The Antibiotic Epidemic Antibiotic Resistance

Antibiotic Resistance: Surviving An Uncertain Future

Antibiotic use can damage and weaken a healthy immune system and our reliance on them has been a double-edged sword. In fact, there are many, many powerful plant-based antimicrobials, scientifically tested, that can step up to the plate and help us face the growing threat of resistant bacteria. And you'll find them in this new eBook: The Antibiotic Epidemic: How to Fight Superbugs and Emerging Bacteria with Miracles from Mother Earth. This Ebook Shows You The Many Powerful Plant-based Antimicrobials And Provides Recipes To Help Diminish The Need For Antibiotics. ebooThis can be your guide during the coming antibiotic apocalypse.

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Antibiotic Resistance Plasmids

Plasmids were first discovered in Japan just after World War II, inhabiting the bacterium Shigella, which causes dysentery. The type of dysentery due to bacteria was originally treated with sulfonamides, the earliest type of antibiotic. Suddenly, strains of Shigella appeared that were resistant to sulfonamide treatment. The genes for resistance to sul-fonamide proved to reside on plasmids, rather than the bacterial chromosome. Plas-mids that confer antibiotic resistance are called R-plasmids or R-factors (Fig. 16.10). Worse, the plasmids carrying the sulfonamide resistance genes were able to transfer copies of themselves from one bacterial cell to another. Consequently, the sulfon-amide resistance spread rapidly from Shigella to Shigella. Although the resistance plasmid allowed the Shigella to survive, transferable antibiotic resistance is highly dangerous from the human medical viewpoint. By 1953, the year Watson and Crick discovered the double helix, 80 percent of the...

Mechanisms of Antibiotic Resistance

Antibiotic resistant mutants of bacteria may be easily isolated in the laboratory. However, the mechanism of resistance in such chromosomal mutants is usually quite distinct from that of plasmid-borne resistance. The chromosomal mutations usually alter the cell component that is the target of antibiotic action, which often causes detrimental side effects. Plasmid-borne resistance generally avoids altering vital cell components. Instead the antibiotic may be inactivated or pumped out of the cell. Occasionally plasmids do provide an altered (but still functional) target component. Several of the resistance genes originally found on plasmids have been used in genetic engineering. Antibiotic resistance allows scientists to screen for cells that contain a plasmid, and kill all the cells that do not (see Ch. 22). Chloramphenicol, kanamycin neomycin, tetracycline and ampicillin resistance genes are the most widely used in laboratories.

Antibiotic resistance

Studies of antibiotic resistance in children are small in number an US network for antibiotic resistance that tracked the national incidence rate of H. pylori and microbial resistance reported 340 clinical H. pylori isolates collected over a period of 4 years that demonstrated a 29 rate of antibiotic resistance to at least one antimicrobial agent and 5 resistant to two or more antimicrobial agents 25 were resistant to Metronidazole and 12.9 were resistant to Clarithromycin. Only a very small number of cases (0.9 ) were resistant to Amoxicillin 82 . However, more recently, the results of a larger prospective multi-center study from Europe on the rate of antibiotic resistance in H. pylori strain in 1233 children have been reported. These patients came mostly from western and southern Europe. Most of the isolates were obtained prior to any treatment, and overall the resistance rate for Clarithromycin was 24 . This increased to 42 in those who had previously received treatment that had...

Detection of Antibiotic Resistance in Microbial Pathogens Using Microarray Technology

Recently, the application of the microarray-based single-base-mutation identification assay for detection of resistance against fluoroquinolones in clinical diagnostics has been demonstrated by identifying prevalent gyrA mutations in 30 clinical E.coli isolates 65 . Moreover, oligonucleotide microarrays offer an attractive option for the identification and epidemiologic monitoring of TEM b-lactamases in the routine clinical diagnostic laboratory. Using these DNA arrays Grimm et al. describe the identification of the single nucleotide polymorphisms (SNPs) of 96 of the TEM b-lactamase variants known to date which are related to extended-spectrum beta-lactamase (ESBL) and or inhibitor-resistant TEM (IRT) phenotype 66 . High-density oligonucleotide arrays have also been applied to simultaneous species identification and detection of mutations that confer rifampicin and pyrazina-mide resistance in mycobacteria 54, 67 . Consequently, the DNA microarray strategy could be expanded to include...

Microbial Forensic Tools

Clearly DNA typing methodologies will figure prominently in the attribution of a pathogenic weapon. First, the DNA must be successfully extracted from a sample. Extraction procedures that are used for pristine samples may not be adequate for more challenging environmental samples. Efficiency of recovery may be low, and polymerase chain reaction (PCR) inhibitors may not be removed effectively. The BFAC and its partners will have to promote the development of better DNA extraction methods. Once DNA has been recovered, molecular biology tools and biomarkers exist to assist analyses these include sequencing, microarray analyses, pathogenicity array analyses, single-nucleotide polymorphism (SNP) characterizations, 16S rRNA sequencing, variable number tandem repeat analysis, and antibiotic resistance gene characterizations. This list of analyses is impressive. However, more research is needed regarding the selection of the genomic region of pathogen agents to determine the severity of...

