Lead tolerance capacity of clinical bacterial isolates and change in their antibiotic susceptibility pattern after exposure to a heavy metal

Introduction: Heavy metal pollutions of soil and wastewater are a signifi cant environmental problem as they are not degraded or destroyed. Several metal resistance mechanisms have been identifi ed which is responsible for alteration of normal cell physiology leading to development of drug resistance in microorganisms. Heavy metals used in industry and in household products are, along with antibiotics, creating a selective pressure in the environment that leads to the mutations in microorganisms. The present study was carried out to study the heavy metal lead tolerance by bacteria and change in antibiotic-sensitivity pattern after its exposure. Materials and Methods: 30 clinical isolates from various samples received in the Department of Microbiology, Government Medical College, Surat, were included in the study. To check the lead tolerance capacity, isolates were exposed to graded concentration of lead nitrate by plate dilution method, starting from 50 up to 1000 μg/ml strength. Antibiotic susceptibility was performed by the Kirby Bauer disc diffusion method. A change in antibiotic susceptibility pattern was studied before and after lead exposure. Result: 30 clinical isolates were included in the study, 25 Gram negative (83.3%) and 5 Gram positive (16.7%). MIC to lead was higher in Acinetobacter spp. and Pseudomonas spp. (600-1000 μg/ml) as compared to E. coli, Klebsiella spp., S. aureus (50-150 μg/ml). Multiple antibiotic resistance indexes were changed signifi cantly after lead exposure. Conclusion: Bacteria exposed to high levels of heavy metals in their environment have adapted to this stress by developing various resistance mechanism. Infection with antibiotic-resistant organisms create problem in treatment and management of patients. We should take efforts to prevent environmental pollution with such heavy metals and transmission of antibiotic-resistant microorganism from environment to health care set up.


INTRODUCTION
Heavy metal pollutions of soil and wastewater are a signifi cant environmental problem as they are not degraded or destroyed. [1] Some heavy metals are essential which are required by the organisms as micro nutrients and are known as "trace elements", while some heavy metals do not play any role in metabolism so they are required in low amount. In nature, there are about 50 heavy metals of special concern because of their toxicological effect to human beings and other living organisms. Many agricultural and industrial practices led to environmental pollution by heavy metals. Heavy metals infl uence the organism's population as well as it affects the growth, morphology, biochemical activities and leading to a decrease in biomass and diversity. Heavy metals can damage the cell membranes, alter enzymes specifi city, disrupt cellular functions and damage the structure of the DNA. [2][3][4] Heavy metals can be accumulated and transferred to higher organisms in food chain and lead to serious ecological and health problem. [5,6] To combat with heavy metal perfusion in the environment, bacteria have evolved several resistance mechanisms that lead to persist them in the environment or to grow. [5] Several metal resistance mechanisms have been identifi ed which include exclusion by permeability barrier, intra and extra cellular sequestration, active transport, effl ux pumps, enzymatic detoxifi cation and reduction in the sensitivity of the cellular targets to metal ions. [4] There are many known mechanisms by which resistant traits are retained and propagated in the presence of elevated chemical stressors like quaternary ammonium compounds and heavy metals, which locally infl uence resistance markers in exposed microbial populations. [7] As man further contaminates his own environment, he alters the milieu of those organisms for whom he is the host. Heavy metals used in industry and in household products are, along with antibiotics, creating a selective pressure in the environment that leads to the mutations in microorganisms that will allow them better survive and multiply. There is also evidence to indicate that there may be a correlation between the emergence of resistance to antibiotics and heavy metals. [4,8] The present study was carried out to study the heavy metal lead tolerance by bacteria and change in antibiotic sensitivity pattern after its exposure.

Sample collection
The present study was conducted during April-May 2013, in microbiology laboratory at New Civil Hospital, Surat, after having ethical permission from institutional ethical committee. Clinical samples like pus, swab, urine, CSF, blood culture and ET secretion received from various ICUs, wards, OPDs were included.

