PemK's Arg24 is a crucial residue for PemIK toxin-antitoxin system to induce the persistence of Weissella cibaria against ciprofloxacin stress.

The toxin-antitoxin (TA) system plays a key role in bacteria escaping antibiotic stress with persistence, however, the mechanisms by which persistence is controlled remain poorly understood. Weissella cibaria, a novel probiotic, can enters a persistent state upon encountering ciprofloxacin stress. Conversely, it resumes from the persistence when ciprofloxacin stress is relieved or removed. Here, it was found that PemIK TA system played a role in transitioning between these two states. And the PemIK was consisted of PemK, an endonuclease toxic to mRNA, and antitoxin PemI which neutralized its toxicity. The PemK specifically cleaved the U↓AUU in mRNA encoding enzymes involved in glycolysis, TCA cycle and respiratory chain pathways. This cleavage event subsequently disrupted the crucial cellular processes such as hydrogen transfer, electron transfer, NADH and FADH2 synthesis, ultimately leading to a decrease in ATP levels and an increase in membrane depolarization and persister frequency. Notably, Arg24 was a critical active residue for PemK, its mutation significantly reduced the mRNA cleavage activity and the adverse effects on metabolism. These insights provided a clue to comprehensively understand the mechanism by which PemIK induced the persistence of W. cibaria to escape ciprofloxacin stress, thereby highlighting another novel aspect PemIK respond for antibiotic stress.

HigBA toxin-antitoxin system of Weissella cibaria is involved in response to the bile salt stress.

BACKGROUND: Toxin-antitoxin (TA) systems are prevalent adaptive genetic elements in bacterial genomes, which can respond to environmental stress. While, few studies have addressed TA systems in probiotics and their roles in the adaptation to gastrointestinal transit (GIT) environments. RESULTS: The Weissella cibaria 018 could survive in pH 3.0-5.0 and 0.5-3.0 g L-1 bile salt, and its HigBA system responded to the bile salt stress, but not to acid stress. The toxin protein HigB and its cognate antitoxin protein HigA had 85.1% and 100% similarity with those of Lactobacillus plantarum, respectively, and they formed the stable tetramer HigB-(HigA)2 -HigB structure in W. cibaria 018. When exposed to 1.5-3.0 g L-1 bile salt, the transcriptions of higB and higA were up-regulated with 4.39-19.29 and 5.94-30.91 folds, respectively. Meanwhile, W. cibaria 018 gathered into a mass with 48.07% survival rate and its persister cells were found to increase 8.21% under 3.0 g L-1 bile salt. CONCLUSION: The HigBA TA system of W. cibaria 018 responded to the bile salt stress, but not to acid stress, which might offer novel perspectives to understand the tolerant mechanism of probiotics to GIT environment. © 2022 Society of Chemical Industry.

Tetracycline residue alters profile of lactic acid bacterial communities and metabolites of ginger pickle during spontaneous fermentation.

Antibiotic residue, an emerging contaminant, has been an increasing concern worldwide in vegetables. However, the focus is still limited to its accumulation in vegetables and its adverse health effect on humans, with little mention of its impact on the vegetable process. Fermentation is an important method of the vegetable process. Tetracycline (TC) is the most widely used antibiotic, and its residue is often higher than other antibiotics in ginger. Therefore, current research was to characterize how the TC residue affected the lacticacidbacterial (LAB) communities and metabolites during the salted fermentation of ginger. The TC residue, organic acids, volatile compounds and LABs were analyzed during the fermentation, and the correlations between the LABs and compounds were also revealed. The results suggested that the hetero-lactic fermentation did not happen under the TC residue although the TC residue decreased from 4 mg/kg to 2.56 mg/kg in salt brine of ginger fermentation. Meanwhile, TC residue affected the occurrence, propagation and succession of LABs. The LAB biomarkers shifted to Lab. parafarraginis, Lab. buchneri and Lab. kisonensis under TC residue. Responded to LAB communities, the organic acids and volatile compounds were also markedly changed under the TC residue. And the important volatile compound variables shifted to citronellol, allylsenevol, geranyl acetate, vinylstearylether, isothiocyanic acid phenethyl, 3-octanol, geraniol, bingpian, citral and camphor. The transformation of LAB biomarkers induced by TC residue was the main cause of the change of important volatile compound variables, but interestingly, not all of them had significant positive or negative correlations. These results indicated that the antibiotic residue has an adverse ecological effect on the vegetable fermentation process.Therefore, the antibiotic residue should be listed as a quality control index in fermented vegetable raw materials.

