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In vitro and In vivo characterization of Amyloliquecidin, a novel two-component lantibiotic produced by Bacillus amyloliquefaciens

Van Staden, Anton Du Preez (2015-04)

Thesis (PhD)--Stellenbosch University, 2015.


ENGLISH ABSTRACT: Antimicrobial resistance is one of the major problems faced by the medical industry today. The ability of bacteria to rapidly acquire resistance against antibiotics and the over prescription and inappropriate use of antibiotics further exacerbate this crisis. Few new antimicrobials are, however, making it through the drug discovery pipeline. The search and development of novel and effective antimicrobials is therefore of the utmost importance. Lantibiotics are ribosomally synthesized cationic antimicrobial peptides with extensive post-translational modifications. They are active against a wide range of Gram-positive bacteria, including antibiotic-resistant strains. They are characterized by the presence of lanthionine and methyllanthionine rings and have been suggested as alternatives or for use in conjunction with antibiotics against resistant pathogens. Staphylococcus aureus is the most common bacteria isolated from skin and soft tissue infections (SSTIs). Strains of S. aureus have emerged with resistance against antibiotics with the most common being methicillin-resistant S. aureus (MRSA). Several lantibiotics are active against MRSA in vivo and have even shown superior activity to traditional antibiotics. Lantibiotics therefore show much promise for the treatment of SSTIs caused by resistant- and non-resistant S. aureus. In this study the bacterially diverse soil of the Fynbos in the Western Cape was screened for novel antimicrobials. Two antimicrobial producing Bacillus strains were isolated, Bacillus clausii AD1 and Bacillus amyloliquefaciens AD2. Both of these strains produce lantibiotics with B. clausii AD1 producing a known lantibiotic, clausin. B. amyloliquefaciens AD2 produces a novel two-component lantibiotic which was designated amyloliquecidin. The lantibiotic operon of amyloliquecidin was sequenced and annotated. All the genes required for successful production of amyloliquecidin are present in the operon. Amyloliquecidin was characterized in vitro and along with clausin is active against clinical strains of S. aureus (including MRSA), Enterococcus spp., Listeria spp. and beta-haemolytic streptococci. Amyloliquecidin has remarkable stability at physiological pH compared to nisin and clausin. A comparative in vivo murine infection model was used to evaluate the effectiveness of amyloliquecidin, nisin, clausin and Bactroban (commercial S. aureus topical treatment) in treating wound infections caused by S. aureus. All the lantibiotics proved to be just as effective as the Bactroban treatment. Furthermore, the tested lantibiotics did not have a negative influence on the wound closure rates of infected and non-infected wounds. Bactroban had a negative effect on wound healing compared to the lantibiotics. To our knowledge amyloliquecidin is the third two-component lantibiotic isolated from Bacillus. This study represents the first to test the effectiveness of amyloliquecidin in vivo and is one of a handful to test lantibiotics as topical treatments.

AFRIKAANSE OPSOMMING: Antimikrobiese weerstandbiedende bakterieë is op die oomblik een van die grootste probleme in die mediese veld. Die antibiotika krisis word vererg deur die vermoë van bakterieë om vinnig weerstand op te bou teen antibiotika, asook die alledaagse misbruik van antibiotika. Daar is ook ʼn tekort in die hoeveelheid antibiotika wat na die finale fases van ontwikkeling gaan. Om die oorhand teen antibiotika-weerstandige bakterieë te kry is dit van uiterste belang dat meer effektiewe antibiotika ontdek word. Lantibiotika is kationiese antimikrobiese peptiede wat deur die ribosoom gesintetiseer word en bevat ʼn verskeidenheid van modifikasies wat na translasie ingebou word. Hulle word gekarakteriseer deur lanthionien en metiellanthionien ringe. Lantibiotika is aktief teen ʼn verskeidenheid Gram-positiewe bakterieë en kan in kombinasie met antibiotika, of as alternatief gebruik word. Staphylococcus aureus is die mees algemene bakterium wat geassosieer word met vel en sagte weefsel infeksies (VSWIs). Staphylococcus aureus met weerstand teen antibiotika is ook al geïsoleer, die mees algemene weerstandige ras is methisillien-weerstandige S. aureus (MWSA). Lantibiotika is wel aktief teen MWSA in vitro en in vivo, met van hulle wat tot beter aktiwiteit as die voorgeskrewe antibiotika het. Lantibiotika kan dus gebruik word as behandeling vir VSWIs wat veroorsaak word deur weerstandige S. aureus, asook teen nie-weerstandige rasse. In hierdie studie was die bakteriese diverse grond van die Fynbos in die Wes-kaap ondersoek vir bakterieë wat antimikrobiese middels produseer. Twee Bacillus rasse, Bacillus clausii AD1 en Bacillus amyloliquefaciens AD2, wat antimikrobiese middels produseer, is geïsoleer. Bacillus clausii AD1 produseer ʼn bekende lantibiotikum, naamlik clausin. Bacillus amyloliquefaciens AD2 produseer ʼn nuwe twee-komponent lantibiotikum, amyloliquecidin. Die lantibiotikum operon wat verantwoordelik is vir die produksie van amyloliquecidin is geïdentifiseer en geannoteer. Die operon bevat al die gene benodig vir die biosintese van amyloliquecidin. Amyloliquecidin is in vitro gekarakteriseer en het aktiwiteit teen ʼn verskeidenheid Gram-positiewe bakterieë. Amyloliquecidin en clausin is aktief teen S. aureus (insluitend MWSA), Enterococcus spp., Listeria spp. en beta-hemolitiese streptococci wat vanaf infeksies geïsoleer is. Amyloliquecidin is baie stabiel by filologiese pH en aansienlik meer stabiel as nisin en clausin. Die effektiwiteit van nisin, clausin en amyloliquecidin in die behandeling van muis vel infeksies veroorsaak deur S. aureus was vergelyk met die kommersiële behandeling Bactroban. Al drie lantibiotika het die verspreiding van S. aureus met die selfde effektiwiteit as Bactroban belemmer. Geen van die lantibiotika het ʼn negatiewe effek op wond genesing nie. Bactroban, inteendeel, belemmer wond genesing. So ver ons weet is amyloliquecidin die derde twee-komponent lantibiotikum wat uit Bacillus geïsoleer is. Die studie is ook die eerste om die effektiwiteit van amyloliquecidin in vivo te rapporteer, asook ook een van die min studies wat kyk na lantibiotika as behandeling vir topikale infeksies.

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