The A to Z of A/C Plasmids

Plasmids belonging to incompatibility groups A and C (now A/C) were among the earliest to be associated with antibiotic resistance in Gram-negative bacteria. A/C plasmids are large, conjugative plasmids with a broad host range. The prevalence of A/C plasmids in collections of clinical isolates has revealed their importance in the dissemination of extended-spectrum β-lactamases and carbapenemases. They also mobilize SGI1-type resistance islands. Revived interest in the family has yielded many complete A/C plasmid sequences, revealing that RA1, designated A/C1, is different from the remainder, designated A/C2. There are two distinct A/C2 lineages. Backbones of 128-130 kb include over 120 genes or ORFs encoding proteins of at least 100 amino acids, but very few have been characterized. Genes potentially required for replication, stability and transfer have been identified, but only the replication system of RA1 and the regulation of transfer have been studied. There is enormous variety in the antibiotic resistance genes carried by A/C2 plasmids but they are usually clustered in larger regions at various locations in the backbone. The ARI-A and ARI-B resistance islands are always at a specific location but have variable content. ARI-A is only found in type 1 A/C2 plasmids, which disseminate blaCMY-2 and blaNDM-1 genes, whereas ARI-B, carrying the sul2 gene, is found in both type 1 and type 2. This review summarizes current knowledge of A/C plasmids, and highlights areas of research to be considered in the future

IS26-Mediated Precise Excision of the IS26-aphA1a Translocatable Unit

We recently showed that, in the absence of RecA-dependent homologous recombination, the Tnp26 transposase catalyzes cointegrate formation via a conservative reaction between two preexisting IS26, and this is strongly preferred over replicative transposition to a new site. Here, the reverse reaction was investigated by assaying for precise excision of the central region together with a single IS26 from a compound transposon bounded by IS26. In a recA mutant strain, Tn4352, a kanamycin resistance transposon carrying the aphA1a gene, was stable. However, loss of kanamycin resistance due to precise excision of the translocatable unit (TU) from the closely related Tn4352B, leaving behind the second IS26, occurred at high frequency. Excision occurred when Tn4352B was in either a high- or low-copy-number plasmid. The excised circular segment, known as a TU, was detected by PCR. Excision required the IS26 transposase Tnp26. However, the Tnp26 of only one IS26 in Tn4352B was required, specifically the IS26 downstream of the aphA1a gene, and the excised TU included the active IS26. The frequency of Tn4352B TU loss was influenced by the context of the transposon, but the critical determinant of high-frequency excision was the presence of three G residues in Tn4352B replacing a single G in Tn4352. These G residues are located immediately adjacent to the two G residues at the left end of the IS26 that is upstream of the aphA1a gene. Transcription of tnp26 was not affected by the additional G residues, which appear to enhance Tnp26 cleavage at this end

A large conjugative Acinetobacter baumannii plasmid carrying the sul2 sulphonamide and strAB streptomycin resistance genes

