Biofilm Formation and Antimicrobial Resistance of Staphylococcus aureus and Streptococcus uberis Isolates from Bovine Mastitis

Simple Summary Staphylococcus (Staph.) aureus and Streptococcus (Strep.) uberis are key causes of intra-mammary infection in dairy cows, and their ability to form biofilms is recognized as a significant virulence factor influencing mastitis pathogenesis and the response to antimicrobial treatment. This study aimed to evaluate (a) the biofilm producer ability and antimicrobial resistance of Staph. aureus (n = 197) and Strep. uberis (n = 119) isolated from cows with clinical and subclinical mastitis, and (b) the association between biofilm formation and antimicrobial resistance. Both Staph. aureus and Strep. uberis exhibited high biofilm formation ability. However, no correlation was found between the form of mastitis presentation (clinical or subclinical) and the biofilm-forming capacity. Moreover, a significant proportion of Staph. aureus and Strep. uberis isolates demonstrated resistance to penicillin, ampicillin, and tetracycline. Interestingly, we observed no association between biofilm formation ability and antimicrobial resistance. Abstract This study aimed to assess (a) the biofilm producer ability and antimicrobial resistance profiles of Staphylococcus (Staph.) aureus and Streptococcus (Strep.) uberis isolated from cows with clinical mastitis (CM) and subclinical mastitis (SCM), and (b) the association between biofilm producer ability and antimicrobial resistance. We isolated a total of 197 Staph. aureus strains (SCM = 111, CM = 86) and 119 Strep. uberis strains (SCM = 15, CM = 104) from milk samples obtained from 316 cows distributed in 24 dairy herds. Biofilm-forming ability was assessed using the microplate method, while antimicrobial susceptibility was determined using the disk diffusion method against 13 antimicrobials. Among the isolates examined, 57.3% of Staph. aureus and 53.8% of Strep. uberis exhibited the ability to produce biofilm, which was categorized as strong, moderate, or weak. In terms of antimicrobial susceptibility, Staph. aureus isolates displayed resistance to penicillin (92.9%), ampicillin (50.8%), and tetracycline (52.7%). Conversely, Strep. uberis isolates exhibited resistance to penicillin (80.6%), oxacillin (80.6%), and tetracycline (37.8%). However, no significant correlation was found between antimicrobial resistance patterns and biofilm formation ability among the isolates.


Introduction
Bovine mastitis is one of the prevailing diseases of dairy cows globally, leading to significant economic losses in dairy herds and the dairy industry [1].Among the major causative agents, Staphylococcus aureus and Streptococcus uberis have been described as major causes of mastitis [2], and their transmission mechanisms, genetic diversity, virulence factors, and antimicrobial resistance profile have been extensively studied [3].
Staph.aureus is recognized as a contagious pathogen associated with chronic intramammary infections (IMI) [4].This pathogen poses a challenge for antibiotic therapy owing to its antimicrobial resistance characteristics, including its ability to survive within phagocytes and form biofilms [5,6].Conversely, molecular studies showed that Strep.uberis, initially considered an environmental reservoir pathogen, has also the potential of contagious transmission [7,8].Similarly, to Staph.aureus, managing Strep.uberis IMI proves challenging, with low cure rates [9].This difficulty may be attributed, in part, to virulence factors of Strep.uberis, including its biofilm-forming capability [10].
Biofilm, which is characterized by an extracellular polysaccharide matrix, serves as a protective layer for microorganisms, allowing their proliferation within and subsequent release into the environment [11].The initiation of biofilm formation starts with a small number of bacterial cells adhering to a substrate.Subsequently, these bacteria release an extracellular polymeric substance (EPS), which, in conjunction with host components, constructs the extracellular matrix.While primarily composed of polysaccharides, proteins, nucleic acids, and lipids, the structure and composition of biofilms exhibit significant variability [12].Biofilms have effects on the public health and industrial considerations pertaining to their influence on the economy, energy utilization, equipment deterioration and the occurrence of infections [13].
Bacteria within biofilms exhibit enhanced survival in adverse environments and innate resistance to antibiotics, disinfectants, and host defense mechanisms [14].This high antimicrobial resistance can be attributed to a modified chemical microenvironment, spore formation, reduced growth rate, antibiotic inactivation by the extracellular matrix, and the occurrence of horizontal gene transfer [11].In additional, biofilm plays a significant role in inefficient wound healing and contributes to the persistence of chronic wounds [15].Thus, biofilm production may significantly impact disease progression and treatment outcomes and may contribute to the proliferation of antimicrobial resistance [16].
The escalation of antimicrobial resistance (AMR) is marked by the emergence and global spread of novel resistance mechanisms.With the diminishing efficacy of antibiotics, specific infections are becoming increasingly challenging, and in some cases, impossible to treat.Public health concerns are, therefore, increasing with the AMR growing challenge [17].
While antimicrobial treatment remains a primary strategy for mastitis treatment in dairy cows, excessive antibiotic use may escalate antibiotic resistance [18][19][20].Bacterial resistance not only undermines the efficacy of current therapies but also amplifies crossresistance to antimicrobials used in both veterinary and human medicine [21].Staphylococcus spp.isolated from bovine mastitis have been reported as developing resistance to multiple antimicrobial classes, including β-lactams, tetracyclines, aminoglycosides, amphenicols, macrolides, trimethoprim, lipopeptides, and lincosamides [17].For Strep.uberis, AMR are linked mainly to gentamicin and tetracycline [22].Consequently, the dynamic nature of antimicrobial resistance necessitates ongoing vigilance and monitoring.
Studies evaluating the relationship between biofilm production and antimicrobial resistance can offer deeper insights into the mechanisms that can influence the efficacy of antimicrobial therapy against Staph.aureus and Strep.uberis in dairy herds.Thus, this study aimed to evaluate (a) the biofilm production and antimicrobial resistance of Staph.aureus and Strep.uberis isolates from cows with clinical mastitis (CM) and subclinical mastitis (SCM), and (b) the potential association between biofilm production and antimicrobial resistance.

