Isolation Of Strains Research Articles

Potential of training of anti-Staphylococcus aureus therapeutic phages against Staphylococcus epidermidis multidrug-resistant isolates is restricted by inter- and intra-sequence type specificity.

Phage therapy appears to be a promising approach to tackle multidrug-resistant bacteria, including staphylococci. However, most anti-staphylococcal phages have been characterized in Staphylococcus aureus, while a limited number of studies investigated phage activity against S. epidermidis. We studied the potential of phage training to extend the host range of two types of anti-S. aureus phages against S. epidermidis isolates. The Appelmans protocol was applied to a mixture of Kayvirus and a mixture of Silviavirus phages repeatedly exposed to seven S. epidermidis strains representative of nosocomial-associated sequence types (ST), including the world-wide disseminated ST2. We observed increased activity only for the Kayvirus mixture against two of these strains (ST2 or ST35). Phage subpopulations isolated from the training mixture using these two strains (five/strain) exhibited different evolved phenotypes, active only against their isolation strain or strains of the same ST. Of note, 16/47 ST2 strains were susceptible to one of the groups of trained phages. A comparative genomic analysis of ancestral and trained phage genomes, conducted to identify potential bacterial determinants of such specific activity, found numerous recombination events between two of the three ancestors. However, a small number of trained phage genes had nucleotide sequence modifications impacting the corresponding protein compared to ancestral phages, two to four of them per phage genome being specific of each group of phage subpopulations exhibiting different host range. The results suggest that anti-S. aureus phages can be adapted to S. epidermidis isolates but with inter- and intra-ST specificity.ImportanceS. epidermidis is increasingly recognized as a threat for public health. Its clinical importance is notably related to multidrug resistance. Phage therapy is one of the most promising alternative therapeutic strategies to antibiotics. Nonetheless, only very few phages active against this bacterial species have been described. In the present study, we showed that phage training can be used to extend the host range of polyvalent Kayvirus phages within the Staphylococcus genera to include S. epidermidis species. In the context of rapid development of phage therapy, in vitro forced adaptation of previously characterized phages could be an appealing alternative to fastidious repeated isolation of new phages to improve the therapeutic potential of a phage collection.

Coat Proteins of the Novel Victoriviruses FaVV1 and FaVV2 Suppress Sexual Reproduction and Virulence in the Pathogen of Fusarium Head Blight.

Fusarium head blight (FHB), a disease inflicted by Fusarium graminearum and F. asiaticum, poses a growing threat to wheat in China, particularly in the face of climate change and evolving agricultural practices. This study unveiled the discovery of the victorivirus FgVV2 from the F. asiaticum strain F16176 and comprehensively characterized the function of the two victoriviruses FaVV1 and FaVV2 in virulence. Through comparative analysis with a virus-free strain, we established that these mycoviruses markedly repress the sexual reproduction and pathogenicity of their fungal hosts. Furthermore, we synthesized the coat protein (CP) genes CP1 from FaVV1 and CP2 from FaVV2, which were fused with the green fluorescent protein (GFP) gene and successfully expressed in Fusarium strains in wild-type isolates of F. asiaticum and F. graminearum. Similar to virus-infected strains, the transformed strains expressing CPs showed a significant decrease in perithecia formation and pathogenicity. Notably, CP2 exhibited a stronger inhibitory effect than CP1, yet the suppression of sexual reproduction in F. graminearum was less pronounced than that in F. asiaticum. Additionally, the pathogenicity of the F. asiaticum and F. graminearum strains expressing CP1 or CP2 was substantially diminished against wheat heads. The GFP-tagged CP1 and CP2 revealed distinct cellular localization patterns, suggesting various mechanisms of interaction with the host. The findings of this study provide a significant research foundation for the study of the interaction mechanisms between FaVV1 and FaVV2 with their hosts, as well as for the exploration and utilization of fungal viral resources.

Transmission dynamics of ESBL/AmpC and carbapenemase-producing Enterobacterales between companion animals and humans.

