Diversity and Local Coadaptation of Escherichia coli and Coliphages From Small Ruminants

Abstract

Bacteriophages are highly specific predators that drive bacterial diversity through coevolution while striking tradeoffs among preserving host populations for long-term exploitation and increasing their virulence, structural stability, or host range. Escherichia coli and other coliform bacteria present in the microbiota of milk and during early ripening of raw milk cheeses have been linked to the production of gas, manifested by the appearance of eyes, and the development of off-flavors; thus, they might cause early blowing and cheese spoilage. Here, we report the characterization of coliphages isolated from manure from small ruminant farms and E. coli strains isolated from goat and sheep raw milk cheese. Additionally, the virulence and host range of locally isolated and laboratory collection phages were determined by comparing the susceptibility of E. coli strains from different sources. In agreement with the high genetic diversity found within the species E. coli, clustering analysis of whole-cell protein revealed a total of 13 distinct profiles but none of the raw milk cheese isolates showed inhibition of growth by reference or water-isolated coliphages. Conversely, 10 newly isolated phages had a broad host range (i.e., able to lyse ≥50% of bacterial hosts tested), thus exhibiting utility for biocontrol and only one cheese-isolated E. coli strain was resistant to all the phages. Whereas there was a high positive correlation between bacterial susceptibility range and lysis intensity, the phages virulence decreased as range increased until reaching a plateau. These results suggest local gene-for-gene coevolution between hosts and phages with selective tradeoffs for both resistance and competitive ability of the bacteria and host-range extension and virulence of the phage populations. Hence, different phage cocktail formulations might be required when devising long-term and short-term biocontrol strategies.

A New Pipeline for Designing Phage Cocktails Based on Phage-Bacteria Infection Networks

“…To design a phage cocktail, we will use a previously obtained quantitative host range matrix ( Molina et al, 2020 ) comprising reference E. coli strains ( Table 1 ) and isolates from goat and ewe raw milk cheeses and coliphages isolated from sheep feces ( Figure 2B ). To evaluate both modularity and nestedness, the heatmap was transformed into bipartite values (lysis/no lysis).…”

“…In contrast to the algorithms that determine modularity and nestedness ( Figure 2B ), hierarchical clustering does not require bipartite matrices and allows us to consider virulence apart from the host range of the phages. To formulate a phage cocktail, we used a quantitative host range matrix comprising E. coli from different sources ( Table 2 ) and coliphages isolated from sheep feces ( Molina et al, 2020 ) that includes the same bacteria and phages depicted in Figure 3A . The hierarchical clustering of the coliphages by Ward’s method generated seven clusters ( Figure 7A ), each exhibiting a different host range (R) and virulence (V).…”

“…(A) Hierarchical clustering of an E. coli and coliphage matrix ( Figure 2B ) by Ward’s method. Each phage-host interaction was analyzed several times (6 > N > 2), and the average virulence (growth inhibition) is represented by the clearing intensity (0 = no lysis, 3 = complete lysis) of cross-streaks ( Molina et al, 2020 ). The host range (R) and the average virulence (V) for each phage and the average susceptibility of each host strain to phages are shown (S).…”

“…(B) Evaluation of growth inhibition by the phage cocktails. The reference E. coli strain MG1655 and seven cheese-isolated E. coli strains with different proteomic ( Molina et al, 2020 ) and phage sensitivity profiles (A) were used as controls. Two cocktails of phages are compared: the control (λ, T4, T6, and P1) and that designed from ewe feces isolates (A) .…”

“…The phage cocktail was tested by inoculation of the selected phages into a combination of seven E. coli isolates. Host strains were selected according to their distinct proteomic profiles, which show a moderate positive correlation with their sensitivity to the phages ( Molina et al, 2020 ). The assays were performed on reconstituted milk ( Figure 7B ) and LB medium (not shown), producing similar results.…”