Intraoperative application of photodynamic therapy as a method of inactivation of uropathogenic flora (pilot study)

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BACKGROUND

In modern urology, endoscopic techniques, including intraluminal surgery, are rapidly developing, which offer advantages such as low trauma and shorter patient recovery periods. However, the complex design of the equipment and instruments used, labor-intensive process after use, and human factors at all stages of work are risk factors for postoperative complications. Other risk factors for urinary infection include the need for drainage of urinary organs, such as the introduction of a foreign body, and the need to visualize the surgical field, which is achieved by fluid irrigation. These can increase the pressure in the system with pyelointerstitial reflux. In addition, preoperative and/or intraoperative antibiotic prophylaxis may be ineffective because of uropathogens resistant to antibiotics. According to the literature, between 30% [1] and 51% [2] of urinary stones are infected. Contemporary lithotripsy principles involve crushing concretions into small fragments, which are then removed through small-diameter accesses. However, in infection-related stones, small fragmentation inevitably releases large amounts of toxins and bacteria contained in the concrement [3]. Postoperative complications can be serious, such as systemic inflammatory response syndrome (SIRS), pyelonephritis, and urosepsis. The incidence of postoperative SIRS can reach 27.4%, whereas urosepsis can occur in up to 7.9% of cases [4–6]. Importantly, postoperative fever can occur even if the preoperative urine culture is sterile and prophylactic antibiotic therapy is administered because the source of infection may be the bacteria present in the nodule [7–9]. In the urological departments of medical and preventive institutions in the Russian Federation, as well as in many other countries worldwide, nosocomial infections (healthcare-associated infections [HCAIs]) are a significant problem. Despite adherence to sanitary, hygienic, and antiepidemic policies in medical institutions, the frequency of HCAIs remains high [10].

The prevention of postoperative infectious and inflammatory complications is a pressing issue. An area of scientific research is exploring the potential of photodynamic therapy (PDT), which is traditionally used to treat patients with cancer, for the treatment of localized bacterial infections, including antibiotic-resistant strains [11]. Furthermore, PDT [12] has been used in experimental and clinical studies to treat purulent wounds and purulent-septic complications of ENT organs. A previous study showed the promising potential of PDT application in dentistry [13]. Antibacterial PDT is a promising technology for combating chronic infections [14–17]. Our research group previously presented an analysis of the effects of PDT on the urinary “planktonic” microflora in patients with nephrolithiasis (NL) [18]. However, currently, no data exist on the use of PDT for preventing infectious complications in urological practice, specifically for treating NL.

This study analyzed the microbial landscape of patients’ urine and washes from various surfaces in a regional urology hospital over 10 years and the sensitivity of isolated microorganisms to antibiotics. The results of this study suggest the need for new methods of antibacterial therapy. To this end, the safety of the intraoperative use of antibacterial PDT was evaluated.

This study aimed to investigate the possibility of intraoperative photodynamic inactivation of uropathogenic microorganisms. The study objectives were as follows: (1) analyze the microflora of patients’ urine and environmental surfaces in the urological clinic and (2) evaluate the biocidal effect on uropathogenic bacteria and the potential tissue damage caused by intraoperative photodynamic exposure on the animal kidney pelvis.

MATERIALS AND METHODS

Analysis of microbial species specificity

In this study, a retrospective analysis of observational data on the urine microbial landscape of patients in the urology clinic of a large hospital (120 beds) from 2010 to 2019 was conducted. This study investigated the results of washes from different working surfaces of environmental objects in the clinic, including hands, linen, overalls, medical instruments, furnishings, medical equipment, and dressing tables. The primary uropathogens found in native urine samples from patients with complicated NL were also analyzed. This included patients with established bacteriuria, long-term urostomies, recurrent NL, and episodes of exacerbation of infectious-inflammatory processes.

Working with animals

The pelvic ducts of seven 4- to 4.5-month-old Landrace pigs, weighing 40–45 kg, were exposed to analyze the response of the pelvic tissue to PDT. In addition, four kidneys were obtained from different pigs that had not been exposed to serve as normal controls. The pigs were anesthetized during surgical interventions using a mixture of Zoletil and Rometar. The surgical area was disinfected twice using a 0.5% aqueous–alcoholic chlorhexidine bigluconate solution. Access to the kidney was achieved through an oblique incision in the subcostal region. The kidney and upper third of the ureter were isolated retroperitoneally. A soft tourniquet was used to clamp the ureter, and a catheter was inserted into the pelvis through an incision in the upper third.

