The dynamic architecture of Map1- and NatB-ribosome complexes coordinates the sequential modifications of nascent polypeptide chains.

Cotranslational modification of the nascent polypeptide chain is one of the first events during the birth of a new protein. In eukaryotes, methionine aminopeptidases (MetAPs) cleave off the starter methionine, whereas N-acetyl-transferases (NATs) catalyze N-terminal acetylation. MetAPs and NATs compete with other cotranslationally acting chaperones, such as ribosome-associated complex (RAC), protein targeting and translocation factors (SRP and Sec61) for binding sites at the ribosomal tunnel exit. Yet, whereas well-resolved structures for ribosome-bound RAC, SRP and Sec61, are available, structural information on the mode of ribosome interaction of eukaryotic MetAPs or of the five cotranslationally active NATs is only available for NatA. Here, we present cryo-EM structures of yeast Map1 and NatB bound to ribosome-nascent chain complexes. Map1 is mainly associated with the dynamic rRNA expansion segment ES27a, thereby kept at an ideal position below the tunnel exit to act on the emerging substrate nascent chain. For NatB, we observe two copies of the NatB complex. NatB-1 binds directly below the tunnel exit, again involving ES27a, and NatB-2 is located below the second universal adapter site (eL31 and uL22). The binding mode of the two NatB complexes on the ribosome differs but overlaps with that of NatA and Map1, implying that NatB binds exclusively to the tunnel exit. We further observe that ES27a adopts distinct conformations when bound to NatA, NatB, or Map1, together suggesting a contribution to the coordination of a sequential activity of these factors on the emerging nascent chain at the ribosomal exit tunnel.

Simultaneous single-molecule detection of the acetyltransferase and crotonyltransferase activities of histone acetylation writer p300.

We demonstrate for the first time the simultaneous measurement of the acetyltransferase (HAT) and crotonyltransferase (HCT) activities of histone acetylation writer p300 by integrating antibody-based fluorescence labeling with single molecule detection. This methods exhibits good specificity and high sensitivity. Moreover, it can accurately evaluate the kinetic parameters of both the HAT and HCT activities of p300 and screen inhibitors.

Exploring antimicrobial resistance to beta-lactams, aminoglycosides and fluoroquinolones in E. coli and K. pneumoniae using proteogenomics.

Antimicrobial resistance is mostly studied by means of phenotypic growth inhibition determinations, in combination with PCR confirmations or further characterization by means of whole genome sequencing (WGS). However, the actual proteins that cause resistance such as enzymes and a lack of porins cannot be detected by these methods. Improvements in liquid chromatography (LC) and mass spectrometry (MS) enabled easier and more comprehensive proteome analysis. In the current study, susceptibility testing, WGS and MS are combined into a multi-omics approach to analyze resistance against frequently used antibiotics within the beta-lactam, aminoglycoside and fluoroquinolone group in E. coli and K. pneumoniae. Our aim was to study which currently known mechanisms of resistance can be detected at the protein level using liquid chromatography-mass spectrometry (LC-MS/MS) and to assess whether these could explain beta-lactam, aminoglycoside, and fluoroquinolone resistance in the studied isolates. Furthermore, we aimed to identify significant protein to resistance correlations which have not yet been described before and to correlate the abundance of different porins in relation to resistance to different classes of antibiotics. Whole genome sequencing, high-resolution LC-MS/MS and antimicrobial susceptibility testing by broth microdilution were performed for 187 clinical E. coli and K. pneumoniae isolates. Resistance genes and proteins were identified using the Comprehensive Antibiotic Resistance Database (CARD). All proteins were annotated using the NCBI RefSeq database and Prokka. Proteins of small spectrum beta-lactamases, extended spectrum beta-lactamases, AmpC beta-lactamases, carbapenemases, and proteins of 16S ribosomal RNA methyltransferases and aminoglycoside acetyltransferases can be detected in E. coli and K. pneumoniae by LC-MS/MS. The detected mechanisms matched with the phenotype in the majority of isolates. Differences in the abundance and the primary structure of other proteins such as porins also correlated with resistance. LC-MS/MS is a different and complementary method which can be used to characterize antimicrobial resistance in detail as not only the primary resistance causing mechanisms are detected, but also secondary enhancing resistance mechanisms.

Visualizing RNA Cytidine Acetyltransferase Activity by Northern Blotting.