Creation And Maintenance Of Cell Lines Stably And Heterologously Expressing nAChR Of Defined Subunit Composition

To introduce nAChR subunit cDNA(s) into host cells, no consensus exists in the literature as to whether electroporation, any of a number of homemade or commercially available lipofection aids, or calcium-phosphate precipitation is superior. The latter two techniques give higher initial survival of cells and are likely to be preferred for transient transfection studies (involving study of cells and nAChR that they make within a week of transfection). Electroporation is a more harsh approach (lower initial viability) that has to be custom-designed to optimize transfection efficiency balanced against cytotoxicity for every type of host cell, but requires an equipment investment rather than recurring costs for transfection reagents. In the laboratory, good and comparable success has been experienced using each of these approaches, but studies have not been done systematically. Typically, a 24- to 48-hour period of recovery from transfection is allowed, thus permitting transgenes of...

The Postgenome Era of S aureus

With the long experience in Bacillus proteomics, this expertise has been transferred to a closely related pathogenic gram-positive bacterium, Staphylococcus aureus. S. aureus is a human pathogen of increasing importance, mainly as a result of the spread of antibiotic resistance. The pathogenicity of this species is very complex and involves the strongly regulated production of cell-wall-associated and extracellular proteins forming a changing set of virulence factors. Due to the great variety of these proteins, S. aureus causes a broad spectrum of infectious diseases ranging from superficial abscesses to endocarditis, osteomyelitis, and toxic shock syndrome 21 . Methicillin-resistant S. aureus (MRSA) strains are currently predominant and dangerous nosocomial pathogens, since infections caused by these strains have become difficult to treat. Vancomycin has become the drug of choice for treating MRSA infections. However, the emergence of vancomycin-resistant MRSA strains is leading to...

Urinary Tract Infection

The use of fluoroquinolones as first-line therapy for uncomplicated UTIs is recommended for patients who cannot tolerate sulfonamides or trimethoprim, who have a high frequency of antibiotic resistance because of recent antibiotic treatment, or who reside in an area with significant resistance to trimethoprim-sulfamethoxazole. Treatment should consist of a three-day regimens of one of the following

Isolation And Characterization Of Recombinant Proteins

Recombinant protein can be obtained in large amounts via overexpression of the cDNA of interest in, for example, the bacterium Escherichia coli, the yeasts Saccharomyces cerevisiae and Pichia pastoris, or the Sf9 insect cell line of Spodoptera frugiperda. This is typically achieved by the introduction of a plasmid vector containing the cDNA. He plasmid is a circular DNA replicon that is maintained because it confers a selective advantage, such as an antibiotic resistance. The use of an appropriate promoter results in high expression levels of the cDNA. As a consequence, the abundance of recombinant proteins in these systems is typically much higher than that of the native protein in a crude plant extract. The higher abundance facilitates the purification of the protein to apparent homogeneity. Purification of the recombinant protein can be achieved through traditional biochemical separation techniques, including various types of chromatography (see Section 2). Many of the expression...

Evaluation Of The Etiologic Agent

Microbes are dynamic organisms, and are therefore constantly changing in response to natural selection pressures in their environment. It is often possible to determine the laboratory or geographic origin and character of microbes by use of strain typing, molecular biology tools, and antibiotic resistance patterns. For example, there are seven major serotypes of foot-and-mouth disease virus, and these have definite geographic distributions throughout the world. Ribotyping of Yersinia pestis was useful in ruling out a terrorist attack by Kashmir separatists in the pneumonic plague epidemic in Surat in 1994. Similarly, when parties to the war in Northern Bosnia accused each other of

Expression Vectors 5221 Plasmid vectors

Cho Protein Production

The bacterial backbone always includes an origin of replication (ori) and its associated cis-acting control elements, the whole genetic unit being termed a replicon. Replicons derived from the pMB1 (or ColE1) plasmid do not require plasmid-encoded functions for replication, and can function in the absence of ongoing protein synthesis, leading to the accumulation of several thousand copies of the plasmid in the cell (Stadenbauer 1978). One or more antibiotic resistance genes (i.e ampr-ampicillin, kanr-kanamycin, tetr-tetracycline or camr-chloramphenicol) for selection in E.coli must also be present. Inclusion of elements of the lacZ operon permits blue white screening of recombinant plasmids, while a multiple cloning site (MCS) with a wide range of single-cutting RE sites for insertion of the gene to be expressed is essential. The MCS can be designed to permit the cloning of blunt or sticky-ended polymerase chain-reaction-derived cDNA and to allow insertion in a specific orientation....

Composite Transposons

Many of the well-known bacterial transposons that carry genes for antibiotic resistance or other useful properties are composite transposons. Three of the best known are Tn5 (kanamycin resistance), Tn9 (chloramphenicol resistance) and Tn10 (tetracycline resistance). Usually the pair of insertion sequences at the ends of the transposon are inverted relative to each other, as in Tn5 and Tn10. The IS elements of Tn5 and Tn10 have not been found alone and are named IS50 and IS10 respectively. Less often

Conjugative Transposons

Tn916 jumps by the cut-and-paste mechanism. However, it differs in two respects from typical conservative transposons. First, the target sequence is not duplicated when Tn916 inserts itself. Second, it can excise itself precisely, leaving the host cell DNA intact.When moving from one bacterial cell to another,Tn916 is thought to excise itself temporarily from the DNA of the original cell. It then transfers itself into the recipient and, once inside, it transposes into the DNA of the new host cell (Fig. 15.24). Tn916 and related elements can enter many different groups of bacteria, both gram-positive and gram-negative. Because the host range of conjugative transposons is so broad they are partly to blame in the spread of antibiotic resistance genes among diverse groups of bacteria.

