A kinetic analysis of the inhibition of FOX-4 ß-lactamase, a plasmid-mediated AmpC cephalosporinase, by monocyclic ß-lactams and carbapenems.

OBJECTIVES: Class C ß-lactamases are prevalent among Enterobacteriaceae; however, these enzymes are resistant to inactivation by commercially available ß-lactamase inhibitors. In order to find novel scaffolds to inhibit class C ß-lactamases, the comparative efficacy of monocyclic ß-lactam antibiotics (aztreonam and the siderophore monosulfactam BAL30072), the bridged monobactam ß-lactamase inhibitor BAL29880, and carbapenems (imipenem, meropenem, doripenem and ertapenem) were tested in kinetic assays against FOX-4, a plasmid-mediated class C ß-lactamase (pmAmpC). METHODS: The FOX-4 ß-lactamase was purified. Steady-state kinetics, electrospray ionization mass spectrometry (ESI-MS) and ultraviolet difference (UVD) spectroscopy were conducted using the ß-lactam scaffolds described. RESULTS: The K(i) values for the monocyclic ß-lactams against FOX-4 ß-lactamase were 0.04 ± 0.01 µM (aztreonam) and 0.66 ± 0.03 µM (BAL30072), and the Ki value for the bridged monobactam BAL29880 was 8.9 ± 0.5 µM. For carbapenems, the Ki values ranged from 0.27 ± 0.05 µM (ertapenem) to 2.3 ± 0.3 µM (imipenem). ESI-MS demonstrated the formation of stable covalent adducts when the monocyclic ß-lactams and carbapenems were reacted with FOX-4 ß-lactamase. UVD spectroscopy suggested the appearance of different chromophoric intermediates. CONCLUSIONS: Monocyclic ß-lactam and carbapenem antibiotics are effective mechanism-based inhibitors of FOX-4 ß-lactamase, a clinically important pmAmpC, and provide stimulus for the development of new inhibitors to inactivate plasmidic and chromosomal class C ß-lactamases.

Involvement of the AcrAB-TolC efflux pump in the resistance, fitness, and virulence of Enterobacter cloacae.

Multidrug efflux pumps have emerged as important mechanisms of antimicrobial resistance in bacterial pathogens. In order to cause infection, pathogenic bacteria require mechanisms to avoid the effects of host-produced compounds, and express efflux pumps may accomplish this task. In this study, we evaluated the effect of the inactivation of AcrAB-TolC on antimicrobial resistance, fitness, and virulence in Enterobacter cloacae, an opportunistic pathogen usually involved in nosocomial infections. Two different clinical isolates of E. cloacae were used, EcDC64 (multidrug resistance overexpressing the AcrAB-TolC efflux pump) and Jc194 (basal AcrAB-TolC expression). The acrA and tolC genes were deleted in strains EcDC64 and Jc194 to produce, respectively, EcΔacrA and EcΔtolC and JcΔacrA and JcΔtolC knockout (KO) derivatives. Antibiotic susceptibility testing was performed with all isolates, and we discovered that these mechanisms are involved in the resistance of E. cloacae to several antibiotics. Competition experiments were also performed with wild-type and isogenic KO strains. The competition index (CI), defined as the mutant/wild-type ratio, revealed that the acrA and tolC genes both affect the fitness of E. cloacae, as fitness was clearly reduced in the acrA and tolC KO strains. The median CI values obtained in vitro and in vivo were, respectively, 0.42 and 0.3 for EcDC64/EcΔacrA, 0.24 and 0.38 for EcDC64/EcΔtolC, 0.15 and 0.11 for Jc194/JcΔacrA, and 0.38 and 0.39 for Jc194/JcΔtolC. Use of an intraperitoneal mouse model of systemic infection revealed reduced virulence in both E. cloacae clinical strains when either the acrA or tolC gene was inactivated. In conclusion, the structural components of the AcrAB-TolC efflux pump appear to play a role in antibiotic resistance as well as environmental adaptation and host virulence in clinical isolates of E. cloacae.

Expression of OXA-type and SFO-1 ß-lactamases induces changes in peptidoglycan composition and affects bacterial fitness.

