PREVALENCE AND ANTIBIOTIC SENSITIVITY OF ENTEROTOXIGENIC ESCHERICHIA COLI ISOLATES IN SOUTH AFRICAN PIG POPULATION Zizile Emelda Lilly Sikhosana Submitted in fulfillment of the requirements for the degree of Master of Science (Genetics) in the School of Life Sciences, University of KwaZulu-Natal February 2015 Supervisor: Mr E.F. Dzomba Signature:………………… Co-supervisor: Dr E. Madoroba Signature:………………… Co-supervisor: Dr F.C. Muchadeyi Signature:………………… i ABSTRACT Escherichia coli (E. coli) are among the leading bacterial causes of diarrhoea and edema in newborn and weaned pigs. Pathogenic strains of E. coli are classified into enterotoxigenic E. coli (ETEC), enterohemorrhagic E. coli (EHEC), enteroaggreagative E. coli (EAEC), enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC) and diffusely adherent E. coli (DAEC) based on virulence factors. Infection with E. coli is achieved by adherence of fimbrial and/or non-fimbrial adhesins to the intestines and the release of toxins thereafter. The increasing rates of antimicrobial resistance are posing a threat in the treatment of porcine E. coli infections. The aim of this study was to determine the prevalence of pathogenic Escherichia coli virulence genes and antibiotic sensitivity of enterotoxigenic Escherichia coli isolates from neonatal and post-weaning pigs in Limpopo and Eastern Cape provinces of South Africa. For this purpose, 325 rectal swabs were collected from pigs from the Eastern Cape and Limpopo provinces of South Africa to investigate the prevalence of ETEC relative to other E. coli strains. Classical microbiological tests were conducted for confirmation of E. coli and PCR was used for the detection of fimbrial, non-fimbrial adhesins and toxin genes. In addition, antimicrobial susceptibility of ETEC positive isolates was determined by the Kirby-Bauer disk diffusion method. Of the 325 swabs collected, 303 isolates were identified as E. coli with 67% (205/303) harboring at least one of the tested virulence genes (LT, STa, STb, EAST-1, Stx1, Stx2, Stx2e, VT1, VT2, hlyA, F4, F5, F6, F18, F41, AIDA-1, EAE and PAA) and categorized into 48 pathotypes. A total of 36 (11.9%) isolates was classified as ETEC, having heat-labile (LT) enterotoxin as the most prevalent. Only a single isolate (2.8%) carried fimbriae (F4/F5). Instead, non-fimbrial adhesins PAA, AIDA-1 and EAE were detected. The ETEC positive isolates displayed 47.2%, 38.9% and 36.1% resistance to oxytetracycline, ampicillin and trimetroprim respectively. Most of the ETEC isolates were sensitive to florphenicol (100%), cefotaxime (97.2%) and enrofloxacin (77.8%). Multi- drug resistance was detected in 50% of the isolates. The study demonstrated that there are various E. coli pathotypes in South Africa. The detection of non-fimbrial adhesins reinforces existing knowledge that fimbriae are not the only colonization factors associated with ETEC. Based on the antimicrobial i susceptibility patterns observed, florphenicol, cefotaxime and enrofloxacin could be used for the treatment of ETEC infections in South African pigs. ii DECLARATION I, Zizile Emelda Lilly Sikhosana, declare that: i. The research reported in this thesis is my original work except where acknowledged. ii. This thesis has not been submitted for any degree or examination at any other university but the University of KwaZulu-Natal. iii. Information and pictures obtained from other sources have been acknowledged. iv. Assistance received while conducting the research and writing this thesis has been acknowledged. v. This work was conducted under the supervision of Mr E.F. Dzomba, Dr E. Madoroba and Dr F.C. Muchadeyi. Signature: .............................. Date: .................... Zizile Emelda Lilly Sikhosana Approved in terms of style and content by: Mr E.F. Dzomba: ……………………. Date: ………………. Dr E. Madoroba: ……………………. Date: ……………….. Dr F.C. Muchadeyi: …………………. Date: ………………. iii ACKNOWLEDGEMENTS My most profound gratitude goes to my Almighty God for courage and strength that kept me going throughout this journey. To my supervisors Mr E.F. Dzomba, Dr E. Madoroba and Dr F. Muchadeyi thank you for your constant support, guidance and believing in me. I am very grateful to you all for the valuable guidance and constructive input in compiling this thesis. I would like to thank my colleagues Mr K. Khanyile, Miss N. Chaora and Miss P. Sarela for assisting with sample collection and laboratory work. Special thanks to the Agricultural Research Council-Onderstepoort Veterinary Institute (ARC-OVI), Bacteriology Department for providing the laboratory space and equipment to carry out this study. I am thankful to the University of KwaZulu-Natal and the National Research Foundation for funding this research. Lastly, I would like to pass my sincere gratitude to my family and friends for the love, support and encouragement. iv TABLE OF CONTENTS ABSTRACT ............................................................................................................................................. i DECLARATION ................................................................................................................................... iii ACKNOWLEDGEMENTS ................................................................................................................... iv TABLE OF CONTENTS ........................................................................................................................ v LIST OF FIGURES ............................................................................................................................. viii LIST OF TABLES ................................................................................................................................. ix LIST OF ABBREVIATIONS ................................................................................................................. x CHAPTER 1 ........................................................................................................................................... 1 General Introduction ............................................................................................................................... 1 1.1 Introduction ................................................................................................................................... 1 1.2 Justification ................................................................................................................................... 3 1.3 Objectives ..................................................................................................................................... 3 1.4 References ..................................................................................................................................... 4 CHAPTER 2 ........................................................................................................................................... 