Urinary or ureteral catheter insertion remains one of the most common urological procedures, yet is considered a predisposing factor for urinary tract infection. Diverse bacterial consortia adhere to foreign body surfaces and create various difficult to treat biofilm structures. We analyzed 347 urinary catheter- and stent-related samples, treated with sonication, using both routine culture and broad-range 16S rDNA PCR followed by Denaturing Gradient Gel Electrophoresis and Sanger sequencing (PCR-DGGE-S). In 29 selected samples, 16S rRNA amplicon Illumina sequencing was performed. The results of all methods were compared. In 338 positive samples, from which 86.1% were polybacterial, 1,295 representatives of 153 unique OTUs were detected. Gram-positive microbes were found in 46.5 and 59.1% of catheter- and stent-related samples, respectively. PCR-DGGE-S was shown as a feasible method with higher overall specificity (95 vs. 85%, p < 0.01) though lower sensitivity (50 vs. 69%, p < 0.01) in comparison to standard culture. Molecular methods considerably widened a spectrum of microbes detected in biofilms, including the very prevalent emerging opportunistic pathogen Actinotignum schaalii. Using massive parallel sequencing as a reference method in selected specimens, culture combined with PCR-DGGE was shown to be an efficient and reliable tool for determining the composition of urinary catheter-related biofilms. This might be applicable particularly to immunocompromised patients, in whom catheter-colonizing bacteria may lead to severe infectious complications. For the first time, broad-range molecular detection sensitivity and specificity were evaluated in this setting. This study extends the knowledge of biofilm consortia composition by analyzing large urinary catheter and stent sample sets using both molecular and culture techniques, including the widest dataset of catheter-related samples characterized by 16S rRNA amplicon Illumina sequencing.
- Publikační typ
- časopisecké články MeSH
Complex samples are a challenge for sequencing-based broad-range diagnostics. We analysed 19 urinary catheter, ureteral Double-J catheter, and urine samples using 3 methodological approaches. Out of the total 84 operational taxonomic units, 37, 61, and 88% were identified by culture, PCR-DGGE-SS (PCR denaturing gradient gel electrophoresis followed by Sanger sequencing), and PCR-DGGE-RM (PCR- DGGE combined with software chromatogram separation by RipSeq Mixed tool), respectively. The latter approach was shown to be an efficient tool to complement culture in complex sample assessment.
- MeSH
- Bacteria klasifikace genetika izolace a purifikace MeSH
- denaturační gradientová gelová elektroforéza metody MeSH
- diagnostické techniky molekulární metody MeSH
- diagnostické techniky urologické MeSH
- DNA bakterií MeSH
- lidé MeSH
- moč chemie mikrobiologie MeSH
- močové katétry mikrobiologie MeSH
- polymerázová řetězová reakce metody MeSH
- software * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
More than 40% of nosocomial infections are those of the urinary tract, most of these occurring in catheterized patients. Bacterial colonization of the urinary tract and catheters results not only in infection, but also various complications, such as blockage of catheters with crystalline deposits of bacterial origin, generation of gravels and pyelonephritis. The diversity of the biofilm microbial community increases with duration of catheter emplacement. One of the most important pathogens in this regard is Proteus mirabilis. The aims of this study were to identify and assess particular virulence factors present in catheter-associated urinary tract infection (CAUTI) isolates, their correlation and linkages: three types of motility (swarming, swimming and twitching), the ability to swarm over urinary catheters, biofilm production in two types of media, urease production and adherence of bacterial cells to various types of urinary tract catheters. We examined 102 CAUTI isolates and 50 isolates taken from stool samples of healthy people. Among the microorganisms isolated from urinary catheters, significant differences were found in biofilm-forming ability and the swarming motility. In comparison with the control group, the microorganisms isolated from urinary catheters showed a wider spectrum of virulence factors. The virulence factors (twitching motility, swimming motility, swarming over various types of catheters and biofilm formation) were also more intensively expressed.
