In vitro assessment of the antibacterial effect of watery date extracts (Mozafati, Zahedi and Piaroum) on Staphylococcus aureus and Escherichia coli

The present research aimed at investigating the antibacterial effects of date extracts (Mozafati, Zahedi and Piaroum) on the stationary and exponential growth phases of Staphylococcus aureus and Escherichia coli O157: H7 in vitro. The methods used in this study included ow cytometry and culture. Carboxyl uorescein diacetate was utilized to determine the effects of extracts and estimate bacterial survivability. Under the impacts of Mozafati, Zahedi and Piaroum extracts, the bacterial elimination percentages recorded in the log phase of S. aureus were 30.90, 86.35 and 97.18% respectively. However, the effect of exposure of E. coli to date extracts in log phase resulted in the destruction of 8.76, 24.69 and 38.97% of these bacteria respectively. The effect of the extracts on the log phase of S. aureus showed a signicant increase compared to that of E.coli (p<0.05). The extracts had the maximum impacts on the stationary phase of both S. aureus and E. coli within 3h exposure time. Mozafati and Piaroum extracts yielded the highest bacterial elimination effect within 3h while Zahedi extract had the maximum effect at zero time on stationary phase of E.coli. The effect of these extracts on the stationary and logarithmic phases of growth of S. aureus and E. coli is bacteriostatic. The result of ow cytometry showed an increase as compared with that of culture. The antibacterial effect of Zahedi extract on S. aureus and E. coli proved to be meaningfully higher than those of other extracts. In order to destroy the examined bacteria in both stationary and logarithmic phase, the use of Zahedi, Piaroum and Mozafati extracts is recommended respectively.


Background
Food borne diseases have been on the rise in recent years, causing major public health problems around the world. The pathogenic bacteria are the main cause of food spoilage that creates serious illnesses in humans. Since food borne pathogens such as Staphylococcus aureus (S. aureus) and Escherichia coli O157: H7 (E. coli) bacteria are commonly found in various food stuffs, it is highly important to identify them. The current methods used for identifying food borne pathogens are both di cult and time consuming. Thus a number of quick diagnostic methods of food pathogens have been devised that are required in any investigation related to food borne disease.
In general these quick identi cation methods are e cient, sensitive and exclusive that saves in time and energy (Woan-Fei Law, 2012). The necessity to develop quick identi cation methods in early prevention and treatment of food borne illnesses as well as the upsurge of interest in applying quick and automated techniques in food microbiology during recent decades gave rise to the use of uorogenic substrates along with uorescence measurement techniques including ow cytometry for quick diagnosis of bacteria (Shapiro, 2003). Given the time required for carrying out the routine tests which can, at times, lead to delayed entry of products into the market, a strong relationship between the results of ow cytometry and shelf life of products might provide a real picture of their quality, allowing their early supply in the market. Meanwhile, the use of ow cytometry enables direct assessment of bacterial growth so as to maintain a controlled microbial biomass during production process. In addition, considering the time needed to obtain the results and con rm the links between ow cytometry and conventional counting method in the industry, it may provide a suitable predictive means of controlling the quality of product and production process. The advantages of ow cytometry include high precision, no need for enrichment, qualitative and quantitative deliverability, live/dead cell distinction and identi cation of uncultivable living organisms. In addition, it is possible, via ow cytometry to make distinctions between microbial cells against other bacteria or nonbacterial particles (Endo et al., 2002). So far, ow cytometry has made it feasible to identify, S. aureus, E. coli (Tanaka et al., 2000)and Lactococcus lactis (Bunthof et al., 1999) bacteria present in natural water bodies (Yamaguchi and Nasu, 1997).
Cultivation of date trees goes back to some 4000 Before Christ (BC). Date constitutes one of the most signi cant species within date palm family which is also the biggest and most ancient tree ever planted by human kind. Date embrace some 200 genera along with 2500 species. There are about 400 species of dates growing in Iran (Ashraf and Hamidi Esfahani, 2011). The country is the second major producer of dates with 14% of total world date production. By-products from dates have, until now, included juice, jam, jelly, and liquid sugar. The fermented products are alcohol, syrup, Tarooneh, wine, organic acids, soft drinks made through fermentation and single cell proteins. Nevertheless, there has, so far, been no extracts made from dates (El Hadrami and Al-Khayri, 2012).
Whilst enjoying common nutritional value and valuable qualities, the various kinds of dates such as Mozafati (soft), Piaroum and Zahedi (semi dry) have also certain biogenic characteristics that distinct them from one another. Although, dates have been consumed as staple food by millions of people throughout centuries, it had not caught the attention of health specialists as a health food and thus, their important antimicrobial properties were remained unnoticed or not fully grasped. Considering that dates are recognized mainly as a phenolic compound and the antibacterial properties of phenolic compounds have already been con rmed in a number of researches (Pereira et al., 2009, ;Rauha, 2000;Fernandez-Panchon et al., 2008) they can therefore, be regarded as a dietary, functional and natural compound with antibacterial effects. The antimicrobial properties of dates have also been assessed through various means including culture and minimum inhibitory concentrations (MIC) methods (Saleh and Otaibi, 2013).
However, up to now there has not been any attempt to con rm the antibacterial effects of date extracts on S. aureus and E. coli bacteria through ow cytometry technique.
The present research aimed at investigating the bactericidal effects of date extracts (Mozafati, Zahedi and piaroum dates) on the stationary phase and logarithmic (exponential) growth phase of S. aureus and E. coli O157: H7 in vitro.

