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Abstract Tuberculosis (TB) remains one of the leading infectious disease killers in the world. The ongoing development of novel anti-TB medications has yielded potent compounds that often target single sites with well-defined mechanisms of action. However, despite the identification of resistance-associated mutations through target deconvolution studies, comparing these findings with the diverse Mycobacterium tuberculosis populations observed in clinical settings is often challenging. To address this gap, we constructed an open-access database encompassing genetic variations from > 50,000 clinical isolates, spanning the entirety of the M. tuberculosis protein-encoding genome. This resource offers a valuable tool for investigating the prevalence of target-based resistance mutations in any drug target within clinical contexts. To demonstrate the practical application of this dataset in drug discovery, we focused on drug targets currently undergoing phase II clinical trials. By juxtaposing genetic variations of these targets with resistance mutations derived from laboratory-adapted strains, we identified multiple positions across three targets harbouring resistance-associated mutations already present in clinical isolates. Furthermore, our analysis revealed a discernible correlation between genetic diversity within each protein and their predicted essentiality. This meta-analysis, openly accessible via a dedicated dashboard, enables comprehensive exploration of genetic diversity pertaining to any drug target or resistance determinant in M. tuberculosis.

ObjectiveTo compare genetic characteristics of Bordetella pertussis isolates from child patients in Xishuangbanna, Yunnan province with those of vaccine isolate and Bordetella pertussis isolates from patients of other regions for providing evidence to pertussis prevention and control. MethodsFour Bordetella pertussis strains (named BP-L1, BP-L2, BP-L3, BP-L4) were isolated from nasopharyngeal swab specimens of child pertussis patients in the People′s Hospital of Xishuangbanna Prefecture, Yunnan province during October – December 2019 and the whole genome of the isolated strains were sequenced; the ptxP, ptxA, prn, fim3 genotype of the isolated strains were determined with MegAlign 7.1.0. According to the gene sequences published in National Center for Biotechnology Information (NCBI) database, the genotypes of the isolated strains were compared with those of CS strain of acellular pertussis vaccine innoculated in China and of the Bordetella pertussis strains prevalent in other countries and regions. Phylogenetic trees were constructed using RAxML software based on single nucleotide polymorphism (SNP) of 28 Bordetella pertussis strains. ResultsGene rearrangement due to 33 – 36 insertion sequences of the four isolated strains were identified in comparison with those of the CS strain. The similarity between ptxP, ptxA, prn, fim3 gene sequence of the isolated strains and CS strain was 99.2% – 100%. The results of phylogenetic tree showed that CS strain and reference strain TohamaⅠ clustered into a branch and formed an independent evolutionary branch. In recent years, Bordetella pertussis, which is prevalent in the world, can be divided into two clusters; the isolated BP-L1 and BP-L4 were clustered with the Bordetella pertussis prevalent in most European and American countries, which mainly included ptxP3/ptxA1/prn2 strains; while, the isolated BP-L2 and BP-L3 were clustered with Bordetella pertussis inducing endemic in Asia and mainly including ptxP1/ptxA1/prn1 strains. ConclusionCompared with the vaccine strains, the main virulence protein genes and evolutionary branches of the Bordetella pertussis strains clinically isolated in Xishuangbanna, Yunnan province changed significantly and the changes are consistent with the genetic evolution trend of the Bordetella pertussis prevalent globally in recent years.

