CC BY
17УДК 57.086.3:628.353.153:615.472.5
scanning electron microscopy of biofilm
on urethral and venous catheters
stepanova A.A.* (head of the laboratory), vasilyeva N.v. (director of the institute, head of the chair), Pinegina o.N. (assistant lecturer of the chair)
North-Western State Medical University named after I.I. Mechnikov: Kashkin Research Institute of Medical Mycology and Chair of Medical Microbiology, St. Petersburg, Russia
©Collective of authors, 2014
The data about the ultrastructural organization of the biofilms formed on different type of catheters (5 urethral and 3 venous) of the patients with various disease and during different exposition (time of using) have been presented in this article. Catheters were examined by microbiological methods for revealing the bacterial and fungal biodiversity and electron microscopycally. We demonstrated, that in the surfaces of the urethral catheters, in comparison with venous, were formed more massive biofilms. Assumed, that the functionally biofilms «may scan and repro-duce» the character of texture of the solid catheter surface.
Key words: biofilm, Candida species, catheter-associated infections in vivo, scanning electron microscopy, ultrastructure
сканирующая
электронная
микроскопия биопленок
уретральных и венозных катетеров
степанова А.А. (зав. лаб.), Васильева н.В.
(директор института, зав. кафедрой),
Пинегина о.н. (ассистент кафедры)
Северо-Западный государственный медицинский университет им. И.И. Мечникова: НИИ медицинской микологии им. П.Н. Кашкина и кафедра медицинской микробиологии, Санкт-Петербург, Россия
©Коллектив авторов, 2014
В статье представлены данные об ультраструктурной организации биопленок, формируемых на различных типах катетеров (5 уретральных и 3 венозных) у пациентов с различными заболеваниями и длительностью катетеризации. Изучали видовой состав микроорганизмов, образующих биопленки, а также мор-фобиологические особенности биопленок методом сканирующей электронной микроскопии. Нами показано, что на поверхности уретральных катетеров, по сравнению с венозными, были сформированы более массивные биопленки; предположено, что функ-
* Контактное лицо: Степанова Амалия Аркадьевна, тел.: (812) 303-51-40
ционально биопленки, возможно, «могут сканировать и воспроизводить» характер текстуры твердой поверхности катетера.
Ключевые слова: биопленки, in vivo, Candida species, катетер-ассоциированные инфекции, сканирующая электронная микроскопия, ультраструктура
introduction
Catheter biofilms represent the highly organized association of the microorganisms and the extracellular products of their vital functions, which form a structured community on the solid surface. The biofilms formation dangerous with catheter-associated infections. Bacteria and fungi which pass into the urinary tract and the blood circulatory system from the lumen of the catheter cause complications in the patients disease. The medical consequences of device-related infections can be disastrous; they include potentially life-threatening systemic infections and device malfunction that may require device removal [1]. Data about the micromorphology of biofilms, which in vivo formed on human catheters, few in number [2, 3; Ganderton L., et al. //Eur. J. Clin. Microbiol. Infect. Dis. - 1992. - Vol. 11, №9; Marrie T.J., Costerton J.W. //J. of Clin. Microbiol. - 1984. - Vol. 19, №5; Gorman S.P., et al. //Epidemiol. Infect. - 1994. - Vol. 112. and etc.]. The aim of the present work was the trace the relationship between ultrastructural and microbiological variability of clinical catheter-related infections.
materials and methods
A total of 8 catheter tips from 7 patients were included in the present study. All data about investigated samples, time of the catheter standing and isolated species have been included in the table. The urethral catheters were produced from siliconized latex (Unomedical, Denmark) and the venous from polyurethane (BBraun, Germany).
Table
The description of investigated samples
№ Patients, their age and disease The time of the catheter using Isolated microorganisms
urethral catheter
1 C., 54 years; spontaneous esophageal perforation 7 days Candida albicans, Klebsiella pneumoniae
2 K., 63 years; biliary peritonitis 7 days C. albicans, K. pneumoniae
3 N, 82 years; gastrointestinal passage of blood 3 days C. albicans, Pseudomonas aeruginosa
4 C., 56 years; fecal peritonitis 7 days K. pneumonia, Corinebacterium amylolatum
5 B., 57 years; soldering disease 6 days K. pneumonia, Staphylococcus haemoliticus
venous catheter
1 I., 60 years; heart attack 10 days C. albicans, K. pneumoniae
2 N., 82 years; gastrointestinal passage of blood 3 days Grows absent
3 S., 57 years; acute peritonitis 25 days K. pneumoniae
Isolation of microorganisms from catheters.