- MeSH
- bakteriální adheze MeSH
- biofilmy růst a vývoj MeSH
- faktory virulence metabolismus MeSH
- infekce bakteriemi rodu Proteus mikrobiologie MeSH
- infekce močového ústrojí mikrobiologie MeSH
- katétrové infekce mikrobiologie MeSH
- lidé MeSH
- lokomoce MeSH
- Proteus mirabilis izolace a purifikace patogenita fyziologie MeSH
- ureasa metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Nozokomiální infekce spojené s tvorbou biofilmu představují v posledních letech závažný problém. Až 32 % těchto infekcí představují infekce močového traktu u pacientů s dlouhodobě zavedeným katétrem. Katétry tvoří vhodný povrch pro přisednutí bakterií a umožňují snazší kolonizaci močových cest. Mezi významné původce těchto infekcí patří bakterie rodu Proteus, které ke kolonizaci katétrů využívají nejen tvorby biofilmu, ale také další faktory virulence. Práce byla zaměřena na vybrané faktory virulence, mezi které byly zařazeny tři typy motility, swarming motility přes různé typy močových katétrů, produkce biofilmu v různých médiích, na katétru, produkce hemolyzinů a ureázy. Celkem bylo vyšetřeno 102 izolátů z močových katétrů a 50 izolátů ze stolice. U twitching motility byl statisticky prokázán rozdíl mezi izoláty z katétrů a ze stolic (p = 0,012), u swimming a swarming motility nebyl tento rozdíl signifikantní (p = 0,074; resp. p = 0,809). V pohyblivosti přes různé typy katétrů byl prokázán statisticky signifikatní rozdíl u izolátů z katétrů i ze stolic (v obou případech p << 0,01). Pro test na tvorbu biofilmu byla použita mozkosrdcová infúze (BHI) a BHI s 4 % glukózy. V BHI tvořily biofilm všechny izoláty, 65 % izolátů z katétrů a 88 % izolátů ze stolic bylo silnými producenty. Všechny izoláty tvořily biofilm také v BHI s 4 % glukózy, kdy bylo silnými producenty 94 % izolátů z katétrů a 92 % izolátů ze stolic. Ve tvorbě biofilmu na katétru byl statisticky prokázán rozdíl mezi izoláty z katétrů a ze stolic (p = 0,00008). Všechny izoláty z katétrů i ze stolic produkovaly ureázu, statisticky nebyl v produkci ureázy prokázán rozdíl (p = 0,653). Na agaru s propíranými beraními erytrocyty nebyla prokázána produkce hemolyzinů u žádného z izolátů. Kvantitativní metodou, při které byly použity koňské erytrocyty, byla prokázána tvorba hemolyzinů u tří izolátů z katétrů.
Nosocomial infections associated with biofilm formation have been a serious problem in recent years. Up to 32% of them are urinary tract infections in patients with long-dwelling catheters. Catheters represent an ideal surface for bacterial adhesion, facilitating easier colonization of the urinary tract. Important pathogens causing these infections are bacteria of the genus Proteus that colonize catheters not only by biofilm formation but also using other virulence factors. Those were developed for survival in the host organism and are also used by bacteria to infect the host or fight the defence mechanisms. The study focused on the following selected virulence factors: swimming, swarming and twitching motility, swarming motility across various types of urinary catheters, biofilm formation in various media, formation of biofilm on catheters, haemolysin and urease production. A total of 102 strains isolated from urinary catheters and 50 strains isolated from stools were analyzed. In twitching motility, a difference between strains isolated from catheters and stools was statistically significant (p = 0.012). In swimming and swarming motility, the difference was not significant (p = 0.074 and p = 0.809, respectively). In motility across various catheter types, a statistically significant difference was found in strains isolated from both catheters and stools (p << 0.01 in both cases). For biofilm formation analyses, BHI and BHI with 4% glucose were used. In BHI, biofilm was produced by all strains, with 65% of catheter strains and 88% of strains from stools being strong producers. Similarly, all strains produced biofilm in BHI with 4% glucose, with strong producers in 94% and 92% of strains isolated from catheters and stools, respectively. In formation of biofilm on catheters, there was a statistical difference between strains from catheters and stools (p = 0.00008). All strains isolated from both catheters and stools produced urease; no difference in urease production was statistically significant (p = 0.653). On agar with washed sheep erythrocytes, haemolysin production was not detected in any of the isolated strains. The quantitative method using horse erythrocytes revealed haemolysis production in three strains isolated from catheters.
- Klíčová slova
- infekce močového traktu, biofilm,
- MeSH
- biofilmy MeSH
- faktory virulence analýza MeSH
- infekce močového ústrojí mikrobiologie MeSH
- infekce spojené se zdravotní péčí mikrobiologie MeSH
- katetrizace močového měchýře škodlivé účinky MeSH
- katétrové infekce MeSH
- kontaminace zdravotnického vybavení MeSH
- lidé MeSH
- Proteus mirabilis fyziologie izolace a purifikace patogenita MeSH
- Check Tag
- lidé MeSH
