Heavy Metals And Antibiotic Resistance Biology Essay

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The increasing growth of the world population also technological development has added the wastewater containing heavy metals to the environment. Pollution due to the heavy metals is a problem that may have negative consequences on the hydrosphere. Heavy metals such as Copper, Cobalt, Manganese, Nickel, and Zinc, in trace amount, are essential for the growth of microorganisms and serve as the components of enzymes, pigments, structural proteins and maintain ionic balance of cells, but at high concentrations they have noxious effects on various organisms and on human health. Also they can change the ecological balance of environment (Fillali et al., 2000; Sabry et al., 1997; Spain and Alm, 2003; Yann-Chen et al., 2006; Nwuche and Ugoji, 2008). Other heavy metals such as Cadmium, Lead and metalloids are extremely toxic because of their relative accessibility to biological systems (Sabry et al., 1997).

The presence of toxic levels of heavy metal in the environment has an inhibitory effect on most microorganisms. However they have developed resistance mechanisms that lead to the selection of resistant variants that can tolerate metal toxicity (Hassen et al., 1998, Neis, 2003 and Verma et al., 2001). As a response to the resulting metal toxicity, metal resistance determinants evolved which are mostly plasmid-encoded in bacteria.

Microbial resistance to antibiotics and metal ions is a potential health hazard because these traits are generally associated with transmissible plasmids. Since resistance genes to various heavy metals and antibiotics locate nearly on the same plasmid (Hassen et al., 1998; Neis, 2003 , Spain and Alm, 2003 ), the resistant isolate for one could be tolerate the other (Verma et al.; 2001). However, the use of microbial biomass of fungi , algae (Brinza et al., 2009) and bacteria (Ansari and Malik, 2007) for removal of heavy metals from aqueous solutions is gaining increasing attention due to low cost and safety.

Therefore, in this study, we report the natural metal tolerance levels of the bacterial community isolated from a polluted industrial wastewater. Their resistance patterns through MIC, and the multi metal resistance (MMR) to these metals are also examined. Then, the most resistant bacteria with clinical importance are selected and tested for their response to therapeutic agents.

2. MATERIALS AND METHODS

2.1. Sampling and bacterial analysis

The effluent samples from weaving factory located in Esfahan, Iran were collected in sterile glass bottles and transported on ice to the laboratory (Sabry et al., 1997; Verma et al., 2001). The amount of pollution as BOD, COD and heavy metals concentrations such as Cd , Pb , Zn and Cu (by Buck Scientific atomic absorption) were measured.

These samples were also enumerated for bacteria within 5-6h of collection, employing heterotrophic plate count by pour plate method and serial dilution technique (spread plate method) and Replica plating method for resistant bacteria. Heterotrophs and resistant bacteria were isolated and enumerated on nutrient agar and PHG II agar plates (containing: peptone, yeast extract, glucose and agar supplemented with different concentration of metals), respectively. The plates were incubated at 35 oC for 3-5 days and the total numbers of bacteria were determined as colony forming units per ml (CFU/ml) (Fillali et al., 2000, Hassen et al., 1998, Teitzel and Parsek, 2003, Yann-Chen et al., 2006).

Further isolation of bacterial colonies were done by streak plate method, then bacterial identification was done based on gram staining , morphological , cultural and biochemical characteristics following Bergey`s manual of determinative bacteriology (Noel and Holt, 1989).

2.2. Chemicals

Heavy metals tested in this study were sulfate and nitrate salts: ZnSO4.7H2O, CuSO4.5H2O,( Fillali et al., 2000, Hassen et al., 1998, Teitzel and Parsek, 2003, Verma et al., 2001; Yann-Chen et al., 2006) , Cd(NO3)2.4H2O , Pb(NO3)2 (Teitzel and Parsek, 2003 ) .The range of concentration for heavy metals were as follows (mM/L) : 0.5 , 1 , 2 , 4, 8 , 12 , 16, 24, 32 .Stocks were prepared in distilled water and sterilized with milli pore filters with 0.22 µm pore size.

