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Levels of heavy and other metals in tilapia species raised in different ponds and resistant pattern of associated Enterococcus species.

Introduction

Heavy metals are widely distributed in the environment and are not biodegradable but can be transformed into different chemical forms, often with different valance states [40]. Heavy metals pollution causes serious problems for human health and other forms of life. Some of the transformation processes result from activities related to industrialization, including combustion of fuels, or other temperature driven reactions associated with motor vehicle performance [33]. Cadmium (Cd), lead (Pb), copper (Cu), and zinc (Zn) are fuel additives [40], that are released into the atmosphere and carried to the soil through rain and wind. Chemical manufacturing, painting and coating, mining, extractive metallurgy, nuclear and other industries also release heavy metals to the environment [13].

The majority of the heavy metals are toxic to the living organisms and even those considered as essential can be toxic if present in excess. The heavy metals can impair important biochemical processes posing a threat to human health, plant growth and animal life [13,25,35]. Studies have shown that such pollutants can be harmful to life [8,38].

Bacterial diseases and infections of fish are very common and are probably one of the hardest health problems to deal with effectively to prevent losses [17]. Pathogenic bacteria are normally absorbed through the gills or gut, or gain entry through the skin and can lead to systemic infections. Bacterial infections usually resulting from environmental stress, lesions on the fish's body, gill infections, invade the fish's body and damage of internal organs [17,3].

Diseases of fish can be caused by both primary (and obligate) and opportunistic pathogens. Primary pathogens which are not part of the normal aquatic flora and are capable of causing diseases in healthy individuals include Aeromonas salmonicida. Aeromonas hydrophilia, Pseudomonas spp., Enterococcus spp. and Vibrio spp. have been reported to cause opportunistic infections. These pathogens (opportunistic) are free-living, either in the water or on the fish [22,3,31].

Enterococci are widely distributed, found mostly in air, marine and freshwater, sewage, soil, and vegetation. The primary source of enterococci is the intestine of humans and warm-blooded animals. Enterococci survive in environmental conditions that destroy other microorganisms of sanitary significance. Contrary to other faecal bacteria, enterococci can survive for a long time also outside their natural intestinal hosts.

It was reported that these bacteria can remain in seawater and mud around farms throughout the year, with seasonal changes in their distribution[41]. They cause diseases in different aquatic animals [19,20,31]. Infections caused by Enterococcus sp. strains have been described among commercially important fish species [3]. The disease has caused heavy economic losses to the fish farming industry worldwide. The transmission is thought to be horizontal by direct contact or by contaminated fish food [16,21]. The ingestion of fecal material containing the pathogen from infected fish can also be a significant route for the horizontal transmission of these pathogens [19,22].

Many studies indicate food animals as reservoirs of resistant enterococci which might be transmitted to humans through the food chain. This represents a potential risk for the consumers [5,4,18,11] as they cause various human diseases.

The aim of this study was to investigate the occurrence of enterococci in different kinds of agricultural and municipal waste waters and to test their sensitivity to selected antimicrobial agents.

Materials and Methods

Five tilapia fishes weighing between 250 to 300g were collected from each of the five ponds sampled between January and June 2009. The animals were euthanized by a standard procedure of Petrino et al. [29]. One gram each was aseptically weighed from intestine of each of the samples and homogenized in 9ml of 0.1% sterile peptone water for 5 mins in a Colwwarth Stomacher (A.J. Seward and Co. London). Thereafter, 10 fold serial dilutions were carried out. One ml of appropriate dilution was aseptically plated, using pour plate technique, on Slanetz and Bartney agar (Oxoid) and incubated at 37[degrees]C for 24h. The identification of Enterococcus spp. was carried out on 18h old culture using standard methods of Olutiola et al. [27], Fawole and Oso [10] and Schleifer and Kilpper-Balz [34].

Antibiotic Sensitivity Testing:

The isolates were grown at 37[degrees]C in Mueller-Hilton broth (Oxoid) for 16-18h and diluted to an optical density of 0.1 (0.5 McFarland Standard) at a wavelength of 625nm and stored at 4[degrees]C. The disc diffusion method was used for susceptibility testing as described by Clinical and Laboratory Standard Institute (2008). The isolates were tested against eight commercial antibiotic disks (Abtek Biologicals Limited) with their concentrations (in mg): amoxicillin (25), gentamicin (10), cotrimoxazole (25), augmentin (30), tetracycline (30), erythromycin (5),

Mineral Analyses of Pond Water, Sediment and Fish Samples:

Minerals were analyzed in the pond water, sediment and fish samples using the method of AOAC [2] and standardized by the method of Techtron [37]. All the mineral values were reported in mg/100g.

