Global distribution of epidemic-related Shiga toxin 2 encoding phages among enteroaggregative Escherichia coli.

Since the Shiga toxin-producing enteroaggregative Escherichia coli (Stx-EAEC) O104:H4 strain caused a massive outbreak across Europe in 2011, the importance of Stx-EAEC has attracted attention from a public health perspective. Two Stx-EAEC O86 isolates were obtained from patients with severe symptoms in Japan in 1999 and 2015. To characterize the phylogeny and pathogenic potential of these Stx-EAEC O86 isolates, whole-genome sequence analyses were performed by short-and long-read sequencing. Among genetically diverse E. coli O86, the Stx-EAEC O86 isolates were clustered with the EAEC O86:H27 ST3570 subgroup. Strikingly, there were only two loci with single nucleotide polymorphisms (SNPs) between the Stx2a phage of a Japanese O86:H27 isolate and that of the European epidemic-related Stx-EAEC O104:H4 isolate. These results provide evidence of global distribution of epidemic-related Stx2a phages among various lineages of E. coli with few mutations.

Isolation and identification of specific bacteriophage against enteropathogenic Escherichia coli (EPEC) and in vitro and in vivo characterization of bacteriophage.

In recent years, the increasing resistance of enteropathogenic Escherichia coli (EPEC) to commonly used antibiotics has made it difficult to choose the best treatment option. Bacteriophage therapy could be a potent alternative to antibiotic therapy for antibiotic-resistant bacteria. The aim of the present study was to isolate and identify a specific bacteriophage against EPEC and characterize bacteriophage in vitro and in vivo. The specific bacteriophage was isolated, and the effect of phage therapy on 48 mice (Balb/c) was investigated. Animals were divided into six groups, including A: PBS (negative control); B: bacteria (positive control); C: bacteria + ciprofloxacin (after 24 h); D: bacteria + bacteriophage (after 24 h); E: bacteria + ciprofloxacin + bacteriophage (after 24 h) and F: bacteriophage + bacteria (after 24 h). Specific bacteriophage against EPEC was isolated from hospital sewage. The bacteriophage had an icosahedral head (120 nm) and a tail (138 nm). The single dose of the bacteriophage (2 × 109 pfu ml-1) was able to control the infection. Unfortunately, because of the misuse of antibiotics by EPEC infected patients, the antibiotic resistant bacteria will become prevalent in the future and the treatment of EPEC infection is going to become more difficult than ever.

Characterization of biofilm-forming capacity and resistance to sanitizers of a range of E. coli O26 pathotypes from clinical cases and cattle in Australia.

BACKGROUND: The formation of biofilms and subsequent encasement of bacterial cells in a complex matrix can enhance resistance to antimicrobials and sterilizing agents making these organisms difficult to eradicate and control. The aim of this study was to evaluate and compare the capacity of 40 E. coli O26 isolates of enterohemorrhagic E. coli (EHEC, n = 27), potential EHEC (pEHEC, n = 3), atypical enteropathogenic E. coli (aEPEC, n = 8) and non-toxigenic E. coli (NTEC, n = 2) from human and cattle sources to form biofilms on different surfaces, and determine whether extracellular matrix (ECM) components (cellulose, curli), motility, prophage insertion in mlrA and cell surface hydrophobicity could influence biofilm formation. Finally, the influence of biofilm formation on the sensitivity of isolates to quaternary ammonium compounds (QACs; Profoam, Kwiksan 22) and peracetic acid-based sanitizer (Topactive Des.) for 2 min on polystyrene plate were also evaluated. RESULTS: Biofilm production on one surface may not indicate biofilm formation on a different surface. Biofilm was formed by different pathotypes on polystyrene (70%), stainless steel (87.5%) and glass slides (95%), however only 50% demonstrated pellicle formation. EHEC isolates were significantly more likely to form a pellicle at the air-liquid interface and biofilms on polystyrene surface at 48 h than aEPEC. Strains that don't produce ECM (curli or cellulose), harbor a prophage insertion in mlrA, and are non-motile have lower biofilm forming capacities than those isolates possessing combinations of these attributes. Hydrophobicity had no impact on biofilm formation. After 2 min exposure, none of the disinfectants tested were able to completely inactivate all cells within a biofilm regardless of pathotypes and the amount of biofilm formed. CONCLUSION: Pathotypes of E. coli O26 showed varying capacities to form biofilms, however, most EHEC strains had the capacity to form biofilm on all surfaces and at the air-liquid interface under the conditions used in this study. Biofilms provided a protective effect to E. coli O26 strains against the three sanitizers, previously shown to successfully control the growth of their planktonic counterparts. Whether the characteristics of biofilm forming and non-biofilm forming strains observed in this study reflect their attributes within the food and meat-processing environments is unknown. Further studies that represent the food and meat-processing environments are required.

