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. Read more here...

Antibiotic Resistance Surviving An Uncertain Future Overview

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Concern About the Insertion of Genes Coding for Antibiotic Resistance

There is a general concern about the increase of antibiotic resistant pathogenic bacteria. In that perspective, the introduction of antibiotic resistance selectable marker genes in GMOs is worrying. So far, most concern has referred to agricultural crops due to the risk for horizontal gene transfer, even though that risk is likely to be small or theoretical.10 The risk, even though small, is probably more realistic with GMMs. The significance for gene transfer of antibiotic resistance genes is very difficult to determine. The risk includes the transfer, the expression of the gene and the consequences followed from that event. In general, Swedish authorities involved in GMO regulation are of the opinion that the risks with using antibiotic resistance as marker genes are very small.11 Nevertheless, due to the following arguments Swedish authorities recommend a development to the use of alternative marker genes techniques exist and are being developed to remove unwanted genes, such as...

Use of Antibiotic Resistance Genes to Monitor Gene Transfer in Soil

Antibiotic resistance markers have been used to study gene mobility and dissemination of antibiotic resistance genes has been the subject of a large number of studies due to the problem of drug resistance in bacterial pathogens. Resistance genes are highly mobile under intense selection pressure in clinical environments and have been found associated with chromosomes, plasmids, integrons and bacteriophages.38,39 The application of antibiotics such as streptomycin to treat bacterial rots in soft fruit has resulted in the spread of selected streptomycin resistance genes in Pseudomonas species.40 In addition, genes involved in streptomycin production by streptomycetes are more widely distributed than was previously thought.41,42 There is evidence for the actual presence of antibiotics in soil. Phenazine43 and thiostrepton16 have been detected in soils following the introduction of the respective producer strain. Such antibiotics produced in situ could provide selection pressure for...

Choice of Antibiotic Resistance Gene Markers

Amner et al19 suggested that examination of the antibiotic resistance profiles of indigenous populations and resistance genes within the gene pool of microbial populations from different environments could be useful in the development of marker systems. The fate of thermophilic actinomycetes in compost was monitored,20 where all microbial constituents of the microflora were sensitive to tetracycline. This resistance gene was thus suitable for introduction into host vector systems to allow the detection of host organisms.21 A plasmid encoding tetracycline resistance was used to monitor Bacillus subtilis in compost. The use of just a single resistance marker was feasible due to the low level (103) of tetracycline resistant isolates in the indigenous bacteria.21 Conversely, Smalla et al22 examined the distribution of kanamycin resistance in bacteria from different habitats and demonstrated that the resistance phenotype was widely distributed among indigenous bacteria in most habitats...

The Use of Antibiotic Resistance Gene Markers for Studying Bacterial Populations in Natural Environments

Antibiotic resistance genes have been used to mark bacteria by providing a readily selectable phenotype, which can be detected using selective growth media. Detection and monitoring is therefore culture-dependent. A wide range of resistance genes have been characterised (Tables 2.1 and 2.2) which confer resistance to commercially available, inexpensive antibiotics. In addition, resistance genes have provided a valuable tool for cloning and genetic manipulation procedures in both prokaryotes and eukaryotes. Many industrial strains carry resistance genes which have resulted from cloning procedures, enabling selective maintenance of a construct or plasmid carrying the resistance gene by inclusion of the respective antibiotic in the g-owth medium (For details see ref. 1). The main environmental application for antibiotic resistance gene markers has been the monitoring of gene transfer associated with plasmid or transposon mobility. In addition, the tracking of bacterial populations in...

Marking of Bacteria with Antibiotic Resistance Genes

Antibiotic resistance gene markers have been inserted on multiple and single copy plas-mids for monitoring the fate of inoculants and gene transfer in natural environments. The choice of chromosomal or pasmid location is dependent on the requirements of the study, but gene dose effects with multicopy plasmids will greatly improve molecular detection of both DNA and mRNA. In addition, enhanced phenotypic resistance from multiple copies of the resistance gene marker will aid selection.9 Both naturally occurring R-plasmids and constructed plasmids with one or multiple resistance genes are readily available. However, R-plasmids are mainly found in gram-negative hosts and many will not be able to replicate in gram positive backgrounds. Plasmids from gram-negative and gram positive bacteria have very different conjugation systems and genes involved in plasmid transfer, replication and maintenance are very diverse.36 For gram positive bacteria, resistance plasmids have been constructed (see...

