AbstractResistance to antimicrobial agents and heavy metals is inevitable as a result of natural consequence of bacterial cell adaptation to exposure to antimicrobials and heavy metals in the environment. Heavy use of bioactive substances in the medical and agricultural sectors has led to increasing resistance to those substances. This study was carried out to demonstrate antibiotic and heavy metal tolerance in bacteria isolated from drinking water supplies in Nsukka, Southeast, Nigeria. Fresh water samples were collected from different sources in different locations in Nsukka metropolis from where bacterial isolates were obtained. The isolates were assessed for their antibiotic and heavy metal tolerance by the agar disc diffusion assay. Determination of the maximum tolerable concentrations (MTC) of the metal salts was carried out by the broth dilution method. Six bacterial isolates obtained and identified tentatively based on their cultural, morphological and biochemical characteristics were Escherichia coli, Citrobacter spp., Bacillus cereus, Micrococcus varians, Proteus mirabilis and Salmonella spp. The antibiotic resistance assay revealed that Salmonella spp. and Proteus mirabilis were resistant to all antibiotics tested and fifty percent of all the isolates were sensitive to sulphafurazole and furazolidone. It was observed that Bacillus cereus had MTC values for Pb2+ (800 µg/mL), Cu2+ (700 µg/mL) and Cr6+ (200 µg/mL) while Citrobacter spp. had the least MTC value for Cr6+. There was a significant difference (p ≤ 0.05) in the effect of heavy metal concentration on the sensitive isolates. We, therefore, conclude that the water environment in Nsukka, Southeast Nigeria is a potential reservoir of antibiotic and heavy metal tolerance determinants.
Antibiotic products and heavy metals have become agents of environmental pollution as these agents affect water quality and soil health. This is attributed to industrialization and heavy use of agricultural products with high metal content in current agricultural practices . Owing to the recalcitrance or indestructibility of heavy metals occasioned by their inability to undergo chemical or biological degradation, they are difficult to eliminate from polluted sites . Naturally, the environment comprises heavy metals, but careless use by humans has changed their biochemical balance and geochemical cycles. This leads to heavy release of too many heavy metals into environments such as the water bodies and soil . Heavy metals inundate the environment through metallurgical activities, disposal of wastes, corrosion of metals, petroleum exploration and agriculture, among other anthropogenic activities. Essential metals such as Zn, Fe, Pb, Ni, Cd, Cr, Cu, Co function in osmoregulation and stabilization of molecules through electrostatic interactions and as cofactors in various enzyme-catalysed reactions . However, at high concentrations, these metals become toxic to cells. Heavy metals also affect both the biochemical activities and growth of organisms, thereby leading to decrease in biomass. High levels of metals can be found naturally in some locations that are not polluted by human activity and the metal levels at these sites can pose a potential threat to humans .
Metal tolerance reflects the capacity of an organism to survive at a high metal concentration or accumulate them without dying . Heavy metal tolerance is a phenomenon which describes the ability of bacteria and other organisms to accommodate or tolerate the effect of heavy metals on them with respect to their concentrations. Some bacteria are very sensitive to low metal concentrations while others are sensitive to high concentrations. Incidentally, products such as heavy metals, sterilants, disinfectants and antibiotics used in industries make room for the environmental pressure that selects for organisms which are better genetically endowed to survive and multiply .