ObjectiveThis study aims to investigate the distribution and drug resistance of bacteria in clinical blood culture specimens from children in Jiangxi province in recent years and to provide a foundation for preventing and treating bloodstream infection diseases in children.MethodsThe study involved a statistical analysis of the isolation and drug resistance of bacterial strains obtained from blood culture specimens of children in Jiangxi province between 2017 and 2021. The analysis was performed using the WHONET 5.6 software.ResultsA total of 7,977 bacterial strains were isolated from the blood samples of children between 2017 and 2021. Of these, 2,334 strains (29.3%) were identified as Gram-negative bacteria, and 5,643 strains (70.7%) were identified as Gram-positive bacteria. The most commonly isolated pathogens were coagulase-negative Staphylococcus, Escherichia coli, and Staphylococcus aureus. Among the Gram-negative bacteria, Escherichia coli (840 strains, 36.0%), Klebsiella pneumoniae (385 strains), Salmonella (283 strains), Acinetobacter baumannii (137 strains), and Pseudomonas aeruginosa (109 strains) were the most prevalent. Among the Gram-positive bacteria, coagulase-negative Staphylococcus (3,424 strains, 60.7%), Staphylococcus aureus (679 strains), Streptococcus pneumoniae (432 strains), Enterococcus sp. (292 strains), and Streptococcus agalactiae (192 strains) were the most common. Resistance to third-generation cephalosporins (cefotaxime/ceftriaxone) was observed in 45.9% and 56.0% of Escherichia coli and Klebsiella pneumoniae strains, respectively, while resistance to carbapenems was observed in 4.6% and 20.3% of these strains, respectively. Resistance to third-generation cephalosporins (cefotaxime/ceftriaxone) was observed in 15.5% of Salmonella strains, while resistance to imipenem was absent. Carbapenem resistance was observed in 17.1% (20/117) and 13% (14/108) of Acinetobacter baumannii and Pseudomonas aeruginosa strains, respectively. Methicillin-resistant Staphylococcus aureus (MRSA) was detected in 32.7% of the strains, while methicillin-resistant coagulase-negative Staphylococcus was detected in 64.3% of the coagulase-negative Staphylococcus strains. No Staphylococcus bacteria resistant to vancomycin were detected. Four strains of vancomycin-resistant Enterococcus faecium were detected over the 5-year period, and one strain of linezolid-resistant Enterococcus faecalis was detected.ConclusionGram-positive cocci were the most commonly isolated clinical pathogens in blood specimens from children in Jiangxi province. The composition of the pathogen species showed a slight change over the years. The detection ratios of pathogens varied with age group and season. Although the isolation rate of common carbapenem-resistant Enterobacter bacteria has decreased, it remains high. It is necessary to monitor the antimicrobial resistance of pathogens causing bloodstream infections in children more closely, and antimicrobial agents should be used with caution.

Abstract Background Leishmania infantum is the most important etiological agent of visceral leishmaniasis in the Americas and Mediterranean region, and the dog is the main host. Miltefosine was authorized to treat canine leishmaniasis (CanL) in Brazil in 2017, but there is a persistent fear of the emergence of parasites resistant not only to this drug but, through cross-resistance mechanisms, also to meglumine antimoniate and amphotericin B. Additionally, the literature shows that acquisition of resistance is followed by increased parasite fitness, with higher rates of proliferation, infectivity and metacyclogenesis, which are drivers of parasite virulence. In this context, the aim of this study was to analyze the impact of treating a dog with miltefosine and allopurinol on the generation of parasites resistant to miltefosine, amphotericin B and meglumine antimoniate. Methods In vitro susceptibility tests were conducted against miltefosine, amphotericin B and meglumine antimoniate with T0 (parasites isolated from a dog before treatment with miltefosine plus allopurinol), T1 (after 1 course of treatment) and T2 (after 2 courses of treatment) isolates. The rates of cell proliferation, infectivity and metacyclogenesis of the isolates were also evaluated. Results The results indicate a gradual increase in parasite resistance to miltefosine and amphotericin B with increasing the number of treatment courses. An increasing trend in the metacyclogenesis rate of the parasites was also observed as drug resistance increased. Conclusion The data indicates an increased L. infantum resistance to miltefosine and amphotericin B after the treatment of a dog with miltefosine plus allopurinol. Further studies with a larger number of L. infantum strains isolated from dogs with varied immune response profiles and undergoing different treatment regimes, are advocated. Graphical Abstract

Integrons are genetic elements that store, express and exchange gene cassettes. These elements are characterized by containing a gene that codes for an integrase (intI), a cassette integration site (attI) and a variable region holding the cassettes. Using bioinformatics and molecular biology methods, a functional integron found in Aeromonas sp. 3925, a strain isolated from diarrheal stools, is described. To confirm the integron class, a phylogenetic analysis with amino acid sequences was conducted. The integrase was associated to class 4 integrases; however, it is clearly different from them. Thus, we classified the associated element as a class 4-like integron. We found that the integrase activity is not under the control of the SOS or catabolic repression, since the expression was not increased in the presence of mitomycin or arabinose. The class-4-like integron is located on the chromosome and contains two well-defined gene cassettes: aadA1 that confers resistance to streptomycin and lpt coding for a lipoprotein. It also includes eight Open Reading frames (ORFs) with unknown functions. The strain was characterized through a Multilocus Phylogenetic Analyses (MLPA) of the gyrB, gyrA, rpoD, recA, dnaJ and dnaX genes. The phylogenetic results grouped it into a different clade from the species already reported, making it impossible to assign a species. We resorted to undertaking complete genome sequencing and a phylogenomic analysis. Aeromonas sp. 3925 is related to A. media and A. rivipollensis clusters, but it is clearly different from these species. In silico DNA-DNA hybridization (isDDH) and Average Nucleotide Identity (ANI) analyses suggested that this isolate belongs to the genomospecies paramedia. This paper describes the first class 4-like integron in Aeromonas and contributes to the establishment of genomospecies paramedia.