Results and Discussion

Determining the quality of the water samples from different fish ponds by measuring of dissolved heavy metals (Zn, Pb, Mn, Fe, Cu, Cd, Co and Cr) concentration in the various fish ponds, the results of the analysis are summarized in Table 1. The water from the ponds differed slightly in the concentrations metals. Water samples from pond 4 contained the highest amounts of Na, K and Ca. Concentration Pb was higher in ponds 2 and 3 with 0.25 and 0.28 mg/1001 than in the other sampling locations. The amount of Fe and Mn in the ponds were not exceeded the range of 0.05-1.00 mg/l and 0.01 -1.00 mg/l respectively set by Stone and Thomforde [36]. Heavy metal pollution and chemical profile of ponds can be reduced by Phytoplanktons which are the bioindicators of the presence of metals in an aquatic ecosystem [14,]. High water temperature, oxygen concentration, basic pH and hardness of water increase heavy metal toxicity [42]. The observed (increasing order of) concentrations of metals in the ponds are Zn > Pb > Mn > Cu > Cd. The occurrence of higher concentrations of metals in the ponds is attributed to the geology and higher concentrations in the sediments [6].

Metal mobilization in the sediment environment is dependent on physicochemical changes in the water at the sediment-water interface. The precipitation of lead, copper, manganese, chromium and zinc might be the result of alkaline pH in the form of insoluble hydroxides, oxides and carbonates [12]. Metals such as chromium, copper and nickel have interacted with organic matter in the aqueous phase and settled, resulting in a high concentration of these metals in the sediment. Mobilization of zinc and lead is also effected by higher concentrations of manganese in the sediment concentrations of metals. According to the USEPA [39] categorization all the ponds are polluted.

Co and Cr were not detected in all the samples. Na was the most abundant mineral detected in the fish samples. It ranged between 243.8 mg/100g and 278.2 mg/100g in Pond 4 and 3 respectively. Pond 4 had the highest values of K and Ca, and the least values of Zn and Fe. The amount of heavy metals in the fish gives insight to the amount that can finally get to man. Bioaccumulation and biomagnification must have responsible for the high amount of the metals in the fish compared to the levels in the water and in the sediment. Concentrations of metals in fish muscles that were observed during the study are shown in Table 3. The highest level of total trace metals ions were found in the fish muscles compare to water and sediment of the ponds. Mineral deposit in the catchment area of the studied ponds has moderate levels of trace element except Zn and Fe with 91.88 mg/100g and 74.66 respectively. Copper is an essential nutrient, but at high doses it has been shown to cause stomach and intestinal distress, liver and kidney damage, and anemia [39]. The highest iron level was found in the fish sample varied from 84.8 and 72.4 in Pond 5 and 1respectively. Magnesium is an important mineral element in connection with circulatory diseases such as ischemic heart diseases and with calcium metabolism in bone [15]. The Mg values in the fish samples ranged between 85.98 and 347.16mg/100g. The highest value was recorded in Pond. Iron content of the fish samples varied considerably. Iron is an essential trace element for haemoglobin formation, normal functioning of the central nervous system (CNS) and the oxidation of carbohydrate, protein and fat [1].

The Na/K of the fish from the ponds ranged between 1.01 and 1.29. Nieman et al. [26] had set the standard of Na/K as 0.60 to avoid the two cardiac diseases. Decreased Na/K ratio might be important for protection against hypertension and arteriosclerosis since K depresses and Na enhances blood pressure [15]. Nieman et al. [26] had set the standard of Na/K as 0.60 to avoid the cardiac diseases. The ratio of 3:4 (0.75) is considered most adequate for normal retention of protein during growth stage [15].

The resistant pattern of the enterococci isolated from water and the intestine of fish samples were shown in the Tables 4 and 5 respectively. The isolates showed varying resistant patterns. The highest resistance was observed in penincillin while the isolates had the least resistance to gentamicin. The majority of enterococci (74%) were resistant to more than one antibiotic tested. There was no significant difference in the resistance of the isolates from the water and the fish intestine (at p [less than or equal to] 0.5).

Enterococcus spp. isolated were resistant to most antibiotics used in clinical practice. This result, however, was lower than the resistance of Enterococcus spp. isolated by Ruiz-Garbajosa et al. [32] and Calva et al. [7] from clinical samples. The isolates recovered from this study were resistant to common (first hand) antibiotics. These 'first hand' antibiotics are often used in disease prevention, and their widespread use has likely contributed to high rates of resistance [7]. Since most resistance genes of large number of handy antimicrobials are located on mobile genetic elements, they are easily transmissible between bacteria [24,30]. Antimicrobials and antimicrobial residues enter the pond and may establish a selective pressure in favour of antimicrobial-resistant bacteria.

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(1) David, O.M., (1) Falegan, C.R. and (2) Ogunlade, J.T.

Departments of (1) Microbiology, (2) Animal Production and (3) Zoology, University of Ado-Ekiti, P. M. B. 5363, AdoEkiti, Nigeria.

