MiSeq Platform Research Articles

Analysis of the genetic features of the structural organization of integrative conjugative (ICE) elements of Vibrio cholerae strains of various origins

BACKGROUND: Integrative conjugative elements (ICE) make a significant contribution to the spread of bacterial antibiotic resistance genes among cholera pathogen strains. However, at the moment there are no unified methods for typing ICE elements that allow analyzing a large number of genomes. AIM: The aim of the study was to conduct a comparative analysis of the genetic organization and identify features in the structure of integrative conjugative (ICE) elements with the development of new methods for automating their rapid typing in genome-wide sequencing data, followed by the study of a representative collection of genomes of V. cholerae strains of various origins. METHODS: The work uses data from genome-wide sequencing (WGS) of toxigenic (ctxAB+tcpA+) 120 strains of V. cholerae O1 El Tor obtained on the MiSeq platform (Illumina, USA) and MinIon (Oxford Nanopore, UK), and from the NCBI databases (1887 strains) and the European Nucleotide Archive (441 strains). The software for detecting and typing the ICE element is developed in the Java programming language, version 11.0.13 and is located at http://antiplague.ru/ice-genotyper/. RESULTS: A comparative analysis of ICE elements in toxigenic strains of cholera vibrions was carried out. An algorithm for typing ICE elements based on their genetic composition is proposed. Analysis of the genome collection of V. cholerae strains revealed three previously undescribed ICE elements, designated as ICEVchRus1, ICEVchHai3 and ICEVchLaos. Discussion. The distribution of various types of ICE elements in Russia and the world has been studied. It is shown that some variants of ICE elements, despite their almost identical composition, have different designations in the world literature.

Long-term adaptation to dietary shifts of gut microbiota in gilthead seabream (Sparus aurata)

In many meta-analyses and literature reviews on fish microbiota, the provenance of the animals (farmed vs. wild) is often overlooked. Given the well-established role of diet as a key factor in shaping gut microbiota, this study investigates the impact of dietary nature by comparing the microbiota of gilthead seabream (Sparus aurata) fed a commercial diet versus a wild-type diet, all reared within a recirculating aquaculture system. Over a 60-day period, we tracked changes in gut bacterial diversity, structure, and composition following a shift from a commercial feed to a diet exclusively based on pink shrimp (Parapenaeus longirostris). Gut bacterial communities were assessed using 16S rRNA gene sequencing (Illumina MiSeq platform) with primers targeting the V3-V4 hypervariable regions. Twenty days after the dietary change, microbial diversity (Shannon index) increased in fish fed the shrimp diet compared to those fed the commercial diet, while Dominance index values decreased. Additionally, inter-individual (beta-) diversity based on Bray-Curtis distances also differed between dietary treatments. These results support further that microbiota comparisons between farmed/captive and wild fish are challenging due to the unpredictable feeding regimes and dietary variations in wild fish. However, the diet impact on microbiota diminished over time, with the differences in intra- and inter-individual diversity being reduced after 40 days, which suggests an adaptation of microbial communities to dietary changes. At this point, gut microbial communities also showed a similar taxonomical composition. Moreover, a core microbiota consisting of species belonging to the genera Ralstonia, Paraburkholderia, Fulvimonas, Pseudomonas, and Cutibacterium was maintained in all sampling times under both dietary treatments. Overall, this study serves as a conceptual approach that shows a long-term adaptation of the gut microbiota after a radical dietary change, probably driven by host-inherent factors. Furthermore, these results may be a valuable insight for feed manufacturers aiming to develop sustainable and cost-effective ingredients since they suggest that some alternative feeds and ingredients do not have adverse long-term effects on fish gut microbiota.

Genomic analysis, culturing optimization, and characterization of Escherichia bacteriophage OSYSP, previously studied as effective pathogen control on fresh produce

