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Microbiology

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    Evaluation of the larvicidal potential of Bacillus velezensis strain PHP1601 as a viable biological control agent against selected fly species = Ukuhlolwa kwamandla elarvicidal yeBacillus velezensis strain PHP1601 njenge-ejenti yokulawula yebhayoloji esebenzayo ngokumelene nezinhlobo zezimpukane ezikhethiwe
    (2024) Ramesar, Danvir Rajesh.; Hunter, Charles Haig.
    Flies are one of the most abundant and prevalent insect pests posing a growing threat to various sectors of the economy. In response to this, a study was undertaken to evaluate Bacillus spp. strain PHP1601 as a candidate biocontrol agent against Lucilia cuprina larvae as a proxy for fly species of biocontrol significance. The identity of PHP1601 was confirmed as B. velezensis using MLSA and species-specific PCR. Bioassays demonstrated a larvicidal effect of cell, endospore (102 – 1010 cells/endospores g -1 ) and cell-free supernatant (1 – 30% v w -1 ) treatments on second instar larvae of L. cuprina. Studies were directed to the larvicidal effect of extracellular compounds, namely lipopeptides. Crude lipopeptide extract (CLP) was acquired using organic extraction from Landy broth. Bioassays with CLP extract (5 – 1000 μg g -1 ) resulted in a dose-dependent larvicidal response. Lipopeptides in the CLP extract were purified by TLC and characterised using UPLC ESI-TOF MS. This indicated the presence of iturin, fengycin and surfactin homologues of which, the purified surfactin fraction (Rf 0.91) was the most larvicidal. Bioassays were repeated with commercial surfactin, confirming its larvicidal potency, exhibiting an LC50 of 9.87 μg g -1 at 240 h. Larvae scent choice tests using TSB and MG bioassay medium fermented by PHP1601 showed that resulting VOCs were attractive to fly larvae, which was considered a viable trait of a fly biocontrol agent. CG-MS of the VOCs produced indicated that ketones were the dominant VOC class and, presumably, the major contributor to this larvae attraction effect. Field performance evaluation using pig manure trials demonstrated successful inhibition of several fly species of agricultural and veterinary importance using endospore treatments (105 and 1010 endospores g -1 ) of PHP1601. qPCR and REP-PCR fingerprinting confirmed that PHP1601 could grow in the manure slurries and was amiable to recovery and monitoring. Zebrafish embryo toxicity bioassays of the CLP produced by PHP1601 indicated that they achieved an LC50 of 22.77 µg ml-1, which characterised these metabolites as slightly toxic. Genome mining detected no genes associated with pathogenicity or virulence and presented no apparent pathogenic threat. The investigation demonstrated that B. velezensis PHP1601 is a viable fly biocontrol candidate and constitutes the first report of a B. velezensis antagonist of Brachycera flies. Iqoqa: Izimpukane izinambuzane ezivamile kakhulu nezibanga ubungozi obukhulayo emikhakheni eyahlukahlukene kwezomnotho. Ukubhekana nalokhu, lolu cwaningo lwenziwa ukuhlola ingcindezi yeBacillus sp. i-PHP1601 njengesinye sezilawulo semvelo ukubhekana nezimpethu zeLucilia cuprina njengesimeleli sohlobo lokundizayo lokwengamela okuphilayo okubalulekile. Ubunjalo bePHP1601 batholakala njengeB. velezensis kusetshenziswa i-MLSA kanye nohlobo oluthile lwe-PCR. Izikali zokuphilayo zaveza ukukhephuka kwezimpethu kweseli i-endospore (102 – 1010 cells/endosporesꞏg-1) kanye nokubhekelelwa kwe-cell-free supernatant (1 – 30% vꞏw-1) esigabeni sesibili sokukhula kwezimpethu ze-L. cuprina. Ucwaningo lwalubhekiswe emitheleleni yokuphethuka kwezimpethu endaweni ezungeze amaseli, nokuyi-lipopeptides. Ukuhluzwa kwe-crude lipopeptide (CLP) kwatholakala ngokusebenzisa ukudonswa kwendabuko kwi-Landy broth. Izikali zokuphilayo nokukhanywa kwe-CLP (5 – 1000 μgꞏg-1) kwaveza ekuncikeni kokusetshenzwa kwempendulo enezimpethu. Ama-Lipopeptides ekukhanyweni kwe-CLP acwengisiswa nge-TLC futhi abukwa ngokusebenzisa i-UPLC ESI-TOF MS. Lokhu kwaveza ubukhona be-iturin, i-fengycin kanye ne- surfactin homologues