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The use of cinnamon oil as antibacterial agent to eliminate some antibiotic-resistant bacteria isolated from water sources

© Slovak University of Agriculture in Nitra Faculty of Agrobiology and Food Resources


Introduction
Some microorganisms, including Escherichia coli and Klebsiella species, have the ability to produce large amounts of extended-spectrum β-lactamases (ESBLs) (Al Yousef et al., 2016). These enzymes can cause multidrug resistance and lead to urinary tract infections, which are difficult to treat (Zorc et al., 2005). Members of the Enterobacteriaceae family, especially E. coli and Klebsiella species harboring ESBLs, have been recognized since the 1980's as the main causes of hospital-acquired infections.
Many bacteria, which are responsible for urinary tract infections and loose stools, have the capacity to be ESBL producers. These bacteria include E. coli, Salmonella, Shigella, and Vibrio cholerae (Doi et al., 2007). ESBLs and AmpC β-lactamases are a cause of increasing clinical concern. ESBLs are commonly produced by Klebsiella species and other Gram-negative bacteria. Clavulanate enzymes are known to break down penicillin, extendedspectrum cephalosporins, and aztreonam (Bradford, 2001). For the evaluation of ESBLs, susceptibility to cefotaxime, ceftazidime, ceftriaxone, and cefpodoxime was examined, followed by phenotypic corroborative testing (Moland et al., 2002).
ESBLs are competent enzymes, responsible for pathogen resistance to penicillin and first-, second-, and thirdgeneration cephalosporins. They are not active against cephamycin but are rather susceptible to clavulanic acid (Rosenthal et al., 2010). Because of antibiotic overuse, the antimicrobial resistance of bacteria has increased around the world (Gaspari et al., 2005;Stürenburg and Mack, 2003). Bacteria responsible for urinary tract infections (UTIs) show resistance to conventional medications. The effectiveness of ESBL screening may alter according to the type of antimicrobial agent (Mackenzie et al., 2002), as indicated by the Clinical and Laboratory Standards Institute (CLSI) M100-S11 guidelines (Patel et al., 2001).
Antibacterial resistance monitoring is essential for identifying problems and choosing an appropriate antimicrobial drug for therapy. Therefore, the development of new active antimicrobials, with the fewest side effects, is essential (Reddy and Yang, 2015). Different studies have demonstrated the positive effect of oils on different types of microorganisms, such as Staphylococcus aureus, Listeria monocytogenes, E. coli, Bacillus cereus, and Salmonella typhimurium (Burt, 2004;Brenes and Roura, 2010;Pesavento et al., 2015). Evidence shows that phenolic mixtures are the most dynamic against these bacteria. Consequently, cinnamon, thyme, and rosemary oils are considered as the most successful agents against microorganisms (Dorman et al., 2000;Ojagh et al., 2010). The antimicrobial effects of cinnamon oil on different microbial agents, such as E. coli, Pseudomonas aeruginosa, Enterococcus faecalis, S. aureus, Salmonella species, and Vibrio parahaemolyticus, have been reported in the literature (Chang et al., 2008).
This study aimed to define the current trend of ESBL production in bacterial isolates collected from the water. Also, the antimicrobial activity of cinnamon essential oil was examined alone and in combination with antibiotics against pathogenic microorganisms in water.

Study samples
This study was performed at the Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Hafr Al-Batin University (Saudi Arabia) from October 2016 to October 2019.

Samples collection and prosessing
In Hafr Al-Batin, Saudi Arabia, approximately 100% of water supply originates from groundwater sources. In this city, water is transferred from a vehicle to the client's house and is stored in wells, which may be situated next to sewage tanks. Under aseptic conditions, 250 mL sterile bottles were used to collect water for the tests. The samples were transferred to the research center on ice and examined for four hours. A total of 227 samples were collected from the wells. Water samples were centrifuged at 6,000 rpm for 12 minutes at 4 °C. The pellets were streaked on the CLED medium agar and incubated at 37 °C for 48 hours. Out of 227 samples collected, the culture of only 170 isolates was positive on the CLED medium.

MicroScan analysis
The CLED-positive isolates (170) were subjected to MicroScan analysis. A MicroScan WalkAway-96 SI system was used for detecting antibiotic sensitivity. Antimicrobial susceptibility testing was performed using Negative BP/Combo 30-B1017-306E combination panels. The methodology was performed according to the manufacturer's instructions. The integrated LabPro version 1.12, which incorporates the Alert expert system, uses growth in the presence of cefpodoxime (4 µg mL -1 ) and ceftazidime (1 µg mL -1 ), i.e., at concentrations proposed by the National Committee for Clinical Laboratory Standards (NCCLS) for ESBL screening (Jones, 1986) as the primary indicators of potential ESBL production. The minimum inhibitory concentrations (MICs) of ceftriaxone and cefotaxime were determined according to the CLSI breakpoints (Patel et al., 2001). It should be noted that screening with this system is restricted to E. coli, K. pneumoniae, and K. oxytoca (i.e., primary species in the CLSI guidelines).

