Inactivation of polyketide synthase and related genes results in the loss of complex lipids in Mycobacterium tuberculosis H37Rv.

AIMS: Phthiocerol dimycocerosate (PDIM) waxes and other lipids are necessary for successful Mycobacterium tuberculosis infection, although the exact role of PDIM in host-pathogen interactions remains unclear. In this study, we investigated the contribution of tesA, drrB, pks6 and pks11 genes in complex lipid biosynthesis in M. tuberculosis. METHODS AND RESULTS: Four mutants were selected from M. tuberculosis H37Rv transposon mutant library. The transposon insertion sites were confirmed to be within the M. tuberculosis open reading frames for tesA (a probable thioesterase), drrB (predicted ABC transporter), pks11 (putative chalcone synthase) and pks6 (polyketide synthase). The first three of these transposon mutants were unable to generate PDIM and the fourth lacked novel polar lipids. CONCLUSIONS: Mycobacterium tuberculosis can be cultivated in vitro without the involvement of certain lipid synthesis genes, which may be necessary for in vivo pathogenicity. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of transposon mutants is a new functional genomic approach for the eventual definition of the mycobacterial 'lipidome'.

Expression, characterization and subcellular localization of the Mycobacterium tuberculosis PPE gene Rv1917c.

SETTING: The PPE gene family of Mycobacterium tuberculosis is thought to be of immunological significance. One member, Rv1917c, is highly polymorphic in clinical isolates. OBJECTIVE: To characterize Rv1917c gene polymorphism and expression, and to determine the cellular location and glycosylation status of the encoded protein. DESIGN: Tandem repeat regions of Rv1917c were amplified and sequenced to determine the molecular basis for the gene polymorphism. RT-PCR analysis was utilized to detect expression of Rv1917c mRNA in liquid cultures of M. tuberculosis H37Rv. The gene was cloned as a 3'-terminal green fluorescent protein (GFP) fusion, downstream of an acetamide-inducible promoter, and expressed in Mycobacterium smegmatis and Mycobacterium bovis BCG. The expression product was characterized in terms of cellular location and glycosylation status. RESULTS: PCR and sequence data demonstrated that variable numbers of tandem repeats within Rv1917c contribute to gene polymorphism. RT-PCR analysis demonstrated that Rv1917c mRNA is expressed in liquid cultures of M. tuberculosis H37Rv. Expression of the recombinant protein in M. smegmatis and M. bovis BCG was visualized by fluorescence microscopy and flow cytometry. A protein of the predicted size (166 kDa) was confirmed by Western blotting. Cell fractionation studies demonstrated that the recombinant protein is hydrophobic, suggestive of cell wall-association, while flow cytometric data derived from antibody binding experiments suggested that it is surface exposed. Analysis of the glycosylation status of the expressed protein failed to demonstrate glycosylation. CONCLUSION: Rv1917c mRNA is expressed in M. tuberculosis H37Rv, and Rv1917c gene polymorphism is associated with variable numbers of tandem repeats. The recombinant Rv1917c protein is surface exposed.

Imaging of the oral cavity using optical coherence tomography.

Optical coherence tomography is a new method for noninvasively imaging internal tooth and soft tissue microstructure. The intensity of backscattered light is measured as a function of depth in the tissue. Low coherence interferometry is used to selectively remove the component of backscattered signal that has undergone multiple scattering events, resulting in very high resolution images (< 15 microns). Lateral scanning of the probe beam across the biological tissue is then used to generate a 2-D intensity plot, similar to ultrasound images. This imaging method provides information that is currently unobtainable by any other means, making possible such diverse applications as diagnosis of periodontal disease, caries detection, and evaluation of restoration integrity. This chapter presents an overview of this exciting new imaging technique and its current application to dental diagnosis.

Dental optical coherence tomography: a comparison of two in vitro systems.

OBJECTIVE: To compare the imaging results obtained with two different in vitro prototype dental optical coherence tomography (OCT) systems. METHODS: Two prototypes were evaluated: an 850 nm wavelength, 700 microW OCT system with a relatively low numerical aperture (0.03) and a 1310 nm wavelength, 140 microW system with a higher numerical aperture (0.20). RESULTS: Using the 850 nm system a characteristic scattering signal was observed that correlated with the depth of a periodontal probe. There was, however, insufficient light penetration to create images with adequate resolution. Improved image quality was achieved with the 1310 nm OCT system; these images had sufficient resolution to allow identification of anatomical structures important for the diagnostic assessment of oral structures. CONCLUSIONS: These results illustrate the improvement in imaging dental structures that can be obtained with a prototype 1310 nm OCT system. The feasibility of OCT as a dental imaging technique is verified.

Optical coherence tomography: a new imaging technology for dentistry.

