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Prevalence and risk assessment of nosocomial associated pathogens in environmental water samples
Thesis (MSc)--Stellenbosch University, 2023.
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Stellenbosch : Stellenbosch University
2023
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| access_status_str | Open Access |
| author | Denissen, Julia Katherine |
| author2 | Khan, Wesaal |
| author_browse | Denissen, Julia Katherine Khan, Wesaal |
| author_facet | Khan, Wesaal Denissen, Julia Katherine |
| author_sort | Denissen, Julia Katherine |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MSc)--Stellenbosch University, 2023. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/128485 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:44:09.875Z |
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| 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:10019.1/128485 Prevalence and risk assessment of nosocomial associated pathogens in environmental water samples Denissen, Julia Katherine Khan, Wesaal Reyneke, Brandon Stellenbosch University. Faculty of Science. Dept. of Microbiology. Drug resistance in microorganisms. Gram-positive bacterial infections Antibiotics Virulence (Microbiology) -- Genetic aspects ESKAPE -- Pathogens Water -- Environmental aspects Pathogenic bacteria Water -- South Africa -- Western Cape Risk assessment Water quality management UCTD Thesis (MSc)--Stellenbosch University, 2023. ENGLISH ABSTRACT: Enterococcus faecium (E. faecium), Klebsiella pneumoniae (K. pneumoniae), and Pseudomonas aeruginosa (P. aeruginosa), are notorious for their increased antibiotic resistance (AR) and virulence, and role in the onset of hospital-acquired infections. While extensive research has been conducted on the identification and characterisation of these species in clinical settings, limited research is available on the phenotypic and genotypic comparison of clinical and environmental isolates. In Chapter 1 (published in the International Journal of Hygiene and Environmental Health, 2022), a comprehensive overview of literature pertaining to the AR and virulence mechanisms of the ESKAPE (E. faecium, Staphylococcus aureus, K. pneumoniae, Acinetobacter baumannii, P. aeruginosa, and Enterobacter spp.) pathogens, was thus outlined. Thereafter, their environmental reservoirs, and subsequent role in the onset of community-acquired (CA) disease outbreaks were discussed, while risk assessment analysis was also outlined. The aim of Chapter 2 was then to compare the phenotypic, genotypic, AR, and virulence characteristics of environmental E. faecium, K. pneumoniae, and P. aeruginosa isolates obtained from surface runoff, stormwater runoff, stream water, and marsh water in the Vlottenburg informal settlement and Sir Lowry’s Pass Village (Western Cape, South Africa), to clinical counterparts. Culture-based analysis on selective media was used to isolate 307 strains, whereafter conventional polymerase chain reaction (PCR) analysis confirmed that 42.3% (52/123), 27.7% (36/130), and 66.7% (36/54) of the presumptive environmental isolates were E. faecium, K. pneumoniae, and P. aeruginosa, respectively. A high frequency of detection of E. faecium and K. pneumoniae isolates was obtained for the Vlottenburg surface runoff, Sir Lowry’s Pass stream, and Sir Lowry’s Pass stormwater sites, while P. aeruginosa had an increased detection at the Sir Lowry’s Pass stormwater and surface runoff (periphery), and Vlottenburg marsh sites. Representative environmental isolates were selected for further analysis based on the sampling sites and sessions from which they were obtained. Therefore, 19 E. faecium (12 environmental, 7 clinical), 13 K. pneumoniae (9 environmental, 3 clinical, 1 reference), and 16 P. aeruginosa (12 environmental, 3 clinical, 1 reference) isolates, were further analysed. Cluster analysis using Repetitive Extragenic Palindromic Sequence-based PCR indicated a low genetic relatedness between the clinical and environmental isolates of each species, respectively, with only one clinical K. pneumoniae (P2) and one clinical P. aeruginosa (PAO1) exhibiting a high genetic similarity (≥ 85%) to certain environmental strains. All the P. aeruginosa isolates then displayed increased antibiotic susceptibility, while the E. faecium and K. pneumoniae isolates displayed a range of AR profiles. The clinical