Microbiological, Immunological and Genomic Characterization of Nontyphoidal Salmonella Isolates From Nairobi County

Abstract

Nontyphoidal Salmonellae (NTS) of major clinical significance in sub-Saharan Africa (SSA) consist mainly of Salmonella enterica serovars Typhimurium and Enteritidis. No vaccine is currently available. In SSA, NTS cause both invasive and gastrointestinal disease and contribute a huge burden of illness especially among young children under five years and HIV-infected adults. In Kenya, this includes sick children presenting to rural district and urban-based facilities. Invasive NTS (iNTS) disease has an associated case-fatality rate of around 20%. Previous reports indicate the importance of the O-antigen of Salmonella lipopolysaccharide (LPS) for virulence and resistance to antibody-mediated killing. The objective of the study was to immunochemically and genomically characterize S. Typhimurium and S. Enteritidis isolates from Kenya implicated with invasive and gastrointestinal NTS disease in order to understand if immunochemical and genomic features correlate with different clinical presentations. The work drew from an existing culture collection of 193 NTS isolates from studies at the Centre for Microbiology Research (CMR), Kenya Medical Research Institute (KEMRI) between 2000 and 2011. These included 114 S. Typhimurium and 79 S. Enteritidis, 70 of which were from blood, 56 from stool, 12 from both blood and stool, three from urine, two from cerebral spinal fluid, and 31 were from stools of healthy carriers. The clinical isolates were mostly from children aged below 10 years admitted to four main hospitals in Kenya. Additional isolates included one from soil, one from a sewer and 17 isolates whose records of their sources of isolation were not available. A complete database detailing clinical metadata of each study isolate was created. Isolates were assigned as invasive (from blood, CSF, urine), gastro-intestinal (from stool), mixed infection (from both blood and stool) or healthy carrier for comparative analysis of results. xx The susceptibility of all 193 isolates to antibody-mediated killing was tested by serum bactericidal assay (SBA). Additionally, LPS O-antigen was extracted from each of the 193 isolates for further characterization to evaluate if the different categories of NTS isolates express O-antigen with different features. To estimate the molecular size distribution of O-antigen populations in the samples, all the isolates were analysed by size-exclusion chromatography. Similarly, to confirm the identity of the extracted O-antigen samples and to verify the presence of O-acetyl groups along the O-antigen chain, nuclear magnetic resonance spectroscopy analysis was performed on a subset of isolates and glycosylation levels were determined. Differences in O-antigen profiles were correlated with clinical presentation and serum susceptibility. To genetically characterize the samples, whole-genome sequencing was performed. The population structure of NTS isolates was compared with the available clinical metadata. Antimicrobial Susceptibility Testing (AST) was done and associations between the antibiograms and genomic data were looked for. S. Typhimurium isolates were found to be susceptible to antibody-mediated killing, but S. Enteritidis showed reduced susceptibility and expressed more O-antigen than Typhimurium (p<0.0001 for both comparisons). For S. Typhimurium, but not Enteritidis, O-antigen expression correlated with reduced sensitivity to killing (r=0.29, 95% CI=0.10 - 0.45, p=0.002). Both serovars expressed O-antigen populations ranging 21 - 33 kDa average molecular weight. O-antigen from most Typhimurium were O-acetylated on rhamnose and abequose residues, while Enteritidis O-antigen had low or no O-acetylation. Both Typhimurium and Enteritidis O-antigen were approximately 20-50% glucosylated. Glucosylation levels and amount of O-antigen of S. Typhimurium were inversely related. There was no clear association between clinical presentation and antibody susceptibility, O-antigen level or other O-antigen features. A majority (77%, 88/114) of S. Typhimurium and 30% (24/79) of S. Enteritidis isolates were MDR, with S. Typhimurium isolates having a higher proportion of isolates resistant to at least one antibiotic (97%, 111/114) compared to S. Enteritidis isolates (92%, 73/79). Whole genome sequences showed that a majority 78%, (89/114) of S. Typhimurium isolates analysed belonged to the sequence type ST313 which also fell within the two previously described lineages, designated lineage I (36%, 41/114) & II (42%, 48/114) while 22% (25/114) isolates belonged to two ST19 lineages, designated lineage IIIA (18/114) & IIIB (7/114). The novel lineage ST19 IIIB has not previously been described. For S. Enteritidis, all the 79 isolates belonged to ST11 and fell into two lineages, designated lineage I (30%, 24/79) & lineage II (70%, 55/79). The phylogenetic structure of S. Typhimurium isolates was found to be similar to a previously published population structure of invasive S. Typhimurium across SSA. A dominant S. Typhimurium pathotype, designated ST313, rarely reported outside of Africa, was found to dominate among the MDR S. Typhimurium isolates. A majority of these were primarily associated with invasive disease and febrile illness. Clinical records showed that diarrheal patients of S. Typhimurium isolates had febrile invasive illness symptoms (fever, vomiting, cough), implying that other S. Typhimurium pathotypes (ST19 lineages), previously associated mainly with gastroenteritis are capable of establishing invasive disease. In conclusion, Kenyan S. Typhimurium and Enteritidis clinical isolates are susceptible to antibody-mediated killing, with resistance correlating with amount of O-antigen among S. Typhimurium (r=-0.51, 95% CI - 0.69 to -0.27, p<0.0001). This supports the development of an antibody-inducing vaccine against NTS for SSA. No clear differences were found in the phenotype of isolates from blood and stool, suggesting that the same isolates can cause invasive disease and gastroenteritis. Phylogenetic structure of Kenyan S. Typhimurium was similar to published structures of NTS in SSA hence NTS disease in Kenya mirrors what is seen in many different SSA countries. Multi-drug resistance observed in a majority of S. Typhimurium isolates and a substantial proportion of S. Enteritidis isolates confirms widespread MDR among Kenyan NTS as also reported in many other SSA countries. Genomically, invasive and gastroenteric isolates appear to be indistinguishable and so more in-depth genome studies are required to understand whether invasive and gastrointestinal isolates differ.