Antimicrobial Resistance Properties of Staphylococcus Aureus Isolates From Powdered Packaged Medicinal Plants and Bottle Herbal Distillates

Background Human involvement in the production and processing of medicinal plants and herbal distillates caused a potential risk of microbial contamination, particularly with Staphylococcus aureus. The present research was performed to assess the prevalence and phenotypic and genotypic properties of antibiotic resistance of S. aureus bacteria isolated from diverse kinds of powdered packaged medicinal plant and bottle herbal distillate samples. Three-hundred different powdered packaged medicinal plant and bottle herbal distillate samples produced in traditional conditions were collected and examined by the culture method. Phenotypic and genotypic patterns of antibiotic resistance of S. aureus isolates were examined using disk diffusion and PCR techniques. Thirty out of three-hundred (10%) powdered packaged medicinal plant and bottle herbal distillate samples were contaminated with S. aureus. The prevalence of S. aureus amongst the powdered packaged medicinal plant and bottle herbal distillate samples were 8.33% and 11.11%, respectively. A. citrodora (10%) and R. damascene (10%) powdered packed medicinal plants and A. maurorum (16.66%) bottle herbal distillate had the highest contamination rate with S. aureus. S. aureus isolates harbored the highest prevalence of resistance toward penicillin (93.33%), tetracycline (90%), gentamicin (86.66%), erythromycin (70%), trimethoprim-sulfamethoxazole (63.33%) and ciprooxacin (53.33%). Totally, 13.33% of the S. aureus isolates harbored resistance toward more than 7 antibiotic agents. blaZ (63.33%), tetK (60%), ermA (46.66%), msrA (43.33%), aacA-D (43.33%), and mecA (43.33%) were the most frequent antibiotic resistance genes. is substantial to assess the microbial quality of medicinal plants and herbal distillates. Thus, the present survey was conducted to assess the prevalence rate and phenotypic and genotypic assessment of S. aureus bacteria's antibiotic resistance isolated from different powdered packaged medicinal plant and bottle herbal distillate samples produced in traditional producing units in Iran. doxycycline and vanB (P <0.05), vatA and vatB (P <0.05), ermA and ermB (P <0.05), msrA and msrB (P <0.05) and tetK and tetM (P <0.05) antibiotic resistance genes. There was no signicant difference between the distribution of uoroquinolones resistance genes (P >0.05). tetK, such as the Hazzard Analysis Critical Control Points (HACCP) system are essential to minimize the risk to the consumer. Furthermore, using high quality raw materials in production of powdered packaged medicinal plants and bottle herbal distillate samples, prevention from cross-contamination and antibiotic prescription based on disk diffusion outcomes can diminish the risk of transmission of multidrug resistant-S. aureus bacteria from powdered packaged medicinal plants and bottle herbal distillate samples to the human population. On the basis of these observations, we recommend that attention should be paid by governments and individuals to prevent the further spread of multidrug resistant-S. aureus. However, supplementary surveys are essential to determine more epidemiological features of the multidrug resistant-S. aureus bacteria in powdered packaged medicinal plants and bottle herbal distillate samples.


Materials And Methods
Sampling From May 2019 to January 2020, a total of 300 diverse kinds of powdered packaged medicinal plants including Z. multi ora (n= 30), S.bachtiarica (n= 30), A.citrodora (n= 30) and R.damascene (n= 30) and bottle herbal distillates including L.angustifolia (n= 30), A. maurorum (n= 30), C. intybus (n= 30), M. o cinalis (n= 30), M.piperita (n= 30) and F.o cinalis (n= 30) were randomly collected from shopping centers, Tehran, Iran. A total of 50 g samples were collected from each powdered packaged medicinal plant and bottle herbal distillate sample using a sterile laboratory tube. All bootle herbal distillates were produced conventionally in small traditional producing units. Additionally, all collected powdered packaged medicinal plants were dried, powdered, and packed conventionally in traditional production units. Speci cations about samples were recorded according to their labels. All samples were directly transferred to the laboratory at 4 °C.
