ABSTRACT
The atlD gene from an Enterococcus faecalis strain isolated from a Mexican artisanal cheese was cloned, sequenced and expressed in Escherichia coli in order to perform a biochemical characterization. A partial amino acid sequence of the heterologous protein was obtained by LC-MS/MS, and it corresponded to a novel peptidoglycan hydrolase designated AtlD. Its molecular mass was 62-75 kDa, as determined by SDS-PAGE, zymography, Western blot, and exclusion chromatography. Electrofocusing rendered an isoelectric point (pI) of 4.8. It exhibited N-acetylglucosaminidase activity, with an optimal pH and temperature between 6-7 and 50°C, respectively. It retained 85% activity with NaCl at 1,000 mM, but it was susceptible to divalent ions, particularly Zn2+. It showed antibacterial activity against Listeria monocytogenes, Staphylococcus aureus, and enterococcal strains of clinical origin. Due to the fact that it showed activity versus pathogenic bacteria, and because of its capabilities under ionic strength, temperature, and pH values present in food matrices, it could be applied as an additive in the food industry. This study will aid in the design of new antibacterial agents of natural origin to combat food-borne diseases, and it could be used as an industrial or hospital hygiene agent as well.
Subject(s)
Acetylglucosaminidase/chemistry , Acetylglucosaminidase/genetics , Cloning, Molecular/methods , Enterococcus faecalis/enzymology , Enterococcus faecalis/genetics , Enterococcus faecalis/metabolism , Acetylglucosaminidase/pharmacology , Amino Acid Sequence , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Cheese/microbiology , Chromatography, Liquid , Enterococcus/drug effects , Enzyme Stability , Escherichia coli/genetics , Food Industry , Food Microbiology , Foodborne Diseases , Gene Expression Regulation, Bacterial , Genes, Bacterial/genetics , Hydrogen-Ion Concentration , Isoelectric Point , Listeria monocytogenes/drug effects , Molecular Weight , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Staphylococcus aureus/drug effects , Substrate Specificity , Tandem Mass Spectrometry , TemperatureSubject(s)
Drug Resistance, Bacterial , Enterococcus faecalis/enzymology , Enterococcus faecalis/isolation & purification , Gram-Positive Bacterial Infections/veterinary , tRNA Methyltransferases/genetics , Animals , Anti-Bacterial Agents/pharmacology , Brazil , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Electrophoresis, Gel, Pulsed-Field , Enterococcus faecalis/classification , Enterococcus faecalis/genetics , Gram-Positive Bacterial Infections/microbiology , Molecular Typing , Polymerase Chain Reaction , Ribosomal Proteins/genetics , Sequence Analysis, DNA , SwineABSTRACT
Although the agmatine deiminase system (AgDI) has been investigated in Enterococcus faecalis, little information is available with respect to its gene regulation. In this study we demonstrate that the presence of exogenous agmatine induces the expression of agu genes in this bacterium. In contrast to the homologous and extensively characterized AgDI system of S. mutants, the aguBDAC operon in E. faecalis is not induced in response to low pH. In spite of this, agmatine catabolism in this bacterium contributes by neutralizing the external medium while enhancing bacterial growth. Our results indicate that carbon catabolic repression (CCR) operates on the AgDI system via a mechanism that involves interaction of CcpA and P-Ser-HPr with a cre site found in an unusual position considering the aguB promoter (55 nt upstream the +1 position). In addition, we found that components of the mannose phosphotransferase (PTS(Man)) system also contributed to CCR in E. faecalis since a complete relief of the PTS-sugars repressive effect was observed only in a PTS(Man) and CcpA double defective strain. Our gene context analysis revealed that aguR is present in oral and gastrointestinal microorganisms. Thus, regulation of the aguBDAC operon in E. faecalis seems to have evolved to obtain energy and resist low pH conditions in order to persist and colonize gastrointestinal niches.
