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Climo, Michael W., Kerstin Ehlert, and Gordon L. Archer. "Mechanism and Suppression of Lysostaphin Resistance in Oxacillin-Resistant Staphylococcus aureus." Antimicrobial Agents and Chemotherapy 45, no. 5 (May 1, 2001): 1431–37. http://dx.doi.org/10.1128/aac.45.5.1431-1437.2001.
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ABSTRACT The potential for the development of resistance in oxacillin-resistant Staphylococcus aureus (ORSA) to lysostaphin, a glycylglycine endopeptidase produced byStaphylococcus simulans biovar staphylolyticus, was examined in vitro and in an in vivo model of infection. Following in vitro exposure of ORSA to subinhibitory concentrations of lysostaphin, lysostaphin-resistant mutants were idenitifed among all isolates examined. Resistance to lysostaphin was associated with a loss of resistance to β-lactams and a change in the muropeptide interpeptide cross bridge from pentaglycine to a single glycine. Mutations in femA, the gene required for incorporation of the second and third glycines into the cross bridge, were found following PCR amplification and nucleotide sequence analysis. Complementation of lysostaphin-resistant mutants with pBBB31, which encodes femA, restored the phenotype of oxacillin resistance and lysostaphin susceptibility. Addition of β-lactam antibiotics to lysostaphin in vitro prevented the development of lysostaphin-resistant mutants. In the rabbit model of experimental endocarditis, administration of a low dose of lysostaphin for 3 days led predictably to the appearance of lysostaphin-resistant ORSA mutants in vegetations. Coadministration of nafcillin with lysostaphin prevented the emergence of lysostaphin-resistant mutants and led to a mean reduction in aortic valve vegetation counts of 7.5 log10 CFU/g compared to those for untreated controls and eliminated the isolation of lysostaphin-resistant mutants from aortic valve vegetations. Treatment with nafcillin and lysostaphin given alone led to mean reductions of 1.35 and 1.65 log10 CFU/g respectively. In ORSA, resistance to lysostaphin was associated with mutations in femA, but resistance could be suppressed by the coadministration of β-lactam antibiotics.
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Gründling, Angelika, Dominique M. Missiakas, and Olaf Schneewind. "Staphylococcus aureus Mutants with Increased Lysostaphin Resistance." Journal of Bacteriology 188, no. 17 (September 1, 2006): 6286–97. http://dx.doi.org/10.1128/jb.00457-06.
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ABSTRACT Staphylococcus simulans secretes lysostaphin, a bacteriolytic enzyme that specifically binds to the cell wall envelope of Staphylococcus aureus and cleaves the pentaglycine cross bridges of peptidoglycan, thereby killing staphylococci. The study of S. aureus mutants with resistance to lysostaphin-mediated killing has revealed biosynthetic pathways for cell wall assembly. To identify additional genes involved in cell wall envelope biosynthesis, we have screened a collection of S. aureus strain Newman transposon mutants for lysostaphin resistance. Bursa aurealis insertion in SAV2335, encoding a polytopic membrane protein with predicted protease domain, caused a high degree of lysostaphin resistance, similar to the case for a previously described femAB promoter mutant. In contrast to the case for this femAB mutant, transposon insertion in SAV2335, herein named lyrA (lysostaphin resistance A), did not cause gross alterations of cell wall cross bridges such as truncations of pentaglycine to tri- or monoglycine. Also, inactivation of LyrA in a methicillin-resistant S. aureus strain did not precipitate a decrease in β-lactam resistance as observed for fem (factor essential for methicillin resistance) mutants. Lysostaphin bound to the cell wall envelopes of lyrA mutants in a manner similar to that for wild-type staphylococci. Lysostaphin resistance of lyrA mutants is attributable to altered cell wall envelope properties and may in part be due to increased abundance of altered cross bridges. Other lyr mutants with intermediate lysostaphin resistance carried bursa aurealis insertions in genes specifying GTP pyrophosphokinase or enzymes of the purine biosynthetic pathway.
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Batool, Nayab, Kwan Soo Ko, Akhilesh Kumar Chaurasia, and Kyeong Kyu Kim. "Functional Identification of Serine Hydroxymethyltransferase as a Key Gene Involved in Lysostaphin Resistance and Virulence Potential of Staphylococcus aureus Strains." International Journal of Molecular Sciences 21, no. 23 (November 30, 2020): 9135. http://dx.doi.org/10.3390/ijms21239135.
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Gaining an insight into the mechanism underlying antimicrobial-resistance development in Staphylococcus aureus is crucial for identifying effective antimicrobials. We isolated S. aureus sequence type 72 from a patient in whom the S. aureus infection was highly resistant to various antibiotics and lysostaphin, but no known resistance mechanisms could explain the mechanism of lysostaphin resistance. Genome-sequencing followed by subtractive and functional genomics revealed that serine hydroxymethyltransferase (glyA or shmT gene) plays a key role in lysostaphin resistance. Serine hydroxymethyltransferase (SHMT) is indispensable for the one-carbon metabolism of serine/glycine interconversion and is linked to folate metabolism. Functional studies revealed the involvement of SHMT in lysostaphin resistance, as ΔshmT was susceptible to the lysostaphin, while complementation of the knockout expressing shmT restored resistance against lysostaphin. In addition, the ΔshmT showed reduced virulence under in vitro (mammalian cell lines infection) and in vivo (wax-worm infection) models. The SHMT inhibitor, serine hydroxymethyltransferase inhibitor 1 (SHIN1), protected the 50% of the wax-worm infected with wild type S. aureus. These results suggest SHMT is relevant to the extreme susceptibility to lysostaphin and the host immune system. Thus, the current study established that SHMT plays a key role in lysostaphin resistance development and in determining the virulence potential of multiple drug-resistant S. aureus.
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Kusuma, Caroline, Anna Jadanova, Tanya Chanturiya, and John F. Kokai-Kun. "Lysostaphin-Resistant Variants of Staphylococcus aureus Demonstrate Reduced Fitness In Vitro and In Vivo." Antimicrobial Agents and Chemotherapy 51, no. 2 (February 2007): 475–82. http://dx.doi.org/10.1128/aac.00786-06.
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ABSTRACT Lysostaphin is under development as a therapy for serious staphylococcal infections. During preclinical development, lysostaphin-resistant Staphylococcus aureus variants have occasionally been reported in vitro and in vivo. The acquisition of resistance to this drug, however, leads to a significant increase in β-lactam antibiotic susceptibility, rendering methicillin-resistant S. aureus (MRSA) strains functionally methicillin susceptible. In this study, we have demonstrated that the development of lysostaphin resistance by two strains of MRSA also led to a loss of fitness in the variants. Consistent with the mutations found in previously reported lysostaphin-resistant S. aureus variants, these two variants had mutations in their femA genes, resulting in nonfunctional FemA proteins and, thus, monoglycine cross bridges in the peptidoglycan. The diminished fitness of the lysostaphin-resistant variants was reflected by (i) a reduced logarithmic growth rate, with the variants being outcompeted in cocultures by their wild-type parental strains; (ii) increased susceptibility to elevated temperatures; and (iii) at least fivefold less virulence of the lysostaphin-resistant variants than their wild-type strains in a mouse kidney infection model, with the lysostaphin-resistant variants being outcompeted in coinfections with their wild-type parental strains. During a 14-day serial passage without selective pressure, the lysostaphin-resistant variants failed to develop compensatory mutations which restored their fitness. These results suggest that should lysostaphin resistance due to an alteration in the FemA function emerge in S. aureus during therapy with lysostaphin, the resistant variants would be less fit and less virulent, and, in addition, infections with these strains would be easily treatable with β-lactam antibiotics.
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Heath Farris, M., Lucie S. Heath, Harry E. Heath, Paul A. LeBlanc, Robin S. Simmonds, and Gary L. Sloan. "Expression of the genes for lysostaphin and lysostaphin resistance in streptococci." FEMS Microbiology Letters 228, no. 1 (November 2003): 115–19. http://dx.doi.org/10.1016/s0378-1097(03)00743-2.
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Gargis, Shaw R., Harry E. Heath, Paul A. LeBlanc, Linda Dekker, Robin S. Simmonds, and Gary L. Sloan. "Inhibition of the Activity of Both Domains of Lysostaphin through Peptidoglycan Modification by the Lysostaphin Immunity Protein." Applied and Environmental Microbiology 76, no. 20 (August 20, 2010): 6944–46. http://dx.doi.org/10.1128/aem.01066-10.
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ABSTRACT Resistance to lysostaphin, a staphylolytic glycylglycine endopeptidase, is due to a FemABX-like immunity protein that inserts serines in place of some glycines in peptidoglycan cross bridges. These modifications inhibit both binding of the recombinant cell wall targeting domain and catalysis by the recombinant catalytic domain of lysostaphin.
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Hertlein, Tobias, Volker Sturm, Udo Lorenz, K. Sumathy, Peter Jakob, and Knut Ohlsen. "Bioluminescence and19F Magnetic Resonance Imaging Visualize the Efficacy of Lysostaphin Alone and in Combination with Oxacillin against Staphylococcus aureus in Murine Thigh and Catheter-Associated Infection Models." Antimicrobial Agents and Chemotherapy 58, no. 3 (December 23, 2013): 1630–38. http://dx.doi.org/10.1128/aac.01422-13.
