Plasmid Size Research Articles

Screening of Metal and Antibiotic Resistance in Beta-lactamase Producing Coliform Bacteria from Hospital Wastewater of Northern India.

Our exploration work has uncovered the different anti-toxin/metal tolerance and patterns against the heavy metal resistant coliform microscopic organisms from the aquatic waste of the hospital. It might give new routes for the treatment of irresistible ailments particularly by coliform and critical for hazard evaluation as well as hazard management associated with the effluents of the hospital. The higher use of pharmaceuticals, Radionuclides, and other antimicrobial solvents are the major source of metals in hospital wastewater. The hospital aquatic environment has a high content of both organic and inorganic matter with living organisms. Bacteria can resist an antimicrobial agent by producing extracellular enzymes that eliminate antibiotics and metal toxicity. In this study, we covered the existing patent literature in this area. New patents in the areas of topically applied antibiotics and agents that can potentiate the achievement of existing antibiotics may extend their helpful lifetime. Samples were collected from three different Departments of King George Medical University, Lucknow during the month of December to May (2015-16). Isolation and metal tolerance of coliform isolates were done on metal amended plates. The antibiotic sensitivity test was done by disc diffusion method. The plasmid DNA of bacterial isolates was done by the alkaline lysis method. The conjugation study was also performed in wastewater as well as a nutrient medium. Maximum isolates demonstrated their MICs at 400, 800 and 1600 μg/ml against all the metals, respectively. The high level of resistance was observed against Methicillin (88.32%, 80.60%) followed by penicillin (75%, 76%), Cephradin (59.52%, 28.84%) and least to Gentamycine (1.92%, 5.76) in E. coli and Enterobacter, respectively. Of 70%, 78% E. coli and Enterobacter isolates produce beta-lactamase activity. Six amino acid residues namely, Glu104, Tyr105, Asn132, Asn170, Ala237, and Gly238 of the beta-lactamase were found in the common interaction with the selected drugs. Plasmid DNA size ranged between 48-58.8 kb. The conjugation experiments showed a higher transfer frequency (5.5×10-1 and 3.6×10-1) rate among antibiotics and metals tested. The finding of this study presents a potential health problem as the predominant coliform species have increasingly been associated with outbreaks of hospital infections. It is recommended that hospital waste must be properly treated before its release into the environment.

Antimicrobial resistance and phenotypic and molecular detection of extended-spectrum β-lactamases among extraintestinal Escherichiacoli isolated from pneumonic and septicemic sheep and goats in Rajasthan, India

The present study was planned to isolate Escherichia coli from pneumonic and septicemic sheep and goats and to determine antimicrobial resistance (AMR) patterns of the isolates and evaluate them for the presence of extended-spectrum s-lactamase (ESBL) using the phenotypic and molecular methods. Pneumonic lung tissues and heart blood samples were collected by performing necropsy on sheep (n = 96) and goats (n = 08). The samples were processed and used for bacterial isolation. E. coli isolates (n = 58) including 53 from 34 sheep and 5 from 3 goats were recovered and identified by the cultural and biochemical characteristics and 16S rRNA sequencing. The isolates were tested to ascertain AMR pattern, plasmid features, and ESBL production. The highest rate of resistance (65.5%) was recorded against amoxicillin, enrofloxacin, norfloxacin, and ofloxacin. Eighteen (31%) out of 58 isolates showed the ESBL phenotype by combination disk assay. There was a variability in the number (1-5) and size (2 to >20 kb) of plasmids among isolates. Polymerase chain reaction (PCR) amplification revealed the presence of blaH$_{TEM}$ (70.6%) and blaH$_{SHM}$(1.7%) genes. PCR specificity was confirmed by nucleotide sequencing. The present study indicates that the extraintestinal and multidrug-resistant (MDR) E. coli strains were associated with pneumonia and/or septicemia in sheep and goats. Therefore, thorough surveillance and monitoring of MDR bacteria are urgently needed to implement the infection control strategies and to restore the efficacy of available antibiotics.

Microvesicle-Mediated Delivery of Minicircle DNA Results in Effective Gene-Directed Enzyme Prodrug Cancer Therapy.

