Looking for alternative treatments for bovine and caprine mastitis: Evaluation of the potential of Calliandra surinamensis leaf pinnulae lectin (CasuL), both alone and in combination with antibiotics

Abstract This work aimed to evaluate the effects of CasuL on growth and viability of 15 mastitis isolates from cows and goats, to determine the synergistic potential between CasuL and antibiotics, and to investigate the effects on bacterial ultrastructure and antibiofilm activity. The lectin inhibited the growth of Staphylococcus isolates from either bovine (Ssp6PD and Sa) or caprine (Ssp5D and Ssp01) mastitis. The minimal inhibitory concentrations were ranged from 3.75 to 15 µg/ml. Synergistic effect was observed for CasuL‐tetracycline against Sa and Ssp6PD and CasuL‐ampicillin against Ssp01. No structural damage was observed under the scanning electron microscope in CasuL treatments. Flow cytometry analysis using thiazol orange and propidium iodide demonstrated that CasuL was unable to reduce the cell viability of the isolates tested. At sub‐inhibitory concentrations, CasuL reduced biofilm formation by the isolates Sa and Ssp5D. However, CasuL‐tetracycline and CasuL‐ampicillin combinations inhibited biofilm formation by Ssp6PD and Ssp01, respectively. In conclusion, CasuL is a bacteriostatic and antibiofilm agent against some mastitis isolates and displayed a synergistic potential when used in combination with either ampicillin (against one isolate) or tetracycline (against two isolates). The results stimulate the evaluation of CasuL for the treatment of mastitis, particularly when used in conjunction with antibiotics.


| INTRODUC TI ON
Mastitis is an infection that is caused by certain microorganisms that are present in the mammary glands. It leads to functional impairment resulting from the destruction of milk-producing tissues (Mushtaq et al., 2018;Schroeder, 1997). Reduced milk production caused by cases of mastitis in bovines and caprines has an enormous economic impact on the dairy industry (Guimarães et al., 2017). The presence of enterotoxins in milk and the spread of antibiotic-resistant microorganisms are further problems associated with cases of mastitis (Scali, Camussone, Calvinho, Cipolla, & Zecconi, 2015).
Although fungi, viruses, and algae can cause mastitis, bacteria are responsible for the highest infection rates in the mammary glands of cows and goats (Costa, 1991;Spanamberg, Sanches, Santurio, & Ferreiro, 2009). Staphylococcus aureus is one of the main causes of clinical and subclinical mastitis (Klein et al., 2015;Peixoto, França, Souza Júnior, Veschi, & Costa, 2010;Zadoks & Fitzpatrick, 2009). However, other bacteria such as streptococci, Escherichia coli, and Klebsiella pneumoniae are also responsible for this infection (Contreras & Rodríguez, 2011). Mastitis treatment and prevention consists mainly of the use of antibiotics and proper animal handling to prevent the spread of the disease to healthy animals. However, the use of antibiotics requires caution in order to avoid the emergence of resistant bacteria (Costa et al., 2013;Moritz & Moritz, 2016). Krewer et al. (2013) found simultaneous resistance to three or more antibiotics in 65.6% of Staphylococcus isolates that cause mastitis.
Biofilms are complex and structured communities of microorganisms enclosed in a self-produced polymeric matrix that contains exopolysaccharides, proteins, teichoic acids, enzymes, and extracellular DNA (Klein et al., 2015). Biofilms give these microorganisms protection against environmental adversities and a higher tolerance (10-1,000 times) to antibiotics as compared to planktonic forms (Cerca et al., 2005;Kumar, Alam, Rani, Ehtesham, & Hasnain, 2017). It is believed that biofilm development may contribute to the low efficacy of certain therapies used in bovine mastitis treatment, as well as to the difficulties of treating recurrent infections (Martins et al., 2016;Melchior, Vaarkamp, & Fink-Gremmels, 2006).
Lectins are proteins of non-immunological origin that bind specifically and reversibly to free or conjugated carbohydrates. These proteins have significant antibacterial potential which is attributed to their ability to bind molecules present in the surface of gram-positive and gram-negative cells, leading to damage to the cell wall, loss of metabolic stability, inhibition of cell growth, and reduction in cell viability . Lectins can also interfere with adhesion and invasion of host cells by bacteria (Silva et al., 2016). Finally, lectins have been reported to be able to both prevent biofilm formation and eradicate already established biofilms (Moura et al., 2015;Moura, Trentin, et al., 2017).
In view of a previous report on the antibacterial effects of CasuL and the problems associated with bovine and caprine mastitis, this work aimed to evaluate the bacteriostatic and bactericide effects of CasuL on fifteen mastitis isolates, to determine the synergistic potential between CasuL and commercially available antibiotics, and to investigate the effects of CasuL alone or combined with antibiotics on bacterial ultrastructure and antibiofilm activity.

