Biochemistry and Pharmacology - Research Publications

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    Detection of Klebsiella pneumoniae human gut carriage: a comparison of culture, qPCR, and whole metagenomic sequencing methods
    Lindstedt, K ; Buczek, D ; Pedersen, T ; Hjerde, E ; Raffelsberger, N ; Suzuki, Y ; Brisse, S ; Holt, K ; Samuelsen, O ; Sundsfjord, A (TAYLOR & FRANCIS INC, 2022-12-31)
    Klebsiella pneumoniae is an important opportunistic healthcare-associated pathogen and major contributor to the global spread of antimicrobial resistance. Gastrointestinal colonization with K. pneumoniae is a major predisposing risk factor for infection and forms an important hub for the dispersal of resistance. Current culture-based detection methods are time consuming, give limited intra-sample abundance and strain diversity information, and have uncertain sensitivity. Here we investigated the presence and abundance of K. pneumoniae at the species and strain level within fecal samples from 103 community-based adults by qPCR and whole metagenomic sequencing (WMS) compared to culture-based detection. qPCR demonstrated the highest sensitivity, detecting K. pneumoniae in 61.2% and 75.8% of direct-fecal and culture-enriched sweep samples, respectively, including 52/52 culture-positive samples. WMS displayed lower sensitivity, detecting K. pneumoniae in 71.2% of culture-positive fecal samples at a 0.01% abundance cutoff, and was inclined to false positives in proportion to the relative abundance of other Enterobacterales present. qPCR accurately quantified K. pneumoniae to 16 genome copies/reaction while WMS could estimate relative abundance to at least 0.01%. Quantification by both methods correlated strongly with each other (Spearman's rho = 0.91). WMS also supported accurate intra-sample K. pneumoniae sequence type (ST)-level diversity detection from fecal microbiomes to 0.1% relative abundance, agreeing with the culture-based detected ST in 16/19 samples. Our results show that qPCR and WMS are sensitive and reliable tools for detection, quantification, and strain analysis of K. pneumoniae from fecal samples with potential to support infection control and enhance insights in K. pneumoniae gastrointestinal ecology.
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    COVID-19 vaccine hesitancy among pregnant women: a systematic review and meta-analysis.
    Bhattacharya, O ; Siddiquea, BN ; Shetty, A ; Afroz, A ; Billah, B (BMJ, 2022-08-18)
    OBJECTIVE: The aim of this study was to perform a systematic review and meta-analysis to estimate the vaccines' acceptance level and to find the factors influencing pregnant women's vaccination decisions, with the goal of assisting in the development of interventions and promoting more research in this area. DESIGN: Systematic review and meta-analysis. DATA SOURCES: MEDLINE, Embase, CINAHL and PubMed. ELIGIBILITY CRITERIA: Studies providing any kind of quantitative assessment of overall COVID-19 vaccination acceptance among pregnant women in any country or region across the globe. DATA EXTRACTION AND SYNTHESIS: The pooled prevalence of COVID-19 vaccine acceptance among pregnant women was calculated using the random-effects model. Subgroup (sensitivity) analysis was performed to determine the overall COVID-19 vaccine acceptance level to understand the sources of substantial heterogeneity. RESULTS: Out of the 375 studies identified, 17 studies from four continents assessing 25 147 participants (pregnant women) were included in this study. Among the participants, only 49% (95% CI 42% to 56%, p<0.001) had COVID-19 vaccine acceptance. High-income countries (47%; 95% CI 38% to 55%, p<0.001), participants with fewer than 12 years of education (38%; 95% CI 19% to 58%, p<0.001) and multiparous women (48%; 95% CI 31% to 66%, p<0.001) had lower COVID-19 vaccine acceptance. Overall heterogeneity was high (I2 ≥98%), and publication bias was present (p<0.001). A very weak positive correlation between COVID-19 knowledge and COVID-19 vaccine acceptance was observed (r=0.164; 95% CI -0.946 to 0.972; p=0.8359). CONCLUSION: Overall, COVID-19 vaccine acceptance among pregnant women was low across the studies and considerably low among some specific subgroups of participants. These research findings have implications for the development of effective interventions that could increase the COVID-19 vaccine acceptance level among pregnant women to attain herd immunity. PROSPERO REGISTRATION NUMBER: CRD42021277754.
