CS Strains Research Articles

Haplotypes at the sorghum ARG4 and ARG5 NLR loci confer resistance to anthracnose.

Sorghum anthracnose caused by the fungus Colletotrichum sublineola (Cs) is a damaging disease of the crop. Here, we describe the identification of ANTHRACNOSE RESISTANCE GENES (ARG4 and ARG5) encoding canonical nucleotide-binding leucine-rich repeat (NLR) receptors. ARG4 and ARG5 are dominant resistance genes identified in the sorghum lines SAP135 and P9830, respectively, that show broad-spectrum resistance to Cs. Independent genetic studies using populations generated by crossing SAP135 and P9830 with TAM428, fine mapping using molecular markers, comparative genomics and gene expression studies determined that ARG4 and ARG5 are resistance genes against Cs strains. Interestingly, ARG4 and ARG5 are both located within clusters of duplicate NLR genes at linked loci separated by ~1 Mb genomic region. SAP135 and P9830 each carry only one of the ARG genes while having the recessive allele at the second locus. Only two copies of the ARG5 candidate genes were present in the resistant P9830 line while five non-functional copies were identified in the susceptible line. The resistant parents and their recombinant inbred lines carrying either ARG4 or ARG5 are resistant to strains Csgl1 and Csgrg suggesting that these genes have overlapping specificities. The role of ARG4 and ARG5 in resistance was validated through sorghum lines carrying independent recessive alleles that show increased susceptibility. ARG4 and ARG5 are located within complex loci displaying interesting haplotype structures and copy number variation that may have resulted from duplication. Overall, the identification of anthracnose resistance genes with unique haplotype stucture provides a foundation for genetic studies and resistance breeding.

ANTHRACNOSE RESISTANCE GENE2 confers fungal resistance in sorghum.

Sorghum is an important food and feed crop globally; its production is hampered by anthracnose disease caused by the fungal pathogen Colletotrichum sublineola (Cs). Here, we report identification and characterization of ANTHRACNOSE RESISTANCE GENE 2 (ARG2) encoding a nucleotide-binding leucine-rich repeat (NLR) protein that confers race-specific resistance to Cs strains. ARG2 is one of a cluster of several NLR genes initially identified in the sorghum differential line SC328C that is resistant to some Cs strains. This cluster shows structural and copy number variations in different sorghum genotypes. Different sorghum lines carrying independent ARG2 alleles provided the genetic validation for the identity of the ARG2 gene. ARG2 expression is induced by Cs, and chitin induces ARG2 expression in resistant but not in susceptible lines. ARG2-mediated resistance is accompanied by higher expression of defense and secondary metabolite genes at early stages of infection, and anthocyanin and zeatin metabolisms are upregulated in resistant plants. Interestingly, ARG2 localizes to the plasma membrane when transiently expressed in Nicotiana benthamiana. Importantly, ARG2 plants produced higher shoot dry matter than near-isogenic lines carrying the susceptible allele suggesting an absence of an ARG2 associated growth trade-off. Furthermore, ARG2-mediated resistance is stable at a wide range of temperatures. Our observations open avenues for resistance breeding and for dissecting mechanisms of resistance.

551. Burden of Illness in Carbapenem-Resistant Acinetobacter baumannii Infections in US Hospitals (2014 to 2018)

BackgroundInfections caused by Acinetobacter baumannii present a challenge for treating physicians due to the high level of antimicrobial resistance. The current analysis compared the burden of illness in patients infected with carbapenem-resistant (CR) vs. -susceptible (CS) strains of A. baumannii.MethodsHospitalized adult patients with microbiologically confirmed A. baumannii infections (defined as a positive culture and receipt of antibiotics between 2 days prior to 3 days after the culture) included in the Premier Healthcare Database were retrospectively evaluated. Patient characteristics including demographics, comorbidities, time of infection onset and site of infection were assessed. Comparative outcomes between CR and CS patients assessed included in-hospital mortality, length of hospital stay (LOS), ICU LOS, and discharge status. Outcomes were also stratified by site of infection.ResultsA total of 3,471 patients admitted between January 1, 2014 and June 30, 2018 were included. Patients with CR strains of A. baumannii were older (62 vs. 59 years), more likely to have Charlson Comorbidity Index ≥3 (63.4% vs. 56.1%), more likely admitted from a healthcare origin (30.3% vs. 11.4%) and less likely to have the onset of infection within first 48 hours of hospitalization (58% vs. 69%) than those with CS strains. CR patients had increased inpatient mortality compared with CS patients (16.3% vs. 11.0%), driven primarily by patients with bloodstream infections (42.6% vs. 12.4%, respectively, P < 0.001). CR patients had a non-significantly increased median overall LOS from the onset of infection (9 vs. 8 days, P = 0.068), were more likely to be admitted to the ICU, and were significantly less likely to be discharged home (16% vs. 47%, P < 0.001). Hospitalization charges were considerably higher for CR patients (table). Readmission rates were high among CR patients and were similar to patients with CS infections.ConclusionPatients with CR strains of A. baumannii face a greater burden of illness compared with CS patients, experiencing increased mortality, ICU admission and LOS, and incur higher hospitalization charges. Furthermore, CR patients were less likely to be discharged home after admission. Disclosures All authors: No reported disclosures.