The Ethical Committee of the Volga Region Research Medical University of the Ministry of Health of Russia approved all animal studies (Minutes No. 13, dated July 7, 2021), which were conducted in compliance with international legal acts on laboratory animal work.

Assessment of photosensitizer accumulation in kidney tissues

The study used 5 mg/mL photoditazine in a 0.9% sodium chloride solution supplemented with the nonionic surfactant Triton X 100 to a concentration of 10%. The solution was injected through a catheter into the renal pelvis and left for 10 min. Subsequently, the pelvis was washed with 0.9% sodium chloride solution, and the kidney was removed. The accumulation of photosensitizer in kidney tissues was evaluated by measuring the fluorescence intensity using an IVIS surface fluorescence imaging unit.

In vivo PDT

Fifteen minutes after filling the pelvis with the photosensitizer solution, a light guide with a cylindrical diffuser, 5 mm in length at the end (Polironik LLC), connected to the Latus-K laser (Actus LLC), was introduced into the pelvis. The renal pelvic cavity was irradiated in continuous or pulsed modes with an output power of 150 and 300 mW and a wavelength of 662 nm. After irradiation, the ureter was sutured using an atraumatic polyglycolide suture, and the wound was closed layer by layer. The skin was treated with antiseptic and covered with an aseptic sticker. Histological examination of renal tissues was performed 3 h, 1 day, and 3 days after PDT. The temperature in the pelvis was measured using a portable multifunctional multimeter equipped with a thermocouple to exclude thermal effects during irradiation.

Antibacterial PDT in vivo

The microorganisms most frequently found in patient stones, Escherichia coli and Staphylococcus aureus, were used to evaluate the effectiveness of intraoperative PDT [18]. Microorganisms (1 · 10⁸ colony forming units [CFU]/mL) were injected into the kidney pelvis and incubated for 15 min, which corresponded to the average duration of crushing a single stone in the kidney pelvis in humans. Photoditazine was injected into the pelvis and irradiated for 10 min with an output power of 300 mW. The kidney was then removed, and washes were taken from the inner surface of the pelvis. The number of grown colonies was counted 24 h after seeding. The efficiency of PDT was estimated by the value of logarithmic reduction of CFU, where 1, 2, 3, 4, 5, and 7 represent 90%, 99%, 99.9%, 99.99%, 99.999%, and 99.9999% of dead microorganisms, respectively.

Biochemical blood analysis

Blood was collected from the vein located on the outer surface of the auricle of the animals before surgery and on the day of withdrawal from the study. The collection was performed using vacuum tubes containing ethylenediaminetetraacetic acid and vacuum tubes containing a coagulation activator. Biochemical blood tests, including urea, creatinine, and C-reactive protein concentrations, were performed using photometric tests on an automatic biochemical analyzer HumaStar 600 (Human, Germany). Blood samples were analyzed for cystatin C using the immunoturbodimetric method. Cystatin C is a reliable and sensitive marker of the glomerular filtration rate (GFR) and a cysteine protease inhibitor that is independent of the patient’s sex and age characteristics [19]. General blood analysis was performed using a Mindray 6800 analyzer (Mindray, China).

Histological examination

After the animals were removed from the study, their kidneys were collected in a buffered formalin solution. The samples were washed after 48 h, and the sections of the pelvis were dissected. The preparations were fixed in 10% formalin solution, embedded in paraffin blocks, and 5 µm thick slices were prepared using a Leica CM 2000R microtome (Germany). Then, the slices were stained with hematoxylin and eosin using the standard method and studied light-optically (Leica DM 1000, ×40–400) with photofixation of images (Leica DFC290).

Statistical analysis

The data obtained were subjected to statistical analysis using SPSS Statistica version 26. The normality of the parameter samples was determined using the Kolmogorov–Smirnov test. For samples that did not meet the normality criterion, their general characteristics are presented as the quartile range, i. e., Me [Q₁; Q₃], where Me is the median (50%), Q₁ is 25%, and Q₃ is 75%. The standard deviation of σf% percentages was calculated using the following formula:

${\sigma }_f\%=\sqrt{f\cdot (1-f)/n},$

where f is the percentage and n is the total number of sample items.

