Resistance to antimicrobials and sanitizers of bacterial isolates causing caprine mastitisABSTRACT
This study was conducted aiming to analyze bacterial isolates that cause mastitis in dairy goats and their resistance capacity, and was conducted in the goat farming sector of the Federal University of Paraíba (UFPB), campus III, where 60 milk samples were collected from 30 goats and the following tests were carried out: bacterial culture, Maldi-tof technique, evaluation of resistance to the disinfectants used in post-dipping: 0.6% iodine, 2.0% chlorhexidine and 2.0% lactic acid in three different exposure times, assessment of the sensitivity profile to the antimicrobials: gentamicin, sulfadiathrin, ceftiofur, ciprofloxacin, marbofloxacin, neomycin, penicillin and tetracycline. Bacterial growth occurred in 15 samples, 100% of which were isolates from the genus Staphylococcus spp. where 46.7% were non-aureus Staphylococcus (NAS) and 53.3% were Staphylococcus aureus (SA). For disinfectants, at times of 15”, 30” and 60”, respectively, the sensitivity obtained was: 80.0%, 86.7% and 93.3% for iodine, 86.7%, 93.3% and 100% for chlorhexidine and 40%, 53.3% and 73.3% for lactic acid. Regarding the antimicrobials, it was observed that ceftiofour, gentamicin and marbofloxacin obtained 100% sensitivity for SA and NAS respectively, ciprofloxacin obtained 100% and 85.7%, neomycin 62.5% and 85.7%, sulfadiathrin 62.5% and 71.4%, tetracycline 62.5% and 42.8% and penicillin 12.5% and 28.6%.
Keywords:
dairy goat; mammary gland; MALDI-TOF; Staphylococcus
RESUMO
Com o objetivo de analisar os isolados bacterianos causadores de mastite em caprinos leiteiros e sua capacidade de resistência, este estudo foi realizado no setor de caprinocultura da Universidade Federal da Paraíba (UFPB), campus III, onde foram coletadas 60 amostras de leite de 30 cabras e realizados os testes: cultura bacteriana, técnica MALDI-TOF, avaliação de resistência aos desinfetantes utilizados no pós-dipping: iodo a 0,6%, clorexidina a 2,0% e ácido lático a 2,0% em três diferentes tempos de exposição, avaliação do perfil de sensibilidade a antimicrobianos: gentamicina, sulfadiatrina, ceftiofur, ciprofloxacina, marbofloxacina, neomicina, penicilina e tetraciclina. Houve crescimento bacteriano em 15 amostras, sendo 100% dos isolados do gênero Staphylococcus spp. dividindo-se em 46,7% Staphylococcus não aureus (SNA) e 53,3% Staphylococcus aureus (SA). Para os desinfetantes, nos tempos de 15”, 30” e 60”, respectivamente, obteve-se sensibilidade de: 80,0%, 86,7% e 93,3% para o iodo, 86,7%, 93,3% e 100% para a clorexidina e 40%, 53,3% e 73,3% para o ácido lático. Quanto aos antimicrobianos, observou-se que ceftiofur, gentamicina e marbofloxacina obtiveram 100% de sensibilidade para SA e SNA, respectivamente; ciprofloxacina obteve 100% e 85,7%, neomicina 62,5% e 85,7%, sulfadiatrina 62,5% e 71,4%, tetraciclina 62,5% e 42,8% e penicilina 12,5% e 28,6%.
Palavras-chave:
caprinocultura; glândula mamária; MALDI-TOF; Staphylococcus
INTRODUCTION
Mastitis is the name given to the inflammation of the mammary gland, it is the main disease that affects dairy herds, with a high prevalence. The impacts of this disease are related to the decrease in milk production and the quality of its derivatives, early disposal of animals, as well as costs with labor, professionals and medicines (Ordoñez et al., 2022).
