Adenoid reservoir for pathogenic biofilm bacteria

Biofilms of pathogenic bacteria are present on the middle ear mucosa of children with chronic otitis media (COM) and may contribute to the persistence of pathogens and the recalcitrance of COM to antibiotic treatment. Controlled studies indicate that adenoidectomy is effective in the treatment of COM, suggesting that the adenoids may act as a reservoir for COM pathogens. To investigate the bacterial community in the adenoid, samples were obtained from 35 children undergoing adenoidectomy for chronic OM or obstructive sleep apnea. We used a novel, culture-independent molecular diagnostic methodology, followed by confocal microscopy, to investigate the in situ distribution and organization of pathogens in the adenoids to determine whether pathogenic bacteria exhibited criteria characteristic of biofilms. The Ibis T5000 Universal Biosensor System was used to interrogate the extent of the microbial diversity within adenoid biopsy specimens. Using a suite of 16 broad-range bacterial primers, we demonstrated that adenoids from both diagnostic groups were colonized with polymicrobial biofilms. Haemophilus influenzae was present in more adenoids from the COM group (P = 0.005), but there was no significant difference between the two patient groups for Streptococcus pneumoniae or Staphylococcus aureus. Fluorescence in situ hybridization, lectin binding, and the use of antibodies specific for host epithelial cells demonstrated that pathogens were aggregated, surrounded by a carbohydrate matrix, and localized on and within the epithelial cell surface, which is consistent with criteria for bacterial biofilms.     

Bioconjugated nanoparticles for attachment and penetration into pathogenic bacteria

As an antimicrobial agent, silver nanoparticles functionalized with both bacitracin A and polymyxin E (AgNPs-BA&PE) were designed and synthesized with complementary antibacterial functions to act against gram-positive and gram-negative bacteria. AgNPs-BA&PE could easily get attached and penetrate into the bacterial cell membrane through surface-immobilized BA and PE with a membrane target, resulting in up to 10-fold increase in the antibacterial activity, without the emergence of bacterial resistance. Analysis of the antimicrobial mechanism confirmed that the synthesized nanoparticles caused disorganization of the bacterial cytomembrane and leakage of cytoplasmic contents. This antimicrobial agent with better biocompatibility can promote healing of infected wounds, and has promising and useful applications in biomedical devices and antibacterial control systems.     

Actinic apparatus of phagocytes is a target for pathogenic bacteria

The interaction between pathogenic microorganisms and eukaryotic cells is a topical area of research in microbiology and molecular medicine. This problem has been the subject of much study both in this country and abroad since early nineties; legionella, listeria, clostridium and other bacteria pathogenic to man have been used as model organisms. The results of the authors' research and literature data analysis underlie the hypothesis of the important role of bacterial enzymes, which modify components of eukaryotic cell actinic apparatus, in the virulence of infectious pathogens. Change in the activity of actinic cytoskeleton under the influence of microbe products has been demonstrated to be an effective mechanism, promoting bacterial proliferation in human organs and tissues. Here actinic apparatus components acts as a target for both "intracellular" and "extracellular" pathogens.     

Transferrins and heme-compounds as iron sources for pathogenic bacteria.

The low concentration of free iron in body fluids creates bacteriostatic conditions for many microorganisms and is therefore an important defense factor of the body against invading bacteria. Pathogenic bacteria have developed several mechanisms for acquiring iron from the host. Siderophore-mediated iron uptake involves the synthesis of low molecular weight iron chelators called siderophores which compete with the host iron-binding glycoproteins lactoferrin (LF) and transferrin (TF) for iron. Other ways to induce iron uptake, without the mediation of siderophores, are the possession of outer membrane protein receptors that actually recognize the complex of TF or LF with iron, resulting in the internalization of this metal, and the use of heme-compounds released into the circulation after lysis of erythrocytes. In this review, the nonsiderophore-mediated iron-uptake systems used by certain pathogenic bacteria are emphasized. The possible contribution of these iron-uptake systems to the virulence of pathogens is also discussed.     

HIV抗体纳米免疫磁珠快速检测试剂的研制

目的 研制一种用纳米免疫磁珠层析技术用以检测HIV抗体的快速诊断试剂.方法 运用碳二亚（EDC）将重组的HIV抗原gp41、gp36偶联到200 nm的超顺磁纳米颗粒上,在硝酸纤维素（NC）膜上包被gp41、gp36抗原,制备成免疫层析检测卡,然后对检测卡进行性能分析评估.结果 对HIV抗体国家参考品血清盘（胶体金类）检测,符合要求;对20份HIV抗体阳性和600份抗体阴性临床血清检测,灵敏度为100%,特异性为98.5%.检测卡室温保存12个月性能稳定.结论 研制出一种纳米免疫磁珠HIV抗体检测试剂,具有检测简便、快速、稳定性好和适于现场检测等特点.     