Characterization and Antioxidant Activity of Released Exopolysaccharide from Potential Probiotic Leuconostoc mesenteroides LM187.

A released exopolysaccharide (rEPS)-producing strain (LM187) with good acid resistance, bile salt resistance, and cholesterol-lowering properties was isolated from Sichuan paocai and identified as Leuconostoc mesenteroides subsp. mesenteroides. The purified rEPS, designated as rEPS414, had a uniform molecular weight of 7.757 × 105 Da. Analysis of the monosaccharide composition revealed that the molecule was mainly composed of glucose. The Fourier transform-infrared spectrum showed that rEPS414 contained both α-type and ß-type glycosidic bonds. 1H and 13C nuclear magnetic resonance spectra analysis showed that the purified rEPS contained arabinose, galactose, and rhamnose, but less uronic acid. Scanning electron microscopy demonstrated that the exopolysaccharide displayed a large number of scattered, fluffy, porous cellular network flake structures. In addition, rEPS414 exhibited strong in vitro antioxidant activity. These results showed that strain LM187 and its rEPS are promising probiotics with broad prospects in industry.

Microbial Diversity and Succession in the Chinese Luzhou-Flavor Liquor Fermenting Cover Lees as Evaluated by SSU rRNA Profiles.

The microbial diversity and the community succession in the fermenting cover lees of Chinese Luzhou-flavor liquor were investigated by small-subunit rRNA (SSU rRNA) culture independent method. All sequences retrieved from the 1, 7 and 60 days fermented cover lees were respectively assigned into the genera of Streptococcus, Acetobacter, Arthrobacter, Bacillus, Staphylococcus, Serratia, Nocardia, Methanoculleus, Clostridium, Aneurinibacillus, Corynebacterium, Lactobacillus, Microbacterium, Trichosporon, Saccharomycopsis, Sagenomella, Talaromyces, Eurotium, Issatchenkia, Zygosaccharomyces, Saccharomyces and TM7 phylum. The fungal Issatchenkia, Saccharomycopsis and Talaromyces and the bacteria Staphylococcus and Lactobacillus were most abundant in the 1 day fermented cover lees, the fungal Issatchenkia, Saccharomyces and Talaromyces and the bacteria Bacillus and Streptococcus were dominant in the 7 days cover lees, the archaea Methanoculleus and the fungal Eurotium and Talaromyces were prevalent in the 60 days cover lees. When the microbial community profiles in three samples were compared at species level, the prokaryotic community similarity coefficient was from 0.4042 to 0.5703 and descended to 0.2222, and that of eukaryotic community was from 0.3000 to 0.6000 and followed to 0.5215. These results suggested that microbial diversity variability and community succession have happened in the cover lees associated with fermentation proceeding and such variability and succession respond for the appearance of some unique flavor of Luzhou-flavor liquor.

Mycobacterial interspersed repetitive unit typing in Mycobacterium tuberculosis isolates from Sichuan province in China.

BACKGROUND & OBJECTIVES: Emergence and spread of drug resistant Mycobacterium tuberculosis is a serious threat to tuberculosis (TB) control programme. Therefore, the objective of this study was to genotype drug-resistant M. tuberculosis strains isolated from patients in Sichuan, China, using Mycobacterial Interspersed Repetitive Units (MIRU) for epidemiological analysis. METHODS: Drug-resistance testing of M. tuberculosis isolates from pulmonary TB patients was confirmed by proportion method. Twelve MIRU loci were analyzed on 80 drug-resistant and 9 susceptible isolates by polymerase chain reaction and agarose gel electrophoresis. Hunter-Gaston discriminatory index (HGI) values were determined for each 12 MIRU loci for the evaluation of their discrimination power. RESULTS: Among 12 MIRU loci examined, polymorphic bands could be generated on 11 loci. Sixty five isolates had distinct MIRU patterns, while other 24 belonged to 8 clusters and resistant to at least one anti-TB drug tested. The association between the MIRU patterns and the mutation patterns of drug-resistance relevant target genes was not significant among the drug-resistant isolates. INTERPRETATION & CONCLUSIONS: The results showed that with a satisfactory discrimination power exhibited, the 12 loci based MIRU typing could be a valuable tool for epidemiological studies in M. tuberculosis isolates from Sichuan.