Introduction Nosocomial infections are a major global problem. Using metallic oxide nanoparticles to counter bacterial infections as an alternative to antibiotics can be effective. The aim of this study was to evaluate the effect of iron and silver nanoparticles on biofilm formation in bacteria isolated from clinical specimens and the possibility of using these nanoparticles on laboratory surfaces. Materials and Methods This study was performed on three bacterial strains isolated from different wards of Imam Reza, Imam Hossein, and Taleghani hospitals in Kermanshah. The biochemical tests of the bacteria were identified and their antibiotic resistance was determined by the disk method. The microtiter plate method was used to investigate the disinfecting effect of silver and iron nanoparticles on biofilm formation. Data were analyzed by SPSS software. Results Results showed 9 blood samples (30%), 5 skin samples (16.67%), and 16 urine samples (53.33%). The highest rate of inhibition of Escherichia coli biofilm formation by silver and iron nanoparticles was 93% and 64%, respectively. The inhibition of biofilm formation in S. epidermidis by silver and iron nanoparticles was 86 and 64%, respectively, and the highest inhibition of biofilm formation by Pseudomonas aeruginosa by silver and iron nanoparticles was 96 and 88%, respectively. Conclusion The use of silver and iron nanoparticles has a high potential to kill the bacteria in the biofilm of P.aeruginosa.

Introduction Staphylococcus aureus (S. aureus) has been recognized as a powerful pathogen that causes numerous infections. Today, S.aureus has acquired multiple resistance to a wide range of antibiotics, including beta-lactams. Beta-lactamases hydrolyze beta-lactam antibiotics and are expressed through activation of the blaZ gene. The aim of this study was to evaluate the frequency of blaZ gene in clinical isolates of S.aureus isolated from Hamadan hospitals. Materials and Methods In this descriptive cross-sectional study, all S.aureus strains were randomly selected and transferred to the laboratory. In clinical isolates confirmed by biochemical tests of genomic DNA extraction procedures using CinnaGen extraction kit) was performed according to the manufacturerchr('39')s protocol and 1% agarose gel was used to view the PCR product. Data were analyzed by SPSS software. Results From 50 hospitalized patients in Fatemieh and Sina hospitals, 32 men (64%), and 18 women (36%), isolates of S.aureus were isolated. The pattern of antibiotic resistance obtained was that penicillin (10 µg) with 100% resistance and cefazolin (30 µg) with 5% resistance had the highest and lowest resistance, respectively. Also, 94% of isolates were resistant to cefoxitin (30 µg) and 90% of isolates were resistant to oxacillin (1 µg). Of 50 S.aureus isolated from clinical isolates, 44 (88 %) contained the blaZ beta-lactamase gene. Conclusion Based on the findings of this study, isolates isolated from clinical specimens showed the same pattern of abundance. Therefore, it is likely that more or more drug resistance will be transmitted to hospitals.

