Aims: Protein S-bacillithiolations are mixed disulfides between protein thiols and the bacillithiol (BSH) redox buffer that occur in response to NaOCl in Bacillus subtilis. We used BSH-specific immunoblots, shotgun liquid chromatography (LC)-tandem mass spectrometry (MS/MS) analysis and redox proteomics to characterize the Sbacillithiolomes of B. subtilis, B. megaterium, B. pumilus, B. amyloliquefaciens, and Staphylococcus carnosus and also measured the BSH/oxidized bacillithiol disulfide (BSSB) redox ratio after NaOCl stress. Results: In total, 54 proteins with characteristic S-bacillithiolation (SSB) sites were identified, including 29 unique proteins and eight proteins conserved in two or more of these bacteria. The methionine synthase MetE is the most abundant Sbacillithiolated protein in Bacillus species after NaOCl exposure. Further, S-bacillithiolated proteins include the translation elongation factor EF-Tu and aminoacyl-tRNA synthetases (ThrS), the DnaK and GrpE chaperones, the two-Cys peroxiredoxin YkuU, the ferredoxin-NADP + oxidoreductase YumC, the inorganic pyrophosphatase PpaC, the inosine-5¢-monophosphate dehydrogenase GuaB, proteins involved in thiamine biosynthesis (ThiG and ThiM), queuosine biosynthesis (QueF), biosynthesis of aromatic amino acids (AroA and AroE), serine (SerA), branched-chain amino acids (YwaA), and homocysteine (LuxS and MetI). The thioredoxin-like proteins, YphP and YtxJ, are S-bacillithiolated at their active sites, suggesting a function in the de-bacillithiolation process. Sbacillithiolation is accompanied by a two-fold increase in the BSSB level and a decrease in the BSH/BSSB redox ratio in B. subtilis. Innovation: Many essential and conserved proteins, including the dominant MetE, were identified in the S-bacillithiolome of different Bacillus species and S. carnosus using shotgun-LC-MS/MS analyses. Conclusion: S-bacillithiolation is a widespread redox control mechanism among Firmicutes bacteria that protects conserved metabolic enzymes and essential proteins against overoxidation.

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BackgroundBlood cultures are commonly employed to identify bacterial pathogens causing sepsis. PCR assays to diagnose septicemia require extraction of bacterial DNA from blood samples and thus, delay the initiation of appropriate antimicrobial treatment. The presence of abundant human DNA may hamper the sensitivity of PCR in the detection of bacteria.MethodsWe used serial dilutions of E. Coli spiked pseudo-blood-sepsis samples to develop a simple method that combines the use of a polar detergent solvent and adjustment of the basic pH to remove human DNA. A 16S rRNA gene-based screening algorithm was established to differentiate Gram-positive and Gram-negative groups of bacteria and the family of Enterobacteriaceae. A stringent validation with appropriate controls was implemented. The method of human DNA removal was then applied on 194 sepsis blood samples and 44 cerebrospinal fluid (CSF) samples by real-time PCR.ResultsThis uncomplicated and straightforward approach allows to remove up to 98 % of human DNA from peripheral blood of septic patients. The inhibitory effect of human DNA is efficiently prevented and the detection limit of real-time PCR is increased to 10 E. Coli CFUs/ml. This sensitivity is 10 times higher compared to conventional real-time PCR assays. The classical blood culture detected 58/194 (30 %) of sepsis and 9/44 (21 %) of CSF samples. Out of the 194 blood samples tested, the conventional real-time PCR targeting 13 common sepsis causing pathogens correctly detected the bacterial DNA in 16/194 (8 %) only and 14/44 (32 %) in cerebrospinal fluid samples. Our newly established approach was able to provide correct diagnoses in 78 (40 %) of the 194 blood samples and in 14 (32 %) of the CSF samples. The combination of both blood cultures and our technique raised the rate of sepsis diagnoses to 112/194 (58 %). Of the total group tested positive, 46 (24 %) cases showed overlap with the classical methodology.ConclusionWe report a simple optimized in-house protocol for removal of human DNA from blood sepsis samples as a pre-analytical tool to prepare DNA for subsequent PCR assays. With the detection increase of our in-house DNA removal approach, subsequent PCR assays can reach detection limits of 10 E. coli CFUs/ml and significantly improve the diagnostic rate in blood sepsis cases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-016-1568-1) contains supplementary material, which is available to authorized users.

Surgical site infection (SSI) is common in Vietnamese post-operative patients. It contributes to increased morbidity, mortality, hospitalization time and health care expenditure. Bacterial culture is considered the gold standard procedure to identify SSI pathogens and antibiotic resistant properties; however, it can detect microbes that can readily grow and is time-consuming. We propose optimized multiplex PCR assays to diagnose the most relevant microbes and associated genes encoding for acquired extended spectrum betalactamases (ESBL) or carbapenemases from Vietnamese patients with SSI in a hospital setting in Hanoi.MethodsNinety-one patients (n = 91) were collected in order to identify microbial pathogens and associated genes encoding for acquired extended spectrum betalactamases (ESBL) or carbapenemases by both conventional bacterial culture and in-house multiplex PCR assays.Result and conclusionThe novel in-house multiplex PCR assays are comparable to the bacterial culture approach in screening for common pathogens causing SSI and for relevant genotypes conferring betalactam/carbapenem resistance for bacteria. This is the first report of Turkey-specific ESBL gene (PER-1) and two Oxacilinase families (Oxa23 and Oxa 58) in Vietnam.

BACKGROUND: Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are severe cutaneous adverse drug reactions. Human leukocyte antigens (HLA) may play an important role in the pathogenesis of SJS/TEN. AIMS: This study aims to identify HLA-B alleles in Vietnamese patients with SJS/TEN and to investigate the possible link between HLA-B alleles and causative drugs. MATERIALS AND METHODS: Sixty patients including SJS (30 patients) and TEN (30 patients) were enrolled in a cross-sectional descriptive study at two hospitals in Hanoi, Vietnam, from July 2018 to July 2019. Clinical features and laboratory findings were noted, HLA-B alleles were analyzed by the polymerase chain reaction (PCR)-sequence-specific oligonucleotide assay and LuminexTM Multiplex Technology. RESULTS: The most common HLA-B allele was HLA-B*15:02 (41.7%) followed by HLA-B*58:01 (25%) and HLA-B*46:01 (15%). Of the 25 patients possessing HLA-B*15:02 allele, culprit medicines were carbamazepine (13 patients; 52%), traditional medicine (two patients; 8%), and unknown drugs (seven patients; 28%). Of the 15 patients carrying HLA-B*58:01 allele, there were 13 patients whose offending medicine was allopurinol. Of the eight patients whose culprit drug was traditional medicine, there were 6 patients (75%) carrying HLA-B*51:02. Patients who carry HLA-B*15:02 were found to have 4 times higher risk of developing carbamazepine-induced SJS/TEN as compared with the tolerant control group (OR=4.17; 95% CI=2.07–8.37; p < 0.001). CONCLUSION: HLA-B*15:02 was the most common HLA-B allele in Vietnamese patients with SJS/TEN. In traditional medicine-induced SJS/TEN patients, HLA-B*51:02 allele might play an important role. The link between the HLA-B genotypes and causative drugs may suggest physicians to avoid risk medications for certain patients.