Directly repeated insertion of 9-nucleotide sequence detected in penicillin-binding protein 2B gene of penicillin-resistant Streptococcus pneumoniae

We investigated the molecular mechanism of 50 penicillin-resistant Streptococcus pneumoniae strains (penicillin: MIC, > or = 0.125 microgram/ml) having neither class A nor class B mutations in the penicillin-binding protein 2B gene (pbp2b). An analysis of the nucleotide sequences of the pbp2b genes from seven strains revealed an unique direct repeat of 9 nucleotides (TGGTATACT) between active-site serine (residue 385) and Ser-X-Asn (residues 442 to 444) motifs. The same insertion was detected in 13 strains.     

Stability of insertion sequence IS1245, a marker for differentiation of Mycobacterium avium strains

Recently a novel insertion element, IS1245, has been described and suggested for use as a probe in restriction fragment length polymorphism studies of Mycobacterium avium strains. An important issue in this context is the stability of the insertion element. We analyzed single colonies of M. avium cultures and found frequent small one- to two-band changes. However, following repeated in vitro passages over 1 year, similar one- to two-band changes were observed in the IS1245 patterns of only six M. avium strains investigated.     

Identification and characterisation of IS1383, a new insertion sequence isolated from Pseudomonas putida strain H

This is the first report of postcolumn amperometric reaction detection for capillary electrophoresis and dual-electrode detection. Bromide present in the run buffer is oxidized to bromine at the first electrode and subsequently detected at a second electrode downstream. Analytes that react with bromine cause a decrease in signal at the downstream electrode that is proportional to analyte concentration. Bromine is known to react with a variety of compounds, including thiols, thioethers, disulfides, amines, and unsaturated organic compounds. In this paper, the development of a new wire--wire on-capillary dual electrode that is well suited to bromine-based post-column reaction detection is described. System performance was evaluated using glutathione, cysteine, and methionine as test analytes. The final optimized system could be operated continuously for 24 h and was stable for day-to-day use for at least two weeks. The response for cysteine was linear from 0.5 to 20 microM with a limit of detection of approximately 80 nM.     

Target choice and orientation preference of the insertion sequence IS903

We have examined the targeting preference of the bacterial insertion element IS903 by determining the sites of insertion of a large number of transposition events into the 55-kb conjugative plasmid pOX38. Despite the large target size, all the insertions were clustered in four small distinct regions associated with conjugal DNA transfer. Within these regions, many different sites were used for insertion; however, there were a few sites that IS903 inserted into more than once. Alignment of the insertion sites showed that there was no consensus sequence within the 9-bp target duplication but that there were preferred sequences located symmetrically on either side of the target. This is consistent with target recognition by a dimer or multimer of transposase, with either sequence-specific or structure-specific interactions on both sides of the target. We show further that when one of these preferred regions was cloned into a second conjugative plasmid, pUB307, it was still a preferred target, implying that all the sequences necessary for target selection are contained within this DNA segment. Also, we observed a very strong preference for insertion in a single orientation in pUB307. We examined the possibility that either DNA replication from the origin of vegetative replication, oriV, or the origin of transfer, oriT, might determine this orientation effect. We find that reversing the direction of vegetative replication had no effect on the orientation of transposon insertions; however, reversing the direction of DNA transfer abolished the orientation effect. This supports the idea that conjugal DNA transfer imparts a polarity on the target that is sensed by the transposon.     

Identification of an insertion sequence located in a region encoding virulence factors of Streptococcus pyogenes

An insertion sequence, IS1562, was identified in a Streptococcus pyogenes strain of the clinically important M1 serotype. IS1562 is located in the mga regulon between the genes coding for the M protein and the C5a peptidase, both important virulence factors. The same or similar insertion sequences were found in most S. pyogenes strains, but the chromosomal location differed among isolates.     

