Antimicrobial Activity of the Quinoline Derivative HT61 against Staphylococcus aureus Biofilms.

Staphylococcus aureus biofilms are a significant problem in health care settings, partly due to the presence of a nondividing, antibiotic-tolerant subpopulation. Here we evaluated treatment of S. aureus UAMS-1 biofilms with HT61, a quinoline derivative shown to be effective against nondividing Staphylococcus spp. HT61 was effective at reducing biofilm viability and was associated with increased expression of cell wall stress and division proteins, confirming its potential as a treatment for S. aureus biofilm infections.

Mycobacterium tuberculosis chaperonin 10 stimulates bone resorption: a potential contributory factor in Pott's disease.

Pott's disease (spinal tuberculosis), a condition characterized by massive resorption of the spinal vertebrae, is one of the most striking pathologies resulting from local infection with Mycobacterium tuberculosis (Mt; Boachie-Adjei, O., and R.G. Squillante. 1996. Orthop. Clin. North Am. 27:95-103). The pathogenesis of Pott's disease is not established. Here we report for the first time that a protein, identified by a monoclonal antibody to be the Mt heat shock protein (Baird, P.N., L.M. Hall, and A.R.M. Coates. 1989. J. Gen. Microbiol. 135:931-939) chaperonin (cpn) 10, is responsible for the osteolytic activity of this bacterium. Recombinant Mt cpn10 is a potent stimulator of bone resorption in bone explant cultures and induces osteoclast recruitment, while inhibiting the proliferation of an osteoblast bone-forming cell line. Furthermore, we have found that synthetic peptides corresponding to sequences within the flexible loop and sequence 65-70 of Mt cpn10 may comprise a single conformational unit which encompasses its potent bone-resorbing activity. Our findings suggest that Mt cpn10 may be a valuable pharmacological target for the clinical therapy of vertebral tuberculosis and possibly other bone diseases.

Comparison of the sterilising activities of the nitroimidazopyran PA-824 and moxifloxacin against persisting Mycobacterium tuberculosis.

OBJECTIVES: To measure the bactericidal activity of the nitroimidazopyran PA-824 against Mycobacterium tuberculosis, strain H37Rv, in the three Hu/Coates models of bacterial persistence, in comparison with the activity of moxifloxacin (MXF), a drug shown to be likely to shorten treatment in current clinical trials. METHODS: The bactericidal activity of a wide range of PA-824 and MXF concentrations was tested against a 100-day static, starved, anaerobically-adapted culture in Model 1. In Models 2 and 3, rifampicin (RMP) 100 mg/ml was added to the 100-day culture for 5-7 days and bactericidal activities against surviving tolerant bacilli were tested by addition of the test drugs after removal of RMP in Model 2, and during exposure to RMP in Model 3. RESULTS AND DISCUSSION: PA-824 exhibited little bactericidal activity at low concentrations up to 1.25 microg/ml in each of these models, but high concentrations of >or=10 microg/ml showed considerable bactericidal activity, sufficient to kill all bacilli in Model 3, and appreciably greater than with MXF. However, as PA-824 is 94% plasma bound, concentrations of free drug sufficient to reach the zone of high bactericidal activity may not be obtained in cavities of pulmonary tuberculosis.

Novel approaches to developing new antibiotics for bacterial infections.

Antibiotics are an essential part of modern medicine. The emergence of antibiotic-resistant mutants among bacteria is seemingly inevitable, and results, within a few decades, in decreased efficacy and withdrawal of the antibiotic from widespread usage. The traditional answer to this problem has been to introduce new antibiotics that kill the resistant mutants. Unfortunately, after more than 50 years of success, the pharmaceutical industry is now producing too few antibiotics, particularly against Gram-negative organisms, to replace antibiotics that are no longer effective for many types of infection. This paper reviews possible new ways to discover novel antibiotics. The genomics route has proven to be target rich, but has not led to the introduction of a marketed antibiotic as yet. Non-culturable bacteria may be an alternative source of new antibiotics. Bacteriophages have been shown to be antibacterial in animals, and may find use in specific infectious diseases. Developing new antibiotics that target non-multiplying bacteria is another approach that may lead to drugs that reduce the emergence of antibiotic resistance and increase patient compliance by shortening the duration of antibiotic therapy. These new discovery routes have given rise to compounds that are in preclinical development, but, with one exception, have not yet entered clinical trials. For the time being, the majority of new antibiotics that reach the marketplace are likely to be structural analogues of existing families of antibiotics or new compounds, both natural and non-natural which are screened in a conventional way against live multiplying bacteria.

Sterilising action of pyrazinamide in models of dormant and rifampicin-tolerant Mycobacterium tuberculosis.

