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Background Bacterial genotyping is a crucial process in outbreak investigation and epidemiological studies. Several typing methods such as pulsed-field gel electrophoresis, multilocus sequence typing (MLST) and whole genome sequencing are currently used in routine clinical practice. However, these methods are costly, time-consuming and have high computational demands. An alternative to these methods is mini-MLST, a quick, cost-effective and robust method based on high-resolution melting analysis. Nevertheless, no standardized approach to identify markers suitable for mini-MLST exists. Here, we present a pipeline for variable fragment detection in unmapped reads based on a modified hybrid assembly approach using data from one sequencing platform. Results In routine assembly against the reference sequence, high variable reads are not aligned and remain unmapped. If de novo assembly of them is performed, variable genomic regions can be located in created scaffolds. Based on the variability rates calculation, it is possible to find a highly variable region with the same discriminatory power as seven housekeeping gene fragments used in MLST. In the work presented here, we show the capability of identifying one variable fragment in de novo assembled scaffolds of 21 Escherichia coli genomes and three variable regions in scaffolds of 31 Klebsiella pneumoniae genomes. For each identified fragment, the melting temperatures are calculated based on the nearest neighbor method to verify the mini-MLST’s discriminatory power. Conclusions A pipeline for a modified hybrid assembly approach consisting of reference-based mapping and de novo assembly of unmapped reads is presented. This approach can be employed for the identification of highly variable genomic fragments in unmapped reads. The identified variable regions can then be used in efficient laboratory methods for bacterial typing such as mini-MLST with high discriminatory power, fully replacing expensive methods such as MLST. The results can and will be delivered in a shorter time, which allows immediate and fast infection monitoring in clinical practice.

Bacillus cereus sensu lato is a common contaminant of improperly stored food and human milk, capable of causing severe emetic and diarrheal diseases, particularly in infants and immunocompromised individuals. In this in vitro study, the effects of various temperatures and carbohydrate substrates on the growth of B. cereus ( pacificus ) strain ATCC 10987 and its expression of enterotoxin genes were explored. Bacterial growth at several temperatures and with six different carbohydrate substrates was evaluated. In cultures grown with various substrates at 37 °C, selected metabolites were analyzed using capillary electrophoresis and RNA-seq transcriptomic profiling was performed. At the same temperature, glucose, fructose, sucrose, and xylitol-enriched environments were preferable for the bacterial growth to lactose and galactose-enriched media. Lactate production markedly increased in bacterial cultures grown with glucose, fructose, or sucrose, accompanied by a drop in pH and increased substrate consumption. The lowest expression of non-hemolytic enterotoxin genes ( nhe ) was observed in glucose and lactose-enriched media, while downregulation of enterotoxin FM gene ( entFM ) expression was found in bacteria cultured with lactose. Other known enterotoxin genes, such as hemolysin BL ( hbl ) and cytotoxin K ( cytK ), were not expressed by this bacterial strain under any of the tested conditions. In conclusion, a lactose-rich environment slowed the growth of B. cereus ( pacificus ) ATCC 10987 and was associated with a downward trend in the expression of the enterotoxin genes nhe and entFM . These findings indicate that the presence of lactose, a dominant carbohydrate of human milk, may influence the pathogenic potential of B. cereus s. l.

Background Human milk harbors diverse bacterial communities that contribute to infant health. Although pumping and storing milk is a common practice, the viable bacterial composition of pumped milk and the impact of storage practice on these bacteria remains under-explored. This metagenomic observational study aimed to characterize viable bacterial communities in freshly pumped human milk and its changes under different storage conditions. Methods In 2023, twelve lactating mothers from the CELSPAC: TNG cohort (Czech Republic) provided freshly pumped milk samples. These samples were stored under various conditions (refrigeration for 24 h, 48 h, or freezing for six weeks) and treated with propidium monoazide (PMA) to selectively identify viable cells. The DNA extracted from individual samples was subsequently analyzed using 16S rRNA amplicon sequencing on the Illumina platform. Results The genera Streptococcus, Staphylococcus, Diaphorobacter, Cutibacterium, and Corynebacterium were the most common viable bacteria in fresh human milk. The median sequencing depth and Shannon index of fresh human milk samples treated with PMA (+ PMA) were significantly lower than in untreated (-PMA) samples ( p  < 0.05 for all), which was true also for each time point. Also, significant changes in these parameters were observed between fresh human milk samples and their paired frozen samples ( p  < 0.05), while no differences were found between fresh human milk samples and those refrigerated for up to 48 h ( p  > 0.05). Of specific genera, only + PMA frozen human milk samples showed a significant decrease in the central log-ratio transformed relative abundances of the genera Diaphorobacter and Cutibacterium ( p  < 0.05) in comparison to + PMA fresh human milk samples. Conclusions The study demonstrated that the bacterial profiles significantly differed between human milk samples treated with PMA, which represent only viable bacteria, and those untreated. While storage at 4 °C for up to 48 h did not significantly alter the overall diversity and composition of viable bacteria in human milk, freezing notably affected both the viability and relative abundances of some bacterial genera.

