Detection of Nocardia by 16S Ribosomal RNA Gene PCR and Metagenomic Next-Generation Sequencing (mNGS)

In this study, the aim was to investigate the discriminatory power of molecular diagnostics based on mNGS and traditional 16S ribosomal RNA PCR among Nocardia species. A total of fourteen clinical isolates from patients with positive Nocardia cultures and clinical evidence were included between January 2017 and June 2020 in HeNan Provincial People’s Hospital. DNA extraction and 16S rRNA PCR were performed on positive cultures, and pathogens were detected by mNGS in these same samples directly. Among the 14 Nocardia isolates, four species were identified, and N. cyriacigeorgica (8 cases) is the most common species. Twelve of the 14 Nocardia spp. isolates were identified by the two methods, while two strains of N. cyriacigeorgica were not identified by mNGS. All tested isolates showed susceptibility to trimethoprim-sulfamethoxazole (SXT), amikacin and linezolid. Apart from Nocardia species, other pathogens such as Acinetobacter baumannii, Klebsiella pneumonia, Aspergillus, Enterococcus faecalis, Human herpesvirus, etc., were detected from the same clinical samples by mNGS. However, these different pathogens were considered as colonization or contamination. We found that it is essential to accurately identify species for determining antibiotic sensitivity and, consequently, choosing antibiotic treatment. 16S rRNA PCR was useful for identification of nocardial infection among species, while this technique needs the clinicians to make the pre-considerations of nocardiosis. However, mNGS may be a putative tool for rapid and accurate detection and identification of Nocardia, beneficial for applications of antimicrobial drugs and timely adjustments of medication.

Ursodeoxycholic Acid Treatment Restores Gut Microbiota and Alleviates Liver Inflammation in Non-Alcoholic Steatohepatitic Mouse Model

Gut microbiota dysbiosis plays an important role in the progression of non-alcoholic fatty liver disease (NAFLD), and no approved drugs are available for NAFLD treatment. In this study, we aimed to explore the dynamic changes of gut microbiota at the different stages of NAFLD and determine whether ursodeoxycholic acid (UDCA) could improve liver histopathological features of non-alcoholic steatohepatitis (NASH) mice induced by a high-fat high-cholesterol (HFHC) diet and its impact on gut microbiota. 6-week-old male C57BL/6 mice were fed with a HFHC or normal diet for 12, 18, and 24 weeks, respectively, to simulate the different stages of NAFLD. 16s ribosomal RNA genes from mice fecal samples at the different time points were sequenced to evaluate the dynamic changes of the gut microbiota. Then, C57BL/6 mice were fed with a HFHC diet for 24 weeks to establish the NASH model. Different doses of UDCA were administered intragastrically for additional 4 weeks. Normal diet–fed mice were taken as control. Serum samples, liver, and intestine tissues were harvested for biochemical tests and histopathological examinations. 16s ribosomal RNA genes from mice fecal samples were sequenced to assess the structural changes of gut microbiota. HFHC diet–fed mice developed simple steatosis, steatohepatitis, and fibrosis at 12, 18, and 24 weeks, respectively. The profile of gut microbiota dynamically changed with the different stages of NAFLD. NASH mice had significantly higher abundance of Fecalibaculum, Coriobacteriaceae_UCG-002, and Enterorhabdus, and lower abundance of norank_f_Muribaculaceae, Bacteroides, and Alistipes, which were partially restored by UDCA treatment. UDCA treatment significantly attenuated hepatic inflammation of NASH mice as indicated by the sum of ballooning and lobular inflammation of the NALFD activity score (3.2 ± 0.8 vs 1.8 ± 0.8, p = 0.029), and partially restored gut microbiota dysbiosis, and increased the expression of Claudin-1 and ZO-1 in the intestine, but did not activate the suppressed Farnesoid X receptor signal pathway. Conclusions: The gut microbiota dynamically changes with the different stages of NAFLD. UDCA treatment (120 mg/kg) could partially restore gut microbiota, repair gut barrier integrity, and attenuate hepatic inflammation in the NASH mouse model.

Endodontic Recontamination and Retreatment: The Concise Systematic Review

Introduction: It is necessary to know the nature of the endodontic microbiota within the root canal system of teeth with necrotic pulp tissues. There are several methods of microbial identification, including techniques based on culture or non-predominance of facultative anaerobes and Gram-positive species, especially Enterococcus faecalis. The 16S ribosomal RNA (rRNA) gene sequencing approach has become the reference method. Polymerase chain reaction (PCR) is a molecular technique also used. A condition for successful endodontic retreatment is proper cleaning of the root canals. Objective: Evaluate through a systematic literature review the main contaminations, recontaminations, and endodontic retreatments in root canals. Methods: The present study was followed by a systematic literature review model, according to the PRISMA rules. Clinical studies included case reports, retrospective, prospective and randomized trials. The quality of the studies was based on the GRADE instrument. The risk of bias was analyzed according to the Cochrane instrument. Results: A total of 94 articles were found. A total of 58 articles were evaluated in full, and 34 were included and discussed in this study. The overall assessment did not result in significant risks that could compromise the science of the present study. According to the GRADE classification, the studies were of moderate quality. Conclusion: It was concluded that it is essential to characterize the microbiota of root canals with failed endodontic treatment through 16S ribosomal RNA (GS) gene sequencing and PCR. Furthermore, it can be stated that the root canal instrumentation system with rotating files maintains the quality of root preparation, reducing the operative time and also the risk of a torsional fracture within the root canal.