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ABSTRACTStaphylococci are the leading cause of hospital-acquired infections worldwide. Increasingly, they resist antibiotic treatment owing to the development of multiple antibiotic resistance mechanisms in most strains. Therefore, the activity and efficacy of recombinant lysostaphin as a drug against this pathogen have been evaluated. Lysostaphin exerts high levels of activity against antibiotic-resistant strains ofStaphylococcus aureus, including methicillin-resistantS. aureus(MRSA). The therapeutic value of lysostaphin has been analyzed in two different clinically relevantin vivomodels, a catheter-associated infection model and a thigh infection model. We infected mice with luciferase-expressingS. aureusXen 29, and the efficacies of lysostaphin, vancomycin, oxacillin, and combined lysostaphin-oxacillin were investigated by determining numbers of CFU, detecting bioluminescent signals, and measuring the accumulation of perfluorocarbon emulsion at the site of infection by19F magnetic resonance imaging. Lysostaphin treatment significantly reduced the bacterial burden in infected thigh muscles and, after systemic spreading from the catheter, in inner organs. The efficiency of lysostaphin treatment was even more pronounced in combinatorial therapy with oxacillin. These results suggest that recombinant lysostaphin may have potential as an anti-S. aureusdrug worthy of further clinical development. In addition, both imaging technologies demonstrated efficacy patterns similar to that of CFU determination, although they proved to be less sensitive. Nonetheless, they served as powerful tools to provide additional information about the course and gravity of infection in a noninvasive manner, possibly allowing a reduction in the number of animals needed for research evaluation of new antibiotics in future studies.
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Wu, Julie A., Caroline Kusuma, James J. Mond, and John F. Kokai-Kun. "Lysostaphin Disrupts Staphylococcus aureus and Staphylococcus epidermidis Biofilms on Artificial Surfaces." Antimicrobial Agents and Chemotherapy 47, no. 11 (November 2003): 3407–14. http://dx.doi.org/10.1128/aac.47.11.3407-3414.2003.
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ABSTRACT Staphylococci often form biofilms, sessile communities of microcolonies encased in an extracellular matrix that adhere to biomedical implants or damaged tissue. Infections associated with biofilms are difficult to treat, and it is estimated that sessile bacteria in biofilms are 1,000 to 1,500 times more resistant to antibiotics than their planktonic counterparts. This antibiotic resistance of biofilms often leads to the failure of conventional antibiotic therapy and necessitates the removal of infected devices. Lysostaphin is a glycylglycine endopeptidase which specifically cleaves the pentaglycine cross bridges found in the staphylococcal peptidoglycan. Lysostaphin kills Staphylococcus aureus within minutes (MIC at which 90% of the strains are inhibited [MIC90], 0.001 to 0.064 μg/ml) and is also effective against Staphylococcus epidermidis at higher concentrations (MIC90, 12.5 to 64 μg/ml). The activity of lysostaphin against staphylococci present in biofilms compared to those of other antibiotics was, however, never explored. Surprisingly, lysostaphin not only killed S. aureus in biofilms but also disrupted the extracellular matrix of S. aureus biofilms in vitro on plastic and glass surfaces at concentrations as low as 1 μg/ml. Scanning electron microscopy confirmed that lysostaphin eradicated both the sessile cells and the extracellular matrix of the biofilm. This disruption of S. aureus biofilms was specific for lysostaphin-sensitive S. aureus, as biofilms of lysostaphin-resistant S. aureus were not affected. High concentrations of oxacillin (400 μg/ml), vancomycin (800 μg/ml), and clindamycin (800 μg/ml) had no effect on the established S. aureus biofilms in this system, even after 24 h. Higher concentrations of lysostaphin also disrupted S. epidermidis biofilms.
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Kokai-Kun, John F., Scott M. Walsh, Tanya Chanturiya, and James J. Mond. "Lysostaphin Cream Eradicates Staphylococcus aureus Nasal Colonization in a Cotton Rat Model." Antimicrobial Agents and Chemotherapy 47, no. 5 (May 2003): 1589–97. http://dx.doi.org/10.1128/aac.47.5.1589-1597.2003.
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ABSTRACT The anterior nares are a primary ecologic niche for Staphylococcus aureus, and nasal colonization by this opportunistic pathogen increases the risk of development of S. aureus infection. Clearance of S. aureus nasal colonization greatly reduces this risk. Mupirocin ointment is the current standard of care for clearance of S. aureus nasal colonization, but resistance to this antibiotic is emerging. Lysostaphin is a glycylglycine endopeptidase which specifically cleaves the cross-linking pentaglycine bridges in the cell walls of staphylococci. Lysostaphin is extremely staphylocidal (MIC at which 90% of isolates are inhibited, 0.001 to 0.064 μg/ml) and rapidly lyses both actively growing and quiescent S. aureus. This study demonstrates that a single application of 0.5% lysostaphin (actual dose, ∼150 μg of lysostaphin), formulated in a petrolatum-based cream, dramatically reduces S. aureus nasal colonization in 100% of animals tested and eradicates S. aureus nasal colonization in 93% of animals in a cotton rat model. A single dose of lysostaphin cream is more effective than a single dose of mupirocin ointment in eradicating S. aureus nasal colonization in this animal model. The lantibiotic peptide nisin, which has potent in vitro antistaphylococcal activity, was ineffective in reducing staphylococcal nasal carriage in this model. Nasal colonization was not reduced after three treatments with 5% nisin (∼1,500 μg/dose) in any of the treated animals. Lysostaphin formulated in cream may prove to be a superior alternative to mupirocin ointment for clearance of S. aureus nasal colonization.
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Heath, H. E., Lucie S. Heath, James D. Nitterauer, Karen E. Rose, and Gary L. Sloan. "Plasmid-encoded lysostaphin endopeptidase resistance of staphylococcussimulans biovar staphylolyticus." Biochemical and Biophysical Research Communications 160, no. 3 (May 1989): 1106–9. http://dx.doi.org/10.1016/s0006-291x(89)80117-2.
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Wu, Xia, Jian Zha, Mattheos A. G. Koffas, and Jonathan S. Dordick. "Reducing Staphylococcus aureus resistance to lysostaphin using CRISPR‐dCas9." Biotechnology and Bioengineering 116, no. 12 (September 3, 2019): 3149–59. http://dx.doi.org/10.1002/bit.27143.
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Sundarrajan, Sudarson, Junjappa Raghupatil, Aradhana Vipra, Nagalakshmi Narasimhaswamy, Sanjeev Saravanan, Chemira Appaiah, Nethravathi Poonacha, et al. "Bacteriophage-derived CHAP domain protein, P128, kills Staphylococcus cells by cleaving interpeptide cross-bridge of peptidoglycan." Microbiology 160, no. 10 (October 1, 2014): 2157–69. http://dx.doi.org/10.1099/mic.0.079111-0.
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P128 is an anti-staphylococcal protein consisting of the Staphylococcus aureus phage-K-derived tail-associated muralytic enzyme (TAME) catalytic domain (Lys16) fused with the cell-wall-binding SH3b domain of lysostaphin. In order to understand the mechanism of action and emergence of resistance to P128, we isolated mutants of Staphylococcus spp., including meticillin-resistant Staphylococcus aureus (MRSA), resistant to P128. In addition to P128, the mutants also showed resistance to Lys16, the catalytic domain of P128. The mutants showed loss of fitness as shown by reduced rate of growth in vitro. One of the mutants tested was found to show reduced virulence in animal models of S. aureus septicaemia suggesting loss of fitness in vivo as well. Analysis of the antibiotic sensitivity pattern showed that the mutants derived from MRSA strains had become sensitive to meticillin and other β-lactams. Interestingly, the mutant cells were resistant to the lytic action of phage K, although the phage was able to adsorb to these cells. Sequencing of the femA gene of three P128-resistant mutants showed either a truncation or deletion in femA, suggesting that improper cross-bridge formation in S. aureus could be causing resistance to P128. Using glutathione S-transferase (GST) fusion peptides as substrates it was found that both P128 and Lys16 were capable of cleaving a pentaglycine sequence, suggesting that P128 might be killing S. aureus by cleaving the pentaglycine cross-bridge of peptidoglycan. Moreover, peptides corresponding to the reported cross-bridge of Staphylococcus haemolyticus (GGSGG, AGSGG), which were not cleaved by lysostaphin, were cleaved efficiently by P128. This was also reflected in high sensitivity of S. haemolyticus to P128. This showed that in spite of sharing a common mechanism of action with lysostaphin, P128 has unique properties, which allow it to act on certain lysostaphin-resistant Staphylococcus strains.
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Cheleuitte-Nieves, Christopher E., Leslie L. Diaz, Maria Pardos de la Gandara, Alejandra Gonzalez, Winrich A. Freiwald, Hermínia M. de Lencastre, Alexander Tomasz, and Chad W. Euler. "Evaluation of Topical Lysostaphin as a Novel Treatment for Instrumented Rhesus Macaques (Macaca mulatta) Infected with Methicillin-Resistant Staphylococcus aureus." Comparative Medicine 70, no. 5 (October 1, 2020): 335–47. http://dx.doi.org/10.30802/aalas-cm-19-000102.
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Lytic enzymes are novel antimicrobial agents that degrade bacterial cell walls, resulting in cell rupture and death. We tested one enzyme, the bacteriocin lysostaphin, for treatment of nonhuman primates (Macaca mulatta) with persistent methicillinresistant Staphylococcus aureus (MRSA) infection of their cranial implant margins. The goal of this study was to determine if topical lysostaphin, either alone or as an adjunct therapy, could eliminate MRSA. Lysostaphin had in vitro lytic activity against all 4 previously identified NHP MRSA clones, as well as against 12 MRSA isolates of the same clonal type (MLST ST3862 and spa type t4167) before and after treatment, with no resistance discovered. In an in vivo pilot study, a 2-d application of lysostaphin alone reduced MRSA in the implant margins by 3-logs during treatment of one animal; however, MRSA titers had returned to control levels by 1 wk after treatment. In the main study, all animals (n = 4) received 10 d of systemic antibiotic treatment and both the animals and their environment (cages, equipment, room) underwent 5-d of decontamination. The experimental animals (n = 2) received 5 doses of topical lysostaphin (15 mg, every other day) applied onto their implant margins. Daily cultures showed that MRSA counts decreased significantly (≤ 25 colony-forming units/mL; P < 0.05). However, sampling of the cranial implant margin 7 d after last treatment showed that MRSA counts had returned to control levels. Our study suggests that lysostaphin, coupled with other treatment modalities, can decrease MRSA infection short-term but do not completely eradicate MRSA in the long-term. This reappearance of MRSA may be due to cross-contamination or reinfection from other infected areas, an inability of the treatment to reach all colonized areas, or insufficient dosing or length of treatment. Topical lysostaphin may be more useful clinically for superficial nonimplant associated wounds in which the lytic enzyme has better access to the infected tissue.
