Objective. Hepatitis B virus (HBV) spontaneous mutations may impact the severity of liver disease. This study aimed to assess the mutations in the pre-core (PC) region in HBV-HIV (human immunodeficiency virus) co-infected patients. Additionally, we explored its association with genotypes and examined the clinical implications.Methods. A total of 100 HBV-HIV co-infected patients and 50 HBV mono-infected patients were included in the study. We focused on the PC region of the HBV genome, sequencing it to identify PC mutant variants. PCR products were quantified via spectrophotometry and sequenced using the Sanger method. The resulting sequences were assembled, annotated and aligned in a single reading frame. Subsequent mutational and phylogenetic analyses were performed using UGENE software to determine the genotypes of the isolates.Results. The PC region was successfully amplified and sequenced in 27 samples, comprising 16 from HBV-HIV co-infected patients and 11 from HBV mono-infected patients. Phylogenetic analysis identified two HBV genotypes: genotype D, which was predominant and found in 24 samples (88.9%), and genotype A, present in 3 samples (11.1%). A T-to-C mutation at nucleotide position 1912 was detected in 48.1% of the patients. Furthermore, several additional PC mutations were observed, including A1850T, C1858T, G1899A, G1862T, G1951T, T1812C and T1809G, along with novel mutations such as C1936T, A2011G, T2020A and C2044T. Notably, the prevalence of these PC mutations did not significantly differ between the HBV mono-infected and HBV-HIV co-infected groups.Conclusion. This study underscored the prevalence of PC mutations in HBV-HIV co-infected patients. Although several of these mutations have been previously reported, our findings also revealed novel variants. Further research is needed to elucidate the clinical significance of these new mutations.

Interactions between environmental protists and bacteria play a crucial role in shaping bacterial survival strategies and pathogenic potential. Certain bacteria have evolved mechanisms to resist predation by protists such as Acanthamoeba, allowing them to persist intracellularly and, in some cases, enhance their virulence. We hypothesize that Acanthamoeba species may also play a role in promoting antimicrobial resistance (AMR) in amoeba-resistant bacteria. This study investigated whether Acanthamoeba castellanii enhanced AMR development in Pseudomonas putida under lethal ciprofloxacin concentrations. P. putida was co-incubated with A. castellanii and maintained in ciprofloxacin concentrations starting at 2 µg ml-1, four times the planktonic MIC, which was incrementally increased as resistance emerged. The survival of the co-incubated P. putida and the development of resistance were monitored, and antimicrobial susceptibility tests were conducted using multiple antibiotics. P. putida co-incubated with A. castellanii in the presence of ciprofloxacin became increasingly resistant in a dose-dependent manner, with the MIC increasing from 0.5 to 20 µg ml-1 after 17 days. Contrastingly, the naïve strain did not survive sustained exposure at 2 µg ml-1. Co-incubated bacteria maintained under ciprofloxacin pressure developed resistance to ciprofloxacin, chloramphenicol, azithromycin and enrofloxacin while retaining susceptibility to streptomycin and tetracycline. Co-incubation in the absence of ciprofloxacin did not promote resistance in P. putida, suggesting that the combination of extracellular drug pressure and intracellular survival is important in driving resistance. These findings indicate that intracellular survival within Acanthamoeba can significantly accelerate AMR development in P. putida under fluoroquinolone pressure. Further research into the molecular mechanisms involved is warranted to inform strategies for mitigating AMR emergence in clinical and environmental contexts.

