Antibiotic resistance and the presence of bla CfxA and bla CSP genes in β-lactamase-producing clinical Capnocytophaga isolates from a university hospital in Japan

Introduction . Capnocytophaga species are common inhabitants of the oral cavity and can be responsible for systemic diseases in immunocompromised patients with granulocytopenia. Furthermore, it has been reported that some clinical isolates of Capnocytophaga species produce extended-spectrum β-lactamases (ESBLs). Gap statement. Information is lacking about the types of β-lactamase genes possessed by Capnocytophaga spp. and the antimicrobial susceptibility of Capnocytophaga spp. possessing each β-lactamase gene. Aim. The aim of this study was to investigate the presence of β-lactamase genes in clinical strains of β-lactamase-producing Capnocytophaga species isolated from clinical samples acquired at Shinshu University Hospital and examine the antimicrobial susceptibility of those strains. Methodology. The β-lactamase-producing Capnocytophaga species (n=49) were obtained from clinical specimens. PCR assays were used to detect bla CfxA, bla CSP, bla TEM, bla CepA/CblA and transposon Tn4555 genes. Southern hybridization assays were used to detect bla CfxA and bla CSP. The minimum inhibitory concentration of some β-lactams was determined using the E-test method. Results. PCR analysis indicated that the bla CfxA gene was present in 15 (30.6 %) and the bla CSP gene in 35 (69.3 %) of the 49 Capnocytophaga strains investigated, . Both bla CfxA and bla CSP genes were detected in a Capnocytophaga gingivalis strain. The PCR results were confirmed by Southern hybridization assays. Transposon Tn4555 was only detected in Capnocytophaga spp. harbouring the bla CfxA gene. All the β-lactamase-producing Capnocytophaga isolates were susceptible to ceftazidime–clavulanic acid, cefoxitin and imipenem. In contrast, most of the isolates were resistant to amoxicillin. Conclusions. The clinical isolates of Capnocytophaga spp. showed a high prevalence of the bla CSP gene in Japan. The presence of the bla CSP gene was distributed in Capnocytophaga sputigena as well as other Capnocytophaga spp. These results seem to suggest the dissemination of bla CfxA and bla CSP β-lactamase genes among Capnocytophaga species.

Large-scale comparative analysis of cytogenetic markers across Lepidoptera

Fluorescence in situ hybridization (FISH) allows identification of particular chromosomes and their rearrangements. Using FISH with signal enhancement via antibody amplification and enzymatically catalysed reporter deposition, we evaluated applicability of universal cytogenetic markers, namely 18S and 5S rDNA genes, U1 and U2 snRNA genes, and histone H3 genes, in the study of the karyotype evolution in moths and butterflies. Major rDNA underwent rather erratic evolution, which does not always reflect chromosomal changes. In contrast, the hybridization pattern of histone H3 genes was well conserved, reflecting the stable organisation of lepidopteran genomes. Unlike 5S rDNA and U1 and U2 snRNA genes which we failed to detect, except for 5S rDNA in a few representatives of early diverging lepidopteran lineages. To explain the negative FISH results, we used quantitative PCR and Southern hybridization to estimate the copy number and organization of the studied genes in selected species. The results suggested that their detection was hampered by long spacers between the genes and/or their scattered distribution. Our results question homology of 5S rDNA and U1 and U2 snRNA loci in comparative studies. We recommend the use of histone H3 in studies of karyotype evolution.

Controlled Over-Expression of AtDREB1A Enhances Tolerance against Drought and Salinity in Rice

Engineering transcription factors (TF) hold promise in enhancing abiotic stress tolerance in plants. In this study, one of the popular rice varieties of South India, namely ADT 43, was engineered with a TF AtDREB1A driven by a stress-inducible rd29A promoter. PCR and Southern hybridization were employed to confirm the integration and copy number of the transgene. Transgenic lines (T1) of ADT 43 showed enhanced tolerance to drought and salinity compared to the non-transgenic ADT 43. Transgenic lines were found to maintain higher RWC %, lower leaf temperature, and partially closed stomata, enabling better survival under stress conditions. qRT-PCR analysis revealed the strong induction of AtDREB1A transcripts during drought. Transgenic lines of ADT 43 exhibited increased germination and retention of chlorophyll in their leaves under salinity. Evaluation of transgenic lines under transgenic screen house conditions revealed that line # A16 exhibited on par agronomic performance against its non-transgenic counterpart under normal conditions. Under drought, non-transgenic ADT 43 showed >20% reduction in the total number of spikelets per panicle, whereas transgenic line # A16 registered only a 2% reduction. Non-transgenic ADT 43 recorded 80% yield reduction under drought, whereas line # A16 recorded only 54% yield loss. The above results demonstrated the effectiveness of controlled expression of DREB1A in regulating dehydration responses in rice.