Genomic evidence for antibiotic resistance genes of actinomycetes as origins of antibiotic resistance genes in pathogenic bacteria simply because actinomycetes are more ancestral than pathogenic bacteria

Although in silico analysis have suggested that the antibiotic resistance genes in actinomycetes appear to be the origins of some antibiotic resistance genes, we have shown that recent horizontal transfer of antibiotic resistance genes from actinomycetes to other medically important bacteria have not taken place. Although it has been speculated in Benveniste and Davies' attractive hypothesis that antibiotic resistance genes of actinomycetes are origins of antibiotic resistance genes in pathogenic bacteria because the actinomycetes require mechanisms such as metabolic enzymes (encoded by the antibiotic resistance genes) to degrade the antibiotics they produce or to transport the antibiotics outside the bacterial cells, this hypothesis has never been proven. Both the phylogenetic tree constructed using 16S rRNA gene sequences and that constructed using concatenated amino acid sequences of 15 housekeeping genes extracted from 90 bacterial genomes showed that the actinomycetes is more ancestral to most other bacteria, including the pathogenic Gram-negative bacteria, Gram-positive bacteria, and Chlamydia species. Furthermore, the tetracycline resistance gene of Bifidobacterium longum is more ancestral to those of other pathogenic bacteria and the actinomycetes, which is in line with the ancestral position of B. longum. These suggest that the evolution of antibiotic resistance genes of antibiotic-producing bacteria in general parallels the evolution of the corresponding bacteria. The ancestral position of the antibiotic resistance genes in actinomycetes is probably unrelated to the fact that they produce antibiotics, but simply because actinomycetes are more ancestral than pathogenic bacteria.     

HIV抗体纳米免疫磁珠快速检测试剂的研制

目的 研制一种用纳米免疫磁珠层析技术用以检测HIV抗体的快速诊断试剂.方法 运用碳二亚(EDC)将重组的HIV抗原gp41、gp36偶联到200 nm的超顺磁纳米颗粒上,在硝酸纤维素(NC)膜上包被gp41、gp36抗原,制备成免疫层析检测卡,然后对检测卡进行性能分析评估.结果 对HIV抗体国家参考品血清盘(胶体金类)检测,符合要求;对20份HIV抗体阳性和600份抗体阴性临床血清检测,灵敏度为100%,特异性为98.5%.检测卡室温保存12个月性能稳定.结论 研制出一种纳米免疫磁珠HIV抗体检测试剂,具有检测简便、快速、稳定性好和适于现场检测等特点.     

HIV抗体纳米免疫磁珠快速检测试剂的研制

目的 研制一种用纳米免疫磁珠层析技术用以检测HIV抗体的快速诊断试剂.方法 运用碳二亚(EDC)将重组的HIV抗原gp41、gp36偶联到200 nm的超顺磁纳米颗粒上,在硝酸纤维素(NC)膜上包被gp41、gp36抗原,制备成免疫层析检测卡,然后对检测卡进行性能分析评估.结果 对HIV抗体国家参考品血清盘(胶体金类)检测,符合要求;对20份HIV抗体阳性和600份抗体阴性临床血清检测,灵敏度为100%,特异性为98.5%.检测卡室温保存12个月性能稳定.结论 研制出一种纳米免疫磁珠HIV抗体检测试剂,具有检测简便、快速、稳定性好和适于现场检测等特点.     

A fermenter dialysis cultivation technic for encapsulated pathogenic bacteria. II. Group B streptococci

For mass cultivation of group B streptococci (B-III and B-variant streptococci), a fermenter dialysis culture technique is described and compared to conventional shaking culture and fermenter batch culture techniques. The influence of two kinds of cultivation media on the bacterial yield is demonstrated. The growth rate of the bacteria and the yield of the microbes is higher in modified POPE medium than that observed with N?hrbouillon I. The type-specific polysaccharide of B-III-streptococci prepared by phenol-water extraction followed by gel-chromatography can be used as a screening antigen for the production of monoclonal antibodies against B-III-streptococci.     