Abstract Background The only licensed live Bacille Calmette-Guérin (BCG) vaccine used to prevent severe childhood tuberculosis comprises genetically divergent strains with variable protective efficacy and rates of BCG-induced adverse events. The whole-genome sequencing (WGS) allowed evaluating the genome stability of BCG strains and the impact of spontaneous heterogeneity in seed and commercial lots on the efficacy of BCG-vaccines in different countries. Our study aimed to assess sequence variations and their putative effects on genes and protein functions in the BCG-1 (Russia) seed lots compared to their progeny isolates available from immunocompetent children with BCG-induced disease (mainly, osteitis). Results Based on the WGS data, we analyzed the links between seed lots 361, 367, and 368 used for vaccine manufacture in Russia in different periods, and their nine progeny isolates recovered from immunocompetent children with BCG-induced disease. The complete catalog of variants in genes relative to the reference genome (GenBank: CP013741) included 4 synonymous and 8 nonsynonymous single nucleotide polymorphisms, and 3 frameshift deletions. Seed lot 361 shared variants with 2 of 6 descendant isolates that had higher proportions of such polymorphisms in several genes, including ppsC, eccD5, and eccA5 involved in metabolism and cell wall processes and reportedly associated with virulence in mycobacteria. One isolate preserved variants of its parent seed lot 361 without gain of further changes in the sequence profile within 14 years. Conclusions The background genomic information allowed us for the first time to follow the BCG diversity starting from the freeze-dried seed lots to descendant clinical isolates. Sequence variations in several genes of seed lot 361 did not alter the genomic stability and viability of the vaccine and appeared accumulated in isolates during the survival in the human organism. The impact of the observed variations in the context of association with the development of BCG-induced disease should be evaluated in parallel with the immune status and host genetics. Comparative genomic studies of BCG seed lots and their descendant clinical isolates represent a beneficial approach to better understand the molecular bases of efficacy and adverse events during the long-term survival of BCG in the host organism.

Abstract Background Genome sequencing and genetic polymorphism analysis of clinical isolates of M. tuberculosis is carried out to gain further insight into molecular pathogenesis and host-pathogen interaction. Therefore the functional evaluation of the effect of single nucleotide variation (SNV) is essential. At the same time, the identification of invariant sequences unique to M. tuberculosis contributes to infection detection by sensitive methods. In the present study, genome analysis is accompanied by evaluation of the functional implication of the SNVs in a MDR clinical isolate VPCI591. Result By sequencing and comparative analysis of VPCI591 genome with 1553 global clinical isolates of M. tuberculosis (GMTV and tbVar databases), we identified 141 unique strain specific SNVs. A novel intergenic variation in VPCI591 in the putative promoter/regulatory region mapping between embC (Rv3793) and embA (Rv3794) genes was found to enhance the expression of embAB, which correlates with the high resistance of the VPCI591 to ethambutol. Similarly, the unique combination of three genic SNVs in RNA polymerase β gene (rpoB) in VPCI591 was evaluated for its effect on rifampicin resistance through molecular docking analysis. The comparative genomics also showed that along with variations, there are genes that remain invariant. 173 such genes were identified in our analysis. Conclusion The genetic variation in M. tuberculosis clinical isolate VPCI591 is found in almost all functional classes of genes. We have shown that SNV in rpoB gene mapping outside the drug binding site along with two SNVs in the binding site can contribute to quantitative change in MIC for rifampicin. Our results show the collective effect of SNVs on the structure of the protein, impacting the interaction between the target protein and the drug molecule in rpoB as an example. The study shows that intergenic variations bring about quantitative changes in transcription in embAB and in turn can lead to drug resistance.

Colistin is frequently a last resort treatment for Pseudomonas aeruginosa infections caused by multidrug-resistant (MDR) and extensively drug resistant (XDR) strains, and detection of colistin resistance is essential for the management of infected patients. Therefore, we evaluated the recently developed MALDIxin test for the detection of colistin resistance in P. aeruginosa clinical strains using the routine matrix-assisted laser desorption ionization (MALDI) Biotyper Sirius system. The test is based on the detection by mass spectrometry of modified lipid A by the addition of 4-amino-l-arabinose (l-ara4N) molecules on one or two phosphate groups, in strains resistant to colistin. Overproduction of l-Ara4N molecules is mainly due to the constitutive activation of the histidine kinase (PmrB) or the response regulator (PmrA) following an amino-acid substitution in clinical strains. The performance of the test was determined on a panel of 14 colistin-susceptible and 14 colistin-resistant P. aeruginosa clinical strains, the reference strain PAO1 and positive control mutants PmrB (V28G), PmrB (D172), PhoQ (D240–247), and ParR (M59I). In comparison with the broth microdilution (BMD) method, all the susceptible strains (n=14) and 8/14 colistin-resistant strains were detected in less than 1h, directly on whole bacteria. The remaining resistant strains (n=6) were all detected after a short pre-exposure (4h) to colistin before sample preparation. Validation of the method on a larger panel of strains will be the next step before its use in diagnostics laboratories. Our data showed that the MALDIxin test offers rapid and efficient detection of colistin resistant P. aeruginosa and is thus a valuable diagnostics tool to control the spread of these emerging resistant strains.

