Relationship between Biofilm Formation and Antibiotic Resistance and Adherence Genes in Staphylococcus aureus Strains Isolated from Raw Cow Milk in Shahrekord, Iran

The production of biofilms by S. aureus contributes significantly to treatment failures. The present study aims to establish the relationship between biofilm formation and antibiotic resistance and adhesion genes in Staphylococcus aureus strains isolated from raw cow milk in Shahrekord, Iran. A total of 90 samples of raw cow's milk were collected. Presumptive S. aureus strains were obtained using Baird-Parker plates after enrichment in tryptone soy broth, and final colonies were selected from brain heart infusion. Additional tests such as coagulase were done, and the identification was confirmed by the detection of the aroA gene. Biofilm producing strains were screened using a spectrophotometry method applied to microplates. Crystal violet staining was used to quantify the formation of biofilm. An antibiotic susceptibility test was performed using the Kirby–Bauer disc diffusion method. PCR was used to detect several biofilm and antibiotics resistance related genes. The chi-square test and Fisher's exact test were used to establish a statistically significant relationship between biofilm reaction and antibiotic resistance (p value <0.05). Results show a moderate (38.88%) recovery rate of S. aureus in milk and 65.71% of the isolates were strong biofilm producers. Antibiotic susceptibility tests show an alarming rate of resistance to beta-lactam antibiotics, especially penicillin (100%), ampicillin (91.42%), and oxacillin (71.42%). This finding correlates with antibiotic resistance gene detection, in which the gene blaZ was most found (71.42%), followed by mecA and Aac-D (42.85%). Detection of biofilm-related genes shows that all the genes targeted were found among S. aureus isolates. Statistical tests show a significant correlation between biofilm production and antibiotic resistance in S. aureus. This study revealed that there is a significant correlation between biofilm production and antibiotic resistance in S. aureus isolated from raw milk. These results highlight the need for regular surveillance of the occurrence of S. aureus strains in milk and milk products in Iran.


Introduction
Staphylococcus aureus is a Gram-positive facultatively anaerobic cocci [1,2]. Staphylococcus aureus is well known to cause zoonotic diseases and is one of the main agents of food poisoning [3,4]. Indeed, its presence in food represents a serious health problem as it can produce a wide range of virulence factors such as enzymes and exotoxins that cause food poisoning [5]. Handling, close contact, and In addition, the pasteurisation step does not inhibit the activity of staphylococcal enterotoxins, as they can be very heat-resistant [9]. Many studies have reported the presence of S. aureus strains producing staphylococcal toxins in milk across the world [10]. Consumption of milk poisoned with staphylococcal enterotoxins can cause nausea, vomiting, and abdominal cramps [11].
Apart from the production of enterotoxin, the ability of S. aureus to form a biofilm is essential for its long existence in a harsh environment [12,13]. Biofilms are structured clusters of bacterial cells embedded in a polymeric matrix and attached to a surface [14]. Biofilm formation in S. aureus is not a simple process and is encoded by many genes, such as rbf [15], mgrA [16], and icaR [17]. Biofilm formation defends bacteria against desiccation, the host's immune defences, and the action of oxidising biocides and antibiotics [18]. S. aureus strains can be resistant to one or more antibiotics and can cause serious and difficult-to-treat infections [11,19].
As many pathogenic bacteria produce biofilms, there is growing interest in studying the correlation between biofilm production and antimicrobial susceptibility profile [20].
us, the role of biofilm has been studied on S. aureus strains isolated from humans [21], pork [22], dairy products [23], cows [24], and milk [25]. e study of multidrug-resistant S. aureus in dairy production is of great concern as it has a negative impact on milk production and may represent a public health problem for workers involved in food production [26].
Dairy production is one of the main high-income sectors in the world [6]. In Iran, the dairy sector is one of the main traditional and economic activities, and milk production has increased to a level of about 9 billion kg of milk per year [27]. With the high demand, the sale of raw milk for direct consumption may have increased human exposure to zoonotic agents [28]. Numerous studies conducted in Iran recovered S. aureus from dairy products [1,[29][30][31]. However, data on the role of biofilm formation of S. aureus recovered from dairy products in Iran and the antimicrobial susceptibility profile are scarce.
is information is important to better understand the evolution of S. aureus and to assess the risk to those involved in dairy production. In this regard, the present study aims to establish the relationship between biofilm formation and antibiotic resistance and adhesion genes in Staphylococcus aureus strains isolated from raw cow milk in Shahrekord, Iran.

