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Antibiotics 2020, 9, 98 7 of 12 Although the bactericidal effect of the treatment is consistent across all strains, there are statistically significant differences in susceptibility amongst them (Figure 1). These results are consistent with those found on S. aureus [20]. It has been proposed that some of the resistance factors present in the strains have weight in the susceptibility of S. aureus to PDT [31], but no clear correlation has been found between the strains’ response to aPDT and antibiotic resistance. The situation is less clear in E. coli. While some studies had found that the reference strain ATCC 25922 was slightly more susceptible to photoinactivation than multidrug resistant clinical isolates [21,31], our results show that this is the case only for a few isolates (strains 11, 13, and 24), while the majority are significantly more susceptible (Figure 1). Since MB is internalized by E. coli [32,33] and is known to be a substrate for efflux pumps in E. coli as well as in other bacteria [19,34,35], one could reasonably expect some tolerance to aPDT in the resistant strains. We can speculate that resistance mechanisms such as reduced permeability to antimicrobial agents, active efflux of the antimicrobial from the cell, enzymatic alterations, or degradation of the antimicrobial agent [36] may contribute to modulate the photodynamic activity of MB, but they are not determinants for aPDT efficiency. Biofilms play a major role in bacterial infections and strongly affect the susceptibility of the bacteria to antibiotics and also to aPDT [37]. E. coli biofilms evolve from early stage to mature within 16 h [38], concomitantly increasing the thickness of the extracellular matrix. In our study, biofilms were allowed to grow for at least 16 h to let them reach the mature state. Figure 2 shows the aPDT results obtained for the 24 clinical isolates growing in biofilms. The main finding is that aPDT was able to produce an antimicrobial effect (99.9% kill) on all strains, irrespective of their antibiotic resistance profile. Comparing with the results in planktonic state (Figure 1), it is apparent that photoinactivation is not as efficient and no disinfecting effect (>99.999% kill) could be achieved for any isolate despite increasing the concentration of MB by 2.5-fold. It is well known that photoinactivation of cells living in biofilms require higher concentration of MB compared to their planktonic counterparts [39,40], owing mainly to the mechanical barrier to MB diffusion posed by the extracellular matrix and to alterations in gene expression. As in planktonic cells, statistically significant differences were detected among the strains: Firstly, none of the multidrug-resistant strains appear to be more tolerant to aPDT than the reference ATCC 25922. Thus, as in the planktonic state, the antibiotic-resistance mechanisms do not confer tolerance to aPDT in the biofilm state. In fact, some of the clinical isolates were found more susceptible to aPDT than the reference ATCC 25922 also in biofilm state. Secondly, the differences among strains are less marked than in planktonic state, which indicates that the major determinant of photoinactivation efficacy must be a factor common to all strains, likely the existence of an extracellular matrix. Thus, not surprisingly, no clear correlation is observed between the planktonic and biofilm results. 4. Materials and Methods 4.1. Bacterial Strains Twenty-four strains of uropathogenic E. coli and two strains obtained from the American Type Culture Collection (E. coli ATCC 35218 and E. coli ATCC 25922) were tested in this study. The clinical specimens were isolated from twenty-four different anonymized urine samples with pyuria obtained from twenty-four patients on different days after receiving their consent for the use of microorganisms obtained from their samples in investigational work. The patients had received prior instruction in the proper methods for the collection of midstream urine specimens. Specimens were cultured quantitatively for bacteria on brolacin agar (C.L.E.D. agar, Merck, Darmstadt, Germany) and tryptic soy agar (TSA, Merck, Darmstadt, Germany) within one hour of collection unless they were refrigerated. Species identifications were determined after obtaining a pure culture. All the isolated strains were maintained at −80 ◦C at our premises.PDF Image | Effective Photodynamic Inactivation of 26 E coli Strains
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