Full Text Available
Note: Clicking the button above will open the full text document at the original institutional repository in a new window.
Chemical and biological control of acinetobacter baumannii
Thesis (PhD)--Stellenbosch University, 2023.
Saved in:
| Main Author: | |
|---|---|
| Other Authors: | |
| Format: | Thesis |
| Language: | en_ZA |
| Published: |
Stellenbosch : Stellenbosch University
2023
|
| Subjects: | |
| Tags: |
No Tags, Be the first to tag this record!
|
| _version_ | 1867614075242938368 |
|---|---|
| access_status_str | Open Access |
| author | Havevenga, Benjamin |
| author2 | Khan, Wesaal |
| author_browse | Havevenga, Benjamin Khan, Wesaal |
| author_facet | Khan, Wesaal Havevenga, Benjamin |
| author_sort | Havevenga, Benjamin |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (PhD)--Stellenbosch University, 2023. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/128416 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:46:15.146Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2023 |
| publishDateRange | 2023 |
| publishDateSort | 2023 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/128416 Chemical and biological control of acinetobacter baumannii Havevenga, Benjamin Khan, Wesaal Ndlovu, Thando Reyneke, Brandon Stellenbosch University. Faculty of Science. Dept. of Microbiology. Acinetobacter baumannii -- Molecular genetics Drug resistance in microorganisms Acinetobacter -- Environmental aspects Bdellovibrio bacteriovorus Biosurfactants Acinetobacter infections -- Prevention Molecular epidemiology Thesis (PhD)--Stellenbosch University, 2023. ENGLISH ABSTRACT: Multidrug (MDR) and extensively drug resistant (XDR) Acinetobacter baumannii (A. baumannii) have emerged as the leading causes of nosocomial infections worldwide and are characterised as critical- priority pathogens by the World Health Organisation. Chapter one (abbreviated version published in Microorganisms) thus focused on the extensive antibiotic resistance and virulence mechanisms employed by A. baumannii as well as potential chemical (i.e., biosurfactants) and biological (i.e., predatory bacteria and bacteriophages) control agents which could be applied to target this opportunistic pathogen. Additionally, limitations associated with these alternative methods, and potential mitigation strategies, including curtailing resistance development using combination therapies, product stabilisation, and large-scale (up-scaling) production, were outlined. The primary focus of this study was thus to determine the efficacy of chemical and biological control strategies to combat or control reference, clinical and environmental MDR and XDR A. baumannii. While the research and development of alternative or novel strategies to combat infections caused by MDR and XDR A. baumannii is crucial, genotypic, and phenotypic characteristics, which may influence therapeutic potential, are often overlooked. The primary aim of Chapter two (published in Microbial Pathogenesis) was then to characterise and compare the genotypic and phenotypic characteristics of clinical (n = 13) and environmental (n = 7) A. baumannii isolates. Genotyping using Repetitive Extragenic Palindromic Sequence-based polymerase chain reaction (REP-PCR) analysis, indicated a low genetic relatedness between the clinical and environmental A. baumannii. Multilocus sequence typing (MLST, Oxford scheme) assigned the clinical A. baumannii isolates to three sequence types (i.e., ST231, ST945 and ST848), while the environmental A. baumannii were assigned to the novel ST2520 (except AB 14 which was assigned to ST945). While the majority of the clinical and environmental A. baumannii isolates were capable of phase variation, with both the translucent and opaque colony phenotypes detected, the clinical isolates exhibited significantly higher biofilm