Project description:Background. Pneumococcus is a major human pathogen and the polysaccharide capsule is considered its main virulence factor. Nevertheless, strains lacking a capsule, named non-typeable pneumococcus (NT), are maintained in nature and frequently colonise the human nasopharynx. Interest in these strains, not targeted by any of the currently available pneumococcal vaccines, has been rising as they seem to play an important role in the evolution of the species. Currently, there is a paucity of data regarding this group of pneumococci. Also, questions have been raised on whether they are true pneumococci. We aimed to obtain insights in the genetic content of NT and the mechanisms leading to non-typeability and to genetic diversity. Methods. A collection of 52 NT isolates representative of the lineages circulating in Portugal between 1997 and 2007, as determined by pulsed-field gel electrophoresis and multilocus sequence typing, was analysed. The capsular region was sequenced and comparative genomic hybridisation (CGH) using a microarray covering the genome of 10 pneumococcal strains was carried out. The presence of mobile elements was investigated as source of intraclonal variation. Results. NT circulating in Portugal were found to have similar capsular regions, of cps type NCC2, i.e., having aliB-like ORF1 and aliB-like ORF2 genes. The core genome of NT was essentially similar to that of encapsulated strains. Also, competence genes and most virulence genes were present. The few virulence genes absent in all NT were the capsular genes, type-I and type-II pili, choline-binding protein A (cbpA/pspC), and pneumococcal surface protein A (pspA). Intraclonal variation could not be entirely explained by the presence of prophages and other mobile elements. Conclusions. NT circulating in Portugal are a homogeneous group belonging to cps type NCC2. Our observations support the theory that they are bona-fide pneumococcal isolates that do not express the capsule but are otherwise essentially similar to encapsulated pneumococci. Thus we propose that NT should be routinely identified and reported in surveillance studies.

Project description:Background: The rapid evolution and dissemination of mobilized colistin resistance gene (mcr) family has revealed as a severe threat to the global public health. Nevertheless, dramatic reduction in the prevalence of mcr-1, the major member of mcr family, was observed after the withdrawal of colistin in animal fodder in China since 2017, demonstrating that colistin acts as a selective stress to promote the dissemination of mcr-1. As the second largest lineage, mcr-3 was firstly discovered in 2017 and has been identified from numerous sources. However, whether the spreading of mcr-3 is driven by colistin remains unknown. Methods: To this end, we investigated the global prevalence of mcr-3 from 2005 to 2022 by an up-to-date systematic review, along with a nation-wide epidemiological study to establish the change of mcr-3 prevalence in China before and after 2017. To investigate the fitness cost imposed by MCR-3 upon bacterial host, in vitro and in vivo competitive assays were employed, along with morphological study and fluorescent observation. Moreover, by replacing non-optimal codons with optimal codons, synonymous mutations were introduced into the 5’-coding region of mcr-3 to study mechanisms accounting for the distinct fitness cost conferred by MCR-1 and MCR-3. Furthermore, by combining AlphaFold and molecular dynamics (MD) simulation, we provided a complete characterization on the putative lipid A binding pocket localized at the linker domain of MCR-3. Crucially, inhibitors targeting at the putative binding pocket of MCR-1 or MCR-3 were identified from small molecules library using the pipeline of virtual screening. Findings: The global prevalence of mcr-3 increased continuously from 2005 to 2022. The average prevalence was 0.18% during 2005-2014 and rapidly increased to 3.41% during 2020-2022. The prevalence of mcr-3 in China increased from 0.79% in 2016 to 5.87% in 2019. We found that the fitness of mcr-3-bearing E. coli and empty plasmid control was comparable but higher than that of mcr-1-positive strain. Although the putative lipid A binding pocket of MCR-3 was similar to that of in MCR-1, mcr-3 occupies remarkable codon bias at the 5’-end of coding region that disrupted the stability of mRNA, further reduced its protein expression in E. coli, resulting in the low fitness burden of bacterial host. Moreover, the 5’-end codon usage frequency appeared as a critical factor related with the evolution of mcr family. Furthermore, based on the similar lipid A binding pocket among MCR family protein, we identified three novel MCR inhibitors targeting at such pocket by screening from small-molecule library, which effectively restored the colistin susceptibility of mcr-bearing E. coli. Interpretation: For the first time, we found that the prevalence of mcr-3 increased continuously during 2016-2019 in China, demonstrating that the withdrawal of colistin in husbandry failed to prevent the dissemination of mcr-3. Our study evidenced that the 5’-end codon bias appeared as a crucial regulator upon the fitness cost conferred by horizontally transferred genes. Most importantly, the putative lipid A binding pocket verified from current study was a promising target site for designing inhibitors against mcr-positive strains.

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