In Vitro Effect on Piglet Gut Microbiota and In Vivo Assessment of Newly Isolated Bacteriophages against F18 Enterotoxigenic Escherichia coli (ETEC).

Enterotoxigenic Escherichia coli (ETEC) causing post-weaning diarrhea (PWD) in piglets have a detrimental impact on animal health and economy in pig production. ETEC strains can adhere to the host's small intestinal epithelial cells using fimbriae such as F4 and F18. Phage therapy could represent an interesting alternative to antimicrobial resistance against ETEC infections. In this study, four bacteriophages, named vB_EcoS_ULIM2, vB_EcoM_ULIM3, vB_EcoM_ULIM8 and vB_EcoM_ULIM9, were isolated against an O8:F18 E. coli strain (A-I-210) and selected based on their host range. These phages were characterized in vitro, showing a lytic activity over a pH (4-10) and temperature (25-45 °C) range. According to genomic analysis, these bacteriophages belong to the Caudoviricetes class. No gene related to lysogeny was identified. The in vivo Galleria mellonella larvae model suggested the therapeutic potential of one selected phage, vB_EcoS_ULIM2, with a statistically significant increase in survival compared to non-treated larvae. To assess the effect of this phage on the piglet gut microbiota, vB_EcoS_ULIM2 was inoculated in a static model simulating the piglet intestinal microbial ecosystem for 72 h. This study shows that this phage replicates efficiently both in vitro and in vivo in a Galleria mellonella model and reveals the safety of the phage-based treatment on the piglet microbiota.

Impact Assessment of vB_KpnP_K1-ULIP33 Bacteriophage on the Human Gut Microbiota Using a Dynamic In Vitro Model.

New control methods are needed to counter antimicrobial resistances and the use of bacteriophages as an alternative treatment seems promising. To that end, the effect of the phage vB_KpnP_K1-ULIP33, whose host is the hypervirulent Klebsiella pneumoniae SA12 (ST23 and capsular type K1), was assessed on intestinal microbiota, using an in vitro model: the SHIME® system (Simulator of the Human Intestinal Microbial Ecosystem). After stabilization of the system, the phage was inoculated for 7 days and its persistence in the different colons was studied until its disappearance from the system. The concentration of short chain fatty acids in the colons showed good colonization of the bioreactors by the microbiota and no significant effect related to the phage treatment. Diversity (α and ß), the relative abundance of bacteria, and qPCR analysis targeting different genera of interest showed no significant variation following phage administration. Even if further in vitro studies are needed to assess the efficacy of this phage against its bacterial host within the human intestinal ecosystem, the phage ULIP33 exerted no significant change on the global colonic microbiota.

In Vitro Characterization and In Vivo Efficacy Assessment in Galleria mellonella Larvae of Newly Isolated Bacteriophages against Escherichia coli K1.

Extra-intestinal Escherichia coli express several virulence factors that increase their ability to colonize and survive in different localizations. The K1 capsular type is involved in several infections, including meningitis, urinary tract, and bloodstream infections. The aims of this work were to isolate, characterize, and assess the in vivo efficacy of phages targeting avian pathogenic E. coli (APEC) O18:K1, which shares many similarities with the human strains responsible for neonatal meningitis. Eleven phages were isolated against APEC O18:K1, and four of them presenting a narrow spectrum targeting E. coli K1 strains were further studied. The newly isolated phages vB_EcoS_K1-ULINTec2 were similar to the Siphoviridae family, and vB_EcoP_K1-ULINTec4, vB_EcoP_K1-ULINTec6, and vB_EcoP_K1-ULINTec7 to the Autographiviridae family. They are capsular type (K1) dependent and present several advantages characteristic of lytic phages, such as a short adsorption time and latent period. vB_EcoP_K1-ULINTec7 is able to target both K1 and K5 strains. This study shows that these phages replicate efficiently, both in vitro and in vivo in the Galleria mellonella model. Phage treatment increases the larvae survival rates, even though none of the phages were able to eliminate the bacterial load.

New Bacteriophages against Emerging Lineages ST23 and ST258 of Klebsiella pneumoniae and Efficacy Assessment in Galleria mellonella Larvae.

Klebsiella pneumoniae is a bacterial pathogen of high public health importance. Its polysaccharide capsule is highly variable but only a few capsular types are associated with emerging pathogenic sublineages. The aim of this work is to isolate and characterize new lytic bacteriophages and assess their potential to control infections by the ST23 and ST258 K. pneumoniae sublineages using a Galleria mellonella larvae model. Three selected bacteriophages, targeting lineages ST258 (bacteriophages vB_KpnP_KL106-ULIP47 and vB_KpnP_KL106-ULIP54) and ST23 (bacteriophage vB_KpnP_K1-ULIP33), display specificity for capsular types KL106 and K1, respectively. These podoviruses belong to the Autographivirinae subfamily and their genomes are devoid of lysogeny or toxin-associated genes. In a G. mellonella larvae model, a mortality rate of 70% was observed upon infection by K. pneumoniae ST258 and ST23. This number was reduced to 20% upon treatment with bacteriophages at a multiplicity of infection of 10. This work increases the number of characterized bacteriophages infecting K. pneumoniae and provides information regarding genome sequence and efficacy during preclinical phage therapy against two prominent sublineages of this bacterial species.

Assessment of two selective agar media to isolate colistin-resistant bovine Escherichia coli: Correlation with minimal inhibitory concentration and presence of mcr genes.

The identification of colistin-resistant enterobacteria in veterinary medicine is impaired by the absence of first-line reliable phenotypic assay. The purpose of this study was to assess two selective agar media for the detection of colistin-resistant bovine pathogenic Escherichia coli. A total of 158 E. coli (46 R , 96 I and 16 S at the disk diffusion assay) isolated between 2013 and 2018 from <3 month-old calves suffering enteritis or septicaemia, were (i) tested by the broth dilution assay to determine colistin Minimal Inhibitory Concentrations (MIC); (ii) streaked on CHROMID® Colistin_R and CHROMagar™ COL-APSE agar plates; (iii) submitted to a pentaplex PCR to identify the presence of mcr-1 to mcr-5 genes. Of the 92 E. coli growing on both agar media, 90 had a MIC > 2.0 µg/ml as had the 3 E. coli that grew only on the CHROMID® Colistin_R agar medium and one E. coli that grew on neither agar media. Therefore, the positive predictive values of the CHROMID® Colistin_R and CHROMagar™ COL-APSE agar media were both 0.98 whereas their negative predictive values were 0.98 and 0.94, respectively. Also noteworthy 43 of the 46 R isolates had a MIC > 2.0 µg/ml and grew on both selective media as did half of the 96 I isolates and only 1 of the S isolates. Conversely, only 30 of the 90 isolates that grew on both agar media and with a MIC > 2.0 µg/ml tested positive for the mcr-1 or mcr-2 genes with the pentaplex PCR. These two selective agar media can be used to reliably detect colistin-resistant E. coli. Positive growth was highly correlated with R results at the disk diffusion assay, but not with the presence of mcr genes.