Formaldehyde effects on kanamycin resistance gene of inactivated recombinant Escherichia coli vaccines.

OBJECTIVES: Earlier studies have demonstrated the use of inactivated recombinant E. coli (bacterins), to protect against Clostridium spp. in vaccinated animals. These bacterins have a simpler, safer, and faster production process. However, these bacterins carry expression plasmids, containing antibiotic resistance gene, which could be assimilate accidentally by environmental microorganisms. Considering this, we aimed to impair this plasmids using formaldehyde at different concentrations. RESULTS: This compound inactivated the highest density of cells in 24 h. KanR cassette amplification was found to be impaired with 0.8% for 24 h or 0.4% for 72 h. Upon electroporation, E. coli DH5α ultracompetent cells were unable to acquire the plasmids extracted from the bacterins after inactivation procedure. Formaldehyde-treated bacterins were incubated with other viable strains of E. coli, leading to no detectable gene transfer. CONCLUSIONS: We found that this compound is effective as an inactivation agent. Here we demonstrate the biosafety involving antibiotic resistance gene of recombinant E. coli vaccines allowing to industrial production and animal application.

Evaluation of the antibacterial and anticancer activities of some South African medicinal plants.

BACKGROUND: Several herbs are traditionally used in the treatment of a variety of ailments particularly in the rural areas of South Africa where herbal medicine is mainly the source of health care system. Many of these herbs have not been assessed for safety or toxicity to tissue or organs of the mammalian recipients. METHODS: This study evaluated the cytotoxicity of some medicinal plants used, inter alia, in the treatment of diarrhoea, and stomach disorders. Six selected medicinal plants were assessed for their antibacterial activities against ampicillin-resistant and kanamycin-resistant strains of Escherichia coli by the broth micro-dilution methods. The cytotoxicities of methanol extracts and fractions of the six selected plants were determined using a modified tetrazolium-based colorimetric assay (3-(4, 5-dimethylthiazol)-2, 5-diphenyl tetrazolium bromide (MTT) assay). RESULTS: The average minimum inhibitory concentration (MIC) values of the plants extracts ranged from 0.027 mg/mℓ to 2.5 mg/mℓ after 24 h of incubation. Eucomis autumnalis and Cyathula uncinulata had the most significant biological activity with the least MIC values. The in vitro cytotoxicity assay on human hepatocarcinoma cell line (Huh-7) revealed that the methanol extract of E. autumnalis had the strongest cytotoxicity with IC(50) of 7.8 µg/mℓ. Ethyl acetate and butanol fractions of C. uncinulata, Hypoxis latifolia, E. autumnalis and Lantana camara had lower cytotoxic effects on the cancer cell lines tested with IC(50) values ranging from 24.8 to 44.1 µg/mℓ; while all the fractions of Aloe arborescens and A. striatula had insignificant or no cytotoxic effects after 72 h of treatment. CONCLUSIONS: Our results indicate that the methanol fraction of E. autumnalis had a profound cytotoxic effect even though it possessed very significant antibacterial activity. This puts a query on its safety and hence a call for caution in its usage, thus a product being natural is not tantamount to being entirely safe. However, the antibacterial activities and non-cytotoxic effects of A. arborescens and A. striatula validates their continuous usage in ethnomedicine.

Structure-Guided Optimization of Inhibitors of Acetyltransferase Eis from Mycobacterium tuberculosis.

The enhanced intracellular survival (Eis) protein of Mycobacterium tuberculosis (Mtb) is a versatile acetyltransferase that multiacetylates aminoglycoside antibiotics abolishing their binding to the bacterial ribosome. When overexpressed as a result of promoter mutations, Eis causes drug resistance. In an attempt to overcome the Eis-mediated kanamycin resistance of Mtb, we designed and optimized structurally unique thieno[2,3-d]pyrimidine Eis inhibitors toward effective kanamycin adjuvant combination therapy. We obtained 12 crystal structures of enzyme-inhibitor complexes, which guided our rational structure-based design of 72 thieno[2,3-d]pyrimidine analogues divided into three families. We evaluated the potency of these inhibitors in vitro as well as their ability to restore the activity of kanamycin in a resistant strain of Mtb, in which Eis was upregulated. Furthermore, we evaluated the metabolic stability of 11 compounds in vitro. This study showcases how structural information can guide Eis inhibitor design.

Reversal of heavy metal-induced antibiotic resistance by dandelion root extracts and taraxasterol.

