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.     

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.     

Human complement regulators: a major target for pathogenic microorganisms

The C3 convertases of the human complement system are controlled by fluid-phase and membrane proteins in the RCA (regulators of complement activation) family. Accumulated data show that many pathogenic microorganisms interact with these complement regulators. Recent advances in this field include determination of the crystal structure of the binding domains in the measles virus receptor CD46 and identification of a CD46 transgenic mouse line that is sensitive to measles virus. Moreover, recent findings support the hypothesis that pathogenic bacteria binding fluid-phase RCA proteins exploit these proteins to escape complement attack. These studies provide novel insight into the interplay between pathogens and the innate immune system and may have implications for the plans to use animals expressing an RCA protein for xenotransplantation.     

Genomics of pathogenic bacteria

The general principles of structural and functional organization of genomes in pathogenic bacteria are considered. Main data on the specific features of genomes of Chlamydia trachomatis, Rickettsia prowazekii, Treponema pallidum, Helicobacter pylori, Haemophilus influenzae, Neisseria meningitidis, Vibro cholerae and pathogenic strains of Escherichia coli are summarized. Particular attention is paid to the problems of genetic control of pathogenicity, intraspecies variations in bacterial genomes, to the environmental and evolutionary meaning of horizontal gene transfer. Whether methods for genotyping bacterial strains can be used is discussed.     

Detection of pathogenic bacteria by magneto-immunoassays: a review

Magnetic particle-based immunoassays are widely used in microbiology-related assays for both microbial capture, separation, analysis, and detection. Besides facilitating sample operation, the implementation of micro-to-nanometer scale magnetic beads as a solid support potentially shortens the incubation time (for magnetic immuno capture) from several hours to less than an hour. Analytical technologies based on magnetic beads offer a rapid, effective and inexpensive way to separate and concentrate the target analytes prior to detection. Magneto-immuno separation uses magnetic particles coated with specific antibodies to capture target microorganisms, bear the corresponding antigens, and subsequently separate them from the sample matrix in a magnetic field. The method has been proven effective in separating various types of pathogenic bacteria from environmental water samples and in eliminating background interferences. Magnetic particles are often used to capture target cells (pathogenic bacteria) from samples. In most commercially available assays, the actual identification and quantitation of the captured cells is then performed by classical microbiological assays. This review highlights the most sensitive analytic methods (i.e., long-range surface plasmon resonance and electrochemical impedance spectroscopy) to detect magnetically tagged bacteria in conjunction with magnetic actuation.     

Population structure of pathogenic bacteria revisited

This minireview summarizes the historical development of bacterial population genetic concepts since the early 1980s. Initially multilocus enzyme electrophoresis was used to determine population structures but this technique is poorly portable between laboratories and was replaced in 1998 by multilocus sequence typing. Diverse population structures exist in different bacterial species. Two distinctive structures are described in greater detail. "Young" organisms, such as Yersinia pestis, have evolved or undergone a severe bottleneck in recent millennia and have not yet accumulated much sequence diversity. "genoclouds" in subgroup III Neisseria meningitidis arise because of the accumulation of diversity due to herd immunity, which is then purified during subsequent epidemic spread.     

Immunosuppressive effects of pathogenic gram-negative bacteria

The review deals with the Immunosuppressive effects of virulent gram-negative bacteria (Salmonella, Shigela, Pseudomonas aeruginosa), with the importance of these effects for the bacteria to survive in the infected body. The above bacteria affect the immunity system in a different way, yet have common features. They are characterized by the occurrence of endotoxin shock, by the suppression of the phagocytic system and cell-mediated immunity. A significant role in suppressing a cellular immune response is played by the lipopolysaccharide of virulent bacteria that greatly differs from that of nonvirulent strains. The immunosuppressive activity of the bacteria and their lipopolysaccharide is closely related to their virulent properties.     

Neurofilament light as an immune target for pathogenic antibodies

Antibodies to neuronal antigens are associated with many neurological diseases including paraneoplastic neurological disorders, epilepsy, amyotrophic lateral sclerosis and multiple sclerosis. Immunization with neuronal antigens such as neurofilament light (NF‐L), a neuronal intermediate filament in axons, has been shown to induce neurological disease and spasticity in mice. Also, although antibodies to NF‐L are widely used as surrogate biomarkers of axonal injury in amyotrophic lateral sclerosis and multiple sclerosis, it remains to be elucidated if antibodies to NF‐L contribute to neurodegeneration and neurological disease. To address this, we examined the pathogenic role of antibodies directed to NF‐L in vitro using spinal cord co‐cultures and in vivo in experimental autoimmune encephalomyelitis (EAE) and optic neuritis animal models of multiple sclerosis. Here we show that peripheral injections of antibodies to NF‐L augmented clinical signs of neurological disease in acute EAE, increased retinal ganglion cell loss in experimental optic neuritis and induced neurological signs following intracerebral injection into control mice. The pathogenicity of antibodies to NF‐L was also observed in spinal cord co‐cultures where axonal loss was induced. Taken together, our results reveal that as well as acting as reliable biomarkers of neuronal damage, antibodies to NF‐L exacerbate neurological disease, suggesting that antibodies to NF‐L generated during disease may also be pathogenic and play a role in the progression of neurodegeneration.     

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.     

