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Article

Epidemiological Investigation on Pathogenic Bacteria of Buffalo Subclinical Mastitis and Their Antibiotic Resistance and Virulence Characteristics in Guangxi, China

by
Ling Li
1,2,
Jiaping Zhang
2,
Xingqi Wei
1,
Ruimin Wang
2,
Xia Dan
2,
Jianfeng Li
1,
Enghuan Hau
2,
Qingkun Zeng
2,
Qingyou Liu
1,3,
Jiafeng Ding
1,* and
Kuiqing Cui
1,3,*
1
College of Animal Science and Technology, Guangxi University, Nanning 530004, China
2
Guangxi Zhuang Autonomous Region Buffalo Milk Quality and Safety Control Technology Engineering Research Center, Guangxi Buffalo Research Institute, Chinese Academy of Agricultural Sciences, Nanning 530001, China
3
Guangdong Provincial Key Laboratory of Animal Molecular Design and Precise Breeding, School of Life Science and Engineering, Foshan University, Foshan 528225, China
*
Authors to whom correspondence should be addressed.
Animals 2025, 15(22), 3321; https://doi.org/10.3390/ani15223321
Submission received: 23 September 2025 / Revised: 3 November 2025 / Accepted: 14 November 2025 / Published: 18 November 2025
(This article belongs to the Section Cattle)
Browse Figures
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Simple Summary

Subclinical mastitis (SCM) is a major but often neglected issue in dairy farming, affecting milk yield and quality. Identification of pathogenic bacteria of subclinical mastitis (PSM) in dairy cows is crucial for implementing effective prophylactic and control measures. This paper highlights the first systematic study to investigate the PSM in buffalo farms in Guangxi, China. It also analyzes the antibiotic resistance and virulence characteristics of typical PSM. A total of 1659 bacterial strains were isolated from 132 milk samples with SCM, among which 1058 were identified as PSM. Coagulase-negative Staphylococci (55.30%), Enterococcus faecalis (51.52%), Escherichia coli (31.82%), and Klebsiella pneumoniae (28.03%) were frequently isolated PSM in total samples. All PSM strains showed multiple-antibiotic resistance. E. faecalis and Lactococcus garvieae were resistant to all 12 antibiotics. E. coli exhibited the strongest mortality of Galleria mellonella. These results highlight that the prevention and control of PSM in buffalo farms should be strengthened.

Abstract

Subclinical mastitis (SCM) is one of the most common and detrimental diseases affecting dairy cows, causing lower milk yield and quality. Identification of pathogenic bacteria of subclinical mastitis (PSM) in dairy cows is crucial for selecting suitable antibiotic treatments and implementing effective prophylactic measures. This paper highlights the first systematic study to investigate the PSM in buffalo farms in Guangxi, China. It also analyzes the antibiotic resistance and virulence characteristics of typical PSM. The bacteriological characteristics of 132 milk samples collected from buffaloes with SCM across 3 representative buffalo farms in Guangxi, China were investigated. A total of 1659 bacterial strains were isolated and classified into 46 genera and 183 species, where 1058 bacterial strains were identified as PSM, representing 64% of the total isolates. The frequently isolated PSM in total samples were coagulase-negative Staphylococci (55.30%), Enterococcus faecalis (51.52%), Escherichia coli (31.82%), and Klebsiella pneumoniae (28.03%). All PSM strains showed multiple antibiotic resistance. Notably, E. faecalis and Lactococcus garvieae were resistant to all 12 antibiotics, whereas Staphylococcus chromogenes (95.24%), E. coli (89.19%), K. pneumoniae (83.87%), and Staphylococcus epidermidis (83.33%) were sensitive to levofloxacin (LEV). Additionally, E. coli exhibited the strongest mortality of Galleria mellonella. This study concluded that multiple PSM are present in the milk of buffaloes suffering from SCM in Guangxi, China. LEV may be a suitable antibiotic for the treatment of PSM. In the future, it is necessary to monitor the prevalence of PSM in buffalo farms and develop control strategies to prevent their spread.

