The role of conjugative transposons in spreading antibiotic resistance between bacteria that inhabit the gastrointestinal tract

There is huge potential for genetic exchange to occur within the dense, diverse anaerobic microbial population inhabiting the gastrointestinal tract (GIT) of humans and animals. However, the incidence of conjugative transposons (CTns) and the antibiotic resistance genes they carry has not been well studied among this population. Since any incoming bacteria, including pathogens, can access this reservoir of genes, this oversight would appear to be an important one. Recent evidence has shown that anaerobic bacteria native to the rumen or hindgut harbour both novel antibiotic resistance genes and novel conjugative transposons. These CTns, and previously characterized CTns, can be transferred to a wide range of commensal bacteria under laboratory and in vivo conditions. The main evidence that gene transfer occurs widely in vivo between GIT bacteria, and between GIT bacteria and pathogenic bacteria, is that identical resistance genes are present in diverse bacterial species from different hosts.     

The clostridial mobilisable transposons

Mobilisable transposons are transposable genetic elements that also encode mobilisation functions but are not in themselves conjugative. They rely on coresident conjugative elements to facilitate their transfer to recipient cells. Clostridial mobilisable transposons include Tn4451 and Tn4452 from Clostridium perfringens, and Tn4453a and Tn4453b from Clostridium difficile, all of which are closely related, and Tn5398 from C. difficile. The Tn4451 group of elements encodes resistance to chloramphenicol and is unusual in that transposition is dependent upon a large resolvase protein rather than a more conventional transposase or integrase. This group of elements also encodes the mobilisation protein TnpZ that, by acting at the RS_A or oriT site located on the transposon, and in the presence of a coresident conjugative element, promotes the movement of the nonreplicating circular intermediate and of plasmids on which the transposon resides. The erythromycin resistance element Tn5398 is unique in that it encodes no readily identifiable transposition or mobilisation proteins. However, the element is still capable of intraspecific transfer between C. difficile isolates, by an unknown mechanism. The detailed analysis of these mobilisable clostridial elements provides evidence that the evolution and dissemination of antibiotic resistance genes is a complex process that may involve the interaction of genetic elements with very different properties. RID="*" ID="*"Corresponding author.     

The role of conjugative transposons in the <Emphasis Type="Italic">Enterobacteriaceae</Emphasis>

Although widely studied in Gram-positive Streptococci and in the Gram-negative Bacteroides, there is a scarcity of information on the occurrence and nature of conjugative transposon-like elements in the well-studied Enterobacteriaceae. In fact, some of the major reviews on conjugative transposons prior to 1996 failed to mention their occurrence in this group. Recently, their presence has been reported in Salmonella, Vibrio and Proteus species, and in some cases such as the SXT element in Vibrio and the IncJ group element CTnR391, there has been some molecular characterization. The elements thus far examined appear to be larger than the common Gram-positive conjugative transposons and to be mosaic in structure, with genes derived from several sources. Recent evidence suggests that in the Enterobacteriaceae the elements may be related to enteric pathogenicity islands. The evolution, distribution and role of these elements in the Enterobacteriaceae is discussed.     

Spread of antibiotic resistance with food-borne pathogens

This short review summarizes data on antibiotic resistance profiles of common food-borne pathogens like Salmonella sp., Escherichia coli, Campylobacter sp., Listeria monocytogenes, Clostridium perfringens, Staphylococcus aureus, and coagulase-negative staphylococci. As a flashlight on the literature of the last few years, it provides ample evidence that antibiotic resistance traits have entered the microflora of farm animals and the food produced from them. Molecular analysis of the resistance genes, where available, shows that the food microflora is not separated from its human counterpart and conjugative transfer of resistance genes has been demonstrated in vitro and in a few cases in vivo. For example, for Salmonella typhimurium, resistance towards tetracyclines has increased from zero in 1948 to a 98% level in certain epidemic populations of S. typhimurium DT104 in 1998. The high incidence of food-borne pathogens in raw meat and milk together with a high level of therapeutic, prophylactic and nutritional application of antibiotics in agriculture reveals an antibiotic resistance problem of global dimensions. The resistance problem in human medicine will not be solved if there is a constant influx of resistance genes into the human microflora via the food chain.     

