Causative Agent Of Bovine Mastitis Research Articles

In vitro antimicrobial and antibiofilm activity of phage cocktail against Mammaliicoccus sciuri, a causative agent of bovine mastitis.

In this research paper the in vitro antimicrobial and antibiofilm activity of phage cocktail against the coagulase negative Mammaliicoccus sciuri was investigated. Three M. sciuri isolates obtained from clinical bovine mastitis samples were characterized and identified by 16S rRNA gene sequencing. Bacteriophages with lytic activity against M. sciuri isolates were isolated from dairy farm effluents. Two typical phages were isolated using standard enrichment and plaque assay techniques, purified by polyethylene glycol precipitation, and morphologically characterized based on shape and size using transmission electron microscopy. This was followed by determination of host range using spot tests and stability to varying temperature, pH and UV treatment. The phage cocktail suppressed bacterial activity within 30 min of exposure. Crystal violet assay showed that the tested phages and their cocktail significantly reduced the biofilm biomass of all three M. sciuri strains compared to the untreated control in vitro within 24 h with a single dosing. Transmission electron micrography of the purified phage particle revealed an icosahedral head and a rigid contractile tail, characteristic of the class Caudoviricetes. The findings open new avenues in phage-based antimicrobial approaches for controlling contagious and teat skin opportunistic bacteria causing bovine mastitis.

Pathogens Associated with Bovine Mastitis: The Experience of Bosnia and Herzegovina.

To obtain improved insights into the complex microbial aetiology of bovine mastitis, this study investigated the pathogens involved in cattle mastitis in Bosnia and Herzegovina. A total of 179 milk samples from cows with clinical mastitis (CM) and subclinical mastitis (SCM), as well as eight bulk tank milk (BTM) samples from 48 dairy farms, were analysed by standard bacteriological and mycological methods. Mycoplasma detection and identification were performed using culture techniques and real-time polymerase chain reaction (PCR). A total of 88 (49.2%) mastitis samples were positive for known mastitis pathogens at 32 of 47 farms (68.1%). Mycoplasma bovis was a predominant pathogen (25/187; 13.4%) in the majority of herds (14/48; 29.2%) and accounted for 48.9% of positive CM samples. Escherichia coli was the second most dominant CM pathogen (34%), followed by Streptococcus agalactiae (10.6%), whereas Staphylococcus aureus and coagulase-negative staphylococci were the most common in SCM samples (17.1%). Other mastitis pathogens included Candida spp. and Prototheca zopfii. Two BTM samples were positive for M. bovis only, and one was positive for a mixed culture of S. aureus and Streptococcus uberis. The finding of various causative agents of bovine mastitis, with M. bovis emerging as the main pathogen, emphasizes the significance of comprehensive testing that includes not only common mastitis pathogens but also mycoplasmas, fungi, and algae.

In silico analysis of virulence factors of Streptococcus uberis for a chimeric vaccine design

Streptococcus uberis is one of the causative agents of bovine mastitis, which has detrimental effects on animal health and the dairy industry. Despite decades of research, the requirement for effective vaccines against the disease remains unmet. The goal of this study was to create a multi-epitope vaccine using five virulence factors of S. uberis through the reverse vaccinology approach, which has been employed due to its high efficiency and applicability. Plasminogen activator A (PauA), glyceraldehyde-3-phosphate dehydrogenase C (GapC), C5a peptidase, S. uberis adhesion molecule (SUAM), and sortase A (SrtA) were selected for the T cytotoxic (CTL) and B cell epitope analyses as they were extensively studied in S. uberis or other pathogens. Eighteen CTL and ten B cell epitopes that were antigenic, non-toxic, and non-allergenic were selected in order to design a chimeric vaccine candidate that in silico analysis revealed to be potentially immunogenic, non-allergenic, and stable. Molecular docking analysis of the vaccine candidate with Toll-like receptor (TLR) 2 and TLR 4 revealed stable interactions between the candidate and the immune receptors. Meanwhile, the stability of the docked complexes was confirmed using normal mode analysis. Additionally, in silico immune simulation of the vaccine candidate demonstrated the stimulation of primary immune responses, indicating that the chimeric protein can hold promise as a viable vaccine candidate for preventing S. uberis mastitis. Moreover, the current study can provide a background for designing epitope-based vaccines based on the explored epitopes.

