Resistance of Bacillus cereus and E. coli Towards Lead, Copper, Iron, Manganese and Arsenic
Abstract: In present study, drinking ground water of different sectors of Islamabad was analyzed. Twenty-three water samples were collected in sterile bottles from different tube wells located in various sectors of Islamabad. These water samples were observed microbiologically for total viable count (TVC) and coliform count by heterotrophicplate count and MPN method, respectively. Different pathogenic bacteria were isolated and identified. Water samples of tube well number 101 and 138 were found to be contaminated and showed the presence of E.coli while the all other tube wells were contaminated with B. cereus. These bacterial species were isolated and then the tolerance of these isolated strains were checked in the presence of metals like Pb, Cu, Fe, Mn and As at different concentration (from 0.5 to 10 ppm). Results revealed that these bacteria successfully survive in these toxic conditions. As shown in results that E.coli has the ability to survive at 10 ppm of As but B. cereus can survive at 10 ppm of Mn and As. While no growth of E.coli and B. cereus was observed at 10 ppm of other metals like Pb, Cu and Fe.
Resistance of Bacillus cereus and E. coli Towards Lead, Copper, Iron, Manganese and Arsenic. Pakistan Journal of Biological Sciences, 7: 6-9.
INTRODUCTION
Microbial resistance to heavy metals is widespread. This is not surprising, as early in evolutionary history microorganism would have been in contact with toxic concentrations of heavy metals. Geo-chemical events caused the release of heavy metals from earth’s crust into the biosphere and still do. The change from a nontoxic to a toxic biosphere altered the redox state and biological availability of number of heavy metals. Resistance to a wide range of toxic metal ions has been reported in bacteria. These include resistances to metals that are purely toxic. Most heavy metals are transition elements with incompletely filled d orbitals. These d orbitals provide heavy-metal cations with the ability to form complex compounds, which may or may not be redox-active. Thus, heavy metal cations play an important role as “trace elements” in sophisticated biochemical reactions. At higher concentrations, however, heavy Bmetal ions form unspecific complex compounds in the cell, which leads to toxic effects. Some heavy-metal cations, e.g. Hg (II), Cd (II) and Ag(I) form strong toxic complexes, which makes them too dangerous for any physiological function. Even highly reputable trace elements like Zn (II) or Ni(II) and especially Cu(II) are toxic at higher concentrations. Thus, the intracellular concentration of heavy-metal ions has to be tightly Copper resistance in E.coli was first identified in enteric bacteria isolated from Australian pig farms. The plasmid born copper resistance genes (pco) was found on the conjugative plasmid and resistance was inducible by cupric salts.
Manganese exists in various oxidation states; from Mn (II) to Mn (VII) every state is possible with the Mn 2+ cations being the predominant form. Therefore it seems logical that managanese is used by bacteria as an electron acceptor in anaerobic respiration processes. Iron is the only mico-bioelement of the heavy metals. In addition siderophore-mediated uptake of Fe 3+, Fe 2+ is also transported into bacterial cells. Fe 2+ is similar in ionic diameter and charge to Mg 2+ Thus it is also accumulated by the fast and unspecific Magnesium transport system in E.coli.
Radical character of copper makes it very toxic and many organisms are more sensitive to copper than E.coli. Copper toxicity is based on the production of hydrperoide radicals and on interaction with the cell membrane. Because of toxicity, arsenic has no function in trace elements; however bacteria may use it as an electron acceptor for anaerobic respiration. Aerobic bacteria are able to oxidize arsenite again; thus a geomicrobial redox cycle of arsenic exists, similar to the iron and sulfur cycles. Lead (Pb) is not as bad as its reputation. Lead is no transition element, but belong to the element group Iva, C, Si, Ge. Sn. Pb. In seawater it is more rare than mercury (Weast, 1984). Owing to its low solubility its biologically available concentration is low. Thus lead is not extraordinary toxic for microorganisms. Lead tolerant bacteria have been isolated and the precipitation of lead phosphate within the cells of these bacteria. Thus lead resistance may also be based predominantly on metal ion efflux.
The main objective of this study to analyze the ground drinking waters for the bacterial contamination. In this study, an attempt was made to check the ability of microorganisms (E. coli and B.cereus) to degrade different concentrations of metals like Pb, Cu, Fe, Mn and As.
MATERIALS AND METHODS
Isolation procedures: Approximately 23 water samples were collected from different tube wells of different sectors of Islamabad. Samples were collected in sterile leak proof bottles following the standard methods of water collection for microbiological analysis between October and January 2003, which are given in Table 1.
Isolation and identification: Isolated colonies were picked up randomly from media plates and pure culture was obtained by further streaking. The isolated cultures were then identified by different bio-chemical tests as given in the earlier literature (Collins and Lyne, 1980, Cappuccino and Sherman, 1986 and Anonymous, 1974, 2001).
The water samples were then examined for total viable count (TVC) by serial dilutions on the Nutrient media plates and Coliform count by Most Probable Number (MPN) method.
