Alpha-Toxin Contributes to Biofilm Formation among Staphylococcus aureus Wound Isolates
"> Figure 1
<p><b>Ex vivo biofilm formation and alpha-toxin production.</b> The degree of biofilm formed on porcine vaginal mucosa (PVM) explants was highly variable. (<b>A</b>) All methicillin-resistant <span class="html-italic">S. aureus</span> (MRSA) isolates formed biofilms; scores ranged from 0.67 to 3.67 (grey bars, right axis). Similar to the MRSA ex vivo biofilms, alpha-toxin (AT) was highly variable, ranging from <1.0 ng to >10 ng per explants (black circles, left axis). (<b>C</b>) In contrast, several methicillin-susceptible <span class="html-italic">S. aureus</span> (MSSA) isolates did not form biofilms on the PVM; overall, the scores were lower than for MRSA (grey bars, right axis). AT was not detected by ELISA (<60 pg/mL) in MSSA isolates that failed to form biofilm (black circles, left axis). Increased ex vivo alpha-toxin production corresponds with ex vivo biofilm formation (black circles, left axis). (<b>B</b>,<b>D</b>) Correlation analysis of ex vivo alpha-toxin production vs. ex vivo biofilm formation showed a strong direct correlation for both MRSA (<span class="html-italic">n</span> = 18, r<sub>s</sub> = 0.67, <span class="html-italic">p</span> = 0.002) and MSSA (<span class="html-italic">n</span> = 20, r<sub>s</sub> = 0.67, <span class="html-italic">p</span> = 0.001).</p> "> Figure 2
<p><b>Scoring system for <span class="html-italic">S. aureus</span> biofilm images.</b> Biofilm formation by <span class="html-italic">S. aureus</span> (SA) isolates on biological substrate (porcine vaginal mucosa (PVM) explants) at 72 h was evaluated by LIVE/DEAD<sup>®</sup> staining (SYTO<sup>®</sup> 9 [green = live] plus propidium iodide [red = dead]), which does not discriminate eukaryotic from prokaryotic cells, and confocal laser scanning microscopy. Ex vivo biofilms were scored on a 5-point scale. <b>0:</b> no biofilm, few if any attached bacteria, live epithelial cells observed throughout image (large, green, punctate staining) with some interspersed dead cells (red punctate staining). <b>1+</b>: biofilm covers >0–25% of surface area; live, adherent bacteria observed as small, bright green punctate staining interspersed among live (green) and dead (red) epithelial cells. <b>2+</b>: biofilm covers 26–50% of surface area; microcolonies observed as clusters of live bacteria in the lower right quadrant, more mature biofilm observed in the upper right quadrant (continuous green staining), many dead (red) epithelial cells apparent, some of which have sloughed off (black areas). <b>3+</b>: biofilm covers 51–75% of surface area; large, cloudlike, live (green) biofilm observed and no epithelium evident. <b>4+</b>: biofilm covers 76–100% of surface area; thick, dense biofilm (large proportion of image covered in green, small red punctate staining in the middle to lower left quadrant are dead bacteria). Isolates (<span class="html-italic">n</span> = 3) individually scored by two blinded observers. This was done twice for a total of 6 observations per isolate. Scale bar within each image is equal to 30 μm.</p> "> Figure 3
