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Ribosomal RNA gene amplicon sequencing is commonly used to evaluate microbiome profiles in health and disease and document the impact of interventional treatments. Nanopore sequencing is attractive since it can provide greater classification at the species level. However, optimized protocols to target marker genes for bacterial and fungal profiling are needed. To achieve an increased taxonomic resolution, we developed extraction and full-length amplicon PCR-based approaches using Nanopore sequencing. Three lysis conditions were applied to a mock microbial community, including known bacterial and fungal species: ZymoBIOMICS lysis buffer (ML) alone, incorporating bead-beating (MLB) or bead-beating plus MetaPolyzyme enzymatic treatment (MLBE). In profiling of bacteria in comparison to reference data, MLB had more statistically different bacterial phyla and genera than the other two conditions. In fungal profiling, MLB had a significant increase of Ascomycota and a decline of Basidiomycota, subsequently failing to detect Malassezia and Cryptococcus. Also, a principal coordinates analysis plot by the Bray-Curtis metric showed a significant difference among groups for bacterial (P=0.033) and fungal (P=0.012) profiles, highlighting the importance of understanding the biases present in pretreatment. Overall, microbial profiling and diversity analysis revealed that ML and MLBE are more similar than MLB for both bacteria and fungi; therefore, using this specific pipeline, bead-beating is not recommended for whole gene amplicon sequencing. However, ML alone was suggested as an optimal approach considering DNA yield, taxonomic classification, reagent cost and hands-on time. This could be an initial proof-of-concept study for simultaneous human bacterial and fungal microbiome studies.

Introduction. Hepatitis B infection causes a spectrum of clinical diseases varying from asymptomatic infection to severe or fulminant acute hepatitis, chronic liver disease, cirrhosis and hepatocellular carcinoma. Hepatitis B virus (HBV) genotypes appear to influence transmission dynamics, clinical outcomes and responses to antiviral therapy. However, hepatitis B genotyping has been poorly investigated in Sri Lanka. This study intended to determine hepatitis B genotypes in a group of HBV-infected people in central and northern Sri Lanka. Methodology. The study was a laboratory-based descriptive cross-sectional study. Initial detection of HBV DNA in 100 EDTA blood samples was done by using a commercially validated quantitative real-time PCR kit. Hepatitis B genotyping was performed by in-house conventional semi-nested multiplex PCR using genotype-specific primers (for genotypes A-F). The serological profile was determined using a commercially validated ELISA/chemiluminescence immunoassay. The results were evaluated for genotype prevalence, viral load association and hepatitis B e antigen (HBeAg) expression in the study population. Results and conclusion. The study detected that genotype C (n=38) is most prevalent and infections with multiple genotypes (n=52, 52%) were commoner than mono-genotype (n=23, 23%) infections. In total, 25% of patients had no detectable genotype among genotypes A-F. The mean viral load in asymptomatic patients with a single genotype was 3.28 log₁₀ copies ml^-1 and in multiple genotypes was 4.18 log₁₀ copies ml^-1 before treatment. Statistical significance was not detected in mean viral loads and HBeAg expression in these two groups. In the future, chronic HBV infection may be effectively treated and managed according to the infected genotype.

Lactiplantibacillus pentosus is a probiotic bacterium reported to be present in various fermented foods, such as fermented olives, and it significantly influences human health. The present study concerns a lactic acid bacterial strain designated L. pentosus krglsrbmofpi2, isolated from traditional fermented rice, and which has been shown to have an assortment of beneficial attributes. Using Illumina technologies, we have sequenced and investigated the whole genome sequence of L. pentosus krglsrbmofpi2 to understand its functionality and safety. The chromosomal genome was 3.7 Mb in size with 46% GC content and 3192 protein-coding genes. Additional extensive bioinformatics investigations were carried out involving whole genome sequence assembly and annotation.