Introduction. Metal exposure is an important factor for inducing antibiotic resistance in bacteria. Dandelion extracts have been used for centuries in traditional Chinese and Native American medicine.Aim. We assessed the effects of dandelion water extracts and taraxasterol on heavy metal-induced antibiotic resistance in Escherichia coli as well as the underlying mechanisms.Methodology. Dandelion extracts were obtained through 4 h of boiling in distilled water. Bacterial growth was monitored with a spectrophotometer. Biochemical assays were performed to assess the activities and gene transcriptions of ß-lactamase and acetyltransferase. Oxidative stress was determined using an oxidation-sensitive probe, H2DCFDA.Results. The present study demonstrated that higher concentrations of nickel (>5 µg ml-1), cadmium (>0.1 µg ml-1), arsenic (>0.1 µg ml-1) and copper (>5 µg ml-1) significantly inhibited the growth of E. coli. Lower concentrations of nickel (0.5 µg ml-1), cadmium (0.05 µg ml-1) and arsenic (0.05 µg ml-1) had no effect on bacterial growth, but helped the bacteria become resistant to two antibiotics, kanamycin and ampicillin. The addition of dandelion root extracts and taraxasterol significantly reversed the antibiotic resistance induced by these heavy metals. The supplements of antibiotics and cadmium generated synergistic effects on the activities of ß-lactamase and acetyltransferase (two antibiotic resistance-related proteins), which were significantly blocked by either dandelion root extract or taraxasterol. In contrast, oxidative stress was not involved in the preventative roles of dandelion root extracts and taraxasterol in heavy metal-induced antibiotic resistance.Conclusion. This study suggests that heavy metals induce bacterial antibiotic resistance and dandelion root extracts and taraxasterol could be used to help reverse bacterial resistance to antibiotics.

Potent 1,2,4-Triazino[5,6 b]indole-3-thioether Inhibitors of the Kanamycin Resistance Enzyme Eis from Mycobacterium tuberculosis.

A common cause of resistance to kanamycin (KAN) in tuberculosis is overexpression of the enhanced intracellular survival (Eis) protein. Eis is an acetyltransferase that multiacetylates KAN and other aminoglycosides, rendering them unable to bind the bacterial ribosome. By high-throughput screening, a series of substituted 1,2,4-triazino[5,6 b]indole-3-thioether molecules were identified as effective Eis inhibitors. Herein, we purchased 17 and synthesized 22 new compounds, evaluated their potency, and characterized their steady-state kinetics. Four inhibitors were found not only to inhibit Eis in vitro, but also to act as adjuvants of KAN and partially restore KAN sensitivity in a Mycobacterium tuberculosis KAN-resistant strain in which Eis is upregulated. A crystal structure of Eis in complex with a potent inhibitor and CoA shows that the inhibitors bind in the aminoglycoside binding site snugly inserted into a hydrophobic cavity. These inhibitors will undergo preclinical development as novel KAN adjuvant therapies to treat KAN-resistant tuberculosis.

Diploid potato (Solanum tuberosum L.) as a model crop to study transgene expression.

This paper presents a method of Agrobacterium-mediated transformation for two diploid breeding lines of potato, and gives a detailed analysis of reporter gene expression. In our lab, these lines were also used to obtain tetraploid somatic hybrids. We tested four newly prepared constructs based on the pGreen vector system containing the selection gene nptII or bar under the 35S or nos promoter. All these vectors carried gus under 35S. We also tested the pDM805 vector, with the bar and gus genes respectively under the Ubi1 and Act1 promoters, which are strong for monocots. The selection efficiency (about 17%) was highest in the stem and leaf explants after transformation with pGreen where nptII was under 35S. About half of the selected plants were confirmed via PCR and Southern blot analysis to be transgenic and, depending on the combination, 0 to 100% showed GUS expression. GUS expression was strongest in multi-copy transgenic plants where gus was under Act1. The same potato lines carrying multi-copy bar under Ubi1 were also highly resistant to the herbicide Basta. The suggestion of using Agrobacterium-mediated transformation of diploid lines of potato as a model crop is discussed herein.