Background. Nanopore‐based sequencing by Oxford Nanopore Technologies (ONT) offers rapid, cost‐effective and portable sequencing. As an emerging technology, ONT must be evaluated for efficacy and practical application in both high‐ and low‐resource settings. This scoping review (SR) aimed to (1) describe how nanopore technology is used in Africa for surveillance and diagnosis of human infectious diseases, (2) describe how nanopore technology aids in the real-time detection of infectious pathogens in Africa and (3) identify challenges and opportunities for utilizing nanopore technology in Africa to study infectious diseases.Methods. This SR followed the Joanna Briggs Institute Reviewer’s Manual framework for SRs. English language studies published from 1 January 2008 to 30 April 2024 that used ONT on human specimens collected in Africa and targeted ≥1 microbial agent were included. Searches were performed in Embase, Medline, PubMed, CINAHL and the Cochrane Library. The protocol was publicly available on the Open Science Framework Bastug et al. (Nanopore Sequencing for Infectious Diseases Surveillance and Diagnostics in Africa: a Scoping Review 2024) prior to data collection. Two independent reviewers screened studies using Covidence, and data was extracted using a custom REDCap instrument. Descriptive statistics and data visualization were performed in Microsoft Excel.Results. One thousand one hundred sixty-two studies were identified and 93 (8%) underwent full-text review. The portable MinION Mk1B was the most common ONT device (65% of studies). Eighty-eight studies analysed specimens from a single African country. Of these, 45% were sequenced in the same country, 7% in a different African country and 11% in a non-African country, while 32% did not specify the location. Specimen types included direct patient specimens (62%) and cultured isolates (35%), or a combination of both. Blood, serum or plasma was most common (35%), followed by naso- or oropharyngeal specimens (27%). Forty-four studies used ONT during an active infectious disease outbreak, 25 of which studied severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Seventy-two studies used ONT for genomic surveillance of infectious pathogens or antibiotic resistance genes, and one study used ONT for a direct clinical application. African-affiliated authors were included as first, middle and last authors in 46% of studies, and 15% were published by entirely African-affiliated teams. Ten studies published information on workflow timeline, and five studies published the per-specimen cost.Conclusions. ONT can enable timely and affordable sequencing in African countries as demonstrated through a small number of studies that accomplished these goals individually. Most studies used ONT for genomic surveillance of pathogens or antimicrobial resistance genes, while only one study used ONT directly for a real-time clinical application. A small number of studies described a short interval between specimen collection and sequence result, supporting that clinical applications are possible. There is a need for improved reporting of ONT methodology including pipeline timelines, cost, use of barcoding, flow cell models and the use of negative controls. Publications that provide these details will enhance reproducibility and support the development of new studies using ONT for the diagnosis and surveillance of infectious diseases in low-resource settings.

Little is known regarding the vaginal microbiota of sheep that undergo spontaneous abortions. The aim of this pilot study was to characterize, using 16S rRNA gene sequencing and shotgun metagenomics, the vaginal microbiota throughout the gestation of two ewes (Ewe1 and Ewe2) that spontaneously aborted. To achieve this, weekly vaginal swabs were collected from the ewes prior to breeding until pregnancy testing; thereafter, biweekly swabs were collected until the spontaneous abortion occurred. Based on the 16S rRNA sequencing data, Ewe1’s vaginal microbiota, overall, contained high abundances of Histophilus (12.9% relative abundance), Staphylococcus (10.8% relative abundance) and Unclassified Pasteurellaceae (8.7% relative abundance). Most notable was the high abundance of Campylobacter following the abortion in Ewe1’s vaginal microbiota. Ewe2’s vaginal microbiota was characterized by high abundances of Pasteurella (41.7% relative abundance) throughout gestation. Shotgun metagenomic sequencing produced two high-quality metagenome-assembled genomes (MAGs), identified as Campylobacter jejuni and Histophilus somni. The C. jejuni MAG had 99.95% average nucleotide identity to the most abundant sheep abortive C. jejuni clone in the USA. The H. somni MAG was most similar to a pathogenic H. somni strain and contained genes that contribute to serum resistance and sialic acid utilization. The results presented here demonstrate the need for continued research into the vaginal microbiota, specifically to identify potential predictors of spontaneous abortion.

