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Unlabelled: The data on the incidence of Cronobacter spp. was collated from hospital records for the seven-year period 2005-2011. The majority of Cronobacter spp. isolates (n = 91) were from throat swabs (61), followed by urine (5), tracheal aspirates (5), bronchoalveolar lavage (4), cannulae (4), and sputum (3) samples. This is the first study which profiles the carriage of Cronobacter spp. according to patient age, based on seven years of clinical data from 2005-2011. It reveals a high recovery (63.7% of strains, n = 91) of the organism from children, 1-14 years in age., Keywords: Cronobacter spp. - meningitis - nosocomial infection.

Resistance of bacteria to antibiotics is a global medical problem requiring close cooperation between veterinary and human physicians. Raw materials and foods of animal origin may be not only a source of pathogenic bacteria causing alimentary tract infections but also a source of bacteria with a dangerous extent of resistance to antibiotics, potentially entering the human food chain. This article presents results of the first study in the Czech Republic detecting the presence of Enterobacteriaceae-producing extended-spectrum b -lactamases (ESBLs) in swabs collected in slaughterhouses from surfaces of healthy animal carcasses. In 2012, swabs taken from pig (n = 166) and cattle (n = 140) carcass surfaces were analyzed. In 17 % of 53 studied slaughterhouses, ESBL-producing Escherichia coli strains were isolated. ESBLs were found in 11 and 4 % of porcine and bovine samples, respectively. Swabs collected from pigs yielded 18 ESBL-producing E. coli strains. The bla genes were found to encode production of CTX-M-1 group enzymes in 16 strains, SHV in one case, and both CTX-M-1-like and TEM in another case. In swabs taken from cattle, five ESBL-producing E. coli strains were isolated. In three cases, the bla genes for CTX-M-1-like production were identified; in two cases, genes for both CTX-M-1-like and TEM production were found. The similarity/identity of ESBL-positive isolates was compared by pulsed-field gel electrophoresis. This is the first report and characterization of the presence and nature of ESBL-producing E. coli in swabs collected from surfaces of healthy pig and cattle carcasses in slaughterhouses in the Czech Republic.

Objectives: 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., Material and Methods: 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., Results: 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., Conclusions: 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.

The strains belonging to Burkholderia cepacia complex are important opportunistic pathogens in immunocompromised patients and cause serious diseases. It is possible to obtain isolates from soil, water, plants and human samples. Taxonomy of this group is difficult. Burkholderia cepacia complex consists of seventeen genomic species and the genetic scheme is based on recA gene. Commonly, first five genomovars occurre in humans, mostly genomovars II and III, subdivision IIIA. Within this study we tested identification of first five genomovars by PCR with following melting analysis and RFLP. The experiments were targeted on eubacterial 16S rDNA and specific gene recA, which allowed identification of all five genomovars. RecA gene appeared as more suitable than 16S rDNA, which enabled direct identification of only genomovars II and V; genomovars I, III and IV were similar within 16S rDNA sequence.

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.

Objective: The study aimed at determining the presence of qnr genes in ESBL-positive strains of Klebsiella pneumoniae isolated from clinical material obtained from patients hospitalized in several intensive care units in the University Hospital Olomouc., Material and Methods: The study comprised 100 ESBL-producing Klebsiella pneumoniae isolates from clinical samples obtained from patients hospitalized between 1 January 2008 and 31 January 2011. blaTEM, blaSHV, blaCTX-M and qnr genes were detected by polymerase chain reaction (PCR). To compare the similarity or identity of the isolates, pulsed-field gel electrophoresis (PFGE) of DNA fragments was used., Results and Conclusion: The blaSHV gene was detected in 99 from 100 isolates of Klebsiella pneumoniae, which is in accordance with the published data that suppose the chromosomal position of the blaSHV-1 gene in this species. The blaCTX-M gene was detected in 77 isolates. The qnr genes were revealed in 56 isolates and majority (76.8 %) of these qnr-positive bacteria carried also the genes encoding the beta-lactamases CTX-M and TEM. In all cases, the qnrB variant was detected. Plasmids from nine different incompatibility groups were identified, in most cases from the IncFII group (52 isolates). Antibiotic susceptibility tests revealed high frequency of resistance not only to beta-lactams, but also to aminoglycosides, tetracycline and sulfamethoxazole/trimethoprim. PFGE detected 65 different strains and several groups of isolates with the same restriction profile. Although the clinical significance of the qnr genes is not well established, their presence in ESBL-positive Enterobacteriaceae is not negligible and it should be kept in mind.

Background: The aim was to assess the epidemiology of Burkholderia cepacia complex strains isolated at the Department of Microbiology, Faculty of Medicine and Dentistry, Palacky University and University Hospital Olomouc, determine the most frequent strains and confirm or rule out potential clonal spread of the strains., Material and Methods: Over a period of eight months, all strains classified as Burkholderia cepacia complex were collected. Susceptibility to selected antimicrobial agents was determined and adequate molecular genetic methods were used to assess their genetic relationship., Results: A total of 52 isolates were tested, with the most frequent (88.5 %) being genomovar II (Burkholderia multivorans). More than 46 % of them were genetically related; 58.3 % of them were detected in intensive care units. All isolates were highly resistant to antimicrobial agents. In four cases, deaths associated with Burkholderia multivorans infection were reported., Conclusion: It may be assumed that genetically related strains of Burkholderia multivorans spread from the hospital setting. As yet, the source of infection has not been determined and further investigations are needed.

