413 Veterinaria Italiana 2022, 58 (4), 413‑423. doi: 10.12834/VetIt.2601.17023.2 Accepted: 11.02.2022 | Available on line: 31.12.2022 1Health and Animal Production Master's Degree, Pitágoras-Unopar University. 2UNOPAR. *Corresponding author at: Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise "G. Caporale". E-mail: rafaelfagnani@hotmail.com. Josiane Ito Eleodoro1, Rafael Fagnani2* Keywords Dairy herd, Cow, Resistance, Antibiotic, Pathogen. Summary Considering the high prevalence of subclinical mastitis and its impacts on milk production, thematic studies are need to provide strategic data for its control. This study aimed at in‑ vestigating the most frequent microorganisms associated with subclinical mastitis in dairy cows in Brazil through compiling the occurrence of the etiological agents and their sensitivi‑ ty to antibiotics. The systematic review includes articles published between 2009 and 2019. Fifty‑seven articles evaluating 22,287 milk samples were selected. The number of publica‑ tions and the sample size were not homogeneous among Brazilian regions. Most of the stu‑ dies and sampling were conducted in Rio Grande do Sul, whereas no studies were found in some states in the north and mid‑west regions. The most frequent group of pathogens was Staphylococcus spp. It was isolated in all studies and had an average prevalence of 49% in the analyzed samples. Resistance to penicillin was the most frequent microbial resistance found in Brazil, with an average of 66% among the isolates evaluated. Moreover, bacterial resistan‑ ce to cephalexin, cefoperazone, erythromycin, gentamicin, neomycin, penicillin, tetracycli‑ ne, and trimethoprim increased over the research period. Given the territorial extension, the etiological diversity, and the lack of studies with a representative sample, the compilation of scientific data must be interpreted with caution. Regions where a greater number of studies were conducted and with numerous samples, such as the South, provided a comprehensive scenario that is closer to reality. Nevertheless, although decision making on the farm cannot be replaced by scientific studies, it can be supported by such efforts. Please refer to the forthcoming article as: Ito Eleodoro et al. 2022. Etiological agents and bacterial sensitivity in subclinical mastitis in Brazil: a ten‑year systematic review. Vet Ital. doi: 10.12834/VetIt.2601.17023.2 Etiological agents and bacterial sensitivity in subclinical mastitis in Brazil: a ten-year systematic review treatment period (9%), increased labor (1%), and the premature disposal of animals (14%) (Sharma et al., 2012). Moreover, there are losses for dairy products owing to the decrease in the quality of the final product, the decrease in the industrial yield for the manufacture of derivatives, and changes in the composition of mastitic milk (Ruegg, 2017). Antimicrobial drugs are used to treat several diseases that affect dairy cows, and clinical mastitis is one of the main diseases that require the use of these drugs (Gomes and Henriques, 2016). Despite the many benefits of these drugs, from the perspective of public health and food safety, there is concern regarding antibiotic residues in food intended for human consumption from animals treated with Introduction The dairy herd occupies a prominent position within the Brazilian economic scenario, with milk being one of the main products of national agriculture. In fact, the agribusiness of milk and dairy products plays an important role in the supply of food and the social issue, with the generation of jobs and income for the population, mainly in southern region (Beber et al., 2019). However, mastitis is the most frequent and costly infection of dairy farming. This is because intramammary infections result in significant economic losses associated with several factors including the reduction in milk production (more than 70% of cases), cost of treatment and veterinary medical charges (7%), disposal of milk during the Subclinical mastitis in Brazil Ito Eleodoro et al. 414 Veterinaria Italiana 2022, 58 (4), 413-423. doi: 10.12834/VetIt.2601.17023.2 to 2019 in SciELO, Capes Periodical