Wild-type Isolates Research Articles

The point mutation A1387G in the 16S rRNA gene confers aminoglycoside resistance in Campylobacter jejuni and Campylobacter coli.

Thermotolerant Campylobacter spp. are the most frequent cause of foodborne bacterial diarrhea and high-priority antibiotic-resistant pathogens, according to the World Health Organization (WHO). Monitoring revealed current low prevalence of gentamicin resistance in European Campylobacter spp. isolates but substantial presence of gentamicin modifying genes circulating globally. Using a combined approach of natural transformation and whole-genome sequencing, we revealed a novel gentamicin resistance mechanism, namely the point mutation A1387G in the 16S rRNA gene, originally identified in a C. coli isolate from turkey caecal content. The transformation rate of the resistance using genomic DNA of the resistant donor to sensitive recipient C. jejuni and C. coli was ~2.5 log10 lower compared to the control rpsL-A128G point mutation conferring streptomycin resistance. Antimicrobial susceptibility tests showed cross-resistance to apramycin, kanamycin, and tobramycin, with transformants exhibiting more than 4- to 8-fold increased MICs to apramycin and tobramycin and over 64-fold higher MICs to kanamycin compared to wild-type isolates. Although transformants showed 177-1,235 variations relative to the recipient, only the A1387G point mutation in the 16S rRNA was in common. This mutation was causal for resistance, as transformation of a 16S rRNA_A1387G PCR fragment into susceptible isolates also led to resistant transformants. Sanger sequencing of the 16S rRNA genes and Oxford nanopore whole-genome sequencing of transformants identified clones harboring either all three copies with A1387G or a mixed population of wild-type and mutated 16S rRNA gene alleles. Within 15 passages on non-selective medium, transformants with mixed populations of the 16S rRNA gene copies partially reverted to wild type, both geno- and phenotypically. In contrast, transformants harboring the A1387G point mutation in all three 16S rRNA gene copies kept full resistance within at least 45 passages. We speculate that partial acquisition and rapid loss of the point mutation limited its spread among C. spp. isolates. In-depth knowledge on resistance mechanisms contributes to optimal diagnosis and preventative measures.

Frequency of chloroquine-resistant haplotype of Plasmodium falciparum (CVIET) in Ibadan, Southwest Nigeria 17 years post-chloroquine withdrawal

The replacement of chloroquine with artemisinin-based combination therapies (ACTs) for over a decade has had varying impacts on the ability of the malaria parasite to sustain its chloroquine resistance prowess in different malaria-endemic regions. We evaluated the frequency of Plasmodium falciparum chloroquine resistance transporter (PfCRT) mutations in Ibadan, Nigeria 17 years after the replacement of chloroquine with ACTs for malaria treatment. Fragments of PfCRT gene from genomic DNA of microscopically confirmed P. falciparum-infected patients were amplified and sequenced. There were 19% CVIET mutant and 81% CVMNK wild-type haplotypes on residues 72–76. A220S change were found in 16.7% of samples occurring concurrently with the CVIET haplotype, while a Q271E mutation occurred in a PfCRT wild-type isolate. The reduced prevalence of the PfCRT mutant alleles in this study compared to previous reports suggests a gradual disappearance of chloroquine-resistant malaria parasites following reduced drug pressure. It may also be a result of fitness demand on the parasites in attempts to evolve resistance against the current first-line regimen. However, evaluating the prevalence of other chloroquine resistance markers such as Plasmodium falciparum multidrug resistance 1 gene mutations in this population, and a more robust sample size will help to consolidate these findings.

