Isolation of S. aureus

Thirty (30) isolates of S. aureus from cases of subclinical mastitis of cows located in dairy farms located in localities in the states of Guanajuato and Michoacán, Mexico, which use semi-intensive production and backyard production systems, were analyzed. The sampling, isolation and characterization of the isolates was already reported by Varela et al¹²⁾ in 2018.

Antibiotic multi-resistance profile

An antibiogram was performed by the Kirbi-Bauer disc-diffusion method¹³, using Biorad^® sensidiscs with the following antibiotics and concentrations: penicillin (PE) 6 μg, oxacillin (Oxa) 6 μg, dicloxacillin (DC) 30 μg, pefloxacin (PEF) 5 μg, cefuroxime (CXM) 30 μg, gentamycin (GE) 120 μg, cefotaxime (CTX) 30 μg, sulfamethoxazole + trimethoprim (SXT) 1.25 and 23.75 μg, tetracycline (TE) 30 μg, ampicillin (AM) 10 μg, erythromycin (E) 15 μg, ceftazidime (CAZ) 30 μg and cephalotin (CF) 30 μg. The results are reported as sensitive, intermediate and resistant based on the parameters established in Performance Standards for Antimicrobial Susceptibility Testing, 2019¹⁴. Once the resistance profile was established, the 30 isolates were classified as described below: Group 1: High resistance (resistant to 11-13 antibiotics), Group 2: Medium resistance (9-10 antibiotics), Group 3: Low resistance (4-8 antibiotics).

Biofilm formation

To measure the biofilm-forming capacity of S. aureus isolates, the violet crystal staining protocol¹¹ was used, as described below: the bacterial isolate was cultured in LB medium and incubated 24 h at 37 ⁰C. The tests were performed on sterile plates of 96 wells and placed ≈ 1 x 10⁶ CFUs in a final volume of 100 μL in each well. Isolates were incubated for 48 h at 37 °C.

Once the incubation time had elapsed, the supernatant was discarded, the wells were washed with 100 μL of PBS solution (137 mM NaCl, 2.7 mM KCl, 8 mM Na₂HPO₄ and 2 mM KH₂PO₄) and the wells were dried. Subsequently, 100 μL of violet crystal solution (0.5 % weight/volume) was added to each well and it was left to stand for 15 min. The dye was then removed, and it was washed twice with 100 μL of PBS. Then, 125 μL of 95 % ethanol (volume/volume) was added and it was vigorously resuspended to dissolve the dye. The absorbance reading was taken at 495 nm in an ELIREAD microelisa analyzer (Kontrolab^®, Guidonia, Italy). Once the data were obtained, the percentage of biofilm formation was plotted using as 100 % the registered absorbance of the certified strain of S. aureus (ATCC 27543). Three independent experiments with three repetitions were conducted.

Analysis of resistance genes and biofilm formation

To evaluate the presence of genes related to antibiotic resistance and biofilm formation, it was carried out from genomic DNA, which was obtained by chopping a bacterial colony from a fresh culture plate to later place it in the PCR reaction mixture. The oligonucleotides used in this study are shown in Table 1. The reaction was performed in a final volume of 20 μL containing 0.4 μM oligonucleotides, 200 μM deoxynucleotides triphosphates (Invitrogen, Carlsbad, California, United States), 2 mM magnesium chloride (Invitrogen, Invitrogen, Carlsbad, California, United States) and 1 U of Taq polymerase (Invitrogen, Invitrogen, Carlsbad, California, United States). The amplification conditions were as follows: initial denaturation temperature at 95 °C for 10 min, followed by 30 denaturing amplification cycles for 10 min at 94 °C, alignment for 1 min at the specific temperature of oligonucleotides (Table 1), polymerization for 30 sec at 72 °C, and a final extension cycle for 7 min at 72 °C. Amplicons (5 μL) were analyzed by 1 % agarose gel electrophoresis (weight/volume) and stained with ethidium bromide. It was considered positive for the gene, the presence of an amplification band corresponding to the size of the expected product.

To analyze the genetic basis of bacterial resistance mechanisms, the presence of the blaZ and mecA genes for beta-lactam antibiotics was analyzed^15,16; tetK and, tetM for tetracyclines¹⁶ and gyrA and gyrB for quinolones¹⁷.

Statistical analysis

Three independent experiments were carried out in which the absorbance produced by the staining of the formed biofilm was measured. The experiments were done in triplicate. The difference between the highest absorbance minus the largest amount of biofilm was obtained, this difference is defined in this study as absorbance.

The 30 isolates of S. aureus were classified into three groups according to the level of antibiotic resistance: high, medium and low, of 10 isolates in each and were subsequently evaluated according to their absorbance. The results of the positive controls for each resistance level of each experiment were included in the analyses.

The normality of the dependent variable absorbance was evaluated using the Chi-square goodness of fit test, resulting in normal (P>0.05). Data were analyzed with an analysis of variance (ANOVA) with a factorial design with a completely randomized arrangement. The model used is shown below:

Yijk=μ + EXi + GRj + EXixGRj + eijk, where:

Yijk= is the k-th observation of absorbance, of the i-th experiment and the j-th degree of resistance,

μ=general mean as a constant parameter,

EXi= i-th experiment, j=1, 2 and 3,

GRj= j-th degree of resistance, i= 1, 2 and 3,

GRixEXj= interaction between the i-th degree of resistance and the j-th experiment,

eijk= experimental error.

Additionally, the Spearman rank correlation between degree of resistance and absorbance was estimated.

