Satish Walia

Self-Transmissible Antibiotic Resistance to Ampicillin, Streptomycin, and Tetracyclin Found in Escherichia coli Isolates from Contaminated Drinking Water

Abstract Presence and survival of cultivable bacteria in drinking water can act as a vehicle to disseminate virulence genes (adherence, enterotoxigenic and antibiotic resistance) to other bacteria. This can result in high morbidity and mortality, and the failure of the treatment of life threatening bacterial infections in humans and animals. In this study, antibiotic resistance (ABR) patterns and transferability of the ABR markers was investigated in Escherichia coli isolates obtained from drinking water and human urine samples. The ABR in E. coli isolates was determined against 15 antibiotics commonly used in human and veterinary medicine. A high frequency of ABR to carbenicillin (56%), tetracycline (53%) and streptomycin (49%) and a low frequency of cefizoxime (5%), amikacin (8%), cefazidine, (5%), chloramphenicol (9%), and kanamycin (18%) was found in the tested E. coli isolates. ABR to kanamycin (0% vs. 35%) and moxalactam (4% vs. 30%) was higher in drinking water isolates whereas resistance to streptomycin (92% vs. 15 %), ampicillin (24% vs. 10%), and nalidixic acid (12% vs. 0%) was higher in human urine isolates. A large number of E. coli isolates (93%) exhibited resistance to two or more antibiotics. Two of E. coli isolates from drinking water showed resistances to six (Cb Cm Cx Ip Mx Tc and An Cb Km Mx Sm Tc) and one was resistant to seven antibiotics (Am An Cb Km Mx Sm Tc). A majority of the multiple antibiotic resistant E. coli isolates contained one or more plasmids (size ranged ˜1.4 Kb to ˜40 Kb). The ABR traits (Am and Tc) were transferable to other bacteria via conjugation. These data raise an important question about the impact of E. coli containing self-transmissible R-plasmids as a potential reservoir of virulence genes in drinking water.

Detection of metapyrocatechase homologous genes in petroleum hydrocarbon contaminated groundwater by polymerase chain reaction

PCR assay was developed and used for rapid detection of metapyrocatechase (MPC) homologous gene sequences among hydrocarbon-degrading bacterial populations present in contaminated environments. The primers for the PCR assay were selected from the DNA sequence of MPC gene that has been cloned in Escherichia coli pAW313 after aligning with other published sequences. The primary primers, PF313 and PR313 were located 882 bp apart and were able to amplify the conserved region of the MPC homologous gene sequences under standardized PCR conditions. The nested primers, NF313 and NR313 amplified a confirmatory internal fragment of 506 bp from within the 882 bp region of MPC gene. Specific amplification of the unique 506 bp nested fragment was also obtained using DNA extracted from nine naturally occurring hydrocarbon degrading bacteria while, no amplification was observed when the DNAs extracted from 18 unrelated bacterial strains were used as template. The specificity and identity of the amplified 506 bp nested DNA fragment from hydrocarbon-degrading bacterial strains was confirmed by restriction digestion with EcoRI and by southern hybridization using digoxigenin-labeled internal probe. The MPC homologous gene sequences were also amplified when DNA directly extracted from petroleum hydrocarbon contaminated groundwater samples was used as template. Humic substances present in the groundwater samples did not inhibit the amplification reaction when the DNA extracted directly from groundwater was used for the PCR assay without further purification.

pH-dependent modulation of alkaline phosphatase activity in Serratia marcescens.

