Better Cardiorespiratory Fitness Defined as VO2max Increases the Chance of Partial Clinical Remission and Prolongs Remission Duration in People with Newly Diagnosed Type 1 Diabetes.

Introduction: An essential process affecting the course of type 1 diabetes (DM1) is the appearance and duration of clinical remission. One of the most important factors promoting the occurrence of remission is physical activity, due to increased activity of antioxidants, reduces insulin resistance and improves glucose transport. Maximal oxygen capacity (VO2max) is an objective measure of the body's aerobic capacity. To assess VO2max, oxygen uptake should be measured directly during the exercise test. The aim of the study was to evaluate the physical capacity in adults with DM1 and its relationship with the occurrence of partial clinical remission (pCR) during 2 years follow-up. Methods: The pCR was assessed by the following mathematical formula: A1c (%) + [4 × insulin dose (U/kg/d)]. The result ⩽9 indicates pCR. VO2max was assessed between 6th and 24th month of diabetes duration using an ergospirometer (COSMED K5 System), during an exercise test carried out on a cycloergometer (RAMP incremental exercise test). Results: The study group consisted of 32 adults with DM1. People with pCR were proved to have higher VO2max level [36.0 (33.0-41.5) vs 30.9 (26.5-34.4) ml/min/kg, P = .009. Univariate and multivariate regression confirmed a significant association between VO2max and presence of pCR [AOR 1.26 (1.05-1.52), P = .015]. Duration of remission was longer among group with higher VO2max results [15 (9-24) vs 9 (0-12) months, P = .043]. The positive relationship was observed between diabetes duration and VO2max (rs = 0.484, P = .005). Multivariate linear regression confirms a significant association between remission duration and VO2max (ml/min/kg) (ß = 0.595, P = .002). Conclusion: The higher VO2max, the better chance of partial clinical remission at 2 years of DM1 and longer duration of remission.

Better cardiorespiratory fitness increases the chance of partial clinical remission and prolongs remission duration in people with newly diagnosed type 1 diabetes. Introduction An essential process affecting the course of type 1 diabetes (DM1) is the appearance and duration of clinical remission. One of the most important factors promoting the occurrence of remission is physical activity, due to increased activity of antioxidants, reduces insulin resistance and improves glucose transport. Maximal oxygen capacity (VO₂max) is an objective measure of the body's aerobic capacity. To assess VO₂max, oxygen uptake should be measured directly during the exercise test. The aim of the study was to evaluate the physical capacity in adults with DM1 and its relationship with the occurrence of partial clinical remission (pCR) during 2 years follow-up. Methods The pCR was assessed by the following mathematical formula: A1c (%) + [4 × insulin dose (U/kg/d)]. The result ⩽9 indicates pCR. VO₂max was assessed between 6th and 24th month of diabetes duration using an ergospirometer (COSMED K5 System), during an exercise test carried out on a cycloergometer (RAMP incremental exercise test). Results The study group consisted of 32 adults with DM1. People with pCR were proved to have higher VO₂max level [36.0 (33.0-41.5) vs 30.9 (26.5-34.4) ml/min/kg, P = .009. Univariate and multivariate regression confirmed a significant association between VO₂max and presence of pCR [AOR 1.26 (1.05-1.52), P = .015]. Duration of remission was longer among group with higher VO₂max results [15 (9-24) vs 9 (0-12) months, P = .043]. The positive relationship was observed between diabetes duration and VO₂max (rs = 0.484, P = .005). Multivariate linear regression confirms a significant association between remission duration and VO₂max (ml/min/kg) (ß = 0.595, P = .002). Conclusions The higher VO₂max, the better chance of partial clinical remission at 2 years of DM1 and longer duration of remission.

Methods of Assessment of Physical Capacity in People with Diabetes Mellitus Type 1.

