- Volume 4, Issue 6, 2022

Modification of DNA bases plays important roles in the epigenetic regulation of eukaryotic gene expression. Among the different types of DNA methylation, 5-methylcytosine (5mC) is common in higher eukaryotes. Although bisulfite sequencing is the established detection method for this modification, newer methods, such as Oxford nanopore sequencing, have been developed as quick and reliable alternatives. An earlier study using sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) indicated the presence of 5mC at very low concentration in Saccharomyces cerevisiae. More recently, a comprehensive study of the yeast genome found 40 5mC sites using the computational tool Nanopolish on nanopore sequencing output raw data. In the present study, we are trying to validate the prediction of the 5mC modifications in yeast with Nanopolish and two other nanopore software tools, Tombo and DeepSignal. Using publicly available genome sequencing data, we compared the open-access computational tools, including Tombo, Nanopolish and DeepSignal, for predicting 5mC. Our results suggest that these tools are indeed capable of predicting DNA 5mC modifications at a specific location from Oxford nanopore sequencing data. We also predicted that 5mC present in the S. cerevisiae genome might be located predominantly at the RDN locus of chromosome 12.

Introduction. Group B Streptococcus (GBS) is a human commensal bacterium that is also associated with infection in pregnant and non-pregnant adults, neonates and elderly people.

Gap Statement. The authors hypothesize that knowledge of regional GBS genetic patterns may allow the use of prevention and treatment measures to reduce the burden of streptococcal disease.

Aim. The aim was to report the genotypic diversity and antimicrobial sensitivity profiles of invasive, noninvasive urinary and colonizing GBS strains, and evaluate the relationships between these findings.

Methodology. The study included consecutive and non-duplicated GBS isolates recovered in southern Brazil from 2015 to 2017. We performed multiple-locus variable-number tandem repeat analysis (MLVA) and PCR analyses to determine capsular serotypes and identify the presence of the resistance genes mefA/E, ermB and ermA/TR, and also antibiotic susceptibility testing.

Results. The sample consisted of 348 GBS strains, 42 MLVA types were identified, and 4 of them represented 64 % of isolates. Serotype Ia was the most prevalent (42.2 %) and was found in a higher percentage associated with colonization, followed by serotypes V (24.4 %), II (17.8 %) and III (7.8 %). Serotype V was associated with invasive isolates and serotypes II and III with noninvasive isolates, without significant differences. All isolates were susceptible to penicillin. GBS 2018/ hvgA was observed in 17 isolates, with 11 belonging to serogroup III. The Hunter–Gaston diversity index was calculated as 0.879. The genes mefA/E, erm/B and erm/A/TR were found in 45, 19 and 46 isolates.

Conclusion. This report suggests that the circulating GBS belong to a limited number of genetic lineages. The most common genotypes were Ia/MT12 and V/MT18, which are associated with high resistance to macrolides and the presence of the genes mefA/E and ermA/TR. Penicillin remains the antibiotic of choice. Implementation of continuous surveillance of GBS infections will be essential to assess GBS epidemiology and develop accurate GBS prevention, especially strategies associated with vaccination.

Introduction. Increased plasma cell-free DNA (cfDNA) has been reported for various diseases in which cell death and tissue/organ damage contribute to pathogenesis, including sepsis.

Gap Statement. While several studies report a rise in plasma cfDNA in bacteraemia and sepsis, the main source of cfDNA has not been identified.

Aim. In this study, we wanted to determine which of nuclear, mitochondrial or bacterial cfDNA is the major contributor to raised plasma cfDNA in hospital subjects with bloodstream infections and could therefore serve as a predictor of bacteraemic disease severity.

Methodology. The total plasma concentration of double-stranded cfDNA was determined using a fluorometric assay. The presence of bacterial DNA was identified by PCR and DNA sequencing. The copy numbers of human genes, nuclear β globin and mitochondrial MTATP8, were determined by droplet digital PCR. The presence, size and concentration of apoptotic DNA from human cells were established using lab-on-a-chip technology.

Results. We observed a significant difference in total plasma cfDNA from a median of 75 ng ml−1 in hospitalised subjects without bacteraemia to a median of 370 ng ml−1 (P=0.0003) in bacteraemic subjects. The copy numbers of nuclear DNA in bacteraemic also differed between a median of 1.6 copies µl−1 and 7.3 copies µl−1 (P=0.0004), respectively. In contrast, increased mitochondrial cfDNA was not specific for bacteraemic subjects, as shown by median values of 58 copies µl−1 in bacteraemic subjects, 55 copies µl−1 in other hospitalised subjects and 5.4 copies µl−1 in healthy controls. Apoptotic nucleosomal cfDNA was detected only in a subpopulation of bacteraemic subjects with documented comorbidities, consistent with elevated plasma C-reactive protein (CRP) levels in these subjects. No bacterial cfDNA was reliably detected by PCR in plasma of bacteraemic subjects over the course of infection with several bacterial pathogens.

Conclusions. Our data revealed distinctive plasma cfDNA signatures in different groups of hospital subjects. The total cfDNA was significantly increased in hospital subjects with laboratory-confirmed bloodstream infections comprising nuclear and apoptotic, but not mitochondrial or bacterial cfDNAs. The apoptotic cfDNA, potentially derived from blood cells, predicted established bacteraemia. These findings deserve further investigation in different hospital settings, where cfDNA measurement could provide simple and quantifiable parameters for monitoring a disease progression.

A 38 year old male HIV positive patient with a history of intravenous drug use presented with chest pains, cough, sputum and weight loss and radiology demonstrated the evolution of a right basal lung abscess. A lung biopsy sent for 16S rRNA analysis and sputum cultured about the same time demonstrated Raoultella ornithinolytica . No other causative pathogens were clearly identified. He gradually improved with a 4 week course of intravenous cefazolin. R. ornithinolytica is a rare, but recognised pathogen.

Invasive infections caused by Capnocytophaga canimorsus , a Gram-negative rod found in the oral cavity of healthy dogs and cats, are rare but they are increasing worldwide. We report a case of septic arthritis in a native knee joint due to this micro-organism. A 57-year-old man, with a well-controlled chronic HIV infection, attended the Emergency Department because of left knee pain and shivering without measured fever. A knee arthrocentesis and a computed tomography scan were performed, revealing septic arthritis with collections in the left leg posterior musculature. He was admitted to the Infectious Diseases Department for antibiotic treatment. Initial synovial fluid was inoculated in blood culture bottles, and the anaerobic one was positive after 63 h. Gram stain revealed fusiform Gram-negative rods, identified as C. canimorsus by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) directly from the bottle. Identification was confirmed by 16S rRNA sequencing and serotyping was performed by PCR, with serovar A as the outcome. Due to an unfavourable clinical course, the patient required two surgical cleanings and after appropriate antibiotic treatment he was discharged 2 months later.