Cytidine acetyltransferases are an emerging class of nucleic-acid-modifying enzymes responsible for the establishment of N4 -acetylcytidine (ac4C) in RNA. In contrast to histone acetyltransferases, whose activity is commonly studied by western blotting, relatively few methods exist for quickly assessing the activity of cytidine acetyltransferases from a biological sample of interest or the distribution of ac4C across different RNA species. In this protocol, we describe a method for analysis of cellular cytidine acetyltransferase activity using dot- and immuno-northern-blotting-based detection. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Detection of N4 -Acetylcytidine in RNA by dot blotting Basic Protocol 2: Visualizing N4 -Acetylcytidine Distribution in RNA by northern blotting.

Hepatic lipid metabolism in adult rats using early weaning models: sex-related differences.

Non-pharmacological early weaning (NPEW) induces liver damage in male progeny at adulthood; however, pharmacological early weaning (PEW) does not cause this dysfunction. To elucidate this difference in liver dysfunction between these two models and determine the phenotype of female offspring, de novo lipogenesis, ß-oxidation, very low-density lipoprotein (VLDL) export, and gluconeogenesis in both sexes were investigated in the adult Wistar rats that were weaned after a normal period of lactation (control group) or early weaned either by restriction of access to the dams' teats (NPEW group) or by reduction of dams' milk production with bromocriptine (PEW group). The offspring received standard diet from weaning to euthanasia (PN180). NPEW males had higher plasma triglycerides and TyG index, liver triglycerides, and cholesterol by de novo lipogenesis, which leads to intracellular lipids accumulation. As expected, hepatic morphology was preserved in PEW males, but they showed increased liver triglycerides. The only molecular difference between PEW and NPEW males was in acetyl-CoA carboxylase-1 (ACC-1) and stearoyl-CoA desaturase-1 (SCD-1), which were lower in PEW animals. Both early weaning (EW) females had no changes in liver cholesterol and triglyceride contents, and the hepatic cytoarchitecture was preserved. The expression of microsomal triglyceride transfer protein was increased in both the female EW groups, which could constitute a protective factor. The changes in hepatic lipid metabolism in EW offspring were less marked in females. EW impacted in the hepatic cytoarchitecture only in NPEW males, which showed higher ACC-1 and SCD-1 when compared to the PEW group. As these enzymes are lipogenic, it could explain a worsened liver function in NPEW males.

Reduction of Tat-interacting Protein 30 Expression Could be a Prognostic Marker in Bladder Urothelial Cancer.

BACKGROUND: Tat-interacting protein 30 (TIP30) has been reported to be a tumor suppressor, with reduced or absent expression in various tumors. However, its role in bladder urothelial cancer (BUC) has not been investigated. Therefore, herein, we investigated the expression of TIP30 protein in BUC and normal bladder mucosa and the clinical significance of TIP30 expression in the prognosis of BUC. METHODS: We reviewed data from 79 cases of BUC and 15 adjacent tissue samples from 79 patients treated at our institution between 2004 and 2007. TIP30 expression was examined by immunohistochemistry. The relationship between TIP30 expression and tumor stage, histological grade, and survival was analyzed. Differences between groups were evaluated using the t-test or matched-pairs test, and differences in the survival rates were analyzed with the log-rank test. RESULTS: TIP30 protein expression was significantly reduced in BUC tissue (t = -6.91, P < 0.05) compared with normal tissue samples, and in invasive bladder cancer (t = 10.89, P < 0.05) compared with superficial bladder cancer. TIP30 protein expression differed significantly among different differentiated groups classified either according to the World Health Organization (2004, F = 17.48, P < 0.01) or World Health Organization (1973, F = 10.68, P < 0.01). TIP30 protein expression was significantly reduced in high-grade papillary urothelial carcinoma compared with papillary urothelial neoplasm of low malignant potential (P < 0.05) and low-grade papillary urothelial carcinoma (P < 0.05). Meanwhile, TIP30 protein expression was significantly reduced in Grade III BUC, compared with Grade I (P < 0.05) and Grade II (P < 0.05). Patients with low TIP30 expression showed a higher incidence of disease progression than those with high TIP30 expression (t = 2.63, P < 0.05). Kaplan-Meier survival analysis showed a strong positive relationship between TIP30 expression and overall survival (OS) (χ2 = 17.29, P < 0.05). CONCLUSIONS: TIP30 expression was associated with clinical tumor stage in BUC, suggesting that it might play an important role in disease progression. Furthermore, TIP30 might predict postoperative OS. Thus, its evaluation might be useful for predicting prognosis.