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Ehlert, Kerstin, Martin Tschierske, Claudio Mori, Werner Schröder, and Brigitte Berger-Bächi. "Site-Specific Serine Incorporation by Lif and Epr into Positions 3 and 5 of the Staphylococcal Peptidoglycan Interpeptide Bridge." Journal of Bacteriology 182, no. 9 (May 1, 2000): 2635–38. http://dx.doi.org/10.1128/jb.182.9.2635-2638.2000.
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ABSTRACT The FemAB-like factors Lif and Epr confer resistance to glycylglycine endopeptidases lysostaphin and Ale-1, respectively, by incorporating serine residues into the staphylococcal peptidoglycan interpeptide bridges specifically at positions 3 and 5. This required the presence of FemA and/or FemB, in contrast to earlier postulations.
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Schmelcher, Mathias, Anne M. Powell, Stephen C. Becker, Mary J. Camp, and David M. Donovan. "Chimeric Phage Lysins Act Synergistically with Lysostaphin To Kill Mastitis-Causing Staphylococcus aureus in Murine Mammary Glands." Applied and Environmental Microbiology 78, no. 7 (January 27, 2012): 2297–305. http://dx.doi.org/10.1128/aem.07050-11.
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ABSTRACTStaphylococci cause bovine mastitis, withStaphylococcus aureusbeing responsible for the majority of the mastitis-based losses to the dairy industry (up to $2 billion/annum). Treatment is primarily with antibiotics, which are often ineffective and potentially contribute to resistance development. Bacteriophage endolysins (peptidoglycan hydrolases) present a promising source of alternative antimicrobials. Here we evaluated two fusion proteins consisting of the streptococcal λSA2 endolysin endopeptidase domain fused to staphylococcal cell wall binding domains from either lysostaphin (λSA2-E-Lyso-SH3b) or the staphylococcal phage K endolysin, LysK (λSA2-E-LysK-SH3b). We demonstrate killing of 16 differentS. aureusmastitis isolates, including penicillin-resistant strains, by both constructs. At 100 μg/ml in processed cow milk, λSA2-E-Lyso-SH3b and λSA2-E-LysK-SH3b reduced theS. aureusbacterial load by 3 and 1 log units within 3 h, respectively, compared to a buffer control. In contrast to λSA2-E-Lyso-SH3b, however, λSA2-E-LysK-SH3b permitted regrowth of the pathogen after 1 h. In a mouse model of mastitis, infusion of 25 μg of λSA2-E-Lyso-SH3b or λSA2-E-LysK-SH3b into mammary glands reducedS. aureusCFU by 0.63 or 0.81 log units, compared to >2 log for lysostaphin. Both chimeras were synergistic with lysostaphin againstS. aureusin plate lysis checkerboard assays. When tested in combination in mice, λSA2-E-LysK-SH3b and lysostaphin (12.5 μg each/gland) caused a 3.36-log decrease in CFU. Furthermore, most protein treatments reduced gland wet weights and intramammary tumor necrosis factor alpha (TNF-α) concentrations, which serve as indicators of inflammation. Overall, our animal model results demonstrate the potential of fusion peptidoglycan hydrolases as antimicrobials for the treatment ofS. aureus-induced mastitis.
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Boyle-Vavra, Susan, Sarah K. Berke, Jean C. Lee, and Robert S. Daum. "Reversion of the Glycopeptide Resistance Phenotype in Staphylococcus aureus Clinical Isolates." Antimicrobial Agents and Chemotherapy 44, no. 2 (February 1, 2000): 272–77. http://dx.doi.org/10.1128/aac.44.2.272-277.2000.
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ABSTRACT The recent identification of glycopeptide intermediate-resistantStaphylococcus aureus (GISA) clinical isolates has provided an opportunity to assess the stability of the glycopeptide resistance phenotype by nonselective serial passage and to evaluate reversion-associated cell surface changes. Three GISA isolates from the United States (MIC of vancomycin = 8 μg/ml) and two from Japan (MICs of vancomycin = 8 and 2 μg/ml) were passaged daily on nutrient agar with or without vancomycin supplementation. After 15 days of passage on nonselective medium, vancomycin- and teicoplanin-susceptible revertants were obtained from each GISA isolate as determined by broth dilution MIC. Revertant isolates were compared with parent isolates for changes in vancomycin heteroresistance, capsule production, hemolysis phenotype, coagulase activity, and lysostaphin susceptibility. Several revertants lost the subpopulations with intermediate vancomycin resistance, whereas two revertants maintained them. Furthermore, although all of the parent GISA isolates produced capsule type 5 (CP5), all but one revertant tested no longer produced CP5. In contrast, passage on medium containing vancomycin yielded isolates that were still intermediately resistant to vancomycin, had no decrease in the MIC of teicoplanin, and produced detectable CP5. No consistent changes in the revertants in hemolysis phenotype, lysostaphin susceptibility, or coagulase activities were discerned. These data indicate that the vancomycin resistance phenotype is unstable in clinical GISA isolates. Reversion of the vancomycin resistance phenotype might explain the difficulty in isolating vancomycin-resistant clinical isolates from the blood of patients who fail vancomycin therapy and, possibly, may account for some of the difficulties in identifying GISA isolates in the clinical laboratory.
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Boyle-Vavra, S. "Development of vancomycin and lysostaphin resistance in a methicillin-resistant Staphylococcus aureus isolate." Journal of Antimicrobial Chemotherapy 48, no. 5 (November 1, 2001): 617–25. http://dx.doi.org/10.1093/jac/48.5.617.
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Pipiya, Sofiya O., Yuliana A. Mokrushina, Alexander G. Gabibov, Ivan V. Smirnov, and Stanislav S. Terekhov. "Selective Eradication of Staphylococcus aureus by the Designer Genetically Programmed Yeast Biocontrol Agent." Antibiotics 9, no. 9 (August 19, 2020): 527. http://dx.doi.org/10.3390/antibiotics9090527.
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Staphylococcus aureus is a common human pathogen that is particularly often associated with antibiotic resistance. The eradication of this ubiquitous infectious agent from its ecological niches and contaminated surfaces is especially complicated by excessive biofilm formation and persisting cells, which evade the antibacterial activity of conventional antibiotics. Here, we present an alternative view of the problem of specific S. aureus eradication. The constitutive heterologous production of highly specific bacteriolytic protease lysostaphin in yeast Pichia pastoris provides an efficient biocontrol agent, specifically killing S. aureus in coculture. A yeast-based anti-S. aureus probiotic was efficient in a high range of temperatures and target-to-effector ratios, indicating its robustness and versatility in eliminating S. aureus cells. The efficient eradication of S. aureus by live lysostaphin-producing P. pastoris was achieved at high scales, providing a simple, biocompatible and cost-effective strategy for S. aureus lysis in bioproduction and surface decontamination. Future biomedical applications based on designer yeast biocontrol agents require evaluation in in vivo models. However, we believe that this strategy is very promising since it provides highly safe, efficient and selective genetically programmed probiotics and targeted biocontrol agents.
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Peschel, Andreas, Cuong Vuong, Michael Otto, and Friedrich Götz. "The d-Alanine Residues ofStaphylococcus aureus Teichoic Acids Alter the Susceptibility to Vancomycin and the Activity of Autolytic Enzymes." Antimicrobial Agents and Chemotherapy 44, no. 10 (October 1, 2000): 2845–47. http://dx.doi.org/10.1128/aac.44.10.2845-2847.2000.
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ABSTRACT Recently, Staphylococcus aureus strains with intermediate resistance to vancomycin, the antibiotic of last resort, have been described. Multiple changes in peptidoglycan turnover and structure contribute to the resistance phenotype. Here, we describe that structural changes of teichoic acids in the cell envelope have a considerable influence on the susceptibility to vancomycin and other glycopeptides. S. aureus cells lackingd-alanine esters in teichoic acids exhibited an at least threefold-increased sensitivity to glycopeptide antibiotics. Furthermore, the autolytic activity of the d-alanine mutant was reduced compared to the wild-type, and the mutant was more susceptible to the staphylolytic enzyme lysostaphin. Vancomycin inhibited autolysis at very high concentrations but neither in the wild-type nor in the mutant was the autolytic activity influenced in the range of the MIC. Mutant cells had a considerably higher capacity to bind vancomycin.
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Septimus, Edward J., and Marin L. Schweizer. "Decolonization in Prevention of Health Care-Associated Infections." Clinical Microbiology Reviews 29, no. 2 (January 27, 2016): 201–22. http://dx.doi.org/10.1128/cmr.00049-15.
