Waste Water Bacterial Isolates Resistant to Heavy Metals and Antibiotics
B.K. Filali,
1J. Taoufik,
1Y. Zeroual,
1F.Z. Dzairi,
2M. Talbi,
2M. Blaghen
11
Laboratoire de Biotechnologie et Environnement, Universite´ Hassan II, Faculte´ des Sciences Aı¨n chock, Km 8 route d’El Jadida, B.P. 5366 Maˆarif, Casablanca, Morocco
2
Laboratoire de Chimie Analytique, Universite´ Hassan II, Faculte´ des Sciences Ben M’sik, Casablanca, Morocco Received: 23 December 1999 / Accepted: 6 April 2000
Abstract. Sewage water of Casablanca, an industrial city in Morocco, was studied for microorganisms resistant to heavy metals. Isolates were purified and collected on agar slants to be screened for resistance to heavy metals, including mercury in vitro. The strains that showed high resistance to heavy metals were also studied for their resistance to antibiotics and aromatic hydrocarbons. Results indicated that the strains most resistant to all tested products belonged to Ps. fluorescens, Ps. aeruginosa, Klebsiella pneumoniae, Proteus mirabilis, and Staphylococcus sp. These strains exhibit high minimal inhibitory concentrations for heavy metals such as cadmium (2 m M ) or mercury (1.2 m M ). Growth of Ps. fluorescens and Klebsiella pneumoniae in the presence of heavy metals was also determined, and the growth curves indicated that mercury, copper, and zinc present a slight inhibitory action, while cadmium and silver could have a potent inhibitory action on growth compared with the controls. These studies also investigated growth in media containing aromatic compounds as the sole source of carbon. The results demonstrate that these strains could be good candidates for remediation of some heavy metals and aromatic compounds in heavily polluted sites.
Pollution due to chemicals, including heavy metals, is a problem that may have negative consequences on the hydrosphere. The most abundant pollutants in waste wa- ters and sewage are heavy metals [16]. Cobalt, copper, manganese, nickel, and zinc in trace amounts are essen- tial for growth of microorganisms, but at high concen- trations they have noxious effects on various organisms and on human health [10, 12, 20], such as emissions arising from smelting of metals, combustion of fuels, and presence in common antiseptics and disinfectants used for several years [19,21]. Various metal-resistant bacte- ria have been previously reported. They were isolated from contaminated soils, waters, and sediments [6,14].
Bacterial resistance to metal ions is frequently deter- mined by plasmids, which in many cases encode resis- tance to antibiotics [2,17]. This resistance could also be chromosomal, as reported by Smith [18] and Nakahara et al. [13]. In this report, we present several strains that show resistance to heavy metals, antibiotics, and aro-
matic hydrocarbons. The resistance to these products was also studied. These strains were isolated from metal- contaminated water.
Materials and Methods
Sample collection. Sewage samples were collected aseptically from 16 sites in Casablanca city. The samples were sown in Trypticase Soy Agar (TSA, Bio-born) and incubated at 37°C for 48 h; then a pure culture was obtained by successive isolation of colonies in TSA. Bacterial identification was done by biochemical analysis according to the standardized micromethod API 20 E and 20 NE (Biomereux).
Minimal inhibitory concentrations (MICs). The solid media Tryp- ticase Soya Agar or liquid media non-amended (controls) or amended with the respective metal element at different concentrations from stock solutions were inoculated with 100 l of cell suspensions from pre- cultures of one night diluted to 1%. The following concentrations, in
M of metal, were tested:
HgCl
2: 150 –300 – 600 –1200 CMB: 50 –100 –200 – 400 – 800 EMTA: 5–10 –20 – 40 – 80 –160 AgNO
3: 150 –30 – 600 –120 Correspondence to: M. Blaghen; e-mail: Blaghen@facsc-achok.ac.ma
Microbiology
An International Journal
© Springer-Verlag New York Inc. 2000
LaCl
3: 320 – 640 –1280 –2560 –5120 –10240 Co(NO
3)
2: 400 – 800 –1600 –3200 – 6400 BaCl
2: 80 –160 –320 – 640 –1280 ZnSO
4: 500 –1000 –2000 – 4000 – 8000 FeSO
4: 500 –1000 –2000 – 4000 – 8000
CdCl
2: 500 –1000 –3000 – 4000 – 6000 – 8000 –12000 –16000 BLiO
2: 100 –200 – 400 – 800 –1600 –3200
All plates were incubated at 37°C for 48 h.
