Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology最新文献

Yersinia pestis, the causative agent of plague, is a highly virulent bacterium that poses a significant threat to human health. Preserving this bacterium in a viable state is crucial for research and diagnostic purposes. This paper presents and evaluates a simple lyophilization protocol for the long-term storage of Y. pestis strains from Fiocruz-CYP, aiming to explore its impact on viability and long-term stability, while replacing the currently used methodologies. The lyophilization tests were conducted using the non-virulent Y. pestis strain EV76, subjected to the lyophilization process under vacuum conditions. Viability assessment was performed to evaluate the effects of lyophilization and storage conditions on Y. pestis under multiple temperature conditions (- 80 °C, - 20 °C, 4-8 °C and room temperature). The lyophilization protocol employed in this study consistently demonstrated its efficacy in maintaining high viability rates for Y. pestis samples in a up to one year follow-up. The storage temperature that consistently exhibited the highest recovery rates was - 80 °C, followed by - 20 °C and 4-8 °C. Microscopic analysis of the post-lyophilized cultures revealed preserved morphological features, consistent with viable bacteria. The high viability rates observed in the preserved samples indicate the successful preservation of Y. pestis using this protocol. Overall, the presented lyophilization protocol provides a valuable tool for the long-term storage of Y. pestis, offering stability, viability, and functionality. By refining the currently used methods of lyophilization, this protocol can improve long-term preservation for Y. pestis strains collections, facilitating research efforts, diagnostic procedures, and the development of preventive and therapeutic strategies against plague.

The microbial diversity associated with terrestrial groundwater seepage through permafrost soils is tightly coupled to the geochemistry of these fluids. Terrestrial alkaline methane seeps from Lagoon Pingo, Central Spitsbergen (78°N) in Norway, with methane-saturated and oxygen-limited groundwater discharge providing a potential habitat for methanotrophy. Here, we report on the microbial community's comparative analyses and distribution patterns at two sites close to Lagoon Pingo's methane emission source. To target methane-oxidizing bacteria from this system, we analysed the microbial community pattern of replicate samples from two sections near the main methane seepage source. DNA extraction, metabarcoding and subsequent sequencing of 16S rRNA genes revealed microbial communities where the major prokaryotic phyla were Pseudomonadota (42-47%), Gemmatimonadota (4-14%) and Actinobacteriota (7-11%). Among the Pseudomonadota, members of the genus Methylobacter were present at relative abundances between 1.6 and 4.7%. Enrichment targeting the methane oxidising bacteria was set up using methane seep sediments as inoculum and methane as the sole carbon and energy source, and this resulted in the isolation of a novel psychrophilic methane oxidizer, LS7-T4A^T. The optimum growth temperature for the isolate was 13 °C and the pH optimum was 8.0. The morphology of cells was short rods, and TEM analysis revealed intracytoplasmic membranes arranged in stacks, a distinctive feature for Type I methanotrophs in the family Methylomonadaceae of the class Gammaproteobacteria. The strain belongs to the genus Methylobacter based on high 16S rRNA gene similarity to the psychrophilic species of Methylobacter psychrophilus Z-0021^T (98.95%), the psychrophilic strain Methylobacter sp. strain S3L5C (99.00%), and the Arctic mesophilic species of Methylobacter tundripaludum SV96^T (99.06%). The genome size of LS7-T4A^T was 4,338,157 bp with a G + C content of 47.93%. The average nucleotide identities (ANIb) of strain LS7-T4A^T to 10 isolated strains of genus Methylobacter were between 75.54 and 85.51%, lower than the species threshold of 95%. The strain LS7-T4A^T represents a novel Arctic species, distinct from other members of the genus Methylobacter, for which the name Methylobacter svalbardensis sp. nov. is proposed. The type of strain is LS7-T4A^T (DSMZ:114308, JCM:39463).

