Punjab Univ. J. Zool., Vol. 26(2), pp. 113-124, 2011

0079-8045/011/0113-0124 $ 03.00/0 Copyright 2011, Dept. Zool., P.U., Lahore, Pakistan

STUDY OF ANTIBIOTIC RESISTANCE IN LOCALLY ISOLATED BACTERIAL AND FUNGAL STRAINS

ASMA PERVEEN, SAIQA ANDLEEB, SHAUKAT ALI*,

FOUZIA AZIZ AND HAFIZ ABDULLAH SHAKIR

Biotechnology lab., Department of Zoology, The University of Azad Jammu and Kashmir (AP, SA, SA, FA), Muzaffarabad; Department

of Zoology, University of the Punjab, Lahore (HAS), Pakistan.

Abstract: In this study some antibiotics were selected to control the mycorhizae fungus (SAZAK2010) and other bacterial species including Escherichia coli, Klebsiella pneumoniae and Enterococcus faecalis through standard agar discs diffusion method. By antifungal activity it was shown that SAZAK2010 was sensitive against tobramycin, gentamicin, tetracycline, ciprofloxacin, streptomycin, and kanamycin and resistance against ampicillin, amoxicillin and penicillin G. On the other hand, the antibacterial activity of antibiotics against E. faecalis indicated that it was susceptible to various antibiotics such as ciprofloxacin, streptomycin, kanamycin, gentamicin, tetracycline, and amoxicillin, whereas resistant to tobramycin and tetracycline. Interesting results were observed in case of biotechnological bacterial 10b strain E. coli, which indicated sensitivity against all used antibiotics on both MacConky agar as well as Nutrient agar medium. Whereas, it was observed that K. pneumoniae was susceptible to tetracycline, ciprofloxacin, kanamycin, gentamicin, tetracycline, and amoxicillin but showed resistance against penicillin G. The genomic DNA was extracted from SAZAK2010, E. coli, K. pneumoniae and E. faecalis through modified techniques for finding disease controlling genes. . Key words: Antibacterial activity; fungi, agar disc diffusion method.

INTRODUCTION

Pathogens associated with food products are very harmful for human and animal health. These pathogens penetrate into body, cause diseases to host and lead to the opportunistic infections. The various

pathogens like bacteria, fungus and viruses are transmitted through the primary pathway by eating and drinking sewerage contaminated food and water, respectively. Today many medical advances have been made to

*corresponding author: shaukatali134@yahoo.com

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control the infections caused by pathogens. The infections can be treated through the use of antibiotics, vaccination, bactericides and fungicides, but the pathogens continue to threaten the human life.

In previous studies it was shown that essential oil had great importance to control the important fungal F. culmorum and F. solani infections and diseases (Morris et al., 1979; Bagci and Digrak, 1996; Lis-Balchin et al., 1998; Mirosława et al., 2002). Dynowska (1998) assessed the antifungal activity of the essential oils from Pinus ponderosa, Pinus resinosa and Pinus strobus against three species of genus Fusarium such as F. culmorum, F. poae and F. solani, respectively. Dynowska (1998) demonstrated that F. solani may be the etiological agent of numerous skin lesions, inflammation of the internal structures in eye and joints. On the other hand, the antimicrobial activity of essential oils has also been determined (Morris et al., 1979) and some plant extracts were also used against fungus (Eloff, 1998; Kone et al., 2004; Umadevi et al., 2003; Valiollah et al., 2004). Mahesh and Satish (2008) illustrated that Acacia nilotica and Sida cordifolia leaf extracts showed highest antibacterial activity against Bacillus subtilis while Ziziphus mauritiana leaf extract showed significant activity against X. campestris. They also showed that root and leaf extract of S. cordifolia exhibited significant activity against bacteria. A. nilotica bark and leaf extract showed significant antifungal activity against Aspergillus flavus, Z. mauritiana and T. cordifolia recorded significant antifungal activity against D. turcica.

The aim of present research was to evaluate the antimicrobial activity of selected antibiotics against isolated fungus and some bacterial species.

MATERIALS AND METHODS

Sample collection

The samples were collected early in the morning from the ground of the Department of Zoology, The University of Azad Jammu and Kashmir. Samples having roots with rhizospheric soil were stored in four separate, properly capped, labeled, and sterile plastic jars containing Ringer’s solution. These jars were stored in cool, dark and dry atmosphere for further processing like standard agar disc diffusion method and genomic DNA isolation. The other samples like 10b strain of E. coli, K. pneumoniae

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and E. faecalis were taken from the National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, the Microbiology Lab of Combined Military Hospital (CMH), Muzaffarabad and Microbiology Lab., Department of Zoology, The University of Azad Jammu and Kashmir, Pakistan. The samples were brought to the laboratory for further analysis. Rhizospheric suspensions and dilutions

