Comparison of biofilm formation by Staphylococcus aureus and Staphylococcus epidermidis strains isolated from sheep milk using three diagnostic methods (original) (raw)
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Brazilian Journal of Microbiology, 2013
Biofilm formation is considered to be a selective advantage for Staphylococcus aureus mastitis isolates by facilitating bacterial persistence in the udder. It requires attachment to mammary epithelium, proliferation and accumulation of cells in multilayers. The objective of this study was to determine the sensitivity and specificity of three techniques for the detection of S. aureus biofilm-positive strains. Two phenotypic tests, including growth on microtitre plates and Congo red agar, were compared with a PCR technique using 94 S. aureus strains obtained from cows with subclinical mastitis from two farms in the state of São Paulo. These strains were characterised by in vitro slime production on Congo red agar, biofilm formation on microtitre plates and the presence of the icaA and icaD genes. The results revealed that 85% of the isolates tested produced slime on the Congo red agar, 98.9% of the isolates produced biofilms in vitro by adhering to sterile 96-well "U" bottom polystyrene tissue culture plates, and 95.7% of the isolates carried the icaA and icaD genes. The results of the phenotypic tests for biofilm formation were compared with those of the molecular analysis, and the sensitivity and specificity of the Congo red agar test were 88.9% and 100%, respectively, while those of the microtitre plate test were 100% and 25%, respectively. When the phenotypic methods for the detection of biofilm producers, namely growth on microtitre plates and Congo red agar, were compared, the sensitivity and specificity were 86% and 100%, respectively. Therefore, growth on Congo red agar and the microtitre plate test are methods that could be used to determine whether an isolate has the potential for biofilm production.
Veterinary sciences, 2018
Biofilm formation is of growing concern in human and animal health. However, it is still unclear how biofilms are related to mastitis infections in dairy cattle. In this study, a comparison between two tests for biofilm formation and the association between biofilm and the presence of genes associated with biofilm formation were investigated for 92 isolates from clinical mastitis cases. Congo red agar (CRA) and microtitre test assay (MTA) in vitro phenotypic tests were used to evaluate biofilm formation. The presence of , , and genes associated with biofilm formation was confirmed using the polymerase chain reaction. Results show that most of the isolates, though not possessing one of the biofilm-forming genes, were able to produce biofilms. MTA was more frequently positive in identifying biofilm-forming isolates than CRA.
Biofilm-Producing Ability of Staphylococcus aureus Obtained from Surfaces and Milk of Mastitic Cows
Veterinary Sciences
This study was conducted to evaluate the incidence of mastitis in 153 dairy cows and to evaluate the kinetics of adhesion of isolates obtained from surfaces and milk in comparison with the reference strain (RS), CCM 4223. The surfaces of the floor, teat cup, and cow restraints were aseptically swabbed in three replicates (n = 27). Of the total number of infected cows (n = 43), 11 samples were found to be positive for Staphylococcus aureus, 12 samples tested positive for non-aureus staphylococci, 6 samples tested positive for Streptococcus spp., and 11 samples tested positive for other bacteria (Escherichia coli, Pseudomonas spp.) or a mixed infection. The most represented pathogen in milk (11/43) and on surfaces (14/27) was S. aureus. The kinetics of adhesion of the reference strain and isolates of S. aureus on stainless steel surfaces were determined after 3, 6, 9, 12, 24, and 48 h, and 3, 6, 9, 12, and 15 days of incubation. All strains reached counts higher than 5 Log10 CFU/cm2 n...
Animal Science Journal, 2014
Biofilm formation and antimicrobial resistance of Staphylococcus aureus are important virulence factors in cases of mastitis in dairy cows. However, few studies have investigated mastitis strains isolated from heifers. Within this context, the objective of the present study was to investigate biofilm formation on Congo red agar, the presence of the icaA and icaD genes by polymerase chain reaction (PCR), and the percentage of in vitro antimicrobial resistance of 110 S. aureus isolates from mammary gland secretions of heifers and cows with mastitis. PCR detected the icaA and icaD genes in 98% and 100% of isolates, respectively. However, only 55.5% of all isolates produced a biofilm on Congo red agar. Antimicrobial susceptibility testing revealed that 47.0% of isolates from heifers and 70.4% of isolates from cows were resistant to at least one of the antimicrobial agents tested. Resistance to penicillin and/or ampicillin was the most frequent (44.5%). These results indicate the need to implement prophylactic and control measures of mastitis for heifers. Heifers and cows can carry resistant strains with the capacity of biofilm production, a fact representing a threat to public health and animal well-being and generating losses to dairy farmers.
Polish Journal of Veterinary Sciences, 2013
The aim of this study was to examine virulence factors and the ability of S. aureus and CNS species isolated from milk of cows with mastitis to form biofilm, and to compare them with virulence factors of staphylococci from milk of cows without mastitis and cowshed environment. Most of S. aureus strains from cows with mastitis showed haemolytic activity (93.9%), among them 72.7% and 21.2% produced α- and β-haemolysin, respectively. S. aureus from cows with mastitis symptoms produced proteases (above 48%) and esterase (42.4%). The highly significant relationship between the number of S. xylosus strains producing haemolysins (62%) and the origin of these strains from milk of cows with mastitis was observed. The ability to produce proteases was significantly associated with S. sciuri from milk of cows with mastitis. The ability of biofilm formation by staphylococcal strains from milk of cows with mastitis was greater than in strains from milk of cows without mastitis and the difference ...