Antibiotic resistance is a phenomenon used to describe a situation where a drug fails or loses its capacity to inhibit bacterial growth . However, this ability to resist antibiotics can be acquired through mutation or by transfer of antibiotic-resistance plasmids . Multidrug resistance (MDR) can also be due to the clustering of multiple antibiotic resistance genes in which one gene codes for resistance of a single antibiotic on (R) plasmids . Antimicrobial resistance in bacteria has become a problem of concern in recent times; for example, the cost of medical care is now assuming an alarming proportion with the heavy use of current antibiotics against resistant pathogens . An earlier study revealed a direct relationship between human activities (e.g. pollution) and spread; persistence of resistance genes and bacteria resistant to antibiotics within the resident bacterial communities . Furthermore, a direct relationship between antibiotic and metal resistance has been widely documented owing to the tendency that genes coding for resistance to both heavy metals and antibiotics may be clustering together on the same region of plasmids and therefore can be transferred together in the environment by one or more processes of horizontal gene transfer (8, 11]. Moreover, bacterial transposons and plasmids can carry multiple genes that encode antibiotic and metal resistance . Survival of microorganisms in polluted environments is dependent on genetic adaptation, physiological adaptation and cell morphological alterations, as well as environmental modifications of metal speciation, among other factors . The current phenomenon of multiple antibiotic/metal resistance among bacterial populations increases public health risk. The objective of the present study was, therefore, to isolate and identify some bacterial species from drinking water supply samples and assess their tolerance levels to some heavy metals and antibiotics.
2 Materials and methods
2.1 Collection of samples
Water samples were aseptically collected in sterile 20 ml bijou bottles from several locations within Nsukka metropolis. Sources of the samples included bore-holes and pipe-borne water which were immediately transported to the laboratory for the study.
2.2 Isolation and identification of bacteria
The water samples which were first serially diluted (10-fold) were then plated out on sterile nutrient agar and incubated for 24 - 48 hr at 37 oC. A number of morphologically different colonies were randomly selected and sequentially sub-cultured for purification on the same medium. The isolates were characterized and identified tentatively using standard bacteriological methods .
2.3 Preparation of heavy metal salt-impregnated paper discs
Whatman number 4 filter paper was cut into discs of uniform size (6mm, diameter) using a perforator and were sterilized in the autoclave at 121 ⁰C for 15 min. The sterile discs were soaked in each of the heavy metal salt solutions of different concentrations (500, 200, 100, 50, 20 µg/mL) for 30 min and the excess salt solution was drained off. The salts of Cu2+, Cr6+, Pb2+ and Zn2+ were used in the form of CuSO4, K2Cr2O7, Pb(NO3)2 and ZnCl2, respectively.
2.4 Heavy metal tolerance test
Heavy metal tolerance assay was carried out by the disc diffusion method. The heavy metal-impregnated discs of different concentrations were placed on nutrient agar inoculated with overnight cultures of the test bacteria. The plates were incubated at 37 ⁰C for 24 hr. After incubation, tolerance or sensitivity to a heavy metal was determined by the absence or presence of clear zone around the colonies on the plates. Isolates that showed no clear zones around them were employed in the maximum tolerable concentration assay of the tested heavy metal.
2.5 Determination of maximum tolerable concentration (MTC) of heavy metal
The maximum tolerable concentrations of the heavy metals under study were determined for the bacterial isolates using Tris-minimal salts (TMS) agar medium . The medium was composed of (g/L): KCl (1.49), D-glucose (10), NH4Cl (1.07), Tris-HCl (6.06), Na2SO4 (0.43), CaCl2.2H2O (0.03), NaCl (4.68), MgCl2.2H2O (0.2), pH 7. The tested concentrations were increased to 550-800 µg/mL for Cu2+, 600-850 µg/mL for Pb2+, 1200-1400 for Zn2+. The concentration of Cr6+ was not increased in the MTC assay since the MTC of Cr6+ for all the heavy metal-tolerant isolates were already gotten from the heavy metal tolerance test preceding MTC determination. Isolates that exhibited relatively higher sensitivities were not employed in the determination of MTC. The highest concentration of heavy metal that allowed for the growth of the bacterial isolates after incubation for 48 hr at 37 ⁰C was designated the maximum tolerable concentration (MTC) of that particular heavy metal.
