Emerging pathogens — Streptococcus uberis isolates share some pathogenicity features with human disease-causing streptococci

Streptococcus uberis, which is a major cause of bovine mastitis in the dairy industry, is commonly treated with antimicrobials. The objective of this study was to investigate antimicrobial resistance (AMR) patterns of S. uberis isolates recovered from bovine mastitis. Totally 120 milk samples were collected in 2025 from cows with mastitis on a farm. S. uberis isolates were identified using standard bacteriological analyses and confirmed by sequence analysis of the 16S rRNA gene. Six different antimicrobials (gentamicin, streptomycin, penicillin, vancomycin, erythromycin, and tetracycline) were used for AMR test by the Kirby-Bauer disc diffusion method. Eighteen S. uberis isolates (15.0%, 18/120) were recovered, none of which were susceptible to all the tested antimicrobials. All isolates (100.0%, 18/18) were resistant to aminoglycosides, gentamycin, and streptomycin. In contrast, all isolates (100.0%, 18/18) were susceptible to penicillin and vancomycin. This finding supports the use of beta lactams as first-line antimicrobials to treat S. uberis infections. Additionally, 27.8% (5/18) and 61.1% (11/18) of the isolates were resistant to erythromycin and tetracycline, respectively. The high resistance rate for tetracycline indicates that it is not an effective treatment option for S. uberis infections. Four different AMR patterns were determined. Isolates with the CN-S-E-TE pattern were defined as multidrug-resistant (MDR) (16.7%, 3/18). The identification of MDR isolates emphasizes the necessity of conducting AMR testing prior to initiating antimicrobial treatment for bovine mastitis. In conclusion, these findings align with existing literature and demonstrate the significance of regular surveillance to guide evidence-based and sustainable approaches in bovine mastitis management.

Bovine mastitis is an infectious disease of the cow’s udder, which is usually treated with antibiotics. The overuse and misuse of antibiotics has led to the widespread development of bacterial resistance. Restrictions on the use of antibiotics create a need for alternative treatments and prophylactic measures to reduce the incidence and spread of mastitis. Antioxidant properties of plants – ability to scavenge free radicals, help animals recover and heal faster. Herbal medicine has been used traditionally by farmers for centuries to treat infectious and chronic diseases. However, there is little scientific evidence on the veterinary use of plant extracts. The Thesis aims to provide information on the use of plant extracts against bovine mastitis pathogens, their antiradical activity and to characterise their polyphenolic content. The dried plant material was extracted with 30 %, 50 %, 70 % ethanol and acetone and then freeze-dried for further analysis. The total phenolic, flavonoid, and tannin contents were determined by spectroscopic methods. Fourier transform infrared spectroscopy and high-performance liquid chromatography were used to characterise the chemical composition of the extracts. Clinical isolates inducing bovine mastitis (Escherichia coli, Streptococcus agalactiae, Streptococcus uberis, Serratia liquefaciens, Staphylococcus aureus) and reference cultures of S. aureus and E. coli were used for antibacterial tests. All extracts were screened for their antibacterial activity using a disc diffusion test, and the minimum inhibitory concentration and the minimum bactericidal concentration were determined for the most effective extracts. The antiradical activity was determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. The type of solvent affected the amount of phenolics extracted and the pharmacological effects of the extracts. The plants contained comparable amounts of tannins, which are not often reported in studies but have pharmacological activities. The acetone extracts had a higher total phenolic content and a higher total flavonoid content. All the plant extracts analysed were effective against S. aureus, but the effect against other bovine mastitis pathogens varied by the type of extract used. The types of phenolic compounds present in these plant extracts have a significant impact on the antioxidant activity. The bark of Q. robur and the herbs of C. vulgaris, and the flower and leaf of T. vulgare had good antioxidant and antibacterial potential. Plant extracts have promising pharmacological effects in vitro for the development of a product for bovine mastitis. The Doctoral Thesis was developed at Rīga Stradiņš University, Latvia. Defence: at the public session of the Promotion Council of Basic Medicine on 4 April 2025 at 12.00 in the Hippocrates Lecture Theatre, 16 Dzirciema Street, Rīga Stradiņš University and remotely via online platform Zoom. EU Structural Funds, European Agricultural Fund for Rural Development and Ministry of Agriculture and Rural Support Service Republic of Latvia “Development of herbal plants containing medical extracts with anti-parasitic effect”, Project No 18-00-A01620-000028; EU Structural Funds, European Agricultural Fund, Ministry of Agriculture and Rural Support Service Republic of Latvia “Development of medicinal forms of the leaf extract of the Latvian traditional medicinal plant tansy and its effect on the digestive tract microbiome and anti-parasitic control in sheep”, Project No: 22-00-A01612-000007. 3.1.Basic Medicine; Sub-Sector – Pharmacognosy. Keywords: antibacterial; antioxidant; bovine mastitis; herbal medicine; polyphenols; tannins.