Antimicrobial resistance mediated by extended-spectrum beta-lactamase (ESBL)- and plasmid-mediated cephalosporinase (AmpC)-producing Enterobacterales, as well as carbapenemase-producing Enterobacterales have globally increased among companion animals, posing a potential health risk to humans in contact with them. This prospective longitudinal study investigates the transfer of ESBL/AmpC- and carbapenemase-producing Enterobacterales between companion animals and their cohabitant humans in Portugal (PT) and the United Kingdom (UK) during animal infection. Fecal samples and nasal swabs collected from dogs and cats with urinary tract infection (UTI) or skin and soft tissue infection (SSTI), and their cohabitant humans were screened for resistant strains. Relatedness between animal and human strains was established by whole-genome sequencing (WGS). ESBL/AmpC-producing Enterobacterales were detected in companion animals (PT = 55.8%; UK = 36.4%) and humans (PT = 35.9%; UK = 12.5%). Carbapenemase-producing Enterobacterales carriage was observed in one dog from Portugal (2.6%) and another dog from the UK (4.5%). Transmission of index clinical ESBL-producing Escherichia coli and Klebsiella pneumoniae strains to cohabitant humans was observed in three Portuguese households (6.9%, n = 43), with repeated isolation of the index strains on fecal samples from the animals and their cohabiting humans. In addition, longitudinal sharing of E. coli strains carried by companion animals and their owners was observed in other two Portuguese households and two households from the UK. Furthermore, a multidrug-resistant ACT-24-producing Enterobacter hormaechei subsp. hoffmannii strains were also shared within another Portuguese household. These results highlight the importance of the household as an epidemiological unit in the efforts to mitigate the spread of antimicrobial resistance, further emphasizing the need for antimicrobial surveillance in this context, capable of producing data that can inform and evaluate public health actions.

Distribution and species composition of potato viruses in the Novosibirsk region.

Among the many diseases that affect potato plants, viral infections are the most common and cause significant damage to farms, affecting both the yield and quality of potatoes. In this regard, an important condition for preserving the potato seed fund in Russia is systematic monitoring and early highly specific detection of potato viral infections. The purpose of the work is to study samples of potato varieties collected in the Novosibirsk region for the presence of viral infections using RT-PCR. 130 potato plants from three districts of the Novosibirsk region (NR) were studied. As a result of monitoring, the following viruses were identified: PVY (potato virus Y), PVS (potato virus S), PVM (potato virus M) and PVX (potato virus X). The quarantine pathogen potato spindle tuber viroid (PSTVd) was not detected in any of the samples analyzed. The maximum frequency of occurrence in the region was noted for three viruses: PVY, PVM and PVS. A significant proportion of the samples were mixed viral infections: the occurrence of the combination of infection PVY + PVM in plants was 25.0 %, and PVY + PVS, 22.6 %. To develop methods for determining the strain affiliation of the studied samples, the nucleotide sequences of the capsid protein genes of 10 Y-virus isolates were sequenced. Phylogenetic analysis of the studied sequences of NR isolates was carried out with a set of sequences of reference strains 261-4, Eu-N, N:O, NE-11, NTNa, NTNb, N-Wi, O, O5, SYR_I, SYR_II and SYR_III retrieved from GenBank. As a result of phylogenetic analysis, it was established that NR viral samples fell into two groups of strains: group 1, which also includes isolates of the reference strains 261-4/SYR_III, and group 2, NTNa. The obtained results of the strain affiliation of NR samples lay the basis for the development of DNA and immunodiagnostic systems for identifying PVY circulating in NR, as well as for elucidating the source and routes of entry of specific virus strains.Key words: Solanum tuberosum; viral infections; RT-PCR; potato Y virus; phylogenetic analysis.

Prevalence of Influenza B/Yamagata Viruses From Season 2012/2013 to 2021/2022 in Italy as an Indication of a Potential Lineage Extinction.