The results obtained on TMS agar were confirmed in TMS broth by inoculating 0.1 mL of overnight broth culture (OD620= 0.8) of each isolate in 10 mL sterile TMS broth supplemented with respective concentrations of the metals under study. A negative control (culture medium supplemented with the same concentrations of metals without inoculation) and blank (culture medium neither with inoculation with bacteria nor heavy metal supplementation) were set up. After incubation for 48 hr at 37 ⁰C, bacterial growth was measured spectrophotometrically (UNICO 2100 UV) at 620 nm.
2.6 Antibiotic susceptibility test
Susceptibility of the bacteria to the different antibiotics used was performed by the agar-disc diffusion method  using antibiotic-impregnated discs (multi discs). The antibiotics used in the present study were oxytetracycline (50 µg), chlortetracycline (50 µg), chloramphenicol (50 µg), furazolidone (100 µg), sulphafurazole (500 µg), penicillin G (5 µg) and streptomycin (25 µg).
The antibiotic-impregnated discs were placed over freshly prepared Mueller Hinton agar seeded with overnight incubated bacterial isolates under study. Plates were incubated at 37 °C for 24 hr. The mean zones of inhibition were obtained by measuring in millimeters. The isolates were classified as resistant or sensitive by the mean diameter of inhibition zone given in standard antibiotic disc chart .
2.7 Statistical analysis
Results obtained were reported as the mean ± standard deviation of triplicate determinations. All data were subjected to one-way analysis of variance (ANOVA) using the Statistical Package for The Social Sciences (SPSS), version 20.0. Significant difference between means was established at p value of ≤ 0.05.
3 Results and discussions
3.1 Bacteria isolated
A total of six bacteria, namely, Bacillus cereus, Citrobacter spp., Micrococcus varians, Escherichia coli, Proteus mirabilis and Salmonella spp. (Table 1) were isolated from the different water samples and were identified tentatively on the basis of their cultural, morphological and biochemical characteristics. Some of these isolates have been reported  as drinking water isolates.
3.2 Heavy metal tolerance by the isolates
Isolates that were resistant to various concentrations of the different heavy metals were Bacillus cereus, Citrobacter spp, Proteus mirabilis and Salmonella spp. The above organisms have been reported by several researchers [1, 18] as having metal-tolerant attributes. Heavy metal tolerance was found in both Gram positive and Gram negative bacteria. Devika et al.  noted the same trend in a similar study in which they isolated both Gram negative and Gram positive multiple antibiotic and heavy metal tolerant bacteria from equatorial Indian ocean. This result revealed that drinking water sources are potential reservoirs for heavy metal-tolerant bacteria.
3.3 Maximum tolerable concentrations (MTC) of isolates to heavy metals
Isolates that were resistant from the heavy metal tolerance assay were subjected to MTC determination. The MTC profile of isolates is shown inTable 2.