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SUMMARYColonization with health care-associated pathogens such asStaphylococcus aureus, enterococci, Gram-negative organisms, andClostridium difficileis associated with increased risk of infection. Decolonization is an evidence-based intervention that can be used to prevent health care-associated infections (HAIs). This review evaluates agents used for nasal topical decolonization, topical (e.g., skin) decolonization, oral decolonization, and selective digestive or oropharyngeal decontamination. Although the majority of studies performed to date have focused onS. aureusdecolonization, there is increasing interest in how to apply decolonization strategies to reduce infections due to Gram-negative organisms, especially those that are multidrug resistant. Nasal topical decolonization agents reviewed include mupirocin, bacitracin, retapamulin, povidone-iodine, alcohol-based nasal antiseptic, tea tree oil, photodynamic therapy, omiganan pentahydrochloride, and lysostaphin. Mupirocin is still the gold standard agent forS. aureusnasal decolonization, but there is concern about mupirocin resistance, and alternative agents are needed. Of the other nasal decolonization agents, large clinical trials are still needed to evaluate the effectiveness of retapamulin, povidone-iodine, alcohol-based nasal antiseptic, tea tree oil, omiganan pentahydrochloride, and lysostaphin. Given inferior outcomes and increased risk of allergic dermatitis, the use of bacitracin-containing compounds cannot be recommended as a decolonization strategy. Topical decolonization agents reviewed included chlorhexidine gluconate (CHG), hexachlorophane, povidone-iodine, triclosan, and sodium hypochlorite. Of these, CHG is the skin decolonization agent that has the strongest evidence base, and sodium hypochlorite can also be recommended. CHG is associated with prevention of infections due to Gram-positive and Gram-negative organisms as well asCandida. Conversely, triclosan use is discouraged, and topical decolonization with hexachlorophane and povidone-iodine cannot be recommended at this time. There is also evidence to support use of selective digestive decontamination and selective oropharyngeal decontamination, but additional studies are needed to assess resistance to these agents, especially selection for resistance among Gram-negative organisms. The strongest evidence for decolonization is for use among surgical patients as a strategy to prevent surgical site infections.
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Fuller, Elizabeth, Catherine Elmer, Fiona Nattress, Richard Ellis, Glenda Horne, Peter Cook, and Tony Fawcett. "β-Lactam Resistance in Staphylococcus aureus Cells That Do Not Require a Cell Wall for Integrity." Antimicrobial Agents and Chemotherapy 49, no. 12 (December 2005): 5075–80. http://dx.doi.org/10.1128/aac.49.12.5075-5080.2005.
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ABSTRACT Staphylococcus aureus ATCC 9144 cells with defective cell walls were generated on a medium with elevated osmolality in the presence of sublethal levels of penicillin G. On removal of antibiotic pressure, the cells exhibited stable penicillin and methicillin resistance. The resistance was homogeneous and its acquisition was enhanced following transient cell wall-defective growth. The resistant cells were mecA negative, β-lactamase negative and did not contain any mutations in the coding regions of pbp genes. When penicillin was added back to resistant cells, they continued to grow and produced a diffuse cell wall that was resistant to the action by lysostaphin but was very sensitive to lysis with Triton X-100. These data indicate that the resistant cells are not dependent upon an intact cell wall for osmotic stability and they are able to switch readily to this mode of growth in the presence of penicillin G.
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Rossi, Jutta, Markus Bischoff, Akihito Wada, and Brigitte Berger-Bächi. "MsrR, a Putative Cell Envelope-Associated Element Involved in Staphylococcus aureus sarA Attenuation." Antimicrobial Agents and Chemotherapy 47, no. 8 (August 2003): 2558–64. http://dx.doi.org/10.1128/aac.47.8.2558-2564.2003.
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ABSTRACT A novel membrane-associated protein, MsrR, was identified in Staphylococcus aureus which affects resistance to methicillin and teicoplanin, as well as the synthesis of virulence factors. MsrR belongs to the LytR-CpsA-Psr family of cell envelope-related transcriptional attenuators and was shown to be inducible by cell wall-active agents, such as β-lactams, glycopeptides, and lysostaphin. The expression of msrR peaked in the early exponential growth phase and decreased sharply thereafter. msrR mutants showed increased sarA transcription and an earlier and higher expression of RNAIII, resulting in altered expression of virulence factors such as alpha-toxin and protein A. These observations suggest that MsrR is a new component involved in sarA attenuation and the regulatory network controlling virulence gene expression in S. aureus.
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Arroyo-Moreno, Sara, Máire Begley, Kornelia Dembicka, and Aidan Coffey. "Engineering of the CHAPk Staphylococcal Phage Endolysin to Enhance Antibacterial Activity against Stationary-Phase Cells." Antibiotics 10, no. 6 (June 16, 2021): 722. http://dx.doi.org/10.3390/antibiotics10060722.
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Bacteriophage endolysins and their derivatives have strong potential as antibacterial agents considering the increasing prevalence of antibiotic resistance in common bacterial pathogens. The peptidoglycan degrading peptidase CHAPk, a truncated derivate of staphylococcal phage K endolysin (LysK), has proven efficacy in preventing and disrupting staphylococcal biofilms. Nevertheless, the concentration of CHAPk required to eliminate populations of stationary-phase cells was previously found to be four-fold higher than that for log-phase cells. Moreover, CHAPk-mediated lysis of stationary-phase cells was observed to be slower than for log-phase cultures. In the present study, we report the fusion of a 165 amino acid fragment containing CHAPk with a 136 amino acid fragment containing the cell-binding domain of the bacteriocin lysostaphin to create a chimeric enzyme designated CHAPk-SH3blys in the vector pET28a. The chimeric protein was employed in concentrations as low as 5 μg/mL, producing a reduction in turbidity in 7-day-old cultures, whereas the original CHAPk required at least 20 μg/mL to achieve this. Where 7-day old liquid cultures were used, the chimeric enzyme exhibited a 16-fold lower MIC than CHAPk. In terms of biofilm prevention, a concentration of 1 μg/mL of the chimeric enzyme was sufficient, whereas for CHAPk, 125 μg/mL was needed. Moreover, the chimeric enzyme exhibited total biofilm disruption when 5 μg/mL was employed in 4-h assays, whereas CHAPk could only partially disrupt the biofilms at this concentration. This study demonstrates that the cell-binding domain from lysostaphin can make the phage endolysin CHAPk more effective against sessile staphylococcal cells.
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DeHart, H. P., H. E. Heath, L. S. Heath, P. A. LeBlanc, and G. L. Sloan. "The lysostaphin endopeptidase resistance gene (epr) specifies modification of peptidoglycan cross bridges in Staphylococcus simulans and Staphylococcus aureus." Applied and environmental microbiology 61, no. 4 (1995): 1475–79. http://dx.doi.org/10.1128/aem.61.4.1475-1479.1995.
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Yan, Xianghe, Yanping Xie, Charles Li, David M. Donovan, Andrew Gehring, Peter Irwin, and Yiping He. "Comparative Transcriptome Analysis Reveals Differentially Expressed Genes Related to Antimicrobial Properties of Lysostaphin in Staphylococcus aureus." Antibiotics 11, no. 2 (January 18, 2022): 125. http://dx.doi.org/10.3390/antibiotics11020125.
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Comparative transcriptome analysis and de novo short-read assembly of S. aureus Newman strains revealed significant transcriptional changes in response to the exposure to triple-acting staphylolytic peptidoglycan hydrolase (PGH) 1801. Most altered transcriptions were associated with the membrane, cell wall, and related genes, including amidase, peptidase, holin, and phospholipase D/transphosphatidylase. The differential expression of genes obtained from RNA-seq was confirmed by reverse transcription quantitative PCR. Moreover, some of these gene expression changes were consistent with the observed structural perturbations at the DNA and RNA levels. These structural changes in the genes encoding membrane/cell surface proteins and altered gene expressions are the candidates for resistance to these novel antimicrobials. The findings in this study could provide insight into the design of new antimicrobial agents.
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Moreira, B., S. Boyle-Vavra, B. L. deJonge, and R. S. Daum. "Increased production of penicillin-binding protein 2, increased detection of other penicillin-binding proteins, and decreased coagulase activity associated with glycopeptide resistance in Staphylococcus aureus." Antimicrobial Agents and Chemotherapy 41, no. 8 (August 1997): 1788–93. http://dx.doi.org/10.1128/aac.41.8.1788.
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The mechanism of glycopeptide resistance in the genus Staphylococcus is unknown. Since these antimicrobial compounds act by binding the peptidoglycan precursor terminus, the target of transglycosylase and transpeptidase enzymes, it was hypothesized that resistance might be mediated in Staphylococcus aureus by increased production or activity of these enzymes, commonly called penicillin-binding proteins (PBPs). To evaluate this possibility, glycopeptide-resistant mutants were prepared by passage of several clinical isolates of this species in nutrient broth containing successively increasing concentrations of the glycopeptide vancomycin or teicoplanin. Decreased coagulase activity and increased resistance to lysostaphin were uniformly present in the vancomycin-resistant mutants. Peptidoglycan cross-linking increased in one resistant isolate and decreased in two resistant isolates. The amounts of radioactive penicillin that bound to each PBP in susceptible and resistant strains were compared; PBP2 production was also evaluated by Western blotting. Increased penicillin labeling and production of PBP2 were found in all resistant derivatives selected by either vancomycin or teicoplanin. Moreover, the increase in PBP2 penicillin labeling occurred early in a series of vancomycin-selected derivatives and was strongly correlated (r > 0.9) with the increase in vancomycin and teicoplanin MIC. An increase in penicillin labeling also occurred, variably, in PBP1, PBP3, and/or PBP4. These data demonstrate a strong correlation between resistance to glycopeptides and increased PBP activity and/or production in S. aureus. Such an increase could allow PBPs to better compete with glycopeptides for the peptidoglycan precursor.
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Vacek, Lukáš, Šárka Kobzová, Richard Čmelík, Roman Pantůček, and Lubomír Janda. "Enzybiotics LYSSTAPH-S and LYSDERM-S as Potential Therapeutic Agents for Chronic MRSA Wound Infections." Antibiotics 9, no. 8 (August 15, 2020): 519. http://dx.doi.org/10.3390/antibiotics9080519.
