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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Development And Transfer Of Antibiotic Resistance

These mutations occur at a background mutation rate in the absence of selection pressure. Organisms can also become resistant by acquiring a resistance gene or genes from other organisms by horizontal transfer. DNA carrying the gene(s) can be acquired from other microbes by 1) conjugation, direct cell-to-cell transfer of DNA through a membrane protein complex 2) transduction with a bacteriophage and 3) transformation, the uptake of naked DNA from the environment. Conjugative DNA elements that carry antibiotic resistance genes include plasmids and conjugative transposons. These genetic elements can carry more than one antibiotic resistance gene, contributing to multidrug resistance. In animal agriculture, these plasmids containing multiple antibiotic resistance genes are often seen in E. coli, and the plasmids can often be very large (> 50 kb). Tetracycline, florfenicol, and p-lactamase resistance genes are often found on these plasmids. Integrons are another genetic element...

Antibiotic Resistance

Element could be conjugally transferred from V. cholerae O139 to V. cholerae O1 and Escherichia coli strains, where it integrated into recipient chromosomes in a site-specific rec-A-independent manner. Considering the rapidly changing patterns of antibiotics observed among V. cholerae strains, it appears that there is substantial mobility in genetic elements encoding antibiotic resistance in V. cholerae.

Antibiotic resistance of nonenterococcal LAB 2041 Lactobacillus

Lactobacilli possess a wide range of naturally antibiotic resistances (Charteris 1998b Danielsen 2003). For instance L. plantarum, L. rhamnosus, L. fermentum, L. casei or L. paracasei are naturally resistant to vancomycin and teicoplanin. However, species of the L. acidophilus taxonomic group (L. acidophilus, L. gasseri, L. crispatus) or L. delbrueckii are susceptible to the glycopeptides (Danielsen 2003 Felten 1999 Illot-Klein 1994 Nicas 1989 Swenson 1990). Charteris et al. (1998b) found that L. acidophilus was resistant to vancomycin, which could reflect methodological problems (antibiotic susceptibility testing was done by agar disc diffusion, which is not recommended for glycopeptides). Rare acquired antibiotic resistances for tetracyclines, macrolides, chloramphenicol, and fluoroquinolones have been described (Felten 1999 Mandar 2001 Vescovo 1982 Zarazaga 1999). Finally, transfer of antibiotic resistance genes between phylogenetically distant bacteria (by mobile elements that...

Antibiotic Resistance In Bacteria

The genetic changes associated with the evolution of antibiotic resistance are well documented (21) and can be classified according to whether they involve lateral gene transfer or chromosomal mutagenesis. However, some pathogenic bacteria may be incapable of exogenous DNA uptake (121) or may occupy specific environments within the host that restrict the opportunities for the acquisition of novel, transmissible genetic elements. For example, the sterility of the ecological niche occupied by M. tuberculosis, coupled with the apparent absence of natural competence in mycobacteria (23), implies increased roles for rearrangement and point mutation, rather than lateral gene transfer, in the evolution of resistance within this organism - factors which probably apply to most pathogens colonising locations other than such g

Dormancy and Low Growth States in Microbial Disease

Organisms multiply only when the conditions are beneficial, and when not multiplying, they concentrate on survival of environmental stress. Many bacteria that harm humans survive for most of the period of infection in a low-growth state. This book addresses the basic scientific aspects of microbial dormancy and low-growth states, and places them in the context of human medicine. The book introduces basic scientific aspects of bacterial growth, non-growth, culturability, and viability. Later chapters cover the crucial relationship between low-growth states and survival of stress, the survival of the immune response, and interbacterial signalling. This is followed by chapters on aspects that are of direct importance to medicine, namely antibiotic resistance arising in stationary phase, biofilms, tuberculosis, and the bacteria, which cause gastric ulcers.

Section Iii Food Contamination and Safety

Principle 2 Determine Critical Control Points (CCPs) Principle 3 Establish Critical Limits for Preventive Measures Principle 4 Establish Procedures to Monitor CCPs Principle 5 Corrective Action When a Critical Limit Is Exceeded Principle 6 Effective Record-Keeping Systems Principle 7 Verify That the HAACP System Is Working Antibiotic Resistance

General Modes Of Resistance

Antibiotic resistance mechanisms fall into various categories for both resistance to specific antibiotics and for multidrug resistance. 1,2 In general, the mechanisms for resistance to specific antibiotics include 1) mutations that prevent the antibiotic from affecting the target, 2) porins or other cytoplasmic membrane proteins that actively pump the antibiotic out of the cell, 3) enzymatic inac-tivation of the antibiotic, 4) modification or protection of the antibiotic target, 5) circumvention of the effects of the antibiotic, and 6) failure to activate the antibiotic. The mechanisms used for multidrug resistance include 1) efflux pumps in the cytoplasmic membrane that actively pump multiple antibiotics out of the cell, 2) alterations to the cytoplasmic membrane or cell wall such that the antibiotics do not enter the cell, and 3) formation of protective biofilms that restrict exposure of the microbes to antibiotics.

Glycopeptide Antibiotics

Glycopeptide antibiotics such as vancomycin and teicho-planin bind the D-alanine dipeptide and inhibit the transglycosylation and transpeptidation steps in peptido-glycan synthesis of the cell wall. Gram-negative bacteria are generally not affected by glycopeptide antibiotics as these antibiotics cannot transverse the outer membrane and gain access to the peptidoglycan in the cell wall. Glycopeptide antibiotic resistance in Gram-positive bacteria is most often mediated by changes in the pep-tidoglycan side-chain thus circumventing the effects of

Emerging Approaches To Finding And Identifying Contaminants

The advance of genetic-based identification DNA fingerprinting (100) may come to aid the resolution of sterility-test failure ambiguity in that genomic characterization makes it possible, in theory, to determine the origin of contaminants (i.e., a true product contaminant or an artifact of testing) based on an organism's genetic relatedness to environmental isolates, either of production or lab origin. Genetic methods are being developed for analogous epidemiological purposes in other disciplines including diagnosing, identifying, and tracking the origin and progress of infectious agents (101) and food-borne disease without the concomitant need for microbial enrichment (102), in some cases supplanting traditional, culture dependent serotyping (103) tracking antibiotic resistance genes (104) and tracking the origin of organisms used for bioterrorism (i.e., anthrax) (105,106). This latter field has been referred to as microbial forensics (107).