1. Introduction

Buffalo milk is the second most consumed milk globally, second only to dairy cow milk [1]. It contains higher levels of protein, total solids, and vitamins compared to dairy cow milk [2]. Subclinical mastitis (SCM) is one of the most prevalent and damaging diseases affecting dairy animals. Apart from reducing milk yield and quality, SCM also increases the cost of breeding and processing, impedes the development of the dairy sector and poses potential risks to public health [3,4,5]. SCM is defined as an elevated level of milk somatic cell count (SCC), at which raw milk from healthy dairy cows has less than 1.5 × 105/mL SCC [6,7].
The pathogenesis of SCM in dairy cows is complex, with pathogenic bacterial infections serving as its main contributing factors [8]. Many bacterial species have been linked to bovine mastitis (Supplementary Table S1), including coagulase-negative Staphylococci (CoNS) [9], Staphylococcus aureus [10], Klebsiella pneumoniae [11], Escherichia coli, Macrococcus caseolyticus [12], Lactococcus garvieae [13] and others. CoNS is the primary pathogen causing SCM in dairy cows, including Staphylococcus chromogenes, Staphylococcus haemolyticus, Staphylococcus epidermidis, Staphylococcus simulans, and Staphylococcus xylosus [9,14,15]. It is worth noting that not all CoNS are pathogenic bacteria of subclinical mastitis (PSM), and no studies have shown that Staphylococcus cohnii, Staphylococcus capitis, and Staphylococcus edaphicus belong to PSM.
There are obvious regional differences in the distribution of buffalo PSM. Staphylococcus spp. and E. coli are the most commonly isolated buffalo PSM, followed by Streptococcus spp. and Klebsiella spp. in northwestern Pakistan [4]. On the other hand, Staphylococcus spp. are the most prevalent and commonly isolated PSM strains, followed by E. coli, Pseudomonas spp., and Bacillus spp. in the Punjab province of Pakistan [16]. A survey in 2007 showed that the detection ratio of E. coli and S. aureus in raw buffalo milk samples from Guangxi and Yunnan provinces of China was high, but PSM was not isolated for targeted research [17]. China is one of the world’s leading producers of buffalo milk, with the primary production area in Guangxi Province [17]. Nevertheless, there is limited data on the specific pathogens affecting buffaloes in this region.
Antibiotic resistance in bacteria becomes more and more challenging [4,18,19]. Most buffalo PSM were resistant to sulfamethoxazole (99.3%), lincomycin (98%), oxytetracycline (89.3%), and ampicillin (AMP, 86.1%) in Northwest Pakistan [4]. An antibiotic resistance test of E. coli and K. pneumoniae from buffalo in Bangladesh showed that these strains revealed significant resistance to AMP, amoxicillin-clavulanic acid, and aminoglycosides, with 31.5% of E. coli and 39.3% of K. pneumoniae isolates exhibiting multiple antibiotic resistance [18]. It is worth noting that the virulence of PSM is also severe. The mortality of Galleria mellonella larvae was 60% after 48 h of infection with E. coli [20]. On the other hand, 75% of G. mellonella larvae died after 24 h of infection with K. pneumoniae, while the remaining survivors died within 72 h [21]. Hence, it is essential to develop effective strategies to reduce the spread of pathogens in cattle herds within particular geographic areas by understanding the population structure, spread, virulence traits, and antibiotic resistance of PSM. It is worth noting that there are few systematic studies on the antibiotic resistance and virulence of Chinese buffalo PSM.
This study aimed to investigate the prevalence of PSM in buffalo farms in Guangxi, China. The antibiotic resistance and virulence of typical PSM were further analyzed by the disk diffusion method and G. mellonella larvae infection test. This study will provide an important basis for the prevention and treatment of buffalo SCM in Guangxi.

2. Materials and Methods

2.1. Collection of Samples

Three representative buffalo farms were investigated in Guangxi, China. The farms were labeled as Herd A, Herd B, and Herd C. Herd A is the major buffalo breeding farm in China, comprising purebred Murrah buffalo, purebred Nile/Rafi buffalo, and a variety of hybrid dairy buffalo. Herds B and C were large commercial buffalo farms that primarily raised hybrid dairy buffaloes.
Previous studies have shown that SCM increases the SCC in milk [6,7]. In healthy cows, milk SCC is typically below 1.5 × 105/mL, and some even below 1 × 105/mL [6,22]. Based on this standard, milk samples with SCC greater than 1.5 × 105/mL are classified as coming from SCM cows.
Raw buffalo milk samples were collected randomly from three representative buffalo farms in the morning and afternoon from March 2023 to January 2024. Sample collection and procedures were performed according to the steps of Ranasinghe et al. [23]. The udder was disinfected with 1.0% (m/v) povidone iodine before sampling. Then the first three streams of milk were discarded, followed by buffalo milk samples collected in sterile tubes and stored at 4 °C. The SCC analysis was conducted within 2 h using BacsomaticTM (FOSS, Hillerød, Denmark), strain isolation was performed on SCM milk samples within 24 h of sampling.

2.2. Isolation of Bacteria

There were two methods applied to separate PSM: (a) direct spread [11,24] and (b) enrichment before spread [25,26,27]. The separation process is shown in Figure 1.
(a)
Direct spread: 0.1 mL of milk samples was directly spread on chromogenic S. aureus agar, mannitol salt agar, Baird Parker agar, blood agar, and MacConkey agar. Then, plates were incubated at 37 °C for 24 h to 48 h.
(b)
Enrichment before spread: 5 mL of milk samples was added to 45 mL of brain heart infusion, azide dextrose broth, and Luria broth, respectively, and cultured at 37 °C for 18 h to 24 h. One milliliter of cultures was added into a sterile test tube having 9 mL of sterile water. After mixing, the culture was serially diluted up to 1: 105. Then, 0.1 mL of each dilution was spread to corresponding agar plates (Figure 1) and incubated at 37 °C for 24 h to 48 h. The culture enriched by brain heart infusion was spread on chromogenic S. aureus agar, mannitol salt agar, Baird Parker agar, and blood agar. The culture enriched by azide dextrose broth was spread on Baird Parker agar and blood agar. The culture enriched by Luria broth was spread on Baird Parker agar, blood agar, and MacConkey agar.
Single colonies picked from the above plates were streaked on tryptic soy agar to prepare pure cultures. All culture media were purchased from Qingdao Haibo Biology Company (Qingdao, China).

