Intrammary infections are a major problem for dairy sheep farms, and Streptococcus uberis is one of the main etiological agents of ovine mastitis. Surveys on antimicrobial resistance are still limited in sheep and characterization of isolates is important for acquiring information on resistance and for optimizing therapy. In this study, a sampling of 124 S. uberis isolates collected in Sardinia (Italy) from sheep milk was analysed by multilocus-sequence typing (MLST) and pulsed field gel electrophoresis (PFGE) for genetic relatedness. All isolates were also subjected to antimicrobial susceptibility analysis by the disk diffusion test using a panel of 14 antimicrobials. Resistance genes were detected by PCR assays. MLST analysis revealed that the isolates were grouped into 86 sequence types (STs), of which 73 were new genotypes, indicating a highly diverse population of S. uberis. The most frequently detected lineage was the clonal complex (CC)143, although representing only 13.7% of all characterized isolates. A high level of heterogeneity was also observed among the SmaI PFGE profiles, with 121 unique patterns. Almost all (96.8%) isolates were resistant to at least one antimicrobial, while all exhibited phenotypic susceptibility to oxacillin, amoxicillin-clavulanic acid and ceftiofur. Of the antimicrobials tested, the highest resistance rate was found against streptomycin (93.5%), kanamycin (79.8%) and gentamicin (64.5%), followed by novobiocin (25%) and tetracycline-TE (19.3%). Seventy-four (59.7%) isolates were simultaneously resistant to all aminoglycosides tested. Seventeen isolates (13.7%) exhibited multidrug resistance. All aminoglycosides-resistant isolates were PCR negative for aad-6 and aphA-3’ genes. Among the TE-resistant isolates, the tetM gene was predominant, indicating that the resistance mechanism is mainly mediated by the protection of ribosomes and not through the efflux pump. Three isolates were resistant to erythromycin, and two of them harboured the ermB gene. This is the first study reporting a detailed characterization of the S. uberis strains circulating in Sardinian sheep. Further investigations will be needed to understand the relationships between S. uberis genotypes, mastitis severity, and intra-mammary infection dynamics in the flock, as well as to monitor the evolution of antimicrobial resistance.

In the present study, we aimed to determine the antimicrobial resistance and genetic structure of a population of S. aureus recovered from transient and persistent intramammary infections and nares/muzzles. We investigated the antimicrobial resistance of 189 S. aureus strains using a broad antimicrobial susceptibility profile. Furthermore, 107 S. aureus isolates were strain-typed using staphylococcal protein-A (spa) typing. Here, a great proportion of strains exhibited multidrug resistance to antimicrobials, including resistance to critically important antimicrobials, although no methicillin-resistant S. aureus strains were found. Our study did not strengthen the idea that extramammary niches (i.e., nares/muzzles) are an important source for S. aureus. A discrepancy in the antimicrobial resistance between S. aureus strains isolated from nasal/muzzles and milk samples was observed. Furthermore, S. aureus isolates from transient and persistent IMIs did not differ by spa typing, suggesting that the persistence of bovine IMIs was determined by cow factors. Thus, the high level of multidrug-resistant S. aureus found in the two herds studied together with the predominance of a well udder-adapted S. aureus strain may contribute to the history of the high prevalence of mastitis caused by S. aureus, leading to great animal and public health concerns.

Globally, the use of whole-cell matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) in clinical microbiology laboratories has increased considerably. With the rapid growth of artificial intelligence technology, there is an increasing number of areas devoted to solving problems by implementing machine learning-based methods. Applying the use of artificial intelligence to the analysis of the massive amount of information obtained in the mass spectrum derived from MALDI-TOF MS should be a breakthrough in clinical microbiology. Specifically, it would facilitate a more rapid and accurate strain typing or antibiotic susceptibility test (AST) based on whole-cell MALDI-TOF MS spectra, and hence, it is a promising solution. Even though the reproducibility of peaks could result in some critical problems, computational methods have been developed to address this issue. Therefore, clinical microbiologists are able to perform strain typing or an AST as smoothly and easily as identifying species by using MALDI-TOF MS and artificial intelligence.