Routes Of Host Invasion And Types Of Organisms Implicated

Sepsis is a modern epidemic created by the success of medical technology (19). The use of antibiotic therapy, invasive medical procedures and devices, immunosup-pressive therapy for transplant patients, radiation and chemotherapy in cancer patients, an increased elderly population and those with serious underlying diseases, as well as the gathering of patients into critical care units and nursing homes have all contributed to increasing the pool of potential sepsis victims (20). The use of antibiotics has paralleled the rise in the number of fatal infections due to gram-negative bacteria as a result of (i) the fact that antibiotics select for gram-negative organisms (due in part to LPS provided protection from antibiotic efficacy) and (ii) the rise of increased bacterial antibiotic resistance (21).

Mechanisms of Resistance

The phenomenon of antibiotic resistance in some cases is innate to the microbe. For instance, penicillin directly interferes with the synthesis of bacterial cell walls. Therefore, it is useless against many other microbes such as fungi, viruses, wall-less bacteria like Mycoplasma (which causes walking pneumonia), and even many Gram negative bacteria whose outer membrane prevents penicillin from penetrating them. Other bacteria change their genetic programs, which allows them to circumvent the antibiotic effect. These changes in the genetic programs can be in the form of chromosomal mutations, acquisition of R (resistance) plasmids, or through transposition of pathogenicity islands. An example of a chromosomal mutation is the increasing number of cases of penicillin-resistant Neisseria gonorrhae. This bacterium mutated the gene coding for a porin protein in its outer membrane, thereby halting the transport of penicillin into the cell. This is also termed vertical evolution, meaning...

Resistance and Public Health

The effects of antibiotic resistance are reflected in the agriculture, food, medical, and pharmaceutical industries. Livestock are fed about half of the antibiotics manufactured in the United States as a preventative measure, rather than in the treatment of specific diseases. Such usage has resulted in hamburger meat that contains drug-resistant and difficult-to-treat Salmonella Newport, which has led to seventeen cases of gastroenteritis including one death. Some MDR-tuberculoid strains arise because patients are reluctant to follow the six-months or more of treatment needed to effectively cure tuberculosis. If the drug regimen is not followed, less sensitive bacteria have the chance to multiply and gradually emerge into resistant strains. In other cases the shotgun method of indiscriminately prescribing taking several antibiotics runs the risk of creating super MDR-germs. Moreover, millions of antibiotic prescriptions are written by physicians each year for viral infections, against...

Microbial Populations And Resistance To Antibiotics

Antimicrobial resistance is an example of how bacterial traits are selected by the environment and incorporated into the prokaryotic genome. LPS, as a necessary constituent of the bacterial cell, cannot be eliminated from viable cells as can antibiotic resistance genes, but as we have seen, LPS can be presented in alternating alternative forms. In similar fashion, the introduction of antibiotics into a population of bacteria creates an arena in which those mutations that can best survive, thrive, and perpetuate their survival traits and often do so due to an acquired resistance to the antibiotic in question. This topic fits into the subject of biodiversity because antibiotic resistance is a prime and significant example of such genomic sharing via plasmids that bring about and perpetuate the diversity that is generated.

Cloning and Recombinant DNA

Recall that bacteria possess a single circular chromosome. In addition, they, as well as some eukaryotes (e.g., yeast) and some plants, can also contain an additional piece of circular double-stranded DNA called a plasmid. In bacteria, the plasmid often contains genes for antibiotic resistance. Bacteria can exchange plasmids, and plasmids can exist outside the cell. Their possession of antibiotic genes makes it easy to select for bacteria that have been transfected by recombinant plasmid DNA. Whichever vector is used, it is transfected into an appropriate host cell using chemical and or physical techniques. It is somewhat of a random process only a fraction of the cells in a culture will be transfected successfully. Antibiotic resistance or other marker genes are included in the transfection to help select the desired cells. These are then cultured for further application.

Transformation of mES cells for selective enrichment of EBderived cardiomyocytes

The requisite mES-cell modification involves transfection with a plasmid containing (i) a gene allowing positive selection of transformed, undifferentiated mES-cell clones, and (ii) a gene conferring antibiotic resistance (or, alternatively, a fluorescence cell marker such as EGFP) controlled by a muscle-restricted promoter that is active in the differentiated cells. For (i), the relatively position-independent, constitutive promoters for phosphoglycerate kinase (PGK-1), RNA polymerase II, b-actin, and elongation factor 1 alpha (EF-1a) are most commonly used (30). For (ii), several tissue-restricted promoters are effective for isolation of specific muscle-cell types lineages, e.g. the cardiac-restricted NCX1 and aMhc promoters, and the ventricular-associated Mlc2v promoter. For positive selection in (i) and (ii), the most commonly used genes are those conferring resistance to the antibiotics, neomycin (neo), puromycin (pac), and hygromycin (hph), for which the in vitro selective...

Escherichia Coli As A Carotenoid Production System

Escherichia coli is a very convenient host for heterologous carotenoid production. Because of its fast and easy cultivation in substantial quantity, it can be transformed simultaneously with several plasmids as long as they belong to different incompatibility groups, i.e., possessing different origins of replication. For their stable maintenance in the cells, it is essential that each plasmid carries a different antibiotic resistance. Only when this selection pressure is maintained spontaneous plasmid loss prevented. Several useful plasmids for cotransformation are available. Convenient vectors are pUC-related plasmids with the pMB1 origin of replication and ampicillin resistance, pACYC184 with a p15A origin of replication and chloramphenicol resistance, pRK404 with an RK2 origin of replication and tetracycline resistance, and pBBR1MCS2 with a SC101 origin of replication and kanamy-cin resistance. These plasmids can be used for expression of individual genes or groups of genes which...

