Ustav mikrobiologie

Czech Republic

Ustav mikrobiologie

Czech Republic
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PubMed | GeneProof a. s. and Ustav mikrobiologie
Type: Journal Article | Journal: Klinicka mikrobiologie a infekcni lekarstvi | Year: 2014

A new method has been developed for detecting genes determining the extended-spectrum beta-lactamase (ESBL) phenotype directly from patients clinical material. The method enables detection of the bla(CTX-M) gene encoding CTX-M beta-lactamases and the bla(SHV) gene variants with real-time PCR technology using locked nucleic acid oligonucleotides.In this pilot study, tracheal aspirates obtained from patients with mechanical ventilation hospitalized at Department of Anaesthesiology and Resuscitation of the University Hospital in Olomouc between 1st March and 30th December 2010 period were tested. Each sample was identified with standard microbiological procedures including phenotypic determination of ESBL-positive enterobacteria. At the same time, each sample was analyzed for the presence of nucleic acids (DNA) which encode CTX-M and SHV ESBL using real-time PCR.150 samples of tracheal aspirates from 71 patients were included into testing. In the set, 13 (8.7%) ESBL-positive samples were identified by culture methods while 27 (18 %) positive samples were identified by the real-time PCR method. Of the 27 PCR-positive samples, 24 were positive for the bla(CTX) gene; in 2 samples, the ESBL bla(SHV) gene was detected, and both genes were present in 1 sample. All culture-positive samples were also PCR-positive for the presence of bla(CTX) and/or bla(SHV) sequences.The new real-time PCR assay is likely to shorten the time for detection of enterobacteria producing SHV and CTX-M beta-lactamases from 48 to 6 hours. It enables ESBL-positive enterobacteria determination in tracheal aspirates of patients suffered from life-threatening nosocomial pneumonia where the early introduction of adequate antimicrobial treatment plays the important role.


Kolar M.,Ustav Mikrobiologie | Htoutou Sedlakova M.,Ustav Mikrobiologie | Suchankova H.,Ustav Klinicke Farmakologie | Hanulik V.,Ustav Mikrobiologie
Klinicka Mikrobiologie a Infekcni Lekarstvi | Year: 2013

As a result of high resistance of bacterial pathogens to broad-spectrum penicillins and cephalosporins, carbapenems have been increasingly used recently. The presented study aimed at analyzing the association between carbapenem consumption and resistance of selected Gram-negative pathogens to meropenem. Using linear regression analysis, a statistically significant association was found between carbapenem consumption and resistance of Pseudomonas aeruginosa.


Koudela Jr. K.,Klinika Ortopedie a Traumatologie Pohyboveho Ustroji | Geigerova L.,Ustav Mikrobiologie | Hes O.,Sikluv Patologicko Anatomicky Ustav | Koudela Sr. K.,Klinika Ortopedie a Traumatologie Pohyboveho Ustroji
Acta Chirurgiae Orthopaedicae et Traumatologiae Cechoslovaca | Year: 2010