First genetically confirmed records of the little gulper shark Centrophorus uyato (Squaliformes: Centrophoridae) from Cypriot waters

The taxonomy within the genus Centrophorus has been controversial almost since its origin, raising uncertainties about the identification, the phylogenetic placement and the geographical distribution of several species. The partial nucleotide sequences of two mitochondrial DNA gene regions, the cytochrome c oxidase subunit I and the 16S ribosomal RNA, genetically confirmed the presence of the little gulper shark in Cypriot waters. The species presence in the Mediterranean Sea is revised and discussed.

HyperEx: A Tool to Extract Hypervariable Regions from 16S rRNA Sequencing Data

The 16S ribosomal RNA gene is one of the most studied genes in biology. This 16S ribosomal RNA importance is due to its wide application in phylogenetics and taxonomic elucidation of bacteria and archaea. Indeed, 16S ribosomal RNA is present in almost all bacteria and archaea and has, among many other useful characteristics, a low mutation rate. The 16S ribosomal RNA is composed of nine hypervariable regions which are commonly targeted by high throughput sequencing technologies in identification or community studies like metabarcoding studies. Unfortunately, the hypervariable regions do not have the same taxonomic resolution among all bacteria taxa. This requires a preliminary in silico analysis to determine the best hypervariable regions to target in a particular study. Nevertheless, to the best of our knowledge, no automated primer-based open-source tool exists to extract hypervariable regions from complete or near-complete 16S rRNA sequencing data. Here we present HyperEx which efficiently extracts the hypervariable region of interest based on embedded primers or user-given primers. HyperEx implements the Myers algorithm for the exact pairwise sequence alignment. HyperEx is freely available under the MIT license as an operating system independent Rust command-line tool at https://github.com/Ebedthan/hyperex and https://crates.io.

Molecular characterization of oral bacteria isolated from human saliva

Periodontitis is an inflammation of gums and bones that supporting the teeth which caused by Staphylococcus intermedius. The saliva from a patient of periodontitis or suspect to periodontitis will have higher levels of Staphylococcus intermedius. Hence, human saliva is clinically informative in diagnosing oral disease and the oral health of an individual. In this study, oral bacteria in human saliva were identified using 16S ribosomal RNA. 16S ribosomal RNA (rRNA) genes from the isolated colonies were amplified through the colony Polymerase Chain Reaction (PCR) method. 16S rRNA genes were used to determine species identity by sequencing and generating the phylogenetic tree. The results showed that Streptococcus sp. and Staphylococcus sp. were the most prevalent oral bacteria found from all the saliva samples, while Lactobacillus sp. was found from two samples. From the constructed phylogenetic trees, bacteria strains B1 and B2 clustered with the Staphylococcus sp. database. Bacteria strains B9 and B10 were categorized as Streptococcus sp. as the confidential level between Streptococcus sp. database is 100% in Neighbour-Joining tree. Sample B15 and B16 clustered with Lactobacillus sp. database. Oral bacteria species typically associated with periodontitis was detected in all saliva samples. Therefore, it is important to fully understand the nature of the oral bacteria before further research on drug design and administration of oral treatment is executed.

Rapid and accurate detection of aminoglycoside modifying enzymes and 16S ribosomal RNA methyltransferases by targeted LC-MS/MS

New and rapid diagnostic methods are needed for the detection of antimicrobial resistance to aid in the curbing of drug-resistant infections. Targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a method that could serve this purpose, as it can detect specific peptides of antimicrobial resistance mechanisms with high accuracy. In the current study, we developed an accurate and rapid targeted LC-MS/MS assay based on parallel reaction monitoring for detection of the most prevalent aminoglycoside modifying enzymes and 16S ribosomal RNA methyltransferases in E. coli and K. pneumoniae that confer resistance to aminoglycosides. Specific tryptic peptides needed for detection were selected and validated for AAC(3)-Ia, AAC(3)-II, AAC(3)-IV, AAC(3)-VI, AAC(6’)-Ib, AAC(6’)-Ib-cr, ANT(2”)-I, APH(3’)-VI, ArmA, RmtB, RmtC and RmtF. In total, 205 isolates containing different aminoglycoside resistance mechanisms that consisted mostly of E. coli and K. pneumoniae were selected for assay development and evaluation. Mass spectrometry results were automatically analyzed and were compared to whole genome sequencing results. Of the 2460 isolate and resistance mechanism combinations tested, 2416 combinations matched. Discrepancies were further analyzed by repeating LC-MS/MS analysis and performing additional PCRs. Mass spectrometry results were also used to predict resistance and susceptibility to gentamicin, tobramycin and amikacin in only the E. coli and K. pneumoniae isolates (n=191). The category interpretations were correctly predicted for gentamicin in 97.4% of the isolates, for tobramycin in 97.4% of the isolates, and for amikacin in 82.7% of the isolates. Targeted LC-MS/MS can be applied for accurate and rapid detection of aminoglycoside resistance mechanisms.