Detection of rare carbapenemases in Enterobacterales-comparison of two colorimetric and three CIM-based carbapenemase assays.

Rapid and reliable detection of carbapenemase-producing Enterobacterales (CPE) is crucial for prompt treatment and infection control. Most assays target the primary four enzymes (KPC, OXA-48-like, VIM, and NDM), often missing less common variants (e.g., GES, IMI, OXA-23, and OXA-58). Therefore, assays based on the hydrolysis of carbapenems are recommended in addition to differentiation tests such as PCR or immunochromatographic assays. The aim of this study was to compare the currently Clinical and Laboratory Standards Institute (CLSI)-recommended tests mCIM (modified carbapenem inactivation method) and Carba NP with new colorimetric tests (NitroSpeed-Carba NP) and novel variations of the carbapenem inactivation method (CIM) such as simplified CIM (sCIM) or modified zinc-supplemented CIM (mzCIM). The challenge collection included 205 clinical isolates, 139 CPE vs 66 non-CPE. Among all 205 isolates, the sensitivity/specificity of mCIM was 81.3%/98.5%, Carba NP 76.3%/100%, NitroSpeed-Carba NP 86.3%/78.8%, sCIM 100%/94%, and mzCIM 97.8%/98.5%. For rare carbapenemases (n = 48), the sensitivity of mzCIM (98.3%) and sCIM (100%) was higher than that of mCIM (60.4%), Carba NP (50%), or NitroSpeed-Carba NP (70.2%). Most indeterminate results occurred for mCIM (14.4%), Carba NP (8.2%), and sCIM (6.3%). The detection of rare carbapenemases remains challenging with the currently recommended assays. The CIM-based tests demonstrated superior sensitivity, with sCIM and mzCIM outperforming the currently recommended mCIM and Carba NP, especially among isolates with weakly hydrolyzing carbapenemases (e.g., OXA-23 and OXA-58). Although colorimetric assays provide more rapid results, laboratories have to be aware of the low sensitivity for rare carbapenemases. Both sCIM and the new mzCIM performed well, are cost-effective, and can easily be implemented in any laboratory.IMPORTANCEDetection of so-called rare carbapenemases (e.g., GES, IMI, OXA-23, and OXA-58) in Enterobacterales is challenging, and data on the performance of currently available assays are scarce. This study systematically assessed the performance of currently recommended and novel hydrolysis-based assays on a set of molecularly characterized isolates. It demonstrates that the currently recommended assays mCIM and Carba NP perform well on isolates producing common carbapenemases such as KPC, VIM, NDM, and OXA-48, but have only a moderate sensitivity in the detection of rare carbapenemases. In contrast, the newer CIM-based variants, sCIM and mzCIM, are equally capable of detecting frequent and uncommon carbapenemases. These assays could potentially help to improve our knowledge on the epidemiology of these "rare" enzymes.

Proteus mirabilis - analysis of a concealed source of carbapenemases and development of a diagnostic algorithm for detection.

OBJECTIVES: To analyse carbapenemases in Proteus mirabilis and assess the performance of carbapenemase detection assays. METHODS: Eighty-one clinical P. mirabilis isolates with high-level resistance at least to ampicillin (>32 mg/L) or previous detection of carbapenemases were selected and investigated by three susceptibility testing methods (microdilution, automated susceptibility testing, and disk diffusion), six phenotypic carbapenemase assays (CARBA NP, modified carbapenemase inactivation method [CIM], modified zinc-supplemented CIM, simplified CIM, faropenem, and carbapenem-containing agar), two immunochromatographic assays, and whole-genome sequencing. RESULTS: Carbapenemases were detected in 43 of 81 isolates (OXA-48-like [n = 13]; OXA-23 [n = 12]; OXA-58 [n = 12]; New Delhi metallo-ß-lactamase (NDM) [n = 2]; Verona integron-encoded metallo-ß-lactamase (VIM) [n = 2]; Imipenemase (IMP) [n = 1]; Klebsiella pneumoniae carbapenemase (KPC) [n = 1]). Carbapenemase-producing Proteus were frequently susceptible to ertapenem (26/43; 60%), meropenem (28/43; 65%), ceftazidime (33/43; 77%), and some even to piperacillin-tazobactam (9/43; 21%). Sensitivity/specificity of phenotypic tests were 30% (CI: 17-46%)/89% (CI: 75-97%) for CARBA NP, 74% (CI: 60-85%)/82% (CI: 67-91%) for faropenem, 91% (CI: 78-97%)/82% (CI: 66-92%) for simplified CIM, and 93% (CI: 81-99%)/100% (CI: 91-100%) for modified zinc-supplemented CIM. An algorithm for improved detection was developed, which demonstrated sensitivity/specificity of 100% (CI: 92-100%)/100% (CI: 91-100%) on the 81 isolates, and 100% (CI: 29-100%)/100% (CI: 96-100%) in a prospective analysis of additional 91 isolates. Interestingly, several OXA-23-producing isolates belonged to the same clonal lineage reported previously from France. DISCUSSION: Current susceptibility testing methods and phenotypic tests frequently fail to detect carbapenemases in P. mirabilis, which could result in inadequate antibiotic treatment. In addition, the non-inclusion of blaOXA-23/OXA-58 in many molecular carbapenemase assays further impedes their detection. Therefore, the prevalence of carbapenemases in P. mirabilis is likely underestimated. With the herein proposed algorithm, carbapenemase-producing Proteus can be easily identified.

