In Vitro Activity of Plazomicin Compared to Amikacin, Gentamicin, and Tobramycin against Multidrug-Resistant Aerobic Gram-Negative Bacilli.

The worldwide spread of multidrug-resistant Enterobacterales is a serious threat to public health. Here, we compared the MICs of plazomicin, amikacin, gentamicin, and tobramycin against 303 multinational multidrug-resistant Gram-negative bacilli. We followed Clinical and Laboratory Standards Institute (CLSI) guidelines and applied CLSI breakpoints as well as those of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) for amikacin, gentamicin, and tobramycin and of the U.S. Food and Drug Administration for plazomicin. Overall, the highest percentage of susceptible isolates (80.2%) was demonstrated for plazomicin, which had the lowest MIC50 (1 µg/ml) of the aminoglycosides studied. Of the 42 isolates resistant to plazomicin, 34 had MICs of ≥128 µg/ml, with 33 of the 34 having MICs of >128 µg/ml for amikacin, gentamicin, and tobramycin. Among the 42 blaNDM-positive isolates, 35.7% were plazomicin susceptible, with the percentage of isolates susceptible to amikacin being 38.1% or 35.7% when applying the CLSI or EUCAST breakpoint, respectively. The 20 blaOXA-48-like-positive isolates showed 50.0% susceptibility to plazomicin. Among 35 isolates with blaCTX-M as their only characterized resistance mechanism, 68.6% were plazomicin susceptible, while the percentage susceptible to amikacin was 74.3% or 62.9% when applying the CLSI or EUCAST breakpoint, respectively. Among the 117 blaKPC-positive isolates, 94.9% were susceptible to plazomicin, whereas when the CLSI and EUCAST breakpoints were applied, 43.6% and 25.6%, respectively, were susceptible to amikacin; 56.4% and 44.4%, respectively, were susceptible to gentamicin; and 5.1% and 4.3%, respectively, were susceptible to tobramycin.

Biochemical Properties and Phylogeny of Hydroxypyruvate Reductases from Methanotrophic Bacteria with Different C1-Assimilation Pathways.

In the aerobic methanotrophic bacteria Methylomicrobium alcaliphilum 20Z, Methylococcus capsulatus Bath, and Methylosinus trichosporium OB3b, the biochemical properties of hydroxypyruvate reductase (Hpr), an indicator enzyme of the serine pathway for assimilation of reduced C1-compounds, were comparatively analyzed. The recombinant Hpr obtained by cloning and heterologous expression of the hpr gene in Escherichia coli catalyzed NAD(P)H-dependent reduction of hydroxypyruvate or glyoxylate, but did not catalyze the reverse reactions of D-glycerate or glycolate oxidation. The absence of the glycerate dehydrogenase activity in the methanotrophic Hpr confirmed a key role of the enzyme in utilization of C1-compounds via the serine cycle. The enzyme from Ms. trichosporium OB3b realizing the serine cycle as a sole assimilation pathway had much higher special activity and affinity in comparison to Hpr from Mm. alcaliphilum 20Z and Mc. capsulatus Bath assimilating carbon predominantly via the ribulose monophosphate (RuMP) cycle. The hpr gene was found as part of gene clusters coding the serine cycle enzymes in all sequenced methanotrophic genomes except the representatives of the Verrucomicrobia phylum. Phylogenetic analyses revealed two types of Hpr: (i) Hpr of methanotrophs belonging to the Gammaproteobacteria class, which use the serine cycle along with the RuMP cycle, as well as of non-methylotrophic bacteria belonging to the Alphaproteobacteria class; (ii) Hpr of methylotrophs from Alpha- and Betaproteobacteria classes that use only the serine cycle and of non-methylotrophic representatives of Betaproteobacteria. The putative role and origin of hydroxypyruvate reductase in methanotrophs are discussed.