The determination of MIC(s) was also made for various antibi- otics. Tubes containing 5 ml of nutrient broth and various antibiotics at different concentrations were inoculated with 100 l of preculture of each isolated strain diluted to 1%. Antibiotic concentrations tested were:
Ampicillin and Amoxicillin: 20 – 400 – 800 –1600 g/ml
Ceftriaxone, Cefotaxione and Rifampycine: 0.031– 0.062– 0.125– 0.25–
0.5–1–2–25–50 –100 –200 – 400 g/ml
Ciprofloxacine: 0.0625– 0.125– 0.25– 0.5–1–2– 4 – 8 g/ml
Chloramphenicol and tetracycline: 6.25–12.5–25–50 –100 –200 – 400
g/ml
Spiramycine: 0.25– 0.5–1–2– 4 g/ml
The CMI for anthracen and naphthalene were also determined.
The concentrations tested were: 0.025– 0.05– 0.1– 0.2– 0.5–1–2– 4%.
The minimal inhibitory concentration (MIC) is defined as the lowest concentration that causes no visible growth.
Growth rate of the isolates. The growth rate of each isolate in the presence of heavy metals was also determined. A set of Erlenmeyer flasks, containing 200 ml of nutrient broth and heavy metal, was inoculated with 2 ml of preculture of each isolated strain diluted to 1%.
A control for each isolate was carried out under the same conditions but without heavy metal.
Erlenmeyer flasks were incubated at 37°C with agitation of 100 rpm on a shaker. Cell growth was followed by measuring the optical density (OD
600nm) with a Cecil CE 303 spectrophotometer.
The bacterial growth in the presence of aromatic hydrocarbons as sole source of carbon was also studied by sowing Erlenmeyer flasks containing a minimum medium composed of MgSO
4(0.1 g/L);
KH
2PO
4(1.36 g/L); NH
4SO
4(0.6 g/L); CaCl
2(0.02 g/L); MnSO
4(1.1 mg/L); CuSO
4(0.2 mg/L); ZnSO
4(0.2 mg/L); FeSO
4(0.14 mg/L);
NaCl (0.5 g/L), in the presence of 2% benzene, 2% toluene, or 0.05%
naphthalene. A control blank contained the minimum medium supplied
with 2% glucose. Cultures were shaken at 100 rpm during incubation at 37°C for 24 hs. Optical density was examined at 600 nm.
Results and Discussion
Water samples sown on TSA containing heavy metal showed the presence of five bacterial strains with differ- ent macroscopic appearances. The API system identifi- cation has revealed that the strains were: Pseudomonas fluorescens, Pseudomonas aeruginosa, Klebsiella pneu- moniae, Proteus mirabilis, and Staphylococcus sp.
Minimal inhibitory concentrations (MICs) of heavy metals and organomercurials registered on solid culture medium are represented in Table 1. The tests obtained in broth media were essentially the same. The results indi- cate that these strains are strongly multiresistant. Indeed, each strain showed resistance to different compounds tested, with great MICs compared with results reported by several author [1, 3, 8].
Klebsiella pneumoniae revealed itself the most re- sistant species to all of the metals tested, followed by the species Ps. fluorescens, Ps. aeruginosa, P. mirabilis, and Staphylococcus sp. For example, the MIC in solid me- dium of K. pneumoniae to AgNO
3is over 1200 M and that of Staphylococcus sp. to LaCl
3is 5120 M , and the MICs of 2.4 dichloro-phenoxyl acetic acid were 6, 6, 4, 6 and 6 m M respectively for K. pneumoniae, P. mirabilis, and Staphylococcus sp, Ps. fluorescens, and Ps. aerugi- nosa. Resistance of the five bacterial strains to the tested organomercurials was relatively weak compared with other metals. This may be explained by their difficult breakdown by the microbial strains.
Typical growth curves of Ps. fluorescens and K.
pneumoniae in the presence of different concentrations of metal ions are shown in Figs. 1 and 2. These two strains could grow at higher concentrations (500 M ) of
Table 1. Minimal inhibitory concentrations (
M) of some heavy metals
Metal compounds Ps. fluorescens Ps. aeruginosa S. Sp K. pneumoniae P. mirabilis
CMB 400 200 400 200 200
EMTA 20 40 80 40 80
Hg
2⫹1200 1200 1200 1200 1200
Ag
2⫹1200 300 600 1200 600
La
3⫹1280 1280 5120 2560 2560
Co
2⫹3200 3200 1600 3200 3200
Ba
2⫹320 320 160 160 320
Zn
2⫹ND
aND ND 2000 2000
Fe
2⫹ND ND ND 2000 2000
Cu
2⫹6000 6000 6000 6000 6000
Cd
2⫹2000 2000 2000 4000 2000
Li
⫹800 800 400 400 800
a
ND: Not determined.
