Biological Control of Foodborne Pathogenic Enterobacter sp. and Bacillus sp. Using Different Spice Extracts Available in Bangladesh

MATERIALS AND METHODS

Sample collection: Egg and meat samples were obtained from poultry egg and meat retailers of local market of katakhali, Rajshahi, Bangladesh. Samples were collected aseptically in sterile pack and transported to the Microbiology Laboratory, Department of Genetic Engineering and Biotechnology, Rajshahi University for further experiment. All the samples were processed within 6 h of its collection.

Isolation and growth optimization of pathogenic bacterial strains: In the present study, peptone water (peptone 10.0 g, sodium chloride 5.0 g) was used as a non-selective enrichment media for sample inoculation. For the isolation of bacteria from the surface of the egg shell, cotton swab stick was moistened in 0.1% peptone water and used to swab the surface of the egg shell and was transferred to the peptone water for incubating at 37°C for 24 h. Approximately 5 g of meat (from thigh muscle) was excised from collected sample, minced and placed in 100 mL of peptone water as non selective pre-enrichment media for incubating at 37°C for 24 h. After the non-selective enrichment, MacConkey and Mannitol agar media were used as selective media for the isolation of Gram-negative and Gram-positive pathogenic bacterial strains, respectively from enriched food samples. Inoculated plates were incubated aerobically at 37°C and growth were examined after 18-24 h of incubation. The single colonies from the selective media were sub cultured and eventually transferred into LB agar plates. Then the plates were incubated at 37°C for 24 h and a single bacterial colony was isolated for further study.

Different pH (5, 6, 7 and 8) and temperature (25, 30, 35 and 40°C) were used for determining the optimum bacterial growth. Bacterial cell density was measured at 660 nm using double beam spectrophotometer (ANALYTK JENA AG, SPOKOL 1500/1, GERMANY. All the experiments were done in triplate.

Morphological and biochemical characterization of isolated bacteria: Bacterial isolates were characterized by several morphological and biochemical tests like gram staining¹⁸, motility¹⁹, methyl red²⁰, simmons citrate²¹, catalase²², urease²³, triple sugar iron²⁴, starch agar²⁵ tests etc.

Molecular identification of the bacterial isolates: Molecular identification has been used as a worldwide acceptable technique to identify any bacterium up to genus level across all major phyla. At first, the genomic DNA of the isolated bacterial strains was extracted (about 1465 bp) using phenol/chloroform method²⁶. The 16S rRNA genes were amplified by PCR using 16S rRNA specific primer forward primer 27F 5-AGAGTTTGATCMTGGCTCAG-3 and reverse primer 1492R 5 -GGTTACCTTGTTACGACTT-3. The PCR reactions were carried out in thermal cycler (Applied Biosystem 9700, USA) using following amplification conditions: An initial denaturation step at 95°C for 5 min, followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 1 min and the final extension at 72°C for 10 min. The PCR products were purified and were sequenced for both strands on genetic analyzer (Prism 310, USA). The sequences were then edited by bioinformatics software Chromas. The homology of the 16S rRNA gene sequences were checked with the 16S rRNA gene sequences of other organisms using the BLASTN (https://blast.ncbi.nlm.nih.gov/Blast.cgi) algorithm.

Cytotoxicity evaluation of isolated bacterial strains: Cytotoxic activities of bacterial strains were evaluated through brine shrimp (A. salina) lethality assay²⁷ at different concentrations of 50, 100, 150 and 200 μL. The mortality (%) of the brine shrimp nauplii was calculated for every concentration for each sample. A plot of log concentration of the sample dose levels versus percent of mortality showed an approximate linear correlation between them.

Antibacterial activity test: In this study, the antibacterial screening of spice extracts (methanolic and aqueous) were undertaken with well diffusion method²⁸ as well as C. verum (Cinnamon) oil with disc diffusion method²⁹.

