Topical Delivery of Lactobacillus Culture Supernatant Increases Survival and Wound Resolution in Traumatic Acinetobacter baumannii Infections

  • Josh Stanbro
  • Ju Me Park
  • Matthew Bond
  • Michael G. Stockelman
  • Mark P. Simons
  • Chase WattersEmail author


Species of Lactobacillus have been proposed as potential candidates for treating wound infections due to their ability to lower pH, decrease inflammation, and release antimicrobial compounds. This study investigated the impact of lactobacilli (Lactobacillus acidophilus ATCC 4356, Lactobacillus casei ATCC 393, Lactobacillus reuteri ATCC 23272) secreted products on wound pathogens in vitro and in a murine wound infection model. Evaluation of 1–5 day lactobacilli conditioned media (CM) revealed maximal inhibition against wound pathogens using the 5-day CM. The minimum inhibitory concentration (MIC) of 5-day Lactobacillus CMs was tested by diluting CM in Mueller-Hinton (MH) broth from 0 to 25% and was found to be 12.5% for A. baumannii. Concentrating the CM to 10× with a 3 kDa centrifuge filter decreased the CM MIC to 6.25–12.5% for A. baumannii planktonic cells. Minimal impact of 5-day CMs was observed against bacterial biofilms. No toxicity was observed when these Lactobacillus CMs were injected into Galleria melonella waxworms. For the murine A. baumannii wound infection studies, improved survival was observed following topical treatment with L. acidophilus ATCC 4356 or L. reuteri ATCC 23272, while L. reuteri ATCC 23272 treatment alone improved wound resolution. Overall, this study suggests that the topical application of certain Lactobacillus species byproducts could be effective against gram-negative multi-drug resistant (MDR) wound pathogens, such as A. baumannii.


Lactobacillus Acinetobacter baumannii Wound infections Lactic acid bacteria 



Special thanks to the Army Wound Infections department at WRAIR for their support and assistance with the animal studies, specifically Yonas Alamneh, Rania Abu-Taleb, Jonathan Shearer, Samandra Demons, Anna Jacobs, Kathleen Umayam, Natalie-Makenna Gingras, Daniel Zurawski, and Yuanzheng Si.

Funding Information

The support for this work was provided by the Office of Naval Research under work unit number A1601.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflicts of interest.

Ethical Approval

All procedures were performed in accordance with protocols approved by the Walter Reed Army Institute of Research (WRAIR)/Naval Medical Research Center (NMRC) Institutional Animal Care and Use Committee in compliance with all applicable Federal regulations governing the protection of animals in research.


The views expressed are those of the authors and do not necessarily reflect the official policy or position of the Department of the Navy, Army, Department of Defense, nor the US Government. Some authors are service members of the US Government. I am a military service member or federal/contracted employee of the US government. This work was prepared as part of my official duties. Title 17 U.S.C. 105 provides that “copyright protection under this title is not available for any work of the United States Government.” Title 17 U.S.C. 101 defines a US Government work as work prepared by a military service member or employee of the US Government as part of that person’s official duties.

Supplementary material

12602_2019_9603_MOESM1_ESM.docx (192 kb)
Fig. S1 (DOCX 49 kb)


