Abstract
Family farming is central to Brazilian milk production, yet smallholder dairy systems in agrarian settlements often have limited access to veterinary assistance and microbiological diagnosis. This study describes the bacteria recovered from 2432 quarter milk samples with California Mastitis Test (CMT) scores of 2+ or 3+, collected in 2020 from dairy herds in eight agrarian settlements of the Pontal do Paranapanema region, São Paulo State, Brazil, and the antimicrobial susceptibility of the resulting isolates. Bacterial growth was obtained from 1296 samples (53.3%), whereas 1121 (46.1%) yielded no growth. Staphylococcus spp. (26.3% of samples, pure culture), Bacillus spp. (12.6%) and Corynebacterium spp. (3.8%) were the most frequently recovered genera. Susceptibility was determined by disk diffusion. Among 648 Staphylococcus isolates tested, resistance was most frequent to ampicillin (13.7%), penicillin (12.8%) and cephalothin (11.6%); among 363 Bacillus isolates, to cephalothin (22.0%), amoxicillin (19.8%) and penicillin (19.3%); and among 115 Corynebacterium isolates, to cephalothin (46.1%). Resistance was unevenly distributed and concentrated in a few settlements. β-Lactams accounted for the most frequent resistance phenotypes among the main genera recovered from CMT-positive quarters. These descriptive data support the use of bacterial culture and susceptibility testing to guide mastitis treatment in smallholder herds of the region.
1. Introduction
Milk is an essential component of the human diet and is produced worldwide. Good hygienic practices during milking, storage, and cooling are essential to ensure milk quality and safety. However, these practices remain inadequate in many production systems, particularly in regions with limited technological development. The implementation of good dairy farming practices contributes to improved productivity, product quality, food safety, and sustainability throughout the dairy production chain [1].
Brazil is one of the world’s largest milk producers, with annual production exceeding 34 billion L. Despite its importance, dairy production is characterized by marked heterogeneity in productivity and management practices, particularly among smallholder production systems, where limited technological resources may compromise milk quality and animal health [2]. Family-based dairy farming plays an important socioeconomic role in Brazil, particularly in rural settlements. However, limited access to technology and technical support may impair milk quality, disease control, and farm profitability [3].
Herd health is a key determinant of dairy production efficiency, directly influencing animal productivity and farm economic performance [4]. Mastitis is one of the most significant diseases of dairy cattle and causes substantial economic losses through reduced milk yield, treatment costs, and discarded milk. It also compromises milk quality and may increase the risk of pathogen transmission and antimicrobial residues, representing an animal and public health concern [5].
Bovine mastitis is caused by both contagious and environmental pathogens, particularly Staphylococcus aureus, coagulase-negative Staphylococcus (CNS), Streptococcus spp., Escherichia coli, Klebsiella spp., and other opportunistic bacteria. Intramammary infections are strongly associated with inadequate milking hygiene and poor environmental sanitation [5].
The increasing occurrence of antimicrobial-resistant mastitis pathogens has become a major challenge for dairy production. Inappropriate antimicrobial use and limited microbiological diagnosis contribute to the selection of multidrug-resistant bacteria, compromising treatment efficacy and representing a concern for both animal and public health within the One Health framework [5].
Local data on mastitis-associated bacteria and their antimicrobial susceptibility in smallholder herds of agrarian settlements are scarce. The present study was conducted in family-based dairy herds of agrarian settlements in the Pontal do Paranapanema region, São Paulo State, Brazil. Its objectives were to describe the bacteria recovered from milk samples of CMT-positive mammary quarters and to determine the antimicrobial susceptibility profiles of the isolates obtained.
2. Materials and Methods
2.1. Study Area and Sampling Design
The study was conducted in the Pontal do Paranapanema region, located in the westernmost part of São Paulo State, southeastern Brazil. The region comprises 32 municipalities and is characterized by a tropical climate with a dry season (Aw), with a mean annual temperature of 21.9 °C and mean annual rainfall of 1370 mm [6].
A total of 181 dairy farms located in eight agrarian settlements across different municipalities in the Pontal do Paranapanema region were included in the study, and 724 animals were sampled. A two-stage sampling design (farms, then cows within farms) was used, and sample sizes were calculated with the Epitools epidemiological calculators (Ausvet, Canberra, Australia), assuming diagnostic sensitivity and specificity of at least 80%, an expected prevalence of 10% at the herd level and 50% at the animal level, and a 95% confidence level. All quarters with a CMT score of 2+ or 3+ in the sampled cows were collected, so that more than one quarter per cow could enter the dataset. This calculation was designed for prevalence estimation within the parent project [7] and was not powered for the antimicrobial resistance proportions reported here; these are therefore presented with 95% confidence intervals to convey their precision.