Advances in bacteriophage genome sequencing and regulatory approvals of some bacteriophages in various applications have renewed interest in these antibacterial viruses as a potential solution to persistent food safety challenges. Here, we analyzed in depth the genome of the previously studied Escherichia bacteriophage OSYSP (phage OSYSP), revealed its application-related characteristics, and optimized its enumeration techniques for facilitating industrial implementation. We previously sequenced phage OSYSP genome completely by combining results from Illumina Miseq and Ion Torrent sequencing platforms and completing the remaining sequence gaps using PCR. Based on the genomics analysis completed herein, phage OSYSP was confirmed as an obligate lytic phage of the Caudoviricetes class. The genome encodes 81 proteins of identifiable functions, including two endolysins and 45 proteins that support host-independent DNA replication, transcription, and repair. Despite its similarities to T5-like phages, unique genome arrangements confirm phage OSYSP’s novelty. The genomic analysis also confirmed the absence of DNA sequences encoding virulence or antibiotic resistance factors. For optimizing phage detection and quantification in the conventional plaque assay, it was observed that decreasing the concentration of agar or agarose, when used as a medium gelling agent, increased phage recovery (p < 0.05), but using agarose resulted in smaller plaque diameters (p < 0.05). Phage OSYSP inactivated pathogenic and non-pathogenic strains of E. coli and some Salmonella enterica serovars, with more pronounced effect against E. coli O157:H7. Phage titers remained fairly unchanged throughout a 24-month storage at 4°C. Incubation for 30 min at 4°C−47°C or pH 4–11 had no significant detrimental effect (p > 0.05) on phage infectivity. In vitro application of phage OSYSP against E. coli O157:H7 EDL933 decreased the pathogen’s viable population by >5.7-log CFU/mL within 80 min, at a multiplicity of infection as low as 0.01. The favorable genome characteristics, combined with improved enumeration methodology, and the proven infectivity stability, make phage OSYSP a promising biocontrol agent against pathogenic E. coli for food or therapeutic applications.

Genetic Variability in Neisseria meningitidis Strains Isolated in A Japanese Hospital

Neisseria meningitidis is a significant pathogen causing invasive meningococcal disease, posing clinical and public health concerns worldwide. This study aimed to investigate the genetic characteristics of N. meningitidis clinical isolates at Okayama University Hospital in Japan. Between 2018 and 2023, five clinical strains were isolated, of which three were subjected to the antimicrobial susceptibility testing and whole genetic analysis using MiSeq platform (Illumina, San Diego, CA, USA). One non-groupable isolate, belonging to sequence types (STs)-11026 (ST-32 complex), exhibited non-susceptibility to penicillin G, with a five-mutation pattern (F504L, A510V, I515V, H541N, I566V) in the penA amino acid sequence and additional mutations (XXXIV, N513Y) characteristic of a mosaic penA gene. The other two isolates, ST-1655 (ST-23 complex) with serogroup Y and ST-2057 with serogroup B, were susceptible to penicillin G, neither of which contained the five-mutation pattern. Levofloxacin resistance was observed in two isolates carrying T91I mutation in the gyrA protein. Our findings suggest the presence of antimicrobial-resistant N. meningitidis in Japan, underscoring the necessity for continuous local surveillance. Additional research is crucial for clarifying the ongoing spread of resistance mechanisms and for establishing effective countermeasures to reduce the clinical burden of invasive meningococcal disease.

Whole-Genome Analysis of Multidrug-Resistant Klebsiella pneumoniae Kp04 Reveals Distinctive Antimicrobial and Arsenic-Resistance Genomic Features: A Case Study from Bangladesh.

Multidrug-resistant bacteria, particularly extended-spectrum-beta-lactamase-producing (ESBL) bacteria, pose a significant global public health challenge. Klebsiella pneumoniae (KPN) is frequently implicated in cases of this resistance. This study aimed to investigate the presence of drug and metal resistance genes in clinical K. pneumoniae isolate Kp04 and comparative genomics of clinical KPN isolates characterized from Bangladesh. A total of 12 isolates were collected. Disk-diffusion assay showed that all five isolates were resistant to 14 out of 21 tested antibiotics and sensitive to only three-tigecycline, imipenem, and meropenem. KPN Kp04 was positive for both blaSHV and blaCTX-M ESBL genes in PCR. All five isolates produced PCR amplicons of the correct size for ampicillin (ampC), tetracycline (tetC), fluoroquinolone (qnrS), and aminoglycoside (aadA) resistance genes. The whole genome of Kp04 was sequenced using the MiSeq Platform (V3 kit, 2 × 300 cycles). We utilized different databases to detect Antibiotic-Resistant Genes (ARGs), virulence factor genes (VFGs), and genomic functional features of the Kp04 strain. Whole-genome sequencing identified 75 ESBL, virulence, and multiple drug-resistant (MDR) genes including blaSHV, tetA, oqxA, oqxB, aadA, sul1-5, and mphA in KPN Kp04 isolate. Pan-genomic analysis of 43 Bangladeshi KPN isolates showed similarities between Dhaka and Chattogram isolates regarding virulence and antibiotic-resistant genes. Our results indicate the transmission of similar virulent KPN strains in Dhaka and Chattogram. This study would provide valuable information about drug sensitivity, antibiotic, and metal resistance features of K. pneumoniae circulated among hospitalized patients in Bangladeshi megacities.

