Metal tolerance analysis of Gram negative bacteria from hospital effluents of Northern India

Article history: Received on: 31/12/2016 Accepted on: 25/02/2017 Available online: 30/04/2017 Effluents from different hospitals were analysed to Nickel, Chromium, Cobalt, Copper, Mercury, Cadmium and Zinc resistance among Gram negative bacteria. The resistance among the Gram negative bacterial population varied considerably in different metal and water sampling sites. Gram negative bacteria showed lower metal resistant viable count range 4.01 x10 4 -1.3 x10 3 at 50-100 μg/ml in site-IV as compared to 11.03x105-1.03x10 4 , 12.02x10 5 -1.4x10 3 and 12.33x10 5 -2.7x10 3 in site-I, II and III against all metal tested, respectively. Viable counts of Gram negative bacterial population were recorded higher against Nickel and Zinc from sampling siteIII as compared to other sites tested. Lower viable counts of Gram negative bacteria were recorded against Mercury in all sites tested. All the isolates of Gram negative bacteria showed their tolerance level (Minimum inhibitory concentration) in the range of 50-1600μg/ml against all the metal tested. Of 88%, 76% and 86% isolates exhibited their MIC at 50-100 μg/ml against Mercury, Cadmium and Cobalt in all the sites tested, respectively. Maximum 60% and 32% of the isolates demonstrated their MIC at 1200-1600 μg/ml against Cr 2+


INTRODUCTION
Wastewater released from hospitals could be loaded with antimicrobial resistant micro-organism and toxic chemicals.Improperly treated hospital wastewater is hazardous for reuse or for releasing into natural water source (Rutala and Mayhall, 1992;Blumenthal et al., 2001).Metal pollution remains a major challenge in environmental biotechnology.Some industrial processes results in the discharge of metals into aquatic systems.The concentration of metal pollutants in the environment is usually low excluding in specific areas, which are polluted by various hospitals and industrial wastes.The concentration of heavy metals is very high in ore containing and mining areas (Roane et al., 1996).This has led to growing concern about the consequence of toxic metals as environmental pollutants.This kind of contamination presents a challenge, as the presence of metals in soils and aqueous effluents leads to severe trouble because they cannot be biodegraded.Unlike many other pollutants, metals are complicated to remove from the environment (Ren et al., 2009).Some heavy metals such as nickel, iron, copper and zinc are necessary to metabolic reactions and are required as trace elements by the organisms.Others like mercury, silver and cadmium have no biological role and are injurious to the organisms, even at very low concentrations (Hughes et al., 1989).Many bacteria have precise genetic mechanisms of resistance to toxic metals (Silver and Misra, 1988;Mindlin et al., 2001).In the environment metals, may select these resistant variants in a manner similar to the selection of antibiotic resistant strains.Indeed, it is relatively frequent the association of metal and antimicrobial resistance, since both resistance genes are commonly located on the same mobile genetic elements (Foster, 1983;McIntosh et al., 2008).Accordingly, it can be assumed that the selective pressure exerted by heavy metals contribute to the indirect co-selection of antibiotic resistance, particularly in environments polluted with the two elements.
Microorganisms resistant to both antibiotics and metals have been isolated commonly from the environments, and this has led to proposition that the combined expression of antibiotic resistance & metal resistance is caused by selection, consequential from metals present in particular environment (Bell et al., 1983;Viti and Giovannetti, 2003).The occurrence of antibiotic-resistant bacteria in the natural habitats can pose a public health risk (Nies, 1999).The study was aimed to explore the threat of wastewater generated from hospitals to the public.Wastewater treatment requires suitable methods and constant monitoring to ensure that the treated effluent be not hazardous to the environment.

Sample collection
Water samples were collected from three sites of hospital wastewater along with King George's Medical University Site -I untreated, Sanjay Gandhi Post Graduate Institute of Medical Sciences Site-II treated, Sanjay Gandhi Post Graduate Institute of Medical Sciences Site-III untreated and Dr. Ram Manohar Lohia Hospital Site -IV untreated at Lucknow city as shown in Figure 1.Samples were collected in sterile 250-ml polypropylene bottles, according to internationally recommended methodology (Lösch et al., 2008).Samples were kept at 4ºC until their arrival to laboratory.

Isolation and identification of metal tolerant Gram negative bacterial population
Isolation of metal resistant Gram negative bacteria from water samples were done on metal amended Mac conky agar plates at varying concentration (25-1200μg/ml).Serial dilutions of the water samples were plated by spreading 0.1 ml on medium for the count of total metal resistant Gram negative bacteria.Plates incubated at 37ºC for 24 hours and Gram negative bacterial counts were expressed as CFU/ml on Mac conky agar medium.The selected isolates were finally identified on the basis of biochemical characterization as described elsewhere (Cappuccino and Sherman, 1995).