Principles of Nosocomial Infections

These are infections acquired in hospitals and are associated with multiple factors, including immunosuppression (either iatrogenic or due to disease), the presence of infected or colonized patients nearby, transmission by personnel from patient to patient (as fomites or as carriers), invasive procedures that bypass host defense barriers, and the high frequency of antibiotic resistance in the hospital environment. These diseases usually have a more serious outcome than diseases occurring in the community. Some of the etiologic agents are Pseudomonas aeruginosa, nearly all Enterobacteriaceae, C. difficile, Enterococcus, and S. aureus.

Transgenic Trees for Improvement of Forestry 131 Northern America

Even though a country with traditionally strong research in forest biology, the share of field releases of transgenic trees in Canada is small. The Canadian database lists for 1997 a poplar with an antibiotic resistance released in Quebec (the only one found also in the OECD database), for 1998 a submission for herbicide tolerant poplar in Alberta, and from 2000-2004 two submissions for black spruce with selectable marker genes, an insect-resistant white spruce and a poplar with a selectable marker. These trials are well covered in the non-technical media. A National Post article of 2003, for example, covered a planned field trial with transgenic trees (Jack 2003). The trial comprises 400 transgenic spruces and poplars planted out in a forest near Val Cartier, Quebec. The article pointed out that there were as yet no commercial plantings of transgenic trees in Canada, but that the development by now had reached a point at which use in commercial plantations was within reach. This work...

Cultivation Based Assays

The traditional approach for monitoring bacteria using antibiotic resistance is selective agar plate counting, although the most probable number technique (MPN) can be used for selective broth culture. Agar antibiotic gradient plates, in which the antibiotic concentration increases linearly from zero to an established level, are produced by making an agar gradient containing the antibiotic and covering a basal agar layer without antibiotic. Such gradients are difficult to replicate and have not generally been used for isolation and counting although they are useful for characterisation of isolates.5 Many antibiotics are unstable, and biological activity can therefore decrease with time or due to inappropriate handling. For example, tetracycline is light sensitive and p-lactams and aminoglycosides, such as neomycin are heat sensitive. Some antibiotics, such as thiostrepton, are only soluble in organic solvents and so will be insoluble in agar forming a suspension, thus affecting...

Eva Tas and Kristina Lindstrom 41 Introduction

Classical microbial identification methods are primarily based on morphology, growth characteristics and metabolic properties of the oiganism under study. For pathogens or symbiotic organisms, host specificity is also an important feature. In addition, bacteriophage typing, antibiotic resistance, lipopolysaccharide and protein profiles, serological properties, and plasmid profiles of bacteria can be characterized. More advanced immunological techniques indude detection of antigens by enzyme-linked immunosorbent assay (ELISA) or by fluorescent antibody labeling. The problem with most of these techniques is that they require microbiological cultivation. Thus, for example, detection of released organisms may not be possible when their numbers are low relative to those of the indigenous population.1 In addition, disadvantages of the immunological methods lay in possible cross-reactions and nonspecific binding. The use of monodonal antibodies can be a solution,2,3 but the antigens to be...

Detection of Specific Genes

An antibiotic resistance marker carried by Tn5 was not expressed in some cells under high temperature stress. Another example is when the marker is not a fully functional gene but a recombinant fragment detectable by DNA techniques. Van Elsas et al28 used a small eukary-otic DNA fragment, pat from Solanum tuberosum, as a marker to study the fate of Pseudomonas fluorescens introduced into soil. This DNA sequence was not present in indigenous soil bacteria, so problems with background were eliminated.31 Although not expressed as a phe-notypic marker, the pat fragment was readily detected by DNA hybridization and by PCR.

Prevention And Control

Those traveling to the tropics should be advised to eat only those foods that are thoroughly cooked and served piping hot, or fresh fruits that can be peeled. In general, chemo-prophylaxis for traveler's diarrhea is not recommended. People who are at high risk of complications from traveler's diarrhea, such as those with diabetes, inflammatory bowel disease, advanced AIDS, or those on immunosuppressive drugs, should be warned that agents such as Cryptosporidium are not prevented by antibiotics and may cause far worse disease than ETEC or EAEC. Moreover, the increasing antibiotic resistance of certain pathogens (e.g., Campylobacter in Thailand) makes chemoprophylaxis less attractive.

Choice of Expression System

Integration can result in multiple insertion events as demonstrated with the C-terminal fragment of TeNT (14 copies per cell). These multicopy transformants can be selected by growing transformants at increasing concentrations of aminoglycoside antibiotics such as kanamycin or geneticin (G418), because the antibiotic resistance is encoded on the pHIL-D4 plasmid (Figure 4.1). These multiple insertion events occur at a frequency of 1 to 10 of HIS+ transformants. For the cell banks generated for Antigen A and Antigen B, these genes are present in the genome at frequencies of three and five copies per cell, respectively. In this expression system, further use of the antibiotics to maintain either single or multicopy insertions is not required.