formation capabilities. Moreover, the clinical isolates exhibited significantly higher antibiotic resistance (first line and last resort) profiles, with 92.3% (12/13) classified as XDR, and five clinical A. baumannii isolates exhibiting colistin resistance (38.5%; 5/13). In contrast, while the environmental A. baumannii were predominantly characterised as MDR (57.1%; 4/7), the AB 14 isolate was characterised as XDR. Thus, while the differences in the genotypic, phenotypic, and antibiotic resistance profiles of clinical and environmental A. baumannii were highlighted, the environmental strains were assigned to the novel ST2520, which confirms the existence of this opportunistic pathogen in extra-hospital reservoirs. The primary aim of Chapter three was then to investigate the efficacy of lipopeptides and glycolipids against MDR and XDR strains of A. baumannii. Lipopeptides produced by Bacillus amyloliquefaciens (B. amyloliquefaciens) ST34 and Serratia marcescens (S. marcescens) NP1, and glycolipids produced by Pseudomonas aeruginosa (P. aeruginosa) SB24, were solvent extracted and partially characterised using ultra-performance liquid chromatography (UPLC) coupled to electrospray ionisation mass spectrometry (ESI-MS). The presence of surfactin (C13 to C16) and bacillomycin-L were detected in the B. amyloliquefaciens ST34 crude extract; serratamolides A, B, C, and glucosamine derivatives A, B, C, E and K in the S. marcescens NP1 crude extract, and di- and mono- rhamnolipids in the P. aeruginosa SB24 crude extract. Overall, in comparison to the NP1 and SB24 crude extracts, the ST34 crude extract exhibited increased efficacy against the MDR and XDR clinical and environmental A. baumannii, based on the disc diffusion results. Broth microdilutions in combination with the redox dye resazurin, then confirmed that the ST34 crude extract exhibited antimicrobial activity, with a minimum inhibitory concentration ranging from 5 to 20 mg/mL recorded. Using reverse-phase high-performance liquid chromatography (RP-HPLC), the surfactin fractions (Srf1 to Srf4) were purified with results indicating that specifically the Srf2 and Srf4 fractions exhibited potent antimicrobial activity of 60% and 80%, respectively, against representative A. baumannii STs, including the reference MDR AB 1 (ST931), clinical XDR AB 3 (ST231), environmental XDR AB 14 (ST945), and environmental MDR AB 16 (ST2520). In addition, the Srf1 to Srf4 fractions did not exhibit haemolytic activity and in vivo toxicity was not recorded for the ST34 crude extract and Srf2 and Srf4 fractions using the Galleria mellonella model. The ST34 crude extract, Srf2 and Srf4 fractions could thus be employed as novel or alternative strategies to control MDR and XDR A. baumannii. The primary aim of Chapter four was then to investigate the predation capabilities of a Bdellovibrio bacteriovorus (B. bacteriovorus) strain HW 1 (isolated from hospital wastewater) against the reference MDR AB 1 (ST931), clinical XDR AB 3 (ST231), clinical XDR CAC 8 (ST848), environmental XDR AB 14 (ST945), and environmental MDR AB 16 (ST2520). Culture-dependant and ethidium monoazide bromide quantitative polymerase chain reaction (EMA-qPCR) analysis of the co-culture assays indicated that the B. bacteriovorus HW 1 predated on all the MDR and XDR A. baumannii isolates, with log reductions ranging from 2.47 to 3.98 [colony forming units (CFU)/mL] and 2.42 to 4.54 [gene copies (GC)/mL] recorded for the first 24 hours. However, the cell counts and GC for all the A. baumannii strains in co-culture with B. bacteriovorus HW 1, increased at 48 hours and continued to increase until the end of the co-culture trial (i.e., 96 hours). Correspondingly, B. bacteriovorus HW 1 cell counts [plaque forming units (PFU)/mL] increased by 1.92 to 3.24 log at 24 hours, which was further confirmed with