Isolation and Characterization of Bacterial Isolates from Psidium guajava Obtained from Local Markets of Patna and Their Antibiotic Sensitivity Test

The study was conducted to investigate the presence of antibiotic resistant bacteria in guava, sold in the local markets of Patna. A total of twenty five fresh samples of guava were collected from five different market places in Patna city. Several microbiological tests were performed to assess the growth and type of bacteria. The colonies were isolated and identified as isolate 1 and 3 (identical to Staphylococcus spp.), isolate 2 (identical to Escherichia spp.) and isolate 4 and 5 (identical to Bacillus spp.) based on their cultural, morphological, Gram’s staining and biochemical characteristics. Antibiotic susceptibility tests were conducted to detect their actual resistance capability. In the present study, S1 and S3 were found resistant to ampicillin, chloramphenicol, ofloxacin and intermediate to gentamycin while S2 was found resistant to ampicillin, chloramphenicol, gentamycin, ciprofloxacin, cephalexin and intermediate to ofloxacin. The isolates S4 and S5 were found sensitive to gentamycin, intermediate to chloramphenicol and resistant to ciprofloxacin. Results of this study showed that the guava samples obtained from different markets of Patna possess multidrug resistant bacteria.


INTRODUCTION
Guava (Psidium guajava) is widely distributed throughout the regions of India and also considered as popular indigenous fruit. It is most often called the "apple of the tropics" belonging to the family Myrtaceae (Shruthi et al. 2013). It possesses excellent medicinal properties with a pleasant aroma. Guava is a popular and important fruit consumed all over India for its easy availability, low cost and high nutritive value (Kaneria & Chanda 2011). Fruits like guava, which are mostly eaten raw and are highly perishable, and can also be a source of several bacterial diseases due to their improper handling during storage and transportation. It exhibits antioxidant properties, recognized for its anti-allergic, antimicrobial, antidiabetic, anti-inflammatory significance, and is a rich source of vitamin C (Shruthi et al. 2013). It can also be used for the treatment of gastroenteritis, diarrhoea and dysentery as well as green mature guava is a rich source of pectin, calcium and phosphorus (Morton et al. 1987). India, due to its diverse climatic conditions and rich biodiversity is the natural habitat for varieties of fruit. Extending from the Himalayan belt in the north to the southern tropical belt, India offers much in terms of fruit diversity. Among Indian states, Bihar is one of the leading producers of guava (Dinesh & Vasugi et al. 2010).
Improper manipulation and exposure to the environment such as soil, water and air can harbour a heterogeneous range of microorganisms (Sarker et al. 2018). Microorganisms, especially bacteria, thrive on the fresh guava fruits making them prone to several diseases. Consumption of such guava by humans can result in varieties of infection and associated health risks. Most microbes which are initially observed on whole fruit or vegetables are soil inhabitants (Andrews & Harris 2000, Janisiewicz & Korsten 2002. The worldwide use of antibiotics in different fields has created pressure to select resistance among infectious bacterial pathogen (Balotescu et al. 2003, Sharma et al. 2005. Public organization illustrates resistant bacteria as a disaster or a nightmare scenario that could have deleterious effects on human health (Viswanathan 2014).
The emergence of antibiotic resistant bacteria is a major concern of public health nowadays. The widespread misuse of antibiotics has led to an alarming increase of antibiotic resistant bacteria such as Staphylococcus spp., Pseudomonas spp., Streptococcus spp., Escherichia coli.
It is evident that not much attention has been paid to guava fruit safety, especially, in context of Patna (Bihar). Therefore, the main objective of the present study was to isolate bacterial spp. from different guava samples collected from different areas of Patna and to investigate their antibiotic resistant properties which are a major cause of several health issues.

Sample Collection
Guava samples were collected from different regions of Patna (Table 1). The selected market places for sample collection were Patliputra colony, Kankarbagh, Boring road, Agamkuan and Bazar Samiti. The samples were collected in a sterilized zip lock bag with appropriate labelling.

Isolation of Bacteria
For the isolation of bacteria, samples were washed thoroughly with 10mL of sterile PBS (phosphate buffer saline) solution. Inoculation through spreading was done on a nutrient agar plate (Sanders 2012) with different serially diluted sample (up to 10 -4 dilution) and the plates were incubated at 37°C for 24 hours. The strains that were obtained on nutrient agar plates were again streaked on eosin methylene blue (EMB) selective media and incubated at 37°C for 24-48 hours (Islam et al. 2014). The obtained colonies were pured by streaking on nutrient agar plates for further analysis.