Herpes simplex virus type 1 (HSV-1) is the only FDA- and EMA- approved oncolytic virus, and accordingly, many potential oncolytic HSVs (oHSV) are in clinical development. The utilized oHSV parental strains are, however, mostly based on laboratory reference strains, which may possess a compromised cytolytic capacity in contrast to circulating strains of HSV-1. Here, we assess the phenotype of thirty-six circulating HSV-1 strains from Finland to uncover their potential as oHSV backbones. First, we determined their capacity for cell-to-cell versus extracellular spread, to find strains with replication profiles favorable for each application. Second, to unfold the differences, we studied the genetic diversity of two relevant viral glycoproteins (gB/UL27, gI/US7). Third, we examined the oncolytic potential of the strains in cells representing glioma, lymphoma, and colorectal adenocarcinoma. Our results suggest that the phenotype of a circulating isolate, including the oncolytic potential, is highly related to the host cell type. Nevertheless, we identified isolates with increased oncolytic potential in comparison with the reference viruses across many or all of the studied cancer cell types. Our research emphasizes the need for careful selection of the backbone virus in early vector design, and it highlights the potential of clinical isolates as backbones in oHSV development.

Histoplasma capsulatum is a dimorphic, thermal, and nutritional fungus. In the environment and at an average temperature of 28 °C, it develops as a mold that is composed of infecting particles. Once in the host or in cultures at 37 °C, it undergoes a transition into the parasitic form. In the present work, we performed chemical extraction and characterization using chromatography techniques of the associated lipid composition of the external surface of the cell wall of the mycelial phase of two isolates of the H. capsulatum: one clinical and one environmental. Several differences were evidenced in the fatty acids in the phospholipid composition. Surface pressure–area isotherms and compression module curves of the Amphotericin B and lipid extract monolayers, as well as (AmB)-lipid extract mixed monolayers were recorded. Results show a high affinity of AmB towards lipid extracts. The most stable monolayers were formed by AmB + environmental with a mass ratio of 1:3 and AmB + clinical with a mass ratio of 1:2. Knowledge of the AmB aggregation processes at a molecular level and the characterization of the lipid extracts allows the possibility to understand the interaction between the AmB and the lipid fractions of H. capsulatum.

The tuberculosis (TB) disease is still widely found even though BCG vaccine given to many people. Ineffectiveness of the BCG vaccine is one of causes that make the difficulties in preventing TB transmission. Objective of the research was to determine the immunogenicity of KatG protein of M. tuberculosis clinical isolate L19 in mice. The KatG protein as antigen was prepared by expression of the katG gene of M. tuberculosis clinical isolate L19 in Escherichia coli BL21 using pColdII-DNA vector. After purification by affinity chromatography, the KatG was vaccinated to mice to detect its immunogenicity. The expression of katG in E. coli BL21 could result in KatG protein with molecular weight 80 kDa in sodium dodecyl sulfate gel electrophoresis (SDS-PAGE). The pure KatG protein could significantly stimulate the immune response of mice by triggering the antibodies production of IgG1, IgG2a, IgG2b, IgG2c, IgG3, and IgM. The highest antibody level was obtained when the mice were vaccinated by KatG L19 with the dose of 45 μg/ml. Of the antibodies, the IgG2c isotype was dominantly produced in the blood serum. The KatG protein exhibited a high immunogenici­ty in mice, so it is possible to develop as a vaccine candidate for TB. A clinical test should be performed in a future to ensure its safety as a therapeutic protein.

Abstract Background The sexual stages (gametocytes) of Plasmodium falciparum do not directly contribute to the pathology of malaria but are essential for transmission of the parasite from the human host to the mosquito. Mature gametocytes circulate in infected human blood for several days and their circulation time has been modelled mathematically from data of previous in vivo studies. This is the first time that longevity of gametocytes is studied experimentally in vitro. Methods The in vitro longevity of P. falciparum gametocytes of 1 clinical isolate and 2 laboratory strains was assessed by three different methods: microscopy, flow cytometry and reverse transcription quantitative real-time PCR (RT-qPCR). Additionally, the rate of gametocytogenesis of the used P. falciparum strains was compared. Results The maximum in vitro lifespan of P. falciparum gametocytes reached almost 2 months (49 days by flow cytometry, 46 days by microscopy, and at least 52 days by RT-qPCR) from the starting day of gametocyte culture to death of last parasite in the tested strains with an average 50% survival rate of 6.5, 2.6 and 3.5 days, respectively. Peak gametocytaemia was observed on average 19 days after initiation of gametocyte culture followed by a steady decline due to natural decay of the parasites. The rate of gametocytogenesis was highest in the NF54 strain. Conclusions Plasmodium falciparum mature gametocytes can survive up to 16–32 days (at least 14 days for mature male gametocytes) in vitro in absence of the influence of host factors. This confirms experimentally a previous modelling estimate that used molecular tools for gametocyte detection in treated patients. The survival time might reflect the time the parasite can be transmitted to the mosquito after clearance of asexual parasites. These results underline the importance of efficient transmission blocking agents in the fight against malaria.