Oral Phage Therapy of Acute Bacterial Diarrhea With Two Coliphage Preparations: A Randomized Trial in Children From Bangladesh.

BACKGROUND: Antibiotic resistance is rising in important bacterial pathogens. Phage therapy (PT), the use of bacterial viruses infecting the pathogen in a species-specific way, is a potential alternative. METHOD: T4-like coliphages or a commercial Russian coliphage product or placebo was orally given over 4 days to Bangladeshi children hospitalized with acute bacterial diarrhea. Safety of oral phage was assessed clinically and by functional tests; coliphage and Escherichia coli titers and enteropathogens were determined in stool and quantitative diarrhea parameters (stool output, stool frequency) were measured. Stool microbiota was studied by 16S rRNA gene sequencing; the genomes of four fecal Streptococcus isolates were sequenced. FINDINGS: No adverse events attributable to oral phage application were observed (primary safety outcome). Fecal coliphage was increased in treated over control children, but the titers did not show substantial intestinal phage replication (secondary microbiology outcome). 60% of the children suffered from a microbiologically proven E. coli diarrhea; the most frequent diagnosis was ETEC infections. Bacterial co-pathogens were also detected. Half of the patients contained phage-susceptible E. coli colonies in the stool. E. coli represented less than 5% of fecal bacteria. Stool ETEC titers showed only a short-lived peak and were otherwise close to the replication threshold determined for T4 phage in vitro. An interim analysis after the enrollment of 120 patients showed no amelioration in quantitative diarrhea parameter by PT over standard care (tertiary clinical outcome). Stool microbiota was characterized by an overgrowth with Streptococcus belonging to the Streptococcus gallolyticus and Streptococcus salivarius species groups, their abundance correlated with quantitative diarrhea outcome, but genome sequencing did not identify virulence genes. INTERPRETATION: Oral coliphages showed a safe gut transit in children, but failed to achieve intestinal amplification and to improve diarrhea outcome, possibly due to insufficient phage coverage and too low E. coli pathogen titers requiring higher oral phage doses. More knowledge is needed on in vivo phage-bacterium interaction and the role of E. coli in childhood diarrhea for successful PT. FUNDING: The study was supported by a grant from Nestlé Nutrition and Nestlé Health Science. The trial was registered with Identifier NCT00937274 at ClinicalTrials.gov.

[Resistance of clinical strains of pathogenic E. coli to antibiotics and bacteriophage in hospitalized children with escherichiosis in St. Petersburg].

The data on the resistance frequency of the diarrheagenic E. coli isolates to antibacterial agents and a specific bacteriophage are presented. The strains were isolated from hospitalized children in St. Petersburg in 2011-2013 and belonged to three groups, i.e. enteropathogenic (EPE), enterotoxigenic (ETE) and enteroinvasine (EIE). It was shown that in the children aged from 1 month to 18 years the average antibiotic resistance was maximum in the EIE isolates and decreased in the following order: EIE (15.2%), EPE (6.0%), ETE (3.3%). The clinical EIE isolates showed no resistance to the new generation cephalosporins (cefuroxime, cephepime) and nitrofurans. The E. coli isolates of the three groups were characterized by high resistance to a specific bacteriophage which decreased in the following order: ETE (44.8%), EIE (37.0%), EPE (28.8%). The multiple resistance of the diarrheagenic E. coli isolates of the three groups to the antibacterials averaged 2.8%. The maximum frequency of resistance of the clinical isolates of the three groups to nalidixic acid was observed: EIE - 28.6%, EPE - 26.3%, ETE - 9.1%. The results of the study may be useful in the tactics of therapy of diarrheagenic E. coli infection in children.

