شناسایی توانایی تولید بیوفیلم، عوامل حدت و الگوی مقاومت آنتی‌بیوتیکی استافیلوکوکوس اورئوس جدا شده از ورم پستان تحت بالینی

Introduction

Bovine mastitis causes an economic loss to the dairy industry and Staphylococcus spp. play an important role in this etiology (Pacha et al., 2020). Of these, S. aureus, stands out among the prevalent etiological agents in this type of infection with subclinical prevalence and poor response to the treatments (Pacha et al., 2020). The improper use of antimicrobials and formation of biofilms undermines the effectiveness of mastitis therapy. The biofilm structures are made up of surface attached bacteria in the organic matrix (Bolte et al., 2020). The Staphylococcus aureus, can producea series of virulence factors that contribute to the bacterium invading the host's phagocytic defense, facilitating its adherence to the epithelial cells and colonization in the tissue, favoring its extracellular persistence and thus guaranteeing its successful installation and maintenance in the host tissues (Bolte et al., 2020). Among these factors is the production of a mucopolysaccharide extracellular “slime”, which seems to help the adherence and colonization of the microorganism to the mammary glandular epithelium. The ability of S. aureus to adhere to the surface of the epithelium has been associated with the production of biofilms, which are described as agglomeration of the cells embedded in an extracellular heterogeneous matrix, resulting in three-dimensional structures with specific physiological characteristics (Hathroubi et al., 2017). Several researches have studied on S. aureus mastitis.

Biofilm is a multi-step process involved in the formation and adherence to the host surface by adhesion factors, followed by the growth to form a matrix (Schiffer et al., 2019). The microbial surface components recognizing the adhesive matrix molecules (MSCRAMMs) are adhesion proteins of the staphylococcal families, such as fibronectin-binding proteins (FnbA and FnbB), and biofilm-associated protein (Bap) (Kıvanç, 2018; Schiffer et al., 2019). An intercellular polysaccharide adhesion molecule has been found that mediates the intracellular adhesion (icaADBC) and controls the biofilm production (Uribe-García et al., 2019; Zhao et al., 2021). Previous studies have not evaluated the antibiotic resistance in planktonic and biofilm conditions in the subclinical mastitis of bovine S. aureus, which can detect the trend in the biofilm formation ability, and the genes encoding biofilm and antibiotic resistance pattern. Thus, due to the necessity of this research, data obtained from the pattern of antibiotic resistance and virulence genes can gather more information in this regard for the possibility of developing more effective strategies for the treatment and control strategies. This study aimed to characterize the biofilm formation ability in the antibiotic resistance pattern of S. aureus isolates from the subclinical bovine mastitis.

Materials and Methods

Phenotyping S. aureus

Forty primary samples of the cows' milk belonging to the five farms located in the Tehran province were collected. The samples were subjected to the primary isolation and subsequent experiments for the phenotypic identification of the species. The 1-9 parities of lactating dairy cows were screened for the subclinical mastitis using the CMT and SCC determinations. The SCC cutoff value (200.000-500.000 cells/mL) of the diagnostic subclinical mastitis was appointed on the herd prevalence of S. aureus. The positive quarters were defined; sampling was done and the samples were transported to the laboratory on ice-pack. Classical microbiological, biochemical, and coagulase tests were conducted using the methods described previously by Hogan (Hogan et al., 1986). The isolates were confirmed as S. aureus by PCR on the nuc gene. The genomic DNA was extracted as described before (Fatholahzadeh et al., 2009). The primers sequences were synthesized according to Sahebekhtiari and colleagues study (Sahebekhtiari et al., 2011). The S. aureus ATCC 29213 was included as control strain. Finally, a total of 30 isolates were defined as S. aureus. For the next experiments, S. aureus inoculum was prepared from each isolate in TSB (MERCK, Germany) including 1% glucose broth (Baldassarri et al., 2001). All assays were performed in triplicate (Figure 1 Step-A).

Biofilm Formation Study

The S. aureus biofilm forming and quantification was described before (Stepanović et al., 2007). Each S. aureus inoculum was diluted 2:200 in TSB + 1% glucose and poured into the wells of the sterile tray (Tissue culture 96-wells plate, JET BIOFIL, Canada) and incubated aerobically for 24 h (37°C); after which the supernatant was discarded, and the wells were washed thrice. The precipitates were fixed by Bouin’s reagent, dried by air (60°C, 1 h), and stained with crystal violet. The bound dye was re-solubilized with 95% ethanol. The S. aureus ATCC 25923 and broth (TSB + 1% glucose) were used as positive and negative controls, respectively. The optical density was measured at 570 nm by a microplate reader (Epoch™ Microplate Spectrophotometer, BioTek). The cut-off value was established as ODc= average OD of negative control + (3SD of negative control). The biofilm formation was categorized as follow: OD≤ODc = no; ODc<OD≤2ODc = weak; 2ODc<OD≤4ODc = moderate; 4ODcFigure 1 Step-B).