BACKGROUND: The article aims to present the most popular methods of assessing physical capacity. Moreover, the article sheds light on the beneficial impact of improving physical capacity in people with Diabetes Mellitus type 1 (DM1). METHODS: A computer-based literature search of PubMed, SCOPUS and Web of Science included studies up to September 2022. RESULTS: The significant role of regular physical exertion could be observed in the group of people suffering from DM1, which implicates a positive correlation between the activity and the remission time. A suitable and objective indicator of sport influence on the organism is physical capacity (PC), which describes the efficiency of the cardiovascular system and its correlation between BMI, sex, and age. PC is mostly shown as VO2max. Well metabolically controlled DM1 is not a contraindication to stress test. Even though physical activity is closely related to human history, the range of research into the importance of PC is still limited to particular groups of patients, which presents an opportunity for further research and future conclusions. CONCLUSIONS: Undertaking physical activities has a multidirectional influence on the organism. According to up-to-date knowledge, various methods of PC assessment are available. Patients can choose more easily accessible, simpler, and cheaper options like CRT, RT, and HST which do not need specialized equipment and skills. They can also decide on more advanced examinations like ergospirometry, where direct measurements of VO2max and other cardiorespiratory parameters are made.

Helicobacter pylori infection is associated with increased accumulation of advanced glycation end products in the skin in patients with type 1 diabetes: A preliminary study.

BACKGROUND: Helicobacter pylori infection (HPI) is more frequently diagnosed in patients with diabetes. Insulin resistance in patients with type 1 diabetes (DMT1) is associated with the accumulation of advanced glycation end products (AGEs) in the skin and progression of chronic complications. OBJECTIVES: Assessment of the relationship between the incidence of HPI and skin AGEs in patients with DMT1. MATERIAL AND METHODS: The study included 103 Caucasian patients with a DMT1 duration >5 years. A fast qualitative test was performed to detect the HP antigen in fecal samples (Hedrex). The content of AGEs in the skin was estimated using an AGE Reader device (DiagnOptics). RESULTS: The HP-positive (n = 31) and HP-negative (n = 72) groups did not differ in terms of age, gender, duration of diabetes, fat content, body mass index (BMI) and lipid profile, metabolic control, and inflammatory response markers. The studied groups differed in the amount of AGEs in the skin. The relationship between HPI and increased AGEs in the skin was confirmed in a multifactor regression model taking into account age, gender, DMT1 duration, glycated hemoglobin A1c (HbA1c), BMI, low-density lipoprotein cholesterol (LDL-C) and the presence of hypertension, and tobacco use. The studied groups also differed in serum levels of vitamin D. CONCLUSIONS: Increased accumulation of AGEs in the skin of patients with DMT1 with coexisting HPI suggests that eradication of HP may significantly improve DMT1 outcomes.

Indirect insulin resistance markers are associated with nonalcoholic fatty liver disease in type 1 diabetes.

INTRODUCTION: Insulin resistance (IR) in type 1 diabetes mellitus (T1DM) is associated with increased insulin dose requirements, poor glycemic control, and elevated risk of chronic complications. IR increases lipid synthesis and hepatic lipid content. Disruption in hepatic lipid accumulation and export leads to liver steatosis resulting in nonalcoholic liver disease (NAFLD). OBJECTIVES: The aim of the study was to explore the relationship between indirect IR markers and NAFLD in T1DM. PATIENTS AND METHODS: We analyzed 151 patients with T1DM (59 men, 92 women), with a median (interquartile range [IQR]) age of 40 (33-47) years and a median (IQR) diabetes duration of 19 (13-21)years. The median (IQR) value of glycated hemoglobin (HbA1c) was 7.5% (6.8%-8.%; 58 [51-66] mmol/mol). The following indirect IR markers were evaluated: estimated glucose distribution rate (eGDR), visceral adiposity index (VAI), and the triglyceride to high­density lipoprotein cholesterol ratio (TG/HDL­C). Fatty infiltration of the liver was quantified using transient elastography. Presence of NAFLD was defined as a controlled attenuation parameter value of 238 dB/m or greater. RESULTS: NAFLD was observed in 65 patients (43%). The participants with NAFLD were less insulin­sensitive (eGDR, 8.93 [6.39-9.97] vs 9.94 [8.09-11.13] mg/kg/min; P = 0.001; VAI, 1.52 [1.2-2.64] vs 1.34 [0.92-1.74]; P = 0.014; TG/HDL­C ratio, 1.35 [0.95-2.11] vs 1.11 [0.77-1.6]; P = 0.02) and were characterized by higher HbA1c values (7.75% [7.2%-8.4%] vs 7.3% [6.5%-8.1%]; 61 [55-68] vs 56 [48-65] mmol/mol; P = 0.02) than the patients without the disease. In a multivariable regression analysis adjusted for sex, diabetes duration, and HbA1c level, indirect IR markers were independently associated with NAFLD (eGDR: odds ratio [OR], 0.86; 95% CI, 0.77-0.97; P = 0.01; VAI: OR, 1.61; 95% CI, 1.05-2.49; P = 0.03, TG/HDL­C ratio: OR, 1.88; 95% CI, 1.11-3.18; P = 0.02). CONCLUSIONS: In T1DM, NAFLD is more likely to be found in individuals with lower insulin sensitivity.

