The CTX-M beta-lactamase pandemic

In the past decade CTX-M enzymes have become the most prevalent extended-spectrum beta-lactamases, both in nosocomial and in community settings. The insertion sequences (ISs) ISEcp1 and ISCR1 (formerly common region 1 [CR1] or orf513) appear to enable the mobilization of chromosomal beta-lactamase Kluyvera species genes, which display high homology with blaCTX-Ms. These ISs are preferentially linked to specific genes: ISEcp1 to most blaCTX-Ms, and ISCR1 to blaCTX-M-2 or blaCTX-M-9. The blaCTX-M genes embedded in class 1 integrons bearing ISCR1 are associated with different Tn402-derivatives, and often with mercury Tn21-like transposons. The blaCTX-M genes linked to ISEcp1 are often located in multidrug resistance regions containing different transposons and ISs. These structures have been located in narrow and broad host-range plasmids belonging to the same incompatibility groups as those of early antibiotic resistance plasmids. These plasmids frequently carry aminoglycoside, tetracycline, sulfonamide or fluoroquinolone resistance genes [qnr and/or aac(6')-Ib-cr], which would have facilitated the dissemination of blaCTX-M genes because of co-selection processes. In Escherichia coli, they are frequently carried in well-adapted phylogenetic groups with particular virulence-factor genotypes. Also, dissemination has been associated with different clones (CTX-M-9 or CTX-M-14 producers) or epidemic clones associated with specific enzymes such as CTX-M-15. All these events might have contributed to the current pandemic CTX-M beta-lactamase scenario.     

Tn916-like genetic elements: a diverse group of modular mobile elements conferring antibiotic resistance

Antibiotic-resistant Gram-positive bacteria are responsible for morbidity and mortality in healthcare environments. Enterococcus faecium, Enterococcus faecalis, Staphylococcus aureus and Streptococcus pneumoniae can all exhibit clinically relevant multidrug resistance phenotypes due to acquired resistance genes on mobile genetic elements. It is possible that clinically relevant multidrug-resistant Clostridium difficile strains will appear in the future, as the organism is adept at acquiring mobile genetic elements (plasmids and transposons). Conjugative transposons of the Tn916/Tn1545 family, which carry major antibiotic resistance determinants, are transmissible between these different bacteria by a conjugative mechanism during which the elements are excised by a staggered cut from donor cells, converted to a circular form, transferred by cell-cell contact and inserted into recipient cells by a site-specific recombinase. The ability of these conjugative transposons to acquire additional, clinically relevant antibiotic resistance genes importantly contributes to the emergence of multidrug resistance.     

ISCR elements: novel gene-capturing systems of the 21st century?

"Common regions" (CRs), such as Orf513, are being increasingly linked to mega-antibiotic-resistant regions. While their overall nucleotide sequences show little identity to other mobile elements, amino acid alignments indicate that they possess the key motifs of IS91-like elements, which have been linked to the mobility ent plasmids in pathogenic Escherichia coli. Further inspection reveals that they possess an IS91-like origin of replication and termination sites (terIS), and therefore CRs probably transpose via a rolling-circle replication mechanism. Accordingly, in this review we have renamed CRs as ISCRs to give a more accurate reflection of their functional properties. The genetic context surrounding ISCRs indicates that they can procure 5' sequences via misreading of the cognate terIS, i.e., "unchecked transposition." Clinically, the most worrying aspect of ISCRs is that they are increasingly being linked with more potent examples of resistance, i.e., metallo-beta-lactamases in Pseudomonas aeruginosa and co-trimoxazole resistance in Stenotrophomonas maltophilia. Furthermore, if ISCR elements do move via "unchecked RC transposition," as has been speculated for ISCR1, then this mechanism provides antibiotic resistance genes with a highly mobile genetic vehicle that could greatly exceed the effects of previously reported mobile genetic mechanisms. It has been hypothesized that bacteria will surprise us by extending their "genetic construction kit" to procure and evince additional DNA and, therefore, antibiotic resistance genes. It appears that ISCR elements have now firmly established themselves within that regimen.     

