The effect of the nisin-based pharmaceutical formulation used in the treatment plan for cows with subclinical mastitis on the milk microbiota

INTRODUCTION

Mammary gland inflammation in cows is most often caused by a bacterial infection [1-3]. The quantity of bacteria detected depends on the form of mastitis and its severity, as well as the pathogen type [4-7]. The most often detected microorganisms in milk of cows suffering from mastitis are Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Escherichia coli, Enterococcus faecium, Enterococcus faecalis [1-4]. The subclinical form of mastitis, which is more difficult to diagnose due to the lack of visible changes in the mammary gland and milk is the major danger for dairy farming, though it gives higher somatic cell counts and total bacterial counts in milk. Microbial metabolites and toxins accumulate in the milk from subclinically mastitic cows affecting milk taste, nutritional value of raw milk and dairy products and decreasing their shelf life [8-11]. The prevalence of subclinical mastitis in high yielding cows in developing countries is high [12-16]. That is why to reduce milk rejection and prevent antimicrobial resistance (AMR), the use of antimicrobials is minimized in treatment plans, and vaccines, bacteriophages, phage lysines, bacteriocins are used as an alternative [17-24]. Pursuant to the “Strategy to Prevent the Spread of Antimicrobal Resistance in the Russian Federation to 2030”, approved by the Russian Federation Government Decree on 25 September 2017 No. 2045-r, we tested the drugs based on antimicrobial peptides of microbial origin used to treat infectious mammary gland diseases of high yielding cows.

The work is relevant because the formulation is based on bacteriocin nisin to be used in the treatment regimen for cows with subclinical mastitis as an alternative to known antimicrobial drugs.

The novelty of the work is that for the first time, data on the effect of a new nisin-based formulation used in the treatment regimen of cows with subclinical mastitis on the milk microbiota were obtained.

Practical significance: in order to prevent AMR, the use of the nisin-based formulation makes it possible to reduce the use of antimicrobial drugs for mastitis treatment.

The purpose of this study was to evaluate the effect of the nisin-based pharmaceutical formulation used in the treatment plan for cows suffering from subclinical mastitis on the milk microbiota. For this purpose the following tasks were set: to study the microbiota composition of milk from high yielding cows with subclinical mastitis; to analyze the comparative AMR profiles of microorganisms isolated from milk of subclinically mastitic cows; to study the effect of the nisin-based formulation used in the treatment regimen of cows with subclinical mastitis on the milk microbiota composition.

MATERIALS AND METHODS

Objects of the study: high yielding cows with subclinical mastitis, microorganisms isolated from milk, a nisin-based formulation.

The effect of the nisin-based formulation, used in the treatment regimen of cows with subclinical mastitis, on the milk microbiota composition was studied in 35 high yielding cows with a milk yield of more than 8,000 kg per year, kept at the nucleus farm in the Polevsky Raion of the Sverdlovsk Oblast. According to the treatment regimen of subclinical mastitis, the animals received 10 mL of a new pharmaceutical formulation intra-cisternally into the affected quarter daily for five days.

All experiments were carried out in strict accordance with the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (ETS No. 123).

During the study in 2023, the following isolates were recovered: Staphylococcus aureus (n = 32), Streptococcus dysgalactiae (n = 23), Staphylococcus haemolyticus (n = 20), Enterococcus faecium (n = 19), Aerococcus viridans (n = 17), Staphylococcus epidermidis (n = 13), coliform bacteria of Escherichia and Enterobacter genera (n = 8), Corynebacterium pseudotuberculosis (n = 5), Mucor (n = 4) and Penicillium spp. (n = 3) fungi.

A previously developed formulation containing nisin and water-based excipients was used with the following weight ratio percentage: nisin A – 0.3; silicon glycerolates in a 6-mole excess of glycerol Si(C₃H₇O₃)₄ × 6C₃H₈O₃ – 3.0; boron bisglycerolates H[B(C₃H₆O₃)₂] – 2.0; glycerol – 10.0; distilled water – up to 100 [25][26].