Efficiency and resistance risk of flusilazole against northern corn leaf blight caused by Setosphaeria turcica

Northern corn leaf blight (NCLB) infected by Setosphaeria turcica is a devastating disease of corn worldwide. Flusilazole is a broad-spectrum triazole fungicide. However, its resistance risk and field efficiency in controlling NCLB are still unknown. The present research evaluated the antifungal activity of flusilazole against 101 S. turcica isolates, and their EC50 values ranged from 0.0013 to 0.0466 μg/mL, with a mean of 0.0157 μg/mL. Seven S. turcica mutants resistant to flusilazole were obtained from two wild-type isolates by fungicide adaptation. After 10 consecutive transfers on PDA medium without fungicide, their resistance decreased. Cross-resistance was not existed between flusilazole and fluazinam, pyraclostrobin, amobam, epoxiconazole, or fluxapyroxad. Compared to the wild-type isolates, seven flusilazole-resistant mutants showed reduced biological fitness. No point mutation was detected, however, over-expression of StCYP51 and StatrD genes were detected in the resistant mutants. In addition, in the field experiment, flusilazole exhibited over 85 % efficacy against NCLB, significantly higher than amobam. In summary, these results suggested that the resistance risk of S. turcica to flusilazole was low, and the over-expression of StCYP51 and StatrD might be related to the flusilazole resistance against S. turcica. Flusilazole showed great potential as an alternative fungicide for controlling NCLB.

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

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

Mechanistic insights into antifungal potential of Alexidine dihydrochloride and hexachlorophene in Candida albicans: a drug repurposing approach.

Candida albicans has been listed in the critical priority group by the WHO in 2022 depending upon its contribution in invasive candidiasis and increased resistance to conventional drugs. Drug repurposing offers an efficient, rapid, and cost-effective solution to develop alternative therapeutics against pathogenic microbes. Alexidine dihydrochloride (AXD) and hexachlorophene (HCP) are FDA approved anti-cancer and anti-septic drugs, respectively. In this study, we have shown antifungal properties of AXD and HCP against the wild type (reference strain) and clinical isolates of C. albicans. The minimum inhibitory concentrations (MIC50) of AXD and HCP against C. albicans ranged between 0.34 and 0.69 µM and 19.66-24.58 µM, respectively. The biofilm inhibitory and eradication concentration of AXD was reported comparatively lower than that of HCP for the strains used in the study. Further investigations were performed to understand the antifungal mode of action of AXD and HCP by studying virulence features like cell surface hydrophobicity, adhesion, and yeast to hyphae transition, were also reduced upon exposure to both the drugs. Ergosterol content in cell membrane of the wild type strain was upregulated on exposure to AXD and HCP both. Biochemical analyses of the exposed biofilm indicated reduced contents of carbohydrate, protein, and e-DNA in the extracellular matrix of the biofilm when compared to the untreated control biofilm. AXD exposure downregulated activity of tissue invading enzyme, phospholipase in the reference strain. In wild type strain, ROS level, and activities of antioxidant enzymes were found elevated upon exposure to both drugs. FESEM analysis of the drug treated biofilms revealed degraded biofilm. This study has indicated mode of action of antifungal potential of alexidine dihydrochloride and hexachlorophene in C. albicans.

Comprehensive database of HIV mutations selected during antiretroviral in vitro passage experiments

BackgroundIn vitro passage experiments are crucial to the development of antiretroviral (ARV) drugs. MethodsWe created an online database containing data from 102 published studies in which HIV-1 or HIV-2 was cultured with increasing concentrations of the FDA-approved nucleoside RT inhibitors (NRTIs), nonnucleoside RT inhibitors (NNRTIs), integrase strand transfer inhibitors (INSTIs), protease inhibitors (PIs), capsid inhibitor (CAI) lenacapavir, and nucleoside RT translocation inhibitor (NRTTI) islatravir. We summarized the mutations selected in the subset of passage experiments with NRTIs lamivudine (3TC), emtricitabine (FTC), abacavir (ABC), tenofovir (TFV), and zidovudine (AZT), NNRTIs doravirine (DOR), efavirenz (EFV), and rilpivirine (RPV), INSTIs bictegravir (BIC), cabotegravir (CAB), and dolutegravir (DTG), and PIs atazanavir (ATV), darunavir (DRV), and lopinavir (LPV). Mutations selected in vitro were compared with those selected in persons receiving the same ARV. ResultsTwenty-seven studies described 89 experiments of wildtype isolates passaged with 3TC, FTC, ABC, TFV, or AZT; sixteen studies described 89 experiments passaged with EFV, RPV, or DOR; eleven studies described 76 experiments passaged with the INSTIs BIC, CAB, or DTG; six studies described 33 experiments passaged with ATV, LPV, or DRV. With several exceptions, mutations selected in two or more experiments were among the most common mutations selected in persons receiving the same ARV. ConclusionsWe created a database of published ARV in vitro selection experiments. Mutations emerging from these experiments generally predict those observed in persons receiving the same ARV. However, there are notable differences in mutation frequencies between in vitro and in vivo settings.

