Assessment of the reversibility of resistance in the absence of antibiotics and its relationship with the resistance gene's fitness cost: a genetic study with mcr-1.

BACKGROUND: The intensive use of antibiotics has resulted in strong natural selection for the evolution of antimicrobial resistance (AMR), but whether, and under what circumstances, the removal of antibiotics would result in a rapid reduction in AMR has been insufficiently explored. We aimed to test the hypothesis that in the simple, yet common, case of AMR conferred by a single gene, removing antibiotics would quickly reduce the prevalence of resistance if the AMR gene imposes a high fitness cost and costless resistance is extremely rare among its proximal mutants. METHODS: In this genetic study, to test our hypothesis, we used the mcr-1 gene in Escherichia coli, which confers resistance to the last-resort antibiotic colistin, as a model. A high-throughput reverse genetics approach was used to evaluate mcr-1 variants for their fitness cost and resistance levels relative to a non-functional construct, by measuring relative growth rates in colistin-free media and at 2 µg/mL and 4 µg/mL colistin. We identified costless resistant mcr-1 mutants, and examined their properties within the context of the sequential organisation of mcr-1's functional domains as well as the evolutionary accessibility of these mutations. Finally, a simple population genetic model incorporating the measured fitness cost was constructed and tested against previously published real-world data of mcr-1 prevalence in colonised inpatients in China since the 2017 colistin ban in fodder additives. FINDINGS: We estimated the relative growth rates of 14 742 mcr-1 E coli variants (including the wild type), 3449 of which were single-nucleotide mutants. E coli showed 73·8% less growth per 24 h when carrying wild-type mcr-1 compared with the non-functional construct. 6252 (42·4%) of 14 741 mcr-1 mutants showed colistin resistance accompanied by significant fitness costs, when grown under 4 µg/mL colistin selection. 43 (0·3%) mcr-1 mutants exhibited costless resistance, most of which contained multiple mutations. Among the 3449 single mutants of mcr-1, 3433 (99·5%) had a fitness cost when grown in colistin-free media, with a mean relative growth of 0·305 (SD 0·193) compared with the non-functional variant. 3059 (88·7%) and 1833 (53·1%) of 3449 single mutants outgrew the non-functional mcr-1 in the presence of 2 µg/mL and 4 µg/mL colistin, respectively. Single mutations that gave rise to costless mutants were rare in all three domains of mcr-1 (transmembrane domain, flexible linker, and catalytic domain), but the linker domain was enriched with cost-reducing and resistance-enhancing mutations and depleted with cost-increasing mutations. The population genetics model based on the experimental data accurately predicts the rapid decline in mcr-1 prevalence in real-world data. INTERPRETATION: Many identified costless resistant variants that consist of multiple mutations are unlikely to evolve easily in nature. These findings for colistin and mcr-1 might be applicable to other cases in which AMR entails a substantial fitness cost that cannot be mitigated in proximal mutants. FUNDING: National Natural Science Foundation of China, and National Key Research and Development Program of China.

Performance of high-efficiency UV-C LEDs in water disinfection: Experimental, life cycle assessment, and economic analysis of different operational scenarios.

The widespread use of low or medium pressure mercury lamps in UV-C water disinfection should consider recent advances in UV-C LED lamps that offer a more sustainable approach and avoid its main drawbacks. The type of water and the mode of operation are critical when deciding on the treatment technology to be used. Therefore, this study investigates the potential application of UV-C LED disinfection technology in terms of kinetics, environmental assessment, and economic analysis for two scenarios: the continuous disinfection of a wastewater treatment plant (WWTP), and disinfection of harvested rainwater (RWH) in a residential household that operates intermittently. Experiments are conducted using both the new UV-C LED system and the conventional mercury lamp to disinfect real wastewater. Removal of total coliforms and Escherichia coli bacteria, with concentrations of approximately 105 and 104 CFU per 100 mL has been followed to assess the performance of both types of UV-C lamps. The experimental study provides kinetic parameters that have been further used in the environmental assessment conducted from a life cycle perspective. Additionally, considering the significant role of electricity consumption, a preliminary economic analysis has been conducted. The results indicate that first-order kinetic constants of pathogens removal with UV-C LEDs achieve 1.4 times higher values than Hg lamp. Regarding the environmental and economic assessment, for disinfection systems operating continuously, LEDs result in environmental impacts 5 times higher than Hg lamp in most categories, indicating that Hg lamps offer a viable option both from economic and environmental point of view. However, for installations with intermittent operation, LEDs emerge as the most competitive alternative, due to their ability to be turned on and off without affecting their lifespan. This study shows that UV-C LED lamps hold promise to replace conventional mercury lamps in a near future.

