VIM metallo-␤-lactamase-producing serotype O11 or O12 Pseudomonas aeruginosa isolates infecting or colonizing 19 patients from seven hospitals in Hungary were characterized between October 2003 and November 2005. Macrorestriction analysis revealed the involvement of hospitals from three different towns in northwest Hungary in an outbreak caused by VIM-4-producing P. aeruginosa.The worldwide spread of acquired metallo-␤-lactamase (MBL)-producing gram-negative pathogens was observed in the past decade, with the bla VIM -type acquired MBL genes currently being the most prevalent in Europe (16,23). The first VIM-producing Pseudomonas aeruginosa clinical isolates in Hungary (isolates PA396 and PA450) were characterized at the National Center for Epidemiology in 2003 (7). We established routine screening of carbapenem-resistant Pseudomonas sp. isolates provided by collaborating regional laboratories for acquired MBL genes. In 2005, MBL-positive isolates from six towns in Hungary were detected (Fig. 1). Our aim was to characterize these isolates and to examine the clonal relationships between them and between the major European multiresistant serotype O12 P. aeruginosa clone, clone P12 (4, 8, 10).The VIM-positive clinical and environmental isolates tested in this study are listed in Table 1, together with the previously published control isolates PA396 and PA450 (7). Isolates P12-Q and P12-E were from French patients "Q" and "E," respectively, who participated in a previous clone P12-related study (8). MICs were determined by the agar dilution method (1) for ␤-lactam antibiotics and by the Etest (AB Biodisk, Solna, Sweden) for other antibiotics. The MBL Etest and the imipenem-EDTA, ceftazidime-EDTA, and cefepime-EDTA double-disk methods were used for phenotypic screening (21,22).bla VIM genes and class 1 integrons were detected by PCR (7). The variable regions of the integrons from isolates PA555 and MB197 were sequenced by using the following primers, together with those described previously (7): primer 197F (5Ј-AAT CGC TCA GTC GCC GAG-3Ј), primer 197R1 (5Ј-TAG TGC TTC TCC GTC GGG-3Ј), primer 197R2 (5Ј-AAT TCC GCA TTG CTG ATC G-3Ј), and primer 197R3 (5Ј-AGG TAT TGC TCC TGC ACT T-3Ј). Isolate PA555 was selected for full integron sequencing in 2003, as it was the first VIM-positive isolate from Pécs, Hungary, while isolate MB197 was selected as an invasive isolate from a cluster of clonally closely related VIMpositive isolates from northwest Hungary. For the other VIMproducing isolates, the integron structures were determined by PCR mapping and partial sequencing.Pulsed-field gel electrophoresis (PFGE) was performed as described earlier (11), with modifications, and the patterns were interpreted by using Fingerprinting II Informatix software (Bio-Rad, Madrid, Spain). Pseudomonas aeruginosa antisera (Bio-Rad, Marnes-la-Coquette, France) were used for serotyping.Conjugation experiments were carried out with strains Escherichia coli J5-3 Rif r and P. aeruginosa PAO4089Rp (6, 7) as the recipients. Transconjugants were selec...

A fast and reproducible method was developed to characterize cell lysates by their electrophoretic profiles using capillary electrophoresis (CE). Characteristic and reproducible patterns were recorded for each bacterial strains when "dynamic sieving" CE, using a polymer solution in the capillary, was applied to distinguish four strains of the Enterobacteriaceae family. The electropherograms showed distinct differences when comparing them to the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protein profiles. This is certainly a result of the differences in the separation principles and in the detection methods of the two techniques.

Outer membrane proteins are indispensable components of bacterial cells and participate in several relevant functions of the microorganisms. Changes in the outer membrane protein composition might alter antibiotic sensitivity and pathogenicity. Furthermore, the effects of various factors on outer membrane protein expression, such as antibiotic treatment, mutation, changes in the environment, lipopolysaccharide modification and biofilm formation, have been analyzed. Traditionally, the outer membrane protein profile determination was performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Converting this technique to capillary electrophoresis format resulted in faster separation, lower sample consumption and automation. Coupling capillary electrophoresis with mass spectrometry enabled the fast identification of bacterial proteins, while immediate quantitative analysis permitted the determination of up- and downregulation of certain outer membrane proteins. Adapting capillary electrophoresis to microchip format ensured a further ten- to 100-fold decrease in separation time. Application of different separation techniques combined with various sensitive detector systems has ensured further opportunities in the field of high-throughput bacterial protein analysis. This review provides an overview using selected examples of outer membrane proteins and the development and application of the electrophoretic and microchip technologies for the analysis of these proteins.

No abstract

Pituitary adenylate cyclase activating polypeptide (PACAP) is widely distributed in ocular tissues, including the lacrimal gland. PACAP has been shown to influence the activity of several exocrine glands, but its effects on the composition of the tear film are not known yet. Similarly, the presence of PACAP has already been shown in the inner ear, but it is not known whether PACAP influences the composition of the endolymph. The aim of the present study was to investigate whether systemic injection of PACAP has any modulatory effects on the protein composition of the tear film and endolymph using chip electrophoresis and mass spectrometry analysis. Tear and endolymph samples were collected from rats and chickens, respectively, at various time points after systemic injection of PACAP. Fluid samples were further processed for chip electrophoretic studies. No difference was found in the protein composition of the endolymph between control and PACAP-treated animals. In contrast, tear samples showed a marked difference after PACAP treatment. Proteins in the molecular range 50-70 kDa, which showed a different chip electropherogram profile in every PACAP-treated sample, were further analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. PACAP treatment induced a repression in certain keratins, while others were induced after PACAP injection. Furthermore, PACAP treatment decreased aldehyde dehydrogenase expression. The present study provides a base for further studies on the in vivo effects of PACAP on the composition of tear film. These investigations may have important clinical relevance because of the noninvasive sample collection, the correlation between tear proteins and ocular diseases, and the possible presence of biomarkers for both ophthalmological and systemic pathological conditions.