Combined Antimicrobial Blue Light and Antibiotics as a Tool for Eradication of Multidrug-Resistant Isolates of Pseudomonas aeruginosa and Staphylococcus aureus: In Vitro and In Vivo Studies.

Increased development of resistance to antibiotics among microorganisms promotes the evaluation of alternative approaches. Within this study, we examined the efficacy of antimicrobial blue light (aBL) with routinely used antibiotics against multidrug-resistant isolates of Pseudomonas aeruginosa and Staphylococcus aureus as combined alternative treatment. In vitro results of this study confirm that both S. aureus and P. aeruginosa can be sensitized to antibiotics, such as chloramphenicol, linezolid, fusidic acid or colistin, fosfomycin and ciprofloxacin, respectively. The assessment of increased ROS production upon aBL exposure and the changes in cell envelopes permeability were also goals that were completed within the current study. Moreover, the in vivo experiment revealed that, indeed, the synergy between aBL and antibiotic (chloramphenicol) occurs, and the results in the reduced bioluminescence signal of the S. aureus Xen31 strain used to infect the animal wounds. To conclude, we are the first to present the possible mechanism explaining the observed synergies among photoinactivation with blue light and antibiotics in the term of Gram-positive and Gram-negative representatives.

Priming effect with photoinactivation against extensively drug-resistant Enterobacter cloacae and Klebsiella pneumoniae.

In this study, we present antimicrobial blue light (aBL) and antimicrobial photoinactivation with green light in the presence of Rose Bengal (aPDI) to modulate the susceptibility of extensively drug-resistant (XDR) Enterobacter cloacae and Klebsiella pneumoniae clinical isolates to antimicrobials. This process can be considered a photodynamic priming tool that influences other therapeutic options, such as antibiotics. The current study evaluated the different environments to estimate the most effective priming conditions by testing a broad spectrum of antimicrobials (including antimicrobials with different targets and mechanisms of action). The susceptibility of the E. cloacae and K. pneumoniae clinical isolates to various antibiotics after aBL and green light (with rose bengal) as aPDI treatment was examined with multiple methods of synergy testing (e.g., diffusion methods, checkerboard assay, postantibiotic effect), and most effective photoinactivation conditions were implemented for each environment. When Enterobacteriaceae were exposed to aBL, the most efficient reduction in survival rate under TSB conditions was observed. Similar results were observed when rose bengal, as a photosensitizer, was present during the exposure to green light in PBS. aBL and aPDI led to an increased susceptibility of K. pneumoniae and E. cloacae isolates to chloramphenicol and colistin or fosfomycin and colistin antibiotics, respectively. However, among the 4 tested isolates, we observed synergies between different antimicrobial agents and photoinactivation conditions. Thus, it may suggest that the sensitization process may be considered a strain dependent priming tool.

Is it time to change the goals of lipid management in type 1 diabetes mellitus? Changes in apolipoprotein levels during the first year of type 1 diabetes mellitus. Prospective InLipoDiab1 study.