The glass used was leached in 2N HNO3 and rinsed several times with distilled water to avoid metal contamination.

2.3. MIC determination

The minimum inhibitory concentration (MIC) of metals at which no colony growth occurred was determined by the agar dilution method. PHG II agar plates supplemented with different concentration of each heavy metal were inoculated aseptically with a culture of bacterial isolates in exponential growth phase. The plates were incubated for 36-48h at 35 oC. Minimum concentration of metal allowing growth of the isolates was an indication of positive tolerance.

2.4. Multi-metal resistance (MMR) determination

To determining multi-metal resistance, PHG II agar plates supplemented with heavy metals were used. Resistant colonies to one metal were chosen, and then four strains were inoculated in radial streaks (Hassen et al., 1998) on PHG II agar supplemented with the other metal and in duplicate. Plates were then incubated at 35 oC for 48h. Growth was investigated.

2.5. Antibiotic susceptibility

Susceptibility to different antibiotics for the most isolated resistant bacteria was determined by the disk diffusion method. The antibiotic saturated discs (padtan teb co. Iran) were placed on freshly prepared lawns of each isolate on Muller Hinton Agar plates, incubated at 35 oC for 24 h and examine for inhibition zones.

Discs containing the following antibiotics (µg/disc): Gentamycin (10), Carbencilin(100), Vancomycin(30), Ampicilin(10), Erithromycin(15), Cefalothin(30) Clindamycin (2), Penicillin G (10 U).

3. RESULTS

3.1. Pollution assessment:

The amount of BOD, COD, EC and pH of the effluents are shown in (table 1). The range of BOD and COD of the effluents in comparison with the standard amounts and the average of BOD and COD of Tehran`s weaving factories are shown in (fig 1). It is evident that the studied effluents were much polluted. The amounts of heavy metals in the effluents are also shown in (fig 2).

Table 1: Some of the physicochemical characteristics of industrial wastewaters

BOD COD TOC pH EC

Effluents mg/L mg/L mg/L ds/m

1: Acrilic & human 284.1 767.76 119.86 6.8 2.2

effluent

2: Acrilic effluent 654.04 1767.67 460 6.5 2.2

Fig. 1: BOD and COD of the effluents in comparison with the standard amounts and the average of BOD& COD of Tehran`s weaving factories

.

Fig 2: The amount of heavy metals in the effluents

3.2. Bacterial analysis

The average numbers of hterotrophic and resistance bacteria are given in table 2 and 3 respectively. There was not a significant difference between the effluents in the number of heterotrohs and resistant bacteria

Table 2: The average of heterotrophic bacteria (CFU/ml) in the Nutrient Agar plates.

CFU/ml

Effluent

1.33* 106

1.2*106

1

2

Table 3: The average no. of heavy metal resistant bacteria in the effluents (CFU/ml) on PHG II Agar supplemented with 0.5 mM/L of each heavy metal.

________ metal ions __________

Effluent Cd Pb Zn Cu

1 1.33*105 6.6 * 105 7.5*105 1.2*105

2 2 * 105 4 * 105 8 * 105 1 * 105

3.3. Resistance to the heavy metals

The MIC and the percentage of the isolates susceptible to various concentrations of heavy metals are shown in table 4.

The greatest resistance has been related to Zinc. It`s maximum MIC in the effluents was 24 mM/L which is related to such bacteria as Staphilococus aureus (fig3), Corynebacterium and Enterococus. It`s minimum MIC was 8 mM/L that`s of Citrobacter .

The maximum MIC for cadmium resistant bacteria was 16 mM/L that`s of Corynebacterium and the minimum was 8 mM/L that`s of some genus of Bacilus and Corynebacterium. The MIC of all of the lead resistant bacteria was 8 mM/L. The minimum degree of resistance in this study was related to Cu resistant bacteria. It`s maximum MIC was 4mM/L of Moraxella and Pseudomonas and it`s minimum is arranged as 1mM/L related to Klebsiella, and 2 mM/L related to the genus of Bacillus, Providencia and Staphilococcus.

