Persistent Infections by Nontyphoidal Salmonella in Humans: Epidemiology and Genetics

Abstract

Background: Although chronic infections by typhoidal Salmonella are well-known, prolonged human infections by nontyphoidal Salmonella (NTS) are poorly characterized.

Methods: We retrospectively analyzed 48 345 culture-confirmed NTS infections that occurred in Israel 1995-2012. A case-control study was performed to identify risk factors associated with persistent infections. Whole-genome-sequencing, pulsed-field gel electrophoresis (PFGE), and a mouse infection model were used to study genetic and phenotypic differences between same-patient persistent, recurring isolates.

Results: In total, 1047 cases of persistent NTS infections, comprising 2.2% of all reported cases of salmonellosis, were identified. The persistence periods ranged between 30 days to 8.3 years. The majority (93%) of the persistently infected patients were immunocompetent, and 65% were symptomatic with relapsing diarrhea, indicating a distinct clinical manifestation from the asymptomatic carriage of typhoidal Salmonella. Four NTS serovars (Mbandaka, Bredeney, Infantis and Virchow) were found to be significantly more frequently associated with persistence than others. Comparative genomics between early and later isolates obtained from the same patients confirmed clonal infection and showed 0 to 10 SNPs between persistent isolates. A different composition of mobile genetic elements (plasmids and phages) or amino acid substitutions in global regulators was identified in multiple cases. These changes resulted in differences in phenotype and virulence between early and later same-patient isolates.

Conclusions: These results illuminate the overlooked clinical manifestation of persistent salmonellosis that can serve as a human reservoir for NTS infections. Additionally, we demonstrate mechanisms of in-host microevolution and exhibit their potential to shape Salmonella pathogenicity, antimicrobial resistance and host-pathogen interactions.

Keywords: Salmonella enterica; WGS; epidemiology; persistent infection; salmonellosis.

Figure 1.

Recurrent NTS isolates obtained from the same patients are genetically related. 42 recurrent NTS isolates from 16 different patients were analyzed by PFGE using XbaI endonuclease. Isolate number, NTS serovar, isolation date and the minimal duration of persistence are indicated on the right. A dendogram showing the percentage of similarity between isolates is shown on the left. Abbreviations: NTS, nontyphoidal Salmonella; PFGE, pulsed-field gel electrophoresis.

Figure 2.

Plasmids gained and lost in related persistent isolates. Nine S. Typhimurium persistent isolates obtained from patients B, D, I, and K were subjected to an S1 nuclease digest followed by PFGE. Plasmids that were found in the later isolates, but not in the early isolates are indicated by a blue arrow. Plasmids that were found in the early, but not the later, isolates are indicated by a red arrow. The prevalent approximately 95 kilobase (Kb) virulence S. Typhimurium plasmid, pSLT, is indicated by a black arrow. Abbreviation: PFGE, pulsed-field gel electrophoresis.

Figure 3.

Related persistent isolates differ in their pathogenicity in vivo. Groups of 5 female C3H/HeNHsd mice were treated with streptomycin and 24 hours later were infected orally with a 1:1 mixed inoculum of early and later S. Typhimurium isolates (5 × 10⁶–1 × 10⁷ CFU from each isolate) obtained from patients A (isolate 85982 and 87541; panel A), D (102261 and 102923; panel B), E (128781 and 130302; panel C), and K (135497 and 137309; panel D). Four days post infection mice were sacrificed, and the colonization of the later isolates relative to the early isolate was determined in the cecum and colon as explained in “Materials and Methods” section. Each point shows the competitive index (C.I.) value in a single mouse, whereas the geometrical mean of each group is indicated by the horizontal line. Two-tailed 1-sample t-test against a theoretical mean of 1.00 was used to determine statistical significance and is shown in brackets. Abbreviation: CFU, colony-forming unit.