SETTING: Pyrazinamide (PZA) is an effective sterilising drug in tuberculosis, but its mode of action is controversial. OBJECTIVE: To test the bactericidal activity of 1.56-100 microg/ml PZA in Hu/Coates models of dormant and rifampicin (RMP) tolerant Mycobacterium tuberculosis. METHODS: In model 1, bactericidal activity was tested in pH 5.5 medium against 4-day, 30-day or 100-day static, hypoxic cultures. In models 2 and 3, 100 microg/ml RMP was added to a 100-day culture and PZA was added either during incubation with RMP in model 3, or after resuspension in RMP-free medium in model 2. RESULTS: Model 1: cfu counts on the 100-day and 30-day cultures fell by a maximum of about 1.6 log cfu/ml with increasing culture age, PZA concentration and incubation period, while counts on the 4-day culture showed little change. Model 2: cfu counts at the end of 7 days of recovery showed little bactericidal activity. Model 3: viable bacilli were almost completely eliminated. Bactericidal activity in these models increased with decreasing metabolic bactericidal activity, as measured by the uptake of [3H] uridine into bacterial RNA. CONCLUSION: PZA differs from other anti-tuberculosis drugs in showing greater bactericidal activity the slower the bacillary metabolic activity, hence its great value as a sterilising drug, likely to remain as an effective companion drug with newer sterilising drugs.

Overproduction and purification of Mycobacterium tuberculosis chaperonin 10.

The overexpression of the Mycobacterium tuberculosis chaperonin 10 (Cpn10)-encoding gene was accomplished using baculovirus expression vectors. The product was immunoreactive with a Cpn10 monoclonal antibody (mAb) and had an electrophoretic mobility identical to authentic Cpn10. The baculovirus system was most successful in terms of reaching nearly the full expression potential of the system. Recombinant Cpn10 was purified from recombinant baculovirus-infected Spodoptera frugiperda cells by isoelectrofocussing and size-exclusion chromatography. The baculovirus vector and purification methodology described represent a very powerful system for the large-scale production of the M. tuberculosis Cpn10 which may allow us to undertake structure-function analysis.

Predictions of linear T-cell and B-cell epitopes in proteins encoded by HIV-1, HIV-2 and SIVMAC and the conservation of these sites between strains.

An important consideration in the design of vaccines to prevent HIV-1 infection effective against different strains is the amino acid sequence conservation of antigenic determinants. Even one amino acid change can destroy the antigenicity of a site for the antibody or T-cell receptor. The comparisons of predicted T- and B-cell epitopes between human HIV-1, HIV-2 and monkey SIVMAC AIDS viruses are presented. The three major gene products (env, gag and pol) were examined. A number of epitopes were identical between strains of HIV-1. Our analysis highlights the problem of designing an effective HIV-1 and HIV-2 vaccine and also the problem of testing human vaccines in monkey models.

Prediction of antigenic determinants and secondary structures of the major AIDS virus proteins.

Criteria for the design of peptide vaccines to prevent AIDS are presented. The best vaccine candidates contain both B and T lymphocyte-defined epitopes in regions conserved in sequence between viral isolates. We propose that attention should focus on proteins specified by the gag and, possibly, pol genes in addition to the env gene envelope glycoproteins being actively studied. The predictions of B- and T-epitopes are refined by consideration of secondary structure prediction and inter-isolate sequence variability to suggest peptides from env, gag and pol that would be the best vaccine candidates.

Chaperonins are cell-signalling proteins: the unfolding biology of molecular chaperones.

The chaperonins are a subgroup of oligomeric molecular chaperones; the best-studied examples are chaperonin 60 (GroEL) and chaperonin 10 (GroES), both from the bacterium Escherichia coli. At the end of the 20th century, the paradigm of chaperonins as protein folders had emerged, but it is likely that during the 21st century these proteins will come to be viewed as intercellular signals. Indeed, it is possible that the chaperonins were among the first intercellular signalling proteins to evolve. During the past few years, it has emerged that chaperonin 10 and chaperonin 60 can be found on the surface of various prokaryotic and eukaryotic cells, and can even be released from cells. Secreted chaperonins can interact with a variety of cell types, including leukocytes, vascular endothelial cells and epithelial cells, and activate key cellular activities such as the synthesis of cytokines and adhesion proteins. Much has been made of the high degree of sequence conservation among the chaperonins, particularly in terms of the immunogenicity of these proteins. However, different chaperonin 60 proteins can bind to different cell-surface receptors, including the Toll-like receptors, suggesting that this family of proteins cannot be treated as one biological entity and that several subfamilies may exist. Chaperonins have been implicated in human diseases on the basis of their immunogenicity. The finding that chaperonins can also induce tissue pathology suggests that they may play roles in infections and in idiopathic diseases such as atherosclerosis and arthritis.

A novel semi-automated technique for measuring inhibition of intracellular mycobacterial growth by macrophage activating factor.