Introduction: Apolipoprotein complement is a critical determinant of lipoprotein function and metabolism. The relation between exogenous insulin and apolipoproteins (apos) in newly diagnosed type 1 diabetes mellitus (T1DM) has not yet been studied extensively. The aim of this study was to prospectively observe the changes in serum apos AI (apo AI) and AII (apo AII) in patients with newly diagnosed T1DM and their association with the daily insulin requirement. Material and methods: Thirty-four participants of the InLipoDiab1 study aged 26 (IQR: 22-32) were enrolled in this analysis. Apolipoprotein AI and AII concentrations were assessed at diagnosis and at follow-up after 3 weeks, 6 months, and 1 year of insulin treatment. The daily dose of insulin (DDI) was calculated as the amount of short- and long-acting insulin at discharge from the hospital and at follow-up visits. Results: The changes in apo AI concentration were observed after 3 weeks of insulin treatment (p = 0.04), with the largest increase between 3 weeks and 6 months of observation (p < 0.001). Apolipoprotein AII level did not change significantly after 3 weeks, while a significant increase was observed between 3 weeks and 6 months of treatment (p < 0.001). The correlations between DDI and apo concentration were not statistically significant. Conclusions: In the first year of T1DM, there is a significant increase in apos concentration. Due to the significant deviation of apos concentration from accepted norms, changes in the recommendations of lipid control criteria in T1DM may be considered.

Association between physical activity before diagnosis and the presence of clinical remission in type 1 diabetes - InlipoDiab1 study.

AIM: To investigate whether physical activity is associated with the occurrence of remission in adults with type 1 diabetes. METHODS: Ninety nine adult participants with newly diagnosed type 1 diabetes were enroled into a prospective, observational study. The participants were advised to exercise 2-3 times a week with moderate intensity for a one-year period. Physical activity was assessed by a self-administrated questionnaire on every fourth visit. We counted the months in which participants fulfiled a partial-remission criteria: HbA1c < 6.5%, C-peptide > 0.5 ng/ml, and daily dose of insulin <0.3 U/kg/day. We assigned the participants to two groups: MORE EFFORT and LESS EFFORT, depending on the median value of physical activity in the studied population. RESULTS: The occurrence of the remission achieved statistical significance at 6th month with a greater prevalence in MORE EFFORT group (55% vs. 35% p = 0.047). In multivariate logistic regression analysis for the occurrence of remission at 12th month, physical activity before the diagnosis was the only variable that influences the occurrence of the remission (adjusted odds ratios = 3.32 [95% confidence intervals 1.25-8.80]; p = 0.02). CONCLUSION: In adults with newly diagnosed type 1 diabetes physical activity before the diagnosis is associated with higher occurrence of remission.

Development and In Vitro Evaluation of Biocompatible PLA-Based Trilayer Nanofibrous Membranes for the Delivery of Nanoceria: A Novel Approach for Diabetic Wound Healing.

The attempts to explore and optimize the efficiency of diabetic wound healing's promotors are still in progress. Incorporation of cerium oxide nanoparticles (nCeO2) in appropriate nanofibers (NFs) can prolong and maximize their promoting effect for the healing of diabetic wounds, through their sustained releases, as well as the nanofibers role in mimicking of the extra cellular matrix (ECM). The as-prepared nCeO2 were analyzed by using UV-Vis spectroscopy, XRD, SEM-EDX, TEM and FTIR, where TEM and SEM images of both aqueous suspension and powder showed spherical/ovoid-shaped particles. Biodegradable trilayer NFs with cytobiocompatibility were developed to sandwich nCeO2 in PVA NFs as a middle layer where PLA NFs were electrospun as outer bilayer. The nCeO2-loaded trilayer NFs were characterized by SEM, XRD, FTIR and DSC. A two-stage release behavior was observed when the nanoceria was released from the trilayer-based nanofibers; an initial burst release took place, and then it was followed by a sustained release pattern. The mouse embryo fibroblasts, i.e., 3T3 cells, were seeded over the nCeO2-loaded NFs mats to investigate their cyto-biocompatibility. The presence and sustained release of nCeO2 efficiently enhance the adhesion, growth and proliferation of the fibroblasts' populations. Moreover, the incorporation of nCeO2 with a higher amount into the designed trilayer NFs demonstrated a significant improvement in morphological, mechanical, thermal and cyto-biocompatibility properties than lower doses. Overall, the obtained results suggest that designated trilayer nanofibrous membranes would offer a specific approach for the treatment of diabetic wounds through an effective controlled release of nCeO2.

Can Gram-Negative Bacteria Develop Resistance to Antimicrobial Blue Light Treatment?