2.3. Identification of Pathogenic Bacteria of Subclinical Mastitis

The biochemical and 16S ribosomal ribonucleic acid (16S rRNA) of the strain were determined based on the microbial identification procedure described in Figure 1 [11], in order to determine the PSM species. The biochemical tests flowchart in Figure 1 illustrates the identification process for coagulase-positive Staphylococci, CoNS, E. coli, and K. pneumoniae. In detail, coagulase-positive Staphylococci, CoNS were confirmed by gram stain, catalase test, and coagulase test [28]. E. coli and K. pneumoniae were confirmed by Gram-stain, capsular staining, lactose fermentation, indole, Voges-Proskauer, methyl red, and citrate utilization tests [24,29].
The well-established method was utilized to extract deoxyribonucleic acid (DNA) from PSM as previously described by de Boer et al. [30]. Further, the extracted DNA was used for polymerase chain reaction (PCR) amplification by the ProFlex PCR System (Life Technologies, Foster City, CA, USA). The reaction was prepared as follow; 12.5 µL of 2× Super Flash Master Mix (Kangwei Century, Taizhou, China), 0.5 µL of 27F (AGAGTTTGATCMTGGCTCAG), 0.5 µL of 1492R (GGTTACCTTGTTACGACTT), 0.5 µL of extracted DNA, and 11 µL of double distilled water. Primers 27F and 1492R were biosynthesized by Beijing Qingke Biotechnology Co., Ltd. (Beijing, China). Amplification consisted of one cycle of initial denaturation at 95 °C for five minutes, followed by 34 cycles of denaturation at 94 °C for 60 s, annealing at 55 °C for 45 s, and extension at 72 °C for 90 s; and a final extension cycle at 72 °C for ten minutes. The 16S rRNA was determined by Beijing Qingke Biotechnology Co., Ltd. (Beijing, China) and compared by BLAST in NCBI website (https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 30 January 2024)).
A list of reported PSM isolates has been collected (Supplementary Table S1). The strains isolated in this study were compared with the Supplementary Table S1 to determine whether our isolates were PSM.

2.4. Screening Principles for Strains Used in Antibiotic Resistance and Galleria mellonella Larvae Infection Tests

The typical PSM were selected as the research subjects to investigate the antibiotic resistance and virulence characteristics of buffalo PSM in Guangxi, China. Total sample isolation ratios of typical PSM were more than 20% (Table 1), including Enterococcus faecalis, K. pneumoniae, E. coli, L. garvieae, and CoNS (Staphylococcus chromogenes and Staphylococcus epidermidis). The 16S rRNA phylogenetic tree of 6 typical PSM was constructed by MEGA 11, and the clustering was carried out according to the standard of 16S rRNA gene sequence similarity ≥ 99%. In each cluster, only individual strains isolated from the milk samples of the same buffalo at the same time were retained. After the above screening, 147 representative strains were finally screened from 6 typical PSM for subsequent antibiotic resistance and G. mellonella larvae infection tests. The numbers of strains tested were as follows: 29 E. faecalis, 31 K. pneumoniae, 37 E. coli, 21 S. chromogenes, 11 L. garvieae, and 18 S. epidermidis (Table 1).

2.5. Analysis on Antibiotic Resistance of Typical PSM

The disk diffusion method was used to perform antibiotic resistance testing on Mueller-Hinton agar plates in accordance with the guidelines established by the Clinical and Laboratory Standards Institute [31].
Based on the veterinary antibiotic list of the World organization for Animal Health (https://www.woah.org/en/document/list-of-antimicrobial-agents-of-veterinary-importance/ (accessed on 29 July 2024)), twelve veterinary antibiotics spanning nine different antibiotic classes were selected for antibiotic resistance test. The following antibiotics purchased from Macklin (Shanghai, China) were used: penicillin (ampicillin 10 µg, penicillin 30 IU), aminoglycosides (amikacin 30 µg, gentamicin 30 µg), quinolones (levofloxacin 30 µg, ciprofloxacin 5 µg), cephalosporins (ceftriaxone 30 µg), tetracycline (tetracycline 30 µg), β-lactam (amoxicillin 20 µg), macrolides (azithromycin 15 µg), chloramphenicol (chloramphenicol 30 µg), and folate pathway antagonists (trimethoprim 10 µg). The antibiotic resistance of the strain was categorized as sensitive (S), intermediate (I), or resistant (R), based on the inhibition zone diameters on the plate.

2.6. Analysis on Virulence of Typical PSM

G. mellonella larvae have emerged as a useful insect model in research on host–pathogen interactions [32], and the reliability of the larvae infection tests on PSM (E. faecalis, K. pneumoniae, E. coli) virulence have been validated by several investigations [33,34,35]. Hence, the current study also applied the similar method to test on the virulence of typical PSM (Laughing Monkey Information Technology Company, Chongqing, China).
The appropriate colony concentration was selected according to the previous research and pre-experimental results [21,36,37,38], injected into the larvae of G. mellonella. The experimental concentrations of E. coli [36] and K. pneumoniae [21] were 103 CFU/larvae, those of S. chromogenes [37], L. garvieae [37], and S. epidermidis [37] were 104 CFU/larvae, and that of E. faecalis [38] was 105 CFU/larvae. Bacterial suspensions (10 µL) were inoculated on the last left worm proleg, and the G. mellonella larvae were incubated at 37 °C (negative controls were only inoculated with 10 µL saline). Each strain was inoculated into 10 G. mellonella larvae, the mortality of G. mellonella larvae were observed and recorded every 12 h.