Isolation and identi cation of S. aureus bacteria Twenty-ve grams of each collected powdered packaged medicinal plant and bottle herbal distillate samples were blended with 225 mL of buffered peptone water (Merck, Germany). At that time, solutions were homogenized using Stomacher (Interscience, Saint-Nom, France). At that point, ve milliliters of the achieved solution was transferred into 50 mL Trypticase Soy Broth (TSB, Merck, Germany) supplemented with 10% NaCl and 1% sodium pyruvate and incubated for 18 h at 35 °C. At that moment, a loopful of the culture was transferred into Baird-Parker agar supplemented with egg yolk tellurite emulsion (Merck, Germany) and incubated at 37 °C for about 24 h. Black shiny colonies enclosed with signi cant zones identi ed using biochemical tests including Gram staining, oxidase test, catalase activity, resistance to bacitracin (0.04 U), coagulated test (rabbit plasma), urease activity, glucose O/F test, vogesproskaver (Merck, Germany) test, nitrate reduction, phosphatase, deoxyribonuclease (DNase, Merck, Germany) test, mannitol fermentation, hemolysis activity on blood agar (Merck, Germany) and carbohydrate (xylose, sucrose, trehalose and maltose, fructose, lactose, mannose) fermentation tests [19].
DNA extraction and quality examination S. aureus isolates were sub-cultured on TSB media (Merck, Germany) and incubated for 48 h at 37 o C. Genomic DNA was extracted from MRSA colonies using the DNA extraction kit (Thermo Fisher Scienti c, St. Leon-Rot, Germany). Guidelines of the producing company were performed for this purpose. Purity (A260/A280) of extracted DNA was examined by the NanoDrop device (NanoDrop, Thermo Scienti c, Waltham, MA, USA). The quality of extracted DNA was examined using electrophoresis on 2% agarose gel.

Statistical analysis
Statistical analysis was done using the SPSS 21.0 statistical software (SPSS Inc., Chicago, IL, USA). Chi-square test and Fisher's exact two-tailed test were used to assess any signi cant relationship between the phenotypic and genotypic properties of antibiotic resistance of the S. aureus bacteria isolated from powdered packaged medicinal plant and bottle herbal distillate samples. P-value <0.05 was considered as signi cant statistical level.

Results
Contamination rate of examined samples with S. aureus The present survey was conducted to assess the prevalence and phenotypic and genotypic examination of S. aureus bacteria's antibiotic resistance isolated from diverse kinds of powdered packaged medicinal plant and bottle herbal distillate samples. Table 2 shows the contamination rate of diverse powdered packaged medicinal plant and bottle herbal distillate samples with S. aureus. Thirty out of threehundred (10%) medicinal plant and herbal distillate samples were contaminated with S. aureus. The contamination rates of powdered packaged medicinal plant and bottle herbal distillate samples were 8.33% and 11.11%, respectively. A.citrodora (10%) and R.damascene (10%) had the highest contamination rate with S. aureus amongst all examined powdered packaged medicinal plants, while S.bachtiarica (6.66%) and Z.multi ora (6.66%) had the lowest. A. maurorum (16.66%) had the highest contamination rate with S. aureus amongst all examined bottle herbal distillates, while M.o cinalis (6.66%) and M.piperita (6.66%) had the lowest. Statistically signi cant difference was obtained for the contamination rate of S. aureus between powdered packaged medicinal plants and bottle herbal distillates (P <0.05). Additionally, statistically signi cant differences were obtained between examined samples and the contamination rate with S. aureus (P <0.05).
Prevalence of multidrug resistant-S. aureus Figure 1 shows the prevalence of multidrug resistant-S. aureus bacteria amongst all examined samples. All of the S. aureus bacteria isolated from powdered packaged medicinal plant and bottle herbal distillate samples harbored resistance to at least one of the examined antibiotic agents. The prevalence of resistance toward at least ve antibiotic agents was 46.66%. Findings revealed that 13.33% of the S. aureus isolates harbored resistance toward more than seven antibiotic agents.

Discussion
Extensive diversity of microbial contaminants might accompany medicinal plants. Unavoidably, the microbial background relies on numerous ecological factors and employs an imperative in uence on herbal products' safety and quality. The microbial contaminants of herbal products are simply transferred through air, soil, animal-and human-based fertilizers, and nally infected staff and workers producing units [10]. Otherwise, a host of agricultural, environmental, industrial, and urban factors, together with less than good harvesting, storage, and processing procedures, are additional reasons for contamination in herbal products [10]. In these cases, medicinal plants and herbal products with con rmed therapeutic effects not only do not improve the patient's condition, but also lead to diverse kinds of foodborne diseases and disorders. Thus, assess the microbial quality of herbal products is essential as an imperative public health matter.
