Protecting the piglet gut microbiota against ETEC-mediated post-weaning diarrhoea using specific binding proteins.

Post-weaning diarrhoea (PWD) in piglets presents a widespread problem in industrial pig production and is often caused by enterotoxigenic E. coli (ETEC) strains. Current solutions, such as antibiotics and medicinal zinc oxide, are unsustainable and are increasingly being prohibited, resulting in a dire need for novel solutions. Thus, in this study, we propose and evaluate a protein-based feed additive, comprising two bivalent heavy chain variable domain (VHH) constructs (VHH-(GGGGS)3-VHH, BL1.2 and BL2.2) as an alternative solution to manage PWD. We demonstrate in vitro that these constructs bind to ETEC toxins and fimbriae, whilst they do no affect bacterial growth rate. Furthermore, in a pig study, we show that oral administration of these constructs after ETEC challenge reduced ETEC proliferation when compared to challenged control piglets (1-2 log10 units difference in gene copies and bacterial count/g faeces across day 2-7) and resulted in week 1 enrichment of three bacterial families (Prevotellaceae (estimate: 1.12 ± 0.25, q = 0.0054), Lactobacillaceae (estimate: 2.86 ± 0.52, q = 0.0012), and Ruminococcaceae (estimate: 0.66 ± 0.18, q = 0.049)) within the gut microbiota that appeared later in challenged control piglets, thus pointing to an earlier transition towards a more mature gut microbiota. These data suggest that such VHH constructs may find utility in industrial pig production as a feed additive for tackling ETEC and reducing the risk of PWD in piglet populations.

Orally active bivalent VHH construct prevents proliferation of F4+ enterotoxigenic Escherichia coli in weaned piglets.

A major challenge in industrial pig production is the prevalence of post-weaning diarrhea (PWD) in piglets, often caused by enterotoxigenic Escherichia coli (ETEC). The increased use of antibiotics and zinc oxide to treat PWD has raised global concerns regarding antimicrobial resistance development and environmental pollution. Still, alternative treatments targeting ETEC and counteracting PWD are largely lacking. Here, we report the design of a pH, temperature, and protease-stable bivalent VHH-based protein BL1.2 that cross-links a F4+ ETEC model strain by selectively binding to its fimbriae. This protein inhibits F4+ ETEC adhesion to porcine epithelial cells ex vivo and decreases F4+ ETEC proliferation when administrated as a feed additive to weaned F4+ ETEC challenged piglets. These findings highlight the potential of a highly specific bivalent VHH-based feed additive in effectively delimiting pathogenic F4+ ETEC bacteria proliferation in piglets and may represent a sustainable solution for managing PWD while circumventing antimicrobial resistance development.

Application of Whole-Genome Sequencing Data for O-Specific Antigen Analysis and In Silico Serotyping of Pseudomonas aeruginosa Isolates.

Accurate typing methods are required for efficient infection control. The emergence of whole-genome sequencing (WGS) technologies has enabled the development of genome-based methods applicable for routine typing and surveillance of bacterial pathogens. In this study, we developed the Pseudomonas aeruginosa serotyper (PAst) program, which enabled in silico serotyping of P. aeruginosa isolates using WGS data. PAst has been made publically available as a web service and aptly facilitates high-throughput serotyping analysis. The program overcomes critical issues such as the loss of in vitro typeability often associated with P. aeruginosa isolates from chronic infections and quickly determines the serogroup of an isolate based on the sequence of the O-specific antigen (OSA) gene cluster. Here, PAst analysis of 1,649 genomes resulted in successful serogroup assignments in 99.27% of the cases. This frequency is rarely achievable by conventional serotyping methods. The limited number of nontypeable isolates found using PAst was the result of either a complete absence of OSA genes in the genomes or the artifact of genomic misassembly. With PAst, P. aeruginosa serotype data can be obtained from WGS information alone. PAst is a highly efficient alternative to conventional serotyping methods in relation to outbreak surveillance of serotype O12 and other high-risk clones, while maintaining backward compatibility to historical serotype data.

A Bacteriophage-Acquired O-Antigen Polymerase (Wzyß) from P. aeruginosa Serotype O16 Performs a Varied Mechanism Compared to Its Cognate Wzyα.