Broad host range gene transfer: plasmids and conjugative transposons

Abstract Conjugation is the primary route of broad host range DNA transfer between different genera of bacteria. Plasmids are the most familiar conjugative elements, but there are also self-transmissible integrated elements called conjugative transposons. Conjugative transposons have been found in many genera of gram-positive bacteria, in mycoplasmas and in gram negative bacteria such as Bacteriodes spp. and Moraxella spp., and they have a very broad host range. The best-studied conjugative transposons are: the ones related to Tn 916 , a 16 kb conjugative transposon found originally in Gram-positive bacteria; Tn 5276 , a 70 kb conjugative transposon from Lactococcus lactis ; and a group of large (> 70 kb) conjugative transposons found in Bacteroides spp. Transfer of conjugative transposons takes place in three steps: excision to form a circular intermediate, transfer of one strand of the circular intermediate to a recipient, and integration into the recipient genome. Some conjugative transposons integrate almost randomly, whereas other integrate site-specifically. Conjugative transposons not only transfer themselves but also mobilize co-resident plasmids, either by providing transfer functions in trans or by inserting themselves into the plasmid. In addition, the conjugative transposons found in Bacteroides spp. can excise and mobilize unlinked integrated elements, called NBUs. Transfer of many of the Bacteroides conjugative transposons is regulated by tetracycline, whereas transfer of Tn 916 and other conjugative transposons appears to be constitutive. The conjugative transposons are clearly widespread in clinical isolates, but their distribution in environmental isolates remains to be determined.     

插入序列共同区元件：细菌中新出现的一种基因捕获系统

摘要：插入序列共同区（Insertion sequence common region,ISCR）元件是一类在结构和功能上与IS91家族相似的特殊插入序列,特点是缺少了末端反向重复序列（Inverted repeats, IRs）,在插入位点不产生直接重复序列,并通过滚环式（Rolling circle, RC）进行转座。ISCR元件作为一种新的基因捕获系统,它可以移动邻近的任何DNA序列,为耐药基因在不同种属细菌间水平传播提供了高效的媒介。世界各地多种革兰氏阴性病原菌中已发现有19种ISCR元件,大部分ISCR元件同时携带了多种耐药基因,提示ISCR有可能会造成细菌多重耐药性的快速传播。本文就ISCR结构特征、类型、移动方式、起源及进化的研究进展进行了综述。     

Conjugative transposons: an unusual and diverse set of integrated gene transfer elements.

Conjugative transposons are integrated DNA elements that excise themselves to form a covalently closed circular intermediate. This circular intermediate can either reintegrate in the same cell (intracellular transposition) or transfer by conjugation to a recipient and integrate into the recipient's genome (intercellular transposition). Conjugative transposons were first found in gram-positive cocci but are now known to be present in a variety of gram-positive and gram-negative bacteria also. Conjugative transposons have a surprisingly broad host range, and they probably contribute as much as plasmids to the spread of antibiotic resistance genes in some genera of disease-causing bacteria. Resistance genes need not be carried on the conjugative transposon to be transferred. Many conjugative transposons can mobilize coresident plasmids, and the Bacteroides conjugative transposons can even excise and mobilize unlinked integrated elements. The Bacteroides conjugative transposons are also unusual in that their transfer activities are regulated by tetracycline via a complex regulatory network.     

Acquired antibiotic resistance in lactic acid bacteria from food

Acquired antibiotic resistance, i.e. resistance genes located on conjugative or mobilizable plasmids and transposons can be found in species living in habitats (e.g. human and animal intestines) which are regularly challenged with antibiotics. Most data are available for enterococci and enteric lactobacilli. Raw material from animals (milk and meat) which are inadvertantly contaminated with fecal matters during production will carry antibiotic resistant lactic acid bacteria into the final fermented products such as raw milk cheeses and raw sausages. The discovered conjugative genetic elements of LAB isolated from animals and food are very similar to elements studied previously in pathogenic streptococci and enterococci, e.g. -type replicating plasmids of the pAM1, pIP501-family, and transposons of the Tn916-type. Observed resistance genes include known genes like tetM, ermAM, cat, sat and vanA. A composite 29'871 bp resistance plasmid detected in Lactococcus lacti s subsp. lactis isolated from a raw milk soft cheese contains tetS previously described in Listeria monocytogenes, cat and str from Staphylococcus aureus. Three out of five IS elements on the plasmid are almost or completely identical to IS1216 present in the vanA resistance transposon Tn1546. These data support the view that in antibiotic challenged habitats lactic acid bacteria like other bacteria participate in the communication systems which transfer resistance traits over species and genus borders. The prevalence of such bacteria with acquired resistances like enterococci is high in animals (and humans) which are regularly treated with antibiotics. The transfer of antibiotic resistant bacteria from animals into fermented and other food can be avoided if the raw substrate milk or meat is pasteurized or heat treated. Antibiotic resistance traits as selectable markers in genetic modification of lactic acid bacteria for different purposes are presently being replaced, e.g. by metabo lic traits to generate food-grade vectors.     