The present survey was conducted to assess the prevalence and antibiotic resistance properties of the S. aureus bacteria isolated from diverse kinds of powdered packaged medicinal plant and bottle herbal distillate samples produced in traditional circumstances in Iran. Findings of the current investigation disclosed that the contamination rate of examined raw medicinal plants and herbal distillates with S. aureus was 10%. Some of the examined samples, such as Z.multi ora (6.66%) and S.bachtiarica (6.66%) powdered packaged medicinal plants and M.o cinalis (6.66%), and M.piperita (6.66%) bottle herbal distillates had the lower contamination rate with S. aureus. One of the probable reasons for this nding is the high antimicrobial effects of Z.multi ora, S.bachtiarica, M.o cinalis, M.piperita, and C. intybus against diverse kinds of bacteria [29][30][31][32]. Thus, S. aureus' growth and survival have been decreased and even stopped in these medicinal plants and their derived products.
S. aureus is most expected to originate from herbal products' contact with food handlers throughout harvesting, processing, and storage, and its absence re ects the acceptable hygiene practices. Our ndings also revealed that herbal distillates had a higher contamination rate with S. aureus than medicinal plants. The probable reason for this nding is maybe the extinction of S. aureus bacteria during medicinal plants' drying process. Additionally, the processing of herbal distillates requires more human involvement and manipulation. Thus, the transmission of S. aureus bacteria from the infected staff and workers producing units to the herbal distillates may be another reason for the high prevalence of S. aureus in these samples. Despite the high importance of the topic, many limited surveys have been conducted in this eld. A survey concocted by Sousa Lima et al. (2020) [33] disclosed that the prevalence of S. aureus bacteria amongst the homemade and commercial herbal medicine samples (Lippia alba, Peumus boldus Molina, Cymbopogon citratus, Carapa guianensis, Copaifera langsdor i, Stryphnodendron adstringens, Costus spicatus, and Arrabidaea chica) was 88.50% and 23.50%, respectively. Kaume et al. (2012) [34] described that the prevalence of S. aureus amongst the medicinal plants marketed to patients suffered from the HIV-infection in Kenya was 71.40%, which was entirely higher than our ndings. Esimone et al. (2007) [35] also reported that the prevalence of S. aureus amongst the medicinal plants sold in Nigeria was 8.70%. Reversely, No S. aureus strains bacteria were recovered from the medicinal plants in the study conducted in South-Africa [36]. Ideh et al. (2019) [37] described that the Staphylococcal microbial load of some kinds of medicinal plants in Nigeria had ranged between 1.50 × 10 5 to 6.75 × 10 6 Colony Forming Unit (CFU)/g, which was entirely higher than the limit microbial load introduced by the World Health Organization (WHO) (10 4 CFU/g) [38]. Similarly, a high contamination rate of herbal products with S. aureus and other Staphylococcal species has been reported previously from Bangladesh [39], Korea [40], Nigeria [41,42], Germany [43], Sudan [44], Saudi Arabia [45], Benin [46], and Ethiopia [47]. Likewise, high microbial contamination of some kinds of medicinal plants other than S. aureus has been reported in diverse researches conducted on Iran [48,49], Bangladesh [50], Tanzania [51], Kenya [52,53], Malaysia [54], Iraq [55], Poland [56,57], Italy [58], Saudi Arabia [59], Thailand [60], South Africa [61], Pakistan [62], and United States [63]. One possible reason for the presence of diverse bacteria, particularly S. aureus, in powdered packaged medicinal plant and bottle herbal distillate samples in their proper intrinsic factors such as pH. The pH levels of examined powdered packaged medicinal plant and bottle herbal distillate samples ranged from 3.5 to 9.5, facilitating luxuriant growth and survival of most bacterial species [64]. The contamination rate of herbal product samples with S. aureus vary between diverse researches. The difference in data advises that time, season, place of sampling, method of sampling, types of samples, and even laboratory techniques applied in research may affect surveys' outcomes. Moreover, difference hygienic levels of producing units of herbal products may affect the prevalence of S. aureus in diverse investigations. Compared to the results of other scientists, the comparatively low rate of S. aureus isolation was reported in our survey. Relatively low contamination rate may be due to natural antimicrobials and possibly to a commonly good hygiene situation, however high contamination rate may specify less favourable hygienic circumstances.