Multivalent artificial opsonin for the recognition and phagocytosis of Gram-positive bacteria by human phagocytes

Hospital-acquired infections (HAIs) remain a leading cause of death in the United States. Unfortunately, treatment of HAIs is complicated by the emergence of antibiotic-resistant bacterial strains. In an effort to enhance the body's natural immune response to infection, we have developed an artificial opsonin to promote the recognition, phagocytosis, and destruction of pathogenic bacteria by human phagocytes. The artificial opsonin is constructed from multivalent conjugates of poly(L-lysine)-graft-poly(ethylene glycol) with vancomycin and human IgG-Fc. Our approach utilizes vancomycin's inherent ability to bind to D-Ala-D-Ala terminated peptides present in the cell wall of Gram-positive bacteria. Here, we show that conjugation of vancomycin to PLL-g-PEG prevents its action as an antibiotic and allows vancomycin to function solely as a recognition molecule. Human IgG-Fc antibody fragment serves as a phagocyte recognition molecule and is recognized by the Fcγ cell surface receptors expressed on professional human phagocytes. Using flow cytometry, we found that a polysaccharide-encapsulated, methicillin-resistant strain of Staphylococcus epidermidis is efficiently recognized by the artificial opsonin (nearly 100% of cells were opsonized) and that opsonin binding is specific since it can be inhibited by the soluble cell wall peptide analog acetyl-Lys-D-Ala-D-Ala. Opsonization of S. epidermidis resulted in an approximate 2-fold increase in phagocytosis by a human neutrophil cell line. Notably, Enterococcus faecalis VanB, a bacterial strain with inducible vancomycin resistance, was used to show that the artificial opsonin does not unintentionally induce antibiotic resistance mechanisms.     

Phagocytosis and killing of bacteria by professional phagocytes and dendritic cells

Dendritic cells (DC) represent a class of professional antigen-presenting cells whose primary function is to alert the immune system, not to clear invading microorganisms. The objective of our study was to compare the abilities of polymorphonuclear neutrophilic leukocytes (PMN), monocytes, monocyte-derived macrophages (MDM), monocyte-derived immature DC (imDC), and mature DC (maDC) to ingest and destroy Staphylococcus aureus and Escherichia coli. Acridine orange staining and fluorescence microscopy demonstrated that MDM, followed by monocytes, imDC, and PMN, internalized bacteria well but that maDC exhibited less pronounced phagocytic activity. PMN, monocytes, and MDM exhibited a much higher capacity to kill ingested bacteria than both imDC and maDC. In summary, these data are in agreement with the generally accepted idea that different types of leukocytes fulfill specialized tasks in antigen presentation and killing of pathogens.     

Survival of pathogenic bacteria in crude oil

The survival of three strains of Staphylococcus aureus and one strain each of Pseudomonas aeruginosa, Bacillus subtilis and Clostridium sporogenes has been investigated in samples of seven different crude oils obtained from Alberta oil fields. One strain each of Staphylococcus aureus, Pseudomonas aeruginosa, Clostridium sporogenes and Bacillus subtilis survived for longer than three years in three of the seven crude oils tested. The majority (four out of six) of the species of test micro-organisms remained viable for over two years in six out of the seven crude oils tested. Paraffinic or asphaltic-naphthenic base crude oils were better suited for the survival of the bacteria employed in these tests than were naphthenic-paraffinic or aromatic-paraffinic base oils. Only Clostridium sporogenes survived longer than three days in one of the crude oils (No. 7) which contained considerably more aromatic material than any of the other oils tested. The public health implications of the experimental results are discussed.     

Multiple sample flow through immunomagnetic separator for concentrating pathogenic bacteria

The standard method of immunomagnetic separation for isolating pathogenic bacteria from food and environmental matrices processes 1 ml volumes. Pathogens present at low levels (<0.5 pathogenic bacteria/g) will not be consistently detected by this method. Here a multiple sample flow through immunomagnetic separator has been designed and tested to process large volume samples (50 to 250 ml). Preliminary results show >97% recovery of polydisperse magnetic particles (diameter range 1 to 8 microm) containing 29-33% w/w Fe3O4 content. Between 70 and 130 times more of the pathogenic bacteria Escherichia coli O157 is recovered from PBS compared with the standard 1 ml method. Also, the recovery of E. coli O157 from beef mince homogenates, after a 4 h incubation at 42 degrees C, is between 80 and 180 times higher than the standard 1 ml method.     

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.     

Recovery of pathogenic bacteria from cerebrospinal fluid.

We studied the conditions necessary for optimal recovery of bacteria from cerebrospinal fluid. Our results indicated that Streptopcoccus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae can be quantitatively recovered in the sediment after centrifugation at 1,500 X g for 15 min. Equivalent numbers of bacteria were recovered by centrifugation or filtration of antibiotic-free cerebrospinal fluid; however, bacterial recovery by filtration was less effective with antibiotic-supplemented cerebrospinal fluid.     

Mucosal immunology of vaccines against pathogenic nasopharyngeal bacteria

The introduction of Haemophilus influenzae type b conjugate vaccines during the 1990s was followed by dramatic decreases both in the incidence of Haemophilus influenzae type b related invasive disease and in nasopharyngeal carriage of the organism. The extent of this effect has been influenced by the fact that Haemophilus influenzae type b conjugate vaccines reduce nasopharyngeal carriage and induce herd immunity. Based on the success of Haemophilus influenzae type b conjugate vaccines, chemical conjugation has been applied to the development of pneumococcal and meningococcal polysaccharide conjugate vaccines. Evidence has begun to accumulate that these new polysaccharide based conjugate vaccines can also reduce nasopharyngeal carriage and can induce immune responses at the local mucosal level, which may be responsible for these effects. This article reviews recent studies on mucosal immune responses induced by polysaccharide based vaccines and some protein vaccine antigens against several pathogenic nasopharyngeal bacteria, and discusses the mechanisms and functions of these immune responses that may help our understanding of mucosal immune responses to both immunisation and infection.