Detection of Luminescence by Eye and by Photographic and Xray Film

Viable cell enumeration of luciferase-marked strains can be achieved using traditional dilution plate counting and luminescent colonies can usually be detected by the unaided eye in a darkened room.32 Greater sensitivity can be achieved using photographic film (with long exposure), X-ray film or photon imaging (see below). The majority of luciferase-marked strains are also marked with antibiotic resistance (see Chapter 2), enabling selectivity against indigenous populations by enumeration on media supplemented with the appropriate antibiotic. In the absence of antibiotic resistance markers, selective enumeration depends on the competitive ability of the marked strain on enumeration media, the nutrients required for luminescence and the sensitivity of detection. Nevertheless, visual detection of a single luminescent colony of lux-marked Erwinia carotovora is possible in the presence of 3000 nonluminescent colonies.32 Similar techniques may be used for in situ detection of luminescent...

Stable Tagging of Bacteria with Firefly Luciferase Gene for Environmental Monitoring

The XPR promoter and the luc genes was isolated and cloned into pUC18Not. The NotI fragment with the luc gene was cloned into the unique NotI site of pUT mini-Tn5 Sm Sp and into pUTKm. The resulting plasmids, pTCR210 and pTCR240, were selected for studies of transposition and luciferase expression in various gram-negative bacteria.36 To tag bacteria without the use of antibiotic resistance genes, the Km resistance gene may be replaced with the lPR luc fusion. To attenuate the expression in the environment, where the marked strain must survive, and to allow sensitive detection, a DNA fragment containing the repressor gene laclq and a PR lucOR fusion has been introduced on a suicide plasmid. PTEB510 (Fig. 5.5) is a representative of constructs able to express high luciferase levels only when induced by IPTG. Transposition frequencies were suitable for marking purposes and the levels of luminescence were sufficient for detection by the standard methods.

Construction of P fluorescens SBW25EeZY6KX and Detection and Monitoring Methods

To facilitate detection, marker genes, for antibiotic resistance (aph1, kanamycin), lactose utilization (lacZY, lactose permease and p-galactosidase also converts X-gal to a blue pigmented product) and colorimetric detection (xylE, 2,3,catechol dioxygenase, converts catechol to a soluble yellow pigmented semi-aldehyde), were selected to provide a unique set of marker phenotypes for highly sensitive plate isolation and enumeration assays.14 These marker genes were introduced, by site-directed homologous recombination, to predetermined, nonessential sites in the choromosome,16 for the construction of P fluorescens SBW25EeZY6KX.14,17 PCR based methods were then used for the detection of the introduced genes,18 and plate isolation methods were used for detecting mobile genetic elements acquired by the GMM from the indigenous microbial community.4,18

Environmental Risk and Deployment Strategies for Genetically Engineered Insectresistant Trees

Field-growth and performance of GM trees need to be evaluated over several years in order to assess their potential impact on human health and environment and obtain information relevant to commercial use. Small-scale trials are not recommended due to the long life cycle of perennial plants. Field trials of transgenic poplars expressing Bt and proteinase inhibitor genes are currently performed, as described in the following paragraph. The environmental safety of GM trees is considered a primary issue for their commercial use. For this reason, the potential risks associated with their release should be compared to the actual benefits and carefully assessed according to a concept of substantial equivalence with the non-GM strategies (Shelton 2004). Some risks are of general concern (gene flow to wild populations, invasiveness of GM trees, food safety issues, spread of marker genes responsible for antibiotic resistance) whilst some others specifically refer to the insect-resistant...

High Yield Facts

A chart highlighting the similarities and differences among the various agents can be a helpful tool The charts included in this section are simple examples More elaborate charts can be constructed that would include how the drug is administered, its pharmacological effects, its adverse effects, its mechanism of toxicity (if known), and significant drug-drug interactions. Tor infectious disease agents, the spectrum of antimicrobial activity and the basis of antibiotic resistance can be added.

