Antibiotic resistance pattern of the allochthonous bacteria isolated from commercially available spices

Abstract Spices are often used in dried form, sometimes with significant microbial contamination including pathogenic and food spoilage bacteria. The antibiotic resistance represents an additional risk for food industry, and it is worthy of special attention as spices are important food additives. During our work, we examined the microbiological quality of 50 different spices with cultivation methods on diverse selective media. The identification of the most representative bacteria was carried out using 16S rDNA gene sequence analysis. Antibiotic resistance profiling of twelve identified Bacillus species (B. subtilis subsp. stercoris BCFK, B. licheniformis BCLS, B. siamensis SZBC, B. zhangzhouensis BCTA, B. altitudinis SALKÖ, B. velezensis CVBC, B. cereus SALÖB isolate, B. tequilensis KOPS, B. filamentosus BMBC, B. subtilis subsp. subtilis PRBC2, B. safensis BMPS, and B. mojavensis BCFK2 isolate) was performed using the standard disk‐diffusion method against 32 antibiotics. The study showed that the majority resistance was obtained against penicillin G (100%), oxacillin (91.67%), amoxyclav (91.67%), rifampicin (75%), and azithromycin (75%). Our findings suggest that spices harbor multidrug‐resistant bacteria.

Bacillus species are among the most usually detected bacteria species from diverse spices samples with different geographical origin (Antai, 1988;Banerjee & Sarkar, 2003;Chakraborty et al., 2020;Hariram & Labbé, 2015). Contamination of spices with molds and spore-forming B. cereus is attributed to environmental conditions of manufacturing and distribution (Fogele et al., 2018). The survival of these microorganisms in spices and herbs is attributed to the high resistance of spores to different stress conditions including excessive pH values, heat, and low water activity . The result of improper food handling is that B. cereus, originating from herbs and spices, can reach the 10 5 -10 6 CFU/g causing food poisoning (Sagoo et al., 2009). Some strains are capable of producing nonhemolytic enterotoxin, hemolysin BL, and cytotoxin K responsible for diarrhea and emesis causing heat stable cereulide .
Among the spore-forming bacteria also, Bacillus subtilis and Clostridium perfringens are often present (Sagoo et al., 2009).

Other bacteria strains like Staphylococcus aureus, Escherichia coli,
and Shigella spp. were detected in different spices (Banerjee & Sarkar, 2003). In contrast to Bacillus spores, S. aureus has a short life cycle even though it supported by the low water activity conditions .
The herbs in many cases are used as a fresh cut product without any or with improper treatment to combat microbial contamination.
The contaminated plant material can take part in the transmission of pathogens to foods that provide suitable growth conditions and toxin production . This increases the health hazard to consumers. The risk is increased in the case of multidrugresistant strains present on the plant surface.
The overuse of antibiotics selected the antimicrobial resistance of bacteria, and it represents a virulence factor resulting in a worldwide health threat to humans. Bacterial strains with drug resistance are listed as causes of human mortality (Bennani et al., 2020;Thapa et al., 2020). Bacterial strains with antibiotic resistance can enter the food supply chain from different environments (Dutta & Ramamurthy, 2020;Thapa et al., 2020). Diverse food commodities may act as a reservoir and transmission vector of antibiotic resistance due to contamination by bacteria (Mercimek Takci et al., 2020;Navaneethan & Effarizah, 2021). In food producing animals, bacterial strains like Campylobacter jejuni and Campylobacter coli resistant to quinolones and fluoroquinolones were determined. It was shown that E. coli, Salmonella spp., and C. jejuni harbor resistance to tetracyclines. Another association was found between macrolides and E. coli, Salmonella enterica serovar Heidelberg, and C. coli (Bennani et al., 2020).
The spread of antibiotic resistance genes is an increasing problem, and it is also indicated that clinically relevant resistance could be detected in nonpathogenic bacterial strains as in the case of different Bacillus spp. strains originating from natural ecosystems (Berić et al., 2018). The occurrence of the B. cereus group as opportunistic pathogen has been associated with vegetables. These bacterial groups display resistance to commonly used antibiotics. Multidrugresistant strains were also identified among them. These bacteria can contribute to the transfer of antibiotic resistance genes in the food chain (Fiedler et al., 2019;Navaneethan & Effarizah, 2021).
According to Zarzecka et al., (2020), there is a risk of transmission of antibiotic resistance genes to pathogenic bacteria like S. aureus, B. cereus and others.
The utilization of antimicrobials exceeding the therapeutic use in food production and agriculture may promote the transmission of antibiotic genes . To prevent the dissemination of antibiotic resistance, the knowledge and estimation of allochthonous bacteria and their antibiotic pattern are necessary.
This study aimed to evaluate the microbiological quality of different spices and to determine the antibiotic resistance of the most representative bacterial isolates originating from the studied spice.
With this study, we attempt to find an answer to the question of whether spices could represent a reservoir of multidrug-resistant strains and whether their spread in the food supply chain is possible.

