Chronic wounds represent a “silent epidemic” that affects a large fraction of the world population [5]. In developed countries, 1–2% of the population is estimated to experience a chronic wound during its lifetime [10]. Despite the fact that a variety of therapeutic modalities and multidisciplinary approaches have been developed, antimicrobial treatments are often ineffective and impaired wound healing remains a major challenge for both health professionals and patients.

Wound healing is a natural biological multi-layered process that can be affected by many factors, among which the interaction of the wound with the skin microflora is one of the most important [11]. At present, 19 bacterial phyla and over 1000 bacterial species have been identified within the most superficial level of skin, including in wounds [12, 13]. Systemic or topic antimicrobial treatments may reduce beneficial bacterial populations and contribute to the emergence of an antibiotic resistant flora, with detrimental effects on wound healing [14, 15].

In about 60% of chronic wounds [2], bacterial biofilm is associated with impaired wound healing. Biofilm is constituted by bacteria embedded in an exo-polymeric substance consisting of polysaccharides, lipids, and proteins [16]. This extracellular matrix acts as a physical barrier that protects the embedded bacteria from antibiotics as well as host defense mechanisms. At the same time, since space and resources are not unlimited, different bacterial species are forced to develop the capability to counteract proliferation of other microorganisms by secreting toxic metabolites or by interfering with competitors’ quorum-sensing [17,18,19,20]. This concept, known as ‘bacterial interference’, has been transposed to practice by administering alpha streptococci with the intent to reduce the recurrences of acute and secretory otitis media in children [21].

Probiotics are defined by the World Health Organization as “live microorganisms which, when administered in adequate amounts, confer a health benefit to the host” [22]. Bacteria with probiotic properties have been already reported to be beneficial in the management of diabetes, respiratory tract infections, gastro-intestinal disorders, and uro-genital infections [23,24,25,26,27,28,29]. Oral ingestion of probiotics has also been reported to be advantageous in skin inflammatory diseases and chronic wounds [30].

To the knowledge of the authors, this is the first time that a probiotic formulation was applied topically on a chronic ischemic wound infected by three MDR bacteria.

Obviously, the probiotic treatment might exert its positive effects on wound healing not only by counteracting pathogenic bacteria proliferation as evidenced by the results of the in vitro MBD against K. pneumoniae, E. faecalis and P. mirabilis, but also through modulation of host immune response (e.g. by inducing cytokine production: tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-4, IL-6, transforming growth factor (TGF)-β and matrix metalloproteinases (MMPs) [31]) and by stimulating keratinocyte proliferation and migration [32, 33].

Chronic wounds produce exudates containing high levels of proteases, i.e. metalloproteases and elastase, as well as reduced concentrations of tissue protease inhibitors [34]. The unbalanced activity of proteases and their inhibitors leads to chronic degradation of the extracellular matrix with consequent release of “waste amino acid products”, which can be identified by metabolomic analysis. In our patient, the 1H-NMR delineated that metabolic pattern of wound exudate drastically changed after the topical probiotic treatment. Seventeen molecules, out of the 63 overall identified, showed a change in their concentration of at least 30% after the treatment (Figs. 2 and 3). In particular, proprionate, 2-hydroxyisovalerate, 2-oxoisocaproate, 2,3-butanediol, putrescine, thymine, and trimethylamine significantly differed between the infected phase and sterile phase of the wound. Pathway enrichment analysis based on the molecules identified and quantified, suggested that three pathways were differently expressed before and after the treatment (Fig. 3). By means of 1H-NMR, we could quantify 5 molecules pertaining to the pyrimidine metabolism and the concentration of three of them, namely cytidine, thymine and malonate, was found to decrease by more than 30% in connection to the treatment. A similar decrease characterized also lysine and N6-acetyllysine, two of the three molecules involved in lysine degradation that we were able to quantify. Finally, a decrease of more than 30% characterized also pyroglutamate and putrescine, two of the four molecules involved in glutathione metabolism that we could quantify. The differential expression of each of the three pathways seems to be connected to the modification in bacterial activity in connection to the resolution of infection.