RESULTS
Thirty clinical bacterial isolates were included in the present study, out of which 25 were Gram negative (83.3%) and 5 were Gram positive (16.7%). Among Gram-negative isolates Klebsiella spp. When the organisms were exposed to lead, it was observed that Acinetobacter spp. and Pseudomonas spp. had higher MIC to lead (Pb) ranging from 750 to 1000 μg/ml and 600 to 750 μg/ml as compared to E. coli, Klebsiella spp. and Staphylococcus aureus which had lower MIC; ranging between 60 to 100, 60 to 150 and 50 to 100 μg/ml, respectively. Single isolate of S.typhi, E. cloacae and Citrobacter spp. showed MIC of 50, 270 and 672 μg/ml, respectively.
There was no change observed in morphology and biochemical reactions of the organisms after exposure of lead (Pb). Antibiotic susceptibility pattern was changed in all isolates after lead exposure. After exposure to lead, resistance to amoxicillin/clavulanic acid, co-trimoxazole, cefoxitin and amoxicillin/clavulanic acid was developed in S. aureus isolates. In Enterobacteriaceae family, third-and fourth-generation cephalosporins, gentamycin, ciprofl oxacin, co-trimoxazole and tetracycline became resistant after lead exposure. Single isolate of S. typhi was sensitive to all tested antibiotics before exposure, while resistance developed to nalidixic acid, levofloxacin and ampicillin-sulbactam after exposure to lead (Pb). In Pseudomonas spp., after lead exposure colistin, levofl oxacin and cefepime showed resistance. In Acinetobacter spp., resistance to cefepime, tetracycline, co-trimoxazole and ampicillin-sulbactam was reported after lead exposure. Change in antibiotic resistance was statistically significant for gentamycin, ciprofloxacin, cefoxitin, amoxicillin-clavulanic acid, tetracycline, co-trimoxazole and cefepime (P value < 0.05). Changes in resistance pattern of various antibiotics after lead exposure is shown in Chart 1. International Journal of Medicine and Public Health | Jul-Sep 2014 | Vol 4 | Issue 3 The multiple antibiotics resistance (MAR) index was calculated among isolates before and after lead exposure by the formula given by Kawane et al [1] According to that, MAR index for S. aureus was 0.

DISCUSSION
The present study deals with the change in antibiotic susceptibility pattern in clinical isolates after exposure to heavy metal (i.e. Pb). Resistance to number of antibiotics was increased after exposure of clinical bacterial isolates to lead. Thus, there was a signifi cant change observed in multiple antibiotic resistance index in clinical isolates before and after lead exposure.
In a study by Kawane et al, [1] 60 isolates of E. coli (30 from drinking water and 30 from clinical isolates) were included; antibiotic and heavy metal resistance patterns were studied by disc diffusion and cup method. The study revealed that 57% of clinical isolates had tolerance to heavy metal like Pb, Cd and Cu. Also, the antibiotic resistance noted in clinical isolates (64%) was more as compared to drinking water isolates (46%). The metal tolerance was 62% in water sample isolates and 57% in clinical isolates. According to the study, the incidence of high level of metal tolerance among bacteria was due to release of metal ions in water bodies due to geochemical processes. Nath et al [11] have reported MIC of soil and sewage isolates to lead as 1400-1800 μg/ml for Pseudomonas spp., 1600 μg/ml for Klebsiella spp. and 800 μg/ml for S. aureus and Proteus spp. each., which was more as compared to MIC of clinical isolates of the present study.
A study by Raja et al [12] has also reported high degree of heavy metals resistance associated with multiple antibiotic resistances in sewage bacteria. In a study by Ug et al, [13] 22 Staphylococcus spp. isolates recovered from clinical sources were studied for antibiotic and heavy metal resistance patterns and plasmid profi les. The study has observed association between the occurrence of plasmids and resistance to antibiotics and heavy metals.
In antibiotic susceptibility, it was observed that aminoglycosides, fl uroquinolones, beta-lactam-lactamse combinations, cephalosporins, tetracycline, co-trimoxazole groups of antibiotics showed more resistance after lead exposure. In the MAR index, a change after lead exposure was noted signifi cantly in Enterobacteriaceae family. The study by Nath et al [11] has also reported more resistance to aminoglycosides, beta-lactams, tetracycline, cephalosporins, fl uroquinolones and co-trimoxazole after heavy metal exposure, though the isolates were from soil. The study has reported that multiple tolerances to antibiotics are common phenomenon among heavy metalresistant isolates. The study by Atieno et al [6] also has reported higher antibiotic resistance to tetracycline, chloramphenicol, cotrimoxazole and aminoglycosides after lead exposure in isolates from wastewaters of abattoirs. The study has shown that the combined expression of antibiotic and heavy metal resistance may not be a chance phenomenon but rather a result of selection by heavy metal presence in an environment.
The transfer of heavy metals and antibiotics from agriculture and animal husbandry to the environment may cause a combined effect of selection and co-selection toward antibiotic-resistant bacteria. It has been also reported that soil and water bodies impacted by agriculture and aquaculture act as hot spots for evolution of antibiotic-resistant bacteria which require special scientifi c consideration. [14] It has been reported that even low levels of metal in soil and water may be associated with co-selection of antibiotic resistance in microorganisms. [7] Therefore, getting infection with such antibiotic-resistant bacteria will lead us in post antibiotic era, where treatment of infectious disease would be diffi cult.
The limitation of the present study was that only one heavy metal was studied. Also, the numbers of clinical isolates studied were less. Further studies must be carried out with common heavy metals of the environment and change in antibiotic resistance after their exposure.