| Lectin purification
Calliandra surinamensis leaves were collected at Recife (Pernambuco, Brazil) and dried for 2 weeks at 28ºC. Plant collection was performed under authorization (36,301) of the Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio). The access was recorded (A2E872B) in the Sistema Nacional de Gestão do Patrimônio Genético e do Conhecimento Tradicional Associado (SisGen). The pinnulae were then detached and ground using a blender. CasuL was then purified from the pinnulae powder according to the protocol described by Procópio, Patriota, et al. (2017). Briefly, an extract was prepared by suspending 10 g of the powder in 100 ml of 0.15 mol/L NaCl with magnetic stirring for 16 hr, followed by filtration and centrifugation (12,000 g, 15 min, 4ºC). The extract was then treated with ammonium sulfate at 60% saturation (Green & Hughes, 1955) and the precipitated fraction obtained was dialyzed against distilled water (4 hr) and 0.15 mol/L NaCl (4 hr), and then loaded onto a Sephadex G-75 column (30.0 × 1.0 cm) equilibrated with 0.15 mol/L NaCl. Elution was monitored by absorbance at 280 nm and CasuL was recovered in fractions 9-15. Isolated CasuL was exhaustively dialyzed (6 hr, two liquid changes) against distilled water before use in the antibacterial assays.

| Hemagglutinating activity
The hemagglutinating activity (HA) assay was used to determine the carbohydrate-binding ability of CasuL. A 2.5% (v/v) suspension of glutaraldehyde-treated rabbit erythrocytes in 0.15 mol/L NaCl was used. The Ethics Committee on Animal Use of the Universidade Federal de Pernambuco approved the method that was used to collect erythrocytes (process 23076.033782/2015-70). The HA was determined as described by Procópio, Patriota, et al. (2017) and the number of HA units was determined as the reciprocal of the highest dilution of the lectin that was able to agglutinate erythrocytes.
Specific HA was calculated by determining the ratio of HA to protein concentration (mg/ml). An HA inhibitory assay was performed by incubating CasuL for 15 min with fetuin prior to the addition of erythrocyte suspension.

| Bacterial isolates
Fifteen mastitis bacterial strains isolated from goats and cows (Table 1) were obtained from the collection maintained by the Hinton Agar (MHA) overnight at 37°C and the culture density was adjusted turbidimetrically at 600 nm (OD 600 ) to 1 × 10 8 colony forming units (CFU) per ml in sterile 0.15 mol/L NaCl. This suspension was subsequently diluted in saline solution to 1 × 10 6 CFU/ml to yield approximately 1 × 10 5 CFU/ml as the final concentration used in the antibacterial assay described below.

| Determination of minimal inhibitory and bactericidal concentrations
The broth microdilution assay was used to determine minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) values. First, a twofold serial dilution of either CasuL (37.50 µg/ml) or antibiotic (8.00 µg/ml) in 80 μl of distilled water was performed in a row of a 96-well microplate. Distilled water (80 μl) was used in the 100% growth control. Next, 40 μl of MHB and 80 µl of bacterial inoculum (in saline) was added. The final concentrations of CasuL in the wells ranged from 0.03 to 15.00 µg/ml. A sterility control contained only medium was also made. The OD 600 was measured at time zero and following incubation at 37ºC for 24 hr.
The MIC was determined as the lowest sample concentration that was able to promote a reduction of OD 600 by 50% or higher in comparison with the 100% growth control (Amsterdam, 1996). Each assay was performed in duplicate and three independent experiments were performed.
To determine the MBC, the supernatants from each well containing CasuL at concentration ≥ MIC were smeared onto MHA medium and the plates were then incubated for 24 hr at 37ºC. The MBC corresponded to the lowest sample concentration that was able to reduce the number of CFU in 99.9% in comparison with the initial inoculum.

| Synergism assay
Possible synergistic effects between CasuL and antibiotics (ampicillin or tetracycline) were evaluated using the method described by Pillai, Moellering, and Eliopoulos (2005). Lectin-susceptible isolates (Sa, Ssp6PD, Ssp5D, and Ssp01) were tested in the assays. Each experiment corresponded to two rows of a 96-well microplate. CasuL was added (80 µl) to the fourth well of the first row and a serial twofold dilution in sterile Milli-Q water was performed until the penultimate well of the second row. Next, the antibiotic was added (80 µl) to the penultimate well of the second row and a two-fold serial dilu-

| Growth curves
Six-hour growth curves were determined for CasuL-sensitive isolates using either the lectin alone, or synergic combinations of CasuL with antibiotics. This assay was performed in 96-well microtiter plates according to Gaidamashvili and Van Staden (2002). Eighty microlitre of the inoculums (10 6 CFU/ml) in the exponential growth phase were incubated with 40 µl of MHB and 80 µl of the lectin, ampicillin, or tetracycline (at MIC), or with a synergic combination. In the 100% growth control, sterile distilled water (negative control) was used instead of CasuL. The plates were incubated at 37ºC and the OD 600 was measured every hour.