Helicobacter pylori infection

There are not many studies on the attenuation of microbiota disturbances following an anti-Helicobacter triple treatment by probiotics. In a pilot study, Madden et al. (2005) found that probiotic combination including two strains of L. acidophilus (CLT60 and CUL21) and two strains of B. bifidum (CUL17 and Rhodia) suppressed the rise in the numbers of facultative anaerobes seen in the placebo group. Later, the same probiotic product was also able to suppress a rise in the amount of antibiotic-resistance among enterococci, the effect of which was seen in the placebo group during and after the triple therapy (Plummer et al., 2005). However, despite the probiotic supplementation the microbiota in both studies were susceptible to the effects of the antibiotics administered to eradicate H. pylori.

Pathogenically Dormant Bacteria

In some cases bacteria can be present and metabolically active but not actively virulent until circumstances favour pathogenesis. Put simply, some bacteria will lie low and try to proliferate while evading host defences until such a time as the population has gathered sufficient numbers to produce a pathogenic insult capable of overwhelming the host and its defences. These bacteria can be considered pathogenically dormant but not metabolically dormant. The roles of population size, evasion of host defence and quorum sensing are entwined in the control of pathogenicity of at least two important pathogens, Staphylococcus aureus and Pseudomonas aeruginosa. Both organisms are common in our environment and are responsible for a wide range of infections (see 2, 15, 55, 56, 101 for reviews). Often these infections are hospital-acquired and are difficult to treat because of antibiotic resistance (reviewed by 15, 55, 106). The pathogenesis of both species relies upon the coordinated expression...

Phylum Firmicutes The low GC Grampositive bacteria

The cells of staphylococci occur in irregular bunches rather than ordered chains. They also produce lactic acid but can additionally carry out aerobic respiration involving cytochromes, and lack the complex nutritional requirements of the lactic acid bacteria. They are resistant to drying and able to tolerate relatively high concentrations of salt. These properties allow Staphylococcus aureus to be a normal inhabitant of the human skin, where it can sometimes give rise to dermatological conditions such as acne, boils and impetigo. It is also found in the respiratory tract of many healthy individuals, to whom it poses no threat, but in people whose immune system has been in some way compromised, it can cause serious respiratory infections. S. aureus can also cause a type of food poisoning and is the causative agent of toxic shock syndrome. Widespread antibiotic use has been largely responsible for the development of resistant forms of S. aureus, which have become ubiquitous inhabitants...

Safety Of Antimicrobials

Complete ban would likely have little effect on antibiotic resistance levels or patterns, according to some long-term, antibiotic withdrawal studies. 10 Three years after the European ban on growth-promoting antibiotics, there was little effect on resistance levels in humans, whereas the health of pigs and chickens markedly deteriorated. 16

The special case of probiotics

When SCAN guidelines for the evaluation of micro-organisms used as feed additives were formulated, no exemption was made for the species and strains that have a history of human use. it was soon recognized that this led to an anomalous situation where a strain is subjected to a much more stringent safety evaluation, if it is intended as feed additive, than would be the case if it were used as a food starter or human probiotic. The large number of species and strains already used in food production further makes their safety evaluation strain by strain a practical impossibility. Consequently a position paper 'Safety assessment and regulatory aspects of micro-oranisms in feed and food applications' was published by SCAN in 2002 (http europa.eu.in comm. food fs sc Scan outcome_en.html). In this paper it was suggested that specific micro-organisms with a history of safe use or otherwise established safety, could be compiled in order to allow for a generic approval of microorganisms for...

Sources of further information and advice

Respectively) and direct links to various EU regulations and directives can be found at http europa.eu.int eur-lex . Partial lists of GRAS micro-organisms and their uses in foods can are presented at http www.cfsan.fda.gov dms opa-micr.html. The ACE-ART project, which has relevance to the question of the presence of transmissible antibiotic resistance markers in starters or probiotics, and might also have regulatory implications, is presented at http www.aceart.net asp .

Transfection of mES cells with selectable marker genes for neuronal and oligodendroglial lineage selection

Lineage selection involves the isolation of a desired phenotype from a heterogeneous cell population. This can be facilitated by transfecting undifferentiated mES cells initially with selectable marker genes that are expressed under control of a cell type-specific promoter. Following cell differentiation, and if the marker is an antibiotic-resistance gene, selection is performed by adding the respective antibiotic to the culture medium. Alternatively, reporter genes such as EGFP permit enrichment of the desired, differentiated phenotype by FACS . Protocols 1-5 encompass controlled differentiation and lineage selection mES cells are first transfected with lineage-selection constructs (Protocol 2), differentiated according to Protocol 1, and subsequently subjected to lineage-selection strategies such as FACS , immunopanning or antibiotic selection (see Protocols 3, 4 and 5). To enable the isolation of transfected clones, mES cells should be cotransfected with constructs carrying an...

Nature of Genetic Variation

We can use the study of genetic variation to examine differences between members of the same species, ranging from the study of bacterial characteristics (such as antibiotic resistance) to investigation of human genetic diseases, or to differentiate between individuals (for example in forensic analysis). Alternatively, we can compare the genetic composition of members of different species - even over wide taxonomic ranges - which can throw invaluable light on the processes of evolution as well as helping to define the taxonomic relationship between species. Many of the concepts involved are similar, but of course the differences are (usually) much greater when we are comparing widely different species.

Single nucleotide polymorphisms

However, it is much easier to obtain a mutation that results in the loss of function than one which changes the characteristics of the product, so although we can study the products of changes that have accumulated over a long time scale, we rarely see these changes in action. The main exception to this is where there is a strong selective pressure in favour of such a change - the best example being some types of antibiotic resistance in bacteria. For example, rifampicin resistance can be caused by specific mutations in the gene (rpoB) for one of the subunits of RNA polymerase. Although this is an essential enzyme, and therefore there are constraints on the mutations that can be tolerated, the use of rifampicin as an antibiotic provides a selective pressure under which only those rare mutants with an appropriate rpoB mutation can survive.