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Antibacterial antibiotic therapy has played an important role in the treatment of bacterial infections for almost a century. The increasing resistance of pathogenic bacteria to antibiotics leads to an attempt to use previously neglected antibacterial therapies. Here we provide information on the two recombinantly modified antistaphylococcal enzymes derived from lysostaphin (LYSSTAPH-S) and endolysin (LYSDERM-S) derived from kayvirus 812F1 whose target sites reside in the bacterial cell wall. LYSSTAPH-S showed a stable antimicrobial effect over 24-h testing, even in concentrations lower than 1 µg/mL across a wide variety of epidemiologically important sequence types (STs) of methicillin-resistant Staphylococcus aureus (MRSA), especially in the stationary phase of growth (status comparable to chronic infections). LYSDERM-S showed a less potent antimicrobial effect that lasted only a few hours at concentrations of 15 μg/mL and higher. Our data indicate that these antimicrobial enzymes could be of substantial help in the treatment of chronic MRSA wound infections.
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Rosy, R. C. "Influence of variation in the sequence(s) of factors essential for methicillin resistance (fem genes) on the expression of resistance to Lysostaphin and secretion of DNAse." International Journal of Infectious Diseases 45 (April 2016): 113. http://dx.doi.org/10.1016/j.ijid.2016.02.288.
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Beltramini, Amanda M., Chitrangada D. Mukhopadhyay, and Vijay Pancholi. "Modulation of Cell Wall Structure and Antimicrobial Susceptibility by a Staphylococcus aureus Eukaryote-Like Serine/Threonine Kinase and Phosphatase." Infection and Immunity 77, no. 4 (February 2, 2009): 1406–16. http://dx.doi.org/10.1128/iai.01499-08.
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ABSTRACT It is well established that prokaryotes and eukaryotes alike utilize phosphotransfer to regulate cellular functions. One method by which this occurs is via eukaryote-like serine/threonine kinase (ESTK)- and phosphatase (ESTP)-regulated pathways. The role of these enzymes in Staphylococcus aureus has not yet been examined. This resilient organism is a common cause of hospital-acquired and community-associated infections, infecting immunocompromised and immunocompetent hosts alike. In this study, we have characterized a major functional ESTK (STK) and ESTP (STP) in S. aureus and found them to be critical modulators of cell wall structure and susceptibility to cell wall-acting β-lactam antibiotics. By utilizing gene knockout strategies, we created S. aureus N315 mutants lacking STP and/or STK. The strain lacking both STP and STK displayed notable cell division defects, including multiple and incomplete septa, bulging, and irregular cell size, as observed by transmission electron microscopy. Mutants lacking STP alone displayed thickened cell walls and increased resistance to the peptidoglycan-targeting glycylglycine endopeptidase lysostaphin, compared to the wild type. Additionally, mutant strains lacking STK or both STK and STP displayed increased sensitivity to cell wall-acting cephalosporin and carbapenem antibiotics. Together, these results indicate that S. aureus STK- and STP-mediated reversible phosphorylation reactions play a critical role in proper cell wall architecture, and thus the modulation of antimicrobial resistance, in S. aureus.
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Pfeltz, Richard F., Vineet K. Singh, Jennifer L. Schmidt, Michael A. Batten, Christopher S. Baranyk, Matthew J. Nadakavukaren, Radheshyam K. Jayaswal, and Brian J. Wilkinson. "Characterization of Passage-Selected Vancomycin-Resistant Staphylococcus aureus Strains of Diverse Parental Backgrounds." Antimicrobial Agents and Chemotherapy 44, no. 2 (February 1, 2000): 294–303. http://dx.doi.org/10.1128/aac.44.2.294-303.2000.
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ABSTRACT A series of 12 Staphylococcus aureus strains of various genetic backgrounds, methicillin resistance levels, and autolytic activities were subjected to selection for the glycopeptide-intermediate S. aureus (GISA) susceptibility phenotype on increasing concentrations of vancomycin. Six strains acquired the phenotype rapidly, two did so slowly, and four failed to do so. The vancomycin MICs for the GISA strains ranged from 4 to 16 μg/ml, were stable to 20 nonselective passages, and expressed resistance homogeneously. Neither ease of acquisition of the GISA phenotype nor the MIC attained correlated with methicillin resistance hetero- versus homogeneity or autolytic deficiency or sufficiency. Oxacillin MICs were generally unchanged between parent and GISA strains, although the mec members of both isogenic methicillin-susceptible and methicillin-resistant pairs acquired the GISA phenotype more rapidly and to higher MICs than did their susceptible counterparts. Transmission electron microscopy revealed that the GISA strains appeared normal in the absence of vancomycin but had thickened and diffuse cell walls when grown with vancomycin at one-half the MIC. Common features among GISAs were reduced doubling times, decreased lysostaphin susceptibilities, and reduced whole-cell and zymographic autolytic activities in the absence of vancomycin. This, with surface hydrophobicity differences, indicated that even in the absence of vancomycin the GISA cell walls differed from those of the parents. Autolytic activities were further reduced by the inclusion of vancomycin in whole-cell and zymographic studies. The six least vancomycin-susceptible GISA strains exhibited an increased capacity to remove vancomycin from the medium versus their parent lines. This study suggests that while some elements of the GISA phenotype are strain specific, many are common to the phenotype although their expression is influenced by genetic background. GISA strains with similar glycopeptide MICs may express individual components of the phenotype to different extents.
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Hiba Muneer Al-Khafagi. "EFFECT OF TEMPERATURE AND SODIUM CHLORIDE CONCENTRATION ON GROWTH OF Staphylococcus aureus MUTANTS." journal of the college of basic education 17, no. 72 (June 17, 2019): 117–29. http://dx.doi.org/10.35950/cbej.v17i72.4504.
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The study included the identification of Staphylococcus aureus (wild type and mutant) isolates. All these isolates were identified as coagulase and catalase positive and able to ferment mannitol.
Strain number 24 was chosen & undergone mutation with NTG to isolate temperature sensitive osmotically fragile (TOF) mutants. TOF mutants were successfully isolated by exposing the cell suspension to 200 Mg/ml of NTG for 75 minutes. The results of characterization of TOF mutants, showed that these mutants were identical to their mother strain morphologically and biochemically. The result of this mutation is to make the lysozyme able to lyse the cell wall of the TOF mutants in pattern similar to lysostaphin .
Antibiotic sensitivity pattern was demonstrated for all the isolates and multiple resistance of some isolates were detected. The obtained results showed that the two mutant isolates were resistant to Methicillin, Benzathin, Penicillin, Cloxacillin Sodium and Lincomycin, wherease these isolates were sensitive to Amoxicillin, Cefotaxine Sodium, Cephalexin Sodium and Tetracyclin but intermediate in resistance to Clindamycin.
The effect of different temperature showed that the growth of the mother strain (strain No.24) increased with the increase of temperature.The mutant strains (3M,10M) are not stable in the different temperature and their growth is very low at 40C.
The effect of different NaCl concentration showed that the growth of the mother strain (strain No.24) and the 2 mutant strain (3M,10M) would increase with the increase of the NaCl concentration but decrease at NaCl concentration = 1.5 M .
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Willing, Stephanie, Emma Dyer, Olaf Schneewind, and Dominique Missiakas. "FmhA and FmhC of Staphylococcus aureus incorporate serine residues into peptidoglycan cross-bridges." Journal of Biological Chemistry 295, no. 39 (August 5, 2020): 13664–76. http://dx.doi.org/10.1074/jbc.ra120.014371.
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Staphylococcal peptidoglycan is characterized by pentaglycine cross-bridges that are cross-linked between adjacent wall peptides by penicillin-binding proteins to confer robustness and flexibility. In Staphylococcus aureus, pentaglycine cross-bridges are synthesized by three proteins: FemX adds the first glycine, and the homodimers FemA and FemB sequentially add two Gly-Gly dipeptides. Occasionally, serine residues are also incorporated into the cross-bridges by enzymes that have heretofore not been identified. Here, we show that the FemA/FemB homologues FmhA and FmhC pair with FemA and FemB to incorporate Gly-Ser dipeptides into cross-bridges and to confer resistance to lysostaphin, a secreted bacteriocin that cleaves the pentaglycine cross-bridge. FmhA incorporates serine residues at positions 3 and 5 of the cross-bridge. In contrast, FmhC incorporates a single serine at position 5. Serine incorporation also lowers resistance toward oxacillin, an antibiotic that targets penicillin-binding proteins, in both methicillin-sensitive and methicillin-resistant strains of S. aureus. FmhC is encoded by a gene immediately adjacent to lytN, which specifies a hydrolase that cleaves the bond between the fifth glycine of cross-bridges and the alanine of the adjacent stem peptide. In this manner, LytN facilitates the separation of daughter cells. Cell wall damage induced upon lytN overexpression can be alleviated by overexpression of fmhC. Together, these observations suggest that FmhA and FmhC generate peptidoglycan cross-bridges with unique serine patterns that provide protection from endogenous murein hydrolases governing cell division and from bacteriocins produced by microbial competitors.
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Qin, Zhiqiang, Xiaomei Yang, Lei Yang, Juan Jiang, Yuanzhu Ou, Soeren Molin, and Di Qu. "Formation and properties of in vitro biofilms of ica-negative Staphylococcus epidermidis clinical isolates." Journal of Medical Microbiology 56, no. 1 (January 1, 2007): 83–93. http://dx.doi.org/10.1099/jmm.0.46799-0.