E. faecium isolate, Ef CD1, was subsequently classified as extensively drug resistant, while all the clinical and environmental K. pneumoniae isolates were classified as multidrug resistant. The tetM gene (tetracycline resistance) was detected in 47.4% (9/19) of the E. faecium isolates, while 52.6% (10/19) and 15.4% (2/13) of the E. faecium and K. pneumoniae isolates, respectively, possessed the blaKPC gene (carbapenem resistance). Analysis of phenotypic and genotypic virulence characteristics revealed that the E. faecium isolates were predominantly poor biofilm formers, lacked haemolytic activity and gelatinase production, with the esp (enterococcal surface protein) and gelE (gelatinase production) genes not detected. The K. pneumoniae isolates displayed a range of biofilm formation capabilities (weak to strong) and all possessed the fimH (fimbriae adhesion) and ugE (uridine diphosphate galacturonate 4-epimerase synthesis) genes however, none displayed haemolytic activity or hypermucoviscosity. All the P. aeruginosa isolates however, possessed the phzM (phenazine production) and algD (alginate biosynthesis) genes, as well as displayed strong biofilm forming capabilities, haemolytic activity, and gelatinase production. The results of Chapter 2 thus highlight the differences and similarities in the phenotypic, genotypic, AR, and virulence characteristics of clinical and environmental E. faecium, K. pneumoniae, and P. aeruginosa isolates, and illustrates the threat posed by highly resistant and virulent strains of these species in environmental reservoirs. The primary aim of Chapter 3 was to determine the prevalence of E. faecium, K. pneumoniae, and P. aeruginosa in the environmental water samples, and to estimate the potential human health risks associated with the use of these water sources for various potable and non-potable purposes. However, as the issue of AR is augmented by the dissemination of AR genes in the environment, a secondary aim of Chapter 3 was to apply surrogate risk assessment models for aminoglycoside- resistant Gram-positive [aac(6’)-aph(2’’) gene] and Gram-negative [aac(6’)-Ib gene] pathogens within these water sources. Overall, using ethidium monoazide bromide (EMA) with quantitative PCR (qPCR) analysis, E. faecium, K. pneumoniae, P. aeruginosa, and the aac(6’)-Ib and aac(6’)-aph(2’’) genes were detected in 88.9%, 100%, 93.3%, 95.6%, and 100% of the water samples (n = 45), respectively. Of the target bacterial species, K. pneumoniae was detected at the highest mean concentration (7.83 × 10⁴ cells/100 mL), followed by E. faecium (3.53 × 10² cells/100 mL), and P. aeruginosa (9.04 × 10¹ cells/100 mL). The aac(6’)-Ib gene was then detected at a higher mean concentration (7.07 × 10⁶ GC/100 mL) than the aac(6’)-aph(2’’) gene (6.68 × 10⁵ GC/100 mL). Risk assessment analysis subsequently indicated that P. aeruginosa, K. pneumoniae and Gram-negative pathogens containing the aac(6’)-Ib gene were found to pose a significant health risk [i.e., exceeded the acceptable benchmark limit of one infection per 10 000 persons per year (1 × 10⁻⁴)] if water from the marsh, surface runoff, stream, and stormwater runoff at both the Vlottenburg informal settlement and Sir Lowry’s Pass Village is used for intentional drinking, washing/bathing, swimming, and cleaning of the home, amongst other scenarios. In contrast, there were no risks associated with E. faecium or Gram-positive pathogens containing the aac(6’)-aph(2’’) gene for any of the exposure scenarios at any of the sampling sites. The results of Chapter 3 thus highlight the persistence of E. faecium, K. pneumoniae, and P. aeruginosa, as well as the aac(6’)-Ib and aac(6’)-aph(2’’) resistance genes in environmental water samples, which may pose a significant threat to the residents of the informal settlements, potentiating CA disease outbreaks. Moreover, results indicated that the combination of risk assessment input parameters for different bacterial species may be beneficial for establishing the risks associated with AR genes and multiple pathogens in environmental waters, simultaneously, using surrogate dose-response models. AFRIKAANSE OPSOMMING: Enterococcus faecium (E. faecium), Klebsiella pneumoniae (K. pneumoniae) en Pseudomonas aeruginosa (P. aeruginosa), is berug vir hul verhoogde antibiotika weerstandbiedendheid (AW) en virulensie, en die rol wat hul speel in die aanvang van hospitaal-verworwe