Sequencing and antimicrobial resistance profiling of clinical isolates from Nairobi, Kenya, in a resource-limited setting

Meenakshi Iyer (National Centre for Biological Sciences, TIFR, India) began her breakout talk by describing how antimicrobial resistance (AMR) is projected to grow year-on-year worldwide and, without action, could ‘become a major global threat by 2050’. Neisseria gonorrhoeae is a bacterium which causes the sexually transmitted infection gonorrhoea. Meenakshi described how antibiotic-resistant gonorrhoea is a major threat across the world, with a particularly high number of cases reported in Africa. She displayed a timeline showing the accumulation of resistance by the bacterium to numerous antibiotics, from the late 1930s to the present day, and stressed that resistance levels were slowly approaching ‘untreatable’ cases of gonorrhoea.

Meenakshi then described how she and her colleagues employed nanopore sequencing to sequence and assemble N. gonorrhoeae bacterial genomes from clinical research samples, in resource-limited settings in Nairobi, Kenya. N. gonorrhoeae bacteria were cultured from vaginal swab samples which had been confirmed positive via a clinical test, at the University of Nairobi sexually transmitted infection clinic. DNA was then extracted from the bacteria and prepared for sequencing on the MinION. Samples were sequenced in multiplex via sample barcoding, with 14 samples sequenced on one MinION Flow Cell. Basecalling was performed offline via Guppy, demultiplexed, and the sequencing adapter sequences trimmed via Porechop. De novo genome assembly was then performed via a workflow using the tools Minimap, Canu, Miniasm, BWA-mem, Nanopolish, and Racon. Species identification revealed N. gonorrhoeae for 12 genomes and, for the remaining two, Neisseria meningitidis. Meenakshi explained that the latter is implicated in cerebrospinal fluid infections and brain fever and, in recent years, sexually-transmitted infections, and highlighted their surprise at this result. The team used the corresponding genomes to generate a reference-based genome assembly, and took these forward for further analysis along with the plasmids generated via de novo assembly.

Meenakshi and her colleagues then focused on the AMR genes present, and mutations within them. She described how existing databases capture only a subset of these mutations, and so they decided to carry out a thorough literature survey to identify all the genes and mutations implicated in AMR; this produced a dataset of ~100 mutations and genes. These comprised four types: proteins which mediate the entry of drugs into the cells, modified drug targets, over-expression of efflux pumps, and plasmid-mediated drug resistance. They then carried out phylogenetic analysis of their genomes alongside other strains of N. gonorrhoeae from Kenya, Africa, and other geographical regions, assessing the clustering of strains and the AMR types present. The sequenced strains clustered more closely to those from Kenya and Africa, but also showed inter-mixing of different strains across the world, indicating high genomic similarity.

Following this, Meenaskhi and her team investigated the structural basis of AMR via homology modelling of different complexes of drugs and their targets. Using the template Staphylococcus aureus GyrA-GyrB complex with Ciprofloxacin and DNA, she showed how a GyrA mutant abolished a contact and hydrogen bonding between the antibiotic and an amino acid versus the wildtype. This confirmed that most of the AMR mutations were in the drug-binding category. The study was also carried out with other drug targets.

Summarising, Meenakshi highlighted how ‘nanopore-only sequencing can be used for sequencing and analysis of clinical isolates in resource-limited settings’. She described how the developed pipeline can be used in settings without a next-generation sequencing facility, without need to export samples or DNA for sequencing in other countries.