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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Natural Gene Transfer and Antibiotic Resistance

Antibiotic resistance in bacteria frequently results from the action of genes located on R plasmids, small circular The results of recent studies demonstrate that R plas-mids and their resistance genes are transferred among bacteria in a variety of natural environments. In one study, plasmids carrying genes for resistance to multiple antibiotics were transferred from a cow udder infected with E. coli to a human strain of E. coli on a hand towel a farmer wiping his hands after milking an infected cow might unwittingly transfer antibiotic resistance from bovine- to human-inhabiting microbes. Conjugation taking place in minced meat on a cutting board allowed R plasmids to be passed from porcine

Antibiotic resistance in mycobacteria

Multiple drug resistance in mycobacteria compromises the use of antibiotics. Although the genetic and biochemical bases of antibiotic resistance in mycobacteria are largely understood, a number of questions remain to be addressed. This chapter discusses the potential roles of hypermutability and compensatory mutations in establishing stable resistant phenotypes in the pathogenic mycobacteria. The appearance of antibiotic resistance during the course of treatment of M. tuberculosis infections has been well documented. Streptomycin, the first effective antibiotic available for tuberculosis therapy, was discovered in 1944 and introduced into general practice in 1947 within a year reports of streptomycin-resistant strains of M. tuberculosis were frequent. The same scenario has unfortunately been repeated for every antibiotic since introduced for tuberculosis, and indeed for any bacterial infection in humans.

Mechanisms and development of antibiotic resistance

Treatment of mycobacterial infections, there are several mechanisms of resistance that have been well characterized on a genetic and biochemical basis. For some agents whose mode of action remains incompletely characterized (such as pyrazinamide), the detailed resistance mechanisms have proved difficult to assign. A number of efflux pump systems have been identified in mycobacteria, and the role of some level of increased drug efflux in the overall development of antibiotic resistance appears to be important (Takiff et al 1996). It is possible that an initial low level resistance due to increased drug efflux during therapy provides a selective advantage (survival), which then increases the possibility of the appearance of mutations leading to resistance by other mechanisms, such as target modification. In this brief review I would like to discuss and speculate on several aspects of antibiotic resistance and its phenotypic expression in pathogenic mycobacteria which set them apart from...

Outlook for Further Improvements of Expression Vectors Employing the trc Promoter

Et al. (1996). (1) Replacement of the p-lactamase antibiotic resistance gene with the tetracycline resistance gene (as in the pK series described by Andrews et al., 1996), as traces of the potentially allergenic penicillin derivatives should be avoided during bacterial growth of strains overexpressing therapeutic proteins. (2) The lacl gene carrying the wild-type promoter can be exchanged with alleles producing greater amounts of the lac repressor. This measure, as shown in pKBiq-Vp5.3, improves regulation of the trc promoter but reduces the levels of recombinant protein after induction by a factor of five (Andrews et al., 1996). (3) The lacl allele located on the expression vector can be exchanged with a temperature-sensitive allele, which eliminates the need to use IPTG, a substance that is toxic and expensive. The use of protease-deficient strains (Goff and Goldberg, 1987 Gottesman, 1990) with this thermoinducible system minimizes the proteolysis of recombinant proteins due to the...

Plant Microbe Interaction

Many GM plants harbour antibiotic-resistance genes,since these marker genes were used in the process of the construction of the transgenic plant. There is concern that GM crop plants could render pathogenic bacteria unresponsive to antibiotics after receiving transgenes from GM plant materials. However, it must be realised that antibiotic-resistance genes in GM plants originate from microorganisms in the first place. The traits are so widely spread among soil microbes that transfer of these genes from transgenic plants to soil microbes would not create novel gene combinations. Nevertheless, the revised EU regulation on the deliberate release into the environment of GMOs (Directive 2001 18 EC) includes the gradual phasing out of antibiotic-resistance marker genes by the end of 2004 for GMOs to be placed on the market and by end of 2008 in the case of field trials.

Genome Overview at the Level of Chromosomes

In some bacteria essential genes are located on two genetic elements. For instance, in Rhodobacteria sphaeroides the genes encoding the ribosomal RNAs (rRNAs) are located on a 0.9 Mb chromosome, whereas the remainder of the genome is located on a 3 Mb chromosome. In Pseudomonas species, many genes that code for catabolic (degradative) enzymes are located on large extrachromosomal plasmids. Plasmids containing antibiotic resistance genes have been isolated from many species. In part, it is the rapid transfer of plasmids, sometimes even between different genera, that accounts for the rapid development of large populations of antibiotic resistant bacteria.