Novel F0F1-ATPase molecular motor biosensors for rapid detection of foodborne pathogenic bacteria

F₀F₁-ATPase molecular motor biosensors were constructed by connecting specific probes to F₀F₁-ATPase’s ε-subunit through the avidin–biotin system. The test sample and negative sample were combined with the biosensors, respectively, and the DNA of the pathogenic bacteria can be tested by the comparison of the production of catalytically synthesized ATP after 10 min. The amount of synthesized ATP can be indirectly determined by the fluorescence intensity of luciferase–luciferin testing reagent. Through the comparison of the detection results of actual samples using three different methods, it was found that the method described in this study showed excellent agreement with the traditional detection method and polymerase chain reaction (PCR) detection method. The results also indicated that the F₀F₁-ATPase molecular motor biosensor could specifically detect bacterial DNA at a very low concentration level, and it would be a convenient, quick, and promising tool for pathogens detection.     

Multiple selective media for the isolation of anaerobic bacteria from clinical specimens.

Using fresh clinical material, a comparison of a number of anaerobic selective media was made. For Gram-negative anaerobes nalidixic acid tween agar (NAT), neomycin agar (NA), and neomycin-vancomycin agar (NVA) all performed equally well. Kanamycin-containing media were more inhibitory to all Gram-negative anaerobes other than Bacteroides fragilis and B. melaninogenicus. When the recovery of Gram-positive anaerobes was examined NAT performed better than any of the other selective media used. No single selective medium could recover all anaerobes. Better isolation was achieved using a combination of two selective media (the best combinations being NAT and NVA or NAT and NA). Only a combination of three selective media gave the maximum recovery of anaerobes in this study (NAT, NVA, and NA or KA).     

Techniques for measurement of blood flow through intact vessels

Flowmeters which operate by coupling a field through a blood vessel and measuring resultant externally-available velocity-dependent variables are particularly attractive, since they are applicable in chronic animal preparations, in humans where a vessel is exposed at surgery, and conceivably through the unopened skin for measurement of flow through superficial vessels. Two types of flowmeter which operate in this way are the electro-magnetic induction flowmeter and the ultrasonic flowmeter. This paper will discuss the theoretical and practical applications and limitations of these, emphasizing the ultrasonic flow-measuring techniques, since excellent reviews and critical appraisals of electro-magnetic flowmeters are readily available in the literature.     

PCR primers and probes for the 16S rRNA gene of most species of pathogenic bacteria, including bacteria found in cerebrospinal fluid.

A set of broad-range PCR primers for the 16S rRNA gene in bacteria were tested, along with three series of oligonucleotide probes to detect the PCR product. The first series of probes is broad in range and consists of a universal bacterial probe, a gram-positive probe, a Bacteroides-Flavobacterium probe, and two probes for other gram-negative species. The second series was designed to detect PCR products from seven major bacterial species or groups frequently causing meningitis: Neisseria meningitidis, Haemophilus influenzae, Streptococcus pneumoniae, S. agalactiae, Escherichia coli and other enteric bacteria, Listeria monocytogenes, and Staphylococcus aureus. The third series was designed for the detection of DNA from species or genera commonly considered potential contaminants of clinical samples, including cerebrospinal fluid (CSF): Bacillus, Corynebacterium, Propionibacterium, and coagulase-negative Staphylococcus spp. The primers amplified DNA from all 124 different species of bacteria tested. Southern hybridization testing of the broad-range probes with washes containing 3 M tetramethylammonium chloride indicated that this set of probes correctly identified all but two of the 102 bacterial species tested, the exceptions being Deinococcus radiopugnans and Gardnerella vaginalis. The gram-negative and gram-positive probes hybridized to isolates of two newly characterized bacteria, Alloiococcus otitis and Rochalimaea henselii, as predicted by Gram stain characteristics. The CSF pathogen and contaminant probe sequences were compared with available sequence information and with sequencing data for 32 different species. Testing of the CSF pathogen and contaminant probes against DNA from over 60 different strains indicated that, with the exception of the coagulase-negative Staphylococcus probes, these probes provided the correct identification of bacterial species known to be found in CSF.     