Moving Genes between Species

The next step in the process is the introduction of the cloning vector with its segment of new DNA into a living cell. Bacteria have the ability to transport DNA into their cells in a process called transformation, and this ability is commonly exploited to achieve this goal. Getting the DNA into the cell, however, is only the beginning. No transformation is 100 percent efficient, and so the bacteria that receive the gene(s) of interest must be separated from those that did not. One of the best studied and most commonly used cloning vectors, pBR322, is especially useful for this purpose, as it contains several genes for antibiotic resistance. Hence, any cell transformed with DNA containing pBR322 will be antibiotic resistant, and thus can be isolated from similar cells that have not be so transformed by merely growing them in the presence of the appropriate drugs. All that remains is to identify bacteria that are producing the product of the desired gene(s), and cloning is a success.

Use in Research and Technology

Under certain circumstances, recombinant DNA experiments using plasmids are considered to be hazardous, and the ease with which plasmids are acquired by bacteria has led them to be classed as biohazards. They are therefore subject to guidelines and may require registration and approval. A publication produced by the National Institutes of Health, titled Guidelines for Research Involving Recombinant DNA Molecules, is the definitive reference for recombinant DNA research in the United States and should be consulted when considering research, particularly biomedical research, involving plasmids. see also Antibiotic Resistance Cloning Genes Conjugation Inheritance, Extranuclear Marker Systems Restriction Enzymes Transformation.

Molecular Biology Microbial

Polymerase chain reaction methods can be used to detect a variety of specific genes in environmental samples without prior cultivation and isolation of microorganisms. They have been applied particularly to virulence determinants (e.g., toxin genes) providing information on pathogen contamination in the food chain. PCR tracking of antibiotic resistance genes in the environment and in human and animal gut bacteria has implications for the debate over the impact of antibiotic use in animal husbandry, which centers on resistance to the antibiotics used in clinical and veterinary medicine. Microorganisms, especially bacteria, frequently carry genetic elements that are capable of transfer between cells independently of the main chromosome. Such elements, which include extrachromosomal plasmids and chromosomally located conjugative transposons, possess genes that promote their own transfer by cell cell contact (conjugation), or that allow them to be mobilized by other elements. In addition...

Transposon Mutagenesis

A further feature of transposons that is relevant here is that they generally carry antibiotic resistance genes. Indeed they play, together with plasmids, a major role in the spread of antibiotic resistance genes amongst pathogenic bacteria. However, transposition not only moves genes between different sites insertion of a transposon within a coding sequence will usually inactivate that gene, thus producing a mutation. The site of that mutation is now marked by the presence of the resistance gene, which makes it relatively easy to clone, and thus to identify, the affected portion of the DNA. Assuming we can identify a number of mutants that may be of interest, the next step is straightforward. We can extract genomic DNA from those cells, digest it with a restriction enzyme, and ligate these fragments with a suitable vector. In effect, we create a genomic library, but we do not need the complete library we are only interested in those fragments that carry the transposon. We can...

Allelic Replacement and Gene Knockouts

A typical procedure for allelic replacement in a bacterial host would be to manipulate the cloned gene so as to replace the central part of the gene with an antibiotic resistance gene (see Figure 14.7). We would do this using a suicide plasmid (as described above, for transposon mutagenesis) so that when we transform the bacteria with the construct, only those cells in which the resistance gene has become incorporated into the chromosome will become antibiotic resistant. We can select these on agar containing the antibiotic. If things go well, incorporation into the chromosome will have occurred by homologous recombination at the required position, thus inactivating the gene concerned.

Pseudomonas Aeruginosa Infection And Immunity

Pseudomonas aeruginosa is a major cause of infections in Western society, being the causative agent of about 200 000 nosocomial infections per year in North America. Such infections tend to be rapidly progressing and pose serious risks to patients. However, P. aeruginosa can also cause long-term (up to 10 years or more) chronic infections in the lungs of patients with cystic fibrosis, and the onset of P. aeruginosa infection signals the eventual deterioration of lung function in such patients, leading to death from respiratory failure. P. aeruginosa shows high intrinsic antibiotic resistance. While there are antibiotics that show initial success against Pseudomonas infections, even modest mutations lead to clinically significant resistance, making development of antibiotic resistance common and successful treatment difficult. For this reason considerable effort has been expended in seeking vaccine or immunotherapeutic solutions to such infections. At the same time, diagnosis and...

Genetic markers detection of particular strains in the aquatic environment

Bacteria that have been labelled with an introduced novel gene (the genetic marker) can be identified either on the basis of a specific phenotypic characteristic (e.g., antibiotic resistance) in culture, or directly in the environmental sample using a specific molecular probe. Studies attempting to assess the survival and transfer of recombinant organisms in aquatic environments have typically relied on plas-mids which contain antibiotic resistance markers. Attempts to follow the transfer and survival of such plasmids in aquatic systems can be severely limited by the presence of such genes in the natural population, resulting in a high background resistance level. The development of marker systems which avoid the use of antibiotic resistance circumvents this problem, and has been used by Winstanley et al. (1992) to detect pseudomonads released into lake water. The system developed by these workers involves the detection of a marker gene (xylE) and its product catechol 2,3 dioxygenase....

Resistance to antibiotics

Box 14.6 Where did antibiotic resistance come from Have genes responsible for antibiotic resistance always existed in nature, or have they arisen since the development and widespread use of antibiotics The answer, almost certainly, is the former. A sample of an E. coli strain, freeze-dried in 1946, was revived many years later, and found to have plasmid-encoded genes for resistance to streptomycin and tetracycline, neither of which were in clinical use until some years after the culture was preserved. It seems likely that bacteria possessed these genes to protect against naturally occurring antibiotics, an idea supported by the fact that R-plasmids have been found in non-pathogenic soil bacteria. Also, resistance to a number of antibiotics has been demonstrated in soil and water bacteria from sources sufficiently remote to be free from anthropogenic influence.