Antimicrobial blue light (aBL) treatment is considered low risk for the development of bacterial resistance and tolerance due to its multitarget mode of action. The aim of the current study was to demonstrate whether tolerance development occurs in Gram-negative bacteria. We evaluated the potential of tolerance/resistance development in Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa and demonstrated that representative Gram-negative bacteria may develop tolerance to aBL. The observed adaption was a stable feature. Assays involving E. coli K-12 tolC-, tolA-, umuD-, and recA-deficient mutants revealed some possible mechanisms for aBL tolerance development.

Antimicrobial Photodynamic Inactivation Affects the Antibiotic Susceptibility of Enterococcus spp. Clinical Isolates in Biofilm and Planktonic Cultures.

Enterococcus faecium and Enterococcus faecalis are opportunistic pathogens that can cause a vast variety of nosocomial infections. Moreover, E. faecium belongs to the group of ESKAPE microbes, which are the main cause of hospital-acquired infections and are especially difficult to treat because of their resistance to many antibiotics. Antimicrobial photodynamic inactivation (aPDI) represents an alternative to overcome multidrug resistance problems. This process requires the simultaneous presence of oxygen, visible light, and photosensitizing compounds. In this work, aPDI was used to resensitize Enterococcus spp. isolates to antibiotics. Antibiotic susceptibility testing according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommendations was combined with synergy testing methods recommended by the American Society for Microbiology. Two clinical isolates, E. faecalis and E. faecium, were treated with a combination of aPDI utilizing rose bengal (RB) or fullerene (FL) derivative as photosensitizers, antimicrobial blue light (aBL), and 10 recommended antibiotics. aPDI appeared to significantly impact the survival rate of both isolates, while aBL had no significant effect. The synergy testing results differed between strains and utilized methods. Synergy was observed for RB aPDI in combination with gentamycin, ciprofloxacin and daptomycin against E. faecalis. For E. faecium, synergy was observed between RB aPDI and gentamycin or ciprofloxacin, while for RB aPDI with vancomycin or daptomycin, antagonism was observed. A combination of FL aPDI gives a synergistic effect against E. faecalis only with imipenem. Postantibiotic effect tests for E. faecium demonstrated that this isolate exposed to aPDI in combination with gentamycin, streptomycin, tigecycline, doxycycline, or daptomycin exhibits delayed growth in comparison to untreated bacteria. The results of synergy testing confirmed the effectiveness of aPDI in resensitization of the bacteria to antibiotics, which presents great potential in the treatment of infections caused by multidrug-resistant strains.

Factors Determining the Susceptibility of Bacteria to Antibacterial Photodynamic Inactivation.

Photodynamic inactivation of microorganisms (aPDI) is an excellent method to destroy antibiotic-resistant microbial isolates. The use of an exogenous photosensitizer or irradiation of microbial cells already equipped with endogenous photosensitizers makes aPDI a convenient tool for treating the infections whenever technical light delivery is possible. Currently, aPDI research carried out on a vast repertoire of depending on the photosensitizer used, the target microorganism, and the light delivery system shows efficacy mostly on in vitro models. The search for mechanisms underlying different responses to photodynamic inactivation of microorganisms is an essential issue in aPDI because one niche (e.g., infection site in a human body) may have bacterial subpopulations that will exhibit different susceptibility. Rapidly growing bacteria are probably more susceptible to aPDI than persister cells. Some subpopulations can produce more antioxidant enzymes or have better performance due to efficient efflux pumps. The ultimate goal was and still is to identify and characterize molecular features that drive the efficacy of antimicrobial photodynamic inactivation. To this end, we examined several genetic and biochemical characteristics, including the presence of individual genetic elements, protein activity, cell membrane content and its physical properties, the localization of the photosensitizer, with the result that some of them are important and others do not appear to play a crucial role in the process of aPDI. In the review, we would like to provide an overview of the factors studied so far in our group and others that contributed to the aPDI process at the cellular level. We want to challenge the question, is there a general pattern of molecular characterization of aPDI effectiveness? Or is it more likely that a photosensitizer-specific pattern of molecular characteristics of aPDI efficacy will occur?