Ethical Concerns

Although there are obvious advantages in the use of antibiotic resistance gene markers, their value for field releases has been limited. This is probably due to the ethical questions raised about deliberately releasing resistance genes into the environment, which presents the risk of spread into the indigenous bacterial population. However, transfer is known to be limited in conditions of low nutrients24,16 and is also affected by the activity and spatial distribution of bacteria in addition to physical conditions in the soil.50 The problem of transfer has been addressed with the development of modified vectors which inhibit the movement of resistance genes into new hosts51,52 There is widespread resistance in indigenous bacteria to selected antibiotics in current use in agriculture such as streptomycin. However, there will soon be restrictions on the us of antibiotics in animal growth promotion to reduce the future development of resistance in enteric bacteria. There is a clear link...

Review Questions

A pharmaceutical firm is interested in the bacterial production of thymidylate synthase in large quantities for drug-targeting studies. An important step in the overall cloning strategy involves the ligation of synthase cDNA into a plasmid vector containing a replication origin, an antibiotic resistance gene, and a promoter sequence. Which additional nucleotide sequence should be included in this vector to ensure optimal production of the thymidylate synthase

Diagnosis

After isolation, the organism should be tested for antimicrobial sensitivity. If it is resistant to chloramphenicol, it should be checked for the presence of R plasmids encoding for multiple antibiotic resistance. So-called quinolone-resistant strains, which generally have minimum inhibitory concentrations of the fluoroquinolones within the susceptible range of the interpretive criteria of the NCCLS, are characterized by resistance to nalidixic acid.

Chemoprophylaxis

In endemic areas or epidemic areas where secondary transmission within households is shown to be a frequent event, a short (3-day) course of tetracycline (or another cited antibiotic) administered to household contacts can diminish transmission within households.81 However, such use of antibiotics must be strictly controlled because indiscriminate use in the community can rapidly lead to antibiotic resistance. This was the situation in Guayaquil, Ecuador, in 1991 following the introduction of cholera into that community. An attempt at mass chemoprophylaxis rapidly led to the appearance of V cholerae O1 strains that had acquired resistance to tetracycline as well as several other antibiotics.82

Relapses

Up to 20 of patients treated for C. difficile-associated disease will have relapse after cessation of therapy. These relapses usually occur within 2 weeks after initial therapy is discontinued. Relapse is probably due to the persistence of antibiotic-resistant C. difficile spores rather than the development of antibiotic resistance of the vegetative organism. In patients with relapse, re-treatment with the initial agent will result in a greater than 90 cure rate.5,70 Multiple empirical regimens have been evaluated in patients who suffer two or more recurrences. Most of these regimens focus on efforts to repopulate the normal colonic flora. Perhaps most encouraging is the use of Saccharomyces boulardii in combination with standard antimicrobial therapy in patients with recurrent infection. A blinded, controlled study demonstrated a reduced rate of

Other GUS Constructs

Five gusA cassettes, uidAl, uidA2, uidA2-cat, uidA2-aadA and uidA2-aph, suitable for constructing transcriptional fusions,were described by Metcalf and Wanner.28 Three of them contain additional antibiotic resistance genes (see Table 6.3). uidAl, uidA2 and uidA2-aph cause nonpolar mutations after double homologous recombination into the host genome. Non-polar mutation after allele replacement 28 in the host genome no antibiotic resistance

Conclusion

Antibiotic resistance genes have a long history of reliability for use as highly selective, versatile markers allowing detection and enumeration of bacteria in environmental samples. Resistance genes found in non-antibiotic producing bacteria are readily expressed in a wide range of host backgrounds. Examples include the neomycin resistance gene, nptII, which has been used in gram-positive (high and low GC) and gram-negative bacteria, yeasts, plant and animal cell cultures. The resistance genes are highly mobile between bacterial groups and this may be a reason for their versatility and often near universal expression. A wide range of genes have been cloned and frequently used with their own promoter in diverse hosts. Levels of expression and strength of resistance may vary slightly from strain to strain but this is also related to the nature of the construct used. Multiple copies of a resistance gene secure high levels of resistance but only under defined conditions of growth and...

David Markie

In contrast to bacterial cloning systems, where the use of dominant antibiotic resistance determinants is common, selective markers used in YAC manipulation are based mainly on complementation of biosynthetic mutations. Such mutations, producing a growth requirement in the yeast strain that can be satisfied by the addition of a nutritional supplement to the media, are termed auxotrophies, whereas the wild-type state is the absence of that requirement (prototrophy). When using biosynthetic genes as selectable markers, choice is therefore limited to those genes that are mutant in the proposed host, and for which there is a source of a functional copy of the gene. The genotypes of some commonly used YAC host strains can be found in Table 2 in Chapter 17. To apply selection for a functional gene that complements an auxotrophic mutation in the host, the appropriate supplement is omitted from the media. However, toxic metabolite even in the presence of a nonfunctioning mutant copy), in...

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