2.7. Data Processing

Excel spreadsheets were used for meticulous compilation and classifying the data. SPSS version 26 was used to perform all the statistical analyses. Origin 2025 was used to draw all figures. Adobe Illustrator 2024 was used for figures retouching.

3. Results

3.1. Prevalence and Isolation of Buffalo PSM in Different Farms

Bacteriological examination was performed on 132 milk samples collected from buffaloes with SCM across 3 representative buffalo farms in Guangxi, China. A total of 1659 bacterial strains encompassing 183 species across 46 genera were identified based on biochemical and 16S rRNA analysis (Supplementary Table S2). As shown in Figure 2A, Enterococcus spp. (31.65%) was the most isolated genera in total samples, followed by Staphylococcus spp. (15.97%). The dominant genera were Enterococcus spp. (34.18%) and Staphylococcus spp. (12.27%) in Herd A, while Staphylococcus spp. (31.25%), Escherichia spp. (18.75%), and Aerococcus spp. (17.71%) in Herd B. In contrast to Herd A, Staphylococcus spp. (40.52%) was more dominant, followed by Enterococcus spp. (27.45%) in Herd C.
A total of 1058 pathogenic bacteria of subclinical mastitis (PSM) were identified (Supplementary Tables S1 and S2). PSM accounted for over 62% of all the isolates across the three herds, with Herd C having the highest PSM isolation ratio, representing 80% of all the isolates (Figure 2B).
Strains with an isolation ratio of more than 20% in total samples were regarded as typical PSM (Table 1), including Enterococcus faecalis, Klebsiella pneumoniae, Escherichia coli, Staphylococcus chromogenes, Staphylococcus epidermidis, and Lactococcus garvieae. Figure 3 shows the colony morphology and Gram-staining results of typical PSM. As shown in Table 1, E. faecalis was the most frequently isolated strains. A total of 386 E. faecalis strains were isolated from 68 samples, which were mainly distributed in the samples of Herd A (52.17%) and Herd C (88.89%). In addition, 85 strains of K. pneumoniae were isolated from 37 samples, which were isolated solely from samples of Herd A (32.17%). A total of 75 strains of E. coli were isolated from 42 samples, which were distributed in the samples of Herd A (24.35%), Herd B (75.00%), and Herd C (88.89%). Next, 58 strains of L. garvieae were isolated from 31 samples, which were isolated from samples of Herd A (26.09%) and Herd C (11.11%). Notably, 257 strains of CoNS were isolated from 73 samples, and 197 CoNS strains were PSM. No studies have shown that another 60 CoNS strains are associated with subclinical mastitis in dairy cows. The main CoNS were S. chromogenes, and a total of 59 S. chromogenes strains were isolated from 25 samples from Herd A (13.04%), Herd B (25.00%), and Herd C (88.89%).
Macrococcus caseolyticus (n = 59), Enterobacter cloacae (n = 33), Mammaliicoccus sciuri (n = 29), Staphylococcus haemolyticus (n = 28), Staphylococcus borealis (n = 24), and Aerococcus viridans (n = 20) were also frequently isolated PSM. In addition, Acinetobacter baumannii (n = 42), Enterococcus gallinarum (n = 40), Streptococcus macedonicus (n = 25), Kurthia gibsonii (n = 25), and Staphylococcus cohnii (n = 20) were not PSM, but their isolated strains were also greater than 20.

3.2. Antibiotic Resistance of Typical PSM

As shown in Figure 4, significant resistance was observed in six typical PSM against ciprofloxacin (CIP, 100%), amikacin (AMI, 97.96%), and azithromycin (AZI, 95.92%). In contrast (Figure 4), K. pneumoniae (12.90%), E. coli (5.41%), S. chromogenes (4.76%), and S. epidermidis (11.11%) showed low resistance to levofloxacin (LEV). Among 147 representative PSM strains (Figure 5), all of them showed multiple antibiotic resistance (resistance to ≥3 antibiotic classes). As shown in Figure 5, E. faecalis and L. garvieae were resistant to all 12 antibiotics, whereas K. pneumoniae (26/31, 83.87%), E. coli (33/37, 89.19%), and S. chromogenes (20/21, 95.24%) were sensitive to LEV. S. epidermidis was sensitive to gentamicin (16/18, 88.88%) and LEV (15/18, 83.33%). Figure 6A–C shows the experimental phenomenon of antibiotic resistance in typical PMS.