Influenza B/Yamagata viruses exhibited weak antigenic selection in recent years, reducing their prevalence over time and requiring no update of the vaccine component since 2015. To date, no B/Yamagata viruses have been isolated or sequenced since March 2020. The antibody prevalence against the current B/Yamagata vaccine strain in Italy was investigated: For each influenza season from 2012/2013 to 2021/2022, 100 human serum samples were tested by haemagglutination inhibition (HAI) assay against the vaccine strain B/Phuket/3073/2013. In addition, the sequences of 156 B/Yamagata strains isolated during the influenza surveillance activities were selected for analysis of the haemagglutinin genome segment. About 61.9% of the human samples showed HAI antibodies, and 21.7% had protective antibody levels. The prevalence of antibodies at protective levels in the seasons between the isolation of the strain and its inclusion in the vaccine was between 11% and 25%, with no significant changes observed in subsequent years. A significant increase was observed in the 2020/2021 season, in line with the increase in influenza vaccine uptake during the pandemic. Sequence analysis showed that from 2014/2015 season onward, all B/Yamagata strains circulating in Italy were closely related to the B/Phuket/2013 vaccine strain, showing only limited amino acid variation. A consistent prevalence of antibodies to the current B/Yamagata vaccine strain in the general population was observed. The prolonged use of a well-matched influenza vaccine and a low antigenic diversity of B/Yamagata viruses may have facilitated a strong reduction in B/Yamagata circulation, potentially contributing to the disappearance of this lineage.

Phytopathogenic Fungi and Their Active Metabolites With Bioherbicidal Potential Against the Invasive Alligator Weed, Alternanthera philoxeroides

ABSTRACTAlligator weed (Alternanthera philoxeroides), originally from South America, is a notorious semi‐aquatic invasive species in many countries, including India. Due to its amphibious nature, it not only damages the aquatic environment but is also detrimental to the terrestrial ecosystem. To seek an environmentally sound control option for alligator weed, this study focused on selecting an appropriate mycoherbicidal agent. A series of steps from field survey‐based collection, isolation and selection of promising fungal strains, followed by host range determination to biocontrol potential studies against the weed were undertaken. Twenty‐three phytopathogenic fungi were isolated, and among these, the fungi with the highest herbicidal activity (> 90% disease severity) were subjected to morphological and molecular characterisation and identified as Alternaria alternantherae (= Nimbya alternanthera). The fungus and its crude metabolites were tested against 76 plant species, showing a relatively narrow host range. This fungal species is being reported from West Bengal, India, for the first time. Although the fungus has been considered as a potential biocontrol tool against alligator weed in countries such as Australia, Brazil and China, long‐term, detailed research for A. alternantherae and its active metabolites to be developed as bioherbicides against alligator weed remains indispensable.

Efficient and robust reverse genetics system for bovine rotavirus generation and its application for antiviral screening

Unveiling the molecular mechanisms underlying rotavirus replication and pathogenesis has been hampered by the lack of a reverse genetics (RG) system in the past. Since 2017, multiple plasmid-based RG systems for simian, human, and murine-Like rotaviruses have been established. However, none of the described methods have supported the recovery of bovine rotaviruses (BRVs). Here, we established an optimized plasmid-based RG system for BRV culture-adapted strain (BRV G10P [15] BLR) and clinical isolates (BRV G6P[1] C73, G10P[11] HM26) based on a BHK-T7 cell clone stably expressing T7 polymerase. Furthermore, using this optimized RG system, we successfully rescued the reporter virus BRV rC73/Zs, rHM26/Zs and rBLR/Zs, harboring a genetically modified 1.8-kb segment 7 encoding full-length nonstructural protein 3 (NSP3) fused to ZsGreen, a 232-amino acid green fluorescent protein. Analysis of the stability of genomic insertions showed that the rC73/Zs and rBLR/Zs replicated efficiently and were genetically stable in seven rounds of serial passaging, while rHM26/Zs can be stabilized only up to the third generation, indicating that the BRV segment composition may influence the viral fitness. In addition, we adopted the recombinant reporter viruses for high-throughput screening application and discovered 12 candidates out of 1440 compounds with potential antiviral activities against rotavirus. In summary, this improved RG system of BRVs represents an important tool with great potential for understanding the molecular biology of BRV and facilitates the development of novel therapeutics and vaccines for BRV.