<p><b>MRSA and MSSA isolates form biofilm in vitro (<span class="html-italic">n</span> = 8 per isolate).</b> (<b>A</b>) OD<sub>600</sub> of crystal violet-stained methicillin-resistant <span class="html-italic">S. aureus</span> (MRSA) <span class="html-italic">n</span> = 18, and (<b>C</b>) methicillin-susceptible <span class="html-italic">S. aureus</span> (MSSA) <span class="html-italic">n</span> = 20 biofilms following 24 h incubation (grey bars). (<b>B</b>) We performed a correlation analysis with Spearman correction of in vitro vs ex vivo biofilm formation. There was a weak correlation between microtiter biofilm density and degree of porcine vaginal mucosa biofilm for MRSA (<span class="html-italic">n</span> = 18, r<sub>s</sub> = 0.28, <span class="html-italic">p</span> = 0.25). (<b>D</b>) For MSSA isolates, there was a very weak correlation between the in vitro biofilms and those grown on a biological substrate (<span class="html-italic">n</span> = 20, r<sub>s</sub> = 0.07, <span class="html-italic">p</span> = 0.78).</p> "> Figure 4
<p><b>Biofilm formation is prevented by anti-alpha-toxin monoclonal antibody.</b> Porcine vaginal mucosa (PVM) explants were pretreated with 10 uL of MEDI4893* (MedImmune, Fredricksburg, MD, USA) or isotype control antibody (c-IgG) for 1 h prior to infection. Three days post-infection, explants were stained and imaged by confocal laser scanning microscopy. (<b>A</b>) MEDI4893* completely abrogated MRSA’s ability to form a biofilm on PVM epithelium. (<b>B</b>) C-IgG-treated MRSA developed a 4+ biofilm. Scale bar equals 30 μm.</p> "> Figure 5
<p><b>Wild-type alpha-toxin rescues biofilm defect for MRSA LAC Δhla mutant and clinical isolates.</b> Porcine vaginal mucosa (PVM) explants were pretreated with 0.1–5 µg/explant of wild-type (WT) alpha-toxin (AT) or AT H35L for 10 min prior to infection. Three days post-infection, explants were stained with LIVE/DEAD and imaged by confocal laser scanning microscopy. (<b>A</b>) Uninfected control; explants infected with MRSA LAC show extensive biofilm formation, while explants infected with MRSA LAC Δhla exhibit very little to no biofilm. Explants pretreated with WT AT prior to addition of MRSA LAC Δhla exhibit biofilm rescue only at high doses. (<b>B</b>) Explants pretreated with AT H35L show no biofilm rescue at any dose. (<b>C</b>) WT AT rescues the biofilm defect of both MSSA12 and MSSAE2, while AT H35L does not. Scale bar equals 50 μm.</p> "> Figure 6
<p><b>Toxicity of wild-type and H35L mutant alpha-toxins.</b> Purified wild-type (WT) alpha-toxin (AT) or AT H35L mutant was added to porcine vaginal mucosa explants at varying doses and viability was determined by an MTT-based assay. WT AT was cytotoxic at higher concentrations (1 and 5 μg/explant), while the AT H35L mutant (non-pore forming) was non-toxic. Data (<span class="html-italic">n</span> = 3) are represented as mean ± SD; * denotes significance from non-AT treated control explants; <span class="html-italic">p</span> < 0.05 as determined by one-way analysis of variance followed by Dunnett’s multiple comparisons post-test.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Strain Genotyping
2.2. Ex Vivo AT Production on PVM Explants
2.3. Ex Vivo Biofilm Production on PVM Explants
2.4. In Vitro Biofilm Production
2.5. Neutralization of AT Prevents MRSA Biofilm Formation on Mucosal Tissue
2.6. Rescue of Biofilm Defect with Exogenous AT
3. Discussion
4. Materials and Methods
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
References
- Hall-Stoodley, L.; Costerton, J.W.; Stoodley, P. Bacterial biofilms: From the natural environment to infectious diseases. Nat. Rev. Microbiol. 2004, 2, 95–108. [Google Scholar] [CrossRef] [PubMed]