Table 4: The number and percentage of heavy metal resistant strains in specified MIC

Total No.

24

16

12

8

4

2

*1

metal

Metal conce.

9

Ù€

Ù€

2

7

Ù€

Ù€

Ù€

No of strain

Cd

100

Ù€

Ù€

)22.2)

)77.8)

Ù€

Ù€

Ù€

Percentage of isolated strain

14

Ù€

Ù€

Ù€

14

Ù€

Ù€

Ù€

No of strain

Pb

100

(100)

Percentage of isolated strain

20

3

5

10

2

Ù€

Ù€

Ù€

No of strain

Zn

100

(15)

(25)

(50)

(10)

Percentage of isolated strain

10

Ù€

Ù€

Ù€

Ù€

3

6

1

No of strain

Cu

100

30

60

10

Percentage of isolated strain

Fig. 3: Zinc resistant colonies of Staphylococcus aureus on PHG II agar plates supplemented with 24 mM/L of zinc after 24 h incubation in 35oC

3.4. Multiple metal resistances

In examining multiple metal resistances, all isolates but one out of the 54 isolates were resistant to Zn, Pb, Cd ions and most of them were gram positive. Non of them were shown resistance to higher concentrations of Cu, but Cu resistant bacteria (with MIC of 2 & 4 mM/L) were resistant to 8 mM/L of other metals (Zn, Pb, Cd) (table 5). Most of the copper resistant bacteria in the studied effluents were gram negative.

Table 5: Multiple metal resistance pattern of the effluents bacterial isolates

Type of Isolates Resistance pattern

Multiple __________ _______________________

Resistance No % Zn Pb Cd Cu

Tetra - R 9 16.66 + + + +

Tri - R 44 81.49 + + + -

Tri - R 1 1.85 + + - +

3.5. Resistance to antibiotics

Metal resistant bacteria were tested for susceptibility to antibiotics and the results are presented in table 6. According to the results, antibiotic resistances between the tested isolates were very high.

Table 6: Susceptibility of some of the heavy metals resistant bacteria against antibiotic discs on Muller Hinton Agar plates.

antibiotic

Bacterial tested

Cf

)30)

CB

(100)

E

(15)

PnG

(10U)

V

(30)

Gm

(10)

AMP

(10µg)

Bacillus (cereus)

R

R

R

R

R

S

D=20mm

R

Enterococcus( faecalis)

R

R

S

D=28mm

R

R

S

D=15mm

R

Klebsiella

R

R

R

R

R

ND

R

Moraxella

R

R

S

D=15mm

R

R

S

D=15mm

R

Pseudomonas

R

R

R

R

R

S

D=25mm

R

Micrococcus (luteus)

R

R

R

R

R

S

D=20mm

R

Staphylococcus aureus

R

R

S

D=12mm

R

R

S

D=22mm

R

S: Sensitive R: Resistant

4. DISCUSSION

The heavy metals concentration analysis in wastewater revealed a high level of Zn, Pb, Cd and Cu, respectively. Some of the detected metals are considered to be toxic to biological systems. The concentrations of metals were comparable to those reported by earlier workers in different regions (Sabry et al., 1997). According to the results of heavy metals concentrations and the amount of BOD and COD, it is clear that the studied effluents were much polluted.

The number of resistant bacteria was less than heterotrophs. It may be because of the presence of heavy metals in high concentrations especially cadmium and lead which is known for it's high toxicity and it might have been due to the environmental factors of the study area.

Similar results have been showed by Sabry et al. (1997). Verma et al. (2000) also showed low count of resistant bacteria against heterotrophs in the presence of chromate in tannery effluent. Yann-chen et al. (2006) in their study established the toxicity of heavy metals such as Co (II), Mn (II), Zn (II) and Cd (II) to P. aeruginosa PU21. They found that, metal tolerance of PU21 to be strongly related to environmental conditions such as the type of existing metals and medium composition. The present results of metal resistance in the effluents agree with Sabry et al. (1997) in that, a positive correlation exists between heavy metals concentrations and the percentage of heavy metal resistant bacteria. They also reported the MIC of Pb and Zn between 2.5- 5 mM/L and 1.5-2.5 mM/L respectively.