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Coagulase-negative Staphylococcus epidermidis has become the leading cause of foreign-body infections due to its biofilm formation on all kinds of medical-device surfaces. The biofilm development of S. epidermidis includes two steps: the initial attachment phase and the accumulative phase. In the accumulative phase, the polysaccharide intercellular adhesin (PIA), encoded by the icaADBC locus, is the major component mediating intercellular adhesion. However, recent studies have revealed the emergence of biofilm-positive/ica-negative staphylococcal clinical isolates. In this report, two ica-negative S. epidermidis clinical strains, SE1 and SE4, exhibited their heterogeneity in biofilm architecture under static and flow conditions, compared with the biofilm-positive/ica-positive RP62A strain. Strains with this type of absence of PIA from biofilms also displayed intermediate resistance to vancomycin. More importantly, the cells of both SE1 and SE4 strains were more tolerant than those of RP62A to exposure to lysostaphin and vancomycin. Based on the results, it is suggested that the biofilm-positive/ica-negative strain represents a newly emergent subpopulation of S. epidermidis clinical strains, arising from selection by antibiotics in the nosocomial milieu, which displays a survival advantage in its host environment. Recent epidemiological data support this suggestion, by showing a tendency towards an increasing proportion of this subpopulation in staphylococci-associated infections.
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Tamber, Sandeep, Joseph Schwartzman, and Ambrose L. Cheung. "Role of PknB Kinase in Antibiotic Resistance and Virulence in Community-Acquired Methicillin-Resistant Staphylococcus aureus Strain USA300." Infection and Immunity 78, no. 8 (June 14, 2010): 3637–46. http://dx.doi.org/10.1128/iai.00296-10.
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ABSTRACT The regulation of cellular processes by eukaryote-like serine/threonine kinases is widespread in bacteria. In the last 2 years, several studies have examined the role of serine/threonine kinases in Staphylococcus aureus on cell wall metabolism, autolysis, and virulence, mostly in S. aureus laboratory isolates in the 8325-4 lineage. In this study, we showed that the pknB gene (also called stk1) of methicillin-resistant S. aureus (MRSA) strain COL and the community-acquired MRSA (CA-MRSA) strain USA300 is involved in cell wall metabolism, with the pknB mutant exhibiting enhanced sensitivity to β-lactam antibiotics but not to other classes of antibiotics, including aminoglycosides, ciprofloxacin, bactrim, and other types of cell wall-active agents (e.g., vancomycin and bacitracin). Additionally, the pknB mutant of USA300 was found to be more resistant to Triton X-100-induced autolysis and also to lysis by lysostaphin. We also showed that pknB is a positive regulator of sigB activity, resulting in compromise in its response to heat and oxidative stresses. In association with reduced sigB activity, the expression levels of RNAII and RNAIII of agr and the downstream effector hla are upregulated while spa expression is downmodulated in the pknB mutant compared to the level in the parent. Consistent with an enhanced agr response in vitro, virulence studies of the pknB mutant of USA300 in a murine cutaneous model of infection showed that the mutant was more virulent than the parental strain. Collectively, our results have linked the pknB gene in CA-MRSA to antibiotic resistance, sigB activity, and virulence and have highlighted important differences in pknB phenotypes (virulence and sigB activity) between laboratory isolates and the prototypic CA-MRSA strain USA300.
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Levinger, Oren, Tamar Bikels-Goshen, Elad Landau, Merav Fichman, and Roni Shapira. "Epigallocatechin Gallate Induces Upregulation of the Two-Component VraSR System by Evoking a Cell Wall Stress Response in Staphylococcus aureus." Applied and Environmental Microbiology 78, no. 22 (August 31, 2012): 7954–59. http://dx.doi.org/10.1128/aem.02253-12.
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ABSTRACTWe previously found that a short exposure ofStaphylococcus aureusto subinhibitory (SI) doses of epigallocatechin gallate (EGCG) results in increased cell wall thickness, adaptation, and enhanced tolerance to cell-wall-targeted antibiotics. In this study, the response to EGCG ofsigBandvraSRtranscription factor mutants was characterized. We show that in contrast to the results observed for wild-type (WT) strains, anS. aureus315vraSRnull mutant exposed to SI doses of EGCG did not exhibit increased tolerance to EGCG and oxacillin. A diminished increase in tolerance to ampicillin (from 16-fold to 4-fold) and no change in the magnitude of resistance to vancomycin were observed. Preexposure to EGCG enhanced the tolerance of wild-type andsigBnull mutant cells to lysostaphin, but this enhancement was much weaker in thevraSRnull mutant. Marked upregulation (about 60-fold) ofvraRand upregulation of the peptidoglycan biosynthesis-associated genesmurA,murF, andpbp2(2-, 5-, and 6-fold, respectively) in response to SI doses of EGCG were determined by quantitative reverse transcription-PCR (qRT-PCR). EGCG also induced the promoter ofsas016(encoding a cell wall stress protein of unknown function which is not induced invraSRnull mutants) in a concentration-dependent manner, showing kinetics comparable to those of cell-wall-targeting antibiotics. Taken together, our results suggest that the two-component VraSR system is involved in modulating the cell response to SI doses of EGCG.
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Mohamed, Mohamed F., Maha I. Hamed, Alyssa Panitch, and Mohamed N. Seleem. "Targeting Methicillin-Resistant Staphylococcus aureus with Short Salt-Resistant Synthetic Peptides." Antimicrobial Agents and Chemotherapy 58, no. 7 (May 5, 2014): 4113–22. http://dx.doi.org/10.1128/aac.02578-14.
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ABSTRACTThe seriousness of microbial resistance combined with the lack of new antimicrobials has increased interest in the development of antimicrobial peptides (AMPs) as novel therapeutics. In this study, we evaluated the antimicrobial activities of two short synthetic peptides, namely, RRIKA and RR. These peptides exhibited potent antimicrobial activity againstStaphylococcus aureus, and their antimicrobial effects were significantly enhanced by addition of three amino acids in the C terminus, which consequently increased the amphipathicity, hydrophobicity, and net charge. Moreover, RRIKA and RR demonstrated a significant and rapid bactericidal effect against clinical and drug-resistantStaphylococcusisolates, including methicillin-resistantStaphylococcus aureus(MRSA), vancomycin-intermediateS. aureus(VISA), vancomycin-resistantS. aureus(VRSA), linezolid-resistantS. aureus, and methicillin-resistantStaphylococcus epidermidis. In contrast to many natural AMPs, RRIKA and RR retained their activity in the presence of physiological concentrations of NaCl and MgCl2. Both RRIKA and RR enhanced the killing of lysostaphin more than 1,000-fold and eradicated MRSA and VRSA isolates within 20 min. Furthermore, the peptides presented were superior in reducing adherent biofilms ofS. aureusandS. epidermidiscompared to results with conventional antibiotics. Our findings indicate that the staphylocidal effects of our peptides were through permeabilization of the bacterial membrane, leading to leakage of cytoplasmic contents and cell death. Furthermore, peptides were not toxic to HeLa cells at 4- to 8-fold their antimicrobial concentrations. The potent and salt-insensitive antimicrobial activities of these peptides present an attractive therapeutic candidate for treatment of multidrug-resistantS. aureusinfections.
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Donovan, David M., Shengli Dong, Wes Garrett, Geneviève M. Rousseau, Sylvain Moineau, and David G. Pritchard. "Peptidoglycan Hydrolase Fusions Maintain Their Parental Specificities." Applied and Environmental Microbiology 72, no. 4 (April 2006): 2988–96. http://dx.doi.org/10.1128/aem.72.4.2988-2996.2006.
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ABSTRACT The increased incidence of bacterial antibiotic resistance has led to a renewed search for novel antimicrobials. Avoiding the use of broad-range antimicrobials through the use of specific peptidoglycan hydrolases (endolysins) might reduce the incidence of antibiotic-resistant pathogens worldwide. Staphylococcus aureus and Streptococcus agalactiae are human pathogens and also cause mastitis in dairy cattle. The ultimate goal of this work is to create transgenic cattle that are resistant to mastitis through the expression of an antimicrobial protein(s) in their milk. Toward this end, two novel antimicrobials were produced. The (i) full-length and (ii) 182-amino-acid, C-terminally truncated S. agalactiae bacteriophage B30 endolysins were fused to the mature lysostaphin protein of Staphylococcus simulans. Both fusions display lytic specificity for streptococcal pathogens and S. aureus. The full lytic ability of the truncated B30 protein also suggests that the SH3b domain at the C terminus is dispensable. The fusions are active in a milk-like environment. They are also active against some lactic acid bacteria used to make cheese and yogurt, but their lytic activity is destroyed by pasteurization (63°C for 30 min). Immunohistochemical studies indicated that the fusion proteins can be expressed in cultured mammalian cells with no obvious deleterious effects on the cells, making it a strong candidate for use in future transgenic mice and cattle. Since the fusion peptidoglycan hydrolase also kills multiple human pathogens, it also may prove useful as a highly selective, multipathogen-targeting antimicrobial agent that could potentially reduce the use of broad-range antibiotics in fighting clinical infections.
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Dubrac, Sarah, Ivo Gomperts Boneca, Olivier Poupel, and Tarek Msadek. "New Insights into the WalK/WalR (YycG/YycF) Essential Signal Transduction Pathway Reveal a Major Role in Controlling Cell Wall Metabolism and Biofilm Formation in Staphylococcus aureus." Journal of Bacteriology 189, no. 22 (September 7, 2007): 8257–69. http://dx.doi.org/10.1128/jb.00645-07.