3.3. Virulence of Typical PSM

Galleria mellonella larvae were used as an in vivo model to assess the pathogenicity of typical PSM strains. Healthy G. mellonella larvae was pale yellow (Figure 6D), whereas the body color gradually blackened after infection with pathogenic bacteria (Figure 6E). The body color of the dead larvae was dark black (Figure 6F). All six typical PSM showed pathogenicity against G. mellonella larvae (Figure 7). E. coli (103 CFU/larvae) exhibited the strongest mortality against G. mellonella larvae, at which 10 out of 37 E. coli strains induced a mortality rate of more than 90% at 12 h post-injection, and 29 out of 37 E. coli strains induced a mortality rate exceeding 90% at 36 h post-injection. Moreover, 18 out of 31 K. pneumoniae (103 CFU/larvae) strains, 4 out of 11 L. garvieae (104 CFU/larvae) strains, and 6 out of 29 E. faecalis (105 CFU/larvae) strains induced a mortality rate exceeding 90% in G. mellonella larvae at 36 h post-injection. However, only 1 out of 21 S. chromogenes (104 CFU/larvae) strains and 1 out of 18 S. epidermidis (104 CFU/larvae) strains induced a mortality rate exceeding 90% in G. mellonella larvae at 36 h post-injection.

4. Discussion

SCM is an important issue in the buffalo breeding industry, which will reduce milk yield and quality [4]. The occurrence of mastitis in dairy cows is complex, and pathogenic bacterial infection is one of the influencing factors [8]. In our study, Enterococcus faecalis, CoNS, Klebsiella pneumoniae, and E. coli were the most frequently isolated PSM, and their total sample isolation ratios were all greater than 28% (Table 1). Staphylococcus spp. were the most commonly isolated PSM in buffalo farms, followed by K. pneumoniae and E. coli in Pakistan [4,16], whereas K. pneumoniae and E. coli were the most commonly isolated PSM in some dairy farms in China [11,39]. Additionally, Enterococcus spp. showed the highest average relative abundance, followed by Staphylococcus spp. and Lactococcus spp. in a metagenomic analysis of mastitic buffalo milk samples from India [40]. Existing studies have shown that the differences in the geographical distribution of PSM are related to variety of factors, including internal factors (age, parity, lactation stage and health status) and extrinsic factors (udder hygiene, padding, milking machine, management, climate and region) [39].
E. faecalis is one of the most isolated PSM and is ubiquitous in the gastrointestinal tract of mammals and the environment [41,42]. In this study, 386 strains of E. faecalis were isolated from 132 milk samples (Table 1). Różańska et al. isolated 360 strains of E. faecalis from 426 milk samples suspected of mastitis [43]. However, the isolation ratios of E. faecalis in milk samples from the Czech Republic and northeastern Brazil were 20.9% and 26.3%, respectively [44,45]. In addition, the isolation ratio of E. faecalis was only 4.5% in dairy cow PSM from 62 commercial farms in 15 provinces of China (few buffalo samples) [46]. The difference in the distribution of E. faecalis may be associated with feces and equipment in contact with feces, the study of Lee et al. supported this speculation [47].
CoNS is the main pathogen causing SCM in dairy cows [9,14]. The mechanism of CoNS causing SCM is complex and species-specific, primarily leading to the occurrence of SCM through a multi-faceted synergistic action involving adhesion colonization, immune evasion, destruction of mammary tissue, and interference with host metabolism [48]. A total of 257 strains of CoNS were isolated in this study, of which 197 were PSM (Table 1). The CoNS species differ significantly depending on the region. Staphylococcus chromogenes, S. simulans, S. xylosus, and Staphylococcus cohnii were the major PSM associated with bovine mastitis in three dairy farms in Flanders, Belgium [49]. However, the isolation ratios of S. chromgenes, S. epidermidis, and Staphylococcus haemolyticus were higher in Bangladesh [50]. An extensive survey of the prevalence of CoNS in major dairy cows in China revealed that S. chromogenes (33%) exhibited the highest isolation ratio, followed by S. epidermidis (8%) [51]. Aligned to the previous literature, the current study also found that S. chromogenes, S. epidermidis, and S. haemolyticus were still the major CoNS in Guangxi Province, China (Table 1). Additionally, the isolation count of Staphylococcus borealis (n = 24) in this region was at an alarming level. Relevant literature shows that the use of milking machine will increase the risk of mastitis in cows [52]. This is because the milking machine will cause the nipple tube to remain open after milking, and the Staphylococcus attached to the pipe of the breast pump can easily enter the cow ‘s breast [53].
E. coli and K. pneumoniae were also typical PSM isolates in this study, with isolation ratios of 31.82% and 28.03% of total samples (Table 1), respectively. These sample isolation ratios were higher than an epidemiological survey of E. coli (13.38%) and K. pneumoniae (13.96%) in buffalo SCM samples in Bangladesh [18]. However, our results were comparable to those samples in Xinjiang, China where the isolation ratio of K. pneumoniae in SCM samples was 23.2%, and those samples in Romania where the isolation ratio of E. coli in mastitis samples was 27.51% [54]. Both E. coli and K. pneumoniae are environmental pathogens, and padding, milking machines, feces and equipment in contact with feces are their sources of pollution [55].
In the current study, Aerococcus spp. was the characteristic genus in Herd B (Figure 2A), where Aerococcus viridans was the most isolated strain of Aerococcus spp. (Table 1). A total of 20 A. viridans strains were isolated from all three herds. A. viridans is considered an emerging etiological agent of bovine SCM, wherein it exerts a negative effect on somatic cell count, milk yield, and composition [56]. A total of 69 strains of A. viridans were isolated from 1774 mastitis milk samples collected in Korea from 2016 to 2021, accounting for 3.9% of isolation counts [57]. This study is the first report on the prevalence of Aerococcus viridans in Chinese buffaloes.
Antibiotic resistance has emerged as an alarming concern in PSM from buffaloes [57]. In our study, 147 representative PSM strains from buffalo exhibited multiple antibiotic resistance (Figure 5), surpassing the levels of resistance in cattle reported in previous studies [4,45]. Previous literature reported that 72 strains of PSM were resistant to penicillin (PEN, 79.1%), AMP (77.7%), and tetracycline (TET, 63.8%), with 83.4% of PSM showed multidrug resistance in northeastern Brazil [46]. In contrast, most of the PSM were resistant to sulphamethoxazole (99%), lincomycin (98%), oxytetracycline (89%), AMP (86%), and doxycycline (85%) in the cattle and buffalo farms of northeast Pakistan [4].
In this study (Figure 4), E. faecalis and Lactococcus garvieae exhibit high resistance to multiple antibiotics because they are prone to transferring resistance genes, that cause widespread resistance to antibiotics [58,59]. All E. coli strains exhibited strong antibiotic resistance, with 100% resistance to AMP, PEN, AMI, and CIP (Figure 4), aligned to the antibiotic resistance in cow mastitis [19,60,61]. Similarly, K. pneumoniae also exhibited 100% resistance to AMI, CIP, and AZI, while its resistance towards AMP, PEN, and amoxicillin (AMOX) had exceeded 93% (Figure 4). Consistent with earlier findings, K. pneumoniae from dairy cows was typically resistant to AMP [62], chloramphenicol [63], TET [63], and AMOX [64]. What’s more, the total antibiotic resistance of S. chromogenes and S. epidermidis was similar, although some differences between individual strains were observed (Figure 4 and Figure 5). Previous studies also reported the resistance of CoNS (including S. chromogenes and S. epidermidis) to antibiotics such as PEN [65], TET [66] and AMP [67].
It is worth noting that LEV showed the highest antibiotic sensitivity to 147 representative strains (Figure 5). LEV is the third generation of quinolones, exhibiting greater activity against pathogens compared to the first two generations of quinolones [68]. LEV has been used as a veterinary drug in China, Russia, the United States, Argentina, and India [69]. A previous study revealed that E. coli strains isolated from cattle and buffalo farms in Bangladesh were sensitive to LEV [70]. However, 52% (n = 44) of E. coli isolated from buffalo mastitis samples in Karachi, Pakistan were resistant to LEV [71]. The current study reported that only 5.41% (n = 33) of E. coli strains were resistant to LEV (Figure 5). At present, no study has systematically evaluated the sensitivity of LEV to buffalo PSM, hence this study serves as the first report. This suggests that we need to carry out targeted research to evaluate the therapeutic effect of LEV in buffalo mastitis.
The Galleria mellonella larvae infection test is ideal for evaluating the virulence of pathogenic microorganisms [72,73]. Among the six typical PSM, E. coli showed the strongest mortality of G. mellonella larvae (Figure 7). E. coli is one of the leading pathogens of bovine mastitis, which can cause subclinical and clinical mastitis characterized by systemic changes, abnormal milk appearance, and udder inflammation [74]. K. pneumoniae also exhibited a high mortality rate in G. mellonella larvae (Figure 7), aligned with the findings of Mai et al. [34]. K. pneumoniae contains capsular polysaccharides, lipopolysaccharides, pili, siderophores, and other pathogenic factors [75]. It is an environmental pathogen that can enter the breast through the nipple, causing inflammation of the breast [76,77]. Both S. chromogenes (4.76%) and Staphylococcus epidermidis (5.55%) exhibited low mortality for G. mellonella larvae. Because their pathogenicity is weak in a short period of time, their pathogenicity to G. mellonella larvae is persistent. When they undergo long-term and mass reproduction, they will cause serious consequences [78].