| Isolation of bacterial strains
The microbial contamination of 50 different commercially available spices (allspice, Cayenne pepper, marjoram, coriander, basil, cinnamon, granulated garlic, ground garlic, bay leaf, ground pepper, Provence spice mix, rosemary, chili peppers, ginger, star anise, savory, oregano, caraway seeds, ground cumin, tarragon, pepper mix, white mustard, parsley leaf, ground hot peppers, juniper berries, ground turmeric, sweet peppers, cloves, lovage, nutmeg, ground sage, crushed sage, ground vanilla, spice mixture, pepper+garlic mixture, curry, ground anise, green pepper, peppermint, cardamom, mustard seeds, ground cloves, dried celery, dill, parsley) was determined with cultivation methods on different selective media. During the microbiological assay, the total mesophilic aerobic bacteria on Nutrient agar (Himedia) medium were first determined. The incubation was performed at 37℃ for 48 hr. Thereafter, we determined the presence of aerobic spore-forming B. cereus, E. coli, Salmonella spp., and Pseudomonas species on selective agar mediums. The detection of C.
perfringens was carried out in Clostridial Differential Broth (Biolab).
In the case of a positive result, a confirmation test (with regenerated milk) was conducted and the enumeration of C. perfringens was determined with the most probable number method. In 5 test tubes containing 10 ml of double concentrated Clostridial Differential Broth, it was added 10 ml of stock suspension from the samples. In 5 test tubes containing 10 ml of Clostridial Differential Broth, it was added 1 ml of stock suspension. 1-1 ml from 10 -1 serial dilutions was transferred in 5 test tubes containing 10 ml of standard Clostridium selective broth. In the inoculated tubes, it was added paraffin and incubated for 10 min at 80℃ in water bath. After cooling, it was incubated for 48 hr at 44℃. The positive tubes were counted, and the number of Clostridium perfringens was determined from the MPN

| Identification of the isolated bacterial strains
The identification of these bacteria was done using 16S rDNA gene sequence analysis. Genomic DNA isolation was carried out with AccuPrep ® Genomic DNA Extraction Kit from Bioneer according to the manufacturer's protocol. An universal primer set 27f and 1492r (5' AGAGTTTGATCMTGGCTCAG 3', 5' TACGGYTACCTTGTTACGACTT 3') was used to amplify one part of the bacterial 16S rDNA gene. The amplification reaction was carried out with an initial denaturation at 94℃ for 5 min, followed by 30 cycles consisting of denaturation at 94℃ for 30 s, primer annealing at 55℃ for 30 s, and primer extension at 72℃ for 1 min, and a final extension at 72℃ for 7 min. Sequencing of the amplified PCR products of the isolated strains was performed by commercial service of Biomi KFT (Hungary) (György et al., 2020). The resulted sequences were edited and aligned with Chromas (Technelysium Pty. Ltd., South Brisbane, Australia); Molecular Evolutionary Genetics Analysis 4 system (www.megas oftwa re.net) was used forphylogenetic analyses. The isolated bacteria were identified through comparison of the sequences using the EzTaxon server based on the 16SrDNA sequence data (www.ezbio cloud.net/eztaxon).

