Antibiotic resistance in Staphylococcus aureus strains isolated from cows with mastitis in eastern Poland and analysis of susceptibility of resistant strains to alternative non-antibiotic agents: Lysostaphin
, Nisin and Polymyxin.
This was done following manufacturer instructions with modifications such as the addition of 20 [micro]L of 1 mg/mL lysostaphin
Pellet was resuspended in 50 [micro]L of dd water, followed by addition of 5 [micro]L lysostaphin
solution (Sigma-Aldrich Corp.) and incubated at 37[degrees]C for 30 min, followed by DNA extraction processes using PureLink[TM] genomic DNA mini Kit (Thermo Fisher Scientific) according to manufacturer's instructions.
aureus pellets were used to extract chromosomal DNA with TIANamp Bacteria DNA kit (Tiangen, China) supplemented with 1mg/mL lysostaphin
(Sigma, China) according to the manufacturer's instructions.
For both plasmid and genomic DNA, we applied lysostaphin
(20 [micro]gImL) (Wakchemie, Steinbach, Germany) for bacterial cell lysis.
Industrial-scale production and purification of a heterologous protein in Lactococcus lactis using the nisin-controlled gene expression system NICE: the case of lysostaphin
. Microb Cell Fact 2005;4:15.
Zinc-finger nickase-mediated insertion of the lysostaphin
gene into the beta-casein locus in cloned cows.
Abtahi, "Cloning, expression, and purification of recombinant Lysostaphin
from Staphylococcus simulans," Jundishapur Journal of Microbiology, vol.
PFGE was performed with Smal (20U, New England, Ipswich, USA), as described  with the following modifications: (i) bacterial suspensions were prepared in 500 [micro]L PIV buffer [NaCl 1 M and Tris HCl 10 mM (pH 7,6)] so that an aliquot of the 1:10 diluted suspension in distilled water exhibited an OD of 1.5 in a spectrophotometer with 600 nm wavelength; (ii) after solidification, blocks were transferred to lysing solution (Tris HCl 6 mM [pH 7,6], NaCl 1 M, EDTA 100 mM [pH 7,5], Brij 58 at 0.5%, sodium lauroyl sarcosinate at 0.5%, 0.5 mg/ml of lysozyme [Sigma], and 0.05 mg/ml lysostaphin
[Sigma]) for 18-24 h at 37[degrees]C, under gentle agitation.
van Swamet al., "Industrial-scale production and purification of a heterologous protein in Lactococcus lactis using the nisin-controlled gene expression system NICE: the case of lysostaphin
," Microbial Cell Factories, vol.
The CTAB (cetyltrimethylammonium bromide) genomic DNA extraction method was modified to use lysostaphin
and the methodology described by Johnson et al (23).
Approximately [10.sup.8]-[10.sup.9] bacterial cells were treated with 100 mg/mL lysostaphin
(500 U/mg; Sigma-Aldrich) at 37[degrees]C for 10 min.