*3.2. Antimicrobial Activity*

In order to analyze the antimicrobial activity of sumac, a comparative study was conducted by evaluating the antibacterial potential of six different extracts against foodborne pathogens. The isolates involved one *S. aureus* isolate, one *K. pneumoniae* isolate, and five *E. coli* isolates.


**Table 4.** Polyphenolic compounds in *Rhus coriaria* SM extract.

The antimicrobial susceptibility test showed that the Enterobacteriaceae isolates from poultry (*E coli* and *K. pneumoniae* strains) were resistant to AML and N. The Enterobacteriaceae isolates tested were found to be resistant to three or more antimicrobial agents that belong to dissimilar antibiotic classes and are multidrug resistant (MDR). The antimicrobial profile of the tested microorganisms is reported in Table 5.

**Table 5.** Antimicrobial resistance profile.


NA, nalidixic acid; CIP, ciprofloxacin; AML, amoxicillin; AUG, amoxicillin-clavulanic acid; SXT, trimethoprimsulfamethoxazole; TE, tetracycline; N, neomycin; C, chloramphenicol.

These results, showing the multi-resistance of the majority of the isolates, confirm previous studies that reported a high level of multidrug resistant Enterobacteriaceae isolates from poultry in Algeria [23,32]. The causes of this antibiotic resistance phenomenon are very different, and the important cause is the massive and inappropriate use of antibiotics. Currently, antimicrobial resistance represents a growing global concern, and the development of effective therapeutic options against MDR bacteria is a public health priority.

Plant extracts can be valuable alternatives to antibiotics [33,34]. Recently, sumac has gained more attention due to its high amount of polyphenols. Numerous studies have been

conducted to study the in vitro antimicrobial effectiveness of *Rhus coriaria* extracts against several bacterial species [14,35,36], while limited data are available on their effect against MDR bacteria. In this study, the antibacterial activity of *Rhus coriaria* extracts was tested against five MDR Enterobacteriaceae isolates, *S. aureus* ATCC 6538, and *E. coli* ATCC 25922. The results of the antibacterial activity of *Rhus coriaria* extracts are summarized in Table 6, which indicates that almost all the extracts exerted antibacterial activity against all the tested strains.


**Table 6.** Antibacterial activity of extracts from *Rhus coriaria* evaluated by disk diffusion assay.

SA, acetone extract; SE, ethanol extract; SM, methanol extract; SAW, acetone + water extract; SEW, ethanol + water extract; SMW, methanol + water extract; SW, water extract. Means sharing the same capital letters in the raw are not significant at *p* < 0.05 according to Tukey's HSD test.

The higher antimicrobial activity was obtained by using SM and SE. In particular, SE presents growth inhibition zones ranging from 14 to 16 mm and SM ranging from 22 to 25 mm. Given the higher antimicrobial activity of the SM, its MIC was evaluated, and the results are reported in Table 7. These results are in line with a study on Syrian sumac antimicrobial activity, which reports that the antimicrobial activity of the methanol extract was the most effective [37]. In addition, further studies reported on the higher amount of phenolics and flavonoids contained in the methanolic extract compared with the ethanolic one [37,38]. Several studies have described the antibacterial activity of the fractions of sumac extract, highlighting how some compounds, due to their polarity, can act only on Gram positive bacteria, while some others, such as gallic acid, can affect both Gram positive and Gram negative bacteria; nevertheless, their antibacterial activity is not so strong [38].

**Table 7.** Antibacterial activity of SM from *Rhus coriaria* evaluated by serial double dilution method.


Several studies have also reported on the efficacy of the total extract and attribute its biological activity to its content in phenolics, which are the major extract fraction [39–41].

The maximum inhibitory action was observed at a concentration of 9.37 μg/mL for all strains except for *E coli* (S2/15), which presented an MIC of 4.68 μg/mL. This finding supports the use of *Rhus coriaria* in traditional medicine as a bactericide agent. A study reported that the water extract of *R. coriaria* had an effective in vitro antibacterial power against *S. aureus*, *P. aeruginosa*, and *S. aureus* (MRSA) [35]. Another study reported that the extract showed strong antibacterial activity against Gram positive and Gram negative bacteria,

with MIC < 0.78% [42]. A similar study assessed sumac's methanolic extract antibacterial power as having the highest inhibitory activity. In all sumac extracts, increasing the concentration of sumac causes an increase in antibacterial power [14]. In addition, the methanolic extract of sumac leaves revealed antibacterial activity against *E. coli* and *S. aureus.* A MIC of 312 μg/mL was reported, although the inhibitory effect was only bacteriostatic, and the bactericidal effect was observed at a concentration of 2500 μg/mL [36].

The results of our study, highlighting the higher activity of the methanol extract, are therefore in line with previous comparative studies that showed that methanolic extracts of sumac contain a higher content of flavonoids and phenolics when compared with other extracts [13,14].

#### *3.3. Fish Embryo Acute Toxicity (FET)*

The evaluation of the toxicological profile of medicinal plant extract is of utmost importance. Zebrafish (*Danio rerio*) is one of the main study models [43]. Given that the embryo develops quickly outside the mother and that this is visually evident, it is certainly usable for testing and observation. Being that the ZFET is a valid alternative method to animal tests [44,45], the non-toxicity of the *R. coriaria* genotype from Sicily was assessed and confirmed by analyzing the effect of the extract on zebrafish larvae. To date, there is no evidence of the toxicity of Sicilian sumac.

In this study, fertilized zebrafish embryos were exposed to SM extract from *R. coriaria* at a concentration of 9.37 μg/mL. According to our results, the extract was found to be non-toxic using zebrafish FET assay. During the 96 h of exposure, no visible toxic effects of this extract on the development of embryos were observed (Figure 1). The mortality was 5% (one larva) for the whole test period. During the observation under the stereomicroscope, it was observed that at 48 hpf (hours post-fertilization), the hatched larvae were 95%.

**Figure 1.** Development of embryos. Hpf: hours post-fertilization.

According to the OECD guidelines, the *R. coriaria* extract obtained from the Sicilian genotype did not induce any toxic effect on zebrafish embryos and larval development. These results are in line with other findings reporting the safe and even beneficial effects of the *R. coriaria* extract on both humans and animals [5]. Other studies evidenced no acute toxicity of the extract in rat model experiments and, in addition, showed beneficial cardioprotective and hepatoprotective properties under hypercholesterolemic conditions [46]. Another study using a diabetic rats model and testing 250, 500, and 1000 mg/kg of the plant extract reported good tolerance and a non-lethal oral uptake of this extract, even at 1000 mg/kg, showing not only no signs of toxicity and mortality after 3 days of daily extract administration but also a positive effect on diabetes and diabetes-related complications [47]. Taken together, these results sugges<sup>t</sup> the safety of this plant.