Spice extracts preparation using methanol and aqueous solvent: The fresh spice samples (Cinnamomum verum, Syzygium aromaticum, Piper chaba, Zingiber officinale) and C. verum (Cinnamon) oil used in the present study were obtained from the local market of Rajshahi during August, 2017. The collected spice samples were washed, surface sterilized with 0.1% HgCl₂, unwanted materials were discarded and were dried properly in room temperature. The dried spices were grinded into small fine particles by a grinder machine, the powder was transferred to an air tight container and kept in a cool and dark place until the screening of their bioactive potentialities. For extraction, about 10 g powdered of each material was taken in 1 L conical flasks. Each sample was soaked in 50-60 mL of methanol and water. The conical flasks with its contents were then sealed and kept on orbital shaker for continuous shaking at 150 rpm for 2 days. The mixtures were then filtered through Whatman No.1 filter paper. Using rotary evaporator the methanolic and aqueous extract of each spice was evaporated at 55-60°C temperatures at 160-180 rpm. After 30 min of drying process, a slurry concentration was obtained, which was kept in small vial for further drying. After 20-30 days, the solvents were completely evaporated and the extracts became ready for experiment. In case of antibacterial activity, 5 mg of each extract was taken in separate vials and 5 mL of solvent (methanol and water) were carefully added in each vial. The extracts were then dissolved well in the solvent by inverting the tube. Each of the stock solution with concentration of 1 μg μL^–1 was labeled and used for sensitivity test.

Antibacterial screening of spice extracts: Agar well diffusion method is widely used to evaluate the antimicrobial activity of plants or microbial extracts³⁰. Nutrient agar was prepared according to the manufacturer’s instructions, autoclaved and dispensed into sterile petri dishes and allowed to set before use. Usually, 20 mL nutrient agar was placed into 10 mL petri dishes and 0.1 mL of the active bacterial cultures were spread over the plate using a sterile glass spreader in order to get a uniform microbial growth for all plates. A well was done using a 8 mm diameter cork borer in the agar plate. The wells were filled with 25, 50, 75 and 100 μg mL^–1 specific spice extracts (aqueous and methanolic). The plates were left for 30 min at room temperature to allow the diffusion of extracts. Then the plates were incubated at 37°C for 24 h. After 24 h, the inhibition zones were measured using millimeter scale. Values were performed in triplicate and the mean value was recorded. In this case, the antibiotic gentamicin (10 μg disc^–1) was used as control.

Antibacterial screening of C. verum oil: The discs (6 mm diameters) were made by punching the Whatman No. 1 filter paper. For the testing of the antibacterial efficiency of C. verum oil against one bacterial sample, paper discs were soaked with different concentrations of oil (10, 20 and 30 μL). In the present study, laboratory supplied antibiotic gentamicin (10 μg disc^–1) was used as standard. By means of a pair of sterile forceps, the dried discs and standard disc (gentamicin) were placed gently on the solidified agar plates seeded with the tested bacteria to ensure contact with the medium. The plates were then kept in an incubator at 37.5°C temperature for 24 h. After incubation, the antibacterial activities of the tested samples were determined by measuring the diameter of inhibitory zones with a millimeter scale.

RESULTS

Isolation and optimization of growth characteristics: Pathogenic bacterial strains were isolated from egg and meat sample through screening on MacConkey and Mannitol salt agar media respectively and the results are shown in Table 1. The optimum pH and temperature for the growth of both isolates was 7.0 (Fig.1a, b) and 35°C (Fig. 2a, b).

Morphological and biochemical characterization of isolated bacteria: Morphologically both the isolates were motile and rod shaped but isolate E1 was Gram-negative while isolate M2 was Gram-positive. The biochemical tests results are shown in Table 2.

Molecular identification of the bacterial isolates: After gene sequencing, the sequences were checked against the 16S rRNA gene sequences of other organisms that had already been submitted to NCBI Genebank database. Isolate E1 showed 98% identity with Enterobacter sp., while isolate M2 showed 93% identity with Bacillus sp. The band of PCR products of isolated bacteria was shown in Fig. 3.

Brine shrimp lethality assay: The present study explored the cytotoxic effect of isolated bacteria at concentrations of 50, 100, 150 and 200 μL. The LC₅₀ value for Enterobacter sp. and Bacillus sp. was 143.2504 μL mL^–1 (Fig. 4a) and 95.6205 μL mL^–1 (Fig. 4b), respectively.

Fig. 1(a-b):	Effect of pH on growth characteristics of isolate (a) E1 and (b) M2
	Data were recorded after 24 h of incubation

Table 1:	Microscopic and macroscopic morphological observation of bacterial isolates

Fig. 2(a-b):	Effect of temperature on growth characteristics of isolate (a) E1 and (b) M2
	Data were recorded after 24 h of incubation

Fig. 3:	Band of PCR products from isolated bacterial strains
	“M”: 1 kb DNA ladder (Marker), E1, M2: PCR products of isolated bacterial sample

Antibacterial activity of spice extracts: All the spice extracts (methanolic and aqueous) showed their antibacterial activity against the isolated foodborne bacterial pathogens with 4 different doses like 25, 50, 75 and 100 μg mL^–1.