Evaluation of colistin stability in agar and comparison of four methods for MIC testing of colistin

Susceptibility testing for colistin remains challenging primarily due to its inherent properties. We evaluated colistin stability in agar and reproducibility of colistin MICs obtained by agar dilution, broth macro- and micro-dilution and MIC gradient strips on 3–7 iterations of each method using clinical Klebsiella pneumoniae (susceptible-CS, and resistant-CR, n = 2 each), mcr-harboring Escherichia coli (n = 2), and reference strains E. coli ATCC25922 and Pseudomonas aeruginosa ATCC27853. MICs for reference strains were not in the given range using Etest and broth microdilution (ATCC25922, 0.125 and 4 μg/ml, respectively). MICs of CR-1 and CR-2, and of the mcr-harboring E. coli showed high concordance between agar and broth dilution varying up to one 2-fold dilution. However, remarkable variations were observed on broth dilution with CS-1 and CS-2 (MIC range 0.25–32 and 0.5–64 μg/ml, respectively); whereas for agar dilution the MIC for both CS strains was 0.5 μg/ml in all the runs. MICs obtained by MIC gradient strips were lower than those obtained by dilution methods (1–2 dilutions for CS and mcr strains, and up to five dilutions for CR strains). To confirm uniform distribution of colistin in agar, a single strain was spotted in five different regions of the same plate. All spots showed concordant growth with maximum one dilution difference. No effect on MIC was found due to storage of colistin-containing agar plates for 7 days at 4 °C. In our hands, agar dilution was superior in terms of reproducibility and robustness, compared to broth dilution methods, for colistin MIC determination.

Carboxylesterase genes in pyrethroid resistant house flies, Musca domestica

Carboxylesterases are one of the major enzyme families involved in the detoxification of pyrethroids. Up-regulation of carboxylesterase genes is thought to be a major component of insecticide resistant mechanisms in insects. Based on the house fly transcriptome and genome database, a total of 39 carboxylesterase genes of different functional clades have been identified in house flies. In this study, eleven of these genes were found to be significantly overexpressed in the resistant ALHF house fly strain compared with susceptible aabys and wild-type CS strains. Eight up-regulated carboxylesterase genes with their expression levels were further induced to a higher level in response to permethrin treatments, indicating that constitutive and inductive overexpression of carboxylesterases are co-responsible for the enhanced detoxification of insecticides. Spatial expression studies revealed these up-regulated genes to be abundantly distributed in fat bodies and genetically mapped on autosome 2 or 3 of house flies, and their expression could be regulated by factors on autosome 1, 2 and 5. Taken together, these results demonstrate that multiple carboxylesterase genes are co-upregulated in resistant house flies, providing further evidence for their involvement in the detoxification of insecticides and development of insecticide resistance.

Tetracycline Selective Pressure and Homologous Recombination Shape the Evolution of Chlamydia suis: A Recently Identified Zoonotic Pathogen.

Species closely related to the human pathogen Chlamydia trachomatis (Ct) have recently been found to cause zoonotic infections, posing a public health threat especially in the case of tetracycline resistant Chlamydia suis (Cs) strains. These strains acquired a tet(C)-containing cassette via horizontal gene transfer (HGT). Genomes of 11 Cs strains from various tissues were sequenced to reconstruct evolutionary pathway(s) for tet(C) HGT. Cs had the highest recombination rate of Chlamydia species studied to date. Admixture occurred among Cs strains and with Chlamydia muridarum but not with Ct. Although in vitro tet(C) cassette exchange with Ct has been documented, in vivo evidence may require examining human samples from Ct and Cs co-infected sites. Molecular-clock dating indicated that ancestral clades of resistant Cs strains predated the 1947 discovery of tetracycline, which was subsequently used in animal feed. The cassette likely spread throughout Cs strains by homologous recombination after acquisition from an external source, and our analysis suggests Betaproteobacteria as the origin. Selective pressure from tetracycline may be responsible for recent bottlenecks in Cs populations. Since tetracycline is an important antibiotic for treating Ct, zoonotic infections at mutual sites of infection indicate the possibility for cassette transfer and major public health repercussions.