How does resistance arise

Occasionally, mutations occur spontaneously in bacteria, which render them resistant to one antibiotic or another. Usually the mutation leads to a change in a receptor or binding site such as those just described, rendering the antibiotic ineffective. The changes are usually brought about by point mutations (see Chapter 11) occurring at very low frequency on chromosomal DNA. Bacteria can, however, become resistant much more rapidly by acquiring the mutant resistance-causing gene from another bacterium. This is called transmissable antibiotic resistance it occurs mainly as a result of bacterial conjugation, and is the cause of most of the resistance problems we presently face. Transmissable resistance was first reported in Japan in the late 1950s, when multi-drug resistance in Shigella was shown to have been acquired by conjugation with resistant E. coli in a patient's large intestine. E. coli is known to transfer R (resistance) plasmids to several other gut bacteria including...

Use Of Antimicrobials To Enhance Fermentation Efficiency

However, the use of antibiotics as animal growth promotants has come under increased scrutiny due to problems associated with antibiotic resistance (discussed elsewhere in this encyclopedia). In response to this issue, the European Union has recently (2003) enacted a ban on the use of all antimicrobial feed additives in animal rations it remains to be seen if the United States will follow suit. Therefore, the use of antibiotics, especially those used in human medicine, to enhance the efficiency of the rumen fermentation is not widespread or encouraged.

Emerging Food Safety Issues in a Modern World

In addition to the concerns previously expressed about emerging foodborne pathogens, because of the ever-changing nature of the area, food safety is often faced with other new or reemerging issues. Science-based solutions should prevail for such issues however, it is likely that the road to such solutions is long and tedious. The following discussion regarding issues of HACCP (Hazard Analysis of Critical Control Points), antibiotic resistance, and genetically modified organisms (GMOs) provides just a few examples of issues that have challenged, and continue to challenge, scientists in the field.

Understanding microbial adaptation to stress

Next to these valuable studies in model organisms, the advent of fast full genome sequencing has also made it possible to come to a comparison of the genomes of various strains (isolates) of non-pathogenic and pathogenic bacteria and fungi. In this way, the study of interspecies heterogeneity has become experimentally accessible. A comparison of sequenced genomes has shown for instance that there is substantial variation in genes present among pathogenic strains of Escherichia coli and Staphylococcus aureus as discussed in Fitzgerald and Musser (2001). In various isolates horizontal gene-transfer has been shown. Gene-based variation in physiology among strains of a given species has recently been illustrated as a key mechanism operative in soil where antibiotic resistance distribution was shown to be prominently mediated by horizontal gene transfer among a variety of micro-organisms as discussed in Nwosu (2001).

Differentiation Of Human Embryonic Stem Cells

To study the biology of human ES cell derivatives and to generate cells for cellular based transplantation therapies, pure populations of progenitor or somatic cell types need to be derived. Different strategies can be used to achieve this objective. First, the addition of soluble inducing factors to human ES cells may direct their differentiation and or facilitate growth of particular lineages. Second, using markers expressed by lineage-committed progenitors during embryogenesis, isolation or depletion of particular cells from mixed cell populations can be achieved with antibodies raised against cell-surface markers and subsequent fluorescence-activated cell sorting (FACS) or immunomagnetic bead separation. Third, through genetic selection using selectable markers under the control of lineage-specific promoters, specific cell types can be isolated, generating pure cell populations. Thus, for example, using homologous recombination, Li and colleagues (1998) genetically modified mouse...

Genetically Manipulated Organisms

Another area of major concern is that that many of the constructs released into the environment carry genes for antibiotic resistance. In Chapter 9, the rationale for including such genes was described but, briefly, its function is during the construction of the recombinant and, especially in eukaryotic recombinants, serves no function in the final GMO. This being the case, it can be argued that it is reasonable to require the removal of all such selector and reporter genes before release of the GMO. Accordingly, it is especially true because of the limited number of selector and reporter genes in current use. Consequently, it is likely that while the gene or genes of interest may be unique, or almost so, to that construct, it will probably be carrying one of a very limited number of selector or reporter genes. As a result, the total number of GMOs released worldwide in one year, carrying one particular selector or reporter gene could be very high indeed. Given that true figures for...

Construction of Mutants by Recombination Using Linearized Targeting Vectors

In many cases, the construction of the targeting vector makes use of standard recombinant DNA technology that is widely available (1). We therefore restrict our discussion to some general remarks. Gene deletion is performed by exchanging the gene of interest against an antibiotic resistance cassette (e.g., against kanamycin or tetracyclin). In favorable cases, the gene of interest will contain restriction sites that allow its complete or nearly complete excision in a single step (Fig. 1A). A subclone of the viral genome containing the gene of interest and its flanking sequences can then be digested with these restriction enzymes and ligated to the antibiotic resistance cassette. If it is not possible to delete the gene of interest entirely by this approach, ligation introduction of the antibiotic resistance cassette should be performed in such a way that it leads to a frame shift in the remaining sequences of the deleted gene. In addition, it is essential that the final version of the...

Live recombinant vaccines

Live Recombinant Vaccines

Other candidate carriers, notably attenuated strains of Salmonella and also the TB vaccine BCG. These are very safe vaccines, and the manipulations involved are relatively straightforward. At a basic level, you simply clone the genes you want on a shuttle vector, including appropriate expression signals, and transform the chosen host. Ultimately there are further considerations we usually use antibiotic resistance genes as a selectable marker for this for practical use as a vaccine, the inclusion of resistance genes is unlikely to be acceptable, and alternative strategies have to be adopted. Furthermore, plasmids are generally not completely stable, and insertion into the chromosome is preferred. One way of achieving this is to use a suicide plasmid with a bacteriophage integration system. The expression of the phage integrase then recombines the plasmid with the attachment site on the chromosome. There is a wide range of such potential vaccines at different stages of development.

