HEV) variant only shares 50%-60% genomic identity with Paslahepevirus balayani [species HEV balayani (b HEV)] variants, which are the main causes of hepatitis E infection in humans. Here, we report antigen diagnoses for r-1 HEV and b HEV using an enzymatic immunoassay (EIA) method. We detected recombinant virus-like particles protein (HEV 239) of r HEV and b HEV using a collection of hepatitis E virus (HEV)-specific monoclonal antibodies. Two optimal candidates, the capture antibody P#1-H4 and the detection antibodies C145 (P#1-H4*/C145#) and C158 (P#1-H4*/C158#), were selected to detect antigen in infected rat samples and r-1 HEV- or b HEV-infected human clinical samples. The two candidates showed similar diagnostic efficacy to the Wantai HEV antigen kit in b HEV-infected clinical samples. Genomic divergence resulted in low diagnostic efficacy of the Wantai HEV antigen kit (0%, 0 of 10) for detecting r-1 HEV infection. Compared with the P#1-H4*/C145# candidate (80%, 8 of 10), the P#1-H4*/C158# candidate had excellent diagnostic efficacy in r-1 HEV-infected clinical samples (100%, 10 of 10). The two candidates bind to a discrete antigenic site that is highly conserved across r HEV and b HEV. P#1-H4*/C145# and P#1-H4*/C158# are efficacious candidate antibody combinations for rat HEV antigen detection.

Detection of Over Expression of Adeb and Acrb –Tolc Efflux Systems Confers Ceftriaxone Resistance in Clinical Isolates of Acinetobacter Bumannii and Klebsiella Pneumoniae

Ceftriaxone’s widespread utilisation in treating infections brought on by bacteria with multidrug resistance (MDR) has led to an increase in clinical resistance to the antibiotic in recent years. This research seeks to extract and characterise K. pneumoniae and A. baumannii bacteria, as well as examine the expression of Acr-B and Ade-B resistance genes for anti-Ceftriaxone resistance. Due to the collection of 170 human clinical samples from a diversity of sources. After being cultured, inspected microscopically, subjected to biochemical analysis, and put through the Vitec device, the bacteria revealed that 35 of the isolates were K. pneumoniae and 16 were A. baumannii. The MIC of ceftriaxone was then measured using the nutrient broth technique, and efflux pump gene expression was analysed using quantitative reverse transcription-PCR. Gene expression results for the Acr-B and Ade-B genes indicated that there was a change in the levels of expression of these genes after ceftrazone treatment of the bacterial isolates; the value of gene expression for the AcrB-Ab gene was 0.8 relative to the control gene, the amount of gene expression for the AcrB-gene Kp was (0.02), and the gene expression of the Ade-B-Kp gene was (10.33), i.e., an increase of (9. No gene expression was identified for the AdeB-Ab gene (the value was 0).

5′ copyback defective viral genomes are major component in clinical and non-clinical influenza samples

Clinical samples from people with influenza disease have been analyzed to assess the presence and abundance of Defective Viral Genomes (DVGs), but these have not been assessed using the same bioinformatic pipeline. The type of DVG most described for influenza infections (deletion DVGs) differs from the most commonly described DVGs from non-segmented negative stranded viruses (5′ copyback). This could be attributed to either differences between viruses or the tools used to detect and characterize DVGs. Here we analyze several NGS datasets from people infected with different types of influenza virus using the same bioinformatic pipeline. We observe that 5′ copyback DVGs are prevalent in all human clinical samples but not in the cultured samples. To address this discrepancy between clinical and laboratory cultures, we infected cell culture and ferrets with an H5N8 influenza A virus (FLUAV) and analyzed the DVG composition. The results demonstrate that the DVG population is skewed toward 5′ copyback DVGs in the in vivo infections and deletion DVGs in the in vitro infections. This demonstrates that there are differences in vivo genome production and in vitro genome production, and this has implications for how the role of DVGs in clinical disease is studied. We also investigate the role the host cofactor ANP32B has in DVG production.

From farm to fork: Spread of a multidrug resistant Salmonella Infantis clone encoding blaCTX-M-1 on pESI-like plasmids in Central Italy