RESULTS

Observational data on the microflora of urology clinic patients

The analysis showed that NL was the most common pathology among patients treated, with percentages of 37.2% (1351/3626) in 2010, 41.7% (1293/3101) in 2013, 43.2% (1435/3318) in 2016, and 43.3% (1458/3363) in 2019. This represents an increase of 5.8% over 10 years. Most patients admitted had stones in the ureters (N20.1 [57.9%]), followed by the kidneys (N20.0 [22.1%]), and both kidneys and ureters (N20.2 [20%]). The mean age was 47.0 ± 0.8 years for men and 53.1 ± 0.9 years for women. Most patients received surgical treatment at the clinic, with surgical activity ranging from 77% to 80% over the 5-year interval, specifically 77.2% in 2021 (2509/3248). Among patients with NL, 52.5% ± 1.3% were treated surgically and 46.5% ± 1.3% were treated conservatively. Endoscopic procedures were performed in 35.2%, shock wave lithotripsy in 14.4%, and open surgery in 3.9% of the cases.

In the last decade, urologists have increasingly committed to urinary drainage after surgery, resulting in a clear prevalence of two-stage interventions. Consequently, in 2019, 95.1% (467/491) of the patients underwent endoscopic surgeries for NL and had various drains (such as nephrostomies, stents, ureteral and urethral catheters, and cystostomies) placed, which can contribute to hospital infections.

Table 1 presents the results of a 10-year observational analysis of the urine microflora of patients in the urology clinic. Data are presented as relative indices, indicating the detection rate per 1000 urine samples. This study traced the long-term dynamics of the microbial landscape of patients’ urine, considering HCAIs and other infections. In addition, washes collected from environmental surfaces in the urological clinic were analyzed for 2010–2019. The most commonly found bacteria in urine samples were Escherichia coli, Enterobacter cloacae, and Staphylococcus epidermidis. Enterococcus faecalis, Staphylococcus saprophyticus, Acinetobacter baumannii, and Klebsiella pneumoniae were also widely distributed. The data obtained were consistent with the results of the analysis of microorganisms found on the working surfaces of the urological clinic. Over 10 years, the abundance of Escherichia, Enterobacter, and Enterococcus genera increased. In a previous study, multidrug resistance was found in most microorganisms isolated from stones of patients with NL [18]. The constant flow of patients passing through a urologic hospital and the use of endoscopic manipulations and surgeries, which are frequently accompanied by drainage of the urinary system, can lead to microbial contamination of the premises in a urologic clinic.

 

Table 1. Dynamics of changes in the species composition of microorganisms isolated from urine (detectability per 1000 samples)

Таблица 1. Динамика изменений видового состава микроорганизмов, выделенных из мочи (выявляемость на 1000 образцов)

Name of the microorganism	2010	2013	2016	2019	Over 10 years, 50% [Q1; Q3]
Escherichia coli	102.8	77.4	116.9	137.9	105.2 [82.7; 123.8]
Enterobacter cloacae	49.3	70.9	99.4	95.7	75.2 [58.4; 95.3]
Staphylococcus epidermidis	96.9	33.8	70.2	62.1	62.8 [46.6; 75.8]
Enterococcus faecalis	40.7	20.6	64.4	89.5	43.2 [34; 63.44]
Staphylococcus saprophyticus	29.6	21.6	10.5	26.7	23.6 [11.8; 28.8]
Acinetobacter baumannii	16.1	9.6	0	5.9	15.5 [10; 17.1]
Klebsiella pneumoniae	11.5	16.7	0	8.3	13.9 [8.3; 17.9]
Pseudomonas aeruginosa	10.5	6.1	8.4	9.2	8.2 [7.1; 9.4]
Candida spp.	9.5	3.2	7.5	11.0	6.3 [4; 9]
Staphylococcus aureus	2.6	2.9	6.9	10.4	3.4 [3; 6.5]
Proteus mirabilis	6.9	3.2	4.5	0	3.7 [2; 4.7]
Citrobacter freundii	3.3	1.9	0.6	0	1.7 [0.1; 1.9]

 

The factors listed above became predictors in experimental animal studies on the use of antimicrobial PDT in urology to neutralize antibiotic-resistant strains of microorganisms.

Safety assessment of antimicrobial PDT in an animal model

The pig renal pelvis volume was measured by injecting fluid into it while clamping the pelvic–ureter segment. The normal volume was 1.1 ± 0.1 cm³. The accumulation of photoditazine by kidney tissues was evaluated, and no photosensitizer was found in the area of pelvic fluorescence (Fig. 1). The fluorescence signal detected in the ureter and on the lateral kidney surfaces was likely caused by the incomplete removal of the photoditazine solution from the organ during sample preparation. The histological study revealed that the normal pelvis was lined with epithelium consisting of 5–6 layers of cells with smooth contours of the outer layer. Underneath, loose connective tissue and some thin-walled vessels were observed. No changes in the state of the lining epithelium were observed after exposure to the photosensitizer (Fig. 2).