Novel sequence organization and insertion specificity of IS605 and IS606: chimaeric transposable elements of Helicobacter pylori

We have used PKH26 dye, which is incorporated stably into the membrane of cells, to determine, using flow cytometry, lymphocyte proliferative responses to the antigen tetanus toxoid in fresh and cryopreserved samples. Measuring cell proliferation with this dye has advantages over either 3H-thymidine or Bromodeoxyuridine (BrdU). Whereas the existing methods measure proliferation at a single time point, PKH26 gives a cumulative measure of cell proliferation. As PKH26 is incorporated into the cell membrane, cells do not have to be permeabilised to allow dye incorporation into a cytoplasmic compartment. Most importantly, PKH26 can be used in combination with monoclonal antibodies to surface markers on mixed populations of cells, to determine the proliferation of individual subpopulations, without the need for prior cell fractionation. We also show that PKH26 can be used with similar efficacy in both fresh and cryopreserved samples. In addition since PKH26 is a cumulative measure of proliferative responses we were able to show that restimulation of the dividing population in vitro with fresh antigen presenting cells (APC) and antigen permits characterisation of a further proliferating cell population. The use of PKH26 dye in combination with cell phenotyping and measurement of cytokine production at the single cell level will prove a powerful tool for multiparameter analyses of cellular responses to antigen.     

High-frequency transposition of IS1373, the insertion sequence delimiting the amplifiable element AUD2 of Streptomyces lividans

IS1373 is the putative insertion sequence delimiting the amplifiable unit AUD2 of Streptomyces lividans. Two IS1373-derived thiostrepton-resistant transposons, Tn5492 and Tn5494, transposed into multiple sites of the S. lividans chromosome at frequencies as high as 0.4 and 1%, respectively. Hence, IS1373 is a functional insertion sequence and its unique open reading frame, insA, encodes the transposase.     

Identification and characterization of IS1411, a new insertion sequence which causes transcriptional activation of the phenol degradation genes in Pseudomonas putida

A new insertion sequence (IS element), IS1411, was identified downstream of the phenol degradation genes pheBA that originated from plasmid DNA of Pseudomonas sp. strain EST1001. According to sequence analysis, IS1411 belongs to a new family of IS elements that has recently been named the ISL3 family (J. Mahillon and M. Chandler, Microbiol. Mol. Biol. Rev. 62:725-774, 1998). IS1411 generates 8-bp duplication of the target DNA and carries 24-bp inverted repeats (IRs), highly homologous to the IRs of other IS elements belonging to this family. IS1411 was discovered as a result of insertional activation of promoterless pheBA genes in Pseudomonas putida due to the presence of outward-directed promoters at the left end of IS1411. Both promoters located on the IS element have sequences that are similar to the consensus sequence of Escherichia coli sigma70. IS1411 can produce IS circles, and the circle formation is enhanced when two copies of the element are present in the same plasmid.     

Capsular transformation of a multidrug-resistant Streptococcus pneumoniae in vivo

OBJECTIVE: To evaluate the effects of a mandibular advancement device on apneas and sleep in mild, moderate, and severe obstructive sleep apnea. DESIGN: Prospective study. SUBJECTS: Forty-four of 47 patients included. INTERVENTION: Individually adjusted mandibular advancement devices. MEASUREMENTS: Polysomnographic sleep recordings for 1 night without the device and 1 night with it, with a median of 1 day and no changes in weight, medication, or sleep position between the recordings. RESULTS: The device reduced the median obstructive apnea-hypopnea index from 11 (range, 7 to 19) to 5 (range, 0 to 17) (p<0.001) in 21 patients with mild sleep apnea, from 27 (range, 20 to 38) to 7 (range, 1 to 19) (p<0.001) in 15 patients with moderate sleep apnea, and from 53 (range, 44 to 66) to 14 (range, 2 to 32) (p<0.05) in 8 patients with severe sleep apnea. The arousal index decreased and the sleep stage patterns improved in all severity groups. Twenty-eight of 44 patients were successfully treated with an obstructive apnea-hypopnea index of below 10 and a subjective reduction in snoring. Nine of 16 patients with treatment failure still reported a reduction in snoring. The success rate correlated inversely to the disease severity (r=-0.41; p<0.01). CONCLUSIONS: A mandibular advancement device reduces apneas and improves sleep quality in patients with obstructive sleep apnea, especially in those with mild and moderate disease. A follow-up sleep recording during treatment is necessary because of the risk of silent obstructive apneas without subjective snoring with the device.     