Fig. 4(a-b):	Regression line and LC50 value of isolate (a) E1 and (b) M2

Antibacterial activity of methanolic spice extracts: Enterobacter sp. was susceptible at doses of 75 and 100 μg mL^–1 while Bacillus sp. was susceptible to the concentration of 100 μg mL^–1 in case of C. verum methanolic extract (Table 3). For S. aromaticum methanolic extract, both Enterobacter sp. and Bacillus sp. were susceptible at 50, 75 and 100 μg mL^–1 (Table 3). During the study of antibacterial activity of P. chaba methanolic extract against the isolated bacterial strains, Enterobacter sp. was susceptible at a doses of 75, 100 μg mL^–1 as Bacillus sp. was susceptible at a dose of 100 μg mL^–1 (Table 3). In case of Zingiber officinale, Enterobacter sp. was susceptible at doses of 75 and 100 μg mL^–1 but no susceptible zones of inhibition was found against Bacillus sp. (Table 3).

Antibacterial activity of aqueous spice extracts: In case of C. verum, P. chaba and Z. officinale aqueous extract, both Enterobacter sp. and Bacillus sp. were resistant at the prospective doses (Table 4). But, in case of S. aromaticum aqueous extract isolated bacteria were intermediate resistant at dose 25 μg mL^–1 and were susceptible to the remaining doses (Table 4).

Table 2:	Summarized results of biochemical tests used for the characterization of the isolates

Table 3:	Antibacterial activity test of methanolic extracts of different spices against the isolated bacterial strains

Table 4:	Antibacterial activity test of aqueous extracts of different spices against the isolated bacterial strains
Resistant = <13 mm, Intermediate resistant = 13-14 mm, Susceptible = >14 mm

Table 5:	Antibacterial activity test of C. verum oil against the isolated bacterial strains
Antibacterial activity of C. verum oil: When the antibacterial activity of C. verum oil was tested, both bacterial isolates were found to be susceptible (Table 5) at all the three doses (10, 20 and 30 μL).

DISCUSSION

In this study, pathogenic bacteria were isolated and characterized from food samples (poultry eggs and meat) collected from local market of Rajshahi district, Bangladesh. Several workers have isolated pathogenic bacteria from different food sources. Jambalang et al.³¹ isolated Enterobacter cloacae from retailed poultry eggs. Fayaz et al.³² isolated and characterized enterotoxigenic Bacillus cereus from poultry meat. Optimum pH and temperature for the bacterial growth was 7 and temperatures 35°C, respectively as reported earlier by Frazier and Westhoff³³. Molecular identification confirmed that isolate E1 had 98% similarity with Enterobacter sp. while isolate M2 had 93% similarity with Bacillus sp. All the morphological, physiological and biochemical characteristics were found similar with the respective genus for the bacterial isolates according to Bergey’s Manual of Systematic Bacteriology. Through cytotoxicity test it was revealed that, Bacillus sp. (LC₅₀ 95.6205 μL mL^–1) showed more toxicity than Enterobacter sp. (LC₅₀ 143.2504 μL mL^–1).