Nosocomial outbreak due to a novel Sequence Type of carbapenem resistant Acinetobacter seifertii

BackgroundAcinetobacter seifertii, a member of A.baumannii-calcoaceticus complex, can be considered a pathogen of concern due to the presence of resistance genes. The aim of the study was to describe an outbreak of carbapenem-resistant A. seifertii (CRAS) among neonates admitted to the Neonatal Intensive Care Unit (NICU) at a tertiary care hospital. MethodsAll patients with CRAS diagnosed and admitted to the NICU from June 2023 to October 2023 were included. The presence of carbapenemase genes (blaIMP, blaVIM, blaNDM, blaKPC, blaGES, blaOXA-48-like and blaOXA-23) was investigated by qPCR. Whole Genome Sequencing (WGS) was performed by MiSeq (Illumina) and MinION (Nanopore) platforms. FTIR (IR Biotyper®) was applied for microbial strain typing. ResultsEleven patients were affected and a set of measures were implemented at NICU to reduce the risk of transmission. The isolates exhibited identical resistance patterns, additionally, all isolates presented the blaNDM-1 gene and were grouped in the same cluster by IR Biotyper. The WGS revealed that the isolates belonged to a novel ST assigned as ST2712, and the blaNDM-1 was carried by the same plasmid type. DiscussionOur study has identified a novel strain of A. seifertii carrying blaNDM-1 responsible for the outbreak, indicating its emergence in the institution. ConclusionThis is the first report of carbapenem resistant A. seifertii ST2712. The use of WGS for genomic surveillance allowed understanding the dissemination of CRAB which is crucial in outbreak scenarios.

Genome drafts of Lotmaria passim strains C2 and C3 isolated from honey bees in Spain.

Lotmaria passim is a highly prevalent parasite of honey bees. Herein is reported the draft genome sequences of L. passim C2 and C3 strains of 27.15 Mbp and 26.94 Mbp, respectively. The genomes were sequenced using Illumina MiSeq platform and will allow for further comparative and functional genomics studies.

Delayed Sowing Reduced Verticillium Wilt by Altering Soil Temperature and Humidity to Enhance Beneficial Rhizosphere Bacteria of Sunflower

Sunflower Verticillium Wilt (SVW) caused by Verticillium dahliae is a significant threat to sunflower production in China. This soilborne disease is difficult to control. It has been observed that delayed sowing reduces the severity of SVW on different varieties and across various locations. Soil was collected from multiple locations with different sowing dates to understand the underlying biological mechanisms driving this phenomenon. The soil bacterial community was characterized through 16S rRNA gene amplicon sequencing performed on the Illumina MiSeq platform, followed by comprehensive bioinformatics analysis. Microsclerotia numbers in soil were detected using both NP-10 selective medium and quantitative polymerase chain reaction (qPCR). By delaying the sowing date, the number of microsclerotia in soil and the biomass of V. dahliae colonized inside sunflower roots were reduced during the early developmental stages (V2–V6) of sunflowers. Amplicon sequencing revealed an increased abundance of bacterial genera, such as Pseudomonas, Azoarcus, and Bacillus in soil samples collected from delayed sowing plots. Five bacterial strains isolated from the delayed sowing plot exhibited strong antagonistic effects against V. dahliae. The result of the pot experiments indicated that supplying two different synthetic communities (SynComs) in the pot did increase the control efficiencies on SVW by 19.08% and 37.82% separately. Additionally, soil temperature and humidity across different sowing dates were also monitored, and a significant correlation between disease severity and environmental factors was observed. In conclusion, delayed sowing appears to decrease microsclerotia levels by recruiting beneficial rhizosphere bacteria, thereby reducing the severity of SVW.

Genomic Analysis of Carbapenem-Resistant Acinetobacter baumannii Isolated from Bloodstream Infections in South Korea

