Genotype Strains Research Articles

Draft genome sequence of Exiguobacterium sp. from whole cantaloupe, with inhibition capacity against Listeria monocytogenes.

We report the draft genome sequence of a novel species, Exiguobacterium sp., isolated from a freshly harvested and untreated cantaloupe in North Carolina. The strain Exiguobacterium wild type exhibited inhibitory activity against the foodborne pathogen Listeria monocytogenes, including strains of diverse serotypes and genotypes, both on agar media and in biofilms.

Metagenomic identification of pathogens and antimicrobial-resistant genes in bacterial positive blood cultures by nanopore sequencing.

Nanopore sequencing workflows have attracted increasing attention owing to their fast, real-time, and convenient portability. Positive blood culture samples were collected from patients with bacterial bloodstream infection and tested by nanopore sequencing. This study compared the sequencing results for pathogen taxonomic profiling and antimicrobial resistance genes to those of species identification and phenotypic drug susceptibility using traditional microbiology testing. A total of 37 bacterial positive blood culture results of strain genotyping by nanopore sequencing were consistent with those of mass spectrometry. Among them, one mixed infection of bacteria and fungi was identified using nanopore sequencing and confirmatory quantitative polymerase chain reaction. The amount of sequencing data was 21.89 ± 8.46 MB for species identification, and 1.0 MB microbial strain data enabled accurate determination. Data volumes greater than or equal to 94.6 MB nearly covered all the antimicrobial resistance genes of the bacteria in our study. In addition, the results of the antimicrobial resistance genes were compared with those of phenotypic drug susceptibility testing for Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus. Therefore, the nanopore sequencing platform for rapid identification of causing pathogens and relevant antimicrobial resistance genes complementary to conventional blood culture outcomes may optimize antimicrobial stewardship management for patients with bacterial bloodstream infection.

Insidious transmission of Mycobacterium tuberculosis in Ordos, China: a molecular epidemiology study.

In this study, we conducted this population-based study to evaluate the genetic diversity and clustering rate of Mycobacterium tuberculosis (MTB) strains using the whole-genome sequencing (WGS), to better understand its transmission in Ordos. All patients with culture-positive TB notified in Ordos from January 2021 to December 2022 were recruited. WGS was performed to analyze single-nucleotide polymorphism (SNP) and to identify genotypic drug susceptibilities of MTB isolates. Overall, a total of 186 patients were included in the present study, of whom 35 (18.8%) had no symptoms suggestive of active TB. Lineage 2 was the predominant MTB sublineage, accounting for 186 of isolates tested. When the pairwise SNP difference ≤ 12 was used as the cutoff for WGS-based clusters, we identified 17 genotypic clusters, and 38 isolates belonged to these 17 clusters, resulting in a clustering rate of 20.4%. The Beijing genotype was an independent factor associating with genomic-clustering (adjusted OR 4.219, 95% CI 0.962-18.502). The overall sensitivity on WGS-based resistance prediction was 85.7% for rifampicin, 73.1% for isoniazid, 60.0% for Ethambutol, 72.7% for streptomycin, and 72.7% for fluoroquinolones. To conclude, the present study demonstrates the extensive recent transmission of Beijing genotype strains in the community of Ordos. The failure to provide a comprehensive pattern of transmission indicated the missed diagnosis of active TB within the community. A substantial proportion of subclinical TB cases are recognized in the bacteria-positive cases, emphasizing that we must interrupt transmission by finding people with active TB before they infect others.

Effects of Elevated Temperature on Pisum sativum Nodule Development: I-Detailed Characteristic of Unusual Apical Senescence.

Despite global warming, the influence of heat on symbiotic nodules is scarcely studied. In this study, the effects of heat stress on the functioning of nodules formed by Rhizobium leguminosarum bv. viciae strain 3841 on pea (Pisum sativum) line SGE were analyzed. The influence of elevated temperature was analyzed at histological, ultrastructural, and transcriptional levels. As a result, an unusual apical pattern of nodule senescence was revealed. After five days of exposure, a senescence zone with degraded symbiotic structures was formed in place of the distal nitrogen fixation zone. There was downregulation of various genes, including those associated with the assimilation of fixed nitrogen and leghemoglobin. After nine days, the complete destruction of the nodules was demonstrated. It was shown that nodule recovery was possible after exposure to elevated temperature for 3 days but not after 5 days (which coincides with heat wave duration). At the same time, the exposure of plants to optimal temperature during the night leveled the negative effects. Thus, the study of the effects of elevated temperature on symbiotic nodules using a well-studied pea genotype and Rhizobium strain led to the discovery of a novel positional response of the nodule to heat stress.