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    Genomic population structures of microbial pathogens
    Holt, KE ; Aanensen, DM ; Achtman, M (ROYAL SOC, 2022-10-10)
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    Influenza A virus elicits peri-vascular adipose tissue inflammation and vascular dysfunction of the aorta in pregnant mice.
    Oseghale, O ; Liong, S ; Coward-Smith, M ; To, EE ; Erlich, JR ; Luong, R ; Liong, F ; Miles, M ; Norouzi, S ; Martin, C ; O'Toole, S ; Brooks, RD ; Bozinovski, S ; Vlahos, R ; O'Leary, JJ ; Brooks, DA ; Selemidis, S ; Klein, SL (Public Library of Science (PLoS), 2022-08)
    Influenza A virus (IAV) infection during pregnancy initiates significant aortic endothelial and vascular smooth muscle dysfunction, with inflammation and T cell activation, but the details of the mechanism are yet to be clearly defined. Here we demonstrate that IAV disseminates preferentially into the perivascular adipose tissue (PVAT) of the aorta in mice. IAV mRNA levels in the PVAT increased at 1-3 days post infection (d.p.i) with the levels being ~4-8 fold higher compared with the vessel wall. IAV infection also increased Ly6Clow patrolling monocytes and Ly6Chigh pro-inflammatory monocytes in the vessel wall at 3 d.p.i., which was then followed by a greater homing of these monocytes into the PVAT at 6 d.p.i. The vascular immune phenotype was characteristic of a "vascular storm"- like response, with increases in neutrophils, pro-inflammatory cytokines and oxidative stress markers in the PVAT and arterial wall, which was associated with an impairment in endothelium-dependent relaxation to acetylcholine. IAV also triggered a PVAT compartmentalised elevation in CD4+ and CD8+ activated T cells. In conclusion, the PVAT of the aorta is a niche that supports IAV dissemination and a site for perpetuating a profound innate inflammatory and adaptive T cell response. The manifestation of this inflammatory response in the PVAT following IAV infection may be central to the genesis of cardiovascular complications arising during pregnancy.
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    Repurposing the mitotic machinery to drive cellular elongation and chromatin reorganisation in Plasmodium falciparum gametocytes
    Li, J ; Shami, GJ ; Cho, E ; Liu, B ; Hanssen, E ; Dixon, MWA ; Tilley, L (NATURE PORTFOLIO, 2022-08-27)
    The sexual stage gametocytes of the malaria parasite, Plasmodium falciparum, adopt a falciform (crescent) shape driven by the assembly of a network of microtubules anchored to a cisternal inner membrane complex (IMC). Using 3D electron microscopy, we show that a non-mitotic microtubule organizing center (MTOC), embedded in the parasite's nuclear membrane, orients the endoplasmic reticulum and the nascent IMC and seeds cytoplasmic microtubules. A bundle of microtubules extends into the nuclear lumen, elongating the nuclear envelope and capturing the chromatin. Classical mitotic machinery components, including centriolar plaque proteins, Pfcentrin-1 and -4, microtubule-associated protein, End-binding protein-1, kinetochore protein, PfNDC80 and centromere-associated protein, PfCENH3, are involved in the nuclear microtubule assembly/disassembly process. Depolymerisation of the microtubules using trifluralin prevents elongation and disrupts the chromatin, centromere and kinetochore organisation. We show that the unusual non-mitotic hemispindle plays a central role in chromatin organisation, IMC positioning and subpellicular microtubule formation in gametocytes.
