Staphylococcus aureus genetic loci impacting growth and survival in multiple infection environments

The Gram-positive bacterium Staphylococcus aureus infects diverse tissues and causes a wide spectrum of diseases, suggesting that it possesses a repertoire of distinct molecular mechanisms promoting bacterial survival in disparate in vivo environments. Signature-tag transposon mutagenesis screening of a 1520-member library identified numerous S. aureus genetic loci affecting growth and survival in four complementary animal infection models including mouse abscess, bacteraemia and wound and rabbit endocarditis. Of a total of 237 in vivo attenuated mutants identified by the murine models, less than 10% showed attenuation in all three models, emphasizing the advantage of screening in diverse disease environments. The largest gene class identified by these analyses encoded peptide and amino acid transporters, some of which were important for S. aureus survival in all animal infection models tested. The identification of staphylococcal loci affecting growth, persistence and virulence in multiple tissue environments provides insight into the complexities of human infection and on the molecular mechanisms that could be targeted by new antibacterial therapies.     

Evidence for the existence of independent chloromethane- and S-adenosylmethionine-utilizing systems for methylation in Phanerochaete chrysosporium.

O methylation of acetovanillone at 4 position by C2H3Cl and S-adenosyl[methyl-2H3]methionine was monitored in whole mycelia of Phanerochaete chrysosporium in the presence and absence of S-adenosylhomocysteine. Both the amount of the methylation product, 3,4-dimethoxyacetophenone, and the percent C2H3 incorporation into the 4-methoxyl group of the compound were determined. The results strongly suggest the presence of biochemically distinct systems for O methylation of acetovanillone utilizing S-adenosylmethionine and chloromethane, respectively, as the methyl donor. The S-adenosylmethionine-dependent enzyme is induced early in the growth cycle, with activity attaining an initial maximum after 55 h of incubation. Methylation by this enzyme is totally suppressed by 1 mM S-adenosylhomocysteine over almost the entire growth cycle. S-Adenosylmethionine-dependent O-methyltransferase activity is detectable in cell extracts, and the purification and characterization of the enzyme are described elsewhere (C. Coulter, J. T. Kennedy, W. C. McRoberts, and D. B. Harper, Appl. Environ. Microbiol. 59:706-711, 1993). The chloromethane-utilizing methylation system is absent in early growth but attains peak activity in the mid-growth phase after 72 h of incubation. The system is not significantly inhibited by S-adenosylhomocysteine at any stage of growth. No chloromethane-dependent O-methyltransferase activity is detectable in cell extract, suggesting that the enzyme is membrane bound and/or part of a multienzyme complex. Although the biochemical role of the chloromethane-dependent methylation system in metabolism is not known, one possible function could be the regeneration of veratryl alcohol degraded by the attack of lignin peroxidase.     

Effect of NHS reforms on general practitioners' referral patterns.

OBJECTIVE--To compare outpatient referral patterns in fundholding and non-fundholding practices before and after the implementation of the NHS reforms in April 1991. DESIGN--Prospective collection of data on general practitioners'' referrals to specialist outpatient clinics between June 1990 and March 1992 and detailed comparison of two time periods: October 1990 to March 1991 (phase 1) and October 1991 to March 1992 (phase 2). SETTING--10 fundholding practices and six non-fundholding practices in the Oxford region. SUBJECTS--Patients referred to consultant outpatient clinics. RESULTS--After implementation of the NHS reforms there was no change in the proportion of referrals from the two groups of practices which crossed district boundaries. Both groups of practices increased their referral rates in phase 2 of the study, the fundholders from 107.3 per 1000 patients per annum (95% confidence interval 106 to 109) to 111.4 (110 to 113) and the non-fundholders from 95.0 (93 to 97) to 112.0 (110 to 114). In phase 2 there was no difference in overall standardised referral rates between fundholders and non-fundholders. Just over 20% of referrals went to private clinics in phase 1. By phase 2 this proportion had reduced by 2.2% (1.0% to 3.4%) among the fundholders and by 2.7% (1.2% to 4.2%) among the non-fundholders. CONCLUSIONS--Referral patterns among fundholders and non-fundholders were strikingly similar after the implementation of the NHS reforms. There was no evidence that fundholding was encouraging a shift from specialist to general practice care or that budgetary pressures were affecting general practitioners'' referral behaviour.     

