Distribution of Virulence Genes in Campylobacter spp. Isolated from Agaricus Mushrooms in Iran

The white button mushroom (Agaricus) is a significant nutritional and therapeutic species utilized in the human diet and could transmit various bacterial infections. Campylobacter species are the most common cause of foodborne illness across the world. The present study has been planned to determine the frequency of virulence genes and antibiotic susceptibility test in Campylobacter spp. recovered from Agaricus mushroom. In this study, 740 Agaricus mushroom samples were gathered randomly from various markets from June 2020 to December 2020. Confirmation of Campylobacter spp. using biochemical analyses and 23S rRNA-based PCR was performed. The agar dilution technique was used to determine resistance to antibiotics using gentamicin (GM10μg), ciprofloxacin (CIP5μg), nalidixic acid (NA30μg), tetracycline (TE30μg), ampicillin (AM10μg), amoxicillin+ clavulanic acid (AMC30μg), erythromycine (E15μg), azithromycin (AZM15μg), clindamycin (CC2μg), and chloramphenicol (C30μg). Multiplex PCR was utilized to determine the prevalence of the recR, dnaJ, wlaN, virBll, cdtC, cdtB, cdtA, flaA, cadF, pidA, ciaB, ceuE, and cgtB genes. Campylobacter spp. were detected in 74 out of 740 Agaricus mushroom samples (10%). According to the data, Agaricus mushroom samples included 32 (4.32%) C. jejuni, 11 (1.48%) C. coli, and 31 (4.18%) other Campylobacter spp. Antimicrobial resistance was most common in C. jejuni isolates. C. jejuni isolates also had the lowest resistance rate to gentamycin, ciprofloxacin, and nalidixic acid. C. coli isolates were reported to have the highest antimicrobial resistance to ciprofloxacin, ampicillin, and erythromycine. Resistance to gentamycin and amoxicillin+ clavulanic acid was likewise lowest among C. coli strains. The flaA and ciaB genes were found in 100% of B-lactams-susceptible C. jejuni and C. coli strains. When examining the relationship between antibiotic resistance and the existence of virulence genes, it was observed that there is a statistically significant relationship (p < 0.001) between bacterial resistance and virulence genes. Our findings indicated that changes in resistance patterns in Campylobacter strains have emerged from multiple treatment approaches in Agaricus mushrooms.