Biofilm-Encoding Genes Detection

The S. aureus biofilm genes, icaAD, fnbAB, and bap, were targeted by PCR. The primers sequences and amplification cycles were described before (Vancraeynest et al., 2004). The S. aureus ATCC 25923 and S. epidermidis ATCC 12228 were included as positive and negative reference strains, respectively (Figure 1 Step-C).

Antimicrobial Susceptibility/ Disk Diffusion Agar (DAD)

The Antimicrobial susceptibility of the isolates was performed by DAD method (Pfaller et al., 2001; Weinstein & Lewis, 2020). Briefly, the assay was done with, penicillin, gentamicin, ceftiofur, ampicillin, erythromycin, trimethoprim/sulfamethoxazole, tetracycline, chloramphenicol, ciprofloxacin, and enrofloxacin (Mastdiscs®, UK), on Mueller59 Hinton BBLII agar (Becton Dickinson, Heidelberg, Germany). The S. aureus ATCC 25923 was included as quality control (Figure 1 Step-D).

Antimicrobial Susceptibility/ Broth Microdilution (MIC)

The Antimicrobial susceptibility of the isolates was also evaluated using designation of MIC method (Pfaller et al., 2001; Weinstein & Lewis, 2020). Briefly, Mueller-Hinton broth containing ceftiofur was poured into a 96-well tray. Half McFarland density of S. aureus isolates were diluted to 5 x 10⁵ CFU/mL, inoculated to the 96-well tray, and incubated for 24 h at 37°C. The MIC was construed as the lowest antimicrobial agent preventing the visible growth. The susceptibility thresholds and resistance breakpoints were based on the CLSI guidelines as ≤2 and ≥8 μg/mL for ceftiofur, respectively. The S. aureus ATCC 29213 was included as quality control (Figure 1 Step-E).

Determination of the Minimum Biofilm Eliminating Concentrations (MBECs)

All isolated strains were susceptible to ceftiofur in the planktonic state, thus, the antibiotic susceptibility and MBECs for the bacteria embedded in biofilms were determined by colorimetric assay according to (Amorena et al., 1999) study. The biofilms formation was performed as described previously; After biofilms formation in the 96-well tray, with 100 μL of ceftiofur serial dilutions for 20 h (37°C) incubation, 50 μL XTT (Roche, Germany) was added, then tray was covered, and incubated for 1 h at 37°C (Pettit et al., 2005). The MBECs values were construed as the lowest antimicrobial agent preventing the visible growth (Sepandj et al., 2004). These assays were performed in triplicate (Figure 1 Step-F).

Figure 1. The total of 30 isolates of S. aureus were introduced into the experiment A to F (Application Source: Paint)

Results

A total number of 30 S. aureus isolates from the subclinical mastitis were studied to estimate the role and ability of biofilm formation in the antibiotic resistance pattern. The results of biofilm formation demonstrated that all isolates (100%) were biofilm producers, in which 77.4% of them produced strong biofilms, 12.9% and 9.7% produced moderate and weak biofilms, respectively. The biofilm-encoding genes frequency were as; bap (25%), icaA (9.4%), icaD (75%), fnbA (43.8%) and fnbB (31.2%) (Table 1). The rate of resistance to penicillin (74.4%), gentamicin (2.3%), ceftiofur (0%), ampicillin (57.5%), erythromycin (33.3%), trimethoprim/sulfamethoxazole (10%), tetracycline (70.3%), chloramphenicol (2.30%), ciprofloxacin (0%), and enrofloxacin (6.6%) were detected by DAD test. The highest resistance rate was detected against ceftiofur and ciprofloxacin; and the penicillin had the lowest resistance rate (Table 1). The MIC₅₀ of ceftiofur was found 1 and 2 μg/mL for ATCC 29213 and isolated strains, respectively. Based on the CLSI guidelines, the percentages of sensitive, intermediate, and resistant S. aureus to ceftiofur were 96.67, 3.33, and 0%, respectively (Table 1). The MBEC results for the bacterial biofilm are listed in Table 1. Among the isolates, 28 strains were resistant to ceftiofur in biofilm state; however, these strains were susceptible to this agent in the planktonic state.

Table 1. S. aureus Isolates Frequency of The Genotypic Patterns, Biofilm Formation Type and Antimicrobial Susceptibility

* S: Strong, M: Moderate and W: Weak; ** F1: Farm1, F2: Farm2, F3: Farm3, F4: Farm4 and F5: Farm5; ⁺ Se: Sensitive and in: Intermediate; ⁺⁺ Su: Susceptible and Re: Resistant.