Evaluación de test rápidos y diseño de una estrategia para la detección y caracterización de carbapenemasas en cepas de bacilos gramnegativos / Test evaluation and strategy proposal to detect and to characterize carbapenemase-producing gram negative bacilli

Resumen Introducción: La detección de bacilos gramnegativos productores de carbapenemasas es compleja, existiendo actualmente varios test disponibles. La confirmación mediante la caracterización molecular de la enzima no está disponible en todos los laboratorios del país. Objetivo: Plantear una estrategia rápida, eficiente y sencilla para la detección y confirmación de carbapenemasas en cepas de bacilos gramnegativos. Material y Métodos: Se utilizaron 39 aislados productores y ocho no productores de carbapenemasas para evaluar los test fenotípicos Carba NP, CarbAcineto NP, Blue-Carba y validar el test molecular Xpert® Carba-R directo de la colonia en comparación con RPC convencional. Resultados: La sensibilidad para Carba NP, CarbAcineto NP y Blue-Carba fue de 79,5; 87,2 y 84,6%, respectivamente; mientras que la especificidad fue de 100; 100 y 87,5%, respectivamente. La concordancia entre RPC convencional y Xpert® Carba-R fue de 100%. El límite de detección para Xpert® Carba-R fue diferente según el tipo de carbapenemasa: 40,8 ufc/reacción par KPC y NDM y 30,6 ufc/reacción para VIM. Discusión: En aislados con susceptibilidad disminuida a carbapenémicos se propone realizar un tamizaje con CarbAcineto NP, para luego caracterizar la carbapenemasa con Xpert® Carba-R y adoptar las medidas de contención específica: para cada caso.

Introduction: The detection of carbapenemase-producing gram negative bacilli is complicated, because there are available multiple options of test. The confirmation of the enzyme by molecular characterization is not available in all laboratories in our country. Objective: To propose a fast, efficient and simple strategy to detect and confirm CPB. Materials and Methods: 39 CPB isolates and 8 non-producing were used to evaluate the phenotypic test Carba NP, CarbAcineto NP and Blue-Carba, validating the test Xpert® Carba-R, to be used directly with bacterial colonies with conventional PCR. Results: The sensitivity of Carba NP, CarbAcineto NP and Blue-Carba was 79,5; 87,2 y 84,6%, respectively; and specificity was 79.5; 87.2 and 84.6%, respectively. The limit of detection of Xpert® Carba-R was different for each carbapenemasa: 40.8 ufc/reaction to KPC and NDM and 30.6 ufc/reaction to VIM. Discussion: On isolates with decreased susceptibility to carbapenems we propose to use as screening the test CarbAcineto NP, follow by Xpert®Carba-R to characterize the carbapenemase and adopt specific infection control measures.

Factores asociados a infecciones urinarias producidas por enterobacterias productoras de β -lactamasas de espectro extendido: una cohorte prospectiva / Factors associated with extended-spectrum betalactamases-producing organisms among patients with urinary tract infections: a prospective cohort study

Background: Urinary tract infections (UTIs) caused by extended-spectrum betalactamases (ESBL) are an increasingly common problem. Aim: To develop an association model to allow an early detection of ESBL-producing microorganisms. Methods: A prospective observational cohort study was undertaken among patients admitted with a diagnosis of culture-proven UTI to the Internal Medicine Ward of the Hospital Naval Almirante Nef between February and November, 2011. Patients with polimicrobial cultures were excluded from analyses, which was undertaken using multiple logistic regression. Results: Two-hundred and forty-nine patients were analysed and 35 (14%) presented an ESBL-producing microorganism. Seventy-one percent were female and the mean age was 70,7 ± 16,9 years. A history of a recent hospitalization (< 3 months) or institutionalization (p = 0.027), previous infections by an ESBL-producing bacteria (p < 0.001), recent antimicrobial use (p = 0.013) and metastatic cancer (p = 0.007) were independently associated with a current UTI with an ESBL-producing pathogen. Discussion: Our findings are similar to those found in other populations. This tool offers assistance to clinicians who need to choose an appropriate antimicrobial therapy. This model needs to be validated prior to implementation.

