Microbiological
Communication
Biosci. Biotech. Res. Comm. 9(2):
Antibiotic susceptibility and plasmid profile of heavy metal resistant Pseudomonas species
S. Rajasekar and A. Mohankumar*
PG and Research Department of Zoology, Chikkanna Government Arts College,
ABSTRACT
Pseudomonas spp. has become increasingly recognized as an emerging opportunistic pathogen. A total of 80 isolates of Pseudomonas spp. were isolated from blood cancer patient from three tertiary hospitals in and around Tirupur and Erode (Dts). The pathogenic strains were subjected to antibiotic susceptibility testing by disk diffusion method. Ten different antibiotic discs were used to determine the drug resistance pattern of the isolates. The antibiotic resistant pattern showed that Pseudomonas spp. had high resistant against Imipenem (65%),
KEY WORDS: ANTIBIOTICS, BLOOD CANCER, HEAVY METALS, PSEUDOMONAS SPP.
INTRODUCTION
Cancer patients with numerous types of malignancies often become infected with Pseudomonas spp. A total of 25% of all gram negative bacillary infection in cancer patients are due to Pseudomonas spp. the majority occur- ring in patients with neutropenia (Rodriguex and Bodey, 1979). Pseudomonas spp. is an epitome of opportunistic nosocomial pathogen, which caused a wide spectrum of
ARTICLE INFORMATION:
*Corresponding Author: moniver65@gmail.com Received 20th May, 2016
Accepted after revision 22nd June, 2016 BBRC Print ISSN:
Thomson Reuters ISI SCI Indexed Journal NAAS Journal Score : 3.48
©A Society of Science and Nature Publication, 2016. All rights reserved.
Online Contents Available at: http//www.bbrc.in/
infection and leads to substantial morbidity in immune compromised patients despite therapy the mortality due to nosocomial Pseudomonas spp. is approximately 70%. Most of the genetic determinants that confer resistance to antibiotics are located on plasmids. These
211
Rajasekar and Mohankumar
isolates have showed maximum microbial tolerance to Cr and minimum tolerance to Pb and Cu (Singh and Lal, 2015).
The heavy metal and radioactive pollution from nuclear power plants, mining industries, electroplating industries and agricultural runoffs is a major cause of concern to public health, animals and ecosystem (Reh- man et al., 2007). Lead is hazardous waste and highly toxic to human plants animals (Low et al., 2000). One of the major mechanisms by which heavy metals including zinc cause toxicity is through the generation of free rad- icals and oxidative (Krulwich et al., 2005). Resistance to antibiotics can be conferred by chromosomal or mobile genetic elements (e.g. plasmids) and achieved using four main strategies: reduction of membrane permeability to antibiotics, drug inactivation, and rapid efflux of the antibiotic and mutation of the cellular target (Giller and Witter, 1988). Some heavy metal resistance determinants move from plasmid to chromosome.
The Pseudomonas spp. ever growing multi drug resist- ance has been widely reported (Agarwal et al., 2005). This makes plasmid encoding heavy metal resistance an important aspect of environmental research. Therefore, this present investigation has made an attempt to point out the antibiotic resistance of carcinogenic organism which is isolated from different blood cancer patients from different tertiary hospitals in and around Tirupur and Erode district, Tamilnadu and the multidrug resist- ant organism link with heavy metal resistant plasmid of cancer causing Pseudomonas spp.
MATERIALS AND METHODS
BACTERIAL STRAINS AND CULTURE CONDITIONS
A total of 80 Pseudomonas spp. isolates, isolated from blood cancer patients in and around Tirupur District. Isolation was made by adding 5ml blood obtained from peripheral veins of the patients to brain heart infusion (BHI) broth. It was incubated at 37°C and regular sub- cultures were done. Identification of the Pseudomonas spp. isolates was done by morphological and standard biochemical tests according to the manual of clinical microbiology (Cheesbrough, 1984). Identified Pseu- domonas spp. strains were stored as glycerol stocks at
Antibiotic susceptibility Test 10 antibiotics were used to determine antibiotic resistance of carcinogenic stains. These antibiotic discs were Imipenem (10mcg), Amikacin (10mcg,)
and Levofloxacin (5mcg). The susceptibility tests for each isolates were performed using Kirby Bauer disk dif- fusion method. The disk were placed on the solidified agar surface. The plates were incubated aerobically for 24 h at 37°C. The resistance were determined according to the zone of inhibition by the isolates.