ESBL conformatory tests
Out of 170 CLED-positive isolates, 104 were found positive in the MicroScan analysis. The collected isolates were subjected to ESBL confirmatory tests, which are described below.

Double disc synergy test (DDST)
The colonies were collected and recultured in peptone water at 37 °C for 2-6 hours. The 0.5 McFarland standard was also set, and cultures were prepared on Mueller-Hinton agar. Augmentin (20/10 μg) was placed in the middle of the dish. The cefotaxime (30 μg) and ceftazidime (30 μg) discs were placed at a 15 mm distance on two sides of the augmentin disc, and the dish was grown at 37 °C. The ESBL production was determined according to a study by Ramesh et al. in 2019(Ramesh et al., 2019.

Phenotypic confirmatory disc diffusion test (PCDDT)
ESBL production was confirmed in potential ESBLproducing isolates by phenotypic tests. The culture of microbes was prepared, and third-generation cephalosporin, ceftazidime (30 μg), and ceftazidime + clavulanic acid (30 μg + 10 μg) discs were placed at 25 mm distances; the dish was kept at 37 °C. ESBL production was confirmed according to a study by Lal et al. in 2007(Lal et al., 2007.

Extraction and fractionation
The cinnamon bark was chopped into small pieces and distilled using a Clevenger-type apparatus. Oil was extracted, dried in sodium sulfate anhydrous, and stored at 4 °C in sealed dark bottles until further bioassays and investigations. Oil was fractionated into oxygen and hydrocarbon fractions by flash chromatography on silica gel using a glass column. The fractions were assayed against each test organism.

Gas chromatography (GC) and GC-mass spectroscopy (GC-MS)
The oxygenated and hydrocarbon fractions of cinnamon oil were analyzed via GC, using a DB-S capillary column. The oven temperature was set at 50 °C. The GC-MS conditions were similar to GC. The electron ionization was set at 70 eV, the particle source temperature was set at 275 °C, and the output rate was 29-400 atomic mass units sec -1 .

Identification of cinnamon oil components
Authentic components were injected, and comparisons were made in terms of the retention time, Kovats index, and mass spectra. The latter parameter was compared with the mass spectra of the National Bureau of Standards (NBS) library and spectra.

Antibacterial activity of cinnamon essential oil
The antibacterial activity of oxygenated and hydrocarbon fractions was examined in air space. The preliminary examination of the activity of oxygenated and hydrocarbon fractions against ESBL-resistant E. coli and K. pneumoniae isolates was carried out. The oil concentration ranged from 1 to 16 μL per 0.4l L of air space, based on the overturned Petri dish method. The paper discs (5 mm diameter) were impregnated with oil concentrations and placed on a Petri dish. The plate dimensions were 140 × 23 mm, providing 0.4l mL of air space after pouring the medium. Next, a loop from a 24-hour-old culture of tested bacteria was streaked on the CLED medium. The plates were sealed with parafilm and grown at 36 °C for 24 hours. Finally, colony formation was observed. For the controls, distilled water was used rather than oil.

MIC of oxygenated fraction
MIC was expressed as the lowest concentration of oil, preventing the visible growth of microbial agents. It was determined using the lysogeny broth (Sigma, St. Louis, MO, USA). Bacterial concentration was determined based on the McFarland standards. The bacterial isolates were two-fold dilutions of the oxygenated fraction. Cell viability was examined after 24 hours of incubation, using a colony-forming assay on a nutrient agar (Kohanski et al., 2007).