Introducción: La infección del tracto urinario (ITU) por microorganismos productores de β-lactamasas de espectro extendido (BLEE) es un problema infectológico creciente. Objetivo: Determinar factores de riesgo predisponentes a infecciones por microorganismos productores de BLEE. Pacientes y Método: Cohorte prospectiva de pacientes > 18 años ingresados al Servicio de Medicina Interna del Hospital Naval Almirante Nef de Viña del Mar desde febrero a noviembre de 2011 con diagnóstico de ITU confirmado en un urocultivo. Se excluyeron pacientes con urocultivos polimicrobianos. El análisis se hizo mediante una regresión logística múltiple. Resultados: Se analizaron 249 pacientes, 35 (14%) presentaron un microorganismo productor de BLEE. El 71% fueron mujeres y la edad promedio 70,7 ± 16,9 años. El antecedente de hospitalización en los últimos tres meses o el vivir institucionalizado (p = 0,027), la infección por bacteria productora de BLEE previa (p < 0,001), el uso de antimicrobianos recientes (p = 0,013) y el antecedente de cáncer metastásico (p = 0,007) se asociaron a la producción de BLEE. Discusión: Los factores encontrados en la presente cohorte están de acuerdo a lo descrito en otras poblaciones. Esta herramienta ofrece asistencia para el médico clínico en la selección de la antibioterapia más apropiada. Es necesario validar este modelo previo a su implementación.

Structural insights into enzymatic degradation of oxidized polyvinyl alcohol.

The ever-increasing production and use of polyvinyl alcohol (PVA) threaten our environment. Yet PVA can be assimilated by microbes in two steps: oxidation and cleavage. Here we report novel α/ß-hydrolase structures of oxidized PVA hydrolase (OPH) from two known PVA-degrading organisms, Sphingopyxis sp. 113P3 and Pseudomonas sp. VM15C, including complexes with substrate analogues, acetylacetone and caprylate. The active site is covered by a lid-like ß-ribbon. Unlike other esterase and amidase, OPH is unique in cleaving the CC bond of ß-diketone, although it has a catalytic triad similar to that of most α/ß-hydrolases. Analysis of the crystal structures suggests a double-oxyanion-hole mechanism, previously only found in thiolase cleaving ß-ketoacyl-CoA. Three mutations in the lid region showed enhanced activity, with potential in industrial applications.

Ceftolozane/tazobactam activity tested against aerobic Gram-negative organisms isolated from intra-abdominal and urinary tract infections in European and United States hospitals (2012).