The distal (5 cm) part of the catheters were cut from the entire catheter length and the one segment (up to 2 cm) was placed in a tube with 1 ml of 0,9% NaCl,
which was then vortexed for 1 min. Afterwards, 0.1 ml of the suspension was spread onto blood agar and Sab-ouraud dextrose agar plates. Plates were incubated for 48 h at 37 °C and 28 °C aerobically. Isolated bacteria and yeasts were identified with MALDI-TOF-MS (Bruker DaltonikGmbH).
Scanning electron microscopic investigations
(SEM).
The distal part of the catheter was further cut perpendicular to the catheter length with the blade into 2- to 3 mm-long «doughnut» segments. The catheter segments was fixed in 3% glutaraldehyde (on 061 M cacodylate buffer) for 3 hour, post-fixed overnight in 1% osmium tetroxide in same buffer, dehydrated by ethanol series, critical-point dried (HCP-2) for 15 min, coated with gold and observed in JSM 35 (JEOL, Tokyo, Japan).
results and discussion
From 8 investigated catheters in 4 (50%) we isolated fungi and bacteria, but in remain samples - only bacteria. In all cases fungi was present only one species - C. albicans. In 2 cases of urethral catheters C. albicans was isolated in combination with K. pneumonia and in one - with P. aeruginosa. The venous catheters were a major reservoir and source for Candida species bloodstream infections in hospitalized patients [4]. According the date in literature the urethral catheters can be colonized also by single bacterial species [Ganderton L., et al. // Eur. J. Clin. Microbiol. Infect. Dis. - 1992. - Vol. 11, №9]. We isolated P. aeruginosa only in 1 from 5 studied ure-thral catheters. As reported Ohkawa M. with co-authors [Ohkawa M., et al. //J. of Urol. - 1990. - Vol. 143], this last species often isolated from urethral silicone catheters.
From all studied catheters surfaces, with the exception of 1 venous, we isolated K. pneumonia. Among the investigated samples dominate the 2 mixed-species biofilms with K. pneumonia.
Urethral catheters. Notice, that under SEM the outer surface of control sterile samples (before using, without biofilms) of the urethral catheter have the stable slightly folded specific tiled texture (Fig. 1 a). The surface of catheter's lumen was smooth. On the longitudinal catheter section we may see presence of 2-wo distinct layers: upper more thin (from 1,8 to 2,7 |m) covering (Fig. 1 b, 1) and the remaining more thick part (Fig. 1 b, 2). The material of upper part with more lower contrast, irregular in form and lower - with more high electron density and horizontally localized layers.
Catheter 1. On the outer surface of catheter was visible the presence of biofilm (from 5 to 10 |m) with specific «tiled picture» (Fig. 1 c, arrow) look like the same tiled texture of control one, but more relief. Under higher magnification it was obvious the presence of crack (Fig. 1 d, double arrows) and single spherical cells (Fig. 1 e, arrow), which according its dimensions (5,0 x 6,0 |m) were similar with C. albicans. At the smooth sur-
face (Fig. 1 e, arrow) of internal gleam of analyzed catheter we revealed thick (20,0 | m) good developed biofilm (Fig. 1 f), with 2 good distinguishable borders. The «picture» of the outer surface of this biofilm not similar with outer one and have tiled character (Fig.1 f, double arrows). On the internal catheter's surfaces of biofilm we revealed only massive aggregation of cells (Fig. 1, arrow), which according of its morphology (Fig. 1 g, arrows) and sizes (2,5 x 4,5 |m) possible identify as Malassezia spp.
Catheter 2. On this catheter we revealed biofilm (2.5 |m) only on upper surface (Fig. 1 h, arrow), which sculpture (Fig. 1 i) was similar with 2nd samples, which described before. The individual elements of this biofilm was distinguishable due to the fact that they perimeter were lined with cracks (Fig. 1 j, arrows). Under the higher magnification of SEM on the surface of this biofilm we observed the presence of irregular slime-like fine-spumous surface texture, in which possible recognized bac-uliform cells, typical for K. pneumonia (Fig. 1 k, arrow).