- Elgharably, H.; Mann, E.; Awad, H.; Ganesh, K.; Ghatak, P.D.; Gordillo, G.; Sai-Sudhakar, C.B.; Roy, S.; Wozniak, D.J.; Sen, C.K. First evidence of sternal wound biofilm following cardiac surgery. PLoS ONE 2013, 8, e70360. [Google Scholar] [CrossRef] [PubMed]
- James, G.A.; Swogger, E.; Wolcott, R.; Pulcini, E.; Secor, P.; Sestrich, J.; Costerton, J.W.; Stewart, P.S. Biofilms in chronic wounds. Wound Repair Regener. 2008, 16, 37–44. [Google Scholar] [CrossRef] [PubMed]
- Percival, S.L.; Hill, K.E.; Williams, D.W.; Hooper, S.J.; Thomas, D.W.; Costerton, J.W. A review of the scientific evidence for biofilms in wounds. Wound Repair Regener. 2012, 20, 647–657. [Google Scholar] [CrossRef] [PubMed]
- Landrum, M.L.; Neumann, C.; Cook, C.; Chukwuma, U.; Ellis, M.W.; Hospenthal, D.R.; Murray, C.K. Epidemiology of Staphylococcus aureus blood and skin and soft tissue infections in the US military health system, 2005–2010. JAMA 2012, 308, 50–59. [Google Scholar] [CrossRef] [PubMed]
- Tracy, L.A.; Furuno, J.P.; Harris, A.D.; Singer, M.; Langenberg, P.; Roghmann, M.C. Staphylococcus aureus infections in US veterans, Maryland, USA, 1999–2008. Emerg. Infect. Dis. 2011, 17, 441–448. [Google Scholar] [CrossRef] [PubMed]
- Aiello, A.E.; Lowy, F.D.; Wright, L.N.; Larson, E.L. Meticillin-resistant Staphylococcus aureus among US prisoners and military personnel: Review and recommendations for future studies. Lancet Infect. Dis. 2006, 6, 335–341. [Google Scholar] [CrossRef]
- Diekema, D.J.; Richter, S.S.; Heilmann, K.P.; Dohrn, C.L.; Riahi, F.; Tendolkar, S.; McDanel, J.S.; Doern, G.V. Continued emergence of USA300 methicillin-resistant Staphylococcus aureus in the United States: Results from a nationwide surveillance study. Infect. Control Hosp. Epidemiol. 2014, 35, 285–292. [Google Scholar] [CrossRef] [PubMed]
- Montgomery, C.P.; Boyle-Vavra, S.; Adem, P.V.; Lee, J.C.; Husain, A.N.; Clasen, J.; Daum, R.S. Comparison of virulence in community-associated methicillin-resistant Staphylococcus aureus pulsotypes usa300 and usa400 in a rat model of pneumonia. J. Infect. Dis. 2008, 198, 561–570. [Google Scholar] [CrossRef] [PubMed]
- Berube, B.J.; Bubeck Wardenberg, J. Staphylococcus aureus alpha-toxin: Nearly a century of intrigue. Toxins 2013, 5, 1140–1166. [Google Scholar] [CrossRef] [PubMed]
- Inoshima, I.; Inoshima, N.; Wilke, G.A.; Powers, M.E.; Frank, K.M.; Wang, Y.; Wardenburg, J.B. A Staphylococcus aureus pore-forming toxin subverts the activity of ADAM10 to cause lethal infection in mice. Nat. Med. 2011, 17, 1310–1314. [Google Scholar] [CrossRef] [PubMed]
- Nygaard, T.K.; Pallister, K.B.; DuMont, A.L.; DeWald, M.; Watkins, R.L.; Pallister, E.Q.; Malone, C.; Griffith, S.; Horswill, A.R.; Torres, V.J.; et al. Alpha-toxin induces programmed cell death of human T cells, B cells, and monocytes during USA300 infection. PLoS ONE 2012, 7, e36532. [Google Scholar] [CrossRef] [PubMed]
- Sengers, R.C. Hemolytic action of staphylococcal alpha-hemolysin on human erythrocytes in a Na plus-and K plus-containing suspending fluid. Antonie Van Leeuwenhoek 1970, 36, 57–65. [Google Scholar] [CrossRef] [PubMed]
- Tabor, D.E.; Yu, L.; Mok, H.; Tkaczyk, C.; Sellman, B.R.; Wu, Y.; Oganesyan, V.; Slidel, T.; Jafri, H.; McCarthy, M.; et al. Staphylococcus aureus alpha-toxin is conserved among diverse hospital respiratory isolates collected from a gobal surveillance study and is neutralized by monoclonal antibody MEDI4893. Antimicrob. Agents Chemother. 2016, 60, 5312–5321. [Google Scholar] [CrossRef] [PubMed]