Acinetobacter baumannii is an important nosocomial pathogen that often complicates treatment because of its high level of resistance to antibiotics. Though plasmids can potentially introduce various genes into bacterial strains, compared to other Gram-negative bacteria, information about the unique A. baumannii plasmid repertoire is limited. Here, whole genome sequence data was used to determine the plasmid content of strain A297 (RUH875), the reference strain for the globally disseminated multiply resistant A. baumannii clone, global clone 1(GC1). A297 contains three plasmids. Two known plasmids were present; one, pA297-1 (pRAY*), carries the aadB gentamicin, kanamycin and tobramycin resistance gene and another is an 8.7kb cryptic plasmid often found in GC1 isolates. The third plasmid, pA297-3, is 200kb and carries the sul2 sulphonamide resistance gene and strAB streptomycin resistance gene within Tn6172 and a mer mercuric ion resistance module elsewhere. pA297-3 transferred sulphonamide, streptomycin and mercuric ion resistance at high frequency to a susceptible A. baumannii recipient, and contains several genes potentially involved in conjugative transfer. However, a relaxase gene was not found. It also includes several genes encoding proteins involved in DNA metabolism such as partitioning. However, a gene encoding a replication initiation protein could not be found. pA297-3 includes two copies of a Miniature Inverted-Repeat Transposable Element (MITE), named MITE-297, bracketing a 77.5kb fragment, which contains several IS and the mer module. Several plasmids related to but smaller than pA297-3 were found in the GenBank nucleotide database. They were found in different A. baumannii clones and are wide spread. They all contain either Tn6172 or a variant in the same position in the backbone as Tn6172 in pA297-3. Some related plasmids have lost the segment between the MITE-297 copies and retain only one MITE-297. Others have segments of various lengths between two MITE-297 copies, and these can be derived from the region in pA297-3 via a deletion adjacent to IS related to IS26 such as IS1007 or IS1007-like. pA297-3 and its relatives represent a third type of conjugative Acinetobacter plasmid that contributes to the dissemination of antibiotic resistance in this species.NHMR

p39R861-4, a type 2 A/C2 plasmid carrying a segment from the A/C1 RA1

The largest plasmid in the strain 39R861, which is used as a plasmid size standard, was recovered by conjugation and sequenced to determine its exact size. Plasmid p39R861-4 transferred at high frequency. Though reported to be the A/C1 plasmid RA1, p39R861-4 is a 155794 bp Type 2 A/C2 plasmid in which a 39 kb segment derived from RA1 that includes a relative of the RA1 resistance island replaces 26.5 kb of the Type 2 backbone. p39R861-4 includes a single copy of IS10 and two resistance islands with a CR2-sul2 region in each of them. The 84 kb of backbone between the resistance islands is inverted relative to other known A/C plasmids and this inversion has arisen via recombination between the CR2-sul2 regions that are inversely oriented. The resistance islands prior to inversion were one related to but longer than that found in RA1, and a form of the ARI-B island identical to one found in the A/C2 plasmid R55. They contain genes conferring resistance to tetracycline (tetA(D)), sulphonamides (sul2) and florfenicol and chloramphenicol (floR). The tet(D) determinant is flanked by two IS26 in a transposon-like structure named Tntet(D). Both resistance islands contain remnants of the two ends of the integrative element GIsul2, consistent with the sul2 gene being mobilized by GIsul2 rather than by CR2

The implementation of chlamydia screening: a cross-sectional study in the south east of England

Background England's National Chlamydia Screening Programme (NCSP) provides opportunistic testing for under 25 year-olds in healthcare and non-healthcare settings. The authors aimed to explore relationships between coverage and positivity in relation to demographic characteristics or setting, in order to inform efficient and sustainable implementation of the NCSP. Methods The authors analysed mapped NCSP testing data from the South East region of England between April 2006 and March 2007 inclusive to population characteristics. Coverage was estimated by sex, demographic characteristics and service characteristics, and variation in positivity by setting and population group. Results Coverage in females was lower in the least deprived areas compared with the most deprived areas (OR 0.48; 95% CI 0.45 to 0.50). Testing rates were lower in 20 1324-year-olds compared with 15 1319-year-olds (OR 0.69; 95% CI 0.67 to 0.72 for females and OR 0.67; 95% CI 0.64 to 0.71 for males), but positivity was higher in older males. Females were tested most often in healthcare services, which also identified the most positives. The greatest proportions of male tests were in university (27%) and military (19%) settings which only identified a total of 11% and 13% of total male positives respectively. More chlamydia-positive males were identified through healthcare services despite fewer numbers of tests. Conclusions Testing of males focused on institutional settings where there is a low yield of positives, and limited capacity for expansion. By contrast, the testing of females, especially in urban environments, was mainly through established healthcare services. Future strategies should prioritise increasing male testing in healthcare settings