An emerging approach for cancer treatment employs the use of extracellular vesicles, specifically exosomes and microvesicles, as delivery vehicles. We previously demonstrated that microvesicles can functionally deliver plasmid DNA to cells and showed that plasmid size and sequence, in part, determine the delivery efficiency. In this study, delivery vehicles comprised of microvesicles loaded with engineered minicircle (MC) DNA that encodes prodrug converting enzymes developed as a cancer therapy in mammary carcinoma models. We demonstrated that MCs can be loaded into shed microvesicles with greater efficiency than their parental plasmid counterparts and that microvesicle-mediated MC delivery led to significantly higher and more prolonged transgene expression in recipient cells than microvesicles loaded with the parental plasmid. Microvesicles loaded with MCs encoding a thymidine kinase (TK)/nitroreductase (NTR) fusion protein produced prolonged TK-NTR expression in mammary carcinoma cells. In vivo delivery of TK-NTR and administration of prodrugs led to the effective killing of both targeted cells and surrounding tumor cells via TK-NTR-mediated conversion of codelivered prodrugs into active cytotoxic agents. In vivo evaluation of the bystander effect in mouse models demonstrated that for effective therapy, at least 1% of tumor cells need to be delivered with TK-NTR-encoding MCs. These results suggest that MC delivery via microvesicles can mediate gene transfer to an extent that enables effective prodrug conversion and tumor cell death such that it comprises a promising approach to cancer therapy.

Carbapenemase and ESBL genes with class 1 integron among fermenting and nonfermenting bacteria isolated from water sources from India.

The present study was aimed to detect the carbapenemase, extended-spectrum β-lactamase (ESBL), and intI1 gene of class 1 integron among fermenting (n=61) and nonfermenting (n=10) bacterial isolates recovered from water samples (n=128). Isolates were identified by 16S rRNA sequencing. These isolates showed reduced-susceptibility to third-generation cephalosporins and carbapenems. The isolates varied in number and size of plasmids (2kb to >20kb). Plasmid DNA screening showed 5·6, 7, 11·2 and 26·7% prevalence of blaKPC , blaNDM , blaSHV and blaTEM genes respectively. Diverse blaNDM (blaNDM-1 and blaNDM-4 ) and blaSHV subtypes (blaSHV-2 and blaSHV-11 ) were recorded, unlike the single allelic blaKPC (blaKPC-2 ) and blaTEM (blaTEM-1 ) gene. Of the total 27 bla-gene-producing bacterial isolates, seven isolates co-harboured the carbapenemase genes (blaNDM or blaKPC or the both) along with the ESBL genes (blaSHV or blaTEM ). The intI1 gene of class 1 integron was detected among 12 (44·4%) of ESBL- and/or carbapenemase-harbouring isolates. Gene transferability was seen among four of the 10 Enterobacteriaceae donors. Carbapenemases and ESBLs with class 1 integron among aquatic environmental isolates raise the serious issue of the biosecurity and health of the ecosystem. SIGNIFICANCE AND IMPACT OF THE STUDY: Anthropologically affected and polluted environment harbours the resistance threats, where a diverse bacterial species maintain, develop and exchange genetic determinants that constitute a risk to human and ecological health. The antimicrobial resistance (AMR) in Enterobacteriaceae and non-Enterobacteriaceae bacteria caused the failure of the therapy of last resort (carbapenems) and thus lead to life-threatening infections affecting public health. Surveillance and monitoring of AMR could be important for epidemiological, diagnostic testing and control of pathogens. This is a point-prevalence study reporting the comparative occurrence and co-occurrence of carbapenemase and extended-spectrum β-lactamase genes among fermenting and nonfermenting bacteria isolated from the aquatic environment in India.

Selective culture enrichment and sequencing of feces to enhance detection of antimicrobial resistance genes in third-generation cephalosporin resistant Enterobacteriaceae.

