Effects of photobiomodulation on the growth of intestinal bacteria

Necrotizing enterocolitis is an inflammatory bowel disease that occurs in newborns, more commonly in preterm infants. It is the leading cause of death from gastrointestinal diseases in neonates, and is characterized by the development of diffuse intestinal necrosis in premature infants subjected to stress. The high incidence and lack of effective treatment strategies suggest that new approaches to treating the disease are needed. It is in this context that the possibility of using photobiomodulation as a therapeutic modality arises. However, studies on the use of photobiomodulation in intestinal bacteria are scarce. To study the effect of photobiomodulation used in clinical parameters on the growth of bacteria commonly present in the newborn microbiota. Four strains of bacteria were chosen to be studied, two belonging to healthy intestinal microbiota, Lactobacillus acidophilus and Lactobacillus reuteri, and two pathogenic bacteria, Escherichia coli and Staphylococcus aureus. These bacteria were cultivated in planktonic growth and irradiated with LED at a wavelength of 660 nm and a power density of 0.025 W/cm, at fluences of 1, 5 and 10 J/cm. The evaluation of cell growth was performed through absorbance readings in the periods of 4h, 24h and 48h after irradiation. The growth of L. acidophilus, L. reuteri and S. aureus did not undergo biomodulation in any of the fluences. The growth of E. coli was stimulated at 1 J/cm when compared to the Control group, with statistical significance (p<0.005). In the other fluencies there was no biostimulation for the E. coli bacteria. The use of a wavelength of 660 nm in the fluences of 5 J/cm and 10 J/cm in the studied bacteria did not lead to a significant change in the growth rate.


Introduction
Necrotizing enterocolitis (NEC) is an inflammatory bowel disease that occurs in the newborn, more commonly in the premature infant, with a high mortality and morbidity rate. It has been one of the most difficult diseases to eradicate and has become a priority for research. The mortality rate varies from 20 to 30%. The intense inflammatory process that begins in the highly immunoreactive intestine extends the disease systemically, affecting other organs, such as the brain. Despite many studies, prevention strategies do not present a satisfactory result, showing the lack of clear definitions of what constitutes the diagnosis of classic NEC (Obladen, 2009).
The pathophysiology of the disease is not fully understood. Epidemiological observations suggest that it is a multifactorial disease. The condition is probably related to the newborn's gastrointestinal tract's immaturity, which facilitates the multiplication of pathogenic bacteria and their translocation. It happens through mixed colonization of the child's intestine, and pathogens frequently found are Staphylococcus aureus and Escherichia coli. Treatment consists of antibiotic therapy and fasting. The condition is associated with an intense inflammatory process and mesenteric vasoconstriction, leading to a state of intestinal ischemia and necrosis with extremely short and long-term sequelae (Sueh et al., 2002;Samuels et al., 2017).
Photobiomodulation by light in the red to the spectrum's infrared region (630-1000 nm) modulates numerous cellular functions. The clinical and experimental applications of photobiomodulation have expanded over the past 30 years. Low-power lasers and light-emitting diodes (LEDs) are well-accepted therapeutic tools for treating infected, ischemic, and hypoxic wounds and other soft tissue injuries. The positive effects of photobiomodulation include accelerated healing, better recovery from ischemic lesions in the heart, and attenuated degeneration of the injured optic nerve (Desmet et al., 2006). At the cellular level, photobiomodulation can modulate fibroblasts' proliferation, the fixation and synthesis of collagen and procollagen, promote angiogenesis and stimulate macrophages and lymphocytes, improving energy metabolism in mitochondria. Besides, photobiomodulation has demonstrated the ability to increase the production of growth factors, such as keratinocyte growth factor (KGF), transforming growth factor (TGF), and platelet-derived growth factor (PDGF) (Desmet et al., 2006;Hamblin, 2017).
The advances in the clinical use of photobiomodulation and its anti-inflammatory, neovascularization, and cell regeneration effects, transform this therapeutic modality into an excellent option for the treatment of this pathology, being noninvasive and easy to apply (Desmet et al., 2006). The possibility of a new therapeutic modality, without side effects, reducing antibiotic therapy time, the need for surgical intervention, decreased use of antibiotic therapy, parenteral nutrition, and shorter hospital stay is very positive in treating these children. The prospect of a satisfactory biomodulation response in this disease could revolutionize the treatment and survival of premature babies and the quality of life of these children in the future.
The limiting factor at the moment for the use of laser therapy in intestinal pathologies is the potential biostimulating effect on intestinal bacteria, which would make its use unfeasible. Both an increase in pathogenic bacteria and a massive increase in flora bacteria in immunocompromised patients is not tolerable. The number of photobiomodulation studies in intestinal pathologies is scarce, but with a potential for promising clinical application. In in vitro studies on the influence of photobiomodulation on bacterial growth, the results are diverse. Depending on the dose and parameters used, bacterial growth can be stimulated or inhibited, varying with bacteria species (Nussbaum et al., 2003).
In order to study the effect of photobiomodulation on the growth of some bacteria that colonize the newborn's intestine, we conducted this study.