Carbapenemase-producing Gram-negative bacteria in hospital wastewater, wastewater treatment plants and surface waters in a metropolitan area in Germany, 2020.

Carbapenemase-producing bacteria (CPB) such as Klebsiella pneumoniae and Escherichia coli are causing hospital outbreaks worldwide. An important transfer route into the aquatic environment is the urban water cycle. We aimed to determine the presence of CPB in hospital wastewater, wastewater treatment plants (WWTPs) and surface waters in a German metropolitan area and to characterise these bacteria by whole-genome comparisons. During two periods in 2020, 366 samples were collected and cultivated on chromogenic screening media. Bacterial colonies were selected for species identification and PCR-based carbapenemase gene screening. Genomes of all detected CPB were sequenced and analysed for resistance gene content, followed by multilocus sequence typing (MLST) and core genome MLST (cgMLST) for K. pneumoniae and E. coli isolates. Carbapenemase genes were detected in 243 isolates, most of which belonged to genera/species Citrobacter spp. (n = 70), Klebsiella spp. (n = 57), Enterobacter spp. (n = 52) and E. coli (n = 42). Genes encoding KPC-2 carbapenemase were detected in 124 of 243 isolates. K. pneumoniae produced mainly KPC-2 and OXA-232 whereas E. coli harboured various enzymes (KPC-2, VIM-1, OXA-48, NDM-5, KPC-2 + OXA-232, GES-5, GES-5 + VIM-1, IMP-8 + OXA-48). Eight and twelve sequence types (STs) were identified for K. pneumoniae and E. coli, respectively, forming different clusters. The detection of numerous CPB species in hospital wastewater, WWTPs and river water is of concern. Genome data highlight a hospital-specific presence of distinct carbapenemase-producing K. pneumoniae and E. coli strains belonging to "global epidemic clones" in wastewater samples representing local epidemiology. The various detected CPB species including E. coli ST635, which is not known to cause human infections, could serve as reservoirs/vectors for the spread of carbapenemase genes in the environment. Therefore, effective pretreatment of hospital wastewater prior to discharge into the municipal wastewater system may be required, although swimming lakes do not appear to be a relevant risk factor for CPB ingestion and infection.

The prediction values of carbapenemase detection methods and carbapenem susceptibility testing for clinical outcomes of patients with Acinetobacter bacteremia under carbapenem treatment.