Centralized or Onsite Testing? Examining the Costs of Water Quality Monitoring in Rural Africa.

Rural water systems in Africa have room to improve water quality monitoring. However, the most cost-effective approach for microbial water testing remains uncertain. This study compared the cost per E. coli test (membrane filtration) of four approaches representing different levels of centralization: (i) one centralized laboratory serving all water systems, (ii) a mobile laboratory serving all systems, (iii) multiple semi-centralized laboratories serving clusters of systems, and (iv) decentralized analysis at each system. We employed Monte Carlo analyses to model the costs of these approaches in three real-world contexts in Ghana and Uganda and in hypothetical simulations capturing various conditions across rural Africa. Centralized testing was the lowest cost in two real-world settings and the widest variety of simulations, especially those with water systems close to a central laboratory (<36 km). Semi-centralized testing was the lowest cost in one real-world setting and in simulations with clustered water systems and intermediate sampling frequencies (1-2 monthly samples per system). The mobile lab was the lowest cost in the fewest simulations, requiring few systems and infrequent sampling. Decentralized testing was cost-effective for remote systems and frequent sampling, but only if sampling did not require a dedicated vehicle. Alternative low-cost testing methods could make decentralized testing more competitive.

Endotoxin, a novel biomarker for the rapid risk assessment of faecal contamination of coastal and transitional waters.

Current methods for testing water for faecal contamination rely on the culture of faecal indicator bacteria (FIB; Escherichia coli and Enterococci) that take 24-48 h, which leads to delays in taking proactive measures and poses a risk to public health. More rapid methods are therefore required. Here, we have tested a rapid, portable assay (Bacterisk) that detects the bacterial biomarker endotoxin in 30 min to quantify the bacterial biomass present, to evaluate 159 coastal water samples and to compare the results with the traditional culture of FIB. There was a significant correlation between the Bacterisk data given in endotoxin risk (ER) units and FIB culture that could accurately distinguish between poor and sufficient or good quality bathing water using the EU bathing directive values. Receiver operating characteristic analysis was used to determine the optimal ER threshold for coastal water samples, and the area under the curve was 0.9176 with a p-value of <0.0001. The optimal threshold was 7,300 ER units with a sensitivity of 95.45% and a specificity of 83.48%. In conclusion, we have shown that the Bacterisk assay provides a rapid and easy-to-use in situ method to assess bathing water quality.

Customizable Fabrication of Photothermal Microneedles with Plasmonic Nanoparticles Using Low-Cost Stereolithography Three-Dimensional Printing.

Photothermal microneedle (MN) arrays have the potential to improve the treatment of various skin conditions such as bacterial skin infections. However, the fabrication of photothermal MN arrays relies on time-consuming and potentially expensive microfabrication and molding techniques, which limits their size and translation to clinical application. Furthermore, the traditional mold-and-casting method is often limited in terms of the size customizability of the photothermal array. To overcome these challenges, we fabricated photothermal MN arrays directly via 3D-printing using plasmonic Ag/SiO2 (2 wt % SiO2) nanoaggregates dispersed in ultraviolet photocurable resin on a commercial low-cost liquid crystal display stereolithography printer. We successfully printed MN arrays in a single print with a translucent, nanoparticle-free support layer and photothermal MNs incorporating plasmonic nanoaggregates in a selective fashion. The photothermal MN arrays showed sufficient mechanical strength and heating efficiency to increase the intradermal temperature to clinically relevant temperatures. Finally, we explored the potential of photothermal MN arrays to improve antibacterial therapy by killing two bacterial species commonly found in skin infections. To the best of our knowledge, this is the first time describing the printing of photothermal MNs in a single step. The process introduced here allows for the translatable fabrication of photothermal MN arrays with customizable dimensions that can be applied to the treatment of various skin conditions such as bacterial infections.