Metagenomic sequencing of fecal DNA can usefully characterise an individual's intestinal resistome but is limited by its inability to detect important pathogens that may be present at low abundance, such as carbapenemase or extended-spectrum beta-lactamase producing Enterobacteriaceae. Here we aimed to develop a hybrid protocol to improve detection of resistance genes in Enterobacteriaceae by using a short period of culture enrichment prior to sequencing of DNA extracted directly from the enriched sample. Volunteer feces were spiked with carbapenemase-producing Enterobacteriaceae and incubated in selective broth culture for 6 hours before sequencing. Different DNA extraction methods were compared, including a plasmid extraction protocol to increase the detection of plasmid-associated resistance genes. Although enrichment prior to sequencing increased the detection of carbapenemase genes, the differing growth characteristics of the spike organisms precluded accurate quantification of their concentration prior to culture. Plasmid extraction increased detection of resistance genes present on plasmids, but the effects were heterogeneous and dependent on plasmid size. Our results demonstrate methods of improving the limit of detection of selected resistance mechanisms in a fecal resistome assay, but they also highlight the difficulties in using these techniques for accurate quantification and should inform future efforts to achieve this goal.

Trans-Kingdom Conjugation within Solid Media from Escherichia coli to Saccharomyces cerevisiae.

Conjugation is a bacterial mechanism for DNA transfer from a donor cell to a wide range of recipients, including both prokaryotic and eukaryotic cells. In contrast to conventional DNA delivery techniques, such as electroporation and chemical transformation, conjugation eliminates the need for DNA extraction, thereby preventing DNA damage during isolation. While most established conjugation protocols allow for DNA transfer in liquid media or on a solid surface, we developed a procedure for conjugation within solid media. Such a protocol may expand conjugation as a tool for DNA transfer to species that require semi-solid or solid media for growth. Conjugation within solid media could also provide a more stable microenvironment in which the conjugative pilus can establish and maintain contact with recipient cells for the successful delivery of plasmid DNA. Furthermore, transfer in solid media may enhance the ability to transfer plasmids and chromosomes greater than 100 kbp. Using our optimized method, plasmids of varying sizes were tested for transfer from Escherichia coli to Saccharomyces cerevisiae. We demonstrated that there was no significant change in conjugation frequency when plasmid size increased from 56.5 to 138.6 kbp in length. Finally, we established an efficient PCR-based synthesis protocol to generate custom conjugative plasmids.

Vancomycin resistance plasmids affect persistence of Enterococcus faecium in water

Vancomycin resistant enterococci (VRE) cause 20,000 infections annually in the United States, most of which are nosocomial. Recent findings of VRE in sewage-contaminated surface waters demonstrate an alternate route of human exposure, and a possible setting for horizontal gene exchange facilitated by plasmids and other mobile genetic elements. Maintenance of antibiotic resistance genes and proteins may, however, present a fitness cost in the absence of selective pressure, particularly in habitats such as environmental waters that are not optimal for gut-associated bacteria. Nutrient levels, which are transiently elevated following sewage spills, may also affect survival. We tested the hypotheses that nutrients and/or plasmids conferring vancomycin resistance affect Enterococcus faecium survival in river water by measuring decay of strains that differed only by their plasmid, under natural and augmented nutrient conditions. In natural river water, decay rate (log10 reduction) correlated directly with plasmid size; however, plasmid presence and size had no effect on decay rate when nutrients levels were augmented. Under natural nutrient levels, the vancomycin-resistant strain with the largest plasmid (200 kb) decayed significantly more rapidly than the plasmid-less, susceptible parent strain, in contrast to similar decay rates among strains under augmented nutrient conditions. This work is among the first to show that plasmids conferring antibiotic resistance affect fitness of Enterococcus species in secondary habitats such as surface water. The nutrient-dependent nature of the fitness cost suggests that conveyance of VRE to environmental waters in nutrient-rich sewage may prolong survival of these pathogens, providing greater opportunity for host infection and/or horizontal gene transfer.

Characteristic of plasmids of <i>Bacillus pumilus</i> isolated in Belarus

In current study plasmids (size from 6.2 to 8.5 kb) copied with the “rolling circle” mechanism (RCR type) of the pC194 family have been identified in environmental bacteria Bacillus pumilus. It is shown that these extrachromosomal elements are widely distributed in B. pumilus bacteria circulating on the territory of Belarus (19 strains from 41 contain RCRplasmids) and they are characterized by genetic polymorphism. The most common extrachromosomal genetic elements (7.7 kb size) are identical to the plasmid pBp15.1S from the entomopathogenic strain B. pumilus 15.1. The remaining 6 type of plasmids differ from each other and from the known extrachromosomal genetic elements of genus Bacillus. In the investigated bacteria no replicons similar to pLS20 were detected.