Dissemination of the strA-strB streptomycin-resistance genes among commensal and pathogenic bacteria from humans, animals, and plants

Gene transfer within bacterial communities has been recognized as a major contributor in the recent evolution of antibiotic resistance on a global scale. The linked strA-strB genes, which encode streptomycin-inactivating enzymes, are distributed worldwide and confer streptomycin resistance in at least 17 genera of gram-negative bacteria. Nucleotide sequence analyses suggest that strA-strB have been recently disseminated. In bacterial isolates from humans and animals, strA-strB are often linked with the sulII sulfonamide-resistance gene and are encoded on broad-host-range nonconjugative plasmids. In bacterial isolates from plants, strA-strB are encoded on the Tn3-type transposon Tn5393 which is generally borne on conjugative plasmids. The wide distribution of the strA-strB genes in the environment suggests that gene transfer events between human, animal, and plant-associated bacteria have occurred. Although the usage of streptomycin in clinical medicine and animal husbandry has diminished, the persistence of strA-strB in bacterial populations implies that factors other than direct antibiotic selection are involved in maintenance of these genes.     

Sequence of the 68,869 bp IncP-1alpha plasmid pTB11 from a waste-water treatment plant reveals a highly conserved backbone, a Tn402-like integron and other transposable elements

To analyse the significance of conjugative broad-host-range IncP-1alpha plasmids for the spread of antibiotic resistance determinants in waste-water treatment plants we isolated and characterised five different IncP-1alpha plasmids from bacteria of activated sludge and the final effluents of a municipal waste-water treatment plant. These plasmids mediate resistance to ampicillin, cefaclor, cefuroxime, gentamicin, kanamycin, spectinomycin, streptomycin, tetracycline, tobramycin, and trimethoprim. The complete 68,869 bp DNA-sequence of the IncP-1alpha plasmid pTB11 was determined. The pTB11 backbone modules for replication (Rep), mating pair formation (Trb), multimer resolution (Mrs), post-segregational killing (Psk), conjugative DNA-transfer (Tra), plasmid control (Ctl), and stable maintenance and inheritance (KilA, KilE, and KilC) are highly conserved as compared to the 'Birmingham' IncP-1alpha plasmids. In contrast to the 'Birmingham' plasmids pTB11 carries an insert of a Tn402-derivative integrating a class 1 integron in the intergenic region between the multimer resolution operon parCBA and the post-segregational killing operon parDE. The integron comprises the resistance gene cassettes oxa2 (beta-lactamase), aacA4 (aminoglycoside-6'N-acetyltransferase), and aadA1 (aminoglycoside-3'-adenylyltransferase) and a complete tniABQR transposition module. Integron-specific sequences were also identified on other IncP-1alpha plasmids analysed in this work. In contrast to the 'Birmingham' plasmids the pTB11 tetracycline resistance module carries a pecM- and a pncA-like gene downstream of the tetracycline resistance gene tetA and contains an insertion of the new insertion sequence element ISTB11. The transposable elements IS21 and Tn1 which disrupted, respectively, orf7 and klcB on the 'Birmingham' plasmids are not present on pTB11. Identification of IncP-1alpha plasmids in bacteria of the waste-water treatment plant's final effluents indicates that bacteria carrying these kind of plasmids are released into the environment.     

The ICESt1 element of Streptococcus thermophilus belongs to a large family of integrative and conjugative elements that exchange modules and change their specificity of integration

The 34,734-bp element ICESt1 from Streptococcus thermophilus CNRZ368 is site-specifically integrated into the 3(') end of the gene fda. ICESt1 encodes integrative functions and putative transfer functions. Six proteins of the putative conjugative system of ICESt1 are related to those encoded by the conjugative transposon Tn916 from Enterococcus faecalis. A comparison of these proteins with those encoded by the complete or partial genome sequences of various low G+C bacteria including Bacillus subtilis, Clostridium difficile, E. faecalis, Listeria monocytogenes, Staphylococcus aureus, and Streptococcus mutans revealed the presence of numerous putative site-specific integrative conjugative elements and/or conjugative transposons within these genomes. Sequence comparisons revealed that these elements possess a modular structure and that exchanges of unrelated or distantly related modules and genes have occurred between these elements, and also plasmids and prophages. These exchanges have probably led to modifications in the site specificity of integration of these elements. Therefore, a distinction between low specificity integrative conjugative elements (i.e., conjugative transposons) and site-specific integrative conjugative elements does not appear to be relevant. We propose to call all the conjugative elements that excise by site-specific recombination and integrate by recombination between a specific site of a circular intermediate and another site, "Integrative and Conjugative Elements" (ICEs), irrespective of the integration specificity.     