Multiplex PCR detection of the classical enterotoxin genes of Staphylococcus aureus in humans and animal isolates

Staphylococcus aureus is a prevalent bacterial causative agent of bovine mastitis and human food illness by secreting enterotoxins. This study aims to identify the seven classic Staphylococcus aureus enterotoxin genes (SEA to SEH) isolated from humans and animals. A total of 173 human isolates and 14 mastitis (goat and cow) isolates were amplified with a 23S rRNA genes. The presence of classical enterotoxins genes (sea-seh) was amplified using multiplex polymerase chain reaction (m-PCR). Amplification of the SEs genes singly and in combination was observed from both isolates. Of 173 human isolates, the sec genes showed the highest prevalence of 112 isolates (65%), followed by the seh gene were 89 isolates (51%), 61 (35%) for seg gene and one (1%) for sea and see genes. The two genes in combination were also detected se (c, h), se (c, g), se (g, h)] and the three combination genes were se (c, g, h). Furthermore, all 14 animal isolates were positive for seh gene (100%), 11 (78%) for sec gene and 11 (78%) for combination se (c, h). Five classical enterotoxin genes (seh, sea, seg, sec, see) isolated from Staphylococcus aureus in humans were identified. Still, only seh and sec genes were identified from the animal origin. In the present study, identifying the genes encoding SE will contribute to recognize the prevalent of enterotoxins distribution among Staphylococcus aureus strains in samples of animals and human origin.

Priming of the murine mammary gland with Staphylococcus chromogenes IM reduces bacterial growth of Streptococcus uberis: a proof-of-concept study

Streptococcus uberis is a major causative agent of bovine mastitis, an inflammation of the mammary gland with substantial economic consequences. To reduce antibiotic use in animal agriculture, alternative strategies to treat or prevent mastitis are being investigated. Bovine-associated non-aureus staphylococci are proposed in that respect due to their capacity to inhibit the in vitro growth of S. uberis. We demonstrate that priming the murine mammary gland with Staphylococcus chromogenes IM reduces S. uberis growth in comparison with non-primed glands. The innate immune system is activated by increasing IL-8 and LCN2, which may explain this decreased growth.

Genome sequencing and comparative genomic analysis of bovine mastitis-associated Staphylococcus aureus strains from India

BackgroundBovine mastitis accounts for significant economic losses to the dairy industry worldwide. Staphylococcus aureus is the most common causative agent of bovine mastitis. Investigating the prevalence of virulence factors and antimicrobial resistance would provide insight into the molecular epidemiology of mastitis-associated S. aureus strains. The present study is focused on the whole genome sequencing and comparative genomic analysis of 41 mastitis-associated S. aureus strains isolated from India.ResultsThe results elucidate explicit knowledge of 15 diverse sequence types (STs) and five clonal complexes (CCs). The clonal complexes CC8 and CC97 were found to be the predominant genotypes comprising 21 and 10 isolates, respectively. The mean genome size was 2.7 Mbp with a 32.7% average GC content. The pan-genome of the Indian strains of mastitis-associated S. aureus is almost closed. The genome-wide SNP-based phylogenetic analysis differentiated 41 strains into six major clades. Sixteen different spa types were identified, and eight isolates were untypeable. The cgMLST analysis of all S. aureus genome sequences reported from India revealed that S. aureus strain MUF256, isolated from wound fluids of a diabetic patient, was the common ancestor. Further, we observed that all the Indian mastitis-associated S. aureus isolates belonging to the CC97 are mastitis-associated. We identified 17 different antimicrobial resistance (AMR) genes among these isolates, and all the isolates used in this study were susceptible to methicillin. We also identified 108 virulence-associated genes and discuss their associations with different genotypes.ConclusionThis is the first study presenting a comprehensive whole genome analysis of bovine mastitis-associated S. aureus isolates from India. Comparative genomic analysis revealed the genome diversity, major genotypes, antimicrobial resistome, and virulome of clinical and subclinical mastitis-associated S. aureus strains.