Temperature Sensitive Plasmids

Plasmids carrying a temperature-sensitive origin of replication for Bacillus have been isolated and used successfully to integrate the plasmid into the chromosome via a cloned insert DNA that bears homology to a region on the chromosome (Youngman, 1990). Unlike nonreplicating integrative plasmids, temperature-sensitive integrative plasmids can be easily introduced into Bacillus by protoplast transformation. After the introduction into the cell of a temperature-sensitive plasmid bearing a homologous cloned region of DNA and an antibiotic resistance gene, the culture temperature is raised above the permissive temperature, which will force the integration of the plasmid into the chromosome and allow the easy selection of such integrants in the presence of antibiotic. Temperature-sensitive plasmids constructed for Bacillus typically use the replication origin from pE194. Although this origin naturally ceases replication above 45 C, a useful mutant has been isolated that is sensitive at...

Investigations At The Subspecies Level

McBurney and colleagues (48) examined the perturbation of the enterobacterial populations of the initial long-term study by McCartney and colleauges (52). Similar to the bifidobacterial populations of these two individuals, the enterobacterial population of subject one was relatively simple and stable (predominated by a single strain) whilst subject two harbored a diverse and dynamic enterobacterial biota (27 distinct strains were identified over 12 months). As mentioned previously, enterobacterial levels were below detection for subject two during weeks 17 and 20. This individual undertook a 7-day course of amoxicillin for a respiratory infection during weeks 21 and 22, after which time the enterobacterial population re-emerged. Most interesting, though, was the antibiotic-resistance profiles of this bacterial group before and after treatment. Strains isolated prior to antibiotic administration were susceptible to a wide range of antibiotics tested, whereas strains isolated following...

Functional Expression Of G Proteincoupled Receptors In Yeast

The state of the art in pharmaceutical drug discovery requires mechanism-based screening assays of high selectivity, sensitivity, and throughput. This is achieved by using cloned gene targets in a robust and miniaturizable system with low background (i.e., a high signal-to-noise ratio). Given these criteria, yeast strains that functionally express heterologous GPCRs are ideal for HTS applications. The diversity of GPCRs successfully expressed in yeast so far indicates that the technology will have applicability to a broad range of therapeutic targets. Beyond this, the yeast system has also proven to be flexible with regard to important practical considerations in pharmaceutical drug discovery. For instance, yeast screening assays can be performed on agar plates or in microtiter trays (liquid format), each with specific advantages. Test compounds spotted on yeast cells imbedded in agar will diffuse radially, thus effectively displaying a response over a large concentration gradient. On...

Antifungal And Antibacterial Screens

Many screens for antibacterial agents have been developed by exploiting the induction properties of antibiotic resistance genes including screens for P-lactam-like, tetracycline-like, and erythromycin-like compounds 135-137 . New screens are developed as additional regulated antibiotic resistance genes are identified. The vanA gene cluster was recently used by three groups to design screening assays for cell wall acting antibiotics 138-140 . While these assays have

Vertical and Horizontal Gene Transfer

The term horizontal gene transfer describes non-sexual gene transfer e.g. from plant to micro-organisms. Micro-organisms, especially bacteria have the ability to take up DNA from other organisms or their environment and to integrate the DNA into their genome. Horizontal gene transfer has been discussed as a risk of gene escape into the environment without any control. During evolution, horizontal gene transfer has taken place, but it is considered to be a very rare event. Still, it cannot be ruled out and in the context of antibiotic-resistance marker genes this possibility has attracted a lot of attention. According to Directive 2001 18 EC antibiotic-resistance marker genes should be phased out for GMOs to be placed on the market until the end of 2004. Of course, alternative marker genes such as those conferring the possibility of metabolising new substrates, have to undergo new risk assessments.

The Most Primitive Immune Systems

Even simple organisms like bacteria are food for viruses and other parasites (sometimes including other bacteria). Not unexpectedly, bacteria have immune responses of their own. One response is the ability to resist natural antibiotics that can exist in their environment. Naturally occurring antibiotic resistance genes from a number of bacteria have been cloned and are routinely used in laboratory microbiology and molecular genetics research. Often, these genes are found on plasmids, simple circular DNA molecules that can be exchanged between bacteria (this exchange along with a rapid life cycle explains the ability of bacteria under antibiotic siege to develop resistance rapidly). Resistance genes on plasmids work in conjunction with other chromosomal genes in bacteria.

