Predominance of multidrug-resistant Salmonella Typhi genotype 4.3.1 with low-level ciprofloxacin resistance in Zanzibar

Abstract

Background: Typhoid fever is a common cause of febrile illness in low- and middle-income countries. While multidrug-resistant (MDR) Salmonella Typhi (S. Typhi) has spread globally, fluoroquinolone resistance has mainly affected Asia.

Methods: Consecutively, 1038 blood cultures were obtained from patients of all age groups with fever and/or suspicion of serious systemic infection admitted at Mnazi Mmoja Hospital, Zanzibar in 2015-2016. S. Typhi were analyzed with antimicrobial susceptibility testing and with short read (61 strains) and long read (9 strains) whole genome sequencing, including three S. Typhi strains isolated in a pilot study 2012-2013.

Results: Sixty-three S. Typhi isolates (98%) were MDR carrying blaTEM-1B, sul1 and sul2, dfrA7 and catA1 genes. Low-level ciprofloxacin resistance was detected in 69% (43/62), with a single gyrase mutation gyrA-D87G in 41 strains, and a single gyrA-S83F mutation in the non-MDR strain. All isolates were susceptible to ceftriaxone and azithromycin. All MDR isolates belonged to genotype 4.3.1 lineage I (4.3.1.1), with the antimicrobial resistance determinants located on a composite transposon integrated into the chromosome. Phylogenetically, the MDR subgroup with ciprofloxacin resistance clusters together with two external isolates.

Conclusions: We report a high rate of MDR and low-level ciprofloxacin resistant S. Typhi circulating in Zanzibar, belonging to genotype 4.3.1.1, which is widespread in Southeast Asia and African countries and associated with low-level ciprofloxacin resistance. Few therapeutic options are available for treatment of typhoid fever in the study setting. Surveillance of the prevalence, spread and antimicrobial susceptibility of S. Typhi can guide treatment and control efforts.

Fig 1. The structure of MDR composite transposon.

The red arrows show antimicrobial resistance genes, MDR genes are sul1/sul2, dfrA7, bla_TEM-1 and catA1. Green arrows show insertion sequences (IS) or transposases and orange arrow show the replicons. BC04 is study isolate ZNZ13L78, BC06 is study isolate ZNZ55M142.

Fig 2. Phylogenetic tree of S. Typhi isolates from this study using the reference genome ERL 12960 as midpoint root.

The inner circle shows three clusters of our S. Typhi: purple highlighted is a monophyletic clade (MDR/cipR), these are MDR and have reduced susceptibility to ciprofloxacin due to gyrA-D87G; yellow highlighted are strains which are MDR but sensitive to ciprofloxacin (MDR/cipS); one strain in green highlight is not MDR and has low-level ciprofloxacin resistance due to single gyrA-S83F QRDR mutation. The second circle shows the year of isolation of S. Typhi; black represents S. Typhi isolated in 2016, grey in 2015, light grey and very light grey were isolated in 2013 and 2012 respectively. The next circle shows the season when S. Typhi were isolated, green represents isolates collected during the rainy season while orange highlighted S. Typhi were collected during the dry season. The outer circle shows the outcome of the patients; red represents S. Typhi from patients who died, blue represents S. Typhi from patients who survived while grey represents S. Typhi from patients with unknown outcome.

Fig 3. Phylogenetic tree of S. Typhi isolates from this study and of isolates from other publications using the reference genome ERL 12960 as midpoint root.

From inside out, the inner circle shows the year of isolation of S. Typhi. The second circle shows the country where the strains were isolated. The next circle shows the genotype of the S. Typhi strains. The outer circle shows the AMR profile in the current study. The monophyletic MDR/cipR is highlighted in purple, the MDR/cipS cluster is yellow, and the only nonMDR/cipR strain is highlighted in green.