Serratia marcescens is an opportunistic pathogen responsible for causing nosocomial infections, corneal ulcer, necrotizing fasciitis, cellulites, and brain abscess. Alkaline phosphatase (APase) is believed to play an important role in the survival of several intracellular pathogens and their adaptation. We have studied the effect of low phosphate concentration and acid pH on the APase activities of S. marcescens. In a low phosphate medium, some strains of S. marcescens synthesize two different types of APases, a constitutive (CAPase) and an inducible (IAPase). Both the CAPase and IAPase isoenzymes completely lost their enzyme activities at pH 2.3, within 10 min of incubation at 0°C. Acid-treated IAPase isoenzymes I, II, III, and IV solutions when adjusted to pH 7.8 showed recovery of 70%, 52%, 72%, and 60% of the lost activities, respectively. When the pH of the CAPase reaction mixture was raised to pH 7.8, the enzyme activity regained only 5% of its initial activity. Variations in protein concentration also affected the pH-dependent reversible changes of the IAPase activity. The higher the protein concentration, the faster the inactivation of enzyme activity observed at acidic pH at 0°C. Conversely, the lower the protein concentration, the higher the rate of reactivation of enzyme activity observed for IAPase at alkaline pH. Protein interaction studies revealed a lack of similarity between CAPase and IAPase, suggesting separate genetic origin of these potentially virulent genes of S. marcescens.

Sequence analysis and molecular characterization of a nitrocatechol dioxygenase gene from Pseudomonas putida.

A Pseudomonas putida capable of degrading polychlorinated biphenyl was also found to transform 4-nitrocatechol to 3-nitro-2-hydroxy-6-oxo-hexa-2,4-dienoic acid (NHODA). Crude cell extract of this bacterium exhibited an enzyme (nitrocatechol dioxygenase, Ndo) activity catalyzing this transformation. The gene encoding Ndo was cloned in E. coli. The cloned gene (ndo) expressed in E. coli had enzyme activity that degraded not only 4-nitrocatechol but also 4-chlorocatechol, 4-methylcatechol, 2,3-dihydroxybiphenyl, and 4′-chloro-2,3-dihydroxybiphenyl. Nucleotide sequence analysis of the cloned ndo exhibited an open reading frame of 939 base pairs. This sequence can encode a 313 amino acids protein of approximately molecular weight of 35 kd, which was confirmed by in vitro transcription and translation assay and SDS-PAGE analysis. A putative ribosomal binding site (GAGGAGA) was present 7 base pairs upstream from the AUG start codon and a promotor site homologous to E. coli ‘−10’ and ‘−35’ regulatory region was located at ‘−123’ and ‘−174’ area of our clone with sequences of TTGAAG and GTGACA, respectively. The deduced amino acid sequence showed 69% homology with Cdo from Burkholderia cepacia AAI. A unique insertion of 21 amino acids was found towards the N-terminal of the Ndo. Expression of ndo in strain OU83 was repressed in presence of 3-chlorobenzoic acid as judged by the decrease in the expression of ndo specific transcript.

Progesterone binding in a clinical isolate of Pseudomonas aeruginosa

We have undertaken the characterization of progestin binding component(s) in the cytosol prepared from Pseudomonas aeruginosa isolated from an immunocompromised patient. Incubation of P. aeruginosa cytosol aliquots at 0 degrees C with 20 nM [3H]R5020 (a synthetic progestin) revealed the presence of saturable binding. The [3H]R5020 binding reached an equilibrium after 1 h at 0 degrees C and showed saturation at 30-50 nM with a Kd value of 7.7 nM. At 0 degrees C, beta-mercaptoethanol increased the [3H]R5020 binding by 20% but sodium molybdate had no effect. The [3H]R5020-macromolecular complex was stable for up to 4 h at 37 degrees C. Steroid binding specificity analysis revealed that [3H]R5020 binding could be eliminated in the presence of 2 microM progesterone, estradiol, or dihydrotestosterone but that the synthetic glucocorticoid, triamcinolone acetonide, did not compete. Postlabeling of the cytosol fractions obtained after 10-30% glycerol gradient analysis demonstrated association of the radioactivity with a molecule that sedimented as a 6-8 S protease-sensitive moiety which was unaltered in the presence of RNase or DNase. When cells were grown in the presence of 100 nM progesterone, a 50% inhibition in the number of resulting colonies was observed. In addition to its evolutionary significance, the presence of this steroid binding molecule suggests a potential in the endocrine manipulation in the treatment of infections caused by P. aeruginosa.