Methods
Flow cytometry and culture methods were used to assess the effects of date extracts (Mozafati, Zahedi and piaroum) on the stationary phase and exponential (log) growth phase of S. aureus and E. coli.

Features of ow cytometry
The samples were examined using Partec PASS, Flow Max software, FL1 channel, Argon laser beam, -20, 100, ultraviolet lamp 100w, diode laser, green uorescence 488nm from Argon laser with a wavelength of 515-530nm.

Date watery extraction
In order to carry out the watery extraction, the dates (100 g) were rst immersed in distilled water (200 ml) for 72 hours in the dark at the refrigeration temperature. Next, the solution was mixed with a mixer and ltered using lter paper No. 1. The suspension was centrifuged using a refrigerated centrifuge at 3000 rpm for 15 minutes. The supernatant was used as extract and was pasteurized at 65°C for 30 minutes.
The extracts were refrigerated storage until use (Mehdi pour et al., 2017).

Dye preparation from carboxyl uorescein diacetate
Carboxyl uorescein diacetate (CFDA) was used as a bio indicator for live bacteria. To prepare the dye, 0.05g of the dye powder was added to DMSO 1mm.

Preparing positive/negative control for ow cytometry analysis
In order to obtain positive control, 1mm from each bacterial suspension of S. aureus and E. coli produced in exponential (log) phase was exposed 2h to 1mm of isopropanol. After incubation period, the samples were centrifuged 2800rpm for 15min followed by removal of supernatant. The bacteria pellet was rinsed two times with phosphate buffered saline solution and later dissolved in equal volume of the original phosphate saline solution. Next,1µl of Staphylococcus suspension was mixed with 10µl of dye and 1µl E. Coli suspension was added to 20µl of dye. The mix was put to rest in ambient temperature for 15min.
To obtain negative control, 1ml from each suspension of live S. aureus and E. coli bacteria in the log phase were dyed with 10 and 20µl CFDA respectively. In addition, 1 ml from each solution of live and dead bacteria was mixed with CFDA (Sigma 8166 C). The mix was analyzed using ow cytometry.
The analysis of stationary phase S. aureusand E. coli bacteria through ow cytometry technique In order to make synthetic assessment of these bacteria, the stationary phase of growth was used after 6-9h and 6-10h of culture for S. aureus and E. coli respectively. A 6% date extract was obtained of all the three date species (Mozafati, Zahedi and Piaroum). Then, 2ml suspension of stationary phased bacteria and 2ml of the examined watery extracts were separately exposed. The solutions were next incubated at 37 o C, 120rpm for 48h.
For ow cytometric analysis, 1ml of S. aureus and E. coli suspension was exposed to the examined date extracts and the bacterial culture pellet was dissolved again in phosphate buffered saline following centrifugal procedures and removal of supernatant. Samplings for ow cytometric analysis were carried out at 0, 3, 15, 24 and 48h.
Flow cytometric analysis of S. aureusand E. coli in log phase In order to culture log phase S. aureus and Escherichia coli, OD of 1.45 and 1.42 were applied. The extract preparation stages, the exposure and ow cytometry were similar to those of in stationary phase.
Counting S. aureusand E. coli in the stationary and log phases of growth via culture method Preparing samples for the intended bacterial culture in the stationary and log phase of growth was conducted similar to ow cytometry. The culture of S. aureus was done in Mannitol salt agar medium and E. coli was cultured in Sorbitol MacConkey agar.