Introduction
Mushrooms have been utilized in food and medical supplies for decades and have become a vital element of human nutrition. Due to their high-quality nutrients, carbohydrates, enzymes, essential fatty acids, dietary fibers, and low-calorie content, these substances have an appealing taste, fragrance, and nutritive quality [1]. Their production and sales have increased dramatically in recent years all over the globe.
Mushrooms are regarded as a regular diet and a supplier of nutritional supplements because of their favorable impact on humans due to their presence of bioactive components and nutritional ingredients [2]. The white button (Agaricus) mushroom is among the essential edible mushrooms that have long been sought by people searching for food. The Chinese recognized the advantages of mushrooms, believing that mushrooms enhance the physical organs and prolong vitality and vigor [3]. This mushroom is being utilized as a diet and medicinal plant in Iran and other nations. Agaricus mushrooms are particularly fragile because they lack a defensive cuticle, have a rapid breathing function, and contain much wetness. Consequently, they are subjected to mechanical degradation, pathogen assault, weight loss, and caramelization, resulting in a fast-postharvest reduction in quality. Agaricus mushrooms have a storage life of one to three days at room temperature (20-25°C) and five to seven days under a refrigerator (4°C) [4].
Mushrooms are ideal hosts for a wide range of pathogens. Various microorganisms grow on the surface of mushrooms that might infect humans. One of the most significant bacteria is Campylobacter [5]. Campylobacter spp., a primary cause of bacteremia gastroenteritis in mammals, are frequently found in animal digestive tracts and, through fecal pollution, in animal-derived products. Raw milk, chickens, and beef have all been related epidemiologically to epidemics of Campylobacter infection in humans [6,7]. Campylobacter can be identified in 1 to 1.5% of agricultural packaged food. As a result, several items that are epidemiologically linked to Campylobacter outbreaks have been recognized as responsible for the transmission of the Campylobacter genera [8]. The Seattle-King Health Center researched to quantify the migration of Salmonella spp. and Campylobacter spp. from animals to humans through the food supply chain and discovered that people with Campylobacter enteritis ate mushrooms [9]. As a result, there seemed to be an increased relative incidence of Campylobacter infection in humans in people who consumed mushrooms. Our goal was to see if raw mushrooms are a reservoir of Campylobacter spp. [10]. Freshly Agaricus mushroom packets sold at local supermarket shops are high in Campylobacter [11].
Campylobacter can cause bladder infections, pneumonia, or neuropathies, including septic arthritis, Guillain-Barre syndrome (GBS), inflammatory bowel disease, and Miller-Fisher syndrome (MFS). The Agaricus mushroom population causes approximately 40% of people's problems. Recent genetic investigations have shed insight on the primary virulence components involved in the Campylobacter strain's virulence. Furthermore, the capacity of Campylobacter to attach via the cadF, racR, virB11, pldA, and dnaJ proteins infiltrates intestinal mucosa cells by the ciaB and ceuE genes and manufactures toxins via the cdtA, cdtB, and cdtC genes [12]. Even though Campylobacter diseases are typically self-limiting and do not require treatment, antimicrobial therapy is not necessary in most cases of protracted disease in humans and septicemia. Also, it is essential in some types of sepsis. The selection medications for treating human campylobacteriosis include macrolides (erythromycin and azithromycin), fluoroquinolones (ciprofloxacin), and tetracyclines. The primary reasons for rising susceptibility patterns are the overuse of antibiotics in human illnesses and abusing antibacterial medications in animal farming to treat animal diseases or boost growth by adding antibiotics to food within the Campylobacter genus [13]. Antimicrobial resistance is caused by several biological processes that have been well-documented. The chloroquine inhibition domain of the DNA gyrase gene, gyrA, is primarily responsible for fluoroquinolone resistance. The acquired tet(O) gene, which codes for a defensive ribosomal polypeptide, is usually linked to high tetracycline resistance. Mutations in the V motif of the 23S rRNA gene and stimulation of the CmeABC multidrug efflux system are commonly linked to antibiotic resistance to macrolides. Antibiotic-resistant genes like erm(B), aadE, or sat4 (streptomycin/streptothricin resistance), blaOXA-61 (b-lactams resistance), and aphA-3 (aminoglycosides resistance) have also been linked to multidrug resistance in Campylobacter isolates [14]. Numerous studies have found a relationship between virulence and antibiotic susceptibility in bacterial infections, implying a correlation between antibiotic resistance and the bacteria's ability to colonize or invasion. This link has been investigated, and some studies have found that infection with antibiotic-resistant Campylobacter isolates in people is linked to a prolonged length of diarrhea [15,16]. In this research, we focused on the incidence of Campylobacter infection in Agaricus mushrooms in Iran and the infection distribution and antimicrobial resistance in collected isolates. As a result, this analysis is aimed at looking into the genetics of antimicrobial resistance (AMR) and finding virulence indicators in Campylobacter spp. samples from Agaricus mushrooms.

Biochemical Identification of Campylobacter spp.
In this investigation, 740 Agaricus mushroom samples were gathered randomly from various markets from June 2020 to December 2020 and transferred to the laboratory. The selection represented the two varieties of Agaricus mushroom brands produced in Iran. Samples were placed below 4°C during transportation, and testing was performed immediately after receiving the samples. When the Agaricus mushroom trends turned up in the lab, they were torn into pieces and blended in a sterile folder with a sterilized swab stick, and roughly 1 g of each template was homogenized in enrichment broth and incubated for 24 hours at 37°C. Homogenized solution was then scrubbed onto (predried) Campylobacter agar base (Sigma-Aldrich, Germany) plates supplemented with Karmali selective complement SR0167E (Sigma-Aldrich, Germany). Samples were incubated for 48 hours at 42°C in an anaerobic jar with a gas-generating sachet (Oxoid-CampyGen™) to establish a microaerophilic environment for the growth of Campylobacter spp.
On 24-48 h Karmali agar plates, colonies with a clean, flat, colorless, transparent to the grey appearance of diameters of 1 mm were chosen. Since colonies are frequently mixed with other microorganisms, the mobility of suspected bacteria was examined using a section contrast microscope. Colonies exhibiting exceptional mobility were isolated on 5% horse blood agar plates and incubated for 48 hours in an anaerobic jar with a fuel-producing sachet to generate microaerophilic conditions. Gram-negative, catalase-positive, and oxidase-positive samples were snap-frozen in glycerol broth at -70°C for further molecular characterization (PCR), antibiotic sensitivity testing, and resistance gene identification [17]. In an anaerobic jar with a gasgenerating pouch, frozen Campylobacter isolates were grown on 5% horse blood agar plates and incubated for 48 hours at 42°C. The bacterial cells were taken from the plates and put into Eppendorf vials containing 200 μL of sterile water when they had grown sufficiently. The suspensions were heated for 8 minutes at 98°C on a boiling tube. The supernatant was collected and was then placed into sterile microcentrifuge tubes and centrifuged at 17000 g for 5 minutes to serve as a genetic DNA material for the following polymerase chain reaction (PCR). The quality (A260/A280) and amount of the extracted DNA were next measured at an optical density of 260/280 nm using a spectrophotometer (NanoDrop, Thermo Scientific, Waltham, MA, USA

