Legionella species persistence mechanisms in treated harvested rainwater

Dobrowsky, Penelope Heather (2017-03)

Thesis (PhD)--Stellenbosch University, 2017.

Thesis

ENGLISH ABSTRACT: The persistence of Legionella spp. at high pasteurization temperatures poses a threat to human health as a number of Legionella spp. are known to cause Legionnaires’ disease. Research has then indicated that the primary factors that allow Legionella to proliferate and persist in water distribution systems are: the accessibility to nutrients in a water source, water temperature, the presence of free-living amoebae (FLA) and other aquatic bacteria. The focus of the current study was thus to investigate and functionalise selected persistence mechanisms displayed by Legionella spp. that aid in their survival in pasteurized and unpasteurized harvested rainwater. The overall aim of Chapter two was to isolate and identify the dominant Legionella spp. persisting in a domestic rainwater harvesting tank and a solar pasteurization (SOPAS) system and to identify possible FLA vectors of Legionella that remain viable at high pasteurization temperatures (>60°C). For this, pasteurized and unpasteurized tank water samples were screened for the dominant Legionella spp. using culture based techniques. In addition, as FLAs including Acanthamoeba spp., Naegleria fowleri and Vermamoeba (Hartmannella) vermiformis are the most frequently isolated from hot water systems, ethidium monoazide polymerase chain reaction (EMA-qPCR) was utilised for the quantification of viable Legionella spp., Acanthamoeba spp., V. vermiformis and N. fowleri. Eighty-two Legionella spp. were isolated from the unpasteurized tank water samples, where L. longbeachae (35 %) was the most frequently isolated, followed by L. norrlandica (27 %) and L. rowbothamii (4 %). This information provides pertinent knowledge on the occurrence and dominant species of Legionella present in the South African environment. In addition, the SOPAS system was effective in reducing the gene copies of viable N. fowleri (5-log) and V. vermiformis (3-log) to below the lower limit of detection at temperatures of 68–93°C and 74–93°C, respectively. In contrast, as gene copies of viable Legionella and Acanthamoeba were still detected after pasteurization at 68–93°C, it could be concluded that Acanthamoeba spp. primarily act as vectors for Legionella spp. in solar pasteurized rainwater. The primary objective of Chapter three was to determine the resistance of three Legionella species isolated from unpasteurized rainwater [L. longbeachae (env.), L. norrlandica (env.) and L. rowbothamii (env.)], two Legionella reference strains (L. pneumophila ATCC 33152 and L. longbeachae ATCC 33462) and Acanthamoeba mauritaniensis ATCC 50676 to heat treatment (50–90°C). In addition, the resistance of L. pneumophila ATCC 33152 and L. longbeachae (env.) in co-culture with A. mauritaniensis ATCC 50676, respectively, to heat treatment (50–90°C) was determined using EMA-qPCR. The interaction mechanisms exhibited between Legionella and Acanthamoeba during heat treatment (50–90°C) were also elucidated by monitoring the relative expression of genes associated with metabolism and virulence of L. pneumophila ATCC 33152 (lolA, sidF, csrA) and L. longbeachae (env.) (lolA) in co-culture with A. mauritaniensis ATCC 50676, respectively. Legionella longbeachae (env.) and L. pneumophila ATCC 33152 were the most resistant to heat treatment as both organisms were still culturable (CFU/mL) following treatment at 50 and 60°C. However, the sensitivity of detection of viable cells was increased when using EMA-qPCR as all Legionella spp. and A. mauritaniensis ATCC 50676 were detected following heat treatment (50–90°C). In addition, while the heat resistance of L. pneumophila ATCC 33152 in co-culture with A. mauritaniensis ATCC 50676 improved, it is postulated that L. longbeachae (env.) is unable to replicate in A. mauritaniensis ATCC 50676 as L. longbeachae (env.) in co-culture was not detected following heat treatment at 80°C and 90°C. Results also showed a clear trend between genes with related function and differential expression during heat treatment (50-90°C). For example, relative to the untreated samples, the expression of lolA remained constant while the expression of sidF increased and the expression of csrA decreased significantly during L. pneumophila ATCC 33152 co-culture with A. mauritaniensis ATCC 50676. Results thus confirm that while heat treatment may reduce the number of viable Legionella spp., L. pneumophila is able to interact with A. mauritaniensis and persist during heat treatment. The overall aim of Chapter four was to elucidate other microbial and physico-chemical characteristics that may be associated with the incidence of Legionella spp. and Acanthamoeba spp. in rainwater harvested from different roofing materials. Overall results indicated that the roofing materials did not influence the incidence of Legionella and Acanthamoeba spp. as these organisms were detected in all tank water samples collected from the Chromadek®, galvanized zinc and asbestos roofing materials. However, significant (p < 0.05) positive Spearman (ρ) correlations were noted between Legionella spp. vs. nitrites and nitrates and between Acanthamoeba spp. vs. barium, magnesium, sodium, silicon, arsenic and phosphate, respectively. In addition, while no significant correlations were observed between Legionella spp. vs. the indicator bacteria (p > 0.05), positive correlations were established between Acanthamoeba spp. vs. total coliforms and Escherichia coli, respectively. Results thus indicated that the incidence of Legionella and Acanthamoeba spp. in harvested rainwater may primarily be due to external pollutants such as dust and animal faecal matter present on the catchment system.