Methodology
That experimental study, in vitro, used a deductive hypothetical method, with a quantitative approach through statistical analysis of the collected data (Pereira et al., 2018).
Four species of bacteria were selected to be studied. Two belonging to healthy intestinal flora (Lactobacillus acidophilus and Lactobacillus reuteri) and two pathogenic bacteria that commonly infect newborns' intestines (Escherichia coli and Staphylococcus aureus). Strains of Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, grown in BHI medium, and Lactobacillus reuteri DSM17938, Lactobacillus acidophilus LA14, grown in MRS medium were used to carry out the research. After bacterial growth in 24 hours, the cells were added to a culture plate and irradiated by the Irrad LED 5 660 Biopdi equipment (São Carlos Brasil) with diodes at 660 nm wavelengths and a power of 0.025 W/cm 2 , in doses of 1, 5 and 10 J/cm 2 for 40 seconds, 3 minutes 20 seconds and 6 minutes and 40 seconds, respectively. The readings were taken at 4h, 24h, and 48h.
In each experiment, four groups were prepared: initial reading, control, 1J/cm 2 , 5 J/cm 2 , and 10 J/cm 2 , and four experiments were repeated. The initial number of bacteria was read by optical spectrophotometry, using the SpectraCount® Spectrophotometer (Packard) with a 570nm filter to measure the optical density of bacterial suspensions (Aacos et al., 2004 ).

Results
The growth of L. acidophilus (Table1) and L. reuteri (Table 2) did not undergo biomodulation in any of the fluences.

