Infections of wounds by drug resistant microbes is global medical problem including India [[1], [2], [3]]. Bacterial infection interferes with almost all phases of wound healing and increases the probability of development chronic wounds. More seriously, there is possibility of systemic infection, sepsis or even multiorgan failure [[1], [2], [3]]. Traditionally, infection diagnosis and therapeutic assessment are based on visual examination followed by microbiological culture [[4], [5], [6]]. However, classical signs of infection often lead to diagnostic uncertainty [7]. Microbial culture also requires significant waiting time which increases the risk of infection spreading to deeper organs and systemic circulation. Hence, there is considerable need to develop platforms for rapid detection of wound bioburden.

A prerequisite for wound infection management is simultaneous elimination of pathogenic microbe, along with infection detection. While wound infections is treated using antibiotics, emergence of antibiotic resistant pathogens such as methicilin resistant Staphylococcus aureus(MRSA) is a big impediment to wound infection management. MRSA infection of wounds is associated with a higher rate of treatment failure and morbidity etc.[2].Although large efforts have been made to evolve novel antibacterial treatment modalities, such as antibacterial peptides, efflux pump inhibitors, phage therapy etc., possibility of resistance generation and safety concerns may limit the potential application [[8], [9], [10]].

Antimicrobial photodynamic therapy (aPDT), employing interaction of an exogenously applied photosensitizer (PS) with visible light to generate reactive oxygen species (ROS), has considerable promise for inactivation drug resistant micorbes like MRSA and microbial biofilms, virulent factors[11,12].aPDT has been shown to improve of angiogenesis, collagen remodeling and attenuation of hyperinflammation in bacteria infected wounds of mice[[12], [13], [14], [15], [16]]. Moreover, aPDT has shown positive outcomes in treatment of chronic wounds of humans [[17], [18], [19], [20], [21], [22]]. Despite of the immense promise, wide scale use of aPDT for wound infection management is hampered by various biological challenges. One crucial reason is lack of accurate, objective methodologies to detect bioburden and provide rapid feedback on effectiveness of aPDT in absence of which there is possibility of large variabilities in aPDT protocol and outcome.

Excitation of endogenous porphyrins using blue light is an exciting prospect for noninvasive detection, photoinactivation (anti-microbial blue light therapy) and treatment assessment [[23], [24], [25], [26], [27]]. However, limited penetration depth of blue light, large variation in endogenous porphyrin level in microbes, presence of non-porphyrin producing bacteria are the potential challenges [27]. Alternatively, bioburden detection and photoinactivation can also be elicited by use of fluorescent dyes and PS [28,29]. A recent novel approach combines colorimetric dye, nanostructures and PS to enable simultaneous detection of MRSA mediated wound infection and “on-demand aPDT” [29]. However, leakage of fluorescent dyes leading to a subsequent decline in imaging effectiveness, potential “off-target” toxicity and the necessity to conjugate the fluorescent dye and PS onto the same polymer are the potential drawbacks. Clearly, there are unmet needs to integrate bioburden detection, selective photoinactivation and treatment feedback on a single platform.

Prodrug based theranostics for wound infection is one step forward and are designed to minimize the potential “off-target” toxicity. Delta-Aminolevulinic acid (5-ALA), a prodrug, is a precursor of heme and protoporphyrin (PpIX). Exogenously administrated 5-ALA leads to rapid synthesis and accumulation of PpIX in cells with high metabolic activity; bacteria. As light exposure of PpIX leads to emission of red fluorescence, and ROS formation, ALA can be an attractive agent for bioburden detection and aPDT [30]. Previously, ALA has been used for simultaneous visualization and treatment of acne [31,32] and dental carries [33]. However, utility of ALA -PpIX for simultaneous visualization and treatment of wound infections remain largely unexplored. In a study by Lopez et al, biofilms pretreated in vitro with ALA were implanted to wounds followed by fluorescence imaging using a hand held, point-of-care imaging device [34]. However, the protocol does not mimic in vivo scenario and 4 h incubation time with ALA is not suitable for aPDT. Very recently, a combination of ALA-carvacrol-blue light trio therapy for simultaneous detection of infection and aPDT was employed in a murine wound, wherein aPDT was carried out at 6 h post P.aeruginosa and K.pneumoniae application, using 405 nm light [35]. The study has certain limitations which needed to be overcome. First, 6 h inoculation of wounds with bacteria does not encompass an established infection. Further, for aPDT, use of 405 nm restricts the applicability to only superficial layers. Therefore, integrating ALA based simultaneous bioburden detection and aPDT into wound care would require much optimization in terms of selectivity, ALA incubation time, use of appropriate wavelength of light for aPDT. To the best of our knowledge there is no previous study addressing these aspects.

In this paper, we report potentiality of ALA induced PpIX for bioburden detection, photodynamic inactivation, assess aPDT efficacy in MRSA infected wounds of Swiss albino mice. ALA application time, bacteria load dependent PpIX fluorescence of wounds was monitored with help of an in-house fabricated LED based, USB powered, hand-held device. The intra-wound heterogeneity in PpIX fluorescence was also monitored to ascertain localized bioburden. Loss of PpIX fluorescence during the progress of aPDT was visualized. Besides, red fluorescence alteration following AgNO3 treatment and physical removal of biofilms were also evaluated to further ascertain the selectivity. A graphic user interface was used for fluorescence analysis and quantification.