Malonyl-proteome profiles of Staphylococcus aureus reveal lysine malonylation modification in enzymes involved in energy metabolism.

BACKGROUND: Protein lysine malonylation, a novel post-translational modification (PTM), has been recently linked with energy metabolism in bacteria. Staphylococcus aureus is the third most important foodborne pathogen worldwide. Nonetheless, substrates and biological roles of malonylation are still poorly understood in this pathogen. RESULTS: Using anti-malonyl-lysine antibody enrichment and high-resolution LC-MS/MS analysis, 440 lysine-malonylated sites were identified in 281 proteins of S. aureus strain. The frequency of valine in position - 1 and alanine at + 2 and + 4 positions was high. KEGG pathway analysis showed that six categories were highly enriched, including ribosome, glycolysis/gluconeogenesis, pentose phosphate pathway (PPP), tricarboxylic acid cycle (TCA), valine, leucine, isoleucine degradation, and aminoacyl-tRNA biosynthesis. In total, 31 malonylated sites in S. aureus shared homology with lysine-malonylated sites previously identified in E. coli, indicating malonylated proteins are highly conserved among bacteria. Key rate-limiting enzymes in central carbon metabolic pathways were also found to be malonylated in S. aureus, namely pyruvate kinase (PYK), 6-phosphofructokinase, phosphoglycerate kinase, dihydrolipoyl dehydrogenase, and F1F0-ATP synthase. Notably, malonylation sites were found at or near protein active sites, including KH domain protein, thioredoxin, alanine dehydrogenase (ALD), dihydrolipoyl dehydrogenase (LpdA), pyruvate oxidase CidC, and catabolite control protein A (CcpA), thus suggesting that lysine malonylation may affect the activity of such enzymes. CONCLUSIONS: Data presented herein expand the current knowledge on lysine malonylation in prokaryotes and indicate the potential roles of protein malonylation in bacterial physiology and metabolism.

Molecular characteristics of Staphylococcus aureus isolates from food surveillance in southwest China.

BACKGROUND: Staphylococcal food poisoning (SFP) is one of the most common food-borne diseases in the world. Pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and spa typing methods were used to characterize Staphylococcus aureus isolates from food surveillance during 2013-2015 in southwest China, and Staphylococcal cassette chromosome mec (SCCmec) typing was used for methicillin-resistant S. aureus (MRSA). Isolates were also examined for their antibiotic resistance and carriage of virulence genes. RESULTS: Isolation rate of S. aureus was 2.60% during the three years' surveillance and 29.50% of them were MRSA. All the S. aureus had hla genes (100%), 14.34% of the strains had tst, and 16.73% had PVL. 163 PFGE-SmaI patterns, 41 ST types and 36 spa types were obtained for all the S. aureus. Among them, ST6-t701 (13.15%), ST7-t091 (12.75%), ST59-t437 (9.96%) and ST5-t002 (7.57%) were the prevalent genotypes. Most of MRSA in this study belonged to SCCmec IV and V, accounted for 74.32% and 20.27% respectively. ST6-SCCmec IV-t701 (36.50%) was the most prevalent clone among isolates from food, followed by ST59-SCCmec V-t437 (20.30%), ST5-SCCmec IV-t002 (12.20%) and ST59-SCCmec IV-t437 (12.20%). Some strains had the identical PFGE patterns, ST and spa types with isolates from patients. CONCLUSIONS: S. aureus isolated from food in southwest China displayed heterogeneity. Isolates had the same genotype profiles with isolates from patients, indicating high homology.

Characterization of three Bacillus cereus strains involved in a major outbreak of food poisoning after consumption of fermented black beans (Douchi) in Yunan, China.

Three Bacillus cereus strains isolated from an outbreak of food poisoning caused by the consumption of fermented black beans (douchi) containing B. cereus is described. The outbreak involved 139 persons who had nausea, vomiting, and diarrhea. The strains were isolated from vomit and the unprepared douchi. Two of the strains produced the emetic toxin cereulide, as evidenced by polymerase chain reaction analysis for the presence of the nonribosomal synthetase cluster responsible for the synthesis of cereulide and by chemical analysis by high-performance liquid chromatography-mass spectrometry. These two strains belong to genetic group III of B. cereus, and multiple locus sequence typing revealed that the type was ST26, as a major part of B. cereus emetic strains. One of these strains produced significantly more cereulide at 37°C than the type cereulide producer (F4810/72), and it was also able to produce the toxin at 40°C and 42°C. The third strain belongs to genetic group IV, and it is a new multiple locus sequence type closely related to strains that are cytotoxic and enterotoxigenic. It possesses genes for hemolysin BL, nonhemolytic enterotoxin, and cytotoxin K2; however, it varies from the majority of strains possessing genes for hemolysin BL by not being hemolytic. Thus, two B. cereus strains producing the emetic toxin cereulide and a strain producing enterotoxins might have been involved in this food-poisoning incident caused by the consumption of a natural fermented food. The ability of one of the strains to produce cereulide at ≥37°C makes it possible that it is produced in the human gut in addition to occurring in the food.