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    Ultrastructural Characteristics of DHA-Induced Pyroptosis
    Herr, DR ; Yam, TYA ; Tan, WSD ; Koh, SS ; Wong, WSF ; Ong, W-Y ; Chayaburakul, K (HUMANA PRESS INC, 2020-01-04)
    Microglial cells are resident macrophages of the central nervous system (CNS) that respond to bioactive lipids such as docosahexaenoic acid (DHA). Low micromolar concentrations of DHA typically promote anti-inflammatory functions of microglia, but higher concentrations result in a form of pro-inflammatory programmed cell death known as pyroptosis. This study used scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to investigate the morphological characteristics of pyroptosis in BV-2 microglial cells following exposure to 200 µM DHA. Vehicle-treated cells are characterized by extended processes, spine-like projections or 0.4 to 5.2 µm in length, and numerous extracellular vesicles (EVs) tethered to the surface of the plasma membrane. In contrast to vehicle-treated cells, gross abnormalities are observed after treating cells with 200 µM DHA for 4 h. These include the appearance of numerous pits or pores of varying sizes across the cell surface, structural collapse and flattening of the cell shape. Moreover, EVs and spines were lost following DHA treatment, possibly due to release from the cell surface. The membrane pores appear after DHA treatment initially measured ~ 30 nm, consistent with the previously reported gasdermin D (GSDMD) pore complexes. Complete collapse of cytoplasmic organization and loss of nuclear envelope integrity were also observed in DHA-treated cells. These processes are morphologically distinct from the changes that occur during cisplatin-induced apoptosis, such as the appearance of apoptotic bodies and tightly packed organelles, and the maintenance of EVs and nuclear envelope integrity. Cumulatively, this study provides a systematic description of the ultrastructural characteristics of DHA-induced pyroptosis, including distinguishing features that differentiate this process from apoptosis.
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    Ribosomal Protein S3 Gene Silencing Protects Against Cigarette Smoke-Induced Acute Lung Injury
    Dong, J ; Liao, W ; Peh, HY ; Tan, WSD ; Zhou, S ; Wong, WSF (CELL PRESS, 2018-09-07)
    Chronic obstructive pulmonary disease (COPD) is estimated to be the third leading cause of death by 2030. Transcription factor NF-κB may play a critical role in COPD pathogenesis. Ribosomal protein S3 (RPS3), a 40S ribosomal protein essential for executing protein translation, has recently been found to interact with the NF-κB p65 subunit and promote p65 DNA-binding activity. We sought to study whether RPS3 gene silencing could protect against cigarette-smoke (CS)-induced acute lung injury in a mouse model. Effects of an intratracheal RPS3 siRNA in CS-induced lung injury were determined by measuring bronchoalveolar lavage (BAL) fluid cell counts, levels of inflammatory and oxidative damage markers, and NF-κB translocation. Lung RPS3 level was found to be upregulated for the first time with CS exposure, and RPS3 siRNA blocked CS-induced neutrophil counts in BAL fluid. RPS3 siRNA suppressed CS-induced lung inflammatory mediator and oxidative damage marker levels, as well as nuclear p65 accumulation and transcriptional activation. RPS3 siRNA was able to disrupt CS extract (CSE)-induced NF-κB activation in an NF-κB reporter gene assay. We report for the first time that RPS3 gene silencing ameliorated CS-induced acute lung injury, probably via interruption of the NF-κB activity, postulating that RPS3 is a novel therapeutic target for COPD.
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    ESBL plasmids in Klebsiella pneumoniae: diversity, transmission and contribution to infection burden in the hospital setting.
    Hawkey, J ; Wyres, KL ; Judd, LM ; Harshegyi, T ; Blakeway, L ; Wick, RR ; Jenney, AWJ ; Holt, KE (Springer Science and Business Media LLC, 2022-08-23)
    BACKGROUND: Resistance to third-generation cephalosporins, often mediated by extended-spectrum beta-lactamases (ESBLs), is a considerable issue in hospital-associated infections as few drugs remain for treatment. ESBL genes are often located on large plasmids that transfer horizontally between strains and species of Enterobacteriaceae and frequently confer resistance to additional drug classes. Whilst plasmid transmission is recognised to occur in the hospital setting, the frequency and impact of plasmid transmission on infection burden, compared to ESBL + strain transmission, is not well understood. METHODS: We sequenced the genomes of clinical and carriage isolates of Klebsiella pneumoniae species complex from a year-long hospital surveillance study to investigate ESBL burden and plasmid transmission in an Australian hospital. Long-term persistence of a key transmitted ESBL + plasmid was investigated via sequencing of ceftriaxone-resistant isolates during 4 years of follow-up, beginning 3 years after the initial study. RESULTS: We found 25 distinct ESBL plasmids. We identified one plasmid, which we called Plasmid A, that carried blaCTX-M-15 in an IncF backbone similar to pKPN-307. Plasmid A was transmitted at least four times into different Klebsiella species/lineages and was responsible for half of all ESBL episodes during the initial 1-year study period. Three of the Plasmid A-positive strains persisted locally 3-6 years later, and Plasmid A was detected in two additional strain backgrounds. Overall Plasmid A accounted for 21% of ESBL + infections in the follow-up period. CONCLUSIONS: Here, we systematically surveyed ESBL strain and plasmid transmission over 1 year in a single hospital network. Whilst ESBL plasmid transmission events were rare in this setting, they had a significant and sustained impact on the burden of ceftriaxone-resistant and multidrug-resistant infections. If onward transmission of Plasmid A-carrying strains could have been prevented, this may have reduced the number of opportunities for Plasmid A to transmit and create novel ESBL + strains, as well as reducing overall ESBL infection burden.
