Bacterial Genomics for National Antimicrobial Resistance Surveillance in Cambodia.

BACKGROUND: Antimicrobial resistance (AMR) surveillance in low- and middle-income countries (LMICs) often relies on poorly resourced laboratory processes. Centralized sequencing was combined with cloud-based, open-source bioinformatics solutions for national AMR surveillance in Cambodia. METHODS: Blood cultures growing gram-negative bacteria were collected at six Cambodian hospitals (January 2021 - October 2022). Isolates were obtained from pure plate growth and shotgun DNA sequencing performed in-country. Using public nucleotide and protein databases, reads were aligned for pathogen identification and AMR gene characterization. Multilocus sequence typing was performed on whole genome assemblies and haplotype clusters compared against published genomes. FINDINGS: Genes associated with acquired resistance to fluoroquinolones were identified in 59%, TMP/SMX in 45%, and aminoglycosides in 52% of 715 isolates. Extended-spectrum beta-lactamase encoding genes were identified in 34% isolates, most commonly blaCTX-M-15, blaCTX-M-27, and blaCTX-M-55 in E. coli sequence types 131 and 1193. Carbapenemase genes were identified in 12% isolates, most commonly blaOXA-23, blaNDM-1, blaOXA-58 and blaOXA-66 in Acinetobacter species. Phylogenetic analysis revealed clonal strains of A. baumannii, representing suspected nosocomial outbreaks, and genetic clusters of quinolone-resistant typhoidal Salmonella and ESBL E. coli cases suggesting community transmission. INTERPRETATION: With accessible sequencing platforms and bioinformatics solutions, bacterial genomics can supplement AMR surveillance in LMICs. FUNDING: Research was supported by the National Institute of Allergy and Infectious Diseases and the Bill and Melinda Gates Foundation [OPP1211806].

Regional centromere configuration in the fungal pathogens of the Pneumocystis genus.

Centromeres are constricted chromosomal regions that are essential for cell division. In eukaryotes, centromeres display a remarkable architectural and genetic diversity. The basis of centromere-accelerated evolution remains elusive. Here, we focused on Pneumocystis species, a group of mammalian-specific fungal pathogens that form a sister taxon with that of the Schizosaccharomyces pombe, an important genetic model for centromere biology research. Methods allowing reliable continuous culture of Pneumocystis species do not currently exist, precluding genetic manipulation. CENP-A, a variant of histone H3, is the epigenetic marker that defines centromeres in most eukaryotes. Using heterologous complementation, we show that the Pneumocystis CENP-A ortholog is functionally equivalent to CENP-ACnp1 of S. pombe. Using organisms from a short-term in vitro culture or infected animal models and chromatin immunoprecipitation (ChIP)-Seq, we identified CENP-A bound regions in two Pneumocystis species that diverged ~35 million years ago. Each species has a unique short regional centromere (<10 kb) flanked by heterochromatin in 16-17 monocentric chromosomes. They span active genes and lack conserved DNA sequence motifs and repeats. These features suggest an epigenetic specification of centromere function. Analysis of centromeric DNA across multiple Pneumocystis species suggests a vertical transmission at least 100 million years ago. The common ancestry of Pneumocystis and S. pombe centromeres is untraceable at the DNA level, but the overall architectural similarity could be the result of functional constraint for successful chromosomal segregation.IMPORTANCEPneumocystis species offer a suitable genetic system to study centromere evolution in pathogens because of their phylogenetic proximity with the non-pathogenic yeast S. pombe, a popular model for cell biology. We used this system to explore how centromeres have evolved after the divergence of the two clades ~ 460 million years ago. To address this question, we established a protocol combining short-term culture and ChIP-Seq to characterize centromeres in multiple Pneumocystis species. We show that Pneumocystis have short epigenetic centromeres that function differently from those in S. pombe.

Whole-genome sequence of Mucor velutinosus NIH1002, a strain associated with disseminated disease.

The draft genome of Mucor velutinosus NIH1002, a 2011 isolate from a case of disseminated disease, was sequenced using PacBio long-read and HiSeq short-read technologies. The genome has 43 contigs, an N50 of 2.65 Mb, and 13,295 protein-coding genes. It is the most complete M. velutinosus genome to date.