Comparison of SXT and R391, two conjugative integrating elements: definition of a genetic backbone for the mobilization of resistance determinants

The SXT element (SXT) is becoming an increasingly prevalent vector for the dissemination of antibiotic resistances in Vibrio cholerae. SXT is a member of a larger family of elements, formerly defined as IncJ plasmids, that are self-transmissible by conjugation and integrate site-specifically into the host chromosome. Comparison of the DNA sequences of SXT and R391, an IncJ element from Providencia rettgeri, indicate that these elements consist of a conserved backbone that mediates the regulation, excision/integration and conjugative transfer of the elements. Both elements have insertions into this backbone that either confer the element-specific properties or are of unknown function. Interestingly, the conserved SXT and R391 backbone apparently contains hotspots for insertion of additional DNA sequences. This backbone represents a scaffold for the mobilization of genetic material between a wide range of Gram-negative bacteria, allowing for rapid adaptation to changing envi ronments. RID="*" ID="*"Corresponding author.     

Mechanism of integration and excision in conjugative transposons

Translocation of conjugative transposons proceeds via excision of the element to generate a circular molecule that can then integrate into a new site, which can be in the same or a different cell. This review summarises some of the different mechanisms used for excision and integration of conjugative transposons. RID="*" ID="*"Corresponding author.     

Association of different mobile elements to generate novel integrative elements

Among the more important problems in modern hospitals is the prevalence of bacterial pathogens expressing resistance to multiple antimicrobial agents. The frequency of multiresistance suggests mechanisms by which bacterial species can concentrate and efficiently exchange a variety of resistance determinants. Mechanisms by which this occurs include insertion of transposons within transposons, coalescence through the activity of insertion sequences and the employment of integrons. In some instances, more than one of these mechanisms is involved in creating large multiresistance genetic elements. The association of the elements with transferable elements or transposons may promote rapid dissemination among clinical strains, and create further opportunities for inclusion of additional resistance determinants.     

Antibiotic resistance in microbes

The treatment of infectious disease is compromised by the development of antibiotic-resistant strains of microbial pathogens. A variety of biochemical processes are involved that may keep antibiotics out of the cell, alter the target of the drug, or disable the antibiotic. Studies have shown that resistance determinants arise by either of two genetic mechanisms: mutation and acquisition. Antibiotic resistance genes can be disseminated among bacterial populations by several processes, but principally by conjugation. Thus the overall problem of antibiotic resistance is one of genetic ecology and a better understanding of the contributing parameters is necessary to devise rational approaches to reduce the development and spread of antibiotic resistance and so avoid a critical situation in therapy--a return to a pre-antibiotic era.     

Mobile genetic elements of Staphylococcus aureus

Bacteria such as Staphylococcus aureus are successful as commensal organisms or pathogens in part because they adapt rapidly to selective pressures imparted by the human host. Mobile genetic elements (MGEs) play a central role in this adaptation process and are a means to transfer genetic information (DNA) among and within bacterial species. Importantly, MGEs encode putative virulence factors and molecules that confer resistance to antibiotics, including the gene that confers resistance to beta-lactam antibiotics in methicillin-resistant S. aureus (MRSA). Inasmuch as MRSA infections are a significant problem worldwide and continue to emerge in epidemic waves, there has been significant effort to improve diagnostic assays and to develop new antimicrobial agents for treatment of disease. Our understanding of S. aureus MGEs and the molecules they encode has played an important role toward these ends and has provided detailed insight into the evolution of antimicrobial resistance mechanisms and virulence.     

