Investigating the efficacy of hydrogen peroxide agaisnt isolated environmental Escherichia coli strains

Thesis (MSc Food Sc)--Stellenbosch University, 2015.

ENGLISH ABSTRACT: Surface water used for irrigation is often highly contaminated on a microbial level. Using contaminated surface water for the irrigation of fresh produce can lead to foodborne disease outbreaks and Escherichia coli has been a major cause of foodborne outbreaks associated with fresh produce over the past few years. There are many possible on-farm treatment options available to decrease the high microbial loads present in surface water, one of these is H2O2 and various factors can influence its use. The aim of this study was to determine the efficacy of H2O2 on different E. coli strains. Water from the Plankenburg River was sampled and treated with (250, 300 and 350 mg.L-1) H2O2 and the impact at 0, 30, 60, 90 and 120 min was then evaluated. It was found that the log reductions differed between samples. Log reductions ranged between 1.60 – 2.63 for Aerobic colony counts (ACC), total coliforms and Escherichia coli. The water was not considered safe for irrigation use although it had been treated with H2O2. Reference (ATCC) and environmental E. coli strains were individually treated with H2O2 (250, 300 and 350 mg.L-1) at 0, 30, 60, 90 and 120 min. Log reductions for the ATCC strains ranged between 2.13 – 5.48. This indicated a variation in H2O2 resistance between the different reference strains tested. Log reductions for the environmental E. coli strains ranged between 2.17 – 3.93. Escherichia coli M53 and MJ56 were the most resistant and most sensitive environmental strains to the H2O2 treatment, respectively. Once again it was observed that variations existed between the log reductions achieved for different strains. Overall, it was observed that the ATCC E. coli strains were more sensitive to the H2O2 treatments when compared the environmental strains. This indicates that ATCC strains should not be used for H2O2 treatment optimisation. Certain factors can influence the efficacy of H2O2 such as concentration and organic matter (chemical oxygen demand) present in the water. Different H2O2 concentrations were evaluated (50, 350, 700 and 1 000 mg.L-1) on two E. coli strains (M53 and W1371). Results indicated that 50 mg.L-1 was not effective as less than 1 log reduction was achieved after 120 min. When 350 and 700 mg.L-1 were used similar log reductions were achieved (1.78 – 2.27), which was not expected. Using 1 000 mg.L-1 was considered an effective concentration that resulted in no growth present after 120 min. Escherichia coli strain W1371 carried EPEC virulence factors (potential pathogen). This was included in the study in order to determine how a strain carrying virulence factors would react to H2O2. Escherichia coli W1371 was considered resistant to the H2O2 treatment and log reductions were similar to that achieved for M53. The catalase activity of the E. coli strains was studied to determine if a link existed between catalase activity and H2O2 resistance. Although a trend was observed between heat-stable catalase activity and H2O2 resistance, there were exceptions. It was concluded that high catalase activity does not always coincide with H2O2 resistance and that other mechanisms might also contribute to E. coli survival. Overall, it was observed that there are certain factors that influence the efficacy of H2O2 as a treatment option. It can be concluded that environmental E. coli strains are generally more resistant to the H2O2 treatment compared to ATCC E. coli strains, this needs to be considered when using H2O2 or other chemical disinfectants as a treatment option.