Epidemiological Characteristics and Drug-Resistance Genotype of CRE in Patients with Hematological Diseases in a Single Center of China

Patients with blood diseases have a significantly higher risk of infections and mortality rates compared with other type of patients. Referring to the 2019 CDC criteria for CRE, we examined the carbapenemase producing genotype and resistance characteristics of CRE strains (760 strains) in patients with hematological diseases. Among these, carbapenemase producing CRE (CP-CRE) accounts for 60.39% (459/760), non-carbapenemase producing CRE (non-CP-CRE) accounts for 39.61% (301/760). The data indicated that the most common bacteria in patients with hematologic disorders were Escherichia coli, Klebsiella pneumoniae and Enterbater cloacae, which primarily produced NDM-type metallo-β-lactamases. The rates of drug resistance in CP-CRE strains with MIC≥16 to Meropenem and Imipenem were 92.51% (420/454) and 86.12% (391/454), respectively. The drug resistance rates of non-CP-CRE strain with MIC≥16 to Meropenem and Imipenem were only 22.22% and 15.34%, respectively. Furthermore, we discovered that 36.88% (111/301) of non-CP-CRE strains were resistant to Ertapenem alone but still susceptible to Meropenem and Imipenem. The therapy of such CRE may differ significantly from that of other CRE strains, ETP-CRE may not be a typical CRE. In conclusion, Our data determined that the CRE colonized or infected in patients with hematological diseases was dominated by Escherichia coli and metallo-carbapease-producing types, among which the NDM genotypes was predominant. This is quite different from the domestic statistics, indicating that the CRE types infected by hematological disease patients are special and not consistent with other patients. We discovered that some patients with hematological diseases carried or were infected with ETP-CRE, which was sensitive to Meropenem and Imipenem. In terms of therapy, ETP-CRE strains may differ from other CRE in patients with hematological diseases，it may not be a typical CRE.

Enhanced infectivity of bovine viral diarrhoea virus (BVDV) in arginase-producing bovine monocyte-derived macrophages

ABSTRACT Macrophages are important cells of the innate immunity that play a major role in Bovine Viral Diarrhea Virus (BVDV) pathogenesis. Macrophages are not a homogenous population; they exist in different phenotypes, typically divided into two main categories: classically (pro-inflammatory) and alternatively activated (anti-inflammatory) or M1 and M2, respectively. The role of bovine macrophage phenotypes on BVDV infection is still unclear. This study characterized the interaction between BVDV, and monocyte-derived macrophages (Mo-Mφ) collected from healthy cattle and polarized to an M1 or M2 state by using LPS, INF-γ, IL-4 or azithromycin. Arginase activity quantitation was utilized as a marker of the M2 Mo-Mφ spectrum. There was a significant association between arginase activity and the replication rate of BVDV strains of different genotypes and biotypes. Inhibition of arginase activity also reduced BVDV infectivity. Calves treated with azithromycin induced Mo-Mφ of the M2 state produced high levels of arginase. Interestingly, azithromycin administered in vivo increased the susceptibility of macrophages to BVDV infection ex vivo. Mo-Mφ from pregnant dams and calves produced higher arginase levels than those from non-pregnant adult animals. The increased infection of arginase-producing alternatively activated bovine macrophages with BVDV supports the need to delve into a possible leading role of M2 macrophages in establishing the immune-suppressive state during BVDV convalescence.

Origin and spread of leprosy in Suriname. A historical and biomedical study.