The Homologous Recombination Process

A modified version of the target gene replaces it in the chromosome. The target gene is removed and degraded. In this example, the gene is modified by insertion of an antibiotic resistance gene, which both inactivates the gene and allows efficient selection of transformed cells. Homologous recombination of a DNA vector into a gene of interest can be done in almost any cell type but occurs at a very low frequency, and it is therefore important to detect the few cells that have taken up the gene. Gene targeting vectors are designed with this in mind. The simplest strategy is to include an antibiotic resistance gene on the vector, which interrupts the sequence homologous to the gene of interest and thus makes the inserted gene nonfunctional. This selectable marker gene makes the cells that possess it resistant to antibiotics, and can then be used to eliminate cells that are not genetically modified.

Prokaryotic Biodiversity And Prokaryotic Phylogenetics

After all, it is in their sheer numbers, not as individuals, that they are overwhelmingly effective, and it is by virtue of their numbers that their attributes are so quickly selected for, as demonstrated by an example of antibiotic resistance, briefly discussed in section Heterogeneity Conclusions. Therefore, the historical model of the prokaryote as a static, freestanding being is, more often than not, misleading as an aide to understanding prokaryotes and points to the fact that their boundaries and borders with one another maybe more dynamic and in flux than previously believed. The fortress format of bacterial growth can provide a formidable defense against both pharmaceutical manufacturing contaminant containment (i.e., in water systems) and against host-defense mechanisms.

Dairy products and probiotics in childhood disease

The mass market for supplementation of dietary products with probiotics carries some risk of transmission of pathogenicity or antibiotic resistance between different bacteria and the probiotics used. This would be in particularly relevant when genetically modified bacteria enter the use in humans.

Safety testing of probiotics with regard to antimicrobial susceptibility testing

The determination of epidemiological MIC breakpoints seems to be more relevant than the definition of clinical breakpoints to recognize acquired and potentially transferable antibiotic resistances in non-enterococcal LAB species. Epidemiological cut-offs should be defined at the species level (not genus level). However, there are no published breakpoints for non-enterococcal LAB, and several authors recommend to take the MIC breakpoints of streptococci other than S. pneumoniae according to the CLSI documents. The PROSAFE project proposes tentative epidemiological cut-off values. Natural (intrinsic or inherent) antibiotic resistance is (probably) nontransferable and present in the wild type population of a given taxonomic group. It is a species- or genus-specific property of all (or nearly all) members of the corresponding taxonomic group. In contrast, bacterial strains with acquired antibiotic resistances are characterized by MICs that are higher than the normal range of the MIC...

Plasmid Vectors 677 Properties of plasmid vectors

The most notorious property of plasmids lies in their ability to disseminate antibiotic resistance genes. They are responsible to a large extent for the spread of antibiotic resistance - although it should be noted that the plasmids used for gene cloning are nearly always unable to spread from one bacterium to another, and there are restrictions on experimental protocols to ensure that these experiments do not add new antibiotic resistance genes to clinically important pathogenic bacteria. Antibiotic resistance is not the limit of the ability of plasmids, nor the reason for their existence. Interest tends to focus on antibiotic resistance because of its importance in medical microbiology, and because of the ease with which resistance genes can be isolated and studied. However, many naturally occurring plasmids code for other properties, or even for none So we have a plasmid with a replication origin and one or more restriction sites. One further feature is essential for a functionally...

Treatment of acute uncomplicated cystitis in young women

Trimethoprim-sulfamethoxazole remains the antibiotic of choice in the treatment of uncomplicated UTIs in young women. Fluoroquinolones are recommended for patients who cannot tolerate sulfonamides or trimethoprim or who have a high frequency of antibiotic resistance. Three days is the optimal duration of treatment for uncomplicated cystitis. A seven-day course should be considered in pregnant women, diabetic women and women who have had symptoms for more than one week.

Comment and clinical implications

Common practice to prescribe a short course of oral antibiotics in acute infected eczema. There is some evidence to support this, and no evidence of a detrimental effect. In contrast, there is no evidence to support the use of longer-term antibiotics in people with atopic eczema whose skin is colonised with S. aureus, and there is some evidence that use of such antibiotics may promote antibiotic resistance.

Hematologic malignancy

Fever in severely neutropenic patients must be treated promptly. Blood and other appropriate cultures must be taken and the patient given intravenous antibiotics without waiting for the results. The choice of suitable antibiotics is normally determined by local policy in the light of knowledge of typical pathogens and patterns of antibiotic resistance. A typical approach is to use ceftazidime or a combination of an aminoglycoside with a semisynthetic

Stem Cell Therapy

Potential Hurdles One major interest in ESL cells is the prospect of exploiting them therapeutically to repair damage to tissues or organs resulting from disease or injury. This poses a host of new challenges, not all of which have received the attention they deserve. Perhaps the most obvious challenge is whether it will be possible to obtain efficient directed differentiation of stem cells to yield pure cultures of the desired type of more differentiated cells as opposed to a mixed population. If the latter proves to be the case, the rigorous purging of cultures of residual undifferentiated or inappropriately differentiated cells will be necessary. How this is approached will depend on whether any contamination of grafts is acceptable and, if so, how much. One way in which this particular problem has been circumvented in murine model systems for in vitro differentiation of ES cells is to transfect them with the coding region of a gene for an antibiotic resistance or fluorescent...

Pathogen Factors

The virulence of the organism may influence the development of infection. Gram-negative bacterial endotoxin contributes to sepsis syndrome. Exotoxin from flesh eating bacteria such as clostridia and streptococci allow rapid spread of infection through tissue. Postoperative fever should prompt a wound check to avoid dire consequences of missing such an infection. Encapsulated organisms such as Klebsiella and Streptococcus pneumoniae evade host defenses by inhibiting phagocytosis. Numerous mechanisms of antibiotic resistance continue to evolve.

