Lysostaphin

MO M23 M23.004 Lysostaphin Use in treatment of endophthalmitis mediated by methicill in-resistant Staphylococcus aureus... 

Oldham, E.R. and Daley, M.J. (1991), Lysostaphin use of a recombinant bactericidal enzyme as a mastitis therapeutic . Journal of Dairy Science, 74, 4175 -182. 

Warfield, R., Bardelang, P., Saunders, H., Chan, W. C., Penfold, C., James, R. and Thomas, N. R. (2006). Internally quenched peptides for the study of lysostaphin An antimicrobial protease that kills Staphylococcus aureus. Org. Biomol. Chem. 4, 3626-3638. 

J. Sjoquist, B. Meloun and H. Hjelm, Protein A isolated from Staphylococcus aureus after digestion with lysostaphin, Eur. J. Biochem., 29 (1972) 572-578. 

Lysostaphin Peptidase liberates iV-terminal glycine and alanine... 

Walsh, S., A. Shah, and J. Mond. 2003. Improved pharmacokinetics and reduced antibody reactivity of lysostaphin conjugated to polyethylene glycol. Antimicrob. Agents Chemother. 47 554—558. 

Schindler CA, Schuhardt VT (1964) Lysostaphin - new bacteriolytic agent for Staphylococcus. Proc Natl Acad Sci USA 51 414—421... 

Lysostaphin is a potent peptidoglycan hydrolase naturally secreted by... 

Kerr, D.E., Plaut, K., Bramley, A.J., Williamson, C.M., Lax, A.J., Moore, K., Wells, K.D., and Wall, R.J. (2001). Lysostaphin expression in mammary glands confers protection against staphylococcal infection in transgenic mice. Nat. Biotechnol. 19, 66-70. 

About 100 g of wet bacteria are suspended in I 1 50 mM Tris-HCI buffer, pH 7.5, containing 145 mM NaCl and placed in a shaking water bath at 37°C. After equilibration, about 5 mg lysostaphin (Sigma) and 1 mg DNase are added. The rate of digestion is moni-... 

The Kingfisher was programmed to mix sample and beads in well A for 15 min, collect the beads, perform successive bead washes taking a total of 150 s each in wells B to D, mix the beads for 1 min (detergent extraction) or 10 min (lysostaphin-mediated cell lysis) in well E, and then collect the beads and dump them in well D so they did not interfere with light measurements. 

When the assay was transferred to testing of clinical samples, only 50% of positives by the standard test were picked up and the number of false positives was unacceptably high. The monoclonal antibody used was found to be affected by the high salt in the antibiotic broth and to cross-react with Staph, epidermidis. The capture agent was therefore switched to fibrinogen, and lysostaphin was introduced to add compensating specificity. As the results in Table 1 for test set 2 show, this reduced the number of false positives, but did not help with false negatives. 

Lytic agent used to release AK detergent lysostaphin lysostaphin... 

The work reported here has provided valuable lessons. For example, inclusion of high salt levels in the initial enrichment phase can be deleterious both to antibody binding and lysis of non-target organisms (penicillin-based antibiotics only lyse growing cells), and lysostaphin is not totally specific, but is cost effective and does not inhibit AK as detergent-based extractants can. 

Tire expression of lysostaphin protein in mammary glands of transgenic mice confers protection against staphylococcal infection and shows the potential of genetic engineering to combat the disease in dairy cattle . ... 

C -(2-Acetamido-2-deoxy 3-D-glucosyl)-(l 4)-iV-acetyl muramoyl-L-alanyl-D-isoglutamine (31), the dipeptide derivative of the disaccharide isolated from lysozyme digests of bacterial cell walls, has been prepared in a fourteen-step sequence from 2-acetamido-2-deoxy-D-glucose. The dipeptide disaccharide derivative (32) isolated from lysostaphin endo-l3-D-2-acetamido-2-deoxy-glucan-ase digests of cell walls has also been synthesized (Scheme 5). ... 

Scott, P.T. and Rood, J.I. (1989) Electroporation-mediated transformation of lysostaphin-treated Clostridium perfringens. Gene, 82, 327-333. 

For cell lysis, add 10 pL of lysostaphin solution (10 mg/mL) to the cell suspension. After incubation on ice for 10 min, disrupt cells by sonication. For this step, place a beaker of ice water around the sample tube to keep it cold. Sonicate the samples for 1 min (0.5/s, low) followed by a 1-min cooling break. Repeat this process three times. Sonication is complete when the solution appears noticeably less cloudy than the starting solution. 

Lysostaphin (AMBI products, Lawrence, NY) lyophilized powder is resuspended in nuclease-free water (Stock 5 mg/mL). Store at -20 °C. 

Perform genomic DNA isolation from S. aureus test and reference strains using a modification of the Edge Biosystems genomic DNA isolation kit. The modification consists of addition of lysostaphin (100 pg/mL final concentration) to bacterial cells and incubation at 37 °C for 10 min prior to DNA extraction. 

Dialyze PlA-containing extracts against distilled water for 2 x 12 h at 4°C and subsequently digest with DNase (0.5 mg/mL final concentration), RNase (0.5 mg/mL final concentration), lysostaphin (0.5 mg/mL final concentration), and lysozyme (0.5 mg/mL final concentration) at 37 °C for 16 h, followed by incubation with proteinase K (4 mg/mL final concentration) at 37 °C for 16 h. 

The cotton rat (Sigmodon hispidus) is a well-established model for a number of human pathogens, especially viral respiratory pathogens (14,15). The model detailed in this chapter is an adaptation of a model of S. aureus nasal colonization originally described in mice (16) but now adapted to cotton rats. The cotton rat s nasal histology is comparable to that of humans (17) and pretreatment of cotton rats with antibiotics like streptomycin is not required as it is in mice (16) to establish nasal colonization by S. aureus. We have successfully used the cotton rat S. aureus nasal colonization model to demonstrate the efficacy of lysostaphin as a therapy for S. aureus nasal colonization (18) as well as to study the roles of wall teichoic acid (19) and IsdA and IsdH (20) of S. aureus in nasal colonization. 

Kokai-Kun, J. F., Walsh, S. M., Chanturiya, T., and Mond, J. J. (2003) Lysostaphin cream eradicates Staphylococcus aureus nasal colonization in a cotton rat model. Antimicrob. Agents Chemother. 47, 1589-1597. 

Aliquot 100 pL of a 100 pg/mL lysostaphin-water mixture into each well inoculate with 96 isolates using pipet tips. Mix well. 

There are other areas where opportunities exist to improve allograft and xenograft performance. For example, device-related infections are a costly complication in soft tissue repair procedures such as abdominal wall repair. The local delivery of antibiotics may reduce postsurgical infections. The TYRX mesh now marketed by Medtronic/ Covidien delivers antibiotics to the subcutaneous pocket following implantation of a pacing device. The incorporation of antibiotics or other antimicrobial agents such as lysostaphin, as by Stishkumar et al., may represent an innovative approach to infection control particularly in contaminated and infected wounds. 

Belyansky I, Tsirline VB, Martin TR, KilmaDA, Heath J, Lincourt AE, etal. The addition of lysostaphin dramatically improves survival, protects porcine biomesh from infection, and improves graft tensile shear strength. J Surg Res 2011 17(2) 409-15. 

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