ß-Lactamases and penicillin-binding proteins (PBPs) have evolved from a common ancestor. ß-Lactamases are enzymes that degrade ß-lactam antibiotics, whereas PBPs are involved in the synthesis and processing of peptidoglycan, which forms an elastic network in the bacterial cell wall. This study analyzed the interaction between ß-lactamases and peptidoglycan and the impact on fitness and biofilm production. A representative set of all classes of ß-lactamases was cloned in the expression vector pBGS18 under the control of the CTX-M promoter and expressed in Escherichia coli MG1655. The peptidoglycan composition of all clones was evaluated, and quantitative changes were found in E. coli strains expressing OXA-24, OXA-10-like, and SFO-1 (with its upstream regulator AmpR) ß-lactamases; the level of cross-linked muropeptides decreased, and their average length increased. These changes were associated with a statistically significant fitness cost, which was demonstrated in both in vitro and in vivo experiments. The observed changes in peptidoglycan may be explained by the presence of residual DD-endopeptidase activity in these ß-lactamases, which may result in hydrolysis of the peptide cross bridge. The biological cost associated with these changes provides important data regarding the interaction between ß-lactamases and the metabolism of peptidoglycan and may provide an explanation for the epidemiology of these ß-lactamases in Enterobacteriaceae.

Distant and new mutations in CTX-M-1 beta-lactamase affect cefotaxime hydrolysis.

The CTX-M ß-lactamases are an increasingly prevalent group of extended-spectrum ß-lactamases (ESBL). Point mutations in CTX-M ß-lactamases are considered critical for enhanced hydrolysis of cefotaxime. In order to clarify the structural determinants of the activity against cefotaxime in CTX-M ß-lactamases, screening for random mutations was carried out to search for decreased activity against cefotaxime, with the CTX-M-1 gene as a model. Thirteen single mutants with a considerable reduction in cefotaxime MICs were selected for biochemical and stability studies. The 13 mutated genes of the CTX-M-1 ß-lactamase were expressed, and the proteins were purified for kinetic studies against cephalothin and cefotaxime (as the main antibiotics). Some of the positions, such as Val103Asp, Asn104Asp, Asn106Lys, and Pro107Ser, are located in the (103)VNYN(106) loop, which had been described as important in cefotaxime hydrolysis, although this has not been experimentally confirmed. There are four mutations located close to catalytic residues-Thr71Ile, Met135Ile, Arg164His, and Asn244Asp-that may affect the positioning of these residues. We show here that some distant mutations, such as Ala219Val, are critical for cefotaxime hydrolysis and highlight the role of this loop at the top of the active site. Other distant substitutions, such as Val80Ala, Arg191, Ala247Ser, and Val260Leu, are in hydrophobic cores and may affect the dynamics and flexibility of the enzyme. We describe here, in conclusion, new residues involved in cefotaxime hydrolysis in CTX-M ß-lactamases, five of which are in positions distant from the catalytic center.

Role of changes in the L3 loop of the active site in the evolution of enzymatic activity of VIM-type metallo-beta-lactamases.

OBJECTIVES: The new metallo-beta-lactamase VIM-13 has been recently characterized. In comparison with the VIM-1 enzyme, VIM-13 showed 19 amino acid differences, 2 of which were located in the active site centre. The main objective of the present study was to assess whether differences between VIM-1 and VIM-13 beta-lactamases in the active site, at His224Leu and Ser228Arg, are necessary and sufficient to explain the microbiological and biochemical differences between the two enzymes. METHODS: Single mutants VIM-13 (Leu224His) and VIM-13 (Arg228Ser) and double mutant VIM-13 (Leu224His, Arg228Ser) were created by site-directed mutagenesis with the bla(VIM-13) gene as template. VIM-1, VIM-13 and VIM-13 (Leu224His, Arg228Ser) were purified by affinity chromatography, and kinetic parameters for these enzymes were obtained with ceftazidime, cefepime and ampicillin. RESULTS: Ceftazidime and cefepime MICs (mg/L) for Escherichia coli TG1 expressing VIM-1, VIM-13, VIM-13 (Leu224His), VIM-13 (Arg228Ser) and VIM-13 (Leu224His, Arg228Ser) were >256 and 64, 6 and 4, 8 and 1, >256 and 8, and >256 and 48, respectively. VIM-1, VIM-13 and VIM-13 (Leu224His, Arg228Ser) revealed k(cat)/K(m) values (M(-1)s(-1)) for ceftazidime of 3.7 E(4), 1.9 E(4) and 10 E(4), respectively, and revealed k(cat)/K(m) values for cefepime of 3.5 E(5), 3 E(4) and 1.5 E(5), respectively. CONCLUSIONS: Overall, the results showed that the two residues located in the L3 loop are sufficient to confer the substrate specificity of each enzyme, thus highlighting the importance of the L3 loop of the active site in the evolution of VIM-type metallo-beta-lactamases.