Sampling.
In this cross-sectional study, a total of 90 raw cow milk samples (Holstein Friesian) were collected randomly from May to November 2019 in Shahrekord, Iran. Samples were randomly collected from various herds through Shahrekord. e herds were selected by convenience (i.e., the owners of the herds agreed to participate in the study), as all invited herd owners had an existing relationship with the research team. e animals from whose milk samples were collected for this study were clinically healthy and the milk samples showed normal physical characteristics. e cows in each herd that have shown obvious changes in milk, heat or udder swelling, and/or heat and mammary gland swelling (i.e., clinical mastitis) were not selected. Samples were collected under sterile hygienic conditions according to the International Dairy Federation guidelines and were immediately transported to the microbiology and biotechnology laboratories of Islamic Azad University, Shahrekord Branch, Iran [32].

Bacterial
Isolation. Isolation of S. aureus was performed following the method described by Cenci-Goga et al. [33]. e first isolation medium was tryptose blood agar base containing washed bovine red blood cells (HIMEDIA); 1 ml of milk was spread on this medium and incubated at 37°C for 48 h. Creamy grayish white or golden yellow colonies 3 to 5 mm in diameter with distinct zones of hemolysis were considered presumptive S. aureus colonies. e tests performed to identify the S. aureus isolates included growth characteristics on blood agar, Gram staining, catalase test, growth on Mannitol salt agar base, slide and tube coagulase tests, and the presence of black clony on Bird-Parker agar.

Biofilm
Formation. S. aureus ATCC25923 (biofilmforming) and S. epidermidis ATCC12228 (not biofilmforming) were respectively used as positive and negative controls. As specified by Pajohesh et al. [34], spectrophotometry was applied in microplates using crystal violet staining to quantify the formation of biofilm. For this purpose, a mixture was reached by adding 20 ml of bacterial log phase culture to 200 ml of fresh 1% glucose BHI using 96well flat-bottom microtiter plates. BHI without bacteria was used as empty. e plates were put for incubation at 37°C for 48 hr. Using aerobic conditions after each sampling, 300 mL of sterile phosphate-buffered saline was used to wash the wells three times; then, they were inverted for drainage. After that, 200 mL of methanol was added to each well, and the plates were dried for 15 minutes. 150 mL of 0.1% crystalline violet solution was used for staining of sticky cells for 15 minutes and then sterile water was used twice to wash. 150 mL of 95% ethanol was used for 10 minutes to dissolve the purple crystal violet. e optical density of each well was measured at 570 nm (OD570) using the Multiskan FC ( ermo Fisher Scientific Inc., Madison, WI). e interpretation of the results concerning biofilm formation was made according to the following rule: (OD570 ≥ 1) as strong, (0.1 ≤ OD570 < 1) weak positive, (OD570 < 0.1) as negative. S. aureus (ATCC 25923), and S. epidermidis (ATCC 12228) were applied as positive and negative controls, respectively.

Antibiotics Susceptibility Test.
e Kirby-Bauer disc diffusion method was applied by applying Mueller-Hinton agar (Merck), following the Clinical and Laboratory Standards Institute guidelines to carry out the antimicrobial susceptibility tests. As suggested by CLSI [35], the discdiffusion method on Mueller-Hinton agar was applied to examine the susceptibility of all antibiotics. International Journal of Microbiology e procedure is as follows; S. aureus isolates were put to grow during the night on blood agar. e sterile saline water equivalent to a 0.5-McFarland standard was used to achieve the colony suspension; then, 100 µl of suspension was spilled over the media plate and antibiotic disc was put aseptically on the surface of the protected media plate. Next, these plates were put for incubation at 30°C for methicillin and 35 C for other antibiotics for 24 hr. e following antibiotic disks were used; beta-lactam antibiotics such as methicillin (MET 5 µg), penicillin G (P 10 µg), ampicillin (AMP 25 µg), amoxycillin (AMS 30 µg), oxacillin (OX 5 µg), macrolides such as erythromycin (E 10 µg), aminoglycoside antibiotics such as gentamycin (GEN 20 μg), kanamycin (K 20 µg), streptomycin (S 20 µg), lincosamides such as: lincomycin (L 15 µg), clindamycin (CC 2 µg). Glycopeptide antibiotics such as vancomycin (V 10 µg). Chloramphenicol (C 30 µg), tetracycline (TE 30 µg), and rifampicin (R 30 µg).

