#89: Lytic Bacteriophage Against an Emergent Biofilm-Forming Enteroinvasive Escherichia coli
Authors: Yu K O A , Gómez-Duarte O G 
Published: March 2021

Abstract

Background
Bacteriophage are viruses that infect and parasitize bacteria. In the pre-antibiotic era, phage therapy was one of the few tools available against bacterial infection. After penicillin was discovered, phage therapy fell into disfavor in Western medicine, and its use was mostly limited to medical centers in the Soviet bloc. With current issues on multi-drug resistance, especially of Gram-negative bacteria, there is interest in revisiting phage therapy for use in medical and veterinary practice.
This study focuses on the biofilm-forming enteroinvasive Escherichia coli (BF-EIEC) serotype O96:H19. Strain BF-EIEC 52.1 was isolated in a pediatric case–control diarrheal study in Bucaramanga, Colombia, in 2013–2014. This strain is genetically similar to a foodborne outbreak strain isolated in Italy in 2012, and in sporadic outbreaks in the UK, Spain, and Uruguay. BF-EIEC 52.1 produces biofilms in vitro, similarly to enteroaggregative E. coli, and yet invades host cells, similar to enteroinvasive E. coli and Salmonella enterica (J. Iqbal, O. Gómez-Duarte et al., manuscript in preparation), making this a potential emergent pathogen of concern. We hypothesized that lytic bacteriophage derived from wastewater sources are active against BF-EIEC 52.1.

Methods
Environmental wastewater samples were retrieved from the Amherst, New York, wastewater treatment facility. These were filtered against a 0.2 μm membrane and stored at 4°C. An attempt to isolate phage was done by direct plating over E. coli BF-EIEC 52.1 on solid Luria–Bertani (LB) agar. A second strategy involved enrichment for lytic phage by incubating sample water with BF-EIEC 52.1 in LB broth. Cloning was accomplished by re-filtering the preparation (to remove bacteria), then followed by serial limiting dilution of cell-free filtrate in liquid bacterial culture. Characterization of phage preparations included a demonstration of differential lytic activity against BF-EIEC 52.1 and two unrelated laboratory E. coli strains, DH5α and HB101, in both solid agar and liquid medium.

Results
Isolation of phage by direct plating was possible, but cloning phages proved difficult. With the alternative strategy, four of six wastewater specimens incubated with BF-EIEC 52.1 in liquid LB showed decreased turbidity compared with sterile water control. All positive specimens proved to have demonstrable lytic activity against host E. coli by plaque formation in solid medium, and in limiting dilution in liquid medium. Thirty-two putative phage preparations were cloned by repeated limiting dilution from an initial 16 independent wells. Of these, 27 preparations showed visible plaques against BF-EIEC 52.1 in solid agar, while 29 and 23 preparations showed plaques against HB101 and DH5α, respectively. All phage preparations inhibited the growth of low-inoculum BF-EIEC 52.1 in liquid medium for &gt;6 hours. Six virus preparations inhibited growth for &gt;72 hours.

Conclusions
Bacteriophage active against strain BF-EIEC 52.1 were isolated from urban wastewater samples, showing a range of activity against the parental host, as well as unrelated E. coli strains. Further study is needed to demonstrate activity in alternative clinical settings, such as in biofilm-associated infections or in animal models.