Microbial pathogenesis, 2017
Staphylococcus aureus and coagulase-negative staphylococci (CNS) are important causes of intramammary infection in dairy cattle, and their ability to produce biofilm is considered an important virulence property in the pathogenesis of mastitis. However, the published date on mechanisms and factors involved in infection persistence in the mammary gland remains unclear. The aim of this study was to investigate whether the main Staphylococcus species involved in bovine intramammary infections possess specific characteristics that promote colonization of the udder. We evaluated the biofilm-forming ability and distribution of adhesion- and biofilm-associated genes of Staphylococcus spp. isolated from bovine mastitis infected animals in Argentinean dairy farms. For this purpose, the phenotypic biofilm formation ability of 209 Staphylococcus spp. from bovine mastitis was investigated. All isolates produced biofilm in vitro, being 35,0% and 45,0% of the 127 S. aureus or 51,0% and 29,0% of t...
Hassan W. H.a,*, Hatem M. E.b, Elnwary H. A.c, Sediek S. H.c, 2016
In the current study, a total of 20 and 78 milk samples were collected from animals showed signs of clinical and subclinical mastitis, for isolation and identification of different causative pathogens in some dairy farms of Beni-Suef Governorate, and for investigation of in vitro sensitivity. The recovered microorganisms were Staphylococcus species (n=79; 80.61%), Enterococcus spp. (n=28; 28.57%), CAMP negative Streptococci, Pseudomonas aeruginosa (n=7; 7.14%), E. coli (n=3; 3.06%) and Proteus vulgaris (n=1; 1.02%). Antibiogram profile for S. aureus showed that the most effective drug was vancomycin and the least was penicillin. Trials were done to detect biofilm production for recovered isolates of S. aureus (n=23) by the use of a phenotypic method (Congo red agar, CRA) and genotypic methods through determination of some biofilm related genes using PCR. All recovered S. aureus isolates were seeded on the CRA media to detect the biofilm forming ability. It has been found that all tested isolates showed a biofilm forming ability either strong (13; 56.52%) or intermediate (10; 43.48%). The detection of some biofilm associated genes (icaA, icaD and bap genes) using polymerase chain reaction revealed that two (10.53%) isolates out of 19 were negative for all tested genes, 16 (84.21%) isolates harbored both icaA and icaD gene, while only one (5.26%) isolate had all tested genes.
Veterinary World, 2022
Background and Aim: Mastitis is considered a significant disease of lactating animals. There are new attitudes for recognizing genes responsible for causing this disease to overcome and change the manipulation of this problem. This study aimed to isolate and identify Staphylococcus aureus strains from mastitic bovine animals and detect some specific biofilm-forming genes (icaA, icaD, and biofilm-associated protein [bap] genes clfA, fnbA, agrI, agrII, agrIII, agrIV, and cna). Materials and Methods: A total of 121 mastitic milk samples were analyzed using biochemical tests (catalase test, oxidative-fermentative test, and coagulase test) and Gram stain. Multiplex polymerase chain reaction was applied to characterize biofilm genes (icaA, icaD, bap, clfA, and fnbA) in addition to (agrI, agrII, agrIII, agrIV, and cna). Results: Among the 121 milk samples, 35 staphylococci isolates were derived with an incidence of 28.92% (35/121); among them, 19 are coagulase positive. Ninety percent of the isolates had ica genes (icaA and icaD) while bap gene was not recognized in any isolate. In addition, the incidence of fnbA, can, and clfA was 89.5% each. The prevalence of agr specific groups (agrI, agrII, agrIII, and agrIV) was 78.9%, 52.6%, 10.5%, and 15.8%, respectively. Conclusion: This study concluded that S. aureus has variant mechanisms of pathogenicity to form biofilm devoid of carrying a specific gene.
Veterinary Microbiology, 2007
Biofilm formation is considered a selective advantage for staphylococci mastitis isolates, facilitating bacterial persistence in the udder. It requires attachment to mammary epithelium, proliferation and accumulation of cells in multilayers and enclosing in a polymeric matrix, being regulated by several loci. As biofilm formation can proceed through different pathways and time ranges, its detection may differ according to the time of observation. This study aimed at evaluating the time course evolution of biofilm production in Staphylococcus aureus (n=26) and Staphylococcus epidermidis (n=29) mastitis isolates by Fluorescent In Situ Hybridisation. Biofilm-forming ability increased with incubation time for both species: for S. aureus, 34.6%, 69.2% and 80.8% of the isolates were able to produce biofilm at 24, 48 and 72 hours, respectively. For S. epidermidis, 44.8%, 62.1% and 75.9% of the isolates were biofilm-positive at 24, 48 and 72 hours, respectively. No significant difference was found between species at each time point (Friedman's test, p>0.05). For S. aureus, although a significant difference was found between 24 and 48 hours (Wilcoxon matched paired test,