General Properties of Plasmids

The copy number is the number of copies of the plasmid in each bacterial cell. For most plasmids it is one or two copies per chromosome but it may be as many as 50 or more for certain small plasmids such as the ColE plasmids. The number of copies influences the strength of plasmid-borne characteristics, especially antibiotic resistance. The more copies of the plasmid per cell, the more copies there will be of the antibiotic resistance genes, and therefore, higher the resulting level of antibiotic resistance.

[14 Fluorescence Activated Cell Sorting of Hybrid and Transfected Cells

The introduction of chromosomes and DNA into somatic cells by whole cell hybridization and transfection has supported studies on gene mapping, cloning, and function. The selection of host cells containing donor genetic material most often has depended on the complementation of defective genes, antibiotic resistance, or altered phenotype. Genes coding for cell surface exposed molecules also have been introduced into somatic cells, and their expression assessed with specific antibodies and indirect immunofluorescence. Using the fluorescence-activated cell sorter (FACS) individual cells have been isolated based on the expression of these surface antigen genes.

Mechanisms Of Resistance

Efforts to overcome the actions of the p-lactamases have led to the development of such p-lactamase inhibitors as clavulanic acid, sulbactam, and tazobactam. They are called suicide inhibitors because they permanently bind when they inactivate p-lactamases. Among the p-lactamase inhibitors, only clavulanic acid is available for oral use. Chemical inhibition of p-lactamases, however, is not a permanent solution to antibiotic resistance, since some p-lactamases are resistant to clavulanic acid, tazobactam, or sulbactam. Enzymes resistant to clavulanic acid include the cephalosporinases produced by Citrobacter spp., Enterobacter spp., and Pseudomonas aeruginosa. An additional mechanism of antibiotic resistance involves an alteration of PBPs. Resistant bacteria, usually gram-positive organisms, produce PBPs with low affinity for p-lactam antibiotics. The development of mutations of bacterial PBPs is involved in the mechanism for p-lactam resistance in Streptococcus pneumoniae,...

Spinal Cord And Root Compression

In developing countries, spinal TB is mostly a disease of childhood or adolescence. In Britain it usually affects the middle aged and is particularly prevalent in immigrant populations. The incidence is now increasing, probably due to the development of antibiotic resistance.

Mutagenesis

The products of the linear amplification reaction are treated with the restriction enzyme Dpn , which specifically cleaves fully methylated GMe6ATC sequences. Dpn will therefore digest the bacterially generated DNA used as template for amplification but will not digest DNA synthesized during the course of the reaction in vitro. Dpn -resistant molecules, which are rich in the desired mutants, are recovered by transforming E. coli to antibiotic resistance.

Evolving Sideways Horizontal Gene Transfer

For example, when antibiotic resistance genes are carried on plasmids they can be passed between unrelated types of bacteria (see Ch. 16). Since genes carried on plas-mids are sometimes incorporated into the chromosome, a gene can move from the genome of one organism to an unrelated one in a couple of steps. The complete genomes of many bacteria have now been fully sequenced. Estimates using this data suggest that about 5-6 of the genes in an average prokaryotic genome have been acquired by horizontal transfer. The effects of horizontal transfer are especially noticeable in a clinical context. Both virulence factors and antibiotic resistance are commonly carried on transmissible bacterial plasmids.

Problems in Estimating Horizontal Gene Transfer

The ease of horizontal transfer of genetic information by plasmids, viruses, transposons under laboratory conditions is misleading. Under natural conditions there are major barriers to such movements. Furthermore, the results of horizontal transfer are often only temporary. Newly acquired genes, especially those on plasmids, transposons, etc., are easily lost. Such genes tend to be acquired in response to selection such as antibiotic resistance and, conversely, they will be lost when the original selective conditions disappear.

In Vitro Mutagenesis Using Doublestranded DNA Templates Selection of Mutants with Dpnl

The products of the linear amplification reaction are treated with the restriction enzyme Dpnl, which specifically cleaves fully methylated GMe6ATC sequences. Dpnl will therefore digest the bacterially generated DNA used as template for amplification but will not digest DNA synthesized during the course of the reaction in vitro. Dpnl-resistant molecules, which are rich in the desired mutants, are recovered by transforming E. coli to antibiotic resistance.

Claims about potential risks

There are concerns about the impact of GM crops on food variety and quality. If companies invest mainly in the development of a few crops and varieties with the greatest market potential, and those are widely adopted by growers, then the production of more localised varieties and regionally typical produce may decline. Other concerns about GM foods include their implications for human health, for example that they might unexpectedly contain toxins or allergens, or that the antibiotic resistance marker genes sometimes used in the GM process might transfer to gut bacteria and make antibiotic treatment ineffective.

Introduction In Our Hands

The same is true for the genomic structure of antibiotic resistance determinants, which basically undergo a comparable distribution among bacteria using a very similar mechanism of horizontal spreading 7, 8 . The emergence of antibiotic resistance and multidrug resistance in bacterial pathogens underscores the need for the development of novel classes of antibiotics. The availability of complete genome sequence data from many important human pathogens has already provided a wealth of fundamental information from which to identify potential molecular targets for drug discovery. Determining the presence or absence of certain pathogenicity islands or genomic islands harboring multiple antibiotic resistance genes in bacterial isolates may further aid in identifying the cause of a disease, estimating its pathogenic potential, and predicting its antibiotic resistance. Thus, pathogenomic research has contributed to microbial diagnostics, pathotyp-ing of bacteria, and the detection of novel...