GROWTH STUDIES
Growth studies of clinical bacterial isolates were studied in 250 ml flasks containing 50 ml Luria Broth medium supplemented with 0.1mM concentration of different metals such as Copper, lead and Zinc. 0.5 ml of over- night culture was added to conical flask containing cul- ture medium. It was kept in Rotary shaker at 150 rpm, 24 h. The growth of carcinogenic pathogen was checked by absorbance at 620 nm using spectrophotometer (Selvam et al., 2009).
PLASMID DNA ISOLATION
Plasmid DNA was isolated based on alkaline lysis method described by (Kado et al., 1981). Purified DNA prepara- tions were analyzed on 0.8% agarose gels stained with ethidium bromide and visualized using (Maniatis et al., 1982).
RESULTS AND DISCUSSION
Totally 80 multi drug resistant isolates were identified as Pseudomonas spp., according to Bergey’s Manual of sys- tematic bacteriology. The isolated carcinogenic isolates were tested to determine the antibiotic resistant isolates by Disc diffusion assay. The antibiotic discs of Imipenem (10mcg) Amikacin (10mcg),
Among 80 isolates 30 different antibiogram were found and showed resistance against imipenem (65%),
Isolates which are showed (50%), resistant against tested antibiotics were used for plasmid DNA isolation by alkaline lysis method. Two fragment were obtained in strain No. PA 41, PA 43, PA 44 and PA 47 with molecular size of 10,000 bp and 7000 bp respectively.
The highest level of resistance carcinogenic isolates were treated with metal curing agents such as Lead, Zinc
Rajasekar and Mohankumar
PLATE 1. Antibiotic Susceptibility Test
Lane 1: Molecular marker, Lane 2: PA 41,
Lane 3: PA 43, Lane 4: PA 44and Lane 5: PA 47
PLATE 2. Isolation of plasmid from MDR Pseudomonas spp.
Lane 1: Molecular marker, Lane 2: PA 41,
Lane 3: PA 43, Lane 4: PA 44and Lane 5: PA 47
PLATE 2.1. Isolation of heavy metal resist- ant curing plasmid from Pseudomonas spp.
and Copper with different concentration (0.2%, 0.4%, 0.6% and 0.8% mg∕ml). Cured colonies of Pseudomonas spp. were taken for the isolation of plasmid. According to four multidrug plasmid isolates, two isolates PA 41, PA 43 possessed no plasmid it denotes that the cancer causing Pseudomonas spp. was most sensitivity to cur- ing agents such as Zinc, Copper and Lead and other two heavy metals resistant PA 44 and PA 47 harbour two fragments of molecular size between 10, 000 bp and 6, 000 bp respectively.
Akingbade et al. (2012) have showed Pseudomonas spp. had high resistant to Amoxicillin (92.7%), Ampicil-
lin (90%), Cloxacillin (88.2%), Cotrimoxazole (77.3%), Erythromycin (72.7%), Tetracycline (70.9%), Strepto- mycin (65.5%), and Ofloxacin 60% and had low resist- ant to Ceftazidime (20%), Gentamycin (26.4%), Levoxin (30.9%), Ceftriaxone (34.5%) and Ciprofloxacin (35.5%). Further they carried out the plasmid profile on 22 selected multi drug resistant (MDR) isolates that were resistant to three or more classes of antibiotics. Eight (36.4%) strains were found to possess plasmid born. Six of the strains had single plasmid band while two strains possessed two bands with sizes ranging from 662 to 830bp. The sizes of the plasmids among Pseudomonas spp. isolates ranged
Rajasekar and Mohankumar
FIGURE 1. Heavy metal tolerance of Pseudomonas spp.
from 662bp to 830bp. All the strains that had plasmids were resistant to Amoxicillin, Ampicillin, Cloxacillin, Cotrimoxazole, Erythromycin and Tetracycline. In this present investigation also followed the similar method for antibiotic susceptibility test. But dissimilar results were found that the antibiotic resistant pattern showed that Pseudomonas spp. had low resistant to
The antibiotic resistance of clinical pathogen is emerging in hospital settings to adequately diagnosis pseudomonal infections and prescribe the antibiotic treatment most effective in preventing the increase in multidrug resistant organisms (Brown and Anicetus Izundu, 2004). Organisms were cultured from wound swabs (56%), high vaginal swabs (10.5%) and ear swabs (42.5%). Overall, the highest percentage rates of resist- ance were found for cofactor (100% of all isolates), Nalidixic acid (82.4%), Kanamycin (76.5%), and Tri- methoprim/Sulfamethoxazole (56.9%). Resistance rates were (25.5%) or lower for Tobramycin, Gentamicin and Polymyxin B, Cefotaxime, Ciprofloxacin and Norfloxa- cin, Piperacillin, Carbapenems and Amikacin.