Hasen et al. (1998) found the range of cadmium MIC from 0.1 to 1.5 mM/L and it is related to different genus of bacteria such as Staphylococcus, Bacillus, Streptococcus, Citrobacter, Providencia & Klebciella. They also showed the MIC of Zn resistant bacteria between 0.2-1.5mM/L. Other findings also showed different MIC for cadmium from 2 to 4 mM/L, for zinc resistant bacteria about 3mM/L (Fillali et al 2000). Thus our isolates have greater resistance which is because of genetic structure, bacterial location and environmental factors. Ansari and Malik (2007) also reported the maximum MIC 200 µg/ml for Cd, 400 µg/ml for Zn and Cu and 1600 µg/ml for Pb. In the case of Cu resistant bacteria, Sabry et al showed MIC about 2 mM/L for gram negative bacteria. Titzel et al. (2003) and Hasen et al (1998) reported the MIC for Cu about 0.03 - 2 mM/L and 0.02 - 1.8 mM/L, respectively. Our results are in agreement with those results. Sarangi et al. (2008) also isolated chromate resistant and reducing bacterial strains such as Bacillus sp. from chromium contaminated soil and they confirmed that the usage of these different resistant genera is useful for development of bioremediation.

Furthermore, it is evident that the existence of heavy metals significantly affects the microbial activity in soil and other environments. Many reports specify that heavy metals can interfere with the biochemistry of diverse group of microorganisms isolated from their natural habitat (Utgikar, 2004; Nwuche and Ugoji, 2008). Nwuche and Ugoji (2008) also reported the additive or synergistic effects of the metals.

The results revealed that, there is a positive correlation between the tolerance of high concentration of heavy metals and multi-metal resistance in bacteria. The multi-metal resistance in bacteria is in different patterns. MMR characters are often conferred by a single plasmid (Dressler, 1991; Fillali, 2000; Hassen, 1998).

The resistant bacteria in this study also showed high resistance to antibiotics. The adaptive responses of the bacterial community to several stress agents observed in the present investigation seemed to be result of sewage disposals previously stated by other investigators and using antibiotics for the treatment of infectious diseases in humans and also for a number of non-human applications. In last decade a number of studies have reported that antibiotic resistant bacteria may arise in the environment through co- or cross-resistance to metals or co-regulation of resistance pathways (Fillali et al., 2000, Matyar, 2008; Verma , 2001; Berg et al., 2005; Akinbowale et al., 2007).

Qureshi (1992) pointed out that this phenomenon may have possible implication on public health. The public health risk is further stressed by the occurrence of a high frequency of strains that are typically resistant to more than one antibiotic.

The resistance to a particular heavy metal has been correlated to antibiotics and other heavy metal resistance in a variety of organisms and the role of plasmids in conferring resistance to both antibiotics and metals has been previously demonstrated.

This study together with Ansari and Malik`s (2007) indicated that despite these toxic stresses, these bacterial isolates had evolved resistance mechanisms to cope with metal toxicity, which included volatilization, extracellular precipitation and exclusion, binding to cell surface, and intracellular sequestration. Finally we can use these resistant isolates for bioremediation because Abbas et el. (2006) have demonstrated the existence of relationship between tolerance and Cd2+ uptake using tolerant and non-tolerant isolates.

5. CONCLUSION

It is clear that a correlation exists between metal tolerance and antibiotic resistance in bacteria because of the likelihood that resistance genes to both may be located closely together on the same plasmid in bacteria and are thus more likely to be transferred together in the environment .So increasing heavy metal in the environment lead to increase bacterial resistance to these antimicrobial agent & thus prevalence of antibiotic resistant pathogenic bacteria increase and infectious disease are becoming more difficult and more expensive to treat; thus we need to not only be more careful of the drastic overuse of antibiotics in our society but also be more aware of other antimicrobial agents such as heavy metals that we put into the environment.

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