Test methods. The morphology of the recovered isolates was studied by seeding on Hiss growth media with sugars (“motley row”), was referenced by Bergey’s Manual of Determinative Bacteriology [27], and the Guide to Clinically Significant Fungi [28], and then studied by MALDI-TOF mass spectrometry (Matrix-assisted laser desorption ionization time-of-flight mass spectrometry) using VITEK® MS (bioMérieux, France). For bacteriological and mycological testing, the milk samples were seeded on liquid and solid nutrient agars: meat peptone, Streptococcus Selective Agar, Enterococcus Selective Agar, Endo agar, Staphylococcus Selective Agar No. 10, Czapek medium, Sabouraud Dextrose Liquid Medium, bismuth sulfite agar, cetrimide agar, Levin medium, Ploskirev medium (State Research Center for Applied Microbiology and Biotechnology, Russia), 5% sheep blood agar (based on Columbia agar; Bio-Rad, France), defibrinated sheep blood (E&O Laboratories Ltd, Scotland), Salt Egg Yolk Agar Base (nutrient agar for microorganism culture GRM-agar, State Research Center for Applied Microbiology and Biotechnology, Russia), UriSelect4 non-selective chromogenic agar (Bio-Rad Laboratories, Inc., USA) and Sabouraud 2% Glucose Chloramphenicol Agar (SIFIN diagnostics GmbH, Germany).

The resistance of recovered isolates to 34 antimicrobial drugs from 15 groups (tetracyclines, penicillins, carbapenems, macrolides, lincosamides, ansamycins, amphenicols, I, II, III generation aminoglycosides, I, II, III generation cephalosporins, II, III generation fluoroquinolones) was evaluated by the disc diffusion test [29]. Commercially available discs were used (Scientific Research Center of Pharmacotherapy, Russia). The results were interpreted taking into account the recommendations of the European Committee on Antimicrobial Susceptibility Testing (EUCAST).

Statistical data were processed using AMRcloud, Microsoft Excel 2007 and Statistica 6.0. softwares.

RESULTS AND DISCUSSION

The studies were performed in 2023–2024 in the Department of Reproductive Biology and Neonatology, the Laboratory of Microbiological and Molecular Genetic Research of the Ural Federal Agrarian Research Center, Ural Branch of the Russian Academy of Sciences, in the laboratory of the medical center “Quality Med” LLC (Ekaterinburg) funded by the grant of the Russian Science Foundation No. 22-76-00009.

144 microorganisms were isolated from 70 milk samples from cows with subclinical mastitis, among them (Fig. 1): S. aureus (22.2%), S. dysgalactiae (16.0%), S. haemolyticus (13.9%), E. faecium (13.2%), A. viridians (11.8%), S. epidermidis (9.0%), coliforms of Escherichia and Enterobacter species (5.6%), C. pseudotuberculosis (3.5%), as well as Mucor (2.8%) and Penicillium spp. (2.1%) fungi.

In the present study, 81.4% of cows with subclinical mastitis were co-infected, among them 21.4% were co-infected with two pathogens, 28.6 and 17.1% of cows were infected with three and four pathogens. A complex microbiome consisting of five microorganisms was isolated in 14.3% of the samples.

The proportion of S. aureus isolates resistant to I, II and III generation cephalosporins was 53.1, 46.8 and 37.5%, respectively; to macrolides (erythromycin, clarithromycin) – 34.4%. Resistance to tetracyclines and penicillins was found in 31.3 and 28.1% of isolates, respectively. The minimum percentage of resistant S. aureus strains was reported for the following groups of antibiotics: II generation fluoroquinolones (12.5%), III generation fluoroquinolones (9.4%), carbapenems (6.3%). Intermediate resistance was established in 25.0% of isolates to amikacin (III generation aminoglycoside antimicrobial drug).

52.6% of S. dysgalactiae isolated strains showed resistance to tetracyclines (tigecycline, doxycycline). Non-sensitivity to III generation cephalosporins was established in 42.1% of isolates. Comparatively lower resistance was reported against II generation aminoglycosides (31.6%). 57.9% of S. dysgalactiae isolates demonstrated intermediate resistance to II generation cephalosporins (cefuroxim, cefoxitin).

Monitoring of antimicrobial resistance of S. haemolyticus isolates recovered from milk showed their resistance to macrolides (erythromycin, clarithromycin) in 33.3% of cases. A few isolates showed resistance to III generation cephalosporins (cefixime, cefoperazone, ceftriaxone) and ansamycins (rifampicin) – 13.3 and 6.7%, respectively. Intermediate sensitivity was revealed in 6.7% of the isolates to tetracyclines (doxycycline).

Resistance to penicillins, I generation aminoglycosides and III generation cephalosporins (42.1, 36.8 and 26.3%, respectively) was demonstrated by E. faecium isolates. Intermediate sensitivity was found in 21.1% of the isolates to doxycycline from the tetracycline group.