Staph. aureus and Strep. uberis Isolates
A total of 197 Staph.aureus (SCM = 111, CM = 86) and 119 Strep.uberis (SCM = 15, CM = 104) isolates were randomly selected from bovine milk samples obtained from 24 dairy farms from January 2015 to September 2016, and submitted to the Milk Quality Research Laboratory (Qualileite Lab) at the University of São Paulo, Brazil.SCM categorization included cows with somatic cell count (SCC) > 200,000 cells/mL or positive California mastitis test (CMT).CM was designated when cows exhibited visible milk changes, re-gardless of associated systemic inflammation signs [20].Composite samples were collected from cows with SCM, while samples from affected mammary quarters were taken for CM cases.CM severity data were unavailable.Milk collection was performed according to the guidelines outlined by the National Mastitis Council [20].
Staph.aureus identification relied on colony morphology, Gram-positive staining, positive catalase and tube coagulase tests, and a positive latex agglutination test.Strep.uberis identification was based on Gram-positive staining, a negative catalase reaction, Christie-Atkins-Munch-Petersen (CAMP) test negativity (or positivity) along with esculin hydrolysis, and no reaction in the bile esculin test [23].Additionally, all isolates were identified at the species level using Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) with scores > 2.0 [24].The isolates were cryopreserved at −80 • C in sterile tubes containing brain heart infusion broth (BBL-Becton Dickinson and Co., Le Point de Claix, France) supplemented with 10% glycerin until further analysis.