infeksies. Terwyl wesenlike navorsing voltooi is oor die identifikasie en karakterisering van hierdie spesies in kliniese omgewings, is beperkte navorsing beskikbaar oor die fenotipiese en genotipiese vergelyking van kliniese en omgewingsisolate. In Hoofstuk 1 (gepubliseer in die “International Journal of Hygiene and Environmental Health”, 2022), is 'n omvattende oorsig van literatuur met betrekking tot die AW en virulensiemeganismes van die ESKAPE (E. faecium, Staphylococcus aureus, K. pneumoniae, Acinetobacter baumannii, P. aeruginosa, en Enterobacter spp.) patogene uiteengesit. Daarna is hul omgewingsbronne, en hul rol in die aanvang van gemeenskap-verworwe (GV) siekte-uitbrake bespreek, terwyl risiko analise ook uiteengesit is. Die doel van Hoofstuk 2 was dus om die fenotipiese, genotipiese, AW, en virulensie-eienskappe van omgewings E. faecium, K. pneumoniae en P. aeruginosa isolate wat verkry is vanaf oppervlakafloop water, stormwaterafloop, stroomwater en vleiwater in die Vlottenburg informele nedersetting en Sir Lowry's Pass Village (Wes-Kaap, Suid-Afrika), te vergelyk met kliniese eweknieë. Kultuur-afhanklike analise op selektiewe media is gebruik om 307 stamme te isoleer, waarna konvensionele polimerase kettingreaksie (PKR) analise bevestig het dat 42.3% (52/123), 27.7% (36/130) en 66.7% (36/54) van die vermoedelike omgewingsisolate onderskeidelik E. faecium, K. pneumoniae en P. aeruginosa was. Die teenwoordigheid van E. faecium en K. pneumoniae- isolate is teen ‘n hoë opsporingsfrekwensie opgemerk in die Vlottenburg oppervlakafloop water, Sir Lowry's Pass stroomwater en Sir Lowry's Pass stormwater, terwyl P. aeruginosa 'n verhoogde voorkoms by die Sir Lowry's Pass stormwater en oppervlakafloop water (periferie) gehad het, asook by die Vlottenburg vleiland. Verteenwoordigende omgewingsisolate is geselekteer vir verdere ontleding gebaseer op die isolasie terrein en sessie waaruit hulle verkry is. Gevolglik is 19 E. faecium (12 omgewings, 7 klinies), 13 K. pneumoniae (9 omgewings, 3 klinies, 1 verwysing) en 16 P. aeruginosa (12 omgewings, 3 klinies, 1 verwysing) isolate verder ontleed. Groeperingsanalise met behulp van herhalende ekstrageniese palindromiese volgorde-gebaseerde PKR het 'n lae genetiese verwantskap tussen die kliniese en omgewingsisolate van onderskeidelik elke spesie aangedui, met slegs een kliniese K. pneumoniae (P2) en een kliniese P. aeruginosa (PAO1) wat 'n hoë genetiese ooreenkoms (≥ 85%) met sekere omgewingsstamme getoon het. Al die P. aeruginosa isolate het verhoogde vatbaarheid vir antibiotika getoon, terwyl die E. faecium en K. pneumoniae isolate 'n reeks AW profiele getoon het. Die kliniese E. faecium isolaat, Ef CD1, is gevolglik as ekstensief geneesmiddel weerstandig geklassifiseer, terwyl al die kliniese en omgewings K. pneumoniae isolate as multigeneesmiddel weerstandig geklassifiseer is. Die tetM-geen (tetrasiklienweerstand) is in 47.4% (9/19) van die E. faecium isolate waargeneem, terwyl 52.6% (10/19) en 15.4% (2/13) van die E. faecium en K. pneumoniae isolate onderskeidelik die blaKPC-geen (karbapenemweerstand) besit het. Die ontleding van fenotipiese en genotipiese virulensie eienskappe het gevolglik aangedui dat die E. faecium isolate oorwegend swak biofilmvormers was, geen hemolitiese aktiwiteit en gelatienase produksie het nie, en die esp (enterokokkie oppervlak proteïen) en gelE (gelatinase produksie) gene nie teenwoordig was in die isolate nie. Die K. pneumoniae isolate het 'n reeks biofilm vorming vermoëns (swak tot sterk) vertoon en al die isolate het die fimH (fimbriae aanhegting) en ugE (uridien difosfaat galakturonaat 4-epimerase sintese) gene gehad, maar nie een het hemolitiese aktiwiteit of hipermukoviskositeit getoon nie. Al die P. aeruginosa isolate het egter die phzM (fenasienproduksie) en algD (alginaatbiosintese) gene besit, asook sterk biofilmvormende vermoëns, hemolitiese aktiwiteit en gelatienaseproduksie getoon. Die resultate van Hoofstuk 2 beklemtoon dus die verskille en ooreenkomste in die fenotipiese, genotipiese, AW en virulensie eienskappe van kliniese en omgewings E. faecium, K. pneumoniae en P. aeruginosa isolate, en illustreer die gevaar van hoogs weerstandbiedende en virulente stamme van hierdie spesies in omgewingsbronne. Die primêre doel van Hoofstuk 3 was om die teenwoordigheid van E. faecium, K. pneumoniae en P. aeruginosa in die omgewingswatermonsters te bepaal, en om die