Escherichia coli in urban marine sediments: interpreting virulence, biofilm formation, halotolerance, and antibiotic resistance to infer contamination or naturalization

Abstract

Marine sediments have been suggested as a reservoir for pathogenic bacteria, including Escherichia coli. The origins, and properties promoting survival of E. coli in marine sediments (including osmotolerance, biofilm formation capacity, and antibiotic resistance), have not been well-characterized. Phenotypes and genotypes of 37 E. coli isolates from coastal marine sediments were characterized. The isolates were diverse: 30 sequence types were identified that have been previously documented in humans, livestock, and other animals. Virulence genes were found in all isolates, with more virulence genes found in isolates sampled from sediment closer to the effluent discharge point of a wastewater treatment plant. Antibiotic resistance was demonstrated phenotypically for one isolate, which also carried tetracycline resistance genes on a plasmid. Biofilm formation capacity varied for the different isolates, with most biofilm formed by phylogroup B1 isolates. All isolates were halotolerant, growing at 3.5% NaCl. This suggests that the properties of some isolates may facilitate survival in marine environments and can explain in part how marine sediments can be a reservoir for pathogenic E. coli. As disturbance of sediment could resuspend bacteria, this should be considered as a potential contributor to compromised bathing water quality at nearby beaches.

Keywords: antibiotic resistance; bathing water; biofim; environmental Escherichia coli; halotolerance; whole genome sequencing.

Figure 1.

Sampling locations. The filled colour in each square corresponds to the sampling depth in meters. The approximate location of the WWTP effluent DP is at location N27_F and marked with a cross. The initial letter in the sample name indicates its position relative to the DP of the WWTP with N for North, E for east, S for south, and W for west, while the following number indicates the distance to the DP in meters.

Figure 2.

Percentage of entries in EnteroBase associated with different sources and the assigned STs of different isolates as of 19 December 2022. Colours in the heatmap correspond to the colour bar on the right. The corresponding isolates to the STs are displayed on the x-axis. Number of entries in EnteroBase for each ST is displayed in brackets next to STs.

Figure 3.

Geographical distribution, and virulence genes identified, in the different isolate phylogroups. (A) Distribution of assigned phylogroups of isolates in coastal sediment samples. DP of WWTP is marked with a cross. (B) Identification of genes potentially linked to virulence in the different isolates, with coloured dots indicating the gene and phylotype associated with the isolate named along the x-axis. To group isolates by their genome relatedness, isolates are clustered by ANI. Only genes detected in at least 20 isolates are shown. Less frequently detected virulence genes are described in Figure S2.

Figure 4.

(A) Boxplot showing biofilm formation capacity of different isolates grown in LB and LB with 3.5% NaCl. Values above indicate the P-value of the Wilcoxon signed-rank exact test. (B) Linked boxplot of biofilm formation capacity of different isolates grown in LB and LB with 3.5% NaCl. (C) Boxplots of biofilm formation capacity in isolates grown in LB, grouped by assigned phylogroup A, B1, or B2 with ANOVA P-value (P-value = .031) and p-adj of the Tukey HDS test (above the square brackets). (D) Boxplots of biofilm formation capacity in isolates grown in LB with 3.5% NaCl, grouped by assigned phylogroup A, B1, or B2 with ANOVA P-value (P-value = .29).

Figure 5.

(A) Boxplot showing generation times of different isolates grown in LB and LB with 3.5% NaCl. Values above indicate the P-value of the Wilcoxon signed-rank exact test. (B) Linked boxplot of generation times of different isolates grown in LB and LB with 3.5% NaCl. (C) Boxplots of generation time in isolates grown in LB, grouped by assigned phylogroup A, B1, or B2 with ANOVA P-value (P-value = .3). (D) Boxplots of generation time in isolates grown in LB with 3.5% NaCl, grouped by assigned phylogroup A, B1, or B2 with ANOVA P-value (P-value = .021) and p-adj of the Tukey HDS test (above the square brackets).