Adaptation of IncX3 plasmid encoding blaNDM-4 within broad host range: A study from India

Background: Different variants of blaNDM have been reported across Indian subcontinent within diverse host range of gram negative bacilli. Most of their transferability depends on types of plasmid that encodes resistance determinant. These plasmids with external sub-inhibitory stress of antibiotics facilitate their propagation and maintenance within broad host range. IncX type plasmid is more recently known for carrying carbapenem resistance and NDM-4 variant is considered to have better hydrolytic property than other variants. Both of these factors interferes with therapeutic outcome in clinical setting. The current study was aimed to investigate the maintenance and stability of blaNDM-4 within broad host range and transcriptional response.Results: IncX3 plasmid encoding blaNDM-4 was successfully transferred in six different host when imipenem (0.5µg/ml) screen agar was used for selection of transformants. It was also found to coharbour resistance for aminoglycosides and quinolone. When checked for stability, it was observed that the plasmid was successfully expanded within all the six recipients for 55th serial passages. Transcriptional expression with IncX3 was random but at consistent level for wild type and without concentration gradient stress of imipenem. Transcriptional expression with NDM gene was variable for parent isolates though for new hosts it was showing randomly increased pattern in proteus, E.coli, and DH5α Conclusion: the present study could highlight that external carbapenem pressure helps in maintenance and expression of blaNDM-4 within different host range. This study is of epidemiological significance and will help in tracking the genetic vehicle responsible for their transmission theirby restricting their spread.

Adaptation of IncX3 plasmid encoding blaNDM-4 within broad host range: A study from India

Different variants of blaNDM has been reported across Indian subcontinent within diverse host range of gram negative bacilli. Most of their transferability depends on types of plasmid that encodes resistance determinant. IncX type plasmid is more recently known for carrying carbapenem resistance and NDM-4 variant is considered to have better hydrolytic property. This study aims to investigate the maintenance and stability of blaNDM-4 within BHR and transcriptional response. E.coli of clinical origin that harbor blaNDM-4 within IncX3 plasmid were used. Six different hosts were selected for gene transfer experiment, two strains of E.coli, K.pneumoniae, P.mirabilis, A.baumannii and P.aeruginosa. All the transformants and parent isolate were subjected for stability within new host. IncX3 plasmid encoding blaNDM-4 was transferred in host when imipenem (0.5µg/ml) screen agar was used for selection of transformants which are coharbouring resistance for aminoglycosides and quinolone. Plasmid was successfully expanded within all the six recipients till 55th serial passages. Transcriptional expression with NDM gene was variable for parent isolates though for new hosts it was showing random increased pattern in Proteus, E.coli, and DH5α. This could highlight, external carbapenem pressure helps in maintenance and expression of blaNDM-4 within different host range and epidemiological significance and will help in tracking the genetic vehicle responsible for their transmission,restricting their spread.

Genetic Transformation of Musa acuminata cv. Vaibalhla (AAA) using Agrobacterium tumefaciens

Agrobacterium-mediated genetic transformation of Musa acuminata cv. Vaibalhla (AAA) was performed using Agrobacterium tumefaciens strain EHA105 harboring pCAMBIA2301AtNHX1 plasmid. For the transformation efficiency assay, the un-and pre-cultured immature male flower explants were subjected to various methods of injury such as hypodermal needle injury, with and without sonication and vacuum infiltration. The explants were then inoculated in bacterial suspension by occasional shaking for 30 minutes. After inoculation, explants were co-cultivated for 3 days in dark condition at 22° C in a liquid MS basal medium supplemented with 100 µM acetosyringone. For selection of transformants, the treated explants were cultured on Murashige and Skoog (MS) medium supplemented with BAP (2 mg/L), NAA (0.5 mg/L) and ascorbic acid (75 mg/L) along with antibiotics kanamycin (100 mg/L), cefotaxime (300 mg/L) and augmentin (300 mg/L).The putative transformation was analyzed using histochemical GUS assay. 14 and 21 days pre-cultured male flower explants subjected to 4-5 needle point injury resulted in 93.33% and 100% putative transformation respectively. 30s sonication combined with 5 minutes of vacuum infiltration for 7, 14 and 21 days pre-cultured immature male flower explants gave 73.33%, 66.66% and 71.42% putative transformation respectively.

Strategy for In Situ Detection of Natural Transformation-Based Horizontal Gene Transfer Events

ABSTRACT A strategy is described that enables the in situ detection of natural transformation in Acinetobacter baylyi BD413 by the expression of a green fluorescent protein. Microscale detection of bacterial transformants growing on plant tissues was shown by fluorescence microscopy and indicated that cultivation-based selection of transformants on antibiotic-containing agar plates underestimates transformation frequencies.

Deletion and Allelic Exchange of the Aspergillus fumigatus veA Locus via a Novel Recyclable Marker Module

ABSTRACT Detailed evaluation of gene functions in an asexual fungus requires advanced methods of molecular biology. For the generation of targeted gene deletions in the opportunistic pathogen Aspergillus fumigatus we designed a novel blaster module allowing dominant selection of transformants due to resistance to phleomycin as well as dominant (counter)selection of a Cre recombinase-mediated marker excision event. For validation purposes we have deleted the A. fumigatus pabaA gene in a wild-type isolate by making use of this cassette. The resulting pabaA::loxP strain served as the recipient for subsequent targeting of the velvet locus. Homologous reconstitution of the deleted gene was performed by an allele whose expression is driven in a nitrogen source-dependent manner, as validated by Northern analyses. Overexpression of the veA locus in A. fumigatus does not result in any obvious phenotype, whereas the sporulation capacities of the veA null mutant are reduced on nitrate-containing medium, a phenotype that is completely restored in the reconstituted strain.