The insertion sequence IS 26 plays a key role in disseminating antibiotic resistance genes in Gram-negative bacteria, forming regions containing more than one antibiotic resistance gene that are flanked by and interspersed with copies of IS 26 . A model presented for a second mode of IS 26 movement that explains the structure of these regions involves a translocatable unit consisting of a unique DNA segment carrying an antibiotic resistance (or other) gene and a single IS copy. Structures resembling class I transposons are generated via RecA-independent incorporation of a translocatable unit next to a second IS 26 such that the ISs are in direct orientation. Repeating this process would lead to arrays of resistance genes with directly oriented copies of IS 26 at each end and between each unique segment. This model requires that IS 26 recognizes another IS 26 as a target, and in transposition experiments, the frequency of cointegrate formation was 60-fold higher when the target plasmid contained IS 26 . This reaction was conservative, with no additional IS 26 or target site duplication generated, and orientation specific as the IS 26 s in the cointegrates were always in the same orientation. Consequently, the cointegrates were identical to those formed via the known mode of IS 26 movement when a target IS 26 was not present. Intact transposase genes in both IS 26 s were required for high-frequency cointegrate formation as inactivation of either one reduced the frequency 30-fold. However, the IS 26 target specificity was retained. Conversion of each residue in the DDE motif of the Tnp26 transposase also reduced the cointegration frequency. IMPORTANCE Resistance to antibiotics belonging to several of the different classes used to treat infections is a critical problem. Multiply antibiotic-resistant bacteria usually carry large regions containing several antibiotic resistance genes, and in Gram-negative bacteria, IS 26 is often seen in these clusters. A model to explain the unusual structure of regions containing multiple IS 26 copies, each associated with a resistance gene, was not available, and the mechanism of their formation was unexplored. IS 26 -flanked structures deceptively resemble class I transposons, but this work reveals that the features of IS 26 movement do not resemble those of the IS and class I transposons studied to date. IS 26 uses a novel movement mechanism that defines a new family of mobile genetic elements that we have called “translocatable units.” The IS 26 mechanism also explains the properties of IS 257 (IS 431 ) and IS 1216 , which belong to the same IS family and mobilize resistance genes in Gram-positive staphylococci and enterococci.

Extracellular polysaccharides are major immunogenic components of the bacterial cell envelope. However, little is known about their biosynthesis in the genus Acinetobacter , which includes A. baumannii , an important nosocomial pathogen. Whether Acinetobacter sp. produce a capsule or a lipopolysaccharide carrying an O antigen or both is not resolved. To explore these issues, genes involved in the synthesis of complex polysaccharides were located in 10 complete A. baumannii genome sequences, and the function of each of their products was predicted via comparison to enzymes with a known function. The absence of a gene encoding a WaaL ligase, required to link the carbohydrate polymer to the lipid A-core oligosaccharide (lipooligosaccharide) forming lipopolysaccharide, suggests that only a capsule is produced. Nine distinct arrangements of a large capsule biosynthesis locus, designated KL1 to KL9, were found in the genomes. Three forms of a second, smaller variable locus, likely to be required for synthesis of the outer core of the lipid A-core moiety, were designated OCL1 to OCL3 and also annotated. Each K locus includes genes for capsule export as well as genes for synthesis of activated sugar precursors, and for glycosyltransfer, glycan modification and oligosaccharide repeat-unit processing. The K loci all include the export genes at one end and genes for synthesis of common sugar precursors at the other, with a highly variable region that includes the remaining genes in between. Five different capsule loci, KL2, KL6, KL7, KL8 and KL9 were detected in multiply antibiotic resistant isolates belonging to global clone 2, and two other loci, KL1 and KL4, in global clone 1. This indicates that this region is being substituted repeatedly in multiply antibiotic resistant isolates from these clones.

In Gram-negative bacteria, IS 26 recruits antibiotic resistance genes into the mobile gene pool by forming transposons carrying many different resistance genes. In addition to replicative transposition, IS 26 was recently shown to use a novel conservative movement mechanism in which an incoming IS 26 targets a preexisting one. Here, we have demonstrated how IS 26 -bounded class I transposons can be produced from translocatable units (TUs) containing only an IS 26 and a resistance gene via the conservative reaction. TUs were incorporated next to an existing IS 26 , creating a class I transposon, and if the targeted IS 26 is in a transposon, the product resembles two transposons sharing a central IS 26 , a configuration observed in some resistance regions and when a transposon is tandemly duplicated. Though homologous recombination could also incorporate a TU, Tnp26 is far more efficient. This provides insight into how IS 26 builds transposons and brings additional transposons into resistance regions.