BACKGROUND: Carbapenem-resistant Acinetobacter species have emerged as notorious pathogens causing nosocomial infections. Several phenotypic methods have been developed for detecting carbapenemase production in Enterobacteriaceae. The accuracy of these methods in the prediction of carbapenemase production in Acinetobacter species has not been studied well. METHODS: This retrospective study enrolled adult patients with Acinetobacter bacteremia from four medical centers in Taiwan between 2012 and 2016. Their demographics and clinical outcomes were recorded. The carbapenem susceptibility of the Acinetobacter species was determined using the agar diffusion method. The carbapenemase genes were detected by PCR. Four phenotypic methods, including the modified Hodge test (MHT), modified carbapenem inactivation method (mCIM), Carba NP test, and CarbAcineto NP test were carried out to determine the production of carbapenemase. RESULTS: We analyzed 257 adults who received initial carbapenem monotherapy for the treatment of Acinetobacter bacteremia. Shock within three days of bacteremia and acquisition of carbapenem non-susceptible isolates were independently associated with a higher 14-day and 30-day mortality in patients with Acinetobacter bacteremia. Among the four phenotypic tests for carbapenemase detection, MHT using the imipenem disc displayed the greatest sensitivity (94%; 95% confidence interval [CI], 89-97%) and specificity (81%; 95% CI, 73-88%) for predicting imipenem non-susceptibility. CONCLUSION: Carbapenem non-susceptibility and shock were independent risk factors for mortality in patients with Acinetobacter bacteremia. The MHT could predict the carbapenem susceptibility of Acinetobacter isolates. It is a cheap and quick assay, which could be applied in clinical practice.

Enhanced production of Aspergillus niger inulinase from sugar beet molasses and its kinetic modeling.

OBJECTIVE: The fermentation medium contains many complex components (vitamins, minerals, etc.) for better growth of the microorganisms. The increasing purity and number of these components used in the medium seriously affect the cost of the microbial process. This study aimed to further optimize the concentration of the components used in the medium (yeast extract and peptone) for inulinase fabrication by Aspergillus niger from sugar-beet molasses in shake flask fermentation by using Central Composite Design (CCD) and to kinetically identify the fermentation. RESULTS: The results indicated that the optimal medium composition consisted of only 4.2% (w/v) yeast extract. By using the fermentation environment, the inulinase generation, inulinase/sucrase ratio, maximum inulinase generation rate, maximum sugar depletion rate, and substrate utilization yield were determined as 1294.5 U/mL, 1.2, 159.6 U/mL/day, 7.4 g/L/day, and 98.1%, respectively. The kinetic analysis of the fungal development (logistic model) indicated that a specific development rate and initial biomass concentration were 0.89/day and 1.79 g/L, respectively. Inulinase and sucrase productions are mixed-development associated since the α value ≠ 0 (8.46 and 4.31 U/mgX) and the ß value ≠ 0 (5.15 and 4.83 U/mgX day), respectively (Luedeking-Piret model). Besides, the maintenance value (Z) (0.009 gS/gX day) was lower than γ value (1.044 gS/gX), showing that A. niger commonly uses the substrates for enzyme fabrication and fungal development (modified Luedeking-Piret model). CONCLUSIONS: The enzyme activity was increased by optimizing the concentration of the components used. It was demonstrated that the proposed kinetic models can victoriously define fungal development, enzyme fabrication, and sugar depletion.

A Combined Proteomics, Metabolomics and In Vivo Analysis Approach for the Characterization of Probiotics in Large-Scale Production.

The manufacturing processes of commercial probiotic strains may be affected in different ways in the attempt to optimize yield, costs, functionality, or stability, influencing gene expression, protein patterns, or metabolic output. Aim of this work is to compare different samples of a high concentration (450 billion bacteria) multispecies (8 strains) formulation produced at two different manufacturing sites, United States of America (US) and Italy (IT), by applying a combination of functional proteomics, metabolomics, and in vivo analyses. Several protein-profile differences were detected between IT- and US-made products, with Lactobacillus paracasei, Streptococcus thermophilus, and Bifidobacteria being the main affected probiotics/microorganisms. Performing proton nuclear magnetic spectroscopy (1H-NMR), some discrepancies in amino acid, lactate, betaine and sucrose concentrations were also reported between the two products. Finally, we investigated the health-promoting and antiaging effects of both products in the model organism Caenorhabditis elegans. The integration of omics platforms with in vivo analysis has emerged as a powerful tool to assess manufacturing procedures.

Comparison of five methods for detection of carbapenemases in Enterobacterales with proposal of a new algorithm.