Microbiological Diagnostics of Bacterial Pathogens Aims Tasks and Current Limitations

The microbiological diagnostics of bacteria has historically focused on the detection and identification of bacteria by providing a differentiation at the species level. In the case of medical microbiology a further emphasis is on determination of the antibiotic resistance pattern revealed by the isolate under investigation. Three important fields of microbiological bacterial diagnostics are (1) medical and food microbiology, (2) environmental microbiology, and (3) the detection of biological warfare agents. These fields differ considerably with regard to the diversity of bacteria to be detected and the time scale for diagnosis 9 . Methods for the detection and identification of microorganisms applied in medical microbiology and food technology are directed towards the reliable detection and or identification at the species subspecies strain level of one or a few microbes out of many that may be present in the diagnostic sample 10, 11 . The further major aspect of clinical...

Conventional Culture Based Methods in Microbiological Diagnostics

Gens, which is one of the primary functions of a diagnostic microbiology laboratory. Individual results have important therapeutic implications for the patient. The detection of resistant bacteria further provides a fundamental basis for infection control measures and antimicrobial surveillance systems. Antibiotic resistance is routinely determined in diagnostic laboratories using culture-based methods, e.g., disc diffusion assay or broth dilution. These traditional methods, however, are largely confined to rapidly growing bacteria and depend on the expression of antibiotic resistance genes under laboratory culture conditions.

Fluorescence In Situ Hybridization

Of complex microbiological communities in humans, animals, and the environment. Furthermore, rRNA-based antibiotic resistance mechanisms are detectable by means of specific FISH probes 27 . FISH has been successfully used to identify pathogens in smears or tissue preparations from humans and animals however, the number of target organisms has to be above the detection level, for microscopic techniques 28, 29 . FISH using peptide nucleic acid probes (PNA-FISH) is a novel diagnostic technique combining FISH with the unique performance of PNA probes to provide rapid and accurate diagnosis of infectious diseases. Both FISH and PNA-FISH are well suited to routine application and enable clinical microbiology laboratories to report information important for patient treatment within a time frame that is unreachable using classic biochemical methods 30 . However, the FISH technique still remains quite laborious, it is restricted to the sensitivity of light microscopy, and is only to a limited...

PCR Methods for Microbial Diagnostics

Besides the identification and classification of bacterial pathogens, the detection of specific antibiotic resistance in clinical isolates has always been the second major task of microbiological diagnostics. In recent years the molecular mechanisms of antibiotic resistance have been thoroughly characterized, providing the basis for molecular tool to detect a multitude of antibiotic resistance mechanisms on the level of chromosomal or episomal DNA 34, 38, 39 . The introduction of real-time PCR methods offers a cost-effective, user-friendly format for genetic methods that fuels their use for the detection and characterization of antimicrobial resistance determinants in routine diagnostic microbiology. The implementation of these assays to detect resistance in clinically important slow-growing organisms (e.g., Mycobacterium tuberculosis), to rapidly identify clinically important resistance mechanisms, and to overcome laborious and time-consuming culture techniques in the control and...

Bacterial and P1 Artificial Chromosomes

The YAC has two forms, a circular form for growing in bacteria, and a linear form for growing in yeast. The circular form can be manipulated and grown like any other plasmid in bacteria since it has a bacterial origin of replication and an antibiotic resistance gene. In order to use this in yeast, the circular form is isolated and linearized such that the yeast telomere sequences are on each end. This form can accommodate up to 2,000 kb of cloned DNA inserted into its multiple cloning site (MCS).

Introduction Methods for the Detection of mRNA in Soil

Luminescent marker genes luc and luxAB, derived from eukaryotic and bacterial luciferase, respectively, have been used in microbiological studies due to limitations such as energetic demands on cell metabolism, their usefulness for studies of soil microbial populations is limited. Consequently, fluorescent marker genes, mainly those encoding fluorescent proteins such as the green fluorescent protein (GFP), have been used, despite limitations such as a requirement for molecular oxygen and a need for specialised detection equipment. In contrast to luminescent markers, fluorescent markers place less demands on cell energy. Chromogenic reporter genes have also been used in many environmental studies, including xylE encoding catechol 2,3-dioxygenase, lacZ for 3-galactosidase, gusA encoding E. coli glucuronidase, inaZ encoding ice nucleation activity, and different antibiotic resistance genes such as nptII which produces kanamycin resistance. Most bioreporters use an environmentally or...

Current Limitations on the Use of DNA Microarrays in Diagnostics in Medical Microbiological Laboratories

Currently, the limitation on a broader application of pathogenomic tools (DNA microarrays) in microbial diagnostics basically relates to costs and operative overheads. Upfront investment and molecular expertise are required for the development and validation of in-house genetic techniques and may hinder application in smaller laboratories. Aside from their costs and the difficulties associated with designing and making a suitable array, the most obvious problems are those of quality control, because of the difficulties of standardization and reproducibility associated with the large number of probes on an array, and the large number of slides that need to be made. Commercially available arrays may be inflexible because they are based on just one target sequence, whereas in-house arrays of oligonucleotides or PCR amplicons spotted on glass slides may be contaminated if PCR amplicons are used. Additionally, a number of relevant experimental factors must be considered in order to obtain...