BACKGROUND: Optical coherence tomography, or OCT, is a new diagnostic imaging technique that has many potential dental applications. The authors present the first intraoral dental images made using this technology. METHODS: The authors constructed a prototype dental OCT system. This system creates cross-sectional images by quantifying the reflections of infrared light from dental structures interferometrically. RESULTS: We used our prototype system to make dental OTC images of healthy adults in a clinical setting. These OCT images depicted both hard and soft oral tissues at high resolution. CONCLUSIONS: OCT images exhibit microstructural detail that cannot be obtained with current imaging modalities. Using this new technology, visual recordings of periodontal tissue contour, secular depth and connective tissue attachment now are possible. The internal aspects and marginal adaptation of porcelain and composite restorations can be visualized. CLINICAL IMPLICATIONS: There are several advantages of OCT compared with conventional dental imaging. This new imaging technology is safe, versatile, inexpensive and readily adapted to a clinical dental environment.

Comparison of polarized-light propagation in biological tissue and phantoms.

We demonstrate significant differences in the propagation of polarized light through biological tissue compared with two common tissue phantoms. Depolarization of linearly and circularly polarized light was measured versus propagation distance by use of two independent measurement techniques. The measurements were performed on adipose and myocardial tissues and on tissue phantoms that consisted of polystyrene microsphere suspensions and Intralipid. The results indicate that, in contrast with results obtained in tissue phantoms, linearly polarized light survives through longer propagation distances than circularly polarized light in biological tissue.

Evaluation of optical coherence quantitation of analytes in turbid media by use of two wavelengths.

We introduce a novel method for determining analyte concentration as a function of depth in a highly scattering media by use of a dual-wavelength optical coherence tomography system. We account for the effect of scattering on the measured attenuation by using a second wavelength that is not absorbed by the sample. We assess the applicability of this technique by measuring the concentration of water in an Intralipid phantom, using a probe wavelength of 1.53 microm and a reference wavelength of 1.31 microm. The results of our study show a strong correlation between the measured absorption and the water content of the sample. The accuracy of the technique, however, was limited by the dominance of scattering over absorption in the turbid media. Thus, although the effects of scattering were minimized, significant errors remained in the calculated absorption values. More-accurate results could be obtained with the use of more powerful superluminescent diodes and a choice of wavelengths at which absorption effects are more significant relative to scattering.

Birefringence characterization of biological tissue by use of optical coherence tomography.

An improved polarization-sensitive optical coherence tomography (OCT) system is developed and used to measure birefringence in porcine myocardium tissue and produce two-dimensional birefringence mapping of the tissue. Signal-to-noise issues that cause systematic measurement errors are analyzed to determine the regime in which such measurements are accurate. The advantage of polarization-sensitive OCT systems over standard OCT systems in avoiding image artifacts caused by birefringence is also demonstrated.

Imaging of hard- and soft-tissue structure in the oral cavity by optical coherence tomography.

We have developed a prototype optical coherence tomography (OCT) system for the imaging of hard and soft tissue in the oral cavity. High-resolution images of in vitro porcine periodontal tissues have been obtained with this system. The images clearly show the enamel-cementum and the gingiva-tooth interfaces, indicating OCT is a potentially useful technique for diagnosis of periodontal diseases. To our knowledge, this is the first application of OCT for imaging biologic hard tissue.

Mapping of Birefringence and Thermal Damage in Tissue by use of Polarization-Sensitive Optical Coherence Tomography.

We demonstrate cross-sectional birefringence- and polarization-independent backscatter imaging of laser-induced thermal damage in porcine myocardium in vitro, using a polarization-sensitive optical coherence tomography system. We compare the generated images with histological sections of the tissue and demonstrate that birefringence is a more sensitive indicator of thermal damage than is backscattered light. Loss of birefringence in thermally damaged regions is quantified and shown to have significant contrast with undamaged sections of the tissue. A detailed theoretical analysis of the birefringence measurements is provided, including a calculation of the systematic errors associated with background noise, system imperfections, and tissue dichroism.

Non-gyrA-mediated ciprofloxacin resistance in laboratory mutants of Streptococcus pneumoniae.

Two strains of Streptococcus pneumoniae, M4 (NCTC 7465, type strain) and M5 (clinical isolate), and their respective ciprofloxacin-resistant mutants, M4/C1, M5/C1 and M5/C3, were evaluated. All mutants were stable after one year's storage and all grew more slowly in Brain Heart Infusion broth than the parent. The MICs of ciprofloxacin, sparfloxacin and tosufloxacin were increased for the mutants of M4, whereas the mutants of M5 were less susceptible to ciprofloxacin only. The optimal bactericidal concentration (OBC) of each quinolone for all the strains was approximately ten-fold greater than the MIC. The OBCs for the mutants were increased for ciprofloxacin, but not for the other two quinolones. The DNA synthesis IC50 values of all quinolones correlated well with the MIC of each drug. All quinolones accumulated rapidly within all five strains; 10 mM magnesium chloride decreased the concentration of quinolone accumulated, but carbonyl cyanide m-chlorophenyl hydrazone had no effect. Mutant strains M4/C1, M5/C1 and M5/C3 accumulated less quinolone than their respective parent strains. DNA sequencing of those regions of gyrA and gyrB corresponding to the quinolone resistance-determining region in other bacteria did not reveal any differences between the parent and mutant strains.