A tripeptide deletion in the R2 loop of the class C beta-lactamase enzyme FOX-4 impairs cefoxitin hydrolysis and slightly increases susceptibility to beta-lactamase inhibitors.

OBJECTIVES: A natural variant of the AmpC enzyme from Escherichia coli HKY28 with a tripeptide deletion (Gly-286/Ser-287/Asp-288) was recently described. The isolate produced an inhibitor-sensitive AmpC beta-lactamase variant that also conferred higher than usual levels of resistance to ceftazidime in the E. coli host. To demonstrate whether this is true in other class C beta-lactamase enzymes, we deleted the equivalent tripeptide in the FOX-4 plasmid-mediated class C beta-lactamase. METHODS: By site-directed mutagenesis, we deleted the tripeptide Gly-306/Asn-307/Ser-308 of FOX-4, thus generating FOX-4(DeltaGNS). The enzymes (FOX-4 wild-type and DeltaGNS) were purified and kinetic parameters (kcat, Km, kcat/Km) as well as IC50 values of several beta-lactams were assessed. Modelling studies were also performed. RESULTS: FOX-4(DeltaGNS) did not increase the catalytic efficiency towards ceftazidime, although it conferred a slight increase in the susceptibility to beta-lactamase inhibitors. There was also a noteworthy decrease in the cefoxitin MIC with the FOX-4(DeltaGNS) mutant (from 512 to 16 mg/L) as well as a 10-fold decrease in kcat/Km towards imipenem, which revealed specific structural features. CONCLUSIONS: Although deletions in the R2-loop are able to extend the substrate spectrum of class C enzymes, the present results do not confirm this hypothesis in FOX-4. The FOX-4 R2 site would already be wide enough to accommodate antibiotic molecules, and thus any amino acid replacement or deletion at this location would not affect the hydrolytic efficiency towards beta-lactams and would have a less drastic effect on the susceptibility to beta-lactamase inhibitors.

Associating growth-phase-related changes in the proteome of Acinetobacter baumannii with increased resistance to oxidative stress.

Acinetobacter baumannii is an opportunistic pathogen that has been associated with severe infections and outbreaks in hospitals. At present, very little is known about the biology of this bacterium, particularly as regards mechanisms of adaptation, persistence and virulence. To investigate the growth phase-dependent regulation of proteins in this microorganism, we analyzed the proteomic pattern of A. baumannii ATCC 17978 at different stages of in vitro growth. In this study, proteomics analyses were conducted using 2-DE and MALDI-TOF/TOF complemented by iTRAQ LC-MS/MS. Here we have identified 107 differentially expressed proteins. We highlight the induction of proteins associated with signaling, putative virulence factors and response to stress (including oxidative stress). We also present evidence that ROS (O(2)(-) and OH(-)) and RNI (ONOO(-)) accumulate during late stages of growth. Further assays demonstrated that stationary cells survive at high concentrations of H(2)O(2) (30 mM), the O(2)(-) donor menadione (500 muM) or the NO donor sodium nitroprusside (1 mM), and showed a higher survival rate against several bactericidal antibiotics. The growth phase-dependent changes observed in the A. baumannii proteome are discussed within a context of adaptive biological responses, including those related to ROS and RNI stress.

Structure-function studies of arginine at position 276 in CTX-M beta-lactamases.

