Enrichment of bacterial DNA for the diagnosis of blood stream infections.

BACKGROUND: Blood 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. METHODS: We 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. RESULTS: This 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. CONCLUSION: We 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.

Simple multiplex PCR assays to detect common pathogens and associated genes encoding for acquired extended spectrum betalactamases (ESBL) or carbapenemases from surgical site specimens in Vietnam.

UNLABELLED: 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. METHODS: Ninety-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 CONCLUSION: The 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.

Genome-Wide Identification and Analysis of Genes Encoding Putative Heat Shock Protein 70 in Papaya (Carica papaya).

<b>Background and Objective:</b> In high plants, the 70 kDa heat stress proteins (Hsp70-s) have been regarded as one of the vital components of the cellular network of chaperones and folding catalysts that play important roles in numerous biological processes during growth and development. The Hsp70 families have been reported in many plant species, unfortunately, no information on this important protein family in papaya (<i>Carica papaya</i>). The objective of this study was to provide comprehensive information on the CpHsp70 family in papaya. <b>Materials and Methods:</b> The <i>CpHsp70</i> genes in the papaya genome were identified by a basic local alignment search tool against the papaya genome database by using well-known <i>Arabidopsis</i> Hsp70-s. Sequences were then analyzed by various bioinformatics tools to investigate the characteristics of the CpHsp70 family. <b>Results:</b> A total of 12 members of the CpHsp70 family has been identified and characterized in papaya. By using various computational tools, these results revealed that all general characteristics of the CpHsp70 family, like physic-chemical parameters, gene structure, phylogenetic tree and subcellular localization were provided. The transcriptome atlas was applied to re-analyze the expression patterns of genes encoding the CpHsp70 family in major tissues/organs during the growth and development of papaya plants. <b>Conclusion:</b> Results from this work exhibited the characteristics and expression analysis of the <i>CpHsp70</i> genes of this important tropical fruit crop. Taken together, this study could provide a solid foundation of the CpHsp70 family, which will be helpful in the construction of stress tolerance in papaya plants.

SWEET Gene Family in Sugar Beet (Beta vulgaris): Genome-Wide Survey, Phylogeny and Expression Analysis.

<b>Background and Objective:</b> The SWEET (Sugars Will Eventually be Exported Transporter) proteins play important roles in modulating the growth and development processes in plants. However, little information is available on the SWEET family in sugar beet (<i>Beta vulgaris</i>). The objectives of this present study were to genome-wide identify and characterize the BvSWEET family in sugar beet. <b>Materials and Methods:</b> Based on the available genome, proteome and transcriptome databases of sugar beet, various computational tools have been used to analyze the nucleotide and full-length protein sequences of members of the BvSWEET family. <b>Results:</b> A total of 16 members of the BvSWEET family has been identified in sugar beet at the genome-wide scale. Structural analysis indicated that the BvSWEET family exhibited variable characteristics. Furthermore, the BvSWEET family in sugar beet could be categorized into four distinct groups like in other plant species. Of our interest, we found that some <i>BvSWEET</i> genes exhibited strongly preferential expression in major organs/tissues under adverse environmental stimuli. <b>Conclusion:</b> The results provided a comprehensive foundation for further functional characterization of the <i>BvSWEET </i>gene family.

Diagnostic Function of 3-Tesla Magnetic Resonance Imaging for the Assessment of Brachial Plexus Injury.

BACKGROUND: This study aimed to evaluate the diagnostic function of 3-Tesla (T) magnetic resonance imaging (MRI) during the assessment of brachial plexus injury (BPI), in comparison with intraoperative findings. METHODS: A retrospective study was performed on 60 patients (47 men and 13 women), who had clinical manifestations of BPI, underwent 3T MRI of the brachial plexus, and were surgically treated at the Viet Duc and Vinmec Times City hospitals, in Hanoi, Vietnam, from March 2016 to December 2019. Preganglionic and postganglionic lesion features were identified on MRI. The diagnostic function of MRI features for the determination of BPI was evaluated and correlated with intraoperative findings. RESULTS: The root avulsion and pseudomeningocele preganglionic injuries were observed in 57% and 43% of MRIs, respectively, and were commonly observed at the C7 and C8 roots. Nerve disruption and never edema were observed in 47.56% and 33.53% of MRIs, respectively, and were commonly observed at the C5 and C6 roots. The sensitivity, specificity, accuracy, positive prognostic value, and negative prognostic value of 3T MRI were 64.12%, 92.90%, 80.33%, 87.50%, and 76.96%, respectively, for the diagnosis of total avulsion, and 68.52%, 83.33%, 80.67%, 47.44%, and 92.34%, respectively, for the diagnosis of nerve disruption. CONCLUSION: MRI offers valuable details regarding the location, morphology, and severity of both preganglionic and postganglionic injuries during the preoperative diagnosis of BPI. However, this modality played a moderate diagnostic role. Therefore, 3T MRI should be used as a supplemental evaluation, coupled with clinical tests and electromyography, to determine the most appropriate treatment strategies for BPI patients.

S-bacillithiolation protects conserved and essential proteins against hypochlorite stress in firmicutes bacteria.

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 S-bacillithiolomes 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 S-bacillithiolated 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. S-bacillithiolation 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.