The role of oxalic acid in Clarireedia jacksonii virulence and development on creeping bentgrass.

Dollar spot is a destructive foliar disease of amenity turfgrass caused by the fungus Clarireedia spp., and mainly Clarireedia jacksonii on the northern US region's cool-season grass. Oxalic acid (OA) is an important pathogenicity factor in related fungal plant pathogens such as Sclerotinia sclerotiorum, however, the role of OA in the pathogenic development of C. jacksonii remains unclear due to its recalcitrance to genetic manipulation. To overcome these challenges, a CRISPR/Cas9-mediated homologous recombination approach was developed. Using this novel approach, the oxaloacetate acetylhydrolase (Oah) gene that is required for the biosynthesis of OA was deleted from C. jacksonii wild-type stain. Two independent knockout mutants, ΔCjoah-1 and ΔCjoah-2, were generated and inoculated on potted creeping bentgrass along with a wild-type isolate (WT) and a genome sequenced isolate LWC-10. After 12 days, bentgrass inoculated with the mutants ΔCjoah-1 and ΔCjoah-2 exhibited 59.41% lower dollar spot severity compared to the WT and LWC-10 isolates. Oxalic acid production and environmental acidification were significantly reduced in both mutants when compared to the WT and LWC-10. Surprisingly, stromal formation was also severely undermined in the mutants in vitro, suggesting a critical developmental role of OA independent of plant infection. These results demonstrate that OA plays a significant role in C. jacksonii virulence and provide novel directions for future management of dollar spot.

Characterization of the Neurospora crassa Galactosaminogalactan Biosynthetic Pathway.

The Neurospora crassa genome has a gene cluster for the synthesis of galactosaminogalactan (GAG). The gene cluster includes the following: (1) UDP-glucose-4-epimerase to convert UDP-glucose and UDP-N-acetylglucosamine to UDP-galactose and UDP-N-acetylgalactosamine (NCU05133), (2) GAG synthase for the synthesis of an acetylated GAG (NCU05132), (3) GAG deacetylase (/NCW-1/NCU05137), (4) GH135-1, a GAG hydrolase with specificity for N-acetylgalactosamine-containing GAG (NCU05135), and (5) GH114-1, a galactosaminidase with specificity for galactosamine-containing GAG (NCU05136). The deacetylase was previously shown to be a major cell wall glycoprotein and given the name of NCW-1 (non-GPI anchored cell wall protein-1). Characterization of the polysaccharides found in the growth medium from the wild type and the GAG synthase mutant demonstrates that there is a major reduction in the levels of polysaccharides containing galactosamine and N-acetylgalactosamine in the mutant growth medium, providing evidence that the synthase is responsible for the production of a GAG. The analysis also indicates that there are other galactose-containing polysaccharides produced by the fungus. Phenotypic characterization of wild-type and mutant isolates showed that deacetylated GAG from the wild type can function as an adhesin to a glass surface and provides the fungal mat with tensile strength, demonstrating that the deacetylated GAG functions as an intercellular adhesive. The acetylated GAG produced by the deacetylase mutant was found to function as an adhesive for chitin, alumina, celite (diatomaceous earth), activated charcoal, and wheat leaf particulates.