7 Literature Review .................................................................................................................................... 7 2.1 Escherichia coli ............................................................................................................................ 7 2.2 Diarrheagenic E. coli .................................................................................................................... 7 2.2.1 ETEC ...................................................................................................................................... 8 2.2.2 EPEC ...................................................................................................................................... 8 2.2.3 EHEC ..................................................................................................................................... 9 2.2.4 EAEC ................................................................................................................................... 10 2.2.5 EIEC ..................................................................................................................................... 10 2.2.6 DAEC ................................................................................................................................... 11 2.3 Enterotoxigenic E. coli ................................................................................................................ 11 2.4 Pathogenesis ................................................................................................................................ 12 2.4.1 Colonization factors ............................................................................................................. 12 2.4.1.1 F5 ...................................................................................................................................... 12 2.4.1.2 F6 ...................................................................................................................................... 13 2.4.1.3 F4 ...................................................................................................................................... 13 2.4.1.4 F18 .................................................................................................................................... 14 2.4.1.5 F41 .................................................................................................................................... 14 2.4.2 Receptors .............................................................................................................................. 14 v 2.4.3 Enterotoxins ......................................................................................................................... 15 2.4.3.1 Heat-labile enterotoxins .................................................................................................... 15 2.4.3.2 Heat-stable enterotoxins .................................................................................................... 16 2.5 Isolation and identification of E. coli .......................................................................................... 20 2.5.1 Microbiological methods ..................................................................................................... 20 2.5.2 Molecular diagnosis ............................................................................................................. 22 2.5.2.1 Nucleic acid probes ........................................................................................................... 23 2.5.2.2 PCR ................................................................................................................................... 23 2.6 Treatment and prevention of E. coli-induced diarrhoea in pigs .................................................. 24 2.7 Antimicrobial resistance ............................................................................................................. 25 2.8 Summary ..................................................................................................................................... 28 2.9 References ................................................................................................................................... 28 CHAPTER 3 ......................................................................................................................................... 33 Detection of enterotoxigenic Escherichia coli (ETEC) virulence profiles relative to enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enteroaggregative E. coli (EAEC) and diffusely adherent E. coli (DAEC) by multiplex PRC in South African pigs ...................................................... 33 Abstract ................................................................................................................................................. 33 3.1 Introduction ................................................................................................................................. 35 3.2 Materials and Methods ................................................................................................................ 36 3.2.1 Sampling .............................................................................................................................. 36 3.2.2 Classical microbiological methods ...................................................................................... 37 3.2.3 DNA extraction .................................................................................................................... 38 3.2.4 Multiplex PCR ..................................................................................................................... 39 3.2.5 Gel electrophoresis ............................................................................................................... 40 3.2.6 Statistical analysis ................................................................................................................ 41 3.2.7 Pathotype grouping .............................................................................................................. 41 3.3 Results ......................................................................................................................................... 43 3.3.1 Classical microbiological methods ...................................................................................... 43 3.3.2 Genotyping ........................................................................................................................... 45 3.4 Discussion ................................................................................................................................... 53 3.5 Conclusion .................................................................................................................................. 