Immunological detection of sheep experimentally infected with strains of Mycobacterium avium subspecies containing insertion sequence IS901/IS902 and a 40 kDa protein

A monoclonal antibody raised against a 40 kDa protein present in certain M. avium strains (IS901/IS902 positive) was used for developing a blocking ELISA. Sera from experimentally infected sheep were evaluated by indirect ELISA, AGID and blocking ELISA. The blocking assay proved to be highly specific for differentiation of sheep infected with different subspecies of M. avium.     

Nasopharyngeal carriage of community-acquired, antibiotic-resistant Streptococcus pneumoniae in a Zambian paediatric population

The present study was conducted to examine the response of amoeboid microglial cells in the postnatal rat brain to colchicine administration. One-day-old postnatal rats were given intraperitoneal injections of colchicine and sacrificed at 7, 14 and 21 days of age. In rats killed at 7 days age, the number of OX-42, OX-18 and ED1 positive amoeboid microglial cells was considerably reduced when compared with the control rats. At 14 and 21 days, the number of cells immunoreactive with the above antibodies was comparable to that of the control rats. The intensity of the immunoreaction with the various antibodies was also comparable in colchicine injected and control rats. When rhodamine isothiocyanate (RhIC) was administered, amoeboid microglial cells emitted a bright fluorescence in control rats as well as in colchicine-injected rats, although in the latter, the number of RhIC labelled cells was considerably reduced. With the antibody bromodeoxyuridine a large number of stained cells were observed in the control rats. On the other hand, occasional labelled cells were recognized in colchicine-injected rats. Apoptotic amoeboid microglial cells were observed in 4-day-old colchicine-injected rats. At the electron microscopic level, amoeboid microglial cells in colchicine-injected rats killed at 7 days of age showed a large number of phagosomes in their cytoplasm compared with the corresponding control rats. At 14 and 21 days, in colchicine-injected and control rats, amoeboid microglial cells did not display any noticeable differences. It is concluded from the present study that colchicine suppresses the number of amoeboid microglial cells, and that this may be attributed to the antimitotic effect of the drug as well as apoptosis induced by it; the phagocytic activity, however, was not affected. The cells returned to their normal population and morphological features once the drug was discontinued, indicating the reversible nature of the drug effect.     

The molecular mechanism of pneumolysin, a virulence factor from Streptococcus pneumoniae

In order to develop a clinically feasible gene marking approach, we have used the recently described PINCO retroviral expression system, composed of the enhanced green fluorescence protein (EGFP) cDNA driven by Moloney MLV LTR and packaged in the Phoenix amphotropic cell line. Two T, five B, one erythromyeloid and three myeloid cell lines were successfully infected with % GFP+ cells ranging from 4% to 79%, showing a lineage-dependent difference in infection susceptibility, with the myeloid cells being the least efficiently infected. We also infected normal mononuclear peripheral cells cultured in PHA and rhIL-2 for 2 d, and obtained an average of 30% GFP+ cells, all present within the CD3+ population, with CD4+ and CD8+ cells being equally infected. Finally, the tonsillar purified B population showed lower levels of infectivity (6%) whereas high susceptibility was shown by normal human umbilical vein endothelial cells (57%). Highly purified CD34+ cells were also susceptible, varying from 6% to 10% GFP+ cells. Immature myeloid/erythroid progenitors have been infected which stably expressed the GFP protein during further differentiation in culture. The GFP+ T cells were FACS-sorted rapidly upon infection, subsequently cultured and the fluorescence intensity monitored. In all cases the difference in percentage of GFP+ cells did not correlate with the percentage of S/G2/M cycling cells as determined at the moment of infection or with the expression levels of Ram-1 amphotrophic receptor. The improved safety of this retroviral system, the rapidity of the technique, the high efficiency of infection with respect to normal T lymphocytes (in this last case higher than previously reported) and the lack of need for in vitro selection make this system favourable for clinical development.     