5. Conclusions

This study investigated the prevalence of PSM in Guangxi, China. A total of 1058 PSM were identified, which were from 95.45% of total samples with coagulase-negative Staphylococci (55.30%), Enterococcus faecalis (51.52%), Klebsiella pneumoniae (28.03%), and Escherichia coli (31.82%). All PSM strains showed multiple antibiotic resistance. Levofloxacin may be a suitable antibiotic for the treatment of PSM. E. coli and K. pneumoniae showed the high mortality of Galleria mellonella larvae. In this paper, the pathogenic bacteria, drug resistance, and virulence characteristics of subclinical mastitis in Guangxi buffalo were systematically studied for the first time. In the future, it will be necessary to monitor the emergence of PSM in buffalo farms and develop control strategies to prevent their spread.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ani15223321/s1, Table S1: The number of pathogenic bacteria of subclinical mastitis isolated and the basis for judgment; Table S2: Biochemical and 16S rRNA test results of 1659 bacterial strains.

Author Contributions

Conceptualization, L.L., Q.L., J.D. and K.C.; methodology, L.L.; software, J.Z., X.W. and J.L.; validation, J.D. and K.C.; formal analysis, L.L. and J.Z.; investigation, L.L., J.Z., X.W., R.W., X.D. and J.L.; resources, L.L. and Q.Z.; data curation, L.L., J.Z., R.W. and X.D.; writing—original draft preparation, L.L. and J.Z.; writing—review and editing, L.L., J.Z., E.H., J.D. and K.C.; visualization, L.L., J.Z. and E.H.; supervision, Q.L., J.D. and K.C.; project administration, L.L.; funding acquisition, L.L., Q.Z. and K.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Key Research and Development Program of China, grant number 2024YFD1301300; the Science and Technology Major Project of Guangxi, grant number AA22068099.

Institutional Review Board Statement

The animal study protocol was approved by Animal Ethics Committee of Guangxi University (approval no. GXU-2023-270) on 12 February 2023.

Informed Consent Statement

Written informed consent has been obtained from the owner of the animals involved in this study.

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Abbreviations

The following abbreviations are used in this manuscript:
SCMSubclinical mastitis
PSMPathogenic bacteria of subclinical mastitis
CoNSCoagulase-negative Staphylococci
SCCSomatic cell count
16S rRNA16S ribosomal ribonucleic acid
DNADeoxyribonucleic acid
PCRPolymerase chain reaction
SSensitive
IIntermediate
RResistant
AMPAmpicillin
PENPenicillin
AMIAmikacin
GENGentamicin
LEVLevofloxacin
CIPCiprofloxacin
AZIAzithromycin
CHLChloramphenicol
TXCeftriaxone
AMOXAmoxicillin
TETTetracycline
TRTrimethoprim

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Animals 15 03321 g001
Figure 1. Isolation and identification process of PSM from buffalo milk. + and − indicate positive and negative results for the indicators, respectively.
Figure 1. Isolation and identification process of PSM from buffalo milk. + and − indicate positive and negative results for the indicators, respectively.
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Figure 2. (A) Bacterial isolation of milk samples from buffaloes with subclinical mastitis across different farms at genus level. (B) The separation ratio of pathogenic bacteria of subclinical mastitis in each farm. The numbers of strains isolated from total, Herd A, Herd B, and Herd C were 1659, 1410, 96 and 153, respectively.
Figure 2. (A) Bacterial isolation of milk samples from buffaloes with subclinical mastitis across different farms at genus level. (B) The separation ratio of pathogenic bacteria of subclinical mastitis in each farm. The numbers of strains isolated from total, Herd A, Herd B, and Herd C were 1659, 1410, 96 and 153, respectively.
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Figure 3. Colony morphology and Gram-staining (100×) of typical PSM. (A) Colony morphology of E. faecalis, (B) Colony morphology of K. pneumoniae, (C) Colony morphology of E. coli, (D) Gram staining of E. faecalis, (E) Gram staining of K. pneumoniae, (F) Gram staining of E. coli, (G) Colony morphology of S. chromogenes, (H) Colony morphology of L. garvieae, (I) Colony morphology of S. epidermidis, (J) Gram staining of S. chromogenes, (K) Gram staining of L. garvieae, (L) Gram staining of S. epidermidis.
Figure 3. Colony morphology and Gram-staining (100×) of typical PSM. (A) Colony morphology of E. faecalis, (B) Colony morphology of K. pneumoniae, (C) Colony morphology of E. coli, (D) Gram staining of E. faecalis, (E) Gram staining of K. pneumoniae, (F) Gram staining of E. coli, (G) Colony morphology of S. chromogenes, (H) Colony morphology of L. garvieae, (I) Colony morphology of S. epidermidis, (J) Gram staining of S. chromogenes, (K) Gram staining of L. garvieae, (L) Gram staining of S. epidermidis.
