Identifying the primary microbial and chemical source tracking markers in harvested rainwater for the detection of faecal contamination

Waso, Monique (2017-03)

Thesis (MSc)--Stellenbosch University, 2017.

Thesis

ENGLISH ABSTRACT: Rainwater harvesting has been earmarked as an additional source of fresh water. However, research has indicated that the microbiological quality is substandard as pathogens have been detected in this water source. As it is impractical to monitor for the presence of all pathogens in a water source, indicator organisms are routinely utilised to monitor water quality and predict the presence of pathogens in contaminated environmental waters. Various research groups have however indicated that the analysis of indicator organisms in a water source may not be sufficient to accurately identify the source of contamination. Supplementary indicators are therefore required to accurately identify contamination sources, with chemical and microbial source tracking markers currently being investigated and applied to various water sources. The primary focus of the current study was thus to identify a toolbox of microbial source tracking (MST) and chemical source tracking (CST) markers that could be utilised to supplement indicator organism analysis of domestic rainwater harvesting (DRWH) systems. To achieve this aim, harvested rainwater (n = 60) and rooftop debris (n = 60) samples were screened for a range of MST (conventional PCR) and CST (high-performance liquid chromatography tandem mass spectrometry) markers previously utilised in literature to analyse various water sources (Chapter two). All the tank water samples collected at the Kleinmond Housing Scheme site (Kleinmond, Western Cape), were also screened for traditional indicator organisms using culture based techniques. Additionally, Escherichia coli (E. coli) and enterococci were screened for in all tank water and rooftop debris samples using quantitative PCR (qPCR) analysis. Based on the conventional PCR results, Bacteroides HF183, adenovirus, Lachnospiraceae and human mitochondrial DNA (mtDNA) were the most prevalent MST markers. These markers were subsequently quantified in the tank water and rooftop debris samples by qPCR. The HF183 marker was then detected at a mean concentration of 5.1 × 103 and 4.7 × 103 gene copies/μL in the tank water and rooftop debris, respectively. Adenovirus was detected at 3.2 × 102 and 6.4 × 103 gene copies/μL; human mtDNA was detected at 1.1 × 106 and 3.0 × 105 gene copies/μL and Lachnospiraceae was detected at 3.0 × 104 and 6.9 × 103 gene copies/μL in the tank water and rooftop debris samples, respectively. Additionally, E. coli and enterococci were quantifiable in all tank water and rooftop debris samples by qPCR analysis. The CST markers caffeine, salicylic acid, acetaminophen, triclosan, triclocarban and methylparaben were then detected at μg/L levels in all the tank water [except salicylic acid (98%)] and rooftop debris samples. A secondary aim was to establish correlations between the MST and CST markers as well as indicator organisms to ascertain which markers may be employed to supplement indicator organism analysis of DRWH systems. In the tank water samples, significant positive correlations were observed for adenovirus versus E. coli (enumerated with the culturing techniques) (p = 0.000), the HF183 marker versus E. coli (quantified by qPCR) (p = 0.023), Lachnospiraceae versus heterotrophic bacteria (p = 0.000) and human mtDNA versus enterococci (enumerated with the culturing techniques) (p = 0.026). In addition, significant positive correlations were observed for caffeine versus enterococci (quantified by qPCR) (p = 0.000); faecal coliforms (p = 0.001); total coliforms (p = 0.000) and enterococci (enumerated with culturing techniques) (p = 0.002). Salicylic acid also positively correlated with total coliforms (p = 0.024) in the tank water samples. For the rooftop debris samples, significant positive correlations were observed for E. coli (quantified by qPCR) versus methylparaben (p = 0.000) and salicylic acid (p = 0.042), respectively. Based on the results obtained, it is thus evident that faecal contamination and anthropogenic activities may be the primary sources of contamination in the DRWH systems. Moreover, the markers Bacteroides HF183, Lachnospiraceae, human mtDNA, adenovirus, caffeine, salicylic acid and methylparaben may be utilised to supplement traditional indicator organism analysis for the monitoring of harvested rainwater. It is however recommended that future studies focus on correlation analysis of the source tracking markers with pathogens frequently detected in harvested rainwater, in order to determine which source tracking markers may be utilised as surrogates for these pathogens and subsequently as supplementary indicators. Avian species are vectors of microorganisms in the environment and have been identified as major sources of faecal contamination of DRWH systems. The focus of Chapter three was thus to design and validate (on a small-scale) novel MST markers for the detection of avian faecal contamination in the DRWH systems. Three primer sets [AVF1 and AVR (designated AV1); AVF2 and AVR (designated AV2); and ND5F and ND5R (designated ND5)] were subsequently designed to target regions of the NADH dehydrogenase subunit 5 mitochondrial DNA gene of avian species. Mitochondrial DNA is abundant in animal faecal matter and may thus be readily detected. Conventional PCR assays were optimised for each of the three primer sets. Avian and non-avian faecal samples were then screened to validate the host-specificity and host-sensitivity of the mtDNA markers. The mtDNA markers AV1, AV2 and ND5 displayed a host-sensitivity of 1.00, 0.892 and 0.622, respectively. While the host-specificity of each assay was equal to 0.316, 0.0526 and 0.237 for AV1, AV2 and ND5, respectively. Tank water samples (n = 60) and rooftop debris (n = 60) were then screened for the prevalence of the three markers. Overall, AV1 was the dominant marker detected in the tank water (85%) and rooftop debris (90%) samples. Bayes’ theorem then indicated that there was an 89.2% and 92.9% probability that the AV1 marker detected true avian faecal contamination in the tank water and rooftop debris samples, respectively. The AV1 marker thus exhibited the greatest potential as an avian mtDNA marker for the detection of avian faecal contamination in DRWH systems. However, based on the low host-specificity obtained for all three primer sets (AV1, AV2 and ND5), further optimisation should include the use of a Taqman™ probe to increase the specificity of this marker.