The mangrove ecosystems are of great ecological importance found in tropical and subtropical coasts, including Malaysia. The microbial communities in the mangrove sediments play an indispensable role in maintaining homeostasis and supporting biodiversity. However, mangroves are facing various threats due to increasing anthropogenic activities. Thus, it is important to monitor the microbial community to improve our understanding of anthropogenic pressure on reshaping these ecosystems. This study examines the microbial community diversity in mangrove sediments of southern peninsular Malaysia. High-throughput MinION sequencing of the 16S rRNA gene was performed to compare the soil microbiome diversity in 35 samples from 8 different mangroves representing Sungai Sedili Kecil and Sungai Sedili Besar that flow into the South China Sea; Sungai Pulai, Sungai Melayu, Sungai Danga, Sungai Skudai and Sungai Johor that join the Straits of Johor; and Pulau Kukup from the Straits of Malacca. The metagenomic classification performed with 16S rRNA showed 2,573 taxa comprising 32 phyla. Total abundance analysis showed Pseudomonadota (67-69%), Bacteroidota (6-8%), Bacillota (5-8%), Campylobacterota (4-5%), Acidobacteriota (3-4%), Planctomycetota (2-4%) and Actinomycetota (1-2%) as the relatively common phyla. Alpha diversity indices revealed significantly higher richness in samples from mangroves of the South China Sea. Further, the 'Shannon' index showed a significant difference in diversity between Sungai Melayu and Sungai Pulai. Higher abundance of Burkholderiaceae, Bacillaceae and Enterobacteriaceae suggests a difference in the microbial community structure. This study stands as the first comprehensive analysis of microbial communities for future monitoring and conservation in these mangroves.

Antibiotic-resistant infections cause an estimated 2.8 million illnesses and 35,900 deaths annually in the USA. Carbapenems are a class of antibiotics that are generally reserved to treat life-threatening invasive infections including sepsis. Accurate diagnosis of carbapenem-resistant infections is critical for early and appropriate treatment. bla IMP encodes bacterial production of the IMP metallo-beta-lactamase (MBL), which can confer resistance to all the beta-lactams including carbapenems. Zinc is an essential co-factor in the IMP MBL enzymatic hydrolysis of carbapenems. Tests for the presence of IMP carbapenemase, such as the Carba NP, include zinc sulphate (ZnSO4) although broth dilution methods for determining MIC for carbapenems may vary. We hypothesized that ZnSO4 availability would improve the accuracy of carbapenem MIC determination for bacteria expressing bla IMP. Thus, the objective of this study was to determine if supplemental ZnSO4 affects the carbapenem MICs of Enterobacterales, Alteromonadales and Moraxellales expressing bla IMP. Isolates utilized for this study were originally recovered from environmental samples collected at farms, wastewater treatment plants and surface water. They were selected based on phenotypic non-susceptibility to carbapenems and genetic confirmation of bacterial carriage of bla IMP. Cation-adjusted Mueller-Hinton broth suspensions of each isolate standardized to a 0.5 MacFarland standard were tested with and without ZnSO4 added at 0.1 mmol l-1 concentration to determine MICs using standard extended-spectrum beta-lactamase microbroth dilution MIC panels. Although we observed that Morganellaceae imipenem MICs were higher (P<0.001) than those from other bacteria harbouring bla IMP, the inclusion of supplemental ZnSO4 did not influence carbapenem MIC. This suggests that supplemental ZnSO4 will not improve the accuracy of carbapenem MICs in environmental bacteria expressing IMP carbapenemase. Additional research will be required to identify important factors that may influence the expression of carbapenemase including IMP and the accurate determination of clinical MICs, which is critical to appropriate therapeutic decision-making.

Over the past three decades, molecular epidemiological studies have provided new opportunities to investigate the transmission dynamics of Mycobacterium tuberculosis. In most studies, a sizable fraction of individuals with notified tuberculosis cannot be included, either because they do not have culture-positive disease (and thus do not have specimens available for molecular typing) or because resources for conducting sequencing are limited. A recent study introduced a regression-based approach for inferring the membership of unsequenced tuberculosis cases in transmission clusters based on host demographic and epidemiological data. This method was able to identify the most likely cluster to which an unsequenced strain belonged with an accuracy of 35%, although this was in a low-burden setting where a large fraction of cases occurred among foreign-born migrants. Here, we apply a similar model to M. tuberculosis whole-genome sequencing data from the Republic of Moldova, a setting of relatively high local transmission. Using a maximum cluster span of ~40 single nucleotide polymorphisms (SNPs) and a cluster size cutoff of n≥10, we could best predict the specific cluster to which each clustered case was most likely to be a member with an accuracy of 17.2 %. In sensitivity analyses, we found that a more restrictive (~20 SNPs threshold) or permissive (~80 SNPs) threshold did not improve performance. We found that increasing the minimum cluster size improved prediction accuracy. These findings highlight the challenges of transmission inference in high-burden settings like Moldova.