Salmonella enterica subsp. enterica serovar Infantis (S. Infantis) is one of the “top five Salmonella serovars” of clinical significance in the European Union (EU). Antimicrobial resistant and extended spectrum β-lactamase (ESBL) AmpC-producing S. Infantis have been described in food production systems and human clinical samples in Italy. Recently, an increase of MDR S. Infantis carrying blaCTX-M genes involved in 3rd generation cephalosporin resistance was noticed in the EU, including Italy, mainly due to the spread of S. Infantis harboring a pESI-like plasmid.The aim of this study was to investigate the occurrence of the S. Infantis pESI-like plasmid among antibiotic resistant S. Infantis strains isolated at different points of the food chain, and to provide a phylogenetic analysis to gain further insight on their transmission pathways from ‘farm to fork’.MDR S. Infantis strains (n. 35) isolated from 2016 to 2021 at different stages of the food chain (animals, food, food-related environments, and humans) were investigated with in depth molecular characterization using real-time PCR, S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) and whole genome sequencing (WGS).Our study reported the occurrence of S. Infantis strains harboring pESI-like plasmids, carrying blaCTX-M-1 genes, in Central Italy, at different sampling points along the food chain. Results confirmed the presence of a plasmid with a molecular size around 224–310 kb, thus consistent with the pESI-like, in 97 % of the 35 samples investigated. Two variants of S. Infantis pESI-like IncFIB(K)_1_Kpn3 were detected, one associated with the European clone carrying blaCTX-M-1 (21 isolates) and the other associated with U.S. isolates carrying blaCTX-M-65 (2 isolates, pESI-like U.S. variant). The majority was resistant to 3rd generation cephalosporins but none of the strains tested positive for the carbapenemase encoding genes. A total of 118 virulence genes were identified in isolates harboring the pESI-like plasmid. cgMLST and SNP-based analysis revealed the presence of one main cluster, composed by strains isolated from the environment, animals, food and humans.The results of this investigation underline the importance of phylogenetic studies to monitor and understand pathogen and AMR spread in a One Health approach.

Virulence genes and antibiotic resistance assessment of the blaCTX-M-15 gene in ESBL-producing Escherichia coli and Klebsiella pneumoniae from poultry food chain and human clinical cases in Brazil

Brazil is one of the world’s largest exporters of chicken-derived products. Consumer concerns regarding food contamination by multidrug-resistant bacteria capable of causing diseases have increased steadily over the years as they pose a significant public health risk. This study aimed to characterize strains of beta-lactam-resistant Escherichia coli and Klebsiella pneumoniae isolates from the poultry production chain and human clinical samples (chicken cloaca, chicken meat, human feces). A total of 36 isolates were examined, including 28 Escherichia coli and 8 Klebsiella pneumoniae isolates carrying the blaCTX-M-15 gene. These isolates were obtained from chicken cloaca and meat from poultry farms and slaughterhouses, respectively, as well as from human fecal samples from a clinical laboratory. All the establishments were located in São Paulo, Brazil. The isolates were characterized for their virulence genes by polymerase chain reaction, antimicrobial resistance by the disc diffusion method, serotyping by an agglutination test, and pathogenicity by an in vivo day-old chicken mortality test. The isolates exhibited a high frequency of Escherichia coli associated virulence genes such as iutA, iss, hlyF, ompT and iroN. In addition, mrkD was detected in the isolates. All isolates exhibited resistance to at least three different antimicrobial classes, and 21.4% (n = 6) of the Escherichia coli strains exhibited high pathogenicity in the day-old chicken assay in vivo. These results indicate a potential increase in the blaCTX-M-15 gene associated with virulence genes and antimicrobial resistance in Escherichia coli and Klebsiella pneumoniae. Both of these Enterobacteriaceae can be found in poultry feces and possibly contaminate poultry products, thus posing a risk of infection to other animals and humans, raising an alert about the sanitary aspects of food production in Brazil.

Employing the Anchor DSPE-PEG as a Redox Probe for Ratiometric Electrochemical Detection of Surface Proteins on Extracellular Vesicles with Aptamers.

Quantitative analysis of surface proteins on extracellular vesicles (EVs) has been considered to be a crucial approach for reflecting the status of diseases. Due to the diverse composition of surface proteins on EVs and the interference from nonvesicular proteins, accurately detecting the expression of surface proteins on EVs remains a challenging task. While membrane affinity molecules have been widely employed as EVs capture probes to address this issue, their inherent biochemical properties have not been effectively harnessed. In this paper, we found that the electrochemical redox activity of the DSPE-PEG molecule was diminished upon its insertion into the membrane of EVs. This observation establishes the DSPE-PEG molecule modified on the Au electrode surface as a capture and a redox probe for the electrochemical detection of EVs. By utilizing methylene blue-labeled aptamers, the targeted surface proteins of EVs can be detected by recording the ratio of the oxidation peak current of methylene blue and DSPE-PEG. Without complicated signal amplification, the detection limit for EVs is calculated to be 8.11 × 102 particles/mL. Using this platform, we directly analyzed the expression of CD63 and HER2 proteins on the surface of EVs in human clinical plasma samples, demonstrating its significant potential in distinguishing breast cancer patients from healthy individuals.