Whole genome sequencing (WGS) can provide a comprehensive analysis of Mycobacterium tuberculosis mutations that cause resistance to anti-tuberculosis drugs. With the deployment of bench-top sequencers and rapid analytical software, WGS is poised to become a useful tool to guide treatment. However, direct sequencing from clinical specimens to provide a full drug resistance profile remains a serious challenge. This article reviews current practices for extracting M. tuberculosis DNA and possible solutions for sampling sputum. Techniques under consideration include enzymatic digestion, physical disruption, chemical degradation, detergent solubilization, solvent extraction, ligand-coated magnetic beads, silica columns, and oligonucleotide pull-down baits. Selective amplification of genomic bacterial DNA in sputum prior to WGS may provide a solution, and differential lysis to reduce the levels of contaminating human DNA is also being explored. To remove this bottleneck and accelerate access to WGS for patients with suspected drug-resistant tuberculosis, it is suggested that a coordinated and collaborative approach be taken to more rapidly optimize, compare, and validate methodologies for sequencing from patient samples.

Water is considered to play a role in the dissemination of antibiotic-resistant Gram-negative bacteria including those encoding Extended-spectrum beta-lactamases (ESBL) and carbapenemases. To investigate the role of water for their spread in more detail, we characterized ESBL/Carbapenemase-producing bacteria from surface water and sediment samples using phenotypic and genotypic approaches. ESBL/Carbapenemase-producing isolates were obtained from water/sediment samples. Species and antibiotic resistance were determined. A subset of these isolates (n = 33) was whole-genome-sequenced and analyzed for the presence of antibiotic resistance genes and virulence determinants. Their relatedness to isolates associated with human infections was investigated using multilocus sequence type and cgMLST-based analysis. Eighty-nine percent of the isolates comprised of clinically relevant species. Fifty-eight percent exhibited a multidrug-resistance phenotype. Two isolates harbored the mobile colistin resistance gene mcr-1. One carbapenemase-producing isolate identified as Enterobacter kobei harbored blaVIM–1. Two Escherichia coli isolates had sequence types (ST) associated with human infections (ST131 and ST1485) and a Klebsiella pneumoniae isolate was classified as hypervirulent. A multidrug-resistant (MDR) Pseudomonas aeruginosa isolate encoding known virulence genes associated with severe lung infections in cystic fibrosis patients was also detected. The presence of MDR and clinically relevant isolates in recreational and surface water underlines the role of aquatic environments as both reservoirs and hot spots for MDR bacteria. Future assessment of water quality should include the examination of the multidrug resistance of clinically relevant bacterial species and thus provide an important link regarding the spread of MDR bacteria in a One Health context.

ABSTRACT Lassa virus (LASV), a member of the family Arenaviridae, is the causative agent of Lassa fever. Lassa virus is endemic in West African countries, such as Nigeria, Guinea, Liberia, and Sierra Leone, and causes outbreaks annually. Lassa fever onset begins with “flu-like” symptoms and may develop into lethal hemorrhagic disease in severe cases. Although Lassa virus is one of the most alarming pathogens from a public health perspective, there are few licensed vaccines or therapeutics against Lassa fever. The fact that animal models are limited and the fact that mostly laboratory-derived viruses are used for studies limit the successful development of countermeasures. In this study, we demonstrated that the LASV isolate LF2384-NS-DIA-1 (LF2384), which was directly isolated from a serum sample from a fatal human Lassa fever case in the 2012 Sierra Leone outbreak, causes uniformly lethal infection in outbred Hartley guinea pigs without virus-host adaptation. This is the first report of a clinically isolated strain of LASV causing lethal infection in outbred guinea pigs. This novel guinea pig model of Lassa fever may contribute to Lassa fever research and the development of vaccines and therapeutics. IMPORTANCE Lassa virus, the causative agent of Lassa fever, is a zoonotic pathogen causing annual outbreaks in West African countries. Human patients can develop lethal hemorrhagic fever in severe cases. Although Lassa virus is one of the most alarming pathogens from a public health perspective, there are few available countermeasures, such as antiviral drugs or vaccines. Moreover, the fact that animal models are not readily accessible and the fact that mostly laboratory viruses, which have been passaged many times after isolation, are used for studies further limits the successful development of countermeasures. In this study, we demonstrate that a human isolate of Lassa virus causes lethal infection uniformly in Hartley guinea pigs. This novel animal model of Lassa fever may contribute to Lassa fever research and the development of vaccines and therapeutics.