152 C
URRENTM
ICROBIOLOGYVol. 41 (2000)
mercury, with a short lag phase indicating that there is no effect on the growth properties. With the same concen- tration as for mercury, other heavy metals (silver and cad- mium) showed the same pattern with the same strains, but with a delayed lag phase for both strains (Figs. 1 and 2).
This could be explained by the toxicity of these two heavy metals, which cause a retardation in the growth of the strains compared with mercury. All strains could grow in the presence of high concentrations of copper, zinc, and nickel. However, no growth was observed with 250 M CMB [4-(chloromercuril)-benzoic acid] or EMTA.
For more information on the behavior of the micro- bial strains in a medium and the capacity of the strains to survive and grow in unfavorable conditions such as the presence of growth inhibitors, the growth rates of iso- lated strains in the presence of high concentration of heavy metals were also determined. Growth rates of the studied strains with 500 M HgCl
2were respectively 0.13, 0.22, 0.5, and 0.24 for Ps. fluorescens, Ps. aerugi- nosa, K. pneumoniae, and Staphylococcus sp.
The development of new systems for treatment would require a survey of the microorganisms present in these systems and their resistance to highly inhibitory agents. The ability of microbial strains to grow in the
presence of heavy metals would be helpful in the waste water treatment where micro-organisms are directly in- volved in the decomposition of organic matter in biolog- ical processes for waste water treatment, because often the inhibitory effect of heavy metals is a common phe- nomenon that occurs in the biological treatment of waste water and sewage.
Some authors reported that bacteria isolated from natural water showed a net resistance to antibiotics [7, 11, 15]. These bacteria were also resistant to some anti- microbial agents as well as other environmental factors [9, 13]. Under these conditions, these bacteria could survive in aquatic environments [4, 5]. Also, it is as- sumed that strains resistant to heavy metals are also usually resistant to antibiotics [6]. Our isolated strains showed a notable resistance to all tested antibiotics, and more resistance was observed to Ampicillin and amoxi- cillin (1600 g/ml) (Table 2). Spiramycin and penicillin G were tested only for Staphylococcus sp. This strain could grow in the presence of 0.5 for spiramycin and 100
g/ml for penicillin G.
According to these results, Ps. aeruginosa was the most resistant strain, followed by Ps. fluorescens, K.
pneumoniae, P. mirabilis, and Staphylococcus sp. Also,
Fig. 1. Growth curve pattern of Pseudomonas
fluorescens in the presence of 500
Mof heavy
metals and 250
Mof organomercurials.
isolated strains showed resistance to some aromatic com- pounds such as naphtalene and anthracene with MICs respectively of 0.05% and 2%.
On the other hand, the isolated strains have also been shown to utilize aromatic hydrocarbons such as naphthalene, benzene, and toluene as a sole source of
carbon and energy. The results of growth of Ps. fluo- rescens (Fig. 3) and K. pneumoniae (Fig. 4) indicate that these strains and the other three isolated strains should be used in the treatment of metal-polluted water as well as in the decontamination of water polluted by hydrocarbons.
Fig. 2. Growth curve pattern of Klebsiella pneumoniae in the presence of 500 M of heavy metals.
Table 2. Minimal inhibitory concentrations ( g/ml) of the different isolates to antibiotics, Naphthalene and anthracene
Antibiotics Ps. fluorescens Ps. aeruginosa S. Sp K. pneumoniae P. mirabilis
Ampicillin 1600 1600 1600 1600 1600
Amoxicillin 1600 1600 1600 1600 1600
Ceftriaxone 50 400 1 0.0625 ND
aCefotaxione 100 400 1 0.0625 ND
Ciprofloxacine 2 2 1 0.125 2
Rifampycine 200 400 50 2 0.0625
Chloramphenicol 400 400 400 400 12.5
Tetracycline 12.5 100 400 100 12.5
Penicillin G ND ND 200 ND ND
Spiramycine ND ND 1 ND ND
* Naphthalene 0.05 0.2 0.05 0.05 0.05
* Anthracene 2 0.05 2 2 2
* Concentrations were expressed in %.
a