Antimicrobial activities of graphene oxide against biofilm and intracellular Staphylococcus aureus isolated from bovine mastitis

BackgroundS. aureus is one of the causative agents of bovine mastitis. The treatment using conventional antimicrobials has been hampered due to the development of antimicrobial resistance and the ability of the bacteria to form biofilms and localize inside the host cells.ObjectivesHere, the efficacy of graphene oxide (GO), a carbon-based nanomaterial, was tested against the biofilms and intracellular S. aureus invitro. Following that, the mechanism for the intracellular antimicrobial activities and GO toxicities was elucidated.MethodsGO antibiofilm properties were evaluated based on the disruption of biofilm structure, and the intracellular antimicrobial activities were determined by the survival of S. aureus in infected bovine mammary cells following GO exposure. The mechanism for GO intracellular antimicrobial activities was investigated using endocytosis inhibitors. GO toxicity towards the host cells was assessed using a resazurin assay.ResultsAt 100 ug/mL, GO reduced between 30 and 70% of S. aureus biofilm mass, suggesting GO’s ability to disrupt the biofilm structure. At 200 ug/mL, GO killed almost 80% of intracellular S. aureus, and the antimicrobial activities were inhibited when cells were pre-treated with cytochalasin D, suggesting GO intracellular antimicrobial activities were dependent on the actin-polymerization of the cell membrane. At < 250 ug/mL, GO enhanced the viability of the Mac-T cell, and cells were only affected at higher dosages.ConclusionThe in vitro efficacy of GO against S. aureus in vitro suggested the compound could be further tested in Vivo to zrecognize its potential as one of the components of bovine mastitis therapy.

Molecular characterisation of antimicrobial resistance in coagulase negative staphylococci isolated from bovine subclinical mastitis

Coagulase negative staphylococci (CNS) are emerging as the most prevalent causative agent of bovine mastitis. They are resistant to many commonly used antibiotics due to the presence of antimicrobial resistance (AMR) genes. A study was conducted to evaluate the AMR profiling of CNS isolated from bovine subclinical mastitis.Coagulase negative staphylococci were isolated from 49 (44.95 per cent) of the subclinical mastitis samples. Disc diffusion assay revealed that highest resistance was shown against gentamicin (42.85 per cent) followed by methicillin (32.6 per cent), ceftriaxone – tazobactam (24.48 per cent), enrofloxacin (20.4 per cent), tetracycline (16.32 per cent) and least resistance to cotrimoxazole (4 per cent). Genotypic characterisation of AMR genes such as mecA, aacA-aphD and norA by PCR was done for determining resistance to methicillin, gentamicin and fluroquinolone resistance. The CNS carried aacA-aphD, norA and mecA in 44.89 per cent, 32.65 per cent and 14.28 per cent, respectively. Comparison of phenotypic and genotypic characterisation of AMR in CNS was carried out by McNemar test and it was found that there was significant difference between the presence of mecA gene and methicillin resistance. There was no significant difference noticed forcharacterisation of phenotypic and genotypic AMR of CNS for gentamicin and fluroquinolone resistance.

ҚОСТАНАЙ АУДАНЫ СИЫРЛАРЫНЫҢ СҮТІНЕН БӨЛІНІП АЛЫНҒАНSTREPTOCOCCUS AGALACTIAE ИЗОЛЯТТАРЫНЫҢ НЕГІЗГІБИОЛОГИЯЛЫҚ ҚАСИЕТТЕРІ ЖӘНЕ АНТИБИОТИКТЕРГЕ ТӨЗІМДІЛІГІ

One of the most common causative agents of bovine mastitis is Str. agalactiae, which is also potentially dangerous for humans and aquatic animals. To date, there is a trend towards the emergence and spread of Str. agalactiae microorganisms with multidrug resistance. The constant use of antimicrobial drugs contributes to the emergence of polyresistant microorganisms, which shows the relevance of rational pharmacotherapy of animals. The article presents the results of studying the main phenotypic properties of Str. agalactiae isolated from the milk of cows with clinical and subclinical forms of mastitis. For the studies conducted during 2021-2023, 398 milk samples were collected, 26 isolates of Str. agalactiae (6.5%) were isolated and identified. Studies on antibiotic sensitivity were carried out using the disco-diffuse method. The studies results showed that, the largest number of isolates were resistant to the group of beta–lactam antibiotics: benzylpenicillin (76.9%), amoxacillin (65.3%), ampicillin (65.3%); aminoglycosides – kanamycin (61.5%) and macrolides – tylosin (50.0%), less resistant to tetracyclines: doxycycline (42.3%) tetracycline (30.7%). The studied strains of Str. agalactiae demonstrated the lowest resistance to levomycetin – 23.0%. Strains of Str. agalactiae in our studies showed resistance to all antibacterial drugs to different extents. This indicates the urgency of the problem of antibiotic resistance in Kostanay region and the need to find solutions.