Introduction. There are many multidrug-resistant isolates of the nosocomial pathogen, Acinetobacter baumannii, causing severe healthcare-acquired infections in terminally ill patients with high mortality and morbidity rates. Aim. This study aims to retrospectively analyse A. baumannii bacteraemia (ABB) cases in Saudi Arabia, where the information is sparse regarding the prevalence, risk factors, clinical disease, antibiotic regimen, antibiotic susceptibility, treatment outcomes and mortality associated with this infection. Methods. A retrospective chart review was conducted between 1 January 2015 and 31 December 2022 to identify all patients aged 14 years and above with ABB. Demographic and clinical data, as well as results from laboratory analyses, were collected from patients' electronic charts. Statistical analyses were performed on the data to identify factors associated with 90-day mortality. Results. Of the 122 ABB cases, 71 (63.4%) died. The factors that were found to be associated with 90-day mortality were the Charlson Comorbidity Index, Pitt bacteraemia score, quick Sequential Organ Failure Assessment score (P<0.001 for each), hospital ward (P<0.02), short duration of antibiotic treatment (P<0.01) and higher age (P<0.05). The most common source of infection was central line-associated bloodstream infection in 52.7%. Also associated with mortality were inappropriate antimicrobial therapy (P<0.02) and empirical use of colistin (P<0.05). In many patients, ABB was caused by carbapenem-resistant A. baumannii [(CRAB), 69.6%], and 74.4% of those patients died. Conclusion. To prevent ABB-associated mortality, an appropriate regimen and duration of treatment are necessary. Hospitals should also practice proper hygiene to prevent the spread of ABB. CRAB is a growing threat in hospitals in Saudi Arabia, especially in the critical care setting, and carries a very high risk of mortality.

We describe the whole-genome sequences of seven diverse marine bacteria isolated from Portuguese environments that presented the ability to promote the growth of the model diatom, Phaeodactylum tricornutum. The bacterial genome sequences will contribute to the study of genetic and molecular mechanisms involved in diatom-bacteria interactions.

The rat is a useful laboratory model for respiratory diseases. SARS-CoV-2 proteins, such as the spike (S) protein, can induce inflammation. This study has investigated the ability of the Q498Y, P499T (QP-YT) amino acid change, described in the S-protein of the mouse-adapted laboratory SARS-CoV-2 MA strain, to interact with rat angiotensin converting enzyme-2 (ACE2) and stimulate responses in rat lungs. A real-time S-ACE2 quantitative fusion assay shows that ancestral and L452R S-proteins fuse with human but not rat ACE2 expressed on HEK293 (human embryonic kidney-293) cells. The QP-YT S-protein retains the ability to fuse with human ACE2 and increases the binding to rat ACE2. Although lower lung of the rat contains both ACE2 and TMPRSS2 (transmembrane serine protease 2) target cells, intratracheal delivery of ancestral or QP-YT S-protein pseudotyped lentivirus did not induce measurable respiratory changes, inflammatory infiltration or innate mRNA responses. Isolation of primary cells from rat alveoli demonstrated the presence of cells expressing ACE2 and TMPRSS2. Infection of these cells, however, with ancestral or QP-YT S-protein pseudotyped lentivirus was not observed, and the QP-YT S-protein pseudotyped lentivirus poorly infected HEK293 cells expressing rat ACE2. Analysis of the amino acid changes across the S-ACE2 interface highlights not only the Y498 interaction with H353 as a likely facilitator of binding to rat ACE2 but also other amino acids that could improve this interaction. Thus, rat lungs contain cells expressing receptors for SARS-CoV-2, and the QP-YT S-protein variant can bind to rat ACE2, but this does not result in infection or stimulate responses in the lung. Further, amino acid changes in S-protein may enhance this interaction to improve the utility of the rat model for defining the role of the S-protein in driving lung inflammation.