DNA Extraction and Polymerase Chain Reaction (PCR).
e DNA extraction kit PrepMan ® Ultra Reagent (Applied Biosystems, Woolston, Warrington, United Kingdom) was used to extract genomic DNA from S. aureus isolates following the manufacturer's instructions. Total DNA was determined at an optical density of 260 nm.
S. aureus isolates were evaluated by PCR for the presence of the aroA gene, as described by Dastmalchi Saei et al. [36].
e PCR was performed in a 25-µl reaction mixture containing 12.5 µl of 2x master mix (0.04 U/µl Taq DNA polymerase, reaction buffer, 3 mM MgCl 2 0.4 mM of each dNTP), 0.4 µM of each primer, and 2 µl of template DNA. For the negative control, sterile water was added instead of nucleic acids. As a positive control, we used S. aureus ATCC 29213. e molecular amplification was conducted for the detection of the aroA gene by using species specific primers and thermal profile, which is shown in Table 1 [37][38][39][40][41][42]. Analysis of the PCR products for aroA was performed by agarose gel electrophoresis using a 1.2% gel and 0.5 µg/ml ethidium bromide in 0.5x TBE electrophoresis buffer at 80 V for 1 h and photographed under UV light. A single PCR product 1,153-bp was obtained from all S. aureus DNA extracts. e size of the PCR product was determined by comparison to the ΦX174 DNA/HaeIII markers (Fermentas, Germany). e oligonucleotide primers of the biofilm's genes encoding and antibiotic resistance genes, multiplex PCR programs, and the product size are indicated in Table 1. A DNA thermal cycler (Mastercycler Gradient, Eppendorf, Germany) was used to perform the PCR. e ethidium bromide and electrophoresed were used in 1.5% agarose gel at 80 volts for 30 minutes to stain amplifiers. UV doc gel documentation systems (Uvitec, UK) were used to visualize and photograph PCR products. A comparison was run between PCR products and 100 bp DNA markers (Fermentas n, Germany).

Statistical Analysis.
e data were transferred to a Microsoft Excel spreadsheet (version 15; Microsoft Corp., Redmond, WA, USA) for analysis. Using statistical software (version 16; SPSS Inc., Chicago, USA), the chi-square test and Fisher's exact two-tailed test analysis were performed, and differences were considered significant at values of p < 0.05.

