Introduction. The absence of a gold-standard methodology for the microbiological diagnosis of urinary tract infections (UTI) has led to insufficient standardization of criteria for the interpretation of results and processing methods, particularly incubation time and culture media.Hypothesis. 48-hour incubation time period and use of blood agar enhances the sensitivity of microorganisms isolated significantly.Aim. To determine the sensitivity of blood agar and Brilliance UTI chromogenic agar, incubating for different periods (24-48 hours), for the detection of positive urine cultures.Methodoloy. Comparisons were made between all possible combinations of media and incubation times. As the gold-standard reference, we used the routine methodology of our laboratory, which involves prior screening with available clinical data, flow cytometry, sediment analysis and/or Gram staining. Screened samples were then cultured on blood agar and chromogenic agar and incubated for 48 hours. Also, based on the results of Gram staining, additional media were added in selected cases.Results. The most significant difference was found between chromogenic agar incubated for 24 hours and blood agar incubated for 48 hours, with the latter method allowing the recovery of 10.14 % more microorganisms (P < 0.0001). Furthermore, the value of performing Gram staining to guide processing was demonstrated, as it avoided the loss of at least 5.14 % of isolates.Conclusions. At least in urological and nephrological patients it is essential to include enriched culture media (blood agar) or to extend the incubation times due to the improvement of the diagnostic sensitivity of urine cultures. Gram staining also can help detect the presence of fastidious microorganisms or mixed infections, indicating whether rich and/or selective media should be included to enhance the diagnostic sensitivity of cultures. If this methodology is not followed, it should be noted that besides fastidious species, fastidious strains of Escherichia coli, Proteus mirabilis, Pseudomonas aerugniosa and Stenotrophomonas maltophilia will also be missed.

Prokaryotes dominate global oceans and shape biogeochemical cycles, yet most taxa remain uncultured and uncharacterized as of today. Here we present the characterization of 26 novel marine bacterial strains from a large isolate collection obtained from Blanes Bay (NW Mediterranean) microcosm experiments made in the four seasons. Morphological, cultural, biochemical, physiological, nutritional, genomic, and phylogenomic analyses were used to characterize and phylogenetically place the novel isolates. The strains represent 23 novel bacterial species and six novel genera: three novel species pertaining to class Alphaproteobacteria (families Rhodobacteraceae and Sphingomonadaceae), six novel species and three new genera from class Gammaproteobacteria (families Algiphilaceae, Salinispheraceae, and Alteromonadaceae), 13 novel species and three novel genera from class Bacteroidia (family Flavobacteriaceae), and one new species from class Rhodothermia (family Rubricoccaceae). The bacteria described here have potentially relevant roles in the cycles of carbon (e.g., carbon fixation or energy production via proteorhodopsin), nitrogen (e.g., denitrification or use of urea), sulfur (oxidation of sulfur compounds), phosphorus (acquisition and use of different forms of phosphorus and remodeling of membrane phospholipids), and hydrogen (oxidation of hydrogen to obtain energy). We mapped the genomes of the presented strains to the Tara Oceans metagenomes to reveal that these strains were globally distributed, with those of the family Flavobacteriaceae being the most widespread and abundant, while Rhodothermia being the rarest and most localized. While molecular-only approaches are also important, our study stresses the importance of culturing as a powerful tool to further understand the functioning of marine bacterial communities.

Leptospirosis is an infectious disease with a worldwide distribution, which represents a major challenge in animal production across developing countries, mainly in tropical areas. Horses are particularly susceptible to the disease, presenting manifestations ranging from subclinical to the development of uveitis that compromises the visual health of the animals. In recent years, serological studies have been carried out in equid populations from America, demonstrating high exposure. For this reason, the aim of this study was to demonstrate microbiologically and molecularly the presence of the members of the genus Leptospira in urine samples from equids in an endemic state of leptospirosis in Mexico, and to detect the serological presence of anti-Leptospira antibodies in the sampled animals. For this reason, blood and urine samples were collected from 28 horses and one mule from three localities in the state of Veracruz, Mexico. Urine samples were inoculated in Ellinghausen-McCullough-Johnson-Harris (EMJH) medium, and the recovered isolates were typed using a short Multi Locus Sequence Typing scheme. Amplifications of the expected size were subjected to sequencing, and the recovered sequences were compared with those of reference deposited in GenBank using the BLAST tool. To identify their phylogenetic position, we performed a phylogenetic reconstruction using the maximum likelihood method. Additionally, Microscopic Agglutination test was performed on the serum samples to identify anti-Leptospira antibodies. We recovered 16 urine isolates which tested positive for the presence of Leptospira DNA. The phylogenetic reconstruction and the MLST analysis confirmed the presence of several genotypes of Leptospira interrogans and Leptospira santarosai. An overall serological frequency of 97.1 % was detected. Our results represent the first record of the presence of Leptospira through bacteriological isolates in equids from Mexico.