OBJECTIVES: The aim of this study was to evaluate the performance of five different carbapenemase tests and to develop an algorithm which will permit the detection of most common and rare carbapenemases in routine microbiology laboratories. METHODS: The immunochromatographic tests CARBA-5 (NG), RESIST-4 O.K.N.V. (Coris), the colorimetric ß-CARBA (BioRad), a newly developed carbapenem-inactivation method (CIM) supplemented with zinc (zCIM), and the Xpert Carba-R (Cepheid) were challenged with a collection of 189 molecularly characterized Enterobacterales isolates, including 146 carbapenemase producers (CPE): VIM (n = 48), OXA-48-like (n = 40), NDM (n = 29), KPC (n = 13), IMI (n = 9), IMP (n = 9), OXA-58 (n = 2), and GES (n = 2). RESULTS: The overall sensitivity/specificity values for the five carbapenemase detection tests were 84.2% (CI 77.6-89.2%)/100% (CI 91.8-100%) for RESIST-4, 88.2% (CI 82.1-92.4%)/100% (CI 91.8-100%) for CARBA-5, 88.2% (CI 82.1-92.4%)/100% (CI 91.8-100%) for Xpert Carba-R, 73.7% (CI 66.2-80.0%)/100% (CI 93.4-99.0%) for ß-CARBA, and 97.4% (CI 87.9-99.6%)/97.7% (CI 87.9-99.6%) for zCIM. The four common carbapenemases (KPC, OXA-48-like, NDM, and VIM) were detected with ≥97.6% sensitivity by all tests except for ß-CARBA (76.6% (CI 68.4-83.2%)). IMI and GES were only detected by zCIM (sensitivity 90.9% (CI 62.3-98.4%)). Based on these results a new algorithm was developed, consisting of an immunochromatographic assay as the first test followed by zCIM, which allows detection of 99.3% of all carbapenemases assessed. CONCLUSIONS: Except for ß-CARBA, all methods showed excellent sensitivity/specificity for the detection of the four most frequent carbapenemases. With the new algorithm, rare variants can also be detected. It is rapid, simple, and inexpensive and can be performed in any microbiology laboratory, as no PCR equipment is required.

Rapid detection of carbapenemases directly from positive blood cultures by the ß-CARBA test.

The rapid detection of blood stream infections (BSI) by carbapenemase-producing Enterobacterales (CPE) is indispensable to early optimize antibiotic treatment and to improve survival. While phenotypic tests are time-consuming and PCR is expensive and not available in many routine laboratories, colorimetric tests (e.g., Carba NP test) can provide rapid results at moderate cost. However, up to now, the detection of CPE-BSI requires a further 3-h incubation in broth supplemented with zinc sulfate and imipenem after a blood culture has become positive, thereby causing delay and additional hands-on time. The purpose of this study was to develop and evaluate a new method for the detection of CPE directly from positive blood culture without the need for incubation in broth, based on the commercially available colorimetric ß-CARBA test. For the evaluation, blood cultures spiked with 140 different Enterobacterales isolates producing diverse beta-lactamases were tested with the new method. Of these, 70 were CPE (OXA-48-like, NDM, KPC, VIM, and GIM). After blood cultures turned positive, blood culture fluid was drawn, and erythrocytes were hemolyzed with SDS, washed, and equilibrated before the ß-CARBA was performed on the bacterial pellet. All carbapenemases were reliably detected, including weak carbapenemases of the OXA-48 group. The sensitivity was 100% (95% CI 94.9-100) and the specificity 94.3% (95% CI 89.2-99.4). The time to result was 20 to 45 min. Carbapenemases can rapidly and reliably be detected directly from blood cultures using the new method, which could help to improve the outcome of these difficult-to-treat infections.

Rapid detection of OXA-48-like, KPC, NDM, and VIM carbapenemases in Enterobacterales by a new multiplex immunochromatographic test.