Determination of minimum inhibitory concentration (MIC) of metal among different Gram negative isolates
The heavy metal resistance was determined by the minimum inhibitory concentration (MIC) against the test bacterial strain by spot plate method (Malik and Jaiswal, 2000).Nutrient plates of each heavy metal (Chromium K2Cr2O7, Cadmium CdCl2, Cobalt CoCl2, Mercury HgCl2, Copper CuSo4, Zinc ZnCl2 and Nickel NiCl2) of different concentrations (50μg/ml to 1600 μg/ml) were prepared.Inoculums of test strain (3x106 CFU/ml) were spotted on heavy metal amended plates and control plates in duplicate with the help of platinum loop of 5mm diameter.
The plates were incubated at 37 ºC for 24 hr to observe the growth of bacterial strain on the spotted area.The MIC was defined as the minimum inhibitory concentration of the heavy metal that inhibits the visible growth of test strain.Metal concentration range bellow MIC was considered as sub MIC of the isolates.

Multiple metal resistances (MMR) indexing
The MMR index profile based on isolate was evaluated to access the health risk of the environment.MMR index for test isolates was calculated according to the formula: No. of metal to which all isolates were resistant/No. of metal tested x No. of isolates.
In case of site-IV, no MIC was observed at range 50-100μg/ml against Co 2+ , Ni 2+ , Cr 3+ and Cu 2+ , respectively.76% of the isolates showed their MIC at lower concentration (50-100 μg/ml) against Hg 2+ while 46% of the isolates showed their MIC at 1200-1600 μg/ml against Cr 3+ .Maximum 72%, 66%, 42%, 38%, 34%, 16% and 12% of the isolates showed their MIC at 200-400 μg/ml against Zn 2+ , Co 2+ Cu 2+ , Cd 2+ , Ni 2+ , Hg 2+ and Cr 3+ , respectively (Fig- 5).Majority of isolates from all the sites exhibited resistance to multiple metals (Tables 3, 4 and 5).Maximum 42% and 38% of the isolates showed 6 and 5 metal resistance pattern at a time in five different combinations in site-I.Whereas, in site-II 36% and 34% of isolates exhibited resistance pattern among 4 and 6 metal at a time in two and three different combinations.In addition, of 36% and 26% isolates showed metal resistance pattern among five and six metal at a time in five and three different combinations from site-III while 60% and 38% of the isolates exhibited metal resistance pattern among 6 and 7 metals at a time in two and one combination in site-4, respectively.Resistance potential of the isolates was also evaluated in terms of multiple metal resistance indexes.A varied trend of MMR Index was observed among the isolates from the four different sampling sites.Low and high risk MMR were recorded among the Gram negative bacterial isolates from the hospital wastewater.MMR range 0.03-0.42,0.03-0.10,0.03-0.5 and 0.05-0.71were recorded among the isolates from site I (untreated), site II (treated), site III and site IV (untreated), respectively.