| Cell viability analysis
The viability of bacterial cells treated with CasuL was evaluated using the Cell Viability Kit of BD Biosciences (San Jose, CA). The isolates were incubated with the lectin at the MIC as described above.
The negative control was prepared by adding distilled water instead

| Statistical analysis
The data were expressed as the mean or the percent mean ± SD and statistical differences were determined using Tukey's test. A p < 0.05 was considered to be statistically significant.

| RE SULTS
CasuL was isolated according to the protocol previously established by Procópio, Patriota, et al. (2017). The isolated lectin showed a specific HA of 1,420.0 and was inhibited by fetuin, as in the previous report, confirming that the carbohydrate-binding activity of the sample was effective. CasuL was able to inhibit the growth of four isolates. The MIC values are presented in Table 1 Table 1. Eight isolates were found to lack sensitivity to at least one of the antibiotics.
The potential synergy between CasuL and antibiotics was evaluated, and the results are shown in Tables 2 and 3. A synergistic effect was observed for the combination CasuL-ampicillin against the isolate Ssp01 (Table 2) and for the combination CasuL-tetracycline against the isolates Sa and Ssp6PD (Table 3). An additive effect was observed against the isolate Ssp5D, while antagonism was observed for the combinations CasuL-ampicillin and CasuL-tetracycline, against the strains Sa and Ssp01, respectively.
Six-hour growth curves were determined for the lectin-sensitive isolates in the absence and presence of either CasuL or antibiotics ( Figure 1). When incubated with the lectin, all of the isolates grew similar to the negative control (100% growth), indicating that the bacteriostatic effect only appears later on (after the 6-hr period evaluated). The presence of tetracycline and ampicillin led to a reduction in the growth of the Sa isolate at 3 hr of incubation ( Figure 1a), while the isolate Ssp6PD had its growth reduced after 5 hr of incubation with both antibiotics (Figure 1b). For the isolate Ssp01, neither antibiotic showed any inhibitory effect in the first 6 hr of incubation (Figure 1c), similar to CasuL. Finally, for the isolate Ssp5D, tetracycline was shown to be able to inhibit growth from the fourth hour of incubation on ward (Figure 1d). This isolate was not sensitive to ampicillin. Neither of the commercial antibiotics was found to act as antibiofilm compounds against Ssp6PD, also, stimulating biofilm formation (Figure 5f,g). Interestingly, the combination, CasuL-tetracycline, inhibited biofilm formation by almost 60.0% (Figure 5h).
Antibiofilm activity was not observed following treatment of the isolate Ssp01 with CasuL ( Figure 5i). While tetracycline was found to stimulate biofilm development (Figure 5j), ampicillin had no effect  The values correspond to the concentration of CasuL or ampicillin in the microplate well containing both compounds.