Limitations of flow cytometry for the specific detection of bacteria in mixed populations

Flow immunofluorescence (FIF) techniques were established for the specific detection of the bacteria Escherichia coli, Legionella pneumophila and Bacillus anthracis spores after staining with fluorescein-conjugated antibacterial antibody. For each bacterial type, a comparison was made of gating on narrow forward angle (NFA) light scatter and on the red fluorescence (Red Flu) signal available from staining with the nucleic acid dye propidium iodide. No universal gating method was found, since Bacillus spores did not take up propidium iodide and only a part of the Legionella population gave detectable NFA scatter signals. The efficiency of detecting bacteria stained with antibody remained constant with differing concentrations of the specific bacterium, and the estimate of the count for specific bacteria expressed as a fraction of the total cytometer count fell sharply with bacterial concentration. This effect was apparently due to cytometer noise inherent in the high sensitivity of detection needed for particles as small as these bacteria. The noise did not originate in the photomultipliers and was evidently the result either of light scatter from sub-micron particles in the sheath fluid or scatter from optical components. Part of the noise could be removed by selective gating, but there remained a noise component overlapping with the NFA scatter and Red Flu signals from the heterologous bacteria, i.e., those not stained with specific antibody. In consequence, at the low bacterial concentrations used no meaningful cytometer count could be obtained for the excess of the unstained bacteria and the proportion of specific bacteria in the mixed population could not, therefore, be calculated.     

Stem cell collection using the dideco excel continuous flow blood cell separator: parameters for optimal stem cell collection timing

This study evaluates stem cell collection procedures performed with the Dideco Excel blood cell separator, with particular attention given to yields and separator collection efficiencies. Patients' blood precounts and yield parameters related to the harvest capacity of the collection system were investigated. Fifty-five collection procedures were analyzed in 32 patients suffering from hematological malignancies and solid tumors and mobilized with chemotherapy plus G-CSF. The median blood volume processed in each procedure was 15.8 liters (12-19.750), with a blood flow rate of 70 ml/min. Patients had the following median blood precount value: NC 7.81x10(9)/L, CD34+ cells 49.08x10(3)/ml. Leukapheresis procedures gave the following yields: NC 14.95x10(9), MNC 10.83x10(9), CD34+ cells 4.37x10(6); yields/kg, NC 0.21x10(9)kg, MNC 0. 15x10(9)/kg CD34+ cells 4.26x10(6)/kg. Procedures show the following collection efficiencies: NC 10.79%, MNC 29.06%, CD34+ 42.33%, PLT 26.5%. The RBC (red blood cell) contamination of the product was (median value) 20.9 ml for each procedure, and for platelets 1.76x10(11) per procedure. The CD34+ cell precounts strongly correlated with the CD34+ yields/kg (r=0.82. p=0.000). Furthermore the NC and MNC precounts correlated with the CD34+ yields/kg but only the MNC precount correlation is notable (r=0.57, p=0.000). The logistic regression analysis shows that CD34+ (p=0.008) but not NC (po=0.14), MNC (p=0.09), or PLT (p=0.53) precounts significantly influenced the collection of a sufficient dose of CD34+ cells for transplantation (> or =2.5x10(6)/kg). Eleven of the thirty-two patients have been transplanted till now, and all had a prompt and lasting trilineage engraftment NC >1x10(9)/L on day 12 (10-17). Our data show that the collection system analyzed in this report is able to collect large amounts of progenitor cells, harvesting >2.5x10(6)/kg CD34+ cells with a single procedure in 68.8% of patients and assuring complete recovery after stem cell transplantation.     

Long target droplet polymerase chain reaction with a microfluidic device for high-throughput detection of pathogenic bacteria at clinical sensitivity

In this article we present a long target droplet polymerase chain reaction (PCR) microsystem for the amplification of the 16S ribosomal RNA gene. It is used for detecting Gram-positive and Gram-negative pathogens at high-throughput and is optimised for downstream species identification. The miniaturised device consists of three heating plates for denaturation, annealing and extension arranged to form a triangular prism. Around this prism a fluoropolymeric tubing is coiled, which represents the reactor. The source DNA was thermally isolated from bacterial cells without any purification, which proved the robustness of the system. Long target sequences up to 1.3 kbp from Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa have successfully been amplified, which is crucial for the successive species classification with DNA microarrays at high accuracy. In addition to the kilobase amplicon, detection limits down to DNA concentrations equivalent to 10² bacterial cells per reaction were achieved, which qualifies the microfluidic device for clinical applications. PCR efficiency could be increased up to 2-fold and the total processing time was accelerated 3-fold in comparison to a conventional thermocycler. Besides this speed-up, the device operates in continuous mode with consecutive droplets, offering a maximal throughput of 80 samples per hour in a single reactor. Therefore we have overcome the trade-off between target length, sensitivity and throughput, existing in present literature. This qualifies the device for the application in species identification by PCR and microarray technology with high sample numbers. Moreover early diagnosis of infectious diseases can be implemented, allowing immediate species specific antibiotic treatment. Finally this can improve patient convalescence significantly.