Transposase-assisted tagmentation: an economical and scalable strategy for single-worm whole-genome sequencing.

AlphaMissense identifies 23 million human missense variants as likely pathogenic, but only 0.1% have been clinically classified. To experimentally validate these predictions, chemical mutagenesis presents a rapid, cost-effective method to produce billions of mutations in model organisms. However, the prohibitive costs and limitations in the throughput of whole-genome sequencing (WGS) technologies, crucial for variant identification, constrain its widespread application. Here, we introduce a Tn5 transposase-assisted tagmentation technique for conducting WGS in Caenorhabditis elegans, Escherichia coli, Saccharomyces cerevisiae, and Chlamydomonas reinhardtii. This method, demands merely 20 min of hands-on time for a single-worm or single-cell clones and incurs a cost below 10 US dollars. It effectively pinpoints causal mutations in mutants defective in cilia or neurotransmitter secretion and in mutants synthetically sterile with a variant analogous to the B-Raf Proto-oncogene, Serine/Threonine Kinase (BRAF) V600E mutation. Integrated with chemical mutagenesis, our approach can generate and identify missense variants economically and efficiently, facilitating experimental investigations of missense variants in diverse species.

Assessment of elimination efficacy on foodborne pathogenic microbes and foulant precipitates using phytic acid and sulfamic acid.

This research investigated the comparative efficacy of sulfamic acid (SA) and phytic acid (PA), both individually and in combination, for treating potential foodborne pathogens and pre-formed foulants. Pathogens studied included Listeria monocytogenes, E. coli DH5α, Salmonella typhimurium, Staphylococcus aureus, and vegetative Bacillus cereus, in suspended aqueous solutions, as well as Pseudomonas aeruginosa biofilm on quartz glass surfaces. Inactivation kinetics for Listeria monocytogenes revealed concentration-dependent rate constants (k) of 6.6(±0.2) × 10-6 M and 2.8(±0.1) × 10-8 M for single treatments of SA and PA, respectively, and ranged from 6.9(±0.3) to 50.7(±2.3) × 10-6 M for combined treatments with PA pre-treatment concentrations of 75-758 µM. Observable cellular abnormalities in Listeria monocytogenes, such as membrane vesiculation, chelation, cellular disruption, biomolecule leakage, and lipid peroxidation, were identified after exposure to PA or SA, either individually or in combination. The optimized combined treatment of PA and SA achieved significant removal (i.e., >3-log; 99.9%) of potential foodborne pathogens under simulated food-washing process conditions. Additionally, over 90% descaling efficacy was observed for pre-formed foulants such as CaCO3 precipitates and Pseudomonas aeruginosa biofilm on quartz glass surfaces with the combined treatment. These findings provide novel insights into the versatile utility of PA and SA for optimizing combinational water disinfection systems and addressing (in)organic foulant scaling on surfaces in the food processing industry.

Assessment of antibacterial activity, modes of action, and synergistic effects of Origanum vulgare hydroethanolic extract with antibiotics against avian pathogenic Escherichia coli.

This study evaluates the antibacterial effectiveness of Origanum vulgare hydroethanolic extract, both independently and in combination with antibiotics, against Escherichia coli strains associated with avian colibacillosis-a significant concern for the poultry industry due to the rise of antibiotic-resistant E. coli. The urgent demand for new treatments is addressed by analyzing the extract's phytochemical makeup via High-Performance Liquid Chromatography (HPLC), which identified sixteen phenolic compounds. Antibacterial activity was determined through agar diffusion and the measurement of minimum inhibitory and bactericidal concentrations (MIC and MBC), showing moderate efficacy (MIC: 3.9 to 7.8 mg/mL, MBC: 31.2 to 62.4 mg/mL). Combining the extract with antibiotics like ampicillin and tetracycline amplified antibacterial activity, indicating a synergistic effect and highlighting the importance of combinatory treatments against resistant strains. Further analysis revealed the extract's mechanisms of action include disrupting bacterial cell membrane integrity and inhibiting ATPase/H+ proton pumps, essential for bacterial survival. Moreover, the extract effectively inhibited and eradicated biofilms, crucial for preventing bacterial colonization. Regarding cytotoxicity, the extract showed no hemolytic effect at 1 to 9 mg/mL concentrations. These results suggest Origanum vulgare extract, particularly when used with antibiotics, offers a promising strategy for managing avian colibacillosis, providing both direct antibacterial benefits and moderating antibiotic resistance, thus potentially reducing the economic impact of the disease on the poultry industry.