Targeted isolation of Methanobrevibacter strains from fecal samples expands the cultivated human archaeome

Archaea are vital components of the human microbiome, yet their study within the gastrointestinal tract (GIT) is limited by the scarcity of cultured representatives. Our study presents a method for the targeted enrichment and isolation of methanogenic archaea from human fecal samples. The procedure combines methane breath testing, in silico metabolic modeling, media optimization, FACS, dilution series, and genomic sequencing through Nanopore technology. Additional analyzes include the co-cultured bacteriome, comparative genomics of archaeal genomes, functional comparisons, and structure-based protein function prediction of unknown differential traits. Successful establishment of stable archaeal cultures from 14 out of 16 fecal samples yielded nine previously uncultivated strains, eight of which are absent from a recent archaeome genome catalog. Comparative genomic and functional assessments of Methanobrevibacter smithii and Candidatus Methanobrevibacter intestini strains from individual donors revealed features potentially associated with gastrointestinal diseases. Our work broadens available archaeal representatives for GIT studies, and offers insights into Candidatus Methanobrevibacter intestini genomes’ adaptability in critical microbiome contexts.

Epibiotic Bacteria Isolated from the Non-Indigenous Species Codium fragile ssp. fragile: Identification, Characterization, and Biotechnological Potential.

Due to their richness in organic substances and nutrients, seaweed (macroalgae) harbor a large number of epiphytic bacteria on their surfaces. These bacteria interact with their host in multiple complex ways, in particular, by producing chemical compounds. The released metabolites may have biological properties beneficial for applications in both industry and medicine. In this study, we assess the diversity of culturable bacterial community of the invasive alga Codium fragile ssp. fragile with the aim to identify key groups within this epiphytic community. Seaweed samples were collected from the Northern Tunisian coast. A total of fifty bacteria were isolated in pure culture. These bacterial strains were identified by amplification of the ribosomal intergenic transcribed spacer between the 16S and the 23S rRNA genes (ITS-PCR) and by 16S rRNA sequencing. Antimicrobial activity, biochemical, and antibiotic resistance profile characterization were determined for the isolates. Isolated strains were tested for their antimicrobial potential against human and fish bacterial pathogens and the yeast Candida albicans, using the in vitro drop method. About 37% of isolated strains possess antibacterial activity with a variable antimicrobial spectrum. Ba1 (closely related to Pseudoalteromonas spiralis), Ba12 (closely related to Enterococcus faecium), and Bw4 (closely related to Pseudoalteromonas sp.) exhibited strong antimicrobial activity against E. coli. The isolated strain Ba4, closely related to Serratia marcescens, demonstrated the most potent activity against pathogens. The susceptibility of these strains to 12 commonly used antibiotics was investigated. Majority of the isolates were resistant to oxacillin, cefoxitin, tobramycin, and nitrofurantoin. Ba7 and Ba10, closely related to the Vibrio anguillarum strains, had the highest multidrug resistance profiles. The enzymes most commonly produced by the isolated strains were amylase, lecithinase, and agarase. Moreover, nine isolates produced disintegration zones around their colonies on agar plates with agarolitic index, ranging from 0.60 to 2.38. This investigation highlighted that Codium fragile ssp. fragile possesses an important diversity of epiphytic bacteria on its surface that could be cultivated in high biomass and may be considered for biotechnological application and as sources of antimicrobial drugs.

Antilisterial and antioxidant exopolysaccharide from Enterococcus faecium PCH.25 isolated from cow butter: characterization and probiotic potential.

