Impact of Cinnamomum verum against different Escherichia coli strains isolated from drinking water sources of rural areas in Riyadh, Saudi Arabia

2.1 Microorganisms and media

Two isolates were isolated from drinking water bottled, tap and wells’ water from rural areas in Riyadh, Saudi Arabia; namely Escherichia coli DSM 1103 QC DSM and E. coli MB 11,464 1 CHB. Media used: all media were prepared as described by (APHA, 2005). Violet red bile lactose (VRBL) Agar was prepared for E. coli sp. isolation. It has the following composition (g/l): Lactose 10.0, Peptone 7.0, Yeast extract 3.0, Sodium chloride 5.0, Bile salts mixture 1.5, Neutral red 0.03, Crystal violet 0.002, Agar 15.000 with pH adjusted to 7.4 ± 0.2 at 25 °C. For isolate maintenance, Nutrient agar medium (g/l) was prepared. It has the following composition: meat extract 3, peptone 5, pH 7. For testing the inhibitory activities of cinnamon essential oil, Muller and Hinton agar with a composition of (g/l) beef, dehydrated infusion 30.0, casein hydrolysate 17, Starch 1.5, agar 15.0 with pH adjusted to 7.2 ± 0.1 at 25 °C was prepared.

2.2 Preparation of cinnamon extracts

1. Aqueous Extract: Ten Gram of dried and crushed cinnamon barks were soaked in 100 ml of sterilized distilled water for 6 h. at 50 °C. filtration was done every two hours using eight layered muslin cloth then centrifuged at 10000 rpm for 15 min. The supernatant was then collected and concentrated through evaporation at 40 °C to the final volume. The aqueous extract was sterilized by filtration. For aqueous extract maintenance, it was stored at 4 °C in airtight bottles for further studies. (Parekh et al., 2005).

2. Solvent extract: Ten grams of dried and crushed cinnamon barks was added to 100 ml of methanol solvent 90 % (v/v) then, kept on a rotatory shaker at 120 rpm for 24^◦C (Lab-Line Orbital Shaker- USA Lab Equipment) for 24 h. filtration and maintenance were carried out as previously described by (Parekh et al., 2005).

3. Essential oil of cinnamon: The cinnamon essential oil was prepared by extraction of aromatic oil unit. The extraction was done by hydro distillation process following the methods of Narayanankutty et al., (2021b).

2.3 Standard inoculum

The standard inoculum was prepared by inoculating 5 ml of peptone water by 3–5 single colonies of the selected isolate and incubated at 37 °C for 24 h. Thereafter, the Optical density (O.D.) of the grown culture was adjusted to 0.06–0.8 using the spectrophotometer at 625 nm which is equivalent to (14x10⁶ CFU/ml) (Ebrahim et al., 2018).

2.4 Inhibitory effect of Cinnamomum verum extracts against E. Coli sp. Using well diffusion test

Inhibitory activity of cinnamon (aqueous, methanolic and oil) extracts was tested individually using well-diffusion method as recommended by (NCCLS, 1993). Muller Hinton agar medium was poured into petri dishes and inoculated with 1 ml of E. coli isolates (14X10⁶ CFU/ml) using the spreading technique. Agar wells were made using a sterilized 7 mm corkborer and filled with 100 µl of the tested cinnamon essential oil. Incubation of petri dishes was done at 37 °C for 24 h. All experiments were carried out in triplicates. The cinnamon inhibitory activity was expressed as the inhibition zone diameter’s means (NCCLS, 1993; Narayanankutty et al 2021c).

2.5 Effect of different concentrations of cinnamon essential oil against E. Coli sp. By well diffusion method

Serial concentrations of cinnamon oil were prepared by emulsifying (30–40–50–60–70–80 % v/v) in 2 % of tween 80. These concentrations of cinnamon oil were tested for their inhibitory activity against E. coli isolate sp. The inoculum was prepared by inoculating the nutrient broth by 3–5 colonies of E. coli and incubated for 24 h. at 37 °C. Inhibitory activity was determined as described above. All experiments were carried out in triplicates and the cinnamon inhibitory activity was expressed as the inhibition zone diameter's mean (Mahdi et al., 2018).