Compensatory mutations

There is another factor related to the development of significant antibiotic resistance in bacteria that has not been discussed with reference to mycobacteria. When components of essential macromolecular components (such as ribosomes) are altered by mutation, as in antibiotic-resistant strains, the mutants are frequently growth defective, which can lead to reduction in the pathogenicity of the antibiotic-resistant strains as compared to the drug-sensitive wild-type. This may be more apparent in the case of an intracellular pathogen that is exposed to severe growth-limiting stress in the host. Recent research with Gram-negative pathogens has revealed the possibility of additional factors associated with the acquisition of successful antibiotic resistance genotypes these factors warrant study in mycobacteria as well. Several researchers (Lenski 1997, Levin et al 1997, Schrag & Perrot 1996) have studied the effects of the acquisition of an antibiotic resistance plasmid, or a mutation...

Moral Attitudes that Take New Information into Consideration

The rigorous risk analysis is comprised of toxic, allergic and composition tests. Antibiotic-resistance genes present in some of the first generation transgenic crops are being phased out. To date there are no documented cases that anyone has been harmed as a direct consequence of the use of GMO crops or products. At the same time, tens of thousands of people die yearly of food poisoning. The relative statistical danger of GM products to health is much lower than other problems facing the food industry today, a fact that is readily acknowledged by the consumer associations. A different situation is when considering a new functional food, or crops grown to be used as edible vaccines. Here, more rigorous analysis will need to be carried out to ensure that these new and different crops that substantially differ from their non-transformed parents are indeed safe to eat.

Proposed Health Benefits Of Probiotics

Growth and multiplication in the human gastrointestinal tract Well-documented clinical safety, organism must be accurately identified to strain level before recommending its use. It should be non-toxic, non-pathogenic, non-allergenic, non-mutagenic, non-carcinogenic and have no transferable antibiotic resistance Prevention of pathogen colonization through competition for nutrients and binding sites and through production of antimicrobial substances Clinically documented and validated therapeutic effects. Dose-response data for minimum effective dosage of the probiotic organism in different formulations All of the aforementioned desirable characteristics should be maintained during processing and storage of these products organism should be genetically stable, no plasmid transfer Culture should be suitable for production of acceptable quality finished products with desirable viable counts

Symbiosis and modern biology

The recognition of symbiotic relationships has had a revolutionary impact on modern biological thought. The idea that mitochondria and chloroplasts are transformed by symbiotic bacteria provides a common thread to the biological world and raises hope of finding other symbiotic wonders among life's diversity. Plants and animals have acquired new metabolic capabilities through symbioses with bacteria and fungi. Mammalian herbivores and termites digest cellulose with the help of microbial symbionts. The luminescent bacteria contained in the specialized light organs of some fishes and squids produce marine bioluminescence. Diverse animal life around deep-sea vents is based on symbiosis with bacteria that oxidize hydrogen sulfide and chemosynthetically fix carbon dioxide into carbohydrates. Associations between fungi and algae have resulted in unique morphological structures called lichens. Early land plants formed associations with mycorrhizal fungi, which greatly facilitated their...

Adverse Effects of Antibiotics

Children who received antibiotics in the studies reviewed in the Cochrane review were significantly more likely to experience vomiting, diarrhoea or rash (16.6 in antibiotic group vs. 10.5 in placebo group). However in the more recent LeSaux study, there were no statistically significant differences in the occurrence of diarrhoea, rash or vomiting between children in the placebo and control groups. This may be because the majority of children in this latter study had moderate AOM, and hence more likely to have systemic symptoms already. However, these findings are important since the risk of adverse effects from antibiotics may weigh parents' and clinicians' decision against using antibiotics. Emergence of antibiotic resistance in normal as well as pathogenic bacteria is a further potential side effect of using antibiotics, particularly with longer courses or lower doses of antibiotics (Guillemot

Preparation of recombination template vector

The template vectors that we developed (such as pTosca, Figure 1) consisted of the following basic features, in sequence cloning site 1 (for 5' homology arm), reporter gene (without promoter), poly A, FRT, genes providing eukaryotic antibiotic resistance (to either neomycin, hygromycin, or puromycin) and prokaryotic antibiotic resistance (typically kanamycin), preceded by appropriate promoters, second FRT, cloning site 2 (for 3' homology arm). Figure 1 Design of template vector. The template vector consists of the following components a 5' homologous arm, a reporter gene followed by a poly-adenylated region, an FRT-flanked antibiotic resistance cassette including both bacterial and eukaryotic promoters, and a 3' homologous arm.