Ceftolozane/tazobactam is under clinical development for treatment of complicated intra-abdominal infections (IAI), complicated urinary tract infections (UTI) and ventilator-associated pneumonia. We evaluated the in vitro activity of ceftolozane/tazobactam and comparator agents tested against Gram-negative aerobic bacteria causing IAI and healthcare-associated UTI (HCA-UTI). The organisms were consecutively collected from January to December 2012 from 59 medical centers located in the United States (USA) and 15 European countries by the Program to Assess Ceftolozane/Tazobactam Susceptibility (PACTS). The collection included 809 organisms from IAI and 2474 organisms from HCA-UTI, and susceptibility testing was performed by reference broth microdilution methods as described by the Clinical and Laboratory Standards Institute (CLSI) M07-A9 document. Overall, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa were the most frequently isolated pathogens from both infection types. Ceftolozane/tazobactam was very active against E. coli (MIC50/90, 0.25/0.5 mg/L; 98.5-99.9% inhibited at an MIC of ≤8 mg/L) and retained activity against many of the multidrug-resistant (MDR; MIC50/90, 0.5/2->32 mg/L) and ESBL-phenotype strains (MIC50/90, 0.5/2-32 mg/L). Ceftolozane/tazobactam was active against most K. pneumoniae strains (MIC50/90, 0.25/16 mg/L, 88.9-89.6% inhibited at an MIC of ≤8 mg/L), but some ESBL-phenotype (MIC50/90, 4-8/>32 mg/L) and MDR (MIC50/90, 16/>32 mg/L) isolates exhibited elevated MIC values. Ceftolozane/tazobactam was the most active agent tested against P. aeruginosa (MIC50/90, 0.5/4 mg/L; 93.4-95.7% inhibited at ≤8 mg/L) and retained potency against many MDR (MIC50/90, 2-4/>32 mg/L), ceftazidime-nonsusceptible (MIC50/90, 2-4/>32 mg/L) and meropenem-nonsusceptible (MIC50/90, 2/>32 mg/L) strains. Ceftolozane/tazobactam was also active against Klebsiella oxytoca (MIC50/90, ≤0.12-0.25/0.5-1 mg/L), Enterobacter spp. (MIC50/90, 0.25-0.5/4-8 mg/L), Citrobacter spp. (MIC50/90, 0.25/2-32 mg/L), Proteus mirabilis (MIC50/90, 0.5/0.5 mg/L), indole-positive Proteae (MIC50/90, 0.25/0.5-1 mg/L), and Serratia spp. (MIC50/90, 0.5/1-2 mg/L). In summary, ceftolozane/tazobactam demonstrated potent in vitro activity when tested against contemporary aerobic Gram-negative pathogens causing IAI and HCA-UTI in USA and European medical centers.

Intersubunit communication in the dihydroorotase-aspartate transcarbamoylase complex of Aquifex aeolicus.

Aspartate transcarbamoylase and dihydroorotase, enzymes that catalyze the second and third step in de novo pyrimidine biosynthesis, are associated in dodecameric complexes in Aquifex aeolicus and many other organisms. The architecture of the dodecamer is ideally suited to channel the intermediate, carbamoyl aspartate from its site of synthesis on the ATC subunit to the active site of DHO, which catalyzes the next step in the pathway, because both reactions occur within a large, internal solvent-filled cavity. Channeling usually requires that the reactions of the enzymes are coordinated so that the rate of synthesis of the intermediate matches its rate of utilization. The linkage between the ATC and DHO subunits was demonstrated by showing that the binding of the bisubstrate analog, N-phosphonacetyl-L-aspartate to the ATC subunit inhibits the activity of the distal DHO subunit. Structural studies identified a DHO loop, loop A, interdigitating between the ATC domains that would be expected to interfere with domain closure essential for ATC catalysis. Mutation of the DHO residues in loop A that penetrate deeply between the two ATC domains inhibits the ATC activity by interfering with the normal reciprocal linkage between the two enzymes. Moreover, a synthetic peptide that mimics that part of the DHO loop that binds between the two ATC domains was found to be an allosteric or noncompletive ATC inhibitor (K(i) = 22 µM). A model is proposed suggesting that loop A is an important component of the functional linkage between the enzymes.

Crystal structure of LpxK, the 4'-kinase of lipid A biosynthesis and atypical P-loop kinase functioning at the membrane interface.

In Gram-negative bacteria, the hydrophobic anchor of the outer membrane lipopolysaccharide is lipid A, a saccharolipid that plays key roles in both viability and pathogenicity of these organisms. The tetraacyldisaccharide 4'-kinase (LpxK) of the diverse P-loop-containing nucleoside triphosphate hydrolase superfamily catalyzes the sixth step in the biosynthetic pathway of lipid A, and is the only known P-loop kinase to act upon a lipid substrate at the membrane. Here, we report the crystal structures of apo- and ADP/Mg(2+)-bound forms of Aquifex aeolicus LpxK to a resolution of 1.9 Å and 2.2 Å, respectively. LpxK consists of two α/ß/α sandwich domains connected by a two-stranded ß-sheet linker. The N-terminal domain, which has most structural homology to other family members, is responsible for catalysis at the P-loop and positioning of the disaccharide-1-phosphate substrate for phosphoryl transfer on the inner membrane. The smaller C-terminal domain, a substructure unique to LpxK, helps bind the nucleotide substrate and Mg(2+) cation using a 25° hinge motion about its base. Activity was severely reduced in alanine point mutants of conserved residues D138 and D139, which are not directly involved in ADP or Mg(2+) binding in our structures, indicating possible roles in phosphoryl acceptor positioning or catalysis. Combined structural and kinetic studies have led to an increased understanding of the enzymatic mechanism of LpxK and provided the framework for structure-based antimicrobial design.