A total of 78 bacterial strains with known β-lactamases were used to optimize a rapid detection system consisting of multiplex polymerase chain reaction and melting curve analysis to amplify and identify blaTEM, blaSHV, and blaCTX-M genes in a single reaction. Additionally, to evaluate the applicability of this method, 32 clinical isolates of Escherichia coli displaying an extended-spectrum β-lactamase phenotype from patients hospitalized at intensive care units were tested. Results were analyzed by the Rotor-Gene operating software and Rotor-Gene ScreenClust HRM Software. The individual melting curves differed by a temperature shift or curve shape, according to the presence of β-lactamase genes. With the use of this method and direct sequencing, blaCTX-M-15-like was identified as the most prevalent β-lactamase gene. In conclusion, this novel detection system seems to be a suitable tool for rapid detection of present β-lactamase genes and their characterization., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Bacterial infections are an important issue in current clinical medicine. The severity of infectious diseases has increased dramatically in recent years, which is also due to increasing numbers of resistant bacteria, including strains producing broad-spectrum beta-lactamases. The study aimed at determining the prevalence of ESBL- and AmpC-positive Enterobacteriaceae at the Department of Neonatology, University Hospital Olomouc. Enterobacteriaceae were isolated from clinical samples from infants hospitalized at the Department of Neonatology, University Hospital Olomouc over a period of 2 years. ESBL- and AmpC-positive isolates were subjected to basic genetic analysis. In the study period, a total of 1,526 isolates of the Enterobacteriaceae family were identified, including 55 (3.6%) cases of the ESBL phenotype and 17 (1.1%) AmpC-positive isolates. Genetic analysis of ESBL-positive isolates revealed a majority of CTX-M enzymes. Among AmpC beta-lactamases, the EBC, CIT, DHA, and MOX types were detected. An Escherichia coli strain was isolated with mutations in the promoter region of the ampC chromosomal gene that are associated with overproduction of the relevant enzyme.

Background: bacterial infections have become an important issue in current medicine. Recently, their frequency and severity have significantly increased as a result of the rising number of resistant bacteria. One of important mechanisms of resistance is production of broad-spectrum beta-lactamases, namely the ESBL type. The study aimed at determining the frequency of ESBL-positive Enterobacteriaceae in three large hospitals in Moravia, the eastern part of the Czech Republic., Material and Methods: enterobacteriaceae were isolated from clinical material obtained from patients hospitalized in the University Hospital Olomouc, Teaching Hospital Ostrava and Bata Regional Hospital Zlín throughout 2009. Standard microbiology techniques were used for identification. The production of ESBLs was determined by the modified Double-Disk Synergy Test. ESBL-positive isolates of Escherichia coli from ICU patients were subjected to basic genetic analysis., Results and Conclusion: during the study period, a total of 12,922 strains from the Enterobacteriaceae family were detected. The ESBL phenotype was found in 907 cases, i.e. 7 % of all isolates. The most prevalent species of ESBL-producing Enterobacteriaceae were Klebsiella pneumoniae, Klebsiella oxytoca and Escherichia coli. A comparison of general wards and ICUs revealed a higher percentage of ESBL-positive strains of Klebsiella pneumoniae and a lower proportion of ESBL-positive Escherichia coli isolates in intensive care patients. When assessing the patients' clinical material, ESBL-producing strains were most frequently detected in urine. Genetic analysis of ESBL-positive Escherichia coli strains from ICU patients revealed the CTX-M type of ESBL production in most isolates.

Background: Problematic bacteria in the community include enterobacteria which produce extended-spectrum beta-lactamases. As yet there is no description of the prevalence of these bacteria in persons in the community in the Czech Republic. Therefore the main goal of this study was to determine the prevalence of ESBL-positive enterobacteria in the gastrointestinal tracts of subjects in the community in the Czech Republic., Material/methods: Rectal swabs from the investigated subjects were inoculated onto chromID ESBL selective medium and enterobacteria were identified by the Vitek2 automated system. ESBL were detected using a modified DDST test. The results were confirmed by PCR and direct sequencing of CTX-M-positive amplicons., Results: A total of 579 rectal swabs from subjects in the community were analyzed and ESBL production was both phenotypically and genotypically confirmed in 7 isolates. Thus the prevalence of ESBL-positive bacteria in the gastrointestinal tracts of the persons in the community was 1.2%. All the cases were Escherichia coli strains producing the CTX-M-type ESBL. CTX-M-15 was the most prevalent type in this group of isolates., Conclusions: The presented results are in accord with other authors' studies and suggest that the epidemiologic profile of ESBL-positive enterobacteria in the Czech Republic is comparable to that in other European countries.