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ABSTRACT The highly conserved WalK/WalR (also known as YycG/YycF) two-component system is specific to low-G+C gram-positive bacteria. While this system is essential for cell viability, both the nature of its regulon and its physiological role have remained mostly uncharacterized. We observed that, unexpectedly, Staphylococcus aureus cell death induced by WalKR depletion was not followed by lysis. We show that WalKR positively controls autolytic activity, in particular that of the two major S. aureus autolysins, AtlA and LytM. By using our previously characterized consensus WalR binding site and carefully reexamining the genome annotations, we identified nine genes potentially belonging to the WalKR regulon that appeared to be involved in S. aureus cell wall degradation. Expression of all of these genes was positively controlled by WalKR levels in the cell, leading to high resistance to Triton X-100-induced lysis when the cells were starved for WalKR. Cells lacking WalKR were also more resistant to lysostaphin-induced lysis, suggesting modifications in cell wall structure. Indeed, lowered levels of WalKR led to a significant decrease in peptidoglycan biosynthesis and turnover and to cell wall modifications, which included increased peptidoglycan cross-linking and glycan chain length. We also demonstrated a direct relationship between WalKR levels and the ability to form biofilms. This is the first example in S. aureus of a regulatory system positively controlling autolysin synthesis and biofilm formation. Taken together, our results now define this signal transduction pathway as a master regulatory system for cell wall metabolism, which we have accordingly renamed WalK/WalR to reflect its true function.
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SZWEDA, Piotr, Marta SCHIELMANN, Aneta FRANKOWSKA, Barbara KOT, and Magdalena ZALEWSKA. "Antibiotic Resistance in Staphylococcus aureus Strains Isolated from Cows with Mastitis in Eastern Poland and Analysis of Susceptibility of Resistant Strains to Alternative Nonantibiotic Agents: Lysostaphin, Nisin and Polymyxin B." Journal of Veterinary Medical Science 76, no. 3 (2014): 355–62. http://dx.doi.org/10.1292/jvms.13-0177.
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Shaw, Lindsey N., Ewa Golonka, Grzegorz Szmyd, Simon J. Foster, James Travis, and Jan Potempa. "Cytoplasmic Control of Premature Activation of a Secreted Protease Zymogen: Deletion of Staphostatin B (SspC) in Staphylococcus aureus 8325-4 Yields a Profound Pleiotropic Phenotype." Journal of Bacteriology 187, no. 5 (March 1, 2005): 1751–62. http://dx.doi.org/10.1128/jb.187.5.1751-1762.2005.
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ABSTRACT The cytoplasmic protein SspC of Staphylococcus aureus, referred to as staphostatin B, is a very specific, tightly binding inhibitor of the secreted protease staphopain B (SspB). SspC is hypothesized to protect intracellular proteins against proteolytic damage by prematurely folded and activated staphopain B (M. Rzychon, A. Sabat, K. Kosowska, J. Potempa, and A. Dubin, Mol. Microbiol. 49:1051-1066, 2003). Here we provide evidence that elimination of intracellular staphopain B activity is indeed the function of SspC. An isogenic sspC mutant of S. aureus 8325-4 exhibits a wide range of striking pleiotropic alterations in phenotype, which distinguish it from the parent. These changes include a defect in growth, a less structured peptidoglycan layer within the cell envelope, severely decreased autolytic activity, resistance to lysis by S. aureus phages, extensively diminished sensitivity to lysis by lysostaphin, the ability to form a biofilm, and a total lack of extracellular proteins secreted into the growth media. The same phenotype was also engineered by introduction of sspB into an 8325-4 sspBC mutant. In contrast, sspC inactivation in the SH1000 strain did not yield any significant changes in the mutant phenotype, apparently due to strongly reduced expression of sspB in the sigma B-positive background. The exact pathway by which these diverse aberrations are exerted in 8325-4 is unknown, but it is apparent that a very small amount of staphopain B (less than 20 ng per 200 μg of cell proteins) is sufficient to bring about these widespread changes. It is proposed that the effects observed are modulated through the proteolytic degradation of several cytoplasmic proteins within cells lacking the inhibitor. Seemingly, some of these proteins may play a role in protein secretion; hence, their proteolytic inactivation by SspB has pleiotropic effects on the SspC-deficient mutant.
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Soni, Ankit, S. N. Askari, and Shamim Ahmad. "Experimental Lysostaphin therapy in methicillin resistant Staphylococcus aureus induced keratitis in Rabbits: A comparative evaluation." Asian Pacific Journal of Health Sciences 2, no. 2 (April 2015): 105–12. http://dx.doi.org/10.21276/apjhs.2015.2.2.17.
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Batool, Nayab, Kwan Soo Ko, Akhilesh Kumar Chaurasia, and Kyeong Kyu Kim. "Draft Genome Sequences of Lysostaphin-Resistant (K07-204) and Lysostaphin-Susceptible (K07-561) Staphylococcus aureus Sequence Type 72 Strains Isolated from Patients in South Korea." Microbiology Resource Announcements 9, no. 49 (December 3, 2020). http://dx.doi.org/10.1128/mra.01057-20.
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ABSTRACT Methicillin-resistant Staphylococcus aureus sequence type 72 (ST72) is prevalent in South Korea and has shown resistance to multiple antimicrobials. ST72 isolates display different levels of resistance to the antistaphylococcal lysostaphin. Draft genome sequencing of ST72 human isolates exhibiting lysostaphin resistance or susceptibility was performed to better understand the mechanism of lysostaphin resistance using subtractive genomics.
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Sharma, Namita, Anil Kumar Chhillar, Sweety Dahiya, Aruna Punia, Pooja Choudhary, Prity Gulia, Akanksha Behl, and Mehak Dangi. "Chemotherapeutic Strategies for Combating Staphylococcus aureus Infections." Mini-Reviews in Medicinal Chemistry 21 (April 2, 2021). http://dx.doi.org/10.2174/1389557521666210402150325.
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: Staphylococcus aureus is a prominent human pathogen that causes nosocomial and community acquired infections. The accelerating emergence and prevalence of staphylococcal infections have grotesque health consequences which are mostly due to its anomalous capability to acquire drug resistance and scarcity of novel classes of antibacterials. Many combating therapies are centered on primary targets of S. aureus which are cell envelope, ribosomes and nucleic acids. This review describes various chemotherapeutic strategies for combating S. aureus infections which includes monotherapy, combination drug therapy, phage endolysin therapy, lysostaphins and antibacterial drones. Monotherapy has dwindled in due course of time but combination therapy, endolysin therapy, lysostaphin and antibacterial drones are emerging alternatives which efficiently conquer the shortcomings of monotherapy. Combinations of more than one antibiotic agents or combination of adjuvant with antibiotics provide a synergistic approach to combat infections causing pathogenic strains. Phage endolysin therapy and lysostaphin are also presents as possible alternatives to conventional antibiotic therapies. Antibacterial Drones goes a step further by specifically targeting the virulence genes in bacteria giving them a certain advantage over existing antibacterial strategies. But the challenge remains on the better understanding of these strategies for executing and implementing them in health sector. In this day and age, most of the S. aureus strains are resistant to ample number of antibiotics, so there is an urgent need to overcome such multidrug resistant strains for the welfare of our community.
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Barros, Elaine M., Melissa J. Martin, Elizabeth M. Selleck, François Lebreton, Jorge Luiz M. Sampaio, and Michael S. Gilmore. "Daptomycin Resistance and Tolerance Due to Loss of Function inStaphylococcus aureusdsp1andasp23." Antimicrobial Agents and Chemotherapy 63, no. 1 (November 5, 2018). http://dx.doi.org/10.1128/aac.01542-18.
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ABSTRACTLipopeptide daptomycin is a last-line cell-membrane-targeting antibiotic to treat multidrug-resistantStaphylococcus aureus. Alarmingly, daptomycin-resistantS. aureusisolates have emerged. The mechanisms underlying daptomycin resistance are diverse and share similarities with resistances to cationic antimicrobial peptides and other lipopeptides, but they remain to be fully elucidated. We selected mutants with increased resistance to daptomycin from a library of transposon insertions in sequent type 8 (ST8)S. aureusHG003. Insertions conferring increased daptomycin resistance were localized to two genes, one coding for a hypothetical lipoprotein (SAOUHSC_00362, Dsp1), and the other for an alkaline shock protein (SAOUHSC_02441, Asp23). Markerless loss-of-function mutants were then generated for comparison. All transposon mutants and knockout strains exhibited increased daptomycin resistance compared to those of wild-type and complemented strains. Null and transposon insertion mutants also exhibited increased resistance to cationic antimicrobial peptides. Interestingly, theΔdsp1mutant also showed increased resistance to vancomycin, a cell-wall-targeting drug with a different mode of action. Null mutations in bothdsp1andasp23resulted in increased tolerance as reflected by reduced killing to both daptomycin and vancomycin, as well as an increased tolerance to surfactant (Triton X-100). Neither mutant exhibited increased resistance to lysostaphin, a cell-wall-targeting endopeptidase. These findings identified two genes core to theS. aureusspecies that make previously uncharacterized contributions to antimicrobial resistance and tolerance inS. aureus.
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Peignier, Adeline, Paul J. Planet, and Dane Parker. "Differential Induction of Type I and III Interferons by Staphylococcus aureus." Infection and Immunity 88, no. 10 (July 20, 2020). http://dx.doi.org/10.1128/iai.00352-20.
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ABSTRACT Staphylococcus aureus is a leading cause of bacterial pneumonia, and we have shown previously that type I interferon (IFN) contributes to the pathogenesis of this disease. In this study, we screened 75 S. aureus strains for their ability to induce type I and III IFN. Both cytokine pathways were differentially stimulated by various S. aureus strains independently of their isolation sites or methicillin resistance profiles. These induction patterns persisted over time, and type I and III IFN generation differentially correlated with tumor necrosis factor alpha production. Investigation of one isolate, strain 126, showed a significant defect in type I IFN induction that persisted over several time points. The lack of induction was not due to differential phagocytosis, subcellular location, or changes in endosomal acidification. A correlation between reduced type I IFN induction levels and decreased autolysis and lysostaphin sensitivity was found between strains. Strain 126 had a decreased rate of autolysis and increased resistance to lysostaphin degradation and host cell-mediated killing. This strain displayed decreased virulence in a murine model of acute pneumonia compared to USA300 (current epidemic strain and commonly used in research) and had reduced capacity to induce multiple cytokines. We observed this isolate to be a vancomycin-intermediate S. aureus (VISA) strain, and reduced Ifnb was observed with a defined mutation in walK that induces a VISA phenotype. Overall, this study demonstrates the heterogeneity of IFN induction by S. aureus and uncovered an interesting property of a VISA strain in its inability to induce type I IFN production.