2.6 GCMS analysis of essential oil of Cinnamomum verum

A Hewlett Packard gas chromatograph (HP6890) connected to a VG Analytical 70250S mass spectrometer with an HP5MS capillary column (30 m × 0.25 mm, film thickness 0.25 µm). In this system, helium is used as the carrier gas and the flow rate is 1 ml/min. The oven temperature range is adjusted from 50° C in 5 min to 280° C, and the oven temperature increase program is 40° C / 5 min, and finally, the temperature is kept isothermal for 5 min. Inject 1 μl of cinnamon oil sample in split mode. GC–MS detection was performed using a 70-eV ionization energy electron ionization system. A scan rate of 0.6 s (cycle time: 0.2 s) was applied, covering a mass range from 35 to 600 amu. The identification of the essential oil compounds was based on the comparison of retention time and mass spectra of the homolog’s series of (C4-C28) with data generated under identical experimental conditions (Adams, 2017).

2.7 Identification of pathogenic waterborne isolates by MALDI-TOF MS

MALDI-TOF-MS was used to identify the E. coli isolates by picking up a single colony of the selected isolate and transferring it directly using wood backs on MALDI target plate at ambient temperature (25 °C) until dry. Overlaying of samples was done by adding 1 µl of Bruker HCCA solution to all MALDI plates and leaving them until drying.

2.8 Statistical analysis

The determination coefficient (R²) was calculated according to Microsoft office Excel 2016 package.

3.1 Isolation and identification of E. Coli isolates by MALDI-TOF MS

VRBL agar medium was prepared to isolate E. coli isolates from drinking water in the rural areas in Riyadh, Saudi Arabia, which represent 35 % of the bacterial waterborne microorganisms. The medium was inoculated using 1 ml of water sample then incubated at 37 °C for 24 h. Single colonies with lactose-fermenting appearance (pink colonies) were picked up and sub-cultured into nutrient agar for maintenance. The results indicate the high score of the two selected E. coli isolates ranged from 2.41 and 2.46 identified as E. coli DSM 1103 QC DSM and E. coli MB 11,464 1 CHB (Table 1).

3.2 Inhibitory effect of Cinnamomum verum against E. Coli strains

Different (Aqueous, methanolic and oil) cinnamon extracts were prepared and tested for their inhibitory activity against E. coli strains using the well-diffusion method. The current results show that the oil extract of cinnamon inhibited the growth of E. coli DSM 1103 QC DSM and E. coli MB 11,464 1 CHB rather than the aqueous and methanolic extracts that indicates the high sensitivity of E. coli strains towards the oil extract with inhibition zone 4.5 cm and 5.2 cm for E. coli DSM 1103 QC DSM and E. coli MB 11,464 1 CHB (Figs. 1 & 2).

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Fig. 1

Antimicrobial activity of different cinnamon extracts against E. coli DSM 1103 QC DSM and E. coli MB 11,464 1 CHB strains.

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Fig. 2

Antimicrobial activity of different cinnamon extracts against E. coli DSM 1103 QC DSM and E. coli MB 11,464 1 CHB strains using well diffusion method.

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3.3 Effect of different concentrations of cinnamon essential oil against E. Coli DSM 1103 QC DSM and E. Coli MB 11,464 1 CHB strains

Different concentrations of cinnamon oil extracts were prepared from 30 to 80% (v/v) and tested against E. coli DSM 1103 QC DSM and E. coli MB 11,464 1 CHB strains. The results show that oil concentration of 80% (v/v) had the highest inhibitory activity on E. coli strains followed by the 70% and 60% (v/v) concentrations, respectively (Fig. 3). Determination coefficient of R² = 0.9449 and 0.9667.

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Fig. 3

Different concentrations of cinnamon oil extract against E. coli DSM 1103 QC DSM and E. coli MB 11,464 1 CHB strains.

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3.4 Chemical components of cinnamon essential oil extract

The chemical composition of cinnamon essential oil was done using GCMS analysis (Table 2). The major constituents include Propylene glycol, Linalool, Cinnamaldehyde, 3-Allyl-6-methoxyphenol, Methyl cis-cinnamate, Ethyl- cinnamate, Cinnamic acid, phenethyl ester, and Benzyl cinnamate. Cinnamaldehyde was found to have the highest percentage (24.42%) followed by cinnamic acid (20.93 %) and 3-Allyl-6-methoxypheno (m-eugenol) (18.55%).