Antimicrobial Resistance Profiling and Phylogenetic Analysis of Neisseria gonorrhoeae Clinical Isolates From Kenya in a Resource-Limited Setting

Abstract

BackgroundAfrica has one of the highest incidences of gonorrhea. Neisseria gonorrhoeae is gaining resistance to most of the available antibiotics, compromising treatment across the world. Whole-genome sequencing (WGS) is an efficient way of predicting AMR determinants and their spread in the population. Recent advances in next-generation sequencing technologies like Oxford Nanopore Technology (ONT) have helped in the generation of longer reads of DNA in a shorter duration with lower cost. Increasing accuracy of base-calling algorithms, high throughput, error-correction strategies, and ease of using the mobile sequencer MinION in remote areas lead to its adoption for routine microbial genome sequencing. To investigate whether MinION-only sequencing is sufficient for WGS and downstream analysis in resource-limited settings, we sequenced the genomes of 14 suspected N. gonorrhoeae isolates from Nairobi, Kenya.MethodsUsing WGS, the isolates were confirmed to be cases of N. gonorrhoeae (n = 9), and there were three co-occurrences of N. gonorrhoeae with Moraxella osloensis and N. meningitidis (n = 2). N. meningitidis has been implicated in sexually transmitted infections in recent years. The near-complete N. gonorrhoeae genomes (n = 10) were analyzed further for mutations/factors causing AMR using an in-house database of mutations curated from the literature.ResultsWe observe that ciprofloxacin resistance is associated with multiple mutations in both gyrA and parC. Mutations conferring tetracycline (rpsJ) and sulfonamide (folP) resistance and plasmids encoding beta-lactamase were seen in all the strains, and tet(M)-containing plasmids were identified in nine strains. Phylogenetic analysis clustered the 10 isolates into clades containing previously sequenced genomes from Kenya and countries across the world. Based on homology modeling of AMR targets, we see that the mutations in GyrA and ParC disrupt the hydrogen bonding with quinolone drugs and mutations in FolP may affect interaction with the antibiotic.ConclusionHere, we demonstrate the utility of mobile DNA sequencing technology in producing a consensus genome for sequence typing and detection of genetic determinants of AMR. The workflow followed in the study, including AMR mutation dataset creation and the genome identification, assembly, and analysis, can be used for any clinical isolate. Further studies are required to determine the utility of real-time sequencing in outbreak investigations, diagnosis, and management of infections, especially in resource-limited settings.

Comparing Long-Read Assemblers to Explore the Potential of a Sustainable Low-Cost, Low-Infrastructure Approach to Sequence Antimicrobial Resistant Bacteria With Oxford Nanopore Sequencing

“…ONT has been invaluable for direct field sequencing during the Ebola ( Quick et al, 2016 ) and Zika outbreaks ( de Jesus et al, 2019 ). While there are differences between bacterial and viral WGS, notably the size and complexity of the bacterial accessory genome, the increase in publications featuring bacterial AMR WGS solely from LR ONT sequencing highlight increased interest ( Juma et al, 2021 ; Tegha et al, 2021 ). There are now several options for LR assembly/polishing ( Chen et al, 2020 ; Wick and Holt, 2021 ).…”

“…Although the COVID-19 pandemic has led to a favorable re-evaluation of the merit in active genomic surveillance, limited resources in LMICs necessitate particular care when identifying processes/infrastructure to implement and support. While the aforementioned practicalities are not necessarily mentioned, a salient number of publications during the last 12 months promote successful “in house” Oxford Nanopore sequencing in African LMICs ( Juma et al, 2021 ; Tegha et al, 2021 ).…”

Antimicrobial Susceptibility Profile ofNeisseria gonorrhoeaefrom Patients Attending a Medical Laboratory, Institut Pasteur de Madagascar between 2014-2020: Phenotypic and Genomic characterization in a Subset ofNeisseria gonorrhoeaeIsolates

“…Among the most common STs in our study population, ST 7822 was prevalent in France since 2018 (37). The ST 1588 and 1931 have been described in Africa, namely in Burkina Faso, South Africa and Kenya (38)(39)(40). We hypothesize that these STs may have been imported by visitors or persons who travelled to and from Europe or the African continent.…”

The Notable Achievements and the Prospects of Bacterial Pathogen Genomics

“…An additional challenge is to make the sequencing technologies more mobile, thus enabling the collection and processing of data in remote environments or places where resources may be limited or sample transferring is complicated. The recent use of ONT devices in studying the antimicrobial resistance and phylogeny of Neisseria gonorrhoeae clinical isolates in Kenya indicates useful progress in this direction [ 119 ]. For time-critical applications such as those aimed at infection surveillance and tracking to prevent and control the spread of disease, or the determination of virulence serotypes or antimicrobial resistance genotypes in a clinical setting, the speed of generating actionable results from samples taken is vital.…”