Graduate Paper Winner - Irrigation Water as a Source of Antibiotic Resistant and Pathogenic E. coli on Irrigated Lettuce – an extended abstract

Matthew Aijuka and Elna M Buys   

Introduction

Global increase in frequency of foodborne outbreaks associated with fresh produce necessitates investigation into possible sources of environmental pathogenic bacterial contamination. Most South African water requirements are provided by fresh water sources with the bulk used for irrigation. The deteriorating bacteriological quality of surface irrigation water in South Africa has been reported (Ijabadeniyi et al. 2011, Gemmel and Schmidt 2012). Antibiotic resistant and virulent bacterial pathogens present a major risk to food safety and public health (Da Silva and Medonça 2012). Lettuce is popular in South Africa with notable use in salads.

Materials and methods

Characterisation of environmental E. coli from irrigation water and irrigated lettuce to assess whether water was a source of contamination with pathogenic E. coli was done. Previously identified  E. coli (Aijuka et al. 2014) from irrigation water (12) and irrigated lettuce (10) were subjected to 11 antibiotics (Oxoid Ltd, Basingstoke, Hampshire, UK) at single concentrations (multiple antibiotic resistance). Antibiotic susceptibility testing was determined by disc diffusion method on Mueller-Hinton agar (Oxoid). Antibiotics included; amikacin (30 μg), gentamicin (10 μg), chloramphenicol (30 μg), nalidixic acid (30 μg), norfloxacin (10 μg), neomycin (30 μg), nitrofurantoin (300 μg), amoxycillin (25 μg), ampicillin (10 μg), cephalothin (30 μg) and oxytetracycline (30 μg). E. coli was tested for shigatoxin 1 (stx 1), shigatoxin 2 (stx 2) and intimin (eae) genes with iQ-Check™ STEC VirX catalogue # 357-8139 using C1000 Touch ThermalCycler CFX96TM Real Time System, (Bio-Rad, Hercules, California) and analysis software (CFX Manager IDE) (Bio-Rad).  Genetic fingerprinting [(GTG)5 Rep-PCR)] was carried out as reported by Aijuka et al. (2014). Analyses of variance (2 way) was performed to test for significant differences in antibiotic resistance patterns for E. coli isolated from irrigation water and irrigated lettuce at 95% confidence interval. Fingerprints from (GTG)5-Rep-PCR were analysed using GelCompare III version 5.10 (Applied Maths, Saint-Marten-Latem, Belgium). Similarity among digitised bands was calculated using Pearson correlation, and an average linkage (UPGMA or unweighted pair group method with arithmetic averages) dendrogram was derived from the profiles. Full cluster formation was defined at points where complete nodes were formed.

Results

E. coli from irrigation water and irrigated lettuce had multiple resistance to amoxycillin, ampicillin, cephalothin and oxytetracycline . Highest resistance to single antibiotics was to ampicillin and cephalothin in E. coli from both sources. Resistance of isolates to two or more antibiotics was 50% in irrigation water and lettuce. E. coli isolates from irrigation water showed significantly higher resistance to multiple antibiotics than those from irrigated lettuce (p≤0.05). In irrigation water, 4 of 12 of isolates were positive for at least one virulence gene.

Additionally 25% of isolates were positive for tested virulence genes (stx1/stx2 and eae). From lettuce, 20% of isolates were positive for stx1/stx2 genes. From irrigation water, 80% of isolates possessing virulence genes showed antibiotic resistance. From lettuce all isolates possessing virulence genes showed antibiotic resistance. Cluster analysis using (GTG)5-Rep PCR generated  five distinct clusters.  Some E. coli isolates from both sources showed complete (100%) genetic relatedness. There were 33% of isolates from irrigation water having 100% genetic similarity to 60% of isolates from lettuce.
 

Discussion

Antibiotic resistance and virulence genes are either mediated through mutations on chromosomes or horizontal gene exchange via plasmids, transposons, bacteriophage and pathogenicity islands (Bruzuszkiewicz et al. 2011). Virulence in bacterial pathogens such as E. coli may be co-expressed or suppressed by antibiotics. Therefore E. coli may effect genetic change in order to acclimatise to both environmental and host conditions increasing the risk of compromising food safety and public health

Conclusion and Intervention

Surface irrigation water in South Africa can be a reservoir of E. coli possessing virulence and antibiotic resistant genes. Additionally, irrigation water with poor bacteriological quality may contaminate irrigated produce presenting a risk to food safety and public health. This study suggests a collaborative approach to help ensure lettuce eaten in South Africa is microbiologically safe. The suggested approach is be similar to that seen with the National Advisory Committee on Microbiological Criteria for Foods (NACMCF) under the US Department of Agriculture which combines efforts drawn from experts in different fields of society (public and private) in a bid to ensure food safety and public health.

References

Ijabadeniyi, OA, Debusho, LK, Van Der Linde, M and Buys, EM (2011) Irrigation water as a potential pre-harvest source of bacterial contamination of vegetables. J Food Safety 31: 452-461

Gemmell, ME and Schmidt, S (2012) Microbiological assessment of river water used for the irrigation of fresh produce in a sub-urban community in Sobantu, South Africa. Food Res. Int. 47(2): 300-305.

Da Silva, DJ and Medonça, N (2012) Association between antimicrobial resistance and virulence in Escherichia coli. Virulence 3(1): 18-28.

Aijuka, M, Charimba, G, Hugo, CJ and Buys, EM (2014) Characterization of bacterial  pathogens in rural and urban irrigation water. J Water Health (in press). doi:10.2166/wh.2014.228

Brzuszkiewicz, E, Thümer, A, Schuldes, J, Leimbach, A, Liesegang, H,

Meyer,F-D, Boelter, , Petersen, H, Gottschalk, G and Daniel, R (2011) Genome sequence analysis of two isolates from the recent Escherichia coli outbreak in Germany reveal the emergence of a new pathotype: Entero-Aggregative-Haemorrhagic Escherichia coli (EAHEC). Arch Microbiol. 193: 883-891.