Genetic Selection of an Errorprone Variant of Bacteriophage T7 RNA Polymerase


Scheme for the genetic selection of an error-prone RNA polymerase variant based on the reversion of antibiotic resistance. The ''selection plasmid'' carries a mutant tetracycline resistance gene under the control of a T7 promoter. This construct renders the host cell sensitive to tetracycline (tet) unless an Fig. 4.3.2. Scheme for the genetic selection of an error-prone RNA polymerase variant based on the reversion of antibiotic resistance. The ''selection plasmid'' carries a mutant tetracycline resistance gene under the control of a T7 promoter. This construct renders the host cell sensitive to tetracycline (tet) unless an

To How Many Cistrons Do These Mutations Belong

Inactivation of antibiotics and the dissemination of resistance genes. Science 264 275 - 282. Reviews the crisis of antibiotic resistance in bacteria, with particular emphasis on the physiology and genetics of resistance. A very good review of how antibiotic resistance develops and how antibiotics can be developed that are less likely to be resisted by bacteria.

In Vivo Expression Technology Approaches

The original IVET approach involves a tandem set of two promoterless reporter genes, namely purA and lacZ, which were used to identify promoters that are specifically switched on in Salmonella typhimurium during infection (35). Purine auxotroph mutants (DpurA) of Salmonella typhimurium were only able to survive in a mouse model system when complemented in trans with a plasmid encoded purA copy. The promoterless purA gene was thereby utilized as a reporter for the identification of chromosomal fragments that are capable to complement the mutants, thereby strongly selecting for chromosomal fragments which harbor promoter elements that are active in the mouse model system. Subsequently, the in vivo active promoters are tested for the absence of promoter activity in vitro utilizing the second reporter gene (lacZ). The second variation of IVET is based on selection of an antibiotic resistance gene as selectable marker. One obvious disadvantage of this second variation of IVET is that the...

History and Uses of Plant Biotechnology

The story of genetic modification in plants started 1980, when it was demonstrated that a soil bacterium, Agrobacterium, caused tumours in plants after transferring a small but distinct DNA fragment to a plant cell, where it would be incorporated in the nucleus and change the physiology of the local tissue. In 1983 the system was put to use and GM Agrobacteria were used to transfer an antibiotic resistance gene into a tobacco plant. Today advanced methods for the genetic transformation in a wide range of plants have been developed that are based on this natural phenomenon.

Group II Introns as Gene Targeting Vectors

Protein Splicing Images

In addition to disrupting genes, group II introns can be used to integrate cargo genes inserted in DIV at desired chromosomal locations (e.g., Frazier et al. 2003). The insertion of extra DNA in DIV may decrease the integration frequency by making the intron more susceptible to degradation by host nucleases (see Guo et al. 2000). In most cases, however, the frequency remains sufficiently high to detect the desired integration. In the example shown in Fig. 7, this approach was used to engineer lactobac-teria by inserting a commercially important phage resistance gene (abiD) into a specific genomic locus, thereby conferring regulatable phage resistance. The desired insertion was identified at a frequency of 0.5-2 by colony PCR screening. The ability to obtain desired insertions without selection for antibiotic resistance is particularly important in food-grade organisms like lacto-bacteria.

Integrative Vectors

The lack of an efficient transposon system in Bacillus and the availability of an efficient transformation mechanism have prompted the development of integrative plasmids as a tool for genetic analysis. Integrative plasmids are usually shuttle vectors that can replicate in E. coli and carry an antibiotic resistance that allows their selection in B. subtilis (Perego, 1993). On transformation and proper selection, these vectors integrate into the chromosome as long as they carry a fragment of DNA homologous to a region of DNA in the Bacillus chromosome. This allows to target precisely the integration into a specific gene and create gene deletion or interruption (Stahl and Ferrari 1984) and to easily carry out several other genetic manipulation, e.g., creation of partial diploidy (Ferrari and Hoch 1983), deletion of a large fragment of genome (Barilla' et al., 1994), or induction of mini artificial chromosomes (Itaya and Tanaka, 1997). One tremendous advantage of this technique is that...


The backbone plasmid contains most the Ad genome (map units 10-100), except for the extreme left-hand end and a deletion of E3. Recombinants between the shuttle and backbone plasmids containing the whole vector genome are selected by appropriate antibiotic resistance markers.

Gene transfer

The importance of gene transfer in prokaryotic evolution has long been documented (Ochman et al. 2000 Gogarten et al. 2002 Gogarten and Townsend 2005). For example the spread of antibiotic resistance and nitrogen fixation abilities have been linked to gene transfer (Tauxe et al. 1989 Chen et al. 2003). It is estimated that gene transfer has contributed up to 24 of some bacterial genomes (Nelson et al. 1999).