High Resolution Melting Analysis (HRM) has been pointed as a suitable alternative method to detect and identify Leishmania species. Herein, we aimed to evaluate the sensitivity, specificity, accuracy, and limitations of a HSP70-HRM protocol both as a diagnostic scheme applied in clinical samples and as a species typing tool for laboratory research and reference services. Our data reveal the pronounced species-typing potential of the HSP70-HRM in DNA from cultured parasites. For clinical samples, however, we advise caution due to the parasite load-dependent accuracy. In the light of these findings and considering the importance of parasite load determination for clinical and research purposes we recommend the integration of the presented typing scheme and the previously published Leishmania quantifying approach as combined tools for clinicians, surveillance, and research.

Stretching along the border of North Dakota and Minnesota, The Red River Valley (RRV) of the North has the highest frequency of reported blizzards within the contiguous United States. Despite the numerous impacts these events have, few systematic studies exist discussing the meteorological properties of blizzards. As a result, forecasting these events and lesser blowing snow events is an ongoing forecast challenge. This study presents a climatology of atmospheric patterns associated with RRV blizzards for the winter seasons of 1979-1980 to 2017-2018. Patterns were identified using subjective and objective techniques using meteorological fields from the North American Regional Reanalysis (NARR). The RRV experiences on average, 2.6 events per year. Blizzard frequency is bimodal with peaks occurring in December and March. The events can largely be typed into four meteorological categories dependent on the forcing that drives the blizzard: Alberta Clippers, Arctic Fronts, Colorado Lows, and Hybrids. Objective classification of these blizzards using a competitive neural network known as the Self-Organizing Map (SOM) demonstrates that gross segregation of the events can be achieved with a small (8-class) map. This implies that objective analysis techniques can be used to identify these events in weather and climate model output that may aid future forecasting and risk assessment projects.

Identifying or authenticating a computer user are necessary steps to keep systems secure on the network and to prevent fraudulent users from accessing accounts. Keystroke dynamics authentication can be used as an additional authentication method. Keystroke dynamics involves in-depth analysis of how you type on the keyboard, analysis of how long a key is pressed or the time between two consecutive keys. This field has seen a continuous growth in scientific research. In the last five years alone, about 10,000 scientific researches in this field have been published. One of the main problems facing researchers is the small number of public data sets that include how users type on the keyboard. This paper aims to provide researchers with a data set that includes how to type free text on the keyboard by 80 users. The data were collected in a single session via a web platform. The dataset contains 410,633 key-events collected in a total time interval of almost 24 hours. In similar research, most datasets are with texts written by users in English. The language in which the users wrote for this research is Romanian. This paper also provides an extensive analysis of the data set collected and presents relevant information for the analysis of the data set in future research.

Trypanosoma cruzi, the protozoan causative of Chagas disease (ChD), exhibits striking genetic and phenotypic intraspecific diversity, along with ecoepidemiological complexity. Human-pathogen interactions lead to distinct clinical presentations of ChD. In 2009, an international consensus classified T. cruzi strains into six discrete typing units (DTUs), TcI to TcVI, later including TcBat, and proposed reproducible genotyping schemes for DTU identification. This article aims to review the impact of classifying T. cruzi strains into DTUs on our understanding of biological, ecoepidemio-logical, and pathogenic aspects of T. cruzi. We will explore the likely origin of DTUs and the intrinsic characteristics of each group of strains concerning genome organization, genomics, and suscepti-bility to drugs used in ChD treatment. We will also provide an overview of the association of DTUs with mammalian reservoirs, and summarize the geographic distribution, and the clinical implica-tions, of prevalent specific DTUs in ChD patients. Throughout this review, we will emphasize the crucial roles of both parasite and human genetics in defining ChD pathogenesis and chemotherapy outcome.