Phage biocontrol of enteropathogenic and shiga toxin-producing Escherichia coli in meat products.

Ten bacteriophages were isolated from faeces and their lytic effects assayed on 103 pathogenic and non-pathogenic Enterobacteriaceae. Two phages (DT1 and DT6) were selected based on their host ranges, and their lytic effects on pathogenic E. coli strains inoculated on pieces of beef were determined. We evaluated the reductions of viable cells of Escherichia coli O157:H7 and non-O157 Shiga toxigenic E. coli strains on meat after exposure to DT6 at 5 and 24°C for 3, 6, and 24 h and the effect of both phages against an enteropathogenic E. coli strain. Significant viable cell reductions, compared to controls without phages, at both temperatures were observed, with the greatest decrease taking place within the first hours of the assays. Reductions were also influenced by phage concentration, being the highest concentrations, 1.7 × 10(10) plaque forming units per milliliter (PFU/mL) for DT1 and 1.4 × 10(10) PFU/mL for DT6, the most effective. When enteropathogenic E. coli and Shiga toxigenic E. coli (O157:H7) strains were tested, we obtained viable cell reductions of 0.67 log (p = 0.01) and 0.77 log (p = 0.01) after 3 h incubation and 0.80 log (p = 0.01) and 1.15 log (p = 0.001) after 6 h. In contrast, all nonpathogenic E. coli strains as well as other enterobacteria tested were resistant. In addition, phage cocktail was evaluated on two strains and further reductions were observed. However, E. coli bacteriophage insensitive mutants (BIMs) emerged in meat assays. BIMs isolated from meat along with those isolated by using the secondary culture method were tested to evaluate resistance phenotype stability and reversion. They presented low emergence frequencies (6.5 × 10(-7)-1.8 × 10(-6)) and variable stability and reversion. Results indicate that isolated phages were stable on storage, negative for all the virulence factors assayed, presented lytic activity for different E. coli virotypes and could be useful in reducing Shiga toxigenic E. coli and enteropathogenic E. coli viable cells in meat products.

Distribution, functional expression, and genetic organization of Cif, a phage-encoded type III-secreted effector from enteropathogenic and enterohemorrhagic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) inject effector proteins into host cells via a type III secretion system encoded by the locus of enterocyte effacement (LEE). One of these effectors is Cif, encoded outside the LEE by a lambdoid prophage. In this study, we demonstrated that the Cif-encoding prophage of EPEC strain E22 is inducible and produces infectious phage particles. We investigated the distribution and functional expression of Cif in 5,049 E. coli strains of human, animal, and environmental origins. A total of 115 E. coli isolates from diverse origins and geographic locations carried cif. The presence of cif was tightly associated with the LEE, since all the cif-positive isolates were positive for the LEE. These results suggested that the Cif-encoding prophages have been widely disseminated within the natural population of E. coli but positively selected within the population of LEE-positive strains. Nonetheless, 66% of cif-positive E. coli strains did not induce a typical Cif-related phenotype in eukaryotic cells due to frameshift mutations or insertion of an IS element in the cif gene. The passenger region of the prophages carrying cif was highly variable and showed various combinations of IS elements and genes coding for other effectors such as nleB, nleC, nleH, nleG, espJ, and nleA/espI (some of which were also truncated). This diversity and the presence of nonfunctional effectors should be taken into account to assess EPEC and EHEC pathogenicity and tropism.