Enhanced Proliferation of Endothelial Progenitor Cells Post-Ultrasonic Microbubble Transfection Is Plasmid DNA Size Dependent and Contributed by Interleukin-6 Generation

We investigated whether ultrasonic microbubble transfection (UMT) would enhance the transfection of large-sized luciferase plasmids (5.6, 9.2 and 33 kb) and biological impacts. Porcine venous blood endothelial progenitor cells (EPCs) were cultured in a medium containing plasmid DNA (pDNA) of different sizes followed by UMT and functional assays. Real-time polymerase chain reaction was conducted to investigate the effects of transfection of pDNA on multiple molecules central to endothelial function. The results indicated enhanced luciferase expression after UMT but the enhancement declined with increase in the size of the plasmid. UMT of pDNAs sized 5.6 and 9.2 kb into EPCs led to significant enhancement of proliferation. The interleukin-6 (IL-6) secreted from UMT of EPCs also increased in the 5.6- and 9.2-kb pDNA groups. Treatment of the transfected EPCs with anti-IL-6 antibody neutralized the proliferation. In conclusion, UMT of pDNAs sized 5.6 and 9.2 kb into EPCs increased the secretion of IL-6, which in turn enhanced cell proliferation.

Characterization of a small plasmid carrying the carbapenem resistance gene bla OXA-72 from community-acquired Acinetobacter baumannii sequence type 880 in China.

BackgroundAcinetobacter baumannii has emerged as an important pathogen associated with hospital- and community-acquired infections. Community-acquired A. baumannii pneumonia is characterized by a fulminant course and high mortality rates. In this study, we report the identification of a community-acquired carbapenem-resistant A. baumannii strain carrying the blaOXA-72 gene.MethodsThis A. baumannii isolate was recovered from a male patient diagnosed with community-acquired pneumonia, septic shock, and respiratory failure. Antimicrobial susceptibility testing were performed and the minimum inhibitory concentrations were determined by the broth microdilution method. Whole-genome sequencing was performed using both long-read MinION and short-read Illumina platforms to fully characterize the blaOXA-72-carrying plasmid of the A. baumannii A52. The in silico multilocus sequence typing and genomic epidemiological analysis of the closely related isolates were further elucidated by our recently updated BacWGSTdb server.ResultsThe isolate was resistant to meropenem and remained susceptible to several other antimicrobial agents. Whole-genome sequencing and bioinformatics analysis indicated that this A. baumannii isolate belonged to the rare sporadic clone sequence type 880 and the blaOXA-72 gene was located on the 8,493-bp plasmid pA52-OXA-72. This plasmid exhibited only partial similarity to different OXA-72-encoding plasmids (size range: 8,771–12,056 bp) in various Acinetobacter spp. recovered from patients and other reservoirs in different countries.ConclusionThis study described the first case of fulminant carbapenem-resistant community-acquired A. baumannii pneumonia caused by a rare sporadic clone in China. Adequate surveillance is warranted to monitor the emergence of A. baumannii as a community pathogen.

Identification, Antagonistic Potentials and Plasmid Profiling of Micro-Organisms Associated with Termitarium and Macerated Dead Termites from Cashew Trees in Ibule-Soro, Akure Nigeria

This research was carried out to identify microorganisms associated with termitarium on cashew tree barks and macerated dead termites from Ibule-Soro, Akure, Nigeria. Pour plate technique was used for isolation, standard and conventional methods of cultural, morphological and biochemical characteristics were employed in the identification and characterization. Bacterial isolates such as Bacillus sp, Micrococcus sp, Corynebacterium sp, Streptococcus sp were identified, while fungi isolates such as Aspergilus niger, Fusarium solani and Penicillium nonatum were identified. The result of antimicrobial sensitivity patterns of the isolates showed that all the bacteria were susceptible to at least three of the antibiotics. However, Micrococcus sp and Bacillus sp were screened to be multiple antibiotic resistant isolates. Plasmid profiling of these multiple antibiotic resistant bacteria isolates were carried out to determine the size of the bacteria plasmids and genetic basis of their antimicrobial resistance. The isolates were cured of their plasmid and subjected to antibiotic treatments again to determine whether their susceptibility to antibiotic is chromosomal or extra-chromosomal. Antagonistic properties of the isolated bacteria and fungi were determined against known bacterial pathogens such as Staphylococus aureus, Shigella sp, Salmonella sp, and Escherichia coli, the result showed that only the fungus Penicillium notatum showed positive and mild antagonistic potential against the selected pathogens. Findings from this research showed the potentials of termite nest as reservoirs for beneficial microorganisms with great antagonistic properties.

