Taking into account the threat caused by A. baumannii, it seems obvious that it is necessary to use non-antibiotic methods to combat these bacteria. It is important to describe not only effective protocols for destroying this pathogen but also to understand the mechanisms of metabolic responses of bacteria that may determine the long-term success of therapy.
In the first part of this work, we demonstrated the effectiveness of antimicrobial photodynamic therapy in the fight against A. baumannii. It was found that multiple sub-lethal and lethal doses of light in the presence of MB as a photosensitizer slightly increased the sensitivity of these bacteria to phototherapy. We have shown that after the first sub-lethal dose of light, the number of bacteria was reduced by 98.77%, while after the fifteenth phototherapy the number of viable bacteria was reduced by 99.29%. A similar effect was observed with a lethal dose of light. After the first phototherapy, the number of viable bacteria was reduced by 99.92%, while the fifteenth phototherapy resulted in a reduction of the number of living cells by 99.94%.
Surprisingly, it was revealed that multiple sub-lethal and lethal phototherapies did not affect the sensitivity of this pathogen to classical antibiotics (gentamicin, streptomycin, nalidixic acid, and chloramphenicol). These results are inconsistent with the observations of Rapacka-Zdonczyk et al.17. The authors have revealed that S. aureus exposed to multiple sub-lethal photosensitizations was characterized by a higher sensitivity to gentamicin and doxycycline. On the other hand, the multiple exposures of A. baumannii to sub-lethal and lethal doses of light in the presence of MB increased the sensitivity of these bacteria to H2O2 by a decrease in the MIC value for this aseptic agent from 5.0 gL−1 to 1.25 and 0.625 gL−1 (after the fifteen exposures to a sub-lethal and lethal dose of light, respectively). Interesting observations were reported by Green et al.27, who studied GABA deficient mutant encoding a GABA transaminase and showed that this mutant was more sensitive to H2O2. In addition, the aΔpaaJKXYI mutant that lacked part of the phenylacetate degradation pathway was more susceptible to killing by this disinfectant. The authors hypothesized that the MumR-regulated pathways could play the key role in defense against ROS. It was observed that the transcription of a number of catabolic pathways, including phenylacetate and GABA metabolism, was downregulated in the ΔmumR strain growing in a rich medium. Mutants lacking gabT GABA transaminase or paaJKXYI phenylacetate degradation genes showed increased sensitivity to ROS, suggesting that the functions of these pathways may play an important role in the response of A. baumannii to oxidative stress. The paaJKXYI gene cluster encodes part of the operon that converts the aromatic compound phenylacetate, a breakdown product of phenylalanine, into succinyl coenzyme A (succinyl-CoA) and acetyl-CoA and it is possible that the products of GABA and phenylacetate catabolism may be required to repair or regenerate from oxidative damage. It was speculated that the use of these pathways allow A. baumannii to bypass other metabolic pathways impaired by oxidative stress, such as those that require enzymes containing iron-sulfur clusters that are critical for many metabolic functions.
There was also found a slight reduction in the biofilm formation capacity of the studied pathogen. The similar results were obtained by Carmello et al.28. These authors observed that photodynamic therapy reduced adhesion capacity and biofilm formation of Candida albicans from induced oral candidiasis in mice. It is worth noting that although A. baumannii infections have attracted the attention of researchers for several years, the virulence factors of this pathogen, including biofilm formation, are not well understood29. It was proved that biofilm-forming strains are known to have a modified phenotype compared to the corresponding planktonic bacteria and show differences in gene transcription30. The ability of A. baumannii to form biofilm is considered as an effective strategy for increasing the survival of bacteria and staying in a hospital environment under stressful conditions. The following molecules have been identified as factors involved in the formation of biofilm by A. baumannii: protein (Bap) encoded by the bap gene, outer membrane protein A (OmpA) encoded by the ompA gene, chaperone pilus (Csu), extracellular exopolysaccharide (EPS), two-component system (BfmS/BfmR), poly-β-(1,6)-N-acetylglucosamine (PNAG) and the quorum sensing system. At this stage of the research, we are not able to identify the metabolic changes of A. baumanni, which are responsible for the reduced ability of these bacteria to form a biofilm. This phenomenon requires further studies to be carried out in our laboratory.
