[Bacterial pathogens of periprosthetic infections and diagnostic possibilities]. / Bakteriální pùvodci periprotetických infekcí a moznosti jejich diagnostiky.

BACKGROUND: Successful treatment of patients with periprosthetic infection (PPI) requires a correct diagnosis supported by clinical, imaging, microbiological and other laboratory evidence. Equally important is to determine the causative agent of the infection as this may affect the subsequent treatment strategy. The paper aims at presenting cultivation results in a group of PPI patients and their comparison with molecular biology methods. MATERIALS AND METHODS: Material obtained during operations of PPI patients was examined by both the standard culture methods and the PCR technique to identify the etiological agents. The results were statistically compared. RESULTS: In total, the group comprised 49 patients with hip, knee or elbow replacement infection verified during the operation. In 42 cases (85.7 %), the infectious agent was identified by cultivation. The infection was most frequently (62.0 %) caused by coagulase-negative staphylococci and Staphylococcus aureus. Monomicrobial and polymicrobial infections were demonstrated in 35 (71.4 %) and 7 (14.3 %) patients, respectively. The PCR assay aimed at the 16S rRNA segment of bacterial DNA was performed in 35 patients, with positive results in 25 cases (71.4 %). In 82.6 %, the agents detected by specific PCR were consistent with the cultivation results. A statistically non-significant difference in sensitivity between the two methods was found, with higher specificity in the case of PCR. CONCLUSION: Standard cultivation methods remain a highly successful and useful tool for identifying the PPI causative agents.

Prevalence of vancomycin-resistant enterococci in hospitalized patients and those living in the community in the Czech Republic.

Between July 1, 2002 and December 31, 2003, rectal swabs from both hospitalized patients and community subjects in the Czech Republic were taken to ascertain the prevalence of vancomycin-resistant enterococci (VRE). The swabs were used for isolating and identifying enterococci and their susceptibility to antibiotics. Vancomycin resistance phenotypes were verified by PCR detection of vanA, vanB, vanC1 and vanC2 genes. A molecular biology analysis was performed in Enterococcus faecium VanA strains. During the observed period, 2691 rectal swabs from the hospitalized patients and 6529 rectal swabs from the subjects in community setting were examined. In total, 31 VRE of hospital origin and 13 community-population strains were isolated. The prevalence of VRE in the gastrointestinal tract was 1.9% in the hospitalized patients and 0.4% in the community subjects. The prevailing strains were Enterococcus faecium VanA (61.3%) in the VRE of hospital origin and Enterococcus gallinarum VanC (46.2%) in the community VRE. Mutual comparison between the hospital and community Enterococcus faecium VanA strains showed no similarity.

Occurrence of vancomycin-resistant enterococci in humans and animals in the Czech Republic between 2002 and 2004.

In the period between September 2002 and May 2004, a total of 6023 rectal swabs from humans in the Czech Republic were evaluated and 821 Enterococcus spp. strains were isolated. Nine strains (1.1 %) were identified as vancomycin-resistant enterococci (VRE). Two strains were VanA Enterococcus faecium, one strain was VanB Enterococcus faecalis and six strains were VanC Enterococcus casseliflavus. In total, 527 Enterococcus spp. strains were isolated from poultry breeds of which 11 (2.1 %) were VRE. Most (54.5 %) were identified as VanA E. faecium. Cluster analysis of SmaI-generated macrorestriction patterns showed high variability in both human and animal VRE strains and no relatedness between strains from the two sources.

In vitro testing of gentamicin-vancomycin loaded bone cement to prevent prosthetic joint infection.

Sepsis is a greatly feared complication of total joint arthroplasty. One key question is how to prevent perioperative bacterial adherence, and therefore the potential for infectious complications. The objective of our study was to appraise the emerging capacity of staphylococcal survival on prosthetic materials and to analyze the in vitro effects of gentamicin and vancomycin loaded polymethylmethacrylate (PMMA) cement on bacterial adherence and growth. Hospital acquired staphylococcal strains were systematically inoculated on four orthopedic materials (ultrahigh molecular weight polyethylene, PMMA without antibiotic, commercially produced PMMA loaded with gentamicin, and manually mixed PMMA loaded with gentamicin and vancomycin). Staphylococci were identified using culture and biochemical tests. The inoculated material was allowed to incubate in a liquid broth growth media and subsequently prepared for scanning electron microscopy and bacterial growth quantification. Materials without antibiotics showed evidence of staphylococcal growth. PMMA loaded with only gentamicin grew methicillin-resistant Staphylococcus aureus. Gentamicin-vancomycin loaded PMMA completely inhibited any bacterial growth. Low-dose gentamicin-vancomycin loaded PMMA prevents staphylococcal colonization better than commercially manufactured PMMA loaded with gentamicin. We recommend this combination in high-risk procedures and revision surgeries requiring bone cement.

[Prevalence of ESBL-positive strains of Klebsiella pneumoniae in the Czech Republic and their molecular biology analysis]. / Prevalence ESBL-pozitivních kmenu Klebsiella pneumoniae v Ceské republice a jejich molekulárne-biologická analýza.