Aromatic Hydrocarbons Degradation and Plasmid Profile of Marine Bacterial Isolates Obtained from Petroleum Contaminated Marine Environments of Niger Delta Nigeria

Aims: To determine the aromatic hydrocarbons degradability and plasmid profile of the marine bacteria isolated from Rivers State contaminated marine environments of Niger Delta.
 Study Design: Nine treatments and the controls designs were set up in triplicates containing 100 mL of sterile modified mineral basal medium in 500 mL conical flasks supplemented with 1 mg /L of xylene, anthracene and pyrene each; nine marine hydrocarbon degraders and incubated at 24 ºC for 24 days study. The nine treatments and control set ups designated as ANT1, XYL2, PYR3, ANT4, PYR5, ANT6, XYL7, XYL8, PYR9 and CTRL (Without hydrocarbons) were used to determine the aromatic hydrocarbons degradability and plasmid profile of the marine bacteria. 
 Place and Duration of Study: Department of Environmental Sciences, University of South Africa, Pretoria, South Africa between September, 2015 and March, 2018.
 Methodology: A laboratory scale study was carried on six composite samples of the sediment and water samples from the three studied areas using enrichment, screening, selection, morphological, biochemical, degradation and plasmid assays.
 Results: The findings revealed that the three sampling sites harbour a lot of efficient aromatic degrading bacterial strains belonging to the genera: Providencia, Alcaligenes, Brevundimonas, Myroides, Serratia, and Bacillus able to significantly (P = .05) tolerate and grow on the aromatic hydrocarbons. The bacterial strains especially Serratia marcescens XYL7 significantly (P = .05) removed 99.50 ± 0.05 % and 60.00 ± 0.02 % in weights of xylene and pyrene, respectively while Alcaligenes faecalis PYR5 significantly (P =.05) degraded 97.40 ± 0.01 % in weight of anthracene. The degradations of the respective hydrocarbons were found to be plasmid mediated with plasmid sizes between 200 bp - 1.2 kbp.
 Conclusion: Thus, the excellent degradative abilities of these bacterial strains especially Serratia marcescens XYL7 could be exploited for bioremediation purposes in Nigeria.

Plasmid Characteristics Modulate the Propensity of Gene Exchange in Bacterial Vesicles.

Horizontal gene transfer is responsible for the exchange of many types of genetic elements, including plasmids. Properties of the exchanged genetic element are known to influence the efficiency of transfer via the mechanisms of conjugation, transduction, and transformation. Recently, an alternative general pathway of horizontal gene transfer has been identified, namely, gene exchange by extracellular vesicles. Although extracellular vesicles have been shown to facilitate the exchange of several types of plasmids, the influence of plasmid characteristics on genetic exchange within vesicles is unclear. Here, a set of different plasmids was constructed to systematically test the impact of plasmid properties, specifically, plasmid copy number, size, and origin of replication, on gene transfer in vesicles. The influence of each property on the production, packaging, and uptake of vesicles containing bacterial plasmids was quantified, revealing how plasmid properties modulate vesicle-mediated horizontal gene transfer. The loading of plasmids into vesicles correlates with the plasmid copy number and is influenced by characteristics that help set the number of plasmids within a cell, including size and origin of replication. Plasmid origin also has a separate impact on both vesicle loading and uptake, demonstrating that the origin of replication is a major determinant of the propensity of specific plasmids to transfer within extracellular vesicles.IMPORTANCE Extracellular vesicle formation and exchange are common within bacterial populations. Vesicles package multiple types of biomolecules, including genetic material. The exchange of extracellular vesicles containing genetic material facilitates interspecies DNA transfer and may be a promiscuous mechanism of horizontal gene transfer. Unlike other mechanisms of horizontal gene transfer, it is unclear whether characteristics of the exchanged DNA impact the likelihood of transfer in vesicles. Here, we systematically examine the influence of plasmid copy number, size, and origin of replication on the loading of DNA into vesicles and the uptake of DNA containing vesicles by recipient cells. These results reveal how each plasmid characteristic impacts gene transfer in vesicles and contribute to a greater understanding of the importance of vesicle-mediated gene exchange in the landscape of horizontal gene transfer.

