agar and Mastitis--Bovine

This study aimed to examine the antimicrobial susceptibility of bovine mastitis pathogens in Japan and develop criteria for testing antimicrobial susceptibility using the simplified agar disk diffusion (ADD) method that is currently being used in clinical practice. Milk samples from 1,349 dairy cows with clinical mastitis were collected and cultured. The minimum inhibitory concentrations (MICs) of the antimicrobials were determined for 504 strains of 28 bacteria. Of the gram-positive bacteria, most Staphylococcus spp. were susceptible to penicillin G (PCG), kanamycin (KM), oxytetracycline (OTC), cefazolin (CEZ), pirlimycin, enrofloxacin, and marbofloxacin. Streptococcus spp. and Trueperella pyogenes showed resistance to OTC and KM. Most gram-negative bacteria were resistant to OTC and CEZ and particularly susceptible to fluoroquinolones. To develop the criteria for a disk diffusion test of the simplified ADD method, the relationships between MICs and diameters of inhibition zones (DIZs) were analyzed and compared with the conventional method. The susceptibility breakpoints of several antimicrobials were lower for both gram-positive and gram-negative bacteria. Particularly for gram-positive bacteria, the application of the new criteria lowers the breakpoint for PCG, suggesting that the use of PCG instead of CEZ may increase. The results suggest that use of these criteria for the simplified ADD method may lead to appropriate antimicrobial choice and consequently the appropriate use of antimicrobials in clinical practice.

Topics: Agar; Animals; Anti-Bacterial Agents; Anti-Infective Agents; Cattle; Cattle Diseases; Cefazolin; Drug Resistance, Bacterial; Female; Gram-Negative Bacteria; Gram-Positive Bacteria; Japan; Mastitis, Bovine; Microbial Sensitivity Tests

In order to counter the antibiotic resistance phenomenon, a prudent and rational use of antimicrobials should be driven by an accurate clinical diagnosis and, when possible, by the isolation of the etiological agent followed by susceptibility testing, with the aim to select the most suitable molecule for therapy. Cow mastitis is considered the main cause of antibiotic use in the cattle breeding sector. The purpose of this study was to compare the broth microdilution (BMD) method performed with Sensititre Custom Plates and the agar disk diffusion (ADD) method in determining antimicrobial susceptibility of 215 isolates from bovine mastitis, including contagious pathogens (Staphylococcus aureus, Streptococcus agalactiae) and environmental (Streptococcus uberis, Streptococcus dysgalactiae, Enterococcus spp., Escherichia coli, Serratia marcescens, Klebsiella pneumoniae). We compared results of the following antimicrobials: amoxicillin/clavulanic acid, ampicillin, cefazolin, ceftiofur, enrofloxacin, erythromycin, kanamycin, oxacillin, penicillin, pirlimycin, rifampicin and trimethoprim/sulphonamides. We applied MIC breakpoints and zone diameter breakpoints as recommended by CLSI and EUCAST. MIC and disk diffusion diameters were compared for 1839 microorganism/antimicrobial combination and discrepancies between the two methods were classified as very major discrepancy (VMD), major discrepancy (MD) and minor discrepancy (MiD). The overall agreement between the two methods was found to be 80.7% with a Cohen's kappa coefficient of 0.397, thus indicating a fair concordance. BMD method and ADD method demonstrated a satisfactory agreement (89 to 100%) for S. aureus and S. marcescens and all antimicrobial agents tested. Low agreement was observed for S. uberis and rifampicin (20%), enrofloxacin (49%), penicillin (51%) and pirlimycin (52%), E. coli and ampicillin (20%), S. dysgalactiae and enrofloxacin (44%), S. agalactiae and rifampicin (25%). A possible explanation for the discrepancies detected could be found in the breakpoints used which, sometimes, are not specific for the tissue-matrix of isolation/animal species/pathogen agent. The majority of the discrepancies found were MiD and MD, revealing a higher restrictiveness of the BMD method, while VMD represented only 0.2% of the total observations, a comforting fact since this type of error may result in treatment failure.