Foldable low-cost point-of-care device for testing blood coagulation using smartphones

Cardiovascular diseases (CVDs) caused by thrombotic events are a significant global health concern, affecting millions of people worldwide. The international normalized ratio (INR) is the most widely used measure of coagulation status, and frequent testing is required to adjust blood-thinning drug dosage, requiring hospital visits and experts to perform the test. Here we present a low-cost and portable smartphone-based device for screening INR levels from whole blood samples at the point of care. Our device uses a 3D printed platform and light-emitting diode backlight modules to create a uniform optical environment, and its foldable design allows for easy transport. Our device also features an algorithm that allows users to acquire and process video of sample flow in a microfluidic channel on their smartphone, providing a cost-effective and convenient option for blood coagulation monitoring at the point of care. We tested the performance of our smartphone-based INR device using both commercially available control samples and clinical human blood samples, demonstrating high accuracy and reliability. Our device has the potential to improve patient outcomes by enabling more frequent monitoring and, as appropriate, dosage adjustments of blood-thinning drugs, providing an affordable and portable option for screening INR levels at the point of care.

Carbapenem-resistant Escherichia coli in Black-headed gulls, the Danube, and human clinical samples: A One Health comparison of contemporary isolates

Our aim was to characterize and compare contemporary carbapenem-resistant Enterobacterales (CRE) isolates from gulls, the River Danube, and humans in Hungary, Budapest. Multiresistant Enterobacterales were sought for in 227 gull faecal and 24 Danube water samples from 2019 to 2020. Eosin-methylene blue agar containing 2 mg/L cefotaxime and Colilert-test containing 10 mg/L cefotaxime were used for gull and water samples, respectively. Isolates were characterized by polymerase chain reactions (PCRs); acquired carbapenemase producers were further analysed by whole-genome sequencing, together with 21 Hungarian human CR Escherichia coli (CREc) isolates. Gull and water samples exhibited a CRE prevalence of 7.4% (9/122) and 6.7% (7/105), none and 5/12 water samples yielded CRE from 2019 and 2020, respectively; CRE were found only in samples taken downstream of Budapest. The dominant species was Escherichia coli and the most prevalent carbapenemase was blaNDM-1. High-risk CREc clones were found both in gulls (ST224, ST372, ST744) and the Danube (ST10, ST354, ST410); the closest associations were between ST410 from humans and the Danube, among ST1437 among gulls, and between ST1437 in gulls and the Danube (46, 0, and 22-24 allelic distances, respectively). Direct links between human and gull isolates were not demonstrated. The study demonstrates potential epidemiological links among humans, a river crossing a city, and urbanised birds, suggesting a local transmission network. Water bodies receiving influent wastewater, together with animals using such habitats, may serve as a local reservoir system for CRE, highlighting the importance of One Health in CRE transmission, even in a country with a low CRE prevalence in humans.

Rapid inactivation and sample preparation for SARS-CoV-2 PCR-based diagnostics using TNA-Cifer Reagent E.

RT-qPCR remains a key diagnostic methodology for COVID-19/SARS-CoV-2. Typically, nasal or saliva swabs from patients are placed in virus transport media (VTM), RNA is extracted at the pathology laboratory, and viral RNA is measured using RT-qPCR. In this study, we describe the use of TNA-Cifer Reagent E in a pre-clinical evaluation study to inactivate SARS-CoV-2 as well as prepare samples for RT-qPCR. Adding 1 part TNA-Cifer Reagent E to 5 parts medium containing SARS-CoV-2 for 10 min at room temperature inactivated the virus and permitted RT-qPCR detection. TNA-Cifer Reagent E was compared with established column-based RNA extraction and purification methodology using a panel of human clinical nasal swab samples (n = 61), with TNA-Cifer Reagent E showing high specificity (100%) and sensitivity (97.37%). Mixtures of SARS-CoV-2 virus and TNA-Cifer Reagent E could be stored for 3 days at room temperature or for 2 weeks at 4°C without the loss of RT-qPCR detection sensitivity. The detection sensitivity was preserved when TNA-Cifer Reagent E was used in conjunction with a range of VTM for saliva samples but only PBS (Gibco) and Amies Orange for nasal samples. Thus, TNA-Cifer Reagent E improves safety by rapidly inactivating the virus during sample processing, potentially providing a safe means for molecular SARS-CoV-2 testing outside traditional laboratory settings. The reagent also eliminates the need for column-based and/or automated viral RNA extraction/purification processes, thereby providing cost savings for equipment and reagents, as well as reducing processing and handling times.