Exopolysaccharides produced by lactic acid bacteria have gained attention for their potential health benefits and applications in functional foods. This study explores the isolation and characterization of a novel exopolysaccharide-producing strain from dairy products. The aim was to evaluate its probiotic potential and investigate the properties of the produced exopolysaccharide. A strain identified as Enterococcus faecium PCH.25, isolated from cow butter, demonstrated exopolysaccharide production. The study's novelty lies in the comprehensive characterization of this strain and its exopolysaccharide, revealing unique properties with potential applications in food, cosmetic, and pharmaceutical industries. The E. faecium PCH.25 strain exhibited strong acid tolerance, with a 92.24% viability rate at pH 2 after 2h of incubation. It also demonstrated notable auto-aggregation (85.27% after 24h) and co-aggregation abilities, antibiotic sensitivity, and absence of hemolytic activity, suggesting its probiotic potential. The exopolysaccharide produced by this strain showed bactericidal activity (MIC and MBC = 1.8mg/ml) against Listeria monocytogenes and antioxidant properties (22.8%). Chemical analysis revealed a heteropolysaccharide composed of glucose and fructose monomers, with various functional groups contributing to its bioactivities. Physical characterization of the exopolysaccharide indicated thermal stability up to 270°C, a negative zeta-potential (-27 mV), and an average particle size of 235nm. Scanning electron microscopy and energy dispersive X-ray analysis revealed a smooth, nonporous structure primarily composed of carbon and oxygen, with an amorphous nature. These findings suggest that the exopolysaccharide from E. faecium PCH.25 has potential as a natural antibacterial and antioxidant polymer for use in functional foods, cosmetics, and pharmaceuticals.

Insight into Virulence and Mechanisms of Amphotericin B Resistance in the Candida haemulonii Complex.

The Candida haemulonii complex includes emerging opportunistic human fungal pathogens with documented multidrug-resistance profiles. It comprises Candida haemulonii sensu stricto, Candida haemulonii var. vulnera, Candida duobushaemulonii, Candida pseudohaemulonii, and Candida vulturna. In recent years, rates of clinical isolation of strains from this complex have increased in multiple countries, including China, Malaysia, and Brazil. Biofilm formation, hydrolytic enzymes, surface interaction properties, phenotype switching and cell aggregation abilities, extracellular vesicles production, stress response, and immune evasion help these fungi to infect the host and exert pathological effects. Multidrug resistance profiles also enhance the threat they pose; they exhibit low susceptibility to echinocandins and azoles and an intrinsic resistance to amphotericin B (AMB), the first fungal-specific antibiotic. AMB is commonly employed in antifungal treatments, and it acts via several known mechanisms. Given the propensity of clinical Candida species to initiate bloodstream infections, clarifying how C. haemulonii resists AMB is of critical clinical importance. This review outlines our present understanding of the C. haemulonii complex's virulence factors, the mechanisms of action of AMB, and the mechanisms underlying AMB resistance.

Nicoliella lavandulae sp. nov., a novel fructophilic Nicoliella species isolated from flowers of Lavandula angustifolia.

A survey of fructophilic lactic acid bacteria associated with wild and cultivated plants in the metropolitan area of Valencia (Spain) led to the isolation of a novel strain of the genus Nicoliella, named Es01T, from flowers of Lavandula angustifolia. The genus Nicoliella encompasses a single species, Nicoliella spurrieriana. Partial 16S rRNA coding gene sequencing revealed a similarity of 98.8% to N. spurrieriana SGEP1_A5T. Average nucleotide identity (ANI) calculations revealed an ANI value of 80.49% with strain SGEP1_A5T, the only N. spurrieriana strain with an available genomic sequence. A digital DNA-DNA hybridization value of 20% was estimated by the Type Strain Genome Server tool when Es01T was compared with strain SGEP1_A5T. On the basis of these results, strain Es01T represents a novel species, for which the name Nicoliella lavandulae sp. nov. is proposed with Es01T (=CECT 30999T=DSMZ 117325T=CCM 9394T) as type strain.

Whole-genome sequence analysis of canine parvovirus reveals replacement with a novel CPV-2c strain throughout India.

