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Isolation and Quantification of Bacterial Isolates from Citrus Fruits and Determination of Their Anti-bacterial Activity Against Selected Pathogens

Rowshan Akter
Department of Microbiology, Stamford University Bangladesh, 51 Siddeswari Road, Dhaka 1217, Bangladesh

Md. Aftab Uddin
Department of Microbiology, Stamford University Bangladesh, 51 Siddeswari Road, Dhaka 1217, Bangladesh

Nusrat Islam Tanu
Department of Microbiology, Stamford University Bangladesh, 51 Siddeswari Road, Dhaka 1217, Bangladesh

Abstract/ Executive Summary:

The present study was undertaken with the aim of investigating the isolation and quantification of microorganisms from the citrus fruit samples collected from different areas of Dhaka city. Out of ten samples studied, the range of total viable bacterial proliferation was approximately 102 to 107 cfu/g. Among the specific bacterial pathogens, prevalence of Klebsiella spp. was found in five samples, Listeria spp., Staphylococcus spp. and Pseudomonas spp. in four samples and Vibrio spp. in three samples only. The presumptive identification of these isolates was done by the conventional cultural, microscopic and biochemical tests. Fungal growth was also observed in four samples within the range of 1.2×10³ to 3.6×10³ cfu/g. Interestingly, Prunus mume/domestica (plum) showed the anti-bacterial activity against all the laboratory bacterial isolates tested. Among other samples, Tanarindus bacilus (tamarind) was found to exhibit activity against Escherichia coli, Pseudomonas spp., Salmonella spp., Vibrio spp., and Listeria spp. On the other hand, Monifera indica (mango) showed anti-bacterial efficacy against E. coli, Staphylococcus spp. and Listeria spp. and Citrus limon (lemon) only against Pseudomonas spp. and Listeria spp.

Keywords: Citrus fruits; Pathogenic bacteria; fungi; Anti-bacterial activity

Location: Department of Microbiology, Stamford University Bangladesh

Start Date:

End Date:

Program Area: Food Safety and Security

Commodity: Orange

Objectives:

1. In the present investigation emphasized not only on the bacterial contamination extent within the citrus fruits but also attempted to measure the anti-bacterial property of different citrus fruits commonly available and readily consumed in Bangladesh.

Methodology:

Study area and sampling. Samples were collected from different local markets of Dhaka city such as Rampura, Shantinagar, Moghbazar and Mailbagh area in different time intervals and transported to the laboratory as soon as possible according to the method suggested earlier. For the identification and enumeration of pathogenic bacteria and fungi, at first 10g of each sample was taken, then blended with 90 ml normal saline (pH 7.8) & diluted up to 10-5. Total viable bacterial count (TVBC), total fungal load and total coliform count (TCC). The enumeration was performed by spreading 0.1 ml sample from the 10-5 dilution onto nutrient agar (NA) and 0.1 ml from the 10-3 dilution onto Sabouraud Dextrose Agar (SDA) for the determination of total viable bacterial count (TVBC) and total fungal load, respectively (23). Plates were incubated at 37 oC for 24 hours and at 25 oC for 48 hours for TVBC and total fungal load respectively. For the detection of total coliform count (TCC), 0.1 ml sample from the dilution 10-3 was spread onto MacConkey agar media. The plates were incubated at 37 oC for 24 hours. Finally, several biochemical tests were carried out for the final confirmation of coliforms. Isolation of Salmonella spp., Shigella spp., Vibrio spp., Staphylococcus spp. and Pseudomonas spp. An aliquot of 1 ml sample was transferred into each of 9 ml of Selenite Cysteine broth (SCB) and Alkaline Peptone Water (APW) for the enrichment of Salmonella spp., Shigella spp. and Vibrio spp., respectively and incubated at 37 oC for 6 hours. After enrichment, samples were diluted up to 10-4 and then 0.1 ml sample from 10-2 and 10-3 was spread onto Salmonella-Shigella (SS) agar and Thiosulfate Citrate Bile Salt Sucrose (TCBS) agar. Staphylococcus spp. and Pseudomonas spp. were isolated from the Mannitol Salt agar (MSA) and Pseudomonas agar respectively. After incubation at 37⁰C for 24 hours, the typical colony characteristics on these media were observed. For further identification, biochemical traits of these isolates were tested following standard methods. Determination of anti-bacterial activity of the fruit samples. The antibacterial activity of the fruit samples was demonstrated by agar well diffusion method. Briefly, fruit blends were used directly on the Mueller-Hinton agar (MHA) media. At first, the bacterial pathogens (Escherichia coli, Klebsiella spp., Staphylococcus spp., Pseudomonas spp., Salmonella spp., Vibrio spp., and Listeria spp.) were introduced evenly over the MHA separately using cotton swab, followed by making hole (8 mm³) on the MHA by cork borer. Each of the blends was then introduced with the samples (volume of 100 μl with a concentration of 11 μg/μl) separately in the specified hole with a positive control antibiotic disc- Gentamicin (GEN, 10 μg) and the negative control (normal saline). Presence of clear zone around the sample solution (if any) indicated the presence of antibacterial activity.

Source of Information: Stamford Journal of Microbiology, December 2013. Vol. 3, Issue 1, p. 30-33; ISSN: 2074-5346

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

In fine, it can be concluded that the presence of a wide array of microorganisms with a huge load (near about 107 cfu/g) in the different fruit samples is a matter of great concern for the consumers. In addition, a number of pathogenic and indicator bacteria such as Klebsiella spp. in five samples, Listeria spp., Staphylococcus spp. and Pseudomonas spp. in four samples alongwith Vibrio spp. in three samples have been detected. Besides, fungal growth was also observed in a number of samples. Further study is required to detect the antibiotic resistance pattern of the isolates detected from the commonly consumed citrus fruits which is of significance from the view point of public health. Another aspect of the present study was the detection of anti-bacterial activity of the samples against most of the laboratory isolates tested (within a zone of inhibition of minimum 11 mm to a maximum of 16 mm). This indicates that the citrus fruits can be a potential alternative to the conventional commercially available antibiotics found in the market. So different extraction methods need to be standardized to find out the best one before using citrus fruits as therapeutic agents. Every possible measure should be taken by the different regulatory bodies to ensure the safe consumption of fruits.

Knowledge Management: Journal