DNA transposons in vertebrate functional genomics

Genome sequences of many model organisms of developmental or agricultural importance are becoming available. The tremendous amount of sequence data is fuelling the next phases of challenging research: annotating all genes with functional information, and devising new ways for the experimental manipulation of vertebrate genomes. Transposable elements are known to be efficient carriers of foreign DNA into cells. Notably, members of the Tc1/mariner and the hAT transposon families retain their high transpositional activities in species other than their hosts. Indeed, several of these elements have been successfully used for transgenesis and insertional mutagenesis, expanding our abilities in genome manipulations in vertebrate model organisms. Transposon-based genetic tools can help scientists to understand mechanisms of embryonic development and pathogenesis, and will likely contribute to successful human gene therapy. We discuss the possibilities of transposon-based techniques in functional genomics, and review the latest results achieved by the most active DNA transposons in vertebrates. We put emphasis on the evolution and regulation of members of the best-characterized and most widely used Tc1/mariner family.Received 8 June 2004; received after revision 26 October 2004; accepted 18 November 2004     

Epithelial antimicrobial peptides: innate local host response elements

Multicellular organisms have to survive in an environment laden with numerous microorganisms, which represent a potential hazard to life. Different strategies have been developed to ward off infections by preventing microorganisms from entering surfaces and by preventing the attack of microorganisms that have already entered the epithelia. Therefore, it is not surprising that epithelia are equipped with various antimicrobial substances that act rapidly to kill a broad range of microorganisms. This review summarizes our present knowledge about epithelial peptide antibiotics produced in plants, invertebrates, and vertebrates including humans. There is now strong evidence that in addition to constitutively secreted peptide antibiotics, others are induced upon contact with microorganisms or by proinflammatory cytokines. beta-Defensins represent one family of vertebrate antimicrobial peptides, members of which are inducible and have recently been identified in humans. The defensin-characteristic local expression pattern may indicate that specialized surfaces express a characteristic surface antimicrobial peptide pattern that might define the characteristic microflora as well as the density of microorganisms present on the surface.     

Early microbiota,antibiotics and health

The colonization of the neonatal digestive tract provides a microbial stimulus required for an adequate maturation towards the physiological homeostasis of the host. This colonization, which is affected by several factors, begins with facultative anaerobes and continues with anaerobic genera. Accumulating evidence underlines the key role of the early neonatal period for this microbiota-induced maturation, being a key determinant factor for later health. Therefore, understanding the factors that determine the establishment of the microbiota in the infant is of critical importance. Exposure to antibiotics, either prenatally or postnatally, is common in early life mainly due to the use of intrapartum prophylaxis or to the administration of antibiotics in C-section deliveries. However, we are still far from understanding the impact of early antibiotics and their long-term effects. Increased risk of non-communicable diseases, such as allergies or obesity, has been observed in individuals exposed to antibiotics during early infancy. Moreover, the impact of antibiotics on the establishment of the infant gut resistome, and on the role of the microbiota as a reservoir of resistance genes, should be evaluated in the context of the problems associated with the increasing number of antibiotic resistant pathogenic strains. In this article, we review and discuss the above-mentioned issues with the aim of encouraging debate on the actions needed for understanding the impact of early life antibiotics upon human microbiota and health and for developing strategies aimed at minimizing this impact.     

The tetracycline resistome

Resistance to tetracycline emerged soon after its discovery six decades ago. Extensive clinical and non-clinical uses of this class of antibiotic over the years have combined to select for a large number of resistant determinants, collectively termed the tetracycline resistome. In order to impart resistance, microbes use different molecular mechanisms including target protection, active efflux, and enzymatic degradation. A deeper understanding of the structure, mechanism, and regulation of the genes and proteins associated with tetracycline resistance will contribute to the development of tetracycline derivatives that overcome resistance. Newer generations of tetracyclines derived from engineering of biosynthetic genetic programs, semi-synthesis, and in particular recent developments in their chemical synthesis, together with a growing understanding of resistance, will serve to retain this class of antibiotic to combat pathogens.     

Heat shock protein gene expression during Xenopus development

Stress-induced heat shock protein gene expression is developmentally regulated during early embryogen esis of the frog, Xenopus laevis. For example, a number of heat shock protein genes, such as hsp70, hsp90, and ubiquitin are not heat-inducible until after the midblastula stage of embryogenesis. Furthermore, the family of small heat shock protein genes, hsp30, are differentially expressed after the midblastula stage as well as being regulated at the level of mRNA stability. Many of these stress proteins are also synthesized constitutively during oogenesis and embryogenesis during which they may act as molecular chaperones as well as being involved in sequestering proteins in an inactive state until required by the developing embryo. Furthermore the induction of these stress protein genes has been correlated with enhanced thermoresistance. During stressful conditions heat shock proteins probably prevent aggregation or misfolding of damaged protei ns within the embryo.     