Therapeutic options for coronaviruses remain limited. To address this unmet medical need, we screened 5406 compounds, including United States Food and Drug Administration (FDA)-approved drugs and bioactives, for activity against a South Korean Middle East respiratory syndrome coronavirus (MERS-CoV) clinical isolate. Among 221 identified hits, 54 had therapeutic indexes (TI) greater than 6, representing effective drugs. The time-of-addition studies with selected drugs demonstrated eight and four FDA-approved drugs which acted on the early and late stages of the viral life cycle, respectively. Confirmed hits included several cardiotonic agents (TI > 100), atovaquone, an anti-malarial (TI > 34), and ciclesonide, an inhalable corticosteroid (TI > 6). Furthermore, utilizing the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), we tested combinations of remdesivir with selected drugs in Vero-E6 and Calu-3 cells, in lung organoids, and identified ciclesonide, nelfinavir, and camostat to be at least additive in vitro. Our results identify potential therapeutic options for MERS-CoV infections, and provide a basis to treat coronavirus disease 2019 (COVID-19) and other coronavirus-related illnesses.

Aim. Study the prevalence and intensity of anti-hemoglobin and anti-lactoferrin activity in enterococci as representatives of symbiotic intestine microflora and causative agents of infection-inflammatory diseases in children. Materials and methods. 95 strains of enterococci were used in the study, that were isolated from intestines of children during examination for dysbiosis, and 34 clinical isolates. Strain identification was carried out by using multiplex PCR. Anti-lactoferrin and anti-hemoglobin activity were determined by O.V.Bukharin et al. (2005). Results. A higher level of prevalence and intensity of anti-lactoferrin, anti-hemoglobin activity, as well as a combination of those properties was established in clinical isolates of Enterococcus faecalis and E. faecium compared with strains, isolated from intestines of healthy individuals. Conclusion. The studied properties could be used for differentiation of pathogenic and non-pathogenic strains of enterococci and during selection of inhibitory preparations, suitable for therapy of enterococci infections.

The opportunistic fungal pathogen Candida glabrata is the second most common isolate from bloodstream infections worldwide and is naturally less susceptible to the antifungal drug fluconazole than other Candida species. C. glabrata is a haploid yeast that contains three mating-type like loci (MTL), although no sexual cycle has been described. Strains containing both types of mating information at the MTL1 locus are found in clinical isolates, but it is thought that strains containing type a information are more common. Here we investigated if a particular combination of mating type information at each MTLlocus is more prevalent in clinical isolates from hospitalized patients in Mexico and if there is a correlation between mating information and resistance to fluconazole and 5-fluorocytosine. We found that while both types of information at MTL1 are equally represented in a collection of 64 clinical isolates, the vast majority of isolates contain a-type information at MTL2 and α-type at MTL3. We also found no correlation of the particular combination of mating type information at the three MTL loci and resistance to fluconazole.

Objective/Background: The incidence of resistant strains of Mycobacterium tuberculosis (MTB), including multi-drug resistant, extensively drug resistant, or totally drug resistant, is one of the major problems of health policies worldwide. The accumulation of mutations causes multi-drug resistant strains. Mycobacterium abscessus has a plasmid called pMab2401 containing the trfA1 gene in its integron part. The aim of the present study was to investigate the possible existence of the trfA1 gene in clinical strains of MTB for the first time. Methods: Bioinformatics analysis in GenBank revealed an absence of any integrons or internal components in MTB. Several specific primers for different genes and the trfA1 gene of M. abscessus were used in a touch-down (60–52) amplification program and followed by loading on gel electrophoresis. Products were extracted and were sequenced. Sequencing results were analyzed carefully and compared with those in the databank. Results: Bands of 500bp were resulted with pair primers in an amplification reaction by clinical MTB that has 94% identity with a fragment in most plasmids and phages M13. It should be noted that such an independent replication system-like structure has not been reported to date in MTB strains. Conclusion: The trfA1 gene is depends on the replication process. It is necessary to investigate other probable new areas that may be of concern with drug resistance in clinical isolates of MTB.