Antimicrobial Resistance Profile of Salmonella enterica Serovar typhi Isolated from Human Clinical Samples in Ebonyi State, Nigeria

The emergence of resistance to the frequent use of empirical treatment of uncomplicated enteric fever caused by Salmonella enterica serovar Typhi is on the increase. This study was designed to determine the antimicrobial Resistance profile of Salmonella enterica serovar Typhi isolated from human clinical samples in Ebonyi State. A non-duplicated stool culture of Salmonella enterica serovar Typhi of patients diagnosed with typhoid fever at General Hospital Onicha Igboeze were collected from the hospital ward namely: A &amp; E (n = 4), MS (n = 3), FS (n = 3), PD (n = 7), LW (n = 4), ORT (n = 1), LAB (n = 17), THE (n = 9), GOPD (n = 4), MM (n = 3). Antimicrobial studies of Salmonella enterica serovar Typhi were determined using the Kirby–Bauer disk diffusion method. The proportion of resistance ranges from 33 %-100% against colistin, cefepime, nalidixic acid, cefoxitin, amikacin, cefuroxime, and piperacillin-tazobactam but isolates were only susceptible to meropenem 100%. The use of antimicrobial agents for the treatment of Salmonella enterica serovar typhi infection should be guided with antimicrobial susceptibility testing, Nonetheless, the diversity of the Salmonella isolates as a result of the dissemination of these resistant genes is a call for concern and emphasizes a need for an extensive investigation for the presence of these genes in Ebonyi State as well as the implementation of strict antimicrobial policies in a bid to restrict the spread of these resistance genes and prevent the emergence of new resistant strains.

Dissemination of Multidrug-Resistant, Extensively Drug Resistant and Pandrug-Resistant Pseudomonas aeruginosa Isolates among In-Patients and Out-Patients in a Multi-Profile Health Care Settings

Pseudomonas aeruginosa is one of the most life-threatening pathogens, especially in healthcare settings, and a main contributor to multi-drug resistance (MDR), extensive-drug resistance (XDR), and pan-drug resistant (PDR) phenotypes. However, there is limited data on the degree of resistance of these isolates in this region. This study seeks to determine the distribution of MDR, XDR, and PDR Pseudomonas aeruginosa strains from different patient groups. A total of five hundred (500) non-duplicated strains of Pseudomonas aeruginosa of human clinical samples were collected from the Microbiology Laboratory Unit of Alex Ekwueme Teaching Hospital Abakaliki. The isolates were identified and re-characterized by standard microbiology techniques. MDR, XDR, and PDR were determined using the Kirby–Bauer disk diffusion method, and the results were analyzed using the Clinical Laboratory Standard Institute (CLSI) zone diameter breakpoints. The result shows that the overall resistant phenotype was MDR 50. 7%, XDR 20.5%, and PDR 9.6% while in samples from in-patients and out-patients, resistant phenotype proportions were MDR 43.2%, XDR 32.4%, PDR 10.1% and MDR 61.2%, XDR 29.7%, and PDR 18.5% respectively. Worrisomely, only a few tested antimicrobial agents (Amikacin, cefepime) were active against the test organism, presenting a limited therapeutic option. It is therefore imperative to establish strong regulative measures and guidelines that would help in curtailing the increasing dissemination of these superbugs in healthcare institutions in Nigeria.

An integrated public health response to an outbreak of Murray Valley encephalitis virus infection during the 2022-2023 mosquito season in Victoria.

Murray Valley encephalitis virus (MVEV) is a mosquito-borne flavivirus known to cause infrequent yet substantial human outbreaks around the Murray Valley region of south-eastern Australia, resulting in significant mortality. The public health response to MVEV in Victoria in 2022-2023 included a climate informed pre-season risk assessment, and vector surveillance with mosquito trapping and laboratory testing for MVEV. Human cases were investigated to collect enhanced surveillance data, and human clinical samples were subject to serological and molecular testing algorithms to assess for co-circulating flaviviruses. Equine surveillance was carried out via enhanced investigation of cases of encephalitic illness. Integrated mosquito management and active health promotion were implemented throughout the season and in response to surveillance signals. Mosquito surveillance included a total of 3,186 individual trapping events between 1 July 2022 and 20 June 2023. MVEV was detected in mosquitoes on 48 occasions. From 2 January 2023 to 23 April 2023, 580 samples (sera and CSF) were tested for flaviviruses. Human surveillance detected 6 confirmed cases of MVEV infection and 2 cases of "flavivirus-unspecified." From 1 September 2022 to 30 May 2023, 88 horses with clinical signs consistent with flavivirus infection were tested, finding one probable and no confirmed cases of MVE. The expanded, climate-informed vector surveillance system in Victoria detected MVEV in mosquitoes in advance of human cases, acting as an effective early warning system. This informed a one-health oriented public health response including enhanced human, vector and animal surveillance, integrated mosquito management, and health promotion.