Relationship between antibody susceptibility and lipopolysaccharide O-antigen characteristics of invasive and gastrointestinal nontyphoidal Salmonellae isolates from Kenya

Abstract

Background: Nontyphoidal Salmonellae (NTS) cause a large burden of invasive and gastrointestinal disease among young children in sub-Saharan Africa. No vaccine is currently available. Previous reports indicate the importance of the O-antigen of Salmonella lipopolysaccharide for virulence and resistance to antibody-mediated killing. We hypothesised that isolates with more O-antigen have increased resistance to antibody-mediated killing and are more likely to be invasive than gastrointestinal.

Methodology/principal findings: We studied 192 NTS isolates (114 Typhimurium, 78 Enteritidis) from blood and stools, mostly from paediatric admissions in Kenya 2000-2011. Isolates were tested for susceptibility to antibody-mediated killing, using whole adult serum. O-antigen structural characteristics, including O-acetylation and glucosylation, were investigated. Overall, isolates were susceptible to antibody-mediated killing, but S. Enteritidis were less susceptible 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. Amount of S. Typhimurium O-antigen and O-antigen glucosylation level were inversely related. There was no clear association between clinical presentation and antibody susceptibility, O-antigen level or other O-antigen features.

Conclusion/significance: Kenyan S. Typhimurium and Enteritidis clinical isolates are susceptible to antibody-mediated killing, with degree of susceptibility varying with level of O-antigen for S. Typhimurium. This supports the development of an antibody-inducing vaccine against NTS for Africa. 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. Genome studies are required to understand whether invasive and gastrointestinal isolates differ at the genotypic level.

Fig 1. SBA results of STm and SEn isolates with Malawi serum pool.

Bacterial colony forming units (CFU) were counted at time 0 (T0) and after 3 h (T180) incubation of bacteria in 45 μL undiluted serum (final bacterial concentration 1×10⁶ CFU/ml). Killing/growth was determined by Log₁₀ (CFU at T180)-Log₁₀ (CFU at T0). Strains that could grow (Log₁₀ change >0) were considered “resistant”, strains that were killed (Log₁₀ change <0) were considered susceptible. Dashed/continuous line indicates median Log₁₀ change of STm (-2.9) and SEn (-1.2) isolates, respectively.

Fig 2. OAg production of S. Typhimurium (STm) and S. Enteritidis (SEn) isolates after growth and normalization to the same final OD: 35.

Dashed/continuous line indicates median OAg production of S. Typhimurium (STm) (14.4 μg/ml/OD) and S. Enteritidis (SEn) (16.8 μg/ml/OD) isolates, respectively.

Fig 3. a) HPLC-SEC profile of S. Typhimurium Ke212 OAg (similar profiles obtained for all NTS strains; b) ¹H NMR of S. Enteritidis Ke189 OAg and c) S. Typhimurium Ke004 OAg (arrows indicate O-acetyl groups); d) HPAEC-PAD profile of S. Typhimurium Ke012 OAg sample indicating typical sugars of the repeating unit (Rha, Gal, Glc and Man) and of the core region (Gal, Glc and NAcGlc) (similar profiles obtained for all NTS strains).

Fig 4. Correlation of SBA results and OAg production levels in (a) S. Typhimurium isolates (p = 0.002) and (b) S. Enteritidis isolates (p = 0.637).

r is the Spearman correlation coefficient. CI is confidence interval.

Fig 5. Correlation of SBA results (a, c) and OAg production levels (b, d) in S. Typhimurium (a, b) and S. Enteritidis (c, d) isolates with clinical presentations.

Samples for which no clinical presentation was determined were excluded from analysis. Malawi serum pool was used for SBA. Invasive: Salmonella isolates from blood, urine, CSF. Gastrointestinal: Salmonella isolates from stools. Controls: Salmonella isolates from stools of healthy controls.

Fig 6. Correlation of S. Typhimurium (STm) OAg production levels and glucosylation (p<0.0001).

r is the Spearman correlation coefficient. CI is confidence interval.