The new world was considered free of leprosy before the arrival of Europeans. In Suriname, historical migration routes suggest that leprosy could have been introduced from West Africa by the slave trade, from Asia by indentured workers, from Europe by the colonizers, and more recently by Brazilian gold miners. Previous molecular studies on environmental and ancient samples suggested a high variability of the strains circulating in the country, possibly resulting from the various migration waves. However, a current overview of such diversity in humans still needs to be explored. The origin and spread of leprosy in Suriname are investigated from a historical point of view and by strain genotyping of Mycobacterium leprae from skin biopsies of 26 patients with multibacillary leprosy using PCR-genotyping and whole-genome sequencing. Moreover, molecular signs of resistance to the commonly used anti-leprosy drugs i.e. dapsone, rifampicin and ofloxacin, were investigated. Molecular detection was positive for M. leprae in 25 out of 26 patient samples, while M. lepromatosis was not found in any of the samples. The predominant M. leprae strain in our sample set is genotype 4P (n=8) followed by genotype 1D-2 (n=3), 4N (n=2), and 4O/P (n=1). Genotypes 4P, 4N, 4O/P are predominant in West Africa and Brazil, and could have been introduced in Suriname by the slave trade from West Africa, and more recently by gold miners from Brazil. The presence of the Asian strains 1D-2 probably reflects an introduction by contract workers from India, China and Indonesia during the late 19th and early 20th century after the abolition of slavery. There is currently no definite evidence for the occurrence of the European strain 3 in the 26 patients. Geoplotting reflects internal migration, and also shows that most patients live in and around Paramaribo. A biopsy of one patient harbored two M. leprae genotypes, 1D-2 and 4P, suggesting co-infection. A mutation in the dapsone resistance determining region of folP1 was detected in two out of 13 strains for which molecular drug susceptibility was obtained, suggesting the circulation of dapsone resistant strains.

Study of vacuolating cytotoxin A (vacA) genotypes of ulcerogenic and non-ulcerogenic strains of Helicobacter pylori and its association with gastric disease

Globally, Helicobacter pylori (H. pylori), a stomach pathogen, is present in around 50 % of the population. This bacterial infection produces persistent inflammation, which significantly raises the risk of duodenal, gastric ulcer, and stomach cancer. The goal of this study is to identify the vacA genotypes in H. pylori and analyze how they relate to medical conditions brought on by the bacteria and clarithromycin resistance. PCR was used to describe 115 endoscopic stomach samples from infected patients and identify vacA gene. Of the 115 research participants, H. pylori was found in 81 (70.4 %) of them. Of the isolated cultures, only 38 (69.1 %) were resistant to clarithromycin. VacA was discovered in 55 (67.9 %) of the samples that had H. pylori in them. Patients with gastritis were more likely to have s2m2 strains of infection (66.7 %), while those with gastric and duodenal ulcers were more likely to have s1m1 strains (64.7 %). VacA-positive H. pylori strains (60 % n = 33) were more resistantto clarithromycin versus (19.2 % n = 5) for vacA-negative bacteria. Clarithromycin resistance was significantly linked to vacA s2m2 in H. pylori isolates (75.9 %). According to the study's results, the vacA variants s1m1 and s2m2 have a strong connection with the emergence of H. pylori infections that cause peptic ulcer disease in the population of Iraq. Genetic testing is essential in predicting both the course of treatment and the outcome of H. pylori disease.

Nocardioides cremeus sp. nov., Nocardioides abyssi sp. nov. and Nocardioides oceani sp. nov., three actinobacteria isolated from Western Pacific Ocean sediment.

Three Gram-stain-positive, non-motile, short rod-shaped, catalase-positive and oxidase-negative actinomycete strains (SOB44T, SOB72T and SOB77T) were isolated from a deep-sea sediment sample collected from the Western Pacific Ocean. Cells of the three strains showed optimum growth at 30 °C and pH 7.0. Strains SOB44T, SOB72T and SOB77T could tolerate up to 10, 9 and 9 % (w/v) NaCl concentration and grow at pH 5.0-12.0, 5.0-11.0 and 5.0-11.0, respectively. Phylogenetic results based on 16S rRNA gene sequences showed that the three isolates belonged to the genus Nocardioides and were identified as representing three novel species based on 78.0-93.1 % average nucleotide identity and 21.3-50.0 % DNA-DNA hybridization values with closely related reference strains. Strains SOB44T, SOB72T and SOB77T showed highest 16S rRNA gene sequence similarity to Nocardioides salarius CL-Z59T (99.2 %), Nocardioides deserti SC8A-24T (99.2 %) and Nocardioides marmotae zg-579T (98.5 %), respectively. All three strains had MK-8(H4) as the respiratory quinone, iso-C16 : 0 as the major fatty acid, and phosphatidylglycerol, diphosphatidylglycerol and phosphatidylinositol as the major polar lipids. The diagnostic diamino acid in the cell-wall peptidoglycan of all three isolates was ll-diaminopimelic acid. The DNA G+C contents of strains SOB44T, SOB72T and SOB77T were 71.1, 72.9 and 72.9 mol%, respectively. Based on the phenotypic, phylogenetic and genotypic data, strains SOB44T, SOB72T and SOB77T clearly represent three novel taxa within the genus Nocardioides, for which the names Nocardioides cremeus sp. nov. (type strain SOB44T=JCM 35774T= MCCC M28400T), Nocardioides abyssi sp. nov. (type strain SOB72T=JCM 35775T=MCCC M28318T) and Nocardioides oceani sp. nov. (type strain SOB77T=JCM 35776T=MCCC M28544T) are proposed.