Emerging resistant Gram-negative aerobic bacilli in hospital-acquired infections.

The increasing emergence of serious multidrug-resistant (MDR) Gram-negative infections has led to a new health-care crisis. These infections predominately include MDR Pseudomonas aeruginosa, extended-spectrum beta-lactamase-producing Enterobacteriaceae and MDR Acinetobacter baumannii. These organisms are present in a variety of clinical settings, but there is a distinct paucity of antibiotics to effectively treat these infections. The increasing use of broad-spectrum antibiotics and lack of good stewardship have contributed to the increase in these MDR organisms. This review focuses on the main MDR Gram-negative infections contributing to the current crisis in health care, their mechanisms of resistance and various treatment options for empiric therapy.

Aminopeptidase activity by spoilage bacteria and its relationship to microbial load and sensory attributes of poultry legs during aerobic cold storage.

The shelf life of poultry legs stored aerobically and the possible role of the aminopeptidase activity of gram-negative bacteria (p-nitroaniline test) as a predictor of poultry spoilage were evaluated on the basis of microbiological and sensory parameters. Chicken legs (n = 30) obtained immediately after evisceration in a local poultry processing plant were kept under aerobic refrigeration (4 +/- 1 degrees C) for 7 days. Microbiological (counts of aerobic bacteria and psychrotrophs) and sensory (odor, color, and general acceptability on a hedonic scale of 1 to 9) parameters and aminopeptidase activity (absorbance at 390 nm [A(390)]) determinations were performed after 0, 1, 3, 5, and 7 days of storage. Aerobic plate counts of 7 log CFU/g and a score of 6 for general acceptability were used as indicators of the end point of shelf life. Strong correlations (r > or = 0.76; P < 0.001) were obtained between bacterial counts, hedonic scores, and A(390) values. Samples were judged as unacceptable (shelf-life end point) after 2 and 4 days on the basis of sensory and microbiological analyses, respectively. A(390) values of 0.52 and 0.89 (corresponding to p-nitroaniline concentrations of 6.25 and 10.7 microg/ml, respectively) are proposed as the upper limits for acceptability on the basis of sensory and microbiological determinations, respectively. However, these recommendations are based on a small set of samples, and their general application is yet to be verified.

Ertapenem: no effect on aerobic gram-negative susceptibilities to imipenem.

OBJECTIVE: To describe any potential effect on in vitro susceptibility to imipenem for aerobic gram-negative bacteria following the addition of ertapenem to the formulary of a large teaching hospital. METHODS: Changes in imipenem susceptibilities for aerobic gram-negative bacteria were compared among clinical isolates from 2002 to 2007 using a Poisson model. Changes in the susceptibility of imipenem to P. aeruginosa, K. pneumoniae, K. oxytoxa, E. coli, and S. marcescens were compared over time using a chi-squared test for trend. Carbapenem use was measured using a defined daily dose per 1000 patient days. Change in utilization was compared for all years. The prevalence of ESBL-producing K. pneumoniae, K. oxytoxa, and E. coli over time was compared. RESULTS: Susceptibility to imipenem did not change after the addition of ertapenem and this most notably includes P. aeruginosa (p=0.43). Additionally, an increase in the incidence of ESBL K. pneumoniae from 2002 (4%) to 2007 (18%) p<0.0001 occurred with significant increases in both imipenem and ertapenem use in that time frame (p<0.001). CONCLUSION: The continued use of ertapenem over 5 years with predominant use of imipenem did not select for P. aeruginosa resistance to imipenem. The rising rate of community and healthcare associated ESBL K. pneumoniae increased total carbapenem use.

