Study of pandrug and heavy metal resistance among E. coli from anthropogenically influenced Delhi stretch of river Yamuna

Abstract Escalating burden of antibiotic resistance that has reached new heights present a grave concern to mankind. As the problem is no longer confined to clinics, we hereby report identification of a pandrug resistant Escherichia coli isolate from heavily polluted Delhi stretch of river Yamuna, India. E. coli MRC11 was found sensitive only to tobramycin against 21 antibiotics tested, with minimum inhibitory concentration values >256 µg/mL for amoxicillin, carbenicillin, aztreonam, ceftazidime and cefotaxime. Addition of certain heavy metals at higher concentrations were ineffective in increasing susceptibility of E. coli MRC11 to antibiotics. Withstanding sub-optimal concentration of cefotaxime (10 µg/mL) and mercuric chloride (2 µg/mL), and also resistance to their combinatorial use, indicates better adaptability in heavily polluted environment through clustering and expression of resistance genes. Interestingly, E. coli MRC11 harbours two different variants of blaTEM (blaTEM-116 and blaTEM-1 with and without extended-spectrum activity, respectively), in addition to mer operon (merB, merP and merT) genes. Studies employing conjugation, confirmed localization of blaTEM-116, merP and merT genes on the conjugative plasmid. Understanding potentialities of such isolates will help in determining risk factors attributing pandrug resistance and strengthening strategic development of new and effective antimicrobial agents.

Effect of medicinal plants, Heavy metals and antibiotics against P pathogenic bacteria isolated from raw, Boiled and pasteurized milk

Present study has been undertaken to isolate and identify the bacterial flora in raw, boiled and pasteurized milk. Agar disc diffusion method was used to determine their sensitivity using medicinal plants, antibiotics and heavy metals. Methylene blue reduction test was used to test the quality of milk samples. Total 10 pathogenic strains were isolated, five strains were isolated from raw milk, three from boiled milk and 2 two from pasteurized milk. To determine optimum conditions for growth, these pathogenic microorganisms were incubated at various temperatures and pH. Gram's staining and biochemical tests revealed that these pathogenic bacteria include Lactobacillus sp., E. coll, Salmonella sp., Pseudomonas sp., Streptococcus sp. and Staphylococcus. Ribotyping revealed S2 as Pseudomonas fluorescens, S5 as Lactococcus lactis and S9 as Lactobacillus acidophilus. Prevalence of pathogenic organisms provided the evidence that contamination of milk arises during milking, transportation and storage of milk. Raw milk is more contaminated than other two types of milk because it contains highest percentage of pathogenic organisms and pasteurized milk was found to be of best quality among three types. So it is recommended to drink milk after proper boiling or pasteurization. Proper pasteurization and hygienic packing of milk is essential to minimize contamination in milk which can save human beings from many milk borne diseases. Our study suggests that antimicrobial use in animal husbandry should be minimized to reduce the hazard of antibiotic resistance. Plant extracts are better alternative against pathogenic bacteria in milk

Plant Growth Promotion and Root Colonization by EPS Producing Enterobacter sp. RZS5 under Heavy Metal Contaminated Soil.

The heavy metal resistant bacterium isolated from field soil and identified as Enterobacter sp. RZS5 tolerates a high concentration (100-2000 mM) of various heavy metal ions such as Mn2+, Ni2+, Zn2+, Cu2+, CO2+ and Fe2+ when grown in such environment and produces exopolysaccharides (EPS). Here, we have demonstrated EPS production by Enterobacter sp. RZS5 during 60 h of growth in yeast extract mannitol broth (YEMB). The yield increased by two fold after the addition of 60 M of Ca2+; 50 M of Fe2+ and 60 M of Mg2+ ions in YEMB, and the optimization of physico-chemical parameters. EPS was extracted with 30% (v/v) of isopropanol as against the commonly used 50% (v/v) isopropanol method. EPS-rich broth promoted seed germination, shoot height, root length, number of leaves and chlorophyll content of wheat (Triticum aestivum) and peanut (Arachis hypogaea) seeds. The higher colony-forming unit of Enterobacter sp. in soil inoculated with EPS rich broth of Enterobacter sp. indicated the root colonizing potential and rhizosphere competence of the isolate. The FTIR spectra of the EPS extract confirmed the presence of the functional group characteristics of EPS known to exhibit a high binding affinity towards certain metal ions. This overall growth and vigour in plants along with the effective root colonization, reflected the potential of the isolate as an efficient bio-inoculant in bioremediation.