In Silico Design and In Vitro Assessment of Bicyclic Trifluoromethylated Pyrroles as New Antibacterial and Antifungal Agents.

QSAR studies on the number of compounds tested as S. aureus inhibitors were performed using an interactive Online Chemical Database and Modeling Environment (OCHEM) web platform. The predictive ability of the developed consensus QSAR model was q2=0.79±0.02. The consensus prediction for the external evaluation set afforded high predictive power (q2=0.82±0.03). The models were applied to screen a virtual chemical library with anti-S. aureus activity. Six promising new bicyclic trifluoromethylated pyrroles were identified, synthesized and evaluated in vitro against S. aureus, E. coli, and A. baumannii for their antibacterial activity and against C. albicans, C. krusei and C. glabrata for their antifungal activity. The synthesized compounds were characterized by 1H, 19F, and 13C NMR and elemental analysis. The antimicrobial activity assessment indicated that trifluoromethylated pyrroles 9 and 11 demonstrated the greatest antibacterial and antifungal effects against all the tested pathogens, especially against multidrug-resistant strains. The acute toxicity of the compounds to Daphnia magna ranged from 1.21 to 33.39 mg/L (moderately and slightly toxic). Based on the docking results, it can be suggested that the antibacterial and antifungal effects of the compounds can be explained by the inhibition of bacterial wall component synthesis.

Enabling high-throughput enzyme discovery and engineering with a low-cost, robot-assisted pipeline.

As genomic databases expand and artificial intelligence tools advance, there is a growing demand for efficient characterization of large numbers of proteins. To this end, here we describe a generalizable pipeline for high-throughput protein purification using small-scale expression in E. coli and an affordable liquid-handling robot. This low-cost platform enables the purification of 96 proteins in parallel with minimal waste and is scalable for processing hundreds of proteins weekly per user. We demonstrate the performance of this method with the expression and purification of the leading poly(ethylene terephthalate) hydrolases reported in the literature. Replicate experiments demonstrated reproducibility and enzyme purity and yields (up to 400 µg) sufficient for comprehensive analyses of both thermostability and activity, generating a standardized benchmark dataset for comparing these plastic-degrading enzymes. The cost-effectiveness and ease of implementation of this platform render it broadly applicable to diverse protein characterization challenges in the biological sciences.

Costs of antibiotic resistance genes depend on host strain and environment and can influence community composition.

Antibiotic resistance genes (ARGs) benefit host bacteria in environments containing corresponding antibiotics, but it is less clear how they are maintained in environments where antibiotic selection is weak or sporadic. In particular, few studies have measured if the direct effect of ARGs on host fitness is fixed or if it depends on the host strain, perhaps marking some ARG-host combinations as selective refuges that can maintain ARGs in the absence of antibiotic selection. We quantified the fitness effects of six ARGs in 11 diverse Escherichia spp. strains. Three ARGs (blaTEM-116, cat and dfrA5, encoding resistance to ß-lactams, chloramphenicol, and trimethoprim, respectively) imposed an overall cost, but all ARGs had an effect in at least one host strain, reflecting a significant strain interaction effect. A simulation predicts these interactions can cause the success of ARGs to depend on available host strains, and, to a lesser extent, can cause host strain success to depend on the ARGs present in a community. These results indicate the importance of considering ARG effects across different host strains, and especially the potential of refuge strains to allow resistance to persist in the absence of direct selection, in efforts to understand resistance dynamics.