CRISPR–Cas9D10A nickase‐assisted base editing in the solvent producer Clostridium beijerinckii

Clostridium beijerinckii is a potentially important industrial microorganism as it can synthesize valuable chemicals and fuels from various carbon sources. The establishment of convenient to use, effective gene tools with which the organism can be rapidly modified is essential if its full potential is to be realized. Here, we developed a genomic editing tool (pCBEclos) for use in C. beijerinckii based on the fusion of cytidine deaminase (Apobec1), Cas9 D10A nickase and uracil DNA glycosylase inhibitor (UGI). Apobec1 and UGI are guided to the target site where they introduce specific base-pair substitutions through the conversion of C·G to T·A. By appropriate choice of target sequence, these nucleotide changes are capable of creating missense mutation or null mutations in a gene. Through optimization of pCBEclos, the system derived, pCBEclos-opt, has been used to rapidly generate four different mutants in C. beijerinckii, in pyrE, xylR, spo0A, and araR. The efficiency of the system was such that they could sometimes be directly obtained following transformation, otherwise only requiring one single restreaking step. Whilst CRISPR-Cas9 nickase systems, such as pNICKclos2.0, have previously been reported in C. beijerinckii, pCBEclos-opt does not rely on homologous recombination, a process that is intrinsically inefficient in clostridia such as C. beijerinckii. As a consequence, bulky editing templates do not need to be included in the knockout plasmids. This both reduces plasmid size and makes their construction simpler, for example, whereas the assembly of pNICKclos2.0 requires six primers for the assembly of a typical knockout plasmid, pCBEclos-opt requires just two primers. The pCBEclos-opt plasmid established here represents a powerful new tool for genome editing in C. beijerinckii, which should be readily applicable to other clostridial species.

Plasmid-Mediated Transmission of KPC-2 Carbapenemase in Enterobacteriaceae in Critically Ill Patients.

Carbapenem-resistant Enterobacteriaceae (CRE) cause health care-associated infections worldwide, and they are of severe concern due to limited treatment options. We report an outbreak of KPC-2-producing CRE that was caused by horizontal transmission of a promiscuous plasmid across different genera of bacteria and hospitals in Germany. Eleven isolates (8 Citrobacter freundii, 2 Klebsiella oxytoca, and 1 Escherichia coli) were obtained from seven critically ill patients during the six months of the outbreak in 2016. One patient developed a CRE infection while the other six patients were CRE-colonized. Three patients died in the course of the hospital stay. Six of the seven patients carried the same C. freundii clone; one K. oxytoca clone was found in two patients, and one patient carried E. coli and C. freundii. Molecular analysis confirmed the presence of a conjugative, blaKPC-2-carrying 70 kb-IncN plasmid in C. freundii and E. coli and an 80 kb-IncN plasmid in K. oxytoca. All transconjugants harbored either the 70 or 80 kb plasmid with blaKPC-2, embedded within transposon variant Tn4401g. Whole genome sequencing and downstream bioinformatics analyses of all plasmid sequences showed an almost perfect match when compared to a blaKPC-2-carrying plasmid of a large outbreak in another German hospital two years earlier. Differences in plasmid sizes and open reading frames point to the presence of inserted mobile genetic elements. There are few outbreak reports worldwide on the transmission of blaKPC-2-carrying plasmids across different bacterial genera. Our data suggest a regional and supraregional spread of blaKPC-2-carrying IncN-plasmids harboring the Tn4401g isoform in Germany.