The AMR profile of A. viridans isolates recovered from milk demonstrated the highest resistance to penicillins (ampicillin, amoxicillin, penicillin) – 35.3% and I generation aminoglycosides (kanamycin) – 23.5%. Intermediate susceptibility was revealed in 29.4% of the studied isolates to the tetracyclines (tetracycline, doxycycline).

38.5% of S. epidermidis isolates showed resistance to tetracyclines (tetracycline, doxycycline). Minimal resistance (15.4%) was reported against II generation aminoglycosides (gentamicin). 23.1% of the isolates demonstrated intermediate resistance to III generation fluoroquinolones (levofloxacin).

Isolated coliform bacteria had the greatest resistance (62.5%) to penicillins. Resistance to the ansamycins (rifampicin) was found in 37.5% of coliform isolates. Intermediate resistance to penicillins and II generation cephalosporins (cefuroxim, cefoxitin) was reported in 25.0 and 12.5% of coliform bacteria, respectively.

Corynebacterium pseudotuberculosis isolates were found to be equally resistant to penicillin and tetracycline antimicrobials (20.0%). Intermediate sensitivity was observed in 40.0% of the isolates to II generation cephalosporins (cefuroxim).

The obtained AMR profile of the milk microbiota from subclinically mastitic cows revealed the presence of multidrug-resistant coliform bacteria, S. dysgalactiae, A. viridans, S. aureus isolates. 62.5 and 47.1% of the tested coliform and A. viridans isolates respectively, had resistance to two groups of antimicrobials. Resistance of S. dysgalactiae to three antimicrobial groups was found in 43.5% of the isolated strains, to four groups in 26.1% isolates. 62.5% of the tested S. aureus isolates were resistant to four groups of antimicrobials, 46.9% of them were resistant to five groups and resistance to more than six groups of antimicrobials was reported in 15.6% of isolates.

The study showed that milk from subclinically mastitic cows has a complex microbiome, and the isolated microbiota are highly resistant to major antimicrobials used to treat mastitis. In this connection, a new pharmaceutical formulation based on bacteriocin nisin was included in the subclinical mastitis treatment regimen.

In milk samples from cows with subclinical mastitis, before using the nisin-based formulation, the isolated microorganisms were found both as a monoculture (48.6%) and as bacteria and fungi associations (51.4%). S. aureus (22.9%), S. dysgalactiae (11.4%), A. viridans (5.7%), S. epidermidis (2.9%), C. pseudotuberculosis (2.9%), S. haemolyticus (2.9%) were recovered as monocultures.

In the structure of bacterial associations, 11.4% of the samples were represented by S. aureus + coliforms + E. faecium; three-component associations included S. aureus + coliforms + Mucor (5.7%), S. aureus + Streptococcus spp. + Mucor (2.9%). Moreover, four-component associations were most often isolated from milk samples: S. aureus + coliforms + Streptococcus spp. + Penicillium spp. (8.6%), S. aureus + coliforms + Streptococcus spp. + E. faecium (8.6%), S. aureus + coliforms + Streptococcus spp. + Mucor (5.7%), S. aureus + coliforms + E. faecium + Streptococcus spp. (2.9%). The composition of the five-component associations was represented by S. aureus + coliforms + Streptococcus spp. + E. faecalis + Mucor (5.7%). The results are given in Table 1.

Eighty microorganisms in total were isolated from 35 milk samples at the beginning of the experiment, among them 74 bacteria and 6 fungi species (Fig. 2).

Herewith, the number of microbial cells in each sample was different. At the beginning of the experiment, 28.4% of S. aureus isolates were etiologically significant for the inflammation development in the mammary gland; the number of colony-forming units per 1 mL of the tested sample equal to 10³, 10⁶ and 10⁷ CFU/mL was found in 9.5; 8.1 and 10.8% of the isolates, respectively. All 16 isolated Streptococcus spp. cultures (21.6%) were detected in the amount of 10³ CFU/mL; 13.5% of coliform isolates, which can cause mastitis in animals, were in the amount of 10⁵ CFU/mL. 10³ and 10⁵ CFU/mL values were found in 2.7 and 8.1% of E. faecium isolates, respectively. S. epidermidis, C. pseudotuberculosis, and S. haemolyticus were detected in the amount of 10³ CFU/mL in 1.4% of cases. A. viridans were detected in a titer of 10² CFU/mL and from the beginning of the experiment were not an etiologically significant microorganism for mastitis development in cows (Table 2).