Abstract Background Massively parallel sequencing technology is revolutionizing approaches to genomic and genetic research. Since its advent, the scale and efficiency of Next-Generation Sequencing (NGS) has rapidly improved. In spite of this success, sequencing genomes or genomic regions with extremely biased base composition is still a great challenge to the currently available NGS platforms. The genomes of some important pathogenic organisms like Plasmodium falciparum (high AT content) and Mycobacterium tuberculosis (high GC content) display extremes of base composition. The standard library preparation procedures that employ PCR amplification have been shown to cause uneven read coverage particularly across AT and GC rich regions, leading to problems in genome assembly and variation analyses. Alternative library-preparation approaches that omit PCR amplification require large quantities of starting material and hence are not suitable for small amounts of DNA/RNA such as those from clinical isolates. We have developed and optimized library-preparation procedures suitable for low quantity starting material and tolerant to extremely high AT content sequences. Results We have used our optimized conditions in parallel with standard methods to prepare Illumina sequencing libraries from a non-clinical and a clinical isolate (containing ~53% host contamination). By analyzing and comparing the quality of sequence data generated, we show that our optimized conditions that involve a PCR additive (TMAC), produces amplified libraries with improved coverage of extremely AT-rich regions and reduced bias toward GC neutral templates. Conclusion We have developed a robust and optimized Next-Generation Sequencing library amplification method suitable for extremely AT-rich genomes. The new amplification conditions significantly reduce bias and retain the complexity of either extremes of base composition. This development will greatly benefit sequencing clinical samples that often require amplification due to low mass of DNA starting material.

Abstract Background Dengue virus (DENV) infection is the most common arthropod-borne viral disease in man and there are approximately 100 million infections annually. Despite the global burden of DENV infections many important questions regarding DENV pathogenesis remain unaddressed due to the lack of appropriate animal models of infection and disease. A major problem is the fact that no non-human species naturally develop disease similar to human dengue fever (DF) or dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Apart from other risk factors for severe dengue such as host genetics and secondary infection with a heterologous DENV, virus virulence is a risk factor that is not well characterized. Results Three clinical DENV-1 isolates from Cambodian patients experiencing the various forms of dengue disease (DF, DHF, and DSS) were inoculated in BALB/c mice at three different concentrations. The DENV-1 isolates had different organ and cell tropism and replication kinetics. The DENV-1 isolate from a DSS patient infected the largest number of mice and was primarily neurotropic. In contrast, the DENV-1 isolates from milder clinical dengue cases infected predominantly lungs and liver, and to a lesser extent brain. In addition, infection with the DENV isolate derived from a DSS patient persisted for more than two weeks in a majority of mice compared to the other DENV-1 isolates that peaked during the first week. Conclusions These results confirm the in vitro findings of the same DENV-1 isolates, that showed that the isolate derived from a DSS patient can be distinguished based on phenotypic characteristics that differ from the isolates derived from a DF and DHF case 1. We observed in this study that the DSS virus isolate persist longer in vivo with extensive neuroinvasion in contrast to the other DENV-1 isolates originating in milder human cases. Genomic characterization of the three clinical isolates identified six amino acid substitutions unique for the DSS isolates that were located both in structural genes (M and E) and in non-structural genes (NS1, NS3, and NS5). The characterization of these clinically distinct DENV-1 isolates highlight that DENVs within the same genotype may have different in vivo phenotypes. Highlights • Clinical DENV-1 isolates have different organ tropism in BALB/c mice. • The isolate from a DSS patient is primarily neurotropic compared to the other isolates. • The DENV-1 isolates have different in vivo replication kinetics. • The isolate from a DSS patient persists longer compared to the other isolates. • These phenotypic differences confirm our earlier in vitro findings with the same DENV-1 isolates. Thus, DENVs within the same serotype and genotype may differ enough to affect clinical conditions in vivo.