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    Epidemiology and genomic analysis of Klebsiella oxytoca from a single hospital network in Australia
    Stewart, J ; Judd, LM ; Jenney, A ; Holt, KE ; Wyres, KL ; Hawkey, J (BMC, 2022-08-24)
    BACKGROUND: Infections caused by Klebsiella oxytoca are the second most common cause of Klebsiella infections in humans. Most studies have focused on K. oxytoca outbreaks and few have examined the broader clinical context of K. oxytoca. METHODS: Here, we collected all clinical isolates identified as K. oxytoca in a hospital microbiological diagnostic lab across a 15-month period (n = 239). Whole genome sequencing was performed on a subset of 92 isolates (all invasive, third-generation cephalosporin resistant (3GCR) and non-urinary isolates collected > 48 h after admission), including long-read sequencing on a further six isolates with extended-spectrum beta-lactamase or carbapenemase genes. RESULTS: The majority of isolates were sensitive to antimicrobials, however 22 isolates were 3GCR, of which five were also carbapenem resistant. Genomic analyses showed those identified as K. oxytoca by the clinical laboratory actually encompassed four distinct species (K. oxytoca, Klebsiella michiganensis, Klebsiella grimontii and Klebsiella pasteurii), referred to as the K. oxytoca species complex (KoSC). There was significant diversity within the population, with only 10/67 multi-locus sequence types (STs) represented by more than one isolate. Strain transmission was rare, with only one likely event identified. Six isolates had extended spectrum beta-lactamase (blaSHV-12 and/or blaCTX-M-9) or carbapenemase (blaIMP-4) genes. One pair of K. michiganensis and K. pasteurii genomes carried identical blaIMP-4 IncL/M plasmids, indicative of plasmid transmission. CONCLUSION: Whilst antimicrobial resistance was rare, the resistance plasmids were similar to those found in other Enterobacterales, demonstrating that KoSC has access to the same plasmid reservoir and thus there is potential for multi-drug resistance. Further genomic studies are required to improve our understanding of the KoSC population and facilitate investigation into the attributes of successful nosocomial isolates.
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    Archaic chaperone-usher pili self-secrete into superelastic zigzag springs.
    Pakharukova, N ; Malmi, H ; Tuittila, M ; Dahlberg, T ; Ghosal, D ; Chang, Y-W ; Myint, SL ; Paavilainen, S ; Knight, SD ; Lamminmäki, U ; Uhlin, BE ; Andersson, M ; Jensen, G ; Zavialov, AV (Springer Science and Business Media LLC, 2022-09)
    Adhesive pili assembled through the chaperone-usher pathway are hair-like appendages that mediate host tissue colonization and biofilm formation of Gram-negative bacteria1-3. Archaic chaperone-usher pathway pili, the most diverse and widespread chaperone-usher pathway adhesins, are promising vaccine and drug targets owing to their prevalence in the most troublesome multidrug-resistant pathogens1,4,5. However, their architecture and assembly-secretion process remain unknown. Here, we present the cryo-electron microscopy structure of the prototypical archaic Csu pilus that mediates biofilm formation of Acinetobacter baumannii-a notorious multidrug-resistant nosocomial pathogen. In contrast to the thick helical tubes of the classical type 1 and P pili, archaic pili assemble into an ultrathin zigzag architecture secured by an elegant clinch mechanism. The molecular clinch provides the pilus with high mechanical stability as well as superelasticity, a property observed for the first time, to our knowledge, in biomolecules, while enabling a more economical and faster pilus production. Furthermore, we demonstrate that clinch formation at the cell surface drives pilus secretion through the outer membrane. These findings suggest that clinch-formation inhibitors might represent a new strategy to fight multidrug-resistant bacterial infections.