AFRKAANSE OPSOMMING: Geoeste reënwater is geïndentifiseer as ‘n addisionele vars waterbron, maar navorsing het bewys dat die mikrobiese kwaliteit substandaard is aangesien ‘n verskeidenheid patogene al in geoeste reënwater gevind is. Aangesien dit onprakties is om vir alle patogene in ‘n waterbron te toets, word indikator organismes algemeen gebruik om die kwaliteit van waterbronne te monitor en om die teenwoordigheid van patogene in die water te voorspel. Verskeie navorsingsgroepe het egter gewys dat om vir indikator organismes te toets, nie voldoende is om die bron van kontaminasie te identifiseer nie. Daar is dus ‘n behoefte aan aanvullende indikators om die bronne van kontaminasie te identifiseer. Daarom word chemiese en mikrobiese bron spoor merkers deesdae nagevors en toegepas op verskeie waterbronne. Die primêre doel van die huidige studie was dus om ‘n versameling mikrobiese bron spoor (MBS) en chemiese bron spoor (CBS) merkers te identifiseer wat gebruik mag word om die analise van indikator organismes in huishoudelike reënwater oesting (HRWO) sisteme, aan te vul. Hierdie doel is behaal deur geoeste reënwater monsters (n = 60) en detritus monsters vanaf die dakoppervlak (n = 60) te toets vir ‘n paneel MBS (konvensionele PKR) en CBS (hoë-verrigting vloeistof chromatografie tandem massaspektrometrie) merkers, wat voorheen in die literatuur aangewend is om water te analiseer (Hoofstuk twee). Die tenk water monsters wat by die Kleinmond Behuisings-skema (Kleinmond, Wes-Kaap) geneem is, is ook getoets vir tradisionele indikator organismes deur gebruik te maak van groei-gebaseerde tegnieke. Daarby is daar ook vir Escherichia coli ( E. coli) en enterokokkie met kwantitatiewe PKR (kPKR) in die tenk water en detritus monsters getoets. Die konvensionele PKR resultate het getoon dat Bacteroides HF183, adenovirus, Lachnospiraceae en menslike mitokondriale DNS (mtDNS) die mees algemene MBS merkers in die monsters was. Hierdie merkers is dus gekwantifiseer in die tenk water en detritus monsters met behulp van kPKR. Die HF183 merker is toe teen ‘n gemiddelde konsentrasie van 5.1 × 103 en 4.7 × 103 geen kopieë/μL in die tenk water en detritus monsters gekry. Adenovirus is teen 3.2 × 102 en 6.4 × 103 geen kopieë/μL; menslike mtDNS is teen 1.1 × 106 en 3.0 × 105 geen kopieë/μL en Lachnospiraceae is teen 3.0 × 104 en 6.9 × 103 geen kopieë/μL in onderskeidelik die tenk water en detritus monsters gekry. Daarbenewens was die E. coli en enterokokkie ook kwantifiseerbaar in al die tenk water en detritus monsters, onderskeidelik. Die CBS merkers kafeïen, salisielsuur, asetaminofen, metielparabeen, triklosaan en triklokarbaan is teen μg/L vlakke in al die tenk water [behalwe salisielsuur (98%)] en detritus monsters gekry. ‘n Tweede doel van hierdie studie was om korrelasies tussen die MBS en CBS merkers en indikator organismes te ondersoek, om vas te stel watter merkers gebruik mag word om indikator organisme analises aan te vul. In die tenk water monsters is daar beduidende positiewe korrelasies waargeneem vir adenovirus teenoor E. coli (groei-gebaseerd) (p = 0.000), die HF183 merker teenoor E. coli (kPKR) (p = 0.023), Lachnospiraceae teenoor heterotrofiese bakterieë (p = 0.000) en menslike mtDNS