Background/Objectives: Bloodstream infection by carbapenem-resistant Acinetobacter baumannii (CRAB) is a serious clinical problem worldwide. To study its clonal relationship and genetic features, we report the draft genome sequence of CRAB strains isolated from human blood in South Korea. Methods: Among A. baumannii strains isolated from patients at nine general hospitals in 2020, 12 CRAB strains of different genotypes were selected. Genomic DNA was sequenced using a combination of Illumina MiSeq and Oxford Nanopore MinION platforms. Antimicrobial susceptibility testing was performed using the disk diffusion method. Antimicrobial resistance and virulence genes were investigated in silico using the Center for Genomic Epidemiology server and the Virulence Factors Database. Results: The multilocus sequence types of isolates included ST191, ST195, ST357, ST369, ST451, ST469, ST491, ST784, ST862, ST1933, ST2929, and a novel type, ST3326. The predominant sequence type, ST191, demonstrated close genetic relationships with several isolates, including ST469, ST369, ST195, ST784, ST491, and ST3326, with ST3326 classified as a subgroup of ST191. We found 18 antimicrobial resistance genes and one quaternary ammonium compound resistance gene. All examined strains harbored blaOXA-23, which is associated with carbapenem resistance. While variations in antibiotic and disinfectant resistance genes were observed, all isolates exhibited similar virulence factors, with the exception of the biofilm and capsule production genes. Conclusions: This nationwide report of the draft genome sequence of patient-derived strains provides valuable insights into the genomic features associated with clonal relationships and antimicrobial resistance of CRAB in bloodstream infections.

Influence of feeding black soldier fly (Hermetia illucens), cricket (Gryllodes sigillatus), and superworm (Zophobas morio) on the gut microbiota of rainbow trout (Oncorhynchus mykiss).

This study investigates how replacing fishmeal and fish oil with insect meals in feed impacts the gut microbiota in rainbow trout (Oncorhynchus mykiss), a crucial species in aquaculture. Dietary inclusion of black soldier fly (Hermetia illucens), cricket (Gryllodes sigillatus), and superworm (Zophobas morio) were evaluated for their impact on intestinal microbial diversity and community composition following a 12-week feeding trial. Fish were fed one of four isonitrogenous and isoenergetic diets: a control diet without insect meal, and diets with 15% defatted black soldier fly meal, full-fat adult cricket meal, or full-fat superworm meal. The microbiota of intestinal digesta and fish feed was characterized using 16S rRNA gene sequencing on the Illumina MiSeq platform. Results revealed significantly lower alpha diversity indices in the cricket treatment compared to the control. Beta diversity analysis showed Bacillota as the dominant phylum across all treatments, with the initial stock population richer in Mycoplasmatota. A novel genus within Mycoplasmataceae was prevalent at day 0 and in all treatments. Black soldier fly meal increased an unidentified Peptostreptococcaceae genus (bsv123) compared to control and superworm diets, while cricket meal elevated Streptococcus levels. Insect-based diets, particularly with black soldier fly meal, significantly alter beta diversity within the gut microbiota of rainbow trout, with cricket meal reducing alpha diversity and superworm having minimal impact.

Clinical and immunological features of four patients with activation-induced cytidine deaminase deficiency: Renal amyloidosis and other presentations.

Activation-induced cytidine deaminase (AID) deficiency is a rare autosomal recessive inborn error of immunity (IEI) characterized by increased susceptibility to infections, autoimmunity, and/or autoinflammation. AID plays an important role in immunoglobulin class switching and somatic hypermutation. AID deficiency patients have very low or absent levels of IgG, IgA, and IgE, while IgM level is elevated. The disease is designated as type 2 hyperimmunoglobulin M syndrome (HIGM-2). To date, around 130 patients with HIGM-2 have been reported, none from Egypt. Four patients from three different consanguineous families with elevated serum IgM and low IgG and IgA were included in the study. After the exclusion of CD40 and/or CD40L deficiency by flow cytometry, patients' samples were tested by a panel covering 452 genes (four bases PID-Pro) on the Illumina Miseq platform. All patients suffered repeated infections since childhood. Patients 1-3 had inflammatory bowel disease-like (IBD-like) symptoms, while patient 4 did not have autoimmune manifestations. Patient 1 is the first HIGM-2 patient to be reported to have renal amyloidosis as part of the autoinflammation. Patients 1-3 had the same pathogenic variant (NM_020661.4 (AID):c.406del, p.Ile136Ter), while patient 4 had another pathogenic variant (NM_020661.4 (AID):c.374G>A, p.Gly125Glu). The variant p.Ile136Ter was not reported before in any of the documented HIGM-2 patients. HIGM-2 is a rare IEI that can be overlooked; hence, patients' diagnosis is delayed. Autoimmune and autoinflammatory manifestations develop later in the disease course leading to significant morbidities. The diagnosis can be suspected after exclusion of CD40/CD40L deficiencies by flow cytometry, and the diagnosis can be confirmed by genetic testing.

Benefits of Crotalaria juncea L. as Green Manure in Fertility and Soil Microorganisms on the Peruvian Coast.