 

Fig. 1. Longitudinal section on the cavity of the pelvis of the control kidney (а) and the kidney after 10 minutes of exposure to photoditazine solution (b). Images are macrophotographs with superimposed fluorescence signal

 

Fig. 2. Assessment of the condition of the control kidney’s pelvis tissues (a) and after 10 minutes of exposure to a solution of photoditazin 10 % with Triton X-100, 5 vol.% (b). Stained with hematoxylin and eosin. ×100 magnification

 

Then, the state of kidney tissues following PDT was evaluated. The results showed that irradiation of the pelvic cavity containing photoditazine with laser radiation at output powers of 150 and 300 mW resulted in only a focal increase in intercellular contacts of the surface layer of the urothelium when exposed to 150 mW in pulse mode (Fig. 3 a). When using the continuous mode with the same power, loosening and desquamation of the surface layer of cells occur, along with an increase in intercellular contacts of the surface layer of the urothelium (Fig. 3, b). Upon exposure to 300 mW in the pulse mode, the contours of the urothelium surface cells were damaged, and single small foci of desquamation were observed (Fig. 3, c). Continuous exposure with a power of 300 mW resulted in the formation of areas of pronounced loosening of cells because of the destruction of intercellular contacts and multiple merging areas of destruction of the outer rows of the covering urothelium. In some places, the thickness of the preserved rows is 2–3 cells. Thus, the superficial layer of the mucous membrane was determined to have incurred insignificant damage under the influence of 300 mW, whereas the underlying tissue remained unaffected (Fig. 3 d).

 

Fig. 3. Histological preparations: a — power density 150 mW/cm², pulse mode; b — power density 150 mW/cm², continuous mode; c — power density 300 mW/cm², pulse mode; d — power density 300 mW/cm², continuous mode; staining — hematoxylin and eosin, ×100 magnification

 

Determination of the temperature regimen for photodynamic irradiation

During the study, the researchers measured the temperature of the photoditazine solution in the pelvic cavity while being irradiated by a laser in the continuous mode with a power of 300 mW for 20 min. The results showed no increase in the temperature in the pelvis, regardless of whether irrigation was present or not. Irrigation was performed through the nephrostomy at an intra-channel pressure of no more than 30 cm of water.

Determination of microflora in the pelvic contents after photodynamic irradiation

A suspension of daily test cultures (1 · 10⁸ CFU/mL) was injected into the pig kidney pelvis, and PDT was performed after that. Washes from the inner surface of the kidney pelvis were sown on meat-peptone agar, and PDT was found to kill 99.9% of E. coli and 99% of S. aureus, indicating the technique’s effective bactericidal activity.

Assessment of the renal status

Biochemical blood analysis conducted 1 day after surgery revealed slight increases in creatinine and urea levels, which exceeded the acceptable reference interval (Table 2). No adverse effect on GFR was observed, and the cystatin C level decreased postoperatively.

DISCUSSION

A retrospective analysis of microbiological urine cultures from patients at the urology clinic between 2010 and 2019 revealed an increase in the number of patients with positive urine cultures. E. coli, E. cloacae, S. epidermidis, E. faecalis, and K. pneumoniae are most frequently present in urine. These microorganisms were also isolated from the urinary stones of patients in a previous study [18]. In addition, most isolated microorganisms exhibited resistance to multiple drugs, confirming a global trend [20]. Preoperative antibiotic therapy is ineffective against resistant microorganism strains. Furthermore, during surgery, these microorganisms can spread from the stone when it is crushed using instruments. This finding is supported by the similarity in the species composition of microorganisms found in urine and on the working surfaces of urologic hospitals. The specificity of urological operations that require urinary tract drainage and the presence of human factors may serve as additional sources of antibiotic-resistant strains. PDT may serve as an alternative to antibiotic therapy. Unlike antibiotics, PDT targets multiple areas within the bacterial cell and eliminates the possibility of resistance development [21].

Previous studies have shown the effectiveness of antibacterial PDT against various microorganisms. The research team optimized the PDT procedure for Gram-negative uropathogenic microorganisms and tested it on infected urine from patients [18]. This study comprehensively examined the safety of applying the developed technique to animals. Cells of the epithelium lining the renal pelvic ducts do not accumulate photoditazine after a 10-min exposure to the photosensitizer. The results suggest no significant tissue damage during photodynamic action on the renal pelvic tissue. The peculiar structure of the urothelium provides a barrier function that prevents the penetration of various substances, ions, and water into the kidney tissue [22]. The temperature change during PDT was evaluated because of the high-power densities of laser radiation used in the developed technique. The photosensitizer solution administered to the renal pelvis was not heated, which ruled out thermal damage to the organ tissues. A histological examination was conducted to evaluate the condition of the renal pelvic tissues after PDT. The lack of significant damage to the kidney pelvis epithelium in animals subjected to different irradiation regimes is likely due to the absence of photosensitizer in the epithelial cells.

