3. Results and discussion
According to the species identification by the VITEK-2® Compact, Salmonella was isolated from both the knee effusion and stool samples, although not from the blood. Serotype identification showed that the 2 strains (YZU1797 and YZU1798) displayed 6,7,14 :r:- using the Salmonella serotype identification kit (SSI, Denmark). The phase-2 flagella antigen was not detected using the serotype identification kit repeated five times. It was expected that the strains would be identified as S. Infantis (6,7,14 :r:1,5) due to its predominant infection in infants and children (Ranjbar et al., 2018). Nearly 80% of patients infected withS. Infantis were less than 12 years of age, and these isolates were recovered from stools, urine, blood or other biological fluids (Ranjbar et al., 2018).
Whole-genome sequencing analysis was subsequently performed to obtain the genome sequence of YZU1797 and YZU1798. The raw reads were then uploaded to the European Nucleotide Archive database under accession number PRJEB40529. The core genome MLST (cgMLST) was executed to reveal that both strains were S. Virchow (6,7,14:r:1,5). Since thefljAB operon (fljA, fljB , and hin genes) was involved in the synthesis of phase-2 flagella antigen (Lucarelli et al., 2012). Homology analysis was then carried out on the fljAB operon and its surrounding sequences in YZU1797 and YZU1798 with the publishedS. Virchow genomes (Figure 1). The results showed that both strains lost the fragment of fljAB operon, which was subsequently replaced by a ~4.8 kb fragment obtained from E. coli . With nomenclature similar to the of the S. Typhimurium monophasic variant (Salmonella 4,[5],12:i:–) (Lucarelli et al., 2012), the serotype of both strains was named as S. Virchow monophasic variant (Salmonella 6,7,14 :r:-).
Sixteen published genomes of S. Virchow were downloaded and indexed to construct the phylogenic tree using cgMLST analysis to reveal the genomic characteristics of both strains. Figure 2A demonstrates that the 18 strains were divided largely into 2 lineages. Two strains of ST197 belonged to lineage I; the other 16 strains, including YZU1797 and YZU1798, belonged to lineage II. Both strains demonstrated a particular relationship to strain BCW_2815 and BCW_2814 from Denmark and China, respectively (Figure 1, 2A). However, the BCW_2815 and BCW_2814 preserved the fljAB operon with a prophage Entero_P4 inserted at the left side of the fljA gene (Figure 1), indicating expression of H2 flagellar.
CRISPR typing was also performed to demonstrate the serotype of both strains and the phylogenic relationship of the 16 strains (Figure 2B). Twelve S. Virchow CRISPR types (VCTs) were identified amongst 18 strains, with VCT12 shared by both YZU1797 and YZU1798. The majority of spacers identified in the 2 strains were VirN and VirBN (Figure 2B); both strains were identified as S. Virchow as a consequence of this and their close relationship with the other S. Virchow strains of ST16 (Figure 2B), which is considered as the major MLST type for S. Virchow (Bachmann et al., 2014). Additionally, with similarities in the phylogenic tree based on cgMLST, CRISPR typing divided these 18 strains into 2 lineages (Figure 2B, Supplementary Figure S1). BCW_2818 and 82-1040 strain of ST197, located in a separate lineage I, had unique VCT1 and VCT2 types (Figure 2B, Supplementary Figure S1). In lineage II, both Chinese strains of VCT12 showed a relationship to BCW_2814 and BCW_2815 of VCT11 with difference in 2 spacers (Figure 2B). The perfect correspondence between CRISPR typing and cgMLST (Supplementary Figure S1) confirmed that CRISPR typing could be used as an efficient tool to analyze the phylogenic relationship of the isolates belonging to the same Salmonella serotype (Li et al., 2018).
The minimum inhibitory concentration (MIC) of 15 antibiotics for YZU1797 and YZU1798 showed that both strains were multidrug-resistant (MDR) to chloramphenicol, tetracycline, trimethoprim, sulfamethoxazole, and nalidixic acid (Table 1). Genome sequencing analysis revealed that the presence of cmlA9 , sul1 , drfA1 , tetA(G) , andgyrA (S83F) in both isolates were involved in the antimicrobial resistance phenotype. Identification of antimicrobial resistance genes in the other 16 S. Virchow genes confirmed that the BCW_2814 shared the same antimicrobial resistance genotype with YZU1797 and YZU1798 (Figure 2A). Considering genome sequencing results demonstrated that no plasmid existed in either strain, the antimicrobial resistance genes should be located in the chromosome. Further analysis of these genes revealed that both S. Virchow monophasic variants acquired a ~43kb Salmonella genomic island 2 (SGI2), including the antimicrobial resistance gene cassette (drfA1-cmlA9-tetR-tetA(G)-sul1 ) (Figure 3). The SGI2 was first reported in 2005 an S. Emek isolate and was previously considered a variant from SGI1 (SGI1-J) (Levings et al., 2005). The SGI2 or SGI1-J was also detected in 3 clinical S. Virchow isolates from human blood in 1993 and 1994 in China (Doublet et al., 2009). Within the SGI2, the integron carrying the antimicrobial resistance gene cassette was inserted in the S023 reading frame flanked by a 5 bp target site duplication (TSD) (Figure 3), indicating that it was incorporated by transposition (Hall, 2010).