Portal, Google Scholar and PubMed. In each of the databases, the keywords were searched in Portuguese and English. The terms searched were: • Mastitis AND subclinical AND Brazil • Etiology AND mastitis AND subclinical AND Brazil • Antimicrobial resistance AND mastitis AND Brazil • Antimicrobials AND mastitis AND Brazil • Bovine mastitis AND subclinical AND Brazil • Staphylococcus AND subclinical AND mastitis AND Brazil • Streptococcus AND subclinical AND mastitis AND Brazil • Corynebacterium AND subclinical AND mastitis AND Brazil • Milk AND subclinical AND mastitis AND Brazil. The data extracted from each article, when available, included: year of publication; journal; first author; Brazilian region where the study was conducted; number of animals, number of milk samples analyzed, number of culture‑positive milk samples, description of the etiologic agents isolated from subclinical mastitis, antimicrobials tested and number of resistant samples to each antimicrobial class. The data obtained were tabulated and a descriptive statistical analysis of the absolute and relative frequency of the microbiological findings was performed using Microsoft Excel® and the combined Chi‑Square test using BIOSTAT® to compare the prevalence of resistance in each year studied. The heterogeneity of the prevalence estimates between studies was quantified by I2 index for the most frequent microorganisms (Higgins and Thompson, 2002). Results Our search strategy yielded 41,038 records (sum of all database), from which 75 studies were retained after inclusion/exclusion criteria. Following duplicate removal, 69 articles were included in the screening step. During the screening stage, 12 articles were considered as not relevant and were excluded (5 without information regarding the geographic region; 5 without the number of animals and 3 surveys based on interviews). Fifty‑ seven articles, published between 2009 and 2018 with 22,287 milk samples evaluated, were selected according to the inclusion criteria. The sampling technique and representativeness of the 57 articles included can be seen in Supplementary Table 1. antibiotics and the potential development and transmission of antimicrobial resistance which may impact the treatment of diseases (Oliver et al., 2020). The appearance of multidrug‑resistant strains has made it difficult to treat mastitis in cows. Thus, microbiological diagnosis of mastitis needs to be performed routinely, as it is capable of generating fast and safe results that can identify the problems affecting the herd. According to Karach et al., (2015), the isolation and identification of the agent contribute to the most appropriate choice of the drug to be used in therapy, thus avoiding the development of bacterial resistance to antibiotics. One of the strategies to prevent bacterial resistance is knowing the main agents involved in mastitis and their sensitivities. This systematic review aimed at investigating the most frequent microorganisms associated with subclinical mastitis in dairy cows in Brazil, compiling data on the occurrence of the etiological agents causing subclinical mastitis and its sensitivity to antibiotics. A critical analysis of the past ten years is justified as it can provide epidemiological data for better control of subclinical mastitis. The results will allow us to develop an overview of the etiological agents and antimicrobial sensitivity of the main agents that cause mastitis, helping to provide a basis to prevent resistance to the condition and address its chronic nature. Methods The following systematic review with meta‑analysis was planned according to the Preferred Reporting Items for Systematic Reviews and Meta‑Analyses (PRISMA) network meta‑analysis reporting standards using the StArt and Biostat programs (Hutton et al., 2015). Observational studies that assessed etiological agents of subclinical bovine mastitis and its antimicrobial resistance/sensitivity were eligible for inclusion. The inclusion criteria were primary studies: (1) related to the proposed topic and available for online consultation in search engines using keyword strings; (2) published between 2009 and 