| Determination of multiple antibiotic resistance index
The multiple antibiotic resistance (MAR) index of the screened bacterial strains was calculated with the help of the following formula: is the number of antibiotics, to which the bacteria species was resistant, and (y) refers to the number of tested antibiotics defined for multidrug resistance (Costa et al., 2020).

| Statistical analysis
The principal component analysis was performed with Statistica 8.0 (StatSoft, Inc., Oklahoma, USA).

| RE SULTS
During the evaluation of the microbiological quality of spices, it was determined the aerobic mesophilic bacteria count. If this is high, it can be assumed that pathogenic bacteria may also be present in the samples. The highest total plate count was detected in the case of ground pepper 1⋅5•10 7 CFU/g, but a high total colony count was characteristic for turmeric (4⋅10 5 CFU/g), tarragon (3.8⋅10 4 CFU/g), and savory1 (1⋅7.10 4 CFU/g) samples (Table 1). The number of the mesophilic aerobic bacteria was low in green pepper, vanilla, oreg-ano1, spice mixture, and white mustard samples. The plate count of aerobic spore-forming bacteria was highest in the allspice sample (3.5⋅10 4 CFU/g), and the lowest count was detected in vanilla and mustard seed samples.
Within the group of aerobic spore-forming bacteria, typical B. cereus colonies were developed from 15 spice samples, and high count was detected in the case of ground pepper and parsley. Pseudomonas species, that play a role in food spoilage, were generally present in TA B L E 1 Allochthonous bacteria count in studied spices (CFU/g) Ground anise (Hungary) Pepper +garlic ( The multiple antibiotic resistance (MAR) index was taken into account (the antimicrobial categories and agents) used to define multidrug resistance (due to the lack of data for Bacillus strains regarding the antibiotics used to define MAR we applied the antibiotics proposed for S. aureus) (Magiorakos et al., 2012).
When a bacteria strain displays resistance to more than two antibiotics or to at least one antibiotic in three or more categories (Table 3) The lack of or the low microbial load of different spices could be attributed to the bioactive components with antimicrobial properties (Costa et al., 2020;Thanh et al., 2018). Bata-Vidács et al., (2018) determining the microbiological quality of seventy-one spice paprika samples from 10 countries (Hungary, China, Serbia, Spain, India, Bulgaria, Brazil, Peru, Kenya, Thailand, and unknown place) concluded that the dominant microorganisms in spice paprika samples are influenced by different factors such as the climate of cultivation.
It was revealed that bacteria species are related to geographical or- in DNA, and DNA transfer (Breijyeh et al., 2020). It was confirmed that some classes of antibiotic resistance gene can be acquired.
Acquired resistance has confirmed to the most of the standard antibiotics and evaluated in different bacteria (Gold & Moellering, 1996;Kapoor et al., 2017;Mancilla-Becerra et al., 2019). According to WHO priority list of antibiotic resistant bacteria, S. aureus possess high priority, but other bacteria strains are also classified as serious (Breijyeh et al., 2020;Kumar et al., 2020).
Limited scientific information is available regarding the antibiotic resistance of Bacillus strains and the transfer of antibiotic resistance genes. According to Alanber et al., (2020) (Meidong et al., 2017).
A bacterial isolate originating from a river was identified as B.
altitudinis with resistance to three groups of antibiotics beta lactams, lincomycin, and polymyxin B (Lobova et al., 2015). A similar pattern of results was also obtained by Alanber et al., (2020

| CON CLUS ION
The study has revealed that the assayed 50 spices are moderately contaminated. It was shown that among the identified bacteria occur pathogenic and food spoilage bacteria, as B. cereus.   The isolated and identified Bacillus species exhibited different resistance to the tested antibiotics, showing resistance at least against two antimicrobials. All tested strains showed susceptibility to eight antibiotics. The presence of bacteria with multiple antibiotic resistance in the tested products should be considered a health concern. From the results of the present study, it can be concluded that improperly processed or unwashed raw herb spices could contribute to the transfer of multidrug resistance bacteria through the supply chain. Further studies are needed to confirm with accuracy the minimal inhibitory concentration and detection of genetic elements associated with antibiotic resistance in case of bacteria with human health concern.

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.