Protocol optimization for deoxyribonucleic acid (DNA) extraction from dried, fresh leaves, and seeds of groundnut (Arachis hypogaea L.)

Consistent isolation of best quality deoxyribonucleic acid (DNA) from peanut (Arachis hypogaea L.) is particularly problematic due to the presence of phenolic compounds and polysaccharides. Inconsistencies in extraction results can be attributed to the age and growth stages of the plant material analyzed. Mature leaves have higher quantities of polyphenols, tannins and polysaccharides that can contaminate DNA during isolation. In this study, we used fresh and dried leaves as well as seeds for optimization of high quality DNA isolation protocols from A. hypogaea. The DNA extracted with three different methods cetyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS), and cesium chloride (CsCl) density gradient) were comparatively studied by polymerase chain reaction (PCR) analysis in terms of quantity and quality. High quality genomic DNA was obtained from fresh leaves by modified CTAB methods. The DNA obtained ranged from 1 to 2.5 ng/μl. DNA obtained by this method was strong and reliable showing its compatibility for simple sequence repeat (SSR) analyses. The SDS based methodology give large quantities of DNA contaminated with polysaccharides. Fresh leaves also gave best result in SDS method. The quantity and quality of DNA obtained was very poor in all the tested methods in case of dried leaf tissues. The current protocol will probably be useful for the extraction of high-molecular weight DNA from other plant materials containing large amounts of secondary metabolites and essential oils.Key words: Polysaccharides, polyphenols, tannins, cetyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS), cesium chloride (CsCl), secondary metabolites, SSR

Individual and organizational barriers faced by physiotherapists in implementing evidence based practice- an analytical study

Objective: The primary objective of the study was to determine the present barriers in EBP implementation and secondary objective was to find out the routine practices other than evidence-based practice. Methods: It was an analytical cross-sectional study with a sample size of 235 participants. Non-probability convenience sampling technique was used. The data was collected after ethical approval on 01 January 2021, using Barrier Scale by Dr. Funk with written permission via email. The data was encoded and analyzed by SPSS version 16. For descriptive data Mean, Standard Deviation, Frequencies, Percentages, and tables were used to present the results and the Chi-Square test was used to show the association. Results: In this study Male 104 (44.3%) and Females were 131(55.7%) with a mean age of 31.5+9.5 years. 64.3% of Physiotherapists were having 2-5 years and 28.1% had 5-7 years of working experience. This study shows that “Insufficient time on the job to implement new ideas” is 1st barrier at the organizational level and second place, according to Mean value is “time to read research” at the individual level. Conclusion: This study shows that PTs faced barriers at both organizational and individual levels. The top barriers faced at the organizational level were inadequate facilities, not authorized to change the patient care procedure, and at the Individual level were research unawareness, limited time and not beneficial effects of variation in practice, and uncertainty of results. Keywords: Evidence Based Practice, Individual Barriers, Organizational Barriers, Physiotherapists

Genome Analysis of ESBL-Producing <i>Escherichia coli</i> Isolated from Pigs

The resistome, virulome and mobilome of extended spectrum ß-lactamase (ESBL)-producing Escherichia coli (ESBL-Ec) isolated from pigs in Cameroon and South Africa were assessed using whole genome sequencing (WGS). Eleven clonally related phenotypic ESBL-Ec isolates were subjected to WGS. The prediction of antibiotic resistance genes, virulence factors (VFs) and plasmids was performed using ResFinder, VirulenceFinder and PlasmidFinder, respectively. Diverse sequence types (STs) were detected with ST2144 and ST88 being predominant and blaCTX-M-15 (55%) being the principal ESBL gene. All except two isolates harboured various aminoglycoside resistance genes, including aph(3″)-Ib (6/11, 55%) and aph(6)-1d (6/11, 55%), while the qnrS1 gene was identified in four of the isolates. The ESBL-Ec isolates showed a 93.6% score of being human pathogens. The fim, ehaB, ibeB/C were the leading virulence factors detected. All isolates harboured at least three extraintestinal pathogenic E. coli (ExPEC) VFs, with one isolate harbouring up to 18 ExPEC VFs. Five isolates (45.45%) harboured the plasmid incompatibility group IncF (FII, FIB, FIC, FIA). The study revealed that there is an urgent need to implement effective strategies to contain the dissemination of resistant and virulent ESBL-Ec through the food chain in Cameroon and South Africa

