PAA disinfection kinetics of E. coli, and its effects on antibiotic-resistance genes

129 Pages·2013·0.76 MB·English

PAA disinfection kinetics of E. coli, and its effects on
antibiotic-resistance genes

by Ramzi Khairallah

Master of Engineering
Department of Civil Engineering and Applied Mechanics
McGill University, Montreal, Quebec

August 2013

A thesis report submitted in partial fulfillment of the requirements (27 credits out of the
minimum required 45 credits) for the degree of Masters of Engineering

© Ramzi Khairallah
ABSTRACT

The dissemination of antibiotic-resistant bacteria has been a great concern because
of the risk it poses to our health. Although wastewater treatment plants (WWTPs) are
designed to reduce the overall amount of microorganisms and contaminants present in
wastewater, studies suggest that antibiotic-resistant bacteria escape the primary,
secondary and tertiary treatment processes, due partially to the expression of genetic
mechanisms of resistance to disinfectants.

The first objective of this study was to investigate the overall environmental and
intrinsic mechanisms of resistance expressed by Escherichia coli in WWTP effluents
disinfected with peracetic acid (PAA) at low doses and high doses, and over different
time intervals. The second objective of this study was to assess the impact of PAA on the
antibiotic-resistance genetic profile of E. coli isolates that have been pre-screened for
uropathogenic E.coli (UPEC) virulence.

To complete the first objective, samples from a biofiltration WWTP effluent were
exposed to a single PAA dose of 2 mg/L applied once for 1 hour, and intermittent-doses
of 0.5 mg/L applied every 30 minutes for 2 hours, before and after filtration of the
samples in the laboratory. The dose-response curves were plotted and compared for each
experiment using Collins-Selleck’s model. Filtration of the samples led to an overall
reduction of resistance to the disinfectant in the single-dose and intermittent-doses
experiments, probably because of the reduction of COD levels and elimination of
particles that could have shielded the bacteria. There were no significant differences
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between the model parameters of the single-dose and intermittent-doses disinfection
curves, hence no significant adaptive genetic mechanisms of resistance were identified.

To complete the second objective, samples of biofiltration, activated sludge, and
physicochemical effluent were disinfected with PAA. A molecular screening technique
using PCR/ Bioplex was used to detect three of the main UPEC virulence genes (papC,
cnf1, and hlyA) in E. coli isolates obtained before and after disinfection. A DNA
microarray technique was used to detect the presence of 30 antibiotic-resistance genes in
each positively screened isolate. The antibiotic-resistance gene frequency showed a
significant decrease after disinfection. This disinfection was related to a reduction in
isolates carrying resistance genes to multiple classes of antibiotics, while the isolates
carrying resistance genes to a single class increased in frequency. These variations
seemed to be correlated to the presence of a marker for the Tn21 transposon. This
transposon is known in the literature to play a major role in the acquisition of multiple
antibiotic-resistance. Thus, the loss of antibiotic-resistance after PAA disinfection could
be related to SOS oxidative stress responses, which may cause the deletion of DNA
fragments and mobile genetic elements carrying multiple antibiotic-resistance genes such
as Tn21 transposons.

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RÉSUMÉ

La dissémination de bactéries résistantes aux antibiotiques pose un sérieux
problème de santé publique. Des études suggèrent que les microorganismes présents dans
les usines de traitement des eaux usées échappent aux traitements primaires, secondaires
et tertiaires, due en partie à l’expression de gènes résistants aux désinfectants.

Le premier objectif de cette étude fut d’investiguer les mécanismes globaux,
environnementaux et intrinsèques de résistance exprimés par les Escherichia coli présents
dans les effluents d’usines de traitement d’eaux usées, désinfectés avec de l’acide
paracétique à petites et grandes doses, et sur des intervalles de temps différents. Le
deuxième objectif de cette étude fut d’évaluer l’impact de l’acide paracétique sur le profil
génétique de résistance aux antibiotiques des colonies d’E. coli qui furent isolées et
testées positives pour la virulence UPEC.

Pour compléter le premier objectif des échantillons de l’effluent provenant d’une
usine de traitement des eaux usées utilisant un processus de biofiltration furent exposés à
une dose unique d’acide paracétique de 2mg/L appliquée au début de l’expérience, et à
des doses intermittentes de 0.5mg/L appliquées à 30 minutes d’intervalles pendant 2
heures, avant et après la filtration des échantillons en laboratoire. La filtration des
échantillons mena à une réduction de la résistance au désinfectant dans les deux
expériences, probablement à cause de la réduction des niveaux de DCO et l’élimination
des particules pouvant protéger les bactéries. Aucune différence significative ne fut
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observée entre les paramètres du modèle calculés pour l’expérience à dose unique et à
doses intermittentes et donc aucun mécanisme de résistance intrinsèque ne fut identifié.