59 3.6 References ................................................................................................................................... 60 CHAPTER 4 ......................................................................................................................................... 64 Antimicrobial susceptibility of enterotoxigenic Escherichia coli isolates from South African pigs .... 64 Abstract ................................................................................................................................................. 64 4.1 Introduction ................................................................................................................................. 65 vi 4.2 Materials and Methods ................................................................................................................ 67 4.2.1 E. coli isolates ...................................................................................................................... 67 4.2.2 Antimicrobial susceptibility testing ..................................................................................... 67 4.2.3 Statistical analysis ................................................................................................................ 69 4.3 Results ......................................................................................................................................... 70 4.3.1 Sample structure ................................................................................................................... 70 4.3.2 Frequency of susceptible and resistant isolates .................................................................... 70 4.3.3 Effects of province of origin on isolate’s resistance/susceptibility to antimicrobial drugs .. 72 4.3.4 Multiple antimicrobial resistance patterns ........................................................................... 74 4.4 Discussion ................................................................................................................................... 75 4.5 Conclusion .................................................................................................................................. 79 4.6 References ................................................................................................................................... 80 CHAPTER 5 ......................................................................................................................................... 84 5.1 General Discussion ..................................................................................................................... 84 5.2 Conclusion .................................................................................................................................. 87 5.3 Recommendations ....................................................................................................................... 88 5.4 References ................................................................................................................................... 89 vii LIST OF FIGURES Figure 2.1: Pathogenic mechanism of enterotoxigenic E. coli (Croxen and Finlay 2010) .................. 18 Figure 2.2: Mechanisms of action of enterotoxigenic E. coli enterotoxins LT, STa and STb (Nagy and Fekete, 1999)......................................................................................................................................... 19 Figure 3.1: Map illustrating the two provinces of South Africa; Eastern Cape (E) and Limpopo (L) sampled in this study (Fielding and Shields, 2006) .............................................................................. 37 Figure 3.2: Steps involved in the extraction of crude DNA ................................................................. 39 Figure 3.3: Colony morphology observed on a) MacConkey and b) Blood plates after overnight incubation at 37°C to culture E. coli ..................................................................................................... 43 Figure 3.4: Escherichia coli isolates after overnight incubation at 37°C on indole and methyl-red (a) and citrate (b) where C denotes negative control (Klebsiella pneumonia) ........................................... 44 Figure 3.5: A 2% agarose gel illustrating amplification of enterotoxigenic E. coli obtained using primers STa (183 bp), STb (360 bp) and LT (282 bp) for amplification of ETEC enterotoxins where the Lanes were loaded as follows, Lane 1: 100 bp DNA ladder, Lanes 2-12 and Lanes 14-21: ETEC positive isolates, Lanes 22-26: K88 (F4:F5:LT:STb); K99 (F5:STa); 1883-2 (F41:F5:STa), 1883-5 (LT:STb) and E. coli ATCC 25922 (negative control) .......................................................................... 48 Figure 4.1: Frequency of susceptible, intermediate and resistant ETEC isolates to antimicrobial agents .................................................................................................................................................... 72 viii LIST OF TABLES Table 3.1: Virulence genes associated with Escherichia coli categories targeted in this study……...40 Table 3.2: Polymerase chain reaction primers used for amplification of virulence genes of E. coli isolates.......................................................................................................................................42 Table 3.3: Distribution of E. coli strains among 205 isolates harbouring virulence genes...................46 Table 3.4: Proportions of enterotoxins and EAST-1 in the ETEC positive population (n=36)………………………………………………………………………………………….............49 Table 3.5: Distribution of adhesin factors AIDA-1, PAA and EAE in ETEC isolates..........................50 Table 3.6a: Distribution of the different E. coli pathotypes within the 205 isolates containing the tested virulence genes...........................................................................................................................51 Table 3.6b: Distribution of the different E. coli pathotypes within the 205 isolates containing the tested virulence genes ...........................................................................................................................52 Table 4.1: Zone sizes (mm) and interpretation of the 9 tested antibiotics……………………………69 Table 4.2: Frequency of antimicrobial response of enterotoxigenic E. coli positive isolates from neonatal and post-weaning piglets………………………………………………………………71 Table 4.3: Susceptibility patterns in the whole population and between the two provinces, Eastern Cape and Limpopo……………………………………………………………………………73 Table 4.4: Resistance patterns in the whole population and between the two provinces, Eastern Cape and Limpopo……………………………………………………………………………74 Table 4.5: Multi-drug resistance patterns of enterotoxigenic E. coli from rectal swabs of pigs (n=36)…………………………………………………………………………………..75 ix