AFRIKAANSE OPSOMMING: Oppervlakwater wat gebruik word vir besproeiing is dikwels op ‘n mikrobiese vlak hoogs gekontamineer. Die gebruik van oppervlakwater vir die besproeiing van vars produkte kan tot die uitbraak van voedselgedraagde siektes lei. Escherichia coli was een van die hoofoorsake van voedselgedraagde uitbrake geassosieerd met vars produkte gedurende die laaste paar jaar. Daar is verskeie moontlike behandelingsmetodes op plaasvlak beskikbaar om die hoë mikrobiese las in oppervlakwater te verlaag. Een hiervan is waterstofperoksied (H2O2) en verskeie faktore kan die gebruik hiervan beïnvloed. Die doel van hierdie studie was om die doeltreffendheid van H2O2 op verskillende E. coli isolate te bepaal. Watermonsters uit die Plankenburg Rivier is behandel met drie konsentrasies H2O2 (250, 300 en 350 mg.L-1) en die impak is na 0, 30, 60, 90 en 120 minute geëvalueer. Daar is gevind dat die log reduksies tussen monsters verskil het. Log reduksies het gewissel tussen 1.60 en 2.63 vir aerobiese kolonietellings (AKT), totale kolivorme en E. coli. Selfs na H2O2 behandeling, is die water nie as veilig vir besproeiing beskou nie. Verwysingsisolate (ATCC) en omgewingsisolate van E. coli is afsonderlik met H2O2 behandel (250, 300 en 350 mg.L-1) vir 0, 30, 60, 90 en 120 minute. Log reduksies vir die ATCC isolate het gewissel tussen 2.13 en 5.48. Hierdie verskille dui op die variasies wat tussen die getoetste verwysingsisolate voorkom. Log reduksies vir die omgewingsisolate het gewissel tussen 2.17 en 3.93. Escherichia coli M53 en MJ56 was onderskeidelik die mees weerstandbiedende en mees sensitiewe verwysingsisolate wat getoets is. Verskille in log reduksies het daarop gedui dat isolaat variasies voorkom. In geheel is dit gevind dat die ATCC E. coli isolate meer sensitief was vir die H2O2 behandelings vergeleke met die omgewingsisolate. Dit toon dat die ATCC isolate nie gebruik moet word vir H2O2 behandeling optimering nie. Sekere faktore, soos die konsentrasie en organiese materiaal (chemiese suurstof vereiste) in die water, kan die doeltreffendheid van H2O2 behandeling beïnvloed. Verskillende H2O2 konsentrasies is geëvalueer (50, 350, 700 en 1000 mg.L-1) op twee E. coli isolate (M53 en W1371). Resultate dui daarop dat 50 mg.L-1 nie effektief was nie omdat minder as 1 log reduksie behaal is na 120 minute. Toe 350 en 700 mg.L-1 gebruik is, is soortgelyke log reduksies (1.78 – 2.27) teen verwagting in behaal. Die gebruik van 1000 mg.L-1 is as ‘n effektiewe behandeling beskou aangesien daar geen groei na 120 minute teenwoordig was nie. Escherichia coli isolaat W1371 besit EPEC virulensie faktore (potensiële patogeen). Dit is in die studie ingesluit ten einde te bepaal hoe ‘n isolaat met virulensie faktore sou reageer op H2O2. Escherichia coli W1371 is as weerstandbiedend teen die H2O2 behandeling beskou en log reduksies was soortgelyk aan die van M53 . Die katalase aktiwiteit van die E. coli isolate is bestudeer om te bepaal of ʼn skakel bestaan tussen katalase aktiwiteit en H2O2 weerstandbiedendheid. Alhoewel ‘n tendens waargeneem is tussen hitte-stabiele katalase aktiwiteit en H2O2 weerstandbiedendheid, was daar uitsonderings. Die gevolgtrekking was dat hoë katalase aktiwiteit nie altyd saamval met H2O2 weerstandbiedendheid nie en dat ander meganismes ook mag bydra tot E. coli oorlewing. In geheel is dit waargeneem dat daar sekere faktore is wat die doeltreffendheid van H2O2 as ‘n behandelingsmetode beïnvloed. Daar is gevind dat omgewingsisolate van E. coli in die algemeen meer weerstandbiedend is teenoor H2O2 behandeling in vergelyking met ATCC E. coli isolate. Dit moet in ag geneem word wanneer H2O2 of ander chemiese ontsmettingsmiddels oorweeg word as ʼn behandelingsopsie.

Masters