A starch/gelatin-based Halochromic film with black currant anthocyanin and Nanocellulose-stabilized cinnamon essential oil Pickering emulsion: Towards real-time Salmon freshness assessment.

Neoterically, food packaging systems designed solely for prolonging shelf life or monitoring freshness could not fulfil the dynamic demands of consumers. In this current investigation, using the solvent casting method, a versatile halochromic indicator was created by integrating black currant anthocyanin and cinnamon essential oil-loaded Pickering emulsion into a starch/gelatin matrix. The resulting indicator film underwent scrutiny for its structural, pH-sensitive, antioxidant, and antimicrobial attributes. Unexpectedly, the amalgamation of anthocyanin and essential oil led to decreased antioxidant activity, dropping from 73.23 ± 2.17 to 28.87 ± 2.50 mg Trolox equivalent/g sample. Additionally, no discernible antimicrobial properties were detected in the composite film sample against both Staphylococcus aureus and Escherichia coli. Fourier transform infrared analyses unveiled robust intermolecular interactions among the film-forming components, providing insights into the observed antagonistic effect. The indicator film displayed distinctive colour changes corresponding to the fresh (greyish-brown), onset of decomposition (khaki), and spoiled (dark green) stages of the stored fish sample. This highlights its promising potential for providing real-time indications of food spoilage. These findings are important for the efficient design of composite films incorporating anthocyanins and essential oils. They serve as a guide towards their potential use as multifunctional packaging materials in the food industry.

Assessment of three antibiotic combination regimens against Gram-negative bacteria causing neonatal sepsis in low- and middle-income countries.

Gram-negative bacteria (GNB) are a major cause of neonatal sepsis in low- and middle-income countries (LMICs). Although the World Health Organization (WHO) reports that over 80% of these sepsis deaths could be prevented through improved treatment, the efficacy of the currently recommended first- and second-line treatment regimens for this condition is increasingly affected by high rates of drug resistance. Here we assess three well known antibiotics, fosfomycin, flomoxef and amikacin, in combination as potential antibiotic treatment regimens by investigating the drug resistance and genetic profiles of commonly isolated GNB causing neonatal sepsis in LMICs. The five most prevalent bacterial isolates in the NeoOBS study (NCT03721302) are Klebsiella pneumoniae, Acinetobacter baumannii, E. coli, Serratia marcescens and Enterobacter cloacae complex. Among these isolates, high levels of ESBL and carbapenemase encoding genes are detected along with resistance to ampicillin, gentamicin and cefotaxime, the current WHO recommended empiric regimens. The three new combinations show excellent in vitro activity against ESBL-producing K. pneumoniae and E. coli isolates. Our data should further inform and support the clinical evaluation of these three antibiotic combinations for the treatment of neonatal sepsis in areas with high rates of multidrug-resistant Gram-negative bacteria.

Microbiological assessment of ready-to-eat foods and drinking water sources as a potential vehicle of bacterial pathogens in northern India.

Foodborne illnesses caused by the consumption of contaminated foods have frequent occurrences in developing countries. The incorporation of contaminated water in food processes, preparation, and serving is directly linked to several gastrointestinal infections. Keeping in view, this study was conducted to assess the microbial quality of both drinking water sources and commonly consumed fresh ready-to-eat (RTE) foods in the region. The drinking water samples from water sources and consumer points, as well as food samples from canteens, cafes, hotels, and restaurants, were collected for the microbiological analysis. Fifty-five percent (n = 286) of water samples were found to be positive for total coliforms with MPN counts ranging from 3 to 2600 (100 ml) -1. E. coli was detected in nearly 30% of the total water samples. Overall, 65% tap water samples were found unsatisfactory, followed by submersible (53%), filter (40%), and WTP (30%) sources. Furthermore, the examination of RTE foods (n = 80) found that 60% were of unsatisfactory microbial quality with high aerobic plate counts. The salads were the most contaminated category with highest mean APC 8.3 log CFU/g followed by pani puri, chats, and chutneys. Presence of coliforms and common enteropathogens was observed in both water and food samples. The detected isolates from the samples were identified as Enterobacter spp., Klebsiella spp., Pseudomonas aeruginosa, Salmonella spp., Shigella spp., and Staphylococcus spp. Based on these findings, microbiological quality was found compromised and this may pose hazard to public health. This exploratory study in the Punjab region also suggests that poor microbiological quality of water sources can be an important source of contamination for fresh uncooked RTE foods, thus transferring pathogens to the food chain. Therefore, only safe potable drinking water post-treatment should be used at all stages.