Development of Antimicrobial Phototreatment Tolerance: Why the Methodology Matters.

Due to rapidly growing antimicrobial resistance, there is an urgent need to develop alternative, non-antibiotic strategies. Recently, numerous light-based approaches, demonstrating killing efficacy regardless of microbial drug resistance, have gained wide attention and are considered some of the most promising antimicrobial modalities. These light-based therapies include five treatments for which high bactericidal activity was demonstrated using numerous in vitro and in vivo studies: antimicrobial blue light (aBL), antimicrobial photodynamic inactivation (aPDI), pulsed light (PL), cold atmospheric plasma (CAP), and ultraviolet (UV) light. Based on their multitarget activity leading to deleterious effects to numerous cell structures-i.e., cell envelopes, proteins, lipids, and genetic material-light-based treatments are considered to have a low risk for the development of tolerance and/or resistance. Nevertheless, the most recent studies indicate that repetitive sublethal phototreatment may provoke tolerance development, but there is no standard methodology for the proper evaluation of this phenomenon. The statement concerning the lack of development of resistance to these modalities seem to be justified; however, the most significant motivation for this review paper was to critically discuss existing dogma concerning the lack of tolerance development, indicating that its assessment is more complex and requires better terminology and methodology.

Suppression of serum lipid transfer proteins involved in high-density lipoprotein cholesterol metabolism by intensive insulin therapy in the first year of type 1 diabetes mellitus: Prospective InLipoDiab1 study.

BACKGROUND AND AIMS: Cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP) are crucial proteins in reverse cholesterol transport. There are insufficient data on regulating these proteins by insulin therapy in type 1 diabetes mellitus (T1DM). We aimed to assess prospectively the impact of insulin therapy initiation on transfer proteins serum levels in adults with newly diagnosed T1DM. METHODS AND RESULTS: 57 adults with newly diagnosed T1DM were enrolled in the InLipoDiab1 Study. All participants were treated with subcutaneous insulin in the model of intensive insulin therapy since the diagnosis of diabetes. Serum PLTP and CETP concentrations were measured at diagnosis, after three weeks, six months, and after one year of insulin treatment, using the immunoenzymatic method ELISA. A significant decrease in PLTP and CETP concentrations were demonstrated during twelve months of insulin therapy in newly diagnosed T1DM. The dynamics of changes in the level of these proteins varied depending on the occurrence of remission after a year of the disease. In the group without remission, a significant decrease in PLTP and CETP levels appeared after six months of follow-up. The remission group was characterized by a decrease in proteins concentration only after one year of treatment. In the non-remission group, significant negative correlations were found between the daily dose of insulin and levels of PLTP and CETP. CONCLUSION: Exogenous insulin is an inhibitor of lipid transfer proteins involved in high-density lipoprotein cholesterol metabolism in the first year of treatment.

Changes in high-density lipoprotein cholesterol (HDL-C) level and the ratio of triglycerides to HDL-C during the first year of type 1 diabetes.

INTRODUCTION: Patients in an insulin­deficient state show reduced high­density lipoprotein cholesterol (HDL­C) levels. Insulin treatment affects lipid metabolism in this population. There have been no prospective studies evaluating changes in lipid profile after the diagnosis of type 1 diabetes (T1D). OBJECTIVES: We investigated the effect of subcutaneous insulin therapy initiation on quantitative changes in HDL­C levels and other components of lipid profile in patients with newly diagnosed T1D. PATIENTS AND METHODS: A total of 127 patients with newly diagnosed T1D aged 28 years (interquartile range, 23-34 years) were enrolled in the InLipoDiab1 study. The lipid profile was assessed before the first injection of insulin (baseline) and after 3 and 12 months of insulin therapy. The daily dose of insulin (DDI) was defined as the requirement for insulin per kilogram body weight per day. The DDI was calculated at hospital discharge and during visits in an outpatient clinic at 3 and 12 months. RESULTS: We observed a persistent increase in HDL­C levels at 3 and 12 months versus baseline (P <0.001) in men and women. Moreover, a reduction was observed in triglyceride levels (P <0.001) and the ratio of triglycerides to HDL­C (P <0.001) in men and women. In contrast, a decrease was observed in low­density lipoprotein cholesterol and non­HDL­C levels (P <0.001), but only in men. CONCLUSIONS: Subcutaneous insulin therapy reverses the impaired phenotype of lipoproteins during the first year of treatment. Changes in lipoprotein levels in newly diagnosed T1D differ depending on sex.