In the second part of this work, the metabolomic approach was used to investigate the response of A. baumannii to multiple exposures to sub-lethal and lethal doses of laser light in the presence of MB as a photosensitizer. VIP metabolites indicated differences between control samples (designed C10 and C15) and samples after sub-lethal light doses (designed S10 and S15), and after lethal light doses (designed L10 and L15). These metabolites have been grouped into several super pathways, with the majority of the metabolites classified as metabolic reactions of amino acids to oxidative stress. These observations are generally consistent with the findings of other authors25,26, however, in our studies we have not confirmed the significant role of fatty acids in the cell’s response to oxidative stress. Figure 1 presents changes in the accumulation of biomolecules involved in the central metabolism after multiple photosensitization of A. baumannii.
Changes in the concentration of VIP metabolites (Tables 3, 4, Fig. 1) are the result of complex reactions that have occured in the cell due to oxidative destruction of molecules, as well as the regulation of cellular metabolism related to the antioxidant defense. The most significant perturbations after multiple exposures of A. baumannii to aPDT concerned the level of amino acids in bacterial cells. The careful inspection of Tables 3 and 4 revealed two trends in amino acid concentration, depending on the irradiation dose and their multiplication. The first comparison for the sublethal dose shows the difference between the tenth and fifteenth multiplication, where a higher level of major amino acids was observed after the S10 irradiation (Table 3). The exceptions are tyrosine and valine, the levels of which were increased with the S15. The second trend is characteristic for the lethal dose, in which higher repetition of irradiations tended to increase the amino acid concentration (however mostly insignificant) (Table 4).
Central metabolism. Green and red indicate significant decreases and increases in metabolite levels, respectively (p value < 0.05). The metabolites indicated in the dashed boxes are putative.
The increase in amino acid concentration may in part be due to increased protein turnover. One of the reasons may be protein degradation as the result of the elimination of abnormal proteins resulting from oxidative stress or may be interpreted as a way to increase the availability of amino acids needed for the synthesis of proteins important for survival under stress conditions31.
It is known that sulfur-containing amino acids (cysteine and methionine) are particularly susceptible to ROS reactivity. Other amino acids such as arginine, lysine, proline, and threonine can be carbonylated and histidine can be modified to oxo-histidine32. The observed decrease in the concentration of arginine and lysine (Tables 3, 4), regardless of the irradiation dose, may be the result of carbonylation of these amino acids. On the other hand, this change in the concentration of arginine may be related to the easy conversion to ornithine, and then to putrescine. N-acetylputrescine is a part of butanoate metabolism and is used for the biosynthesis of gamma-aminobutanoate (GABA) (https://www.genome.jp/kegg-bin/show_pathway?acb00220). While most of the research on the metabolic role of GABA has focused on its role in carbon and nitrogen metabolism, there is evidence to suggest that it plays a much broader role in bacteria and has a significant impact on survival under environmental stress33. The arginine metabolism is also coupled with glutamate, which in contrary to the arginine level is overproduced (https://www.genome.jp/kegg-bin/show_pathway?acb00220).
It is worth to mentioning that the increased concentration of some amino acids and their derivatives may fulfill the antioxidant functions inside the cells. The levels of tryptophan, tyrosine and long-chain acylated tyrosine (Table 3) increased and it cannot be excluded that this phenomenon was related to the inhibition of lipid peroxidation by these amino acids34,35. Sulfur-containing amino acid—methionine may also be accumulated in response to oxidative stress after ten expositions of A.baumannii cells to the sub-lethal doses of laser light (Table 3). Methionine has antioxidant properties and also acts in important signaling systems36. This amino acid contributes to maintain the redox status in cells by supplying the junction metabolite homocysteine as a substrate to the transsulfuration pathway36. Next homocysteine can be converted to cysteine, which is incorporated into the antioxidant (glutathione). Interestingly, the decreased level of this amino acid indicated the distinction between the two states S10 and S15 and could be the result of the oxidizing properties of ROS produced during light irradiation of bacteria, or decreased cell metabolism which is weakens after light action. The same role is assigned to cysteine, but this amino acid was not detected as a VIP metabolite in our experiments.