Discussion

Studies have shown that S. aureus is the most important microorganism in the bovine subclinical mastitis. In this study, primary milk samples from the subclinical mastitis collected from the five farms in Tehran province were tested for the S. aureus phenotypic identification. For the sensitivity and specificity of the genotypic techniques, S. aureus was confirmed by nuc gene amplification (Fatholahzadeh et al., 2009).

Improper usage of antimicrobials to combat mastitis leads to the selection of resistant strains and undermines the effectiveness of therapies (Pacha et al., 2020). In this study, the isolates showed high resistance rate to tetracycline (70.3%) and penicillin (74.4%). The high resistance rate of S. aureus to penicillin and tetracycline was reported before (Gao et al., 2012), Aslantaş & Demir, (2016), and Jamali et al., (2014). The penicillin resistance rate in this study and Jamali's et al., (2014) study was similar. The tetracycline resistance rate (70.3%) was higher than Aslantaş & Demir, (2016), and Ren et al., (2020) studies and lower than (Jamali et al., 2014) findings. Similarly, erythromycin-resistance (33,3%) was found by Ren et al., (2020) study. The present study showed full susceptibility to ceftiofur (100%) and ciprofloxacin (100%). The rate of resistance to trimethoprim/sulfamethoxazole (10%) was higher than Aslantaş & Demir, (2016) study. Resistance prevalence against enrofloxacin (6.6%) was higher than Aslantaş & Demir, (2016) study. The gentamicin-resistance rate (2.3%) was inconsistent with Ren et al., (2020) study. Our finding of ampicillin-resistance rate (57.5%) was in agreement with Moroni et al., (2006) results. In contrast to these studies, high levels of chloramphenicol-resistance (2.3%) were reported by Liu et al., (2017). The resistance rate to erythromycin (33%) was lower than those from the findings of Liu et al., (2017). According to the multidrug-resistant isolates and inconsistency in the antimicrobial resistance rate in numerous studies, suitable antimicrobial should be district-based.

The rise in multidrug resistant isolates of S. aureus is an important issue in mastitis control and the ability of biofilm formation is a potential role as a virulence factor (Notcovich et al., 2018). The S. aureus ability to produce biofilm is responsible for the establishing a persistent infection (Vasudevan et al., 2003). In S. aureus, the icaA and icaD genes have a significant character in the biofilm formation (Vancraeynest et al., 2004). This study reported the prevalence rate of icaD, fnbA, fnbB, bap and icaA genes at 75, 43.8, 31.2, 25, and 9.4%, respectively. Similarly, the highest frequency of the ica gene was identified in Ahmed et al., (2019); icaA: 58% and icaD: 60% and Salina et al., (2020) studies. However, the prevalence rates of the icaA and icaD genes vary greatly among different studies (Águila-Arcos et al., 2017; Kot et al., 2018; Mahmoudi et al., 2019) and others who found that biofilm formation can be influenced by several aspects (Demir et al., 2020). The icaD gene was the most prevalent among all detected genes, like in the study of Costa et al., (2018), which is in agreement with our study; whereas, these were inconsistent with Ghasemian et al., (2016) finding.

This study expressed that 25% of S. aureus isolates were positive for bap gene, whereas, this was lower than Salina et al., (2020) result. The moderate fnbA gene frequency was reported by Khoramian et al., (2015) and Ghasemian et al., (2016), which were higher compared to our results (43.8%). Zuniga et al., (2015) observed a high frequency of the fnbA gene (87.5%) from the caprine subclinical mastitis. Our reported prevalence rate of fnbB gene was lower than Khoramian et al., (2015) and Ghasemian et al., (2016) studies.

Conclusion

In conclusion, all the strong biofilm-producing isolates were positive for ica gene. The fnbA, fnbB, and bap (MSCRAMM) genes had prevalence in all types of biofilms (strong, moderate, and weak). It may make clear that detection of ica gene is much more important for the biofilm grade prediction than biofilm formation.

The MIC values of the ceftiofur were evaluated on the planktonic cells of S. aureus. The results showed sensitive (96.67%), intermediate (3.3%) and resistant (0%) breakpoints. In conclusion, all isolated S. aureus strains were found biofilm producers and most of them were positive for icaA, and icaD virulence genes; most of the isolated S. aureus strains were sensitive to ceftiofur.

The S. aureus is the most important microorganism in the bovine subclinical mastitis. The high frequency of ica gene, the strong biofilm formation and antibiotic resistance of most of the isolates were related to the antibiotics that are routinely used in the veterinary medicine. Therefore, in order to control, achieve the effective treatment, and prevent the emergence of antibiotic-resistant bacteria, it is necessary to isolate the causative agent and determine the antimicrobial susceptibility.

Acknowledgments

This research has been supported by Tehran University of Medical Sciences and Health Services.

Conflict of Interest

The authors declared no conflict of interest.