Effect of fundholding and indicative prescribing schemes on general practitioners' prescribing costs.

OBJECTIVE--To compare general practitioners'' prescribing costs in fundholding and non-fundholding practices before and after implementation of the NHS reforms in April 1991. DESIGN--Analysis of prescribing and cost information (PACT data; levels 2 and 3) over two six month periods in 1991 and 1992. SETTING--Oxford region. PARTICIPANTS--Three dispensing fundholding practices; five non-dispensing fundholding practices; and seven non-dispensing, non-fundholding practices. MAIN OUTCOME MEASURES--Percentage change in net cost of ingredients, number of items prescribed, average cost per item, and proportion of generic drugs prescribed after NHS reforms. RESULTS--Prescribing costs increased in all practices in the six months after the reforms. The net costs of ingredients increased among dispensing fundholders by 10.2%, among non-dispensing fundholders by 13.2%, and among non-fundholders by 18.7%. The number of items prescribed also increased in all three groups (by 5.2%, 7.5%, and 6.1% respectively). The increase in average cost per item was 4.8% for dispensing fundholders, 5.3% for non-dispensing fundholders, and 11.9% for non-fundholders. Dispensing fundholders increased the proportion of generic drugs prescribed from 26.9% to 34.5% and non-dispensing fundholders from 44.5% to 48.7%; non-fundholders showed no change (47%). Five of the eight fundholding practices made savings in their drugs budgets at the end of the first year of fundholding (range 2.9-10.7%; the three other practices overspent by up to 3.6%). All non-fundholding practices exceeded their indicative prescribing amounts (range 3.2-20.0%). CONCLUSIONS--Fundholding has helped to curb increases in prescribing costs, even among dispensing general practitioners, for whom the incentives are different. Indicative prescribing amounts for non-fundholding practices do not seem to have had the same effect.     

In vivo and in vitro models of demyelinating diseases: tropism of the JHM strain of murine hepatitis virus for cells of glial origin.

Infection of mice with the neurotropic JHM strain of murine hepatitis virus causes demyelinating lesions resulting from an infection of the oligodendroglia. This was most evident in mice inoculated intraperitoneally with JHM. Such CNS lesions were not observed in mice inoculated intraperitoneally with the MHV₃ strain. An in vitro system is described in which the rat glial RN2 cell line functions as a discriminating host for the JHM virus. Shortly after inoculation, this virus establishes a persistent infection in which there is a cyclical rise and fall in titer with an accompanying cytopathology. Furthermore, this host cell confers a thermal lability which the virus does not demonstrate in the fully permissive host cell, L-2. By comparison, infection of RN2 cells with the prototype MHV₃ is aborted immediately. In the persistent infection of RN2 cells with measles virus, Hallé strain, the cell again confers a temperature sensitivity which the virus does not possess when replicating in Vero cells.This appears to be the first instance in which a cloned cell line of glial origin determines the outcome of the infectious process, discriminating in favor of a neurotropic variant which possesses a tropism for the glia in vivo. Systems such as the one described here may now offer a specific screening procedure for selecting, identifying and characterizing the nature of neurotropic viruses.     

Alpha-amanitin-resistant D. melanogaster with an altered RNA polymerase II.

Following EMS mutagenesis we recovered a mutant of D. melanogaster that grows at concentrations of alpha-amanitin lethal to wild-type. To our knowledge this mutant represents the first example of an amanitin-resistant eucaryotic organism. The amanitin resistance of the mutant (AmaC4) is due to an alteration in its DNA-dependent RNA polymerase II, which is approximately 250 times less sensitive to inhibition by amanitin than the wild-type polymerase II whether tested in nuclei, in partially-fractionated extracts or as a highly purified enzyme. While the wild-type enzyme activity is inhibited 50% by 2.1 x 10(-8) M alpha-amanitin, inhibition of 50% of the AmaC4 RNA polymerase II activity requires a toxin concentration of 5.6 x 10(-6) M. The mutation responsible for the amanitin resistance of AmaC4 is on the X chromosome near the vermillion locus.     