Isolation and Determination of Antibacterial Sensitivity Characteristics of Staphylococcus aureus from Lactating Cows in West Shewa Zone, Ethiopia.

Staphylococcus (S.) aureus is one of the etiologies of bovine mastitis, hindering milk production and productivity in dairy farms. This study was aimed at assessing the distribution of bovine mastitis and the isolation rate of S. aureus in milked cows of West Shewa Zone. The clinical mastitis was diagnosed by physical methods including observation and palpation, whereas the subclinical mastitis was tested by the California mastitis test (CMT). All of the cows tested for mastitis were aseptically sampled (teat-milk) for bacteriology. The bacterium was primarily identified based on colony characterization, catalase, coagulase tests, and Gram stain reaction. Finally, MALDI-TOF Biotyper confirmed the species. The antibacterial sensitivity characteristics of the isolates to different antibacterial drugs were tested by the disk diffusion method. The drugs were selected based on the frequent usage in veterinary medicine in the study area. By using particular primers, the presence of the resistance (mecA and blaZ), and thermonuclease (nuc) genes were determined by polymerase chain reaction (PCR). The data were analyzed by R statistical software. The associations between the dependent variables (prevalence of mastitis and S. aureus) and the explanatory variables were analysed by chi-square (χ 2) and logistic regression tests at a 95% confidence interval (CI). Accordingly, 258 lactating cows were examined, of which 97 (37.6%) were mastitis positive. Of these mastitis positive cows, 59 (60.8%) were subclinical and 38 (39.2%) were clinical. Among the 258 milk samples, 43 (16.7%) were positive for S. aureus. According to the results of the current investigation, subclinical mastitis was significantly more prevalent than clinical mastitis (p < 0.05). The disease was found varied with the lactation stage of the animal, milking with washed hand, udder washing before milking, and tick infestation of the teat. In comparison to animals from farms with lower number of lactating cows, the prevalence of the bacteria was significantly higher in animals managed in farms with large (OR = 12.58, 95% CI = 2.33-68.54, and p < 0.05) and medium (OR = 12.58, 95% CI = 2.33-68.54, and p < 0.05) population of lactating cows per herd. The isoation rate of the bacterium was also found significantly higher in tick-infested cows (OR = 27.69, 95% CI = 9.71-93.01, and p < 0.05) than tick free cows. The antibiogram tests revealed that the isolates resisted penicillin G and tetracycline group drugs (oxytetracycline and tetracycline). Moreover, the nuc gene was confirmed to be present in all of the examined isolates. However, they were not found harboring blaZ and mecA genes. We concluded that S. aureus is sustaining as a main causative agent of bovine mastitis, and they were resistant to the frequently used antibiotics in public and veterinary medicines in the study areas. Therefore, research-based interventions need to be taken in action to combat the pathogen.

Immunopathological Responses to the Bovine Mastitis Associated with Staphylococcus Species Infection

Bovine mastitis is a disease that concerns animals' welfare and increases the economic production losses. Bacterial agents such as Staphylococcus species are the main causative agent of bovine mastitis. This bacterial agent expresses some inflammatory cytokines that might enhance the cell-mediated, which may promote the pathogenesis of mastitis. The objective of the current study was to investigate the bovine innate immune response circulating levels of pro-inflammatory and anti-inflammatory cytokines. A total of 10 mL of milk specimens were collected randomly from 100 clinically mastitic cows, and another 20 clinically healthy cows were considered as a control group for the California Mastitis test. The microbiological cultures of milk specimens were performed. The interleukins (ILs)that involved IL-4, IL-6, and IL-10 were detected using the ELISA test for the evaluation of the pro-inflammatory bovine mastitis pathophysiology. The results of this study showed that Staphylococcus aureus detection was in 31.2% of mastitic milk and 8.7% of non-mastitic milk specimens; and the coagulase-negative Staphylococcus was detected in 14.8% and 18.7% in the mastitic and non-mastitic milk specimens, respectively. The IL-6 level was shown significantly higher (P&lt;0.05)in the specimens of mastitic milk (194±12.8 pg/mL) compared to the non-mastitic milk (31±2.9 pg/mL). In conclusion, the elevated level of expression of IL-6 cytokine in the milk of cows with mastitis suggested that IL-6 might be used as a potentially suitable biomarker for early bovine mastitis diagnosis

Chimeric Peptidoglycan Hydrolases Kill Staphylococcal Mastitis Isolates in Raw Milk and within Bovine Mammary Gland Epithelial Cells.