Heavy metal tolerance is a common phenomenon in polluted environments. The MTC results revealed that Bacillus cereus had MTC value of 800 µg/mL for Pb2+, 700 µg/mL for Cu2+and 200 µg/mL for Cr6+ while Salmonella sp had MTC value of 1300 µg/mL for Zn2+. Singh et al.  recorded lower MTC value (100 µg/mL) for Zn2+ among three heavy metal-tolerant Bacillus species isolated from Kanpur, India. Citrobacter spp. had MTC (100 µg/mL) for Cr6+and Pb2+ (650 µg/mL). Proteus mirabilis had MTC (700 µg/mL) for Cu2+. In this study, all the isolates showed tolerance in the order of metal concentrations of Cr6+> Cu2+> Pb2+> Zn2+. The above trend varies slightly with Devika et al.  that had Pb2+> Cu2+ instead. However, Ahemad and Malik  in a study on prevalence of heavy metal and antibiotic resistance among bacterial isolates noted a similar trend of heavy metal resistance in the order Cu2+> Pb2+ among bacteria belonging to the genera Bacillus, Pseudomonas and Staphylococcus. Wani et al.  recorded a higher tolerance to Cr6+ by three Bacillus isolates PSB1, PSB7 and PSB10 which tolerated Cr6+ (K2Cr2O7) 550, 400 and 550 μg mL-1, respectively. Their variance with the present study can be attributed to the report of Hassan et al. , who noted that the differences that exist in the toxicity profile of bacterial isolates could be as a result of the physiological characteristics, nature of bacteria and the conditions surrounding each bacterial cell isolation. Salmonella spp and Proteus mirabilis also demonstrated tolerance to the heavy metals and antibiotics (Table 2 and Table 3). This could be as a result of the fact that the genes coding for both heavy metal and antibiotics tolerance are located on the same region on bacterial operons and are hence likely transferred together in the environment . As can be noted from the present study (Table 3), Salmonella spp. and Proteus mirabilis were the most tolerant in the antibiotic tolerance assay while Bacillus cereus was most tolerant to the varying concentrations of heavy metals except Zn2+ (Table 2).All the heavy metal-tolerant isolates shown in Table 2 have also been reported by several researchers [23, 24, 18] as exhibiting high heavy metal tolerance. These metals, have also been reported widely by several authors [1, 24] as being toxic to microbial cells. Escherichia coli was sensitive to various concentrations of the heavy metals. Ahemad and Malik  noted a similar attribute in a sensitive Escherichia coli strain which they used as the control for heavy metal and antibiotic resistance experiment.
|Probable identity||Bacillus cereus||Micrococcus varians||E. coli||Citrobacter spp||Proteus mirabilis||Salmonella spp|
|Metal ion||Bacillus cereus||Citrobacter spp.||Proteus mirabilis||Salmonella spp.|
|Antibiotic||Conc . (µg/disc)||Bacillus cereus||Micrococcus varians||Escherichia coli||Citrobacter spp.||Proteus mirabilis||Salmonella spp.|
3.4 Antibiotic tolerance of isolates
The antibiotic tolerance/susceptibility profile of the isolates is shown in Table 3. The isolates exhibited varying degrees of tolerance and susceptibility towards the antibiotics at various concentrations. All the isolates showed resistance to penicillin G at the concentrations used. Penicillin is among the first antibiotics produced and as such, organisms may have developed resistance to the natural antibiotics faster because they might have been pre-exposed to these compounds in nature. McDonnell and Trenois  recorded resistance to penicillin among forty coliform isolates tested. Odeyemi  in a similar study reported the highest resistance to penicillin. Furthermore, penicillin is active against Gram-positive bacteria. Meanwhile, the majority (66.7%) of the isolates were Gram negative (Table 1). This might also explain the high penicillin tolerance by all the Gram negative isolates in this study. Also, all the Gram negative isolates were tolerant to five antibiotics including penicillin probably because of heavy use of these antibiotics which are fundamentally intended against them as frequent drinking water enteric Gram negative isolates. Corroborative evidence of this trend was clearly documented .
In the present study, fifty percent of the isolates were susceptible to furazolidone and sulphafurazole. Furazolidone antibiotics are used heavily against diarrhoea and cholera-causing bacteria such as Escherichia coli and Vibrio cholera. Sulphafurazole is also a broad-spectrum bacteriostatic agent. Proteus mirabilis and Salmonella spp. showed high tolerance to all the antibiotics. Poonia et al.  reported a similar trend in which Escherichia, Citrobacter,and Proteusisolated from natural water sources in East Sikkim were shown to be resistant to the antibiotics against which they were tested. Multidrug-resistant strain of Salmonella typhymurium has also been identified in cattle in England and other locations .
In the present study, the bacterial isolates were relatively resistant to some antibiotics and heavy metals employed. Furthermore, it was observed that most of the antibiotic-resistant bacteria were also metal-tolerant. This observation suggests that the genes that code for antibiotic and heavy metal tolerance are horizontally transferred together in the environment.
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