There is a scarcity of published studies on the occurrence of Staphylococcus spp. Among dogs in South Africa. The objective of the study was to characterise the Staphylococcus spp. Isolated from dog samples submitted to a veterinary diagnostic laboratory in South Africa in terms of time, place, and person. This study utilised a dataset of 1627 positive Staphylococcus isolates obtained from a veterinary diagnostic laboratory in South Africa from 2012 to 2017. Out of the 1627 confirmed isolates, 10 different species of Staphylococcus were identified. Among these, 92.0% were classified as coagulase-positive staphylococci (CoPS), 6.0% were coagulase-negative staphylococci (CoNS), and 3.0% were coagulase-variable. Male dogs contributed just over half (53.2%) of the Staphylococcus isolates, while female dogs contributed the remaining 46.8%. The largest proportion of isolates (23.2%) were obtained from dogs aged ≥ 9 years, with the highest number of isolates originating from KwaZulu-Natal Province (45.0%) and the least from Northern Cape Province (0.1%). Out of the total samples included in the records, the majority (46.0%) were skin specimens. The number of Staphylococcus isolates recorded showed limited variation between the seasons (24.3% in autumn, 26.3% in winter, 26.0% in spring, and 24.0% in summer). This study highlighted the diversity of Staphylococcus spp. isolated from dogs, and the burden of staphylococcal carriage among dogs in South Africa. Further research is required to examine the factors that contribute to the observed discrepancies in the proportions of Staphylococcus spp. between the provinces.

UNASSIGNED: Entomopathogenic fungi exists naturally in plants as an asymptote and have the potential to reduce the population of insect pests through indirect interactions. This study was conducted to detect and characterize the endophytic fungi Beauveria bassiana and Metarhizium robertsii from the rhizosphere soil of tomato plants and their virulence effect on Galleria melonella.
UNASSIGNED: From the rhizosphere soil of 40 tomato fields, three Beauveria bassiana and seven Metarhizium robertsii isolates were isolated using the galleria bait method. All fungi isolate were morphologically characterized by their colony color, shape, and surface texture. Isolates with the highest percentages of germination, conidial yield, and radial growth were selected, and their virulence was evaluated on second instar larvae of Galleria melonella under laboratory conditions.
UNASSIGNED: In this study, Beauveria bassiana showed white colony color and aseptate hyphae, whereas Metarhizium robertsii showed dark green to light green colony color and septate hyphal structures. Maximum spore production and conidial length were obtained by Beauveria bassiana isolate APPRC-27 with 2.67x10 7 spores ml -1 and 3.24 µm, respectively. Colony radial growth rates ranged from 1.73 to 3.24 mm day -1. The results revealed that the highest mortality rate of Galleria melonella (100%) was obtained by Metarhizium robertsii isolates K-61 and K-102 at a concentration of 1x10 8 conidial ml -1 at 7 days post-inoculation. The lowest mortality rate was registered by Metarhizium robertsii isolate RST-11.
UNASSIGNED: In the present study, isolates that produced the most spores and had the highest germination rates were the most virulent to Galleria mellonella second instar larvae. Therefore, virulent entomopathogenic fungi, Beauveria bassiana and Metarhizium robertsii, are promising bioagents for the control of insect pests.

Saprolegniosis is a major destructive disease in freshwater aquaculture. The destructive economic impact of saprolegniosis on freshwater aquaculture necessitates further study on the range of Saprolegnia species within Atlantic salmon fish farms. This study undertook a thorough analysis of a total of 412 oomycete and fungal isolates that were successfully cultured and sequenced from 14 aquaculture sites in Scotland across a two-year sampling period. An ITS phylogenetic analysis of all isolates was performed according to whether they were isolated from fish or water samples and during enzootic or epizootic periods. Several genera of oomycetes were isolated from sampling sites, including Achlya, Leptolegnia, Phytophthora, and Pythium, but by far the most prevalent was Saprolegnia, accounting for 66% of all oomycetes isolated. An analysis of the ITS region of Saprolegnia parasitica showed five distinct phylotypes (S2-S6); S1 was not isolated from any site. Phylotype S2 was the most common and most widely distributed phylotype, being found at 12 of the 14 sampling sites. S2 was overwhelmingly sampled from fish (93.5%) and made up 91.1% of all S. parasitica phylotypes sampled during epizootics, as well as 67.2% of all Saprolegnia. This study indicates that a single phylotype may be responsible for Saprolegnia outbreaks in Atlantic salmon fish farms, and that water sampling and spore counts alone may be insufficient to predict Saprolegnia outbreaks in freshwater aquaculture.

The microbiome of Greenland Ice Sheet supraglacial habitats is still underinvestigated, and as a result there is a lack of representative genomes from these environments. In this study, we investigated the supraglacial microbiome through a combination of culturing-dependent and -independent approaches. We explored ice, cryoconite, biofilm, and snow biodiversity to answer: (1) how microbial diversity differs between supraglacial habitats, (2) if obtained bacterial genomes reflect dominant community members, and (3) how culturing versus high throughput sequencing changes our observations of microbial diversity in supraglacial habitats. Genomes acquired through metagenomic sequencing (133 high-quality MAGs) and whole genome sequencing (73 bacterial isolates) were compared to the metagenome assemblies to investigate abundance within the total environmental DNA. Isolates obtained in this study were not dominant taxa in the habitat they were sampled from, in contrast to the obtained MAGs. We demonstrate here the advantages of using metagenome SSU rRNA genes to reflect whole-community diversity. Additionally, we demonstrate a proof-of-concept of the application of in situ culturing in a supraglacial setting.