nobukhona bengxenyana yesimanzisi esicwengekile (Rf 0.91) esasiphethula izimpethu kakhulu. Izikali zokuphilayo zaphindwa ngesimanzisi esikhulu sokwakha inzuzo, ukuqinisekisa ukukhuphuka kokuphethuka kwempethu, okuveza i-LC50 of 9.87 μgꞏg-1 at 240 h. Izikali zokuphilayo ngobungozi besiqalo se-Zebrafish zokukhanywa kwe-CLP kwaveza ukuthi kwakhipha i-LC50 of 22.77 µgꞏml-1, nekhomba ama-metabolites anobungozi obuncane. Ukukhiqiza ufuzo kwaveza ukuthi alukho ufuzo oluhlobene nobugciwane bofuzo noma ubungozi futhi kwaveza ukungabi bikho kokuhlobana kofuzo. Ukuhlola ukusebenza kwendawo kusetshenziswa umanyolo wezingulube kwaveza ukunqamuka okuyimpumelelo kwezinhlobo zezimpukane eziningi zezolimo kanye nokubaluleka kwezilwane kusetshenziswa ukusetshenzwa kwe-endospore (105 and 1010 endosporesꞏg-1) ye-PHP1601. I-qPCR kanye nokuhlotshaniswa kwe-REP-PCR kwavuma ukuthi i-PHP1601 ingakhula ezindaweni zikamanyolo futhi kwakuhlobene nokuthuthuka kanye nokuqapheleka. Iphunga lezimpethu lezikali ezikhethiwe laveza ukuthi ama-VOCs akhiqizwa esimuncwaneni se-PHP1601 eyenomthelela wezimpethu zezimpukane, nokwavela njengenjwayelo eshintshayo yesakhi sokwengamela ukuphila kwezimpukane. Ama-CG-MS e-VOCs akhiqizwa akhomba ukuthi ama-ketones ayewuhlobo olukhulu lwe-VOC futhi, okulindelekile, umthelela omkhulu womphumela wokumema izimpethu. Ukuhlola kwaveza ukuthi i-B. velezensis PHP1601 uwuhlobo olushintshayo lwesengamelo sezimpukane futhi kwakha umbiko wokuqala wokuphikisana kwezimpukane ze-Brachycera.
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    Investigating the role of small RNAS in transcriptome regulation of genetically diverse clinical strains of mycobacterium tuberculosis.
    (2021) Govender, Divenita.; Mvubu, Nontobeko Eunice.
    Tuberculosis (TB), caused by the human adapted members of the Mycobacterium tuberculosis complex (MTBC), is a threat to global health. Understanding the regulatory network of the MTBC members may reveal novel vaccine candidates and drug targets. The small RNAs (sRNAs) have only recently been investigated for their role in Mycobacterium tuberculosis (M. tb) transcriptome regulation with none being explored in clinical strains or within the MTBC lineages. The present study aimed to investigate the regulatory role of sRNAs on the M. tb transcriptome in a lineage-specific manner, with emphasis on the clinical strains most prevalent in South Africa. In silico whole genome sequence alignment of strains belonging to the eight MTBC lineages was performed to identify sRNAs containing lineage-specific mutations and their respective potential targets. To elucidate transcriptome regulation in clinical strains of M. tb belonging to the Beijing and F15/LAM4/KZN lineages, mRNA and sRNA sequencing were performed followed by Hisat-Ballgown Bioinformatics analysis to identify novel sRNAs and their respective targets. The sRNAs discovered from sRNA sequencing were confirmed through real time qPCR. The in silico data revealed several sRNAs that may play a role in transcriptome regulation at a lineage-specific level, such as those involved in macrophage entry, lipid biosynthesis pathway, adaptation mechanisms during antibiotic exposure, and environmental stress. They may also be able to disrupt genes that are detrimental and restore functions to those that are beneficial. The mutated and consensus sRNAs were identified to target the same function, but one pathway may be more efficient than the other. Novel sRNAs were discovered from sRNA sequencing of the Beijing and F15/LAM4/KZN clinical strains, with their predicted targets absent from the mRNA sequencing results, indicating these sRNAs may elicit an inhibitory function. Real time-PCR analysis revealed significant fold change differences between the clinical strains belonging to the Beijing, F15/LAM4/KZN, F11 and Unique families suggesting an underlying regulation of these transcripts at a family level. This data could explain the underlying phenotypic differences observed within the MTBC and understanding of the regulatory function of these sRNAs, may identify novel alternative strategies in the fight against M. tb.