Translation elongation factor P, expressed by the efp gene, is a conserved protein closely related to bacterial virulence and environmental stress regulation responses, however, little is known about the efp gene expression regulations. Here, the strain of Staphylococcus aureus subsp. aureus NCTC 8325 was taken as the research object and cultured under different conditions, including different culture temperatures, pH, and antibiotics, to study the expression of the efp gene in S. aureus by qRT-PCR, the results showed that the expression of the efp gene is upregulated under high temperature (40 °C), acidic (pH 5.4) or alkaline (pH 9.4) culture conditions, but upregulated early and downregulated later under the conditions of 0.5 MIC antibiotics (chloramphenicol at the final concentration of 2 μg/mL and vancomycin at the final concentration of 0.25 μg/mL), indicating that the efp promoter in S. aureus is inducible. The efp promoter sequence and structure in S. aureus were predicted by bioinformatics methods, and the predicted promoter was validated by constructing a promoter-probe vector and a series of promoter mutants, the results showed that the efp promoter sequence in S. aureus, named Pro, located in 1,548,179-1,548,250 of the S. aureus genome (NC_007795.1), and the sequence of - 10 element is CCTTATAGT, - 35 element is TTTACT. The results above could lay a foundation for screening transcription factors involved in the expression of the efp gene and then exploring the transcriptional regulation mechanism of EF-P in S. aureus.

A new member of the family Flavobacteriaceae (termed Hal144^T) was isolated from the marine breadcrumb sponge Halichondria panicea. Sponge material was collected in 2018 at Schilksee which is located in the Kiel Fjord (Baltic Sea, Germany). Phylogenetic analysis of the full-length Hal144^T 16S rRNA gene sequence revealed similarities from 94.3 to 96.6% to the nearest type strains of the genus Maribacter. The phylogenetic tree of the 16S rRNA gene sequences depicted a cluster of strain Hal144^T with its closest relatives Maribacter aestuarii GY20^T (96.6%) and Maribacter thermophilus HT7-2^T (96.3%). Genome phylogeny showed that Maribacter halichondriae Hal144^T branched from a cluster consisting of Maribacter arenosus, Maribacter luteus, and Maribacter polysiphoniae. Genome comparisons of strain Maribacter halichondriae Hal144^T with Maribacter sp. type strains exhibited average nucleotide identities in the range of 75-76% and digital DNA-DNA hybridisation values in the range of 13.1-13.4%. Compared to the next related type strains, strain Hal144^T revealed unique genomic features such as phosphoenolpyruvate-dependent phosphotransferase system pathway, serine-glyoxylate cycle, lipid A 3-O-deacylase, 3-hexulose-6-phosphate synthase, enrichment of pseudogenes and of genes involved in cell wall and envelope biogenesis, indicating an adaptation to the host. Strain Hal144^T was determined to be Gram-negative, mesophilic, strictly aerobic, flexirubin positive, resistant to aminoglycoside antibiotics, and able to utilize N-acetyl-β-D-glucosamine. Optimal growth occurred at 25-30 °C, within a salinity range of 2-6% sea salt, and a pH range between 5 and 8. The major fatty acids identified were C_17:0 3-OH, iso-C_15:0, and iso-C_15:1 G. The DNA G + C content of strain Hal144^T was 41.4 mol%. Based on the polyphasic approach, strain Hal144^T represents a novel species of the genus Maribacter, and we propose the name Maribacter halichondriae sp. nov. The type strain is Hal144^T (= DSM 114563^T = LMG 32744^T).

Antimicrobial peptides (AMPs) are promising cationic and amphipathic molecules to fight antibiotic resistance. To search for novel AMPs, we applied a computational strategy to identify peptide sequences within the organisms' proteome, including in-house developed software and artificial intelligence tools. After analyzing 150.450 proteins from eight proteomes of bacteria, plants, a protist, and a nematode, nine peptides were selected and modified to increase their antimicrobial potential. The 18 resulting peptides were validated by bioassays with four pathogenic bacterial species, one yeast species, and two cancer cell-lines. Fourteen of the 18 tested peptides were antimicrobial, with minimum inhibitory concentrations (MICs) values under 10 µM against at least three bacterial species; seven were active against Candida albicans with MICs values under 10 µM; six had a therapeutic index above 20; two peptides were active against A549 cells, and eight were active against MCF-7 cells under 30 µM. This study's most active antimicrobial peptides damage the bacterial cell membrane, including grooves, dents, membrane wrinkling, cell destruction, and leakage of cytoplasmic material. The results confirm that the proposed approach, which uses bioinformatic tools and rational modifications, is highly efficient and allows the discovery, with high accuracy, of potent AMPs encrypted in proteins.