BACKGROUND: One of the problems of contemporary medicine is an increasing number of bacterial strains with hazardous phenotypes of resistance. The feared bacterial pathogens include Klebsiella pneumoniae strains producing AmpA extended-spectrum beta-lactamases. The study focused on the molecular biological characteristics of ESBL-positive strains of Klebsiella pneumoniae collected in the Czech Republic. MATERIALS AND METHODS: Clinical material from patients hospitalized in 16 Czech hospitals in September and October 2004 was used to isolate and determine Klebsiella pneumoniae strains by standard identification procedures. Their susceptibility to antibiotics was tested using a dilution micromethod. A Double-Disk Synergy Test was used for phenotype determination of ESBL production. The blaTEM, blaSHV and blaOXA genes coding ESBL production were demonstrated by PCR. Molecular biological characteristics of ESBL-positive strains utilized the genomic DNA isolation, XbaI restrictase digestion and PFGE differentiation. The acquired restriction maps of individual isolates were compared using GelCompar II software and their relationship was determined. RESULTS: During the monitored period, 913 Klebsiella pneumoniae strains causing clinically detectable diseases were isolated. Of these, 234 (25.6 %) were determined as ESBL-positive strains. The prevalence of ESBL-positive strains was 38.5 % in ICUs and 15.8 % in standard wards. More than 50 % of ESBL-positive isolates were effectively treated only with meropenem (98 %), cefoperazone/sulbactam (61 %) and amikacin (54 %). Conversely, ESBL-negative strains showed high susceptibility to all tested antibiotics (76-99 %). The molecular biological analysis identified 18 clonal types containing 2-6 identical strains. 17 clones usually contained isolates from one hospital and only in one clone strains from two hospitals were identified. CONCLUSION: Based on the above mentioned results, the prevalence of ESBL-positive strains of Klebsiella pneumoniae in the Czech Republic can be perceived as relatively high, especially in the ICUs. An extensive spread of epidemic clones within Czech hospitals and, to a limited extent, between them can be demonstrated.

[Prevalence of vancomycin-resistant enterococci in hospital and community environment.].

AIM OF THE STUDY: The presented study aimed at determining the prevalence of vancomycin-resistant enterococci (VRE) in rectal swabs taken from both patients in the Teaching Hospital in Olomouc (THO), Czech Republic, and subjects from the community setting of the hospital's catchment area. MATERIALS AND METHODS: Between July 1, 2002 and July 1, 2003, rectal swabs were taken from the THO patients as well as individuals from the community catchment area to be utilized for isolating and identifying enterococci and their suscetibility to antibiotics. Vancomycin resistance phenotypes were verified by PCR detection of vanA, vanB, vanC1 and vanC2 genes. A molecular biology analysis was performed in VanA Enterococcus faecium strains. To determine the relationship of strains, macrorestriction analysis of the total chromosomal DNA digested with SmaI restriction endonuclease was used. RESULTS: During the observed period, 2,157 rectal swabs from the hospitalized patients and 4,874 rectal swabs from the subjects in community setting were examined. In total, 27 VRE of hospital origin and 13 community-population strains were isolated. The prevalence of VRE in the gastrointestinal tract was 2.3 % in the hospitalized patients and 0.6 % in the community subjects. The prevailing strains were Enterococcus faecium VanA (70.4 %) in the VRE of hospital origin and Enterococcus gallinarum VanC (46.2 %) in the community VRE. Mutual comparison between the hospital and community VRE showed no similarity. CONCLUSION: In the Czech Republic, VRE were proved both in community and hospital settings. Their prevalence in rectal swabs is low and does not exceed the values reported in other European countries. The author describes the chief characteristics of the most important quinolone antibiotics, including preparations either in their development stage or whose development has been prematurely interrupted because of adverse side-effects. The list includes all preparations that are or were temporarily registered in the Czech Republic.

Universal primers for detection of common bacterial pathogens causing prosthetic joint infection.

The diagnosis of low grade prosthetic joint infection is difficult and time consuming. Nested-PCR for universal bacterial DNA segments detection of "orthopaedic" bacteria was tested in a laboratory setting. This method is based on amplification of the 16S bacterial ribosomal RNA coding sequences. 11 species of the most frequent bacterial pathogens (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Streptococcus agalactiae, Enterococcus faecium, Enterococcus faecalis, Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Serratia marcescens) involved in prosthetic joint infections were studied. All could be detected rapidly and sensitively by this method.

Molecular-biological analysis of vancomycin-resistant enterococci isolated from a community in the Czech Republic.

OBJECTIVE: To determine the occurrence of vancomycin-resistant enterococci (VRE) in a community of the Czech Republic and a molecular-biological analysis of the VRE isolated. METHODS: Enterococci were isolated from the rectal swabs of healthy people in the Olomouc region (population 300,000), Czech Republic in the period of January-December 2003. The molecular-biological analysis of VRE was performed by analysis of isolated DNA, which was cleaved by restriction enzyme SmaI and separated by pulse field gel electrophoresis (PFGE). RESULTS: A total number of 5,283 swabs were evaluated and 558 Enterococcus sp. strains were isolated during the follow-up period. 9 strains (1.6%) were identified as VRE. Two strains were E. faecium phenotype VanA, one strain was E. faecalis phenotype VanB, two strains were E. gallinarum phenotype VanC and four strains were E. casseliflavus phenotype VanC. PFGE was used to obtain 9 different restriction profiles of VRE strains. The analysis showed closer a similarity of E. casseliflavus strains (80-95%) than between E. faecium strains (41%). CONCLUSIONS: The presence of VRE in a sample community of the Czech population was confirmed. It is clear that it is necessary to take into account the possibility of VRE spreading from the community into health care facilities.