Molecular Characterization and Antibiogram Profiling of the Isolated Food-Borne Pathogenic Bacteria on Fresh-Cut Salad Vegetables

Fresh cut salad vegetables (FCSV) play a significant role in our healthy and balance diet. The consumption of raw salad vegetables are vectors for the transmission of various infectious diseases, which is a public health issue in developing and developed world. Therefore, the aim of this present study was to evaluate the microbial contamination of ready-to-eat salad vegetables sold in different local vendors in Kushtia and Jhenaidah districts in Bangladesh. A total of 21different ready-to-eat FCSV samples were collected aseptically and analyzed immediately in our laboratory. A total viable bacterial count (TVBC) was enumerated up to 10 7 CFU/g in the tested samples. The highest number (3.01±1.4 × 10 7 CFU/g) of TVBC was found in Coriander leaf ( Coriandrum sativum ) and least microbial load (2.33±1.05 × 10 5 CFU/g) was observed in Red spinach. On the basis of morphological differentiation randomly selected bacterial isolates ( Aeromonas sp., Citrobacter sp., Enterobacter sp., Pectobacterium sp., and Staphylococcus sp . ) were identified by 16S rRNA gene sequencing. Moreover, most of the isolates were showed as multidrug-resistant bacteria via the antibiogram profiling testing. The overall antimicrobial susceptibility profile showed that the tested bacterial isolates were resistant against Erythromycin (15μ g), Penicillin G (10 unites), Nalidixic acid (30 μg), Ceftriaxone (30 μg), Ceftazidime (30 μg), susceptible against Kanamycin (30 μg), Polymyxin B (300 units), CO-Trimoxazole (25 μg), Colistin (10 μg), Doxycycline (30 μg) and intermediate against Nalidixic acid (30 μg), CO-Trimoxazole (25 μg), and Kanamycin (30 μg). Pathogenic bacterial contamination of FCSV, hygienic practices should be maintained before consumption.


Introduction
Fresh cut salad vegetables (FCSV) are essential elements for our sound health but they could contaminate via life threatening pathogenic microorganisms (Hamilton et al., 2006).The FCSVincluding cucumber, carrots, tomatoes, green chili, cabbage and lettuce etc. were used to prepare salad recipe.
The FCSV are sold in urban and rural market and it is usually seen the hawker sell these commodities in a public places.However, FCSV have been recognized not only source of pathogenic microbes but also different chemical contaminants (Uzeh et al., 2009).They may be spoiled by a wide variety of pathogenic bacteria, fungi, viruses and parasites (Ahmed et al., 2014).FCSV can also be contaminated from different environmental sources during cultivation from the soil and water.Besides they can expose via air, insects, birds, animal, equipments, and marketing etc. (Hamilton et al., 2006).The most common pathogenic bacteria such as Escherichia coli, Enterobacter spp., Salmonella spp.Pseudomonas spp.Aeromonas spp.Shigella spp.were found on the surface of FCSV samples.So the environmental and food microbiologists have continued to identify and suggest control measures for hazards at all stages in the supply chain from farm to fork (Jongen, 2005).The FCSV generally consumed salad as raw and a number of food-borne outbreaks related to these raw vegetables have increased recently (Buck et al., 2003;Lynch et al., 2009;Olaimat and Holley, 2012).
In developing countries like Bangladesh, the different food-borne outbreaks caused by fecal contaminated vegetables which are frequent.However, foodborne disease surveillance and investigation are limited and most of outbreaks are unrecorded due to lack of the scientific study and government policy.In recent decades, antimicrobial resistance problem not only developing country but also developed world are facinga great challenge (Rabbi et al., 2011).Different scientific studies havebeen reported the presence of multidrug resistant bacteria are exist on the different agricultural produces (Nipa et al., 2011;Osibote et al., 2014).Now-a-days, drug resistance is spreading mainly due to misuse of antibiotics, incomplete use of medications and widespread practice in livestock feeding as growth promotion (Sultana et al., 2014).

Objectives
In the present study, we evaluate the total viable bacterial load, molecular identification and antibiogram profiling of selected bacterial isolates from salad vegetables samples in Kushtia, Jhenaidah city, Bangladesh.

Sample collection and processing
A total (n = 21) consisting of seven category commonly consumed fresh vegetable samples green Chili (Capsicum annuum), Coriander leaf (Coriandrum sativum), Carrot (Daucus carota subsp.sativus), Red spinach (Amaranthus dubius), Cucumber (Cucumis sativus), Pudina leaf (Mentha), and Tomato (Solanum lycopersicum) were collected for analysis.The microbiological tests were carried out in the microbiology laboratory, Dept. of Biotechnology and genetic Engineering, Islamic University, Kushtia.Samples were collected in the sterile polythene zipper bags to avoid any handling contamination and transported to laboratory for microbial analysis.The twenty gram (20g) of each sample was aseptically mixed (1:10) ratio with 180 mL distill water (Muhammad et al., 2017)

Microbiological analysis of fresh cut salad vegetables
The FCSV samples were suspended in distill water and serially diluted up to 10 -6 (Muhammad et al. 2017) and a volume (0.1 mL) of each diluted sample was spread onto the viable cell counting media i.e. nutrient agar (NA) for counting the total viable bacterial count (TVBC).The plates were incubated at 37 ºC for 24-48 h (Muhammad et al., 2017).The distinct morphological colonies on Nutrient agar were preserved for further analysis.On the basis of their morphological difference the distinguished bacterial colonies were isolated for further studies.Pure cultures were stored by glycerol stock at -70 ºC for further studies (Mamun et al., 2016).