  1. 1.
    Huang XZ, Cash DM, Chahine MA, Van Horn GT, Erwin DP, McKay JT, Hamilton LR, Jerke KH, Co EM, Aldous WK, Lesho EP, Lindler LE, Bowden RA, Nikolich MP (2010) Methicillin-resistant Staphylococcus aureus infection in combat support hospitals in three regions of Iraq. Epidemiol Infect:1–4. CrossRefGoogle Scholar
  2. 2.
    Lee BY, Singh A, David MZ, Bartsch SM, Slayton RB, Huang SS, Zimmer SM, Potter MA, Macal CM, Lauderdale DS, Miller LG, Daum RS (2013) The economic burden of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA). Clin Microbiol Infect 19(6):528–536. CrossRefPubMedGoogle Scholar
  3. 3.
    Calhoun JH, Murray CK, Manring MM (2008) Multidrug-resistant organisms in military wounds from Iraq and Afghanistan. Clin Orthop Relat Res 466(6):1356–1362. CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Hospenthal D, Crouch H, English J, Leach F, Pool J, Conger N, Whitman T, Wortmann G, Robertson J, Murray C (2011) Multidrug-resistant bacterial colonization of combat-injured personnel at admission to medical centers after evacuation from Afghanistan and Iraq. J Trauma 71:S52–S57. CrossRefPubMedGoogle Scholar
  5. 5.
    Rice LB (2008) Federal funding for the study of antimicrobial resistance in nosocomial pathogens: no ESKAPE. J Infect Dis 197(8):1079–1081. CrossRefPubMedGoogle Scholar
  6. 6.
    Blackledge MS, Worthington RJ, Melander C (2013) Biologically inspired strategies for combating bacterial biofilms. Curr Opin Pharmacol 13(5):699–706. CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Be NA, Allen JE, Brown TS, Gardner SN, McLoughlin KS, Forsberg JA, Kirkup BC, Chromy BA, Luciw PA, Elster EA, Jaing CJ (2014) Microbial profiling of combat wound infection through detection microarray and next-generation sequencing. J Clin Microbiol 52(7):2583–2594. CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Mohammedsaeed W, McBain AJ, Cruickshank SM, O’Neill CA (2014) Lactobacillus rhamnosus GG inhibits the toxic effects of Staphylococcus aureus on epidermal keratinocytes. Appl Environ Microbiol 80(18):5773–5781. CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Vong L, Lorentz RJ, Assa A, Glogauer M, Sherman PM (2014) Probiotic Lactobacillus rhamnosus inhibits the formation of neutrophil extracellular traps. J Immunol 192(4):1870–1877. CrossRefPubMedGoogle Scholar
  10. 10.
    Sonal Sekhar M, Unnikrishnan MK, Vijayanarayana K, Rodrigues GS, Mukhopadhyay C (2014) Topical application/formulation of probiotics: will it be a novel treatment approach for diabetic foot ulcer? Med Hypotheses 82(1):86–88. CrossRefPubMedGoogle Scholar
  11. 11.
    Karska-Wysocki B, Bazo M, Smoragiewicz W (2010) Antibacterial activity of Lactobacillus acidophilus and Lactobacillus casei against methicillin-resistant Staphylococcus aureus (MRSA). Microbiol Res 165(8):674–686. CrossRefPubMedGoogle Scholar
  12. 12.
    Jebur M (2010) Therapeutic efficacy of Lactobacillus acidophilus against bacterial isolates from burn wounds. N Am J Med Sci 2(12):586–591. CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Tejero-Sarinena S, Barlow J, Costabile A, Gibson GR, Rowland I (2012) In vitro evaluation of the antimicrobial activity of a range of probiotics against pathogens: evidence for the effects of organic acids. Anaerobe 18(5):530–538. CrossRefPubMedGoogle Scholar
  14. 14.
    Onbas T, Osmanagaoglu O, Kiran F (2018) Potential properties of Lactobacillus plantarum F-10 as a bio-control strategy for wound infections. Probiotics Antimicrob Proteins.:1–14.
  15. 15.
    Mohammedsaeed W, Cruickshank S, McBain AJ, O’Neill CA (2015) Lactobacillus rhamnosus GG lysate increases re-epithelialization of keratinocyte scratch assays by promoting migration. Sci Rep 5:16147. CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Al-Mathkhury HJF, Al-Aubeidi HJAR (2008) Probiotic effect of lactobacilli on mice incisional wound infections. J Al-Nahrain Univ 11(3):111–116CrossRefGoogle Scholar