The second part of the present survey was performed on the antibiotic resistance properties of S. aureus isolates. Findings revealed that S. aureus bacteria displayed the highest prevalence of resistance toward penicillin, tetracycline, gentamicin, erythromycin, trimethoprim-sulfamethoxazole, cipro oxacin, and cefoxitin antibiotic agents, which was accompanied by high prevalence of blaZ and mecA, tetK, aacA-D, msrA and ermA, gyrA, dfrA1, and blaCTX-M antibiotic resistance encoding genes, respectively. Thus, the phenotypic presence of antibiotic resistance was con rmed by the genotypic presence of antibiotic resistance encoding genes. Furthermore, the presence of multidrug resistant-S. aureus was found in some isolates. Irregular and unauthorizing antibiotic agents' prescription is the probable reason for the high prevalence of resistance and high distribution of antibiotic resistance genes. Findings disclosed that some S. aureus bacteria exhibited a higher prevalence of resistance toward antibiotic agents used to treat human clinical infections, which can indirectly signify that they may transmit from infected staff and workers of producing units of medicinal plants and herbal distillates. Reversely, some others exhibited a higher prevalence of resistance toward antibiotics used mostly for treatment of animal infections, which can indirectly demonstrate that they may transmit from animal species, particularly in using animal-based fertilizers and polluted water for growth and irrigation of medicinal plants. The presence of resistance toward chloramphenicol (20%), which was assisted with attendance of fexA antibiotic encoding gene (13.33%) may re ect using of poultry-based fertilizers for the growth of medicinal plants since chloramphenicol is a common antibiotic choice in Iranian poultry farms [65,66]. According to the literature, the present survey is the rst report of the phenotypic and genotypic assessments of antibiotic resistance amongst the S. aureus bacteria isolated from powdered packaged medicinal plants and bottle herbal distillate samples globally. Braide et al. (2013) [67] stated that the S. aureus bacteria isolated from herbal remedies were susceptible to o oxacin, chloramphenicol, gentamicin, pe oxacin, cipro oxacin, and erythromycin antibiotic agents. Ngemenya et al. (2019) [68] described that the S. aureus strains isolated from herbal remedies in Cameroon were resistant against ve classes of examined antibiotic agents (amikacin, cefotaxime, cefuroxime, imipenem, trimethoprim, and ceftriaxone). Similarly, Yesuf et al. (2016) [47] reported that the S. aureus strains isolated from medicinal herbal products in Ethiopia harbored the high prevalence of resistance toward ampicillin (80%), penicillin (60%), amoxicillin (40%), amoxicillinclavulanic acid (40%), chloramphenicol (40%), and cloxacillin, (30%). Similar pattern of resistance of S. aureus bacteria has been described toward penicillins [66,[69][70][71][72][73], tetracyclines [66,[69][70][71], aminoglycosides [66,[69][70][71][72][73], macrolides [66,[69][70][71][72][73], cephems [66,[69][70][71][72][73], uoroquinolones [66,[69][70][71][72][73], and folate pathway antagonists [66,[69][70][71][72][73] antibiotic groups. A similar resistance pattern of the S. aureus bacteria was reported previously [74,75]. Differences in the opinion of medical and veterinary practitioners in an antibiotic prescription, observation of ethics and rules in the use of antibiotics, availability or lack of antibiotics, and their prices are probable reasons for differences found in the prevalence of resistance of S. aureus strains in numerous investigations. Diverse researches have been conducted to appraise the antibiotic resistance properties of S. aureus. Most of them reported the con rmation of phenotypic pattern of antibiotic resistance by the presence of diverse antibiotic encoding genes [16,66,76,77] High distribution of blaZ, mecA, tetK, aacA-D, msrA, ermA, gyrA, dfrA1, and blaCTX-M antibiotic resistance encoding genes in the S. aureus bacteria isolated from diverse kinds of food samples and also human clinical infections have been reported from Iran [66,76,78], India [79], Georgia [72], Nigeria [80], Germany [81], Egypt [82], and Switzerland [83]. Comparable to our research, higher prevalence of msrA than msrB [66,76,78,84], ermA than ermB [66,76,78,84,85], tetK than tetM [66,76,78,84] vatA than vatB [66,76,78,84], vanA than vanB [66,76,78,84,85] and gyrA than grlA [66,76,84] antibiotic resistance encoding genes has been reported in recent years. Our ndings were also disclosed a higher prevalence of phenotypic pro le of resistance than genotypic pro le. For instance, all of the penicillin-resistant S. aureus bacteria didn't harbored blaZ and mecA antibiotic resistance genes. This matter was also existed for other antibiotic agents and resistance genes. This nding is maybe owing to the fact that presence of antibiotic resistance genes is one of the known procedures for occurrence of antibiotic resistance in bacteria. In the other hand, several mechanisms have been identi ed to induce antibiotic resistance in bacteria including reduced permeability of bacteria to antibiotics, e ux antibiotic's active pumps to out of the bacterial cell, change in antibiotic target site, inactivation of antibiotics through hydrolysis or changes in their structure, occurrence of genetic mutations and access of bacteria to the secondary metabolic pathways that compensate the antibiotic-inhibited reactions.
