Antimicrobial resistance and genomic investigation of non-typhoidal Salmonella isolated from outpatients in Shaoxing city, China

Abstract

Human non-typhoidal salmonellosis is among the leading cause of morbidity and mortality worldwide, resulting in huge economic losses and threatening the public health systems. To date, epidemiological characteristics of non-typhoidal Salmonella (NTS) implicated in human salmonellosis in China are still obscure. Herein, we investigate the antimicrobial resistance and genomic features of NTS isolated from outpatients in Shaoxing city in 2020. Eighty-seven Salmonella isolates were recovered and tested against 28 different antimicrobial agents, representing 12 categories. The results showed high resistance to cefazolin (86.21%), streptomycin (81.61%), ampicillin (77.01%), ampicillin-sulbactam (74.71%), doxycycline (72.41%), tetracycline (71.26%), and levofloxacin (70.11%). Moreover, 83.91% of isolates were resistant to ≥3 categories, which were considered multi-drug resistant (MDR). Whole-genome sequencing (WGS) combined with bioinformatic analysis was used to predict serovars, MLST types, plasmid replicons, antimicrobial resistance genes, and virulence genes, in addition to the construction of phylogenomic to determine the epidemiological relatedness between isolates. Fifteen serovars and 16 STs were identified, with the dominance of S. I 4, [5], 12:i:- ST34 (25.29%), S. Enteritidis ST11 (22.99%), and S. Typhimurium ST19. Additionally, 50 resistance genes representing ten categories were detected with a high prevalence of aac(6')-Iaa (100%), bla _TEM-1B (65.52%), and tet(A) (52.87%), encoding resistance to aminoglycosides, β-lactams, and tetracyclines, respectively; in addition to chromosomic mutations affecting gyrA gene. Moreover, we showed the detection of 18 different plasmids with the dominance of IncFIB(S) and IncFII(S) (39.08%). Interestingly, all isolates harbor the typical virulence genes implicated in the virulence mechanisms of Salmonella, while one isolate of S. Jangwani contains the cdtB gene encoding typhoid toxin production. Furthermore, the phylogenomic analysis showed that all isolates of the same serovar are very close to each other and clustered together in the same clade. Together, we showed a high incidence of MDR among the studied isolates which is alarming for public health services and is a major threat to the currently available treatments to deal with human salmonellosis; hence, efforts should be gathered to further introduce WGS in routinely monitoring of AMR Salmonella in the medical field in order to enhance the effectiveness of surveillance systems and to limit the spread of MDR clones.

Keywords: antimicrobial resistance; gastroenteritis; non-typhoidal Salmonella; public health; salmonellosis; virulence; whole genome sequencing.

Figure 1

The distribution of different serovars among six counties in Shaoxing city, Zhejiang province, China. (A) The geographical distribution of the Salmonella isolates in Shaoxing with six counties which were examined in current investigations. N.B., The number indicates the numbers of isolates collected from individual county. (B) The distribution of fifteen serovars of Salmonella isolates. The dominant serovars are S. I 4, [5], 12:i:-, S. Enteritidis, and S. Typhimurium. (C) The prevalence of individual serovar with their sequence type (ST) detected in this study.

Figure 2

Antimicrobial resistance of Salmonella isolates. (A) Resistance of isolates grouped by serovars to the tested antimicrobial agents. (B) Prevalence of resistance against 28 antimicrobial agents belonging to 12 categories.

Figure 3

The heatmap of antimicrobial resistance genes (ARGs) and chromosomic mutations in the studied Salmonella isolates.

Figure 4

The combinatorial graph of the wgMLST phylogenomic evolutionary tree and virulence genes in the studied Salmonella isolates. The presence of virulence gene was marked with blue color while the absence was marked with white color. The tree was rooted by using S. Typhimurium SL 1344.

Figure 5

The heatmap of plasmids distribution in different Salmonella isolates. The strength of the colors corresponds to the numerical value of the prevalence of the plasmids. Dark blue color indicates high prevalence and white color for gene absence.

Figure 6

The core genome SNP-based phylogenomic tree by maximum likelihood (TVM+F+ASC) of 87 Salmonella isolates. The tree was rooted by using S. Typhimurium SL 1344.