Heavy Metal Resistance Strategies of Acidophilic Bacteria and Their Acquisition: Importance for Biomining and Bioremediation

Microbial solubilizing of metals in acid environments is successfully used in industrial bioleaching of ores or biomining to extract metals such as copper, gold, uranium and others. This is done mainly by acidophilic and other microorganisms that mobilize metals and generate acid mine drainage or AMD, causing serious environmental problems. However, bioremediation or removal of the toxic metals from contaminated soils can be achieved by using the specific properties of the acidophilic microorganisms interacting with these elements. These bacteria resist high levels of metals by using a few "canonical" systems such as active efflux or trapping of the metal ions by metal chaperones. Nonetheless, gene duplications, the presence of genomic islands, the existence of additional mechanisms such as passive instruments for pH and cation homeostasis in acidophiles and an inorganic polyphosphate-driven metal resistance mechanism have also been proposed. Horizontal gene transfer in environmental microorganisms present in natural ecosystems is considered to be an important mechanism in their adaptive evolution. This process is carried out by different mobile genetic elements, including genomic islands (GI), which increase the adaptability and versatility of the microorganism. This mini-review also describes the possible role of GIs in metal resistance of some environmental microorganisms of importance in biomining and bioremediation of metal polluted environments such as Thiomonas arsenitoxydans, a moderate acidophilic microorganism, Acidithiobacillus caldus and Acidithiobacillus ferrooxidans strains ATCC 23270 and ATCC 53993, all extreme acidophiles able to tolerate exceptionally high levels of heavy metals. Some of these bacteria contain variable numbers of GIs, most of which code for high numbers of genes related to metal resistance. In some cases there is an apparent correlation between the number of metal resistance genes and the metal tolerance of each of these microorganisms. It is expected that a detailed knowledge of the mechanisms that these environmental microorganisms use to adapt to their harsh niche will help to improve biomining and metal bioremediation in industrial processes.

Hormesis response of marine and freshwater luminescent bacteria to metal exposure

The stimulatory effect of low concentrations of toxic chemicals on organismal metabolism, referred to as hormesis, has been found to be common in the widely used luminescence bioassay. This paper aims to study the hormesis phenomenon in both marine and freshwater luminescent bacteria, named Photobacterium phosphorem and Vibrio qinghaiensis. The effects of Cu (II), Zn (II), Cd (II) and Cr (VI) on luminescence of these two bacteria were studied for 0 to 75 minutes exposure by establishing dose- and time-response curves. A clear hormesis phenomenon was observed in all four testing metals at low concentrations under the condition of luminescence assays.

Changes in antioxidant enzyme activities in Eichhornia crassipes (Pontederiaceae) and Pistia stratiotes (Araceae) under heavy metal stress

Whole plants of Eichhornia crassipes and Pistia stratiotes were exposed to various concentrations (0, 0.1, 0.3, 0.5, 1.0, 3.0 and 5.0 mM) of 8 heavy metals (Ag, Cd, Cr, Cu, Hg, Ni, Pb and Zn) hydroponically for 21 days. Spectrometric assays for the total activity of catalase, peroxidase, and superoxide dismutase in the leaves were studied. At the end of the experimental period, data referred to metal treated plants were compared to data of untreated ones (control). Heavy metals increased the activity of catalase, peroxidase and superoxide dismutase in both species and there was differential inducement among metals. Overall, Zn had the least inducement of antioxidant enzymes in both species while Hg had the highest inducement. The increase in antioxidant enzymes in relation to the control plants was more in E. crassipes than P. stratiotes. The results showed that E. crassipes tolerated higher metal concentrations in a greater number of metals than P. stratiotes. Rev. Biol. Trop. 55 (3-4): 815-823. Epub 2007 December, 28.

Plantas completas de Eichhornia crassipes y Pistia stratiotes fueron expuestas a varias concentraciones (0, 0.1, 0.3, 0.5, 1.0, 3.0 and 5.0 mM) de metales pesados (Ag, Cd, Cr, Cu, Hg, Ni, Pb and Zn) utilizando hidroponía, por 21 días. Se realizaron análisis espectrométricos en las hojas para determinar la actividad total de la catalasa, peroxidasa y dismutasa superóxida. Al final del periodo experimental, se comparó con plantas no tratadas (control). Los metales pesados incrementan la actividad de la catalasa, peroxidasa y la dismutasa superóxida para ambas especies y hay diferencias entre los metales. El Zn produce el menor estímulo para enzimas antioxidantes en ambas especies; Hg produce el mayor estímulo. El incremento de las enzimas antioxidantes en relación con las plantas control fue mayor en E. crassipes que P. stratiotes. E. crassipes tolera altas concentraciones de metal en un gran número de ellos, mientras que la tolerancia en P. stratiotes es menor.

Impact of Ni(II), Zn(II) and Cd(II) on biogassification of potato waste.

A study was conducted on anaerobic digestion of potato waste and cattle manure mixture, inoculated with 12% inoculum and diluted to 1:1 substrate water ratio at 37 +/- 1 degrees C. Initially pH of substrate was found to be 4.5 to 5.0. Lime and sodium bicarbonate solutions were employed to adjust the pH to 7.5. Biogas production continued up to 10 and 7 days, when lime and sodium bicarbonate solutions were used to adjust the pH, respectively. Biogassification potential was studied in response to different ratio of waste and cattle manure. Biogas production rate was higher when potato waste and cattle manure were used in 50:50 ratio. Effect of two different concentrations (2.5 and 5.0 ppm) of three heavy metals viz. (Ni (II), Zn (II) and Cd (II)) on anaerobic digestion of substrate (potato waste--cattle manure, 50:50) was studied. At 2.5 ppm, all the three heavy metals increased biogas production rate over the control value. The percentage increase in biogas production over the control was highest by Cd, followed by Ni and Zn. In all the treatments, methane content of biogas increased with increase in time after feeding. Various physico-chemical parameters viz. total solids, total volatile solids, total organic carbon and chemical oxygen demand considerably declined after 7 days of digestion and decline was greater in presence of heavy metals as compared to control. The physico-chemical parameters revealed maximum decrease in the presence of 2.5-ppm concentrations of heavy metals with the substrate. Among all the three heavy metals employed in the study, Cd++ at 2.5 ppm was found to produce maximum biogas production rate. The use of three heavy metals to enhance biogas production from potato and other horticultural waste is discussed.