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Figure 4. The antibiotic resistance of typical PSM. The deeper the red, the stronger the antibiotic resistance. Numbers indicate the percentage (%) of antibiotic-resistant strains in the strain. Blue fonts represent antibiotic classes. AMP, ampicillin; PEN, penicillin; AMI, amikacin; GEN, gentamicin; LEV, levofloxacin; CIP, ciprofloxacin; AZI, azithromycin; CHL, chloramphenicol; TX, ceftriaxone; AMOX, amoxicillin; TET, tetracycline; TR, trimethoprim.
Figure 4. The antibiotic resistance of typical PSM. The deeper the red, the stronger the antibiotic resistance. Numbers indicate the percentage (%) of antibiotic-resistant strains in the strain. Blue fonts represent antibiotic classes. AMP, ampicillin; PEN, penicillin; AMI, amikacin; GEN, gentamicin; LEV, levofloxacin; CIP, ciprofloxacin; AZI, azithromycin; CHL, chloramphenicol; TX, ceftriaxone; AMOX, amoxicillin; TET, tetracycline; TR, trimethoprim.
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Figure 5. Characteristics of antibiotic resistance of 147 representative PSM strains. The abscissa shows the strain number. S, sensitive; I, intermediate; R, resistant; AMP, ampicillin; PEN, penicillin; AMI, amikacin; GEN, gentamicin; LEV, levofloxacin; CIP, ciprofloxacin; AZI, azithromycin; CHL, chloramphenicol; TX, ceftriaxone; AMOX, amoxicillin; TET, tetracycline; TR, trimethoprim.
Figure 5. Characteristics of antibiotic resistance of 147 representative PSM strains. The abscissa shows the strain number. S, sensitive; I, intermediate; R, resistant; AMP, ampicillin; PEN, penicillin; AMI, amikacin; GEN, gentamicin; LEV, levofloxacin; CIP, ciprofloxacin; AZI, azithromycin; CHL, chloramphenicol; TX, ceftriaxone; AMOX, amoxicillin; TET, tetracycline; TR, trimethoprim.
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Figure 6. Phenomenon of antibiotic susceptibility and Galleria mellonella larvae infection tests. (A) resistant strain, (B) intermediate strain, (C) sensitive strain, (D) Normal G. mellonella larvae. (E) Infected G. mellonella larvae. (F) Dead G. mellonella larvae. d indicates the inhibition zone.
Figure 6. Phenomenon of antibiotic susceptibility and Galleria mellonella larvae infection tests. (A) resistant strain, (B) intermediate strain, (C) sensitive strain, (D) Normal G. mellonella larvae. (E) Infected G. mellonella larvae. (F) Dead G. mellonella larvae. d indicates the inhibition zone.
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Figure 7. Characteristics of G. mellonella mortality of 147 representative PSM strains. The abscissa shows the strain number. The deeper the green color, the more mortality is caused.
Figure 7. Characteristics of G. mellonella mortality of 147 representative PSM strains. The abscissa shows the strain number. The deeper the green color, the more mortality is caused.
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Table 1. Isolation of major microorganisms (number of total strains isolated ≥ 20).
Table 1. Isolation of major microorganisms (number of total strains isolated ≥ 20).
BacteriaNo. of Total Strains IsolatedNo. of Total Samples Containing the BacteriaIsolation Ratio of Samples (%) 3
Total Samples
n = 132		Herd A
n = 115		Herd B
n = 8		Herd C
n = 9
Enterococcus faecalis 13866851.5252.17-88.89
Klebsiella pneumoniae 1853728.0332.17--
Escherichia coli 1754231.8224.3575.0088.89
Staphylococcus chromogenes 1,2592518.9413.0425.0088.89
Macrococcus caseolyticus 1591712.8813.91-11.11
Lactococcus garvieae 1583123.4826.09-11.11
Acinetobacter baumannii422216.6719.13--
Enterococcus gallinarum402317.4219.13-11.11
Enterobacter cloacae 1332418.1820.00-11.11
Staphylococcus epidermidis 1,2311511.368.70-55.56
Mammaliicoccus sciuri 1291813.6412.1712.5033.33
Staphylococcus haemolyticus 1,2281712.886.9650.0055.56
Streptococcus macedonicus25129.0910.43--
Kurthia gibsonii25118.339.57--
Staphylococcus borealis 1,2241410.616.96-66.67
Aerococcus viridans 12096.821.7462.5022.22
Staphylococcus cohnii 220107.588.70--
CoNS2577355.3049.57100.00100.00
PSM105812695.4594.78100.00100.00
Total1659132100.00100.00100.00100.00
1 represents pathogenic bacteria of subclinical mastitis (PSM). 2 represents coagulase-negative Staphylococci (CoNS). 3 Percentage of the number of samples isolated from the strain to the total number of samples.
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MDPI and ACS Style