Canine parvovirus (CPV) infection causes severe gastroenteritis in canines, with high mortality in puppies. This virus evolved from feline panleukopenia virus by altering its transferrin receptor (TfR), followed by the emergence of CPV-2 variants in subsequent years with altered immunodominant amino acid residues in the VP2 protein. While previous studies have focused on the VP2 gene, there have been fewer studies on non-structural protein (NS1 and NS2) genes. In the present study, CPV genome sequences from clinical samples collected from canines throughout India in 2023, previous Indian CPV isolates from 2009-2019, and the current Indian CPV vaccine strain were compared. The study showed that the CPV-2c (N426E) variant had almost completely replaced the previously dominant CPV-2a variant (N426) in India. The Q370R mutation of VP2 was the most common change in the recent CPV-2c strain (CPV-2c 370Arg variant). Phylogenetic analysis showed the existence of three clades among the recent CPV-2c strains, and sequence analysis identified several new sites of positive selection in the VP1 (N-terminus), VP2, NS1, and NS2 protein-encoding genes in recent CPV strains, indicating the emergence of new CPV-2c variants with varied antigenic and replication properties. The predominant 'CPV-2c 370Arg variants' were grouped with the Chinese and Nigerian CPV-2c strains but were separate from the CPV vaccine strain and earlier isolates from our repository. VP2 epitope analysis predicted nine amino acid variations (including two new variations) in four potential linear B-cell epitopes in the CPV-2c 370Arg variants that might make vaccine failure more likely. This pan-Indian study lays the foundation for further research concerning the dynamics of virus evolution and understanding genetic mutations.

Isolation, Identification and Molecular Characterization of Lumpy Skin Disease Virus for Development of Vaccine

The highly contagious Lumpy skin disease (LSD) virus has been rapidly spreading across South and East Asia, posing a grave threat to cattle populations in the region. LSD initially emerged in Bangladesh during July 2019, starting in the Chattogram district and rapidly spreading across the entire country. To investigate cases of LSD in cattle, a comprehensive study was conducted in various districts of Bangladesh, including Natore, Kurigram, Dinajpur, Dhaka, and Mymensingh. A total of 58 samples comprising blood, skin scraping, buccal swabs, and nasal swabs were collected for analysis. In order to identify LSDV, the DNA was isolated from the processed samples, and a conentional PCR method was employed utilizing the specific primer designed for the 'P-32 Gene' gene. Out of the blood samples tested, only 29.17 percent yielded a positive result for LSDV, whereas a substantial 80 percent of the skin samples tested positive. This suggests that skin scrapings are a viable specimen for LSD diagnosis using PCR. Five positive samples were partially sequenced utilizing the "ANK Gene" for molecular characterization. Bangladeshi isolates have a strong association with LSDV Kenyan (KSGP 0240, NI-2490) strains and Indian isolates (Ranchi, 2019) strains, based on multiple sequence alignment and phylogenetic analysis. Additionally, the sequencing of all the samples from the five research sites were almost identical, suggesting that a single strain is probably spreading throughout the nation. These findings emphasize the need for continuous monitoring of the genetic makeup and molecular epidemiology of LSDV, which will be useful in the future for the development of vaccines specific to the strains that are then in circulation. J. of Sci. and Tech. Res. 5(1): 31-36, 2023

Physiological reactions of some entomopathogenic nematodes to long-term storage

Entomopathogenic nematodes (EPNs) are commonly used for pest control. Determining the optimal storage duration for EPNs is crucial for their effective utilization. The aim of this study is to determine the efficacy and reproductive capacities of some EPNs stored for different durations. Heterorhabditis bacteriophora Poinar, 1976 (Rhabditida: Heterorhabditidae) HBH Hybrid Strain, HBNL, and HB4 isolates, as well as Steinernema feltiae Weiser, 1955 (Rhabditida: Steinernematidae) SADIÇ and ST5 isolates, were used in the study. The Infective Juveniles (IJs) stored at 4ºC for 6, 12, 18, and 24 months were assessed for their efficacy and reproductive capacities on last instar larvae of Galleria mellonella L., 1758 (Lepidoptera: Pyralidae) at the end of the periods. This study was conducted at Bursa Uludağ University, Plant Protection Department, Nematology Laboratory. The highest mortality rate observed on G. mellonella larvae was 86.67% on the H. bacteriophora HBH Hybrid Strain stored for 6 months. Similarly, the highest reproductive capacity was determined to be 153 000 IJs/G. mellonella larva, also on the H. bacteriophora HBH Hybrid Strain stored for 6 months. This study showed significant results in determining the effects of storage durations on the efficacy and reproductive capacity of the EPNs.

Identification and phylogenetic analysis of bovine herpesvirus 4 isolated from aborted bovine foetuses in Türkiye.