Corresponding Author

David, O.M., Departments of Microbiology, Animal Production and Zoology, University of AdoEkiti, P. M. B. 5363, Ado-Ekiti, Nigeria. E-mail: davidgenerationng@yahoo.com
Table 1: Mineral composition of the water samples from different
fish ponds (mg/100g)

Pond      Na           K            Ca           Mg

A         165.3        125.4        33.21        48.6
B         174.1        130.9        29.66        51.58
C         182.4        143.5        35.85        56.75
D         185.7        150.3        37.5         47.54
E         176.2        142.7        28.72        50.48
Mean      176.74       138.56       32.988       50.99
SD        7.91         10.14        3.80         3.58
CV%       4.48         7.32         11.52        7.02

Pond      Zn           Fe           Cu           Mn

A         3.42         0.01         0.01         0.03
B         4.5          0.02         0.01         0.04
C         5.42         0.03         0.02         0.02
D         4.33         0.02         0.01         0.02
E         4.45         0.03         0.02         0.03
Mean      4.424        0.022        0.014        0.028
SD        0.71         0.01         0.01         0.01
CV%       16.05        45.45        71.42        35.71

Pond      Cd           Pb           Co           Cr

A         0.01         0.18         ND           ND
B         0.01         0.25         ND           ND
C         0.01         0.28         ND           ND
D         0.01         0.20         ND           ND
E         0.02         0.22         ND           ND
Mean      0.012        0.226        ND           ND
SD        0.00         0.04         ND           ND
CV%       0            17.70        ND           ND

ND = Not detected

Table 2: Mineral composition of the sediments from different fish
ponds (mg/100g)

Pond         Na           K            Ca           Mg

A            64.78        74.9         79.5         50.54
B            64.6         89.2         62.45        74.52
C            60.15        85.48        72.6         84.81
D            65.9         75.55        70.5         87
E            49.5         68.74        45.43        50.22
Mean         60.986       78.774       66.096       69.418
SD           6.787        8.363        13.055       18.007
CV%          11.13        10.62        19.75        25.94

Pond         Zn           Fe           Cu           Mn

A            2.54         2.32         0.54         1.12
B            2.35         1.54         0.48         0.95
C            2.42         2.34         0.5          1.08
D            1.98         1.75         0.36         1.25
E            1.45         1.25         0.6          0.55
Mean         2.148        1.84         0.496        0.99
SD           0.443        0.481        0.089        0.268
CV%          20.62        26.14        17.94        27.07

Pond         Cd           Pb           Co           Cr

A            0.04         0.06         ND           ND
B            0.03         0.03         ND           ND
C            0.02         0.04         ND           ND
D            0.05         0.02         ND           ND
E            0.05         0.05         ND           ND
Mean         0.038        0.04         ND           ND
SD           0.013        0.016        ND           ND
CV%          34.21        40.00        ND           ND

ND= Not detected

Table 3: Mineral composition of the fish samples from different fish
ponds (mg/100g)

Pond         Na           K            Ca           Mg

A            258.7        230.3        174.2        218.8
B            274.5        213.4        198.5        174.2
C            278.2        237.8        179.8        251.5
D            243.8        242.4        314.3        178.3
E            254.3        223.5        288.2        209.8
Mean         261.9        229.48       231          206.52
SD           14.32        11.52        65.41        31.72
CV%          5.47         5.02         28.32        15.36

Pond         Zn           Fe           Cu           Mn

A            112.5        72.4         0.01         0.04
B            109.7        74.8         0.36         0.25
C            87.2         72.6         0.21         0.22
D            72.8         68.7         0.08         0.01
E            77.2         84.8         0.11         0.01
Mean         91.88        74.66        0.154        0.106
SD           18.33        6.08         0.14         0.12
CV%          19.95        8.14         88.31        112.17

Pond         Cd           Pb           Co           Cr

A            0.02         0.2          ND           ND
B            0.04         0.18         ND           ND
C            0.11         1.21         ND           ND
D            0.13         0.22         ND           ND
E            0.44         1.12         ND           ND
Mean         0.148        0.586        ND           ND
SD           0.17         0.53         ND           ND
CV%          114.86       90.44        ND           ND

ND= Not detected

Table 4: Percentage resistance of Enterococcus spp. isolated from
water samples from different fish ponds

Antibiotics                           Ponds

               A          B          C          D          E

FUS            64 .00     45.00      62 .00     70.00      54.00
ERY            82.00      72.00      74.00      34.00      44.00
TRM            48.00      82.00      92.00      64.00      82.00
SMX            83.00      64 .00     80.00      32.00      74.00
TET            62.00      92.00      72.00      52.00      62.00
PEN            84.00      74.00      90.00      60.00      54.00
CLN            92.00      74.00      42.00      82.00      62.00
GEN            56.00      39.00      54.00      62.00      44.00

Table 5: Percentage antibiotic resistance of Enterococcus spp.
isolated from intestine fish from different fish ponds

Antibiotics                         Ponds

               A         B         C         D         E

FUS            60.00     40.00     50.00     60.00     85.00
ERY            54.00     60.00     66.00     54.00     65.00
TRM            80.00     62.00     72.00     20.00     75.00
SMX            74.00     80.00     68.00     72.00     70.00
TET            40.00     86.00     18.00     63.00     85.00
PEN            80.00     92.00     64.00     70.00     85.00
CLN            30.00     80.00     62.00     84.00     73.00
GEN            54.00     38.00     40.00     52.00     60.00
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Title Annotation:Original Article
Author:David, O.M.; Falegan, C.R.; Ogunlade, J.T.
Publication:Advances in Environmental Biology
Article Type:Report
Geographic Code:6NIGR
Date:Jan 1, 2010
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