Since lysine is an essential amino acid, the observed decreases of its free form in the wound exudate, which is not regulated endogenously, can be safely considered as a direct consequence of the reduced proteolysis activity locally exerted by bacteria, caused by the resolution of Proteus infection.

Circulating pyrimidine nucleobases are tightly regulated by effective cells’ uptake mechanisms [35], in reason of this observation the lower concentration of cytidine and thymine upon infection resolution may reflect the restore of normal functionality of the cells surrounding the wound.

In addition to the three pathways identified, other metabolites quantified by 1H-NMR may be functionally related to pathogens proliferating in the infected wound of the reported case.

Liu et al. demonstrated that l-serine, l-valine and l-leucine promote macrophage phagocytosis in K. pneumoniae lung infection, thus declining Klebsiella concentration and increasing host survival [36]. The high concentration of, bona fide, microbial related metabolites from branched chain amino acids (e.g. propionate [37]) in the infected wound as well as their conspicuous depletion in the probiotic-treated and pathogen-eradicated healed-wound may indeed reflect infection resolution and reduced systemic stress. Moreover, a high reduction of 2-hydroxyisovalerate and 2-hydroxyisocaproate concentrations after the ulcer treatment with probiotics was also detected. These molecules are hydroxyderivate of the alpha-ketoacid catabolites respectively produced by transamination of valine and leucine [38, 39]. Valine and leucine catabolism may proceed toward propionate synthesis or may shift to 2-hydroxyisovalerate and 2-hydroxyisocaproate production. Marked increase in concentrations of branched-chain amino and 2-oxo acids may have toxic effects for the host, as systemically observed in maple syrup urine disease (MSUD) [40].

In the reported case, the massive decrease of 2,3-butenediol in the second wound exudate collected may reflect the clearance of Klebsiella infection, as confirmed by microbiological analyses. K. pneumoniae, which is one of the most studied microorganisms for the large-scale production of this compound, has in fact an incredible ability to metabolize various substrates to produce high amounts of 2,3-butenediol [41]. A further possible involvement of Klebsiella in conditioning the metabolomic pattern we depicted in the infected wound is suggested by the complementary concentration trend of glycerol, formate and succinate. K. pneumoniae is commonly used and observed in glycerol fermentation under mesophilic conditions [42]. According to its humectant properties, skin endogenous glycerol, which is produced in pilosebaceous units by triglycerides catabolism, plays a key role in skin hydration maintenance [43]. Klebsiella may indeed use skin glycerol as energy source during skin wound infections. According to our data, eradicated Klebsiella infection is associated: (i) to glycerol detection, which was not obtained in infected exudate, and (ii) to a significant decrease of fumarate and succinate, both obtained by glycerol fermentation.

Polyamine putrescine is another product normally derived by amino acid that appeared drastically reduced after probiotic treatment. Putrescine plays a role in several steps of bacterial growth and virulence. It is actually involved in biofilm formation, protection from oxidative and acid stress and escape from phagolysosomes [44,45,46]. Dead tissues are an ideal medium for anaerobic bacteria such as Bacteroides fragilis, Bacteroides prevotella, Clostridium perfrigens and Fusobacterium nucleatum, which produce putrescine and cadaverine. Other bacteria, such as Klebsiella and Proteus, colonize these tissues and contribute to wound smell [47]. Putrescine, which is produced from arginine by the combined activity of SpeA and SpeB proteins, has been reported to be a cell wall component in P. mirabilis, in which it is also known to play a crucial role in control of gene expression, swarming, and tissue colonization [48]. Most likely, the high presence of putrescine in the first exudate sample of our patient was related to the presence of a heterogeneous bacterial community, including both anaerobic and aerobic microorganisms even though the drastic reduction of putrescine and propionate in the second exudate is apparently imputed to the ongoing resolution of Proteus infection.

The main limitation for this study is that it is a case report and therefore the results presented should be considered with extreme caution. Furthermore, a number of studies reported a variability in both microbial counts and isolates obtained from different parts of chronic wounds. In our case, the culture data (obtained from the center and the edge of the wound sampling) may have been influenced by the combination of these locations within one single swab. Finally, the data obtained with the probiotic mixture used for this patient are not automatically extendable to other probiotics and further studies are needed to better define the potential of the products used.