Epidemiological cutoff values and genetic antimicrobial resistance of Lactococcus garvieae and L. petauri

Lactococcus garvieae and L. petauri are etiological agents of lactococcosis, which cause significant losses in trout aquaculture at elevated water temperatures. Optimizing antimicrobial dosage regimens and identifying the minimum inhibitory concentration (MIC) are important prerequisites for determining clinical breakpoints and effective antimicrobial use. In the present study, epidemiological cutoff values (ECV) were established for 14 antimicrobials using L. garvieae (n = 32) and L. petauri (n = 78), isolated from rainbow trout farms in Türkiye, Spain, Italy, and Greece. The provisional ECVs were calculated using the ECOFFinder and normalized resistance interpretation (NRI) approaches. Furthermore, gene cassettes, capsule gene clusters, and the presence of fourteen antibiotic resistance genes (ARGs) that encode resistance against seven antimicrobial agents, were evaluated. The calculated ECVs of antimicrobials ranged from 1.25 μg mL−1 for amoxicillin to 80.25 μg mL−1 for streptomycin. The percentages of wild type (WT) and non-wild type (NWT) isolates differed based on the approach, and ECVs calculated based on ECOFFinder approach were generally higher. Among the ARGs, those encoding resistance against tetracycline (tetA), β-lactamase (blaSHV, blaTEM, and blaOXA), amphenicol (floR), and macrolide (ermA) were detected in <10% of the isolates. The genes encoding resistance against quinolones (gyrA and qnrA) and aminoglycosides (strB) were the most detected genes. While 40.9% of the isolates contained Class 1 integron cassette regions, 33.6% of the isolates contained Class 2 integron cassette regions, and 9.1% of the isolates contained both cassette regions. None of the screened isolates were positive for the capsule gene cluster. Significant but weak correlations were found among and between ARGs and MIC distribution of antibiotics. The results of this study could be useful for identifying WT and non-WT isolates, as well as for susceptibility surveillance.

Comparative pharmacodynamics and dose optimization of liposomal amphotericin B against Candida species in an in vitro pharmacokinetic/pharmacodynamic model.

As comparative pharmacokinetic/pharmacodynamic (PK/PD) studies of liposomal amphotericin B (L-AMB) against Candida spp. are lacking, we explored L-AMB pharmacodynamics against different Candida species in an in vitro PK/PD dilution model. Eight Candida glabrata, Candida parapsilosis, and Candida krusei isolates (EUCAST/CLSI AMB MIC 0.125-1 mg/L) were studied in the in vitro PK/PD model simulating L-AMB Cmax = 0.25-64 mg/L and t1/2 = 9 h. The model was validated with one susceptible and one resistant Candida albicans isolate. The Cmax/MIC-log10CFU/mL reduction from the initial inoculum was analyzed with the Emax model, and Monte Carlo analysis was performed for the standard (3 mg/kg with Cmax = 21.87 ± 12.47 mg/L) and higher (5 mg/kg with Cmax = 83 ± 35.2 mg/L) L-AMB dose. A ≥1.5 log10CFU/mL reduction was found at L-AMB Cmax = 8 mg/L against C. albicans, C. parapsilosis, and C. krusei isolates (MIC 0.25-0.5 mg/L) whereas L-AMB Cmax ≥ 32 mg/L was required for C. glabrata isolates. The in vitro PK/PD relationship followed a sigmoidal pattern (R2 ≥ 0.85) with a mean Cmax/MIC required for stasis of 2.1 for C. albicans (close to the in vivo stasis), 24/17 (EUCAST/CLSI) for C. glabrata, 8 for C. parapsilosis, and 10 for C. krusei. The probability of target attainment was ≥99% for C. albicans wild-type (WT) isolates with 3 mg/kg and for wild-type isolates of the other species with 5 mg/kg. L-AMB was four- to eightfold less active against the included non-C. albicans species than C. albicans. A standard 3-mg/kg dose is pharmacodynamically sufficient for C. albicans whereas our data suggest that 5 mg/kg may be recommendable for the included non-C. albicans species.

