Dr Jai Mehat

In recent years, several plasmids harbouring genes encoding phosphoethanolamine transferases conferring colistin resistance have been described in multiple Enterobacteriaceae species. Avian Pathogenic E. coli (APEC) causes colibacillosis and is responsible for a considerable proportion of the disease burden in commercial poultry flocks, and may be linked to zoonotic infections in humans. Here, we describe the genotypic and phenotypic characteristics of a multidrug-resistant APEC ST69 isolate (APECA2), recovered in 2016 from a diseased broiler at post-mortem examination in Germany. The isolate was resistant to several antibiotics of human and veterinary importance, including colistin. The mcr-1 gene was detected on a mobile genetic element located on an IncHI2/ST4 plasmid, which was characterized using long-read Nanopore and short-read Illumina sequencing of purified plasmid. Isolate APECA2 displayed resistance to chicken serum and harbours numerous virulence genes. This study highlights the public health importance of enhanced antimicrobial resistance surveillance and strict antimicrobial stewardship in human and veterinary healthcare.

Background: Campylobacter jejuni and Campylobacter coli are major global causes of bacterial gastroenteritis. Whilst several individual colonisation and virulence factors have been identified, our understanding of their role in the transmission, pathogenesis and ecology of Campylobacter has been hampered by the genotypic and phenotypic diversity within C. jejuni and C. coli. Autotransporter proteins are a family of outer membrane or secreted proteins in Gram-negative bacteria such as Campylobacter, which are associated with virulence functions. In this study we have examined the distribution and predicted functionality of the previously described capC and the newly identified, related capD autotransporter gene families in Campylobacter. Results: Two capC-like autotransporter families, designated capC and capD, were identified by homology searches of genomes of the genus Campylobacter. Each family contained four distinct orthologs of CapC and CapD. The distribution of these autotransporter genes was determined in 5829 C. jejuni and 1347 C. coli genomes. Autotransporter genes were found as intact, complete copies and inactive formats due to premature stop codons and frameshift mutations. Presence of inactive and intact autotransporter genes was associated with C. jejuni and C. coli multi-locus sequence types, but for capC, inactivation was independent from the length of homopolymeric tracts in the region upstream of the capC gene. Inactivation of capC or capD genes appears to represent lineagespecific gene decay of autotransporter genes. Intact capC genes were predominantly associated with the C. jejuni ST-45 and C. coli ST-828 generalist lineages. The capD3 gene was only found in the environmental C. coli Clade 3 lineage. These combined data support a scenario of inter-lineage and interspecies exchange of capC and subsets of capD autotransporters. Conclusions: In this study we have identified two novel, related autotransporter gene families in the genus Campylobacter, which are not uniformly present and exhibit lineage-specific associations and gene decay. The distribution and decay of the capC and capD genes exemplifies the erosion of species barriers between certain lineages of C. jejuni and C. coli, probably arising through co-habitation. This may have implications for the phenotypic variability of these two pathogens and provide opportunity for new, hybrid genotypes to emerge.

Campylobacter jejuni is recognized as an important causative agent of bacterial gastroenteritis in the developed world. Despite the identification of several factors contributing to infection, characterization of the virulence strategies employed by C. jejuni remains a significant challenge. Bacterial autotransporter proteins are a major class of secretory proteins in Gram-negative bacteria and notably many autotransporter proteins contribute to bacterial virulence. The aim of this study was to characterise the C. jejuni 81116 C8J_1278 gene (capC), predicted to encode an autotransporter protein, and examine the contribution of this factor to virulence of Campylobacter jejuni. The predicted CapC protein has a number of features that are consistent with autotransporters including the N-terminal signal sequence and the C-terminal β-barrel domain and was determined to localise to the outer membrane. Inactivation of the capC gene in C. jejuni 81116 and C. jejuni M1 resulted in reduced insecticidal activity in Galleria mellonella larvae. Furthermore, C. jejuni capC mutants displayed significantly reduced adherence to and invasion of non-polarized, partially differentiated Caco-2 and T84 intestinal epithelial cells. Gentamicin treatment showed that the reduced invasion of the capC mutant is primarily caused by reduced adherence to intestinal epithelial cells, not by reduced invasion capability. C. jejuni capC mutants caused reduced IL-8 secretion from intestinal epithelial cells and elicited a significantly diminished immune reaction in Galleria larvae indicating that CapC functions as an immunogen. In conclusion, CapC is a new virulence determinant of C. jejuni that contributes to the integral infection process of adhesion to human intestinal epithelial cells.

Avian Pathogenic E. coli (APEC) is the causative agent of avian colibacillosis, resulting in economic losses to the poultry industry through morbidity, mortality and carcass condemnation, and impacts the welfare of poultry. Colibacillosis remains a complex disease to manage, hampered by diagnostic and classification strategies for E. coli that are inadequate for defining APEC. However, increased accessibility of whole genome sequencing (WGS) technology has enabled phylogenetic approaches to be applied to the classification of E. coli and genomic characterisation of the most common APEC serotypes associated with colibacillosis O1, O2 and O78. These approaches have demonstrated that that the O78 serotype is representative of two distinct APEC lineages, ST-23 in phylogroup C and ST-117 in phylogroup G. The O1, and O2 serotypes belong to a third lineage comprised of 3 sub-populations in phylogroup B2; ST-95, ST-140 and ST-428/ST-429. The frequency with which these genotypes are associated with colibacillosis implicates them as the predominant APEC populations and distinct from those causing incidental or opportunistic infections. The fact that these are disparate clusters from multiple phylogroups suggests that these lineages may have become adapted to the poultry niche independently. WGS studies have highlighted the limitations of traditional APEC classification and can now provide a path towards a robust and more meaningful definition of the APEC pathotype. Future studies should focus on characterising individual APEC populations in detail and use this information to develop improved diagnostics and interventions.