Characterisation of plasmids purified from Acetobacter pasteurianus 2374

Four cryptic plasmids pAP1, pAP2, pAP3, and pAP4 with their replication regions AP were isolated from Gram-negative bacteria Acetobacter pasteurianus 2374 and characterised by sequence analyses. All plasmids were carrying the kanamycin resistance gene. Three of four plasmids pAP2, pAP3, and pAP4 encode an enzyme that confers ampicillin resistance to host cells. Moreover, the tetracycline resistance gene was identified only in pAP2 plasmid. All plasmids are capable to coexist with each other in Acetobacter cells. On the other hand, the coexistence of more than one plasmid is excluded in Escherichia coli. The nucleotide sequence of replication regions showed significant homology. The nucleotide and protein sequence analyses of resistance genes of all plasmids were compared with transposons Tn3, Tn10, and Tn903 which revealed significant differences in the primary structure, however no functional changes of gene were obtained.     

Integrons,transposons and IS elements promote diversification of multidrug resistance plasmids and adaptation of their hosts to antibiotic pollutants from pharmaceutical companies

Plasmids are important vehicles for the dissemination of antibiotic resistance genes (ARGs) among bacteria by conjugation. Here, we determined the complete nucleotide sequences of nine different plasmids previously obtained by exogenous plasmid isolation from river and creek sediments and wastewater from a pharmaceutical company. We identified six IncP/P-1ε plasmids and single members of IncL, IncN and IncFII-like plasmids. Genetic structures of the accessory regions of the IncP/P-1ε plasmids obtained implied that multiple insertions and deletions had occurred, mediated by different transposons and Class 1 integrons with various ARGs. Our study provides compelling evidence that Class 1 integrons, Tn402-like transposons, Tn3-like transposons and/or IS26 played important roles in the acquisition of ARGs across all investigated plasmids. Our plasmid sequencing data provide new insights into how these mobile genetic elements could mediate the acquisition and spread of ARGs in environmental bacteria.     

Characterization of In0 of Pseudomonas aeruginosa plasmid pVS1, an ancestor of integrons of multiresistance plasmids and transposons of gram-negative bacteria.

Many multiresistance plasmids and transposons of gram-negative bacteria carry related DNA elements that appear to have evolved from a common ancestor by site-specific integration of discrete cassettes containing antibiotic resistance genes or sequences of unknown function. The site of integration is flanked by conserved segments coding for an integraselike protein and for sulfonamide resistance, respectively. These segments, together with the antibiotic resistance genes between them, have been termed integrons (H. W. Stokes and R. M. Hall, Mol. Microbiol. 3:1669-1683, 1989). We report here the characterization of an integron, In0, from Pseudomonas aeruginosa plasmid pVS1, which has an unoccupied integration site and hence may be an ancestor of more complex integrons. Codon usage of the integrase (int) and sulfonamide resistance (sul1) genes carried by this integron suggests a common origin. This contrasts with the codon usage of other antibiotic resistance genes that were presumably integrated later as cassettes during the evolution and spread of these DNA elements. We propose evolutionary schemes for (i) the genesis of the integrons by the site-specific integration of antibiotic resistance genes and (ii) the evolution of the integrons of multiresistance plasmids and transposons, in relation to the evolution of transposons related to Tn21.     

产超广谱β-内酰胺酶细菌中I类整合子的研究进展

产超广谱β-内酰胺酶(Extended-spectrum beta-lactamase, ESBLs)细菌的多重耐药性是临床用药的一大难题, 近年研究发现其耐药性的产生与整合子密切相关, 其中临床最常见、研究最深入的是I类整合子。整合子是一种可移动基因元件, 在整合酶的作用下捕捉外源基因盒并使之表达, 是具有基因整合和切除功能的天然克隆和表达系统。研究表明I类整合子可连续捕捉和整合多种耐药基因, 以质粒或转座子为载体在细菌之间传播耐药性, 使ESBLs细菌多重耐药趋势十分严峻。本文就I类整合子的结构特征、I类整合子对耐药基因盒的整合作用及其与ESBLs细菌耐药性的关系等方面进行综述。     