The rapid detection of carbapenemase-producing Gram-negative bacteria is indispensable to optimize treatment and avoid the further spread of these organisms. While phenotypic tests are time-consuming and PCR is expensive and not available in many routine laboratories, immunochromatographic tests (ICT) can provide rapid results at moderate cost. The aim of this study was to determine the performance of the new ICT RESIST-4 O.K.N.V. K-SeT (Coris BioConcept, Gembloux, Belgium) which can detect the four most prevalent carbapenemases: OXA-48-like, KPC, NDM, and VIM. Additionally, we analyzed the impact of different culture conditions on the sensitivity. The new ICT was challenged with 169 carbapenem-resistant isolates. Of these, 125 were carbapenemase producers: 43 OXA-48-like, 15 KPC, 29 NDM, and 43 VIM. The ICT correctly detected 129 of the 130 carbapenemases resulting in a sensitivity of 99.2% and specificity of 100% when tested from Mueller-Hinton agar (MHA). The sensitivity of the assay increased to 100% when performed from zinc-supplemented MHA and sheep blood agar (SBA) or when the inoculum was harvested from the inhibition zone of an ertapenem disk. All carbapenemase-negative carbapenem-resistant bacteria tested negative and no cross-reaction was observed. The new ICT is an excellent test for rapid diagnostic of carbapenemase-producing Gram-negatives in the routine laboratory. It is easy to handle and provides rapid results with a high sensitivity. For best results, we recommend to obtain the inoculum from a medium with sufficient zinc or from the inhibition zone of an ertapenem disk.

An intrinsic fluorescence method for the determination of protein concentration in vaccines containing aluminum salt adjuvants.

Determination of protein concentration in vaccines containing aluminum salt adjuvant typically necessitates desorption of the protein prior to analysis. Here we describe a method based on the intrinsic fluorescence of tyrosine and tryptophan that requires no desorption of proteins. Adjuvanted formulations of three model Bordetella pertussis antigens were excited at 280 nm and their emission spectra collected from 290 to 400 nm. Emission spectra of protein antigens in the presence of aluminum salt adjuvants were able to be detected, the effects of adjuvants on the spectra were analyzed, and linear regressions were calculated. The fluorescence method proved to be very sensitive with a limit of quantification between 0.4 and 4.4 µg/mL and limit of linearity between 100 and 200 µg/mL, across the formulations tested. The fluorescence method was found to be influenced by adjuvant presence, type of adjuvant, adjuvant concentration, buffer and pH conditions. The method also demonstrated ability to monitor the percent adsorption of antigens to the adjuvants. Furthermore, intrinsic fluorescence showed good correlation with micro-Kjeldahl elemental assay in quantifying protein concentration. Being a non-invasive, quick and sensitive method, intrinsic fluorescence has the potential to be utilized as a high throughput tool for vaccine development and conceivably implemented in-line, using in-line fluorimeters, to monitor antigen concentration during formulation processing.

Vacuole-Targeted Proteins: Ins and Outs of Subcellular Localization Studies.

Accurate and efficient demonstrations of protein localizations to the vacuole or tonoplast remain strict prerequisites to decipher the role of vacuoles in the whole plant cell biology and notably in defence processes. In this chapter, we describe a reliable procedure of protein subcellular localization study through transient transformations of Catharanthus roseus or onion cells and expression of fusions with fluorescent proteins allowing minimizing artefacts of targeting.

Identification of Differentially Regulated Edwardsiella ictaluri Proteins During Catfish Serum Treatment.

Edwardsiella ictaluri is a facultative, intracellular, gram-negative bacterium that causes enteric septicemia of catfish (ESC). Edwardsiella ictaluri is known to be resistant to defense mechanisms present in catfish serum, which might aid in its use of a host's bloodstream to become septicemic. However, the precise mechanisms of the survival of E. ictaluri in host serum are not known. Analysis of the response of E. ictaluri to the host serum treatment at a proteomic level might aid in the elucidation of its adaptation mechanisms against defense mechanisms present in catfish serum. Thus, the objective of this study was to identify differentially regulated proteins of E. ictaluri upon exposure to naïve catfish serum. Two-dimensional difference gel electrophoresis (2D-DIGE) followed by in-gel trypsin digestion and MALDI-TOF/TOF analysis were used for identification of differentially expressed E. ictaluri proteins. A total of 19 differentially regulated proteins (7 up- and 12 downregulated) were identified. Among those were four putative immunogenic proteins, two chaperones and eight proteins involved in the translational process, two nucleic acid degradation and integration proteins, two intermediary metabolism proteins, and one iron-ion-binding protein. Further research focusing on the functions of these differentially expressed proteins may reveal their roles in host adaptation by E. ictaluri.

Proteogenomic insights into uranium tolerance of a Chernobyl's Microbacterium bacterial isolate.