Statistical analysis
The results of microbial tests were analyzed by SPSS Software. Samples were compared to later stages of themselves by Two Way Variance Analysis (culture and ow cytometry). The effect of extracts on the number of S. aureus and E.coli bacteria in the log phase were compared by t-test. The results obtained from ow cytometry were analyzed by Flowing-Software.

Results
Flow cytometry calibration for S. aureus and E. coli were presented in gures 1 and 2.
Bacterial counts related to S. aureus and Escherichia coli affected by exposure to date extracts (Mozafati and Piaroum) showed to be minimal at 3h. However, it increased at other sampling times. Due to being exposed to Zahedi date extract, S. aureus and E. coli showed the least counts at 3h and zero h sampling times respectively whereas their number tended to rise at other sampling times (Figures 3 -12).
As gures (3 -6) show, at zero time, the number of S. aureus exposed to the stress induced by date extracts (Mozafati, piaroum and Zahedi) were 49.62, 55.08 and 72.79%, respectively. The effect of Zahedi extract was meaningfully greater than other extracts on reducing the number of S. aureus. Compared to other date extracts, the effect of Mozafati on these bacteria showed a signi cant decrease. The number of live S. aureus cells exposed to the stress induced by date extracts (Mozafati, piaroum and Zahedi) at 3h was signi cantly lower in comparison to that of zero time. From this point on, the increased exposure duration was mostly associated with an increase in the number of live bacterial cells. These extracts had the highest impact on stationary phase of S. aureus at 3h. Based on the results obtained, the effects of extracts from these date species on stationary phase of the bacteria (S. aureus) proved bacteriostatic.
The results of the present study also showed that the inhibitory effect of date extracts (Mozafati, Piaroum and Zahedi) on S. aureus was more signi cant than that of Escherichia coli in stationary phase.  (Figures 6 -9).
Based on gures 10, 11 and 12, under the impact of Mozafati, piaroum and Zahediextracts, the recorded bacterial elimination percentages of S. aureus in the log phase were 30.90, 86.35 and 97.18% respectively. The gures related to log-passed E. coli cells however, were 8.76, 24.69 and 38.97% respectively. The effect of the examined extracts on bacterial elimination of S. aureus in the log phasewas a signi cant increase in comparison to those of E. coli. However, compared with other date extracts, Zahedi showed a meaningfully higher inhibitory effect on the log phase of S. aureus and E. coli cells whereas the effect of Mozafati extract showed a meaningful decrease on the log phase of S. aureus and E. coli cells. E. coli cells showed less sensitivity to Mozafati, Zahedi and piaroum extracts in the log phase of growth. The effects of all the date extracts on the log phase of S. aureus were more signi cant than those of in the stationary phase. However, the effects of these date extracts on E. coli cells in the stationary phase showed a signi cant increase compared to those of in the log phase. So far, there has been no report on the effects of date extracts on the log-phased S. aureus and E. coli cells. And the present research is the rst attempt in this area.
According to gure 12, the effect of extracts on the number of S. aureus and E. coli cells in the log phase showed signi cant decrease in Zahedi, piaroum and Mozafati treatments. The effects of all three extracts on the log phase of S. aureus were signi cantly higher than those on E. coli. The number of S. aureus in treatments experiencing stress induced by Mozafati, Piaroum and Zahedi extracts were CFU/g 1×10 7 ,18×10 6 , 1×10 3 respectively which proved lower than that of in control. The E. coli number exposed to stress caused by date extracts (Mozafati, piaroum and Zahedi) was lower than control by CFU/g 1×10 3 ,1×10 2 , 1×10 1 respectively.
In each sampling stage for bacterial counting in culture and ow cytometry in both stationary and log phase of bacterial growth, the number of live S. aureus and E. coli cells speci ed in ow cytometry method turned out to be higher than that of culture. Nevertheless, the rising or the downward projection of bacterial count in culture showed to be in conformity with that of ow cytometry technique (Figs 3 -12).

