A multiple antibiotic and serum resistant oligotrophic strain, Klebsiella pneumoniae MB45 having novel dfrA30, is sensitive to ZnO QDs

Abstract

Background: The aim of this study was to describe a novel trimethoprim resistance gene cassette, designated dfrA30, within a class 1 integron in a facultatively oligotrophic, multiple antibiotic and human serum resistant test strain, MB45, in a population of oligotrophic bacteria isolated from the river Mahananda; and to test the efficiency of surface bound acetate on zinc oxide quantum dots (ZnO QDs) as bactericidal agent on MB45.

Methods: Diluted Luria broth/Agar (10-3) media was used to cultivate the oligotrophic bacteria from water sample. Multiple antibiotic resistant bacteria were selected by employing replica plate method. A rapid assay was performed to determine the sensitivity/resistance of the test strain to human serum. Variable region of class 1 integron was cloned, sequenced and the expression of gene coding for antibiotic resistance was done in Escherichia coli JM 109. Identity of culture was determined by biochemical phenotyping and 16S rRNA gene sequence analyses. A phylogenetic tree was constructed based on representative trimethoprim resistance-mediating DfrA proteins retrieved from GenBank. Growth kinetic studies for the strain MB45 were performed in presence of varied concentrations of ZnO QDs.

Results and conclusions: The facultatively oligotrophic strain, MB45, resistant to human serum and ten antibiotics trimethoprim, cotrimoxazole, ampicillin, gentamycin, netilmicin, tobramycin, chloramphenicol, cefotaxime, kanamycin and streptomycin, has been identified as a new strain of Klebsiella pneumoniae. A novel dfr gene, designated as dfrA30, found integrated in class 1 integron was responsible for resistance to trimethoprim in Klebsiella pneumoniae strain MB45. The growth of wild strain MB45 was 100% arrested at 500 mg/L concentration of ZnO QDs. To our knowledge this is the first report on application of ZnO quantum dots to kill multiple antibiotics and serum resistant K. pneumoniae strain.

Figure 1

N-J tree based on 16S rRNA gene sequences showing the position of strain MB45 (Bold face) within the members of genus Klebsiella. Budvicia aquatica DSM 5075^T (AJ233407) used as an outgroup. Bootstrap values (>70%), expressed as a percentage of 1000 replications, are given at branching points. EMBL/GenBank accession numbers are given in parentheses. Bar, 0.005 substitutions per nucleotide position.

Figure 2

Viability and growth in diluted (10^-3) Luria broth. Bars shows standard error.

Figure 3

Effect of ZnO QDs on the growth of MB45. Cultures were setup with an initial cell number 9 × 10⁶ cells in Luria broth amended with different concentration [solid rhombus (♦), Control 0.0 mg/L; open triangle (∆), A 200 mg/L; filled triangle (▲), B 400 mg/L; open rectangle (□), C 500 mg/L] of colloidal solution of ZnO QDs. Cultures were incubated at 37°C with constant shaking at 200 rpm under normal laboratory condition. Bars shows standard error.

Figure 4

Schematic representation of the Int₂F-3'CS amplicon of the strain MB45. CS, conserved segment; intI1, integrase gene; attI1, attachment site; dfr, dihydrofolate reductase; be, base element. Black thick bar shows the distribution of integron features on amplified product. The translation start (GTG) and stop (TAA) codon are in underlined bold face. In the 59 be, the putative integrase binding sites 1L, 2L, 1R and 2R are indicated by arrows. Termination of 59 be is indicated by star (*). Numbers correspond to sequence positions in EMBL Ac. No. AM997279.

Figure 5

Phylogenetic tree showing the position of DfrA30 (Bold face) within the DfrA proteins involved in trimethoprim resistance. The number (>50%) at each major branch point refers to the percentage of times that a particular node was found in 1,000 bootstrap replications. Source and the GenBank/EMBL/DDBJ accession (in parentheses) numbers are given for each DfrA protein. DfrB proteins, which differ distinctly from DfrA proteins in size and structure, are not included in this tree. Common clusters obtained from NJ, MP and UPGMA tree are represented by hash (#).

Figure 6

Sequence alignment of DfrA30 with DfrA5 and sensitive WT-Dfr protein. The residues which form the binding site [43] are shown in red, and the positions of mutations from the wild type (WT) protein are shown in red bold letters. Residues (beyond the active site) which are known to play an important role in trimethoprim binding [44], and which have been mutated are marked in blue.

Figure 7

Surface representation of TMP binding site on DFR (PDB ID: 2W3V). Residues which form the active pocket are shown in red, and the two residues that are mutated in DfrA30 are in magenta; the remaining residues are in yellow. TMP is shown in stick model.