Scanning electron microscopy (SEM)
Changes in the treated bacterial cells were observed using SEM, according to the method proposed by Diao et al. (Diao et al., 2013). Bacterial cells were incubated in nutrient broth (NB) at 37 °C for ten hours, and then, the suspensions were added to the MIC of the oxygenated fraction (80 μl mL -1 and 20 μl mL -1 for E. coli and K. pneumoniae, respectively). The suspension was incubated at 37 °C for four hours; afterward, the suspension was centrifuged at 5,000 rpm for ten minutes. The cells were washed twice with 0.1 M phosphatebuffered saline (PBS; pH = 7.4) and fixed with 2.5% (v/v) glutaraldehyde in 0.1 M PBS at 4 °C. Next, the cells were gradually dried using 30%, 70%, 90%, and 100% ethanol, and ethanol was supplemented with tert-butyl alcohol.
The cells were gold-coated via a cathodic coating, and the morphology of bacterial cells was determined using SEM (INSPECT S50) at the College of Sciences of Hafr Al-Batin University.
A total of 170 isolates were screened for ESBL production, using the MicroScan WalkAway-96 SI system, according to the CLSI guidelines. The results showed that 104 (61.18%) isolates were positive for ESBL production. Among 104 ESBL-positive isolates, 76 were E. coli, and 28 were k. pneumoniae (Table 1).
The ESBL-positive isolates were selected for the corroborative trial of ESBL. Two procedures, i.e., DDST and PCDDT, were performed to confirm ESBL production. Analysis of cinnamon oil indicated that cinnamaldehyde and eugenol were the major components of the oxygenated fraction (57.17% and 15.64% relative to oil, respectively). Camphene was the major component of the hydrocarbon fraction, while cinnamaldehyde was the major component of the oxygenated fraction (Table  2). Nineteen compounds comprising the oxygenated fraction were identified via GC-MS and literature review. Generally, cinnamon oil is characterized by high levels of oxygen-containing monoterpenes, including cinnamaldehyde, which is the main component of cinnamon essential oil.
The susceptibility of ESBL-producing bacteria to the volatiles of oxygenated and hydrocarbon fractions of cinnamon oil was examined using the inverted Petri dish method, as shown in Table 3. Bacterial growth suppression was only observed in the oxygenated fraction. K. pneumoniae showed high sensitivity to the oxygenated fraction. Its growth was suppressed at a concentration of 2 μl 0.4 L -1 air space. E. coli growth was suppressed at a concentration of 8 μl 0.4 L -1 air space (Table 3).
The ESBL-producing E. coli and K. pneumoniae isolates were exposed to two-fold serial dilutions of the oxygenated fraction for 24 hours. The oxygenated fraction influenced the cellular viability of ESBL-producing bacteria in a dose-dependent manner. The bacterial cell viability was monitored to determine the bactericidal effect of different concentrations of the oxygenated fraction on E. coli and K. pneumoniae. The bacterial cell viability was also estimated using a colony-forming assay in a nutrient agar. The E. coli and K. pneumoniae isolates were suppressed at concentrations above 80 and 20 μl mL -1 at 10 5 CFU, respectively, where no bacterial growth was seen in the agar plates at the evaluated oil concentrations (Figure 2).
The study of the morphological changes of microorganisms is essential for distinguishing the  Table 3 Activity of oxygenated and hydrocarbon fraction of cinnamon oil against ESBL-producing bacteria using inverted Petri dish technique.

Bacteria
Oxygenated fraction (μl 0.4 l -1 air space) Hydrocarbon fraction (μl 0.4 l -1 air space) antibacterial properties of cinnamon oil. SEM was used to explore the surface morphology of E. coli and K. pneumoniae after cinnamon oil treatment in the NB medium ( Figure 3). The treated bacterial cells were significantly damaged, as shown in TEM images.
The current study clearly showed that E. coli and K. pneumonia isolates from water were resistant to thirdgeneration cephalosporins and sensitive to carbapenems. The findings also revealed an alarming increase in the prevalence of resistance to commonly used antibiotics.
Similar results were reported by Shakibaie et al., 2014 who isolated E. coli from UTI children in the pediatric unit of a medical clinic in Kerman, Iran. Also, they found that 100% of isolates were sensitive to carbapenems, and 94.4% were sensitive to amikacin; all uropathogenic E. coli isolates were resistant to ampicillin (100%). Likewise, in our study, K. pneumoniae showed complete resistance to ampicillin (100%), followed by mezlocillin (92.5%). On the other hand, it showed the highest sensitivity to both imipenem and fosfomycin (90.7%). Consistent results were reported by Adeyemi et al., 2014 who found an

Figure 2
Colony-Forming Capacity Assay 1 -growth at concentration of 2.5 μl mL -1 of oxy. Fraction, 2 -growth at 5 μl mL -1 , 3 -growth at concentration of 10 μl ml -1 , 4 -growth at 20 μg mL -1 , 5 -growth at concentration of 40 μl ml -1 , 6 -growth at concentration of 80 μl ml -1 , compared with positive and negative controls A standard procedure is necessary for the identification of ESBL-producing microorganisms, based on standard identification methods to control the spread of diseases and propose appropriate treatment plans. The current study showed that PCDDT was more sensitive than DDST in identifying ESBL. DDST identified 67% of K. pneumoniae isolates and 83% of E. coli isolates, whereas PCDDT detected 82% of ESBL-producing K. pneumoniae isolates and 89% of E. coli isolates. According to some studies, the percentage of ESBL-producing bacteria ranges from 28% to 84% in India (Narayanaswamy and Mallika, 2011). The percentage of these bacteria was the lowest in Maharashtra, India, as reported by Rodrigues et al. (Rodrigues et al., 2004). The high prevalence of ESBL-producing K. pneumoniae has also been reported by other researchers (Lal et al., 2007;Ramesh et al., 2019).
Aromatic volatile components of plants exhibit higher antimicrobial activity than non-aromatic volatile oils (Shaaban et al., 2012). Our results are in line with previous studies, which indicated the antimicrobial activity of cinnamon oil (Raeisi et al., 2015;Lin et al., 2017); however, the cell membrane may be injured after treatment. These findings showed that cinnamon oil could destroy the bacterial cell membrane, causing cell lysis and death. Cinnamaldehyde was found to be the main component of cinnamon essential oil ( Figure 4); this result may represent the mechanism of action of the oxygenated fraction.

Conclusions
The results of the present study showed that cinnamon oil and its components could be used as antibacterial agents to prevent bacteria-induced water contamination. Moreover, in vivo studies regarding the mechanism of the antibacterial activity of cinnamon oil should be performed.