Biofilm Formation
Prior to assessing biofilm formation ability, preserved isolates were thawed and streaked onto blood agar plates (BBL-Becton Dickinson and Co., Le Point de Claix, France) supplemented with 5% bovine blood to confirm colony purity.Biofilm formation by Staph.aureus and Strep.uberis isolates followed Stepanovic et al. [25] methodology.Briefly, a single colony was reinoculated onto trypticase soy broth (TSB; BD, Sparks, MD, USA) and incubated at 37 • C for 24 h.Subsequently, bacterial suspensions standardized to a 0.5 McFarland standard were prepared using a DEN-1 McFarland densitometer (Bio-san, Riga, Latvia).
Next, 200 µL aliquots of each suspension were transferred in triplicate to 96-well flatbottomed sterile polystyrene microplates and incubated at 37 • C ± 1 • C for 24 h.Following incubation, microplates were agitated, fixed, stained with crystal violet for 5 min, dried, and resolubilized using 33% (v/v) glacial acetic acid.Biofilm production was quantified using a microtiter-plate reader (Exert Plus UV, Asys Hitech, Seekirchen am Wallersee, Austria) set at 540 nm for Staph.aureus and 620 nm for Strep.uberis.The average OD value of the triplicate was compared with the OD value of the negative control (ODNC) to determine the isolate's capacity to produce biofilm.The negative control was formed only sterile TSB.[25].Each batch incorporated Staph.epidermidis ATCC 12228 (non-producing biofilm) and Staph.epidermidis ATCC 35984 (positive producing biofilm) for biofilm production, ensuring quality control.
Inhibition zone diameters were measured in millimeters, with isolates categorized as susceptible, intermediate, or resistant in accordance with CLSI [26] guidelines.Intermediate isolates were classified as resistant.Antimicrobials were grouped into eight classes: betalactams, cephalosporins, aminoglycosides, macrolides, fluoroquinolones, lincosamides, tetracyclines, and penicillin/novobiocin.Isolates resistant to three or more antimicrobial classes were labeled as multidrug-resistant (MDR) bacteria [27].
The FREQ procedure of SAS 9.4 (SAS Institute Inc., Cary, NC, USA) determined isolate frequencies concerning both biofilm formation and antimicrobial susceptibility.Evaluations focused on the distributions of isolates within the pathogen (Staph.aureus or Strep.uberis) and mastitis presentation form (CM or SCM).
A logistic regression model assessed the impact of biofilm production on antimicrobial resistance in Staph.aureus and Strep.uberis isolated from CM and SCM: In the equation, logit(pi) represents the probability of an isolate displaying resistance or susceptibility to a tested antimicrobial; β 0 denotes the intercept; β 1 stands for the regression coefficient indicating the effect of biofilm production ability (yes or no); β 2 represents the regression coefficient for the effect of bacterial species (Staph.aureus or Strep.uberis); β 3 signifies the regression coefficient for the effect of the mastitis presentation form (CM or SCM); β 4 accounts for the regression coefficient representing the interaction between biofilm production ability and bacterial species; β 5 represents the regression coefficient for the interaction between biofilm production ability and mastitis presentation form.'e' denotes the random residual error.The model included 'herd' as a random effect.Analyses were conducted using PROC GLIMMIX of SAS (version 9.4; SAS Institute Inc., Cary, NC, USA).Statistical significance was declared when p < 0.05.
Table 3 presents the outcomes of the generalized mixed model, assessing the impact of biofilm formation on resistance and multidrug resistance in Staph.aureus and Strep.uberis isolates.A statistical difference was observed between multiresistant Staph.aureus and Strep.uberis isolates (p = 0.016; Table 3), demonstrating that Strep.uberis isolates exhibited a higher likelihood of multidrug resistance in comparison to Staph.aureus isolates.
An individual analysis of antimicrobials revealed significant effects of the presentation form of mastitis (CM or SCM) and resistance to ampicillin.Specifically, SCM isolates exhibited a 2.7-fold higher likelihood of resistance compared to CM isolates.An interaction effect was observed solely between biofilm production ability and the presentation form of mastitis concerning resistance to gentamicin.However, no significant difference was observed between mastitis presentation form and biofilm production ability in the other antimicrobials.  Bac*Bio: interaction between type of causative pathogen and biofilm, 5 Mastitis*Bio: interaction between mastitis presentation form (CM or SCM) and biofilm.
The frequencies of Staph.aureus and Strep.uberis isolates were categorized into three antimicrobial resistance profiles: singular class, two classes, and three or more classes, and were distributed based on their biofilm formation ability (Table 4).Among multidrugresistant Staph.aureus isolates (exhibiting resistance to three or more classes of antimicrobials), 83.3% (5/6) were categorized as strong biofilm producers, while 16.7% (1/6) belonged to the NP category (Table 4).Notably, the beta-lactam class was prevalent among all multidrug-resistant Staph.aureus isolates.