- Anderson, M.J.; Lin, Y.-C.; Gillman, A.N.; Parks, P.J.; Schlievert, P.M.; Peterson, M. Alpha-Toxin Promotes Mucosal Biofilm Formation by Staphylococcus aureus. Front. Cell. Infect. Microbiol. 2012, 2, 64. [Google Scholar] [CrossRef] [PubMed]
- Den Reijer, P.M.; Haisma, E.M.; Toom, N.A.L.; Willemse, J.; Koning, R.A.; Demmers, J.A.; Dekkers, D.H.; Rijkers, E.; El Ghalbzouri, A.; Nibbering, P.H.; et al. Detection of Alpha-Toxin and Other Virulence Factors in Biofilms of Staphylococcus aureus on Polystyrene and a Human Epidermal Model. PLoS ONE 2016, 11, e0145722. [Google Scholar]
- O’Reilly, M.; Kreiswirth, B.; Foster, T.J. Cryptic alpha-toxin gene in toxic shock syndrome and septicaemia strains of Staphylococcus aureus. Mol. Microbiol. 1990, 4, 1947–1955. [Google Scholar] [CrossRef] [PubMed]
- Tenover, F.C.; McAllister, S.; Fosheim, G.; McDougal, L.K.; Carey, R.B.; Limbago, B.; Lonsway, D.; Patel, J.B.; Kuehnert, M.J.; Gorwitz, R. Characterization of Staphylococcus aureus isolates from nasal cultures collected from individuals in the United States in 2001 to 2004. J. Clin. Microbiol. 2008, 46, 2837–2841. [Google Scholar] [CrossRef] [PubMed]
- Anderson, M.J.; Parks, P.J.; Peterson, M.L. A mucosal model to study microbial biofilm development and anti-biofilm therapeutics. J. Microbiol. Methods 2013, 92, 201–208. [Google Scholar] [CrossRef] [PubMed]
- Ando, E.; Monden, K.; Mitsuhata, R.; Kariyama, R.; Kumon, H. Biofilm formation among methicillin-resistant Staphylococcus aureus isolates from patients with urinary tract infection. Acta Med. Okayama 2004, 58, 207–214. [Google Scholar] [PubMed]
- Enright, M.C.; Day, N.P.; Davies, C.E.; Peacock, S.J.; Spratt, B.G. Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus. J. Clin. Microbiol. 2000, 38, 1008–1015. [Google Scholar] [PubMed]
- Kennedy, A.D.; Otto, M.; Braughton, K.R.; Whitney, A.R.; Chen, L.; Mathema, B.; Mediavilla, J.R.; Byrne, K.A.; Parkins, L.D.; Tenover, F.C.; et al. Epidemic community-associated methicillin-resistant Staphylococcus aureus: Recent clonal expansion and diversification. Proc. Natl. Acad. Sci. USA 2008, 105, 1327–1332. [Google Scholar] [CrossRef] [PubMed]
- Tkaczyk, C.; Hamilton, M.M.; Datta, V.; Yang, X.P.; Hilliard, J.J.; Stephens, G.L.; Sadowska, A.; Hua, L.; O’Day, T.; Suzich, J.; et al. Staphylococcus aureus alpha toxin suppresses effective innate and adaptive immune responses in a murine dermonecrosis model. PLoS ONE 2013, 8, e75103. [Google Scholar] [CrossRef] [PubMed]
- David, M.Z.; Taylor, A.; Lynfield, R.; Boxrud, D.J.; Short, G.; Zychowski, D.; Boyle-Vavra, S.; Daum, R.S. Comparing pulsed-field gel electrophoresis with multilocus sequence typing, spa typing, staphylococcal cassette chromosome mec (SCCmec) typing, and PCR for panton-valentine leukocidin, arcA, and opp3 in methicillin-resistant Staphylococcus aureus isolates at a U.S. Medical Center. J. Clin. Microbiol. 2013, 51, 814–819. [Google Scholar] [PubMed]
- Fisher, A.; Webber, B.J.; Pawlak, M.T.; Johnston, L.; Tchandja, J.B.; Yun, H. Epidemiology, microbiology, and antibiotic susceptibility patterns of skin and soft tissue infections, Joint Base San Antonio-Lackland, Texas, 2012–2014. MSMR 2015, 22, 2–6. [Google Scholar] [PubMed]