Bovine herpesvirus 4 (BoHV-4) is responsible for reproductive disorders and abortions in cattle. The aim of this study was to investigate the potential role of BoHV-4 in abortions in dairy cows when the possible aetiological cause could not be determined in Türkiye. The study also provided the molecular characterization of BoHV-4 strains based on the gB gene. In this study, foetuses (n = 188) and serum (n = 50) were retrospectively used for PCR and indirect ELISA assays, respectively. In addition, foetuses with BoHV-4 were tested for the isolation of the BoHV-4 strain in MDBK cell culture. The BoHV-4 gB gene was detected in the brain, liver, lung, kidney, and spleen tissues of three foetuses out of 188 aborted foetuses. The BoHV-4 strain was isolated from the kidney of one foetus in MDBK cell culture. The three aborted foetuses were from different cows in the same herd in 2021. On this dairy farm, the BoHV-4 seroprevalence rate in cattle with a history of abortion was 96% (48/50). Phylogenetic analysis revealed that the detected strains belong to genotype II. The present study shows that the impact of BoHV-4 on abortions in cattle should not be ignored. It can therefore be informative to focus on BoHV-4 in routine laboratory diagnostics for abortions without a proven aetiology or in cattle herds that have previously been infected with BoHV-4.

The Changes of Microbial Diversity and Isolation of Microorganism in Soil for Alleviating the Production Decreasing After Continuous Cultivation of Ganoderma lucidum.

Ganoderma lucidum is a medicinal mushroom usually cultivated in logs and covered with soil. Its production decreases after continuous cultivation. Changes of microbial diversity in soil are suggested to be one of the reasons. This study aims to investigate the changes of microbial diversity and abundance in soil during cultivation, and isolate potential microbial strains that affect the yield of G. lucidum. Soil samples were collected at two different ranges from logs during one complete growth cycle of G. lucidum. The changes in fungi and bacteria were investigated by using high-throughput sequencing and real-time PCR. Results indicated that the relative abundance of Firmicutes in the bacterial community decreased at the short-range site. In the fungal community, the relative abundance of Ganoderma increased to 70% at the long-range site at the end of the cultivation. The abundance of bacteria and fungi decreased significantly at the end of the growth cycle. Recovery of microbial changes in soil should be proceeded separately based on different ranges to logs. The microbial strains in these soil samples were also isolated and identified. Potential strains were assessed in the form of bio-fertilizer. The yield of G. lucidum in the field using bio-fertilizer with isolated bacterial strains from the Firmicutes phylum was about 13% higher than that without using bio-fertilizer, suggesting the possibility of alleviating the production decrease of G. lucidum by this method.

Building microbial consortia to enhance straw degradation, phosphorus solubilization, and soil fertility for rice growth

Straw pollution and the increasing scarcity of phosphorus resources in many regions of China have had severe impacts on the growing conditions for crop plants. Using microbial methods to enhance straw decomposition rate and phosphorus utilization offers effective solutions to address these problems. In this study, a microbial consortium 6 + 1 (consisting of a straw-degrading bacterium and a phosphate-solubilizing bacterium) was formulated based on their performance in straw degradation and phosphorus solubilization. The degradation rate of straw by 6 + 1 microbial consortium reached 48.3% within 7 days (The degradation ability was 7% higher than that of single bacteria), and the phosphorus dissolution rate of insoluble phosphorus reached 117.54 mg·L− 1 (The phosphorus solubilization ability was 29.81% higher than that of single bacteria). In addition, the activity of lignocellulosic degrading enzyme system was significantly increased, the activities of endoglucanase, β-glucosidase and xylanase in the microbial consortium were significantly higher than those in the single strain (23.16%, 28.02% and 28.86%, respectively). Then the microbial consortium was processed into microbial agents and tested in rice pots. The results showed that the microbial agent significantly increased the content of organic matter, available phosphorus and available nitrogen in the soil. Ongoing research focuses on the determination of the effects and mechanisms of a functional hybrid system of straw degradation and phosphorus removal. The characteristics of the two strains are as follows: Straw-degrading bacteria can efficiently degrade straw to produce glucose-based carbon sources when only straw is used as a carbon source. Phosphate-solubilizing bacteria can efficiently use glucose as a carbon source, produce organic acids to dissolve insoluble phosphorus and consume glucose at an extremely fast rate. The analysis suggests that the microbial consortium 6 + 1 outperformed individual strains in terms of both performance and application effects. The two strains within the microbial consortium promote each other during their growth processes, resulting in a significantly higher rate of carbon source consumption compared to the individual strains in isolation. This increased demand for carbon sources within the growth system facilitates the degradation of straw by the strains. At the same time, the substantial carbon consumption during the metabolic process generated a large number of organic acids, leading to the solubilization of insoluble phosphorus. It also provides a basis for the construction of this type of microbial consortium.