Pseudomonas aeruginosa is a Gram-negative bacterium that produces highly varied lipopolysaccharide (LPS) structures. The O antigen (O-Ag) in the LPS is synthesized through the Wzx/Wzy-dependent pathway where lipid-linked O-Ag repeats are polymerized by Wzy. Horizontal-gene transfer has been associated with O-Ag diversity. The O-Ag present on the surface of serotypes O5 and O16, differ in the intra-molecular bonds, α and ß, respectively; the latter arose from the action of three genes in a serotype converting unit acquired from bacteriophage D3, including a ß-polymerase (Wzyß). To further our understanding of O-polymerases, the inner membrane (IM) topology of Wzyß was determined using a dual phoA-lacZα reporter system wherein random 3' gene truncations were localized to specific loci with respect to the IM by normalized reporter activities as determined through the ratio of alkaline phosphatase activity to ß-galactosidase activity. The topology of Wzyß developed through this approach was shown to contain two predominant periplasmic loops, PL3 (containing an RX10G motif) and PL4 (having an O-Ag ligase superfamily motif), associated with inverting glycosyltransferase reaction. Through site-directed mutagenesis and complementation assays, residues Arg(254), Arg(270), Arg(272), and His(300) were found to be essential for Wzyß function. Additionally, like-charge substitutions, R254K and R270K, could not complement the wzy ß knockout, highlighting the essential guanidium side group of Arg residues. The O-Ag ligase domain is conserved among heterologous Wzy proteins that produce ß-linked O-Ag repeat units. Taking advantage of the recently obtained whole-genome sequence of serotype O16 a candidate promoter was identified. Wzy ß under its native promoter was integrated in the PAO1 genome, which resulted in simultaneous production of α- and ß-linked O-Ag. These observations established that members of Wzy-like family consistently exhibit a dual-periplasmic loops topology, and identifies motifs that are plausible to be involved in enzymatic activities. Based on these results, the phage-derived Wzyß utilizes a different reaction mechanism in the P. aeruginosa host to avoid self-inhibition during serotype conversion.

The Widespread Multidrug-Resistant Serotype O12 Pseudomonas aeruginosa Clone Emerged through Concomitant Horizontal Transfer of Serotype Antigen and Antibiotic Resistance Gene Clusters.

UNLABELLED: The O-specific antigen (OSA) in Pseudomonas aeruginosa lipopolysaccharide is highly varied by sugar identity, side chains, and bond between O-repeats. These differences classified P. aeruginosa into 20 distinct serotypes. In the past few decades, O12 has emerged as the predominant serotype in clinical settings and outbreaks. These serotype O12 isolates exhibit high levels of resistance to various classes of antibiotics. Here, we explore how the P. aeruginosa OSA biosynthesis gene clusters evolve in the population by investigating the association between the phylogenetic relationships among 83 P. aeruginosa strains and their serotypes. While most serotypes were closely linked to the core genome phylogeny, we observed horizontal exchange of OSA biosynthesis genes among phylogenetically distinct P. aeruginosa strains. Specifically, we identified a "serotype island" ranging from 62 kb to 185 kb containing the P. aeruginosa O12 OSA gene cluster, an antibiotic resistance determinant (gyrA(C248T)), and other genes that have been transferred between P. aeruginosa strains with distinct core genome architectures. We showed that these genes were likely acquired from an O12 serotype strain that is closely related to P. aeruginosa PA7. Acquisition and recombination of the "serotype island" resulted in displacement of the native OSA gene cluster and expression of the O12 serotype in the recipients. Serotype switching by recombination has apparently occurred multiple times involving bacteria of various genomic backgrounds. In conclusion, serotype switching in combination with acquisition of an antibiotic resistance determinant most likely contributed to the dissemination of the O12 serotype in clinical settings. IMPORTANCE: Infection rates in hospital settings by multidrug-resistant (MDR) Pseudomonas aeruginosa clones have increased during the past decades, and serotype O12 is predominant among these epidemic strains. It is not known why the MDR phenotype is associated with serotype O12 and how this clone type has emerged. This study shows that evolution of MDR O12 strains involved a switch from an ancestral O4 serotype to O12. Serotype switching was the result of horizontal transfer and genetic recombination of lipopolysaccharide (LPS) biosynthesis genes originating from an MDR taxonomic outlier P. aeruginosa strain. Moreover, the recombination event also resulted in acquisition of antibiotic resistance genes. These results impact on our understanding of MDR outbreak strain and serotype evolution and can potentially assist in better monitoring and prevention.