The Pathogens

However, these nosocomial strains of MRSA did not appear to survive well in the community, competing poorly with methicillin-sus-ceptible strains. However, the recent emergence around the world of community-associated methicillin-resistant strains of Staphylococcus aureus (CA-MRSA), apparently associated with fewer antibiotic-resistance genes and often associated with the production of Panton-Valentine leukocidin and perhaps other virulence genes, has occurred at an alarming rate (Holmes et al., 2005 Kaplan, 2005 Kueh-nert et al., 2005 Moran et al., 2005 Robert et al., 2005). These organisms appear well-adapted to a community setting, and have spread quickly in both pediatric and adult populations. While vancomycin (and teichoplanin in several countries) has been the mainstay of parenteral antibiotic therapy of these organisms, vancomycin heteroresistance (decreased susceptibility noted within subpopulations of S. aureus) and complete resistance (due to...


At this point it is worth considering the generation of antibiotic resistance mutations in mycobacteria. For many resistance alleles, the frequency of spontaneous mutation in laboratory studies is relatively low, especially with reference to functions involved in replication (gyrA), transcription (rpoB) or translation (rpsL, rrnA, rrnB), which take place generally at rates of 10_7 per generation (or less) in bacteria. Do mutations conferring antibiotic resistance occur at a more significant rate in vivo While studies with M. tuberculosis infection provide no evidence for such an increase, recent findings with other bacterial pathogens may be significant. LeClerc et al (1996) found that human pathogenic Salmonella enterica and E. coli exhibited an unexpectedly high incidence of the mutator phenotypes commonly associated with methyl-directed mismatch repair. It was suggested that there exists a relationship between hypermutability and pathogenicity such genetic variability may provide...


Bacterial Chromosome Replication Diagram

In addition to having a chromosome, many bacteria possess plasmids, small, circular DNA molecules ( FIGURE 8.6). Some plasmids are present in many copies per cell, whereas others are present in only one or two copies. In general, plas-mids carry genes that are not essential to bacterial function but that may play an important role in the life cycle and growth of their bacterial hosts. Some plasmids promote mating between bacteria others contain genes that kill other bacteria. Of great importance, plasmids are used extensively in genetic engineering (Chapter 18) and some of them play a role in the spread of antibiotic resistance among bacteria.

Genetic Engineering

Shoots, whereas, the cells containing the selectable marker and gene of interest can continue to grow and regenerate shoots. Various selectable markers have been used successfully for tomato transformation. The most commonly used selectable marker for tomato transformation has been the neomycin phosphotransferase gene (nptII), which was isolated from the transposon Tn5 that was present in the bacterium strain Escherichia coli K12. This gene confers resistance to a range of aminoglycoside antibiotics including kanamycin, which is the most commonly used selection agent when nptII is used as the selectable marker gene. Another antibiotic resistance gene that has been used successfully for tomato transformation is the gene for hygromycin phosphotransferase (hpt), which is also from E. coli and confers resistance to the antibiotic hygromycin (Van Eck et al. 2006). Van Eck etal. (2006) also reported the use of the herbicide resistance gene, bar, from Streptomyces hygmscopius as a selectable...


Common cold prevention A 12-week, double-blind randomised study involving 146 people demonstrated that allicin-containing garlic preparations significantly reduce the incidence of colds and accelerate recovery compared with placebo (Josling 2001). More specifically, the number of symptom days in the placebo group was 5.01 compared with 1.52 days in the garlic treated group. Additionally, garlic reduced the incidence of developing a second cold whereas placebo did not. Helicobacter pylori infection It has been suggested that gastrointestinal lesions, such as gastric ulcers, duodenal ulcers and gastric cancers, are strongly associated with H. pylori infection (Scheiman & Cutler 1999). Medical treatment consisting of 'triple therapy' has a high eradication rate, yet is associated with side-effects and has started to give rise to antibiotic resistance. Since garlic intake has been associated with a lowered incidence of stomach cancer, researchers have started investigating whether...

Social Security

I could indeed write here about the issues this disease highlights. I could discuss antibiotic resistance, controversial treatment options, physician care that drives many to self-care, depression, suicide, lack of pain-management options, the physical problems of odor and leakage, lack of diagnosis or misdiagnosis, disability, healthcare costs, loss of self-esteem, disfigurement, secrecy, isolation, lost relationships, cultural taboos and nomenclature, confusion over and between treatment advisors, lack of treatment guidelines and the list goes on. Indeed I could discuss many things including a host of mythologies that have followed this disease for 150 years, but individuals with this disease can speak for themselves. Collectively they have that power now. They are finally free of their long, lonely and painful silence. All that I have done and learned during those 6 years can be stated fairly succinctly living with this disease creates a special human being, one infused with an...