- Volume 9, Issue 4, 2023

The introduction of pneumococcal conjugate vaccines (PCV7, PCV10, PCV13) around the world has proved successful in preventing invasive pneumococcal disease. However, immunization against Streptococcus pneumoniae has led to serotype replacement by non-vaccine serotypes, including serotype 15A. Clonal complex 63 (CC63) is associated with many serotypes and has been reported in association with 15A after introduction of PCVs. A total of 865 CC63 isolates were included in this study, from the USA (n=391) and a global collection (n=474) from 1998–2019 and 1995–2018, respectively. We analysed the genomic sequences to identify serotypes and penicillin-binding protein (PBP) genes 1A, 2B and 2X, and other resistance determinants, to predict minimum inhibitory concentrations (MICs) against penicillin, erythromycin, clindamycin, co-trimoxazole and tetracycline. We conducted phylogenetic and spatiotemporal analyses to understand the evolutionary history of the 15A-CC63 sub-lineage. Overall, most (89.5 %, n=247) pre-PCV isolates in the CC63 cluster belonged to serotype 14, with 15A representing 6.5 % of isolates. Conversely, serotype 14 isolates represented 28.2 % of post-PCV CC63 isolates (n=618), whilst serotype 15A isolates represented 65.4 %. Dating of the CC63 lineage determined the most recent common ancestor emerged in the 1980s, suggesting the 15A-CC63 sub-lineage emerged from its closest serotype 14 ancestor prior to the development of pneumococcal vaccines. This sub-lineage was predominant in the USA, Israel and China. Multidrug resistance (to three or more drug classes) was widespread among isolates in this sub-lineage. We show that the CC63 lineage is globally distributed and most of the isolates are penicillin non-susceptible, and thus should be monitored.

Enterohaemolysin (Ehx) and alpha-haemolysin are virulence-associated factors (VAFs) causing the haemolytic phenotype in Escherichia coli . It has been shown that chromosomally and plasmid-encoded alpha-haemolysin are characteristic of specific pathotypes, virulence-associated factors and hosts. However, the prevalence of alpha- and enterohaemolysin does not overlap in the majority of pathotypes. Therefore, this study focuses on the characterization of the haemolytic E. coli population associated with multiple pathotypes in human and animal infectious diseases. Using a genomics approach, we investigated characteristic features of the enterohaemolysin-encoding strains to identify factors differentiating enterohaemolysin-positive from alpha-haemolysin-positive E. coli populations. To shed light on the functionality of Ehx subtypes, we analysed Ehx-coding genes and inferred EhxA phylogeny. The two haemolysins are associated with a different repertoire of adhesins, iron acquisition or toxin systems. Alpha-haemolysin is predominantly found in uropathogenic E. coli (UPEC) and predicted to be chromosomally encoded, or nonpathogenic and undetermined E. coli pathotypes and typically predicted to be plasmid-encoded. Enterohaemolysin is mainly associated with Shiga toxin-producing E. coli (STEC) and enterohaemorrhagic E. coli (EHEC) and predicted to be plasmid-encoded. Both types of haemolysin are found in atypical enteropathogenic E. coli (aEPEC). Moreover, we identified a new EhxA subtype present exclusively in genomes with VAFs characteristic of nonpathogenic E. coli . This study reveals a complex relationship between haemolytic E. coli of diverse pathotypes, providing a framework for understanding the potential role of haemolysin in pathogenesis.

Malarial parasites exhibit extensive genomic plasticity, which induces the antigen diversification and the development of antimalarial drug resistance. Only a few studies have examined the genome maintenance mechanisms of parasites. The study aimed at elucidating the impact of a mutation in a DNA mismatch repair gene on genome stability by maintaining the mutant and wild-type parasites through serial in vitro cultures for approximately 400 days and analysing the subsequent spontaneous mutations. A P513T mutant of the DNA mismatch repair protein PfMSH2-1 from Plasmodium falciparum 3D7 was created. The mutation did not influence the base substitution rate but significantly increased the insertion/deletion (indel) mutation rate in short tandem repeats (STRs) and minisatellite loci. STR mutability was affected by allele size, genomic category and certain repeat motifs. In the mutants, significant telomere healing and homologous recombination at chromosomal ends caused extensive gene loss and generation of chimeric genes, resulting in large-scale chromosomal alteration. Additionally, the mutant showed increased tolerance to N-methyl-Nʹ-nitro-N-nitrosoguanidine, suggesting that PfMSH2-1 was involved in recognizing DNA methylation damage. This work provides valuable insights into the role of PfMSH2-1 in genome stability and demonstrates that the genomic destabilization caused by its dysfunction may lead to antigen diversification.