Treatment

Axillary infections tend to have a significant anaerobic component. Infections of the oropharynx and below the waist tend to be mixed aerobic and anaerobic flora. Bacteroides species are obligate anaerobes and should be included in the treatment plan when the colon may be involved in the process. Hospital pharmacies review antibiotic resistance patterns specific to their institutions or even in individual intensive care units. These patterns can also help guide antibiotic choices.

Prevention

Evidence points to the importance of improving diet and eradicating H. pylori infection. A diet rich in fresh fruits and vegetables without highly salted, preserved, or smoked food will theoretically offer benefits in primary prevention. In countries where the incidence of stomach cancer is high, screening for H. pylori may be effective for secondary prevention. To this end, programs of mass screening and eradication of H. pylori by antibiotics are being performed in Japan. However, because only a small proportion of individuals with H. pylori colonization develop stomach cancer, concerns have been raised regarding the possibility of antibiotic resistance by a mass H. pylori eradiation program. Use of vaccines against H. pylori may be an alternative approach.

Kennedy Disease

Antibiotic Resistance Antibiotic resistance is the ability of a bacterium or other microorganism to survive and reproduce in the presence of antibiotic doses that were previously thought effective against them. Examples of microbe resistance to antibiotics dot the countryside, plaguing humankind. For instance, in February 1994 dozens of students at La Quinta High School in southern California were exposed to the pathogenic (disease-causing) agent, Mycobacterium tuberculosis, and eleven were diagnosed with active tuberculosis. Many strains of this bacterium are multi-drug resistant (MDR). As for the sexually transmitted pathogen Neisseria gonorrhea, which causes gonorrhea, the antibiotics penicillin and tetracycline that were used against it in the 1980s can no longer be the first lines of defense, again because of antibiotic resistance. If only 2 percent of a N. gonorrhea population is antibiotic resistant, a community-wide infection of this persistent strain can occur.

In vitro packaging

Naked bacteriophage DNA can be introduced into a host bacterial cell by transformation (often referred to as transfection when talking about phage DNA), in much the same way as we described for a plasmid. The big difference is that in this case instead of plating on a selective agar and counting bacterial colonies, we would mix the transfection mix with a culture of a phage-sensitive indicator bacterium in molten soft agar and look for plaques (zones of clearing due to lysis of the bacteria) when overlaid onto an agar plate. Note that in this case we do not need an antibiotic resistance gene as a selective marker.

Adverse effects

Soaps are of alkaline pH and are known irritants, causing itching, dryness and redness acidic soap-free cleansers may therefore be preferential. Aggressive use of abrasives may irritate skin and for that reason common sense suggests that they should not be used in conjunction with topical agents such as benzoyl peroxide, which sensitise the skin, unless tolerance has initially been demonstrated. Dermatological reactions are idiosyncratic and cannot be predicted and the patient should be advised to discontinue use immediately if irritation develops. Like any topical agent, it is possible that antibacterial cleansers and abrasives are less suitable in individuals with sensitive skin. The literature does not support a link between the use of topical antimicrobials and the emergence of antiseptic or antibiotic resistance.22

Gene transfer tools

Sidbis Semliki forest virus, spumavirus and simian virus 40 derived vectors can be foreseen as interesting candidates for transferring genetic information. Similarly, naked DNA, either alone or associated with lipidic particles, proteins or defective viral elements, represents an alternative to viral delivery of a transgene. In these vectors, genes of interest, placed under control of an eukaryotic promoter, are included in a plasmid backbone with a bacterial replication origin and an antibiotic resistance expression unit necessary for large-scale production. Naked DNA vectors have already been considered for clinical trials and rapid evolution in this field is anticipated.

Concluding Comments

Stationary-phase mutagenesis, particularly adaptive mutation mediated by local and environment-dependent mutators, may be implicated in the ascent of antibiotic resistance in clinical bacterial populations. However, resistance generally impacts negatively on bacterial fitness, and the mutations generated to compensate for this cost may entrench antibiotic resistant phenotypes within a population, even after the selective drug is removed. The implication of irreversible evolution is that latent clinical infections, maintained by bacteria in isolated niches in the host, may retain antibiotic resistance for the duration of their existence. Finally, the emergence of antibiotic-resistant mutants within a bacterial population is dependent on the physiology, the genetic complement, and the historical behaviour of bacterial populations, as well as the physical structure of the selective medium, and the in-host antibiotic-bacterium dynamics. Because so many factors are 5 involved, the mutation...

Prokaryotes

The genomes of closely related prokaryotes often have different organizations. These differences arise from rearrangements (such as inversions) between repeated elements, IS elements, and transposons and from the horizontal transfer of nucleotide sequences between cells. The latter phenomenon is mediated most commonly by conjugative plasmids, which are nonessential, autonomous accessory genetic elements that can acquire genes (such as antibiotic resistance genes) and then move them from a donor organism to a recipient. The dynamic character of genomic organization in prokaryotes is often designated as genomic plasticity.

Molecular Tools

The manipulation of commercial baker's yeast is a difficult task because of the special genetic characteristics of these strains. For instance, the high level of ploidy means that multiple disruption and deletion steps are required to obtain a null mutant. Another consequence of this polyploidy is that most of these strains are prototrophic. Therefore, transformation by complementation of auxotrophic mutations, the most common approach in wild-type S. cerevisiae, cannot be employed. On top of this, the molecular tools that could be applied in industrial strains are often limited by legal regulations concerning the commercial exploitation of genetically modified organisms (GMOs). These legal regulations establish, as a criterion of acceptability, the absence of nonessential foreign DNA, and in particular, of vector sequences related to prokaryotic DNAs or antibiotic resistance markers. Additionally, transformants should also (1) keep the same useful properties of the parental strain,...