AFRIKAANSE OPSOMMING: Die voortbestaan van Legionella spp. by hoë pasteurisasie temperature kan 'n bedreiging vir menslike gesondheid inhou deurdat 'n aantal Legionella spp. daarvoor bekend is om die siekte Legionnaires te veroorsaak. Navorsing dui ook aan dat die primêre faktore wat bydra tot die vermeerdering en voortbestaan van Legionella in waterverspreidingsisteme, die toeganklikheid tot voedingstowwe in 'n waterbron, die watertemperatuur, die teenwoordigheid van vrylewende amoeba (VLA) en die teenwoordigheid van ander akwatiese bakterieë, insluit. Die fokus van hierdie studie was dus om die voortbestaansmeganismes te ondersoek wat deur Legionella spp. gebruik word om in gepasteuriseerde en ongepasteuriseerde ge-oeste reënwater te oorleef. Die oorhoofse doel van Hoofstuk twee was om die dominante Legionella spp. te isoleer en te identifiseer wat voorkom en oorleef in 'n huishoudelike reënwater opgaringstenk en 'n sonkrag pasteurisasie (SOPAS) sisteem. Verder was die doel ook om moontlike VLA, wat as vektore vir Legionella kan dien, te identifiseer en om te bepaal of hierdie vektore dan lewensvatbaar bly by hoë pasteurisasie temperature (>60°C). Hiervoor is gepasteuriseerde (45°C, 65°C, 68°C, 74°C, 84°C en 93°C) en ongepasteuriseerde tenkwatermonsters getoets vir die dominante Legionella spp., deur gebruik te maak van groei-gebaseerde tegnieke. Daarbenewens, aangesien VLA insluitend Acanthamoeba spp., Naegleria fowleri en Vermamoeba (Hartmannella) vermiformis die mees algemene amoeba spesies is wat uit watermonsters en warmwatersisteme geïsoleer word, is ethidium monoasied kwantitatiewe polimerase kettingreaksie (EMA-kPKR) aangewend om die lewensvatbare Legionella spp., Acanthamoeba spp., V. vermiformis en N. fowleri in gepasteuriseerde (68°C, 74°C, 84°C en 93°C) en ongepasteuriseerde tenkwatermonsters, te kwantifiseer. Twee-en-tagtig Legionella spp. is vanuit die ongepasteuriseerde tenkwatermonsters geïsoleer, met L. longbeachae (35%) wat die meeste geïsoleer is, gevolg deur L. norrlandica (27%) en L. rowbothamii (4%). Verder is daar bevind dat die die SOPAS sisteem die geen kopieë van die lewensvatbare N. fowleri (5-log) en V. vermiformis (3-log) effektief verminder het tot onder die onderste grens van opsporing by pasteurisasie temperature van 68-93°C en 74-93°C, onderskeidelik. In teenstelling, is daar bevind dat daar steeds lewensvatbare Legionella en Acanthamoeba spp. teenwoordig is by pasteurisasie temperature van 68-93°C, aangesien geen kopieë steeds in hierdie monsters waargeneem is. Daar kon dus afgelei word dat Acanthamoeba spp. hoofsaaklik as vektore dien vir Legionella spp. in son-gepasteuriseerde reënwater. Die primêre doel van Hoofstuk drie was om drie Legionella spp. [L. longbeachae (env.), L. norrlandica (env.) en L. rowbothamii (env.)] geïsoleer vanuit ongepasteuriseerde reënwater; twee Legionella verwysingstamme (L. pneumophila ATKK 33152 en L. longbeachae ATKK 33462) en Acanthamoeba mauritaniensis ATKK 50676 se weerstand teen hittebehandeling (50-90°C), te bepaal. Daarna is die weerstand teen hittebehandeling (50-90°C) van onderskeidelik L. pneumophila ATKK 33152 en L. longbeachae (env.) in samegroeïng met A. mauritaniensis ATKK 50676 bepaal deur gebruik te maak van EMA-kPKR. Verder is die