Catheter 3. On longitudinal section clear visible that the wall of himself catheter consist from tightly localized vertical layers (Fig. 1 l, arrow). The biofilms we observed on outer and internal lumen surfaces. They both very good developed and significantly differ according the ultrastructure. As it was evident, the texture biofilm, which localized on outer surface significantly differ from before described sample (catheter 2). This biofilm more thick (from 13,0 to 20,0 |m), with good expressed and deep parallel about each other crack (Fig. 1 m, n, arrows). Under the higher magnification of SEM, on its surface possible see abundant aggregation of extended cells (Fig. 1 o, arrows), which morphologically were similar with P. aeruginosa. For catheter's lumen (Fig. 1 p) was typical presence of more thin (from 4 to 5 |m) biofilm with specific texture and often crystallic inclusions (Fig. 1 q). Sometimes possible observed semi-submerged spherical cells (Fig. 1 q, arrow) which, perhaps, possible identify as C. albicans according with their morphology, size (4,05,5 x 6,0-6,5) and microbiological data.
Fig. 1. SEM electronnograms of the surfaces of urethral catheters. Numerations on b demonstrated the layers on the catheters wall. Scale : a-f, h-j, l-n, p-v - X10000; g, k, o - X30000
Catheter 4. In this catheter biofilm completely cover upper surface and lumen. Biofilm (from 5 to 10 |m) on outer surface posses with tiled texture (Fig. 2 r). In contrast, the biofilm in catheter's lumen surface was more flat, but with specific reticular organization (Fig. 2 s). We not revealed the profile of bacterial or fungal cells on the on both catheter's surface.
Catheter 5. On these samples, we revealed biofilm on both upper catheter's surface and in lumen. Biofilm on upper catheter's surface was thin (from 2 to 4 |m) and with regular tiled surfaces (Fig. 2 t). On the periphery of the tiled elements clear visible cracks (Fig. 1 v, arrow). The biofilm on lumen surface (Fig. 1 u) with fine-granular (Fig. 1 v) texture. The images of bacterial cells on the biofilm surfaces were absent.
Fig. 2. The general view and fragments of the venous catheters surfaces. Scale: a, b, k - x 10000; c, d, e, g, j - X15000; f - x3000; h - 12000; i, l - X25000
Venous catheters. The outer surface of control
sterile catheter's samples under SEM, contrary with urethral one, was smooth with rare small and parallel oriented each other folders (Fig. 2 a, arrows). The same outer surface texture was revealed Marrie S. J. with coauthors [2] in sterile unused intravenous catheter. On the longitudinal catheter section visible not uniform «fine-vesicular» internal structure of the catheter's wall (Fig. 2 b). In was obvious, that the venous catheter in contrast with urethral, have not special outer clear distinguishable cover.
Catheter 1. On the outer catheter's surface we revealed only one large-sized (40,0 x 150,0 |m) discrete biofilm (Fig. 2 c, arrows) and one small-sizes (12.0x12.0 |m) scales. In both cases the biofilms were very thin (from 2 to 3 |m). The first scale presents the aggregation of the separated small ones. In the lumen of catheter we observed the biofilm-like formation (Fig. 2 d, c) with thickness from 5 to 10 |m, which outside and inside contain the groups of blood's elements. In the biofilms of both surfaces of this catheter the bacterial and fungal cells we not revealed.
Catheter 2. Despite the fact that from this catheter we not isolated microorganisms, the biofilms were good developed on both surfaces. Biofilm on the outer surface of the catheter consist from the slime-like matrix (from 2 to 3 |m, Fig. 2 g) with reticulate texture of surface, in which were immersed the spherical (5,0-6,0 |m) or slightly elongated (5x6 |m) numerous single or in small groups cell (Fig. 2 g, arrows). The surfaces of revealed cells were cover with slime-like material. The similar morphology of the extracellular matrix was revealed in composition of venous catheter's biofilm with other authors [2]. In a whole, the thickness of outer biofilm varied from 6 to 7 |m. We revealed good developed falciform biofilm on the lumen of catheter (Fig. 2 f), which thickness in central part was equal 125,0 |m. Under the higher magnification of SEM, it was clear visible differences in the surface texture of the internal (in lumen) biofilm (Fig. 2 h). The longitudinal surfaces of last composed from tightly localized spherical or slightly elongated profiles of cells (Fig. 2), which according the sizes and form comparable with the same on composition of outer biofilm. The cells were covered with homogeneous slime-like material, in which possible recognized the holes (from 5 to 10 |m, Fig. 2 l, single arrow). Among the cells possible recognized the thin short bands (Fig. 2 i, double arrows) of slime. The biofilm's surface, which reverses in the lumen (Fig. 2 j) be astonished with its texture comparable with pattern of «high architecture»: variable in size and form inclusions «wind» with thing lengthy bands of slime. Thus, in this samples the cellular composition, perhaps, were the same in both catheters surfaces. We suggest that it was the yeast cells of the Candida spp. The affect the fact, that using of blade for cutting of samples not «cut» the cells of biofilm. This fact indicates evidence of protective function of slime in composition of biofilm matrix. Perhaps, the «highly organized architecture» of solid biofilm construction of these samples to prevent its
destruction during the using of shaker and as a result, the absence of growth.