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Raven, Kathy E., Sophia T. Girgis, Asha Akram, Beth Blane, Danielle Leek, Nicholas Brown, and Sharon J. Peacock. "A common protocol for the simultaneous processing of multiple clinically relevant bacterial species for whole genome sequencing." Scientific Reports 11, no. 1 (January 8, 2021). http://dx.doi.org/10.1038/s41598-020-80031-8.
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AbstractWhole-genome sequencing is likely to become increasingly used by local clinical microbiology laboratories, where sequencing volume is low compared with national reference laboratories. Here, we describe a universal protocol for simultaneous DNA extraction and sequencing of numerous different bacterial species, allowing mixed species sequence runs to meet variable laboratory demand. We assembled test panels representing 20 clinically relevant bacterial species. The DNA extraction process used the QIAamp mini DNA kit, to which different combinations of reagents were added. Thereafter, a common protocol was used for library preparation and sequencing. The addition of lysostaphin, lysozyme or buffer ATL (a tissue lysis buffer) alone did not produce sufficient DNA for library preparation across the species tested. By contrast, lysozyme plus lysostaphin produced sufficient DNA across all 20 species. DNA from 15 of 20 species could be extracted from a 24-h culture plate, while the remainder required 48–72 h. The process demonstrated 100% reproducibility. Sequencing of the resulting DNA was used to recapitulate previous findings for species, outbreak detection, antimicrobial resistance gene detection and capsular type. This single protocol for simultaneous processing and sequencing of multiple bacterial species supports low volume and rapid turnaround time by local clinical microbiology laboratories.
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Raven, Kathy E., Sophia T. Girgis, Asha Akram, Beth Blane, Danielle Leek, Nicholas Brown, and Sharon J. Peacock. "A common protocol for the simultaneous processing of multiple clinically relevant bacterial species for whole genome sequencing." Scientific Reports 11, no. 1 (January 8, 2021). http://dx.doi.org/10.1038/s41598-020-80031-8.
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AbstractWhole-genome sequencing is likely to become increasingly used by local clinical microbiology laboratories, where sequencing volume is low compared with national reference laboratories. Here, we describe a universal protocol for simultaneous DNA extraction and sequencing of numerous different bacterial species, allowing mixed species sequence runs to meet variable laboratory demand. We assembled test panels representing 20 clinically relevant bacterial species. The DNA extraction process used the QIAamp mini DNA kit, to which different combinations of reagents were added. Thereafter, a common protocol was used for library preparation and sequencing. The addition of lysostaphin, lysozyme or buffer ATL (a tissue lysis buffer) alone did not produce sufficient DNA for library preparation across the species tested. By contrast, lysozyme plus lysostaphin produced sufficient DNA across all 20 species. DNA from 15 of 20 species could be extracted from a 24-h culture plate, while the remainder required 48–72 h. The process demonstrated 100% reproducibility. Sequencing of the resulting DNA was used to recapitulate previous findings for species, outbreak detection, antimicrobial resistance gene detection and capsular type. This single protocol for simultaneous processing and sequencing of multiple bacterial species supports low volume and rapid turnaround time by local clinical microbiology laboratories.
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Fang, Yongliang, Jack R. Kirsch, Liang Li, Seth A. Brooks, Spencer Heim, Cynthia Tan, Susan Eszterhas, et al. "Deimmunized Lysostaphin Synergizes with Small-molecule Chemotherapies and Re-sensitizes MRSA to β-Lactam Antibiotics." Antimicrobial Agents and Chemotherapy, December 14, 2020, AAC.01707–20. http://dx.doi.org/10.1128/aac.01707-20.
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There is an urgent need for novel agents to treat drug-resistant bacterial infections, such as multidrug-resistant Staphylococcus aureus (MRSA). Desirable properties for new antibiotics include high potency, narrow species selectivity, low propensity to elicit new resistance phenotypes, and synergy with standard of care (SOC) chemotherapies. Here, we describe analysis of the anti-MRSA potential exhibited by F12, an innovative anti-MRSA lysin that has been genetically engineered to evade detrimental antidrug immune responses in human patients. F12 possesses high potency and rapid onset of action, it has narrow selectivity against pathogenic Staphylococci, and it manifests synergy with numerous SOC antibiotics. Additionally, resistance to F12 and β-lactam antibiotics appears mutually exclusive, and importantly we provide evidence that F12 re-sensitizes normally resistant MRSA strains to β-lactams both in vitro and in vivo. These results suggest that combinations of F12 and SOC antibiotics could be a promising new approach to treating refractory S. aureus infections.
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Liu, Pilong, Zehua Hao, Miaomiao Liu, Mingze Niu, Peng Sun, Shunhua Yan, Lixiu Zhao, and Xin Zhao. "Genetic mutations in adaptive evolution of growth-independent vancomycin-tolerant Staphylococcus aureus." Journal of Antimicrobial Chemotherapy, July 23, 2021. http://dx.doi.org/10.1093/jac/dkab260.
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Abstract Background Antibiotic tolerance allows bacteria to overcome antibiotic treatment transiently and potentially accelerates the emergence of resistance. However, our understanding of antibiotic tolerance at the genetic level during adaptive evolution of Staphylococcus aureus remains incomplete. We sought to identify the mutated genes and verify the role of these genes in the formation of vancomycin tolerance in S. aureus. Methods Vancomycin-susceptible S. aureus strain Newman was used to induce vancomycin-tolerant isolates in vitro by cyclic exposure under a high concentration of vancomycin (20× MIC). WGS and Sanger sequencing were performed to identify the genetic mutations. The function of mutated genes in vancomycin-tolerant isolates were verified by gene complementation. Other phenotypes of vancomycin-tolerant isolates were also determined, including mutation frequency, autolysis, lysostaphin susceptibility, cell wall thickness and cross-tolerance. Results A series of vancomycin-tolerant S. aureus (VTSA) strains were isolated and 18 mutated genes were identified by WGS. Among these genes, pbp4, htrA, stp1, pth and NWMN_1068 were confirmed to play roles in VTSA formation. Mutation of mutL promoted the emergence of VTSA. All VTSA showed no changes in growth phenotype. Instead, they exhibited reduced autolysis, decreased lysostaphin susceptibility and thickened cell walls. In addition, all VTSA strains were cross-tolerant to antibiotics targeting cell wall synthesis but not to quinolones and lipopeptides. Conclusions Our results demonstrate that genetic mutations are responsible for emergence of phenotypic tolerance and formation of vancomycin tolerance may lie in cell wall changes in S. aureus.
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Verbree, Carolin T., Steven M. Dätwyler, Susanne Meile, Fritz Eichenseher, David M. Donovan, Martin J. Loessner, and Mathias Schmelcher. "Identification of Peptidoglycan Hydrolase Constructs with Synergistic Staphylolytic Activity in Cow's Milk." Applied and Environmental Microbiology 83, no. 7 (February 3, 2017). http://dx.doi.org/10.1128/aem.03445-16.
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ABSTRACT Peptidoglycan hydrolases (PGHs) have been suggested as novel therapeutics for the treatment of bovine mastitis. However, activity in the presence of cow's milk is an important requirement for drugs administered into the bovine udder. We have screened a library of >170 recombinant PGHs, including engineered bacteriophage endolysins, for enzymes with activity against Staphylococcus aureus in milk, using a microtiter plate-based protocol. Nine suitable PGH constructs were identified by this approach and further compared in time-kill assays for their efficacy against S. aureus in heat-treated milk. The three most active enzymes (lysostaphin, Ami2638A, and CHAPK_CWT-LST) reduced S. aureus in milk to undetectable numbers within minutes at nanomolar concentrations. Due to their different peptidoglycan cleavage sites, these PGH constructs revealed synergistic activity in most combinations, as demonstrated by checkerboard assays, spot assays, and time-kill experiments. Furthermore, they proved active against a selection of staphylococcal mastitis isolates from different geographical regions when applied individually or in synergistic combination. The most effective PGH combination completely eradicated S. aureus from milk, with no more bacteria being detected within 24 h after addition of the enzymes, corresponding to a reduction of >9 log units compared to the control. Efficacy was also retained at different inoculum levels (3 versus 6 log CFU/ml) and when S. aureus was grown in milk as opposed to broth prior to the experiments. In raw cow's milk, CHAPK_CWT-LST showed reduced efficacy, whereas both Ami2638A and lysostaphin retained their activity, reducing bacterial numbers by >3.5 log units within 3 h. IMPORTANCE Staphylococci and S. aureus in particular are a major cause of bovine mastitis, an inflammation of the mammary gland in cows associated with high costs and risks for consumers of milk products. S. aureus-induced mastitis, commonly treated by intramammary infusion of antibiotics, is characterized by low cure rates and increasing antibiotic resistance in bacteria. Therefore, alternative treatment options are highly desirable. PGHs, including bacteriophage endolysins, rapidly and specifically kill selected pathogens by degrading their cell wall and are refractory to resistance development, therefore holding promise as novel antibacterial agents. This study employed a screening approach to identify PGH constructs with high staphylolytic activity in cow's milk within a large collection of enzymes. Our results suggest that the most promising enzymes identified by this strategy hold potential as novel mastitis therapeutics and support their further characterization in animal models.