Genetic ecology: A new interdisciplinary science,fundamental for evolution,biodiversity and biosafety evaluations

Genetic ecology is the extension of our modern knowledge in molecular genetics to studies of viability, gene expression and gene movements in natural environments like soils, aquifers and digestive tracts. In such milieux, the horizontal transfer of plasmid-borne genes between phylogenetically distant species has already been found to be much more frequent than had been expected from laboratory experience. For the study of exchanges involving chromosomally-located genes, more has to be learned about the behaviour of transposons in such environments. The results expected from studies in genetic ecology are relevant for considerations of evolution, biodiversity and biosafety. The role of this new field of research in restoring popular confidence in science and in its biotechnological applications is stressed.     

Evolution of bacterial pathogenesis

The evolution of bacteria is associated with continuous generation of novel genetic variants. The major driving forces in this process are point mutations, genetic rearrangements, and horizontal gene transfer. A large number of human and animal bacterial pathogens have evolved the capacity to produce virulence factors that are directly involved in infection and disease. Additionally, many bacteria express resistance traits against antibiotics. Both virulence factors and resistance determinants are subject to intrastrain genetic and phenotypic variation. They are often encoded on unstable DNA regions. Thus, they can be readily transferred to bacteria of the same species or even to non-related prokaryotes. This review article focuses on the main mechanisms of bacterial microevolution responsible for the rapid emergence of variants with novel virulence and resistance properties. In addition, processes of macroevolution are described with special emphasis on gene transfer and fixation of adaptive mutations in the genome of pathogens.     

2006～2010年革兰阴性菌对碳青霉烯类抗生素耐药性分析

目的调查分析我院20ff6～2010年临床分离的革兰阴性菌对碳青霉烯类抗生素的耐药性。方法采用纸片扩散法进行抗菌药物敏感实验,采用WHONET5．4软件及SPSS13．0软件进行数据分析。结果大肠埃希菌和肺炎克雷伯菌对亚胺培南和美罗培南的耐药率呈上升趋势,但最高不超过4．5％。铜绿假单胞菌对亚胺培南的耐药率2006年和2007年监测显示为21．3％和41％。2008年达到44．8％,在2009年下降为12．4％,但2010年爽迅速增加到30．1％。鲍曼不动杆菌对亚胺培南耐药率的逐年上升更为显著,从2006年9．1％上升到2010年的68％,差异具有统计学意义（P〈0．01）。嗜麦芽窄食单胞菌对碳青霉烯类抗生素天然耐药,2006年对亚胺培南耐药率为30％,但自2007年起就增加到80．8％,最高达100％。结论肠杆菌科细菌对碳青霉烯类敏感性高,近年耐药率有上升趋势。铜绿假单胞菌和鲍曼不动杆菌对碳青霉烯类耐药率高,尤其是鲍曼不动杆菌的分离率逐年增加,耐药形势严峻。临床应合理使用抗生素,减少多重耐药株的产生。     

Bacterial antibiotic efflux systems of medical importance

Multidrug efflux systems endow on bacterial cells the ability to limit the access of antimicrobial agents to their targets. By actively pumping out antibiotic molecules, these systems prevent the intracellular accumulation necessary for antibiotics to exert their lethal activity. Drug efflux appears to be one of the most widespread antibiotic resistance mechanisms among microorganisms, since it has been demonstrated to occur in many Gram-positive and Gram-negative bacteria including medically important species like staphylococci, streptococci, enterobacteria and opportunistic pathogens like Pseudomonas aeruginosa. Efflux pumps can be specific for only one substrate or accommodate a more or less wide range of noxious products. Export of structurally unrelated compounds confers a multidrug-resistance phenotype on bacterial cells. Therapeutically critical levels of resistance can be achieved by overexpression of efflux systems, especially in those species such as P. aeruginosa which possess a low outer membrane permeability. It is suspected that the dual physiological function of active efflux systems is both the secretion of intracellular metabolites and the protection against a variety of harmful substances that the microorganism may encounter in its natural environment.