Results
Of the 90 milk samples, 35 samples (38.88%) were positive for S. aureus and all isolates were approved by PCR for the presence of the aroA gene (this confirms that PCR here is pretty useless). Table 2 shows the results for the detection of biofilm formation. Biofilm formation was strongly and weakly observed in 65.71% and 20% of isolated S. aureus strains, respectively. Table 3 shows the results for the antibiotic resistance pattern of S. aureus strains. All the isolates (100%) exhibited resistance to penicillin and almost all were resistant to ampicillin (91.42%). A high number of isolates (71.42%) exhibited resistance to oxacillin and a significant number of isolates were resistant to methicillin and kanamycin (42.85%). All isolates were sensitive to vancomycin and rifampicin. Table 4 shows the results for the antibiotic resistance pattern based on biofilm reaction. All strong biofilm producer isolates (100%) exhibited resistance to penicillin and almost all were resistant to ampicillin and oxacillin (95.65%). A high number of strong biofilm producer isolates (65.22%) were resistant to methicillin and kanamycin. Based on Fisher's exact test, there is a statistically significant relationship between strong biofilm reaction and resistance to penicillin G, ampicillin, oxacillin, and gentamycin   Table 5 shows the results for the prevalence of antibiotic resistance genes. e gene blaZ was found on a high number (71.42%) of S. aureus isolates, and 42.85% of isolates carry the resistance genes mecA and Aac-D. e same number of isolates (28.57%) carries resistance genes tetK and tetM and 14.28% of isolates carry the genes linA, ermA, and ermC. Table 6 presents the prevalence of antibiotic resistance genes in biofilm-forming and nonforming isolates. According to this table, all strong biofilm-producing strains carry the blaZ genes and a significant number of isolates carry mecA and tetK with a prevalence of 65.22% and 43.48%, respectively. us, the most prevalent genes were mainly found in strong biofilm producing strains. Also, based on the chi-square test, there is a statistically significant relationship between strong biofilm reactions and antibiotic resistance (p value <0.05). Based on Fisher's exact test, there is no statistically significant relationship between any antibiotic and weak and negative biofilm reactions (p value >0.05). Table 7 presents the genotypic evaluation of biofilm production in Staphylococcus aureus isolates. All the 8 genes encoding biofilm production were detected in Staphylococcus aureus isolates. e minimum frequency (71.43%) was found for the gene clfb. Table 8 presents the adherence of attachment factor genes in Staphylococcus aureus isolates based on biofilm reaction. e attachment factor genes icaA, icaB, icaC, icaD, fnbA, and fnbB were present in all strong biofilm producing strains, and a high number of these isolates (86.96%) carry bap, cflA, and cflB. Based on Fisher's exact test, there is a statistically significant relationship between the active genes and strong biofilm reaction (p value <0.05). But there is no statistically significant relationship between any of the active genes and weak or negative biofilm reactions (p value >0.05).

