Biofilms constitute the predominant microbial style of life in natural and engineered ecosystems. Facing harsh environmental conditions, microorganisms accumulate reactive oxygen species (ROS), potentially encountering a dangerous condition called oxidative stress. While high levels of oxidative stress are toxic, low levels act as a cue, triggering bacteria to activate effective scavenging mechanisms or to shift metabolic pathways. Although a complex and fragmentary picture results from current knowledge of the pathways activated in response to oxidative stress, three main responses are shown to be central: the existence of common regulators, the production of extracellular polymeric substances, and biofilm heterogeneity. An investigation into the mechanisms activated by biofilms in response to different oxidative stress levels could have important consequences from ecological and economic points of view, and could be exploited to propose alternative strategies to control microbial virulence and deterioration.

Application of phages as alternative antimicrobials to combat pathogenic bacteria and their association to a healthy gut microbiome has prompted a need for precise methods for detection and enumeration of phage particles. There are many applicable methods, but care should be taken considering the measured object (infectious phage, whole phage particle or nucleic acid and proteins) and the concept behind the technique to avoid misinterpretations. While molecular methods cannot discriminate between viable and non-infectious phages, the traditional techniques for counting infectious phages can be time consuming and poorly reproducible. Here, we describe the methods currently used for phage detection and enumeration and highlight their advantages as well as their limitations. Finally, we provide insight on how to deal with complex samples, as well as future prospects in the field of phage quantification.

This study aimed to characterize in silico Enterotoxigenic E. coli F4- and F18-positive-isolates (n=90) causing swine post-weaning diarrhea, including pathogenic potential, phylogenetic relationship, antimicrobial and biocide resistance (R), prophage content and metal tolerance rates. F4-strains belonged mostly to the O149 and O6 serogroups and ST100 and ST48 MLST-types. F18-strains were mainly assigned to the O8 and O147 serogroups and ST10, ST23 and ST42. The highest rates of antimicrobial-R were found against streptomycin, sulfamethoxazole, tetracycline, trimethoprim and ampicillin. No resistance was found towards ciprofloxacin, cefotaxime, ceftiofur and colistin. Genes conferring tolerance to copper (showing highest diversity), cadmium, silver and zinc were predicted in all genomes. Enterotoxins (ltcA; 100 % F4, 62 % F18, astA; 100 % F4, 38.1 % F18, sta;18.8 % F4, 38.1 % F18, stb; 100 % F4, 76.2 % F18) and fimbriae encoding genes typed as F4ac and F18ac were detected in all the strains, in addition to up to 16 other virulence genes in individual strains. Phage analysis predicted between 7 and 20 different prophage regions in each strain. High diversity of plasmid replicons was shown; IncFII, IncFIB and IncFIC were prevalent among F4-isolates while IncI1, and IncX1 were dominant among F18-strains. Interestingly, F4-isolates from the early 90′s belonged to the same clonal group detected for most of the F4-strains from 2018-2019 (ONT:H10-A-ST100-CH27-0). The low number of SNPs differences between the oldest and recent F4-ST100-isolates suggests a relative stable genome. Overall, the isolates analyzed in this study showed remarkable different genetic traits depending on the fimbria type. IMPORTANCE Diarrhea in the post-weaning period due to Enterotoxigenic E. coli (ETEC) is an economically relevant disease in pig production worldwide. In Denmark, prevention is mainly achieved by Zinc-oxide administration (discontinued by 2022). Besides, a breeding program has been implemented aiming to reduce this illness. Treatment with antimicrobials contributes to the problem of antimicrobial resistance (AMR) development. As a novelty, this study aims to deeply understand the genetic population structure and variation among isolates diarrhea-associated by whole genome sequencing characterization. ST100-F4ac is the dominant clonal group circulating in Danish herds and showed high similarity to ETEC-ST100-isolates from China, USA and Spain. High rates of AMR and high diversity of virulence genes were detected. Characterization of ETEC diarrhea-related is important for understanding the disease epidemiology and pathogenesis, and for implementation of new strategies aiming to reduce the impact of the disease in pig production.