Biodegradation of dinitrotoluene sulfonates and other nitro-aromatic compounds by Pseudomonas sp. X5 isolated from TNT red water contaminated soil

A dinitrotoluene sulfonates degrading bacterium strain Pseudomonas sp. X5 was isolated from 2, 4, 6-trinitrotoluene red water contaminated soil in Gansu, China, which was used for biodegradation of dinitrotoluene sulfonates. The growth experiments were carried out to optimize the growth condition of X5. Effect of inoculation amount was investigated. Plasmid curing tests were conducted to determine whether the dinitrotoluene sulfonates degrading gene is chromosomal or plasmid encoded. The results showed that the optimum growing conditions for X5 were 35 °C, pH 7–9 and 1% (salinity). The biodegradation rates of dinitrotoluene sulfonates were highly affected by inoculation amount. When the inoculation amount was 25%, 100% of 2, 4-dinitrotoluene-3-sulfonate and 2, 4-dinitrotoluene-5-sulfonate could be biodegraded. Liquid chromatography-mass spectrometry analysis showed that the nitro groups in dinitrotoluene sulfonates were transformed to amino groups. Pseudomonas sp. X5 has broad-spectrum degradation characteristics. It can degrade not only dinitrotoluene sulfonates, but also other nitro-aromatic pollutants like 2, 4, 6-trinitrotoluene, dinitrotoluene and mononitrotoluene. The ability of Pseudomonas sp. X5 to degrade dinitrotoluene sulfonates, was found to be plasmid encoded. The size of the relevant plasmid was estimated to be about 20 kb.

Resistance to Azithromycin and β-Lactam Antibiotics by Clinical Isolates of E. coli Isolated from Dhaka, Bangladesh

Antibiotic resistant E. coli are prevalent in Bangladesh. The indiscriminate use of antimicrobials and ready availability of over the counter drugs are responsible for this. This study was conducted to investigate the susceptibility of clinical Escherichia coli to the antibiotics Imipenem, Ceftriaxone, Ceftazidime and Azithromycin. Kirby-Bauer disk diffusion method was used to determine sensitivity to antimicrobials. Agar based assay was employed for the detection of efflux pumps. PCR was used amplify antibiotic resistance genes.All isolates were resistant to Ceftriaxone whereas most were sensitive to Imipenem. The MICs of Ceftazidime and Azithromycin ranged between 128 μg/ml and 256 μg/ml. The prevalence of ²-lactamase producers was 57.89 % with 36.84 % of the isolates exhibiting ESBL activity. No specific correlation could be found between plasmid sizes and antibiotic resistance patterns. Efflux pump was found to be involved in Azithromycin resistance in 63.15% of the isolates. The gene for phosphotransferase, mph(A) was the most common among the macrolide modifying genes, being present in 73.68% (14/19) of the isolates followed by both erm(A) anderm(C) esterases each present in 10.53% (2/19) isolates. This study concluded that clinical isolates of E. coli in Bangladesh could be resistant to multiple classes of antibiotics through different mechanisms of resistance.
 Bangladesh J Microbiol, Volume 34 Number 2 December 2017, pp 61-66

Molecular Additives Significantly Enhance Glycopolymer-Mediated Transfection of Large Plasmids and Functional CRISPR-Cas9 Transcription Activation Ex Vivo in Primary Human Fibroblasts and Induced Pluripotent Stem Cells.

Fast, efficient, and inexpensive methods for delivering functional nucleic acids to primary human cell types are needed to advance regenerative medicine and cell therapies. Plasmid-based gene editing (such as with CRISPR-Cas9) can require the delivery of plasmids that are large (∼9.5-13 kbp) in comparison to common reporter plasmids (∼5-8 kbp). To develop more efficient plasmid delivery vehicles, we investigated the effect of plasmid size on the transfection of primary human dermal fibroblasts (HDFs) and induced pluripotent stem cells (iPSCs) using a heparin-treated trehalose-containing polycation (Tr4-heparin). Transfections with 4.7 kbp to 10 kbp plasmids exhibited high rates of polyplex internalization with both plasmid sizes. However, transfection with the large plasmid was nearly eliminated in HDFs and significantly reduced in iPSCs. Molecular additives were used to probe intracellular barriers to transfection. Chloroquine treatments were used to destabilize endosomes, and dexamethasone and thymidine were used to destabilize the nuclear envelope. Destabilizing the nuclear envelope resulted in significantly increased large-plasmid-transfection, indicating that nuclear localization may be more difficult for large plasmids. To demonstrate the potential clinical utility of this formulation, HDFs and iPSCs were treated with to dexamethasone-Tr4-heparin polyplexes encoding dCas9-VP64, synthetic transcription activator, targeted to collagen type VII. These transfections enhanced collagen expression in HDFs and iPSCs by 5- and 20-fold, respectively, compared to an untransfected control and were the more effective than the Lipofectamine 2000 control. Functional plasmid transfection efficiency can be significantly improved by nuclear destabilization, which could lead to improved development of nonviral vehicles for ex vivo CRISPR-Cas9 gene editing.

