Epidemiological and genomic landscape of antimicrobial resistance in Malawi

Abstract

Antimicrobial resistance (AMR) is a global public health problem, which presents a huge threat to the treatment of all forms of bacterial infections. A wide range of bacterial pathogens across the globe are increasingly developing resistance to multiple classes of antimicrobial agents rendering the agents concerned ineffective for the treatment of infections. Bloodstream infection (BSI) and other bacterial infections in sub-Saharan Africa (SSA) and Malawi in particular, are a common cause of morbidity and mortality. Few facilities in SSA however, are able to conduct long-term surveillance and as such the full burden of drug resistant infection (DRI) remain largely unknown across the region. In this thesis, blood cultures routinely taken from adult and paediatric medical patients admitted to Queen Elizabeth Central Hospital (QECH) in Blantyre, Malawi between 1998 and 2016 were analysed to describe trends in BSI and AMR. The analysis revealed a significant decline of BSI in all major pathogens except S. Typhi. However, the majority of isolates were resistant to the Malawian first-line antimicrobial agents (ampicillin, cotrimoxazole and chloramphenicol). Resistance to all the first line antimicrobial agents was more common in Gram-negative pathogens than Gram-positive pathogens. Non-Salmonellae Enterobacteriaceae that produced extended spectrum beta-lactamase (ESBL) and were fluoroquinolone-resistant were detected, and the proportions of these isolates rose significantly during the surveillance. In contrast, a majority of common Gram-positive pathogens remain susceptible to either penicillin or chloramphenicol. Methicillin resistant S. aureus was first reported in 1998 but became regularly detected in the later years of the surveillance. The analysis of blood culture isolates identified E. coli as one of the common causes of BSI in Blantyre, and the proportion of these isolates that were ESBL producers increased over time. Globally, efforts to treat E. coli infections are increasingly being compromised by the rapid, global spread of ESBL-producing E. coli. In this thesis, a whole genome sequencing (WGS) study was carried out to investigate the genetic population structure and molecular determinants of AMR in E. coli isolates from Malawi. Whole genomes of clinical E. coli isolates from patients admitted to QECH were sequenced and analysed using phylogenetic methods and comparative genomics. It was revealed that the E. coli population in Malawi is highly diverse with isolates belonging to five phylogroups, corresponding to five isolate sequence clusters (SCs) that contained over forty sequence types (STs). A unique sub-lineage of ST131 was identified that was distinct from previously defined sub-lineages of this globally disseminated ST. The most common ESBL gene was blaCT X-M-15. Unlike in other settings where presence of the blaCT X-M-15 gene was strongly linked to ST131, here the gene was not lineage-specific suggesting a distinct genomic landscape of ESBL-producing E. coli in Malawi. This thesis also identified Klebsiella spp. isolates as a common cause of BSI in Blantyre, and an increasing proportion of ESBL-producing and fluoroquinolone resistant isolates were identified. The molecular mechanisms and clones of K. pneumoniae associated with ESBL production and fluoroquinolone resistance were yet to be explored in Malawi. Here, a number of K. pneumoniae isolates were selected for WGS, and placed in a global context by comparison with previously sequenced K. pneumoniae isolates from multiple locations outside SSA, in order to identify the molecular determinants of AMR and determine their relationship with K. pneumoniae population structure. Genomic analysis revealed three main lineages of K. pneumoniae, which corresponded to the previously defined KpI, KpII and KpIII lineages. All three lineages exhibited high genetic diversity. Further phylogenetic analysis revealed a sub-lineage of KpI to be a major cause of CA infections in Malawi. The sub-lineage included the clonally related ST14 and ST15 of K. pneumoniae which cause hospital acquired infection in multiple settings across the globe, A large pool of AMR genes, was identified in the genomes of the Malawian isolates, including multiple ESBL and qnr genes. Plasmid-encoded CTX-M-15 was the most common type of ESBL that was identified. In common with E. coli from Malawi, AMR was not restricted to a particular clade of K. pneumoniae. These findings suggest that dissemination of AMR in the K. pneumoniae population in Malawi was either due to a combination of horizontal gene transfer and clonal expansion, or horizontal gene transfer alone. In conclusion, the thesis has shown that ESBL production and fluoroquinolone resistance is rapidly spreading in Malawi across multiple E. coli and K. pneumoniae lineages that are causing increasing levels of infection. As cephalosporins and fluoroquinolones remain the last resort antimicrobial agents in this setting, urgent action is needed to curb the spread of Gram-negative AMR pathogens.