Evaluación de la resistencia a los antibióticos de cepas de Escherichia coli aisladas en carne de cerdo comercializada en los mercados municipales de la ciudad de Guatemala / Evaluation of antibiotics resistance of Escherichia coli strains isolated in pork sold in municipal markets of Guatemala City

La resistencia a los antimicrobianos es un problema de salud pública a nivel mundial que va en aumento y se ve reflejada en la falta de eficacia de los tratamientos de infecciones bacterianas con antibióticos en humanos y en animales. El presente estudio tuvo como objetivo evaluar la resistencia a los antibióticos de cepas de Escherichia coli aisladas en carne de cerdo expendida en los mercados municipales de la ciudad de Guatemala. Se identificaron los antibióticos que presentaron mayor resistencia y mayor sensibilidad in vitro frente a las cepas de E. coli aisladas a partir de 76 muestras de carne de cerdo. Se realizó un muestreo aleatorio simple con afijación proporcional por mercado. Para la identificación de las cepas de E. coli se utilizó la prueba de IMViC y para evaluar la resistencia a los antimicrobianos se utilizó la prueba de Kirby Bauer empleando 9 antibióticos. Se aisló E. coli en el 55% (42/76) de las muestras. La resistencia en las 42 cepas aisladas fue: tetraciclina (83%) neomicina (50%) y sulfametoxasole + trimetoprim (50%). 83% de las cepas (35/42) fueron resistentes a 2 antibióticos y 50% (21/42) a 3 antibióticos o más. Se obtuvo mayor sensibilidad con ceftriaxona (91%), amikacina (83%), gentamicina (65%) y ácido nalidíxico (65%). Se concluye que existe resistencia a los antibióticos evaluados, lo que constituye un riesgo para la salud pública ya que se encuentra en cepas aisladas en un alimento para consumo humano.

Antimicrobial resistance is a global public health threat that is increasing and is reflected in the lack of efficacy of bacterial infection treatments with antibiotics in humans and animals. The objective of this study was to evaluate the resistance to antibiotics of Escherichia coli strains isolated from pork in the municipal markets of Guatemala City. Antibiotics with the highest resistance and those with the highest sensitivity in vitro against the strains of E. coli were evaluated. A simple random sampling was carried out with proportional allocation by market, and 76 samples were collected. IMViC test was used to identify the E. coli strains, and antibiotics resistance was evaluated using the Kirby Bauer with nine different antibiotics. E. coli was isolated in 55% (42/76) of the samples. Resistance was evaluated in the 42 isolates. Antibiotic resistance was detected to tetracycline (83%), neomycin (50%), and sulfamethoxazole + trimethoprim (50%). All isolates presented resistance to at least one antibiotic; it was determined that 83% (35/42) showed resistance to two antibiotics and 50% (21/42) showed resistance to three antibiotics or more. The sensitivity obtained was higher for ceftriaxone (91%), amikacin (83%), gentamicin (65%), and nalidixic acid (65%). In conclusion, antibiotic resistance was detected, which constitutes a risk to public health since it is found in isolated strains in food for human consumption.

Combating Enhanced Intracellular Survival (Eis)-Mediated Kanamycin Resistance of Mycobacterium tuberculosis by Novel Pyrrolo[1,5-a]pyrazine-Based Eis Inhibitors.

Tuberculosis (TB) remains one of the leading causes of mortality worldwide. Hence, the identification of highly effective antitubercular drugs with novel modes of action is crucial. In this paper, we report the discovery and development of pyrrolo[1,5-a]pyrazine-based analogues as highly potent inhibitors of the Mycobacterium tuberculosis (Mtb) acetyltransferase enhanced intracellular survival (Eis), whose up-regulation causes clinically observed resistance to the aminoglycoside (AG) antibiotic kanamycin A (KAN). We performed a structure-activity relationship (SAR) study to optimize these compounds as potent Eis inhibitors both against purified enzyme and in mycobacterial cells. A crystal structure of Eis in complex with one of the most potent inhibitors reveals that the compound is bound to Eis in the AG binding pocket, serving as the structural basis for the SAR. These Eis inhibitors have no observed cytotoxicity to mammalian cells and are promising leads for the development of innovative AG adjuvant therapies against drug-resistant TB.

Shigatoxin encoding Bacteriophage ϕ24B modulates bacterial metabolism to raise antimicrobial tolerance.

How temperate bacteriophages play a role in microbial infection and disease progression is not fully understood. They do this in part by carrying genes that promote positive evolutionary selection for the lysogen. Using Biolog phenotype microarrays and comparative metabolite profiling we demonstrate the impact of the well-characterised Shiga toxin-prophage ϕ24B on its Escherichia coli host MC1061. As a lysogen, the prophage alters the bacterial physiology by increasing the rates of respiration and cell proliferation. This is the first reported study detailing phage-mediated control of the E. coli biotin and fatty acid synthesis that is rate limiting to cell growth. Through ϕ24B conversion the lysogen also gains increased antimicrobial tolerance to chloroxylenol and 8-hydroxyquinoline. Distinct metabolite profiles discriminate between MC1061 and the ϕ24B lysogen in standard culture, and when treated with 2 antimicrobials. This is also the first reported use of metabolite profiling to characterise the physiological impact of lysogeny under antimicrobial pressure. We propose that temperate phages do not need to carry antimicrobial resistance genes to play a significant role in tolerance to antimicrobials.