Targeting of Virulence Factors and Plasmid Profiling of Klebsiella pneumoniae Causing Urinary Tract Infection in Sylhet City of Bangladesh

ABSTRACT Studies were conducted to characterize Klebsiella pneumoniae isolates from urinary tract infection (UTI) patients in Sylhet city of Bangladesh. At the same time, all isolates were screened for some common virulence genes and four significant isolates were searched for plasmid number and sizes by mini alkaline-lysis method. Among five tested isolates from female UTI patients, gyrase subunit B2 (gyrb2) amplified in all isolates, lipase and nuclease detected in three isolates and serine protease amplifies in two isolates and gave the expected band of 1130 bp, 517 bp, 1055 bp and 211 bp respectively. Two of four isolates showed 9.82 kb plasmid band on agarose gel. Isolates bearing 9.82 kb plasmid were found to be resistant to multiple commercial antibiotics. At the same time all isolates were screened for in-vitro plate assay for proteolytic, lypolytic and hemolytic activity. Isolates with positive plasmid and more than one virulent gene with gyrB2 showed positive result in in-vitro culture plate with clear zone of proteolysis, hemolysis or lipolysis. This study will be helpful for further study in finding correlation or pattern of virulence properties for K. pneumoniae associated UTI in Bangladesh.

Anti- plasmid effect of some plant extracts against multidrug resistant Enterococci.

تعد مشكلة المقاومة المتعددة للعلاجاتMultiple drug resistance (MDR) من أهم التحديات الصحية العالمية التي تتطلب البحث المستمر في معالجتها، وتقع معظم المحددات الوراثية المسؤولة عن هذه المقاومة على البلازميدات. أجري هذا البحث بهدف دراسة التاثير المضاد للبلازميدات في المستخلصات الكحولية لنبات السماق Sumach (RhusCoriaria) ونبات الميرامية Sage (Salvia officinalis) تجاه بكتريا المكورات المعوية Enterococci المقاومة للمضادات الحيوية. أظهرت اختبارات حساسية عزلات هذه البكتريا تجاه المضادات الحيوية المدروسة أن جميع العزلات تمتلك انماط مختلفة من المقاومة المتعددة للمضادات الحيوية وصلت إلى حد تسعة من أصل أربعة عشر مضاداً حيوياً، وأبدت جميع العزلات مقاومة مطلقة بنسبة (100%) للمضادين الحيويين Amikacin وNaldixic acid. وفي دراسة فعالية كل من المستخلص الكحولي للميرامية والسماق المضادة للبلازميدات تجاه العزلات المنتخبة تم إثبات صفة التحييد البلازميدي لهذين المستخلصين ولأول مرة بالتركيز (2000) مايكروغرام/سم3 تجاه عزلات هذه البكتريا عن طريق فقدان صفة المقاومة للمضادات الحيوية مظهرياً (Amikacin وAmpicillin وacid Naldixic وTetracycline) والفقدان المادي للبلازميدات جزيئيا (بلازميدين بحجم 4000bp و3500bp)، مما يثبت وجود الجينات المسؤولة عن مقاومة المضادات المذكورة اعلاه على البلازميدات المُحَيَّدَة، بالتالي فإن هذه النتيجة تفتح الطريق أمام إمكانية استخدام هذين النباتين بعد دراسات إضافية لمعالجة مشكلة المقاومة العلاجية في بكتريا المكورات المعوية والبكتريا المرضية الأخرى.