Identification of biomarkers and pathway-related modules involved in ovarian cancer based on topological centralities.

PURPOSE: The present study was designed to explore the significant biomarkers and pathway-related modules for predicting the effects of eribulin relative to paclitaxel in ovarian cancer. METHODS: The gene expression data E-GEOD-50831 were downloaded from the European Bioinformatics Institute (EBI) database. Differentially expressed genes (DEGs) were screened. Subsequently, differential coexpression network was constructed. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and pathway-related modules mining were conducted. Topological centralities (degree, betweenness, closeness and stress) analyses for coexpression network and pathway-related modules were performed to explore hub genes and the most significant pathways. Then, we verified our findings in an independent sample set via RT-PCR and Western blotting. RESULTS: Centralities results of ESCO1, CDC27and MCM4 ranked the top five. Moreover, among the top 10% hub genes, CDC27, MCM4 and SOS1 were pathway-enriched genes in two networks. A total of 5 and 6 pathway-related modules were obtained under two drugs treatment. Based analyses of degree, betweenness and other centralities, DNA replication pathway-related module was the most significant under paclitaxel treatment, while cell cycle pathway-related module was the most significant under eribulin treatment. RT-PCR and Western blotting results were consistent with the bioinformatics results. The expression level of MCM4 was remarkably decreased under eribulin treatment relative to paclitaxel. CONCLUSIONS: The inhibition of ovarian cancer growth by paclitaxel and eribulin might be connected with downregulation of cell cycle and DNA replication pathway. Moreover, MCM4 signature might be a potential biomarker to predict the effect of eribulin in ovarian cancer.

Homocysteine induces glyceraldehyde-3-phosphate dehydrogenase acetylation and apoptosis in the neuroblastoma cell line Neuro2a.

High plasma levels of homocysteine (Hcy) promote the progression of neurodegenerative diseases. However, the mechanism by which Hcy mediates neurotoxicity has not been elucidated. We observed that upon incubation with Hcy, the viability of a neuroblastoma cell line Neuro2a declined in a dose-dependent manner, and apoptosis was induced within 48 h. The median effective concentration (EC50) of Hcy was approximately 5 mM. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) nuclear translocation and acylation has been implicated in the regulation of apoptosis. We found that nuclear translocation and acetylation of GAPDH increased in the presence of 5 mM Hcy and that higher levels of acetyltransferase p300/CBP were detected in Neuro2a cells. These findings implicate the involvement of GAPDH in the mechanism whereby Hcy induces apoptosis in neurons. This study highlights a potentially important pathway in neurodegenerative disorders, and a novel target pathway for neuroprotective therapy.

Homocysteine induces glyceraldehyde-3-phosphate dehydrogenase acetylation and apoptosis in the neuroblastoma cell line Neuro2a

High plasma levels of homocysteine (Hcy) promote the progression of neurodegenerative diseases. However, the mechanism by which Hcy mediates neurotoxicity has not been elucidated. We observed that upon incubation with Hcy, the viability of a neuroblastoma cell line Neuro2a declined in a dose-dependent manner, and apoptosis was induced within 48 h. The median effective concentration (EC50) of Hcy was approximately 5 mM. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) nuclear translocation and acylation has been implicated in the regulation of apoptosis. We found that nuclear translocation and acetylation of GAPDH increased in the presence of 5 mM Hcy and that higher levels of acetyltransferase p300/CBP were detected in Neuro2a cells. These findings implicate the involvement of GAPDH in the mechanism whereby Hcy induces apoptosis in neurons. This study highlights a potentially important pathway in neurodegenerative disorders, and a novel target pathway for neuroprotective therapy.

Development and Validation of a Multiplexed Protein Quantitation Assay for the Determination of Three Recombinant Proteins in Soybean Tissues by Liquid Chromatography with Tandem Mass Spectrometry.

