We have analyzed 40 metallo-␤-lactamase (MBL)-producing isolates of Pseudomonas aeruginosa (n ‫؍‬ 38), Pseudomonas putida (n ‫؍‬ 1), and Acinetobacter genospecies 3 (n ‫؍‬ 1) from 17 hospitals in 12 cities in Poland that were identified in 2000 to 2004. Pulsed field gel electrophoresis typing classified the P. aeruginosa isolates into eight types, with two types differentiated further into subtypes. Each of the types was specific either to a given center or to several hospitals of the same or neighboring geographic area. Almost all of the organisms produced ␤-lactamase VIM-2; the only exceptions were several P. aeruginosa isolates from two centers which expressed VIM-4. The bla VIM genes resided exclusively within class 1 integrons, and these were located in either chromosomal or plasmid DNA. PCR-restriction fragment length polymorphism study of the variable regions of the integrons, followed by DNA sequencing, revealed the presence of eight different, mostly novel gene cassette arrays, six of which contained bla VIM-2 and two of which contained bla VIM-4 . The occurrence of the integron variants correlated well with the geographic distribution of the MBL-producing organisms, and this suggested that their emergence in particular parts of the country had been likely due to a number of independent events. The following regional dissemination of MBL producers could be attributed to various phenomena, including their clonal spread, horizontal transmission of resistance determinants, or both. All of the data collected in this study revealed that even at this early stage of detection, the epidemiological situation concerning MBL producers in Poland has already been complex and very dynamic.The class B metallo-␤-lactamases (MBLs), which hydrolyze penicillins, cephalosporins, and carbapenems and are not inhibited by site-directed ␤-lactam inhibitors (3), have become a problem with potentially disastrous consequences for therapy of bacterial infections in future (16,34,37). The acquired MBL variants first appeared at the end of the 1980s in Pseudomonas aeruginosa in Japan (35), and since the mid-1990s, they have been observed in many countries all over the world. So far P. aeruginosa has remained the major producer of these enzymes; however, their genes effectively diffuse into other gram-negative bacteria as well. Of the four evolutionary families of acquired MBLs discerned until now, the IMP-and VIM-type

In search of an effective DNA typing technique for hospital epidemiology use, the performance and convenience of a PCR melting profile (PCR MP) technique based on using low denaturation temperatures during ligation-mediated PCR (LM PCR) of bacterial DNA was tested. A number of Escherichia coli isolates from patients of the Clinical Hospital in Gdańsk, Poland, were examined. We found that the PCR MP technique is a rapid method that offers good discriminatory power and excellent reproducibility and may be applied for epidemiological studies. The usefulness of the PCR MP for molecular typing was compared with the pulsedfield gel electrophoresis method, which is currently considered the gold standard for epidemiological studies of isolates recovered from patients and the environment. Clustering of PCR MP fingerprinting data matched pulsed-field gel electrophoresis data. The features of the PCR MP technique are discussed in comparison with conventional methods. Data presented here demonstrate the complexity of the epidemiological situation concerning E. coli that may occur in a hospital.Over the past 20 years, a significant number of DNA-based techniques have been introduced into the field of bacterial characterization and taxonomy. These genomic fingerprinting methods were developed to detect DNA sequence polymorphisms by using general principles, such as restriction endonuclease analysis, molecular hybridization, and PCR amplification. DNA fingerprinting involves the display of a set of DNA fragments from a specific DNA sample. A variety of DNA fingerprinting techniques are presently available (1-7, 11-13), most of which use PCR for detection of fragments. The choice of fingerprinting technique depends on the type of application. Ideally, a fingerprinting technique should require no prior investments in terms of sequence analysis, primer synthesis, or characterization of DNA probes. A number of fingerprinting methods which meet these requirements have been developed. The fingerprints are obtained by visualizing many parts of the genome. Differences in these fingerprints between individuals are interpreted as genetic distances. Obviously, the differences should reflect variations in DNA rather than artifacts due to a nonrobust method. Furthermore, the method should provide the appropriate level of discriminatory power, and it should be relatively rapid and cheap, especially in large-scale population genetic studies. Macrorestriction analysis of genomic DNA followed by pulsed-field gel electrophoresis (REA-PFGE) has become the "gold standard" for molecular typing (10). In recent years, some alternative techniques have been successfully applied for the typing of bacteria below the species level. These include amplification-based methods, such as amplified fragment length polymorphisms (11), random amplification of polymorphic DNA (3, 12, 13), and amplification of DNA fragments surrounding rare restriction sites (5, 6, 8). These techniques are now applied more and more because they involve less time, comparably low cos...

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In patients with leukemia, the portal(s) and reasons for the persistence of an Escherichia coli recurrent bacteremia remain unclear. Adult Hematology Clinic (AHC) databases at the State Clinical Hospital in Gdańsk were reviewed to evaluate the frequency of E. coli bacteremia between 2002 and 2005. Blood and bowel E. coli strains were obtained and the genetic relatedness of the strains was analyzed. The rate of E. coli bacteremia per 1,000 admissions at the AHC was higher (85.0) than in the other clinics of the hospital (2.9), p < 0.001. A higher mortality was observed in patients with a history of E. coli versus non-E. coli bacteremia [30/95 (31 %) vs. 53/430 (12 %), p < 0.001]; 72.8 % of patients with leukemia had an unknown source of bacteremia. In 2005, 6 out of 25 (24 %) patients with leukemia had ≥2 episodes of E. coli-positive blood cultures. These gastrointestinal E. coli isolates were replaced within 3–8 weeks with a new E. coli H genotype. A recurrent episode of bacteremia was usually caused by an infection with a transient E. coli H genotype identical to that found in the subject’s bowel. Consistent with the definition of bowel/blood translocation, the bowel appeared to be a portal for E. coli in these subjects and, hence, a clear source for their recurring bacteremia.