Human RSVA-ON1, The Only Genotype Present During 2019–2020 Winter Season in Riyadh, Saudi Arabia: A Retrospective Study

Aim: The study aimed to analyze human respiratory syncytial virus (hRSV) strains in nasopharyngeal aspirates obtained retrospectively. Materials & methods: Clinical information of ARI patients (n = 53) was accessed and 2nd hypervariable region of G protein gene of RSV was sequenced and analyzed. Results: Cough (98.1%), fever 40 (75.4%) and shortness of breath 32 (60.3%) were the common symptom. Sequence determination (n = 45) categorized all the strains in ON1 genotype. Potential O- and N- glycosylation were observed at some sites and purifying selections were observed at 274 and 320 codon positions. Conclusion: The study reports replacement of other RSVA genotypes with ON1. The future course of studies should emphasize on clinical implications of presence of hRSVA-ON1 genotype in a given population.

Genotyping and traceability analysis of norovirus in Yantai between 2017 and 2019.

To investigate the diversity and evolution of noroviruses in Yantai in recent years, this study focused on the coat protein regions of norovirus-positive samples with nucleic acid detection (cycle threshold) values below 30 between 2017 and 2019. A total of 81 sequences were obtained for genotyping. Initially, a high-throughput sequencing approach was established to perform the whole-genome sequencing of multiple typical diarrheal strains. Using bioinformatics software such as BEAST, recombinant variant analysis was performed for each genotype of the norovirus strains, and genetic evolutionary analysis was conducted for the dominant strain GII.4, as well as the rare variant GII.21. The results showed that there were multiple genotypes such as GI.3, GI.6, GI.7, GII.1, GII.2, GII.3, GII.4, GII.6, GII.13, GII.17, GII.21, and GIX.1 in the positive samples of norovirus from 2017 to 2019. GII.4 is characterized by diverse genotypes, with new changes in antigenic epitopes occurring during the course of the epidemic. This may have led to the emergence of a new pandemic. This suggests a need to strengthen surveillance. The results of this study suggest that attention should be paid to the predominant genotypes prevalent in neighboring countries and regions, and the safety supervision of imported food should be strengthened to aid in the prevention and control of related viruses.

Expression of the transcription factor encoding gene StTCP23 in potato plants infected with the tuber spindle viroid

Background The potato spindle tuber viroid (PSTVd) is the smallest of all known potato pathogens. PSTVd is a circular, single-stranded RNA molecule that does not code for proteins. Symptoms caused by PSTVd lead to a significant reduction in tuber yield or death of the plant. PSTVd infection triggers the silencing of host-plant genes and induces disease symptoms in the plant via vd-sRNA. The StTCP23 potato gene which encodes a transcription factor is one of the targets of PSTVd. The aim of the study was to assess the effect of inoculation of potato plants of cv. ‘Colomba’ with the PSTVd viroid NicTr-3 strain on the StTCP23 expression in infected plants.Materials and methods. Potato plants of cv. ‘Colomba’ were inoculated with leaf sap of the tomato cv. ‘Rutgers’ in which the PSTVd viroid NicTr-3 strain was propagated. RT-PCR with specific primers was used to confirm the infection of potato plants with the viroid. Diversity of RNA molecules in the PSTVd population was revealed using RT, cloning, and sequencing of the viroid. The expression of the StTCP23 gene in infected potato plants was assessed by quantitative PCR with the ef1α gene as a reference.Results. Symptoms of infecting ‘Colomba’ plants with the PSTVd viroid strain NicTr-3 were not detected, while the presence of the viroid in potato leaves was confirmed by molecular methods. Ten mutations were found in individual variants of the PSTVd strain NicTr-3, responsible for reducing the viroid’s aggressiveness. An increase in the expression of StTCP23 was shown at 3 time points in ‘Colomba’ plants inoculated with the NicTr-3 strain compared to the control.Conclusion. The mechanism of interaction between PSTVd and the StTCP23 gene in an infected potato plant is not universal and depends both on the potato genotype and viroid strain. Additional studies are required to prove the existence of specific interaction mechanisms between the host-plant and viroid strain genotypes.