Purification and biochemical characterization of a protease secreted by the Salinivibrio sp. strain AF-2004 and its behavior in organic solvents.

A metalloprotease secreted by the moderately halophilic bacterium Salinivibrio sp. strain AF-2004 when the culture reached the stationary growth phase. This enzyme was purified to homogeneity by acetone precipitation and subsequent Q-Sepharose anion exchange and Sephacryl S-200 gel filtration chromatography. The apparent molecular mass of the protease was 31 kDa by SDS-PAGE, whereas it was estimated as approximately 29 kDa by Sephacryl S-200 gel filtration. The purified protease had a specific activity of 116.8 mumol of tyrosine/min per mg protein on casein. The optimum temperature and salinity of the enzyme were at 55 degrees C and 0-0.5 M NaCl, although at salinities up to 4 M NaCl activity still remained. The protease was stable and had a broad pH profile (5.0-10.0) with an optimum of 8.5 for casein hydrolysis. The enzyme was strongly inhibited by phenylmethyl sulfonylfluoride (PMSF), Pefabloc SC, chymostatin and also EDTA, indicating that it belongs to the class of serine metalloproteases. The protease in solutions containing water-soluble organic solvents or alcohols was more stable than that in the absence of organic solvents. These characteristics make it an ideal choice for applications in industrial processes containing organic solvents and/or salts.

The lipid A palmitoyltransferase PagP: molecular mechanisms and role in bacterial pathogenesis.

Palmitoylated lipid A can both protect pathogenic bacteria from host immune defences and attenuate the activation of those same defences through the TLR4 signal transduction pathway. A palmitate chain from a phospholipid is incorporated into lipid A by an outer membrane enzyme PagP, which is an 8-stranded antiparallel beta-barrel preceded by an amino-terminal amphipathic alpha-helix. The PagP barrel axis is tilted by 25 degrees with respect to the membrane normal. An interior hydrophobic pocket in the outer leaflet-exposed half of the molecule functions as a hydrocarbon ruler that allows the enzyme to distinguish palmitate from other acyl chains found in phospholipids. Internalization of a phospholipid palmitoyl group within the barrel appears to occur by lateral diffusion from the outer leaflet through non-hydrogen-bonded regions between beta-strands. The MsbA-dependent trafficking of lipids from the inner membrane to the outer membrane outer leaflet is necessary for lipid A palmitoylation in vivo. The mechanisms by which bacteria regulate pagP gene expression strikingly reflect the corresponding pathogenic lifestyle of the bacterium. Variations on PagP structure and function can be illustrated with the known homologues from Gram-negative bacteria, which include pathogens of humans and other mammals in addition to pathogens of insects and plants. The PagP enzyme is potentially a target for the development of anti-infective agents, a probe of outer membrane lipid asymmetry, and a tool for the synthesis of lipid A-based vaccine adjuvants and endotoxin antagonists.

Salinity and temperature effects on accessibility of soluble and cross-linked insoluble xylans to endo-xylanases.

Different responses to salinity were observed for an extremely halotolerant endo-xylanase when assayed with soluble birchwood glucoronoxylan and cross-linked dyed insoluble birchwood glucoronoxylan. Shrinking of insoluble xylan particles due to increased ionic strength is proposed as the explanation. Temperature affected the xylanase activity measurement on the insoluble xylan greatly, likely due to increased enzyme accessible surface of the substrate at high temperatures.

Impact of antimicrobial exposure and beta-lactamase-producing bacteria on salivary beta-lactamase activity in infancy.