PURPOSE OF THE STUDY: To make comprehensive diagnoses of the infections associated with revision total knee and hip arthroplasties in our group of patients MATERIAL AND METHODS: From September 2002 till November 2004, a group of 69 patients undergoing revision total joint replacement (65 hips and four knees) were evaluated. The period between primary and revision surgery ranged from 6 months to 25 years. The patients were examined for CRP, erythrocyte sedimentation rate (ESR) and white blood cell (WBC) counts. The samples of their periprosthetic tissue were assessed for biopsy and microbial findings. The removed prosthetic components were sonicated. The samples were cultured in both aerobic and anaerobic conditions for 16 days. A finding of more than 10 neutrophils per viewing field was taken as a positive biopsy result. The definition of an infection was based on the detection of a microorganism with the identical phenotype in two or more samples. RESULTS: Before surgery, 13 patients had a suspected infection which was subsequently diagnosed. A positive culture result in at least one of the collected samples was found in 48 patients; of these, a positive culture finding of a phenotypically identical microorganism in two or three samples was in 32 patients, who thus met the definition of infection. The average values for the whole group were: CRP, 16 mg/L (1-109); ESR, 25 mm/h (3-110); peripheral WBC count, 6.2x109/L (3.6-11.6). The microorganisms most frequently growing in culture were coagulase-negative staphylococci and propionibacteria accounting for 41 % and 29 % of the total isolates obtained, respectively. From the total number of samples, positive culture results were obtained in 36 % of sonicate femoral components; 40 % of sonicate acetabular cups, 51 % of periprosthetic tissues and 48 % of swabs. In these positive microbial cultures strictly anaerobic microorganisms were found in 41 % of femoral component, 49 % acetabular component and 42 % periprosthetic tissue samples and in 27 % of swabs taken at arthrotomy. Prolonged cultivation of the 151 isolates initially obtained yielded 81 (54%) isolates which would have failed to be detected by primary culture. The results of laboratory tests in the patients with negative culture findings, in those with a phenotypically identical microorganism found in one sample, and in those with positive findings in two or more samples were: CRP, 4.3mg/L; 9.8mg/L; and 21.7mg/L, respectively; ERS, 13.5mm/h; 20.1mm/h; and 33.0mm/h, respectively; and WBC counts, 6.27x109/L; 6.25 x109/L; and 6.16 x109/L, respectively. The t-test was used for the statistical analysis of CRP, ESR and WBC count values, and it revealed a significant differences between the patients with negative microbial findings and those with positive microbial findings in two and more samples in all three values, i.e., CRP (p=0.01), ESR (p=0.01) and WBCs (p=0.96). Biopsy findings showed a sensitivity of 62.5 % and a specificity of 91 % in relation to the microbial findings. DISCUSSION: Our results as well as relevant literature data suggest that microorganisms may survive on implant surfaces even in the cases regarded as aseptic. They often grow slowly and, theoretically, can have an adverse effect on the longevity of revision arthroplasty. However, because of current endoprosthetic practices and the ubiquitous presence of microorganisms, contamination of some samples cannot be excluded. CONCLUSIONS: In our group of patients, the CRP and ESR values proved to be useful in making the diagnosis of infection. For this purpose, WBC counts in blood samples were not sensitive enough. Biopsy findings had low sensitivity, but appeared to be a specific marker of infection. Prolonged cultivations of samples and cultivation under anaerobic conditions resulted in a marked increase in isolates obtained, as compared with the routine cultivation technique.


Gallo J.,Ortopedicka klinika LF UP a FN Olomouc | Bogdanova K.,Ustav mikrobiologie | Siller M.,Ustav farmakologie | Svabova M.,Oddeleni klinicke biochemie | And 2 more authors.
Acta Chirurgiae Orthopaedicae et Traumatologiae Cechoslovaca | Year: 2013