Streptococcus uberis: In vitro biofilm production in response to carbohydrates and skim milk

The present study was carried out in an attempt to detect the distribution of antibiotic-resistant genes of Staphylococcus aureus isolates from human infections and bovine mastitis. mecA, msrA, msrB, aacA-D, tetK and tetM genes were selected in order to detect the distribution of antibiotic-resistant genes by multiplex PCR technique. According to the biochemical analysis and detection of 16S-rDNA by PCR method, 108 isolates of 300 human infections samples and 18 strains from 150 bovine mastitis milks were recognized as S. aureus. Distributions of antibiotic-resistant genes in human isolates were as follows: (85.18%) mecA, (46.29%) msrA, (49.07%) msrB, (33.33%) aacA-D, (80.50%) tetK and (66.66%) tetM and in bovine mastitis, isolates were seen to be ranging from: (22.22%) mecA, (66.66%) msrA, (77.77%) msrB, (33.33%) aacA-D, (55.55%) tetKand (50.00%) tetM, respectively. Results indicated that all S. aureus strains have one or more of the antibiotic-resistant genes. Also, multiplex PCR technique is a fast, practical and appropriate technique for determining antibiotic-resistant genes. Hence, it was possible that the treatment and the right antibiotics were used. Key words: Antibiotic-resistant genes, bovine mastitis, human infections, multiplex PCR,Staphylococcus aureus.

