Molecular Techniques Complement Culture-Based Assessment of Bacteria Composition in Mixed Biofilms of Urinary Tract Catheter-Related Samples.

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.

Human native endocarditis caused by Streptococcus canis-a case report.

We report a very rare case of Streptococcus canis native infective endocarditis in a 73-year-old woman living in close contact with her dog. Her echocardiography showed large calcifications in the mitral annulus, massive regurgitation below the posterior leaflet, and adjacent vegetation. Blood culture was positive for Streptococcus Lancefield group G. A coronary artery bypass and mitral valve replacement had to be done. Streptococcus canis was detected in a heart valve using a broad range PCR followed by 16S rRNA and confirmed by tuf gene sequencing, while tissue culture remained negative. The patient was not bitten by her dog nor did she have comorbidities or skin ulcers. She fully recovered.

Contribution of PCR Denaturing Gradient Gel Electrophoresis Combined with Mixed Chromatogram Software Separation for Complex Urinary Sample Analysis.

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.

First report of Sneathia sanguinegens together with Mycoplasma hominis in postpartum prosthetic valve infective endocarditis: a case report.

BACKGROUND: The presence of more than one bacterial agent is relatively rare in infective endocarditis, although more common in prosthetic cases. Molecular diagnosis from a removed heart tissue is considered a quick and effective way to diagnose fastidious or intracellular agents. CASE PRESENTATION: Here we describe the case of postpartum polymicrobial prosthetic valve endocarditis in a young woman. Sneathia sanguinegens and Mycoplasma hominis were simultaneously detected from the heart valve sample using broad range 16S rRNA polymerase chain reaction (PCR) followed by sequencing while culture remained negative. Results were confirmed by independent PCR combined with denaturing gradient gel electrophoresis. Before the final agent identification, the highly non-compliant patient left from the hospital against medical advice on empirical intravenous treatment with aminopenicillins, clavulanate and gentamicin switched to oral amoxycillin and clavulanate. Four months after surgery, no signs of inflammation were present despite new regurgitation and valve leaflet flail was detected. However, after another 5 months the patient died from sepsis and recurrent infective endocarditis of unclarified etiology. CONCLUSIONS: Mycoplasma hominis is a rare causative agent of infective endocarditis. To the best of our knowledge, presented case is the first report of Sneathia sanguinegens detected in this condition. Molecular techniques were shown to be useful even in polymicrobial infective endocarditis samples.

Biofilm formation, antibiotic susceptibility and RAPD genotypes in Pseudomonas aeruginosa clinical strains isolated from single centre intensive care unit patients.

The aim of this study was to analyse genotypes, antimicrobial susceptibility patterns and serotypes in Pseudomonas aeruginosa clinical strains, including the clonal dissemination of particular strains throughout various intensive care units in one medical centre. Using random amplified polymorphic DNA (RAPD-PCR) and P. aeruginosa antisera, 22 different genotypes and 8 serotypes were defined among 103 isolates from 48 patients. No direct association between P. aeruginosa strain genotypes and serotypes was observed. RAPD typing in strains with the same serotype revealed different genotypes and, on the contrary, most strains with a different serotype displayed the same amplification pattern. The resulting banding patterns showed a high degree of genetic heterogeneity among all isolates from the patients examined, suggesting a non-clonal relationship between isolates from these patients. A higher degree of antibiotic resistance and stronger biofilm production in common genotypes compared to rare ones and genetic homogeneity of the most resistant strains indicated the role of antibiotic pressure in acquiring resistant and more virulent strains in our hospital. In conclusion, genetic characterisation of P. aeruginosa strains using RAPD method was shown to be more accurate in epidemiological analyses than phenotyping.

Rapid identification of medically important Candida isolates using high resolution melting analysis.

An increasing trend in non albicans infections and various susceptibility patterns to antifungal agents implies a requirement for the quick and reliable identification of a number of medically important Candida species. Real-time PCR followed by high resolution melting analysis (HRMA) was developed, tested on 25 reference Candida collection strains and validated on an additional 143 clinical isolates in this study. All reference strains and clinical isolates inconclusive when using phenotypic methods and/or HRMA were analysed using ITS2 sequencing. Considering reference and clinical strains together, 23 out of 27 Candida species could be clearly distinguished by HRMA, while the remaining 4 species were grouped in 2 pairs, when applying the mean Tm ± 3 SD values, the shape of the derivative melting curve (dMelt curve) and, in some cases, the normalized and temperature-shifted difference plot against C. krusei. HRMA as a simple, rapid and inexpensive tool was shown to be useful in identifying a wide spectrum of clinically important Candida species. It may complement the current clinical diagnostic approach based on commercially available biochemical kits.

Poly(ethylene glycol) adlayers immobilized to metal oxide substrates through catechol derivatives: influence of assembly conditions on formation and stability.

We have investigated five different poly(ethylene glycol) (PEG, 5 kDa) catechol derivatives in terms of their spontaneous surface assembly from aqueous solution, adlayer stability, and resistance to nonspecific blood serum adsorption as a function of the type of catechol-based anchor, assembly conditions (temperature, pH), and type of substrate (SiO(2), TiO(2), Nb(2)O(5)). Variable-angle spectroscopic ellipsometry (VASE) was used for layer thickness evaluation, X-ray photoelectron spectroscopy (XPS) for layer composition, and ultraviolet-visible optical spectroscopy (UV-vis) for cloud point determination. Polymer surface coverage was influenced by the type of catechol anchor, type of the substrate, as well as pH and temperature (T) of the assembly solution. Furthermore, it was found to be highest for T close to the cloud point (T(CP)) and pH of the assembly solution close to pK(a1) (dissociation constant of the first catechol hydroxy group) of the polymer and to the isoelectric point (IEP) of the substrate. T(CP) turned out to depend on not only the ionic strength of the assembly solution, but also the type of catechol derivative and pH. PEG-coating dry thickness above 10 A correlated with low serum adsorption. We therefore conclude that optimum coating protocols for catechol-based polymer assembly at metal oxide interfaces have to take into account specific physicochemical properties of the polymer, anchor, and substrate.

Protein-resistant surfaces through mild dopamine surface functionalization.

The synthesis and evaluation of new dopamine-based catechol anchors coupled to poly(ethylene glycol) (PEG) for surface modification of TiO(2) are reported. Dopamine is modified by dimethylamine-methylene (7) or trimethylammonium-methylene (8) groups, and the preparation of mPEG-Glu didopamine polymer 11 is presented. All these PEG polymers allow stable adlayers on TiO(2) to be generated through mild dip-and-rinse procedures, as evaluated both by variable angle spectroscopic ellipsometry and X-ray photoelectron spectroscopy. The resulting surfaces substantially reduced protein adsorption upon exposure to full human serum.

Biomimetic surface modifications based on the cyanobacterial iron chelator anachelin.

Siderophores are natural iron chelators that have been evolutionarily selected to bind to Fe ions with very high binding constants. We utilize these unique properties to bind to metal oxide surfaces using a fragment of the cyanobacterial siderophore anachelin. The resulting poly(ethylene glycol) conjugate forms stable adlayers on TiO2 as has been shown by variable angle spectroscopic ellipsometry and X-ray photoelectron spectroscopy. Moreover, these coated surfaces are highly protein-resistant against the adsorption of full human serum.