Population pharmacokinetic meta-analysis of five beta-lactams antibiotics to support dosing regimens in dogs for surgical antimicrobial prophylaxis.

The Pharmacokinetic/Pharmacodynamic (PK/PD) relationship of antimicrobial drugs (AMD) for surgical prophylaxis has been poorly studied, hampering evidence-based decision making around AMD dosing and timing. Our objective is to use PK/PD principles to inform (1) the timing of administration and (2) the interval for re-administration of AMD used peri-operatively in dogs. Raw plasma concentrations of cefazolin, cefuroxime, cefalexin, amoxicillin and ampicillin were retrieved from original intravenous studies performed in dogs. E. coli and methicillin-susceptible staphylococci were identified as possible intraoperative contaminants and their epidemiological cut-offs (ECOFF) were retrieved from the EUCAST database. Individual PK data were refitted with non-linear mixed effect models (Phoenix®). We performed Monte Carlo simulation to compute i) the 95th percentile of time of peak concentration in the peripheral compartment (informing timing between administration and first incision) and ii) the duration for which at least 90% of dogs maintain a free plasma concentration above ECOFF (informing timing of re-administration: 1.5-4 h). Cefazolin (22-25 mg/kg), cefuroxime (20 mg/kg), cefalexin (15 mg/kg) and amoxicillin (16.7 mg/kg) reached peak peripheral concentrations within 30 min, but ampicillin (20 mg/kg) required 82 min, respectively. For methicillin-susceptible staphylococci, cefazolin and cefuroxime require re-administration every 2 h, whereas cefalexin and both amoxicillin and ampicillin can be readministered every 3 and 4 h, respectively. For E. coli, only cefazolin provided adequate perioperative coverage with 2-hourly administration, where cefuroxime and cefalexin failed uniformly. Alternatively, ampicillin and amoxicillin (critically ill dogs) may cover E. coli contaminations, but only if readministered every 1.5 h. These PK-derived conclusions provide a rationale for perioperative AMD administration timing.

Assessment of microorganisms in drinking water disinfected by catalytic ozonation with fluorinated ceramic honeycomb and NaClO disinfectants under laboratory and pilot conditions.

While sodium hypochlorite (NaClO) has long been used to disinfect drinking water, concerns have risen over its use due to causing potentially hazardous byproducts. Catalytic ozonation with metal-free catalysts has attracted increasing attention to eliminate the risk of secondary pollution of byproducts in water treatment. Here, we compared the disinfection efficiency and microbial community of catalytic ozone with a type of metal-free catalyst fluorinated ceramic honeycomb (FCH) and NaClO disinfectants under laboratory- and pilot-scale conditions. Under laboratory conditions, the disinfection rate of catalytic ozonation was 3∼6-fold that of ozone when the concentration of Escherichia coli was 1 × 106 CFU/ml, and all E. coli were killed within 15 s. However, 0.65 mg/L NaClO retained E. coli after 30 min using the traditional culturable approach. The microorganism inactivation results of raw reservoir water disinfected by catalytic ozonation and ozonation within 15 s were incomparable based on the cultural method. In pilot-scale testing, catalytic ozonation inactivated all environmental bacteria within 4 min, while 0.65 mg/L NaClO could not achieve this success. Both catalytic ozonation and NaClO-disinfected methods significantly reduced the number of microorganisms but did not change the relative abundances of different species, i.e., bacteria, viruses, eukaryotes, and archaea, based on metagenomic analyses. The abundance of virulence factors (VFs) and antimicrobial resistance genes (ARGs) was detected few in catalytic ozonation, as determined by metagenomic sequencing. Some VFs or ARGs, such as virulence gene 'FAS-II' which was hosted by Mycobacterium_tuberculosis, were detected solely by the NaClO-disinfected method. The enriched genes and pathways of cataO3-disinfected methods exhibited an opposite trend, especially in human disease, compared with NaClO disinfection. These results indicated that the disinfection effect of catalytic ozone is superior to NaClO, this finding contributed to the large-scale application of catalytic ozonation with FCH in practical water treatment.