| D ISCUSS I ON
Mastitis is the most frequent type of inflammation that occurs in milk-producing animals and the disease that has the greatest impact on dairy farming (Vliegher, Fox, Piepers, McDougall, & Barkema, 2012). S. aureus is the main species of bacteria that causes mastitis and its pathogenesis is attributed to a combination of extracellular Combining antimicrobial phytochemicals with commercial drugs expands the field for the application of these natural compounds and can minimize the impact of pathogen resistance (Lewis & Ausubel, 2006;Mushtaq et al., 2018). Interestingly, synergy between CasuL and tetracycline or ampicillin was observed against some isolates.
The growth of isolate Sa was reduced by treatment with the CasuLtetracycline synergistic combinations after 4 hr. This result demonstrates that the combination was able to affect the bacterial cells within a short incubation period, similar to that was observed for the antibiotic alone, though at a concentration four times greater.
The antibacterial activity of lectins has previously been associated with their ability to bind to peptidoglycans, lipopolysaccharides, F I G U R E 1 Growth curves of the mastitis isolates Sa (a), Ssp6PD (b), Ssp01 (c), and Ssp5D (d) in absence or presence of CasuL, ampicillin, or tetracycline at their respective minimal inhibitory concentrations (MIC). The optical density (OD) at 600 nm was determined every hour for a period of 6 hr. For the negative control, cells were treated with distilled water instead of antibacterial agent. Data are expressed as the mean ± SD. All the MIC values can be seen in Table 1 F I G U R E 2 Growth curves of the mastitis isolates Sa (a), Ssp01 (b), and Ssp6PD (c) in absence or presence of CasuL-tetracyline (a,c) or CasuL-ampicillin (b) synergic combinations. The optical density (OD) at 600 nm was determined every hour for a period of 6 hr. For the negative control, cells were treated with distilled water instead of antibacterial agent. Data are expressed as the mean ± SD. The concentration of CasuL and antibiotics in the synergic combinations can be seen in Table 2 and other molecules present in the cell wall, and by interfering with cell growth and viability and promoting structural damage (Iordache, Ionita, Mitrea, Fafaneata, & Pop, 2015;. Biofilm-forming bacteria are usually highly tolerant to conventional antibiotics and are often resistant to the host immune response (Lebeaux, Ghigo, & Beloin, 2014). Recurrent mastitis infections are often attributed to biofilm growth (Melchior et al., 2006). Therefore, we evaluated the antibiofilm activity of CasuL alone at inhibitory and sub-inhibitory concentrations as well as the antibiofilm activity of CasuL-antibiotic combinations. CasuL was found to be less effective than a C-type lectin from Bothrops jararacussu (3.12-100.0 µg/ml), which inhibited biofilm formation by a S. aureus isolate from bovine mastitis by over 50.0% (Klein et al., 2015).
When used alone, CasuL and commercial antibiotics led to an increase in biofilm development by isolates Ssp6PD and Ssp01. It is believed that biofilm formation can be used as a defensive strategy by bacteria to escape the effects of antimicrobial agents (Moura, Napoleão, Paiva, & Coelho, 2017). On the other hand, the combinations CasuL-tetracycline and CasuL-ampicillin showed antibiofilm effect. These results are interesting as treatment with these combinations not only reduced the amount of CasuL and antibiotic required F I G U R E 3 Scanning electron microscopy of bacterial cells of the isolates Sa, Ssp6PD, Ssp01, and Ssp5D following exposure to either CasuL at the minimal inhibitory concentration (MIC),or to CasuL-antibiotic synergic combination (except for Ssp5D isolate). For the negative control, cells were treated with distilled water instead of antibacterial agent. The synergistic combinations used were as follows: CasuLtetracycline for isolates Sa and Ssp6PD, and CasuL-ampicillin for the isolate, Ssp01. Reduction in cell number and cells under incomplete division can be seen in CasuL treatments, but no bacterial surface alteration was observed following treatments with either lectin or with synergic combinations. The MIC values of CasuL can be seen in Table 1. The concentrations of CasuL and antibiotics in the synergic combinations can be seen in Table 2 to inhibit bacterial growth, but also neutralized the biofilm stimulatory effect of both the antimicrobial agents. A synergistic effect in antibiofilm activity was also detected for the antimicrobial peptide coprisin when it was applied in combination with the antibiotics ampicillin, van-  Table 1 with the absence of bactericidal action and defines it as a bacteriostatic drug. At sub-inhibitory concentrations, CasuL acted as an antibiofilm agent against S. aureus and one Staphylococcus sp. isolate. It is important to highlight that two lectin-sensitive isolates displayed an ability to respond to the presence of an antibacterial compound by forming biofilm. However, the CasuL-antibiotic combinations were able to prevent this response. The results of our work suggest that it would be worthwhile to carry to further studies to evaluate the in vivo effects of CasuL for the treatment of some cases of mastitis, since this lectin does not show a broad spectrum of action. Before this, studies on the toxicity of CasuL to animals should be performed.

ACK N OWLED G M ENTS
The authors express their gratitude to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; 446902/2014-4) for research grants and fellowships (LCBBC, RAM, ALFP, PMGP, and THN). We would also like to thank the Coordenação de Aperfeiçoamento F I G U R E 5 Evaluation of the antibiofilm effect against the isolates Sa (a-d), Ssp6PD (e-h), Ssp01 (i-l), and Ssp5D (m,n) of CasuL(a,e,i,m), tetracycline (b,f,j,n) or ampicillin (c,g,k), all at sub-inhibitory concentrations, as well as of CasuL-tetracycline (d,h), and CasuL-ampicillin (l) combinations. Different letters indicate significant differences (p < 0.05) between the treatments and the negative control. The minimal inhibitory concentrations (MIC) of CasuL and antibiotics are given in Table 1. The concentrations of CasuL and antibiotics in the combinations are given in Table 2 de

CO N FLI C T O F I NTE R E S T S
The authors declare no conflict of interest.

E TH I C S S TATEM ENT
None required.

DATA ACCE SS I B I LIT Y
All data generated or analyzed during this study are included in this published article. Raw data are available from the corresponding author on reasonable request.