After the treatment course of animals with subclinical mastitis using the new nisin-based formulation, no microflora growth was observed in 68.6% of the samples (Table 1). The microbiota isolated from 11 samples of milk was a monoculture in 20.0% of cases represented by S. aureus (11.4%), S. dysgalactiae (5.7%), A. viridans (2.9%). In other samples, microorganism associations were detected: S. aureus + coliforms + E. faecium (5.7%); S. aureus + coliforms (2.9%); E. faecalis + coliforms (2.9%). 10³ and 10⁶ CFU/mL of S. aureus microbial cells were revealed in an equal number of isolates (1.4%). In 6.8% of cases, S. aureus isolated in a diagnostic titer, were not etiologically significant (10² CFU/mL), as well as coliforms, E. faecium, A. viridans, detected at the level of 10¹–10² CFU/mL. In one sample, E. faecium was detected in the amount of 10³ CFU/mL, which accounted for 1.4% in the total structure of isolated microorganisms.

A microbiological testing of milk performed on Day 14 from the beginning of the treatment course showed an increase in the number of microbiota-free samples to 88.6%. In the tested samples, the milk microbiota was represented as a monoculture, where S. aureus and coliforms accounted for 8.6 and 2.8%, respectively (Table 1). 1.4% of S. aureus isolates were revealed in the amount of 10³ CFU/mL. Coliforms and S. aureus isolated in 1.4% (10¹ CFU/mL) and 2.7% (10² CFU/mL) of cases respectively, were not etiologically significant in the diagnostic titer (Table 2).

In the last decade, intensive studies look at the potential of bacteriocins as next-generation therapeutics against drug-resistant bacteria [30-33]. Bacteriocins from lactic acid bacteria are being tested as controlling agents for bacterial and viral infections; they can inhibit biofilm synthesis [33][34][35]. In a number of experiments, high antimicrobial activity of bacteriocin nisin was established against several species of staphylococci, including Staphylococcus saprophyticus, S. aureus, S. epidermidis, S. haemolyticus [36-38], including multi-drug resistant and methicillin-resistant S. aureus [39]. There are studies on clinical isolates of S. agalactiae that have demonstrated different susceptibility to nisin [40]. Pérez-Ibarreche M. et al. [41] described the results of using nisin to effectively control biofilm of S. uberis strains that cause mastitis in cows. The use of nisin, which has antimicrobial activity against major mastitis-causing pathogens, could offer a potential alternative to antibiotics [36][42][43]. The data of our study confirm that it is feasible to include nisin into mastitis treatment regimens. During the experiment, it was found that the mastitis-causing pathogens isolated from milk, such as S. aureus, coliforms, E. faecium, Streptococcus spp., A. viridans, S. epidermidis, C. pseudotuberculosis, S. haemolyticus, are susceptible to the nisin-based formulation. Since the discovery of bacteriocins, researchers have mainly focused on testing their antimicrobial activity in vitro. However, for the use of bacteriocins as antimicrobial drugs, it is necessary to study their clinical efficacy [44]. The effect of the nisin-based formulation on microbiota of milk from high yielding cows proved its effectiveness for treating subclinical mastitis: for example, in 88.6% of the samples, no microorganism growth was observed.

CONCLUSION

The study revealed the milk microbiota composition of high yielding cows with subclinical mastitis. It was found that in 81.4% of cases the disease occurs as a co-infection, among them two pathogens were isolated together in 21.4% of cases and three pathogens were isolated in parallel in 28.6% of cases. S. aureus (22.2%) and S. dysgalactiae (16.0%) were the most frequent isolated species.

A comparative analysis of the AMR of isolates recovered from milk of cows with subclinical mastitis showed the presence of multi-drug resistant strains of coliform bacteria, S. dysgalactiae, A. viridans, S. aureus. 62.5 and 47.1% of the tested coliforms and A. viridans isolates respectively, had resistance to two groups of antimicrobials. Resistance of S. dysgalactiae to three antimicrobial groups was found in 43.5% of the isolated strains, to four groups in 26.1% isolates. 62.5% of the tested S. aureus isolates were resistant to four groups of antimicrobials; 46.9% of them were resistant to five groups and resistance to more than six groups of antimicrobials was reported in 15.6% of isolates.

The study of the effect of the nisin-based formulation on microbiota of milk from cows with clinical mastitis revealed that after treatment 88.6% of the milk samples showed no microorganism growth. The milk microbiota in 8.6% of cases was represented by S. aureus, 2.8% were coliform bacteria. Herewith, in 1.4 and 2.7% of the samples, coliforms and S. aureus were detected in diagnostic titers equal to 10¹ and 10² CFU/mL, respectively, therefore they were not etiologically significant microorganisms for mastitis development.