Assessment of handwashing impact on detection of SARS-CoV-2, Staphylococcus aureus, Escherichia coli on hands in rural and urban settings of Côte d'Ivoire during COVID-19 pandemic.

BACKGROUND: Handwashing is the first line of hygiene measures and one of the oldest methods of preventing the spread of infectious diseases. Despite its efficacy in the health system, handwashing is often inadequately practiced by populations. This study aimed to assess the presence of SARS-CoV-2, Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) on hands as indicators of lack of hand hygiene during COVID 19 pandemic. METHODS: A cross-sectional study was conducted in rural Taabo and urban Abidjan (Côte d'Ivoire) from January to September 2021. A total of 384 participants from 384 households were included in the study. The total households were distributed proportionally within various municipalities in the two study areas according to the number of households in each municipality, based on data of the National Institute of Statistics from the 2014 general population census. Hand swabbing of the 384 participants within households (320 in Abidjan and 64 in Taabo) was performed for the enumeration of E. coli and S aureus, using laboratory standard method and for the detection of SARS-CoV-2 by RT-qPCR. A binary logistic regression model was built with the outcome variable presence of Staphylococcus spp. on hands of respondents that was categorized into binary variables, Staphylococcus spp. (1 = presence, 0 = absence) for the Risk Ratio estimation. Place of living, sex, handwashing, education and age group were used to adjust the model to observe the effects of these explanatory variables. RESULTS: No presence of SARS-CoV-2 virus was detected on the hands of respondents in both sites. However, in urban Abidjan, only Staphylococcus spp. (Coagulase Negative Staphylococci) was found on the hands of 233 (72.8%, 95%CI: 67.7-77.4) respondents with the average load of 0.56 CFU/ Cm2 (95% CI, 0.52-0.60). Meanwhile, in rural Taabo, Staphylococcus spp. (Coagulase Negative Staphylococci) and E. coli were found on the hands of 40 (62.5%, 95%CI: 50.3-73.3) and 7 (10.9%, 95%CI: 5.4-20.9) respondents with the respective average load of 0.49 CFU/ Cm2 (95% CI, 0.39-0.59) and 0.08 CFU/ Cm2 (95% CI, 0.03-0.18). Participants living in rural Taabo were less likely to have Staphylococcus spp. on their hands (RR = 0.811; 95%IC: 0.661-0.995) compared to those living in urban Abidjan. CONCLUSIONS: No SARS-CoV-2 was detected on the hands of participants in both sites, suggesting that our study did not show direct transmission through hands. No E. coli was found in urban Abidjan while E. coli was found on the hands of participants in rural Taabo indicating poor hand washing and disinfection practices in rural Taabo. Living in urban Abidjan is statistically associated to having Staphylococcus spp. on hands. Further studies are necessary especially to understand to what extent the presence of Staphylococcus spp. on hands indicates a higher infection or fecal colonization rates in the case of E. coli.

Low-cost inkjet-printed nanostructured biosensor based on CRISPR/Cas12a system for pathogen detection.