Li, L.; Zhang, J.; Wei, X.; Wang, R.; Dan, X.; Li, J.; Hau, E.; Zeng, Q.; Liu, Q.; Ding, J.; et al. Epidemiological Investigation on Pathogenic Bacteria of Buffalo Subclinical Mastitis and Their Antibiotic Resistance and Virulence Characteristics in Guangxi, China. Animals 2025, 15, 3321. https://doi.org/10.3390/ani15223321

AMA Style

Li L, Zhang J, Wei X, Wang R, Dan X, Li J, Hau E, Zeng Q, Liu Q, Ding J, et al. Epidemiological Investigation on Pathogenic Bacteria of Buffalo Subclinical Mastitis and Their Antibiotic Resistance and Virulence Characteristics in Guangxi, China. Animals. 2025; 15(22):3321. https://doi.org/10.3390/ani15223321

Chicago/Turabian Style

Li, Ling, Jiaping Zhang, Xingqi Wei, Ruimin Wang, Xia Dan, Jianfeng Li, Enghuan Hau, Qingkun Zeng, Qingyou Liu, Jiafeng Ding, and et al. 2025. "Epidemiological Investigation on Pathogenic Bacteria of Buffalo Subclinical Mastitis and Their Antibiotic Resistance and Virulence Characteristics in Guangxi, China" Animals 15, no. 22: 3321. https://doi.org/10.3390/ani15223321

APA Style

Li, L., Zhang, J., Wei, X., Wang, R., Dan, X., Li, J., Hau, E., Zeng, Q., Liu, Q., Ding, J., & Cui, K. (2025). Epidemiological Investigation on Pathogenic Bacteria of Buffalo Subclinical Mastitis and Their Antibiotic Resistance and Virulence Characteristics in Guangxi, China. Animals, 15(22), 3321. https://doi.org/10.3390/ani15223321

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