Currently, traditional immunochemistry technologies such as enzyme-linked immunosorbent assays (ELISA) are the predominant analytical tool used to measure levels of recombinant proteins expressed in genetically engineered (GE) plants. Recent advances in agricultural biotechnology have created a need to develop methods capable of selectively detecting and quantifying multiple proteins in complex matrices because of increasing numbers of transgenic proteins being coexpressed or "stacked" to achieve tolerance to multiple herbicides or to provide multiple modes of action for insect control. A multiplexing analytical method utilizing liquid chromatography with tandem mass spectrometry (LC-MS/MS) has been developed and validated to quantify three herbicide-tolerant proteins in soybean tissues: aryloxyalkanoate dioxygenase (AAD-12), 5-enol-pyruvylshikimate-3-phosphate synthase (2mEPSPS), and phosphinothricin acetyltransferase (PAT). Results from the validation showed high recovery and precision over multiple analysts and laboratories. Results from this method were comparable to those obtained with ELISA with respect to protein quantitation, and the described method was demonstrated to be suitable for multiplex quantitation of transgenic proteins in GE crops.

Chromatic biosensor for detection of phosphinothricin acetyltransferase by use of polydiacetylene vesicles encapsulated within automatically generated immunohydrogel beads.

We developed a simple and sensitive colorimetric biosensor in the form of microparticles by using polydiacetylene (PDA) vesicles encapsulated within a hydrogel matrix for the detection of phosphinothricin acetyltransferase (PAT) protein, which is one of the most important marker proteins in genetically modified (GM) crops. Although PDA is commonly used as a sensing material due to its unique colorimetric properties, existing PDA biosensors are ineffective due to their low sensitivity as well as their lack of robustness. To overcome these disadvantages, we devised immunohydrogel beads made of anti-PAT-conjugated PDA vesicles embedded at high density within a poly(ethylene glycol) diacrylate (PEG-DA) hydrogel matrix. In addition, the construction of immunohydrogel beads was automated by use of a microfluidic device. In the immunoreaction, the sensitivity of antibody-conjugated PDA vesicles was significantly amplified, as monitored by the unaided eye. The limit of detection for target molecules reached as low as 20 nM, which is sufficiently low enough to detect target materials in GM organisms. Collectively, the results show that immunohydrogel beads constitute a promising colorimetric sensing platform for onsite testing in a number of fields, such as the food and medical industries, as well as warfare situations.

Protein restriction in the rat negatively impacts long-chain polyunsaturated fatty acid composition and mammary gland development at the end of gestation.

BACKGROUND AND AIMS: Maternal nutrition during gestation is critical for mammary gland cell proliferation and differentiation and development of optimal delta-6 (Δ6D) and delta-5 (Δ5D) desaturase and elongase 2 and 5 (Elovl 2 and 5) activity for synthesis of the long chain polyunsaturated fatty acids (LC-PUFAs), arachidonic (AA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, important for normal fetal and neonatal brain development. We hypothesized that maternal low protein diet (LPD) impairs mammary gland preparation for lactation and PUFA synthesis. The aim of the study was to evaluate consequences of maternal LPD on mammary gland structure and development and expression of enzymes responsible for LC-PUFA production. METHODS: Pregnant rats were assigned to control or protein restricted, isocaloric diet (R). At 19 days gestation, mammary gland tissue was removed for histological analysis and lipid, AA, EPA and DHA determination by gas chromatography. Gene transcription was quantified by RT-PCR and protein by Western blot. RESULTS: In R mothers, mammary gland lobuloalveolar development was decreased and showed fat cell infiltration. Δ6D, Δ5D, and Elovl 5 mRNA were lower in R, whereas protein levels measured by Western blot were unchanged. This is the first report that detects mammary gland desaturase and elongase protein. Although Elovl 2 mRNA was not detectable by RT-PCR, Elovl 2 protein was not different between groups. AA and DHA were lower and EPA undetectable in the mammary gland of R mothers. CONCLUSIONS: Maternal LPD decreased late gestation mammary gland lobuloalveolar development and LC-PUFAs. Protein restriction negatively impacts maternal mammary gland development prior to lactation.

Relevance of histone marks H3K9me3 and H4K20me3 in cancer.