The data analyzed here was generated within the doctoral research of Reyes [7]. The present study reports the bacteriological and antimicrobial susceptibility data in full, with recalculated totals and confidence intervals that were not presented in [7].
2.2. Milk Sample Collection
A total of 2432 milk samples were collected from dairy farms in the Pontal do Paranapanema region from the mammary quarters with a California Mastitis Test (CMT) score of 2+ or 3+. The teats were disinfected with an antiseptic solution, dried with disposable paper towels; then, the first streams of milk were discarded. Subsequently, 10 mL of milk was aseptically collected into sterile Falcon® tubes labeled with the animal identification number and mammary quarter. Samples were kept refrigerated during transport and stored at −20 °C until microbiological analysis.
2.3. Microbiological Analysis
Aliquots of 10 μL of each milk sample were inoculated onto 5% sheep blood agar and incubated aerobically at 37 °C for 48–72 h. The resulting bacterial colonies were characterized based on their size, shape, color, and presence and type of hemolysis. Smears were subjected to Gram staining and examined for cellular morphology, arrangement, and staining characteristics. Bacterial isolates were identified according to the methods described by Quinn et al. [8] and Winn et al. [9], based on colony morphology, Gram-staining results, and biochemical tests, including catalase, oxidase, indole, nitrate reduction, Voges–Proskauer, motility, and sugar fermentation tests. Gram-positive isolates were identified to the genus level, whereas Gram-negative isolates were identified to the family or species level, including Enterobacteriaceae, Escherichia coli, and Pluralibacter gergoviae, or classified as non-fermenting Gram-negative bacteria (NFGNB). Isolates yielding three or more distinct colony types were classified as contaminated.
2.4. Antimicrobial Susceptibility Testing
Antimicrobial susceptibility testing was subsequently performed using the Kirby–Bauer disk diffusion method, following Bauer et al. [8]. The antimicrobials tested were amoxicillin (20 µg) tetracycline (30 µg), chloramphenicol (30 µg), enrofloxacin (10 µg), ampicillin (10 µg), trimethoprim-sulfamethoxazole (25 µg), ceftiofur (30 µg), gentamicin (10 mg), penicillin (10 units), cephalothin (30 µg), streptomycin (10 µg), and neomycin (30 µg). Isolates were classified as susceptible, intermediate, or resistant according to the inhibition zone diameters recommended by Clinical and Laboratory Standards Institute [10]. Since interpretive breakpoints are not established for every organism–drug combination, results for Bacillus spp. and Corynebacterium spp. should be interpreted as reduced in vitro susceptibility rather than as clinical resistance.
2.5. Definitions and Data Analysis
“Sample” refers to the quarter milk specimen and “isolate” to a bacterial colony type recovered from a sample and submitted to susceptibility testing; a mixed culture could therefore contribute more than one isolate [A5]. Culture results are reported per sample, and susceptibility results per isolate tested. In the tables, “NA” indicates that no isolate of that group was tested in that settlement.
The analyses were descriptive. Proportions are reported as n/N (%), and 95% confidence intervals for overall resistance proportions were calculated with the Wilson score method [11] in R (version 4.4.1; R Foundation for Statistical Computing, Vienna, Austria) [A8]. Because settlement-level denominators were often small and isolates were clustered within cows and farms, no statistical comparisons between settlements were performed; the confidence intervals do not account for this clustering and may therefore be narrower than the true uncertainty.
3. Results
3.1. Microbiological Examination of Milk Samples
Of the 2432 samples, 1121 (46.1%) yielded no growth and 15 (0.6%) were contaminated. Bacterial growth was obtained from the remaining 1296 samples (53.3%): 1174 in pure culture and 122 in mixed culture (Table 1). Staphylococcus spp. were the most frequent genus, recovered in pure culture from 639 samples (26.3%) and in total (pure or mixed culture) from 721 samples (29.6%), followed by Bacillus spp. (306 samples in pure culture, 12.6%; 407 in total, 16.7%) and Corynebacterium spp. (93, 3.8%; 123, 5.1%). Enterobacteriaceae, P. gergoviae, E. coli, NFGNB and Streptococcus spp. were each recovered from fewer than 5% of samples. The distribution of genera differed across settlements; Corynebacterium spp., for instance, were recovered in 56 of the 93 pure-culture samples from the São Paulo settlement.