OBJECTIVES: In order to assess whether or not the Arg-276 of CTX-M-type enzymes is equivalent to the Arg-244 of IRT-TEM-derivative enzymes, we replaced the former with six different amino acids, some of them previously described as involved in resistance to beta-lactamase inhibitors in TEM-IRT derivatives. We also investigated the role of Arg276 in cefotaxime hydrolysis. METHODS: By site-directed mutagenesis and by use of the bla(CTX-M-1) gene as template, Arg-276 was replaced with six different amino acids (Trp, His, Cys, Asn, Gly and Ser). MICs of beta-lactams alone and in combination with beta-lactamase inhibitors were established. The seven enzymes (CTX-M-1 wild-type and six derived mutants) were purified by affinity chromatography, and kinetic parameters (k(cat), K(m), k(cat)/K(m)) towards cefalotin and cefotaxime were determined. Clavulanic acid IC(50) values were also assessed with all enzymes. RESULTS: No increase in MICs of beta-lactam/beta-lactamase inhibitor combination was detected with any of the six CTX-M-1-derived mutants, in agreement with the clavulanic acid IC(50) values. The MICs of cefotaxime were clearly lower for the Escherichia coli harbouring the Trp, Cys, Ser and Gly CTX-M-1 mutant enzymes than for CTX-M-1, and these enzymes showed a clearly reduced catalytic efficiency towards cefotaxime. As regards cefalotin, there was a moderate reduction in catalytic efficiency for Cys and His. CONCLUSIONS: Arg-276 in CTX-M-type beta-lactamases is not equivalent to Arg-244 in IRT-type enzymes. Position Arg-276 appears to be important for cefotaxime hydrolysis in CTX-M-type enzymes, although different effects were obtained regarding the replaced amino acid.

Cloning, nucleotide sequencing, and analysis of the AcrAB-TolC efflux pump of Enterobacter cloacae and determination of its involvement in antibiotic resistance in a clinical isolate.

Enterobacter cloacae is an emerging clinical pathogen that may be responsible for nosocomial infections. Management of these infections is often difficult, owing to the high frequency of strains that are resistant to disinfectants and antimicrobial agents in the clinical setting. Multidrug efflux pumps, especially those belonging to the resistance-nodulation-division family, play a major role as a mechanism of antimicrobial resistance in gram-negative pathogens. In the present study, we cloned and sequenced the genes encoding an AcrAcB-TolC-like efflux pump from an E. cloacae clinical isolate (isolate EcDC64) showing a broad antibiotic resistance profile. Sequence analysis showed that the acrR, acrA, acrB, and tolC genes encode proteins that display 79.8%, 84%, 88%, and 82% amino acid identities with the respective homologues of Enterobacter aerogenes and are arranged in a similar pattern. Deletion of the acrA gene to yield an AcrA-deficient EcDC64 mutant (EcDeltaacrA) showed the involvement of AcrAB-TolC in multidrug resistance in E. cloacae. However, experiments with an efflux pump inhibitor suggested that additional efflux systems also play a role in antibiotic resistance. Investigation of several unrelated isolates of E. cloacae by PCR analysis revealed that the AcrAB system is apparently ubiquitous in this species.

Molecular characterization of the gene encoding a new AmpC beta-lactamase in Acinetobacter baylyi.

OBJECTIVES: The main objective of the present study was to demonstrate the presence of a beta-lactamase ampC gene in the chromosome of the non-pathogenic bacterium Acinetobacter baylyi ADP1. METHODS: beta-Lactam MICs were determined by Etest. The ampC gene was amplified by PCR, with specific oligonucleotides, then cloned into pBGS18 and pAT-RA plasmids and transformed into Escherichia coli TG1 and parental A. baylyi as hosts. The gene was sequenced and analysed. The AmpC protein was expressed, purified by affinity chromatography and the kinetic parameters determined. RESULTS: An ampC gene was amplified from the ADP1 genome. Sequencing of the gene showed typical SVSK and KTG domains and the typical YXN Class C motif. The amplified gene showed significant identity (48.5% to 49.3%) with the AmpC enzymes of Acinetobacter baumannii and AG3 strains, which have recently been renamed ADC-1 to ADC-7. MIC analysis revealed a cephalosporinase profile for the E. coli TG1 clone as well as for the parental A. baylyi strain that overexpressed the ampC gene cloned under the control of an external promoter. Analysis of kinetic parameters of the purified enzyme showed higher catalytic efficiency for cefalotin than for ampicillin. CONCLUSIONS: This study represents the first report of an AmpC beta-lactamase in A. baylyi, which was shown by biochemical and microbiological experiments to have a typical cephalosporinase profile. The presence of the respective gene in the chromosome of A. baylyi ADP1 suggested that this ampC gene is the naturally occurring cephalosporinase in this species, as previously reported for other Acinetobacter spp. We tentatively named the enzyme ADC-8.