Carbapenem resistant Klebsiella pneumoniae causing severe infection resulting in morbidity and mortality have become a global health concern. K. pneumoniae with sequence type ST147 is an international high-risk clonal lineage, genomic studies have been done on K. pneumoniae ST147 isolated from clinical origin but genomic data for environmental K. pneumoniae ST147 is very scarce. Herein, K. pneumoniae IITR008, an extensively drug resistant and potentially hypervirulent bacterium, was isolated from Triveni Sangam, the confluence of three rivers where religious congregations are organized. Phenotypic, genomic and comparative genomic analysis of strain IITR008 was performed. Antibiotic susceptibility profiling revealed resistance to 9 different classes of antibiotics including ß-lactams, ß-lactam combination agents, carbapenem, aminoglycoside, macrolide, quinolones, cephams, phenicol, and folate pathway antagonists and was found to be susceptible to only tetracycline. The strain IITR008 possesses hypervirulence genes namely, iutA and iroN in addition to numerous virulence factors coding for adherence, regulation, iron uptake, secretion system and toxin. Both the IITR008 chromosome and plasmid pIITR008_75 possess a plethora of clinically relevant antibiotic-resistant genes (ARGs) including bla_CTX-M-15, bla_TEM-1, and bla_SHV-₁₁, corroborating the phenotypic resistance. Comparative genomic analysis with other ST147 K. pneumoniae provided insights on the phylogenetic clustering of IITR008 with a clinical strain isolated from a patient in Czech with recent travel history in India and other clinical strains isolated from India and Pakistan. According to the 'One Health' perspective, surveillance of antibiotic resistance in the environment is crucial to impede its accelerated development in diverse ecological niches.

The genus Sporendonema (Gymnoascaceae, Onygenales) was introduced in 1827 with the type species S. casei for a red mould on cheese. Cheese is a consistent niche for this species. Sphaerosporium equinum is another species classified in Gymnoascaceae and has also been reported from cheese. Recently, other habitats have been reported for both Sporendonema casei and Sphaerosporium equinum. The present study aimed to investigate the taxonomy of Sporendonema and Sphaerosporium, as well as a close neighbour, Arachniotus. Two strains of Hormiscium aurantiacum, another related cheese-associated species were also included in the analyses. Strains were evaluated in terms of macro- and micromorphology, physiology including salt tolerance, growth rate at different temperatures, casein degradation, cellulase activity, lipolytic activity, and multi-locus phylogeny with sequences of the nuclear ribosomal internal transcribed spacer region, the D1-D2 region of the large subunit and partial β-tubulin locus sequences. The results showed that the analysed species were congeneric, and the generic names Arachniotus and Sphaerosporium should be reduced to the synonymy of Sporendonema. Therefore, four new combinations as well as one lectotype and one epitype were designated in Sporendonema. Two strains attributed to Sphaerosporium equinum from substrates other than cheese were found to be phylogenetically and morphologically deviant and were introduced as a new species named Sporendonema isthmoides.