Background. Aerococcus urinae was initially considered a commensal of the urinary tract, but there is now increasing evidence for its involvement in urinary tract and systemic infections. A. urinae endocarditis has a non-negligible mortality rate and occurs mainly in patients with underlying conditions or the presence of extraneous material. Case presentation. This report handles the case of a 65-year-old male with cardiac antecedents, who was admitted to the cardiology department after a syncope of unknown origin and diagnosed with severe mixed aortic valve disease and mitral valve sclerosis through the means of transoesophageal echocardiography (TEE). During hospitalization, the patient progressively deteriorated with the development of shortness of breath and an inflammatory syndrome. Both the urine and blood cultures showed growth of A. urinae. Treatment with piperacillin/tazobactam was started empirically. Repeated TEE showed evidence of endocarditis with vegetation and perforation of the mitral valve that required an emergency surgery with mitral valve repair. After surgery, gentamicin and penicillin G were administered for 48 h, followed by combined ceftriaxone/penicillin G treatment for 6 weeks. At first, flucloxacillin was also associated as the culture of the valve was negative. Finally, the 16S rRNA gene PCR on the valve tissue confirmed the A. urinae endocarditis. Conclusion. A. urinae is an underestimated cause of serious infections such as endocarditis. Urinary tract infections mainly in older men can be an entry point for this type of invasive infection.

Blood culture (BC) investigation remains the gold standard for the diagnosis of bloodstream infections. However, BC contamination can have clinical implications for the patient, cost implications for service providers and less well-documented, environmental impacts. Efforts to reduce BC contamination are a long-standing theme in quality improvement initiatives in emergency departments (EDs) and hospitals, prompted by hospital costs, healthcare inefficiencies and antimicrobial stewardship efforts. The WHO's global analysis of healthcare waste in the context of COVID-19 has reported that tens of thousands of tonnes of extra medical waste were produced from the response to the COVID-19 pandemic, basing its estimates on the quantity of personal protective equipment. Additionally, recent literature has also shown increased BC contamination rates during the COVID-19 pandemic. We performed a retrospective review of the trend of BC contamination during the COVID-19 pandemic in our institution's ED. We further discuss some of the potential implications of BC contamination, including potential environmental, economic and efficiency implications.

Riboflavin (vitamin B2) is a water-soluble compound that plays an important role in multiple cellular functions. This study evaluates the probiotic potential of riboflavin-overproducing Bacillus subtilis strains isolated from fermented African locust beans. After strain improvement, B. subtilis ACU-I11MR and ACU-I163MR were selected due to their higher riboflavin production (0.01905±0.0005 mg l-1 to 0.0259±0.0077 mg l-1 and 0.0195±0.0054 mg l-1 to 0.0267±0.0013 mg l-1, respectively). Their safety was confirmed through haemolytic assay, antibiotic susceptibility tests and the absence of gelatinase and biogenic amine activity. Probiotic potential was assessed via in vitro assays including resistance to low pH, bile salts, phenol, temperature and NaCl; auto-aggregation; cell hydrophobicity; biofilm formation; antibacterial activity; and enzyme and exopolysaccharide production. Both strains were non-haemolytic and negative for gelatinase and biogenic amine activity. They showed significant viability at pH 2 (survival 85.05; 87.09%), 1% bile salts (survival 88.82; 87.64%) and 0.5% phenol (survival 48.80; 59.52%), respectively. ACU-I11MR was susceptible to 9 out of 12 antibiotics, while ACU-I163MR was 100% susceptible. The strains demonstrated strong cell surface adhesion and auto-aggregation and inhibited several pathogenic bacteria. They produce amylase, protease and exopolysaccharide and thrive under various temperature and NaCl conditions. B. subtilis ACU-I163MR, showing superior probiotic potential, could be a promising candidate for developing riboflavin-enriched Bacillus-fermented functional foods.