BACKGROUND: Circulating nucleosomes are valuable biomarkers for therapy monitoring and estimation of prognosis in cancer disease. While epigenetic and genetic modifications of DNA have been reported in blood of cancer patients, little is known about modifications of histones on circulating nucleosomes. PATIENTS AND METHODS: Sera of 45 cancer patients (21 colorectal, 4 pancreatic, 15 breast, 5 lung cancer), 12 patients with benign gastrointestinal and inflammatory diseases, and 28 healthy individuals were investigated. Histone modifications were detected by chromatin-immunoprecipitation (ChIP) using antibodies for triple histone methylations at sites H3K9me3 and H4K20me3 and subsequent real-time polymerase chain reaction using primers for the centromeric satellites SAT2. Additionally, the amount of circulating nucleosomes, as well as of carcino-embryonic antigen (CEA) and cancer antigen (CA) 19-9 were measured. RESULTS: Levels of SAT2 on H3K9me3 (median 0.507 ng/ml) and on H4K20me3 (0.292 ng/ml) were elevated in sera of patients with breast cancer when compared with healthy controls (0.049 and 0.035 ng/ml), but were lower in patients with colorectal cancer (0.039 and 0.027 ng/ml). Both histone marks were correlated with each other but did not correlate with CEA or CA 19-9 in cancer patients. When H3K9me3 and H4K20me3 were normalized to nucleosome content in sera, ratios were significantly higher in all types of cancer as well as in colorectal and breast subtypes when compared with healthy controls. Best discrimination was achieved by normalized H4K20me3 reaching areas under the curves (AUC) of 79.1%, 90.4% and 81.2% in receiver operating characteristic (ROC) curves of these three comparisons. CONCLUSION: SAT2 levels on H3K9me3 and H4K20me3 are up-regulated in breast cancer and down-regulated in colorectal cancer. Normalization to total nucleosome content enables better discrimination between cancer and control groups.

Development of an immunochromatographic assay for rapid detection of AAC(6')-Ib-producing Pseudomonas aeruginosa.

To detect aminoglycoside 6'-N-acetyltransferase-Ib [AAC(6')-Ib]-producing, Pseudomonas aeruginosa isolates which are a frequent cause of nosocomial infections in Japan, an immunochromatographic assay was developed using two kinds of monoclonal antibodies (mAbs) recognizing AAC(6')-Ib. The results of the assessment were fully consistent with those of aac(6')-Ib PCR analyses.

Functional and structural characterization of the mammalian TREX-2 complex that links transcription with nuclear messenger RNA export.

Export of messenger RNA (mRNA) from the nucleus to the cytoplasm is a critical step in the gene expression pathway of eukaryotic cells. Here, we report the functional and structural characterization of the mammalian TREX-2 complex and show how it links transcription/processing with nuclear mRNA export. Mammalian TREX-2 is based on a germinal-centre associated nuclear protein (GANP) scaffold to which ENY2, PCID2 and centrins bind and depletion of any of these components inhibits mRNA export. The crystal structure of the GANP:ENY2 complex shows that two ENY2 chains interact directly with GANP, but they have different orientations from those observed on yeast Sac3. GANP is required to recruit ENY2 to nuclear pore complexes (NPCs), but ENY2 is not necessary to recruit GANP, which requires both its CID and MCM3AP domains, together with nucleoporin Nup153. GANP and ENY2 associate with RNA polymerase II and inhibition of mRNA processing redistributes GANP from NPCs into nuclear foci indicating that mammalian TREX-2 is associated with transcription. Thus, we implicate TREX-2 as an integral component of the mammalian mRNA export machinery where it links transcription and nuclear export by facilitating the transfer of mature mRNPs from the nuclear interior to NPCs.

Microbead-assisted PDA sensor for the detection of genetically modified organisms.

A simple and sensitive approach for the detection of marker protein, phosphinothricin acetyltransferase, from genetically modified crops was developed based on the colorimetric transition of polydiacetylene (PDA) vesicles in combination with silica microbeads. PDAs have attracted a great deal of interests as a transducing material due to their special features that allow colorimetric response to sensory signals, as well as their inherent simplicity. However, most PDA-based biosensors require additional analytical equipment such as a fluorescence microscope or UV-Vis spectrometer. In this study, we report a new approach to increase the degree of color transition by coupling antibody-conjugated PDA vesicles with silica microbeads in an effort to monitor the results with the unaided eye or simple RGB analysis. By immobilizing PDA vesicles on silica microbeads, we were able to overcome the disadvantages of colloidal PDA-based sensors and increase the degree of colorimetric changes in response to target molecules to a concentration as low as 20 nM. The additional stresses were given to PDA vesicles by antigen-antibody bridging of PDA vesicles coupled with microbeads, resulting in enhanced blue-red color transition. All the results showed that PDA vesicles in conjunction with silica microbeads will be a promising transducing material for the detection of target proteins in diagnostic and biosensing applications.

N(α)-Acetylation of yeast ribosomal proteins and its effect on protein synthesis.