The antibiotic resistance genes (ARGs) limit the susceptibility of bacteria to antimicrobials, representing a problem of high importance. Current research on the presence of ARGs in microorganisms focuses mainly on humans, livestock, hospitals, or wastewater. However, the spectrum of ARGs in the dust resistome in workplaces and households has gone relatively unexplored. This pilot study aimed to analyze resistome in indoor dust samples from participants’ workplaces (a pediatric hospital, a maternity hospital, and a research center) and households and compare two different approaches to the ARGs analysis; high-throughput quantitative PCR (HT-qPCR) and whole metagenome shotgun sequencing (WMGS). In total, 143 ARGs were detected using HT-qPCR, with ARGs associated with the macrolides, lincosamides, and streptogramin B (MLSB) phenotype being the most abundant, followed by MDR (multi-drug resistance) genes, and genes conferring resistance to aminoglycosides. A higher overall relative quantity of ARGs was observed in indoor dust samples from workplaces than from households, with the pediatric hospital being associated with the highest relative quantity of ARGs. WMGS analysis revealed 36 ARGs, of which five were detected by both HT-qPCR and WMGS techniques. Accordingly, the efficacy of the WMGS approach to detect ARGs was lower than that of HT-qPCR. In summary, our pilot data revealed that indoor dust in buildings where people spend most of their time (workplaces, households) can be a significant source of antimicrobial-resistant microorganisms, which may potentially pose a health risk to both humans and animals.

Historical buildings and monuments are largely made of brickwork. These buildings form the historical and artistic character of cities, and how we look after them is a reflection of our society. When assessing ceramic products, great emphasis is placed on their mechanical properties, whilst their durability is often neglected. However, the durability or resistance to weathering of masonry elements is just as important as their mechanical properties. Therefore, this work deals with predicting the durability of solid-fired bricks before they are used when reconstructing monuments and historical buildings. Durability prediction is assessed by identifying defects in the material’s internal structure. These faults may not be visible on the element’s surface and are difficult to detect. For this purpose, non-destructive electroacoustic methods, such as the resonant pulse method or the ultrasonic pulse method, were used. Based on an analysis of the initial and residual mechanical properties after freezing cycles, four durability classes of solid-fired bricks were determined. This work aimed to find a way to predict the durability (lifetime) of an anonymous solid-fired brick, expressed in terms of the number of freeze cycles the brick would last, based on non-destructive measurements.

Recently, nanopore sequencing has come to the fore as library preparation is rapid and simple, sequencing can be done almost anywhere, and longer reads are obtained than with next-generation sequencing. The main bottleneck still lies in data postprocessing which consists of basecalling, genome assembly, and localizing significant sequences, which is time consuming and computationally demanding, thus prolonging delivery of crucial results for clinical practice. Here, we present a neural network-based method capable of detecting and classifying specific genomic regions already in raw nanopore signals - squiggles. Therefore, the basecalling process can be omitted entirely as the raw signals of significant genes, or intergenic regions can be directly analysed, or if the nucleotide sequences are required, the identified squiggles can be basecalled, preferably to others. The proposed neural network could be included directly in the sequencing run, allowing real-time squiggle processing. Competing Interest Statement The authors have declared no competing interest.

Many participants in microbicide trials remain uninfected despite ongoing exposure to HIV-1. Determining the emergence and nature of mucosal HIV-specific immune responses in such women is important, since these responses may contribute to protection and could provide insight for the rational design of HIV-1 vaccines. We first conducted a pilot study to compare three sampling devices (Dacron swabs, flocked nylon swabs and Merocel sponges) for detection of HIV-1-specific IgG and IgA antibodies in vaginal secretions. IgG antibodies from HIV-1-positive women reacted broadly across the full panel of eight HIV-1 envelope (Env) antigens tested, whereas IgA antibodies only reacted to the gp41 subunit. No Env-reactive antibodies were detected in the HIV-negative women. The three sampling devices yielded equal HIV-1-specific antibody titers, as well as total IgG and IgA concentrations. We then tested vaginal Dacron swabs archived from 57 HIV seronegative women who participated in a microbicide efficacy trial in Southern Africa (HPTN 035). We detected vaginal IgA antibodies directed at HIV-1 Env gp120/gp140 in six of these women, and at gp41 in another three women, but did not detect Env-specific IgG antibodies in any women. Vaginal secretions of HIV-1 infected women contained IgG reactivity to a broad range of Env antigens and IgA reactivity to gp41. In contrast, Env-binding antibodies in the vaginal secretions of HIV-1 uninfected women participating in the microbicide trial were restricted to the IgA subtype and were mostly directed at HIV-1 gp120/gp140.