Introduction: Currently, one of the most serious problems in medicine is the increasing resistance of pathogenic bacteria to antimicrobial drugs. Bacterial resistance may potentially be solved in particular by decreasing the consumption of antibiotics and increasing the quality of their use. Equally important, however, is the development of new antimicrobial drugs and their use in clinical practice. One of the new antibiotic agents is tigecycline of the glycylcycline group. The presented work aimed at assessing its in vitro effect on selected multiresistant bacteria., Material and Methods: Clinical samples were collected from patients hospitalized in the University Hospital Olomouc to isolate ESBL- and AmpC beta-lactamase-producing enterobacteria, methicillin-resistant Staphylococcus aureus (MRSA) strains and vancomycin-resistant enterococci (VRE). In the isolates, susceptibility to tigecycline was determined by the standard microdilution method., Results: A total of 350 isolates were tested (100 MRSA, 10 0 VRE, 100 ESBL-positive and 50 AmpC-positive enterobacteria). In the cases of VRE and MRSA, no resistance to tigecycline was detected and the minimum inhibitory concentrations (MIC) did not exceed 0.25 mg/l and 0.5 mg/l, respectively. In ESBL-positive enterobacteria, 97% susceptibility (MIC range = or <0.06 to 4 mg/l) was detected; in AmpC-positive enterobacteria, the MIC range was = or <0.03-2 mg/l and susceptibility reached 98 %., Conclusion: Tigecycline may be considered a suitable alternative in the treatment of infections caused by the above-mentioned multiresistant strains.

Background: Currently, important bacterial beta-lactamases of increasing clinical significance include AmpC enzymes. The aim was to assess their occurrence in Klebsiella pneumoniae strains isolated from patients with haematological malignancies in a prospective study., Material and Methods: Over a 2-month period, strains of the species were isolated from clinical material obtained from patients hospitalized at the Department of Haemato-Oncology of the University Hospital Olomouc. The strains were identified using standard microbiological techniques and the Vitek 2 automated system. Production of AmpC beta-lactamases was roughly determined by phenotypic tests and subsequently confirmed by PCR detection of genes encoding these enzymes., Results: During the above-mentioned period, a total of 35 K. pneumoniae isolates were collected. In 7 of them, production of AmpC beta-lactamases was preliminarily detected by phenotypic test. The multiplex PCR method confirmed phenotyping and determined DHA types in all the isolates., Conclusions: All AmpC-positive isolates were false-susceptible to at least one of the tested third-generation cephalosporins. In one patient, clinically apparent infection caused by this strain was documented. The reported results suggest the possibility of occurrence of AmpC-beta-lactamases in K. pneumoniae strains with clinical significance.

Objectives: The study aimed at the assessment of the prevalence of ESBL-positive isolates of Klebsiella pneumoniae in intensive care patients and their molecular biology analysis., Material and Methods: Over a 5-month period, Klebsiella pneumoniae strains were isolated from patients hospitalized at the Department of Anaesthesiology and Resuscitation of the University Hospital in Olomouc. For each isolate, an antibiogram was performed by the standard microdilution method and the production of ESBL was determined by the modified double-disk synergy test. PCR was used to demonstrate the presence of the blaTEM and blaSHV genes. The isolates producing SHV- and TEM-types of beta-lactamases were typed using the restriction fragment length polymorphism (RFLP) method to identify the most common mutations responsible for the development of an ESBL phenotype. Similar or identical isolates were determined by pulsed-field gel electrophoresis (PFGE) of DNA fragments cleaved by the XbaI restriction endonuclease., Results: A total of 67 isolates of Klebsiella pneumoniae were obtained. In 13 of them, the production of ESBL was detected and the presence of the blaSHV gene was confirmed by PCR. Restriction cleavage by NheI revealed mutations at position 238 in all SHV-positive PCR products. The restriction analysis did not confirm the presence of the gene encoding TEM-type extended-spectrum beta-lactamase. Molecular biology typing by PFGE detected the presence of 11 different strains., Conclusions: In the observed group of intensive care patients, the prevalence of ESBL-positive strains of Klebsiella pneumoniae reached 19.4 %. The analysis of SHV and TEM products of PCR by the RFLP method showed the prevalence of SHV-type ESBL. Overall, 84.6 % of the strains had unique restriction profiles. The results suggest both high levels of hygienic and epidemiological measures at the monitored department and rational antibiotic policy.

The effect of two phenolic compounds vanillin (4-hydroxy-3-methoxybenzaldehyde) and lignin on the development of drug/antibiotic resistance in Salmonella typhimurium was studied. Using the modified Ames test we have shown that vanillin alone has negligible effect on spontaneous mutability to ciprofloxacin and gentamicin resistance. At the tested concentrations vanillin reduces the toxicity of 4-nitroquinoline-N-oxide (4NQO) and reduces the ability of this compound to induce mutations leading to ciprofloxacin but not to gentamicin resistance. Lignin at higher concentrations increases mutagenicity to ciprofloxacin resistance and possess considerable inhibition effect on the spontaneous and 4NQO induced mutability to gentamicin resistance.