Filip Ruzicka

Quantification of biofilm in microtiter plates: overview of testing conditions and practical recommendations for assessment of biofilm production by staphylococci

The details of all steps involved in the quantification of biofilm formation in microtiter plates are described. The presented protocol incorporates information on assessment of biofilm production by staphylococci, gained both by direct experience as well as by analysis of methods for assaying biofilm production. The obtained results should simplify quantification of biofilm formation in microtiter plates, and make it more reliable and comparable among different laboratories.

Microbial diversity in biofilm infections of the urinary tract with the use of sonication techniques.

Infections of the urinary tract account for >40% of nosocomial infections; most of these are infections in catheterized patients. Bacterial colonization of the urinary tract and catheters causes not only the particular infection but also a number of complications, for example blockage of catheters with crystallic deposits of bacterial origin, generation of gravels and pyelonephritis. Infections of urinary catheters are only rarely single-species infections. The longer a patient is catheterized, the higher the diversity of biofilm microbial communities. The aims of this study were to investigate the microbial diversity on the catheters and to compare the ability to form biofilm among isolated microbial species. The next aim was to discriminate particular causative agents of infections of the urinary tract and their importance as biofilm formers in the microbial community on the urinary catheter. We examined catheters from 535 patients and isolated 1555 strains of microorganisms. Most of the catheters were infected by three or more microorganisms; only 12.5% showed monomicrobial infection. Among the microorganisms isolated from the urinary catheters, there were significant differences in biofilm-forming ability, and we therefore conclude that some microbial species have greater potential to cause a biofilm-based infection, whereas others can be only passive members of the biofilm community.

A case of endocarditis caused by the yeast Pichia fabianii with biofilm production and developed in vitro resistance to azoles in the course of antifungal treatment

Pichia fabianii, a yeast rarely causing human infections, was isolated from the blood of a patient with aortic valve endocarditis. The isolates were initially identified biochemically as Candida pelliculosa, but based on direct sequencing of the ITS2 region of rRNA, they were subsequently reidentified as P. fabianii. Antifungal therapy with fluconazole and later with voriconazole led to the development of resistant variants which had high MIC values to both antifungals. Strong biofilm formation by this yeast could also have played a role in the development of its resistance and allowed for its persistence on the infected valve during antifungal therapy. To our knowledge, this is the first published case of endocarditis and the fourth human infection caused by this yeast species.

Virulence factors in Proteus bacteria from biofilm communities of catheter-associated urinary tract infections

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.

The effectiveness of dried cranberries (Vaccinium macrocarpon) in men with lower urinary tract symptoms.

Lower urinary tract symptoms (LUTS) are a common condition in older men. The objective of the present study was to evaluate the efficacy and tolerability of cranberry (Vaccinium macrocarpon) powder in men at risk of prostate disease with LUTS, elevated prostate-specific antigen (PSA), negative prostate biopsy and clinically confirmed chronic non-bacterial prostatitis. Forty-two participants received either 1500 mg of the dried powdered cranberries per d for 6 months (cranberry group; n 21) or no cranberry treatment (control group; n 21). Physical examination, International Prostate Symptom Score, quality of life (QoL), five-item version of the International Index of Erectile Function (IIEF-5), basic clinical chemistry parameters, haematology, Se, testosterone, PSA (free and total), C-reactive protein (CRP), antioxidant status, transrectal ultrasound prostate volume, urinary flow rate, ultrasound-estimated post-void residual urine volume at baseline, and at 3 and 6 months, and urine ex vivo anti-adherence activity were determined in all subjects. In contrast to the control group, patients in the cranberry group had statistically significant improvement in International Prostate Symptom Score, QoL, urination parameters including voiding parameters (rate of urine flow, average flow, total volume and post-void residual urine volume), and lower total PSA level on day 180 of the study. There was no influence on blood testosterone or serum CRP levels. There was no statistically significant improvement in the control group. The results of the present trial are the first firm evidence that cranberries may ameliorate LUTS, independent of benign prostatic hyperplasia or C-reactive protein level.

Prevalence of Propionibacterium acnes in Intervertebral Discs of Patients Undergoing Lumbar Microdiscectomy: A Prospective Cross-Sectional Study