Findings also revealed the high prevalence of multidrug resistant-S. aureus strains amongst medicinal plants and herbal distillates. Similarly, a high prevalence of multidrug-resistant bacteria has been reported in herbal product samples in Kenya [86], Cameroon [68], and Ethiopia [47]. Additionally, the high distribution of multidrug resistant-S. aureus strains have been described in clinical specimens and foodstuff samples collected from China [87], United States [88], and Kuwait [89]. Altogether, the high prevalence of antibiotic resistance in S. aureus, which was accompanied by the high distribution of antibiotic resistance genes and the presence of multidrug resistance, disclosed a pressing public health issue regarding the consumption of powdered packaged medicinal plants and bottle herbal distillate samples.

Conclusions
Put together, an existing survey is the rst report of prevalence and phenotypic evaluation of antibiotic resistance of S. aureus bacteria isolated from Z. multi ora, S.bachtiarica, A.citrodora and R.damascene powdered packaged medicinal plants and L.angustifolia, A. maurorum, C. intybus, M. o cinalis, M.piperita and F.o cinalis bottle herbal distillates, globally. Additionally, it was the rst report of detection of antibiotic resistance genes amongst the S. aureus strains isolated from diverse kinds of powdered packaged medicinal plants and bottle herbal distillate samples globally. Findings disclose that powdered packaged medicinal plants and particularly bottle herbal distillates are potential sources of multidrug resistant-S. aureus. Bottle herbal distillates harbored a higher prevalence of S. aureus isolates and also higher antibiotic resistance. High prevalence of resistance of S. aureus bacteria toward penicillin, tetracycline, gentamicin, erythromycin, trimethoprim-sulfamethoxazole, cipro oxacin, and cefoxitin antibiotic agents was observed, which was also accompanied by the high prevalence of blaZ and mecA, tetK, aacA-D, msrA ans ermA, gyrA, dfrA1 and blaCTX-M antibiotic resistance encoding genes, respectively. The prevalence of resistance toward human-based antibiotics and animal-based antibiotics can indirectly show S. aureus isolates' origin. It seems that penicillin, tetracycline, gentamicin, erythromycin, trimethoprim-sulfamethoxazole, cipro oxacin, and cefoxitin are not effective therapeutic agents in the cases of S. aureus foodborne diseases at this time in Iran. Types of examined samples had high effects on the prevalence and antibiotic resistance properties of S. aureus strains. Traditional producing and processing centers of powdered packaged medicinal plants and bottle herbal distillates can be severely contaminated with foodborne pathogens, particularly S. aureus; the maintenance of their hygiene, regular microbiological monitoring of these samples, implementation of good manufacturing practices and a food safety system such as the Hazzard Analysis Critical Control Points (HACCP) system are essential to minimize the risk to the consumer. Furthermore, using high quality raw materials in production of powdered packaged medicinal plants and bottle herbal distillate samples, prevention from cross-contamination and antibiotic prescription based on disk diffusion outcomes can diminish the risk of

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Competing interests
The authors declare that they have no competing interests  Tables   Table 1. Figure 1