Brucellosis is a worldwide zoonosis with important public health, animal health and economic implications. Brucella melitensis , commonly associated with small ruminants, is an emerging bovine pathogen in dairy farms. We analysed all B. melitensis outbreaks affecting dairy farms in Israel since 2006, combining traditional and genomic epidemiology to explore the public health implications of this One Health challenge. Whole-genome sequencing was applied to bovine and related human B. melitensis isolates from dairy farm outbreaks. cgMLST-based and SNP-based typing was integrated with epidemiological and investigation data. A secondary analysis combining the bovine-human isolates with endemic human isolates from southern Israel was performed. A total of 92 isolates from dairy cows and related human cases originating from 18 epidemiological clusters were analysed. Most genomic and epi-clusters were congruent, but sequencing showed relatedness between apparently unrelated farm outbreaks. Nine secondary human infections were also genomically confirmed. The bovine-human cohort appeared intermixed with 126 endemic human isolates in southern Israel. We show a persistent and widespread circulation of B. melitensis in dairy farms in Israel with secondary occupational human infection. The genomic epidemiology also uncovered cryptic connections between outbreaks. A regional connection between bovine and endemic human brucellosis cases points to a common reservoir, most probably local small ruminant herds. Control of humans and bovine brucellosis is inseparable. Epidemiological and microbiological surveillance and implementation of control measures across the entire range of farm animals is needed to mitigate this public health challenge.

Bacterial secondary metabolites, synthesized by enzymes encoded in biosynthetic gene clusters (BGCs), can underlie microbiome homeostasis and serve as commercialized products, which have historically been mined from a select group of taxa. While evolutionary approaches have proven beneficial for prioritizing BGCs for experimental characterization efforts to uncover new natural products, dedicated bioinformatics tools designed for comparative and evolutionary analysis of BGCs within focal taxa are limited. We thus developed lineage specific analysis of BGCs (lsaBGC; https://github.com/Kalan-Lab/lsaBGC) to aid exploration of microdiversity and evolutionary trends across homologous groupings of BGCs, gene cluster families (GCFs), in any bacterial taxa of interest. lsaBGC enables rapid and direct identification of GCFs in genomes, calculates evolutionary statistics and conservation for BGC genes, and builds a framework to allow for base resolution mining of novel variants through metagenomic exploration. Through application of the suite to four genera commonly found in skin microbiomes, we uncover new insights into the evolution and diversity of their BGCs. We show that the BGC of the virulence-associated carotenoid staphyloxanthin in   Staphylococcus aureus   is ubiquitous across the genus   Staphylococcus  . While one GCF encoding the biosynthesis of staphyloxanthin showcases evidence for plasmid-mediated horizontal gene transfer (HGT) between species, another GCF appears to be transmitted vertically amongst a sub-clade of skin-associated   Staphylococcus  . Further, the latter GCF, which is well conserved in   S. aureus  , has been lost in most   Staphylococcus epidermidis  , which is the most common   Staphylococcus   species on human skin and is also regarded as a commensal. We also identify thousands of novel single-nucleotide variants (SNVs) within BGCs from the   Corynebacterium tuberculostearicum   sp. complex, a narrow, multi-species clade that features the most prevalent   Corynebacterium   in healthy skin microbiomes. Although novel SNVs were approximately 10 times as likely to correspond to synonymous changes when located in the top five percentile of conserved sites, lsaBGC identified SNVs that defied this trend and are predicted to underlie amino acid changes within functionally key enzymatic domains. Ultimately, beyond supporting evolutionary investigations of BGCs, lsaBGC also provides important functionalities to aid efforts for the discovery or directed modification of natural products.