- Pardos de la Gandara, M.; Garay, J.A.R.; Mwangi, M.; Tobin, J.N.; Tsang, A.; Khalida, C.; D’Orazio, B.; Kost, R.G.; Leinberger-Jabari, A.; Coffran, C.; et al. Molecular Types of Methicillin-Resistant Staphylococcus aureus and Methicillin-Sensitive S. aureus Strains Causing Skin and Soft Tissue Infections and Nasal Colonization, Identified in Community Health Centers in New York City. J. Clin. Microbiol. 2015, 53, 2648–2658. [Google Scholar] [CrossRef] [PubMed]
- Stulik, L.; Malafa, S.; Hudcova, J.; Rouha, H.; Henics, B.Z.; Craven, D.E.; Sonnevend, A.M.; Nagy, E. alpha-Hemolysin activity of methicillin-susceptible Staphylococcus aureus predicts ventilator-associated pneumonia. Am. J. Respir. Crit. Care. Med. 2014, 190, 1139–1148. [Google Scholar] [CrossRef] [PubMed]
- Sanchez, C.J.; Shivshankar, P.; Stol, K.; Trakhtenbroit, S.; Sullam, P.M.; Sauer, K.; Hermans, P.W.; Orihuela, C.J. The pneumococcal serine-rich repeat protein is an intra-species bacterial adhesin that promotes bacterial aggregation in vivo and in biofilms. PLoS Pathog. 2010, 6, e1001044. [Google Scholar] [CrossRef] [PubMed]
- Oganesyan, V.; Peng, L.; Damschroder, M.M.; Cheng, L.; Sadowska, A.; Tkaczyk, C.; Sellman, B.R.; Wu, H.; Dall’Acqua, W.F. Mechanisms of neutralization of a human anti-alpha-toxin antibody. J. Biol. Chem. 2014, 289, 29874–29880. [Google Scholar] [CrossRef] [PubMed]
- Hua, L.; Hilliard, J.J.; Shi, Y.; Tkaczyk, C.; Cheng, L.I.; Yu, X.; Datta, V.; Ren, S.; Feng, H.; Zinsou, R.; et al. Assessment of an anti-alpha-toxin monoclonal antibody for prevention and treatment of Staphylococcus aureus-induced pneumonia. Antimicrob. Agents Chemother. 2014, 58, 1108–1117. [Google Scholar] [CrossRef] [PubMed]
- Tkaczyk, C.; Hua, L.; Varkey, R.; Shi, Y.; Dettinger, L.; Woods, R.; Barnes, A.; MacGill, R.S.; Wilson, S.; Chowdhury, P.; et al. Identification of anti-alpha toxin monoclonal antibodies that reduce the severity of Staphylococcus aureus dermonecrosis and exhibit a correlation between affinity and potency. Clin. Vaccine Immunol. 2012, 19, 377–385. [Google Scholar] [CrossRef] [PubMed]
- Diep, B.A.; Gill, S.R.; Chang, R.F.; Phan, T.H.; Chen, J.H.; Davidson, M.G.; Lin, F.; Lin, J.; Carleton, H.A.; Mongodin, E.F.; et al. Complete genome sequence of USA300, an epidemic clone of community-acquired methicillin-resistant Staphylococcus aureus. Lancet 2006, 367, 731–739. [Google Scholar] [CrossRef]
- Spaan, A.N.; van Strijp, J.A.G.; Torres, V.J. Leukocidins: Staphylococcal bi-component pore-forming toxins find their receptors. Nat. Rev. Microbiol. 2017, 15, 435–447. [Google Scholar] [CrossRef] [PubMed]
- Tawk, M.Y.; Zimmermann-Meisse, G.; Bossu, J.L.; Potrich, C.; Bourcier, T.; Serra, M.D.; Poulain, B.; Prevost, G.; Jover, E. Internalization of staphylococcal leukotoxins that bind and divert the C5a receptor is required for intracellular Ca(2+) mobilization by human neutrophils. Cell Microbiol. 2015, 17, 1241–1257. [Google Scholar] [CrossRef] [PubMed]
- Ortines, R.V.; Liu, H.; Cheng, L.I.; Cohen, T.S.; Lawlor, H.; Gami, A.; Wan, Y.; Dillen, C.A.; Archer, N.K.; Miller, R.J.; et al. Neutralizing alpha-toxin accelerates healing of Staphylococcus aureus-infected wounds in nondiabetic and diabetic mice. Antimicrob. Agents Chemother. 2018, 62, e02288-17. [Google Scholar] [CrossRef] [PubMed]
- Hilliard, J.J.; Datta, V.; Tkaczyk, C.; Hamilton, M.; Sadowska, A.; Jones-Nelson, O.; O’Day, T.; Weiss, W.J.; Szarka, S.; Nguyen, V.; et al. Anti-alpha-toxin monoclonal antibody and antibiotic combination therapy improves disease outcome and accelerates healing in a Staphylococcus aureus dermonecrosis model. Antimicrob. Agents Chemother. 2015, 59, 299–309. [Google Scholar] [CrossRef] [PubMed]