Background The relationship between intervertebral disc degeneration and chronic infection by Propionibacterium acnes is controversial with contradictory evidence available in the literature. Previous studies investigating these relationships were under-powered and fraught with methodical differences; moreover, they have not taken into consideration P. acnes’ ability to form biofilms or attempted to quantitate the bioburden with regard to determining bacterial counts/genome equivalents as criteria to differentiate true infection from contamination. The aim of this prospective cross-sectional study was to determine the prevalence of P. acnes in patients undergoing lumbar disc microdiscectomy. Methods and Findings The sample consisted of 290 adult patients undergoing lumbar microdiscectomy for symptomatic lumbar disc herniation. An intraoperative biopsy and pre-operative clinical data were taken in all cases. One biopsy fragment was homogenized and used for quantitative anaerobic culture and a second was frozen and used for real-time PCR-based quantification of P. acnes genomes. P. acnes was identified in 115 cases (40%), coagulase-negative staphylococci in 31 cases (11%) and alpha-hemolytic streptococci in 8 cases (3%). P. acnes counts ranged from 100 to 9000 CFU/ml with a median of 400 CFU/ml. The prevalence of intervertebral discs with abundant P. acnes (≥ 1x103 CFU/ml) was 11% (39 cases). There was significant correlation between the bacterial counts obtained by culture and the number of P. acnes genomes detected by real-time PCR (r = 0.4363, p<0.0001). Conclusions In a large series of patients, the prevalence of discs with abundant P. acnes was 11%. We believe, disc tissue homogenization releases P. acnes from the biofilm so that they can then potentially be cultured, reducing the rate of false-negative cultures. Further, quantification study revealing significant bioburden based on both culture and real-time PCR minimize the likelihood that observed findings are due to contamination and supports the hypothesis P. acnes acts as a pathogen in these cases of degenerative disc disease.

The differences in the isoelectric points of biofilm-positive and biofilm-negative Candida parapsilosis strains

The isoelectric points of 39 Candida parapsilosis strains were determined by means of capillary isoelectric focusing. The value of the isoelectric point corresponded well with cell surface hydrophobicity, as well as with the ability to form biofilm in these yeasts.

Propionibacterium acnes biofilm is present in intervertebral discs of patients undergoing microdiscectomy.

Background In previous studies, Propionibacterium acnes was cultured from intervertebral disc tissue of ~25% of patients undergoing microdiscectomy, suggesting a possible link between chronic bacterial infection and disc degeneration. However, given the prominence of P. acnes as a skin commensal, such analyses often struggled to exclude the alternate possibility that these organisms represent perioperative microbiologic contamination. This investigation seeks to validate P. acnes prevalence in resected disc cultures, while providing microscopic evidence of P. acnes biofilm in the intervertebral discs. Methods Specimens from 368 patients undergoing microdiscectomy for disc herniation were divided into several fragments, one being homogenized, subjected to quantitative anaerobic culture, and assessed for bacterial growth, and a second fragment frozen for additional analyses. Colonies were identified by MALDI-TOF mass spectrometry and P. acnes phylotyping was conducted by multiplex PCR. For a sub-set of specimens, bacteria localization within the disc was assessed by microscopy using confocal laser scanning and FISH. Results Bacteria were cultured from 162 discs (44%), including 119 cases (32.3%) with P. acnes. In 89 cases, P. acnes was cultured exclusively; in 30 cases, it was isolated in combination with other bacteria (primarily coagulase-negative Staphylococcus spp.) Among positive specimens, the median P. acnes bacterial burden was 350 CFU/g (12 - ~20,000 CFU/g). Thirty-eight P. acnes isolates were subjected to molecular sub-typing, identifying 4 of 6 defined phylogroups: IA1, IB, IC, and II. Eight culture-positive specimens were evaluated by fluorescence microscopy and revealed P. acnes in situ. Notably, these bacteria demonstrated a biofilm distribution within the disc matrix. P. acnes bacteria were more prevalent in males than females (39% vs. 23%, p = 0.0013). Conclusions This study confirms that P. acnes is prevalent in herniated disc tissue. Moreover, it provides the first visual evidence of P. acnes biofilms within such specimens, consistent with infection rather than microbiologic contamination.

Differentiation of Staphylococcus spp. by high- resolution melting analysis

High-resolution melting analysis (HRMA) is a fast (post-PCR) high-throughput method to scan for sequence variations in a target gene. The aim of this study was to test the potential of HRMA to distinguish particular bacterial species of the Staphylococcus genus even when using a broad-range PCR within the 16S rRNA gene where sequence differences are minimal. Genomic DNA samples isolated from 12 reference staphylococcal strains (Staphylococcus aureus, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus intermedius, Staphylococcus saprophyticus, Staphylococcus sciuri, Staphylococcus simulans, Staphylococcus warneri, and Staphylococcus xylosus) were subjected to a real-time PCR amplification of the 16S rRNA gene in the presence of fluorescent dye EvaGreen™, followed by HRMA. Melting profiles were used as molecular fingerprints for bacterial species differentiation. HRMA of S. saprophyticus and S. xylosus resulted in undistinguishable profiles because of their identical sequences in the analyzed 16S rRNA region. The remaining reference strains were fully differentiated either directly or via high-resolution plots obtained by heteroduplex formation between coamplified PCR products of the tested staphylococcal strain and phylogenetically unrelated strain.

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.