Table 1.
Culture results of quarter milk samples (CMT 2+/3+) from dairy herds in eight agrarian settlements of the Pontal do Paranapanema region, São Paulo, Brazil, 2020. Values are number of samples (% of samples in the settlement).
3.2. Antimicrobial Susceptibility
In total, 648 Staphylococcus, 363 Bacillus, 115 Corynebacterium and 101 Enterobacteriaceae isolates were tested. Overall resistance proportions are given in Table 2. Among Staphylococcus isolates, resistance was most frequent to ampicillin (13.7%), penicillin (12.8%), cephalothin (11.6%) and amoxicillin (9.3%), and it was 4.6% or lower for all non-β-lactam antimicrobials. Among Bacillus isolates, the most frequent phenotypes were resistance to cephalothin (22.0%), amoxicillin (19.8%), penicillin (19.3%), and trimethoprim–sulfamethoxazole (13.5%). In Corynebacterium, 46.1% of isolates were resistant to cephalothin.
Table 2.
Overall antimicrobial resistance of the main bacterial groups isolated from quarter milk samples (CMT 2+/3+) from dairy herds in agrarian settlements of the Pontal do Paranapanema region, São Paulo, Brazil, 2020. Values are number of resistant isolates (%; 95% confidence interval, Wilson method).
Resistance was concentrated in a few settlements (Table 3). Among Staphylococcus isolates, 37 of 74 (50.0%) from the São Paulo settlement were resistant to ampicillin, cephalothin and penicillin, compared with 0–29.0% in the other settlements. Resistance in Bacillus was observed only in the Yapinary, São Paulo, Santa Rosa and Asa Branca settlements. All resistant Corynebacterium isolates came from the São Paulo settlement (e.g., cephalothin, 53/78), and the 37 isolates from the other settlements were susceptible to all drugs tested. Likewise, all resistant Enterobacteriaceae came from the Santa Rosa settlement (ampicillin and streptomycin, 13/58 each; penicillin, trimethoprim–sulfamethoxazole and tetracycline, 9/58 each; amoxicillin, 8/58).
Table 3.
Antimicrobial resistance of Staphylococcus spp. and Bacillus spp. isolates by settlement. Values are number of resistant isolates (% of isolates tested in the settlement).
Among the less frequent groups, P. gergoviae isolates (n = 10) showed resistance to penicillin (4/10), amoxicillin (2/10), and ampicillin (2/10), all from the Santa Rosa settlement. All E. coli (n = 26), NFGNB (n = 5) and Streptococcus spp. (n = 6) isolates were susceptible to every antimicrobial tested.
4. Discussion
Nearly half (46.1%) of the CMT-positive quarter samples yielded no bacterial growth. This proportion may reflect previous antimicrobial treatment, low bacterial load, intermittent shedding, or the limitations of conventional culture [12,13]. Storage of samples at −20 °C before culture may also have reduced bacterial recovery. A positive CMT indicates inflammation, but not necessarily an infection detectable by culture.
Staphylococcus spp. were the most frequently recovered genus, in agreement with previous studies of Brazilian dairy herds, including family-based systems, in which staphylococci are consistently the main mastitis-associated bacteria, while Streptococcus spp., Corynebacterium spp. and Gram-negative bacteria occur at lower frequencies [14,15]. Since staphylococci were identified only to the genus level, the present data cannot distinguish S. aureus from non-aureus staphylococci, which differ in epidemiology, clinical relevance, and resistance profiles. The low frequency of Streptococcus spp. contrasts with some Brazilian reports and may reflect regional differences or the culture conditions used.
The distribution of genera varied among settlements. The São Paulo settlement, for example, accounted for most Corynebacterium isolates. Differences in milking hygiene or in the pre- and post-dipping products used are possible explanations, but these management variables were not recorded in the present study and the hypothesis remains to be tested.