Staphylococcus aureus is a major causative agent of bovine mastitis, a disease considered one of the most economically devastating in the dairy sector. Considering the increasing prevalence of antibiotic-resistant strains, novel therapeutic approaches efficiently targeting extra- and intracellular bacteria and featuring high activity in the presence of raw milk components are needed. Here, we have screened a library of eighty peptidoglycan hydrolases (PGHs) for high activity against S. aureus in raw bovine milk, twelve of which were selected for further characterization and comparison in time-kill assays. The bacteriocins lysostaphin and ALE-1, and the chimeric PGH M23LST(L)_SH3b2638 reduced bacterial numbers in raw milk to the detection limit within 10 min. Three CHAP-based PGHs (CHAPGH15_SH3bAle1, CHAPK_SH3bLST_H, CHAPH5_LST_H) showed gradually improving activity with increasing dilution of the raw milk. Furthermore, we demonstrated synergistic activity of CHAPGH15_SH3bAle1 and LST when used in combination. Finally, modification of four PGHs (LST, M23LST(L)_SH3b2638, CHAPK_SH3bLST, CHAPGH15_SH3bAle1) with the cell-penetrating peptide TAT significantly enhanced the eradication of intracellular S. aureus in bovine mammary alveolar cells compared to the unmodified parentals in a concentration-dependent manner.

First Report of Molecular Identification of Prototheca zopfii Genotype 2 as Causative Agent of Bovine Mastitis in Navsari, South Gujarat, India

Prototheca species is emerged as an important causative agent for bovine mastitis worldwide, and is responsible for great economic losses for dairy industry. A 5-year-old HF cow was referred to the Veterinary Clinical Complex of the College, Navsari (India) with a complaint of watery milk with flakes, anorexia and drop in milk production. On physical examination, udder appeared warm and swollen, while milk consistency was thin, watery with flakes. Milk sample obtained aseptically from the cow was inoculated aerobically on Sabouraud dextrose agar and blood agar plates. Prototheca colonies grew in pure and luxuriant form from mastitic milk on both the plates after 48 h of incubation. Prototheca zopfii was identified on the basis of colony morphology, staining characters and biochemical reactions. Molecular confirmation of Prototheca zopfii genotype 2 was performed by employing polymerase chain reaction (PCR). This report presents first isolation and molecular identification of Prototheca zopfii genotype 2 from a case of bovine mastitis from Navsari region, South Gujarat, India.

Knema retusa is antibacterial and antibiofilm against antibiotic resistant Staphylococcus aureus and S. haemolyticus isolated in bovine mastitis.

This study aimed to assess antibacterial activity of Knema retusa wood extract (KRe) against antibiotic resistant staphylococci which are causative agents of bovine mastitis. From 75 cases of intramammary infections in dairy cows, 66 staphylococcal isolates were collected, including 11 Staphylococcus aureus isolates (17%) and 55 coagulase-negative staphylococci (83%). Sixty isolates (91%) formed strong biofilms. KRe had minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) against the isolates ranging 32-256 ug/mL and 64-512 ug/mL, respectively. Two-hour KRe exposures at 4×MIC, viabilities of S. aureus and S. haemolyticus decreased by 3 log10 compared to the control. Scanning EM (SEM) showed that KRe disrupted the bacterial cells of both species. KRe at 1/16×MIC significantly inhibited biofilm formation (P < 0.05) in both S. aureus and S. haemolyticus. At 1/2×MIC, S. aureus and S. haemolyticus biofilm inhibition ranged from 75 to 99%. Cells within established biofilms were disrupted 66-83% by KRe at 32×MIC. Moreover, 1/2×MIC KRe reduced bacterial adhesion to glass surfaces observed by SEM. According to GC-MS analysis, the major compound in KRe was endo-2-hydroxy-9,9-(ethylenedioxy)-1-carbethoxy bicyclo [3.3.1] nonane (E2N). Molecular docking analysis of E2N has a high affinity for staphylococcal accessory regulator A (SarA), binding free-energy - 6.40kcal/mol. The results suggested that KRe may have medicinal benefits by inhibiting the growth, biofilm, and adhesion of antibiotic resistant staphylococci isolated from bovine mastitis.