Pour compléter le deuxième objectif, des échantillons d’effluents provenant
d’usines de traitement des eaux utilisant un processus de boues activées, de biofiltration
et physicochimique furent désinfectés avec de l’acide paracétique. Une technique de
dépistage moléculaire ACP/Bioplex fut utilisée afin de détecter 3 gènes de virulence
UPEC (papC, cnf1 et hlyA) chez les colonies d’E. coli isolées avant et après la
désinfection. Une puce à ADN fut utilisée pour détecter la présence de 30 gènes de
résistance aux antibiotiques, pour chaque colonie d’E.coli qui fut isolée et testée positive
pour la virulence UPEC.

La fréquence des gènes résistant aux antibiotiques diminua de manière significative
après la désinfection. La désinfection contribua à la réduction des E. coli possédant des
gènes de résistance à plusieurs classes d’antibiotiques, tandis que la fréquence des E. coli
possédant des gènes de résistance à une seule classe d’antibiotique augmenta. Ces
variations semblent être liées à la présence du marqueur appartenant au transposon Tn21.
Ce transposon joue un rôle majeur dans l’acquisition de résistances multiples aux
antibiotiques. La diminution de la résistance aux antibiotiques après désinfection pourrait
être liée à des réponses au stress oxydatif, pouvant mener à l’élimination de fragments
d’ADN et d’éléments génétiques porteurs de résistances multiples aux antibiotiques
comme les transposons Tn21.

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ACKNOWLEDGEMENTS

I would like to thank Professor Ronald Gehr for his support and guidance

I would like to thank Professor Dominic Frigon for his help and indispensable input.

I would like to thank Daina Brumelis, Basanta Biswal and Alberto Mazza for their help
and support in the laboratory.

I would like to thanks the plant operators of the four municipal wastewater treatment
plants for their help with the effluent sample collection.

And finally I would like to thank my family and friends for their love and support.

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Table of Contents

ABSTRACT ............................................................... i
RESUME ................................................................ iii
ACKNOWLEDGEMENTS .................................................... v 
Table of Contents ......................................................... vii 
List of Figures ........................................................... viii 
List of Tables .............................................................. ix
List of Abbreviations ......................................................... x
Chapter 1: Introduction and objectives ........................................... 1 
1.1  Impact and mechanisms of antibiotic resistance ............................... 1 
1.2  Prevalence and dissemination of antibiotic-resistant bacteria in WWTPs ............ 1 
1.3  Impact of WWTPs on antimicrobial resistance ................................ 2 
1.4  PAA disinfection in wastewater treatment .................................... 3 
1.5  Model organisms and pathotypes ........................................... 4 
1.6  Objectives ............................................................ 5 
Chapter 2: Literature review.................................................... 6 
2.1  Antibiotics and their effects ............................................... 6 
2.2  The fate of antibiotics in WWTPs .......................................... 7 
2.3  Antibiotic-resistance mechanisms and genes .................................. 8 
2.4  Acquisition and proliferation of antibiotic-resistance genes ..................... 10 
2.5  Prevalence of antibiotic-resistance in WWTPs ............................... 12 
2.6  Impact of WWTP processes on antibiotic-resistant bacteria ..................... 13 
2.7  PAA physicochemical characteristics and mode of action ....................... 15 
2.8  PAA disinfection in WWTPs ............................................. 16 
2.9  Mechanisms of antimicrobial resistance or resistance to disinfectants ............. 19 
2.10  Antibiotic-resistance detection techniques ................................... 21 
Chapter 3: Materials and methods .............................................. 24 
3.1  Sample collection and experimental procedure ............................... 24 
3.2  Stock and working solution concentrations .................................. 26 
3.3  Residual concentrations of PAA .......................................... 26 
3.4  E. coli isolation and purification .......................................... 27 
3.5  Screening of UPEC by PCR amplification and BioPlex detection ................. 28 
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3.6  Gel electrophoresis ..................................................... 31 
3.7  Antibiotic-resistance genes detection by DNA microarray ...................... 32 
3.8  Statistical analysis and disinfection kinetic model ............................. 35 
3.9  PCR/Bioplex screening technique analysis .................................. 36 
Chapter 4: Results ........................................................... 39
4.1  WWTPs effluent quality ................................................ 39 
4.2  PAA degradation ...................................................... 40 
4.3  Disinfection kinetics.................................................... 46 
4.4  Analysis of UPEC screening by PCR/Bioplex ................................ 52
4.5  Antibiotic-resistance genetic profile analysis ................................. 52
Chapter 5: Discussion ........................................................ 61
5.1  The effects of single-dose vs intermittent-doses of PAA on E. coli ................ 61 
5.2  The impact of PAA on the antibiotic-resistance genetic profile of E. coli ........... 65 
Chapter 6: Conclusion and Recommendations .................................... 69
6.1  Conclusions .......................................................... 69 
6.2  Recommendations ..................................................... 69 
References ................................................................ 71 
Chapter 7: Appendices ........................................................ 85 
7.1  Appendix A: Calibration lines ............................................ 85 
7.2  Appendix B: Disinfection kinetics models ................................... 89 
7.3  Appendix C: Antibiotic resistance and virulence genes and probes ............... 110 
7.4  Appendix D: Antibiotic resistance profile .................................. 115 