The suspensions were made from Ringers solution and labeled as 1, 2 and 3. Half samples were incubated at 37ºC and half samples were incubated at room temperature. After 3 days of incubation all samples were diluted. One milliliter solution from the original suspension was transferred to a test tube containing 9ml of Ringer’s solution, resulting in a 10-1 dilution of the spore mass in original material and 1 ml from 10-1 dilution was added to another test tube containing 9ml of Ringer’s solution to have 10-2. The process was repeated to make dilutions up to 10-4 and then placed at 37ºC and room temperature overnight. 200µl of both original and all diluted samples were spread on the Sabouraud agar medium (SAM; DIFCO) plates in laminar flow and half plates were incubated at 37ºC and half plates at 50ºC overnight. For each sample all plates were prepared in duplication. Purification of fungal and bacterial isolates and storage

The test tubes and 250ml flasks having broth were wrapped with aluminum foil and autoclaved for 20min to control contamination. Nutrient broth was poured into the sterile, autoclaved and labeled test tubes. After the growth of colonies, single colony of mycorhizae fungus, SAZAK2010 and other bacterial species such as E. coli, K. pneumoniae and E. faecalis from each incubated plate were picked with sterile yellow tip into NB and all these tubes were incubated in shaking with optimized temperatures 37oC for overnight. Methods of identification of SAZAK2010 isolated fungus

In later experiment, a small portion of each colony was picked with a sterile needle and teased out in a drop of water on a clean slide over-laid with cover glass. Prepared slide was examined under the microscope starting with a low power objective (X10), then the high power dry

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objective (X40) for better field view and magnification. The microscopic examination was done by observing cultural characteristics, asexual and sexual reproductive structures like sporangia, conidia head, arthrospores and vegetative mycelium, septate and non septate (Barnett and Hunter, 1972).

Antimicrobial activity Antibacterial and antifungal assay were performed by using a

standard disc diffusion assay (Acar, 1980) with replication (in triplicate) by using different types of media i.e., nutrient broth medium and MacConky agar medium. 100µl of isolated pathogens that stored at 4oC were inoculated in 100ml flasks containing 70ml nutrient broth at pH 7.0 and fresh cultures were grown at 37oC overnight (12h) by shaking at 200rpm. Next day, the cultures, agar plates, test paper discs of 5mm and antibiotic discs for bacterial and fungal pathogens were prepared. Fresh culture (50ml) of each strain was mixed with separate nutrient agar medium and the plates were poured under control environmental conditions (using laminar flow). The medium was spread equally by glass rod spreader and the inoculated material was allowed to be absorbed for 20min. The antibiotic discs were placed in triplicate on agar plates inoculated with bacterial strains and incubated at 37oC overnight. The antibacterial action of isolates was evaluated by measuring the diameter of zone of inhibition in the middle of the inoculated plate as a positive control. A digital camera (EOS 350D; EF-S 18-55, Kit) was used to photograph in the field as well as in the Lab. Genomic DNA extraction

The genomic DNA was also extracted from SAZAK2010, E. coli, K. pneumoniae and E. faecalis through III modified techniques (Doyle and Doyle, 1990).

RESULTS AND DISCUSSION

Present research indicated that 200µl of original and diluted

SAZAK2010 fungus showed growth on the Sabouraud agar medium (SAM) at 37ºC and 40ºC but they did not show growth at 50ºC (Figure 1). Similar results were obtained in case of nutrient broth. In later experiments

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the fungus (SAZAK2010) showed resistance at 37ºC and 40ºC on SAM plates supplemented with ampicillin (1mg/ml). It was consistent with the previous work that SAM medium was used for the selection of fungi. A small portion of each colony was picked with a sterile needle and teased out in a drop of water on a clean slide over-laid with cover glass. Prepared slides were examined under the microscope. The microscopic examination was done by observing cultural characteristics, asexual and sexual reproductive structures like sporangia, conidia head, arthrospores, and vegetative mycelium, septate and non septate (Barnett and Hunter, 1972). Table I: The antifungal and antibacterial activity of selected antibiotics.

Sr. No.

Name of antibiotic

Conc. of antibiotic (µg/ml)

Mychorihzea fungus

E.coli K. pneumoniae

E. faecalis

1 Tobramycin 10 Sensitive Resistant Sensitive Resistant

2 Penicillin G 10 Resistant Resistant Resistant Sensitive

3 Ciprofloxacin 5 Sensitive Resistant Sensitive Sensitive

4 Streptomycin 10 Sensitive Resistant Sensitive Sensitive

5 Kanamycin 10 Sensitive Resistant Sensitive Sensitive

6 Gentamicin 10 Sensitive Resistant Sensitive Sensitive

7 Tetracycline 10 Sensitive Resistant Sensitive Sensitive

8 Amoxicillin 25 Resistant Resistant Sensitive Sensitive

9 Ampicillin 1 mg/ml Resistant Resistant Sensitive

Sensitive

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Figure 1. Screening of mycorrhizae fungus (A) SAZAK2010 was selected on SAM, with and without ampicillin (1mg/ml) after incubation at both 37°C and 50°C. (B) A single colony of SAZAK2010 was picked and grown in Sabouraud broth with and without ampicillin (1mg/ml). Bi and Bii indicated the growth of fungus SAZAK2010 in Sabouraud broth without ampicillin and Biii and Biv showed the growth of SAZAK2010 in Sabouraud broth medium in the presence of ampicillin.