Isolation and identification of entomopathogenic fungi strains for Colorado potato beetle (Leptinotarsa decemlineata) control.

The Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae) is the most widespread insect pest that causes major economic losses, especially on potatoes. Due to heavy insecticide use, this species now resists most pesticides, posing a significant control challenge. Frequent pesticide application also harms non-target organisms, the environment, and human health. Hence, utilizing biocontrol agents like entomopathogenic fungi (EPF) offers a viable alternative for pest management. The aim of this study was to identify and characterize new EPF strains isolated from soil samples and evaluate their efficacy against adult L. decemlineata under laboratory conditions. Soil samples were collected in potato fields or uncultivated areas adjacent to the field in the Czech Republic and the EPF strains were isolated using a modified Tenebrio bait method. A total of 20 fungal strains were isolated and identified using morphological and molecular markers based on the 28S rRNA, ITS, and elongation factor 1-alpha gene sequences as Beauveria bassiana (Bals.-Criv.) Vuill., Beauveria brongniartii (Sacc.) Petch, and Cordyceps fumosorosea (Wize) Kepler, B. Shrestha & Spatafora (Hypocreales: Cordycipitaceae), Purpureocillium lilacinum (Thom.) Luangsa-ard, Houbraken, Hywel-Jones & Samson (Hypocreales: Ophiocordycipitaceae), Metarhizium brunneum (Petch), and Metarhizium robertsii Bisch., Rehner & Humber (Hypocreales: Clavicipitaceae). The bioassays revealed high variability among virulence of these strains against L. decemlineata with the shortest median time to death (LT50=5 days) in M. robertsii strain MAN3b. Results shown that some EPF strains, particularly those of genera Metarhizium, can be promising biocontrol agents against the Colorado potato beetle.

ANTIMICROBIAL, ANTIOXIDANT, AND OPTIMAL CONDITION FOR EXOPOLYSACCHARIDES SECRATED FROM NEW LOCAL STRAIN-ISOLATE Bifidobacterium longum subsp. infantis strain Iraq-Basrah 3

Metabolic products of exopolysaccharide (EPS) generated by LAB strains have several biomedical effects. Our study focused on the isolation of a new strain of Bifidobacterium spp. From the sources of children's feces, examining them to choose the best EPS-producing strain, studying all the optimal conditions to increase the efficiency of producing more EPS, knowing the stage of their production, and finally introducing them into biomedical applications to test their antimicrobial and antioxidant activities. EPS is secreted by the most efficient new local strain Bifidobacterium longum subsp. infantis strain Iraq-Basrah 3 was extracted and purified, resulting in the highest production (2.08±0.41 g/L) in 1% (w/v) starch, which is the best carbon source with the best inoculum size of 1% (v/v) in 18 h at 37 °C and a pH value of 6.5. Coinciding with the best production phase, the generation time for the new strain was 32.2 min, and maximum production occurred after 16 h (2,45 g/L) to 20 h (2.14 g/L) between the end of the logarithmic phase and the early stationary phase. EPS exhibited antimicrobial activity, where S. aureus (17.24±1.2 mm) and E. coli (16.6±0.94 mm) were immediately resistant to EPS. In contrast, other pathogens were resistant to EPS, whereas strong antioxidant activity was observed at a concentration of 1500 µg/ml (84.43±0.95%). Through this study, we have proven that optimal conditions directly affect the quantity and production of EPS and that it has an inhibitory effect on some microbes in addition to being a natural antioxidant for DPPH free radicals.