Prevalence and risk factors of coliform-associated mastitis and antibiotic resistance of coliforms from lactating dairy cows in North West Cameroon.

Coliform bacteria are major causative agents of bovine mastitis, a disease that has devastating effect on dairy animal health and milk production. This cross-sectional study, carried out in the North West region of Cameroon, sought to determine the prevalence of bovine mastitis, coliforms associated with bovine mastitis, risk factors for infection and the antibiotic resistance pattern of coliform bacterial isolates. A total of 1608 udder quarters were sampled from 411 cows using a questionnaire, clinical examination, California Mastitis Test and milk culture. Primary isolation of coliform bacteria was done on MacConkey agar while identification of coliforms employed Gram-staining and biochemical testing. Each coliform bacterial isolate was challenged with 11 antibiotics using the Kirby-Bauer disc diffusion method. The prevalence of mastitis was 53.0% (218/411) and 33.1% (532/1608) at the cow- and quarter-levels respectively. Overall, 21.9% (90/411) cows and 8.2% (132/1608) udder quarters showed coliform mastitis. Escherichia coli was isolated in 7.0% of mastitis milk, and other coliforms isolated were Enterobacter cloacae (12.6%), Klebsiella pneumoniae (2.4%), Enterobacter sakazakii (1.1%), Klebsiella oxytoca (0.8%), Citrobacter freudii (0.4%), Serratia ficaria (0.4%) and Serratia liquefaciens (0.2%). Lactation stage, breed, history of mastitis and moist/muddy faeces contaminated environment were significantly associated (P-value < 0.05) with coliform mastitis. Coliform isolates (99.0%; 203/205) were resistant to at least one antibiotic tested. Amoxicillin had the highest resistance (88.8%) while norfloxacin had the least resistance (3.4%). Multidrug resistance was exhibited by 52.7% (108/205) of the isolates in a proportion of 27.8% Enterobacter cloacae, 10.7% E. coli, 6.3% Klebsiella pneumoniae, 2.9% Enterobacter sakazakii, 2.0% Klebsiella oxytoca, 1.0% Citrobacter freundii, 1.0% Serratia ficaria, 0.5% Serratia liquefaciens and 0.5% Serratia odorifera. Results indicate a need to educate these dairy farmers about mastitis (particularly subclinical), proper hygiene methods in milking and the public health implications of consuming contaminated raw milk.

Anti-Staphylococcus aureus Single-Chain Fragment Variables Play a Protective Anti-Inflammatory Role In Vitro and In Vivo.

Staphylococcus aureus is a causative agent of bovine mastitis, capable of causing significant economic losses to the dairy industry worldwide. This study focuses on obtaining single-chain fragment variables (scFvs) against the virulence factors of S. aureus and evaluates the protective effect of scFvs on bovine mammary epithelial (MAC-T) cells and mice mammary gland tissues infected by S. aureus. After five rounds of bio-panning, four scFvs targeting four virulence factors of S. aureus were obtained. The complementarity-determining regions (CDRs) of these scFvs exhibited significant diversities, especially CDR3 of the VH domain. In vitro, each of scFvs was capable of inhibiting S. aureus growth and reducing the damage of MAC-T cells infected by S. aureus. Preincubation of MAC-T cells with scFvs could significantly attenuate the effect of apoptosis and necrosis compared with the negative control group. In vivo, the qPCR and ELISA results demonstrated that scFvs reduced the transcription and expression of Tumor Necrosis Factor alpha (TNF-α), interleukin-1β (IL-1β), IL-6, IL-8, and IL-18. Histopathology and myeloperoxidase (MPO) results showed that scFvs ameliorated the histopathological damages and reduced the inflammatory cells infiltration. The overall results demonstrated the positive anti-inflammatory effect of scFvs, revealing the potential role of scFvs in the prevention and treatment of S. aureus infections.

Antibacterial Activity of Ikarugamycin against Intracellular Staphylococcus aureus in Bovine Mammary Epithelial Cells In Vitro Infection Model.

