Development of Bacteriophage Cocktail for bio-control of atypical Escherichia coli O177 strains
Abstract
Atypical enteropathogenic Escherichia coli (aEPEC) strains are emerging pathogens responsible for deadly diarrhoea infections in human in both developing and industrialised countries across the globe. The aEPEC is a heterogeneous group, which shares virulence traits with other E. coli pathotypes from diarrhoeagenic E. coli and extraintestinal E. coli pathogenic groups. In addition, food-producing animals such as cattle are considered as the primary reservoir of aEPEC strain and thus, this may increase the possibilities of food contamination during milking or at slaughter. Although several interventions have been implemented to combat food contamination, some of the strategies have serious side effects, especially in humans. Furthermore, lack of a novel antimicrobial agents coupled with antibiotic resistance precipitate transmission of antibiotic resistant foodborne pathogens from animals to humans via consumption of contaminated food. This warrant a need to search for a novel and practical intervention such as the use of bacteriophages to curb antimicrobial resistance. Therefore, the purpose of this study was to develop phage cocktails to control E. coli O177 strain in food and live animals using in vitro models. Faecal samples were collected from cattle from different farming systems for isolation of E. coli O177 strain and E. coli O177-specific bacteriophages. In addition, genotypic typing and whole genome sequence techniques were employed to determine genetic similarities and genome features of the E. coli O177 isolates. Furthermore, E. coli O177-specific phages were isolated using E. coli O177 host. Phage morphotype, stability against various physical parameters (pH and temperature were assessed) were assessed. In addition, individual phages and phage cocktails were assessed to determine their effectiveness in reducing E. coli O177 cells on artificially contaminated beef and their ability to prevent and destroy pre-formed biofilm structures. Individual phages and phage cocktails were also evaluated for their effectiveness in reducing E. coli O177 cell count in rumen simulation model and complete genome sequence was performed to assess the feature of the phages. A total of 780 cattle faecal samples were collected from different farms. One thousand two hundred and sevety-two presumptive isolates were obtained. Nine hundred and fifteen of the isolates were successfully identified as E. coli isolates through amplification of the uidA genus-specific gene. Out of 915 isolates screened, 376 were confirmed as E. coli O177 strain using multiplex PCR, targeting the rmlB and wzy genes. E. coli O177 isolates harboured hlyA, stx2, stx1, eaeA, stx2a and stx2d (12.74, 11.20, 9.07, 7.25, 2.60 and 0.63%, respectively). Some isolates possessed a combination of the stx1/stx2/hlyA/eaeA and one isolate carried the stx1/stx2/hlyA/eaeA/stx2a/stx2d genes simultaneously. Furthermore, this study revealed that E. coli O177 isolates were resistant to erythromycin, ampicillin, tetracycline, streptomycin, kanamycin, chloramphenicol and norfloxacin (63.84, 21.54, 13.37, 17.01, 2.42, 1.97 and 1.40%, respectively). In addition, 20.7% of the isolates exhibited different phenotypic multi-drug resistance patterns. The Multiple Antibiotic Resistance (MAR) index ranged from 0.29 to 0.86 whereas the average MAR index was 0.65. All 73 isolates harboured at least one antimicrobial resistance gene. The aadA, streA, streB, erm and tetA resistance genes were detected separately and/or concurrently. Genetic typing techniques showed 100% genetic similarities of E. coli O177 isolates obtained from cattle from different farms. Enterobacterial repetitive intergenic consensus (ERIC) typing clustered the isolates into nine clusters made up of mixed isolates from different farms while Random amplified polymorphism deoxyribonucleic acid (RAPD) typing classified the isolates into eight clusters composed of isolates from the various cattle farms. Whole genome sequence (WGS) annotation indicated that the two genomes sequenced showed > 95% similarities to O177 strain with H7 (CF-154) and H21 (CF-335). WGS revealed that E. coli O 177 genomes contained several virulence and antimicrobial resistance genes sequences. The key virulence genes such as intimin (eaeA), haemolysin (hlyA and hlyE) and others, associated with aEPEC group, were found in both genomes. However, genes (stx) encoding for shiga toxins were not found in both genomes. Furthermore, E. coli O 177 genomes possessed six plasmid types, prophages and a cluster of regularly interspaced short palindromic repeats (CRISPR) type I (subtype I-A and I-E) gene sequences. The CRISPR-Cas proteins were found in both genomes. A total of 31 lytic E. coli O177-specific bacteriophages were sucessfully isolated in this study. The spot test revealed that all eight selected phages were capable of infecting different environmental E. coli strains. In addition, Efficiency of plating (EOP) analysis (range: 0.1 to 1.0) showed that phages were capable of infecting a wide range of E. coli isolates. Selected phage isolates had similar morphotype (icosahedral head and contractile tail ranging from 81.2 nm to 110.77 nm and 115.55 nm to 132.57 nm, respectively) and were classified under the order Caudovirales, Myoviridae family. The phages were stable at 37 °C and 40 °C, over 60 minutes of incubation. Furthermore, phages were inactive at pH 3.0. However, quadratic response showed that pH optima ranged between 7.6 and 8.0. Phage latent period ranged from 15 to 25 minutes while burst size ranged from 91 to 522 virion particles (PFU) per infected cell. The current study also showed that eight individual phages and six phage cocktails were capable of reducing E. coli O177 cell count on artificially contaminated beef over a seven-day incubation period at 4 °C. Two individual phages, vB_EcoM_12A1 and vB_EcoM_3A1 and three cocktails, T3, T4 and T6, reduced bacteria cell count to below detection limit (1.0 log10 CFU/mL) over a seven-day incubation period. Relative reduction percentage ranged between 73-100% and 32-100% (for all individual phages and cocktails, respectively). Although E. coli cell counts showed increase at day three and seven in samples treated with individual phages (vB_EcoM_10C2, vB_EcoM_10C3, vB_EcoM_118, vB_EcoM_11B, vB_EcoM_366B, vB_EcoM_366V) and phage cocktails (T1, T2 and T5), viable cell counts were significantly lower than the controls. Individual phages and phage cocktails also revealed potential in inhibiting the growth of E. coli O177 biofilm formation with the later showing greater potency in destroying pre-formed biofilm than the former. This finding suggests that phages cocktails developed in this study can be used for biocontrol of E. coli O177 on meat at storage conditions to improve food safety. Response surface regression analysis revealed significant quadratic responses in the titres of both individual phages and their cocktails over the 48-hour incubation period under simulated rumen fermentation conditions. Individual phage titres were predicted to peak at 50 - 52 hours of in vitro ruminal incubation from response surface regression models with R² values ranging from 0.811 to 0.994 while phage cocktail titres were predicted to peak at 51 and 55 hours of in vitro ruminal incubation from response surface regression models with R2 values ranging from 0.982 to 0.995. When exposed to individual phages, the percent reduction of E. coli O177 cell counts peaked (60.81 - 63.27%) at 47 to 48 hours of incubation as determined from prediction equations with R² values ranging from 0.992 to 0.996. Nevertheless, when treated with phage cocktails, the percentage reduction of E. coli O177 cell counts peaked (63.06 to 73.25%) at 43 to 46 hours of incubation as determined from prediction equations with R² values ranging from 0.970 to 0.993. Over the 48-hour of incubation period, individual phages vB_EcoM_366B and vB_EcoM_3A1 were the most effective (62.31 and 62.74%, respectively) while phage cocktails T1, T3, T4 were the most effective (66.67, 66.92 and 66.42%, respectively) in reducing E. coli O177 cell count at 39 °C, over a 48-hour incubation period. These results indicate that phage cocktail T3, T4, and T6 are the most effective in reducing E. coli O177 cell counts in a simulated ruminal fermentation system. Therefore, these phage cocktails are the most suitable candidates to be used in live animals, particularly cattle, to reduce the level of E. coli O177 load before slaughter. Whole genome sequence annotation showed that Escherichia phage vB_EcoM_11B2-MVA genome was 152,234 bp linear dsDNA with 39.1% GC content. Escherichia phage vB_EcoM_11B2-MVA genome did not contain lysogenic (integrase), virulence or antimicrobial resistance sequences. This indicated that the phage vB_EcoM_11B2 is safe and suitable candidate to be used as a biocontrol agent against the E. coli O177 strain either in food or live animals. In addition, the genome contained 30 genes encoding for phage proteins and 11 tRNA gene sequences coding for 10 amino acids. Based on blast pairwise alignment, phylogenomic and VICTOR analysis, Escherichia phage vB_EcoM_11B2-MVA genome was classified under the order Caudovirales, Myoviridae family and the new genus "Phapecoctavirus".