  17. 17.
    Zahedi F, Nasrabadi MH, Ebrahimi MT, Aboutalebi H (2011) Comparison of the effects of Lactobacillus brevis and Lactobacillus plantarum on cutaneous wound healing in rats. Afr J Microbiol Res 5(24):4226–4233CrossRefGoogle Scholar
  18. 18.
    Huseini HF, Rahimzadeh G, Fazeli MR, Mehrazma M, Salehi M (2012) Evaluation of wound healing activities of kefir products. Burns 38(5):719–723. CrossRefPubMedGoogle Scholar
  19. 19.
    Trabelsi I, Ktari N, Ben Slima S, Triki M, Bardaa S, Mnif H, Ben Salah R (2017) Evaluation of dermal wound healing activity and in vitro antibacterial and antioxidant activities of a new exopolysaccharide produced by Lactobacillus sp.Ca6. Int J Biol Macromol 103:194–201. CrossRefPubMedGoogle Scholar
  20. 20.
    Oryan A, Alemzadeh E, Eskandari MH (2019) Kefir accelerates burn wound healing through inducing fibroblast cell migration in vitro and modulating the expression of IL-1ss, TGF-ss1, and bFGF genes in vivo. Probiotics Antimicrob Proteins Sep 11:874–886. CrossRefGoogle Scholar
  21. 21.
    Vagesjo E, Ohnstedt E, Mortier A, Lofton H, Huss F, Proost P, Roos S, Phillipson M (2018) Accelerated wound healing in mice by on-site production and delivery of CXCL12 by transformed lactic acid bacteria. Proc Natl Acad Sci U S A 115(8):1895–1900. CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Poutahidis T, Kearney SM, Levkovich T, Qi P, Varian BJ, Lakritz JR, Ibrahim YM, Chatzigiagkos A, Alm EJ, Erdman SE (2013) Microbial symbionts accelerate wound healing via the neuropeptide hormone oxytocin. PloS one 8(10):e78898. CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Gudadappanavar AM, Hombal PR, Timashetti SS, Javali SB (2017) Influence of Lactobacillus acidophilus and Lactobacillus plantarum on wound healing in male Wistar rats - an experimental study. Int J Appl Basic Med Res 7(4):233–238. CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Twetman S, Keller MK, Lee L, Yucel-Lindberg T, Pedersen AML (2018) Effect of probiotic lozenges containing Lactobacillus reuteri on oral wound healing: a pilot study. Benef Microbes:1–6. CrossRefGoogle Scholar
  25. 25.
    Gan BS, Kim J, Reid G, Cadieux P, Howard JC (2002) Lactobacillus fermentum RC-14 inhibits Staphylococcus aureus infection of surgical implants in rats. J Infect Dis 185(9):1369–1372. CrossRefPubMedGoogle Scholar
  26. 26.
    Valdez JC, Peral MC, Rachid M, Santana M, Perdigon G (2005) Interference of Lactobacillus plantarum with Pseudomonas aeruginosa in vitro and in infected burns: the potential use of probiotics in wound treatment. Clin Microbiol Infect 11(6):472–479. CrossRefPubMedGoogle Scholar
  27. 27.
    Argenta A, Satish L, Gallo P, Liu F, Kathju S (2016) Local application of probiotic bacteria prophylaxes against sepsis and death resulting from burn wound infection. PloS one 11(10):e0165294. CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Olofsson TC, Butler E, Lindholm C, Nilson B, Michanek P, Vasquez A (2016) Fighting off wound pathogens in horses with honeybee lactic acid bacteria. Curr Microbiol 73(4):463–473. CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Fu T, Liu YM (2017) Antibacterial effect of bacteriocin isolated from Lactobacillus plantarum ATCC 8014 on postoperative infection of mandibular fracture in vivo. J Craniofac Surg 28(3):679–682. CrossRefPubMedGoogle Scholar
  30. 30.
    Satish L, Gallo PH, Johnson S, Yates CC, Kathju S (2017) Local probiotic therapy with Lactobacillus plantarum mitigates scar formation in rabbits after burn injury and infection. Surg Infect (Larchmt) 18(2):119–127. CrossRefGoogle Scholar
  31. 31.
    Peral MC, Martinez MA, Valdez JC (2009) Bacteriotherapy with Lactobacillus plantarum in burns. Int Wound J 6(1):73–81. CrossRefPubMedGoogle Scholar