Leishmaniasis is a disease caused by parasites of Leishmania genus and transmitted by sand fly vectors. Tegumentary leishmaniasis is the most prevalent clinical outcome in Latin America afflicting people from 18 countries. In Panama, leishmaniasis has annual incidence rates as high as 3000 cases, representing therefore one of the major public health problems. In endemic regions, L. panamensis is responsible for almost eighty percent of the human cases that present different clinical outcomes. This differences in disease outcomes could be result of the local interplay between L. panamensis variants and human hosts with different genetic backgrounds. The genetic diversity of L. panamensis in Panama has only been partially explored, and the variability reported for this species is based on few studies restricted to small populations and/or with poor resolutive markers at low taxonomic levels. Accordingly, in this study we explored the genetic diversity of sixty-nine L. panamensis isolates from different endemic regions of Panama using a MLST approach based on four housekeeping genes (Aconitase, ALAT, GPI and HSP70). Two to seven haplotypes per locus were identified, and regional differences in L. panamensis genetic diversity were observed. Genotype analysis evidenced the circulation of thirteen L. panamensis genotypes, a fact that might have important implications for the local control of the disease.

Most computer keyboard layouts (mappings of characters to keys) do not reflect the ergonomics of the human hand, resulting in preventable repetitive strain injuries. We present a set of ergonomics principles relevant to touch typing, introduce a scoring model that encodes these principles, and outline a systematic approach for developing optimized keyboard layouts in any language based on this scoring model coupled with character-pair frequencies. We then create a keyboard layout optimized for touch typing in English by constraining key assignments to reduce lateral finger movements and enforce easy access to high-frequency letters and letter pairs, applying open source software to generate millions of layouts, and evaluating them based on Google’s N-gram data. We use two independent scoring methods to compare the resulting Engram layout against 10 other prominent keyboard layouts based on a variety of publicly available text sources. The Engram layout scores consistently higher than other keyboard layouts.

In this work, we analysed human isolates of nontyphoidal Salmonella enterica subsp. enterica (NTS), which were collected from salmonellosis cases in Armenia from 1996 to 2019. This disease became a leading food-borne bacterial infection in the region, with the younger age groups especially affected. The isolates were characterised by serotyping, Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR) typing, and whole genome sequencing (WGS). The main serotypes were S. Typhimurium, S. Enteritidis, and S. Arizonae. ERIC-PCR indicated a high degree of clonality among S. Typhimurium strains, which were also multidrug-resistant and produced extended spectrum beta-lactamases. During the study period, the frequency of S. Typhimurium and S. Arizonae isolations were decreasing, but with the increase of S. Enteritidis and other NTS. A total of 42 NTS isolates were subjected to WGS and explored for virulence-related traits and the corresponding genetic elements. Some virulence and genetic factors were shared by all NTS serotypes, while the main differences were attributed to the serotype-specific diversity of virulence genes, SPIs, virulence plasmids, and phages. The results indicated the variability and dynamics in the epidemiology of salmonellosis and a high virulence potential of human NTS isolates circulating in the region.

Bacterial cellulose (BC) is receiving great attention due to its unique properties such as high purity, water retention capacity, high mechanical strength, and biocompatibility. However, the production of BC has been limited because of high cost and low productivity. In this light, the isolation of new BC producing bacteria and selection of high productive strains became a promising issue. Kombucha tea is a fermented beverage in which the bacteria fraction of the microbial community is composed mostly by strains belonging to the genus Komagataeibacter. In this study Kombucha tea production trials were performed starting from a previous batch, and bacterial isolation was conducted along cultivation time. From the whole microbial pool, 46 isolates were tested for their ability in producing BC. The obtained BC yield ranged from 0.59 g/L, for the isolate K2G36, to 23 g/L for K2G30 used as the reference strain. The genetic intraspecific diversity of the 46 isolates was investigated using two repetitive-sequence-based PCR typing methods, which are the enterobacterial repetitive intergenic consensus (ERIC) elements and the (GTG)5 sequences, respectively. The results obtained using two different approaches revealed the suitability of the fingerprints techniques, showing a discrimination power, calculated as D index, of 0.94 for (GTG)5 rep-PCR and 0.95 for ERIC rep-PCR. In order to improve the sensitivity of the applied method, a combined model from the two genotyping experiments was performed, allowing to discriminate among strains.