The increased level of glutamate was found in A. baumannii cells induced by multiple oxidative stress (Tables 3, 4). It was previously suggested that the activity of the glutamate catabolic pathway (GAD system and GABA shunt) promotes bacterial resistance to numerous stresses37. This system facilitates intracellular pH homeostasis by consuming protons in the decarboxylation reaction that produces γ-aminobutyrate (GABA) from glutamate. It seems that role of the GAD system in oxidative stress is related to its influence on intracellular pH. It is well known that the oxidation reactions are influenced by pH, and therefore the increase in pH achieved by the GAD system can change the reaction path and the oxidation processes38. It has also been suggested that the NADH or NADPH produced by succinate during GABA catabolism affects redox changes in the cell38.
It is worth emphasizing that the increased concentration of glycine and glutamate may result from the fact that these amino acids (along with cysteine) are components of the tripeptide-glutathione that has a radical scavenging function (https://www.genome.jp/dbget-bin/www_bget?pathway:acb00480).
The increased concentration of leucine in the cells of the studied coccobacillus after multiple sub-lethal and lethal photooxidations was also found. It is known that leucine can be used as an alternative source of carbon and nitrogen38. L-leucine can be assimilated by bacteria when the sugar metabolism pathways may be “inconvenient” for maintaining cell homeostasis. The main product of these subsequent reactions is 3-methylcrotonyl-CoA metabolite, which undergoes successive enzymatic steps towards the production of acetoacetate and acetyl-CoA38. A decreased level of this amino acid indicated a distinction between the two states S10 and S15.
It is assumed that reduced concentration of imidazole after multiple photosensitization of cells may be related to its conversion to glutamate, which plays an important role in protecting the cell against the harmful effects of oxidative stress37. An important antioxidant defense mechanism is the production of various bacterial sensory proteins the recognize the cellular redox state and convert chemical information into structural signals to regulate downstream signaling pathways. For example, redox sensors recognize the redox state using amino acids such as cysteine, methionine, and histidine or related cofactors ([Fe-S], heme, flavin, and pyridine nucleotides of compounds involved in the redox process). It can therefore be assumed that high levels of methionine, histidine, and uracil may be associated with these metabolic functions39.
The results presented above confirm that changes in concentrations of amino acids are closely involved in antioxidant defense, but the key markers characteristic of A. baumannii reaction to multiple photosensitization have not been found. Wirgot et al.24 showed that the S/R ratios of valine and tryptophan were particularly high (4.5 and 6.98, respectively) compared to other amino acids with a VIP score (around 2.0) and it was considered to be key markers of Pseudomonas graminis response to H2O2 stress. It should be emphasized that particularly high amino acid ratios in A. baumannii cells under no circumstances were observed.
A significant decrease in glycolytic compound pyruvate after multiple photooxidation by sub-lethal and lethal doses of laser light was found. Pyruvate is considered a key intermediate in the oxidative or anaerobic glucose metabolism, but it is also a powerful and effective ROS scavenger (https://www.genome.jp/dbget-bin/www_bget?pathway:acb00620)40.
Previously it was revealed that the tricarboxylic acid cycle may be tweaked to limit the formation of NADH. This metabolic redox-balancing act appears to afford a potent tool against oxidative challenge and may be a more widespread ROS-combating tactic than hitherto recognized25. The obtained results demonstrated the significant reduction of fumarate in A. baumannii cells in response to oxidative stress. This metabolic act of redox balancing appears to be a powerful tool against oxidative stress41.