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List of Figures

Figure 3.1: Agarose gel image at the end of the gel electrophoresis experiment.. .................... 32 
Figure 3.2: Scanned image of hybridized DNA microarray chip. ................................ 35
Figure 4.1: Residual PAA concentration vs PAA doses.. ....................................... 40 
Figure 4.2: PAA degradation for P4-a, single-dose experiment .................................. 42
Figure 4.3: PAA degradation for P4-b, single-dose experiment.. ................................. 43 
Figure 4.4: PAA degradation for P4-a, intermittent-doses experiment ............................ 44
Figure 4.5: PAA degradation for P4-b, intermittent-doses experiment.. ........................... 45 
Figure 4.6: Disinfection kinetics P1, P2 and P3 .............................................. 47
Figure 4.7: Disinfection kinetics, single-dose experiment, P4-a and P4-b.. ......................... 49 
Figure 4.8: Disinfection kinetics, intermittent-doses experiment, P4-a and P4-b .................... 50
Figure 4.9: Disinfection kinetics, intermittent-doses and single-dose experiment, P4-a ............... 51
Figure 4.10: Disinfection kinetics, intermittent-doses and single-dose experiment, P4-b.. ............. 52 
Figure 4.11: Proportion of UPECs detected by PCR/Bioplex and DNA microarray .................. 53
Figure 4.12: Proportion of nominal false-positive UPECs and overall false-positive UPECs.. .......... 54 
Figure 4.13: Proportion of isolates screened positive for UPECs, with ABR and with no ABR ......... 55
Figure 4.14: ABR index for P1, P2, P3 and P4-b, before and after disinfection with PAA. ............ 56 
Figure 4.15: Proportion of SABR isolates and MABR isolates, before and after disinfection with PAA .. 57
Figure 4.16: Proportion of isolates with Tn21 transposon, before and after disinfection with PAA.. ..... 58 
Figure 4.17: Proportion of mutltiple antibiotic-resistant isolates with a Tn21 transposon .............. 59
Figure 4.18: Proportion of isolates resistant to beta-lactams, phenicols and erythromycin ............. 60

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List of Tables

Table 4.1: Effluent characteristics ................................................................................................................. 39
Table 4.2: Collins-Selleck model parameters, critical log reduction and critical Ct values .......................... 47
Table B.3: Disinfection kinetics: P4-a single-dose experiment, September 13th 2012 .................................. 89
Table B.4: Disinfection kinetics: P4-a single-dose experiment, September 17th 2012 .................................. 90
Table B.5: Disinfection kinetics: P4-a single-dose experiment, October 2nd 2012 ........................................ 91
Table B.6: Disinfection kinetics: P4-b single-dose experiment, September 13th 2012 .................................. 92
Table B.7: Disinfection kinetics: P4-b single-dose experiment, September 17th 2012 .................................. 93
Table B.8: Disinfection kinetics: P4-b single-dose experiment, October 2nd 2012 ....................................... 94
Table B.9: Disinfection kinetics: P4-a intermittent-doses experiment, September 13th 2012 ....................... 95
Table B.10: Disinfection kinetics: P4-a intermittent-doses experiment, September 17th 2012 ..................... 96
Table B.11: Disinfection kinetics: P4-a intermittent-doses experiment, October 2nd 2012 ......................... 97
Table B.12: Disinfection kinetics: P4-b intermittent-doses experiment, September 13th 2012 ..................... 98
Table B.13: Disinfection kinetics: P4-b intermittent-doses experiment, September 17th 2012 ..................... 99
Table B.14: Disinfection kinetics: P4-b intermittent-doses experiment, October 2nd 2012 ....................... 100
Table B.15: Disinfection kinetics: P1, October 2nd 2011 ............................................................................. 101
Table B.16: Disinfection kinetics: P1, October 7th 2011 ............................................................................. 102
Table B.17: Disinfection kinetics: P1, October 15th 2011 ........................................................................... 103
Table B.18: Disinfection kinetics: P2, August 5th 2011 .............................................................................. 104
Table B.19: Disinfection kinetics: P2, August 9th 2011 .............................................................................. 105
Table B.20: Disinfection kinetics: P2, August 21st 2011 ............................................................................. 106
Table B.21: Disinfection kinetics: P3, October 2nd 2011 ............................................................................. 107
Table B.22: Disinfection kinetics: P3, October 7th 2011 ............................................................................. 108
Table B.23: Disinfection kinetics: P3, October 15th 2011 ........................................................................... 109
Table C.1: Antibiotic-resistance genes and probes: Part 1 .......................................................................... 110
Table C.2: Antibiotic-resistance genes and probes: Part 2 .......................................................................... 111
Table C.3: Antibiotic-resistance genes and mechanisms Part1 ................................................................... 112
Table C.4: Antibiotic-resistance genes and mechanisms Part 2 .................................................................. 113
Table C.5: UPEC-virulence genes and primer sequences ........................................................................... 114
Table D.1: Antibiotic-resistance profile P4-b .............................................................................................. 115
Table D.2: Antibiotic-resistance profile P1 ................................................................................................. 116
Table D.3: Antibiotic-resistance profile P2 ................................................................................................. 117
Table D.4: Antibiotic-resistance profile P3 ................................................................................................. 118

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