In this study, we reported a Gram-stain-negative, ovoid to rod-shaped, atrichous, and facultative anaerobe bacteria strain named YMD61^T, which was isolated from the intertidal sediment of Yangma island, China. Growth of strain YMD61^T occurred at 10.0-45.0 °C (optimum, 30.0 °C), pH 7.0-10.0 (optimum, 8.0) and with 0-3.0% (w/v) NaCl (optimum, 2.0%). Phylogenetic tree analysis based on 16 S rRNA gene or genomic sequence indicated that strain YMD61^T belonged to the genus Fuscovulum and was closely related to Fuscovulum blasticum ATCC 33,485^T (96.6% sequence similarity). Genomic analysis indicated that strain YMD61^T contains a circular chromosome of 3,895,730 bp with DNA G + C content of 63.3%. The genomic functional analysis indicated that strain YMD61^T is a novel sulfur-metabolizing bacteria, which is capable of fixing carbon through an autotrophic pathway by integrating the processes of photosynthesis and sulfur oxidation. The predominant respiratory quinone of YMD61^T was ubiquinone-10 (Q-10). The polar lipids of YMD61^T contained phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, five unidentified lipids, unidentified aminolipid and unidentified aminophospholipid. The major fatty acids of strain YMD61^T contained C_18:1ω7c 11-methyl and summed feature 8 (C_18:1 ω 7c or/and C_18:1 ω 6c). Phylogenetic, physiological, biochemical and morphological analyses suggested that strain YMD61^T represents a novel species of the genus Fuscovulum, and the name Fuscovulum ytuae sp. nov. is proposed. The type strain is YMD61^T (= MCCC 1K08483^T = KCTC 43,537^T).

The current species of Halosegnis and Salella within the class Halobacteria are closely related based on phylogenetic, phylogenomic, and comparative genomic analyses. The Halosegnis species showed 99.8-100.0% 16S rRNA and 96.6-99.6% rpoB' gene similarities to the Salella species, respectively. Phylogenetic and phylogenomic analyses showed that Salella cibi CBA1133^T, the sole species of Salella, formed a single tight cluster with Halosegnis longus F12-1^T, then with Halosegnis rubeus F17-44^T. The average nucleotide identity (ANI), digital DNA-DNA hybridization (dDDH), and average amino acid identity (AAI) values between Salella cibi CBA1133^T and Halosegnis longus F12-1^T were 99.2, 94.2, and 98.6%, respectively, much higher than the thresholds for species demarcation. This genome-based classification revealed that the genus Salella should be merged with Halosegnis, and Salella cibi should be a later heterotypic synonym of Halosegnis longus. Halophilic archaeal strains DT72^T, DT80^T, DT85^T, and DT116^T, isolated from the saline soil of a tidal flat in China, were subjected to polyphasic taxonomic characterization. The phenotypic, chemotaxonomic, phylogenetic, and phylogenomic features indicated that strains DT72^T (= CGMCC 1.18925^T = JCM 35418^T), DT80^T (= CGMCC 1.18926^T = JCM 35419^T), DT85^T (= CGMCC 1.19049^T = JCM 35605^T), and DT116^T (= CGMCC 1.19045^T = JCM 35606^T) represent four novel species of the genera Halorussus, Halosegnis and Haloglomus, respectively, for which the names, Halorussus caseinilyticus sp. nov., Halorussus lipolyticus sp. nov., Halosegnis marinus sp. nov., and Haloglomus litoreum sp. nov., are proposed.

A strictly anaerobic, Gram-stain-negative, catalase-negative, cocci-shaped, and propionate-producing bacterial strain, named Ds1651^T was isolated from the fecal sample collected from a South Korean infant. Through a comparison of 16S rRNA gene sequences, it was revealed that Ds1651^T had the highest phylogenetic affinity with Veillonella nakazawae KCTC 25297 ^T (99.86%), followed by Veillonella infantium KCTC 25370 ^T (99.80%), and Veillonella dispar KCTC 25309 ^T (99.73%) in the family Veillonellaceae. Average nucleotide identity values between Ds1651^T and three reference species were 95.48% for Veillonella nakazawae KCTC 25297 ^T, 94.46% for Veillonella infantium KCTC 25370 ^T, and 92.81% for Veillonella dispar KCTC 25309 ^T. The G + C content of Ds1651^T was 38.58 mol%. Major fermentation end-products were acetic and propionic acids in Trypticase peptone glucose yeast extract broth with 1% (v/v) sodium lactate. The predominant cellular fatty acids that account for more than 10% were summed in Feature 8 (C_17:1 ω8c and/or C_17:2) and C_13:0. Based on the findings from phylogenetic, genomic, phenotypic, and chemotaxonomic studies, we propose that the type strain Ds1651^T (= KCTC 25477 ^T = GDMCC 1.3707 ^T) represents a novel bacterial species within the genus Veillonella, with the proposed name Veillonella faecalis sp. nov.