Discussion
Staph.aureus and Strep.uberis are major causes of intramammary infections among dairy cows, with their capacity for biofilm formation recognized as a crucial virulence trait influencing mastitis pathogenesis and antimicrobial treatments.Our study assessed biofilm production and antimicrobial resistance in these isolates from mastitis-affected cows, which can help to understand the relationship between biofilm formation and antimicrobial resistance.
Our findings revealed that 57.36% of Staph.aureus isolates demonstrated biofilm production, consistent with previous reports (62.5%)[28].However, notably high levels (54.8%) of Staph.aureus classified as NP were detected in Brazilian dairy herds [29].This variance in biofilm-forming potential among Staph.aureus isolates in bovine mastitis highlight the potential chronicity of infections induced by this pathogen.Nonetheless, there remains a need to further investigate factors such as environmental stress, strain diversity, and location, which may influence gene expression related to biofilm production [30].
Discrepancies observed in biofilm evaluation might be attributed to variations in assay methodologies.While the microplate-based biofilm evaluation is considered the gold standard, its reliance on conditions influencing microbial growth and biofilm formation [31,32].
Biofilm formation often confers enhanced antimicrobial resistance [31,33].Contrary to expectations, our study did not find a significant difference in antimicrobial susceptibility between biofilm and non-biofilm-producing Staph.aureus isolates.However, Rychshanova et al. [31] demonstrated that 69.4% of biofilm-producing Staph.aureus exhibited resistance to at least one antimicrobial class evaluated.The biofilm acts as a physical barrier, impeding direct contact between antimicrobials and microorganisms, a condition not mimicked during our in vitro testing.The lack of association between biofilm production and observed antimicrobial resistance might be explained by the absence of environmental conditions during our in vitro antimicrobial susceptibility tests.Additionally, the use of antimicrobials during the biofilm production evaluation can possibly improve the biofilm production capacity.
For Strep.uberis, 53.78% of the isolates demonstrated biofilm formation capacity.However, Dieser et al. [33] observed a higher percentage of isolates (87.5%) classified as weak or non-biofilm producers.Magagula [34] described that all the Strep.uberis isolates evaluated presented biofilm formation capacity, but only 17.8% (n = 30) were classified as strong biofilm producers.As previously mentioned, these variations in biofilm results can be attributed to variations in the strains evaluated, as well as in the expression of genes related to biofilm formation.Moliva et al. [35] demonstrated a correlation between the presence of virulence genes associated with the adhesion process (gapC, hasABC, lbp, pauA, and sua) and distinct biofilm formation patterns observed in Strep.uberis.Greeshma et al. [36] showed that isolates without luxS gene cannot produce a robust biofilm, leading to the inference that additional genes may be involved in regulating biofilm production.Alternatively, the luxS gene might play a regulatory role in one or more genes associated with biofilm formation in Strep.uberis.
The diminished susceptibility to beta-lactams might relate to the presence of resistance genes (e.g., mecA, mecC, and blaz) [40].Penicillin-binding proteins (PBPs) are cell wall transpeptidases that catalyze the assembly of cell wall peptidoglycan.Modification on the pbps can improve the antimicrobial resistance to β-lactams due to the membrane proteins' lower affinities to oxacillin and penicillin.Neelam et al. described that gene mecA, which is responsible for methicillin resistance, was detected in 23.64% (n = 13) of Staph.aureus isolates [40].Aslantas et al. [28] demonstrated that penicillin and oxacillin-resistant Staph.aureus harbored blaz and mecA genes, respectively.For Strep.uberis, modifications in the pbp2x regions are associated with β-lactam resistance [41].The presence of the E381K, Q554E, and G600E substitutions on pbpx was numerically associated with lower bacteriological cure rates following treatment with a β-lactam compared with a non-β-lactam intramammary therapy [41].Molecular characterization analysis could further confirm these findings.However, this was beyond the scope of our study.
In our investigation, Strep.uberis exhibited substantial resistance to oxacillin (80.6%), penicillin (80.6%), tetracycline (37.8%), and pirlimycin (14.2%).This is consistent with varying reports in different regions, which showed high resistance rates to oxacillin in Switzerland (64.7%) and Korea (33.3%) [42].The resistance rates to tetracycline differed notably, ranging from 27.1% to our finding of 37.8% [43].As a result of a meta-analysis, it was determined that the highest levels of resistance for Strep.uberis were observed in gentamicin and tetracycline worldwide [22].Regional or herd disparities, compounded by selective pressure from antimicrobial use, might account for such variations [44,45].Extensive use of specific antimicrobial classes, like aminoglycosides, tetracyclines, and fluoroquinolones in Brazilian herds [46], can influence antimicrobial resistance patterns.For instance, the resistance of Strep.uberis to tetracyclines was linked to their prevalent use in Brazilian dairy herds [47].
Multidrug resistance, characterized by resistance to three or more antimicrobial classes [27], was less common in Staph.aureus (1.52%) but more prevalent in Strep.uberis (15.9%).The number of Strep.uberis that showed multidrug resistance were higher than reported to Magagula et al. (6.4%) [34].These authors concluded that the low overall resistance must be linked to regional differences and prudent use of antimicrobials in the dairy industry [34].For Staph.aureus, resistance to beta-lactams, particularly penicillin and cephalosporins, was consistent among all multidrug-resistant isolates.In contrast, among multidrug-resistant Strep.uberis, the most frequent resistance was observed against beta-lactams and tetracyclines.
Institutional Review Board Statement: All experimentation procedures using animals were carried out under the approval of the Ethical Committee on the Use of Animals of the School of Veterinary Medicine and Animal Science, University of São Paulo (Protocol number: 5301031014).
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Table 2 .
Frequency of in vitro susceptibility to antimicrobials of Staph.aureus and Strep.uberis isolated from bovine mastitis.

Table 3 .
Effect of biofilm formation capacity, type of mastitis (CM or SCM), and causative pathogen (Staph.aureus, Strep.uberis) on resistance and multiresistance to antimicrobials.

Table 4 .
Frequency of Staph.aureus and Strep.uberis isolates, distributed according to the number of different antimicrobial classes with resistance and biofilm formation capacity.