PURPOSE OF THE STUDY Prosthetic joint infection is a feared complication in total hip arthroplasty. The use of antibiotic-impregnated bone cement is the important part of preventive and therapeutic strategies. At present a number of commercial bone cements are available and support of their use by the results of experimental trials and clinical studies has varied. In relation to this issue we studied essential microbiological and pharmacological characteristics of VancogenX in comparison with gentamicin-loaded bone cement used conventionally. MATERIAL AND METHODS Based on a previously described protocol, we tested pellets of four commercial bone cements, namely, Hi-Fatigue G, Palacos R+G, VancogenX, and Paiacos R as a control. Bone cement was prepared in a vacuum-mixing system. The strains used for bacterial load included Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Pseudomonas aeruginosa and Escherichia coli. Each cement was tested for its antimicrobial and antibiofilm activities and the results were evaluated by standard methods. In addition, we investigated time-related release of gentamicin and vancomycin from the bone cements tested. RESULTS All antibiotic-loaded cement pellets were able to prevent growth of the bacterial strains tested. The bactericidal effect lasted for several days in relation to the bacterial species and cement used, with the exception of S. epidermidis whose growth was inhibited by gentamicin-loaded cement only for one day. The antibiotic-loaded pellets also prevented the formation of a biofilm for 24 hours at least. The major amount of vancomycin (32.915 mg/l) was released from VancogenX to MH medium within 24 hours and the last measureable amount (4.327 mg/l) was recorded at 48 hours after the start of the experiment. In physiological saline the highest level of vancomycin was 139.852 mg/ml measured at 24 hours, and the antibiotic was detectable at a level of 2.334 mg/ml as late as 8 days after the experiment started. Release of gentamicin from VancogenX was as follows: the 24-hour level was higher in MH medium than physiological saline (178 versus 131.4 mg/ml); however, gentamicin was still detectable in physiological saline at 192 hours after the start of the experiment while no gentamicin was found in MH medium after 72 hours. DISCUSSION The antimicrobial effect of VancogenX bone cement was not an unexpected finding since both gentamicin and vancomycin have been used with bone cement for a long time and their combination is optimal in terms of preventing prosthetic joint infection. However, there is a disputable issue of poor release of vancomycin from bone cement. In MH medium we were able to detect the vancomycin released from VancogenX only for two days after the initial rapid elution while its release into physiological saline seemed to be slower but much longer. It is possible that more vancomycin is released from bone cement, but this amount is immediately bound to proteins in the cement vicinity and this process is not detectable by any analytical method. CONCLUSIONS The bone cement VancogenX showed excellent antimicrobial and antibiofilm properties and can be recommended for use in orthopaedic practice. Therapy of prosthetic joint infection is the main indication. Extension of the indication to the prevention of prosthetic joint infection in high-risk patients should be preceded by biomechanical studies demonstrating that the cement is appropriate for long-term implant fixation.


Htoutou Sedlakova M.,Ustav Mikrobiologie
Klinická mikrobiologie a infekční lékařství | Year: 2011

Carbapenems are the drugs of choice for the treatment of serious infections caused by ESBL- and AmpC-positive Enterobacteriaceae. An increasing trend of resistance to these antibiotics has been observed recently. The aim of the study was to determine resistance to carbapenems in clinical isolates of the Enterobacteriaceae family and its mechanism. Between 1 April 2009 and 31 August 2010, Enterobacteriaceae were isolated from clinical samples obtained from patients hospitalized in the University Hospital Olomouc, Czech Republic (1,406 beds incl. 155 ICU beds). The strains were identified using the standard microbiological methods and their susceptibility to antibiotics was determined by the microdilution method. The identification of the isolates with the minimum inhibitory concentration (MIC) of meropenem of 2 mg/L or more was confirmed by the Phoenix automated system (Becton Dickinson). The MIC of meropenem was verified by the E-test and also Phoenix automated system. The isolates were tested for carbapenemase production using the modified Hodge test, a combined test with 3-aminophenylboronic acid (3-APB) and EDTA, the CD-test for serine carbapenemases and modified DDST (mDDST) for metallo-beta-lactamases (MBL). ESBL and AmpC production was determined by the mDDST and modified AmpC test, respectively. Genes encoding production of serine carbapenemases, MBL, bla(OXA-23), bla(OXA-48), ESBL and AmpC enzymes were detected with a set of primers that amplify specific segments of individual beta-lactamases. TEM- and SHV-positive PCR products were characterized by restriction analysis. From a total of 12,605 Enterobacteriaceae, 9 strains were isolates with the MIC of meropenem of 2 mg/L or more. Seven isolates were classified as Klebsiella pneumoniae and two as Enterobacter cloacae. The MIC of meropenem for these strains ranged from 2 mg/L to 16 mg/L. The modified Hodge test, the combined test with 3-APB and EDTA, CD-test, mDDST for MBL and a series of PCR analyses did not detect production of class A carbapenemases, MBL, OXA-23 or OXA-48 enzymes in any of the tested strains. In 6 Klebsiella pneumoniae strains and 1 Enterobacter cloacae strain, the mDDST test and genetic analysis revealed ESBL production (CTX-M and SHV types), and in 2 strains, AmpC production was detected (DHA and EBC types). The prevalence of the Enterobacteriaceae with the MIC of meropenem ≥ 2 mg/L in the University Hospital Olomouc was 0.07 %. None of the strains produced either serine carbapenemases or MBL. Borderline resistance of the strains to carbapenems was determined by the ESBL and AmpC production with another associated mechanism of resistance.