Genome analysis of multidrug resistant Enterococcus faecium and Enterococcus faecalis circulating among hospitalized patients in uMgungundlovu District, KwaZulu-Natal, South Africa

Abstract Background Vancomycin-resistant enterococci (VRE) are important pathogens categorized as high-priority bacteria in the Global Priority List of Antibiotic-Resistant Bacteria to Guide Research, Discovery, and Development of New Antibiotics published by the World Health Organization. The aim of this study was to determine the risk factors, resistance, virulence, mobilomes associated with multidrug-resistant and clonal lineages of Enterococcus faecium and faecalis circulating among hospitalized patients following the health system in South Africa, using whole genome sequencing (WGS). Methods A cross-sectional study was conducted during a two-month periods among hospitalized patients in 2017. Rectal swabs were collected from patients admitted to medical and surgical wards in an urban tertiary hospital, and a rural district hospital in uMgungundlovu district, South Africa. Enterococci were screened for vancomycin resistance on bile esculin azide agar supplemented with 6 mg/L of vancomycin and confirmation of VRE was done using ROSCO kits. Conventional and real-time PCR methods were used to ascertain the presence of VanA, VanB, VanC-2/3 and VanC-1 genes. All six multidrug-resistant Enterococcus faecalis and faecium selected were identified using multiplexed paired-end libraries (2 × 300 bp) with the Nextera XT DNA sample preparation kit (Illumina, San Diego, CA, USA) and genome sequencing was done using Illumina MiSeq instrument with 100× coverage at the National Institute of Communicable Diseases Sequencing Core Facility, South Africa. Antibiotic resistance genes, virulence factors, plasmids, integrons and CRISPR were characterized using RAST, ResFinder, VirulenceFinder, PlasmidFinder, PHAST and ISFinder respectively. Results Sequencing analysis revealed that these strains harbouring numerous resistance genes to glycopeptides (vanC[100%], vex3[100%], vex2[83,33%] and vanG[16,66%]), macrolides, lincosamides, sterptogramine B (ermB[33,32%], Isa[16,66%], emeA[16,66%]) and tetracyclines (tetM[33,32%]) in both district and tertiary hospitals. Multidrug efflux pumps including MATE, MFS and pmrA conferring resistance to several classes of antibiotics were also identified. The main transposable elements observed were in the Tn3 family, specifically Tn1546. Four single sequence types (STs) were identified among E. faecium in the district hospital, namely ST822, ST636, ST97 along with a novel ST assigned ST1386, while one lineage, ST29 was detected in the tertiary hospital. Conclusion The study reveals the genetic diversity and high pathogenicity of multidrug-resistant Enterococcus faecalis and faecium circulating among hospitalized patients. It underlines the necessity to implement routine screening of admitted patients coupled with infection control procedures, antimicrobial stewardship and awareness should be strengthened to prevent and/or contain the carriage and spread of multidrug resistant E. faecium and E. faecalis in hospitals and communities in South Africa

Classification of exact Bianchi type V cosmological solutions via conformal vector fields admitting energy conditions in f(T) gravity

The aim of this paper is to discuss exact Bianchi-type V cosmological solutions via conformal vector fields (CVFs) admitting energy conditions within the framework of f(T) gravity. For this study, we first derive the Einstein field equations (EFEs) by considering the Bianchi type V space–times in f(T) gravity. We formulate the exact Bianchi type V metrics in f(T) gravity by imposing certain constraints on space–time components. In particular, we emphasize extracting the power law, exponential, and singular solutions. We plot the dynamical parameters of the obtained solutions versus cosmic time t to observe their behavior graphically. We analyze the metrics via CVFs in order to sort out conservation laws. We also discuss several cosmological constraints for each of the obtained solutions. Furthermore, to check the physical soundness of the resultant metrics, we find several constraints under which the solutions admit energy conditions

A Genomic Snapshot of Antibiotic-ResistantEnterococcus faecalis within Public Hospital Environments in South Africa