We have developed a rapid, in vitro method for measuring T lymphocyte-derived macrophage activating factor (MAF) which inhibits the proliferation of Mycobacterium microti within macrophages. This MAF may be important in the control of mycobacterial disease in vivo. Because MAFs are a heterogeneous group of factors with different activities there is a need for assays which are relevant to specific macrophage effector functions. The existing assays for MAFs which are relevant to killing or inhibition of replication of mycobacteria within macrophages are currently too cumbersome or time consuming for the large scale screening required for their detection and purification. In our assay, monolayers of mouse macrophages were infected with the murine pathogen M. microti and were cultured for 6 days with MAF or control medium. The intracellular bacteria were stained with auramine-O and were quantified using epifluorescence microscopy with television image analysis. Total assay time was one-seventh that of viable count methods, and image analysis estimates of bacterial loads are quicker and more objective than visual counts.

The use of murine monoclonal antibodies without purification of antigen in the serodiagnosis of tuberculosis.

A serum diagnostic test for tuberculosis has been devised on the basis of competitive inhibition by human sera of the binding of 125I-labelled murine monoclonal antibodies (Mabs) to a solid-phase bound pressate of M. tuberculosis. Five monoclonal antibodies binding to distinct antigenic determinants of the organism were used as structural probes which conferred their stringent combining site specificities to the polyclonal mixture of human antibodies. Sera from patients but not from healthy controls competed effectively with the binding of 125I-labelled Mabs to M. tuberculosis-coated polyvinyl plates. This inhibition technique eliminated the need for elaborate purification of antigen used in previous serological methods. Some Mabs gave considerably more positive results than others. The best combination of tests used 2 Mabs and yielded a positive result in 71% of 41 patients with smear-positive pulmonary tuberculosis. This approach is applicable in principle to the serodiagnosis of other human bacterial diseases.

The TB structural genomics consortium: a resource for Mycobacterium tuberculosis biology.

The TB Structural Genomics Consortium is an organization devoted to encouraging, coordinating, and facilitating the determination and analysis of structures of proteins from Mycobacterium tuberculosis. The Consortium members hope to work together with other M. tuberculosis researchers to identify M. tuberculosis proteins for which structural information could provide important biological information, to analyze and interpret structures of M. tuberculosis proteins, and to work collaboratively to test ideas about M. tuberculosis protein function that are suggested by structure or related to structural information. This review describes the TB Structural Genomics Consortium and some of the proteins for which the Consortium is in the progress of determining three-dimensional structures.

Binding of monoclonal anti-DNA and anti-TB glycolipids to brain tissue.

The binding of human lupus anti-DNA antibodies and murine anti-mycobacterial antibodies to human cortical brain tissue sections was assessed by the indirect immunofluorescent technique. Prior adsorption of the reactive monoclonal antibodies on nuclear extracts, ss-DNA, synthetic polynucleotide polymers, histones, mycobacterial glycolipids, and bovine brain extracts abrogated the monoclonal antibodies' binding to the brain. Intermediate blocking activity was conferred also by ribonucleic components as RNP, Ro(SSA), and La(SSB). Specificity to neuronal tissue was demonstrated by failure of non-neuronal tissue extracts to abolish antibodies' reaction to the cortical tissue sections in competition assays. The anti-TB and anti-DNA antibodies seemed to compete on their binding to a common neuronal membranal epitope. These results indicate that mycobacteria share antigens with DNA and human brain tissue. Furthermore, these data support the concept that anti-DNA antibodies may play a pathogenetic role in SLE patients with neuropsychiatric involvement via crossing of a "leaky" blood brain barrier and attachment to brain tissue components.

Molecular specificity of two commercial enzyme linked immunosorbent assays for human immunodeficiency virus antigens.

Only "fair" agreement has been shown between the Abbott and DuPont enzyme linked immunosorbent assays when used for the detection of human immunodeficiency virus (HIV) antigen in serum samples from asymptomatic HIV antibody positive homosexual men. To investigate the discrepancies between the two ELISA results, further experiments were performed. The rabbit detector antibody solutions of both tests were western blotted and showed that the DuPont test was specific for p24; the Abbott detector antibody had bands for p18, p41-43, gp120 as well as p24. By using dilutions of a known amount of HIV antigen, the Abbott test could detect 20 pg/ml p24; the DuPont test could detect 30 pg/ml p24. The DuPont test was also more sensitive than the Abbott test at detecting a synthetic 104mer peptide of p24. Within the 104mer sequence two regions (294-318, 334-348 amino acids) inhibited the binding of the DuPont detector antibody, but no blocking was observed with the Abbott antibody. Although the Abbott test was slightly more sensitive at detecting HIV protein than the DuPont test, the major difference between the tests was in the molecular specificity, in that the Abbott test detected proteins other than p24. This may not be important for detecting antigen in cell culture, but it may affect the detection of antigenaemia in patients' sera.