Quinolone-resistant Salmonella typhi in Viet Nam: molecular basis of resistance and clinical response to treatment.

Nalidixic acid-resistant Salmonella typhi (NARST) was first isolated in Viet Nam in 1993. Analysis of the quinolone resistance-determining region of gyrA in 20 NARST isolates by polymerase chain reaction and single-stranded conformational polymorphism yielded two novel patterns: pattern II corresponding to a point mutation at nucleotide 87 Asp-->Gly (n = 17), and pattern III corresponding to a point mutation at nucleotide 83 Ser-->Phe (n = 3). In trials of short-course ofloxacin therapy for uncomplicated typhoid, 117 (78%) of 150 patients were infected with multidrug-resistant S. typhi, 18 (15%) of which were NARST. The median time to fever clearance was 156 hours (range, 30-366 hours) for patients infected with NARST and 84 hours (range, 12-378 hours) for those infected with nalidixic acid-susceptible strains (P < .001). Six (33.3%) of 18 NARST infections required retreatment, whereas 1 (0.8%) of 132 infections due to susceptible strains required retreatment (relative risk = 44; 95% confidence interval = 5.6-345; P < .0001). We recommend that short courses of quinolones not be used in patients infected with NARST.

Contributions of individual mechanisms to fluoroquinolone resistance in 36 Escherichia coli strains isolated from humans and animals.

Twenty-eight human isolates of Escherichia coli from Argentina and Spain and eight veterinary isolates received from the Ministry of Agriculture Fisheries and Foods in the United Kingdom required 2 to > 128 micrograms of ciprofloxacin per ml for inhibition. Fragments of gyrA and parC encompassing the quinolone resistance-determining region were amplified by PCR, and the DNA sequences of the fragments were determined. All isolates contained a mutation in gyrA of a serine at position 83 (Ser83) to an Leu, and 26 isolates also contained a mutation of Asp87 to one of four amino acids: Asn (n = 14), Tyr (n = 6), Gly (n = 5), or His (n = 1). Twenty-four isolates contained a single mutation in parC, either a Ser80 to Ile (n = 17) or Arg (n = 2) or a Glu84 to Lys (n = 3). The role of a mutation in gyrB was investigated by introducing wild-type gyrB (pBP548) into all isolates; for three transformants MICs of ciprofloxacin were reduced; however, sequencing of PCR-derived fragments containing the gyrB quinolone resistance-determining region revealed no changes. The analogous region of parE was analyzed in 34 of 36 isolates by single-strand conformational polymorphism analysis and sequencing; however, no amino acid substitutions were discovered. The outer membrane protein and lipopolysaccharide profiles of all isolates were compared with those of reference strains, and the concentration of ciprofloxacin accumulated (with or without 100 microM carbony cyanide m-chlorophenylhydrazone [CCCP] was determined. Twenty-two isolates accumulated significantly lower concentrations of ciprofloxacin than the wild-type E. coli isolate; nine isolates accumulated less then half the concentration. The addition of CCCP increased the concentration of ciprofloxacin accumulated, and in all but one isolate the percent increase was greater than that in the control strains. The data indicate that high-level fluoroquinolone resistance in E. coli involves the acquisition of mutations at multiple loci.

Role of gyrA mutation and loss of OprF in the multiple antibiotic resistance phenotype of Pseudomonas aeruginosa G49.

A clinical isolate of Pseudomonas aeruginosa G48, became resistant during fluoroquinolone treatment giving rise to the post-therapy isolate, G49. To determine whether mutation in gyrA gave rise to fluoroquinolone resistance, G49 was transformed with a plasmid encoding gyrA (pNJR3-2); this reduced the MIC of fluoroquinolones for G49 two-fold. DNA sequencing of gyrA of G49 demonstrated a mutation at Thr-83, substituting with isoleucine. The outer membrane of G49 was shown to lack OprF, suggesting that loss of this protein may be involved in the multiple antibiotic resistance phenotype; however, when G49 was transformed with a plasmid encoding oprF (pRW5), expression of oprF was shown to have no effect upon the phenotype.

Induction of the Citrobacter freundii group I beta-lactamase in Escherichia coli is not dependent on entry of beta-lactam into the cytoplasm.

Induction of the cloned ampC Citrobacter freundii beta-lactamase in Escherichia coli by 6-aminopenicillanic acid was prevented by periplasmic TEM beta-lactamase in the same cell. In contrast, cytoplasmic TEM beta-lactamase did not prevent induction. This result implies that entry of the beta-lactam into the cytoplasm is not necessary for induction of ampC.