Succinate is another important intermediate product of the tricarboxylic acid cycle under normoxic conditions and glutamine-dependent anaplerosis or γ-aminobutyric acid leakage under anaerobic conditions. The increase in succinate concentration after multiple expositions of bacterial cells to sub-lethal and lethal doses of laser light in the presence of MB was observed. This compound is usually considered an intermediate product but its accumulation was also observed during metabolic stress42.
Changes in concentrations of two important metabolites-lactate and acetate were also observed. Lactate can be used by cells as an oxidizing substrate, or it can be oxidized to produce ATP. The increase in lactate concentration in A. baumannii cells may be surprising as it has recently been shown to induce ROS43. On the other hand, Tauffenberge et al.44 reported that the increase in lactate concentration promotes stress resistance by activation of pro-oxidative mechanisms. According to the authors, this finding is consistent with the concept of mitohormesis described in many organisms, in which a moderate increase in ROS production promotes stress resistance by the activation of unfolded protein responses and detoxification mechanisms. It should be noted that lactate promotes resistance to oxidative stress and pyruvate restores the lactate-mediated protective effect. An increased level of acetate indicated a distinction between the two states C10 and S10. Acetate is ubiquitous in the environment and is one of the major short-chain fatty acids excreted by cells in a process called acetate overflow45. Acetate can be produced directly from pyruvate by enzymatic decarboxylation or from acetyl-CoA via an intermediate acetylphosphate (reactions catalyzed by the enzymes phosphotransacetylase and acetate kinase). A high concentration of intracellular acetate can reduce the level of the major intracellular anion, i.e. glutamate. It has been noticed that the presence of acetate modulates the expression of genes involved in various biological functions: mobility, biofilm formation, translation, stress response, metabolism, carbohydrate transport, amino acid ions.
The concentration of NAD significantly decreased after ten exposures of A. baumannii to sub-lethal photodynamic therapy, which may have been due to fine-tuning the redox potential of cells (NAD+/NADH ratios). As multiple photosensitization of cells affects glutathione metabolism, glycolysis, the TCA cycle, and the DNA repair system, it seems understandable that it may also indirectly affect the NAD+/NADH coenzymes. This phenomenon may be related to metabolic modulations favoring the production of NADPH, which is involved in many anti-oxidant enzymatic reactions24 and it is worth noting that it did not occur after fifteen sub-lethal light exposures or after exposure to lethal phototherapy.
As can be seen in Tables 3 and 4, the damage caused by ROS may also increase uracil concentration. Liu et al.46 found that oxidative stress had a stronger effect on RNA. Additionally, they showed that highly structured RNAs like tRNA and rRNA are not protected against oxidative damage. This observation is consistent with the findings of other groups that oxidative stress generally inhibits the translation process46.
In summary, we have demonstrated that antimicrobial photodynamic therapy can lead to the effective killing of A. baumannii planktonic culture. It was confirmed that multiple sub-lethal and lethal doses of light in the presence of MB as a photosensitizer slightly increased the sensitivity of these bacteria to phototherapy and decreased their biofilm formation capacity. Moreover, it was revealed that multiple sub-lethal and lethal phototherapies did not affect the sensitivity of this pathogen to classical antibiotics but increased its sensitivity to H2O2. It was clearly shown that multiple photooxidations directly impact the metabolome of A. baumannii and many metabolic pathways are modified. It needs to be highlighted that changes in the level of some metabolites suggest that prolonged exposures of A. baumannii to sub-lethal and lethal doses of light may result in the breakdown of certain defense systems. For example, the concentration of sarcosine (N-methylglycine) significantly decreased in the bacterial cells and it was supposed that a high level of this metabolite was connected with balancing the redox reactions, thus protecting against oxidative damage in cells47.
Understanding the metabolic changes induced by multiple photosensitization of A. baumannii and the cell defense systems remains an open question, requiring further analysis of complex metabolic pathways, also at the level of gene expression.