To evaluate the effectiveness of the integrated schistosomiasis control programme in Wuhan City from 2005 to 2023, so as to provide insights into precision control and elimination of schistosomiasis. The integrated measures for schistosomiasis control implemented by health, agriculture, water resources, and forestry departments of Wuhan City, and the epidemiological data of schistosomiasis in Wuhan City were collected from 2005 to 2023, and the prevalence of human schistosomiasis, prevalence of Schistosoma japonicum infections in humans and bovines, areas of S. japonicum-infected snail habitats, areas of snail habitats in inner embankments, and actual areas of snail habitats were retrieved. In addition, the trends in prevalence of schistosomiasis in humans and livestock and snail status were evaluated in Wuhan City from 2005 to 2023 using Mann-Kendall test and a Joinpoint regression model. Mann-Kendall test revealed a tendency towards a decline in the prevalence of human schistosomiasis (Z = -4.41, P < 0.01), prevalence of S. japonicum infections in humans (Z = -4.89, P < 0.01) and bovines (Z = -4.50, P < 0.01), areas of S. japonicum-infected snail habitats (Z = -3.91, P < 0.01), areas of snail habitats in inner embankments (Z = -2.28, P = 0.02), and actual areas of snail habitats (Z = -5.95, P < 0.01) in Wuhan City from 2005 to 2023. Joinpoint regression analysis showed an average annual reduction of 8.58% in the prevalence of human schistosomiasis in Wuhan City from 2005 to 2023 [average annual percent change (AAPC) = -8.58%, 95% confidence interval (CI): (-10.02%, -6.65%), P < 0.01], with two joinpoints in 2013 and 2016, respectively, and the tendency towards a decline showed statistical significance during the period from 2013 through 2016 [annual percent change (APC) = -34.41%, 95% CI: (-40.36%, -20.01%), P < 0.01]. The prevalence of S. japonicum human infections appeared an average annual reduction of 51.91% in Wuhan City from 2005 to 2023 [AAPC = -51.91%, 95% CI: (-58.12%, -44.25%), P < 0.01], with two joinpoints in 2014 and 2017, respectively, and the tendency towards a decline showed statistical significance during the period from 2014 through 2017 [APC = -98.17%, 95% CI: (-99.17%, -90.87%), P < 0.01]. The prevalence of S. japonicum infections in bovines appeared an average annual reduction of 53.12% in Wuhan City from 2005 to 2023 [AAPC = -53.12%, 95% CI: (-59.65%, -42.44%), P < 0.01], with two joinpoints in 2011 and 2014, respectively, and the tendency towards a decline showed statistical significance during the period from 2014 through 2017 [APC = -98.63%, 95% CI: (-99.44%, -90.93%), P < 0.01]. The areas of S. japonicum-infected snail habitats appeared an average annual reduction of 47.09% in Wuhan City from 2005 to 2023 [AAPC = -47.09%, 95% CI: (-52.92%, -38.26%), P < 0.01], with two joinpoints in 2011 and 2014, respectively, and the tendency towards a decline showed statistical significance during the period from 2011 through 2014 [APC = -97.27%, 95% CI: (-98.65%, -88.06%), P < 0.01]. The areas of snail habitats in inner embankments appeared an average annual reduction of 4.45% in Wuhan City from 2005 to 2023 [AAPC = -4.45%, 95% CI: (-5.18%, -3.82%), P < 0.01], with three joinpoints in 2011, 2015 and 2018, respectively, and statistical significance was seen in the tendency towards a decline during the period from 2005 through 2011 [APC = -16.38%, 95% CI: (-20.15%, -14.25%), P < 0.01]. In addition, the actual areas of snail habitats appeared an average annual reduction of 2.65% in Wuhan City from 2005 to 2023 [AAPC = -2.65%, 95% CI: (-2.89%, -2.40%), P < 0.01], with a joinpoint in 2013, and the tendency towards a decline showed statistical significance during the period from 2013 through 2023 [APC = -4.06%, 95% CI: (-4.66%, -3.58%), P < 0.01]. The integrated schistosomiasis control programme achieved significant effectiveness in Wuhan City from 2005 to 2023, with a tendency towards a decline in morbidity due to schistosomiasis in humans and livestock and snail status. The integrated schistosomiasis control strategy with emphasis on management of the source of S. japonicum infections should continue to be implemented to consolidate the schistosomiasis control achievements and achieve the goal of schistosomiasis elimination in the city.

Background: Antibiotic resistance has negative implications for treating infections in humans and animals. Rapid identification of the pathogen causing the disease is one of the determining factors for effective treatment. This study aimed to use MALDI-TOF MS to investigate multidrug-resistant bacterial strains in bacterial isolates from infectious bovine mastitis. Materials and Methods: Fifty-four milk samples from cows with subclinical mastitis were sown on sheep blood agar, and the bacterial isolates were identified by MALDI-TOF MS. The disk diffusion test verified susceptibility to antimicrobials. Results: The largest number of isolates were resistant to OXA (69%), followed by CFO (52%), AMP (48%), PEN (44%) and AMO (41%). The lowest number of resistant strains was observed against the antibiotic VAN. Although less significant, some bacterial strains were resistant to carbapenems. All the multidrug-resistant strains were identified on the MALDI-TOF MS system, including Staphylococcus aureus and non-aureus, Enterobacter asburiae, Enterobacter cloacae and Enterococcus faecalis. The species Staphylococcus aureus was detected most frequently. Conclusion: The detection of bacteria resistant to two or more antibiotics usually used to treat bovine mastitis indicates the need to look for alternative treatments to control this disease.