Bardon J.,Ustav Mikrobiologie | Husickova V.,Ustav Mikrobiologie | Chroma M.,Ustav Mikrobiologie | Kolar M.,Ustav Mikrobiologie
Klinicka Mikrobiologie a Infekcni Lekarstvi | Year: 2012

Background: The goals of the presented regional study were monitoring the prevalence of Enterobacteriaceae producing broad-spectrum beta-lactamases in slaughter pigs and their basic molecular biology analysis. Material and methods: In the presented study, rectal swabs from 118 slaughter pigs from 5 farms in the Olomouc Region were analyzed. Bacteriological tests aimed at detection and identification of bacteria from the Enterobacteriaceae family producing broad-spectrum beta-lactamases. Suspected isolates were further analyzed using both phenotypic and genotypic methods. Results: From the group of 118 analyzed samples, seven Escherichia coli strains with the presence of the bla genes encoding CTX-M-1 beta-lactamases were isolated. Genes for TEM and SHV enzymes were not detected. This is the first report of ESBL-positive isolates of Escherichia coli in pigs in the Czech Republic.


Svobodova L.,Ustav Mikrobiologie | Lyskova P.,Ustav Mikrobiologie | Hamal P.,Ustav Mikrobiologie
Klinicka Mikrobiologie a Infekcni Lekarstvi | Year: 2015

Vulvovaginal candidiasis is the second most common vaginal infection after bacterial vaginosis. It is caused by yeasts, the vast majority of which belong to the genus Candida. Vulvovaginal candidiasis affects as many as 75% of women during their childbearing years and 40% of them experience recurrences. The most common etiological agent is Candida albicans, which is responsible for nearly 90% of cases. Vulvovaginal candidiasis can be treated with topical and/or systemic antifungals while risk factors for the infection must be eliminated.


Antimicrobial resistance among nosocomial pathogens has emerged as one of the most important health care problems in the new millennium. In this review, we present new methods for bacterial antimicrobial susceptibility testing, based on the detection of antibiotic-mediated cell death markers that could provide valuable alternatives to existing phenotypic approaches in the very near future. © 2016, Czech Medical Association J.E. Purkyne. All rights reserved.


Hrabak J.,Ustav Mikrobiologie | Zemanova A.,Ustav Mikrobiologie | Chudackova E.,Ustav Mikrobiologie
Epidemiologie, Mikrobiologie, Imunologie | Year: 2010

The study of the role of mobile elements and mobilization of resistance genes is crucial for understanding the epidemiology of antibiotic resistance. This review summarizes recent data on the insertion sequences, transposons, integrons and plasmids that are involved in the mobilization of bacterial antibiotic resistance genes.


Hricova K.,Ustav Mikrobiologie | Kolar M.,Ustav Mikrobiologie
Klinicka Mikrobiologie a Infekcni Lekarstvi | Year: 2015

Efflux pumps capable of actively draining antibiotic agents from bacterial cells may be considered one of potential mechanisms of the development of antimicrobial resistance. The most important group of efflux pumps capable of removing several types of antibiotics include RND (resistance - nodulation - division) pumps. These are three proteins that cross the bacterial cell wall, allowing direct expulsion of the agent out from the bacterial cell. The most investigated efflux pumps are the AcrAB-TolC system in Escherichia coli and the MexAB-OprM system in Pseudomonas aeruginosa. Moreover, efflux pumps are able to export other than antibacterial agents such as disinfectants, thus decreasing their effectiveness. One potential approach to inactivation of an efflux pump is to use the so-called efflux pump inhibitors (EPIs). Potential inhibitors tested in vitro involve, for example, phenylalanyl-arginyl-β-naphthylamide (PAβN), carbonyl cyanide m-chlorophenylhydrazone (CCCP) or agents of the phenothiazine class.

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