This study examined in detail the population structure of Escherichia coli from healthy adults with respect to the prevalence of antibiotic resistance and specific resistance determinants. E. coli isolated from the faeces of 20 healthy adults not recently exposed to antibiotics was tested for resistance to ten antibiotics and for carriage of integrons and resistance determinants using PCR. Strain diversity was assessed using biochemical and molecular criteria. E. coli was present in 19 subjects at levels ranging from 2.0×10⁴ to 1.7×10⁸ c.f.u. (g faeces)⁻¹. Strains resistant to one to six antibiotics were found at high levels (>30 %) in only ten individuals, but at significant levels (>0.5 %) in 14. Resistant isolates with the same phenotype from the same individual were indistinguishable, but more than one susceptible strain was sometimes found. Overall, individuals harboured one to four E. coli strains, although in 17 samples one strain was dominant (>70 % of isolates). Eighteen strains resistant to ampicillin, sulfamethoxazole, tetracycline and trimethoprim in 15 different combinations were observed. One resistant strain was carried by two unrelated individuals and a susceptible strain was shared by two cohabiting subjects. Two minority strains were derivatives of a more abundant resistant strain in the same sample, showing that continuous evolution is occurring in vivo. The trimethoprim-resistance genes dfrA1, dfrA5, dfrA7, dfrA12 or dfrA17 were in cassettes in a class 1 or class 2 integron. Ampicillin resistance was conferred by the bla _TEM gene, sulfamethoxazole resistance by sul1, sul2 or sul3 and tetracycline resistance by tetA(A) or tetA(B). Chloramphenicol resistance (cmlA1 gene) was detected only once. Phylogenetic groups A and B2 were more common than B1 and D. Commensal E. coli of healthy humans represent an important reservoir for numerous antibiotic-resistance genes in many combinations. However, measuring the true extent of resistance carriage in commensal E. coli requires in-depth analysis.

SUMMARY: In Gram-negative bacteria, the insertion sequence IS 26 is highly active in disseminating antibiotic resistance genes. IS 26 can recruit a gene or group of genes into the mobile gene pool and support their continued dissemination to new locations by creating pseudo-compound transposons (PCTs) that can be further mobilized by the insertion sequence (IS). IS 26 can also enhance expression of adjacent potential resistance genes. IS 26 encodes a DDE transposase but has unique properties. It forms cointegrates between two separate DNA molecules using two mechanisms. The well-known copy-in (replicative) route generates an additional IS copy and duplicates the target site. The recently discovered and more efficient and targeted conservative mechanism requires an IS in both participating molecules and does not generate any new sequence. The unit of movement for PCTs, known as a translocatable unit or TU, includes only one IS 26 . TU formed by homologous recombination between the bounding IS 26 s can be reincorporated via either cointegration route. However, the targeted conservative reaction is key to generation of arrays of overlapping PCTs seen in resistant pathogens. Using the copy-in route, IS 26 can also act on a site in the same DNA molecule, either inverting adjacent DNA or generating an adjacent deletion plus a circular molecule carrying the DNA segment lost and an IS copy. If reincorporated, these circular molecules create a new PCT. IS 26 is the best characterized IS in the IS 26 family, which includes IS 257 /IS 431 , ISSau10, IS 1216 , IS 1006 , and IS 1008 that are also implicated in spreading resistance genes in Gram-positive and Gram-negative pathogens.

The oxa23 gene encoding the OXA-23 carbapenemase (and several minor variants of it) is widespread in Acinetobacter baumannii clinical isolates and compromises treatment with carbapenem antibiotics. The gene is derived from the chromosome of Acinetobacter radioresistens where it is an intrinsic gene, here designated oxaAr. In A. baumannii and other Acinetobacter species, oxa23 is usually preceded by an IS, ISAba1, which supplies the strong promoter required for the gene to confer clinically relevant levels of resistance. The oxaAr gene appears to have been mobilized twice creating Tn2008 and Tn2008B, both of which consist of a single ISAba1 and an A. radioresistens-derived fragment. Tn2006 and Tn2009 are clearly derived from Tn2008B and are each made up of Tn2008B with an additional segment of unknown origin and an additional ISAba1, creating a compound transposon. Tn2006, Tn2008 and possibly Tn2008B are globally disseminated, while Tn2009 has as yet only been found in China. Of the four ISAba1-associated transposons, Tn2006 has been most frequently observed worldwide and Tn2006 in Tn6022, known as AbaR4, appears to contribute significantly to the dissemination of oxa23. Moreover, AbaR4, Tn2006, Tn2008 and Tn2009 have each been found in conjugative plasmids, further facilitating their spread.