Epidemiologically, it is important to identify the etiology of mastitis, both to track the origin of the infection and to implement adequate treatment and control. Several etiological agents may be involved in cases of mastitis, however, among the bacterial pathogens, Staphylococcus spp. is the most frequent. This bacterial genus causes contagious mastitis and is widely spread from infected to healthy cows during milking. Staphylococcus spp., in most cases, reaches the mammary gland through the surface of inadequately sanitized teats, equipment, milking utensils and contaminated hands of milkers (Sousa et al., 2023).
Containing mastitis in herds is essential to obtain better quality milk, guaranteeing safe food. In this context, sanitary measures must be adopted during the milking process to minimize the transmission of agents that cause mastitis to animals and to milk, as the presence of these agents can harm the microbiological quality of milk (Menezes et al., 2020).
One of the recommended measures is the appropriate use of teat disinfecting agents that are used in pre- and post-dipping, with the aim of reducing the population of pathogenic microorganisms and avoiding the potential spread of infectious agents, which tends to control and even reduce the risks of new mammary gland infections (Soares et al., 2024).
The purpose of this study was to analyze bacterial isolates that cause mastitis in the dairy goat herd of the goat farming sector of the Federal University of Paraíba and the resistance capacity of these bacterial isolates to antimicrobials and sanitizers commonly used to treat and prevent mastitis.
MATERIAL AND METHODS
60 milk samples were collected from 30 Saanen goats at different stages of lactation from the herd belonging to the goat farming sector of the Federal University of Paraíba (UFPB), campus III, located in the municipality of Bananeiras, in the countryside of Paraíba state.
Initially, the dark-bottomed mug test was carried out to identify cases of clinical mastitis, where the first jets of milk collected by manual milking are deposited in a black-bottomed mug to observe whether there are lumps or other changes in the milk that suggested the presence of clinical mastitis, and then the milk was collected for microbiological examination through manual milking after prior cleaning of the teat with a pre-dipping solution based on chlorhexidine, drying with paper towel and antisepsis of the ostium of the tit with alcohol at 70ºGL.
Approximately 5mL of milk was collected per mammary half of each goat, totaling 60 samples, in sterilized tubes and previously identified with the name or number of the animal and the mammary half, being sent under refrigeration in boxes made of isothermal material containing recyclable ice, to carrying out the microbiological examination in the UFPB laboratory.
Milk aliquots were seeded in Petri dishes containing agar base enriched with 5% sheep blood. The plates were incubated in a bacteriological stove at 37º C and readings were taken 24 and 48 hours after incubation. To identify the isolated bacteria, the morphological characteristics of the colonies were observed, such as size, type, color and presence of hemolysis and, under a microscope, the arrangement of the cells and morphotinctorial characteristics were observed using the Gram Test (Carter, 1988).
The isolates were transferred to Muller Hinton agar plates, packed in a thermal box with recyclable ice and sent to Clínica do Leite in the city of Piracicaba - SP to identify the bacterial species using the MALDI - TOF (Matrix- Assisted Laser Desorption Ionization-Time of Flight) as described by Tsuchida and Nakayama (2022).
Bacterial samples were prepared as previously described by Topić Popović et al. (2023). Colonies were transferred directly to a 96-target polished steel plate (Bruker Daltonics) and covered with 1μL of a saturated cyano-4-hydroxycinnamic acid (CHCA) matrix solution (Bruker Daltonics). Mass spectra were obtained and analyzed using a LT microflexo mass spectrometer (Bruker Daltonics) in combination with RUO (Research-use-Only) versions of the MALDI Biotyper software package (version 3.0) and the V reference database. 3.1.2.0 (3,995 entries). The mass spectra of the samples were compared with the reference mass spectra in the database, calculating a value (score) between 0 and 3, reflecting the similarity between the sample and the reference spectrum, displaying the top 10 database records corresponding data.