Rovac is the possible ancestor of the Russian lapinized vaccines LK-VNIVViM and CS strains but not the Chinese strain (C-strain) vaccine against classical swine fever

Classical swine fever (CSF), or hog cholera, is a highly contagious disease that emerged in the first half of the nineteenth century. To fight against the disease and protect pigs, different vaccines were developed, including early generation of lapinized Rovac strain and the later development of the “Chinese” strain (C-strain). However, details of the development of these vaccines are lost in history. In order to investigate the phylogenetic relationship between the Rovac and other lapinized vaccines, this study determined the genome sequence of the Rovac, which comprised 12,304 nucleotides, notably with the 3′untranslated region (3′UTR) containing a 13-nucleotide insertion. The near-complete genome of Russian vaccine strain LK-VNIVViM was determined by next-generation sequencing on Illumina MiSeq platform. Whole genome phylogenetic analysis revealed a closer relationship of the Rovac strain with the Russian LK-VNIVViM, CS strain and its derivative RUCSFPLUM (genotype 1.2), rather than with the C-strain (genotype 1.1). In addition, it demonstrated an ancestry role of the LK-VNIVViM in relation to the CS strain and RUCSFPLUM. The study suggested that the Rovac vaccine is the possible ancestor of the Russian vaccine strains but not the C-strain vaccine.

Assessment of Toxicity and Potential Risk of Butene-fipronil Using Drosophila melanogaster, in Comparison to Nine Conventional Insecticides

The toxicities of butene-fipronil to Drosophila melanogaster were evaluated, in comparison to nine conventional insecticides. According to the mean LD50 values of the larvae from CS, w(1118) and Oregan strains, butene-fipronil,abamectin, spinosad and chlorpyrifos exhibited high levels of toxicity. Imidacloprid, α-cypermethrin and clothianidin showed middle levels of toxicity. And acephate,methomyl and acetamiprid had low levels of toxicity. To the adults, butene-fipronil, α-cypermethrin, spinosad, and chlorpyrifos were relatively more toxic, whereas acephate,methomyl, acetamiprid, imidacloprid, clothianidin and abamectin were relatively less toxic. Butene-fipronil and abamectin were more toxic to larvae. In contrast, spinosad,a-cypermethrin, and clothianidin were more toxic to adults.Moreover, the toxic risk assessment using a risk quotient value revealed that butene-fipronil was safe to D. melanogaster adults. Thus, our results suggested that butene-fipronil is harmful to D. melanogaster larvae, but it is relatively safe to the adults.

Genetic Background Specific Hypoxia Resistance in Rat is Correlated with Balanced Activation of a Cross-Chromosomal Genetic Network Centering on Physiological Homeostasis

Genetic background of an individual can drastically influence an organism’s response upon environmental stress and pathological stimulus. Previous studies in inbred rats showed that compared to Brown Norway (BN), Dahl salt-sensitive (SS) rat exerts strong hypoxia susceptibility. However, despite extensive narrow-down approaches via the chromosome substitution methodology, this genome-based physiological predisposition could not be traced back to distinct quantitative trait loci. Upon the completion and public data availability of PhysGen SS-BN consomic (CS) rat platform, I employed systems biology approach attempting to further our understanding of the molecular basis of genetic background effect in light of hypoxia response. I analyzed the physiological screening data of 22 CS rat strains under normoxia and 2-weeks of hypoxia, and cross-compared them to the parental strains. The analyses showed that SS-9BN and SS-18BN represent the most hypoxia-resistant CS strains with phenotype similar to BN, whereas SS-6BN and SS-YBN segregated to the direction of SS. A meta-analysis on the transcriptomic profiles of these CS rat strains under hypoxia treatment showed that although polymorphisms on the substituted BN chromosomes could be directly involved in hypoxia resistance, this seems to be embedded in a more complex trans-chromosomal genetic regulatory network. Via information theory based modeling approach, this hypoxia relevant core genetic network was reverse engineered. Network analyses showed that the protective effects of BN chromosome 9 and 18 were reflected by a balanced activation of this core network centering on physiological homeostasis. Presumably, it is the system robustness constituted on such differential network activation that acts as hypoxia response modifier. Understanding of the intrinsic link between the individual genetic background and the network robustness will set a basis in the current scientific efforts toward personalized medicine.

Molecular epidemiology of classical swine fever in the Russian Federation.

The ability to discriminate between various classical swine fever virus (CSFV) strains and isolates is a prerequisite for following the spread of the virus after an outbreak. To determine the relatedness between Russian CSFV isolates from different geographical regions, three fragments of the viral genome (5' NTR, the variable region of the E2 gene and a fragment of the NS5B gene) were sequenced and used for genetic typing. Thirty-one field isolates were obtained from CSF outbreaks which occurred between 1994 and 1999. In addition, three attenuated strains were included in the study, namely the LK and CS vaccine strains, and the moderately virulent 238H isolate. The vaccine strains have been used in Russia for more than 30 years. Our results showed that all field isolates are in subgroup 1.1 together with Alfort 187 and with the highly virulent strain Shimen. In contrast, the CS and LK vaccine strains belong to subgroup 1.2. While there is no evidence for the reversion of the two vaccine strains to wild type, it is feasible that the highly virulent Shimen strain, which has been used as a challenge strain for many years, contributed to field strain generation. The Russian field isolates from the 1990s can be distinguished from the CSF virus isolates which occurred in the EU Member States in the same decade, as here all outbreaks were caused by CSF viruses belonging to subgroup 2.