2019; (3) that addressed the bovine species, (4) that used 200.000 cel/mL as somatic cell count threshold for the classification of subclinical mastitis and (5) that were conducted in Brazil. The exclusion criteria were (1) primary studies that did not specifically address the etiological agents and its antimicrobial resistance/sensitivity; (2) primary studies published outside the selected period; (3) primary studies that did not address the bovine species; (4) primary studies conducted outside Brazil, and (5) secondary studies. The research included articles from journals and annals of scientific events published from 2009 Ito Eleodoro et al. Subclinical mastitis in Brazil Veterinaria Italiana 2022, 58 (4), 413-423. doi: 10.12834/VetIt.2601.17023.2 415 Supplementary Table I. Sampling technique and representativeness of 55 articles published between 2009 and 2018 that met the inclusion criteria of the systematic review. Reference State Type of sampling 1 Alencar et al. (2014) Rio de Janeiro Purposive sampling, independent 2 Amorim et al. (2016) Pernambuco Purposive sampling, independent 3 Andrade et al. (2010) Paraná Convenience, random, independent 5 Assis et al. (2017) Espírito Santo Convenience, random, independent 6 Bandeira et al. (2013) R.Grande do Sul Purposive sampling, independent 7 Brito et al. (2014) Maranhão Convenience, random, independent 8 Carvalho et al. 2018 Maranhão Convenience, random, independent 9 Casanova et al. (2016) Santa Catarina Purposive sampling, independent 10 Castro et al. (2012) Rio de Janeiro Quota sampling, independent 11 Chagas et al. (2012) Minas Gerais Convenience, random, independent 12 Costa et al. (2013) Minas Gerais Convenience, random, independent 13 Costa et al. (2013) Santa Catarina Snowball sampling, independent 14 Costa et al. (2015) Santa Catarina Convenience, dependend 15 Cunha et al. (2015) Minas Gerais Convenience, random, independent 16 deSantana Neres et al. (2015) Sergipe Convenience, independent 17 Dias et al. (2011) Minas Gerais Quota sampling, independent 18 Farias et al. (2013) R.Grande do Sul Purposive sampling, independent 19 Ferreira et al. (2010) Piauí Quota sampling, independent 20 Filho et al. (2016) Paraná Convenience, random, independent 21 Freitas et al. (2018) R.Grande do Sul Purposive sampling, independent 22 Gonçalves et al. (2018) São Paulo Purposive sampling, independent 23 Jardim et al. (2014) Paraná Convenience, random, independent 24 Jobim et al. (2010) Paraná Convenience, random, independent 24 Jobim et al. (2010) R.Grande do Sul Convenience, random, independent 24 Jobim et al. (2010) Santa Catarina Convenience, random, independent 25 Junior et al. (2015) São Paulo Purposive sampling, independent 26 Kaiser et al. (2015) R.Grande do Sul Purposive sampling, independent 27 Karach et al. (2016) Paraná Convenience, random, independent 28 Kolling et al. (2011) R.Grande do Sul Purposive sampling, independent 29 Krewer et al. (2013) Bahia Convenience, random, independent 29 Krewer et al. (2013) Pernambuco Convenience, random, independent 30 Lange et al. (2017) Paraná Convenience, random, independent 31 Martins et al. (2010) Mato Grosso Convenience,representative, independent 31 Martins et al. (2014) Piauí Purposive sampling, independent 32 Martins et al. (2015) Goiás Convenience, random, independent 33 Melo et al. (2013) Pernambuco Convenience, random, independent 34 Niero 2018 Santa Catarina Purposive sampling, independent 35 Oliveira et al. 2009 Sergipe Convenience, independent 36 Oliveira et al. (2010) Pará Convenience, random, independent 37 Oliveira et al. (2012) Bahia Convenience, random, independent 38 Oliveira et al. (2013) Paraná Simple random sampling, independent 39 Peters et al. (2016) R.Grande do Sul Purposive sampling, independent 40 Rall et al. (2014) São Paulo Purposive sampling, independent 41 Ribeiro et al. 2009 São Paulo Purposive sampling, independent 42 Ruiz et al. (2011) Pernambuco Convenience, random, independent Subclinical mastitis in Brazil Ito Eleodoro et al. 416 Veterinaria Italiana 2022, 58 (4), 413-423. doi: 10.12834/VetIt.2601.17023.2 The number of publications was not homogeneous (G test = 31.67; P < 0.01) among Brazilian states, with a greater occurrence of studies in Paraná, Rio Grande do Sul, and Santa Catarina. Thus, 49% of the selected studies were conducted in the southern region (Fig. 01). The sample size evaluated in the articles among Brazilian states was not uniform (X2 = 8249.88; P < 0.01). The states with the largest number of milk samples analyzed were Rio Grande do Sul, with 23% of the samples, followed by Minas Gerais (16%), and Paraná (12%) (Fig. 1). homogeneous among the Brazilian states (X2 = 75.40; P < 0.01), with a higher rate in the states of Espírito Santo (80%), Rio de Janeiro (77%), and Bahia (60%). Goiás and São Paulo had the lowest rates at 20% and 28%, respectively (Fig. 2). Figure 1. Publications about subclinical mastitis in dairy cows in Brazil and sample size (milk samples) retrieved from 57 scientific articles published between 2009 and 2018. Figure 2. Occurrence of etiologic agents of subclinical mastitis in dairy herds in Brazil retrieved from 45 scientific articles published between 2009 and 2018.Of the 57 articles selected, 45 studies isolated and identified the etiologic agents that caused subclinical mastitis. The most frequent pathogens were Staphylococcus spp., which was isolated in all studies with an average prevalence of 49% in the samples analyzed. There was no significant heterogeneity between 45 studies (Q=82.03, df=24, p=0.88), with a heterogeneity index I2 of 19.47%. However, when categorized by region, the distribution of Staphylococcus spp. was not The second most frequent pathogens were Streptococcus spp., identified in 76% of the identified articles, with an average occurrence of 14% in the analyzed samples and a significant heterogeneity (Q=337.70, df=98, p<0.001; I2=70.98) between the studies retrieved. The distribution of Streptococcus spp. was not homogeneous among the Brazilian states (X2 = 77.62; P < 0.01), with greater prevalence Reference State Type of sampling 43 Saab et al. (2014) Paraná Convenience, random, independent 44 Saeki et al. (2011) São Paulo Convenience, independent 45 Santos et al. (2010) Paraná Convenience, random, independent 46 Senhorelo et al. (2013) Espírito Santo Convenience, random, independent 47 Silva et al. (2011) Bahia Convenience, random, independent 48 Silva et al. (2012) Pernambuco Purposive sampling, independent 49 Soethe et al. (2015) Paraná Convenience, random, independent 50 Souza et al. (2016) Minas Gerais Convenience, random, independent 51 Ulsenheimer et al. 2018 R.Grande do Sul Purposive sampling, independent 52 Valmorbida et al. (2017) Santa Catarina Purposive sampling, independent 53 Vesco et al. (2017) R.Grande do Sul Purposive sampling, independent 54 Zanette et al. (2010) Santa Catarina Quota sampling, independent 55 Zimermann et al. (2017) Paraná Purposive sampling, independent Ito Eleodoro et al. Subclinical mastitis in Brazil Veterinaria Italiana 2022, 58 (4), 413-423. doi: 10.12834/VetIt.2601.17023.2 417 2010 2011 2012 2013 2015 2016 2017 2018 Amikacin - - - - 0.04a (n=69) 0.04a (n=56) - - Amoxicillin - - - 0.59a (n=453) - 0.71a (n=17) 0.05b (n=313) 0.50a (n=30) Ampicillin 0.68de (n=289) 0.27ab (n=154) 0.78e (n=242) 0.68de (n=805) 0.47c (n=232) 0.67cde (n=39) 0.18a (n=313) 0.48bcd (n=62) Bacitracin 0.41b (n=188) - - 0.08a (n=194) - - 0.07a (n=846) - Cephalexin - 0.00a (n=65) - 0.01a (n=453) - 0.13b (n=56) 0.74c (n=869) 0.19b (n=32) Cephalothin 0.13b (n=188) 0.30b (n=50) 0.00a (n=83) 0.01a (n=546) 0.13b (n=153) - - - Cefoperazone 0.20a (n=101) - - 0.50b (n=352) - - - - Ceftiofur 0.04a (n=101) 0.02a (n=65) - 0.01a (n=546) - - - - Figure 3. Occurrence of bacterial resistance to antibiotics in dairy cows in Brazil from 28 articles published. in the states of Goiás (34%) and Paraná (27%) (Fig. 2). The third most frequent pathogens were Corynebacterium spp., identified in 58% of the studies and with an average prevalence of 8% in the analyzed samples. The heterogeneity index I2 for the prevalence of Corynebacterium spp. between those studies was 80.31% (Q=497.61, df=98, p<0.001). Corynebacterium spp. were not evenly distributed among the Brazilian states (X2 = 140.35; P < 0.01), with greater