potensiële menslike gesondheidsrisiko's wat verband hou met die gebruik van hierdie waterbronne vir verskeie drink- en nie-drinkbare doeleindes te bereken. Aangesien die kwessie van AW egter aangevul word deur die verspreiding van AW gene in die omgewing, was 'n sekondêre doel van Hoofstuk 3 om surrogaat risiko-assesseringsmodelle toe te pas vir aminoglikosied-weerstandige Gram-positiewe [aac(6’)- aph(2’’) geen] en Gram-negatiewe [aac(6’)-Ib gene] patogene binne hierdie waterbronne. Deur gebruik te maak van etidiummonoasiedbromied (EMA) kwantitatiewe PKR (kPKR) analise, is E. faecium, K. pneumoniae, P. eruginosa, en die aac(6’)-Ib en aac(6’)-aph(2’’) gene in onderskeidelik 88.9%, 100%, 93.3%, 95.6% en 100% van die watermonsters (n = 45) aangeteken. Uit al die teikenbakteriespesies is K. pneumoniae teen die hoogste gemiddelde konsentrasie (7.83 × 10⁴ selle/100 mL) aangeteken, gevolg deur E. faecium (3.53 × 10² selle/100 mL), en P. aeruginosa (9.04 × 10¹ selle/100 mL). Die aac(6’)-Ib geen is gevolglik aangeteken teen 'n hoër gemiddelde konsentrasie (7.07 × 10⁶ GK/100 mL) as die aac(6’)-aph(2’’) geen (6.68 × 10⁵ GK/100 mL). Risiko analise het daarna aangedui dat P. aeruginosa, K. pneumoniae en Gram-negatiewe patogene wat die aac(6’)-Ib geen bevat 'n beduidende gesondheidsrisiko inhou [d.w.s., die aanvaarbare maatstaflimiet van een infeksie per 10 000 persone per jaar (1 × 10⁻⁴) oorskry] indien water uit die vlei, oppervlakafloop, stroom en stormwaterafloop by beide die Vlottenburg informele nedersetting en Sir Lowry's Pass Village gebruik word vir drinkwater, was/bad, swem, en skoonmaak van die huis, onder ander gebruike. Daarteenoor was daar geen risiko's verbonde aan E. faecium of Gram- positiewe patogene wat die aac(6’)-aph(2’’) geen bevat vir enige van die huishoudelikegebruike by enige van die toets-gemeenskappe nie. Die resultate van Hoofstuk 3 beklemtoon dus die volharding van E. faecium, K. pneumoniae en P. aeruginosa, sowel as die aac(6’)-Ib en aac(6’)-aph(2’’) weerstandsgene in omgewingswatermonsters, wat 'n beduidende bedreiging vir die inwoners van die informele nedersettings kan inhou, en die uitbraak van GV-siektes versterk. Boonop het resultate aangedui dat die kombinasie van risiko analise insetfaktore vir verskillende bakteriese spesies voordelig kan wees vir die bepaling van die risiko's wat met AW gene en veelvuldige patogene gelyktydig in omgewingswaters geassosieer word, deur gebruik te maak van surrogaat dosis-reaksie modelle. Masters 2023-02-23T16:00:28Z 2023-08-30T13:07:19Z 2023-03 2023-02-23T16:00:28Z 2023-08-31T09:18:51Z 2023-02-23T16:00:28Z 2023-08-31T09:18:51Z 2023-03 Thesis https://scholar.sun.ac.za/handle/10019.1/128485 en_ZA Stellenbosch University application/pdf viii, 207 pages : illustrations (some color) application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Drug resistance in microorganisms. Gram-positive bacterial infections Antibiotics Virulence (Microbiology) -- Genetic aspects ESKAPE -- Pathogens Water -- Environmental aspects Pathogenic bacteria Water -- South Africa -- Western Cape Risk assessment Water quality management UCTD Denissen, Julia Katherine Prevalence and risk assessment of nosocomial associated pathogens in environmental water samples |
| title | Prevalence and risk assessment of nosocomial associated pathogens in environmental water samples |
| title_full | Prevalence and risk assessment of nosocomial associated pathogens in environmental water samples |
| title_fullStr | Prevalence and risk assessment of nosocomial associated pathogens in environmental water samples |
| title_full_unstemmed | Prevalence and risk assessment of nosocomial associated pathogens in environmental water samples |
| title_short | Prevalence and risk assessment of nosocomial associated pathogens in environmental water samples |
| title_sort | prevalence and risk assessment of nosocomial associated pathogens in environmental water samples |
| topic | Drug resistance in microorganisms. Gram-positive bacterial infections Antibiotics Virulence (Microbiology) -- Genetic aspects ESKAPE -- Pathogens Water -- Environmental aspects Pathogenic bacteria Water -- South Africa -- Western Cape Risk assessment Water quality management UCTD |
| url | https://scholar.sun.ac.za/handle/10019.1/128485 |
| work_keys_str_mv | AT denissenjuliakatherine prevalenceandriskassessmentofnosocomialassociatedpathogensinenvironmentalwatersamples |