interaksie meganismes wat uitgevoer word tussen Legionella en Acanthamoeba tydens hittebehandeling (50-90°C) ook geondersoek deur die relatiewe uitdrukking van gene wat geassosieer word met die metabolisme en virulensie van L. pneumophila ATKK 33152 (lolA, sidF, csrA) en L. longbeachae (env.) (slegs lolA) te monitor. Legionella longbeachae (env.) en L. pneumophila ATKK 33152 het die meeste weerstand getoon teen hittebehandeling aangesien beide hierdie organismes steeds op media gegroei het (KVE/ml) by onderskeidelik 50 en 60°C. Verder is bevind dat EMA-kPKR ‘n meer sensitiewe tegniek is om lewensvatbare selle op te spoor, omdat alle Legionella spp. en A. mauritaniensis ATKK 50676 steeds in die monsters opgespoor kon word nadat hittebehandeling (50-90°C) toegepas is. Daarbenewens, terwyl die hitte weerstandigheid van L. pneumophila ATKK 33152 in samegroeïng met A. mauritaniensis ATKK 50676 verbeter het, word daar gepostuleer dat L. longbeachae (env.) nie in staat is om te vermeerder binne in A. mauritaniensis ATKK 50676 nie, aangesien L. longbeachae (env.) in samegroeïng met A. mauritaniensis ATKK 50676 nie met die EMA-kPKR toets opgespoor kon word na hittebehandeling by 80°C en 90°C nie. Verder het die resultate getoon dat daar ‘n defnitiewe tendens tussen gene met verwante funksie en differensiële uitdrukking tydens hittebehandeling (50-90°C) is. Byvoorbeeld, relatief tot die onbehandelde (ongepasteuriseerde) watermonsters, het die uitdrukking van lolA konstant gebly, terwyl die uitdrukking van sidF toegeneem het en die uitdrukking van csrA beduidend afgeneem het tydens die samegroeïng van L. pneumophila ATKK 33152 met A. mauritaniensis ATKK 50676. Resultate bevestig dus dat, terwyl hittebehandeling die aantal lewensvatbare Legionella spp. kan verminder, L. pneumophila en A. mauritaniensis op mekaar inwerk. en kan L. pneumophila dus hittebehandeling oorleef. Die oorhoofse doel van Hoofstuk vier was om vas te stel of ander mikrobiese, fisiese of chemiese eienskappe verband hou met die teenwoordigheid van Legionella spp. en Acanthamoeba spp. in reënwater wat opgevang is vanaf dakke wat uit verskillende materiale gemaak is. Daar is bevind dat die materiaal waarvan die dakke gemaak is nie die voorkoms van Legionella en Acanthamoeba spp in die watermonsters beïnvloed nie. Dit was duidelik omdat hierdie organismes in al die tenkwatermonsters teenwoordig was ongeag die materiaal (Chromadek®, galvaniseerde sink en asbestos) waarvan die dak wat gebruik is om the reënwater te oes, gemaak is. Daar is egter beduidende (p <0.05) positiewe Spearman (ρ) korrelasies opgemerk tussen Legionella spp., nitriete en nitrate, en tussen Acanthamoeba spp. en barium, magnesium, natrium, silikon, arseen en fosfaat. Daarbenewens, terwyl geen beduidende korrelasies waargeneem is tussen Legionella spp. en indikator bakterieë (p> 0.05) nie, is positiewe korrelasies tussen Acanthamoeba spp. en totale kolivorme en Escherichia coli onderskeidelik waargeneem. Resultate dui dus aan dat die teenwoordigheid van Legionella en Acanthamoeba spp. in ge-oeste reënwater hoofsaaklik toegeskryf kan word aan eksterne besoedelingstowwe soos stofdeeltjies en diere fekale materiaal wat op die opvanggebied mag voorkom.

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