Bacterial Enumeration
Colonies were enumerated using a colony counter by placing the plates on the colony counter and visualizing it as well as Total Viable Count (TVC) was calculated (Brugger et al. 2012).

Cultural and Morphological Characterisation
Plates were observed and colony morphology was recorded. The morphological characteristics like shape, colour, texture, margin etc. were qualitatively measured (Bai et al. 2010). Further, identification was done using Gram's staining reaction.

Biochemical Analysis of the Isolated Strains
Various biochemical tests such as catalase, coagulase, mannitol motility, citrate utilization, indole and methyl red were done to identify the bacterial strains (Sarker et al. 2018) Antibiotic Sensitivity Test Three isolates selected from three different genera were subjected to antibiotic sensitivity test using the disk diffusion method (Poonia et al. 2014). After the incubation at 37°C for 24 hours, plates were examined and zones of inhibition were measured with the help of millimetre scale from the edge to the disc.

Isolation of Bacteria from Guava Surface Samples and Their Morphological Characterization
The isolated microorganisms were visualized under the

Isolation of Bacteria from Guava Surface Samples and Their Morphological Characterization
The isolated microorganisms were visualized under the light microscope for morphological characterization. Five different bacterial isolates were identified and further characterization was done. The bacteria isolate S1 and S3 were Gram-positive and coccus in shape while the bacteria S2 was Gram-negative, rod-shaped. The bacterial isolates S4 and S5 were Gram-positive rodshaped (Table 3).  light microscope for morphological characterization. Five different bacterial isolates were identified and further characterization was done. The bacteria isolate S1 and S3 were Gram-positive and coccus in shape while the bacteria S2 was Gram-negative, rod-shaped. The bacterial isolates S4 and S5 were Gram-positive rod-shaped (Table 3).

Biochemical Characterization
The biochemical test results of the isolated bacteria from different guava samples are given in Table 4.

Antibiotic Sensitivity Tests
All the five isolates identical to Staphylococcus spp. (S1 and S3), Escherichia coli (S2), and Bacillus spp. (S4 and S5) were subjected to antibiotic sensitivity test. To detect the sensitivity pattern of all the isolates, the CLSI (Clinical and Laboratory Standard Institute) standards have been used. The present study revealed, S1 and S3 resistant to ampicillin, chloramphenicol, ofloxacin and intermediate to gentamycin while S2 resistant to ampicillin, chloramphenicol, gentamy-

DISCUSSION
Guava is the fourth largest selling or consuming fruits in India after mangoes, bananas and citrus fruits. In the present    study, twenty five samples of guava, all procured from different market places of Patna, were used and isolation, characterization and identification of microbes (bacteria) were made. The identified bacterial isolates were found identical to three genera such as Staphylococcus spp., Escherichia coli, Bacillus spp. Most of the guava samples were found contaminated with foodborne bacteria because of the poor hygiene and handling practices performed by the vendors (Gultie et al. 2013). This might have resulted in the occurrence of antibiotic resistant bacteria which could cause diseases among its consumers.
Three bacterial isolates of guava in this study were found resistant to different antibiotics that were used during the antibiotic sensitivity test. S1 and S3 resistant to ampicillin, chloramphenicol, ofloxacin and intermediate to gentamycin while S2 resistant to ampicillin, chloramphenicol, gentamycin, ciprofloxacin, cephalexin and intermediate to ofloxacin and the isolates S4 and S5 were sensitive to gentamycin, intermediate to chloramphenicol and resistant to ciprofloxacin.
The present investigation further revealed that the bacterial isolates that had been isolated from guava were found almost multidrug resistant. The occurrence of these multidrug resistant bacteria is the consequence of antibiotics either used in agricultural practises (Falkiner 1998) or used as animal manure in the agricultural fields (Boehme et al. 2004). Therefore, it can be suggested that implementation of proper hygienic conditions during pre-and post-harvest stages of production, transportation, storage and selling of the food stuffs for the safeguard of public health (Sarker et al.2018).

CONCLUSION
This study has clearly shown the presence of antibiotic resistant bacteria in the guava samples procured from local markets of Patna, Bihar. These multidrug resistant bacterial isolates could lead to health hazards for the consumers and their presence is of major health concern. ACKNOWLEDGMENTS Financial assistance from the Department of Biotechnology, Government of India under DBT Star College Scheme is thankfully acknowledged.