Comparative anti-microbial and anti-biofilm activities of postbiotics derived from kefir and normal raw milk lactic acid bacteria against bovine mastitis pathogens

Bovine mastitis is a multifactorial disease that produces important losses in livestock and dairy industry. One of the main factors that causes this disease are microorganisms such as Staphylococcus aureus, which has multiresistance to antibiotics and virulence factors such as its ability to internalize into bovine mammary epithelial cells (bMECs), which allows bacteria to evade the host's immune system and persist inside the cell. However, coagulasenegative Staphylococci (CNS) have a significant role as bovine mastitis pathogens. S. agnetis is an emergent CNS pathogen isolated from bovine mastitis, whose capacity to internalize into bMECs is unknown. In the present work we characterized an isolate of S. agnetis from bovine mastitis, which was resistant to penicillin, dicloxacillin and ampicillin; also, the isolate showed resistance to methicillin through a mechanism that may be related to the presence of the mecA gene. In addition, S. agnetis internalized into bMECs (10% relative to S. aureus (ATCC 27543), which could be related to the presence of the fnbpA, clfB and spa genes. This is the first study that demonstrates the ability of internalization of S. agnetis into bMECs, which is a virulence factor that may represent a major problem in the management of bovine mastitis.

Bovine mastitis is a common disease in dairy cows. The majority causes of bovine mastitis disease is an intramammary bacterial infection via the teat orifice. In general, antibiotics are used for bovine mastitis treatment, its inappropriate usage leads to antibiotic-resistance problem and residue in meat and dairy products. One of the alternative ways against pathogen to reduce antibiotic usage is antimicrobial peptides which are innate immune of life. Three cationic peptides as Pm11, Pep64, and L10 peptides were evaluated for an antimicrobial activity to gram-positive and gram-negative bacteria. Pm11 has shown the best potent antimicrobial activity with minimum bactericidal concentration (MBC) range from 2.5-10.0 µM and IC50 0.32-2.07 µM. Time kill kinetics of Pm11 peptide against bovine mastitis pathogens were ascertained, Pm11 is exhibited strong activity to completely killing Streptococcus agalactiae (SCM1084), Streptococcus uberis (SCM1310) within 1 hour and completely killing Staphylococcus aureus (CM967), Escherichia coli (SCM1249) within 4 hour. However, Pm11 peptide at 10.0 µM could not be completely killing Klebsiella spp. (SCM1282), even though 3 log10 CFU/ml of Klebsiella spp. (SCM1282) was decreased within 2 h, but it was returning to grow up within 12 h. Visual observation under the scanning electron microscope of Pm11 peptide attacked to the pathogens shown the distinct morphological changes. The cytotoxicity assay confirmed that Pm11 peptide at the concentration 0 to 160 µM had low hemolytic activity to sheep red blood cells. Nonetheless, the antimicrobial activity of Pm11 peptide was reduced in UHT milk.

Simple SummaryMastitis remains one of dairy cattle’s most perplexing and expensive diseases. This study is the first to look into the virulence traits, antimicrobial and biocide resistance, and epidemiological typing of Streptococcus uberis (S. uberis) isolated from bovine clinical mastitis in dairy farms of diverse hygienic interventions in Egypt. The overall S. uberis infection rate was 20.59%; all were multidrug-resistant (MDR). The sua gene was the most frequent virulence gene (42.02%), followed by pauA (40.57%), cfu (21.73%), skc (20.28%), and opp (11.59%). The erm(B) gene serves as the predominant antimicrobial-resistant gene (75.36%), followed by fexA (52.63%) and tet(M), blaZ, and aac(6′)aph(2″) genes (46.38% each). Of note, 79.71% of S. uberis isolates carried qac genes; among them, 55 (79.71%), 54 (78.26%), and 13 (18.84%) harbored qacED1, qacC/D, and qacA/B genes, respectively. Restriction fragment length polymorphism–polymerase chain reaction (RFLP–PCR) indicated that all analyzed isolates were S. uberis type I by their unique RFLP pattern. This study shows a significant variation in the occurrence of virulent S. uberis in dairy cows with clinical mastitis regarding the prospective hygienic concerns. Furthermore, MDR coupled with the existence of biocide resistance genes indicates the importance of S. uberis surveillance and the prudent use of antimicrobials in veterinary clinical medicine to avoid the dissemination of antimicrobial resistance.Mastitis remains a serious problem for dairy animals. The misappropriation of antimicrobial agents helps accelerate resistance, which poses a serious challenge in controlling environmental S. uberis infection. Here, we study the virulence attributes, antimicrobial and biocide resistance, and epidemiological typing of S. uberis recovered from bovine clinical mastitis in dairy farms of diverse hygienic interventions in Egypt. The overall S. uberis infection rate was 20.59%; all were multidrug-resistant (MDR). The sua gene was the most frequent virulence gene (42.02%), followed by pauA (40.57%), cfu (21.73%), skc (20.28%), and opp (11.59%). The erm(B) gene served as the predominant antimicrobial-resistant gene (75.36%), followed by fexA (52.63%) and tet(M), blaZ, and aac(6′)aph(2″) genes (46.38% each). Of note, 79.71%, 78.26%, and 18.84% of S. uberis isolates harbored qacED1, qacC/D, and qacA/B genes, respectively. All analyzed isolates were S. uberis type I by their unique RFLP–PCR pattern. In conclusion, the sustained presence of pauA and sua genes throughout the investigated farms contributes to a better understanding of the bacterium’s pathogenicity. Furthermore, MDR coupled with the existence of biocide resistance genes indicates the importance of S. uberis surveillance and the prudent use of antimicrobials in veterinary clinical medicine to avoid the dissemination of antimicrobial resistance.