Transposition genes of the Bacteroides mobilizable transposon Tn4555: role of a novel targeting gene

Conjugative transposons have been identified in several bacterial species, most notably the Gram-positive Enterococci and the Gram-negative Bacteroides. In Bacteroides species, these elements encode a complete conjugative machinery, which mediates their own intercellular transfer, and they can mobilize in trans co-resident elements. One such mobilizable element is the antibiotic resistance transposon, Tn4555, which was previously found to integrate into a specific genome target site via a site-specific recombination mechanism. In this work, we demonstrate that three Tn4555 genes were involved in integration of the element. These were int encoding a lambda-type integrase, which was absolutely required for integration of the transposon, and two accessory genes, which increased the frequency of integration. Interestingly, one of these accessory gene products, TnpA, directed the insertion of Tn4555 into the genome target site; in the absence of tnpA, the insertion pattern was essentially random. This is the first example of a site-specific recombinase that uses a specific targeting protein.     

Das bakterielle Mobilom

Mobile elements Meanwhile, we know that mobile elements are present in all forms of life. The genomes of mammals consist of up to 50% of mobile elements, those of plants up to 90%. In bacteria mobile elements establish themselves as autonomously replicating replicons (plasmids and plasmid‐prophages) or are constituents of replicons such as IS elements, transposons, conjugative transposons, integrative and conjugative elements (ICEs), genomic islands and integrons. Why distinct bacterial species still exist? Most probably, still unknown mechanisms ensure that some genes never become mobile.     

Functional identification of conjugation and replication regions of the tetracycline resistance plasmid pCW3 from Clostridium perfringens

Clostridium perfringens causes fatal human infections, such as gas gangrene, as well as gastrointestinal diseases in both humans and animals. Detailed molecular analysis of the tetracycline resistance plasmid pCW3 from C. perfringens has shown that it represents the prototype of a unique family of conjugative antibiotic resistance and virulence plasmids. We have identified the pCW3 replication region by deletion and transposon mutagenesis and showed that the essential rep gene encoded a basic protein with no similarity to any known plasmid replication proteins. An 11-gene conjugation locus containing 5 genes that encoded putative proteins with similarity to proteins from the conjugative transposon Tn916 was identified, although the genes' genetic arrangements were different. Functional genetic studies demonstrated that two of the genes in this transfer clostridial plasmid (tcp) locus, tcpF and tcpH, were essential for the conjugative transfer of pCW3, and comparative analysis confirmed that the tcp locus was not confined to pCW3. The conjugation region was present on all known conjugative plasmids from C. perfringens, including an enterotoxin plasmid and other toxin plasmids. These results have significant implications for plasmid evolution, as they provide evidence that a nonreplicating Tn916-like element can evolve to become the conjugation locus of replicating plasmids that carry major virulence genes or antibiotic resistance determinants.     

IntI2 integron integrase in Tn7

Integrons can insert and excise antibiotic resistance genes on plasmids in bacteria by site-specific recombination. Class 1 integrons code for an integrase, IntI1 (337 amino acids in length), and are generally borne on elements derived from Tn5090, such as that found in the central part of Tn21. A second class of integron is found on transposon Tn7 and its relatives. We have completed the sequence of the Tn7 integrase gene, intI2, which contains an internal stop codon. This codon was found to be conserved among intI2 genes on three other Tn7-like transposons harboring different cassettes. The predicted peptide sequence (IntI2*) is 325 amino acids long and is 46% identical to IntI1. In order to detect recombination activity, the internal stop codon at position 179 in the parental allele was changed to a triplet coding for glutamic acid. The sequences flanking the cassette arrays in the class 1 and 2 integrons are not closely related, but a common pool of mobile cassettes is used by the different integron classes; two of the three antibiotic resistance cassettes on Tn7 and its close relatives are also found in various class 1 integrons. We also observed a fourth excisable cassette downstream of those described previously in Tn7. The fourth cassette encodes a 165-amino-acid protein of unknown function with 6.5 contiguous repeats of a sequence coding for 7 amino acids. IntI2*179E promoted site-specific excision of each of the cassettes in Tn7 at different frequencies. The integrases from Tn21 and Tn7 showed limited cross-specificity in that IntI1 could excise all cassettes from both Tn21 and Tn7. However, we did not observe a corresponding excision of the aadA1 cassette from Tn21 by IntI2*179E.