Potent Inhibitors of Acetyltransferase Eis Overcome Kanamycin Resistance in Mycobacterium tuberculosis.

A major cause of tuberculosis (TB) resistance to the aminoglycoside kanamycin (KAN) is the Mycobacterium tuberculosis (Mtb) acetyltransferase Eis. Upregulation of this enzyme is responsible for inactivation of KAN through acetylation of its amino groups. A 123 000-compound high-throughput screen (HTS) yielded several small-molecule Eis inhibitors that share an isothiazole S,S-dioxide heterocyclic core. These were investigated for their structure-activity relationships. Crystal structures of Eis in complex with two potent inhibitors show that these molecules are bound in the conformationally adaptable aminoglycoside binding site of the enzyme, thereby obstructing binding of KAN for acetylation. Importantly, we demonstrate that several Eis inhibitors, when used in combination with KAN against resistant Mtb, efficiently overcome KAN resistance. This approach paves the way toward development of novel combination therapies against aminoglycoside-resistant TB.

Instability of the Arabidopsis mutant csn5a-2 caused by epigenetic modification of intronic T-DNA.

T-DNA insertion mutants play a crucial role in elucidating Arabidopsis gene function. In some cases, two or more T-DNA mutants are combined to study genetic interactions between homologous genes or genes hypothesized to act in the same pathway. We studied the significance of protein-protein interactions between CSN5A and ROP11 by crossing three independent rop11 T-DNA insertion mutants with csn5a-2, a partial loss-of-function intronic T-DNA insertion mutant. The csn5a-2 single mutant is severely stunted, but double rop11 csn5a-2mutants were rescued and exhibited increased CSN5A transcript and protein levels. The rescued phenotype was maintained in non-Mendelian fashion when the csn5a-2 single mutant was re-isolated from the rop11-1 csn5a-2 double mutant, and was sensitive to two inhibitors of DNA methylation. Loss of kanamycin resistance was also observed in re-isolated csn5a-2. These findings indicate that the rescue of csn5a-2 resulted from a trans T-DNA-mediated epigenetic effect on the csn5a-2 intronic T-DNA, similar to recent reports involving the intronic T-DNA mutants ag-TD, ben1-1, and cob-6. Thus the work reported here provides further support for the recommendation that mutants created through novel combinations of T-DNA alleles should be carefully evaluated for evidence of epigenetic modification of T-DNA before final conclusions are drawn.

Genomic analysis reveals distinct concentration-dependent evolutionary trajectories for antibiotic resistance in Escherichia coli.

Evolution of bacteria under sublethal concentrations of antibiotics represents a trade-off between growth and resistance to the antibiotic. To understand this trade-off, we performed in vitro evolution of laboratory Escherichia coli under sublethal concentrations of the aminoglycoside kanamycin over short time durations. We report that fixation of less costly kanamycin-resistant mutants occurred earlier in populations growing at lower sublethal concentration of the antibiotic, compared with those growing at higher sublethal concentrations; in the latter, resistant mutants with a significant growth defect persisted longer. Using deep sequencing, we identified kanamycin resistance-conferring mutations, which were costly or not in terms of growth in the absence of the antibiotic. Multiple mutations in the C-terminal end of domain IV of the translation elongation factor EF-G provided low-cost resistance to kanamycin. Despite targeting the same or adjacent residues of the protein, these mutants differed from each other in the levels of resistance they provided. Analysis of one of these mutations showed that it has little defect in growth or in synthesis of green fluorescent protein (GFP) from an inducible plasmid in the absence of the antibiotic. A second class of mutations, recovered only during evolution in higher sublethal concentrations of the antibiotic, deleted the C-terminal end of the ATP synthase shaft. This mutation confers basal-level resistance to kanamycin while showing a strong growth defect in the absence of the antibiotic. In conclusion, the early dynamics of the development of resistance to an aminoglycoside antibiotic is dependent on the levels of stress (concentration) imposed by the antibiotic, with the evolution of less costly variants only a matter of time.