Salmonella enterica serovar Senftenberg human clinical isolates lacking SPI-1

Abstract

Nontyphoidal Salmonella species cause gastrointestinal disease worldwide. The prevailing theory of Salmonella enteropathogenesis is that bacterial invasion of the intestinal epithelium is essential for virulence and that this requires the virulence-associated genomic region Salmonella pathogenicity island 1 (SPI-1). Recent studies of Salmonella enterica infection models have demonstrated that enterocolitis and diarrhea in mice and cows can occur independently of SPI-1. In this study, we sought to confirm whether two S. enterica serovar Senftenberg clinical isolates lacked genes essential for SPI-1 function. Two clinical strains were isolated and identified as being S. enterica serovar Senftenberg from four stool samples from a food-borne disease outbreak affecting seven individuals in Shenzhen, Guangdong Province, China, using conventional methods, pulsed-field gel electrophoresis and multilocus sequence typing. The possibility of coinfection with other potential bacteria or usual viruses was excluded. Two isolates were analyzed for the presence of invA, sipA, ssaR, sifA, and sopE2 by PCR and Southern blotting and were then assayed for the presence of SPI-1 by PCR and long-range PCR for fhlA-hilA, hilA-spaP, and spaP-invH and Southern blot analysis. A long-range PCR fragment from fhlA to mutS covering the 5' and 3' flanks of SPI-1 was also amplified from the two clinical isolates and sequenced. In addition, the two clinical isolates were assayed for enteroinvasiveness in vitro. Murine infection models were also examined. Biochemical tests and serotyping confirmed that the two clinical isolates are S. enterica serovar Senftenberg. However, they lacked genes critical for SPI-1 function but contained SPI-2 genes and were attenuated for the invasion of cultured intestinal epithelial cells. In conclusion, clinical S. enterica serovar Senftenberg strains isolated from a food-borne disease outbreak lack the invasion-associated locus SPI-1, indicating that SPI-1 is not essential for human gastroenteritis.

FIG. 1.

PFGE of the clinical isolates from a food-borne disease outbreak in Shenzhen, China. Bacterial DNA from stool samples from diarrhea patients from a single outbreak (CI1 and CI2) or control strains of Salmonella enterica serovar Senftenberg (SC1 to SC5) and S. enterica serovar Typhimurium (STM) were digested with the restriction enzyme XbaI, subjected to PFGE as described in Materials and Methods, and compared to reference Salmonella serovar Braenderup standard strain H9812.

FIG. 2.

Genomic analysis of SPI-1 and SPI-2 of strains isolated from diarrheal patients in Shenzhen, China, compared to control strains. Stool isolates (CI1 and CI2) were compared to S. enterica serovar Senftenberg (Senftenberg-1 and Senftenberg-2), serovar Enteritidis (Enteritidis), and serovar Typhimurium (STM1344 and STMinvA−) strains by PCR for the presence of the SPI-1 genes, including invA, sipA, fhlA-hilA, hilA-spaP, and spaP-invH; the phage-encoded SPI-1 effector gene sopE2; the SPI-2-encoded gene ssaR; and the non-SPI-2-encoded SPI-2 effector gene sifA. SPI-1-encoded genes were PCR negative in both stool isolates. The invA-negative control strain STMinvA− contains an insertional mutation in invA. One strain (Senftenberg-2) of the S. enterica serovar Senftenberg type strains lacked the non-SPI-2-encoded SPI-2 effector gene sifA.

FIG. 3.

Southern hybridization analysis for SPI-1 genes of clinical isolates from Shenzhen, China, and controls. By Southern blot, the presence of the SPI-1 genes invA, sipA, fhlA-hilA, hilA-sicP, sicP-sipD, sipD-spaS, spaS-spaO, and spaP-invH were assessed in clinical isolates (CI1 and CI2) and control strains of S. enterica serovar Senftenberg (Senftenberg-1 and Senftenberg-2) and serovar Typhimurium (STM and STMinvA−). The presence of invA was assessed using two independent probes. SPI-1 genes were absent only in the clinical isolates. STMinvA− contains an insertion within invA, and the gene is consequently larger than wild-type invA.

FIG. 4.

Mapping of the SPI-1 deletion in S. enterica serovar Senftenberg clinical isolates. In order to define the precise deletion site of SPI-1 in the two S. enterica serovar Senftenberg isolates (CI1 and CI2), a long-range PCR was performed using primers annealing to the fhlA and mutS genes located at the 5′ and 3′ flanks of SPI-1, and a 10.255-kb PCR product was obtained and sequenced. Sequence comparison to the known genome sequence of S. enterica serovar Typhimurium LT2 in the GenBank database showed that SPI-1 was deleted at the avrA gene in the two serovar Senftenberg clinical isolates, and there was an IS, about 1 kb in size, present in the deletion site in the two serovar Senftenberg clinical isolates. The positions of the SPI-1 genes are indicated by the black arrow bars, and probes used for Southern hybridization are indicated by the black bars.

FIG. 5.

Murine infections with clinical isolates of S. enterica serovar Senftenberg did not result in intestinal pathology. Although colonization with clinical isolates both containing (Senftenberg-1) and lacking (CI1 and CI2) SPI-1 was evident in murine infection (A), there was no histopathological evidence of inflammation in infected mouse intestines (B).