Simple SummaryAntibiotics are widely used for the treatment and control of bovine mastitis. However, the treatment has only been partially effective, as the cure percentage only ranging from 10–30%. Infection by Staphylococcus aureus (S. aureus) is particularly difficult to treat due to the bacteria’s ability to enter and resides inside the host cells. Most antibiotics are ineffective against intracellular bacterial due to the poor penetration into host cells to achieve optimal intracellular bactericidal bioavailability levels. There is therefore, an increasing need to evaluate candidate active substances and develop novel antibiotics effective against intracellular persistence infection. In this study, we examine the potential antibacterial properties of ikarugamycin compound as an alternative drug candidate to be explored for treating persistent bovine mastitis caused by intracellular S. aureus using bovine mammary cell line as an in vitro infection model. We also assessed the potential cytotoxicity effect of ikarugamycin in the infection model. We found that, the ikarugamycin possessed intracellular killing activity against S. aureus within the mammary epithelial cell. This finding highlights the potential application of ikarugamycin as a novel antimicrobial for the treatment of S. aureus mastitis.Staphylococcus aureus is an ubiquitous and versatile pathogen associated with a wide range of diseases. In animals, this bacterium is one of the causative agents of bovine mastitis, responsible for huge economic losses in the dairy industry. Besides the development of antibiotic resistance, the intracellular survival of S. aureus within udder cells has rendered many antibiotics ineffective, leading to therapeutic failure. Our study therefore aims to investigate the in vitro bactericidal activity of ikarugamycin (IKA) against intracellular S. aureus using a bovine mammary epithelial cells (Mac-T cells) infection model and determine the cytotoxic effect. Minimum inhibitory concentration (MIC) was used to determine the antibacterial activity of IKA, and Mac-T cells were infected with S. aureus using gentamicin protection assay. IKA intracellular antibacterial activity assays were used to determine the bactericidal activity of IKA against intracellular S. aureus. The cytotoxicity of IKA against Mac-T cells was evaluated using the resazurin assay. We showed that, S. aureus is susceptible to IKA with a MIC value of 0.6 μg/mL. IKA at 4 × MIC and 8 × MIC have bactericidal activity by reducing 3 and 5 logs10 CFU/mL of S. aureus in the first six-hour of treatment respectively. In addition, IKA demonstrated intracellular killing activity by killing 90% of intracellular S. aureus at 5 μg/mL. This level is comparatively lower than 9.2 μg/mL determined as the half-maximal inhibitory concentration (IC50) of IKA required to kill 50% of Mac-T cells, highlighting a lower concentration required for bactericidal effect compared to the cytotoxic effect. The study highlighted that importance of IKA as a potential antibiotic candidate to be explored for the in vivo efficacy in treating S. aureus mastitis.

First evidence of Klebsiella pneumoniae infection in Aceh cattle: Pathomorphology and antigenic distribution in the lungs.

Background and Aim:Klebsiella pneumoniae is an emerging zoonotic and foodborne pathogen worldwide. Hypervirulent K. pneumoniae (hvKp) was reported as the causative agent of bovine mastitis. This is the first study in Indonesia that has been conducted to determine the capsular serotype of K. pneumoniae, pulmonary gross pathology and histopathology, and distribution of hvKp in the lungs of Aceh cattle.Materials and Methods:The presence of K. pneumoniae in Aceh cattle was investigated in two slaughterhouses in Banda Aceh and Aceh Besar, Indonesia. Lung tissues with gross pathological lesions were collected from 15 cattle presenting with depression, dehydration, or cachexia. The confirmation and capsular serotyping of K. pneumoniae isolates were performed using polymerase chain reaction. The tissues were stained with hematoxylin-eosin and immunohistochemistry to observe the histopathological lesions and the distribution of the hvKp antigens.Results:The pneumonic lesions identified in the lungs of Aceh cattle included hyperemia, hemorrhage, consolidation, and atelectasis. K. pneumoniae was isolated in all 15 lung tissues with pathological pneumonic lesions. Two patterns of infection were observed histopathologically. Acute infection was characterized by hyperemia, inflammatory cell infiltration, hemorrhage, bronchiolar epithelium hyperplasia, bronchial and bronchiolar obstruction with purulent exudates, edema, and atelectasis. On the other hand, chronic infection was defined by macrophage infiltration, emphysema, bronchial dilatation, pleural fibrosis, and alveolar wall thickening by interstitial fibrosis. Immunohistochemical staining using monospecific antisera induced by the hvKp isolate confirmed the presence of K. pneumoniae-specific antigens in the acute infection, predominantly in the bronchiolar, vascular, and alveolar areas. In contrast, generally diffuse infiltrates were found in the pleura and interstitial alveolar areas in chronic infection.Conclusion:hvKp can be detected in the lungs of Aceh cattle, representing acute and chronic infections. The distribution of Klebsiella antigens in the lung tissue was consistent with the histopathological findings.