Interspecies spread of CTX-M-32 extended-spectrum beta-lactamase and the role of the insertion sequence IS1 in down-regulating bla CTX-M gene expression.

OBJECTIVES: To characterize the extended-spectrum beta-lactamases (ESBLs) as well as their genetic environment in different isolates of Enterobacteriaceae from a patient with repeated urinary tract infections. METHODS: Two isolates of Escherichia coli and one Proteus mirabilis, all with ESBL phenotypes, were studied. Conjugation experiments and restriction fragment length polymorphisms (RFLPs) were performed. Cloning of the bla genes was by plasmid restriction and fragments ligation. Antibiotic susceptibility testing was by Etest. The genetic environment was analysed by direct sequencing of the DNA surrounding the bla gene. RT-PCR was performed to study the differences in the bla(CTX-M) gene expression. RESULTS: The bla gene was transferred by conjugation from the three clinical isolates, which by RFLP showed the same plasmid. The bla gene and surrounding sequences were cloned, an approximately 9 kbp AccI fragment was sequenced and the bla(CTX-M-32) gene was identified. The MICs of ceftazidime for transconjugants and transformants bearing the bla(CTX-M-32) gene were lower than those previously reported. Analysis of the DNA surrounding the ESBL gene revealed a new genetic structure with two insertion sequences, IS5 and IS1, located immediately upstream of the bla(CTX-M-32) gene; IS1 was located between the bla gene and IS5, and within the -10 and -35 promoter boxes of the bla(CTX-M-32) gene. Microbiological and biochemical studies revealed lower bla(CTX-M-32) gene expression in bacterial isolates with IS1 between the promoter boxes. CONCLUSIONS: Data suggest putative in vivo horizontal bla(CTX-M-32) gene transfer between two different genera of Enterobacteriaceae. A new complex structure, IS5-IS1, was detected upstream of the bla gene and IS1 negatively modulated expression of the bla(CTX-M-32) gene because its location modified the bla promoter region.

[Autosomal dominant punctate palmoplantar keratoderma]. / Queratodermia palmoplantar punctata autosómica dominante.

Hereditary punctate palmoplantar keratoderma or Buschke-Fisher-Brauer disease is a rare form of keratoderma that follows a pattern of autosomal dominant inheritance with variable penetrance. The age of onset is usually between 12 and 30 years of age. Clinically, it is characterized by the gradual appearance of multiple punctate hyperkeratotic papules, irregularly distributed on the palms and soles, as well as by its possible association with several diseases, primarily with malignant processes. We present the case of a 43-year-old male patient with this disease, with no other associated symptoms, who had a first-degree relative who was affected and died of colon cancer. We also discuss the differential diagnosis with other nosologic entities.

Langerhans cell histiocytosis of vulva.

Cutaneous involvement of the vulva by Langerhans cell histiocytosis in women older than 70 is a rare phenomenon. A tendency of the disease to involve genital areas, has been described. We present a case of a 72-year-old woman, who had cutaneous vulvar involvement by Langerhans-cell histiocytosis, diagnosed by biopsy. An immunohistochemical study is also described.

Queratodermia palmoplantar punctata autosómica dominante / Autosomal dominant punctate palmoplantar keratoderma

La queratodermia palmoplantar punctata hereditaria o enfermedad de Buschke-Fisher-Brauer es una forma poco frecuente de queratodermia que sigue un patrón de herencia autosómico dominante con penetrancia variable. La edad de inicio suele estar comprendida entre los 12 y los 30 años de edad. Clínicamente se caracteriza por la aparición progresiva de múltiples pápulas hiperqueratósicas puntiformes de distribución irregular a nivel de palmas y plantas así como por su posible asociación con diversas enfermedades, fundamentalmente con procesos malignos. Presentamos un paciente de 43 años con esta enfermedad, sin otra sintomatología asociada, que tenía un familiar de primer grado afectado y fallecido de cáncer de colon, y planteamos el diagnóstico diferencial con otras entidades nosológicas