The soils of the Barranca valley are among the best soils in Peru, but with so many years of application of agrochemicals and other agricultural practices, they are losing their productive capacity. Consequently, it was suggested to assess the impact of Crotalaria juncea L. as a green manure on soil fertility and the populations of bacteria and fungi present in the soil. Crotalaria was cultivated for 75 days and incorporated as green manure. After 90 days, the presence of bacteria and fungi was evaluated. Metabarcoding was employed, and the 16S rRNA and ITS2 amplicons were sequenced using the Illumina MiSeq platform. The sequences were processed using various bioinformatics tools. The results indicate that soils have a high diversity of bacteria and fungi. Likewise, in plots where the presence and action of natural biocontrol is suppressed (C0 and P0), pathogenic fungi increase their population in the fallow period (C1), while in P1, the addition of Crotalaria as a green manure promotes an increase in the population of bacteria and fungi, and at the same time it suppresses pathogenic fungi. The genera (bacterial and/or fungal) that increase due to the effect of Crotalaria are beneficial because they are involved as degraders of organic matter, promotion of plant growth and biological control of pathogens. Crotalaria is an alternative to improve soil fertility, increase the beneficial bacterial population, and reduce pathogenic fungi.

Detection of Molecular Biomarkers in Fanconi Anemia Mucosa

Introduction Fanconi anemia (FA) is the most common cause of inherited bone marrow failure, and a cancer predisposition syndrome caused by impaired DNA interstrand crosslink repair, also known as the FA repair pathway. Hematopoietic stem cell transplantations (HSCT) have improved survival in patients with FA, however, those have also heightened the risk of malignancies. Head and neck squamous cell carcinoma (HNSCC), and specifically oral SCC (oSCC), are the most common solid malignancies and the leading cause of death in adult patients with FA. By 45 years of age, HNSCC has a computed cumulative incidence of 50% and 100% in FA patients who did not and did undergo a HSCT, respectively. Historically, SCC in FA has been mostly diagnosed at an advanced stage of disease conferring poor outcome. FA-associated HNSCC are known to be enriched with p53 and structural variants. We aim to develop molecular screening tools and biomarkers of oSCC development in FA patients, to facilitate measurement of potential prophylactic therapies and allow for earlier detection of premalignant oral lesions and cancer. To that end, we are mapping somatic genetic variants in normal-appearing oral mucosa as well as oral lesions in patients with FA, and we plan to evaluate circulating cancer biomarkers. Methods This is a proof-of-concept study conducted in healthy controls (HC) and FA patients. We use a non-invasive oral brush biopsy to collect oral keratinocytes from six different sites of normal-appearing mucosa: retromolar area, side of the tongue and floor of the mouth, bilaterally. DNA is isolated and the exonic regions of the TP53 gene are sequenced using CleanPlex TP53 Kit on an illumina Miseq platform, to identify single nucleotide variants (SNVs), insertion-deletions (indels) at an allele frequency above 1%. Genome-wide SNP genotyping with Infinium Global Diversity Array (v1.0) is used for copy number variant (CNV) identification. Areas of visible changes/abnormal appearing mucosa are also brushed and are tested for the aforementioned genetic changes, as well as for cytology and DNA ploidy. Circulating plasma and serum biomarkers will be evaluated at the end of the study. Results The study is ongoing. 20 HC and 17 FA patients have completed the study to date. Median age of the HC and FA cohorts were 36.3 years (range 23-61) and 31.8 years (range 15-50), respectively. Sex distribution was similar with males consisting of 44% and 53% in the HC and FA cohorts, respectively. The HC cohort included significantly more tobacco smokers and alcohol consumers than the FA cohort (33.3% vs. 0 for smokers, and 83.3% vs.17.6% for alcohol consumers, in the HC vs. FA cohorts, respectively, p<0.05). Using an optimized DNA isolation protocol, we were able to obtain sufficient amount of high molecular weight DNA, for each of the six sites brushed. Both freshly collected and stored samples were successfully used for molecular studies. Storing of HC samples in SurePath preservation solution, for an average duration of 55 days, did not impair DNA yield or sequencing data. No somatic molecular changes were evident in the oral mucosa of the healthy controls (n=18) in over 100 samples taken. At least one somatic molecular change was evident in most participants with FA. Five out of the 15 participants (33.3%) in the FA cohort tested positive for 10 different clinically significant somatic TP53 SNVs. At least one CNV was detected in samples sequenced from non-lesional oral mucosa in 10 out of 15 FA participants (66.7%), with numerous samples testing positive for multiple CNVs. The most frequently observed CNV was chromosome 9p isodisomy, observed in 6/15 participants (40%). Molecular analysis of 13 brushed lesions demonstrated 2 distinct clinically significant TP53 somatic variants in one lesion and multiple CNVs in 3 lesions, all from one individual with FA. Cytology and DNA ploidy analyses were negative in 2 lesions, the third lesion showed suspicious cytology, resulting in a surgical biopsy, which was negative. Analysis of additional samples and recruitment of FA participants is ongoing. Conclusions This study validates noninvasive screening applications for identification of somatic molecular changes in the normal-appearing mucosa in patients with FA. Further studies will assess if these changes can be used as surrogate biomarkers of a short-term outcome for the evaluation of preventative treatment effectiveness in FA-associated oSCC and early diagnosis.