Microbacterium oleivorans A9 is a uranium-tolerant actinobacteria isolated from the trench T22 located near the Chernobyl nuclear power plant. This site is contaminated with different radionuclides including uranium. To observe the molecular changes at the proteome level occurring in this strain upon uranyl exposure and understand molecular mechanisms explaining its uranium tolerance, we established its draft genome and used this raw information to perform an in-depth proteogenomics study. High-throughput proteomics were performed on cells exposed or not to 10µM uranyl nitrate sampled at three previously identified phases of uranyl tolerance. We experimentally detected and annotated 1532 proteins and highlighted a total of 591 proteins for which abundances were significantly differing between conditions. Notably, proteins involved in phosphate and iron metabolisms show high dynamics. A large ratio of proteins more abundant upon uranyl stress, are distant from functionally-annotated known proteins, highlighting the lack of fundamental knowledge regarding numerous key molecular players from soil bacteria. BIOLOGICAL SIGNIFICANCE: Microbacterium oleivorans A9 is an interesting environmental model to understand biological processes engaged in tolerance to radionuclides. Using an innovative proteogenomics approach, we explored its molecular mechanisms involved in uranium tolerance. We sequenced its genome, interpreted high-throughput proteomic data against a six-reading frame ORF database deduced from the draft genome, annotated the identified proteins and compared protein abundances from cells exposed or not to uranyl stress after a cascade search. These data show that a complex cellular response to uranium occurs in Microbacterium oleivorans A9, where one third of the experimental proteome is modified. In particular, the uranyl stress perturbed the phosphate and iron metabolic pathways. Furthermore, several transporters have been identified to be specifically associated to uranyl stress, paving the way to the development of biotechnological tools for uranium decontamination.

Pestalotiopsis-Like Species Causing Gray Blight Disease on Camellia sinensis in China.

Gray blight of tea, caused by several Pestalotiopsis-like species, is one of the most destructive foliar diseases in tea cultivation yet the characteristics of these pathogens have not been confirmed until now. With morphological and multigene phylogenetic analyses, we have identified the gray blight fungi as Pseudopestalotiopsis camelliae-sinensis, Neopestalotiopsis clavispora, and Pestalotiopsis camelliae. Phylogenetic analyses derived from the combined internal transcribed spacer, ß-tubulin, and translation elongation factor 1-α gene regions successfully resolved most of the Pestalotiopsis-like species used in this study with high bootstrap supports and revealed three major clusters representing these three species. Differences in colony appearance and conidia morphology (shape, size, septation, color and length of median cells, and length and number of apical and basal appendages) were consistent with the phylogenetic grouping. Pathogenicity tests validated that all three species isolated from tea leaves were causal agents of gray blight disease on tea plant (Camellia sinensis). This is the first description of the characteristics of the three species Pseudopestalotiopsis camelliae-sinensis, N. clavispora, and Pestalotiopsis camelliae as causal agents of tea gray blight disease in China.

A Homogeneous Cell-Based Halide-Sensitive Yellow Fluorescence Protein Assay to Identify Modulators of the Cystic Fibrosis Transmembrane Conductance Regulator Ion Channel.

Cystic fibrosis (CF), an inherited genetic disease, is caused by mutation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, which encodes an ion channel involved in hydration maintenance by anion homeostasis. Ninety percent of CF patients possess one or more copies of the F508del CFTR mutation. This mutation disrupts trafficking of the protein to the plasma membrane and diminishes function of mature CFTR. Identifying small molecule modulators of mutant CFTR activity or biosynthesis may yield new tools for discovering novel CF treatments. One strategy utilizes a 384-well, cell-based fluorescence-quenching assay, which requires extensive wash steps, but reports sensitive changes in fluorescence-quenching kinetic rates. In this study, we describe the methods of adapting the protocol to a homogeneous, miniaturized 1,536-well format and further optimization of this functional F508del CFTR assay. The assay utilizes a cystic fibrosis bronchial epithelial (CFBE41o-) cell line, which was engineered to report CFTR-mediated intracellular flux of iodide by a halide-sensitive yellow fluorescence protein (YFP) reporter. We also describe the limitations of quench rate analysis and the subsequent incorporation of a novel, kinetic data analysis modality to quickly and efficiently find active CFTR modulators. This format yields a Z' value interval of 0.61 ± 0.05. As further evidence of high-throughput screen suitability, we subsequently completed a screening campaign of >645,000 compounds, identifying 2,811 initial hits. After completing secondary and tertiary follow-up assays, we identified 187 potential CFTR modulators, which EC50's < 5 µM. Thus, the assay has integrated the advantages of a phenotypic screen with high-throughput scalability to discover new small-molecule CFTR modulators.