Is Streptococcus pneumoniae a nosocomially acquired pathogen?

Streptococcus pneumoniae is most prominently a major cause of community-acquired infections of the respiratory tract, central nervous system, and bloodstream, but there is an increasing interest in its role in the epidemiology of hospital-acquired infections. Penicillin-resistant pneumococcal strains appeared 3 decades ago and now are present worldwide, often displaying multiple resistance due to antibiotic selective pressure. Horizontal spread can cause either sporadic cases or hospital outbreaks, primarily in younger children and elderly patients. Pneumococcal transmission from one patient to another can be documented by polymerase chain reaction or pulsed-field gel electrophoresis typing. Nosocomial acquisition of infection, along with pediatric age, previous hospitalization, and previous beta-lactam therapy, are the main risk factors significantly associated with penicillin-resistant pneumococcal infections. Nosocomial acquisition also is associated with higher mortality from pneumococcal disease. The importance of penicillin resistance as a risk factor significantly associated with higher mortality from pneumococcal infection is found in some studies, but not in others. Mortality from pneumococcal pneumonia is approximately the same for human immunodeficiency virus (HIV)-infected patients without acquired immunodeficiency syndrome (AIDS) as for HIV-negative subjects, but it is significantly higher in AIDS patients. Penicillin-resistant strains are involved in the vast majority of hospital outbreaks, whether presenting as clinically manifest infection or a simple colonization. Pneumococcal vaccination is recommended universally in order to lower the incidence of invasive infection, although a number of problems can limit its effectiveness.     

Multiple antibiotic resistance in Streptococcus pneumoniae

A 40-year-old Libyan male was admitted to the intensive therapy unit of Zliten Central Hospital, Libya after a road traffic accident in which he had been the driver. On physical examination he was irritable, dyspnoeic, cyanotic, had contusions and abrasions on his chest and abdomen (mainly on the left side), a lacerated wound on the forehead, a large haematoma over the left thigh and tenderness over the left side of the chest and abdomen.     

Characterization of IS1547, a new member of the IS900 family in the Mycobacterium tuberculosis complex, and its association with IS6110

Unlike classically defined insertion sequence (IS) elements, which are delimited by their inverted terminal repeats, some IS elements do not have inverted terminal repeats. Among this group of atypical IS elements, IS116, IS900, IS901, and IS1110 have been proposed as members of the IS900 family of elements, not only because they do not have inverted terminal repeats but also because they share other features such as homologous transposases and particular insertion sites. In this study, we report a newly identified IS sequence, IS1547, which was first identified in a clinical isolate of Mycobacterium tuberculosis. Its structure, insertion site, and putative transposase all conform with the conventions of the IS900 family, suggesting that it is a new member of this family. IS1547 was detected only in isolates of the M. tuberculosis complex, where it had highly polymorphic restriction fragment length polymorphism patterns, suggesting that it may be a useful genetic marker for identifying isolates of the M. tuberculosis complex and for distinguishing different strains of M. tuberculosis. ipl is a preferential locus for IS6110 insertion where there are eight known different insertion sites for IS6110. Surprisingly, the DNA sequence of ipl is now known to be a part of IS1547, meaning that IS1547 is a preferential site for IS6110 insertion.     

Primary targets of fluoroquinolones in Streptococcus pneumoniae

Mutants of wild-type Streptococcus pneumoniae IID553 with mutations in parC were obtained by selection with trovafloxacin, levofloxacin, norfloxacin, and ciprofloxacin. All of the parC mutants were cross-resistant to the selecting agents but were not resistant to gatifloxacin and sparfloxacin. On the other hand, gyrA mutants were isolated by selection with gatifloxacin and sparfloxacin. The gyrA mutants were cross-resistant to gatifloxacin and sparfloxacin but were not resistant to the other fluoroquinolones tested. These results suggest that in wild-type S. pneumoniae the primary target of trovafloxacin, levofloxacin, norfloxacin, and ciprofloxacin is topoisomerase IV, whereas the primary target of gatifloxacin and sparfloxacin is DNA gyrase.