Genome-Based Assessment of Antimicrobial Resistance and Virulence Potential of Isolates of Non-Pullorum/Gallinarum Salmonella Serovars Recovered from Dead Poultry in China.

Paratyphoid avian salmonellosis is considered one of the leading causes of poultry death, resulting in significant economic losses to poultry industries worldwide. In China, especially in Shandong province, the leading producer of poultry products, several recurrent outbreaks of avian salmonellosis have been reported during the last decade where the precise causal agent remains unknown. Moreover, the establishment of earlier and more accurate recognition of pathogens is a key factor to prevent the further dissemination of resistant and/or hypervirulent clones. Here, we aim to use whole-genome sequencing combined with in silico toolkits to provide the genomic features of the antimicrobial resistance and virulence potential of 105 regionally representative non-Pullorum/Gallinarum Salmonella isolates recovered from dead poultry between 2008 and 2019 in Shandong, China. Additionally, phenotypic susceptibility to a panel of 15 antibiotics representing 11 classes was assessed by the broth microdilution method. In this study, we identified eight serovars and nine multilocus sequence typing (MLST) types, with Salmonella enterica serovar Enteritidis sequence type 11 (ST11) being the most prevalent (84/105; 80%). Based on their phenotypic antimicrobial resistance, 77.14% of the isolates were defined as multidrug resistant (≥3 antimicrobial classes), with the detection of one S. Enteritidis isolate that was resistant to the 11 classes. The highest rates of resistance were observed against nalidixic acid (97.14%) and ciprofloxacin (91.43%), followed by ampicillin (71.43%), streptomycin (64.77%), and tetracycline (60%). Genomic characterization revealed the presence of 41 resistance genes, with an alarmingly high prevalence of bla TEM-1B (60%), in addition to genomic mutations affecting the DNA gyrase ( gyrA ) and DNA topoisomerase IV ( parC ) genes, conferring resistance to quinolones. The prediction of plasmid replicons detected 14 types, with a dominance of IncFIB(S)_1 and IncFII(S)_1 (87.62% for both), while the IncX1 plasmid type was considered the key carrier of antimicrobial resistance determinants. Moreover, we report the detection of critical virulence genes, including cdtB , rck , sodCI , pef , and spv , in addition to the typical determinants for Salmonella pathogenicity island 1 (SPI-1) and SPI-2. Furthermore, phylogenomic analysis revealed the detection of three intra-farm and five inter-farm transmission events. Overall, the detection of Salmonella isolates presenting high antimicrobial resistance and harboring different critical virulence genes is of major concern, which requires the urgent implementation of effective strategies to mitigate non-Pullorum/Gallinarum avian salmonellosis. IMPORTANCE Avian salmonellosis is one of the leading global causes of poultry death, resulting in substantial economic losses in China (constituting 9% of overall financial losses). In Shandong province, a top poultry producer (30% of the overall production in China, with 15% being exported to the world), extensive outbreaks of avian salmonellosis have been reported in the past decade where the causal agents or exact types remain rarely addressed. From approximately 2008 to 2019, over 2,000 Salmonella strains were isolated and identified from dead poultry during routine surveillance of 95 poultry farms covering all 17 cities in Shandong. Approximately 1,500 isolates were confirmed to be of non-Pullorum/Gallinarum Salmonella serovars. There is an urgent need to understand the mechanisms behind the implication of zoonotic Salmonella serovars in systemic infections of poultry. Here, we analyzed populations of clinically relevant isolates of non-Pullorum/Gallinarum Salmonella causing chicken death in China by a whole-genome sequencing approach and determined that antimicrobial-resistant Salmonella Enteritidis remained the major cause in the past decades.