EMA-qPCR (GC/mL log increased 2.92 to 4.05). Subsequently, three methods [i.e., filtration, sodium dodecyl sulphate (SDS) treatment and kanamycin treatment] were employed to eliminate the B. bacteriovorus HW 1 from the co-cultures, to determine whether the A. baumannii strains initiated plastic phenotypic resistance to survive predation and persist in the co-culture trials. However, the triple filtration steps, SDS treatment and kanamycin treatment only reduced the cell count (PFU/mL) of B. bacteriovorus HW 1 by 2.93 log, 1.28 log and 0.16 log, respectively, in comparison to the unfiltered and untreated controls. Consequently, as the predatory population was not removed, reverse transcription quantitative polymerase chain reaction analysis (RT-qPCR) was applied to investigate the response of the surviving A. baumannii population in terms of virulence gene expression [i.e., lipopolysaccharide biosynthesis (lpsB) gene and the biofilm associated protein (bap) gene]. While variable results were obtained, overall, the expression analysis for the lpsB and bap genes indicated that the XDR CAC 8 (lpsB gene at 48 hours only) XDR AB 3, XDR AB 14 and MDR 16 (lpsB gene at 24 and 48 hours; bap gene at 48 hours only), during co-culture with B. bacteriovorus HW 1, was significantly lower compared to the expression recorded in the predator free controls at both 24 and 48 hours. These results indicated that although A. baumannii persists and survives predation by B. bacteriovorus HW 1, overall, the surviving prey population exhibited decreased virulence, which could be exploited in combination treatment strategies i.e., combining predatory bacteria pre-treatment (24 hours) with solar disinfection or solar pasteurization, to eliminate or eradicate the target pathogen from water sources. AFRIKAANSE OPSOMMING: Multigeneesmiddel (MGW) en ekstensiewe geneesmiddel weerstandige (EGW) Acinetobacter baumannii (A. baumannii) het wêreldwyd na vore getree as die hoofoorsaak van nosokomiale infeksies en word deur die Wêreldgesondheidsorganisasie as ‘n kritieke prioriteit patogeen geklassifiseer. Hoofstuk een (verkorte weergawe gepubliseer in “MDPI Microorganisms”) het dus gefokus op die uitgebreide antibiotika weerstand en virulensie meganismes wat deur A. baumannii aangewend word, sowel as potensiële chemiese (bv. biosurfaktante) en biologiese (d.w.s roofbakterieë en bakteriofage) beheermiddels wat toegepas kan word om hierdie opportunistiese patogeen te teiken. Daarbenewens is beperkings geassosieer met hierdie alternatiewe metodes, en potensiële versagtingstrategieë, insluitend die beperking van weerstand ontwikkeling deur gebruik te maak van kombinasie terapieë, produk stabilisering en grootskaalse (opskaling) produksie, uiteengesit. Die primêre fokus van hierdie studie was dus om die doeltreffendheid van chemiese en biologiese beheerstrategieë, vir die bevegting of beheer, van kliniese en omgewings MGW en EGW A. baumannii, te bepaal. Terwyl die navorsing en ontwikkeling van alternatiewe of nuwe strategieë om infeksies wat deur MGW en EGW A. baumannii veroorsaak word te bestry, deurslaggewend is, word genotipiese en fenotipiese eienskappe wat terapeutiese potensiaal kan beïnvloed, dikwels oor die hoof gesien. Die primêre doel van Hoofstuk twee (gepubliseer in “Microbial Pathogenesis”) was dus om die genotipiese en fenotipiese kenmerke van kliniese (n = 13) en omgewings (n = 7) A. baumannii isolate te karakteriseer en te vergelyk. Genotipering deur gebruik te maak van Herhalende Ekstrageniese Palindromiese Volgorde gebaseerde polimerase kettingreaksie (HEP-PKR) analise, het 'n lae genetiese verwantskap tussen die kliniese en omgewings A. baumannii aangedui. Multilokus volgorde tipering (MLVT, Oxford-skema) het die kliniese A. baumannii isolate in drie volgorde tipes (nl. VT231, VT945 en VT848) groepeer, terwyl die