Germline Variants in DNA Interstrand-Cross Link Repair Genes May Contribute to Increased Susceptibility for Serrated Polyposis Syndrome.

Serrated polyposis syndrome (SPS) is characterized by the development of multiple colorectal serrated polyps and increased predisposition to colorectal cancer (CRC). However, the molecular basis of SPS, especially in cases presenting family history of SPS and/or polyps and/or CRC in first-degree relatives (SPS-FHP/CRC), is still poorly understood. In a previous study, we proposed the existence of two molecular entities amongst SPS-FHP/CRC families, proximal/whole-colon and distal SPS-FHP/CRC, according to the preferential location of lesions and somatic events involved in tumor initiation. In the present study, we aimed to investigate these distinct subgroups of SPS patients in a larger cohort at the germline level and to identify the genetic defects underlying an inherited susceptibility for these two entities. Next-generation sequencing was performed using multigene analysis with a custom-designed panel in a Miseq platform in 60 SPS patients (with and without/unknown FHP/CRC). We found germline pathogenic variants in 6/60 patients (ATM, FANCM, MITF, RAD50, RAD51C, and RNF43). We also found variants of unknown significance (VUS), with prediction of probable damaging effect in 23/60 patients (ATM, BLM, BRCA1, FAN1, ERCC2, ERCC3, FANCA, FANCD2, FANCL, MSH2, MSH6, NTHL1, PALB2, PDGFRA, PMS2, PTCH1, RAD51C, RAD51D, RECQL4, TSC2, WRN, and XRCC5 genes). Most variants were detected in gene coding for proteins of the Fanconi Anemia (FA) pathway involved in the DNA Interstrand-Cross Link repair (ICLR). Notably, variants in ICLR genes were significantly more frequent in the proximal/whole-colon than in the distal subgroup [15/44 (34%) vs 1/16 (6%), p = 0.025], as opposed to the non-ICLR genes that were slightly more frequent in the distal group [8/44 (18%) vs. 5/16 (31%), p > 0.05]. Germline defects in the DNA-ICLR genes may contribute to increased serrated colorectal polyps/carcinoma risk in SPS patients, particularly in proximal/whole-colon SPS. The inclusion of DNA-ICLR genes in the genetic diagnosis of SPS patients, mainly in those with proximal/whole-colon lesions, should be considered and validated by other studies. In addition, patients with germline defects in the DNA-ICLR genes may be more sensitive to treatment with platinum-based therapeutics, which can have implications in the clinical management of these patients.

Nanopore Sequencing for T-Cell Receptor Rearrangement Analysis in Cutaneous T-Cell Lymphoma.

Background: Analysis of T-cell receptor (TCR) clonality is a major diagnostic tool for lymphomas, particularly for cutaneous T-cell lymphomas (CTCL) like Mycosis fungoides and Sézary syndrome. However, a fast and cost-effective workflow is needed to enable widespread use of this method. Methods: We established a procedure for TCR rearrangement analysis via Oxford Nanopore Technology (ONT) sequencing. TCR receptor rearrangements (TCR-gamma and TCR-beta chains) were analyzed in samples from 45 patients with various diagnoses: Mycosis fungoides (37/45), Sézary Syndrome (2/45), folliculotropic CTCL (1/45), and non-CTCL diagnoses as polyclonal controls (5/45). Sample types included formalin-fixed paraffin-embedded (FFPE) samples (27/45), fresh frozen samples (9/45), and CD3-isolated cells (9/45). In addition, DNA of a Jurkat cell line was used as a monoclonal control. TCR amplicons were generated employing an optimized version of the protocol from the Euro Clonality consortium. Sequencing was conducted on the ONT GridION and Illumina MiSeq platforms, followed by similar bioinformatic analysis protocols. The tumor clone frequency (TCF), a crucial prognostic factor for CTCL patients, was used for method comparison. Results: The use of an optimized amplicon protocol and adapted bioinformatic tools demonstrated a strong correlation in TCF values between both sequencing methods across all sample types (range R: 0.992-0.996; range r2: 0.984-0.991). Conclusions: In summary, ONT sequencing was able to detect TCR clonality comparable to NGS, indicating its potential as a faster and more cost-effective option for routine diagnostic use.