Generation of rescued Japanese encephalitis virus genotype 1 from infectious full-size clone using reverse genetics

Japanese encephalitis virus (JEV) is a pathogen responsible for high mortality and morbidity rates among children with encephalitis. Since JEV genotype 1 (GI) is the most prevalent strain in South Korea these days, corresponding research and vaccine development is urgently required. Molecular genetic studies on JEV vaccines can be boosted by obtaining genetically stable full-length infectious JEV complementary DNA (cDNA) clones. Furthermore, the significance of the reverse genetics system in facilitating molecular biological analyses of JEV properties has been demonstrated. This study constructed a recombinant JEV-GI strain using a reverse genetics system based on a Korean wild-type GI isolate (K05GS). RNA extracted from JEV-GI was used to synthesize cDNA, a recombinant full-length JEV clone, pTRE-JEVGI, was generated from the DNA fragment, and the virus was rescued. We performed in vitro and in vivo experiments to analyze the rescued JEV-GI virus. The rescued JEV-GI exhibited similar characteristics to wild-type JEV. These results suggest that our reverse genetics system can generate full-length infectious clones that can be used to analyze molecular biological factors that influence viral properties and immunogenicity. Additionally, it may be useful as a heterologous gene expression vector and help develop new strains for JEV vaccines.

Secreted in Xylem 6 (SIX6) Mediates Fusarium oxysporum f. sp. fragariae Race 1 Avirulence on FW1-Resistant Strawberry Cultivars.

Fusarium oxysporum f. sp. fragariae (Fof) race 1 is avirulent on cultivars with the dominant resistance gene FW1, while Fof race 2 is virulent on FW1-resistant cultivars. We hypothesized there was a gene-for-gene interaction between a gene at the FW1 locus and an avirulence gene (AvrFW1) in Fof race 1. To identify a candidate AvrFW1, we compared genomes of 24 Fof race 1 and three Fof race 2 isolates. We found one candidate gene that was present in race 1, was absent in race 2, was highly expressed in planta, and was homologous to a known effector, secreted in xylem 6 (SIX6). We knocked out SIX6 in two Fof race 1 isolates by homologous recombination. All SIX6 knockout transformants (ΔSIX6) gained virulence on FW1/fw1 cultivars, whereas ectopic transformants and the wildtype isolates remained avirulent. ΔSIX6 isolates were quantitatively less virulent on FW1/fw1 cultivars Fronteras and San Andreas than fw1/fw1 cultivars. Seedlings from an FW1/fw1 × fw1/fw1 population were genotyped for FW1 and tested for susceptibility to a SIX6 knockout isolate. Results suggested that additional minor-effect quantitative resistance genes could be present at the FW1 locus. This work demonstrates that SIX6 acts as an avirulence factor interacting with a resistance gene at the FW1 locus. The identification of AvrFW1 enables surveillance for Fof race 2 and provides insight into the mechanisms of FW1-mediated resistance. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Gamma irradiation induced thermotolerance in Metarhizium spp. enhancing biocontrol of Nilapavarta lugens (Stål)

The Brown Planthopper (BPH), Nilaparvata lugens (Stål), is a devastating pest to rice crops in Southeast Asia, primarily due to its resistance to insecticides. As a result, the fungus Metarhizium spp. is used as an alternative control for BPH. However, their efficiency is limited due to delayed infectivity and sensitivity to environmental variables. In this study, two isolates of Metarhizium spp. MLUBN038 and MNMHN031, were induced for thermotolerance and biocontrol potential against Nilapavarta lugens (Stål). A multilocus phylogeny of four protein-coding genes including Beta-tubulin, rpb1, rpb2, and tef has identified the two isolates to be Metarhizium koreanum. Seventy-nine potential thermotolerant mutants were screened from the two isolates. These mutant isolates demonstrated significant tolerance to vegetative heat at 39 °C and enhanced resistance to wet heat stress at 45 °C after exposure for 60 h, outperforming the wild-type (WT) isolates. The mutant isolates also showed increased mycelial growth, conidial germination, and spore production compared to the WT isolates. Two mutant isolates, ML038_Mt 71 and MN031_Mt 46, exhibited the highest efficacy against BPH nymphs (4th-5th instars), resulting in a maximum mortality of 88.82 % and 83.55 %, respectively, compared to the WT (54.07 % and 47.54 %) after 14 days of inoculation in the greenhouse. These two highly virulent mutant isolates presented the lowest LT50 values of 2.88 and 3.72 days at a concentration of 1 × 109 conidia/mL. RAPD analysis revealed genetic diversity between the mutant and WT isolates, indicating distinct diversity profiles and supporting the successful generation of mutant variants. These findings suggest that the use of gamma irradiation and heat stress to induce mutations is crucial for the successful production of thermotolerant mutant isolates. Our results suggest that ML031_Mt 71 and MN031_Mt 46 have potential as effective biocontrol agents.