Antibiotic Susceptibility Test of Campylobacter Isolates.
Antimicrobial resistance profiles of Campylobacter isolates were recovered from multiple specimens (Tables 2 and 3). C. jejuni isolates exhibited high resistance against tetracycline, ampicillin, amoxicillin+ clavulanic acid, and erythromycine. On the other hand, C. jejuni isolates had the lowest rate of resistance to gentamycin, ciprofloxacin, and nalidixic acid. Furthermore, a significant percentage of resistance to antibiotics azithromycin (40.62%), clindamycin (25%), and chloramphenicol (37.5%) was observed in different isolates. There was a statistical difference between the specimens and antimicrobial resistance incidence (p < 0:05).
C. coli isolates were reported to have the highest antimicrobial resistance against ciprofloxacin (72.72%), ampicillin (72.72%), and erythromycin (72.72%). However, the resistance to gentamycin (0%) and amoxicillin+ clavulanic acid (27.27%) was noticed to be lowest among C. coli strains. The results, on the other hand, revealed that both C. coli and C. jejuni isolates were completely sensitive to antibiotics gentamycin.

Prevalence of Virulence Factors.
The rates of virulence genes among resistant isolates of C. jejuni were as follows for recR, dnaJ, cdtC, cdtB, cdtA, flaA, cadF, and ciaB, respectively. The flaA and ciaB genes were found in 100% (32/32) of C. jejuni strains when these genes were tested in susceptible isolates. The frequency of these genes in C. jejuni was noticed to be the lowest when the wlaN, virbll, and ceuE genes were examined (Table 3). Table 3 shows the genotype distribution of C. coli isolates collected from various types of specimens. FlaA and ciaB were the most prevalent genotypes observed among C. coli recovered from mushroom (100%). DnaJ, wlaN, virbll, and ceuE were the C. coli strains found with the lowest frequency in samples (0%). The recR, cdtC, cdtB, cdtA, cgtB, cadF, and pidA genes were also discovered in a variety of C. coli isolates. These genes were found in between ten and forty percent of the population. There was a significant difference (p < 0:05) between the types of samples and the occurrence of genotypes.

Association of Virulence Genes with the Antibiotic
Resistance Pattern. In terms of the association between the existence of virulence genes and antibiotic susceptibility/ resistance profiles across strains, resistant C. jejuni isolates had more virulence-related genes than sensitive ones. Tetracycline-resistant isolates had more virulence genes than nalidixic acid and gentamycin-resistant strains isolated. The antibiotics GM10 and the virBll and wlaN genes, which  Legend: gentamicin (GM10μg), ciprofloxacin (CIP5μg), nalidixic acid (NA30μg), tetracycline (TE30μg), ampicillin (AM10μg), amoxicillin+ clavulanic acid (AMC30μg), erythromycine (E15μg), azithromycin (AZM15μg), clindamycin (CC2μg), and chloramphenicol (C30μg).   BioMed Research International exhibit no variability (100% resistance), were omitted from the study. When examining the relationship between antibiotic resistance and the existence of virulence genes in C. jejuni isolates, it was shown that there is a statistically significant relationship (p < 0:001) between bacterial resistance and the existence of virulence genes (Figure 1(a)). The medicines GM10 were excluded out of the C. coli investigation, as were the dnaJ, virbll, ceuE, and wlaN genes, which had no variability (100% resistance). When researchers looked at the association between resistance to antibiotics and the presence of genetic variants in C. coli strains, they discovered a positive significant association (p < 0:001) among bacterial resistance and the presence of virulence genes (Figure 1(b)).

