Evaluation of agricultural effluents and irrigation water as sources of antimicrobial resistant Escherichia coli

Romanis, Marco (2013-12)

Thesis (MSc Food Sc)--Stellenbosch University, 2013.

Thesis

ENGLISH ABSTRACT: Food-borne disease outbreaks caused by Escherichia coli have been linked to the use of faecally-polluted irrigation waters. Thus the overall aim of this research was to evaluate irrigation water and agricultural effluents as sources of antibiotic resistant E. coli in the Western Cape. The aim of the first study was to enumerate and characterise E. coli present in irrigation water and in potential contamination sources. Maximum total coliform and E. coli counts for irrigation sites was log 7.862 and log 5.364 MPN.100 mL-1, respectively. Five out of seven irrigation sites had E. coli counts exceeding national and international guidelines for ‘safe’ irrigation water (<1 000 counts.100 mL-1), making it unsafe for the irrigation of fresh produce. In this study, 46.6% of the E. coli strains were characterised in phylogenetic group B1. It has been shown that E. coli in group B1 have the ability to survive and persist in the external environment. Group B1 was also the most common group among isolates from irrigation sites (79.4%), while isolates from environmental sites grouped mainly in group A0 (54.1%). It was concluded that the wide variation of E. coli types present in irrigation water is a concern that should be further investigated. This raises human health implications since the increased exposure to faecal organisms increases the risk of food-borne outbreaks. The E. coli isolates (n = 120) and the marker (n = 37) and reference strains (n = 6), were evaluated for antibiotic resistance to seven medically-important antibiotics from different classes using the Kirby-Bauer disc diffusion method. Thirty-five strains (35/163 = 21.5%) exhibited resistance to one or more antibiotics. Piggery effluent was found to harbour the most antibiotic resistant E. coli isolates (9/35 = 25.7%). Among the resistant E. coli strains, the highest occurrence of antibiotic resistance was to trimethoprim (2.5 μg) (68.6%), tetracycline (30 μg) (57.1%), ampicillin (10 μg) (45.7%) and chloramphenicol (30 μg) (34.3%). Seventy-four percent (26/35) exhibited multiple antibiotic resistances to two or more antibiotics. The antibiotic resistant E. coli strains were evaluated for the presence of pathotypes using Polymerase Chain Reaction analysis to detect Intestinal Pathogenic E. coli (InPEC) and Extra-intestinal Pathogenic E. coli (ExPEC). Five InPEC strains were characterised as four Entero-Pathogenic E. coli (EPEC) strains resistant to three or four antibiotics and one Entero-Aggregative E. coli (EAEC) strain resistant to trimethoprim. The antibiotic resistant EAEC strain also possessed the ExPEC-related gene iutA. Two E. coli isolated from the Mosselbank River were both resistant to chloramphenicol and trimethoprim and also possessed the ExPEC-related gene iutA. It was concluded that the diverse antibiotic resistances of E. coli pathotypes present in irrigation water is a concern that should be further investigated.

AFRIKAANSE OPSOMMING: Voedselverwante siekte uitbrake wat deur Escherichia coli veroorsaak word, is gekoppel aan die gebruik van fekale besoedelde besproeiingswater. Dus was die hoof doel van die navorsing om besproeiingswater en landbou-afvalwater te evalueer as bronne van antibiotika-weerstandbiedende E. coli in die Wes-kaap. Die doel van die eerste studie was om die getalle en eienskappe van E. coli te bepaal wat in besproeiingswater en in ander potensiële besmettingsbronne teenwoordig is. Maksimum totale koliforme en E. coli-tellings vir besproeiingspunte was onderskeidelik log 7.862 en log 5.364 MPN.100 mL-1. Vyf uit sewe besproeiingspunte het E. coli-tellings gehad wat hoër is as die nasionale en internasionale riglyne vir ‘veilige’ besproeiingswater (<1 000 tellings.100 mL-1). Dit maak dit onveilig vir die besproeiing van vars produkte. In hierdie studie was 46.6% van die E. coli-stamme in filogenetiese groep B1 gegroepeer. Dit is reeds bewys dat E. coli in groep B1 oor die vermoë beskik om in die eksterne omgewing te oorleef en voort te bestaan. Groep B1 was ook die mees algemene groep onder die isolate van besproeiingspunte (79.4%), terwyl isolate van omgewingspunte meestal in groep A0 (54.1%) gegroepeer is. Die breë variasie E. coli tipes in die besproeiingswater is bekommerniswaardig en sal gevolglik verder ondersoek moet word. Dit bring gesondsheidsimplikasies mee vir mense aangesien die verhoogde blootstelling aan fekale organismes die risiko van voedselverwante uitbrake verhoog. Die E. coli isolate (n = 120) en die merker (n = 37) en verwysingsstamme (n = 6), is teen sewe medies belangrike antibiotikas uit verskillende klasse getoets vir antibiotika-weerstandbiedendheid. Die Kirby-Bauer skyfie diffusie metode is gebruik. Vyf-en-dertig stamme (35/163 = 21.5%) het weerstand teen een of meer antibiotika getoon. Dit is gevind dat vark-afvalwater die meeste antibiotika-weerstandbiedende E. coli-isolate (9/35 = 25.7%) bevat. Die weerstandbiedende E. coli-stamme het die hoogste antibiotika-weerstandheid getoon teen "trimethoprim" (2.5 μg) (68.6%), tetrasiklien (30 μg) (57.1%), ampisillien (10 μg) (45.7%) en chloramfenikol (30 μg) (34.3%). Vier-en-sewentig persent (26/35) het meervoudige weerstandbiedheid teen twee of meer antibiotikas getoon. Die antibiotika-weerstandbiedende E. Coli stamme is getoets vir die teenwoordigheid van patogene deur van Polimerase Ketting Reaksie analise gebruik te maak om ‘Intestinal Pathogenic’ E. coli (InPEC) en ‘Extra-intestinal Pathogenic’ E. coli (ExPEC) op te spoor. Vyf InPEC-stamme is geklassifiseer as vier ‘Entero-Pathogenic’ E. coli (EPEC)-stamme wat weerstandbiedend teen drie of vier antibiotika getoon het en een ‘Entero-Aggregative’ E. coli (EAEC)-stam wat weerstandbiedendheid getoon het teen "trimethoprim". Die antibiotika-weerstandbiedende EAEC-stam het ook die ExPEC-verwante geen, iutA, besit. Twee E. coli isolate van die Mosselbankrivier het weerstand teen beide chloramfenikol en "trimethoprim" getoon en het ook die ExPEC-verwante geen, iutA, besit. Daar is tot die gevolgtrekking gekom dat die diverse antibiotika-weerstandbiedenheid van E. coli patogene teenwoordig in besproeiingswaters bekommerniswaardig is en verder ondersoek behoort te word.

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