Improving nitrofurantoin resistance prediction in Escherichia coli from whole-genome sequence by integrating NfsA/B enzyme assays.

Nitrofurantoin resistance in Escherichia coli is primarily caused by mutations damaging two enzymes, NfsA and NfsB. Studies based on small isolate collections with defined nitrofurantoin MICs have found significant random genetic drift in nfsA and nfsB, making it extremely difficult to predict nitrofurantoin resistance from whole-genome sequence (WGS) where both genes are not obviously disrupted by nonsense or frameshift mutations or insertional inactivation. Here, we report a WGS survey of 200 oqxAB-negative E. coli from community urine samples, of which 34 were nitrofurantoin resistant. We characterized individual non-synonymous mutations seen in nfsA and nfsB among this collection using complementation cloning and NfsA/B enzyme assays in cell extracts. We definitively identified R203C, H11Y, W212R, A112E, and A112T in NfsA and R121C, Q142H, F84S, P163H, W46R, K57E, and V191G in NfsB as amino acid substitutions that reduce enzyme activity sufficiently to cause resistance. In contrast, E58D, I117T, K141E, L157F, A172S, G187D, and A188V in NfsA and G66D, M75I, V93A, and A174E in NfsB are functionally silent in this context. We identified that 9/166 (5.4%) nitrofurantoin-susceptible isolates were "pre-resistant," defined as having loss of function mutations in nfsA or nfsB. Finally, using NfsA/B enzyme assays and proteomics, we demonstrated that 9/34 (26.5%) ribE wild-type nitrofurantoin-resistant isolates also carried functionally wild-type nfsB or nfsB/nfsA. In these cases, NfsA/B activity was reduced through downregulated gene expression. Our biological understanding of nitrofurantoin resistance is greatly improved by this analysis but is still insufficient to allow its reliable prediction from WGS data.

β-lactam resistant phenotypes reported by VITEK®2 advanced expert systemTM (AES) compared to whole genome sequencing in Enterobacterales from North and Latin America

The VITEK®2 AES β-lactam phenotypes of 488 Enterobacterales from North and Latin America generated by the VITEK®2 were compared to the resistance genotypes provided by whole genome sequencing (WGS). The AES provided phenotypic reports for 447 (91.6 %) isolates, including isolates harbouring carbapenemases (195; 43.6 %), ESBLs (103; 23.0 %) and transferable AmpCs (tAmpC; 28; 6.3 %) genes, as well as wildtype isolates (WT; 121; 27.1 %). Overall, the AES report was accurate for 433/447 (96.9 %) isolates. The AES accurately reported carbapenemase, ESBL, and tAmpC phenotypes for 93.7 %, 93.7 %, and 98.4 % of isolates, respectively, and sensitivity/specificity rates were 96.4 %/91.7 %, 98.1 %/92.4 %, 82.1 %/99.5 %, and 100 %/98.8 %. 14 isolates carrying carbapenemase (7 total; 3 KPC, 2 MBL, 2 OXA-48–like), ESBL (2), and tAmpC-encoding genes (5) were not correctly identified by AES. The AES phenotypic report detected resistance mechanisms among Enterobacterales rapidly and could significantly aid future antimicrobial stewardship initiatives and patient care.