We investigated the prevalence and genetic diversity of Wolbachia pipientis strains in Aedes albopictus populations in Greece. Using a combination of PCR and Sanger sequencing techniques, we genotyped Wolbachia strains in 105 mosquitoes collected across eight different prefectures in 2021. We found a high prevalence of Wolbachia in both male (90%) and female (97%) mosquitoes. Among the infected samples, 84% had double infections with both wAlbA and wAlbB strains, while 16% had infections with only wAlbB. Our comparison of the Multi-Locus Sequence Typing (MLST) profile, employing gatB-coxA-hcpA-ftsZ-fbpA genotyping, revealed a single MLST profile for each wAlbA and wAlbB strain in Greek populations. The same MLST profiles were also reported in populations from China, Russia, and Argentina, suggesting low levels of global diversity in wAlbA and wAlbB strains. Furthermore, our results indicated a significant association between temperature and the prevalence of single infections (P = 6.498E-07), with higher temperatures correlating with an increased likelihood of single infections. Although male-bias showed a tendency towards single infections, the effect was marginally non-significant (P = 0.053). These results were confirmed using a bootstrap-with-replacement analysis approach. Overall, our findings offer novel insights into the distribution and species diversity of Wolbachia strains in Greek Ae. albopictus populations, emphasizing the importance of understanding the short-term plastic and adaptive responses of these organisms to environmental stressors and rapid climate change.

The genus Fusarium includes several agronomically important and toxin-producing species, that are worldwide distributed and can cause a wide range of diseases. Crown and stalk rots and grain infections are among the most severe symptoms that Fusarium spp. can cause on maize. The disease development usually occurs during germination, but it may also affect the later phases of plant growth. The purpose of this study was to investigate the diversity and the patho-genicity of 41 isolates recovered from symptomatic seedlings collected in Northern Italy, and from seeds with five different geographical origins during 2019 and 2020. The pathogenicity was tested and confirmed on 23 isolates causing rotting on maize seedlings. A multi-locus phylogeny analysis, based on four genomic loci (tef1-α, rpb2, calm and tub2), was performed for 23 repre-sentative isolates. Representative isolates were identified as species belonging to three species complexes (SC). Fusarium verticillioides and F. annulatum in the F. fujikuroi SC. Fusarium commune was identified in the F. nisikadoi SC, and three different lineages were found in the Fusarium ox-ysporum SC. This study reports F. annulatum, and two lineages of the Fusarium oxysporum SC as maize pathogens for the first time in Italy.

Patients receiving hemodialysis are at risk of vascular access infections (VAIs) and particularly vulnerable to the opportunistic pathogen Staphylococcus aureus. Hemodialysis patients were also at increased risk of infection during the COVID-19 pandemic. Therefore, this study determined the change in the molecular and antibiotic resistance profiles of S. aureus isolates from VAIs during the pandemic compared with before. 102 S. aureus isolates were collected from VAIs between November 2013 and December 2021. Before the pandemic, 69 isolates were collected, 58%, 39.1%, and 2.9% from arteriovenous grafts (AVGs), tunneled cuffed catheters (TCCs), and arteriovenous fistulas (AVFs), respectively. The prevalence of AVG and TCC isolates changed to 39.4% and 60.6%, respectively, of 33 isolates during the pandemic. Sequence type (ST)59 was the predominant clone in TCC-methicillin-resistant S. aureus (MRSA) and AVG-MRSA before the pandemic, whereas the predominant clone was ST8 in AVG-MRSA during the pandemic. ST59 carrying the ermB gene was resistant to clindamycin and erythromycin. By contrast, ST8 carrying the msrA gene was exclusively resistant to erythromycin. The ST distribution for different VAIs changed from before to during the pandemic. The change in antibiotic resistance rate for different VAIs was closely related to the distribution of specific STs.

