Phenotypic and Genetic Determination of Biofilm Formation in Heat Resistant Escherichia coli Possessing the Locus of Heat Resistance

Abstract

Despite the effectiveness of thermal inactivation processes, Escherichiacoli biofilms continue to be a persistent source of contamination in food processing environments. E. coli strains possessing the locus of heat resistance are a novel food safety threat and raises the question of whether these strains can also form biofilms. The objectives of this study were to determine biofilm formation in heat resistant E. coli isolates from clinical and environmental origins using an in-house, two-component apparatus and to characterize biofilm formation-associated genes in the isolates using whole genome sequencing. Optimal conditions for biofilm formation in each of the heat resistant isolates were determined by manipulating inoculum size, nutrient concentration, and temperature conditions. Biofilm formation in the heat resistant isolates was detected at temperatures of 24 °C and 37 °C but not at 4 °C. Furthermore, biofilm formation was observed in all environmental isolates but only one clinical isolate despite shared profiles in biofilm formation-associated genes encoded by the isolates from both sources. The circulation of heat resistant E. coli isolates with multi-stress tolerance capabilities in environments related to food processing signify that such strains may be a serious food safety and public health risk.

Keywords: Escherichia coli; biofilm; food processing; locus of heat resistance; multi-stress tolerance.

Figure 1

Two-component apparatus for detecting biofilm formation (A). The pegs of the PCR plate are submerged into wells of the flat bottom microplate containing bacterial cells. The PCR plate rests on top of 2 sterile sticks to prevent the pegs from direct contact with the bottom of the microplate. Biofilms form on the pegs that are subsequently stained with 1% crystal violet (B).

Figure 2

Triphenyltetrazolium Chloride (TTC) motility test media of (A) positive control Escherichia coli ATCC 25922 (left) and negative control Klebsiella pneumoniae ATCC 700603 (right) and (B) heat resistant isolates AW1.7 (left) and 128 (right). Results from TTC media of isolates 53, 63, and 111 were identical to isolate AW1.7 and isolate 8354 was identical to isolate 128.

Figure 3

Biofilm formation of environmental and clinical Escherichia coli isolates in 100% LB broth and 8 log CFU/mL inoculum size incubated at temperatures of 37 °C, 24 °C, and 4 °C for optimization of the two-component apparatus. Data presented as means ± standard deviations of triplicate experiments.

Figure 4

Biofilm formation in heat resistant Escherichia coli environmental isolates AW1.7 at 24 °C (A) and 37 °C (B); 53 at 24 °C (C) and 37 °C (D); 63 at 24 °C (E) and 37 °C (F); and clinical isolate 111 at 24 °C (G) and 37 °C (H) under conditions of inoculum size and nutrient concentration manipulation. Data presented as means ± standard deviations of triplicate experiments.