Bhendi yellow vein mosaic virus and bhendi yellow vein mosaic betasatellite cause enation leaf curl disease and alter host phytochemical contents in okra

Whitefly-transmitted begomoviruses cause severe diseases in numerous economically important dicotyledonous plants. In recent years, okra enation leaf curl disease (OELCuD) emerged as a serious threat to okra (Abelmoschus esculentus L. Moench) cultivation in the Indian subcontinent. The present study reports the association of a monopartite begomovirus (bhendi yellow vein mosaic virus - BYVMV) and betasatellite (bhendi yellow vein mosaic betasatellite - BYVB) with OELCuD in the Mau region of Uttar Pradesh, India. The BYVMV alone inoculated N benthamiana and A esculentus cv. Pusa Sawani plants developed mild symptoms. Co-inoculation of BYVMV and BYVB resulted in a reduced incubation period, an increased symptom severity and an enhanced BYVMV accumulation (by Southern hybridization and qPCR). This is the first study which satisfies Koch’s postulates for OELCuD in its natural host. Activities of various antioxidative enzymes were significantly increased in the virus inoculated okra plants. Differential responses in various biochemical components (such as photosynthetic pigments, phenol, proline, sugar) in diseased okra plants were observed. This change in phytochemical responses is of significant importance in understanding its impact on virus pathogenesis and disease development.

Engineering Chickpea Variety HC-1 with OsRuvB Gene for Salt Stress Tolerance

Background: Salinity is a major problem worldwide and is increasing day by day. Salt stress causes severe yield losses in crop plants and the damages in chickpea are up to 100%. To overcome these losses, the present study was undertaken to develop transgenic chickpea plants (var. HC-1) carrying OsRuvB gene for salt stress tolerance. Methods: Transgenic chickpea plants harboring OsRuvB gene were developed using Agrobacterium-mediated transformation. T0 putative transgenic chickpea plants were screened for the presence of OsRuvB gene through PCR using gene specific primers. The stable integration and copy number of transgene in transgenic chickpea plants were confirmed through Southern hybridization and qRT-PCR. T1 generation transgenic chickpea plants were screened for the presence of OsRuvB gene using direct PCR (Phire Direct PCR kit). Result: PCR-based screening of putative transformants using gene-specific primers showed a transformation frequency of 17%. Southern blot and real-time PCR analysis revealed stable and single-copy insertion. In T1 generation a total of 74 plants (out of 170) showed the presence of OsRuvB gene. The engineered lines developed in the present investigation can be further undertaken to develop transgenic chickpea plants for salt stress tolerance.

Molecular characterization of submergence tolerance genes and locus in the deep-water rice cultivars

Most of the rice cultivars exhibit suspension of growth when submerged to overcome the reduced availability of oxygen. When the situation continues, majority of the cultivars unable to recover after the flood recedes. However, there are fortunately some rice genotypes that can withstand such submerged condition for up to two weeks by adapting two totally opposite mechanisms. One type of cultivars elongates enormously at a very short span of time and the leaves come above the water level. In the second type, they remain under water without any growth. Cultivars of both types tolerate the submergence but the first category easily lodges when flood water recede. In those lines, yields are reduced drastically. In this study, we focus on characterize the genetic variation at the Sub1 locus and to associate its relevance, if any, to submergence tolerance among the deep water landraces. As a first step, seeds of some rice cultivars collected from North-east Indian regions were initially selected for the characterization of genetic variation. The PCR based analysis involving several genes known to be associated with submergence tolerance did not reveal much difference. However, Southern hybridization revealed certain differences between submergence tolerant and susceptible cultivars. Although we did not notice major difference with regard to Sub1 genes when tried with EcoRI and BamHI, differences were noticed with adh1 and RAmy3C genes. Representative, Southern analysis showed the genetic variation among the deep-water cultivars as compared to Swarna and Sub1-Swarna. It is possible that deep-water rice cultivars may not differ in their genome at Sub1 locus but they respond through SNORKEL genes under submergence.