Subject(s)
Bacterial Proteins/metabolism , Enterococcus faecalis/metabolism , Hydrolases/metabolism , Mannose/metabolism , Metabolic Networks and Pathways , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Repressor Proteins/metabolism , Agmatine/metabolism , Ammonium Compounds/metabolism , Bacterial Proteins/genetics , Base Sequence , Enterococcus faecalis/enzymology , Enterococcus faecalis/genetics , Enterococcus faecalis/growth & development , Gene Expression Regulation, Bacterial , Genes, Bacterial/genetics , Genetic Loci/genetics , Homeostasis , Hydrogen-Ion Concentration , Metabolic Networks and Pathways/genetics , Models, Biological , Molecular Sequence Data , Operon/genetics , Transcription, Genetic , beta-Galactosidase/metabolismABSTRACT
Despite the increasing importance of Enterococcus as opportunistic pathogens, their virulence factors are still poorly understood. This study determines the frequency of virulence factors in clinical and commensal Enterococcus isolates from inpatients in Porto Alegre, Brazil. Fifty Enterococcus isolates were analysed and the presence of the gelE, asa1 and esp genes was determined. Gelatinase activity and biofilm formation were also tested. The clonal relationships among the isolates were evaluated using pulsed-field gel electrophoresis. The asa1, gelE and esp genes were identified in 38%, 60% and 76% of all isolates, respectively. The first two genes were more prevalent in Enterococcus faecalis than in Enterococcus faecium, as was biofilm formation, which was associated with gelE and asa1 genes, but not with the esp gene. The presence of gelE and the activity of gelatinase were not fully concordant. No relationship was observed among any virulence factors and specific subclones of E. faecalis or E. faecium resistant to vancomycin. In conclusion, E. faecalis and E. faecium isolates showed significantly different patterns of virulence determinants. Neither the source of isolation nor the clonal relationship or vancomycin resistance influenced their distribution.
Subject(s)
Anti-Bacterial Agents/pharmacology , Enterococcus faecalis , Enterococcus faecium , Vancomycin/pharmacology , Virulence Factors/genetics , Biofilms/growth & development , Electrophoresis, Gel, Pulsed-Field , Enterococcus faecalis/drug effects , Enterococcus faecalis/enzymology , Enterococcus faecalis/pathogenicity , Enterococcus faecium/drug effects , Enterococcus faecium/enzymology , Enterococcus faecium/pathogenicity , Gelatinases/metabolism , Microbial Sensitivity Tests , Vancomycin Resistance/geneticsABSTRACT
Despite the increasing importance of Enterococcus as opportunistic pathogens, their virulence factors are still poorly understood. This study determines the frequency of virulence factors in clinical and commensal Enterococcus isolates from inpatients in Porto Alegre, Brazil. Fifty Enterococcus isolates were analysed and the presence of the gelE, asa1 and esp genes was determined. Gelatinase activity and biofilm formation were also tested. The clonal relationships among the isolates were evaluated using pulsed-field gel electrophoresis. The asa1, gelE and esp genes were identified in 38%, 60% and 76% of all isolates, respectively. The first two genes were more prevalent in Enterococcus faecalis than in Enterococcus faecium, as was biofilm formation, which was associated with gelE and asa1 genes, but not with the esp gene. The presence of gelE and the activity of gelatinase were not fully concordant. No relationship was observed among any virulence factors and specific subclones of E. faecalis or E. faecium resistant to vancomycin. In conclusion, E. faecalis and E. faecium isolates showed significantly different patterns of virulence determinants. Neither the source of isolation nor the clonal relationship or vancomycin resistance influenced their distribution.
Subject(s)
Anti-Bacterial Agents/pharmacology , Enterococcus faecalis , Enterococcus faecium , Vancomycin/pharmacology , Virulence Factors/genetics , Biofilms/growth & development , Electrophoresis, Gel, Pulsed-Field , Enterococcus faecalis/drug effects , Enterococcus faecalis/enzymology , Enterococcus faecalis/pathogenicity , Enterococcus faecium/drug effects , Enterococcus faecium/enzymology , Enterococcus faecium/pathogenicity , Gelatinases/metabolism , Microbial Sensitivity Tests , Vancomycin Resistance/geneticsABSTRACT
Enterococcus faecalis encodes a biotin-dependent oxaloacetate decarboxylase (OAD), which is constituted by four subunits: E. faecalis carboxyltransferase subunit OadA (termed Ef-A), membrane pump Ef-B, biotin acceptor protein Ef-D, and the novel subunit Ef-H. Our results show that in E. faecalis, subunits Ef-A, Ef-D, and Ef-H form a cytoplasmic soluble complex (termed Ef-AHD) which is also associated with the membrane. In order to characterize the role of the novel Ef-H subunit, coexpression of oad genes was performed in Escherichia coli, showing that this subunit is vital for Ef-A and Ef-D interaction. Diminished growth of the oadA and oadD single deletion mutants in citrate-supplemented medium indicated that the activity of the complex is essential for citrate utilization. Remarkably, the oadB-deficient strain was still capable of growing to wild-type levels but with a delay during the citrate-consuming phase, suggesting that the soluble Ef-AHD complex is functional in E. faecalis. These results suggest that the Ef-AHD complex is active in its soluble form, and that it is capable of interacting in a dynamic way with the membrane-bound Ef-B subunit to achieve its maximal alkalinization capacity during citrate fermentation.
Subject(s)
Carboxy-Lyases/genetics , Enterococcus faecalis/enzymology , Multienzyme Complexes/genetics , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , Carboxy-Lyases/isolation & purification , Carboxy-Lyases/metabolism , Citric Acid/metabolism , Cytoplasm/enzymology , Enterococcus faecalis/genetics , Enterococcus faecalis/physiology , Escherichia coli/genetics , Escherichia coli/metabolism , Fermentation , Hydrogen-Ion Concentration , Multienzyme Complexes/isolation & purification , Multienzyme Complexes/metabolism , Oxaloacetic Acid/metabolism , Protein Subunits , Recombinant Proteins , Sequence Deletion , TransgenesABSTRACT
The presence of virulence genes (VG) and bacteriocins from different clinical samples was studied in Enterococcus faecalis isolated from urinary tract infections (UTI), bacteremia and endodontitis and was correlated with haemolysin and gelatinase activity. We evaluated the presence of VG by PCR in 150 strains of E. faecalis including cylA, aggA, efaA, eep, gelE, esp, as-48, bac31, entL50A/B, entA, entP, entB, enlA andentl071. Haemolysin and gelatinase activity was studied. gelE and cylA genes expressed hemolysin and gelatinase, respectively. This activity was observed in some strains of bacteremia, UTI and endodontitis. The highest number of VG was detected in bacteremic strains, being aggA and entA genes the most frequent. efaA, esp, entA, entL50A/B were associated with their clinical origin (p < 0.05). The most common genetic profile was aggA-eep-enlA-entL50A/B. E. faecalis from UTI, bacteremia and endodontitis presented different gene combinations. Some of the genes studied were related to their clinical origin. The results obtained in this study are similar to those reported in other countries.
Subject(s)
Bacterial Proteins/genetics , Bacteriocins/genetics , Enterococcus faecalis/genetics , Gelatinases/genetics , Hemolysin Proteins/genetics , Virulence Factors/genetics , Chile , Enterococcus faecalis/enzymology , Enterococcus faecalis/pathogenicity , Female , Gelatinases/biosynthesis , Hemolysin Proteins/biosynthesis , Humans , Male , Oligonucleotide Array Sequence Analysis/methodsABSTRACT
The presence of virulence genes (VG) and bacteriocins from different clinical samples was studied in Enterococcus faecalis isolated from urinary tract infections (UTI), bacteremia and endodontitis and was correlated with haemolysin and gelatinase activity. We evaluated the presence of VG by PCR in 150 strains of E. faecalis including cylA, aggA, efaA, eep, gelE, esp, as-48, bac31, entL50A/B, entA, entP, entB, enlA andentl071. Haemolysin and gelatinase activity was studied. gelE and cylA genes expressed hemolysin and gelatinase, respectively. This activity was observed in some strains of bacteremia, UTI and endodontitis. The highest number of VG was detected in bacteremic strains, being aggA and entA genes the most frequent. efaA, esp, entA, entL50A/B were associated with their clinical origin (p < 0.05). The most common genetic profile was aggA-eep-enlA-entL50A/B. E. faecalis from UTI, bacteremia and endodontitis presented different gene combinations. Some of the genes studied were related to their clinical origin. The results obtained in this study are similar to those reported in other countries.
Desde diferentes muestras clínicas se determinó la presencia de genes codificantes de factores de virulencia (FV) y bacteriocinas en Enterococcus faecalis aislados desde infecciones del tracto urinario (ITU), bacteriemias y endodontitis, correlacionándose con la actividad hemolisina y gelatinasa. En 150 cepas de E. faecalis fue evaluada mediante RPC la presencia de cylA, aggA, efaA, eep, gelE, esp, as-48, bac31, entL50A/B, entA, entP, entB, enlA, y ent1071 determinándose actividad hemolisina y gelatinasa. Los genes cylA y gelE expresaron hemolisina y gelatinasa, respectivamente. Esta actividad fue observada en algunas de las cepas causantes de bacteriemia, ITU y endodontitis. El mayor número de genes estudiados se detectó en cepas bacteriémicas. Los genes aggA y entA, fueron los más frecuentes. Los genes efaA, esp, entL50/AB y entA se asociaron a su origen clínico (p < 0,05). El perfil genético más recurrente fue aggA-eep-enlA-entL50A/B. Enterococcusfaecalis de ITU, bacteriemias y endodontitis presentaron distintas combinaciones génicas. AAlgunos de los genes estudiados se relacionaron con su origen clínico. Los resultados obtenidos son similares a los reportados en otros países.
Subject(s)
Female , Humans , Male , Bacterial Proteins/genetics , Bacteriocins/genetics , Enterococcus faecalis/genetics , Gelatinases/genetics , Hemolysin Proteins/genetics , Virulence Factors/genetics , Chile , Enterococcus faecalis/enzymology , Enterococcus faecalis/pathogenicity , Gelatinases/biosynthesis , Hemolysin Proteins/biosynthesis , Oligonucleotide Array Sequence Analysis/methodsABSTRACT
Objective. To identify infection-causing Enterococcus species in Cuban hospitalsand determine their susceptibility to antimicrobial drugs, as well as their resistance mechanisms. Methods. A total of 687 Enterococcus isolates from 30 Cuban hospitals in nine provinces of the country were studied over the period 20002009. The species were identified using both the conventional method and the automatic API® system.The minimum inhibitory concentration was determined for 13 antimicrobial drugs following the standards recommended by the Clinical Laboratory and Standards Institute. The polymerase chain reaction technique was used to characterize the genes that were resistant to aminoglycosides, erythromycin, tetracycline, andglucopeptides. The presence of beta-lactamase was determined by the chromogenic cephalosporin test. Results. The most prevalent species were Enterococcus faecalis (82.9%) and E. faecium (12.2%). Resistance to glucopeptides (1.0%) was mediated by the vanA and vanB genes. The strains resistant to ampicillin (6%) did not produce beta-lactamases. A high percentage of resistance to aminoglycosides was observed. Gentamicin (31.0%) and streptomycin and amikacin (29.1%) were mediated by the aac(6)Ie-aph(2)Ia, aph(3)-IIIa, ant(6)Ia, and ant(3)(9) genes. A correlation was found between resistance to tetracycline (56.0%) and presence of the tet(M) (75.1%) and tet(L) genes (7.0%), while resistance to erythromycin (34.1%) was due to the erm(B) gene (70.9%). Conclusions. Resistance to vancomycin is infrequent in Cuba, as opposed to a high level of resistance to aminoglycosides, which may be indicative of treatment failures. The microbiology laboratory is a cornerstone of Enterococcus infectionsurveillance, along with ongoing monitoring of the susceptibility of these infections to antimicrobial drugs at a time when resistance of this microorganism is on the rise.
Objetivo. Identificar las especies de Enterococcus causantes de infecciones en hospitales cubanos, su susceptibilidad a los antimicrobianos y sus mecanismos de resistencia.Métodos. Se estudiaron 687 aislamientos de Enterococcus procedentes de 30 hospitalescubanos de nueve provincias del país durante el período de 2000 a 2009. La identificación de las especies se realizó mediante el método convencional y sistema automatizado API®. Laconcentración inhibitoria mínima se determinó para 13 antimicrobianos según las recomendaciones del Instituto de Estándares Clínicos y de Laboratorio. Se determinaron los genes de resistencia a aminoglucósidos, eritromicina, tetraciclina y glucopéptidos mediante reacciónen cadena de la polimerasa. La presencia de betalactamasa se determinó por el método de lacefalosporina cromógena. Resultados. Las especies más prevalentes fueron Enterococcus faecalis (82,9%) y Enterococcus faecium (12,2%). La resistencia a los glucopéptidos (1,0%) estuvo mediada por los genes vanA y vanB y las cepas resistentes a ampicilina (6%) no produjeron betalactamasas. Se observó un alto porcentaje de resistencia a los aminoglucósidos: gentamicina (31,0%) y estreptomicina y amikacina (29,1%) mediada por los genes aac(6)Ie-aph(2)Ia, aph(3)-IIIa, ant(6)Ia, ant(3)(9). Hubo correlación entre la resistencia a tetraciclina (56,0%) y la presencia de los genes tet(M) (75,1%) y tet(L) (7,0%), mientras que la resistencia a eritromicina (34,1%) obedeció al gen erm(B) (70,9%).Conclusiones. La resistencia a vancomicina es infrecuente en Cuba, a diferencia del alto nivel de resistencia a los aminoglucósidos, que sugiere posibles fracasos terapéuticos. El laboratorio de microbiología constituye un pilar fundamental de la vigilancia de las infecciones por cepas de Enterococcus y el monitoreo continuo de su susceptibilidad a los antimicrobianos,dado el incremento de la resistencia de ese microorganismo en el tiempo.
Subject(s)
Humans , Drug Resistance, Microbial , Enterococcus/genetics , Gram-Positive Bacterial Infections/microbiology , Aminoglycosides/pharmacology , Cross Infection/epidemiology , Cross Infection/microbiology , Cuba , Drug Resistance, Microbial/genetics , Drug Resistance, Multiple, Bacterial , Enterococcus faecalis/enzymology , Enterococcus faecalis/genetics , Enterococcus faecalis/isolation & purification , Enterococcus faecium/enzymology , Enterococcus faecium/genetics , Enterococcus faecium/isolation & purification , Enterococcus/enzymology , Enterococcus/isolation & purification , Genes, Bacterial , Gram-Positive Bacterial Infections/epidemiology , Species Specificity , Vancomycin Resistance/geneticsABSTRACT
Iron-sulfur [Fe-S] clusters are inorganic prosthetic groups that play essential roles in all living organisms. In vivo [Fe-S] cluster biogenesis requires enzymes involved in iron and sulfur mobilization, assembly of clusters, and delivery to their final acceptor. In these systems, a cysteine desulfurase is responsible for the release of sulfide ions, which are incorporated into a scaffold protein for subsequent [Fe-S] cluster assembly. Although three machineries have been shown to be present in Proteobacteria for [Fe-S] cluster biogenesis (NIF, ISC, and SUF), only the SUF machinery has been found in Firmicutes. We have recently described the structural similarities and differences between Enterococcus faecalis and Escherichia coli SufU proteins, which prompted the proposal that SufU is the scaffold protein of the E. faecalis sufCDSUB system. The present work aims at elucidating the biological roles of E. faecalis SufS and SufU proteins in [Fe-S] cluster assembly. We show that SufS has cysteine desulfurase activity and cysteine-365 plays an essential role in catalysis. SufS requires SufU as activator to [4Fe-4S] cluster assembly, as its ortholog, IscU, in which the conserved cysteine-153 acts as a proximal sulfur acceptor for transpersulfurization reaction.
Subject(s)
Carbon-Sulfur Lyases/metabolism , Cysteine/metabolism , Enterococcus faecalis/enzymology , Iron-Sulfur Proteins/physiology , Sulfur/metabolism , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Carbon-Sulfur Lyases/chemistry , Carbon-Sulfur Lyases/genetics , Carbon-Sulfur Lyases/isolation & purification , Cloning, Molecular , Cysteine/chemistry , Enterococcus faecalis/chemistry , Enterococcus faecalis/genetics , Enterococcus faecalis/metabolism , Enzyme Activation , Iron-Sulfur Proteins/chemistry , Iron-Sulfur Proteins/genetics , Iron-Sulfur Proteins/metabolism , Models, Molecular , Protein Binding , Substrate Specificity , Sulfur/chemistryABSTRACT
Two paralogous genes, maeE and citM, that encode putative malic enzyme family members were identified in the Enterococcus faecalis genome. MaeE (41 kDa) and CitM (42 kDa) share a high degree of homology between them (47% identities and 68% conservative substitutions). However, the genetic context of each gene suggested that maeE is associated with malate utilization whereas citM is linked to the citrate fermentation pathway. In the present work, we focus on the biochemical characterization and physiological contribution of these enzymes in E. faecalis. With this aim, the recombinant versions of the two proteins were expressed in Escherichia coli, affinity purified and finally their kinetic parameters were determined. This approach allowed us to establish that MaeE is a malate oxidative decarboxylating enzyme and CitM is a soluble oxaloacetate decarboxylase. Moreover, our genetic studies in E. faecalis showed that the citrate fermentation phenotype is not affected by citM deletion. On the other hand, maeE gene disruption resulted in a malate fermentation deficient strain indicating that MaeE is responsible for malate metabolism in E. faecalis. Lastly, it was demonstrated that malate fermentation in E. faecalis is associated with cytoplasmic and extracellular alkalinization which clearly contributes to pH homeostasis in neutral or mild acidic conditions.
Subject(s)
Carboxy-Lyases/genetics , Carboxy-Lyases/metabolism , Enterococcus faecalis/enzymology , Enterococcus faecalis/genetics , Malate Dehydrogenase/genetics , Malate Dehydrogenase/metabolism , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Base Sequence , Carboxy-Lyases/chemistry , Citric Acid/metabolism , Cloning, Molecular , DNA, Bacterial/genetics , Fermentation , Genes, Bacterial , Hydrogen-Ion Concentration , Kinetics , Malate Dehydrogenase/chemistry , Malates/metabolism , Molecular Sequence Data , Phylogeny , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , SolubilityABSTRACT
OBJECTIVE: To identify infection-causing Enterococcus species in Cuban hospitals and determine their susceptibility to antimicrobial drugs, as well as their resistance mechanisms. METHODS: A total of 687 Enterococcus isolates from 30 Cuban hospitals in nine provinces of the country were studied over the period 2000-2009. The species were identified using both the conventional method and the automatic API(®) system. The minimum inhibitory concentration was determined for 13 antimicrobial drugs following the standards recommended by the Clinical Laboratory and Standards Institute. The polymerase chain reaction technique was used to characterize the genes that were resistant to aminoglycosides, erythromycin, tetracycline, and glucopeptides. The presence of beta-lactamase was determined by the chromogenic cephalosporin test. RESULTS: The most prevalent species were Enterococcus faecalis (82.9%) and E. faecium (12.2%). Resistance to glucopeptides (1.0%) was mediated by the vanA and vanB genes. The strains resistant to ampicillin (6%) did not produce beta-lactamases. A high percentage of resistance to aminoglycosides was observed. Gentamicin (31.0%) and streptomycin and amikacin (29.1%) were mediated by the aac(6')Ie-aph(2")Ia, aph(3')-IIIa, ant(6)Ia, and ant(3")(9) genes. A correlation was found between resistance to tetracycline (56.0%) and presence of the tet(M) (75.1%) and tet(L) genes (7.0%), while resistance to erythromycin (34.1%) was due to the erm(B) gene (70.9%). CONCLUSIONS: Resistance to vancomycin is infrequent in Cuba, as opposed to a high level of resistance to aminoglycosides, which may be indicative of treatment failures. The microbiology laboratory is a cornerstone of Enterococcus infection surveillance, along with ongoing monitoring of the susceptibility of these infections to antimicrobial drugs at a time when resistance of this microorganism is on the rise.
Subject(s)
Drug Resistance, Microbial , Enterococcus/genetics , Gram-Positive Bacterial Infections/microbiology , Aminoglycosides/pharmacology , Cross Infection/epidemiology , Cross Infection/microbiology , Cuba , Drug Resistance, Microbial/genetics , Drug Resistance, Multiple, Bacterial , Enterococcus/enzymology , Enterococcus/isolation & purification , Enterococcus faecalis/enzymology , Enterococcus faecalis/genetics , Enterococcus faecalis/isolation & purification , Enterococcus faecium/enzymology , Enterococcus faecium/genetics , Enterococcus faecium/isolation & purification , Genes, Bacterial , Gram-Positive Bacterial Infections/epidemiology , Humans , Species Specificity , Vancomycin Resistance/geneticsABSTRACT
The reporter transposon-based system TnFuZ was used to identify exported proteins of the animal pathogen Corynebacterium pseudotuberculosis. Thirty-four out of 1,500 mutants had detectable alkaline phosphatase (PhoZ) activity. This activity was from 21 C. pseudotuberculosis loci that code for fimbrial and transport subunits and for hypothetical and unknown-function proteins.
Subject(s)
Alkaline Phosphatase/metabolism , Bacterial Proteins/metabolism , Base Sequence , Corynebacterium pseudotuberculosis/genetics , DNA Transposable Elements , Mutagenesis, Insertional/methods , Alkaline Phosphatase/genetics , Animals , Bacterial Proteins/genetics , Biological Transport , Corynebacterium pseudotuberculosis/growth & development , Corynebacterium pseudotuberculosis/metabolism , Enterococcus faecalis/enzymology , Enterococcus faecalis/genetics , Molecular Sequence Data , Sequence Analysis, DNAABSTRACT
A new aspect in the action of ampicillin and gentamicin was detected in Enterococcus faecalis. Reactive oxygen species (ROS) increased in sensitive strains during treatment with each antibiotic up to a certain concentration of antibiotic, above which ROS diminished as a consequence of oxidative stress. Tiron, a scavenger of the superoxide anion O(2)(-), counteracted the effect of the generated ROS. The oxidative stress was a consequence of an increase in ROS in the cytoplasm of bacteria, as observed by the nitroblue tetrazolium reaction. The viability of sensitive strains was significantly reduced at concentrations of antibiotics that increased the ROS, and this increment was parallel to the bactericidal effect. Sensitive E. faecalis strains showed an immediate increase of ATP in the presence of both antibiotics, thus an energy-dependent process had been triggered, indicating a bacterial reaction against the stress. The combination of both antibiotics augmented the effect of ROS, which helps to explain the synergism between ampicillin and gentamicin.
Subject(s)
Ampicillin/pharmacology , Anti-Bacterial Agents/pharmacology , Enterococcus faecalis/drug effects , Gentamicins/pharmacology , Oxidative Stress/drug effects , Oxygen/metabolism , Reactive Oxygen Species/metabolism , Drug Synergism , Enterococcus faecalis/enzymology , Enterococcus faecalis/metabolism , Oxidative Stress/physiologyABSTRACT
Enterococcus gallinarum BM4175 (a vancomycin-susceptible derivative of BM4174 obtained by insertional inactivation of vanC-1) was transformed with plasmid constructs pCA10 (containing the genes necessary for resistance, vanC-1-XYc-T), pJP1 (with a fragment lacking the DNA encoding the transmembrane region of VanT, -vanC-1-XYc-T((Delta))(2-322)-) and with plasmids containing fragments encoding either the transmembrane (mvanT(1-322)) or racemase (svanT(323-698)) domains of VanT under the control of a constitutive promoter. Accumulated peptidoglycan precursors were measured in all strains in the presence of L-Ser, D-Ser (50 mM) or in the absence of any growth supplement. Uptake of 0.1 mM L-[(14)C]serine was also determined in BM4174, BM4175 and BM4175/pCA10. Vancomycin resistance was restored in BM4175 transformed with pCA10(C-1-XYc-T), and the profile of peptidoglycan precursors was similar to wild-type E. gallinarum BM4174. Transformation of E. gallinarum BM4175 with plasmid pJP1(vanC-1-XYc-T((Delta))(2-322)) resulted in: (i) vancomycin MICs remaining within susceptible levels (< or =4 mg/L) in the absence of any growth supplement, but increasing to 8 mg/L when either L-Ser or D-Ser was added to the medium; and (ii) the relative amounts of accumulated UDP-MurNAc-pentapeptide[D-Ser] and tetrapeptide precursors decreasing substantially compared with BM4175/pCA10 and BM4174. The effect on the appearance of tetrapeptide appeared to be host dependent, since a substantial amount was present when the same plasmid construct pJP1(vanC-1-XYc-T((Delta))(2-322)) was electroporated into Enterococcus faecalis JH2-2. The uptake of L-[(14)C]Ser at 240 s was decreased by approximately 40% in BM4175 compared with BM4174. Plasmid pCA10(C-1-XY(C)-T) restored uptake of L-[(14)C]Ser at 180 and 240 s in BM4175. The results suggest that the transmembrane domain of VanT is likely to be involved in the transport of L-Ser, and that in its absence the resistance phenotype is compromised.
Subject(s)
Enterococcus faecalis/enzymology , Enterococcus/enzymology , Membrane Proteins/physiology , Racemases and Epimerases/chemistry , Racemases and Epimerases/physiology , Vancomycin Resistance , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Enterococcus/drug effects , Enterococcus/genetics , Enterococcus faecalis/drug effects , Enterococcus faecalis/genetics , Membrane Proteins/genetics , Protein Structure, Tertiary , Racemases and Epimerases/genetics , Vancomycin Resistance/geneticsABSTRACT
Six beta-lactamase-producing (Bla+) isolates of Enterococcus faecalis recovered over a 17-month period from an Argentinian pediatric hospital were found to have identical or almost identical chromosomal restriction patterns by pulsed-field gel electrophoresis, although the plasmid patterns were different. These isolates, like Bla+ enterococci in the United States, hybridized to a staphylococcal Bla gene probe. The presence of a single strain was somewhat surprising, since all isolates transferred Bla by conjugation.
Subject(s)
Enterococcus faecalis/isolation & purification , Gentamicins/pharmacology , Hospitals, Pediatric , beta-Lactamases/metabolism , Argentina , Drug Resistance, Microbial , Enterococcus faecalis/enzymology , Enterococcus faecalis/genetics , Genome, Bacterial , Humans , Hybridization, Genetic , PlasmidsABSTRACT
Beta-lactamase-producing (Bla+) enterococci have been reported in three state and two countries. Pulsed-field gel electrophoresis was used to compare 14 Bla+ Enterococcus (Streptococcus) faecalis isolated from hospitalized patients in seven states and three continents. The restriction endonuclease digestion patterns of isolates from Connecticut, Massachusetts, Lebanon, and Argentina were all markedly different, indicating that these were different strains. However, isolates from Delaware, Texas, Pennsylvania (Philadelphia and Pittsburgh), Florida, and Virginia were similar, indicating that these isolates were derivatives of a single strain. This conclusion was supported by hybridization using individual fragments as probes. Spread of Bla+ enterococci within the hospital setting was also demonstrated. These findings illustrate the value of pulsed-field gel electrophoresis for epidemiologic analyses and support the importance of identifying and containing organisms with new resistance properties in an effort to decrease their transmission to and from, as well as within, hospitals.
Subject(s)
DNA, Bacterial/analysis , Enterococcus faecalis/classification , Streptococcal Infections/microbiology , beta-Lactamases/biosynthesis , Argentina , Boston , Connecticut , Delaware , Electrophoresis, Agar Gel , Enterococcus faecalis/enzymology , Enterococcus faecalis/genetics , Florida , Humans , Lebanon , Nucleic Acid Hybridization , Pennsylvania , Philadelphia , Plasmids , Restriction Mapping , Streptococcal Infections/transmission , Texas , VirginiaABSTRACT
Of the 90 strains of Enterococcus spp. isolated in an Argentine laboratory in the period from January to July 1989, three were identified as beta-lactamase-producing Enterococcus faecalis. According to the literature reviewed, these appear to be the first beta-lactamase-positive strains of Enterococcus isolated outside the USA. They differed from most beta-lactamase-producing strains already described in that they were susceptible to low concentrations of macrolides. All three strains were resistant to high concentrations of aminoglycosides.