Hereditary punctate palmoplantar keratoderma or Buschke-Fisher-Brauer disease is a rare form of keratoderma that follows a pattern of autosomal dominant inheritance with variable penetrance. The age of onset is usually between 12 and 30 years of age. Clinically, it is characterized by the gradual appearance of multiple punctate hyperkeratotic papules, irregularly distributed on the palms and soles, as well as by its possible association with several diseases, primarily with malignant processes. We present the case of a 43-year-old male patient with this disease, with no other associated symptoms, who had a first-degree relative who was affected and died of colon cancer. We also discuss the differential diagnosis with other nosologic entities

Síndrome de Kindler: aportación de un caso / Kindler syndrome: presentation of a case

El síndrome de Kindler, es una enfermedad muy poco frecuente debida a mutaciones que originan defectos en la unión actina-matriz extracelular. Suele cursar con ampollas acrales desde el nacimiento en zonas más expuestas a los traumatismos, fotosensibilidad marcada que mejora con la edad y desarrollo de poiquilodermia y atrofia cutánea. Con relativa frecuencia se describe afectación de mucosas y degeneración maligna

Kindler syndrome is a very rare disease caused by mutations resulting in defects in the extracellular matrix-actin link. It usually presents with acral blistering from birth in trauma-prone areas, pronounced photosensitivity that improves with age and the development of poikiloderma and cutaneous atrophy. Mucosal involvement and degeneration have been described with relative frequency

[Kindler syndrome: presentation of a case]. / Síndrome de Kindler: aportación de un caso.

Kindler syndrome is a very rare disease caused by mutations resulting in defects in the extracellular matrix-actin link. It usually presents with acral blistering from birth in trauma-prone areas, pronounced photosensitivity that improves with age and the development of poikiloderma and cutaneous atrophy. Mucosal involvement and degeneration have been described with relative frequency.

Micosis fungoide acrómica / Achromic mycosis fungoides

La forma acrómica de micosis fungoide es una variante poco frecuente de linfoma cutáneo de células T que se ha descrito con mayor frecuencia en pacientes de piel oscura y niños. Se presenta un caso de micosis fungoide acrómica en un varón de raza blanca de 23 años cuyas lesiones se caracterizaban por placas hipopigmentadas en la raíz de las extremidades inferiores. Hasta el momento actual se han descrito únicamente 16 casos de micosis fungoide acrómica en pacientes de raza blanca (AU)

Tratamiento de hemangiomas infantiles con corticoides orales / Treatment of hemangiomas in children with oral corticosteroids

Introducción. Actualmente, los corticoides orales son el tratamiento de primera línea en la mayoría de los hemangiomas infantiles, y deben ser utilizados en dosis elevadas. Sin embargo, el tratamiento con corticoides orales en altas dosis en neonatos o lactantes plantea la posibilidad del desarrollo de efectos secundarios inducidos por esta medicación, tanto a corto como a largo plazo. Material y métodos. El objetivo ha sido describir los resultados obtenidos en pacientes con hemangiomas complicados que recibieron altas dosis de corticoides orales y analizar los efectos secundarios de esta medicación a corto y a largo plazo. Para ello se ha realizado estudio retrospectivo de 8 pacientes con hemangiomas tratados con corticoides orales, con edades comprendidas entre los 2 y 4 meses en el momento del inicio del tratamiento. Resultados. Los resultados obtenidos con el tratamiento fueron satisfactorios, con disminución en el tamaño de los hemangiomas en todos los pacientes. Solamente un paciente presentó disminución en la curva de talla hasta niveles inferiores a P10. Solamente en una de las pacientes se observaron facies cushingoide e hirsutismo, que fueron transitorios y reversibles en pocos meses. Conclusión. Los corticoides orales en dosis elevadas y durante periodos prolongados de tiempo son eficaces en el tratamiento de los hemangiomas. Esta medicación es bien tolerada, con escasos efectos secundarios a corto y largo plazo, que son transitorios en la mayoría de los casos (AU)