prevalence in the states of Pernambuco (31%), Mato Grosso (27%), and Bahia and Espírito Santo (both 21%) (Fig. 2). Escherichia coli was the fourth most frequent pathogen, isolated in 47% of the articles and with an average occurrence of 4% in the analyzed samples. A significant heterogeneity was found regarding the prevalence of E. coli among the studies (Q=634.75, df=98, p<0.001; I2=84.56). The states of Goiás (9%) and Rio de Janeiro (8%) had the highest prevalence of E. coli. In 87% of the reviewed articles, other microorganisms were also isolated and associated with subclinical mastitis, such as Candida spp., Micrococcus spp., Proteus spp., Alcaligenes faecalis, Enterobacter aerogenes, Klebsiella spp., Citrobacter spp., Salmonella spp., Yersinia spp., Pseudomonas spp., Nocardia spp., Trueperella spp., and Serratia spp. This wide range of pathogens was more prevalent in isolates from São Paulo (32%) and Goiás (30%) (G test = 44.37; P < 0.01) (Fig. 2). Microbial resistance Among the 57 articles reviewed, 28 investigated the occurrence of resistance and sensitivity of isolated microorganisms against the antibiotics tested. Only Table I. Occurrence of bacterial resistance to antibiotics in dairy cows from 57 articles published between 2009 and 2018 (n= number of isolates analyzed). studies from 11 states of the northeast, southeast, and south regions evaluated microbial resistance. These studies were conducted mainly in Rio Grande do Sul, Minas Gerais, and Santa Catarina, which together accounted for 43% of the retrieved articles. The most comprehensive samples also came from studies conducted in these three states: Rio Grande do Sul (1876 samples), Minas Gerais (552), and Santa Catarina (455) (Fig. 3). The most tested antimicrobials were ampicillin, erythromycin, gentamicin, penicillin, and tetracy‑ cline. Over the years, there has been an increase in the occurrence of bacterial resistance to cephalexin, cefoperazone, erythromycin, gentamicin, neomycin, penicillin, tetracycline, and trimethoprim (Table I). The occurrence of microorganisms resistant to penicillin varied between 34% and 76% between Subclinical mastitis in Brazil Ito Eleodoro et al. 418 Veterinaria Italiana 2022, 58 (4), 413-423. doi: 10.12834/VetIt.2601.17023.2 review, most studies and literature reviews also consider Streptococcus spp. as the second group of microorganisms of importance in the etiological agents causing mastitis in ruminants. In most herds, Streptococcus agalactiae, Streptococcus uberis, and Streptococcus dysgalactiae are the main isolated species (Santos et al., 2018). Streptococcus uberis is an important agent of subclinical infections and clinical episodes of bovine mastitis worldwide (Hillerton, 2020). Santos et al., (2018) reported that the Streptococcus dysgalactiae is one of the most common pathogens of bovine mastitis, causing great economic losses. Regarding Corynebacterium spp., the third most frequent reported in this systematic review, the species isolated the most in bovine mastitis is C. bovis (Karach et al., 2015). They have low pathogenicity and high contagiousness, being transmitted mainly during milking, and are considered one of the causes of contagious mastitis. It is detected mainly in the subclinical form of the disease, which in a certain manner guarantees protection to the mammary gland against other more pathogenic cells. The isolation rates of this pathogen are high in herds with problems related to the cleaning of teats, especially post‑dipping (Gonçalves et al., 2016). The fourth most frequent pathogen was E.coli. Neethan et al., (2017) also indicate E. coli as the main coliform (environmental microorganism) causing subclinical mastitis, with symptoms ranging from mild (with inflammatory signs in the mammary gland) to acute, with systemic signs such as ruminal stasis, dehydration, and shock, which can even lead to the death of the affected animal. Although it mainly causes clinical mastitis, the microorganism has also been investigated in cases of subclinical mastitis. They are usually transient infections and are associated with acute or super‑acute clinical 2010 2011 2012 2013 2015 