Phage Therapies and other Bacteriolytic Approaches

The idea of using bacteriophages as weapons to combat infectious diseases is older than the application of low-molecular-weight antibiotics. In fact, there are a large number of mainly anecdotal reports of the successful use of bacteriophages especially in Eastern Europe and under military conditions 61 . With the advent of the modern antibiotic area, the interest in that approach faded, and, thus, we do not now have access to clinically valid, controlled data that meet today's standards. The increasing threat of rising antibiotic resistance has led to a renewed interest in phage therapy itself as well as in therapeutic approaches derived from the knowledge of modern phage biology.

Analysis of Gene Expression

Suppose that a DNA molecule, such as a cloning plasmid, has been inserted into a new bacterial host cell or a transgene has been inserted into the chromosome of a new animal host. Antibiotic resistance genes are often used to monitor whether the DNA is indeed in the intended location. Thus antibiotic resistance genes may be regarded as reporter genes (Fig. 25.01). As already discussed in Ch. 22, after transformation of the plasmid into the target cells, they are treated with the antibiotic. Those

Selection of Test Strains for Sanitizer Efficacy and Challenge Studies

The use of markers such as antibiotic resistance may be desirable to facilitate the recovery of cells in enrichment broth or counting colonies on selective or nonselective direct plating media. Otherwise, these media may support the growth of large numbers of background microflora which interfere with growth of the test microorganism. Adaptation of Gram-negative pathogens to nalidixic acid (50 ml) has been used to achieve this objective. In a study to determine survival of five strains of nalidixic acid-resistant and refampicin-resistant Salmonella Poona on cantaloupes it was observed that average reductions in the number of control and antibiotic-resistant cells were not significantly different (P 0.05) 44 . Resistance of test cells to rifampicin (80 ml) can also be successfully used as a marker, particularly for isolating pathogens from inoculated fruits and vegetables that have significant adhering soil. Plasmid-borne or chromosomally stabilized markers such as fluorescent proteins...

Linear Plasmids and Evolution

As is often the case for large plasmids, both circular and linear, a relatively high proportion of SCP1 genes (57 ) show no significant database matches, and a further 20 resemble database entries of unknown function. The equivalent numbers for the host chromosome are 23 and 30 . This reinforces the idea that plasmids are an important route by which new genes enter their hosts, though it has been argued elsewhere that fundamentally novel genes probably arise more often in bacteriophages (Chater and Chandra 2006). It is interesting to note that one of the linear plasmids, SCP1, is a vehicle for members of several families of regulatory genes that are particularly important in streptomycetes. One such family is the arpA-like genes referred to above, representatives of which (mmfR and mmyR) function in methylenomycin production. Another comprises genes for ECF (extracytoplasmic function) sigma factors, which are particularly abundant in streptomycetes (e.g. nearly 50 each in S....

Detection of Antibiotic and Heavy Metal Resistance Genes

Exogenous plasmid isolation has been used to detect resistance genes in soil bacteria. This method allows capture of plasmids from the total bacterial fraction of an environmental sample without the necessity to culture the host organism. Smit et al. (1998) investigated mercury resistance plasmids in soil populations using exogenous isolation, and identified plasmids of 10-50 kb carrying resistance to copper, streptomycin and chlorampheni-col. These authors amended soil with mercuric chloride and found this to subsequently increase the recovery of resistance plasmids. Plasmids have also been captured from polluted soils and slurrys (Top et al. 1994 Smalla et al. 2000). The former authors identified multiple antibiotic resistance genes from isolated plasmids. In addition to multi-resistance plasmids antibiotic resistance genes are situated on transposable elements that can associate with other elements such as chromosomes and plasmids. These transposable elements include transposons...

Causative Agent And Classification Of Mutation

Given the mobility of DNA, genes encoding resistance to an antimicrobial drug can spread to different strains, species, and even genera. The most common mechanism of transfer of resistance is through the conjugative transfer of R plasmids. R plas-mids frequently carry several different resistance genes, each one mediating resistance to a specific antimicrobial drug. Thus, when an organism acquires an R plasmid, it acquires resistance to several different medications simultaneously. Many of the resistance genes on R plasmids are carried on transposons that can move from a plasmid to the chromosome, from one plasmid to another, or from the chromosome to a plasmid. Thus, if one organism has two different plasmids, an antibiotic-resistance gene can move from one to the other. In this way a gene could move from a narrow host range plasmid to a wide host range plasmid. Wide host range plasmids, in contrast to narrow host range plasmids, can replicate even if they are transferred to...

Pathogenomic Tools Microarrays in the Diagnosis of Microbiologic Agents as Bioweapons

Current approaches in the detection and differentiation of bioweapons are directed towards the developing of biodefense microarrays that can detect hundreds of top-priority bacterial and viral biological agents, such as anthrax, plague, and smallpox. The new generation of biodefense microarray tests is expected to offer researchers the quickest, most comprehensive single test. Earlier DNA tests required a time-consuming approach, testing for one pathogen at a time. Traditional test methods, which include growing a culture of the bacteria and then identifying it by sight, can overlook genetically engineered organisms expressing unusual toxins or antibiotic resistance. The new arrays are expected to work in as little as 4 h and offer three advantages have been inserted into a pathogen by simultaneously testing for 62 different antibiotic resistance genes. Such a multi-drug-resistant phenotype has been described in a wildtype isolate of Yersinia pestis 81 . If an antibiotic resistance...

