Ultrastructural and morphological studies on variables affecting Escherichia coli with selected commercial antibiotics.

Background: Many studies reported the effects of antibiotic exposure on E. coli bacterial growth and cell modification. However, scarce descriptive information on ultrastructural effects upon exposure of commercial antibiotics. Methods: This study described the morphological and ultrastructural alterations caused by selected antibiotics (amoxicillin-clavulanate, ceftriaxone, polymyxin B, colistin, gentamicin, and amikacin) that targeted cell wall, plasma membrane, and cytoplasmic density, and also proteins synthesis. We determined extracellular morphological changes of exposure through scanning electron microscopy (FESEM) and intracellular activities through transmission electron microscopy (TEM) investigation. Results: FESEM and TEM micrograph of E. coli exposed with selected antibiotics shows ultrastructural changes in beta-lactam class (amoxicillin-clavulanate, ceftriaxone) elongated the cells as the cell wall was altered as it inhibits bacterial cell wall synthesis, polymyxin class (polymyxin B, colistin) had plasmid and curli-fimbriae as it breaking down the plasma/cytoplasmic membrane, and aminoglycoside class (gentamicin, and amikacin) reduced ribosome concentration as it inhibits bacterial protein synthesis by binding to 30 s ribosomes. Conclusion: Morphological and ultrastructural alterations of E. coli's mechanism of actions were translated and depicted. This study could be reference for characterization studies for morphological and ultrastructural of E. coli upon exposure to antimicrobial agents.

Droplet digital PCR application for the detection of SARS-CoV-2 in air sample.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) may transmit through airborne route particularly when the aerosol particles remain in enclosed spaces with inadequate ventilation. There has been no standard recommended method of determining the virus in air due to limitations in pre-analytical and technical aspects. Furthermore, the presence of low virus loads in air samples could result in false negatives. Our study aims to explore the feasibility of detecting SARS-CoV-2 ribonucleic acid (RNA) in air samples using droplet digital polymerase chain reaction (ddPCR). Active and passive air sampling was conducted between December 2021 and February 2022 with the presence of COVID-19 confirmed cases in two hospitals and a quarantine center in Klang Valley, Malaysia. SARS-CoV-2 RNA in air was detected and quantified using ddPCR and real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The comparability of two different digital PCR platforms (QX200 and QIAcuity) to RT-PCR were also investigated. Additionally negative staining transmission electron microscopy was performed to visualize virus ultrastructure. Detection rates of SARS-CoV-2 in air samples using ddPCR were higher compared to RT-PCR, which were 15.2% (22/145) and 3.4% (5/145), respectively. The sensitivity and specificity of ddPCR was 100 and 87%, respectively. After excluding 17 negative samples (50%) by both QX200 and QIAcuity, 15% samples (5/34) were found to be positive both ddPCR and dPCR. There were 23.5% (8/34) samples that were detected positive by ddPCR but negative by dPCR. In contrast, there were 11.7% (4/34) samples that were detected positive by dPCR but negative by ddPCR. The SARS-CoV-2 detection method by ddPCR is precise and has a high sensitivity for viral RNA detection. It could provide advances in determining low viral titter in air samples to reduce false negative reports, which could complement detection by RT-PCR.

Vero CCL-81 and Calu-3 Cell Lines as Alternative Hosts for Isolation and Propagation of SARS-CoV-2 Isolated in Malaysia.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as the etiologic agent for the pneumonia outbreak that started in early December 2019 in Wuhan City, Hubei Province, China. To date, coronavirus disease (COVID-19) has caused almost 6 million deaths worldwide. The ability to propagate the virus into a customizable volume will enable better research on COVID-19 therapy, vaccine development, and many others. In the search for the most efficient replication host, we inoculated three (3) local SARS-CoV-2 isolates of different lineages (Clade L/Lineage B Wuhan, Clade GR/Lineage B.1.1.354, and Clade O/Lineage B.6.2) into various clinically important mammalian cell lines. The replication profile of these isolates was evaluated based on the formation of cytopathic effects (CPE), viral load (Ct value and plaque-forming unit (pfu)), as well as observation by electron microscopy (EM). Next-generation sequencing (NGS) was performed to examine the genomic stability of the propagated SARS-CoV-2 in these cell lines. Our study found that Vero E6 and Vero CCL-81 cell lines posed similar capacities in propagating the local isolates, with Vero CCL-81 demonstrating exceptional potency in conserving the genomic stability of the Lineage B Wuhan isolate. In addition, our study demonstrated the utility of Calu-3 cells as a replication host for SARS-CoV-2 without causing substantial cellular senescence. In conclusion, this study provides crucial information on the growth profile of Malaysian SARS-CoV-2 in various mammalian cell lines and thus will be a great source of reference for better isolation and propagation of the SARS-CoV-2 virus isolated in Malaysia.

Neuroprotective effect of phospholipase A2 from Malaysian Naja sumatrana venom against H2O2-induced cell damage and apoptosis.

Oxidative stress is one of the factors involved in the pathogenesis of several neurodegenerative diseases. It has been reported that a secretory phospholipase A2 known as A2-EPTX-NSm1a has lower cytotoxicity in neuronal cells compared to its crude Naja sumatrana venom. In this study, A2-EPTX-NSm1a was tested for its neuroprotective activity on human neuroblastoma cells (SH-SY5Y) differentiated into cholinergic neurons against oxidative stress induced by hydrogen peroxide (H2O2). H2O2 treatment alone increased the caspase-3 and caspase-8 activities, whereas pre-treatment with A2-EPTX-NSm1a reduced the activity of these apoptosis-associated proteins. Moreover, A2-EPTX-NSm1a protects the morphology and ultrastructure of differentiated SH-SY5Y cells in the presence of H2O2. Oxidative stress increased the number of small mitochondria. Further evaluation showed the size of mitochondria with a length below 0.25 µm in oxidative stress conditions is higher than the control group, suggesting mitochondria fragmentation. Pre-treatment with A2-EPTX-NSm1a attenuated the number of mitochondria in cells with H2O2 Furthermore, A2-EPTX-NSm1a altered the expression of several neuroprotein biomarkers of GDNF, IL-8, MCP-1, TIMP-1, and TNF-R1 in cells under oxidative stress induced by H2O2. These findings indicate that anti-apoptosis with mitochondria-related protection, anti-inflammatory effect, and promote expression of important markers for cell survival may underlie the neuroprotective effect of A2-EPTX-NSm1a in cholinergic rich human cells under oxidative stress, a vital role in the neuronal disorder.