2016 2017 2018 Chloramphenicol 0.26b (n=188) - 0.14b (n=180) 0.05a (n=194) 0.00a (n=69) 0.25b (n=32) - - Enrofloxacin 0.21b (n=101) 0.02a (n=66) 0.00a (n=83) 0.01a (n=453) - - 0.03a (n=36) 0.09a (n=32) Erythromycin 0.25cd (n=227) 0.16bc (n=116) 0.08ab (n=197) 0.04a (n=275) 0.39d (n=163) 0.23abcd (n=26) 0.20bc (n=120) 0.72e (n=32) Streptomycin 0.68b (n=188) - - 0.12a (n=453) - 0.24a (n=46) - - Gentamicin 0.20d (n=101) 0.03ab (n=156) 0.06bc (n=242) 0.02a (n=805) - 0.16cd (n=92) 0.75e (n=2100) 0.87e (n=30) Neomycin 0.39b (n=101) 0.03a (n=118) - 0.03a (n=546) - 0.43b (n=56) 0.73c (n=1880) - Norfloxacin 0.26b (n=188) 0.07a (n=88) - 0.03a (n=282) - 0.12ab (n=26) - 0.09ab (n=32) Data for the Amoxacillin + Clavulanic acid association in 2014 was omitted due to only one entry; a, b,c,d: Proportions followed by equal letters did not differ over the years by the chi-square test with 5% significance. 2010 and 2016 and increased to 88% in 2017 (P < 0.05), reaching the highest level of resistance among the isolates (Table I). Other antimicrobials that started to show increasing values (P < 0.05) of microbial resistance as of 2017 were gentamicin and neomycin, and erythromycin, in 2018. The increase in these three agents may be linked to their widespread use in dairy farms, which results in contributing to the selective pressure of microorganisms resistant to them (Tomazi and dos Santos, 2020). Amikacin and ceftiofur were the antimicrobial drugs with the lowest prevalence of resistance and without variations (P > 0.05) in studies conducted between 2010 and 2016. Resistance to amikacin remained at 4% during 2015 and 2016. Resistance to ceftiofur was 4% in 2010; 2% in 2011; and 1% in 2015. Discussion Regarding the most frequent pathogen reported in this systematic review (pooled prevalence of 49%), Staphylococci are one of the pathogens most frequently isolated in cases of intramammary infection within dairy herds. This estimate is similar to the study done by Ashraf and Imran (2020), who conducted a review about the prevalence of various bacterial species worldwide. Algharib et al., (2020) stated that S. aureus is an agent that is difficult to treat owing to its high resistance in the udder, which consequently, influences the efficiency of the antibiotics administered. This is due to a mechanism used by the pathogen to invade and colonize the animal’s mammary gland; the microorganism invades the mammary gland through the teat canal and colonizes its epithelium, attaching to the epithelial cells of the mammary gland and forming so‑called “bacterial pockets.” As well as the results of this systematic Ito Eleodoro et al. Subclinical mastitis in Brazil Veterinaria Italiana 2022, 58 (4), 413-423. doi: 10.12834/VetIt.2601.17023.2 419 (Dyar et al., 2017). One of the facts that may have led to this result is that after 2013, we found no studies evaluating resistance to ceftiofur. In this review, few studies evaluated the effectiveness of Amikacin on bovine mastitis isolates. However, authors such as Fim Junior et al., (2015) and Souza et al., (2016) reported 92.3% and 96.0% sensitivity of the isolates, respectively, thus demonstrating the effectiveness of Amikacin with their results. The scarcity of studies investigating the use of this antimicrobial drug may be because gentamicin is one of the main aminoglycosides used in veterinary medicine, more specifically in the treatment of mastitis. Limitations It is worth noting that analyzing microbial resistance with information obtained from published scientific articles has its limitations. One of them is the temporal and geographical limitation, since, from an epidemiological point of view, the monitoring of publications over the years does not guarantee a significant sample at the national level, and these data are not from a single region of Brazil. Moreover, the compilation of several studies conducted in one specific period is not representative. Furthermore, some geographical areas with lack of studies pose challenges to obtaining high‑quality survey, contributing with bias that can affect the reliability of the findings, mainly when extrapolating the results to other