Genetic manipulation of animals

Transgenic mice are made by injecting human DNA into fertilized mouse oocytes. Integration is a rare and random event, but almost always involves multiple copies entering at a single site, making interpretation of some transgenic experiments difficult. More specific genetic manipulation is achieved by exploiting homologous recombination in embryonic stem (ES) cells, although the technique is very technically demanding. DNA containing a mutated copy of the gene of interest in tandem with a selectable marker (e.g. an antibiotic resistance gene) is introduced into ES cells where in a small number of cases it recombines with, and consequently replaces, the cell's copy of the gene. ES cells are isolated from embryos and can be grown in flasks while retaining the potential to develop into any tissue. Once they have been genetically manipulated and reinjected back into the pregnant mouse, the resulting embryo is a mixture of mutant and normal cells. If some of the ES cells contribute to the...

Box 93 Bacterial meningitis empirical treatment

Treatment may be modified once pathogens have been identified. For H. influenzae type b infections, ampicillin and chloramphenicol are no longer suitable for empirical treatment, owing to widespread antibiotic resistance. The third generation cephalosporins, cefotaxime or ceftriaxone, have been demonstrated to be effective and are the drugs of choice.80 For meningococcal meningitis, IV benzylpenicillin remains the preferred drug. There have been reports of meningococci resistant to penicillin,106 but this is not associated with clinical failure if the high doses of penicillin used for meningitis are given. Chloramphenicol or a third generation cephalosporin may be used in people who react adversely to penicillin.

Engineering Deletions and Insertions by PCR

PCR is widely used to generate DNA cassettes that can be introduced into chromosomes by homologous recombination. In this procedure, a convenient marker gene, usually an antibiotic resistance gene, is inserted into the chromosome of the host organism where it replaces any chosen gene. In order to target the incoming cassette to the correct location it must first be flanked with DNA sequences homologous to DNA both upstream and downstream of the chosen gene. This is done by using PCR primers that overlap the resistance cassette and also contain about 40-50 bp of DNA homologous in sequence to the target location (Fig. 23.19). The cassette is transformed into the host organism and is inserted into the chromosome by homologous recombination. Antibiotic resistance is then used to select those organisms that have gained the cassette. This approach may be used to generate deletions of any desired chromosomal gene and works especially well in yeast and bacteria. In the first step, a...

Detecting Insertions in Vectors

Once a gene or other fragment of DNA has been cloned into a plasmid vector and transformed into a bacterial cell, we face the problem of detecting its presence. The plasmid itself may be detected by conferring antibiotic resistance on the host cell, but this leaves the question of whether the presumed insert is actually there. If the cloned gene itself codes for a product that is easy to detect, there is no problem. In most cases, however, the presence of the inserted DNA itself must be directly monitored. Rather less laborious is to use a plasmid with two antibiotic resistance genes. One antibiotic resistance gene is used to select for cells, which have received the plasmid vector itself. The second is used for the insertion and detection of cloned DNA (Fig. 22.14). The cut site for the restriction enzyme used must lie within this second antibiotic resistance gene. When the cloned fragment of DNA is inserted this antibiotic resistance gene will be disrupted. This is referred to as...

Integrons Collect Genes for Transposons

Many bacteria that carry multiple drug resistance have emerged since antibiotic use has become widespread. Antibiotic resistance genes are usually carried on plasmids, many of which may be transferred between bacteria. In many cases the antibiotic resistance genes are actually carried within transposons that are inserted into the plasmids. Novel antibiotic resistance genes often appear first in transposons of the Tn21 family found in gram-negative bacteria. This group of transposons possesses an internal element known as an integron that acts as a gene acquisition and expression system. An integron consists of a recognition region, the attI site, into which a variety of gene cassettes may be integrated, plus a gene encoding the enzyme responsible for insertion, the integrase. The attI site is flanked by two 7 bp sequences that act as recognition sites for the integrase (Fig. 15.25). Two promoters are situated upstream of the variable region. One is for the integrase gene the other...

Role of pharmacological management

The development of small intestinal bacterial overgrowth (SIBO) appears to be common in SBS patients and may affect their ability to successfully wean PN because of symptoms that impede oral intake and an exacerbation of malabsorption 67 . The anatomical and physiological changes that occur in SBS together with medications commonly used in these patients facilitate the development of SIBO. Excess bacteria in the small bowel can induce inflammatory changes in the gut impairing nutrient absorption, causing a number of gas-related symptoms and aggravating stool losses 68, 69 . While the identification of an excessive number of bacteria in small bowel fluid is considered the gold standard in the diagnosis of SIBO, it has several limitations that have led some to questions its utility 70 . Nevertheless, the use of the primary noninvasive test to diagnose SIBO, the hydrogen breath test, has greater limitations due to rapid transit in the shortened bowel making it difficult to differentiate...

Human Viral Diseases Are Common

Antibiotics are of no use against viruses they only kill bacteria. So why do doctors often prescribe antibiotics for viral diseases like flu or colds There are two main reasons. The valid reason is that giving antibiotics may help combat secondary or opportunistic infections caused by bacteria, especially in virally-infected patients who are in poor health. However, massive over-prescription of antibiotics occurs because many patients would be upset if faced with the truth. They would rather be given medicine, even if it is of no use, than face the fact that there is no cure. This abuse has in turn contributed to the spread of antibiotic resistance among many infectious bacteria (see Ch. 16) thus creating a major health problem.