The microbiota comparative analysis of the characteristics between colorectal adenomatous polyps and normal mucosal intestinal.

The aim of this study is to systematically examine and compare the characteristics distinguishing colorectal adenomatous polyps from normal mucosal intestinal microbiota. A total of 30 specimens were obtained from patients diagnosed with colorectal adenomatous polyps (adenoma group) who underwent endoscopic removal at Wenzhou People's Hospital between September 2021 and November 2021. Concurrently, 30 normal mucosal specimens were collected from patients without adenomatous polyps (control group). Subsequently, microbiome total DNA extraction was carried out, followed by PCR amplification targeting the V3-V4 region of the 16S rDNA. High-throughput sequencing was conducted using the Illumina MiSeq platform. Subsequent to sequencing, bioinformatics analysis was used to assess the diversity, composition, and functional aspects of the intestinal microbiota in both study groups. A notable dissimilarity in the microbiota structure was identified, specifically within the transverse colon, between these two groups ( P < 0.05). Species composition analysis revealed that Escherichia , Fusobacterium , and Bacteroides were predominant bacteria in both groups, with Escherichia and Enterobacter displaying significant differences at the genera level between the control group and the adenoma group ( P < 0.05). Correlation analysis and functional prediction demonstrated substantial disparities in interactions among dominant intestinal microbial genera within patients from both groups. Additionally, it was discovered that the intestinal microbiomes in patients in the adenoma group exhibited a significantly higher pathogenic potential. Upon conducting a comprehensive analysis, it was discerned that the microbiota present in the transverse colon of the control group exhibited distinctive characteristics that may contribute to the maintenance of intestinal health.

DNA Barcoding Using 18S rRNA Gene Fragments for Identification of Tick-Borne Protists in Ticks in the Republic of Korea.

The objective of this study was to evaluate the diversity and prevalence of tick-borne protists in the Republic of Korea via DNA barcoding using 18S rRNA gene fragments and PCR. Between 2021 and 2022, questing ticks were collected using the flagging method, with a total of 13,375 ticks collected and pooled into 1003 samples. Of these, 50 tick pools were selected for DNA barcoding targeting the V4 and V9 regions of 18S rRNA using the MiSeq platform. A taxonomic analysis of the amplicon sequence variants identified three genera of protozoa, namely Hepatozoon canis, Theileria luwenshuni, and Gregarine sp. However, the number and abundance of protists detected were different depending on the primer sets, and T. gondii was not identified in DNA barcoding. Furthermore, conventional PCR confirmed the presence of H. canis, Toxoplasma gondii, T. luwenshuni, and Theileria sp. in the collected ticks. This study identified H. canis and T. gondii in Ixodes nipponensis for the first time. It demonstrated that the results of DNA barcoding using 18S rRNA gene fragments can vary depending on the primer sets and further optimization is required for library construction to identify tick-borne protists in ticks. Despite these limitations, the findings highlight the potential of DNA barcoding using 18S rRNA gene fragments for screening the diversity of tick-borne protists in ticks.

Severe co-infection caused by difficult-to-diagnose hypermucoviscous Klebsiella pneumoniae K1-ST82 in a patient with COVID-19: a case report