DISCUSSION
Metal resistance is a common process in many microorganisms that deal with toxic compounds in their habitats.In the last few years, metal resistance has increased our knowledge about the cellular mechanisms involved in metal resistance.Mercury resistance has been described in a number of bacterial species (Nakamura and Silver, 1994;Mergeary et al., 2003).In Present study, all the isolates were tested for viable count and their resistance against certain metals (Hg 2+ , Cd 2+ , Cu 2+ , Zn 2+ , Ni 2+ , Co 2+ and Cr 3+ ).The viable count of gram negative bacteria in different concentrations of metal ranged from 11.03x10 5 -8.0x10 2 , 12.02x10 5 -1.0x10 2 , 12.33x10 5 -5.0x10 2 and 4.01x10 4 -1.0 x10 2 cfu/ml of water in site I, II, III and IV, respectively.Out of 50 Gram negative bacteria isolated from all sites, 42% were found to be resistant to six metal ions at a time in five different combinations, while only 2.0% of the isolates were resistant to three metal ions at a time.36% resistance were found in four and five metals at a time in two and five different combinations in both site II and III, respectively.Maximum 60% of the isolates were found to be resistance to six metals at a time in two different combinations and 38% of the isolates were resistance in seven metals at a time in site IV (Table 3,4,5).The frequency of metal resistance in the present study is comparable to those reported elsewhere (Malik and Jaiswal, 2000;Ansari et al., 2008;Alam and Imran, 2014).Malik and Aleem, (2010) reported that the majority of bacterial isolates from metal contaminated soil showed resistance to multiple metal ions.20.8% of the pseudomonas isolates were resistant to eight metal ions at a time, while 12.5% of the isolates from groundwater irrigated soil were resistant to five metal ions at a time in two different combinations.Similar observations have also been reported earlier (Appanna et al., 1996;Malik et al., 2008;Wei et al., 2009).Present results exhibited a high incidence of metal resistance in the isolates from untreated wastewater as compared to treated wastewater (Table 2).Sabry et al, (1997) isolated heterotrophic aerobic metal-resistant bacterial communities from marine water and reported that great portion of the isolates were resistant to lead (94%), nickel (40%), arsenate (35%) and copper (22%).Similarly, Shakoori and Muneer, (2002) also reported that bacteria from wastewater origin exhibited resistance against Ag 2+ (280-350 μg/ml), Co 2+ (200-420 μg/ml), Cr 6+ (280-400 μg/ml), Cd 2+ (250-350 μg/ml), Hg 2+ (110-200 μg/ml), Mn 2+ (300-380 μg/ml), Pb 2+ (300-400 μg/ml), Sn 2+ (480-520 μg/ml) and Zn 2+ (300-450 μg/ml).In the present study, maximum 88%, 76% and 72% bacterial isolates from hospital wastewater exhibited MIC value of 50-100μg/ml for Hg 2+ while 60% and 30% of the isolates showed their MIC range 1200-1600 μg/ml against Cr3 and Cu2+ in entire site tested respectively.High levels of resistance were found in Cu 2+ , Zn 2+ Cr3+, Ni 2+ and Co 2+ respectively.This coherent with other reports where author have found that the multi-resistant strains had higher MIC values compare to the sensitive ones (Karbasizaed et al., 2003;Vajiheh and Naser, 2003;Basu et al., 1997).We also determined the MMR index of Gram negative bacterial isolates from all sampling sites.Isolates exhibited a variation in their MMR index based on sampling sites.Low and high risk MMR were recorded among the Gram negative bacterial isolates from the hospital wastewater.MMR index is ranges between 0.03-0.71among the isolates from entire sites tested.

CONCLUSION
Our observations are contributing to the understanding of metal tolerant among Gram negative bacteria from aquatic environment and underline the importance of describing the succession of bacterial populations indigenously present in such environment due to contamination events.Bacterial resistance to antibiotics and heavy metals is an increasing problem in today's society.Microbes have adapted to tolerate the presence of metals or can even use them to grow.Thus, a number of interactions between microbes and metals have important environmental and health implications.Accordingly, considerable variation in MIC of untreated and treated isolates has been found.Multiple resistances in untreated isolates have also been observed, indicating public health concern.Therefore, there is urgent need of detoxification of untreated sites.
Shakoori A R and Muneer B. Copper-resistant bacteria from industrial effluents and their role in remediation of heavy metals in wastewater.Folia Microbiologica (Praha) 2002;47: 43-50.Karbasizaed V, Badami N, Emtiazi G. Antimicrobial, heavy metal resistance and plasmid profile of coliforms isolated from nosocomial infections in a hospital in Isfahan, Iran.African Journal of Biotechnology 2003; 2: (10).379-383.

How to cite this article:
Alam M, Imran M. Metal tolerance analysis of Gram negative bacteria from hospital effluents of Northern India.J App Pharm Sci, 2017; 7 (04): 174-180.

Fig. 2 :
Fig. 2: Gram negative isolates showing various ranges of MIC of metal from site 1).

Fig. 3 :
Fig. 3: Gram negative isolates showing various ranges of MIC of metal from site 2).

Fig. 4 :
Fig. 4: Gram negative isolates showing various ranges of MIC of metal from site 3).

Fig. 5 :
Fig. 5: Gram negative isolates showing various ranges of MIC of metal from site 4).

Table 1 :
Viable count of metal tolerant Gram negative bacteria from different wastewater sampling sites.

Table 2 :
Multi-metal resistance pattern in 50 gram negative bacterial isolates from KGMU hospital (Untreated).

Table 3 :
Multi-metal resistance pattern in 50 gram negative bacterial isolates from SGPGI hospital (Treated).

Table 4 :
Multi-metal resistance pattern in 50 gram negative bacterial isolates from SGPGI hospital (Untreated).

Table 5 :
Multi-metal resistance pattern in 50 gram negative bacterial isolates from RML hospital (Untreated).