Development of Staphylococcus aureus tolerance to antimicrobial photodynamic inactivation and antimicrobial blue light upon sub-lethal treatment.

Antimicrobial photodynamic inactivation (aPDI) and antimicrobial blue light (aBL) are considered low-risk treatments for the development of bacterial resistance and/or tolerance due to their multitargeted modes of action. In this study, we assessed the development of Staphylococcus aureus tolerance to these phototreatments. Reference S. aureus USA300 JE2 was subjected to 15 cycles of both sub-lethal aPDI (employing an exogenously administered photosensitizer (PS), i.e., rose Bengal (RB)) and sub-lethal aBL (employing endogenously produced photosensitizing compounds, i.e., porphyrins). We demonstrate substantial aPDI/aBL tolerance development and tolerance stability after 5 cycles of subculturing without aPDI/aBL exposure (the development of aPDI/aBL tolerance was also confirmed with the employment of clinical MRSA and MSSA strain as well as other representatives of Gram-positive microbes, i.e. Enterococcus faecium and Streptococcus agalactiae). In addition, a rifampicin-resistant (RIFR) mutant selection assay showed an increased mutation rate in S. aureus upon sub-lethal phototreatments, indicating that the increased aPDI/aBL tolerance may result from accumulated mutations. Moreover, qRT-PCR analysis following sub-lethal phototreatments demonstrated increased expression of umuC, which encodes stress-responsive error-prone DNA polymerase V, an enzyme that increases the rate of mutation. Employment of recA and umuC transposon S. aureus mutants confirmed SOS-induction dependence of the tolerance development. Interestingly, aPDI/aBL-tolerant S. aureus exhibited increased susceptibility to gentamicin (GEN) and doxycycline (DOX), supporting the hypothesis of genetic alterations induced by sub-lethal phototreatments. The obtained results indicate that S. aureus may develop stable tolerance to studied phototreatments upon sub-lethal aPDI/aBL exposure; thus, the risk of tolerance development should be considered significant when designing aPDI/aBL protocols for infection treatments in vitro and in clinical settings.

Antimicrobials Are a Photodynamic Inactivation Adjuvant for the Eradication of Extensively Drug-Resistant Acinetobacter baumannii.

The worldwide emergence of extensively drug resistant (XDR) Acinetobacter baumannii has reduced the number of antimicrobials that exert high bactericidal activity against this pathogen. This is the reason why many scientists are focusing on investigations concerning novel non-antibiotic strategies such as antimicrobial photodynamic inactivation (aPDI) or the use of antimicrobial blue light (aBL). Therefore, the aim of the current study was to screen for antimicrobial synergies of routinely used antibiotics and phototherapies, including both aPDI involving exogenously administered photosensitizing molecules, namely, rose bengal, and aBL, involving excitation of endogenously produced photoactive compounds. The synergy testing was performed in accordance with antimicrobial susceptibility testing (AST) standards, including various methodological approaches, i.e., antibiotic diffusion tests, checkerboard assays, CFU counting and the evaluation of postantibiotic effects (PAEs). We report that combining antimicrobials and aPDI/aBL treatment led to a new strategy that overcomes drug resistance in XDR A. baumannii, rendering this pathogen susceptible to various categories of antibiotics. Sublethal aPDI/aBL treatment in the presence of sub-MIC levels of antimicrobials effectively killed A. baumannii expressing drug resistance to studied antibiotics when treated with only antibiotic therapy. The susceptibility of XDR A. baumannii to a range of antibiotics was enhanced following sublethal aPDI/aBL. Furthermore, 3'-(p-aminophenyl) fluorescein (APF) testing indicated that significantly increased reactive oxygen species production upon combined treatment could explain the observed synergistic activity. This result represents a conclusive example of the synergistic activity between photodynamic inactivation and clinically used antimicrobials leading to effective eradication of XDR A. baumannii isolates and indicates a potent novel therapeutic approach.