A biochemical blood analysis was conducted to evaluate the functional state of the kidneys and the organism as a whole. Determining GFR values is widely accepted for diagnosing and monitoring renal dysfunction [23, 24]. Cystatin C is produced by all nuclear cells in the body at a constant rate. It is freely filtered into the renal tubules, almost completely reabsorbed, and destroyed in the renal tubules. Therefore, an increase in its serum level indicates a decrease in GFR and may signal the preclinical phase of renal dysfunction [23]. In this study, intraoperative PDT did not affect the renal GFR of the animal, and the cystatin C level decreased compared with the initial level.

Thus, this study demonstrates the safety of antibacterial PDT and the possibility of complementing traditional antibiotic therapy in the surgical treatment of NL. An advantage of using this technique is that the simultaneous intraoperative therapeutic action may allow for an antimicrobial effect in a very short time.

ADDITIONAL INFORMATION

Authors’ contribution. Thereby, all authors made a substantial contribution to the conception of the study, acquisition, analysis, interpretation of data for the work, drafting and revising the article, final approval of the version to be published and agree to be accountable for all aspects of the study. Personal contribution of each authors: O.S. Streltsova — concept development, collection of biological material, analysis of the obtained data, writing the text of the manuscript; A.E. Antonyan — collection of biological material, analysis of the obtained data, writing the text of the manuscript; V.V. Elagin — concept development, analysis of the data obtained, editing the manuscript text; N.I. Ignatova — development of the concept, analysis of the data obtained, editing the manuscript text; K.E. Yunusova — preparation and analysis of histological material; T.R. Zhilyaeva-Fomina — preparation and analysis of material on epidemiological observational study; V.F. Lazukin — statistical processing of the material; V.A. Kamensky — general supervision of the study.

Funding source. This work was financially supported by the Russian Science Foundation (grant No. 21-15-00371).

Competing interests. The authors declare that they have no competing interests.

About the authors

Olga S. Streltsova

Privolzhskiy Research Medical University

Author for correspondence.
Email: strelzova_uro@mail.ru
ORCID iD: 0000-0002-9097-0267
SPIN-code: 9674-0382

MD, Dr. Sci. (Medicine)

Russian Federation, Nizhny Novgorod

Artem E. Antonian

Privolzhskiy Research Medical University

Email: 5x5x5@inbox.ru
ORCID iD: 0000-0001-6494-7277
Russian Federation, Nizhny Novgorod

Vadim V. Elagin

Privolzhskiy Research Medical University

Email: elagin.vadim@gmail.com
ORCID iD: 0000-0003-2676-5661
Scopus Author ID: 16515845600

MD, Cand. Sci. (Biology)

Russian Federation, Nizhny Novgorod

Nadezhda I. Ignatova

Privolzhskiy Research Medical University

Email: n.i.evteeva@gmail.com
ORCID iD: 0000-0002-4570-9342
Scopus Author ID: 55648183300

MD, Cand. Sci. (Biology)

Russian Federation, Nizhny Novgorod

Katerina E. Yunusovа

Privolzhskiy Research Medical University

Email: katyayunusova@ya.ru
ORCID iD: 0000-0001-9283-0332
SPIN-code: 9211-4897

MD, Cand. Sci. (Medicine)

Russian Federation, Nizhny Novgorod

Tatiana R. Zhilyaeva-Fomina

N.A. Semashko Nizhny Novgorod Regional Clinical Hospital

Email: zhilyaevatr@yandex.ru
Russian Federation, Nizhny Novgorod

Valery F. Lazukin

Privolzhskiy Research Medical University

Email: valery-laz@yandex.ru
ORCID iD: 0000-0003-0916-0468
SPIN-code: 3400-9905

MD, Cand. Sci. (Biology)

Russian Federation, Nizhny Novgorod

Valadislav A. Kamensky

Privolzhskiy Research Medical University

Email: vlad340@mail.ru
ORCID iD: 0000-0001-6388-2483
Scopus Author ID: 6701639165

MD, Dr. Sci. (Physics and Mathematics)

Russian Federation, Nizhny Novgorod

References

Supplementary files