Beta-lactamase production by oral bacteria is common in infancy and is associated with use of antimicrobial agents in infants. The present longitudinal study aimed to examine the frequency of salivary beta-lactamase activity (SbetaA), to compare SbetaA with the presence of beta-lactamase-producing (beta+) aerobic and anaerobic species in saliva, and to estimate the impact of antimicrobial exposure on the emergence of SbetaA in healthy infants during their first year of life. At 6 months, SbetaA was detected in 46% infants; 89% SbetaA-positive infants and 55% SbetaA-negative infants harboured beta+ species at this time (OR 7.08; CI 1.31-38.34). At 12 months, SbetaA was detected in 54% infants. Exposure to antimicrobials during the first year of life increased the risk (OR 2.60; CI 0.72-9.36) of having SbetaA.

Display of proteins on bacteria.

Display of heterologous proteins on the surface of microorganisms, enabled by means of recombinant DNA technology, has become an increasingly used strategy in various applications in microbiology, biotechnology and vaccinology. Gram-negative, Gram-positive bacteria, viruses and phages are all being investigated in such applications. This review will focus on the bacterial display systems and applications. Live bacterial vaccine delivery vehicles are being developed through the surface display of foreign antigens on the bacterial surfaces. In this field, 'second generation' vaccine delivery vehicles are at present being generated by the addition of mucosal targeting signals, through co-display of adhesins, in order to achieve targeting of the live bacteria to immunoreactive sites to thereby increase immune responses. Engineered bacteria are further being evaluated as novel microbial biocatalysts with heterologous enzymes immobilized as surface exposed on the bacterial cell surface. A discussion has started whether bacteria can find use as new types of whole-cell diagnostic devices since single-chain antibodies and other type of tailor-made binding proteins can be displayed on bacteria. Bacteria with increased binding capacity for certain metal ions can be created and potential environmental or biosensor applications for such recombinant bacteria as biosorbents are being discussed. Certain bacteria have also been employed for display of various poly-peptide libraries for use as devices in in vitro selection applications. Through various selection principles, individual clones with desired properties can be selected from such libraries. This article explains the basic principles of the different bacterial display systems, and discusses current uses and possible future trends of these emerging technologies.

Emerging carbapenemases in Gram-negative aerobes.

Carbapenemases may be defined as beta-lactamases that significantly hydrolyze at least imipenem or/and meropenem. Carbapenemases involved in acquired resistance are of Ambler molecular classes A, B, and D. Class A, clavulanic acid-inhibited carbapenemases are rare. They are either chromosomally encoded (NMC-A, Sme-1 to Sme-3, IMI-1) in Enterobacter cloacae and Serratia marcescens, or plasmid encoded, such as KPC-1 in Klebsiella pneumoniae and GES-2 in Pseudomonas aeruginosa, the latter being a point-mutant of the clavulanic acid-inhibited extended-spectrum beta-lactamase GES-1. The class B enzymes are the most clinically significant carbapenemases. They are metalloenzymes of the IMP or VIM series. They have been reported worldwide but mostly from South East Asia and Europe. Metalloenzymes, whose genes are plasmid and integron located, hydrolyze virtually all beta-lactams except aztreonam. Finally, the class D carbapenemases are increasingly reported in Acinetobacter baumannii but compromise imipenem and meropenem susceptibility only marginally. The sources of the acquired carbapenemase genes remain unknown, as does the relative importance of the spread of epidemic strains as opposed to the spread of plasmid- or integron-borne genes. Because most of these carbapenemases confer only reduced susceptibility to carbapenems in Enterobacteriaceae, they may remain underestimated as a consequence of the lack of their detection.

Purification and characterization of the complex I from the respiratory chain of Rhodothermus marinus.