Spices have been shown to possess medicinal value, in particular, antimicrobial activity. The methanolic and aqueous extracts from four traditionally used spices Cinnamomum verum, Syzygium aromaticum, Piper chaba, Zingiber officinale were prepared and evaluated for their antibacterial activity against isolated foodborne bacterial strains. Puangpronpitag and Sittiwet³⁴ reported antibacterial activity of Cinnamomum verum stem bark aqueous extract against Straphylococcus epidermidis, Krebsilla pneumonia and E. coli at 250 μg mL^–1. In this study, Enterobacter sp. was susceptible to C. verum methanolic extract with zones of inhibition ranging from 14.33±0.58 at concentration of 75 μg mL^–1 to 15.67±0.58 mm at 100 μg mL^–1 while Bacillus sp. was only susceptible at 100 μg mL^–1 with 15.33±0.58 zone of inhibition. In case of C. verum aqueous extract, no susceptible zone of inhibition was found against the isolated bacteria at prospective doses which had similarity with the referenced data. In terms of S. aromaticum methanolic extract, Enterobacter sp. was susceptible with 15.33±0.58, 16.67±0.58 and 18±0 mm zones of inhibition at doses of 50, 75 and 100 μg mL^–1, respectively. Similarly, Bacillus sp. showed susceptibility at doses of 50, 75 and 100 μg mL^–1 with 15±0, 18.33±0.58 and 21±0 mm zones of inhibition, respectively. In case of S. aromaticum aqueous extract, Enterobacter sp., was susceptible with 14.33±0.58, 15.67±0.58 and 16±1 mm zones of inhibition while susceptible zones of inhibition were 15.33±0.5, 16.33±0.58, 19±1 mm for Bacillus sp., at 50, 75 and 100 μg mL^–1 concentration followed by the investigation of Saeed et al.³⁵. In their study, bactericidal effect of methanolic extract of Syzygium aromaticum was found with 17 mm zone of inhibition and aqueous extract with 16 mm zone of inhibition against Bacillus subtilis at dose of 250 μg mL^–1. According to Roy et al.³⁶ antibacterial activity of methanolic extract of P. chaba stem and leaf was observed against some pathogenic bacteria at dose of 400 μg disc^–1. While studying the antibacterial activity of P. chaba methanolic extracts, Enterobacter sp. was susceptible at doses of 75 and 100 μg mL^–1 and zones of inhibition were 15±0 and 16±1 mm, respectively but Bacillus sp. was only susceptible at dose of 100 μg mL^–1 with 15.33±0.58 mm zone of inhibition. On the other hand, Enterobacter sp. and Bacillus sp. were resistant to P. chaba aqueous extract at applied 4 doses. Yassen and Ibrahim³⁷ studied anti-bacterial effect of methanolic and aqueous extracts of root of Zingiber officinale against Escherichia coli and Staphylococcus aureus at dose of 50 μL. In this study, Enterobacter sp. was susceptible to Zingiber officinale methanolic extract with 15±0, 16±0 and 17±0 mm inhibition zones at doses of 50, 75 and 100 μg mL^–1, respectively whereas, Bacillus sp. was resistant at respective 4 doses. However, both Enterobacter sp. and Bacillus sp. were resistant to Zingiber officinale aqueous extract at applied four doses which showed equal (average 8.00 mm) diameter of inhibition zone that means diameter of inhibition zone were not increased due to the increasing of dose from 25-100 μg mL^–1.

This research also studied anti-bacterial activity of C. verum oil against isolated bacteria. In case of Bacillus sp., the highest diameter of inhibition zone was 40±1 mm at the dose of 30 μL mL^–1 and the other 2 inhibition zones were of 25±1, 30±0 mm at the doses of 10 and 20 μL mL^–1. The C. verum oil produced, inhibition zones of 24.33±0.58, 26.67±0.58, 34.33±1.53 mm at the doses of 10, 20 and 30 μL mL^–1, respectively in case of Enterobacter sp. which had similarity with the findings of Gupta et al.³⁸. They investigated that C. verum oil exerted had strongest effect on B. cereus among ten pathogenic bacteria tested and also reported that C. verum oil was more effective than C. verum. In all cases, standard antibiotic Gentamicin (10 μg disc^–1) showed susceptibility with average 17 mm zone of inhibition.

Among different spices extract, Syzygium aromaticum (clove) and C. verum (cinnamon) methanolic extracts were found to possess relatively higher antimicrobial activities than aqueous extracts against isolated bacteria. It was also found that C. verum oil had highest antimicrobial effect than methanolic and aqueous spice extracts.

CONCLUSION

These results suggest that spices represent an alternative source of natural antimicrobial substances and can be used to prevent the growth of foodborne bacteria and extend the shelf-life of the processed food. However, further analysis could be done to isolate the antimicrobial agents present in these spices and to determine their minimal inhibitory concentrations so that they can be used as bio preservatives in various food stuffs.

SIGNIFICANCE STATEMENT

Nutritional value, availability, low price of eggs and meat are well known. Contamination by pathogenic microbes has detrimental effects on human health. This result will help to control those bacteria by using spices extracts in a natural way. The result suggests that spices represent an alternative source of natural antimicrobial substances for inhibiting the growth of foodborne bacteria.