Single amino acid change in tomato brown rugose fruit virus breaks virus-specific resistance in new resistant tomato cultivar.

Tomato cultivation across the world is severely affected by emerging plant viruses. An effective method for protection of commercial crops against viral threats is the use of cultivars harboring resistance genes. Tomato brown rugose fruit virus (ToBRFV), a recently emerged tobamovirus, is able to overcome the dominant Tm-22 resistance that is present in the majority of commercial tomato cultivars. In an effort to alleviate the severe consequences of ToBRFV on tomato production, tomato breeding companies are developing new cultivars with varying levels of resistance against ToBRFV. In the present study, cultivars with a new resistant phenotype against ToBRFV were screened against a wild-type isolate of ToBRFV, and subsequently, their performance under commercial greenhouse conditions was monitored. Following the identification of ToBRFV symptoms in a commercial greenhouse-where both new resistant and susceptible cultivars were interplanted-these cultivars were more closely examined. The presence of ToBRFV was molecularly confirmed on both cultivar types suggesting that the new resistance had been broken. High-throughput sequencing (HTS) was used to study the complete genomes of viral isolates present in the two cultivar types. The analysis revealed a single amino acid change at position 82 of the movement protein of ToBRFV in the isolate present in the new resistant cultivar compared with the isolate identified in the susceptible cultivar. A screening bioassay, that was performed to compare the infectivity of the two ToBRFV isolates, confirmed that only the isolate with this specific amino acid change could successfully infect the resistant cultivar, overcoming the new resistance against ToBRFV.

Role of tcaA, a potential target as a ceftobiprole resistance breaker in MRSA β-lactam resistance

ObjectivesUsing a random forest algorithm, we previously found that teicoplanin-associated gene A (tcaA) might play a role in resistance of methicillin-resistant Staphylococcus aureus (MRSA) to β-lactams, which we have investigated further here. MethodsRepresentative MRSA strains of prevalent clones were selected to identify the role of tcaA in the MRSA response to β-lactams. tcaA genes were deleted by homologous recombination in the selected MRSA strains, and antibiotic susceptibility tests were applied to evaluate the effect of tcaA on the minimum inhibitory concentrations (MICs) of glycopeptides and β-lactams. Scanning electron microscopy, RNA sequencing, and quantitative reverse transcription-polymerase chain reaction were performed to explore the mechanism of tcaA in MRSA resistance to β-lactams. ResultsThe MIC of penicillin plus clavulanate decreased from 3 mg/L to 0.064 mg/L and that of oxacillin decreased from 16 to 0.5 mg/L when tcaA was knocked out in the LAC strain. Compared with wild-type MRSA isolates, when tcaA was deleted, all selected strains were more susceptible to β-lactams. Susceptibility to ceftobiprole was restored in the ceftobiprole-resistant strain when tcaA was deleted. tcaA knockout caused “log-like” abnormal division of MRSA, and tcaA deficiency mediated low expression of mecA, ponA, and murA2. ConclusionsMachine learning is a reliable tool for identifying drug resistance-related genes. tcaA may be involved in S. aureus cell division and may affect mecA, ponA, and murA2 expression. Furthermore, tcaA is a potential resistance breaker target for β-lactams, including ceftobiprole, in MRSA.

Effect of Conformational Variability on the Drug Resistance of Candida auris ERG11p and FKS1.

