Characterization of sediment, sewage and 0157 E. coli: a comparison of their virulence genes, biofilm forming capacities, antibiotic resistance and level of reactive oxygen species (ROS)

Abstract

Even though sediment Escherichia coli is a common source of contamination in freshwater environments, their virulence and physiological characteristics are not well studied. The goal of this study was to compare the virulence genes, biofilm forming capacity, antibiotic resistance and reactive oxygen species (ROS) level of sediment E. coli isolated from three freshwater lakes (Lake Simcoe, Georgian Bay, and Boulevard Lake) with no exposure to sewage effluent to sewage E. coli from two sewage treatment plants (Orillia and Thunder Bay Ontario) and a collection of known O157 E. coli. Multiplex PCR analysis on nine virulence genes (hylA, iroN, papA, hs, hl, ial, bfpA, stx1, and stx2) revealed that no sediment, and very few (3%) sewage E. coli contained diarrheagenic and shiga toxin genes. However, 12.5% of sediment and 42% of sewage isolates contained one or more uropathogenic genes. Interestingly, only the iroN gene was detected in the sediment isolates. The biofilm assays determined that sediment E. coli were significantly better biofilm formers (p<0.001) than the sewage and O157 E. coli, and the sediment E. coli was able to form 2 and 3.5 times as much biofilm as the sewage and O157 E. coli, respectively. The antibiotic resistances of the isolates to eight antibiotics were determined using the Kirby-Bauer disk diffusion method and the antibiotic resistance patterns of the E. coli samples illustrated that the sediment, sewage and O157 isolates belonged to three distinctive populations. Furthermore, there was an overall significant difference between the three sample groups (p<0.05), where sediment was the most susceptible and sewage was the most resistant to the antibiotics tested. It was also determined that the level of ROS in biofilm E. coli cells was significantly lower than their planktonic counterparts (p<0.001). A negative correlation (p=0.066) was observed when comparing the isolates’ biofilm forming capacities with their intrinsic level of ROS, whereby isolates with higher tolerance to oxidative stress (i.e. higher amounts of cellular ROS) were associated with lower biofilm forming capability. Furthermore, isolates with higher resistance to antibiotics in their planktonic state also showed lower biofilm forming capacity. It has been previously determined that the rpoS gene, a crucial regulatory gene in biofilm phase bacteria, is able to improve the survival of E. coli by optimizing the size of the biofilm matrix. The role of stationary phase sigma factor (RpoS) of an E. coli O157:H7 H32 strain in the biofilm phase was examined by comparing the biofilm formation capacity, reactive oxygen species (ROS) level, and antibiotic resistance of the wildtype H32 to its rpoS mutant. The mutant strain formed significantly more biofilm (p<0.05) than the wildtype strain with the mutant strain forming twice as much biofilm as the wildtype strain. To investigate the level of ROS in the two E. coli strains, a DCF-DA assay was conducted and revealed a significant difference between the exponential growth phase and biofilm state of the wildtype H32 strain with biofilm cells illustrating over 100,000 times lower ROS levels (p<0.05) than exponential phase cells (see document)