The relationship between the in vitro detected virulence factors biofilm and capsule formation of Streptococcus (S.) uberis isolates of clinical mastitis in dairy cows and the bacteriological cure rate after antibiotic therapy was investigated in order to better understand the importance of these virulence factors for the bacteriological cure rate. A total of 111 clinical mastitis (CM) cases were collected, in which S. uberis was bacteriologically detected. All mastitis cases were treated in accordance with the approval conditions of the antibiotic udder tubes used. Individual cow information including age, number of lactations, current lactation mastitis and antimicrobial treatment received was recorded. The microtiter plate test was used to detect biofilm formation and Anthony capsule staining was used to detect capsular capacity. Statistical analyses were performed to characterize the correlation between in vitro virulence factors and bacteriological cure (BC) rate. 30.5% (n=29) of the S. uberis isolates of bacteriologically cured cases and 34.5% (n=10) of the isolates of bacteriologically non-cured mastitis cases were found to have the ability to produce capsules in vitro. 70.7% (n=58) of the S. uberis isolates from bacteriologically cured mastitis cases had the ability to produce biofilm in vitro, whereas 58.6% (n=17) of the isolates of non-cured mastitis cases showed ability in producing biofilm. No correlation was found between the in vitro ability of S. uberis to form capsules and biofilms and the BC rate after antibiotic treatment of bovine mastitis. The present work has shown that the investigated in vitro virulence factors are not associated with the BC after antibiotic therapy. Further studies on the role of S. uberis virulence factors are needed to complete the missing knowledge on the difficulties in curing S. uberis mastitis. This study is of great clinical relevance since it enhances the understanding of the occurrence of BC in S. uberis mastitis cases. The investigated virulence factors are often addressed as possible reasons for therapy failure, although respective scientific studies are missing.

In this article, we present the course and results of scientific research aimed at calculation of the concentration of somatic cells in cow's milk to detect subclinical mastitis in individual farms of the Republic of Armenia. Comparison of somatic cell concentrations in milk from healthy and sick cows with subclinical mastitis, analysis and comparison of blood morphological parameters in sick and healthy animals has been implemented and the relationship between somatic cell concentration in milk and morphological abnormalities in sick cow blood has been identified. SC concentrations between 300 and 500 thousand/ml are indicative of mastitis risk. In this case, the type of reaction manifested was positive. It was found that the viscosity of the investigated solution "Somatic plus" + milk increases in proportion to the increasing concentration of somatic cells in the milk sample, which indicates subclinical mastitis. A study of the morphological parameters of blood in healthy and subclinical mastitis animals revealed some deviations from the reference values of blood parameters, leading to the conclusion that these changes in animals' blood are related to an increase in the number of milk's somatic cells. Increased leukocytes were found in sick animals' blood. This was followed by an increase in stab neutrophils, eosinophils, monocytes, lymphocytes, and ESR. As a result of all this, the body is experiencing inflammatory processes mobilizing its defensive mechanisms against pathogens.