To evaluate the disinfectants efficiency used in post-dipping, the following active ingredients were used: iodine (0.6%), chlorhexidine (2.0%), and lactic acid (2.0%), with dilutions being carried out according to the instructions of the manufacturer (Delaval Brasil, Jaguariuna - SP). For analysis, homogeneous bacterial suspensions were prepared in sterile saline solution (5.5ml) corresponding to tube 1 on the McFarland scale. The suspension consisted of the disinfectant solution (0.8ml) diluted according to the manufacturer (Delaval Brasil, Jaguariuna - SP) and sterile milk (0.2mL). Subsequently, the bacterial suspension (1.2ml) was added and the exposure times (15", 30" and 60") were timed and then repeated in BHI broth, as described by Vasconcelos (2023).
The mixture was incubated at 37°C for 24 hours to observe the turbidity of the medium, formation of a film on the surface or precipitate at the bottom of the tubes. After incubation, the suspension was transferred to solid medium (blood agar) to confirm the presence or absence of the microorganism tested against different disinfectants and exposure time, where the absence of bacterial growth on the plates indicates the effectiveness of the product in question (Vasconcelos, 2023).
The isolates were submitted to antibiogram in Petri dishes containing Müller Hinton agar following the methodology described by the Clinical and Laboratory Standards Institute (Performance…, 2006) S. aureus ATCC 25923 was used as control. The following drugs were then tested: gentamicin, sulfadiathrin, ceftiofur, ciprofloxacin, marbofloxacin, neomycin, penicillin and tetracycline by placing discs of these antibiotics on Müller Hinton agar, and consequent measurement of the inhibition halos that appeared around the antimicrobial discs. in accordance with the table provided by the manufacturer (Difco & BBL - BD, New Jersey - USA).
Statistical analysis was performed with GraphPad Prism software version 7.04 for Windows (GraphPad Software, La Jolla, California, USA) and Epi Info™ version 7.2 for Windows (Epi Info™ software, Atlanta, Georgia, USA). The χ2 test was used to verify the statistical significance in the frequencies of disinfectant effectiveness, antimicrobial resistance and Minimum Inhibitory Concentration (MIC) values for the different species studied. Statistically significant differences were considered at P <0.05.
The present work was approved under the ethical aspects of animal experimentation under the CEUA UFPB protocol 6968030221.
RESULTS
Microbiological examinations detected that, of the 60 samples collected, bacterial colony growth was observed in 15 samples. The frequency of detection of organisms in samples sent for the MALDI - TOF examination is described in Table 1.
The MALDI - TOF results demonstrated that 53.3% (8/15) of Staphylococcus spp. isolates. were classified as S. aureus and 46.7% (7/15) were classified as non-aureus (NAS) with three probable species: S. sciuri 20% (3/15), S. epidermidis 20% (3/15) and S. simulans 6.7% (1/15).
The sensitivity profile of the 15 Staphylococcus spp. isolates. against the disinfectants iodine (0.6%), chlorhexidine (2%) and lactic acid (2%) is shown in Table 2.
Sensitivity profile of Staphylococcus spp. isolates. against the disinfectants iodine (0.6%), chlorhexidine (2%) and lactic acid (2%)
The sensitivity profile of Staphylococcus spp. against iodine at times of 15”, 30” and 60” it was 80.0%, 86.7% and 93.3%, respectively. As for chlorhexidine, it was observed that 86.7% were sensitive at a time of 15” and that 93.3% were sensitive at a time of 30” and that at 60” all isolates were sensitive to the action of this disinfectant. Regarding lactic acid, 40%, 53.3% and 73.3% were sensitive at the times previously mentioned.
The antimicrobial sensitivity profile of Staphylococcus spp. isolates is described in Table 3.
The sensitivity profile of Staphylococcus spp. to antimicrobials declared that ceftiofour, gentamicin and marbofloxacin obtained 100% sensitivity for both SA and NAS, however, marbofloxacin and gentamicin were more confident than ceftiofour due to the lower p value, thus demonstrating less chance of error.
The other antimicrobials did not have results as overwhelming as those previously reported where, in decreasing order of effectiveness, we have ciprofloxacin with 100% sensitivity for SA and 85.7% for NAS, neomycin with 62.5% sensitivity for SA and 85.7% for NAS, sulfadiathrin with a sensitivity of 62.5% for SA and 71.4% for NAS, tetracycline with a sensitivity of 62.5% for SA and 42.8% for NAS and, finally, penicillin with a sensitivity of only 12.5% for SA and 28.6% for NAS.