The rotenone sensitive NADH:menaquinone oxidoreductase (NDH-I or complex I) from the thermohalophilic bacterium Rhodothermus marinus has been purified and characterized. Three of its subunits react with antibodies against 78, 51, and 21.3c kDa subunits of Neurospora crassa complex I. The optimum conditions for NADH dehydrogenase activity are 50 degrees C and pH 8.1, and the enzyme presents a KM of 9 microM for NADH. The enzyme also displays NADH:quinone oxidoreductase activity with two menaquinone analogs, 1,4-naphtoquinone (NQ) and 2,3-dimethyl-1,4-naphtoquinone (DMN), being the last one rotenone sensitive, indicating the complex integrity as purified. When incorporated in liposomes, a stimulation of the NADH:DMN oxidoreductase activity is observed by dissipation of the membrane potential, upon addition of CCCP. The purified enzyme contains 13.5 +/- 3.5 iron atoms and approximately 3.7 menaquinone per FMN. At least five iron-sulfur centers are observed by EPR spectroscopy: two [2Fe-2S](2+/1+) and three [4Fe-4S](2+/1+) centers. By fluorescence spectroscopy a still unidentified chromophore was detected in R. marinus complex I.

The succinate dehydrogenase from the thermohalophilic bacterium Rhodothermus marinus: redox-Bohr effect on heme bL.

The succinate dehydrogenase from the thermohalophilic bacterium Rhodothermus marinus is a member of the succinate:menaquinone oxidoreductases family. It is constituted by three subunits with apparent molecular masses of 70, 32, and 18 kDa. The optimum temperature for succinate dehydrogenase activity is 80 degrees C, higher than the optimum growth temperature of R. marinus, 65 degrees C. The enzyme shows a high affinity for both succinate (Km = 0.165 mM) and fumarate (Km = 0.10 mM). It contains the canonical iron-sulfur centers S1, S2, and S3, as well as two B-type hemes. In contrast to other succinate dehydrogenases, the S3 center has an unusually high reduction potential of +130 mV and is present in two different conformations, one of which presents an unusual EPR signal with g values at 2.035, 2.009, and 2.001. The apparent midpoint reduction potentials of the hemes, +75 and -65 mV at pH 7.5, are also higher than those reported for other enzymes. The heme with the lower potential (heme bL) presents a considerable dependence of the reduction potential with pH (redox-Bohr effect), having a pKa(OX) = 6.5 and a pKa(red) = 8.7. This behavior is consistent with the proposal that in these enzymes menaquinone reduction occurs close to heme bL, near to the periplasmic side of the membrane, and involving dissipation of the proton transmembrane gradient.

Crystallization and preliminary X-ray studies of Trp138Phe/Val185Thr xylose isomerases from Thermotoga neapolitana and Thermoanaerobacterium thermosulfurigenes.

Xylose isomerases from Thermotoga neapolitana (TNXI) and Thermoanaerobacterium thermosulfurigenes (TTXI) share 70.4% sequence identity and are thermostable. The double mutants Trp138Phe/Val185Thr of TNXI and TTXI have higher catalytic efficiencies than TNXI and TTXI, respectively. The Trp138Phe/Val185Thr TNXI and TTXI mutants were overexpressed in Escherichia coli strain BL21(DE3) and purified. Crystals of the two proteins were grown with polyethylene glycol 8000 as the major precipitant by the hanging-drop vapour-diffusion method. Crystals of the TNXI mutant were obtained in the absence of substrate, in complex with glucose and in complex with fructose. Crystals of the TTXI mutant were obtained complexed with glucose. Diffraction data were collected at 1.9, 2.1 and 2.1 A resolution for the fructose-TNXI mutant, glucose-TNXI mutant and substrate-unbound TNXI mutant, respectively. The diffraction data for the glucose-TTXI mutant were collected at 2.0 A resolution. The crystals belong to the orthorhombic space groups C222(1) (TNXI mutant) and P2(1)2(1)2(1) (TTXI mutant). The TNXI and TTXI mutant crystals contain two and four monomers in the asymmetric unit, respectively.