BCp12/PLA combination: A novel antibacterial agent targeting Mur family, DNA gyrase and DHFR.

The combination of natural antimicrobial peptide BCp12/phenyllatic acid (BCp12/PLA) presents a more efficient antibacterial effect, but its antibacterial mechanism remains unclear. This study studied the synergistic antibacterial mechanism of BCp12 and PLA against S. aureus. The results demonstrated that the BCp12/PLA combination presented a synergistic antibacterial effect against S. aureus, with a fractional inhibitory concentration of 0.05. Furthermore, flow cytometry and scanning electron microscope analysis revealed that BCp12 and PLA synergistically promoted cell membrane disruption compared with the group treated only with one compound, inducing structural cell damage and cytoplasmic leakage. In addition, fluorescence spectroscopy analysis suggested that BCp12 and PLA synergistically influenced genomic DNA. BCp12 and PLA targeted enzymes related to peptidoglycan and DNA synthesis and interacted by hydrogen bonding and hydrophobic interactions with mur enzymes (murC, murD, murE, murF, and murG), dihydrofolate reductase, and DNA gyrase. Additionally, the combined treatment successfully inhibited microbial reproduction in the storage of pasteurized milk, indicating that the combination of BCp12 and PLA can be used as a new preservative strategy in food systems. Overall, this study could provide potential strategies for preventing and controlling foodborne pathogens.

Prevalence and antimicrobial resistance profiling of Staphylococcus aureus isolated from traditional cheese in Yunnan, China.

The prevalence of staphylococcal infection and the emergence of multidrug resistance of Staphylococcus aureus (S. aureus) are major concerns in food safety and public health. This study aimed to investigate the prevalence of S. aureus isolated from traditional Chinese Rubing and Rushan cheese, antimicrobial resistance profiles, genomic characteristics, and predict antimicrobial resistance genes (ARGs). From 124 samples, 18 of 62 (29.03%) of Rubing and 5 of 62 (8.06%) of Rushan cheese were confirmed to be S. aureus positive by standard culture-based methods. Twenty-three coagulase-positive staphylococci isolates were grouped into 16 clusters by pulsed-field gel electrophoresis and subjected to routine susceptibility testing to 12 antibiotics. Those isolates exhibited high resistance to penicillin (100%), erythromycin, trimethoprim-sulphamethoxazole (34.78%), oxacillin, clindamycin, and cefoxitin (21.74%). Multidrug-resistant (MDR) S. aureus was found in 34.78% (8 of 23) of isolates. Further, S. aureus strain DC.RB_015 isolated from Rubing cheese, recognized as the most resistant to six antibiotics, was selected for whole-genome sequencing (WGS), continued with in silico approaches. S. aureus DC.RB_015 had a single chromosome size of 2,794,578 bp and a plasmid size of 22,961 bp. The strain harbored 18 predicted ARGs, including eight efflux pump genes (mepA, tet(K), arlR, arlS, norA, mgrA, tet(38), LmrS), one peptidoglycan biosynthesis gene (bacA), two ß-lactams resistance genes (mecA, blaZ), and seven genes conferring other antimicrobial resistance (APH(3')-IIIa, aad(6), ErmB, SAT-4, mecR1, GlpT, murA). The results of this study expand the knowledge of S. aureus strain DC.RB_015, increase food safety awareness, and will be helpful in establishing therapeutic therapy. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-03072-4.

Molecular characteristics of Salmonella enterica Paratyphi A in Yunnan Province, southwest China.

Previously, the prevalence of Salmonella enterica Paratyphi A in Yunnan was high; and recently Yunnan was the predominant endemic province in China. To identify the molecular epidemiology, antibiotic resistance profile and genotypic diversity of the S. Paratyphi A isolates from 1995 to 2013 in Yunnan, we performed the study. Antibiotic susceptibility tests, pulse-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used to identify the characteristics of the bacterial isolates. The results showed from 1995 to 2013, 366 S. Paratyphi A were isolated: 295 isolates (80.6%) from Yuxi and 68 isolates (18.58%) from Honghe. All of the strains were resistant to nalidixic acid, and some were resistant to ampicillin and trimethoprim/sulfamethoxazole in different years. All the isolates were sensitive to cefotaxime and ciprofloxacin. Identical PFGE with two enzyme digestion patterns were found for 339 isolates. Some environmental isolates in different years were homologous with the strains isolated from food and patients. MLST showed 349 strains were ST85, only 17 isolates were ST129. S. Paratyphi A isolates from Yunnan showed a high similarity, and we found the pathogen isolated from patients, the environment and food had the close epidemiological relationship, forming a transmission circulation. These findings have important implications for paratyphoid-control strategies.