A New Genotype of Trichophyton quinckeanum with Point Mutations in Erg11A Encoding Sterol 14-α Demethylase Exhibits Increased Itraconazole Resistance.

Trichophyton quinckeanum, the causative agent of mouse favus, has been responsible for several infections of animal owners in recent years and showed an infection peak around 2020 in Jena, Thuringia. The isolated T. quinckeanum strains from Thuringia differ in some positions of the ITS region compared to strains from the IHEM collection as well as to Trichophyton schoenleinii. All T. quinckeanum strains of the new genotype show up to a 100-fold increased itraconazole resistance as measured by microplate laser nephelometry (MLN) assays. Analysis of genes involved in Trichophyton indotineae azole resistance, such as Erg1, which encodes squalene epoxidase, and Erg11B, one of two copies of the sterol 14-α demethylase gene, show a 100% identity between the two T. quinckeanum genotypes. In contrast, Erg11A fragments differ in 15-nucleotide positions between both T. quinckeanum genotypes, resulting in the unique amino acid substitution Ala256Ser in resistant strains. The new T. quinckeanum genotype may have evolved through interspecies mating. Mating type analysis showed a nearly 100% identity of the minus type MAT1-1-1 fragment for all T. quinckeanum isolates. The closely related Trichophyton schoenleinii belongs to the plus mating type and has 100% identical fragments of Erg1 and Erg11B. Erg11A protein sequences of T. schoenleinii and T. quinckeanum showed increased diversity.

Genotypic diversity of staphylococcal enterotoxin B gene (seb) and its association with molecular characterization and antimicrobial resistance of Staphylococcus aureus from retail food

To investigate the expression pattern of staphylococcal enterotoxin B (SEB) in food and the genotypic diversity of SEB-encoding gene in association with molecular characteristics and antimicrobial resistance of S. aureus, 498 isolates from retail food were screened for seb gene and detected for SEB production in S. aureus. In addition, the seb nucleotide sequences, virulence genes, resistance genes, antimicrobial susceptibility and molecular characteristics of S. aureus were examined. A total of 45 (9.0 %) seb-positive S. aureus strains were identified, all of which expressed SEB. The detection rate of SEB-production strains was significantly higher from dairy-related sources than those from other sources (P < 0.05). In vitro simulations showed that S. aureus could grow and express SEB in both milk and pork, with SEB expression exceeding 20 ng/g after 1 day of storage at room temperature. There were 2 distinct SEB genotyping (SEB1 and SEB2) in the SEB amino acid sequences of the 45 isolates, including 4 amino acid differences (Ala-13Val, Ser14Ala, Asn192Ser, and Met222Leu). There was no significant difference (P > 0.05) in SEB production between SEB1 and SEB2 genotyping strains. Based on MLST clustering analysis, the same molecular type strains were found to have the same SEB genotyping, virulence gene profile, resistance gene profile and drug resistance profile. Among them, the dominant molecular types of SEB1 and SEB2 strains were CC1-ST188-t189 and CC59-ST59-t437, respectively. Compared to the CC1-ST188-t189 clonal strain, the CC59-ST59-t437 clonal strain carried a higher number of virulence and resistance genes and exhibited a broader resistance profile. Therefore, understanding the characteristics of the strains and their expression patterns in food can be effective in preventing food poisoning incidents.