Topics: Agar; Ampicillin; Animals; Anti-Bacterial Agents; Anti-Infective Agents; Cattle; Enrofloxacin; Escherichia coli; Female; Mastitis, Bovine; Microbial Sensitivity Tests; Penicillins; Rifampin; Staphylococcus aureus

Bacteriological examination of milk samples is a prerequisite for pathogen-specific therapy and aids in limiting antimicrobial resistance. The aims of this study were to establish a standardized scheme for reliable Streptococcus uberis identification in routine diagnosis and to evaluate the accuracy of conventional tests and growing patterns of Strep. uberis on a selective medium (modified Rambach agar medium, MRAM) using 16S rRNA gene sequencing analysis as a reference method. We obtained isolates of presumptive Strep. uberis (n = 336) from quarter milk samples of dairy cows with intramammary infections and classified the isolates into 2 clusters using biochemical characterization. In cluster 1 (n = 280), cocci grew as non-hemolytic colonies, hydrolyzing esculin, carrying no Lancefield antigen (A/B/C/D/G) or Christie Atkins Munch-Petersen factor, and their growth was inhibited on an Enterococcus agar. Production of β-d-galactosidase on MRAM was shown by 257 of the cluster 1 isolates (91.79%), and 16S rRNA gene sequencing verified 271 (96.79%) of the isolates to be Strep. uberis. In 264 isolates (94.29%), MRAM agreed with the sequencing results. In cluster 2 (n = 56), isolates showed different characteristics: 37 (66.07%) were β-d-galactosidase-positive, and based on 16S sequencing results, 36 (64.29%) were identified correctly as Strep. uberis using biochemical methods. Identification success in this group differed significantly between routine diagnosis and MRAM application: MRAM agreed with sequencing results in 47 isolates (83.93%). To identify Strep. uberis and differentiate it from other lactic acid bacteria in routine diagnosis, we suggest using catalase reaction, hemolysis, esculin hydrolysis, and growth on enterococci agar. Isolates that show a typical biochemical profile can be identified satisfactorily with these tests. For Strep. uberis isolates with divergent patterns, application of MRAM as a follow-up test increased the diagnostic accuracy to 94.64%.

Topics: Agar; Animals; Cattle; Female; Mastitis, Bovine; Milk; RNA, Ribosomal, 16S; Streptococcal Infections; Streptococcus

Bovine mastitis (BM) is a costly disease in dairy cattle production. The prevention and treatment of mastitis is performed by applying antimicrobial products that negatively affect milk quality. In the last years, the use of probiotic microorganisms to prevent infections in humans and animals has being aggressively studied. Samples from teat canal and milk (foremilk and stripping) were taken from healthy and mastitic mammary quarters. A screening of the surface properties and antagonistic substances production of lactic acid bacteria (LAB) isolated from the mammary gland was performed to select potential probiotic strains to prevent mastitis. Somatic cell count, physico-chemical and microbiological studies were carried out. Pre-selected microorganisms were genetically identified. Compared with stripping milk, foremilk showed lower levels of fat and higher levels of pH, density, microorganism numbers, lower percentage of strains with mean and high hydrophobicity and mean autoaggregation and higher number of strains able to produce hydrogen peroxide and bacteriocins. The other parameters analyzed were not statistically significant. One hundred and two LAB strains were isolated. Most of them had low degrees of hydrophobicity and autoaggregation. No correlation between these properties was found. Antagonistic metabolites were mainly produced by strains isolated from healthy quarters. Most of the pre-selected strains were identified as Streptococcus bovis and Weissella paramesenteroides. Three bacteriocin-producers were found and their products partially characterized. The results of this work are the basis for the further design of a specie-specific probiotic product able to prevent BM.