Metagenomics has been transformative in our understanding of the diversity and function of soil microbial communities. Applying long read sequencing to whole genome shotgun metagenomics has the potential to revolutionise soil microbial ecology through improved taxonomic classification, functional characterisation and metagenome assembly. However, optimisation of robust methods for long read metagenomics of environmental samples remains undeveloped. In this study, Oxford Nanopore sequencing using samples from five commercially available soil DNA extraction kits was compared across four soil types, in order to optimise read length and reproducibility for comparative long read soil metagenomics. Average extracted DNA lengths varied considerably between kits, but longer DNA fragments did not translate consistently into read lengths. Highly variable decreases in the length of resulting reads from some kits were associated with poor classification rate and low reproducibility in microbial communities identified between technical repeats. Replicate samples from other kits showed more consistent conversion of extracted DNA fragment size into read length and resulted in more congruous microbial community representation. Furthermore, extraction kits showed significant differences in the community representation and structure they identified across all soil types. Overall, the QIAGEN DNeasy PowerSoil Pro Kit displayed the best suitability for reproducible long-read WGS metagenomic sequencing, although further optimisation of DNA purification and library preparation may enable translation of higher molecular weight DNA from other kits into longer read lengths. These findings provide a novel insight into the importance of optimising DNA extraction for achieving replicable results from long read metagenomic sequencing of environmental samples.

Hydatidosis, also known as cystic echinococcosis, is a widespread zoonosis, caused by a tapeworm of the genus Echinococcus. It presents a significant public health concern, particularly in endemic areas. The occurrence of disseminated hydatid disease is uncommon, even in regions where it is endemic, with an incidence ranging from 1-8%. The definitive diagnosis relies on a parasitological method. In this work, we present an unusual case of disseminated hydatid disease that was diagnosed in the central parasitology-mycology laboratory of 'The Ibn Sina University Hospital'. This is a 21-year-old patient residing in a rural area, who presented with heaviness-type pain in the right hypochondrium, accompanied with nausea and vomiting. During the examination, the patient mentioned the contact with dogs. Abdominal radiography (ultrasound and CT) revealed findings suggestive of multiple hydatid cysts located in the liver and peritoneum. This suspicion was confirmed by positive hydatid serology. After 9 months of treatment with albendazole, the patient underwent surgery for excision of the cysts shown on the x-ray, as well as other cysts incidentally discovered intraoperatively at the pelvic and rectal levels. All of the extracted specimens were sent to the parasitology laboratory. The direct examination, along with the viability test, revealed the presence of hooks and scolex of non-viable Echinococcus granulosus. Disseminated hydatidosis is a rare but serious presentation, and the positive diagnosis relies on several epidemiological, clinical, radiological and parasitological arguments. Medical and surgical treatments play a crucial role in determining the patient's prognosis.

Introduction. Enteric pathogens contribute significantly to morbidity in a developing country such as India. Early and prompt diagnosis of diarrhoeal diseases can reduce the mortality rate, particularly in children. The pattern of sensitivity to antimicrobials for the common pathogens can vary from time to time. The present study was conducted to study the pathogen distribution and antimicrobial susceptibility pattern during the study period (January 2010 to December 2023). Hypothesis/gap statement. Studying the changing trend in the antimicrobial sensitivity pattern of diarrhoeal pathogens over a decade can help to plan future treatment options. Aim. This study was undertaken to provide insights into the changing pattern of pathogen distribution and antimicrobial susceptibility for enteric pathogens over 14 years. Methods. A retrospective observational cohort analysis was conducted on all the stool pathogens isolated from the samples received in the microbiology department of a tertiary care hospital from 2010 to 2023. The demographic details, stool microscopy, culture reports, and antimicrobial susceptibility patterns were noted. Results. A total of 18 336 stool specimens were received in the microbiology laboratory between January 2010 and December 2023, of which 1354 specimens had diarrhoeal pathogens grown in culture. Out of these 1354 specimens, 591 (44%) had Salmonella, 471 (35%) Shigella, 181 (13%) Vibrio cholerae, and 80 (6%) Aeromonas species. Among these pathogens, susceptibility to ceftriaxone was seen in 93% (552 isolates) of Salmonella species, 89% (420 isolates) of Shigella species, and 95% (171 isolates) of Vibrio cholerae; 91% (73 isolates) of Aeromonas species were susceptible to chloramphenicol. Some major parasites were also observed on microscopy. Conclusion. Timely diagnosis of diarrhoeal pathogens can be life-saving for patients at the extremes of age, i.e. in children and the elderly. Pathogens can exhibit a changing susceptibility pattern to antibiotics, which should be regularly observed to plan future therapy.