Blind comparison of Abbott and Dupont HIV antigen ELISA tests for detecting antigenaemia in asymptomatic human immunodeficiency virus antibody positive homosexual men.

Three hundred and ninety eight serum samples from 270 human immunodeficiency virus (HIV) antibody positive asymptomatic homosexual men were tested in the Abbott and Dupont HIV antigen ELISA tests. In the Abbott test 62 (16%) of the sera were positive, according to the manufacturer's instructions, compared with 55 (14%) in the Dupont test. Twenty six sera were positive with the Abbott test but negative with the Dupont test and 19 sera were positive only by the Dupont test. Only 36 (9%) of the sera were positive in both tests. The Abbott confirmatory neutralisation test gave excellent agreement with the initial Abbott HIV antigen ELISA test; the Dupont confirmatory test was only in agreement with the initial positive Dupont antigen ELISA test in one third of the sera tested. Although the overall sensitivity of each of the two commercial assays tested was similar, the Abbott method may be preferable for clinical purposes if confirmation of an initial ELISA positive test result by neutralisation assay is required.

Increased levels of sigJ mRNA in late stationary phase cultures of Mycobacterium tuberculosis detected by DNA array hybridisation.

In order to determine which genes are involved in maintaining viability of 100-day stationary-phase bacteria and persistent bacteria after antibiotic treatment, we used a mini-DNA array to examine the transcription of 82 genes of M. tuberculosis in the 100-day stationary-phase cultures before and after rifampicin treatment. We found that the mRNA level of a sigma factor gene, sigJ, was strongly up-regulated in the late stationary-phase cultures. Other genes were also up-regulated, although to a lesser extent than sigJ. Surprisingly, after rifampicin treatment there was no significant change in sigJ expression, and most of the other 82 genes in the mini-DNA array also maintained expression, some at relatively high levels. These results suggest that SigJ may control gene expression in the quiescent state and may be an important component in the mechanisms by which M. tuberculosis survives prolonged stationary phase even in the presence of sterilising antibiotics.

Protein synthesis is shutdown in dormant Mycobacterium tuberculosis and is reversed by oxygen or heat shock.

Oxygen-limiting conditions are critical to the survival of the bacteria in tuberculosis. Mycobacterium tuberculosis can survive anaerobiosis in vitro for long periods of time only after a gradual transition to a microaerophilic stationary phase. The underlying mechanism behind stationary phase adaption needs to be elucidated. The protein profiles of Mycobacterium tuberculosis during long-term stationary phase growth and under strict anaerobic incubation were monitored by [35S]methionine labelling, SDS-PAGE and fluorography. These experiments have established that protein synthesis gradually decreased over 50 days in the long-term stationary phase cultures which were considered to be microaerophilic. There was an 80% linear decrease in the level of total protein synthesis during the first 40 days of microaerophilic growth and then the rate of protein synthesis faded quickly. For the first time we have shown that total protein synthesis shutdown occurred when bacilli were incubated under further anaerobic conditions. Viability, estimated by cfu counts, remained constant during stationary phase growth and under anaerobic incubation. Furthermore, when oxygen was introduced into the anaerobic culture, protein synthesis restarted. Also heat shock at 45 degrees C, 48 degrees C and 50 degrees C rapidly induced protein synthesis in stationary and anaerobic cultures. These data indicate that dormant bacteria shut down protein synthesis but remain responsive to specific stimuli which restore protein synthesis. In addition the dormant bacilli induced by anaerobiosis developed more heat resistance than nondormant organisms.

The detection of HIV-1 proviral DNA by PCR in clotted blood specimens.

Six hundred and ninety-two specimens, each consisting of a suspension of the residual free cells in samples of clotted blood (but not the clots themselves) from 680 patients at high risk for exposure to human immunodeficiency virus (HIV), were tested by a nested polymerase chain reaction (PCR) procedure which had been optimized to give a sensitivity of detection of one copy of HIV-1 proviral plasmid DNA, and the results were compared to those of testing for antibody to HIV on the same specimens. Fifty-one of the specimens were positive for antibody to HIV and 49 of these were also positive by the PCR; the two samples which gave discordant results were found to be PCR-positive when the test was repeated on DNA extracted from the clots themselves. Two specimens were found to be negative for antibody to HIV but were positive by the PCR (53 positive specimens in all). Direct sequencing of the PCR DNA products confirmed their specificity in all cases and demonstrated that no two patients gave the same predicted amino acid sequence for the V3 loop region. The sequences revealed both European/North American and African motifs at the crown of the V3 loop thus indicating a diversity of HIV strains in the SW Thames Region of South London. The results show that the confirmatory PCR for HIV-1 can be carried out efficiently on the same clotted blood specimens as used for routine HIV serology on patients undergoing diagnostic evaluation.