Amikacin (55%) and low resistant percentage rates were found in Kanamycin (55%), and Tetracycline (55%).
Mathiyazhagan and Natarajan, (2012) have focused on the isolation of metal tolerant and antibiotic sensitive bacteria: A. ferrooxidans from bauxite and P. aeruginosa by using selective medium. These two organisms showed maximum metal resistant potentiality for the selected heavy metals (Mn, Zn, Fe, Cr, Cu and Hg). In this present investigation the cancer causing Pseudomonas spp. isolates were isolated from blood cancer patient using selective medium. The isolates were treated with differ- ent metals Copper, Lead, and Zinc with different con- centrations (0.2, 0.4, 0.6 and 0.8 μg/ml). The antibiotic susceptibility of (50%) resistant strains of were used for metal tolerant.
CONCLUSION
The present study concluded that, there is tremendous multiple drug resistance found to different class of anti- bacterial drugs. There is a quite alarming signal to note that all isolates of antibacterial Pseudomonas spp. were resistance to more antibiotics. All the isolates showed multiple drug resistance characteristics and this organ- ism link with plasmid encoding heavy metal resistant features.
ACKNOWLEDGEMENTS
The authors wish to thank Dr. K. Shanmugasundharam, Principal, Chikkanna Government Arts College, and Tirupur for providing facilities to conduct the research.
REFERENCES
Agarwal G., A. Kapil, S.K. Kabra, Das B.K. and Dwived D. (2005). Characterization of Pseudomonas isolated chronically infected children cystic fibrosis in Indian. BMC Microbiology, Vol. 5, 43.
Akingbade O.A., S.A Balogun, D.A Ojo, R.O. Afolabi, B.O. Motayo, P.O. kerentugba, I.O. and Okonko (2012). Plasmid Pro- file Analysis of Multidrug Resistant Pseudomonas aeruginosa isolated from Wound Infection in South West, Nigeria. World applied science Journal., Vol 20, Issue 6,
Brown P.D., and Izundu A. (2004). Antibiotic resistance in Clinical isolate of Pseudomonas aeruginosa in Jamaica Revm- panam salud publica, Vol. 16, Issue 2,
Edward Raja C., Anbazhagan K and Selvam G.S, (2006). Isola- tion and characterization of a metal resistant Pseudomonas
Rajasekar and Mohankumar
aeruginosa strain. World Journal of Microbiology and Biotech- nology. Vol. 22, 577- 586.
Giller KG., Witter E and McGrath S.P. (1998). Soil Biol Bioch, Vol 30,
Kado C.I., and Liu S.T. (1981). Rapid procedure for detec- tion and isolation of large and small plasmids. J. Bacteriol., Vol.145,
Krulwich TA., and Lewinson O. (2005). Do physiological roles foster persistence of drug/multidrug- efflux transporters? A case study. Nat. Rev. Microbiol. Vol. 36,
Low K.S., Lee C.K. and Liew S.C. (2000). Sorption of Cadmium and Lead form aqueous solution by spent grain. Proc. Bio- chemist., Vol.36, Issue
Maniatis T. E. F., Fritsch and Sambrook J. (1982). “Molecu- lar Cloning, A Laboratory Manual”, Cold Spring Harbor, New York.
Narayanan Mathiyazhagan and Devarajan Natarajan, (2012). Metal and Antibiotic Tolerance Potentiality of Acidithio bacil- lus Spp. and Pseudomonas spp. from waste Dumps of bauxite and magnesite Mines. Archives of Applied Science Research, vol 4, Issue 1,
Rehman A and Shakoori FR. (2007). Multiple metal resistance and uptake by a ciliate, Stylonychia mytilus, isolated from industrial effluents and its possible use in wastewater treat- ment. Bull. Environ. Contam. Toxicol., Vol 79, Issue4,
Rodriguez V and Bodey G. P. (1979). Epidemiology, clinical manifestations and treatment in cancer patients in R. G. Dog- get (ed.), Pseudomonas aeruginosa- clinical manifestations of infection and current therapy. Academic Press, Inc., New York,
Schelz Z., Molnar J and Hohmann. J (2006). Antimicrobial and anti plasmid, activities of essential oils. Fitoterapia, Vol 77,
Yogendra Singh and Nand Lal (2015). Investigation on the heavy metal resistant bacterial isolates in vitro from industrial effluents. World Journal of Pharmacy and Pharmaceutical Sci- ence. Vol 4, Issue 02, 345
ConvertedByBCLTechnologies