Aleutian mink disease (AMD) is one of the most serious diseases in minks worldwide, it brings tremendous financial losses in mink farming. AMD virus (AMDV) has unusually high genetic diversity, its genomic structure remains unclear. In 2014, sudden death of breeding minks was occurred in northeast China. After clinical signs evaluation and virus isolation, AMDV was identified in all sudden death minks, we investigated the complete genomic sequence of AMDV-LM isolated from the sudden death case. The full-genome sequence of AMDV-LM was 7 nucleotides (nts) or 8 nts longer than isolates AMDV-BJ and AMDV-G. AMDV-LM contained two unique nucleotide changes in VP2 (G79T, T710C), which led to two amino acid changes G27W and L237S. For NS1, some unique point mutations, such as A374C, A428C, A463C, and T476A were found and resulted in four unique amino acid mutations at N24V, H125P, V143P, K155Q, and V159N, respectively. The predicted secondary structure of the 5\' terminal of AMDV-LM formed a large bubble formation near the 5\' end, which affected the stability of the U-shaped hairpin. Phylogenetic analysis demonstrated that AMDV-LM was closely related to Chinese isolates and confirmed that AMDV strains circulating in China had different origins of ancestors. This study was first to investigate the association of sudden death of adult breeding minks with AMDV infection. Our findings provide useful suggestions for evaluation of the pathogenic potential of AMDV, additional details on AMDV genome characterization were also presented. Future work should focus on the importance of AMDV-LM strain in mink infection.

Bacterial panicle blight (BPB) caused by Burkholderia glumae (BG) has become significantly more prevalent in the rice-growing regions of North India. Based on virulence screening and in vitro quantification of toxoflavin, the BG strains were classified as hyper- (BG1 and BG3), moderate- (BG2, BG4, BG6, BG8, and BG9), and hypo- (BG5, BG7, and BG10) virulent. Plant inoculation assays with cell-free culture filtrate revealed strains with higher toxoflavin-producing ability had higher virulence. Based on 16S rRNA sequence, 6 isolates from Uttar Pradesh were grouped in clad C1; whereas, clad C2 exhibited 4 isolates, two each from Delhi and Uttar Pradesh. Strain BG1 being the most virulent Indian strain from Uttar Pradesh was further profiled for 11 tox genes. We found all the 11 tox genes present in strain BG1. In toxRABCDE cluster, all tox genes showed high similarity to B. glumae BGR1 except toxB, whereas in toxFGHIJ cluster toxF, toxG, toxH and toxI shared maximum similarity to B. glumae 336gr-1. tox genes of BG1 exhibited homology as well as divergence with B. gladioli. The domain prediction and protein association network analysis indicated the possible involvement of tox genes in the toxoflavin biosynthesis. As per our knowledge, this is the first report in India on characterization of tox genes cluster in B. glumae. Altogether, our study unravels a reliable method for identifying and characterizing B. glumae using tox genes and its relationship with disease production.
UNASSIGNED: The online version contains supplementary material available at 10.1007/s13205-023-03660-6.

Usutu virus (USUV) is a mosquito-borne flavivirus of African origin. Over the past decades, USUV has spread through Europe causing mass die-offs among multiple bird species. The natural transmission cycle of USUV involves Culex spp. mosquitoes as vectors and birds as amplifying hosts. Next to birds and mosquitoes, USUV has also been isolated from multiple mammalian species, including humans, which are considered dead-end hosts. USUV isolates are phylogenetically classified into an African and European branch, subdivided into eight genetic lineages (Africa 1, 2, and 3 and Europe 1, 2, 3, 4, and 5 lineages). Currently, multiple African and European lineages are co-circulating in Europe. Despite increased knowledge of the epidemiology and pathogenicity of the different lineages, the effects of co-infection and transmission efficacy of the co-circulating USUV strains remain unclear. In this study, we report a comparative study between two USUV isolates as follows: a Dutch isolate (USUV-NL, Africa lineage 3) and an Italian isolate (USUV-IT, Europe lineage 2). Upon co-infection, USUV-NL was consistently outcompeted by USUV-IT in mosquito, mammalian, and avian cell lines. In mosquito cells, the fitness advantage of USUV-IT was most prominently observed in comparison to the mammalian or avian cell lines. When Culex pipiens mosquitoes were orally infected with the different isolates, no overall differences in vector competence for USUV-IT and USUV-NL were observed. However, during the in vivo co-infection assay, it was observed that USUV-NL infectivity and transmission were negatively affected by USUV-IT but not vice versa.