Microevolution of Salmonella 4,[5],12:i:- derived from Salmonella enterica serovar Typhimurium through complicated transpositions.

Salmonella enterica subsp. enterica serovar 4,[5],12:i:- (Salmonella 4,[5],12:i:-), derived from S. Typhimurium, has become the dominant serotype causing human salmonellosis. In this study, we define the genetic mechanism of the generation of Salmonella 4,[5],12:i:- from S. Typhimurium through complicated transpositions and demonstrate that Salmonella 4,[5],12:i:- displays more efficient colonization and survival abilities in mice than its parent S. Typhimurium strain. We identified intermediate strains carrying both resistance regions (RRs) and the fljAB operon for the generation of Salmonella 4,[5],12:i:-. The insertion of RR3 into the chromosomal hin-iroB site of S. Typhimurium produced RR3-S. Typhimurium as a primary intermediate. Salmonella 4,[5],12:i:- was then produced by replacing the fljAB operon and/or its flanking sequences through intramolecular transpositions mediated by IS26 and/or IS1R elements in RR3-S. Typhimurium, which was further confirmed both in vitro and in vivo. Overall, we demonstrate the molecular mechanism underlying the origin, generation, and advantage of RRs-Salmonella 4,[5],12:i:- from S. Typhimurium.

Development and application of a multiplex PCR method to differentiate Salmonella enterica serovar Typhimurium from its monophasic variants in pig farms.

Salmonella enterica serovar Typhimurium monophasic variants (Salmonella 4,[5],12:i:-) has increased dramatically, causing human salmonellosis and colonization in pigs. With a difference to S. Typhimurium, the monophasic variants of S. Typhimurium lose the gene cassettes encoding the second phase flagellin. To establish a rapid method to detect and differentiate the two serotypes, we analyzed the published 679 genomes of S. Typhimurium and its monophasic variants and found that no Salmonella 4,[5],12:i:- strains carry both fljB and hin genes. Therefore, we established a novel multiplex PCR method using the fljB-hin region and mdh gene as target sequences to detect and differentiate both serotypes. This method can be used to specifically detect both serotypes with a detection limit for DNA concentration at 10 pg/µL. In addition, the PCR assay successfully differentiated 36 S. Typhimurium isolates from 62 isolates of monophasic variants preserved in our laboratory from 2009 to 2017, which corresponds to the whole-genome-based serotyping results. Application of the multiplex PCR method to 60 fecal samples from a pig farm identified 11.7% (7/60) of S. Typhimurium monophasic variants, which is consistent with the whole-genome-based serotyping results. The multiplex PCR assay is a rapid and precise method for the detection of S. Typhimurium monophasic variants from samples across food production chains.

Preclinical evaluation of OMVs as potential vaccine candidates against Salmonella enterica serovar Enteritidis infection.

Salmonella enterica serovar Enteritidis is the most prevalent serotype that causes human infections worldwide. Consumption of S. Enteritidis-contaminated animal foods is a major source of human infections; however, eradicating bacteria from animals remains difficult. Therefore, it is necessary to develop new measures to prevent and control salmonellosis. Here, we used the outer-membrane vesicles (OMVs) of S. Enteritidis and assessed their protective efficacy and immune response in mice. Deletion of tolR in S. Enteritidis increased the production and size of OMVs compared to those in the wild type (WT) and ΔrfaQ strains. Intramuscular immunization with OMVs conferred greater protection than intraperitoneal and intranasal immunization. Moreover, OMVs extracted from both WT and ΔtolR strains provided an 83.3% protective rate in mice challenged with S. Enteritidis, which was higher than that provided by OMVs extracted from the ΔrfaQ strain. However, compared with OMVs from the ΔtolR strain, OMVs from WT and ΔrfaQ strains rapidly eradicated S. Enteritidis colonizing the liver, spleen, ileum, and cecum of BALB/c mice after immunization. Immunization with OMVs from each of the three strains induced humoral immune responses and showed no side effects on the growth of mice. Our study revealed that OMVs from various S. Enteritidis strains could be developed for use as subunit vaccine candidates against nontyphoidal Salmonella infections in mammals.

Alkaline pretreatment and the synergic effect of water and tetralin enhances the liquefaction efficiency of bagasse.

Bagasse liquefaction (BL) in water, tetralin, and water/tetralin mixed solvents (WTMS) was investigated, and effects of tetralin content in WTMS, temperature, and alkaline pretreatment of bagasse on liquefaction efficiency were studied. At 300°C, bagasse conversion in WTMS with tetralin content higher than 50 wt% was 86-87 wt%, whereas bagasse conversion in water or tetralin was 67 wt% or 84 wt%, respectively. Because the solid conversion from liquefaction in WTMS with tetralin content higher than 50 wt% was always higher than that in water or tetralin at temperatures between 250 and 300°C, a synergic effect between water and tetralin is suggested. Alkaline pretreatment of bagasse resulted in significantly higher conversion and heavy oil yield from BL in water or WTMS. The effect of deoxygenation by the present liquefaction method is demonstrated by lower oxygen contents (16.01-19.59 wt%) and higher heating values (31.9-34.8 MJ/kg) in the produced oils.