Recent studies have shown that the frequency of antibiotic resistance has been the second highest in the genus Klebsiella especially in K. pneumoniae (next to E. coli) within the family Enterobacteriaceae; and the rate of occurrence has been noted to be higher in isolates from developing countries than the developed countries [12, 47–49]. The incidence rates of trimethoprim resistance in Klebsiella spp. and E. coli in particular have been alarming [12, 50–52] in the context of an earlier surveillance study (1987-88) on community isolates revealing an increase in trimethoprim resistance from 15.2% to 24% in Klebsiella/Enterobacter spp . The most frequent mechanism of bacterial trimethoprim-resistance is the production of an additional trimethoprim-resistant Dfr, regularly found on mobile genetic elements (plasmids, transposons, gene cassettes) [14, 17]. Other mechanisms of bacterial resistance to trimethoprim that have been described are impermeability (found in isolates of Serratia, Enterobacter, Klebsiella, Pseudomonas, and Clostridium) and mutational changes in the thymidylate synthase gene [14, 17, 53]. Besides antibiotic resistance, incidence of serum resistance in clinical isolates of pathogenic bacteria is an additional threat. There appears to be a strong correlation between serum resistance and the ability of a variety of gram-negative bacteria to invade and survive in the human blood stream . Earlier authors have developed a simple and rapid assay for determination of serum bactericidal activity using more than hundred clinical isolates of K. pneumoniae and have found that 50% were resistant to 20% normal human serum . Nosocomial septicemia due to extended spectrum β-lactamase producing K. pneumoniae and E. coli are a therapeutic challenge due to resistance . Recently, it was shown that treatment without resistance selection at the infection site with fluoroquinolone treatment can be linked to colonization of the digestive tract by K. pneumoniae (targeted pulmonary bacteria), followed by the emergence of resistance . Complete sequence of K. pneumoniae multidrug resistance plasmid pKP048 has revealed the presence of several important resistance genes, such as bla (KPC-2), bla (DHA-1), qnrB4, and armA, which confer resistance to carbapenems, cephalosporins, fluoroquinolones, and aminoglycosides, respectively . In the present study, the test strain, MB45, isolated from river Mahananda at Siliguri, India, is resistant to serum as well as to antibiotics trimethoprim, cotrimoxazole, ampicillin, gentamycin, netilmicin, tobramycin, chloramphenicol, cefotaxime, kanamycin and streptomycin and could survive in low nutrient condition (oligotrophic).
Characterization of integron-borne cassette arrays in K. pneumonia strains from China revealed a predominance of dfr and aadA cassettes that confer resistance to trimethoprim and aminoglycosides respectively . However, the distribution of dfr genes in K. pneumoniae was not known until recently a study on trimethoprim resistance in 54 trimethoprim resistant K. pneumoniae isolates have revealed the presence of dfrA1, dfrA5, dfrA7, dfrA8, dfrA12, dfrA14, dfrA17 and class 1 and 2 integrons; dfrA1 and dfrA17 being most prevalent and rarest respectively . More than 25 different TMP-resistance-mediating dfr genes isolated from different bacteria, subdivided into two major types, 1 and 2 (referred as dfrA and dfrB), have been observed till date [56, 57]. A novel trimethoprim resistance gene is claimed when the translated Dfr encoded by the gene has <95% identity at the amino acid level compared with known Dfr proteins . Since the degrees of identity between the DfrA30 and the protein sequences of other Dfr(s) ranged between 15.1 and 93%, thus placing DfrA5 and DfrA30 in an indisputable monophyletic group in the phylogenetic analysis (Figure 5).
The strain MB45 showed high level of resistance to TMP (>1500 mg/L). Generally, a single mutation in the active site is enough for TMP-resistance in Dfr, though multiple mutations are common in clinically isolated species. The mutations in the active site residues reduce the binding affinity of the enzyme for the drug. Matthews and co-workers have identified the residues that constitute the TMP-binding site in E. coli Dfr(Figure 6) . Additionally, the mutations in the active site that lead to TMP resistance in E. coli have been enunciated . The mutations in DfrA30 are of the same type as those in DfrA5. In particular, two changes, glutamine for leucine at residue 28 and isoleucine to serine at 94, would change the hydrophobic/polar nature at the two opposite sides of the TMP site (Figure 7), thus possibly weakening the binding. Other mutations (V10, W30 and I94) beyond the active site have also been identified in clinically isolated TMP-resistant genes . Some of these are also found in DfrA30 (mutations V10K and W31L) (Figure 6).
The percent occurrence of integron-positive isolates from clinical samples was much higher compared to environmental samples including fish farms [58–62], irrigation water sources  and other aquatic environments [23, 26, 64–66]. Except one recent study , in all other studies the incidence of class 1 integrons was observed for copiotrophic isolates that grow on rich nutrient medium. The test strain, MB45, is a multiple-antibiotic resistant oligotrophic bacteria recovered on 0.001X LA from river Mahananda. The facultatively oligotrophic strain used in this study was characterized as K. pneumoniae MB45 (ascertained from phenotype as well as from 16S rRNA phylogeny) (Figure 1). Viability assay and growth assessment of K. pneumoniae MB45 cell concentrate in 0.001X Luria broth for more than 72 h by taking viable cell count of the cell suspension on 1X LA at different times (Figure 2) demonstrated its ability to adapt both oligotrophic (ability to survive and grow in extremely poor nutrient conditions) as well as copiotrophic (ability to form colonies in a rich medium) conditions of growth. Such facultative nature of oligotrophy, as shown by the K. pneumoniae MB45, may contribute to the reported adaptation of remaining viable in hospital environment  for several days and cause nosocomial infection. Microbial contamination of working surfaces, clinical materials, and surgical devices poses a major threat in hospitals and intensive care units. With increasing threat because of greater incidence of multiple antibiotic resistant pathogens there is increased demand for novel disinfectants and disinfection methods. Due to exceptional physical and chemical properties of nanostructured materials there have been several attempts to improve the bactericidal activity of metal nanoparticles [31, 67, 68]. Most of these studies were confined to testing the nanoparticles on therapeutically sensitive test strains of E. coli (gram negative representative), Bacillus subtilis and Staphylococcus aureus (gram positive representatives). In an earlier study investigating the role of surface bound anionic species on zinc oxide quantum dots for the antibacterial activity against E. coli, it was shown that ZnO QDs having acetate ions had superior bactericidal activity than those described earlier . In this study, the antibacterial potency of ZnO QDs was tested against the multiple antibiotic and serum resistant K. pneumoniae MB45. The bacterial growth rate was found to be inhibited with the increase in concentration of ZnO QDs under standard cultural conditions (Figure 3). Complete inhibition of growth of MB45 is observed at concentration of 500 mg/L ZnO QDs in the medium. Hence, in future ZnO QDs could be a good nanobiotic candidate for the control of multi-drug resistant pathogens as well as in disinfecting hospital environments, external wounds, medical and surgical devices; and also in suitable format may find application in drinking water treatment plants.