β-Lactams accounted for the most frequent resistance phenotypes in all three predominant genera. In Staphylococcus, overall resistance to ampicillin, penicillin and cephalothin was between 11% and 14%, but it reached 50% in the São Paulo settlement, while resistance to fluoroquinolones and aminoglycosides was low. This pattern is consistent with Brazilian studies reporting resistance of mastitis-associated staphylococci to penicillin and other β-lactams, with preserved susceptibility to ceftiofur, enrofloxacin and several aminoglycosides [14,15]. Streptococcus spp. was rarely isolated and were susceptible to all antimicrobials tested, as was also reported by Lima et al. [14]. Resistance in Bacillus and Corynebacterium should be interpreted cautiously, because validated breakpoints are lacking for several organism–drug combinations and Bacillus spp. are frequently environmental contaminants of milk samples.
Resistant Enterobacteriaceae were found only in the Santa Rosa settlement; they were mainly resistant to ampicillin, streptomycin, amoxicillin, penicillin, trimethoprim–sulfamethoxazole and tetracycline, whereas Lima et al. [14] observed that Gram-negative isolates were susceptible to all the antimicrobials tested. NFGNB and P. gergoviae (formerly Enterobacter gergoviae) were recovered at low frequency. NFGNB are generally regarded as environmental, opportunistic organisms that can reach milk through contaminated water, soil, bedding or equipment [16,17], and P. gergoviae is an uncommon opportunistic organism mainly reported from environmental, food and human clinical sources, whose role in bovine mastitis is poorly understood [16]. Because no environmental sampling or source tracing was performed, the present study can only document that these organisms were recovered infrequently.
Antimicrobial resistance in mastitis pathogens is influenced by antimicrobial exposure, pathogen ecology, farm management and the circulation of resistance genes [17,18]. Since no farm-level data on antimicrobial use were collected, the observed resistance phenotypes cannot be attributed to particular treatment practices. Nevertheless, the concentration of resistance in a few settlements suggests that local susceptibility data are needed to guide pathogen-directed treatment decisions [18,19].
This study has limitations. Only quarters with CMT scores of 2+ or 3+ were sampled, so the results cannot be extrapolated to all cows or to the bulk milk of the participating farms. Gram-positive isolates were identified only to the genus level, and no molecular confirmation of identity or resistance mechanisms was performed. Isolates were clustered within cows and farms, settlement-level denominators were often small, and the confidence intervals do not account for clustering. Finally, the samples were collected in 2020, and resistance patterns and antimicrobial use practices may have changed since then.
5. Conclusions
In CMT-positive quarters from dairy herds in agrarian settlements of the Pontal do Paranapanema region, Staphylococcus spp., Bacillus spp. and Corynebacterium spp. were the bacteria most frequently recovered, and β-lactams accounted for the most frequent resistance phenotypes. Resistance was concentrated in a few settlements, which supports the use of bacterial culture and susceptibility testing to guide mastitis treatment in these herds. Species-level identification and molecular characterization of resistance would be valuable in future research.
Author Contributions
Conceptualization, M.A.R.A., Alessandra Figueiredo de Castro Nassar and Lilian Gregory; methodology, M.A.R.A., A.F.d.C.N., J.S.C., N.C.G., J.M.B.P., G.G.C. and L.G.; investigation, M.A.R.A., A.F.d.C.N., A.C. and D.M.F.V.; formal analysis, M.A.R.A., A.F.d.C.N., J.S.C. and N.C.G.; data curation, G.G.C. and A.C.; writing—original draft preparation, M.A.R.A., A.F.d.C.N., J.S.C. and N.C.G.; writing—review and editing, A.C., D.M.F.V. and L.G.; supervision, L.G. All authors have read and agreed to the published version of the manuscript.
Funding
This study was funded by the São Paulo Research Foundation (FAPESP, Brazil) under grant number 2017/12271-0, for the project “Evaluation of the Milk Production Chain in the Pontal do Paranapanema Region” (“Avaliação da Cadeia Produtiva do Leite no Pontal do Paranapanema”).
Institutional Review Board Statement
This study was approved by the Animal Use Ethics Committee (CEUA) from the School of Veterinary Medicine and Animal Science of the University of São Paulo under protocol number 9737230217. All animal procedures were conducted in accordance with the institutional guidelines for the care and use of animals in research and complied with the applicable ethical standards for animal experimentation.
Informed Consent Statement
Informed consent for participation was obtained from all subjects involved in the study.
Data Availability Statement
The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.
Conflicts of Interest
The authors declare no conflicts of interest.
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