Vectors Used for Phage Display

The first phage displaying a protein fragment was made by inserting a foreign sequence into the otherwise wild-type genome of a filamentous phage (Smith, 1985). However, working with phage has a number of disadvantages, such as low yields of DNA and the need to work with plaques instead of colonies. A very popular alternative is to use a phagemid instead. This is a plasmid carrying a filamentous phage origin of replication that allows it to replicate and be packaged in the presence of helper phage. Generally, a helper phage is tagged with antibiotic resistance and is partially replication deficient, allowing preferential replication and packaging of the phagemid e.g., M13K07, carrying a kanamycin resistance marker (Vieira and Messing, 1987) . pUC119 is a common phagemid on which a large number of current display vectors, including our own, are based (Vieira and Messing, 1987). The vector we use for display, pDISPLAY-B (Fig. 3), was derived from vector pHENl (Hoogenboom et al, 1991)....

Applications 2631 Spatial Distribution

Intestine Xray With White Spots

Normander et al. have reported the significance of bacterial distribution on genetic exchange in the phyllosphere using GFP as an indicator of plasmid transfer 30 . Conjugation was observed under CLSM to occur primarily in the interstitial spaces of epidermal cells and vein cells, and in stomata bacterial aggregation had a great stimulatory effect on plasmid transfer. Such data are pertinent to assessing the risk associated with the dissemination of antibiotic resistance genes among bacterial cells on plants.

Prokaryotic Cells

Range in size from 33bp to 230bp2 and first gained notoriety in the 1950s by being associated with antibiotic resistance in bacteria.3,4 Transposons also may confer antibiotic resistance on the host bacteria. All these genetic elements exist in direct contact with the bacteria's cytoplasm.

Expression vectors

The vectors described thus far have been designed to facilitate the cloning of genomic DNA cDNA sequences, ultimately in order to identify and isolate a gene cDNA coding for a particular polypeptide. The genetic construction of these vectors normally does not support the actual expression (i.e. transcription and translation) of the gene. Once the gene cDNA coding for a potential target protein has been isolated, the goal usually becomes one of achieving high levels of expression of this target gene. This process entails ligation of the gene into a vector that will support high-level transcription and translation. In addition to the basic vector elements (e.g. an origin of replication and a selectable marker, such as an antibiotic resistance gene), expression vectors also contain all the genetic elements required to support transcription and translation, as described earlier in this chapter (e.g. promoters, translational start and stop signals, etc. see Figures 3.7 and 3.8). A wide...

Swgmgf

Information is the key to thwart terrorism and may be our best defense. Databases are required to have ready access to information. For a database to be effective, it must contain the appropriate data entry items, and the data must be retrievable. With proper planning, meaningful and useful databases can be developed. The database(s) criteria need to be defined, and SWGMGF is taking on this function as well. Criteria fields for information databases under consideration are multi-agency threat lists (virus, bacteria, fungi, protozoa, insects, toxins), recognizing synonyms, strain data and virulence, vaccine strains, DNA sequences (whole genomic and partial), bioengineering events, biomarkers, organism sources (laboratory, geographic, and natural), national experts and contact information, laboratory facilities, assays, scientific presentations, literature references and full text, research grants, characteristics (e.g., microscopic morphology, colony morphology), antibiotic resistance,...

Conclusion

Apart from ameliorations of therapeutic measures, broadening the focus on other blood-CNS barrier interfaces could offer new insights in pathophysiological events. In times where antibiotic resistance of microbial pathogens increases and new modalities in treating meningitis patients such as the application of dexamethasone drastically influence the plethora of cellular and molecular events, penetration of the blood-CNS barriers with suitable drugs might gain more attention.

Table 1601

Antibiotic resistance against aminoglycosides, p-lactams, chloramphenicol, sulfonamides, FIGURE 16.10 Antibiotic Resistance Plasmids Plasmids carry genes for replicating their DNA, transferring themselves from one host cell to another, and genes for a variety of phenotypes. Many plasmids carry genes that confer antibiotic resistance on the host cell when the genes are expressed. FIGURE 16.10 Antibiotic Resistance Plasmids Plasmids carry genes for replicating their DNA, transferring themselves from one host cell to another, and genes for a variety of phenotypes. Many plasmids carry genes that confer antibiotic resistance on the host cell when the genes are expressed. by 1970 this had risen to over 30 . These strains often carry resistance genes for different antibiotics on one single plasmid. Today, the transfer of multiple antibiotic resistance plasmids between bacteria has become a major clinical problem. Patients with infections after surgery, with severe burns, or with...

Methods

For biopharming and production of rc- proteins in target tissues, the appropriate DNA expression vectors must be used. In this case, vectors are designed for organ-specific expression and secretion of rc- proteins within the target tissue, the mammary gland of the transgenic animal. The regulatory controlling elements are based on milk specific protein genes. These elements include the promoter (e.g., whey acidic protein, a-lactoglobulin, P-casein, etc.), in order to direct the expression specifically to the mammary gland, and a secretion signal sequence, to direct the secretion of the protein from the cell into the milk. A second expression vector, containing a reporter or selectable marker, is required for the selection of the transgenic stable cell lines to be used as donor cells in NT. Reporter (e.g., green fluorescent protein) or selectable markers based on antibiotic resistance (e.g., neomycin resistance) are used to select transgenic from nontransgenic cells (11,12).