In the current study, 87.5% (7/8) of Staphylococcus aureus and 71.4% (5/7) non-aureus Staphylococcus were resistant to penicillin.
DISCUSSION
Studies have shown an increase in the frequency of NAS as a cause of clinical and subclinical mastitis in the world (Rosa et al., 2022). This increase in the number of cases of mastitis caused by NAS may be associated with the difficult control of breast disorders with this etiology, since there is a great heterogeneity in this group of microorganisms, being composed of several NAS species related to inflammation of the breast gland. (Freu et al., 2024).
Among this group of microorganisms (NAS), the most common antimicrobial resistance occurs through the production of β-lactamases, which can confer resistance to penicillin and some aminoglycosides, tetracyclines and macrolides (Pereira et al., 2021).
The results obtained in this study indicated greater in vitro disinfectant activity of chlorhexidine and iodine, respectively. In a similar study, Fitzpatrick et al. (2021), also found that iodine had a great disinfectant activity against Staphylococcal isolates, especially in combination withs lactic acid. Another study carried out by Silva et al. (2021) pointed out that the use of iodine was still effective against Staphylococcus spp. even thou it’s a widely used disinfectant in Brazil due to its low cost and accessibility, the effectiveness of iodine in the same study particularly higher at concentrations of 0,5% or superior.
Chlorhexidine is widely used for the treatment of superficial teat infections in cows due to its cumulative and continuous effect, remaining on the skin for at least six hours, in addition, it acts in the presence of organic matter, is easy to apply and economical (Mendonça et al., 2020). In this study, iodine showed lower disinfectant action than chlorhexidine.
In this study, the worst sensitivity results observed, among the disinfectants used, were in relation to lactic acid. On the other hand, Soares et al. (2024), observed that lactic acid had excellent reduction rate of developing staphylococcal biofilm, although, for already consolidated biofilm, lactic acid had a much lower efficacy in that study.
It must also be considered that penicillin is an antimicrobial frequently used in the region studied (Santos et al., 2020), especially in mastitis, but also in other infectious diseases of ruminants, resulting in the high use of this medicine, which may contribute for the selection of microorganisms resistant to this drug in herds.
Regarding tetracyclines, NAS demonstrated lower sensitivity (42.8%) than S. aureus (62.5%). A similar study carried out by Silva Júnior et al. (2021) also found NAS with lower sensitivity (74,1) against tetracyclines than S. aureus (83,6), this study also reiterates that high rates of resistance against tetracycline have been observed among different organisms, including pathogenic bacteria.
Due to its antimicrobial action against Gram-positive and Gram-negative bacteria, tetracyclines are often used in the treatment of mastitis. However, a high rate of resistance was observed among the isolates analyzed, especially among NAS. The indiscriminate, continuous and prolonged use of the same antimicrobial favors the emergence of resistant strains within a herd (Moura et al., 2020). About tetracycline, another important fact is that the resistance mechanisms are mediated by mobile genetic elements, such as plasmids, which can carry resistance genes for several antimicrobials simultaneously, in addition to the possibility of transmission of these plasmids between different genera of bacteria, also representing a risk to public health (Souza-Silva et al., 2022).
CONCLUSION
The microbiological agents that cause mastitis in the herds in question are bacteria of the genus Staphylococcus, with great similarity in terms of the number of isolates of S. aureus and non-aureus species, thus demonstrating the possibility of mammary disorders caused by NAS. Regarding sensitivity to disinfectants used in post-dipping, the best in vitro disinfectant activity against Staphylococcus spp. was observed for active ingredients chlorhexidine and iodine, showing better action proportionally to the longer exposure time of the isolates to the reagents. As for antimicrobials, gentamicin and marbofloxacin obtained excellent in vitro results against Staphylococcus spp. isolates Analyzed, and can be used as alternatives for the treatment of mastitis in the herd in question.
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