Legionella pneumophila can cause a large panel of symptoms beside the classic pneumonia presentation. We present here a case of fatal nosocomial cellulitis in an immunocompromised patient followed, a year later, by a second case of legionnaires’ disease in the same ward. While the first one was easily assumed as nosocomial based on the date of symptoms onset, the other case needed to rely on clear typing results to be either assigned as nosocomial and related to the same environmental source as the first one, or community acquired. To untangle this specific question, we applied Core-genome MultiLocus Typing (MLST), whole-genome Single Nucleotide Polymorphism and whole-genome MLST methods to a collection of 36 Belgian and 41 international sequence-type 1 (ST1) isolates using both thresholds recommended in the literature and tailored threshold based on local epidemiological data. Based on the thresholds applied to cluster isolates together, the three methods gave different results and no firm conclusion about the nosocomial setting of the second case could been drawn. Our data highlight that despite promising results in the study of outbreaks and for large scale epidemiological investigations, NGS typing methods applied to ST1 outbreak investigation still need standardization regarding both wet-lab protocols and bioinformatics. A deeper evaluation of L. pneumophila evolutionary clock is also required to increase our understanding of genomic differences between isolates sampled during a clinical infection and in environment.

Trio-based exome sequencing and high-resolution HLA typing were employed to analyze three patients with autoimmune adrenal insufficiency (AAI), including those with autoimmune polyglandular syndrome (APS) type 2, as well as their parents. Benign or likely benign variants of the AIRE gene were identified in all participants of the study. These variants, coupled with clinical data and the results of antibody studies to type I interferons, helped to exclude APS-1. Patients with APS-2, in contrast to patient with AAI, inherited distinct variants of unknown significance in the CLEC16A gene, which is associated with autoimmune diseases including AAI. Various risk alleles in other genes associated with autoimmunity were identified in all patients. HLA typing of second class loci revealed alleles related to APS types 2, 3, and 4. Nevertheless, the frequencies of the haplotypes identified are substantial in the healthy Russian population, precluding from regarding these alleles as genetic determinants linked to APS development. Immunological examination can detect antibody carriers and predict the risk of autoimmune disease development. In the future, to identify genetic predictors of autoimmune endocrinopathies, it is recommended to analyze the whole genome of patients and their relatives, examining clinically relevant variants in non-coding regions.

All empirical water column correction methods have consistently been reported to require existing depth sounding data for the purpose of calibrating a simple depth retrieval model; they yield poor results over very bright or very dark bottoms. In contrast, we set out to (i) use only the relative radiance data in the image along with published data, and several new assumptions, (ii) in order to specify and operate the simplified radiative transfer equation (RTE), (iii) for the purpose of retrieving both the satellite derived bathymetry (SDB) and the water column corrected spectral reflectance over shallow seabeds. Sea truth regressions show that SDB depths retrieved by the method only need tide correction. Therefore it shall be demonstrated that, under such new assumptions, there is no need (i) for formal atmospheric correction, (ii) nor for conversion of relative radiance into calibrated reflectance , (iii) nor for existing depth sounding data, to specify the simplified RTE and produce both SDB and spectral water column corrected radiance ready for bottom typing. Moreover, the use of the panchromatic band for that purpose is introduced. Altogether, we named this process the Self-Calibrated Supervised Spectral Shallow-sea Modeler (4SM). This approach requires a trained practitioner, though, to produce its results within hours of downloading the raw image. The ideal raw image should be a “near-nadir” view, exhibit homogeneous atmosphere and water column, include some coverage of optically deep waters and bare land, and lend itself to quality removal of haze, atmospheric adjacency effect, and sun/sky glint.