N(α)-Acetyltransferases (NATs) cause the N(α)-acetylation of the majority of eukaryotic proteins during their translation, although the functions of this modification have been largely unexplored. In yeast (Saccharomyces cerevisiae), four NATs have been identified: NatA, NatB, NatC, and NatD. In this study, the N(α)-acetylation status of ribosomal protein was analyzed using NAT mutants combined with two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry (MS). A total of 60 ribosomal proteins were identified, of which 17 were N(α)-acetylated by NatA, and two by NatB. The N(α)-acetylation of two of these, S17 and L23, by NatA was not previously observed. Furthermore, we tested the effect of ribosomal protein N(α)-acetylation on protein synthesis using the purified ribosomes from each NAT mutant. It was found that the protein synthesis activities of ribosomes from NatA and NatB mutants were decreased by 27% and 23%, respectively, as compared to that of the normal strain. Furthermore, we have shown that ribosomal protein N(α)-acetylation by NatA influences translational fidelity in the presence of paromomycin. These results suggest that ribosomal protein N(α)-acetylation is necessary to maintain the ribosome's protein synthesis function.

Development of an immunochromatographic assay for the rapid detection of AAC(6')-Iae-producing multidrug-resistant Pseudomonas aeruginosa.

OBJECTIVES: To develop an easy-to-use method for the rapid detection of antibiotic-resistant bacteria. Here, a new immunochromatographic assay specific for aminoglycoside 6'-N-acetyltransferase AAC(6')-Iae was designed. AAC(6')-Iae is a significant marker molecule for multidrug-resistant (MDR) Pseudomonas aeruginosa isolates in Japan. METHODS: Monoclonal antibodies specific for AAC(6')-Iae were used to construct the assay. The assessment of the assay was performed using 116 P. aeruginosa clinical isolates obtained from hospitals in the Kanto area of Japan where little was known about AAC(6')-Iae producers. PCR analyses of the aac(6')-Iae and class 1 integron, antimicrobial susceptibility testing and PFGE analysis were performed to characterize positive strains. RESULTS: The detection limit of the assay was 1.0 x 10(5) cfu. Of 116 clinical isolates, 60 were positive for AAC(6')-Iae using the assay. The results of assessment with clinical isolates were fully consistent with those of aac(6')-Iae PCR analyses, showing no false positives or negatives. All positive strains detected by the assay showed MDR phenotypes that were resistant to several classes of antibiotic. PFGE analysis showed that 59 of 60 positive strains tightly clustered, and these included clonal expansions. CONCLUSIONS: The developed assay is an easy-to-use and reliable detection method for AAC(6')-Iae-producing MDR P. aeruginosa. This approach may be applicable for screening and investigation of antibiotic-resistant bacteria as an alternative to PCR analysis.

[Molecular characterization of Pseudomonas aeruginosa isolates in Cantabria, Spain, producing VIM-2 metallo-beta-lactamase]. / Caracterización molecular de aislados clínicos de Pseudomonas aeruginosa productores de la metalobetalactamasa VIM-2 en Cantabria, España.

INTRODUCTION: Pseudomonas aeruginosa strains producing metallo-beta-lactamases (MbetaL) are uncommon in Spain. This study describes the characterization of 9 new clonally related multiresistant P. aeruginosa isolates possessing the bla(VIM-2) gene in Cantabria (Northern Spain). METHODS: P. aeruginosa clinical strains (1 per patient) were isolated in the Microbiology Service of Marqués de Valdecilla University Hospital between January 2004 and December 2006. Identification and preliminary susceptibility studies were performed with the MicroScan WalkAway system (Dade Behring, Sacramento, CA) and results were verified by a microdilution reference method. RESULTS: MICs of imipenem and meropenem for the 9 isolates ranged from 32 to 128 and 16 to 64 microg/mL, respectively. Nine isolates had a single Rep-PCR pattern and were intermediate or resistant to ceftazidime, cefepime, gentamicin, tobramycin, amikacin and ciprofloxacin. Eight of the 9 isolates were susceptible to aztreonam. Hydrolysis activity of imipenem in MbetaL-positive isolates ranged from 162+/-18 to 235+/-28 pmol/min/microg protein and was abolished in the presence of 5 mM EDTA. All isolates possessed an integron with genes aac(6')32, bla(VIM-2) and a putative transposase-encoding gene, flanked by the conserved 5'CS and 3'CS regions. CONCLUSION: In the clinical isolates studied, the presence of MbetaL VIM-2 sufficed to explain their resistance to carbapenems.