Catheter 3. In this catheter we find the biofilm only on upper surface. According the general macro- and microstructure, the last (Fig. 2 k) was similar with the same, described for previous samples, but they was thicker (from 8 to 10 |m). The biofilm matrix was also thicker, in contrast with the same of previous catheter, and its surfaces was more smooth and with fine-reticular structure (Fig. 2 l). In slime matrix were immersed the numerous spherical (4,5-5,0 |m) or elongated cells (4,5 x 6,0 |m). In a whole, it was more mature type of biofilm (25 days after using of this venous catheter).
resume
Microbiological investigations show, that among the analyzed samples dominate the mixed-species biofilms. Electron microscopic data demonstrate, that in the outer surfaces of the urethral catheters, in comparison with venous, were formed more massive biofilms. This fact we associate with the presence of roughness on its outer surfaces. It was very similar the character of outer surface biofilms texture in 1st and 2nd urethral catheters from which we isolated identical microorganisms (C. albicans and K. pneumonia). The surface texture of 3rd urethral catheter (combination of C. albicans and P. aeruginosa) differ significantly from another one and «reproduce» also the longitudinal irregularities in wall of these samples. It was obvious the special peculiarity in the biofilms of outer surfaces of 4th and 5th urethral catheters from which we have not fungal sowing, but together with K. pneumonia we isolated the another species of bacteria - C. amylolatum and S. haemoliticus. On more smooth, outer venous catheter surface we revealed more small discrete biofilms, composed from several scales. The general pattern of biofilms texture of 1st and 2nd venous catheters was practically the same. This biofilms was thin and occupy the small area in comparison with time of their using (respectively 10 and 25 days). Very indicative the 2nd venous catheter, which demonstrated the good developed biofilms on both samples surfaces in the absence of sowing. Undoubtedly, that on the biofilm microbiological composition effect the differences in the chemical composition of its materials, also internal and external liquids, which surround they and the composition of drugs from which the used. Opinion of Anaissie with co-authors [Anaissie E., et al.//Eur. J. Clin. Microbiol. Infect. Dis. - 1995. - Vol. 14, Iss. 2.], that the extent of biofilm formation was unrelated to the clinical status, was fully conforming to our data.
Inconceivable, but it was obvious, that the fungal-bacterial biofilms «able the scan and reproduce» the character of solid catheter surfaces.
In investigated in present work biofilms bacterial and fungal cells immersed in slime-like matrix. As decided several authors [Nickel J.C., et al. // Antimicrobial Agents and Chemotherapy. - 1985. - Vol. 27; Jensen E.T., et al. // Infect. Imm. - 1990. - Vol. 58 ], this last fact increased the resistance of biofilms to antibiotics, antisep-
tics and host defense mechanisms in contrast with cells growing in suspension. Thus, actually searching the new materials for catheters production. Finally, it was obvious, that during this process exactly necessary preliminary combined microbiological and SEM investigations.
acknowledgements
The authors were grateful to main specialist of the Center of Collective Equipment Using of the Komarov Botanical Institute of the Russian Academy of Sciences Karceva L.A. for technical assistance during samples preparation and SEM exploiting.
references
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2. Ahmad I., Khan M.S.A. Microscopy in mycological research with especial reference to ultrastructures and biofilm studies // Curr. Microscopy Contrib. to Advances and Technology. - 2012. - P. 646-659.
3. Andes D., Nett J., OschelP., et al. Development and characterization of an in vivo central venous catheter Candida albicans biofilm model // Infect. Immun. - 2004. - Vol. 72, №10. - P. 6023-6031.
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Поступила в редакцию журнала 03.12.2014 Рецензент: М.А. Шевяков