Resistance

Such resistance is recognized as a major problem in controlling bacterial infections and may be either chromosome or plasmid mediated. Plasmids (extrachromosomal genetic elements), which code for enzymes that inactivate antimicrobials, can be transferred by conjugation and transduction from resistant bacteria to previously sensitive bacteria. Such a transfer can also occur between unrelated species of bacteria. Enzymes coded by plasmids (e.g., penicillinase, cephalosporinase, and acetylases) that are specific for a given antimicrobial inactivate the drug either by removal or addition of a chemical group from the molecule or by breaking a chemical bond. Transposons are segments of genetic material with insertion sequences at the end of the gene these sequences allow genes from one organism to be easily inserted into the genetic material of another organism. Some of these transposons code for antibiotic resistance.

Treatment

A recent Canadian Helicobacter Study Group Consensus Conference still recommends the use of triple therapy for 2 weeks using a PPI with Clarithromycin and Amoxicillin or Metronidazole given for 14 days. The duration of treatment of 2 weeks is likely to be optimal, but not conclusive there is a 7-9 increase in the eradication rate with 14 days of treatment versus 7 days 87 . Tetracycline should be avoided in children under the age of 12 because it may cause staining of the children's enamel. In addition, treatment failure is increased with antibiotic resistance 88 . A recent Russian study by Nijevitch et al. 89 treated 76 children, who had failed triple therapy, using quadruple therapy. These children were randomized to receive a 2-week course of bismuth subcitrate, Amoxicillin with Nifuratel or Furazolidone plus Omeprazole. The eradication rate was 89 for Nifuratel and 87 for Furazolidone. Nifuratel is preferred because of a lower frequency of side effects. Potentially, this could be...

Resistance Concerns

The use of antimicrobial therapies through aerosolization avoids many of the toxicities associated with systemic therapy with aminoglycosides or polypeptide agents. However, the risk for promoting antibiotic resistance is a concern. The emergence of highly resistant organisms has been reported

With Antibiotic

FIGURE 25.01 Antibiotic Resistance as a Reporter Gene FIGURE 25.01 Antibiotic Resistance as a Reporter Gene Antibiotic resistance genes are included on plasmids in order to determine whether the plasmids are present in a cell. When bacteria are transformed with plasmid DNA those that get a plasmid that carries an antibiotic resistance gene will survive when treated with the antibiotic whereas those cells that fail to get a plasmid will be killed. that receive the plasmid become antibiotic resistant those not getting the antibiotic resistance gene are killed. An antibiotic resistance gene carried on the same fragment of DNA can also report whether a transgene has integrated into another DNA molecule such as a chromosome or a virus.

Chris Good

A similar re-categorization or de-restriction of the proton pump inhibitors can be expected now that they have replaced H2 antagonists as the most expensive treatments for various forms of dyspepsia. With the general acceptance of Marshall's evidence for the pathogenic role of H. pylori in dyspeptic conditions, antibiotics have been added to proton pump inhibitors as combination therapy. So far the licensing authority have been reluctant to de-restrict antibiotics, no doubt for fear of further increasing the already rapid rate of development of antibiotic resistance.

Antibiotic

Antibiotics and Antibiotic Resistance A big worry for modern medicine is antibiotic resistance, the evolution of populations of pathogenic bacteria that antibiotics are unable to kill. Antibiotic resistance may develop in a population of bacteria in several ways. In one case, mutations in bacterial DNA give a bacterium resistance to antibiotics. A cell with such a mutation has a selective advantage when antibiotics are present. Mutant bacteria multiply and take over the population and thus stop the antibiotic's curing power. Figure 23-12 shows how resistance can evolve in a population of bacteria. www.scilinks.org Topic Antibiotic Resistance Keyword HM60081 www.scilinks.org Topic Antibiotic Resistance Keyword HM60081 Present Development of antibiotic resistance spurs more research into antibiotic treatment.

Investigations

Egd Children Antrum Pictures

Tage of being able to detect upper gastrointestinal pathology including the complications of H. pylori infection such as nodular gastritis, peptic ulcer disease, gastric cancer, and MALT lymphoma. In pediatrics, the primary indication for upper GI endoscopy is the presence of persistent, severe upper abdominal symptoms and not simply the presence of H. pylori 33 . It is difficult to differentiate symptoms secondary to the complication of H. pylori infection such as peptic ulcer disease and functional dyspepsia. The most common endoscopic finding in children with H. pylori infection is nodular gastritis, which is seen most commonly in the antrum with an irregular (cobblestone) appearance, which is highlighted with blood from a bleeding biopsy site. When nodular gastritis is found, it has high specificity (98 ) for H. pylori infection, and therefore a high predictive indicator for H. pylori infection, but it has low sensitivities (44 ) 17, 74 . In naive patients, antral biopsy had the...

Pinv-2457t Sequence

S.flexneri harbors a large virulence plasmid, pINV, that contains all of the genes (ipa, mxi-spa, virG lscA, virF) required to express the invasive phenotype. This plasmid is also present in enteroinvasive E. coli strains. Sequence comparison of the four available virulence plasmid sequences of S.flexneri, pINV-2457T, pWR100, pWR501, and pCP301 14, 15, 21, 22 showed that they are essentially identical. Size differences are mainly due to the presence of IS elements. Interestingly, about 50 of all ORFs located on the invasion plasmid are related to putative IS elements indicative of IS-mediated acquisition of gene blocks of various origins. However, the analysis of non-IS-related coding sequences of different pINV variants also indicated that positive selection is also a major driving force involved in the evolution of pINV variants 23 . In contrast to strain 301, strain 2457Talso contains two small multicopy plasmids as well as another 165-kbp plasmid with high homology to the S....