Enterococci are among the most common opportunistic hospital pathogens. This study used whole-genome sequencing (WGS) and bioinformatics to determine the antibiotic resistome, mobile genetic elements, clone and phylogenetic relationship of Enterococcus faecalis isolated from hospital environments in South Africa. This study was carried out from September to November 2017. Isolates were recovered from 11 frequently touched sites by patients and healthcare workers in different wards at 4 levels of healthcare (A, B, C, and D) in Durban, South Africa. Out of the 245 identified E. faecalis isolates, 38 isolates underwent whole-genome sequencing (WGS) on the Illumina MiSeq platform, following microbial identification and antibiotic susceptibility tests. The tet(M) (31/38, 82%) and erm(C) (16/38, 42%) genes were the most common antibiotic-resistant genes found in isolates originating from different hospital environments which corroborated with their antibiotic resistance phenotypes. The isolates harboured mobile genetic elements consisting of plasmids (n = 11) and prophages (n = 14) that were mostly clone-specific. Of note, a large number of insertion sequence (IS) families were found on the IS3 (55%), IS5 (42%), IS1595 (40%), and Tn3 transposons the most predominant. Microbial typing using WGS data revealed 15 clones with 6 major sequence types (ST) belonging to ST16 (n = 7), ST40 (n = 6), ST21 (n = 5), ST126 (n = 3), ST23 (n = 3), and ST386 (n = 3). Phylogenomic analysis showed that the major clones were mostly conserved within specific hospital environments. However, further metadata insights revealed the complex intraclonal spread of these E. faecalis major clones between the sampling sites within each specific hospital setting. The results of these genomic analyses will offer insights into antibiotic-resistantE. faecalis in hospital environments relevant to the design of optimal infection prevention strategies in hospital settings

Impact of Environmental Sub-Inhibitory Concentrations of Antibiotics, Heavy Metals, and Biocides on the Emergence of Tolerance and Effects on the Mutant Selection Window in <i>E. coli</i>

Bacteria’s ability to withstand the detrimental effects of antimicrobials could occur as resistance or tolerance with the minimum inhibitory concentration, the mutant prevention concentration, and the mutant selection window as salient concepts. Thus, this study assessed the impact of exposure to extremely high doses of ampicillin on the level of persistence and tolerance development in isolates previously exposed to different concentrations of selected antibiotics, biocides, and heavy metals. These isolates were previously exposed to oxytetracycline (OXYTET), amoxicillin (AMX), copper (Cu), zinc (Zn), benzalkonium chloride (BAC) 10, dimethylammonium chloride (DADMAC) 12 and a combination of all the individual pollutants (ALL). The isolates were exposed to very high concentrations (25 × MIC) of ampicillin, and their tolerance was calculated as the time required to kill 99.9% of the bacterial population (MDK99.9). The MDK99.9 increased by 30 to 50% in test isolates (DADMAC, OXYTET, Zinc = 28 h; BAC, Copper = 30 h; amoxycillin, ALL = 26 h) compared to the untreated control. BAC-exposed isolates decreased from 2.5 × 108 CFU/mL to 2.5 × 104 CFU/mL on the second day, displaying the highest tolerance increase. The tolerance appeared to originate from two sources, i.e., stochastic persistence and genetic-induced persistence, involving multiple genes with diverse mechanisms. The mutant selection window of the isolates to ampicillin, amoxicillin, and oxytetracycline also slightly increased compared to the control, indicating the selective survival of persister cells during the 30-day exposure. These findings indicate that bacterial exposure to sub-inhibitory concentrations of environmental chemical stressors may not always result in the development of antimicrobial resistance but could initiate this process by selecting persisters that could evolve into resistant isolates

Comparative Pathogenomics of Aeromonas veronii from Pigs in South Africa: Dominance of the Novel ST657 Clone

The pathogenomics of carbapenem-resistant Aeromonas veronii (A. veronii) isolates recovered from pigs in KwaZulu-Natal, South Africa, was explored by whole genome sequencing on the Illumina MiSeq platform. Genomic functional annotation revealed a vast array of similar central networks (metabolic, cellular, and biochemical). The pan-genome analysis showed that the isolates formed a total of 4349 orthologous gene clusters, 4296 of which were shared; no unique clusters were observed. All the isolates had similar resistance phenotypes, which corroborated their chromosomally mediated resistome (blaCPHA3 and blaOXA-12) and belonged to a novel sequence type, ST657 (a satellite clone). Isolates in the same sub-clades clustered according to their clonal lineages and host. Mobilome analysis revealed the presence of chromosome-borne insertion sequence families. The estimated pathogenicity score (Pscore &asymp; 0.60) indicated their potential pathogenicity in humans. Furthermore, these isolates carried several virulence factors (adherence factors, toxins, and immune evasion), in different permutations and combinations, indicating a differential ability to establish infection. Phylogenomic and metadata analyses revealed a predilection for water environments and aquatic animals, with more recent reports in humans and food animals across geographies, making A. veronii a potential One Health indicator bacterium