Molecular identification by polymerase chain reaction (PCR) using 16S rRNA gene sequencing
The template DNA was prepared by a slight modified boiling method described by Rawool et al. (2007).In order to molecular identification, bacterial isolates were sent to Invent Technologies Limited (Dhaka, Bangladesh).PCR protocol was slightly modified according to Rahman et al. (2017) where initial denaturation for 5 min at 94 °C followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 47°C for 30 s and extension at 72 °C for 1 min and 30 s and the final extension was conducted at 72 °C for 10 min.

Isolation, morphological identification and total viable bacterial count
In order to assess the TVBC count (N = 21) on samples consisting of seven types of vegetable samples were tested.The TVBC ranges on FCSV were different from sample types and collection places.The highest number (3.01±1.4× 10 7 CFU/g) of TVBC was found in Coriander leaf (C.sativum) and least microbial load (2.33±1.05× 10 5 CFU/g) was observed in Red spinach (A.dubius).From NA agar media, the distinct morphological colonies were preserved for further molecular analysis.An outbreak of human disease was observed related to the consumption of FCSV not only developing countries but also hasseen frequent in developed countries over the few decades.On the basis of Hazard Analysis and Critical Control Point-Total Quality Management (HACCP-TQM) technical guidelines, the raw produc eranges are containing aerobic plate count of <10 4 CFU/g as "Good", 10 4 -5 × 10 6 CFU/g as "Average", 5 × 10 6 -5 × 10 7 CFU /g as "Poor" and >5 × 10 7 CFU/g as "Spoilt" (Mamun et al. 2016).Moreover, the reference value of per gram vegetable is 10 3 to 10 5 CFU/g.Presence of E. coli, Salmonella spp.and Shigella spp.are also indicate the connection of poor sanitary practices and they might be create a potential risk of food borne illness to human health (Mamun et al., 2016).

Molecular identification
Morphologically distinct identified bacterial isolates were picked from salad vegetables.In order to molecular identification, bacterial isolates were sequenced with 16S rRNA primer and the collected gene sequences were compared for similarity with those of bacteria deposited in GenBank, using the NCBI BLAST.

Antibiogram profiling of the isolated and identified pathogenson fresh cut salad vegetables
A total of nine (n = 9) isolates including six (n = 6) categories bacteria was subjected to antimicrobial susceptibility testing (Fig. 2).Most of the isolates were showed as multidrug-resistant bacteria via the antibiogram profiling testing.The overall antimicrobial susceptibility profile showed that the tested bacterial isolates were resistant against Erythromycin (15 μg), Penicillin G (10 unites), Nalidixic acid (30 μg), Ceftriaxone (30 μg), Ceftazidime (30 μg), susceptible against Kanamycin (30 μg), Polymyxin B (300 units), CO-Trimoxazole (25 μg), Colistin (10 μg), Doxycycline (30 μg) and intermediate against Nalidixic acid (30 μg), CO-Trimoxazole (25 μg), and Kanamycin (30 μg).The isolates (5/10) were resistant to multiple drugs and few (2/10) of them were intermediate (Fig. 3).The isolate No.1 (Aeromonoas sp) and isolate No.8 (Pectobacterium sp.) which confer resistant (40%) to tested different antibiotics, similarly isolate No.4 showed resistant (33%) to tested antibiotics (Figure 3).However, antibiotic resistance caused mainly by using of widely and misuse antimicrobials in humans and other domestic animals, and transmission of acquired resistant strains between humansand animals.Moreover, growing antibiotic resistance has also been related to dumping of incomplete treatment of the pharmaceutical effluents where bulk drugs are manufactured (Gullberg et al. 2011).Antimicrobial resistance is increasing last few decades in the whole world due to greater access to antibiotic drugs.Estimates are that a number of people such as 700,000 to several million deaths result per year (Nordea 2016).

Conclusion
This study indicate that the fresh cut salad vegetables contain higher viable bacterial counts which would be highly risk and threaten to public health.We observed the tested FCSV samples were contaminated with various pathogenic bacteria (Aeromonas sp., Citrobacter sp., Serratia sp., Pectobacterium sp., Enterobacter sp., and Staphylococcus sp.) which can cause serious public health hazards.The pathogenic bacterial isolates from the salad vegetables has been linked to a major risk to public health are not only developing country like Bangladesh but also developed country.However, the pathogenic bacterial isolates present in the commonly consumed salad vegetables showed resistance against the regular antibiotics (Penicillin G-10, CO-Trimoxazole-25) which is threatened to public health in globally.As an extensive research of identification, antibiotic profiling and molecular characterization of pathogenic Fig. 3. Antibiotic resistance profiling (%) of selected bacterial isolates using different antibiotics bacteria, we should analyze more samples for accurate evaluation of this research.Nevertheless, our study contributea significance information of some pathogenic bacterial strains and their antibiotic resistance profile of the fresh cut salad vegetables which would be useful for the all stakeholders.

Fig A . Fig
Fig A. Antibiogram profiling of isolate no.1.as identified species Aeromonas sp.Fig B. Antibiogram profiling of isolate no.3.as identified Serratia marcescens