Isolated and identified bacteria (E. coli and B. cereus from tube well number 101 and 158 respectively) were used to check the effect of different concentration like 0.5, 1, 1.5, 2, 2.5, 3, 5, 7, 8 and 10 ppm of different metals such as Lead (Pb), Copper (Cu), Manganese (Mn), Arsenic (As) and Iron (Fe) was checked. Total Viable cells were also checked by making serial dilutions of different concentration of different metals and effect of different concentration of metals was checked on total viable cells.
Temperature tolerance tests were performed to check the ability of microorganisms to grow at different temperatures like from 10 to 50°C.
RESULTS AND DISCUSSION
All water samples, which were collected according to method given in Anonymous (1989) and were examined for different pathogenic bacteria.
| Table 1: | Total bacterial count |
| Table 2: | Total bacterial count MPN values 100–1ml of sample and 95% confidence limits for various combinations of positive and negative results (when five 10ml, five 1ml and five 0.1 ml test portion are used |
| MPN = most probable number | |
| Table 3: | Biochemical properties of different identified bacteria |
B. cereus = Bacillus. cereus E. coli = Escherichia. coli + = Positive result - = Negative result | |
| Table 4: | Results of sugar test |
| B = Bacillus E. Escherichia + = Positive result - = Negative result | |
| Table 5: | growth of bacteria at different concentrations of different metals |
| ND = Not detected B. cereus = Bacillus. cereus E. coli = Escherichia. coli ppm = Part per million | |
Water samples were tested for TVC by serial dilution on nutrient agar and total coliform by MPN method. Results of TVC and MPN are given in Table 1 and Table 2.
Results showed that all the water samples were found to be contaminated except tube well number SW II, 161, 157, 169, 103 and sample collected from main supply of I-10 Markaz. In all contaminated water samples characteristics growth was observed. Bacterial colonies showed rhizoid growth, which is a characteristic growth of B. cereus, var. mycoides.
All the water samples were analyzed for contamination and different bacteria were also identified. In most cases growth of B. cereus was observed and only in tube well 101, growth of E. coli was observed. All these organisms were identified by different bio-chemical tests, which are mentioned in the material and methods.
The results showed that E. coli were Gram’s negative rods, occurring single or in pairs and catalase positive whereas B. cereus were also Gram’s negative and catalase positive. B. cereus was late Citrate positive. Hydrogen sulphide was not produced by both E. coli. and B. cereus also show a characteristics growth on nutrient media. E. coli were distinguished by confirmed test for E. coli., which is given in Bacteriology Manual. Different bacteria have the different ability to grow at different temperature. All the results related to temperature and growth in NaCl (Table 3).
Sugar tests were also performed for identification purpose. Results showed that B. cereus ferments fructose, glucose and sucrose, which were positive and all other sugars were negative, as these are not fermented by B. cereus. E. coli can ferment all other sugars except sucrose and lactose. Results of sugar test are given in Table 4.
Confirmatory Biochemical tests were done in accordance to Bergey’s manual of determinative bacteriology and Method for examination of water and food by American Public Health Association.
It is clear from this study that microorganisms have the ability to grow in the presence of above-mentioned different concentration of metals. The effect of metals on the growth of bacteria was studied and results are summarized in Table 5.
It is clear from the results that the growth of both E. coli and B. cereus was observed at lower concentration (1-5 ppm) of all different metals but the growth of E.coli and B. cereus was higher in case of Arsenic at 5 ppm and even at 10 ppm of Arsenic.
In case of lead (Pb), the growth of E. coli was not observed at 10 ppm and B. cereus was not observed at 8 ppm of lead (Pb). In case of Copper (Cu), growth of E. coli was not observed at 8 ppm of copper (Cu) and B. cereus was not observed at 10 ppm, but it can grow at 9 ppm of copper (Cu). No growth of E. coli was observed at 8 ppm of copper (Cu).
In case of Iron (Fe), growth of E. coli was not detected at 7 ppm and B. cereus at 10 ppm of Iron (Fe), but B. cereus can grow at 9 ppm of Iron (Fe), concentration. In case of Manganese (Mn), higher growth of B. cereus was observed at 10 ppm and no growth of E. coli was observed at 10 ppm of Manganese (Mn), but it can grow at 9 ppm of Manganese (Mn).
The main objective of this study was to check the quality of drinking ground water, whether it was free of contamination or not. So a complete survey of different sectors of Islamabad was performed. Results indicated that the growth of E. coli was observed in tube well no. 101 and 138 and in most samples of different tube wells, growth of B. cereus was observed. Our complete survey indicated that growth of both these bacteria was observed in water, which was supplied to different sectors of Islamabad, for drinking or other purposes. As these bacterial species are potentially pathogenic and can cause serious diseases, so the water supplied to people, should be properly treated and then supplied. It was also observed that water supplied was without proper treatment with chlorine and tube wells were at contaminated places. By removing these problems, we can get water free of contamination.