The rapid detection of drug resistant mycobacteria

Mycobacterium abscessus is a multidrug resistant pathogen commonly isolated from patients with cystic fibrosis. Currently, there is no rapid diagnostic tool to detect the presence of M. abscessus. Rapid diagnosis followed by appropriate, prompt treatment remains the best curative approach to mitigate disease burden and halt transmission. A major bottle neck in developing a rapid diagnostic assay is DNA extraction. Mycobacterial cells are very difficult to lyse, the existing methods are time consuming resulting in long turnaround time to detect the pathogen. This study employed the use of microwave energy to rapidly release nucleic acids from microorganisms and test the ability to detect the released nucleic acids in a magnetic-bead-based sandwich hybridisation assay using specific DNA probes. Based on published genome sequences, probes targeting the rpoB and erm-41 genes of M. abscessus and M. smegmatis were designed. In a magnetic-bead-based sandwich hybridisation assay using these specific probes, M. abscessus and M. smegmatis were distinguished from non-specific isolates within 70 mins with a lower detection limit of 1 pg/μL. The disruptive effects of microwaves on biological structures has been attributed to the local generation of heat. The contribution, if any, of non-thermal factors is yet to be determined. To study the interaction of microwaves with cell membranes, the structure which represents the major barrier to DNA release, fluorescent microscopy was employed to examine the passage of different sized fluorescent dextran particles into bacterial and yeast cells following microwave exposure. The results show a transient membrane disruption, size dependent permeabilization of dextran particles into cells. In conclusion, a prototype hybridisation assay capable of detecting M. abscessus and M. smegmatis has been developed. The application of microwaves to cells induced membrane disruption allowing internalisation of varying sizes of fluorescent dextran particles and the release of intact DNA for detection in hybridisation assay

Virucidal efficacy of gaseous ozone against type 1 herpes simplex virus (hsv-1)

Viruses represent a major threat to human health and are capable of spreading either via direct exposure or contamination of inanimate surfaces. Certain viruses have been shown to remain viable on surfaces of dental implants and devices, fabrics and on plastics for hours, days and weeks representing a source of continuous viral transmission which must be mitigated. In this study, we compared the effect of varying concentrations of gaseous ozone (40, 20, 10, 5, and 1 ppm) on Herpes Simplex Virus (HSV-1) coated on the surfaces of clear polystyrene tissue culture (TC) plates and stainless-steel disc at varying time intervals (1.5, 3, 6, and 8 h) at >90% relative humidity and at 21–25 ˙C (±2˙C). Test samples were placed at a height of 1 and 1.5 m from ozone source. Viral viability after ozone exposure was determined using Vero cells and the viral titer was quantified using the Spearman–Karber’s method. Overall, inactivation of HSV-1 was dependent on ozone concentration and the duration of exposure. At 20 and 40 ppm, ozone rapidly depleted virus viability after 6 h. At 5 and 10 ppm a time-dependent reduction in viral infectivity was observed. At 10 ppm, viral titer decreased from approximately 5.2 pfu/mL at T = 0 to 3.5 and 2.1 pfu/mL after 1.5- and 6-h exposure on plastic surfaces. Similarly, viral titer decreased from approximately 5.6 pfu/mL at T = 0 to 3.0 to 2.0 pfu/mL at same time frame on steel surfaces. This study builds on earlier research in the field and demonstrates the ability of gaseous ozone to inactivate HSV-1 on two surfaces commonly found in food, medical and dental settings. This study recommends that further work is completed to identify optimum ozone, humidity, and temperature combinations to inactivate such viruses on surfaces found within these environments

The biological effect of 2.45 GHz microwaves on the viability and permeability of bacterial and yeast cells