Ooooooc

It is also quite common for bacterial species to possess extrachromosomal genetic elements called plasmids. These are small, circular DNA molecules which, when present, vary in number from one to about thirty identical copies per cell. Plasmids include the fertility factor (F+ plasmid), described below, as well as plasmids that carry drug-resistance genes. Indeed, these drug-resistance plasmids may be passed from species to species and are a major problem in the spread of antibiotic resistance. Whereas most bacteria that contain plasmids have just a single kind of plasmid, some bacterial species simultaneously possess a number of different plasmids, each of which, in turn, is present in varying numbers within the bacterial cell.

Types of Plasmids

Plasmids are not usually required by their host cell for its survival. Instead, they carry genes that confer a selective advantage on their host, such as resistance to heavy metals or resistance to naturally made antibiotics carried by other organisms. Alternatively, they may produce antibiotics (toxins) that help the host to compete for food or space. For instance, antibiotic resistance genes produced by a plasmid will allow its host bacteria to grow even in the presence of competing bacteria or fungi that produce these antibiotics.

Cloning vectors

A cloning vector is frequently a plasmid or a bacteriophage (bacterial virus) which must be fairly small and fully sequenced, able to replicate itself when reintroduced into a host cell, thus producing large amounts of the recombinant DNA for further manipulation. Also it must carry on it 'selector marker' genes. These are different from the reporter genes described below which are indicators of genomic integrity and activity. A common design of a cloning vector is one which carries two genes coding for antibiotic resistance. The 'foreign' gene is inserted within one of the genes so that it is no longer functional therefore it is possible to discriminate by standard microbiology techniques which bacteria are carrying plasmids containing recombinant DNA and which are not. Selector genes may operate on at least two levels, the first at the level of the bacterium, usually Eschericia coli, in which the manipulations are being performed described above and the second being at the level of...

Campylobacter spp

Ganism is unlikely to grow on raw poultry due to its fastidious nature, initial levels (> 500 mL) are in the range to cause human illness. Most cases of campylobacteriosis are sporadic, and contributing factors typically include cross-contamination and lack of proper handwashing. There is particular concern with campylobacteriosis because of increasing antibiotic resistance and the organisms' connection with Guillain-Barre syndrome.

Gene Technology

The gene multiplication method with plasmid vectors has been applied to the breeding of amino acid producers. When a plasmid vector that can multiply within an amino acid producer is joined enzymatically to a DNA fragment carrying the genetic information from other strains of the same microorganism or other microorganisms, the resultant recombinant DNA is transferred to the amino acid producer to introduce new markers of the donor microorganisms. A plasmid such as pCGll has sites sensitive to various restriction endonucleases and is easily joined to a DNA fragment prepared by digestion with corresponding restriction enzymes. Moreover, because it has the genes determining streptomycin resistance, it is easy to select strains with plasmids carrying these antibiotic resistance markers (40).

Helicobacter Pylori

Diagnosis of H. pylori infection can be made using an invasive endoscopic method or less-invasive breath tests and immunological assays (see Table 2). Endoscopy allows the collection of tissue biopsies that can subsequently be examined by histology, cultured for H. pylori, or measured for urease production. Histology with hematoxylin & eosin stain and special stains (Giemsa, Warthin-Starry) visualizes the bacteria as well as the surrounding inflammation. Culture is highly specific and it permits testing for antibiotic resistance, but its sensitivity is reduced by the risk of contamination. Urease tests on histological samples have a high sensitivity because the entire tissue sample is used to measure urease. Less-invasive tests that do not require gastric mucosal samples include ser-ology, urea breath tests, and stool antigen tests. Serology is used to measure serum IgG antibodies.

Dnadna Hybridization

The use of molecular tests to determine the carriage of specific antibiotic resistance genes began over 20 years ago. 5 Initially, the entire plasmid, or a relatively large fragment (> 600 bp) carrying the gene of interest, was radiolabeled and used as a DNA probe in DNA-DNA hybridization assays to distinguish different tet genes from each other. 6 Then smaller (< 500 bp) intragenic fragments, representing internal segments of the structural gene, were used. These were made by cutting the desired band out of a plasmid after it had been cleaved with restriction enzyme(s) and then by separating the fragments on an agarose gel. Currently, small 20- to 30-bp probes are used for hybridization. The sequences are determined by the gene or genes of interest and are prepared de novo. The advantage is that cross-hybridization is minimized, hybridization time is short, and they are now cheap to purchase.

PCR Assay

Numerous PCR assays have now been developed in a variety of resistance genes discussed in this review. 6-10 Each PCR assay uses a pair of primers, which can be the same as those used for DNA-DNA hybridization. The template may be purified DNA, proteinase-K lysates of whole bacteria, and or direct patient materials. The PCR assay can use direct patient material, if from a normally sterile site, as the source of the template to determine whether a particular antibiotic resistance gene is present in the sample without growing the bacteria. Using nonsterile sites as source of material is not desirable because most of Mutations that upregulate innate efflux pumps can alter the host's susceptibility. The mutation in the marR region of the chromosome enhances intrinsic resistance to a large group of antibiotics including tetracycline. 15 The level of tetracycline resistance increases as the cells are grown continually in the presence of tetracycline. Another example is Neisseria gonorrhoeae,...

Summary

In summary, four public health functions for genetics have been outlined that underscore the complexities involved in public health genetics. All are carried out by efforts among various groups, including partnerships and coordinated efforts among federal, state, and local agencies, the public and private sectors, and the public-health, medical, and academic sectors, with various levels of community and consumer involvement. Annual national meetings on genetics and public health facilitate these efforts. see also Antibiotic Resistance Cancer Diabetes Disease, Genetics of Genetic Discrimination Genetic Testing Hemoglobinopathies Metabolic Disease Prenatal Diagnosis.

Barnard Endocarditis

The management of S. aureus infections in general, and NVE in particular, has become increasingly difficult owing to evolving mechanisms of antibiotic resistance. Penicillin was introduced into clinical practice in 1941 and it was demonstrated to be an effective anti-staphy-