Topics: Agar; Animals; Antigens, Bacterial; Argentina; Bacterial Proteins; Cattle; Dairying; Enterococcus faecalis; Enterococcus faecium; Female; Lactobacillaceae; Listeria monocytogenes; Mammary Glands, Animal; Mastitis, Bovine; Milk; Streptococcus bovis

To investigate the use of mannitol salt agar (MSA) supplemented with acriflavine for selective growth and quantification of Staphylococcus aureus from flushed dairy manure wastewater (FDMW).. Minimal inhibitory concentrations of acriflavine in MSA were determined by comparing the growth of S. aureus subsp. aureus (ATCC 33591) and Staphylococcus epidermidis (ATCC 155) in pure culture. Acriflavine concentrations of 1.3, 1.4 and 1.5 mg l(-1) reduced CFU of S. epidermidis by 43%, 55% and 87%, respectively, while CFU of S. aureus subsp. aureus were only reduced by 15%, 20% and 26% at the respective concentrations of acriflavine. MSA supplemented with 1.5 mg l(-1) acriflavine was tested for selective growth of indigenous S. aureus from three grab samples of FDMW. Acriflavine concentrations of 1.5 mg l(-1) reduced background flora without significantly reducing (P < 0.05) indigenous S. aureus counts.. Acriflavine-supplemented MSA provides an effective media for selective growth and quantification of indigenous S. aureus from FDMW in the presence of high levels of background microflora.. S. aureus is implicated for mastitis infections in dairy cows. Therefore, a reliable means for monitoring and detecting the organism in FDMW provides a tool for measuring the effectiveness of treatment for reducing S. aureus levels and implementing flushwater recycling without affecting herd health.

Topics: Acriflavine; Agar; Animals; Bacteriological Techniques; Cattle; Culture Media; Dairying; Fresh Water; Mannitol; Manure; Mastitis, Bovine; Staphylococcal Infections; Staphylococcus aureus; Waste Disposal, Fluid

Topics: Agar; Animals; Cattle; Diagnosis, Differential; Enzyme Inhibitors; Eosine Yellowish-(YS); Escherichia coli Infections; Female; Food Contamination; Gram-Negative Bacterial Infections; Mastitis, Bovine; Methylene Blue; Sensitivity and Specificity; Specimen Handling

A total of 414 coagulase-positive staphylococcal strains obtained at the mastitis laboratory, National Veterinary Institute, Uppsala, Sweden, were studied. One hundred and seventy seven strains were used for a frequency study. Ninety-seven per cent were identified as Staphylococcus aureus, 2% as Staphylococcus intermedius and 1% as Staphylococcus hyicus. Two hundred and thirty seven strains with atypical hemolysis reactions on bovine blood agar were randomly selected, with the aim to increase the number of S. intermedius and S. hyicus strains available for testing. Eight different characteristics, including physiological, enzymatical and biochemical properties, were used to identify the coagulase-positive Staphylococcus species. The results of this study suggest that the following tests should be included for correct identification of the 3 different species of coagulase-positive staphylococci: P agar supplemented with acriflavin, beta-galactosidase and hemolytic reaction on chocolate agar. These 3 tests are simple and quick to perform and enable accurate for easy differentiation of the 3 coagulase-positive Staphylococcus species.

Topics: Acriflavine; Agar; Animals; beta-Galactosidase; Cattle; Coagulase; Culture Media; Female; Hemolysis; Mastitis, Bovine; Milk; Staphylococcal Infections; Staphylococcus; Staphylococcus aureus

