An Escherichia coli strain, PGB01, isolated from feral pigeon Faeces, thermally fit to survive in pigeon, shows high level resistance to trimethoprim.

In this study, of the hundred Escherichia coli strains isolated from feral Pigeon faeces, eighty five strains were resistant to one or more antibiotics and fifteen sensitive to all the antibiotics tested. The only strain (among all antibiotic-resistant E. coli isolates) that possessed class 1 integron was PGB01. The dihydrofolate reductase gene of the said integron was cloned, sequenced and expressed in E. coli JM109. Since PGB01 was native to pigeon's gut, we have compared the growth of PGB01 at two different temperatures, 42°C (normal body temperature of pigeon) and 37°C (optimal growth temperature of E. coli; also the human body temperature), with E. coli K12. It was found that PGB01 grew better than the laboratory strain E. coli K12 at 37°C as well as at 42°C. In the thermal fitness assay, it was observed that the cells of PGB01 were better adapted to 42°C, resembling the average body temperature of pigeon. The strain PGB01 also sustained more microwave mediated thermal stress than E. coli K12 cells. The NMR spectra of the whole cells of PGB01 varied from E. coli K12 in several spectral peaks relating some metabolic adaptation to thermotolerance. On elevating the growth temperature from 37°C to 42°C, susceptibility to kanamycin (both strains were sensitive to it) of E. coli K12 was increased, but in case of PGB01 no change in susceptibility took place. We have also attempted to reveal the basis of trimethoprim resistance phenotype conferred by the dfrA7 gene homologue of PGB01. Molecular Dynamics (MD) simulation study of docked complexes, PGB01-DfrA7 and E. coli TMP-sensitive-Dfr with trimethoprim (TMP) showed loss of some of the hydrogen and hydrophobic interaction between TMP and mutated residues in PGB01-DfrA7-TMP complex compared to TMP-sensitive-Dfr-TMP complex. This loss of interaction entails decrease in affinity of TMP for PGB01-DfrA7 compared to TMP-sensitive-Dfr

Profile of genome-wide gene expression in <i>C. violaceum</i> MTCC 2656 cells.

<p>Cells were grown in presence (experimental) and absence of 400 µg ml⁻¹ GLE (control).</p

GLE inhibits <i>C. violaceum</i> induced cell-lysis of HepG2 cells.

<p>Phase contrast micrographs at 200X magnification showing (<b>A</b>) lysis of HepG2 cells after 4 h of infection with <i>C. violaceum</i> MTCC 2656 and (<b>B</b>) inhibition of lysis in presence of 400 µg ml⁻¹ GLE. (<b>C</b>) growth of HepG2 cells in presence of GLE alone (to nullify any effect of GLE on HepG2 growth).</p

LDH activity in the culture medium of HepG2 cells.

<p>LDH assay was performed after 4 h treatment with: (A) only water; (B) only 400 µg ml⁻¹ of GLE; (C) only <i>C. violaceum</i>; and (D) <i>C. violaceum</i> along with 400 µg ml⁻¹ GLE.</p

Significantly downregulated <i>C. violaceum</i> genes associated with quorum-sensing and pathogenicity, in presence of GLE.

1<p>gene expression in cells grown for 24 h.</p>2<p>gene expression in cells grown for 24 h in presence of 400 µg ml⁻¹ of GLE.</p><p>Significantly downregulated <i>C. violaceum</i> genes associated with quorum-sensing and pathogenicity, in presence of GLE.</p

Differential expression of genes in <i>C. violaceum</i> in presence of GLE.

<p>The classification was based on Clusters of Orthologous Groups (COG) functional classification (R, General function prediction only; S, Function unknown; and NRF, No results found).</p

Diagram of amplified class 1 integron.

<p>Schematic representation of class 1 integron amplicon of the strain PGB01 amplified with the Int₂F-3′CS primer pair. Numbers correspond to sequence positions in EMBL Ac. No. FN563072.</p

Superimposition of protein active site residues.

<p>Superimposed side chain representation of protein active site residues of TMP-sensitive-Dfr (magenta) and PGB01-DfrA7 (orange).</p

Hydrogen bonding between ligand and protein active site residues.

<p>Dash line shows hydrogen bond between, trimethoprim (magenta) and protein active site residues (green), A) TMP-sensitive-Dfr-TMP complex, B) PGB01-DfrA7-TMP complex.</p

Effect of microwave radiation on cell viability.

<p>A. Thermal effect of microwave radiation: Temperature of LB vs. time of exposure; B. Viability of cells vs. time of microwave exposure.</p