549. First Report for Emergence of Chromosomal Borne Colistin Resistance Gene mcr-1 in a Clinical Acinetobacter Baumannii Isolates from India

Background Efficacy of Colistin the last line agent against infections due to multidrug-resistant (MDR) gram-negative pathogens, has been challenged when Liu et al. reported a plasmid-mediated gene, mcr-1, in 2015. Thereby this plasmid-borne mcr has been reported in bacterial isolates worldwide taken from humans, animals, farms, foods, and the environment. The present work invesitigate the mcr gene among clinical isolates of Acinetobacter Baumannii at our tertiary referral hospital of India. Methods The study was conducted at Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India. MIC values for 100 consecutive non-duplicate MDR isolates of Acinetobacter were checked for Colistin. PCR amplification of mcr gene was performed followed by sequencing of the amplicons. Clinical features of patients infected with mcr positive isolates were unveiled. Clonal relatedness of these isolates was investigated by Pulsed-field gel electrophoresis (PFGE). The mcr-1 localization was checked by conjugation followed by PFGE southern hybridization. Results 20/100 (20%) isolates were colistin resistant with having MIC Values of more than 8≤ µg/mL. The 20 colistin resistances isolates were PCR positive for mcr-1 and had been assigned EMBL/GeneBank nucleotide accession numbers MH730099-MH730118. Oher antibiotic resistance gene like ESBL, NDM-1, VIM, and 16s rRNA methyl transferases like Arm A, rmtC, rmt F were also found in these isolates. Majority of these patients recovered from the infection (65%) after proper antibiotic therapy.The ISApl1 transposable elements were not detected in these isolates. These isolates were found clonally unrelated when analyze by pulsed-field gel electrophoresis. The conjugation attempt to transfer mcr-1 to recipient’s E. coli J53 failed, Southern hybridization showed that mcr-1 was found located on chromosome in multiple copies. Conclusion This is the first case of mcr-1 in a human clinical isolate in Acinetobacter Baumnnii from India. These findings highlight the vertical transferability of colistin resistance by mcr-1 gene in Acinetobacter Baumnnii with the association of known some unknown insertion sequence located on chromosome. Strategies required to contain their spread and evolution of such genes. Disclosures All authors: No reported disclosures.

Emergence of a hybrid plasmid derived from IncN1-F33:A−:B− and mcr-1-bearing plasmids mediated by IS26

Objectives To characterize the complete sequences of four plasmids in MCR-1-producing clinical Escherichia coli strain D72, and to depict the formation mechanism and characteristics of the cointegrate plasmid derived from the pD72-mcr1 and pD72-F33 plasmids. Methods The genetic profiles of plasmids in strain D72 and its transconjugant were determined by conjugation, S1-PFGE, Southern hybridization, WGS analysis and PCR. Plasmid sequences were analysed with bioinformatic tools. The traits of the fusion plasmid were characterized by cointegration, stability and conjugation assays. Results Strain D72, belonging to ST1114, contained four plasmids, including mcr-1-carrying pD72-mcr1, blaCTX-M-55-carrying pD72-F33, blaTEM-238-bearing pD72-IncP and pD72-IncX1 carrying aph(3′)-Ia, qnrS2 and floR. A single plasmid, pD72C, in the transconjugant was found to be larger than any plasmid in the original strain D72. Sequence analysis showed that pD72C was the fusion product of pD72-mcr1 and pD72-F33, and the recombinant event involved an intermolecular replicative mechanism. Plasmid fusion occurred at a frequency of 1.75 × 10−4 cointegrates per transconjugant. The fusion plasmid presented a high stability and conjugation frequency of 8.00 × 10−3. Conclusions To our knowledge, this is the first report of the IS26-mediated fusion of an IncN1-F33:A−:B− plasmid and an mcr-1-carrying phage-like plasmid, providing evidence for the important role of IS26 in the recombination of plasmids. The biological advantages of the fusion plasmid indicated that the fusion event presumably plays a potential role in the dissemination of mcr-1.

Molecular Polymorphism of Pectinase Genes PGU of the Yeast Saccharomyces bayanus var. uvarum

We have conducted a molecular genetic study of the pectinase PGU genes of 74 strains of the yeast Saccharomyces bayanus var. uvarum, isolated from various fermentation processes and natural sources in different regions of Europe and in the USA. Unlike S. cerevisiae, each having a PGU gene, strains of S. bayanus var. uvarum have three divergent genes PGU1b, PGU2b and PGU3b, located respectively on chromosomes X, I and XIV. The high pectinolytic activity of these yeasts appears to be related to the presence of several PGU polymeric genes in their genome. Saccharomyces bayanus var. uvarum, endo-polygalacturonase, yeast pectinase, genes PGU1b, PGU2b and PGU3b, molecular karyotyping, Southern-hybridization This study was supported by Budget-supported project No. 595-00004-18PR. doi: 10.21519/0234-2758-2019-35-2-30-37