Bitrophic and Tritrophic Effects of Transgenic cry1Ab/cry2Aj Maize on the Beneficial, Nontarget Harmonia axyridis (Coleoptera: Coccinellidae).

Harmonia axyridis (Pallas) is a common and abundant predator in China and may be exposed to Cry toxins that are produced in Bt crops either by feeding on plant parts or by feeding on target or nontarget herbivorous insects. A new Bt maize line, expressing the Cry1Ab/Cry2Aj fused protein, has been developed and should be rigorously assessed for the ecological risks on the natural enemy. Laboratory experiments were carried out to study the effects of this Bt maize on nontarget predator H. axyridis via bitrophic interaction of adult H. axyridis feeding on Bt maize pollen and tritrophic interaction of H. axyridis consuming the lepidopteran prey. Spodoptera exigua (Hübner) neonate larvae were used to transfer Bt protein because they could survive after ingesting transgenic cry1Ab/cry2Aj maize kernels in the previous study. ELISA bioassays confirmed that the Bt protein could be transferred, but diluted through Bt maize-prey-predator. Life history parameters such as survival, development, weight, fecundity, and egg hatching rate were not significantly different when H. axyridis consumed prey that had been reared on Bt maize compared with prey reared on a nontransformed parental control. Furthermore, feeding directly on Bt maize pollen also had no detrimental effects on fitness, survival, and weight of female and male adults. In conclusion, our results indicate that transgenic cry1Ab/cry2Aj maize poses no ecological risks on the nontarget predator H. axyridis.

Prevalencia en España de mecanismos de resistencia a quinolonas en enterobacterias productoras de betalactamasas de clase C adquiridas y/o carbapenemasas / Prevalence of quinolone resistance mechanisms in Enterobacteriaceae producing acquired AmpC Beta-lactamases and/or carbapenemases in Spain

Introducción: En los últimos años se ha observado un incremento de la resistencia a fluoroquinolonas en enterobacterias, estando asociado significativamente a la resistencia a betalactámicos. Nuestro objetivo fue conocer la prevalencia de mecanismos cromosómicos y plasmídicos de resistencia a quinolonas en aislados productores de betalactamasas de claseC adquiridas y/o carbapenemasas. Métodos: Se evaluó la presencia de mecanismos cromosómicos y plasmídicos de resistencia a quinolonas [mutaciones en la región determinante de resistencia a quinolonas de gyrA y parCy genes qnr, aac(6')-Ib-cr y qepA] en 289 aislados de enterobacterias productoras de betalactamasas de claseC adquiridas y/o carbapenemasas recogidos entre febrero y julio de 2009 en 35 hospitales españoles. Resultados: Se detectaron determinantes plasmídicos en 92 aislados (31,8%); en 83 aislados (28,7%) se detectó algún gen qnr, y en 20 (7%), la variante aac(6')-Ib-cr. El gen qnr más prevalente fue qnrB4 (20%), asociado en la mayoría de los casos a DHA-1. El 14,6% de los aislados con una CMI de ciprofloxacino superior a 0,25mg/l no presentaban mutaciones en gyrA ni parC, detectándose en el 90% de los mismos algún determinante plasmídico de resistencia a quinolonas. Conclusión: qnrB4 fue el determinante plasmídico más prevalente, claramente asociado a DHA-1. Los mecanismos plasmídicos en asociación con mecanismos cromosómicos diferentes a las mutaciones en los genes de las topoisomerasas (sobreexpresión de bombas de expulsión, alteración del lipopolisacárido o disminución de porinas) pueden dar lugar a valores de CMI de ciprofloxacino que superan los puntos de corte establecidos por los principales comités internacionales de definición de puntos de corte para interpretación de datos de sensibilidad (AU)