Streptococcus agalactiae (GBS) is a major source of human perinatal diseases and bovine mastitis. Erythromycin (Ery) and tetracycline (Tet) are usually employed for preventing human and bovine infections although resistance to such agents has become common among GBS strains. Ery and Tet resistance genes are usually carried by conjugative transposons (CTns) belonging to the Tn916 family, but their presence and transferability among GBS strains have not been totally explored. Here we evaluated the presence of Tet resistance genes (tetM and tetO) and CTns among Ery-resistant (Ery-R) and Ery-susceptible (Ery-S) GBS strains isolated from human and bovine sources; and analyzed the ability for transferring resistance determinants between strains from both origins. Tet resistance and int-Tn genes were more common among Ery-R when compared to Ery-S isolates. Conjugative transfer of all resistance genes detected among the GBS strains included in this study (ermA, ermB, mef, tetM and tetO), in frequencies between 1.10−7 and 9.10−7, was possible from bovine donor strains to human recipient strain, but not the other way around. This is, to our knowledge, the first report of in vitro conjugation of Ery and Tet resistance genes among GBS strains recovered from different hosts.

Bovine mastitis is a worldwide disease affecting dairy cattle and causes major economic losses in the dairy industry. Recently, the emergence of microbial resistance to the current antibiotics complicates the treatment protocol which necessitates antibiotic stewardship and further research to find new active compounds. Recently, phytobiotics have gained interest in being used as an alternative to antibiotics in the poultry industry as an antibiotic stewardship intervention. This study evaluated the in vitro antibacterial activity of 16 flavonoids against bovine mastitis pathogens. Two flavones: 2-(4-methoxyphenyl)chromen-4-one (1) and 2-(3-hydroxyphenyl)chromen-4-one (4) showed inhibition of the growth of Klebsiella oxytoca with MIC values range (25–50 µg mL− 1) followed by a structure-activity relationship (SAR) study indicating that the presence of a hydroxyl group at C-3` or methoxy at C-4` increases the activity against Klebsiella oxytoca while the presence of hydroxyl group at C-7 decreases the activity. Furthermore, a structure-based drug development approach was applied using several in silico tools to understand the interactions of active flavones at the active site of the DNA gyrase protein. Compound (4) showed a higher docking score than quercetin (standard) which is known to have antibacterial activity by inhibiting the DNA gyrase. In addition, the structure-based pharmacophores of compound (4) and quercetin showed similar pharmacophoric features and interactions with DNA gyrase. Based on our findings, compounds (1) and (4) are promising for further study as potential anti-microbial phytochemicals that can have a role in controlling bovine mastitis as well as to investigate their mechanism of action further.

This communication addresses the hypothesis that silver nanoparticles obtained by green synthesis (G-AgNPs), using an aqueous extract of chicory, have antibacterial activity against bovine mastitis-associated strains and may inhibit their adherence capacity. Bovine mastitis is among the diseases with the greatest economic impact worldwide. Multifactorial in nature, it is usually associated with infection caused by bacterial species such as Staphylococcus aureus, coagulase-negative staphylococci, Streptococcus uberis, and Escherichia coli. Antibiotics are currently the main line of treatment, but in order to offset the emergence of resistance, alternative compounds and nanomaterials are being researched for prevention and control. In this study, bacteria that cause bovine mastitis were molecularly identified by groEL gene PCR-RFLP. Then, G-AgNPs were produced using an aqueous extract of chicory as a reducing and stabilizing agent. The nanoparticles showed powerful antibacterial activity, but they did not seem to reduce the adherence of pathogens to MAC-T cells that had been preincubated with a sub-MIC concentration of G-AgNPs. Nevertheless, very low concentrations (21-30 pM) were needed for the antibacterial effect to take place, which makes G-AgNPs promising candidates for the control of bovine mastitis.