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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.

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.

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.

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.

Hanulik V.,Ustav mikrobiologie | Uvizl R.,Klinika Anesteziologie | Husickova V.,Ustav mikrobiologie | Htoutou Sedlakova M.,Ustav mikrobiologie | Kolar M.,Ustav mikrobiologie
Klinicka Mikrobiologie a Infekcni Lekarstvi | Year: 2011

Background: This prospective study aimed at determining etiologic agents causing nosocomial pneumonia in a precisely defined group of patients and resistance of bacterial pathogens to antimicrobial drugs. Material and methods: The study comprised patients hospitalized at the Department of Anesthesiology and Intensive Care Medicine, Faculty of Medicine, Palacký University and University Hospital Olomouc who developed pneumonia. From those patients, secretion samples were collected for microbiological analysis. Results: A total of 77 secretion samples from 51 patients were analyzed. Of 90 isolates, 71 were classified as etiologic agents, with the most frequently isolated strains being those of Klebsiella pneumoniae (32%), Pseudomonas aeruginosa (22%), Burkholderia cepacia complex (10%) and Escherichia coli (8%). The highest proportions of multiresistant strains were found in Pseudomonas aeruginosa (56%), Klebsiella pneumoniae (52%), Escherichia coli (25%) and Burkholderia cepacia complex (100%). Pairs of identical strains were detected in five cases (3x Klebsiella pneumoniae and 2x Pseudomonas aeruginosa). In Burkholderia cepacia complex, the same strain was identified in four out of five cases. Eighteen patients died during hospitalization. Conclusion: Most isolates were single strains and hospital-acquired pneumonia may be characterized as endogenous. Four identical cultures of Burkholderia cepacia complex were classified as Burkholderia multivorans by the MALDI-TOF system and clonal spread of this strain may be assumed.

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