Discussion
Physiological activity of pathogenic bacteria in food, should be investigated to yield useful information pertinent to food hygiene and public health ( Tanaka et al., 2008). As gs 1 and 2 show, in this study dead cells were separated from live ones because enzyme (esterase) is inactive in dead cells According to results, in most cases the number of bacterial cells at 3h experiencing deterred growth due to exposure to the examined date extracts reached its maximum level. From that point onward, increasing the duration of exposure time resulted in the higher increase of live bacterial cells. There has, so far been no research concerning the impact of date extracts on the synthetic growth phase of S. aureus and E. coli cells. Synthetic examinations on the growth of S. aureus (Figs 3, 4, 5 and 6) and E. coli (6, 7, 8 and 9) exposed to watery extracts of Mozafati, piaroumandZahedi suggest their relative inhibiting potentials on the growth of S. aureus and E. coli. The greater increase of bacterial population in certain cases may have its origin in their adaptation to the environment. Despite their antimicrobial effects on S. aureus and E.coli, these extracts possess certain compounds and nutritional contents required for the growth of bacteria and a gradual compromise takes place between bacteria and the environment provided by date extracts (Figs. 3,4,5,6,7,8 and 9). In the present study, ow cytometry was rst time used to assess the effect of date extracts on S. aureus and E. coli, the results of which were in line with the general ndings of bacterial culture method.
Based on diagrams 10, 11 and 12, contrary to Mozafati, the extracts of Piaroumand Zahedi were found to be able to inhibit the growth of log-phased S. aureus but E. coli cells in the log phase were observed to be less affected by exposure to extracts of Mozafati, Piaroum and Zahedi. Up to the present, there has not been any research on the effect of date extracts on the log phase of S. aureus and E. coli cells. This study is the rst in its kind to probe into the issue, though the role of avonoids in deterring bacterial growth has already been investigated (Rauha, 2000). In yet another study by Seifzadeh and Rabbani (2019), the role of bacterial ora was assessed to be of greater signi cance than avonoids in exerting the antibacterial impacts of date extracts. Thus, in addition to avonoids the sensitivity of S. aureus to Piaroum and Zahediextracts and the relative sensitivity toMozafati might be attributed to bacterial ora present in the extracts. The bacterial ora in Zahediextract belonged to Bacillus subtilis strain UD1022, inPiaroum to Lecunostoc mesenteroeides subsp mesenteroeides and in Mozafati to Pediococcus parvalus strain SC8B. Allthese were probiotic bacteria. In addition, it was found in this study that the examined date extracts were unable to fully destroy or rupture the cell walls of S. aureus and E. coli since after sometime S. aureus and E. coli cells reoccurred in date extracts. This was con rmed by the results of ow cytometry and culture obtained at varying sampling times.
According to diagrams (3 -12) bacterial counts revealed in ow cytometry showed higher numbers than those of in cell culture. Such a greater number might stem from lack of growth of injured cells at sub lethal level in culture medium. Having esterase enzymes, the affected cells react to dye and are thus speci able by ow cytometry due to their preserved physiologic functions.