Discussion
Staphylococcus aureus is a common pathogenic bacterium for both humans and animals [43][44][45]. Its pathogenicity depends on the wide range of staphylococcal enterotoxins that it can produce and which have an adverse effect on humans and animal organisms [44,46]. e production of a biofilm enhances its virulence as it resists substances such as antibiotics that can inhibit its growth [47]. e present study aims to establish the relationship between biofilm formation and antibiotic resistance and adhesion genes in Staphylococcus aureus strains isolated from raw cow milk in Shahrekord, Iran.
e results for the presence of S. aureus in raw cow milk show that of the 90 milk samples, 35 samples (38.88%) were positive for S. aureus and all isolates were approved by PCR for the presence of the aroA gene. Previously, several studies have found similar rates of isolation of S. aureus in milk [48,49]. e presence of S. aureus in milk can be explained by poor hygiene conditions during production, handling, and/or distribution [23]. In addition, milk provides good growing conditions for S. aureus, which can survive in products for a long time. Investigation of phenotypic biofilm production showed that biofilm formation was strongly and weakly observed in 65.71% and 20% of isolated S. aureus strains, respectively. is result is consistent with some studies that also reported that most of the S. aureus strains recovered from milk were biofilm producers [50,51]. is result confirms that the majority of S. aureus isolated from raw milk are biofilm producers [52]. According to Shen et al., the presence of milk may play an important role in biofilm production by S. aureus [52]. e sugar (glucose) content of the milk positively influenced biofilm formation [53].
Antibiotic resistance pattern results show that all the isolates (100%) exhibited resistance to penicillin, and almost all were resistant to ampicillin (91.42%). is alarming rate of resistance to beta-lactam antibiotics is corroborated by some recent findings [54,55] and can be explained by the common use of β-lactams in the treatment of bovine mastitis [56]. A study carried out in Kenya by Mbindyo et al. found that 71.4% of S. aureus strains exhibited resistance to ampicillin [57]. Another study reported that the frequency of resistance to penicillin G was 85.2% [58]. A high number of isolates (71.42%) exhibited resistance to oxacillin and a significant number of isolates were resistant to methicillin and kanamycin (42.85%). is pattern of multidrug resistance, particularly methicillin resistance, is increasingly being reported worldwide [23,59,60]. e emergence of resistance observed is associated with the misuse and overuse International Journal of Microbiology 5 of antibiotics in farming [61]. In many developing countries, such as Iran, most of these antibiotics are cheap and easy to find and do not require a veterinary prescription to purchase [62]. e results for antibiotic resistance pattern based on biofilm reaction show that all strong biofilm producer isolates (100%) exhibited resistance to penicillin and almost all were resistant to ampicillin and oxacillin (95.65%). A high number of strong biofilm producer isolates (65.22%) were resistant to methicillin and kanamycin. Similar results were found by Manandhar et al. [63], who reported a high frequency of multiple antibiotics resistance, such as penicillin, cefoxitin, tetracycline, clindamycin, and chloramphenicol, from clinical isolates.
Based on Fisher's exact test, there is a statistically significant relationship between strong biofilm reaction and resistance to penicillin G, ampicillin, oxacillin, and gentamycin antibiotics (p value <0.05). is finding is not in line with the results reported by [63,64], who did not find any significant difference in biofilm production between methicillin-resistant S. aureus and methicillin-sensitive S. aureus. Other studies found that biofilm production correlated well with methicillin resistance [24]. e discrepancies in the findings can be explained by differences in the interpretation of results [25]. Indeed, various methods, such as the Congo red plate assay [65], crystal violet (CV) assay [66], and microtitre plate assay [34], can be used to screen biofilm-producing strains.
e results for the prevalence of antibiotic resistance genes show that the gene blaZ was the most prevalent (71.42%), followed by mecA and Aac-D (42.85%). ese results are in line with the observed antibiotic pattern with a high number of resistances to beta-lactam antibiotics     [67,68]. e significant number of isolates that carry mecA can explain the high rate of resistance to methicillin and multiple antibiotics [25]. e prevalence of antibiotic resistance genes in biofilmforming and nonforming isolates results show that the most prevalent genes were mainly found in strong biofilm-producing strains. Also, based on the chi-square test, there is a statistically significant relationship between strong biofilm reactions and antibiotic resistance (p value <0.05). Based on Fisher's exact test, there is no statistically significant relationship between any antibiotic and weak and negative biofilm reactions (p value >0.05). is result is in line with Marchant et al. [69], who concluded after an in vitro assay that biofilm formation influences antibiotic resistance. But the presence of these resistance genes does not necessarily explain the resistance, as it can manifest itself through other mechanisms. However, in the literature, many authors have found no correlation between biofilm production and antibiotic resistance in S. aureus. Again, the discrepancies are explained by the variety of methods used to screen biofilmproducing strains [21]. To date, no indisputable conclusions have been proposed. e genotypic evaluation of biofilm production in Staphylococcus aureus isolates showed that all the 8 genes encoding biofilm production (fnbA, fnbB, icaA, icaB, icaC, icaD, clfA, clfB, and bap) were detected in Staphylococcus aureus isolates. e attachment factor genes icaA, icaB, icaC, icaD, fnbA, and fnbB were present in all strong biofilmproducing strains, and a high number of these isolates (86.96%) carry bap, cflA, and cflB. Based on Fisher's exact test, there is a statistically significant relationship between the active genes and strong biofilm reaction (p value <0.05). Considering the high number of biofilm-producing strains, these results confirm that strains harbouring the icaADBC cluster [27], clfB, fnbB, clfA, and fnbA [70][71][72] are potential biofilm producers. In addition, the biofilm-associated protein (bap) plays an important role in the adherence of S. aureus and biofilm formation [73,74].

Conclusions
In conclusion, this study revealed that there is a significant correlation between biofilm production and antibiotic resistance in S. aureus isolated from raw milk. A high number of multidrug-resistant strains carrying several biofilm-related genes were found. e presence of potentially biofilm-producing and antibiotic-resistant S. aureus in milk intended for human consumption represents a serious health hazard. ese results indicate that the prevention and management of these infections should be a priority in Iran.

Data Availability
e data supporting the findings of this article are available from the corresponding author upon request. Disclosure e funding agencies had no involvement in the conceptualization, design of the study, data collection and analysis, interpretation of data, or in writing the manuscript.

Conflicts of Interest
e authors declare that they have no conflicts of interest.