The escalating global incidence of infectious diseases caused by pathogenic bacteria, especially in developing countries, emphasises the urgent need for rapid and portable pathogen detection devices. This study introduces a sensitive and specific electrochemical biosensing platform utilising cost-effective electrodes fabricated by inkjet-printing gold and silver nanoparticles on a plastic substrate. The biosensor exploits the CRISPR/Cas12a system for detecting a specific DNA sequence selected from the genome of the target pathogen. Upon detection, the trans-activity of Cas12a/gRNA is triggered, leading to the cleavage of rationally designed single-strand DNA reporters (linear and hairpin) labelled with methylene blue (ssDNA-MB) and bound to the electrode surface. In principle, this sensing mechanism can be adapted to any bacterium by choosing a proper guide RNA to target a specific sequence of its DNA. The biosensor's performance was assessed for two representative pathogens (a Gram-negative, Escherichia coli, and a Gram-positive, Staphylococcus aureus), and results obtained with inkjet-printed gold electrodes were compared with those obtained by commercial screen-printed gold electrodes. Our results show that the use of inkjet-printed nanostructured gold electrodes, which provide a large surface area, in combination with the use of hairpin reporters containing a poly-T loop can increase the sensitivity of the assay corresponding to a signal variation of 86%. DNA targets amplified from various clinically isolated bacteria, have been tested and demonstrate the potential of the proposed platform for point-of-need applications.

Silver nanoparticle biosynthesis utilizing Ocimum kilimandscharicum leaf extract and assessment of its antibacterial activity against certain chosen bacteria.

The use of plants in the biological production of silver nanoparticles for antibacterial applications is a growing field of research. In the current work, we formulated Ocimum kilimandscharicum extracts using silver nanoparticles, and evaluated its potential antibacterial activity. Aqueous and methanol plant extracts were used to reduce silver nitrate at different time intervals (30 to 150 minutes) and pH (2 to 11). The UV-visible absorption spectrum recorded for methanol and aqueous extracts revealed a successful synthesis of AgNPs for methanol and aqueous extracts. The antimicrobial activity of the AgNPs was evaluated against Escherichia coli ATCC 25922, Salmonella choleraesuius ATCC 10708, and Staphylococcus aureus ATCC 25923 The best inhibition zone for the methanol and aqueous-mediated AgNPs, ranging from 12 ± 1 to 16 ± 1mm. Additionally, the methanol and aqueous extract silver nanoparticles had the same Minimum Inhibitory Concentration (6.25 ± 0.00 mg/ml), whereas the Minimum Bactericidal Concentrations were 12.5 ± 0.00 and 25 ± 0.00 mg/ml, respectively. The highest inhibition zone of 16 ± 1 mm was observed against Salmonella choleraesuius with 50 ± 0.00 mg/ml aqueous silver nanoparticles. The results show that the silver nanoparticles made with Ocimum kilimandscharicum have antibacterial action against those microorganisms.

Photostability and photodynamic antimicrobial profile of dye extracts from four (4) plants: prospects for eco-friendly low-cost food disinfection and topical biomedical applications.

In this study, photostability and photodynamic antimicrobial performance of dye extracts from Hibiscus sabdariffa (HS) calyces, Sorghum bicolor (SB) leaf sheaths, Lawsonia inermis (LI) leaves and Curcuma longa (CL) roots were investigated in Acetate-HCl (AH) Buffer (pH 4.6), Tris Base-HCl (TBH) Buffer (pH 8.6), distilled water (dH2O), and Phosphate Buffer Saline (PBS, pH 7.2) using Bacillus subtilis as model for gram positive bacteria, Escherichia coli as model for gram negative bacteria, phage MS2 as model for non-envelope viruses and phage phi6 as model for envelope viruses including SARS CoV-2 which is the causative agent of COVID-19. Our results showed that the photostability of the dye extracts is in the decreasing order of LI > CL > SB > HS. The dye extract-HS is photostable in dH2O but bleaches in buffers-AH, TBH and PBS. The rate of bleaching is higher in AH compared to in TBH and PBS. The bleaching and buffers affected the photodynamic and non-photodynamic antimicrobial activity of the dye extracts. The photodynamic antibacterial activity of the dye extracts is in the decreasing order of CL > HS > LI > SB while the non-photodynamic antibacterial activity is in the decreasing order of LI > CL > HS > SB. The non-photodynamic antiviral activity pattern observed is the same as that of non-photodynamic antibacterial activity observed. However, the photodynamic antiviral activity of the dye extracts is in the decreasing order of CL > LI > HS > SB. Given their performance, the dye extracts maybe mostly suitable for environmental applications including fresh produce and food disinfection, sanitation of hands and contact surfaces where water can serve as diluent for the extracts and the microenvironment is free of salts.