teenoor enterokokkie (groei-gebaseerd) (p = 0.026). Daaropvolglik, is beduidende positiewe korrelasies opgemerk vir kafeïen teenoor enterokokkie (kPKR) (p = 0.000); fekale koliforme (p = 0.001); totale koliforme (p = 0.000) en enterokokkie (groei-gebaseerd) (p = 0.002). Salisielsuur het ook positief gekorreleer met totale koliforme (p = 0.024) in die tenk water monsters. Vir die detritus monsters is beduidende positiewe korrelasies opgemerk vir E. coli (kPKR) teenoor metielparabeen (p = 0.000) en salisielsuur (p = 0.042), onderskeidelik. Hierdie resultate dui dan aan dat fekale kontaminasie en antropogeniese aktiwiteite die primêre bronne van kontaminasie van die HRWO sisteme is. Verder kan Bacteroides HF183, Lachnospiraceae, menslike mtDNS, adenovirus, kafeïen, salisielsuur en metielparabeen gebruik word om tradisionele indikator organisme analises aan te vul om die kwaliteit van geoeste reënwater te monitor. Daar word egter aanbeveel dat toekomstige studies op korrelasies tussen bron spoor merkers en patogene, wat gereeld in geoeste reënwater gevind word, ondersoek word om vas te stel watter bron spoor merkers as surrogate vir hierdie patogene en verder as aanvullende indikators gebruik kan word. Voël spesies is vektore van mikroorganismes in die omgewing en is geïdentifiseer as bronne van fekale kontaminasie in HRWO sisteme. Die fokus van Hoofstuk drie was dus om nuwe MBS merkers, om fekale kontaminasie van voëls in HRWO sisteme op te spoor, te ontwerp en op ‘n klein skaal te verifieer. Drie inleier stelle [AVF1 en AVR (benoem AV1); AVF2 en AVR (benoem AV2); en ND5F en ND5R (benoem ND5)] is dus ontwerp om dele van die NADH dehidrogenase subeenheid 5 mtDNS geen van voëls te teiken. Mitokondriale DNS is vollop in die fekale materiaal van diere en kan dus maklik geamplifiseer word. Konvensionele PKR toetse is vir elke inleier paar geoptimiseer. Fekale monsters van voël spesies en nie-voël spesies is gevolglik geanaliseer om die gasheer-sensitiwiteit en -spesifisiteit van die mtDNS merkers te verifieer. Die gasheer-sensitiwiteit was dus gelyk aan 1.00, 0.892 en 0.622 vir die AV1, AV2 en ND5 merkers, onderskeidelik, terwyl die gasheer-spesifisiteit gelyk was aan 0.316, 0.0526 en 0.237 vir die AV1, AV2 en ND5 merkers, onderskeidelik. Tenk water (n = 60) en detritus (n = 60) monsters is toe getoets vir die teenwoordigheid van die drie merkers. Die AV1 merker is as die dominante merker in die tenk water (85%) en detritus (90%) monsters geïdentifiseer. Bayes se stelling het aangedui dat daar ‘n 89.2% en 92.9% waarskynlikheid is dat die AV1 merker opgespoor is weens ware voël verwante kontaminasie in die tenk water en detritus monsters. Die AV1 merker het dus die grootste potensiaal om as ‘n mtDNS merker, vir die opsporing van voël verwante kontaminasie in HRWO sisteme, gebruik te kan word. As gevolg van die lae gasheer-spesifisiteit wat opgemerk is vir die drie inleier stelle (AV1, AV2 en ND5), word daar egter voorgestel dat hierdie merkers verder geoptimiseer moet word deur gebruik te maak van Taqman™ ondersoekers spesifiek vir voëls, om dan die spesifisiteit van die merkers te verbeter.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/100830
This item appears in the following collections: