ABSTRACT
Enterobacter aerogenes predominates amongst Enterobacteriaceae species that are increasingly reported as producers of extended-spectrum beta-lactamases. Although this mechanism of resistance to beta-lactams is important, other mechanisms bestowing a multidrug-resistant (MDR) phenotype in this species are now well documented. Amongst these mechanisms is the overexpression of efflux pumps that extrude structurally unrelated antibiotics prior to their reaching their targets. Interestingly, although knowledge of the genetic background behind efflux pumps is rapidly advancing, few studies assess the physiological nature of the overall efflux pump system of this, or for that matter any other, bacterium. The study reported here evaluates physiologically the efflux pump system of an E. aerogenes ATCC reference as well as two strains whose MDR phenotypes are mediated by overexpressed efflux pumps. The activities of the efflux pumps in these strains are modulated by pH and glucose, although the effects of the latter are essentially restricted to pH 8, suggesting the presence of two general efflux pump systems, i.e. proton-motive force-dependent and ABC transporter types, respectively.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins/metabolism , Drug Resistance, Multiple, Bacterial , Enterobacter aerogenes/drug effects , Enterobacter aerogenes/physiology , Gene Expression Regulation, Bacterial , ATP-Binding Cassette Transporters/metabolism , Enterobacter aerogenes/genetics , Enterobacter aerogenes/metabolism , Enterobacteriaceae Infections/microbiology , Ethidium/metabolism , Glucose/pharmacology , Humans , Hydrogen-Ion Concentration , Microbial Sensitivity Tests , Proton-Motive Force , Up-RegulationABSTRACT
In this study, we aimed to answer the following question: 'How does a bacterium become so resistant to a given antibiotic even though the levels of antibiotic to which it has become resistant remained constant in the patient?'Escherichia coli AG100 strain induced to high-level resistance due to overexpression of an AcrAB efflux pump was serially cultured in 10mg/L tetracycline for 60 passages. Between each passage it became increasingly resistant to tetracycline, beta-lactams and quinolones with concomitant restoration of wild-type AcrAB activity. Because the multidrug-resistant phenotype could not be reversed with transfer to drug-free medium or with efflux pump inhibitors, it may have resulted from activation of a 'mutator gene' system that reduced the 'energy consumption' associated with an overexpressed efflux pump system.
Subject(s)
Anti-Bacterial Agents/metabolism , Anti-Bacterial Agents/pharmacology , Drug Resistance, Multiple, Bacterial , Escherichia coli Proteins/metabolism , Escherichia coli/drug effects , Escherichia coli/metabolism , Lipoproteins/metabolism , Membrane Transport Proteins/metabolism , Multidrug Resistance-Associated Proteins/metabolism , Adaptation, Biological , Microbial Sensitivity Tests , Quinolones/metabolism , Quinolones/pharmacology , Serial Passage , Tetracycline/metabolism , Tetracycline/pharmacology , beta-Lactams/metabolism , beta-Lactams/pharmacologyABSTRACT
We have developed a number of methods that identify efflux pump mediated multi-drug resistant bacteria, characterize efflux systems and screen for inhibitors of efflux pumps. These approaches were complemented by the quantification of the expression of genes that regulate and code for constituents of efflux pumps. The methods described are easy to use, reproducible and for the most part, require instrumentation normally present in a clinical bacteriology laboratory. Because each method provides good reproducibility, they lend themselves for inter-laboratory use.
Subject(s)
Drug Resistance, Multiple, Bacterial , Membrane Transport Proteins , Microbiological Techniques/methods , Anti-Bacterial Agents/metabolism , Bacteria/genetics , Bacteria/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biological Transport , Gene Expression Regulation, Bacterial , Membrane Transport Proteins/genetics , Reproducibility of ResultsABSTRACT
Total protein, albumin and gamma-globulin values were determined in CSF of 139 children. They had been referred to the emergency department of the Children's Hospital of the University of Berne during the last year because of suspected meningitis which, however, was not confirmed. In newborns and infants the normal range of protein values was wide, 0.1-0.65 g/l. It fell to minimal values at age 3-4 years, and increased gradually thereafter to adult levels which were reached at age 6-8 years. The observed changes during early childhood are mainly due to variations in the albumine fraction. The results are different from the ones reported by Ammon and Richterich [1] who claimed constantly falling protein levels during childhood up to adult values. Similar results in comparison to ours were obtained by other laboratories using the same method [6,7]. Our study therefore supports the usefulness of Bradford's [2] method for routine CSF work-up.
Subject(s)
Cerebrospinal Fluid Proteins/analysis , Adolescent , Albumins/analysis , Child , Child, Preschool , Globulins/analysis , Humans , Infant , Infant, NewbornABSTRACT
Covering a ten-year period (1970-1979), this retrospective study was performed to determine the frequency of unexpected paraproteinemia in hospitalized patients with various internal diseases. The survey was made possible by the fact that serum protein electrophoresis had been routinely performed in all patients upon admission. In 86 cases (0.66% of all admissions) monoclonal gammopathy was an unexpected finding. 5 out of these 86 cases (0.038% of all admissions) had malignant disease (2 patients with Bence Jones myeloma, 3 patients with Waldenström's macroglobulinemia), and 3 more cases had lymphoproliferative syndromes. In all other cases the abnormal serum protein findings corresponded to asymptomatic ("benign") paraproteinemias when first detected. In addition, the following statistical parameters were analyzed: distribution among immunoglobulin classes; sex; and concomitant diseases. A critical evaluation was possible in 67 cases of so-called idiopathic paraproteinemia.
Subject(s)
Hospitals, Special , Internal Medicine , Paraproteinemias/epidemiology , Aged , Bence Jones Protein/urine , Female , Humans , Immunoglobulins/classification , Male , Middle Aged , Multiple Myeloma/complications , Paraproteinemias/complications , Retrospective Studies , Sex Factors , Statistics as Topic , Switzerland , Waldenstrom Macroglobulinemia/complicationsABSTRACT
Pentameric IgM is precipitable by PHA whereas monomeric IgM is not. During electrophoresis in PHA-containing agar gel pentameric IgM is selected by precipitation from concomitant monomeric IgM which may be visualized separately as a precipitation line with specific antibody. This antibody-like use of the lectin in PHA selection electrophoresis provides a quick and simple method of screening macroglobulinemic sera and other biological fluids for the presence of naturally occurring monomeric IgM subunits.
Subject(s)
Immunoglobulin M , Paraproteinemias/diagnosis , Phytohemagglutinins/pharmacology , Waldenstrom Macroglobulinemia/diagnosis , Absorption , Animals , Binding Sites , Concanavalin A/pharmacology , Electrophoresis, Agar Gel , Goats , Humans , Immunoelectrophoresis , Polymers , Precipitins , Rabbits , Ricin/pharmacologyABSTRACT
Phytohemagglutinin P, when used like an antiserum in immunoelectrophoresis, precipitates with pentameric IgM but not with monomeric IgM subunits. In a retrospective study this simple screening method was applied to 180 sera containing monoclonal IgM. Five cases were found to be of the monomeric type of IgM paraproteinemia. Four of these patients had lymphoproliferative diseases with lymphocytic infiltration of the bone marrow. The last case was complicated by acute myelomonocytic leukemia.
Subject(s)
Lymphoproliferative Disorders/complications , Paraproteinemias/complications , Bone Marrow/pathology , Humans , Immunoglobulin M , LymphocytesABSTRACT
HDCS vaccine was given to 20 healthy, not rabies-exposed people on days 0, 3, 7, 14, 28 and 90. Ten of them received in addition 20 IU/kg of human antirabies immunoglobulin. Vaccine potency in NIH tests was 1.8 (WHO). Most individuals did not show any measurable neutralizing antibody titer on days 3 and 7. On days 14, 28, 90 and 300 the mean of titres was above 1:200; with one exception, no titer was lower than 1:50. The sera of days 14 and 90 were separated on "Sephadex G-200" columns. On day 14 at least 46% of the rabies antibody was found in the IgM fraction, while 15% was recovered from the IgG fraction. On day 90 the proportion recovered from the IgM fraction had decreased to 17%; the IgG antibody had increased to 30% of the titres found in whole serum. Almost no antirabies activity was found in the IgG fractions.
Subject(s)
Immunoglobulin G/analysis , Immunoglobulin M/analysis , Rabies Vaccines/immunology , Antibody Formation , Culture Techniques , Humans , Immunization Schedule , Rabies Vaccines/administration & dosageABSTRACT
Cytoplasmic immunoglobulins in human bone marrow plasma cells and lymphoid cells were characterized by direct immunofluorescence with fluorochrome-labelled reagents specific for immunoglobulin heavy and light chains. The percentage distribution of cells containing IgA, IgG or IgM and kappa- or lambda-immunoglobulins was determined in bone marrow samples from 168 immunologically normal individuals, in 11 patients with polyclonal increase of bone marrow plasma cells and in 80 patients with benign or malignant monoclonal gammopathies. A clear differentiation between monoclonal and polyclonal cell populations could be obtained in all cases.
Subject(s)
Bone Marrow Cells , Cytoplasm/immunology , Immunoglobulins/immunology , Plasma Cells/immunology , Adult , Aged , Fluorescent Antibody Technique , Humans , Hypergammaglobulinemia/immunology , Immunoglobulin A/immunology , Immunoglobulin G/immunology , Immunoglobulin M/immunology , Middle Aged , Multiple Myeloma/immunology , Waldenstrom Macroglobulinemia/immunologyABSTRACT
Studies were performed on bone marrow and serum from 28 patients with clinically benign gammopathy and 41 patients with the malignant monoclonal form. In the bone marrow samples, the total number of plasma cells and the monoclonal fraction of these cells were determined by immunofluorescence. Serum samples were analyzed for monoclonal immunoglobulin components and for the total serum protein content. The data could be used for discriminant analysis. The variables had to be transformed and were included in the discriminant function in a stepwise procedure. The resulting function made possible a clear distinction between benign and malignant monoclonal gammopathies.
Subject(s)
Hypergammaglobulinemia/diagnosis , Blood Proteins/analysis , Bone Marrow/pathology , Diagnosis, Differential , Humans , Immunoglobulins/analysis , Mathematics , Plasma Cells/immunologySubject(s)
Serologic Tests , Autoimmune Diseases/diagnosis , Cardiovascular Diseases/diagnosis , Central Nervous System Diseases/diagnosis , Gastrointestinal Diseases/diagnosis , Humans , Hypersensitivity/diagnosis , Immunologic Deficiency Syndromes/diagnosis , Kidney Diseases/diagnosis , Liver Diseases/diagnosis , Neoplasms/diagnosis , Respiratory Tract Diseases/diagnosis , Specimen HandlingABSTRACT
Materials precipitated from an aqueous extract of house dust by saturation with ammonium sulphate showed immunological reaction with antisera to human serum albumin, human alpha1-acid glycoprotein, human IgG, Gm and Inv antigens and to A, B and H antigens. It is concluded that the albumin and alpha1-acid glycoprotein are of human origin. It seems that the apparent IgG activity is due to cross-reactivity since, from the anticipated specificities of the Fab and Fc fragments of human IgG, the former could not be detected, and since Gm (6) activity was present, unexpectedly, in Swiss house dust. The A, B and H blood group antigenic activities were detected in relative concentration different from those expected if they were solely of human origin. It is concluded that they are from both human and non-human sources.