  32. 32.
    Peral MC, Rachid MM, Gobbato NM, Huaman Martinez MA, Valdez JC (2010) Interleukin-8 production by polymorphonuclear leukocytes from patients with chronic infected leg ulcers treated with Lactobacillus plantarum. Clin Microbiol Infect 16(3):281–286. CrossRefPubMedGoogle Scholar
  33. 33.
    Cannon JP, Lee TA, Bolanos JT, Danziger LH (2005) Pathogenic relevance of Lactobacillus: a retrospective review of over 200 cases. Eur J Clin Microbiol Infect Dis 24(1):31–40. CrossRefPubMedGoogle Scholar
  34. 34.
    Gouriet F, Million M, Henri M, Fournier PE, Raoult D (2012) Lactobacillus rhamnosus bacteremia: an emerging clinical entity. Eur J Clin Microbiol Infect Dis 31(9):2469–2480. CrossRefPubMedGoogle Scholar
  35. 35.
    Jacobs AC, Thompson MG, Black CC, Kessler JL, Clark LP, McQueary CN, Gancz HY, Corey BW, Moon JK, Si Y, Owen MT, Hallock JD, Kwak YI, Summers A, Li CZ, Rasko DA, Penwell WF, Honnold CL, Wise MC, Waterman PE, Lesho EP, Stewart RL, Actis LA, Palys TJ, Craft DW, Zurawski DV (2014) AB5075, a highly virulent isolate of Acinetobacter baumannii, as a model strain for the evaluation of pathogenesis and antimicrobial treatments. MBio 5(3):e01076–e01014. CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Vilela SF, Barbosa JO, Rossoni RD, Santos JD, Prata MC, Anbinder AL, Jorge AO, Junqueira JC (2015) Lactobacillus acidophilus ATCC 4356 inhibits biofilm formation by C. albicans and attenuates the experimental candidiasis in Galleria mellonella. Virulence 6(1):29–39. CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Wasfi R, Abd El-Rahman OA, Zafer MM, Ashour HM (2018) Probiotic Lactobacillus sp. inhibit growth, biofilm formation and gene expression of caries-inducing Streptococcus mutans. J Cell Mol Med 22(3):1972–1983. CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Koll-Klais P, Mandar R, Leibur E, Marcotte H, Hammarstrom L, Mikelsaar M (2005) Oral lactobacilli in chronic periodontitis and periodontal health: species composition and antimicrobial activity. Oral Microbiol Immunol 20(6):354–361. CrossRefPubMedGoogle Scholar
  39. 39.
    Stsepetova J, Sepp E, Kolk H, Loivukene K, Songisepp E, Mikelsaar M (2011) Diversity and metabolic impact of intestinal Lactobacillus species in healthy adults and the elderly. Br J Nutr 105(8):1235–1244. CrossRefPubMedGoogle Scholar
  40. 40.
    Stanbro J, Bond M, Moore J, Stockelman M, Watters C (2016) The probiotic biofilm: Lactobacillus and Bifidobacterium survival tactics. In: Henderson J (ed) Biofilms: Characterization, Applications and Recent Advances. Nova Science Publishers, Inc, Hauppauge, NY, pp 121–152Google Scholar
  41. 41.
    McHugh ML (2011) Multiple comparison analysis testing in ANOVA. Biochem Med (Zagreb) 21(3):203–209CrossRefGoogle Scholar
  42. 42.
    Jerome NP, Orton MR, d’Arcy JA, Feiweier T, Tunariu N, Koh DM, Leach MO, Collins DJ (2015) Use of the temporal median and trimmed mean mitigates effects of respiratory motion in multiple-acquisition abdominal diffusion imaging. Phys Med Biol 60(2):N9–N20. CrossRefGoogle Scholar
  43. 43.
    Thompson MG, Black CC, Pavlicek RL, Honnold CL, Wise MC, Alamneh YA, Moon JK, Kessler JL, Si Y, Williams R, Yildirim S, Kirkup BC Jr, Green RK, Hall ER, Palys TJ, Zurawski DV (2014) Validation of a novel murine wound model of Acinetobacter baumannii infection. Antimicrob Agents Chemother 58(3):1332–1342. CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Regeimbal JM, Jacobs AC, Corey BW, Henry MS, Thompson MG, Pavlicek RL, Quinones J, Hannah RM, Ghebremedhin M, Crane NJ, Zurawski DV, Teneza-Mora NC, Biswas B, Hall ER (2016) Personalized therapeutic cocktail of wild environmental phages rescues mice from Acinetobacter baumannii wound infections. Antimicrob Agents Chemother 60(10):5806–5816. CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Servin AL (2004) Antagonistic activities of lactobacilli and bifidobacteria against microbial pathogens. FEMS Microbiol Rev 28(4):405–440. CrossRefPubMedGoogle Scholar