The antagonistic effect of lactic acid bacteria (LAB, including Lactobacillus acidophilus, L. bulgaricus, L. casei and Streptococcus thermophilus) on Staphylococcus aureus and Escherichia coli was evaluated on MRS agar with the deferred and cross-streaking techniques, and in milk with the plate counting method. All LAB were repressive to S aureus and E coli on the agar medium. However, their suppressive activity was significantly reduced when the agar medium was buffered to pH 7.2. In normal milk, L acidophilus strains A and B, S thermophilus and its combinations with L acidophilus A and L bulgaricus 6032 were inhibitory to S aureus, while in mastitic milk, only S thermophilus and its combinations showed inhibition. L acidophilus A and L bulgaricus 34104 were repressive to E coli growth in normal milk. S thermophilus and its combinations were inhibitory to E coli in both the normal and mastitic milk samples. These results indicate that the antagonistic activity of LAB on pathogenic bacteria varied with the type of media in which the tests were done, and that testing of in vitro antagonism in milk would be more informative than that in artificial media for in vivo tests concerning the possible roles of competitive microbiological ecology in mastitis control.

Topics: Agar; Animals; Cattle; Escherichia coli; Female; Lactates; Lacticaseibacillus casei; Lactobacillus; Lactobacillus acidophilus; Mastitis, Bovine; Milk; Reference Values; Staphylococcus aureus; Streptococcus

Coagulase-negative staphylococci from cases of bovine mastitis were identified to species level by using an identification scheme based on a three-plate test system which tested for DNase on DNA agar, for protease on calcium caseinate agar, and for the organism's sensitivity to novobiocin, desferrioxaminine (deferoxamine) and fosfomycin by agar diffusion tests. Testing for the inhibition of Staphylococcus delta haemolysin (Skalka 1991) can replace the protease tests.

Topics: Agar; Animals; Cattle; Coagulase; Deoxyribonucleases; Endopeptidases; Female; Fosfomycin; Mastitis, Bovine; Sensitivity and Specificity; Staphylococcus; Staphylococcus epidermidis

The medium of Rambach was modified to permit differentiation of mastitis streptococci. A total of 377 streptococci isolated from bovine IMI was used in the study. Of the 159 strains identified as Streptococcus uberis, 151 strains (94.9%) were beta-galactosidase-positive and yielded blue colonies on modified Rambach agar. In comparison, Streptococcus agalactiae, Streptococcus dysgalactiae, Enterococcus saccharolyticus, or Enterococcus faecalis strains were negative for propylene glycol utilization (red colonies) or beta-galactosidase production; all strains yielded colorless colonies on modified Rambach agar. However, 5 of 35 (14.3%) Streptococcus equinus strains were also positive for beta-galactosidase. Results indicate that modified Rambach agar is a convenient medium for the differentiation of the Strep. uberis from the other mastitis streptococci. Furthermore, modified Rambach agar could be easily incorporated as a screening medium for Strep. uberis in mastitis bacteriology laboratories.

Topics: Agar; Animals; beta-Galactosidase; Cattle; Female; Mastitis, Bovine; Propylene Glycol; Propylene Glycols; Streptococcal Infections; Streptococcus

Thirty of 104 strains of Staphylococcus aureus isolated from clinical cases of bovine mastitis in England grew as diffuse colonies in serum soft agar (SSA), 45 grew as mixed diffuse and compact colonies and 29 yielded compact colonies only. The compact strains grew as diffuse colonies in SSA after one passage in the mammary gland of mice. However, none of the strains had an unstained halo when examined by the India ink technique and there was a 99.99 per cent reduction in the viable numbers of the bacteria in 30 representative strains 24 hours after inoculation into the peritoneal cavity of mice. By contrast the truly encapsulated strain M had an unstained halo by the India ink technique and resisted phagocytic killing in the peritoneal cavity. It is concluded that these strains from cases of mastitis are not encapsulated and that growth as diffuse colonies in SSA is not a reliable test of encapsulation.