CFDA is applied for specifying the number of active bacterial cells having esterase such as Klebsiella pneumonia (Diaper and Edwards, 1994), Listeria monocytogenes (Nexmann Jacobsen et al., 1997) as well as counting bacterial number in pure water aimed at use in pharmaceutical industry (Kawai et al., 1999) and bacterial survivability (Porter et al., 1995). In this study CFDA was used to dye S. aureus and E. coli cells for ow cytometric analysis. CFDA is used here as substrate for the cell esterase. This enzyme is present in the living organism. It is a non-uorescent substrate, broken up by inner cell enzymes which result in the production of uorescence. Considering that the cell is of inner esterase activity with healthy membrane, a pile up of broken substrate is formed within the cell, creating uorescence (Hoefel et al., 2003). Renggli et al (2013) determined E. coli treated with bactericidal antibiotics by ow cytometry. Malmberg (2013) found that ow cytometry worked equally well as in cell culture in bacterial identi cation and counting of E. coli exposed to antibiotic stressin a way that it could substitute quick counting method used in conventional cell culture. Endo (et.al 2002) gave a report on E. coli number and the total bacterial counts on the surface of surimi products through ow cytometry and parallel method of cell culture. Shariati et al. (2010) evaluated antibacterial effects of date pit extract on multidrug-resistant S. aureus. Alawi et al. (2017) found that the date extract had inhibitory effects on S. aureus and E. coli. The antimicrobial property of date against S. aureus and E. coli was proven by Baliga et al. in 2011. Idris et al. (2017 demonstrated that the date extract had antibacterial effects on S. aureus and E. coli. These results are in line with the results of the current study. Ou (et.al 2017) reported a combined culture trial consisting of live/dead E. coli cells through both ow cytometry and parallel traditional method. However, in this study, the number of cells speci ed in ow cytometry showed a rise compared to that of in culture method. This might be accounted for by different steps in preparing the strain and the bacterial sample. Fröhling and Schlüter (et.al 2015) reported the survivability of negative gram E. coli exposed toPeracetic acid and ozone via ow cytometry. With a view to evaluate the inactivating impacts of heat, Peracetic acid and ozone on E. coli population. Vorgelegt (et.al 2011) acquired the results through ow cytometry and compared them with those of culture. The results were in conformity with ndings of the present study.
Rüger (et. al 2012) investigated the use of ow cytometry in survivability estimates of S. aureus in combined culture method which were similar to the ndings of the present study. Shrestha (et. al 2012) could identify Methicillin-Resistant S. aureus (MRSA) via ow cytometry. These researchers detected live and dead cells using ow cytometry and reported that the results could be considered as equal to those of culture method or dealt with greater sensitivity.

Conclusion
The emergence of microbial strains resistant to antibiotics is one of problems facing pharmaceutical industry. S. aureus and E. coli constitute the major cause of food poisoning and other serious illnesses.
Considering the noticeable effects of date extracts on these bacteria and their potential uses in pharmaceutical and/or food industries and in order to prevent and cure infections, it is recommended to utilize date extracts either separately or in combination with antibiotics.  The effect of Mozafati extract on the stationary phased S. aureus by Flowing-software% The effect of Piaroum extract on the stationary phased S. aureus by Flowing-software% Page 14/16 Figure 5 The effect of Zahedi extract on stationary phased S. aureus by Flowing-software% Figure 6 The effect of Mozafati, Zahedi and Piaroum extracts on stationary phase of Staphylococcus aureus and Escherichia coli via culture method (logCFU/g) Figure 7 The effect of Mozafati extract on stationary phased E.coli by Flowing-software% Page 15/16 Figure 8 The effect of Piaroum extract on stationary phased E.coli by Flowing-software% Figure 9 The effect of Zahedi extract on stationary phased E.coli by Flowing-software% Figure 10 The effect of Mozafati, Piaroum and Zahedi extracts on the log phazed (after 24h) E. coli bacteria by Flowing-software%