Candida auris infection has been recognized as an urgent threat to antifungal drug resistance, and the Eagle effect of C. auris FKS1 (1,3-β-d-glucan synthase) wild-type isolates has also been noted. The Eagle effect, namely, where higher concentrations of antifungals reduce fungicidal activity relative to lower concentrations, is a confounding factor of apparent antifungal resistance, but the detailed mechanism remains unclear. Here, we present the conformational variability of mutation sites for ERG11p (lanosterol 14α-demethylase) and FKS1 from deep neural network-based prediction along with the reported X-ray crystallographic and cryo-electron microscopy (cryo-EM) structures of antifungals. The sequence variability maps provide valuable insights into the inconsistent correlation between azole resistance and the mysterious Eagle effect with the dispersion of minimal inhibitory concentration (MIC) for echinocandin resistance. The conformational variability prediction supports the hypothesis that mutations K143R of clade I, VF125AL of clade III, and Y132F of clade IV for C. auris ERG11p make the corresponding site variable and that an increased population of invisible variable conformations potentially contributes to triazole resistance. In contrast, the predicted rigid conformation by the S639F mutation of hot spot region 1 (HS1) for FKS1 suggests that caspofungin (CAS) is involved in an uncompetitive inhibition, and a decreased population of the CAS-bound state of FKS1 with Rho1 leads to drug resistance. The predicted variable HS1 region for FKS1 WT isolates and the rigid one for FKS1 S639F mutants support the in vivo drug response and the in vitro MIC dispersion. A plausible mechanism of the Eagle effect is hereby proposed, namely, that a high concentration of CAS with a high membrane affinity reduces the population of the CAS-bound state of FKS1 with Rho1, as well as accompanying events such as aggregation or association depending on the conformational variability of HS1.

Saccharomyces cerevisiae strains performing similarly during fermentation of lignocellulosic hydrolysates show pronounced differences in transcriptional stress responses.

The need for sustainable alternatives to oil-based production of biochemicals and biofuels is undisputable. Saccharomyces cerevisiae is the most commonly used industrial fermentation workhorse. The fermentation of lignocellulosic hydrolysates, second-generation biomass unsuited for food and feed, is still hampered by lowered productivities as the raw material is inhibitory for the cells. In order to map the genetic responses of different S. cerevisiae strains, we performed RNA sequencing of a CEN.PK laboratory strain, two industrial strains (KE6-12 and Ethanol Red), and two wild-type isolates of the LBCM collection when cultivated anaerobically in wheat straw hydrolysate. While the response to inhibitors of S. cerevisiae has been studied earlier, this has in previous studies been done in aerobic conditions. The transcriptomic analysis highlights different evolutionary adaptations among the different S. cerevisiae strains and suggests novel gene targets for improving fermentation performance and robustness.

Hybrid capture-based next-generation sequencing of new and old world Orthohantavirus strains and wild-type Puumala isolates from humans and bank voles

Orthohantaviruses, transmitted primarily by rodents, cause hemorrhagic fever with renal syndrome (HFRS) in Eurasia and hantavirus pulmonary syndrome in the Americas. These viruses, with documented human-to-human transmission, exhibit a wide case-fatality rate, 0.5–40 %, depending on the virus species, and no vaccine or effective treatment for severe Orthohantavirus infections exists. In Europe, the Puumala virus (PUUV), carried by the bank vole Myodes glareolus, causes a milder form of HFRS. Despite the reliance on serology and PCR for diagnosis, the three genomic segments of Swedish wild-type PUUV have yet to be completely sequenced.We have developed a targeted hybrid-capture method aimed at comprehensive genomic sequencing of wild-type PUUV isolates and the identification of other Orthohantaviruses. Our custom-designed panel includes >11,200 probes covering the entire Orthohantavirus genus. Using this panel, we sequenced complete viral genomes from bank vole lung tissue, human plasma samples, and cell-cultured reference strains. Analysis revealed that Swedish PUUV isolates belong to the Northern Scandinavian lineage, with nucleotide diversity ranging from 2.8 % to 3.7 % among them. Notably, no significant genotypic differences were observed between the viral sequences from reservoirs and human cases except in the nonstructural protein.Despite the high endemicity of PUUV in Northern Sweden, these are the first complete Swedish wild-type PUUV genomes and substantially increase our understanding of PUUV evolution and epidemiology. The panel's sensitivity enables genomic sequencing of human samples with viral RNA levels reflecting the natural progression of infection and underscores our panel's diagnostic value, and could help to uncover novel Orthohantavirus transmission routes.