regions. Thus, we strengthen the need of more studies at regional level with properly methodologies regarding the sample representativeness. Another limitation is related to the seasons, since the time of year is related to antimicrobial treatment in dairy herds (Tomazi and dos Santos, 2020). The articles selected in this study did not provide enough data to analyze this variable. Conclusions With the etiological diversity found in this review, our results strengthen the knowledge of the microbiological agent and antibiotic‑resistance patterns of pathogens isolated from subclinical mastitis in dairy cows at regional level. The spread of bacterial resistance can be prevented using the culture test and antibiogram, and that although decision making in a farm cannot be replaced by scientific studies, it can be supported by such efforts. Nevertheless, the identification of the microbiological agent is essential to the most appropriate therapy. When possible, etiology should be determined before treatment to avoid microbial resistance. conditions, which can be fatal. It is important to note that older cows, those at the beginning of lactation, and those with higher yields are most susceptible to the severe manifestation of mastitis by coliforms (Byomi et al., 2020; Hamali et al., 2017). We observed a high heterogeneity for Streptococcus spp, Corynebacterium spp and E. coli regarding the prevalence estimates between the retrieved studies, probably due to diversity in farm practices (hygienic milking, dry cow therapy and therapeutic actions) together with herd characteristics (genetic, stage of lactation) and agroclimatic conditions (Bangar et al., 2015). Regarding microbial resistance, it can be inferred that some of the most used agents in intramammary therapies were evaluated by only a few of the selected studies. An example is the third generation cephalosporins, identified as the second most frequently used class of antimicrobials in Brazil (Tomazi and dos Santos, 2020). We recommend that studies on microbial resistance select the agents most used in the geographic region studied so that the results better reflect reality. Penicillin is one of the main antibiotics used for intramammary treatments not only in Brazil but also in other countries (Tomazi and dos Santos, 2020). In the United States, more than 70% of isolates obtained from mastitis caused by S. aureus are resistant to penicillin, whereas in Ireland, the level of resistance is around 85% (Cazoto et al., 2011). The widespread use of an agent is one of the causes of bacterial resistance (Freitas et al., 2018). Another cause of resistance to penicillins is owing to Staphylococcus spp., the main genus associated with subclinical mastitis, being able to develop resistance to most antimicrobials. Resistance to beta‑lactams, as is the case with penicillins, can occur via two main mechanisms: through the production of beta‑lactamases, encoded by the blaZ gene and the change in the antimicrobial action site owing to the production of a modified low‑affinity penicillin‑binding protein (PBP2a or PBP2), encoded by the mecA gene (Soares et al., 2012). Indeed, in a systematic review that addressed article from 5 continents, Molineri et al. (2021) found that the highest overall prevalence of resistant S. aureus was against penicillin. Although belonging to the third generation cephalosporin class, the second most frequently used class in Brazil between 2014 and 2016, among the isolates tested, low microbial resistance was shown to ceftiofur, with values ranging between 4% and 1% (Tomazi and dos Santos, 2020). In line with our results, Molinieri et al. (2021) stated that ceftiofur and cephalothin presented the lowest overall prevalence of antimicrobial‑resistant S. aureus from article retrieved between the years 1969−2020. This result contradicts the common understanding that associates the duration of use with greater resistance Subclinical mastitis in Brazil Ito Eleodoro et al. 420 Veterinaria Italiana 2022, 58 (4), 413-423. doi: 10.12834/VetIt.2601.17023.2 Algharib S.A., Dawood A. & Xie S. 2020. 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