omgewings A. baumannii as ‘n nuwe VT2520 volgorde tipe geïdentifiseer is (behalwe AB 14 wat gegroepeer is aan VT945). Terwyl die meerderheid van die kliniese en omgewings A. baumannii isolate in staat was tot fase variasie, met beide die deurskynende en ondeursigtige kolonie fenotipes wat waargeneem is, het die kliniese isolate aansienlik hoër biofilm vorming vermoëns getoon. Boonop het die kliniese isolate aansienlik hoër antibiotika weerstands (eerste lyn en laaste uitweg) profiele getoon, met 92.3% (12/13) geklassifiseer as EGW, en vyf kliniese A. baumannii isolate wat kolistien weerstand getoon het (38.5%; 5/13). Daarteenoor, terwyl die omgewings A. baumannii hoofsaaklik as MGW (57.1%; 4/7) gekarakteriseer is, is die AB 14 isolaat as EGW gekarakteriseer. Dus, terwyl die verskille in die genotipiese, fenotipiese en antibiotika weerstands profiele van kliniese en omgewings A. baumannii uitgelig is, is die omgewings isolate aan die nuwe VT2520 toegeken, wat die bestaan van hierdie opportunistiese patogeen in buite-hospitaal reservoirs bevestig. Die primêre doel van Hoofstuk drie was dus om die doeltreffendheid van lipopeptiede en glikolipiede teen MGW en EGW isolate van A. baumannii te ondersoek. Lipopeptiede wat deur Bacillus amyloliquefaciens (B. amyloliquefaciens) ST34 en Serratia marcescens (S. marcescens) NP1 geproduseer word, en glikolipiede wat deur Pseudomonas aeruginosa (P. aeruginosa) SB24 geproduseer word, was deur oplosmiddel-ekstraksie onttrek en gedeeltelik gekarakteriseer met behulp van ultraprestasie vloeistofchromatografie (UPVC) wat aan elektronsproei ionisasie massaspektrometrie (ESI-MS) gekoppel is. Die teenwoordigheid van surfaktien (C 13 tot C 16 ) en bacillomisoen-L was bevestig in die B. amyloliquefaciens ST34 kru-ekstrak; serratamoliede A, B, C, en glukosamien afgeleides A, B, C, E en K in die S. marcescens NP1 kru-ekstrak, en di- en mono- rhamnolipiede in die P. aeruginosa SB24 kru-ekstrak. In die algemeen, in vergelyking met die NP1 en SB24 kru-ekstrakte, het die ST34 kru-ekstrak verhoogde doeltreffendheid teen die MGW en EGW kliniese en omgewings A. baumannii getoon, gebaseer op die skyfdiffusie resultate. Antibiotiese mikroverdunnings in kombinasie met die redoks kleurstof, resazurin, het toe bevestig dat die ST34 kru-ekstrak antimikrobiese aktiwiteit vertoon het, met 'n minimum inhiberende konsentrasie wat gewissel het vanaf 5 tot 20 mg/mL. Gevolglik, met behulp van omgekeerde fase hoëprestasie vloeistofchromatografie (OP-HPVC), is die surfaktien fraksies (Srf1 tot Srf4) gesuiwer, met resultate wat aangedui het dat spesifiek die Srf2- en Srf4 fraksies kragtige antimikrobiese aktiwiteit van onderskeidelik 60% en 80% vertoon het, teen verteenwoordigende A. baumannii VT's, insluitend die verwysings MGW AB 1 (VT931), kliniese EGW AB 3 (VT231), omgewings EGW AB 14 (VT945), en omgewings MGW AB 16 (VT2520). Daarbenewens het die Srf1 tot Srf4 fraksies nie hemolitiese aktiwiteit getoon nie en in vivo toksisiteit is nie aangeteken vir die ST34 kru-ekstrak en Srf2 en Srf4 fraksies deur gebruik te maak van die Galleria mellonella model nie. Die ST34 kru-ekstrak, Srf2 en Srf4 fraksies kan dus as nuwe of alternatiewe strategieë aangewend word om MGW en EGW A. baumannii te beheer . Die primêre doel van Hoofstuk vier was om die predasie vermoëns van 'n Bdellovibrio bacteriovorus (B. bacteriovorus ) isolaat HW 1 (geïsoleer uit hospitaal rioolwater) te ondersoek teen die verwysings MGW AB 1 (VT931), kliniese EGW AB 3 (VT231), kliniese EGW CAC 8 (VT848), omgewings EGW AB 14 (VT945), en omgewings MGW AB 16 (VT2520). Kultuur-afhanklike en etidium monoasied bromied kwantitatiewe polimerase kettingreaksie (EMA-kPKR) analise van die ko-kultuur het aangedui dat die B. bacteriovorus HW 1 geprooi het op al die MGW en EGW A. baumannii isolate, met logaritmiese verminderings wat gewissel