Highly diverse dynamics of Pseudomonas aeruginosa colonization from initial detection in cystic fibrosis patients: A 7-year longitudinal genetic diversity study

In cystic fibrosis (CF), Pseudomonas aeruginosa (Pa) is a major pathogen that can persistently colonize patients. Genetic studies showed a high diversity of Pa, the success of widespread or ‘international’ clones and described epidemic clones in CF and Epidemic High-Risk (ERH) clones. Here, we characterized Pa genetic diversity over time after first colonization in CF patients, with the aim of accurately describing the dynamics of colonization in a context of scarce longitudinal studies including the first isolated Pa strain. Results represent the first genotyping data based on multilocus sequence typing (MLST) available for CF Pa in France. Forty-four CF patients with a first Pa colonization were included; 265 strains collected over 7 years in these patients were genotyped by multiplex rep-PCR, MLST, pulsed-field gel electrophoresis and/or whole genome sequencing. Forty-one sequence types were identified: 4 were unknown, 22 never previously reported for CF patients, and 6 corresponded to widespread clones colonizing 16 patients (36%). Unrelated strains were identified in 41 patients (93%). Twenty-six patients (59%) presented a recurrence during the study period. No specific clones were associated with transient, recurrent or persistent colonization. Our longitudinal study revealed that 9 of the 26 patients with recurrence (35%) harbored strains of different genotypes. Great genetic diversity was observed among initial Pa isolates excluding any cross-transmission. Persistent colonization may appear more complex than expected, imitating persistence, with successive colonization events by unrelated Pa.

Expansion of the genetic toolbox for manipulation of the global crop pest Drosophila suzukii: Isolation and assessment of eye colour mutant strains.

Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), commonly called spotted wing Drosophila, is an important agricultural pest recognised worldwide. D. suzukii is a pest of soft-skinned fruits as females can lay eggs in ripening fruit before harvest. While strains for genetic biocontrol of D. suzukii have been made, the development of transgenic D. suzukii strains and their further screening remain a challenge partly due to the lack of phenotypically trackable genetic-markers, such as those widely used with the model genetic organism D. melanogaster. Here, we have used CRISPR/Cas9 to introduce heritable mutations in the eye colour genes white, cinnabar and sepia, which are located on the X, second and third chromosomes, respectively. Strains were obtained, which were homozygous for a single mutation. Genotyping of the established strains showed insertion and/or deletions (indels) at the targeted sites. A strain homozygous for mutations in cinnabar and sepia showed a pale-yellow eye colour at eclosion but darkened to a sepia colour after a week. The fecundity and fertility of some of the cinnabar and sepia strains were comparable with the wild type. Although white mutant males were previously reported to be sterile, we found that sterility is not fully penetrant and we have been able to maintain white-eyed strains for over a year. The cinnabar, sepia and white mutant strains developed in this study should facilitate future genetic studies in D. suzukii and the development of strains for genetic control of this pest.

Evaluation of the Analytical Capabilities of MALDI-TOF Mass Spectrometry in the Molecular Typing of &lt;i&gt;Bacillus anthracis&lt;/i&gt;

The aim of the work was to compare the discriminating capacity of the canSNP13 genotyping and MALDI‑TOF mass spectrometry methods based on the results of the study of anthrax pathogen strains belonging to the two main genetic lines A and B.Materials and methods. 73 Bacillus anthracis strains from the collection of microorganisms of the Stavropol Research Anti-Plague Institute of the Rospotrebnadzor were investigated. Protein profiling was carried out using a Microflex mass spectrometer, data processing – in the environment of the statistical programming language “R”.Results and discussion. It has been experimentally confirmed that the proposed approach for differentiating proteotypes of B. anthracis strains with a discrimination index of 0.952 exceeds the one for the canSNP typing method and is comparable to the discrimination index for the MLVA31 method. The correlation of the results of strain clustering during typing by MALDI-TOF mass spectrometry and canSNP genotyping reaches 95 % as regards the division into the main genetic lines A and B. The studied strains of anthrax microbe, which mostly fall under phylogenetic groups of lineage A, represent more than a dozen protein profiles, which may be due to differences in the level of protein expression in strains of each canSNP genotype. MALDI-TOF mass spectrometry makes it possible to obtain the results comparable with genetic tests, has a better discriminating capacity compared to canSNP typing, and is easier to perform.

Antiviral Efficacy of RNase H-Dependent Gapmer Antisense Oligonucleotides against Japanese Encephalitis Virus.