BackgroundCo-infection with Klebsiella pneumoniae presents a significant concern in hospitalized patients with coronavirus disease (COVID-19), increasing the risk of severe disease progression. Hypervirulent (hv) and hypermucoviscous (hm) K. pneumoniae (Kp) has gained prominence in Asia due to its capacity to cause invasive community-acquired infections. However, recognition of hvKp/hmKp co-infections in the context of COVID-19 remains limited. We report a severe case of rapidly progressing co-infection with hmKp exhibiting “difficult-to-diagnose” phenotypes in a hospitalized patient with COVID-19.Case presentationA 61-year-old woman with COVID-19 initially exhibited mild symptoms resembling the common cold. However, her condition rapidly deteriorated over 7 days, leading to hospital admission with the development of dyspnea. The patient required supplemental oxygen, antibiotic treatment, and mechanical ventilation. Gram-negative bacteria with atypical phenotypes were isolated from alveolar lavage fluid and blood cultures. Both strains formed small, glossy, non-lactose-fermenting colonies on clinically relevant media and were susceptible to ampicillin. Conventional biochemical tests failed to identify the Enterobacteriales strains owing to the urease-negative phenotype. Consequently, the identification of K. pneumoniae was difficult until matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis was performed. A positive string test indicated mucoviscosity, but with variability in the material used for stretching colonies. Whole-genome sequencing performed on the MiSeq and GridION platforms revealed the blood-derived strain JARB-RN-0063 as belonging to serotype K1 and sequence type (ST) 82. The hvKp-associated genes rmpA and iroCD were located on a 5.0-Mb chromosome, and iucABCD-iutA was identified on a 217.9-kb IncFIB(K)/IncR-type plasmid. Therefore, JARB-RN-0063 was genetically classified as hvKp with a Kleborate virulence score of 3. The intrinsic penicillinase gene blaSHV was defective owing to an IS1F element insertion, resulting in the strain being atypically susceptible to ampicillin.ConclusionsThis is the first case of severe COVID-19-associated co-infection with a difficult-to-diagnose K. penummoniae strain. Notably, co-infection by the hmKp K1-ST82 clone exhibited atypical phenotypes, including stunted growth, non-lactose fermentation, urease-negative reaction, ampicillin susceptibility, and abnormal mucoviscosity, posing diagnostic challenges for clinical laboratories and impedes the identification of hvKp/hmKp. Delayed identification may worsen patient outcomes, highlighting the need for increased clinical awareness of such difficult-to-diagnose clones to prevent deterioration.

Circulating miRNAs and Machine Learning for Lateralizing Primary Aldosteronism.

Distinguishing between unilateral and bilateral primary aldosteronism, a major cause of secondary hypertension, is crucial due to different treatment approaches. While adrenal venous sampling is the gold standard, its invasiveness, limited availability, and often difficult interpretation pose challenges. This study explores the utility of circulating microRNAs (miRNAs) and machine learning in distinguishing between unilateral and bilateral forms of primary aldosteronism. MiRNA profiling was conducted on plasma samples from 18 patients with primary aldosteronism taken during adrenal venous sampling on an Illumina MiSeq platform. Bioinformatics and machine learning identified 9 miRNAs for validation by reverse transcription real-time quantitative polymerase chain reaction. Validation was performed on a cohort consisting of 108 patients with known subdifferentiation. A 30-patient subset of the validation cohort involved both adrenal venous sampling and peripheral, the rest only peripheral samples. A neural network model was used for feature selection and comparison between adrenal venous sampling and peripheral samples, while a deep-learning model was used for classification. Our model identified 10 miRNA combinations achieving >85% accuracy in distinguishing unilateral primary aldosteronism and bilateral adrenal hyperplasia on a 30-sample subset, while also confirming the suitability of peripheral samples for analysis. The best model, involving 6 miRNAs, achieved an area under curve of 87.1%. Deep learning resulted in 100% accuracy on the subset and 90.9% sensitivity and 81.8% specificity on all 108 samples, with an area under curve of 86.7%. Machine learning analysis of circulating miRNAs offers a minimally invasive alternative for primary aldosteronism lateralization. Early identification of bilateral adrenal hyperplasia could expedite treatment initiation without the need for further localization, benefiting both patients and health care providers.

Effect of conservation agriculture on soil fungal diversity in rice-wheat-greengram cropping system in eastern Indo-Gangetic plains of South Asia.

Conservation agriculture (CA) is emerging as an eco-friendly and sustainable approach to food production in South Asia. CA, characterized by reduced tillage, soil surface cover through retaining crop residue or raising cover crops, and crop diversification, enhances crop production and soil fertility. Fungal communities in the soil play a crucial role in nutrient recycling, crop growth, and agro-ecosystem stability, particularly in agricultural crop fields. This study investigates the impact of seven combinations of tillage and crop residue management practices of agricultural production systems, including various tillage and crop residue management practices, on soil fungal diversity. Using the Illumina MiSeq platform, fungal diversity associated with soil was analysed. The results show that the partial CA-based (pCA) production systems had the highest number of unique operational taxonomic units (OTUs) (948 OTUs) while the conventional production system had the lowest number (665 OTUs). The major fungal phyla identified in the topsoil (0-15 cm) were Ascomycota, Basidiomycota, and Mortierellomycota, with their abundance varying across different tillage-cum-crop establishment (TCE) methods. Phylum Ascomycota was dominant in CA-based management treatments (94.9±0.62), followed by the partial CA (pCA)-based treatments (91.0 ± 0.37). Therefore, CA-based production systems play a crucial role in shaping soil fungal diversity, highlighting their significance for sustainable agricultural production.