het van 2.47 tot 3.98 [kolonievormende eenhede (KVE)/mL] en 2.42 tot 4.54 [geenkopieë (GK)/mL] oor die eerste 24 uur. Die seltellings en GK vir al die A. baumannii isolate in ko-kultuur met B. bacteriovorus HW 1 het egter na 48 uur toegeneem en het aangehou styg tot aan die einde van die ko-kultuur proef (d.w.s 96 uur). Dienooreenkomstig het B. bacteriovorus HW 1 seltellings [plaakvormende eenhede (PVE)/mL] toegeneem met 1.92 tot 3.24 logaritmes na 24 uur, wat verder bevestig is met EMA-kPKR (GK/mL logaritmies verhoog van 2.92 tot 4.05). Vervolgens is drie metodes [d.w.s filtrasie, natriumdodesielsulfaat (NDS) behandeling en kanamisien behandeling] aangewend om die B. bacteriovorus HW 1 uit die ko-kulture te elimineer, om ten einde te bepaal of die A. baumannii isolate plastiese fenotipiese weerstand geïnisieer het. Die drievoudige filtrasie stappe, NDS behandeling en kanamisien behandeling het egter slegs die seltelling (PVE/mL) van B. bacteriovorus HW 1 met onderskeidelik 2.93 logaritmes, 1.28 logaritmes en 0.16 logaritmes verminder in vergelyking met die ongefiltreerde en onbehandelde kontroles. Gevolglik, aangesien die roof populasie nie verwyder is nie, is omgekeerde transkripsie kwantitatiewe polimerase kettingreaksie (OT-kPKR) analise toegepas om die reaksie van die oorlewende A. baumannii populasie in terme van virulensie-geen uitdrukking te ondersoek [nl. lipopolisakkaried biosintese (lpsB) geen en die biofilm geassosieerde proteïen (bap) geen]. Alhoewel wisselvallige resultate verkry is, het die lpsB en bap geen uitdrukking analise in die algemeen aangedui dat die EGW CAC 8 (slegs lpsB geen op 48 uur) EGW AB 3, EGW AB 14 en MDR 16 (lpsB geen op 24 en 48 uur; bap geen slegs op 48 uur), tydens ko-kultuur met B. bacteriovorus HW 1, aansienlik laer was in vergelyking met die geen uitdrukking wat aangeteken is in die roofdier vrye kontroles op beide 24 en 48 uur. Hierdie resultate het aangedui dat alhoewel A. baumannii voortduur en predasie deur B. bacteriovorus HW 1 oorleef, die oorlewende prooi populasie in die algemeen verminderde virulensie getoon het, wat uitgebuit kan word in kombinasiebehandeling strategieë, d.w.s. die kombinasie van predatoriese bakterieë voorbehandeling (24 uur) met sonkrag ontsmetting of sonkrag pasteurisasie, om die teiken patogeen uit waterbronne uit te skakel of uit te roei. Doctorate 2023-02-07T06:43:56Z 2023-08-30T13:05:43Z 2023-03 2023-02-07T06:43:56Z 2023-08-31T09:18:39Z 2023-02-07T06:43:56Z 2023-08-31T09:18:39Z 2023-03 Thesis https://scholar.sun.ac.za/handle/10019.1/128416 en_ZA Stellenbosch University application/pdf xi, 204 pages : illustrations application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Acinetobacter baumannii -- Molecular genetics Drug resistance in microorganisms Acinetobacter -- Environmental aspects Bdellovibrio bacteriovorus Biosurfactants Acinetobacter infections -- Prevention Molecular epidemiology Havevenga, Benjamin Chemical and biological control of acinetobacter baumannii |
| title | Chemical and biological control of acinetobacter baumannii |
| title_full | Chemical and biological control of acinetobacter baumannii |
| title_fullStr | Chemical and biological control of acinetobacter baumannii |
| title_full_unstemmed | Chemical and biological control of acinetobacter baumannii |
| title_short | Chemical and biological control of acinetobacter baumannii |
| title_sort | chemical and biological control of acinetobacter baumannii |
| topic | Acinetobacter baumannii -- Molecular genetics Drug resistance in microorganisms Acinetobacter -- Environmental aspects Bdellovibrio bacteriovorus Biosurfactants Acinetobacter infections -- Prevention Molecular epidemiology |
| url | https://scholar.sun.ac.za/handle/10019.1/128416 |
| work_keys_str_mv | AT havevengabenjamin chemicalandbiologicalcontrolofacinetobacterbaumannii |