| Summary: | Although numerous studies have investigated diarrhoea aetiology in many sub-Saharan African countries, recent data on <i>Shigella</i> species’ involvement in community-acquired acute diarrhoea (CA-AD) in Malawi are scarce. This study investigated the incidence, antibiotic susceptibility profile, genotypic characteristics, and clonal relationships of <i>Shigella flexneri</i> among 243 patients presenting with acute diarrhoea at a District Hospital in Lilongwe, Malawi. <i>Shigella</i> spp. were isolated and identified using standard microbiological and serological methods and confirmed by identifying the <i>ipaH</i> gene using real-time polymerase chain reaction. The isolates’ antibiotic susceptibility to 20 antibiotics was determined using the VITEK 2 system according to EUCAST guidelines. Genes conferring resistance to sulfamethoxazole (<i>sul1</i>, <i>sul2</i> and <i>sul3</i>), trimethoprim (<i>dfrA1</i>, <i>dfrA12</i> and <i>dfrA17</i>) and ampicillin (<i>oxa-1</i> and <i>oxa-2</i>), and virulence genes (<i>ipaBCD</i>, <i>sat</i>, <i>ial</i>, <i>virA</i>, <i>sen</i>, <i>set1A</i> and <i>set1B</i>) were detected by real-time PCR. Clonal relatedness was assessed using ERIC-PCR. Thirty-four <i>Shigella flexneri</i> isolates were isolated (an overall incidence of 14.0%). All the isolates were fully resistant to sulfamethoxazole/trimethoprim (100%) and ampicillin (100%) but susceptible to the other antibiotics tested. The <i>sul1</i> (79%), <i>sul2</i> (79%), <i>sul3</i> (47%), <i>dfrA12</i> (71%) and <i>dfrA17</i> (56%) sulfonamide and trimethoprim resistance genes were identified; <i>Oxa-1</i>, <i>oxa-2</i> and <i>dfrA1</i> were not detected. The virulence genes <i>ipaBCD</i> (85%), <i>sat</i> (85%), <i>ial</i> (82%), <i>virA</i> (76%), <i>sen</i> (71%), <i>stx</i> (71%), <i>set1A</i> (26%) and <i>set1B</i> (18%) were detected. ERIC-PCR profiling revealed that the <i>Shigella</i> isolates were genetically distinct and clonally unrelated, indicating the potential involvement of genetically distinct <i>S. flexneri</i> in CA-AD in Malawi. The high percentage resistance to ampicillin and sulfamethoxazole/trimethoprim and the presence of several virulence determinants in these isolates emphasises a need for continuous molecular surveillance studies to inform preventive measures and management of <i>Shigella</i>-associated diarrhoeal infections in Malawi.
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