Evaluation of methods of DNA extraction from Staphylococcus aureus in milk for use in real-time PCR

Dibbern, Aline Gerato; Botaro, Bruno Garcia; Viziack, Mariana P.; Silva, Luís Felipe Prada e; Santos, Marcos Veiga dos

doi:10.4238/2015.january.16.6

Cited by 10 publications

(8 citation statements)

References 15 publications

(17 reference statements)

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“…DNA from all presumptive S. aureus isolates was extracted using the conventional boiling method as previously described [57]. All extracted DNA was stored at −20 • C until used for polymerase chain reaction (PCR) amplification.…”

Section: Dna Extraction and Molecular Identification Of S Aureusmentioning

confidence: 99%

Biofilm Production Ability, Virulence and Antimicrobial Resistance Genes in Staphylococcus aureus from Various Veterinary Hospitals

et al. 2020

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Staphylococcus aureus (S. aureus) is one of the most clinically important zoonotic pathogens, but an understanding of the prevalence, biofilm formulation ability, virulence, and antimicrobial resistance genes of S. aureus from veterinary hospitals is lacking. By characterizing S. aureus in different origins of veterinary hospitals in Guangzhou, China, in 2019, we identified with the presence of S. aureus in pets (17.1%), veterinarians (31.7%), airborne dust (19.1%), environmental surfaces (4.3%), and medical device surfaces (10.8%). Multilocus sequence typing (MLST) and Staphylococcus protein A (spa) typing analyses demonstrated methicillin-sensitive S. aureus (MSSA) ST398-t571, MSSA ST188-t189, and methicillin-resistant S. aureus (MRSA) ST59-t437 were the most prevalent lineage. S. aureus with similar pulsed-field gel electrophoresis (PFGE) types distributed widely in different kinds of samples. The crystal violet straining assays revealed 100% (3/3) of MRSA ST59 and 81.8% (9/11) of MSSA ST188 showed strong biofilm formulation ability, whereas other STs (ST1, ST5, ST7, ST15, ST88, ST398, ST3154 and ST5353) showed weak biofilm production ability. Polymerase chain reaction (PCR) confirmed the most prevalent leucocidin, staphylococcal enterotoxins, ica operon, and adhesion genes were lukD-lukE (49.0%), sec-sel (15.7%), icaA-icaB-icaC-icaR (100.0%), and fnbB-cidA-fib-ebps-eno (100.0%), respectively. Our study showed that the isolates with strong biofilm production ability had a higher prevalence in clfA, clfB, fnbA and sdrC genes compared to the isolates with weak biofilm production ability. Furthermore, 2 ST1-MRSA isolates with tst gene and 1 ST88-MSSA isolate with lukS/F-PV gene were detected. In conclusion, the clonal dissemination of S. aureus of different origins in veterinary hospitals may have occurred; the biofilm production capacity of S. aureus is strongly correlated with ST types; some adhesion genes such as clfA, clfB, fnbA, and sdrC may pose an influence on biofilm production ability and the emergence of lukS/F-PV and tst genes in S. aureus from veterinary hospitals should raise our vigilance.

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Section: Dna Extraction and Molecular Identification Of S Aureusmentioning

confidence: 99%

Biofilm Production Ability, Virulence and Antimicrobial Resistance Genes in Staphylococcus aureus from Various Veterinary Hospitals

et al. 2020

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“…This result represents the evolution of the microorganisms with regards to the development of resistance mechanisms. According to Dibbern, Botaro, Viziack, Silva and Santos (2015), the majority of mastitis cases are caused by bacteria, especially S. aureus, a microorganism capable of producing biofilm. Scherr, Heim, Morrison and Kielian (2014) define biofilm as a group of cells attached to a biotic or abiotic surface imbedded in a protective extracellular matrix, thus granting resistance to antimicrobial drugs used during the treatment of illness, as well as to disinfectants and the immune system of the host, allowing the infection to acquire a chronic nature (Arciola, Campoccia, Speziale, Montanaro, & Costerton, 2012;Guimarães et al, 2012;Tremblay, Caron, Blondeau, Messier, & Jacques, 2014).…”

Section: Resultsmentioning

confidence: 99%

Antimicrobial potential of Jurema preta and umburana, native species of the Caatinga biome, on Staphylococcus isolated from small ruminants with mastitis

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et al. 2020

Semina: Ciênc. Agrár.