Uropathogenic and diarrheal Escherichia coli strains expressing adhesins of the Dr family bind to decayaccelerating factor, invade epithelial cells, preferentially infect children and pregnant women, and may be associated with chronic or recurrent infections. Thus far, no fimbrial domain(s) that facilitates cell invasion has been identified. We used alanine scanning mutagenesis to replace selected amino acids in hydrophilic domain II of the structural fimbrial subunit DraE and evaluated recombinant mutant DraE for attachment, invasion, and intracellular compartmentalization. The mutation of amino acids V28, T31, G33, Q34, T36, and P40 of DraE reduced or abolished HeLa cell invasion but did not affect attachment. Electron micrographs showed a stepwise entry and fusion of vacuoles containing Escherichia coli mutants T36A and Q34A or corresponding beads with lysosomes, whereas vacuoles with wild-type Dr adhesin showed no fusion. Mutants T31A and Q34A, which were deficient in invasion, appeared to display a reduced capacity for clustering decay-accelerating factor. Our findings suggest that hydrophilic domain II may be involved in cell entry. These data are consistent with the interpretation that in HeLa cells the binding and invasion phenotypes of Dr fimbriae may be separated.

This review article shows that coagulase-negative staphylococci (CoNS) are widely responsible for laryngological diseases. General characteristics of CoNS infections are shown in the introduction, and the pathogenicity in terms of virulence determinants, biofilm formation and genetic regulation mechanisms of these bacteria is presented in the first part of the paper to better display the virulence potential of staphylococci. The PubMed search keywords were as follows: CoNS and: nares infections, nasal polyps, rhinosinusitis, necrosing sinusitis, periprosthetic joint infection, pharyngitis, osteomyelitis of skull and neck bones, tonsillitis and recurrent tonsillitis. A list of laryngological infections and those related to skull and neck bones was presented with descriptions of the following diseases: rhinosinusitis, necrotizing sinusitis, nasal polyps, nares and nasal skin infections, periprosthetic joint infections, osteomyelitis, pharyngitis, and tonsillitis. Species identification and diagnostic problems challenging for diagnosticians are presented. Concluding remarks regarding the presence of CoNS in humans and their distribution, particularly under the effect of facilitating factors, are mentioned.

The aim of the study was to investigate whether there are unique pathotypes of Escherichia coli capable of transmission from the gastrointestinal tract to the vascular bed. The study included E. coli strains isolated from clinical materials collected from 115 patients suffering from haematologic malignancies diagnosed with bacteraemia. The genotyping techniques established that 89 E. coli isolates from the blood had the same genotype as the E. coli from the patient’s bowel. The presence of 21 genes encoding virulence factors typical of various E. coli pathotypes and their relationship with the phylogenetic group was established. One-dimensional analysis showed that the focG gene occurred more frequently in the control bowel group, while the ampicillin-resistant afa/dr E. coli were associated with bacteraemia. Blood isolates with the highest occurrence of virulence factors belonged to pathogenic group B2 and non-pathogenic group A. The co-occurrence of multiple genes encoding papC, sfa, usp and cnf1 virulence factors probably predisposes E. coli to translocation from the gastrointestinal tract to the vascular bed in the group of patients with haematologic malignancies. Based on clustering analysis, dominance of the most virulent strains assigned to the cluster with seven virulence factors encoded by the following genes, papC, sfaD/E, cnf1, usp, agn43, hlyA and iutA, was found. The obtained results enforce the previously proposed concept of bowel–blood translocation and further expand our hypothesis by defining the unique virulence characteristics of E. coli isolates, which predispose them to bowel colonisation or translocation and bacteraemia in this group of patients.

Significant changes in the frequency of candidaemia and the distribution of causative species have been noted worldwide in the last two decades. In this study, we present the results of the first multicentre survey of fungaemia in Polish hospitals. A total of 302 candidaemia episodes in 294 patients were identified in 20 hospitals during a 2-year period. The highest number of infections was found in intensive care (30.8%) and surgical (29.5%) units, followed by haematological (15.9%), 'others' (19.2%) and neonatological (4.6%) units. Candida albicans was isolated from 50.96% of episodes; its prevalence was higher in intensive care unit and neonatology (61.22% and 73.33%, respectively), and significantly lower in haematology (22%; P < 0.001). The frequency of C. krusei and C. tropicalis was significantly higher (24% and 18%) in haematology (P < 0.02); whereas, the distribution of C. glabrata (14.1%) and C. parapsilosis (13.1%) did not possess statistically significant differences between compared departments. Obtained data indicates that species distribution of Candida blood isolates in Polish hospitals reflects worldwide trends, particularly a decrease in the prevalence of infections due to C. albicans.