Antibiotic-resistance profile of S. aureus isolates from bovine mastitis

The results show that 100 % of the isolates are resistant to penicillin and dicloxacillin, in addition, 86.6, 83.3 and 80.0 % show resistance to ampicillin, cephalotin and ceftazidime, respectively; it was also observed that 36.6 % of the isolates are resistance to oxacillin. Overall, all isolates analyzed were resistant to at least 33.3 % of the antibiotics tested (Table 2).

Antibiotic resistance is a phenomenon that continues to increase and that significantly affects the health sector, both in human medicine and veterinary medicine, since it hinders the proper management of infectious diseases. Such is the case of bovine mastitis; S. aureus, as one of the main bacteria isolated from bovine mastitis, has high resistance rates. In recent years, globally, the selection of bacterial resistance mechanisms continues to increase¹⁸. In this sense, in several countries where the resistance profile of S. aureus from bovine mastitis was analyzed, percentages of resistance to penicillin close to 100 %^19-23 were found, which coincides with the results reported here. Regarding the resistance presented to oxacillin, contrasting results were found, since studies carried out in countries such as India and China showed resistance levels from 48 to 84 %^21,24; however, in Germany, Japan and Colombia, the levels of resistance to oxacillin are minimal (2-7 %)^21,23,25, while these results show intermediate levels of resistance (33.3 %). In Mexico, studies reveal high levels of bacterial resistance to penicillin, amoxicillin and dicloxacillin (100 %)^26,27, which is consistent with what was reported in this study. Likewise, there is a significant increase in resistance to cephalotin, for example, in 2008 it was reported that 30 % of the strains of S. aureus studied had resistance²⁷; however, in this work resistance of up to 83 % was found. These variations may be due to the possible genetic variability of the isolates, climatological differences, as well as geographical discrepancies among other factors²⁸. Based on these differences, the need to carry out works such as the one presented here is highlighted, to define the virulence characteristics of S. aureus isolated from particular regions; to generate the necessary information that allows the implementation of more efficient treatments for subclinical bovine mastitis.

The presence of the blaZ gene was observed in 100 % of the isolates analyzed, while only 36.6 % were positive for the mecA gene (Table 3). These results are consistent with what was previously reported, where the presence of these genes was shown in all the isolates analyzed^19,29. The presence of the tetK, tetM, gyrA and gyrB genes was expressed in a descending way according to the groups analyzed, which is consistent with the phenotype found in the antibiograms. This coherence between the phenotype and the genotype of bacterial resistance has already been demonstrated before^29,30, so that the resistance observed can be attributed to the presence and eventual expression of the genes analyzed³¹.

Biofilm formation of S. aureus isolated from bovine mastitis

The analysis of biofilm formation showed that all isolates had the ability to form biofilm with a range from 20 to 98 %. Several studies around the world report that the strains of S. aureus analyzed have the capacity of biofilm formation (90 to 99 %)^32,33, which coincides with the present study.

Because S. aureus has high levels of antibiotic resistance, it is necessary to analyze the virulence factors and characteristics of this bacterium in order to design more efficient control strategies. In this sense, it has already been reported that S. aureus has the ability to form biofilm³⁴, which may be related to the low effectiveness that conventionally used drugs have³⁵.

To establish the possible relationship between resistance and biofilm formation in the 30 isolates from bovine mastitis, the following strategy was followed. First, the results of biofilm formation were analyzed according to the resistance level of the isolates (Figure 1); for which, these were ordered into three groups as described in the materials and methods section. In the analysis of variance of this study, only the effect of degree of resistance was significant (P<0.01), finding that the mean absorbance levels of the isolates were 1.34 (63.17 %), 0.77 (38.78 %) and 0.66 (26.28 %) for groups 1, 2 and 3, respectively. In the comparison of means, groups 1-3 were different (P<0.05) (Figure 1B). The estimated correlation between the degree of resistance and biofilm formation was positive (0.50) and significant (P<0.01) (Figure 2); in addition, these differences were analyzed microscopically, where the formation of biofilm in the isolates of group 1 were observed to be significantly increased compared to the other two groups (Figure 1A). Because the formation of biofilm by S. aureus induces the development of chronic and recalcitrant infections³², the need to analyze how this virulence characteristic is related to other mechanisms, such as antibiotic resistance, stands out. With reference to the above, this relationship has been studied in both Gram-negative^36-39 and Gram-positive^40,41 bacteria; however, there are discrepancies in defining whether the correlation that occurs is positive^42,43,44 or negative⁴¹.

A) Representative images of the biofilm staining in the three groups (Group 1: High resistance, from 11 to 13 antibiotics; Group 2: Medium resistance, from 9 to 10 antibiotics and Group 3: Low resistance, from 4-8 antibiotics), visualized with light-field microscopy.

B) The graph represents the percentage of biofilm formation of the three groups of isolates. The certified strain of S. aureus ATCC 27543 was used as a positive control, whose absorbance value was standardized as 100 %. The average of three independent experiments in triplicate ± their standard deviation is presented. (*) represents a statistically significant difference between groups (P≤0.001).

Figure 1 Biofilm formation by S. aureus isolates. The violet crystal staining protocol was used to measure the formation of biofilm. 

Figure 2: Relationship between the percentage of resistance and biofilm formation (%) of S. aureus isolates 

In this study, it was observed that isolates with a high multi-resistance present a greater formation of biofilm; establishing a positive correlation, which is consistent with most of the works reported in this regard^42,43,44. Finally, the presence of the icaA and icaD genes was analyzed, which are directly related to the formation of biofilm⁴⁵. Interestingly, it was found that there is a high frequency of both genes in the isolates (86.6 %), regardless of the degree of resistance they present (Table 3). Several authors agree with this result, since they report a frequency close to 100 % of one or both genes in biofilm-forming strains of S. aureus^46,47,48.