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Mastitis is responsible for both damage to animal health and economic losses to the industry. To identify effective treatments for this disease, products extracted from a variety of plants with antimicrobial potential have gained attention. The present study aimed to assess the in vitro antibacterial potential of the ethanolic extract of two plant species from the Caatinga biome against bacteria isolated from small ruminants with subclinical mastitis. The leaves of Mimosa tenuiflora and Commiphora leptophloeos were dried and processed to obtain crude ethanolic extracts and their phenolic composition was evaluated. In total, 33 Staphylococcus spp. isolates from the bacterial collection of the Laboratory of Food Quality Control of IF SERTÃO-PE were used for evaluation of biofilm production. Furthermore, an antimicrobial susceptibility test was conducted using the minimal bactericidal concentration (MBC) method against the two ethanolic extracts. The toxic potential was measured through a toxicity test with Artemia salina. The quantification of the phenolic compounds revealed that the ethanolic extracts of M. tenuiflora and C. leptophloeos possessed higher amounts of myricetin (43.2 and 294.9 mg in 10 g, respectively) in relation to the other compounds. A 39.4% positivity rate was observed in the nuc gene investigation. The biofilm production analysis revealed that 96.9% of the isolates produced biofilm, evidencing the evolution the microorganisms regarding the development of resistance mechanisms. The MBC results showed an inhibition range between 195.30 and 3125.00 µg mL-1 and between 781.20 and 6250.00 µg mL-1 for the extracts of M. tenuiflora and C. leptophloeos, respectively. The M. tenuiflora extract showed the highest activity, suppressing 100% of the bacterial isolates (n=26), whereas the extract of C. leptophloeos showed an inhibition percentage of 69.23%. The crude ethanolic extract (EEB) of M. tenuiflora was found to be toxic, presenting a DL50 of 118.356 µg mL-1. In contrast, the EEB of C. leptophloeos was found to be non-toxic (DL50 = 1527.430 µg mL-1). In conclusion, both native Caatinga species presented antibacterial activity and myricetin was the major compound. These findings highlight the need for further studies regarding the identification of anti-mastitis products from natural extracts.

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“…This lower performance could be due to the presence of fat in which some organisms may get trapped. [20] This sensitivity is on par with the bacterial loading commonly seen in contaminated samples, which ranges from 100 to 50,000 organisms/ml [21]. …”

Section: Resultsmentioning

confidence: 99%

Visual detection of Brucella in bovine biological samples using DNA-activated gold nanoparticles

et al. 2017

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Brucellosis is a bacterial disease, which, although affecting cattle primarily, has been associated with human infections, making its detection an important challenge. The existing gold standard diagnosis relies on the culture of bacteria which is a lengthy and costly process, taking up to 45 days. New technologies based on molecular diagnosis have been proposed, either through dip-stick, immunological assays, which have limited specificity, or using nucleic acid tests, which enable to identify the pathogen, but are impractical for use in the field, where most of the reservoir cases are located. Here we demonstrate a new test based on hybridization assays with metal nanoparticles, which, upon detection of a specific pathogen-derived DNA sequence, yield a visual colour change. We characterise the components used in the assay with a range of analytical techniques and show sensitivities down to 1000 cfu/ml for the detection of Brucella. Finally, we demonstrate that the assay works in a range of bovine samples including semen, milk and urine, opening up the potential for its use in the field, in low-resource settings.

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Evaluation of methods of DNA extraction from Staphylococcus aureus in milk for use in real-time PCR

Cited by 10 publications

References 15 publications

Biofilm Production Ability, Virulence and Antimicrobial Resistance Genes in Staphylococcus aureus from Various Veterinary Hospitals

Biofilm Production Ability, Virulence and Antimicrobial Resistance Genes in Staphylococcus aureus from Various Veterinary Hospitals

Antimicrobial potential of Jurema preta and umburana, native species of the Caatinga biome, on Staphylococcus isolated from small ruminants with mastitis

Visual detection of Brucella in bovine biological samples using DNA-activated gold nanoparticles

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