Background: Quinolone resistance in Enterobacteriaceae species has increased over the past few years, and is significantly associated to beta-lactam resistance. The aim of this study was to evaluate the prevalence of chromosomal- and plasmid-mediated quinolone resistance in acquired AmpC Beta-lactamase and/or carbapenemase-producing Enterobacteriaceae isolates. Methods: The presence of chromosomal- and plasmid-mediated quinolone resistance mechanisms [mutations in the quinolone resistance determining region (QRDR) of gyrA and parC and qnr, aac(6')-Ib-cr and qepA genes] was evaluated in 289 isolates of acquired AmpC Beta -lactamase- and/or carbapenemase-producing Enterobacteriaceae collected between February and July 2009 in 35 Spanish hospitals. Results: Plasmid mediated quinolone resistance (PMQR) genes were detected in 92 isolates (31.8%), qnr genes were detected in 83 isolates (28.7%), and the aac(6')-Ib-cr gene was detected in 20 isolates (7%). qnrB4 gene was the most prevalent qnr gene detected (20%), associated, in most cases, with DHA-1. Only 14.6% of isolates showed no mutations in gyrA or parC with a ciprofloxacin MIC of 0.5mg/L or higher, whereas PMQR genes were detected in 90% of such isolates. Conclusion: qnrB4 gene was the most prevalent PMQR gene detected, and was significantly associated with acquired AmpC Beta -lactamase DHA-1. PMQR determinants in association with other chromosomal-mediated quinolone resistance mechanisms, different to mutations in gyrA and parC (increased energy-dependent efflux, altered lipopolysaccharide or porin loss), could lead to ciprofloxacin MIC values that exceed breakpoints established by the main international committees to define clinical antimicrobial susceptibility breakpoints (AU)

N-Acetylglucosamine-1-Phosphate Transferase, WecA, as a Validated Drug Target in Mycobacterium tuberculosis.

The mycobacterial phosphoglycosyltransferase WecA, which initiates arabinogalactan biosynthesis in Mycobacterium tuberculosis, has been proposed as a target of the caprazamycin derivative CPZEN-45, a preclinical drug candidate for the treatment of tuberculosis. In this report, we describe the functional characterization of mycobacterial WecA and confirm the essentiality of its encoding gene in M. tuberculosis by demonstrating that the transcriptional silencing of wecA is bactericidal in vitro and in macrophages. Silencing wecA also conferred hypersensitivity of M. tuberculosis to the drug tunicamycin, confirming its target selectivity for WecA in whole cells. Simple radiometric assays performed with mycobacterial membranes and commercially available substrates allowed chemical validation of other putative WecA inhibitors and resolved their selectivity toward WecA versus another attractive cell wall target, translocase I, which catalyzes the first membrane step in the biosynthesis of peptidoglycan. These assays and the mutant strain described herein will be useful for identifying potential antitubercular leads by screening chemical libraries for novel WecA inhibitors.

Utilidad de la resistencia a piperacilina/tazobactam como factor predictor de carbapenemasa OXA-48 en enterobacterias / Usefulness of piperacillin/tazobactam resistance as a predictor of OXA-48 carbapenemase in Enterobacteriaceae

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Comparisons of IgA response in saliva and colostrum against oral streptococci species.

The present study compared IgA specificity against oral streptococci in colostrum and saliva samples. Sixty-two mother-and-child pairs were included; samples of colostrum (C) and saliva (MS) were collected from the mothers and saliva samples were collected from babies (BS). The specificity of IgA against Streptococcus mutans and S. mitis were analyzed by western blot. Only 30% of babies' samples presented IgA reactivity to S. mutans, while 74 and 80% of MS and C, respectively, presented this response. IgA reactivity to S. mutans virulence antigens (Ag I/II, Gtf and GbpB) in positive samples showed differences between samples for Gtf and especially for GbpB (p < 0.05), but responses to Ag I/II were similar (p > 0.05). The positive response of Gtf-reactive IgA was different between C (90%) and MS (58%) samples (p < 0.05), but did not differ from BS (p > 0.05). GbpB was the least detected, with 48 and 26% of C and MS, and only 5% of BS samples presenting reactivity (p > 0.05). Eight percent of MS and C samples presented identical bands to SM in the same time-point. In conclusion, the differences of IgA response found between C and MS can be due to the different ways of stimulation, proliferation and transportation of IgA in those secretions. The colostrum has high levels of IgA against S. mutans virulence antigens, which could affect the installation and accumulation process of S. mutans, mainly by supplying anti-GbpB IgA to the neonate.