Effect of water stress and heavy metals on induction of somatic embryogenesis in wheat leaf base cultures.

In vitro cultures of plant tissues are known to mimic the response of field-grown plants when subjected to stress treatments. This investigation on Triticum aestivum explores the effect of drought stress on somatic embryogenesis and endogenous proline content. Leaf bases were cultured on MS medium supplemented with 2,4-D (10 microM) and different concentrations of PEG (2.5, 5, 7.5%) or mannitol (0.25 and 0.5 M) and also subjected to different periods of aerial drying in the laminar flow for one-day and subsequently transferred to MS basal medium. PEG treatment induced a high percentage (up to 50%) of embryoid formation. However, with mannitol and aerial drying, percentage of embryoid formation decreased with increasing concentrations and duration. After ten days, the endogenous proline content of explants treated with different concentrations of PEG, mannitol and different durations of aerial drying increased with increasing concentration and increasing duration of the treatment, thus, corroborating the role of proline as an osmolyte during stress conditions. Similarly, addition of metals such as cadmium and cobalt caused a reduction in percentage explants depicting embryogenesis. However, when cadmium was employed alone, 22% explants displayed somatic embryogenesis as compared to 54% in 2,4-D treated cultures.

Resistência a antibióticos e a metais pesados de Escherichia coli isoladas de agua do mar e galerias pluviais / Resistance to antibiotics and heavy metals from Escherichia coli isolated from sea water and pluvial galeries

OBJECTIVE: Test the resistance to antibiotics and heavy metals of E. coli strains isolated from storm sewer water and adjacent seawater samples from three beaches (Meio, Area Preta and Ponta Negra) in the city of Natal/RN/Brazil, and determine the association among those characteristics. METHODS: A total of 98 strains of E. coli, 50 from storm sewers and 48 from the seawater were analyzed resistance to several antimicrobials by disk diffusion and agar dilution and to heavy metals by dilution in plates with aqueous solutions of CuSO4 incorporated to Mueller Hinton agar in concentrations of 100, 150, 200 and 250 ig/mL and HgCl2 in concentrations of 5, 10, 20, 30, and 50 ig/mL. Standard strains were used as control. RESULTS: Among the twelve antimicrobials tested, 28 (28.5%) of E. coli strains showed resistance to different antimicrobials drugs to seven. The greatest resistance rate was to tetracycline (46.4%), ampicillin (39.3%) and cephalothin (32.1%), with the remainder (nitrophurantoine, nalidixic acid, sulfatomexazol-trimethoprin and chloramphenicol) at lower percentages. Among the heavy metals, all the strains (100%) were resistant to zinc and to copper in the largest concentration (250 ig/mL), and 18.4% were resistant to HgCl the 50 ig/mL. Ten (55.5%) of the E. coli strains resistant to Hg were associated to resistance to antibiotics. CONCLUSION: These results suggest the existence of extra chromosomal genes in E. coli strains isolated from storm sewer water and adjacent seawater, which encoders of the resistance to antibiotics and heavy metals.

R-factor in Proteus vulgaris from ulcerative disease of fish, Channa punctatus.

A Proteus vulgaris isolated from external ulcers of the fresh water fish Channa punctatus showed multidrug resistance and heavy metal tolerance. The isolate from the ulcer showed resistance to chloramphenicol (Ch), nalidixic acid (Nx), streptomycin (Str) and tetracycline (Tet) with minimum inhibitory concentration (MIC) values of 750, 150, 75 and 125 microg/ml, respectively. The isolate showed growth in medium containing cadmium (Cd2+), up to a concentration of 2.5 mM indicating its heavy metal tolerance. Resistance to Ch, Str, Tet and Cd2+ of the isolate was lost after plasmid curing. Presence of plasmid DNA in the wild type and its absence in the cured P. vulgaris suggested that the resistance were plasmid mediated.

Biochemical responses of Camellia sinensis (L.) O. Kuntze to heavy metal stress.

Three heavy metals-mercury (II), copper (II) and nickel (II), each at a concentration of 10 and 100 micrograms/ml, were tested for their effects on various biochemical constituents of tea leaves. Both NI (II) and Hg (II) decreased the phenolic contents, while Cu (II) increased it to some extent. The metal treatments enhanced the activity of phenyl alanine ammonia lyase (PAL), while the activity of poly phenol oxidase (PPO) showed a decline. Heavy metal stress also decreased the chlorophyll content of the leaves, along with a significant reduction in Hill activity. Proline content increased significantly in all treatments.