Synthesis of transferrin and transferrin mRNA in bovine Sertoli cells in culture and in vivo: sequence of partial cDNA clone for bovine transferrin

Techniques were developed for generating enriched cultures of bovine Sertoli cells and indifferent supporting cells (immature Sertoli cells). The [35S]methionine and [35S]sulfate-labeled proteins secreted by cultured cells were analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and fluorography. The electrophoretic pattern of the major Sertoli cell-secreted proteins was distinct from that of the major proteins secreted by cultured peritubular cells (the predominant contaminating cell type). Five major polypeptides ranging in molecular mass from 22 kDa to 77 kDa were resolved by 2D-PAGE in reducing conditions and were assigned numbers for reference purposes. Polypeptides 1 and 2 appeared to be analogous to two rat Sertoli cell-secreted proteins, sulfated glycoprotein-1 and sulfated glycoprotein-2, because of similar molecular mass, isoelectric point, subunit composition, sulfation, and sialation characteristics. Transferrin was detected in conditioned medium by immunoprecipitation using an antibody to bovine serum transferrin. Cultured Sertoli cells isolated from prepubertal bulls secreted higher levels of transferrin than did cells isolated from infant bulls. An 850 bp cDNA corresponding to the 3' portion of bovine transferrin mRNA was cloned and sequenced. Transferrin message was shown to be present in testicular tissue isolated from infant and prepubertal bulls and it increased as bulls matured. Levels of testicular transferrin mRNA were subsequently shown to correlate with daily sperm production in yearling beef bulls.     

Ontogeny of angiotensin II type 1 receptor and cytochrome P450(c11) in the sheep adrenal gland

In the present study we investigated the ontogeny of the expression of the type 1 angiotensin receptor (AT(1)R mRNA) and the zonal localization of AT(1)R immunoreactivity (AT(1)R-ir) and cytochrome P450(c11) (CYP11B-ir) in the sheep adrenal gland. In the adult sheep and in the fetus from as early as 90 days gestation, intense AT(1)R-ir was observed predominantly in the zona glomerulosa and to a lesser extent in the zona fasciculata, and it was not detectable in the adrenal medulla. AT(1)R mRNA decreased 4-fold between 105 days and 120 days, whereas AT(1)R mRNA levels remained relatively constant between 120 days and the newborn period. In contrast, both in the adult sheep and in the fetal sheep from as early as 90 days gestation, intense CYP11B-ir was consistently detected throughout the adrenal cortex and in steroidogenic cells that surround the central adrenal vein. In conclusion, we speculate that the presence of AT(1)R in the zona fasciculata, and the higher levels of expression of AT(1)R at around 100 days gestation, may suggest that suppression of CYP17 is mediated via AT(1)R at this time. The abundant expression of AT(1)R-ir and CYP11B-ir in the zona glomerulosa of the fetal sheep adrenal gland would also suggest that lack of angiotensin II stimulation of aldosterone secretion is not due to an absence of AT(1)R or CYP11B in the zona glomerulosa.     

Quantitation of Bacteroides forsythus in subgingival plaque comparison of immunoassay and quantitative polymerase chain reaction

Our objective was to compare three methods (enzyme-linked immunosorbent assay [ELISA], endpoint and quantitative polymerase chain reaction [E-PCR and Q-PCR]) for detection and quantitation of Bacteroides forsythus in 56 plaque samples from seven subjects with progressive periodontal disease. Samples collected in buffer were pelleted and resuspended in 500 microl of water. Fifty microl aliquots were removed for an ELISA performed on bacteria or plaque immobilized on 96-well plates and probed with B. forsythus specific antibody. An occurrence of 3.7+/-0.6 x 10(4) or more bacteria were detected by ELISA in pure culture; 26 of 54 plaque samples were positive, two samples could not be analyzed. Samples for PCR were autoclaved for 10 min prior to use. The detection level of E-PCR using primers specific for B. forsythus 16S rRNA was 200 cells and 42 out of 56 samples were positive based on ethidium bromide stained agarose gels. Q-PCR using the same primers combined with a nested fluorescent oligonucleotide probe detected 10+/-0.32 bacteria in pure culture; 43 of 56 plaque samples were positive. The ELISA and Q-PCR obtained identical results with 36 of the 54 samples assayed; there were one false positive and 17 false negative ELISA results using Q-PCR as standard. The positive proportions of plaque samples were almost the same for E-PCR and Q-PCR. We conclude that the PCR methods are more appropriate for a multicenter study because of greater sensitivity and convenience of sample transportation from clinics to a central laboratory.