Microwaves are a form of non-ionizing radiation composed of electric (E) and magnetic (H) fields and are absorbed by biological tissues with a high water content. Our study investigated the effect of the E field, H field, and a combination of both (E + H) field’s exposure of structurally diverse micro-organisms, at a frequency of 2.45 GHz. We observed that the exposure to a microwave E field of an amplitude of 9.3 kV/m had no significant effect on cell viability; however, it did increase membrane permeability of Mycobacterium smegmatis to propidium iodide and to a range of different sized dextran particles in Escherichia coli, Staphylococcus aureus, Candida albicans, and M. smegmatis. The permeability of propidium iodide was observed in microwave treated cells (M. smegmatis) but not in heat-treated cells. Permeability of 3 kDa sized fluorescently labeled dextrans was observed across all cell types; however, this was found not to be the case for larger 70 kDa dextran particles. In terms of efflux, DNA was detected following E field exposure of M. smegmatis. In contrast, H field exposure had no effect on cell viability and did not contribute to increase cell’s membrane to dextran particles. In conclusion, this study shows that microwave generated E fields can temporarily disrupt membrane integrity without detrimentally impacting on cell viability. This approach has the potential to be developed as a high efficiency electropermeabilization method and as a means of releasing host DNA to support diagnostic applications

Whole Genome Comparisons Suggest Random Distribution of <i>Mycobacterium ulcerans</i> Genotypes in a Buruli Ulcer Endemic Region of Ghana

<div><p>Efforts to control the spread of Buruli ulcer – an emerging ulcerative skin infection caused by <i>Mycobacterium ulcerans</i> - have been hampered by our poor understanding of reservoirs and transmission. To help address this issue, we compared whole genomes from 18 clinical <i>M</i>. <i>ulcerans</i> isolates from a 30km² region within the Asante Akim North District, Ashanti region, Ghana, with 15 other <i>M</i>. <i>ulcerans</i> isolates from elsewhere in Ghana and the surrounding countries of Ivory Coast, Togo, Benin and Nigeria. Contrary to our expectations of finding minor DNA sequence variations among isolates representing a single <i>M</i>. <i>ulcerans</i> circulating genotype, we found instead two distinct genotypes. One genotype was closely related to isolates from neighbouring regions of Amansie West and Densu, consistent with the predicted local endemic clone, but the second genotype (separated by 138 single nucleotide polymorphisms [SNPs] from other Ghanaian strains) most closely matched <i>M</i>. <i>ulcerans</i> from Nigeria, suggesting another introduction of <i>M</i>. <i>ulcerans</i> to Ghana, perhaps from that country. Both the exotic genotype and the local Ghanaian genotype displayed highly restricted intra-strain genetic variation, with less than 50 SNP differences across a 5.2Mbp core genome within each genotype. Interestingly, there was no discernible spatial clustering of genotypes at the local village scale. Interviews revealed no obvious epidemiological links among BU patients who had been infected with identical <i>M</i>. <i>ulcerans</i> genotypes but lived in geographically separate villages. We conclude that <i>M</i>. <i>ulcerans</i> is spread widely across the region, with multiple genotypes present in any one area. These data give us new perspectives on the behaviour of possible reservoirs and subsequent transmission mechanisms of <i>M</i>. <i>ulcerans</i>. These observations also show for the first time that <i>M</i>. <i>ulcerans</i> can be mobilized, introduced to a new area and then spread within a population. Potential reservoirs of <i>M</i>. <i>ulcerans</i> thus might include humans, or perhaps <i>M</i>. <i>ulcerans</i>-infected animals such as livestock that move regularly between countries.</p></div

Micromolecular epidemiology of BU in the Asante Akim North District revealed by <i>M</i>. <i>ulcerans</i> whole genome sequence comparisons.

<p>(A) Median-joining network graph showing the genetic relationship between 18 <i>M</i>. <i>ulcerans</i> clinical isolates comprising the Agogo-1 and Agogo-2 genotypes (shaded), inferred from whole genome sequence alignments. Node sizes in the graph are proportional to the frequency of genotype occurrence and have been colour-coded accordingly. Edges are labelled in red with the number of mutational steps between each node. (B) Map of Asante Akim North District study area, showing the location of endemic villages and the origin of each of the 18 BU cases, with a coloured circle corresponding with the genotype displayed in the network graph in (A). The number “2” within some coloured circles indicates an Agogo-2 genotype.</p

Genetic relationship among the 33 <i>M</i>. <i>ulcerans</i> isolates used in this study.

<p>A maximum-likelihood consensus phylogeny was inferred based on whole genome alignments of each of the isolates against the <i>M</i>. <i>ulcerans</i> Agy99 reference genome. The alignment file from pairwise comparisons of the resulting 320 variable nucleotide positions was used as input for RaxML. Nodes with less than 70% bootstrap support (1000 replicates) were collapsed.</p