The first outbreak of methicillin-resistant Staphylococcus aureus in dairy cattle in Poland with evidence of on-farm and intrahousehold transmission

Staphylococcus aureus is a widely recognized pathogen responsible for many serious diseases in both humans and animals. It is also one of the major causative agents of bovine mastitis. Methicillin-resistant S. aureus (MRSA), although relatively rare in this pathology, has been increasingly reported in livestock animals, mainly in pigs, but also cattle, sheep, and poultry. The recent emergence of livestock-associated (LA-)MRSA is cause for an immediate public health concern due to the risk of zoonotic transmission to humans, and is of particular concern for people who work in animal husbandry or have prolonged contact with livestock animals. This study reports on the first LA-MRSA outbreak in dairy cattle and the first probable case of MRSA transmission between humans and cows in Poland. A single dairy farm located in Eastern Poland was monitored on a regular basis for the occurrence of mastitis. Over a 1-yr study period, 717 quarter-milk samples from 583 cows were collected and examined microbiologically. A total of 5 MRSA isolates from as many cows with subclinical mastitis were cultured. They all belonged to the same outbreak, given a 2-mo time window in which they were identified. During the outbreak, 24 oral and nasal swabs were voluntarily taken from 6 people: a milker, a veterinarian, and 4 members of the veterinarian's family. Eight swabs from a milker, veterinarian, and 2 family members yielded positive MRSA cultures. All MRSA isolates were genotyped with a combination of multiple-locus variable number tandem repeat analysis, multilocus sequence typing, and staphylococcal protein A gene (spa) typing. Eleven bovine (n = 5; 5 cases) and human (n = 6; 4 cases) isolates showed an identical drug-susceptibility profile and were indistinguishable upon multiple-locus variable number tandem repeat analysis (pattern A), multilocus sequence typing (ST398) and spa (t034) typing. The results of this study provide the evidence of transmission of MRSA between humans and cows, and between humans in the family setting. This work, despite being a preliminary investigation, underscores the risk of intra- and interspecies transmission of LA-MRSA and urges enhancement of the existing biosecurity measures aimed at preventing MRSA (and other milk pathogens) spread at both the farm- and household levels.

Differences in the coinfective process of Staphylococcus aureus and Streptococcus agalactiae in bovine mammary epithelial cells infected by Mycobacterium avium subsp. paratuberculosis

The interactions between Mycobacterium avium subsp. paratuberculosis (MAP) and the causative agents of bovine mastitis are still relatively unknown. Still, it is suspected that they may contribute to the worsening and persistence of mastitis within the mammary epithelial cells. Considering the growing economic implications of paratuberculosis and subclinical mastitis in dairy herds, this study aimed to determine the coinfection interaction between MAP and S. aureus or S. agalactiae in bovine mammary epithelial cells (MAC-T) in an ex-vivo model. For this purpose, internalisation tests of MAP + S. aureus or MAP + S. agalactiae were performed in MAC-T cells for 10, 30 and 120 min. The qPCR was performed to quantify internalised MAP at the time of exposure. Colony-forming units were counted on BHI agar medium for internalised subclinical mastitis bacteria at each time of infection. Viability tests of MAC-T cells, using the lactate dehydrogenase assay, were performed. The results showed that in the MAC-T cells previously infected by MAP and subsequently by S. aureus, there was a rapid internalisation in the first 10 min, maintaining a higher number of internalised bacteria during all exposure times. Regarding MAP + S. agalactiae, there were no changes in the internalisation patterns. The amount of MAP remained constant at all times evaluated, and there was no compromise in the viability of MAC-T cells during the tests. Thus, the results demonstrate the existence of an interaction between MAP + S. aureus, favouring internalisation and being able to contribute to the persistence of subclinical mastitis in dairy herds.