- Kennedy, A.D.; Wardenburg, J.B.; Gardner, D.J.; Long, D.; Whitney, A.R.; Braughton, K.R.; Schneewind, O.; DeLeo, F.R. Targeting of alpha-hemolysin by active or passive immunization decreases severity of USA300 skin infection in a mouse model. J. Infect. Dis. 2010, 202, 1050–1058. [Google Scholar] [CrossRef] [PubMed]
- McDougal, L.K.; Steward, C.D.; Killgore, G.E.; Chaitram, J.M.; McAllister, S.K.; Tenover, F.C. Pulsed-field gel electrophoresis typing of oxacillin-resistant Staphylococcus aureus isolates from the United States: Establishing a national database. J. Clin. Microbiol. 2003, 41, 5113–5120. [Google Scholar] [CrossRef] [PubMed]
- Christensen, G.D.; Simpson, W.A.; Younger, J.J.; Baddour, L.M.; Barrett, F.F.; Melton, D.M.; Beachey, E.H. Adherence of coagulase-negative staphylococci to plastic tissue culture plates: A quantitative model for the adherence of staphylococci to medical devices. J. Clin. Microbiol. 1985, 22, 996–1006. [Google Scholar] [PubMed]
Methicillin Phenotype | Isolate | PFGE USA Type a | MLST b | Spa c | Q113Stop d |
---|---|---|---|---|---|
MRSA | 4 | 100 | ST231 | t002 | ND |
5 | 100 | ST5 | t088 | ND | |
12 | 100 | ST5 | t002 | ND | |
13 | 100 | ST5 | t002 | ND | |
A2 | 100 | ST5 | t002 | ND | |
A3 | 100 | ST5 | t002 | ND | |
B1 | 100 | ST5 | t002 | ND | |
C1 | 100 | ST5 | t105 | ND | |
C2 | 100 | ST5 | t002 | ND | |
C3 | 100 | ST5 | t002 | ND | |
9 | 300 | ST8 | t008 | ND | |
10 | 300 | ST8 | t008 | ND | |
15 | 300 | ST8 | t008 | ND | |
16 | 300 | ST8 | t008 | ND | |
A1 | 300 | ST8 | t008 | ND | |
B2 | 300 | ST8 | t008 | ND | |
2 | Non-typeable | ST5 | t242 | ND | |
B3 | Non-typeable | ST88 | t11140 | ND | |
MSSA | D3 | 100 | ST231 | t548 | no |
D1 | 200 | ST30 | t012 | yes | |
1 | 200 | ST30 | t012 | yes | |
5 | 200 | ST30 | t1577 | yes | |
E2 | 200 | ST30 | t012 | yes | |
13 | 200 | ST45 | t015 | no | |
11 | 300 | ST8 | unknown | no | |
8 | 400 | ST1 | t127 | no | |
9 | 400 | ST1 | t127 | no | |
2 | 600 | ST45 | t073 | no | |
3 | 900 | ST15 | t084 | no | |
E1 | 900 | ST15 | t084 | no | |
12 | Non-typeable | ST30 | t1577 | yes | |
E3 | Non-typeable | ST30 | t012 | yes | |
4 | Non-typeable | ST39 | t2271 | no | |
10 | Non-typeable | ST5 | t010 | no | |
F2 | Non-typeable | ST5 | t002 | no | |
6 | Non-typeable | ST8 | unknown | no | |
F3 | Non-typeable | ST97 | t521 | no | |
D2 | non-typeable | ST97 | t267 | no |
© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
Share and Cite
Anderson, M.J.; Schaaf, E.; Breshears, L.M.; Wallis, H.W.; Johnson, J.R.; Tkaczyk, C.; Sellman, B.R.; Sun, J.; Peterson, M.L. Alpha-Toxin Contributes to Biofilm Formation among Staphylococcus aureus Wound Isolates. Toxins 2018, 10, 157. https://doi.org/10.3390/toxins10040157
Anderson MJ, Schaaf E, Breshears LM, Wallis HW, Johnson JR, Tkaczyk C, Sellman BR, Sun J, Peterson ML. Alpha-Toxin Contributes to Biofilm Formation among Staphylococcus aureus Wound Isolates. Toxins. 2018; 10(4):157. https://doi.org/10.3390/toxins10040157
Chicago/Turabian StyleAnderson, Michele J., Emily Schaaf, Laura M. Breshears, Heidi W. Wallis, James R. Johnson, Christine Tkaczyk, Bret R. Sellman, Jisun Sun, and Marnie L. Peterson. 2018. "Alpha-Toxin Contributes to Biofilm Formation among Staphylococcus aureus Wound Isolates" Toxins 10, no. 4: 157. https://doi.org/10.3390/toxins10040157