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Figure 2. The standard disc diffusion method of antibiotics against fungus and other bacterial species.

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Figure 3. The genomic DNA extraction from SAZAK2010 fungus and other bacterial species. (3.A) indicated the genomic DNA isolation from 8 samples of SAZAK2010 whereas (3.B) showed the DNA extraction from E. coli (Lane 1-5), Klebsiella pneumoniae (Lane 5-10), and Enterococcus faecalis (Lane 11-15) M represents DNA marker phage lambda- HindIII (AppliChem).

The antifungal and antibacterial activity of selected antibiotics

against SAZAK2010 fungus, 10b strain of E. coli, K. pneumoniae and E. faecalis was performed by using the standard disc diffusion method (Perez et al., 1990). Three types of media such as nutrient agar, Sabouraud agar and MacConky agar were used. It was observed that the desired incubation temperature for standard disc diffusion method was 37°C. The antifungal and antibacterial activity was evaluated by measuring zones of inhibition of fungal and bacterial growth surrounding the antibiotics (Fig. 2.A-2.H). The complete antimicrobial analysis was carried out under strict aseptic conditions and the experiment was carried out in duplication. It was shown

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that SAZAK2010 was sensitive against tobramycin, gentamicin, tetracycline, ciprofloxacin, streptomycin, and kanamycin (Fig. 2.A- 2.B). On the other hand, the SAZAK2010 had been shown resistance against ampicillin, amoxicillin and penicillin-G. The results were consistent with the previous literature (Walker et al., 2004; Cheng et al., 2007). Similarly, the antibacterial activity of antibiotics against bacterial species indicated that E. faecalis was susceptible to ciprofloxacin, streptomycin, kanamycin, gentamicin, tetracycline and amoxicillin whereas resistant to tobramycin and tetracycline (Fig. 2.C- 2.D, Table I).

It was further found that E. coli was sensitive against all selected antibiotics as shown on both MacConky agar and Nutrient agar plates (Fig. 2.E-2.F). The results were consistent that ampicillin, tetracycline, tobramycin, sulfisoxazole, gentamycin, penicillin, kanamycin, streptomycin, chloramphenicol, bacitracin, and erythromyasin were effective against E. coli (Andleeb et al., 2008; Andleeb et al., 2010a). Similarly, these antibiotics such as ampicillin, kanamycin, tetracycline, gentamycin, chloroamphenicol, and penicillin were also used against DH 5α, TOP10F strain of E. coli and LBA404, GV3101 strains of Agrobacterium tumefician during cloning and expression of heterologous genes (Andleeb et al., , 2008; Andleeb et al., 2010a; Andleeb et al., 2010b). As compared to the E. coli bacterial pathogen, it was observed that K. pneumoniae was susceptible to tetracycline, ciprofloxacin, streptomycin, kanamycin, gentamicin, tetracycline, and amoxicillin but showed resistance against penicillin G see Fig. 2.G and 2.H. The present findings were in agreement with some other reports (Bush et al., 1989; Sapico et al., 1989).

After the antimicrobial analysis through standard diffusion disc method, SAZAK2010, E. coli, K. pneumoniae and E. faecalis were successfully grown at 37°C and single colonies were grown in Nutrient broth medium for genomic DNA analysis by using three different methods. It was observed that method II displayed better results through 2% agarose gel electrophoresis as indicated in Fig. 3.A and 3.B. In later experiments, the genomic DNA extraction was modified by using proteinase K digestion, phenol/chloroform extraction and ethanol precipitation for sequence analysis and its characterization (Doyle and Doyle, 1990; Millar et al., 2000; Aziz et al., 2003; Badri and Sariah, 2009).

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The present investigation clearly indicates that the antibacterial and antifungal activity of SAZAK2010, E. coli, K. pneumoniae and E. faecalis by using the standard disc diffusion method were vary with the antibiotic used. It has been concluded that these antibiotics could be of considerable interest for the development of new drug. The modified genomic DNA extraction protocols may be useful for sequence analysis and for finding the different genes which play an important role in replication, act as a protein precursors and transporters.

Acknowledgement

The author is grateful to Mr. Muhammad Saleem from CMH, Muzaffarabad for providing bacterial species to continue research work.

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(Received: October 21, 2011; Revised: November 03, 2011)