  46. 46.
    Ramos AN, Sesto Cabral ME, Arena ME, Arrighi CF, Arroyo Aguilar AA, Valdez JC (2015) Compounds from Lactobacillus plantarum culture supernatants with potential pro-healing and anti-pathogenic properties in skin chronic wounds. Pharm Biol 53(3):350–358. CrossRefPubMedGoogle Scholar
  47. 47.
    Onwuakor CE, Nwaugo VO, Nnadi CJ, Emetole JM (2014) Effect of varied culture conditions on crude supernatant (bacteriocin) production from four Lactobacillus species isolated from locally fermented maize (ogi). Amer J Microbiol Res 2(5):125–130. CrossRefGoogle Scholar
  48. 48.
    Sesto Cabral ME, Ramos AN, Macedo AJ, Trentin DS, Treter J, Manzo RH, Valdez JC (2014) Formulation and quality control of semi-solid containing harmless bacteria by-products: chronic wounds pro-healing activity. Pharm Dev Technol:1–8. CrossRefGoogle Scholar
  49. 49.
    Cabrera CA, Ramos AN, Loandos Mdel H, Valdez JC, Sesto Cabral ME (2016) Novel topical formulation for ischemic chronic wounds. Technological design, quality control and safety evaluation. Pharm Dev Technol 21(4):399–404. CrossRefPubMedGoogle Scholar
  50. 50.
    Prince T, McBain AJ, O’Neill CA (2012) Lactobacillus reuteri protects epidermal keratinocytes from Staphylococcus aureus-induced cell death by competitive exclusion. Appl Environ Microbiol 78(15):5119–5126. CrossRefPubMedPubMedCentralGoogle Scholar
  51. 51.
    Sultana R, McBain AJ, O’Neill CA (2013) Strain-dependent augmentation of tight-junction barrier function in human primary epidermal keratinocytes by Lactobacillus and Bifidobacterium lysates. Appl Environ Microbiol 79(16):4887–4894. CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Castiblanco G A, Yucel-Lindberg T, Roos S, Twetman S (2017) Effect of Lactobacillus reuteri on cell viability and PGE2 production in human gingival fibroblasts. Probiotics Antimicrob Proteins 9(3):278–283. CrossRefGoogle Scholar
  53. 53.
    Zimmerman T, Gyawali R, Ibrahim S (2017) Autolyse the cell in order to save it? Inducing, then blocking, autolysis as a strategy for delaying cell death in the probiotic Lactobacillus reuteri. Biotechnol Lett 39(10):1547–1551. CrossRefPubMedGoogle Scholar
  54. 54.
    Rousselle P, Braye F, Dayan G (2018) Re-epithelialization of adult skin wounds: cellular mechanisms and therapeutic strategies. Adv Drug Deliv Rev. CrossRefGoogle Scholar
  55. 55.
    Halper J, Leshin LS, Lewis SJ, Li WI (2003) Wound healing and angiogenic properties of supernatants from Lactobacillus cultures. Exp Biol Med (Maywood) 228(11):1329–1337. CrossRefGoogle Scholar
  56. 56.
    Schneider LA, Korber A, Grabbe S, Dissemond J (2007) Influence of pH on wound-healing: a new perspective for wound-therapy? Arch Dermatol Res 298(9):413–420. CrossRefPubMedGoogle Scholar
  57. 57.
    Percival SL, McCarty S, Hunt JA, Woods EJ (2014) The effects of pH on wound healing, biofilms, and antimicrobial efficacy. Wound Repair Regen 22(2):174–186. CrossRefPubMedGoogle Scholar
  58. 58.
    Nagoba B, Suryawanshi N, Wadher B, Selkar S (2015) Acidic environment and wound healing: a review. Wounds 27(1)Google Scholar
  59. 59.
    Chan AP, Choi Y, Brinkac LM, Krishnakumar R, DePew J, Kim M, Hinkle MK, Lesho EP, Fouts DE (2018) Multidrug resistant pathogens respond differently to the presence of co-pathogen, commensal, probiotic and host cells. Sci Rep 8(1):8656. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply 2019

Authors and Affiliations

  1. 1.Wound Infections DepartmentNaval Medical Research CenterSilver SpringUSA

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