Expression and regulation of CYP6D3 in the house fly, Musca domestica (L.)

Recently, a new cytochrome P450 gene, CYP6D3, was identified from house fly. CYP6D3 was found upstream of a related gene ( CYP6D1) on autosome 1. CYP6D3 cDNA sequences were obtained and compared from insecticide resistant (LPR) and susceptible (CS and Edinburgh) strains. Although each strain had a different CYP6D3 allele, the deduced amino acid sequences revealed no consistent differences between the susceptible and resistant strains. There was approximately 12-fold more CYP6D3 mRNA detected in adult LPR flies compared to CS, and the elevated level of expression in LPR was not due to gene amplification. Northern blots indicate expression of CYP6D3 mRNA is developmentally regulated with no expression in eggs, yet it is readily detectable in larvae as well as male and female adults. Phenobarbital is a well studied inducer of P450s in insects and it induced expression of CYP6D3 mRNA in both the CS (16-fold) and LPR (1.6 fold) strains. The CYP6D3 5′ flanking regions were sequenced from the resistant and susceptible strains. Possible regulatory sequences within this region are discussed.

Genetic complementation groups in cockayne syndrome.

Skin fibroblasts from patients with cockayne syndrome (CS cell) exhibited marked ultraviolet (UV) sensitivity as measured by colony-forming ability. Further, recovery of semiconservative DNA synthesis following UV irradiation was absent in CS cells, as it is in xeroderma pigmentosum cells. We found that the rate of semiconservative DNA synthesis measured at 12 h after 12 J/m2 of UV irradiation had recovered to nearly normal levels in binuclear cells obtained by the fusion of CS strains CS3BE (GM 1856) and CS7SE (GM1428) and of CS3BE (GM1856) and CS1BE (GM1629), but not of C57SE (GM1428) and CS1BE (GM1629). These results indicate that there are at least two genetic complementation groups in CS.

Mitochondrial genetics. XI. Mutations at the mitochondrial locus omega affecting the recombination of mitochondrial genes in Saccharomyces cerevisiae.

1. A series of CS revertants has been selected from various strains (both omega+ and omega-) carrying a CR mitochondrial mutation at the RIB1 locus. The properties of mitochondrial recombination exhibited by these CS revertants in various crosses, have been examined systematically. The omega allele of the CS revertants has been defined in crosses with omega+ and omega- tester strains using two criteria: the polarity of recombination and a new criterium called relative output coefficient. We found that mutations of omega appear frequently associated with the mutations at the RIB1 locus selected from omega- strains but not with those selected from omega+ strains. A new allelic form of omega (omega n) which had not been found amongst wild type yeast strains is characterised. Similarly omega n mutation was found frequently associated with CR mutants at the RIB1 locus selected from omega- CS strains but not with those selected from omega+ CS strains. The omega n mutants, and the omega+ and omega- strains, explain the groups of polarity previously observed by Coen et al. (1970). 2. Main features of mitochondrial crosses with omega n strains (omega+ x omega n, omega- x omega n and omega n x omega n) are analysed. Recombination is possible between the different mitochondrial genetic markers. No high polarity of recombination is observed and the frequency of recombinants are similar to those found in homosexual crosses (omega+ x omega+ and omega- x omega-). A striking property, observed for the first time, exists in crosses between zota+ omega n CS strains and some zota- CREO mutants: the zota- CREO are unable to integrate by recombination their CR allele into the zota+ mit-DNA of omega n CS strains while being capable of integrating it into omega+ CS or omega- CS genomes. 3. It is proposed that the omega locus is the site of initiation of non reciprocal recombination events, the omega+/omega- pairing specifically initiates the non-reciprocal act while omega+/omega n or omega-/omega n pairings do not. 4. The molecular nature of the omega n mutation and its bearing on the structure of the omega locus are discussed. It is suggested that omega n mutations correspond to macrolesions (probably deletions) of a segment of the mit-DNA covering the omega and RIB1 loci. If omega n is a partial deletions of the omega- sequence the omega+ could be an additionnal deletion of the omega n sequence. 5. The occurrence of spontaneous CR and ER mitochondrial mutations has been analysed by the Luria and Delbrück fluctuation test in omega- and omega n isonuclear strains. Results of these tests indicate that an intracellular selection of resistant copies preexisting the action of the anttibiotic occurs.