RNase H-dependent gapmer antisense oligonucleotides (ASOs) are a promising therapeutic approach via sequence-specific binding to and degrading target RNAs. However, the efficacy and mechanism of antiviral gapmer ASOs have remained unclear. Here, we investigated the inhibitory effects of gapmer ASOs containing locked nucleic acids (LNA gapmers) on proliferating a mosquito-borne flavivirus, Japanese encephalitis virus (JEV), with high mortality. We designed several LNA gapmers targeting the 3' untranslated region of JEV genomic RNAs. In vitro screening by plaque assay using Vero cells revealed that LNA gapmers targeting a stem-loop region effectively inhibit JEV proliferation. Cell-based and RNA cleavage assays using mismatched LNA gapmers exhibited an underlying mechanism where the inhibition of viral production results from JEV RNA degradation by LNA gapmers in a sequence- and modification-dependent manner. Encouragingly, LNA gapmers potently inhibited the proliferation of five JEV strains of predominant genotypes I and III in human neuroblastoma cells without apparent cytotoxicity. Database searching showed a low possibility of off-target binding of our LNA gapmers to human RNAs. The target viral RNA sequence conservation observed here highlighted their broad-spectrum antiviral potential against different JEV genotypes/strains. This work will facilitate the development of an antiviral LNA gapmer therapy for JEV and other flavivirus infections.

Antimicrobial resistance and virulence of carbapenem-resistant &lt;i&gt;Klebsiella pneumoniae&lt;/i&gt; strains isolated from children in intensive care and surgical units

Introduction. Infections associated with carbapenem-resistant (Carba-R) Klebsiella pneumoniae (KP) are a serious public health problem because they lead to an increase in hospital stays, treatment costs, and an increase in patient morbidity and mortality.&#x0D; Aim to characterize antimicrobial resistance, virulence factors and genotypes of Carba-R KP strains isolated from patients in intensive care and surgical units.&#x0D; Materials and methods. In total, 455 KP strains that were resistant to meropenem and/or imipenem, or susceptible to meropenem and/or imipenem at increased exposure were collected in 20182020. For further analysis, total 90 KP strains isolated from clinically significant sites blood/сerebrospinal fluid/urine/lower respiratory tract/wounds/abdominal cavity of patients in intensive care and surgical units were selected. Antibiotic susceptibility was determined using the broth microdilution method. Carbapenemase genes were detected by real-time PCR. The virulence genes and K1/K2 capsular serotypes were determined by multiplex PCR. Sequence types (ST) were determined using multilocus sequence typing.&#x0D; Results. Most of the selected isolates (97%) were recognized as extensively drug-resistant pathogens, three isolates were classified as multidrug-resistant pathogens. The major determinant of carbapenem resistance was blaOXA-48-like (53%), blaNDM-group was detected in 13% strains, a combination of blaNDM-group and blaOXA-48-like genes was found in 31% isolates. Two isolates harbored blaKPC-group. Most of the isolates had the virulence genes entB (100%), mrkD (99%), and ybtS (78%). The iutA gene was found in 16% strains. Four isolates had kfu gene and four strains carried rmpA gene. The K2 capsule type was determined in 13% isolates. Four ST dominated in the studied population: ST395 (26%), ST2975 (19%), ST198 (12%) and ST307 (11%).&#x0D; Conclusion. Carba-R KP isolates had a high level of resistance not only to carbapenems, but also to antibiotics of other classes. Carbapenem resistance was mainly associated with OXA-48 carbapenemase. The entB, mrkD and ybtS genes were the main determinants of virulence. The epidemically successful clones CG395 and CG307 were the predominant genetic lines. Resistance to antimicrobials was combined with the presence of various virulence factors. The data obtained are important for the epidemiological surveillance of the spread of KP and have important clinical implications.

Relationships Between Serotype B and The Virulent Type cagE in Aggregatibacter actinomycetemcomitans in Mosul City

The study aimed to determine the frequency of isolates containing the gene cagE within serotype b strains. The study showed the isolation of 35 strains belonging to the species Aggregatibacter actinomycetemcomitans based on biochemical and phenotypical characteristics. 60% of isolates belonged to serotype b, and 68.5% showed a positive result for cagE. The remaining 8.5% of strains carrying the gene cagE were not serotype b, so these isolates most likely may be diagnosed as belonging to JP2 genotype strains. Keywords: Serotype b, cagE, Aggregatibacter actinomycetemcomitans