Discussion
Campylobacter bacteria cause foodborne infections, and MDR variants are a severe health problem. There has been no information on pathogenic molecular features of regional Campylobacter isolates because there have been few investigations on the bacteria in Iran [24]. As a result, the presence of virulence in Campylobacter genera isolated from Agaricus mushrooms was investigated in this work. Several researchers worldwide have found that gene expression related to mobility, colonization, epithelium invasion, and toxin generation is crucial in the development of Campylobacter-related illnesses [25][26][27][28]. Most of the strains in this investigation were found to have associated virulence genes linked to pathogenic adherence, colonization, and invasive features. This was in line with prior research, which had found flaA, ciaB, racR, virB11, and pldA to be often prevalent [23]. Furthermore, as described by various investigations [29,30], the cdtA, cdtB, and cdtC alleles required to produce the CDT toxic substance were found in all Campylobacter strains. Concerning C. jejuni, 59% of the examined strains had the ceuE gene, which confers the ability to chelate iron. The wlaN gene was found in 90% of Campylobacter strains, consistent with research conducted in Iran [31], which found a high incidence of isolated Campylobacter strains (82.22%). Furthermore, the cgtB gene was found in 54% of C. coli and 71% of C. jejuni. As a result, we believe that the high prevalence of these alleles among the tested samples might imply their significant pathogenic capability and high danger to human health. Lipooligosaccharide of Campylobacter (LOS), similar to gangliosides in neurons, is considered a critical factor in the initiation of GBS neuropathies and Miller-Fisher syndrome after C. jejuni infection [31]. The higher prevalence of these genes may be associated with GBS in humans. Antibiotic resistance, a global concern for animal and human health, has received much attention. Because of the extensive utilization of antibiotics in the food sector, antibiotic resistance has become a significant problem [32]. Previous research has found that Campylobacter has a high antimicrobial resistance to various drugs [33][34][35][36][37][38][39][40]. Antimicrobial resistance patterns correspond well with the presence of genes expressing resistance to antibiotics, according to a study examining the genetic basis of antibiotic resistance in isolates tested [34,35].
Our findings demonstrated that multiple virulence factors are related to resistant bacteria when examining the frequency of virulence genes and antibiotic susceptibility. Indeed, the detection of cadF and ciaB in amoxicillin/clavulanic acid-resistant bacteria, ciaB in ampicillin-resistant bacteria, racR in nalidixic acid-resistant isolates, and cadF and ceuE in chloramphenicol-resistant isolates was linked. Although the presence of positive or negative relationships among antibiotic resistance and virulence genes in microorganisms has been demonstrated, the Campylobacter species remains contentious [41][42][43]. In vitro experiments have shown that resistant bacteria invade more than susceptible strains, whereas others have highlighted the tendency of susceptible strains to produce more serious diseases than resistant organisms. In the current investigation, virulence genes and antibiotic resistance among C. jejuni samples were shown to have some favorable relationships (p > 0:05). A few virulence genes linked to antimicrobial-resistant C. jejuni isolates are implicated in bacterial attachment and invasion,  Figure 1: The prevalence of virulence genes in susceptible C. jejuni (a) and C. coli (b) isolates. Legend: virulence genes (recR, dnaJ, wlaN, virbll, cdtC, cdtB, cdtA, flaA, cadF, pidA, ciaB, ceuE, and cgtB). When researchers looked at the association between resistance to antibiotics and the presence of genetic variants, they discovered a positive significant association ( * * * p < 0:001) among bacterial resistance and the presence of virulence genes. 7 BioMed Research International indicating that resistant strains have more adherence and attack potential than susceptible strains. Further research is needed to understand the association between virulent characteristics and antibiotic resistance in greater depth in Campylobacter isolates.

Conclusions
In the present study, a significant recovered rate of Campylobacter was found. Antibiotic resistance in Campylobacter strains isolated from Agaricus mushroom is a growing source of worry and might pose a severe public health danger. Resistance to multiple antibiotics with a significant association with virulent factors has been discovered. Agaricus mushroom Campylobacter strains from Iran have little resistance to antibiotics important to global health.

Data Availability
The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.

Conflicts of Interest
The authors declare that they have no competing interests.