Topics: Agar; Animals; Carbon; Cattle; Coloring Agents; Female; Mammary Glands, Animal; Mastitis, Bovine; Mice; Mice, Inbred Strains; Peritoneal Cavity; Pregnancy; Staining and Labeling; Staphylococcus aureus

Early detection of the CAMP-reactivity of group B streptococci was achieved in 5% sheep or bovine blood agar supplemented with maltose or glucose at a final concentration of 0.04% under aerobic as well as under microaerobic conditions in a candle jar. All 55 cultures of group B streptococci tested on these media yielded distinct CAMP-reactions within 8 to 10 h of incubation at 37 degrees C, allowing a more rapid recognition of these streptococci.

Topics: Agar; Animals; Bacterial Proteins; Bacteriological Techniques; Blood; Cattle; Female; Hemolysin Proteins; Humans; Mastitis; Mastitis, Bovine; Pregnancy; Sheep; Staphylococcus aureus; Streptococcus agalactiae

The elaboration of type-specific capsular antigens by group B streptococci can be demonstrated by the serum-soft agar technique. Group B streptococci isolated from bovine mastitis, namely, strains 9F, 14Mi, 8Mo, 44B, and 4S, were shown to form diffuse and compact types of colony morphology in serum-soft agar. Immunochemical and chemical analyses of antigens isolated from diffuse and compact colonies of strain 9F indicated that the diffuse-type growth of this strain was due to the elaboration of a galactose-rich surface antigen, whereas the compact 9F strain was devoid of this antigen. Specific 9F antiserum was effective in converting the diffuse 9F colonies of the compact type, indicating the presence of capsular material. Preliminary evidence suggests that the serum-soft agar technique could also be used to determine the antigenic diversity of the surface antigens of group B streptococci, thus providing an effective means of typing those organisms.

Topics: Agar; Animals; Antigens, Bacterial; Cattle; Epitopes; Evaluation Studies as Topic; Immunodiffusion; Immunoelectrophoresis; Mastitis, Bovine; Serotyping; Streptococcus agalactiae

Topics: Acute Disease; Agar; Animals; Cattle; Clostridium Infections; Clostridium perfringens; Culture Media; Drug Therapy, Combination; Female; Mastitis, Bovine; Penicillin Resistance; Penicillins; Streptomycin; Sulfonamides

Topics: Agar; Animals; Bacteriological Techniques; Blood; Cattle; Culture Media; Diagnosis, Differential; Escherichia coli; Female; Mammary Glands, Animal; Mastitis, Bovine; Milk; Spinal Nerve Roots; Staphylococcus; Streptococcus

The cellophane-over-agar technique has been shown to give high titers of enterotoxins A, B, and C for routine testing of staphylococci for enterotoxigenicity.

Topics: Agar; Animals; Brain; Caseins; Cattle; Cellophane; Culture Media; Enterotoxins; Female; Heart; Humans; Immunodiffusion; Mastitis, Bovine; Methicillin; Microbial Sensitivity Tests; New Zealand; Penicillin Resistance; Staphylococcal Infections; Staphylococcus

Topics: Agar; Animals; Anti-Bacterial Agents; Cattle; Coagulase; Drug Resistance, Microbial; Female; Genetics, Microbial; Hemolysin Proteins; Humans; Hungary; Immune Sera; Mastitis, Bovine; Microbial Sensitivity Tests; Milk; Nasal Mucosa; Penicillin Resistance; Pigmentation; Rabbits; Staphylococcus

Topics: Agar; Agglutination; Animals; Antigens; Cattle; Hemagglutination; Immunodiffusion; Mastitis, Bovine; Polysaccharides, Bacterial; Precipitin Tests; Research; Streptococcus

Topics: Agar; Animals; Anti-Bacterial Agents; Blood; Cattle; Chlortetracycline; Culture Media; Dihydrostreptomycin Sulfate; Drug Resistance; Drug Resistance, Microbial; Erythromycin; Escherichia coli; Female; Mastitis; Mastitis, Bovine; Micrococcus; Milk; Neomycin; Nitrofurazone; Penicillin G; Polymyxins; Pseudomonas aeruginosa; Research; Staphylococcus; Streptococcus