Discussion
Necrotizing Enterocolitis (NEC) is the leading cause of death from gastrointestinal diseases in neonates and is characterized by the development of diffuse intestinal necrosis in preterm infants subjected to stress. Still, it is the leading cause of intestinal failure in premature infants, which leads to Short Bowel Syndrome. The high incidence of NEC, and the lack of effective treatment strategies suggest that new approaches to the treatment of the disease are needed (Hackam et al., 2005). It is in this context that the possibility of using photobiomodulation as a therapeutic modality arises.
Current treatment for NEC involves antibiotics, fasting bowel rest, total parenteral nutrition, and surgical resection or peritoneal drainage when indicated. Each of these modalities is used to prevent the patient's septic condition from getting worse. However, none of these modalities effectively deals with the biological process involved in ECN. This may explain why the ECN mortality rate has changed so little over the past 40 years (Hackam et al., 2005;Hackam & Caplan, 2018).
Possible new treatment fronts would neutralize the intestinal nitric oxide levels, minimizing the enterocytes' apoptosis, and restoring the epithelial restitution and proliferation. Photobiomodulation has already proven effective in this function, protecting cells against NO-induced apoptosis (Hackam et al., 2005;Farivar et al., 2014;Rizzi et al., 2018). Besides, therapeutic agents that act on RhoA-GTPase in the intestine, or restore basal sodium-proton exchange in the enterocyte, could mitigate endotoxins' effects on the intestinal barrier function. Photobiomodulation proved to be effective in activating RhoA-GTPase, stimulating corneal epithelial cells (Hackam et al., 2005;Rhee et al., 2017).
The fact that enterocytes exposed to bacterial endotoxins express high levels of cyclooxygenase-2 leads us to an intriguing possibility that agents that modify COX-2 activity, or its expression may reduce the extent and severity of NEC in these patients. Tatmatsu The choice of parameters used for irradiation was based on previous data from the literature, showing promising results in endothelial cells and fibroblasts in the red wavelength (Moore et al., 2005). Wavelengths of 500-700 nm are effective on more superficial tissues, while wavelengths of 800 to 1000 nm are indicated for deeper tissues (Kuffler, 2016). For the treatment of abdominal pathologies in newborns, wavelengths in the red band are sufficient. Another aspect is the behavior of different wavelengths in bacteria. While infrared irradiation alters bacteria's growth rate, in some cases stimulating them, irradiation in the red band has little effect on bacterial growth (Liebert et al., 2019;Nussbaum et al., 2002).
Regarding the choice of bacteria, Escherichia coli and Staphylococcus aureus are among the bacteria most commonly associated with NEC, one of which is Gram-negative and the other Gram-positive, allowing the study of the response of different bacterial types to the same treatment with photobiomodulation. Still, Lactobacilli are found in breast milk and the newborn's flora, but when supplemented with probiotic pills, they can lead to sepsis, showing that their increase during photobiomodulation would also be harmful (Hunter et al., 2008).
We observed in previous studies that very high fluences inhibit bacterial growth. Despite the positive result for pathogenic bacteria, irradiation would probably inhibit the growth of commensal bacteria (Sousa et al., 2016;Barboza et al., 2015).
The growth of L. acidophillus was slower compared to the growth of L. reuteri. Ribeiro in 2012 showed that the LA14 strain has a slower growth when compared to other strains of Lactobacillus spp (Ribeiro et al., 2012). Our study demonstrated that there is no influence of photobiomodulation on the growth of Lactobacillus. In 2020, Nemeth et al (2020) showed a decrease in the number of CFU of Lactobacillus irradiated with LEDs at wavelengths 625, 660, and 850 nm and an energy density of 16 mW /cm 2 . The difference found can be explained by the fact that the salivary glands were irradiated by the extra-oral route (Nemeth et al., 2020).  (Sousa et al., 2016).
In our study at 660 nm 0.025 W/cm 2 , at doses of 5 and 10J/cm 2 , there was a decrease in the growth of S. aureus, but without statistical significance. Ranjbar & Takhtfooladi (2016), in his in vivo study using photobiomodulation at parameters 685 nm InGaAlP 15 mW, 3 J/cm 2 , obtained inhibition of the bacterial growth of S. aureus (Ranjbar & Takhtfooladi, 2016).
Sousa in 2012 showed that the infrared spectrum was more effective when compared to red, mainly for 904 nm, since 660 nm did not promote global inhibition (Sousa et al., 2012).
The results obtained were optimistic. If there is no significant change in the intestinal flora, the potential for using photobiomodulation in intestinal pathologies, including necrotizing enterocolitis, begins to appear promising. The use of the 660nm wavelength in the fluences of 5 J/cm 2 and 10 J/cm 2 in pathogenic bacteria did not lead to a significant change in the speed of growth, as well as in Lactobacillus.

Conclusion
Cultures of bacteria Lactobacillus acidophillus, L. reuteri, S. aureus irradiated at a wavelength of 660 nm and a power of 0.025 W/cm 2 , in doses of 1, 5, and 10 J/cm 2 did not undergo biostimulation. E. coli growth was stimulated when we compared the 1 J/cm 2 group to the Control group, with statistical significance (p <0.005). In the other fluencies, there was no biostimulation. The use of the 660nm wavelength in the fluences of 5 J/cm 2 and 10 J/cm 2 in pathogenic bacteria did not lead to a significant change in the speed of growth, as well as in Lactobacillus.
The results obtained were optimistic. If there is no significant change in the intestinal flora, the potential for using photobiomodulation in intestinal pathologies, including necrotizing enterocolitis, begins to appear promising.