A Type 2 A/C2 plasmid carrying the aacC4 apramycin resistance gene and the erm(42) erythromycin resistance gene recovered from two Salmonella enterica serovars

Objective: To determine the relationships between RepA/C2 plasmids carrying several antibiotic resistance genes found in isolates of Salmonella enterica serovars Ohio and Senftenberg from pigs. Methods: Illumina HiSeq was used to sequence seven S. enterica isolates. BLAST searches identified relevant A/C2 plasmid contigs, and contigs were assembled using PCR. Results: Two serovar Ohio isolates were ST329 and the five Senftenberg isolates were ST210. The A/C2 plasmids recovered from the seven isolates belong to Type 2 and contain two resistance islands. Their backbones were closely related, differing by five or fewer single nucleotide polymorphisms. The sul2-containing resistance island ARI-B is 19.9 kb and also contains the kanamycin and neomycin resistance gene aphA1, the tetracycline resistance gene tetA(D), and an erythromycin resistance gene, erm(42), not previously seen in A/C2 plasmids. A second 30.3 kb resistance island, RI-119, is in a unique location in the A/C2 backbone 8.2 kb downstream of rhs. RI-119 contained genes conferring resistance to apramycin, netilmicin, tobramycin (aacC4), hygromycin (hph), sulphonamides (sul1) and spectinomycin and streptomycin (aadA2). In one of the seven plasmids, this resistance region contained two IS26-mediated deletions. A discrete 5.7 kb segment containing the aacC4 and hph genes and bounded by IS26 on one side and the IR of Tn5393 on the other was identified. Conclusions: The presence of almost identical A/C2 plasmids in two serovars indicates a common origin. Type 2 A/C2 plasmids continue to evolve via addition of new resistance regions such as RI-119 and evolution of existing ones

A Type 2 A/C2 plasmid carrying the aacC4 apramycin resistance gene and the erm(42) erythromycin resistance gene recovered from two Salmonella enterica serovars

Objective: To determine the relationships between RepA/C2 plasmids carrying several antibiotic resistance genes found in isolates of Salmonella enterica serovars Ohio and Senftenberg from pigs. Methods: Illumina HiSeq was used to sequence seven S. enterica isolates. BLAST searches identified relevant A/C2 plasmid contigs, and contigs were assembled using PCR. Results: Two serovar Ohio isolates were ST329 and the five Senftenberg isolates were ST210. The A/C2 plasmids recovered from the seven isolates belong to Type 2 and contain two resistance islands. Their backbones were closely related, differing by five or fewer single nucleotide polymorphisms. The sul2-containing resistance island ARI-B is 19.9 kb and also contains the kanamycin and neomycin resistance gene aphA1, the tetracycline resistance gene tetA(D), and an erythromycin resistance gene, erm(42), not previously seen in A/C2 plasmids. A second 30.3 kb resistance island, RI-119, is in a unique location in the A/C2 backbone 8.2 kb downstream of rhs. RI-119 contained genes conferring resistance to apramycin, netilmicin, tobramycin (aacC4), hygromycin (hph), sulphonamides (sul1) and spectinomycin and streptomycin (aadA2). In one of the seven plasmids, this resistance region contained two IS26-mediated deletions. A discrete 5.7 kb segment containing the aacC4 and hph genes and bounded by IS26 on one side and the IR of Tn5393 on the other was identified. Conclusions: The presence of almost identical A/C2 plasmids in two serovars indicates a common origin. Type 2 A/C2 plasmids continue to evolve via addition of new resistance regions such as RI-119 and evolution of existing ones

Movement of IS26-Associated Antibiotic Resistance Genes Occurs via a Translocatable Unit That Includes a Single IS26 and Preferentially Inserts Adjacent to Another IS26

TheinsertionsequenceIS26playsakeyroleindisseminatingantibioticresistancegenesinGram-negativebacteria, forming regions containing more than one antibiotic resistance gene that are flanked by and interspersed with copies of IS26. A model presented for a second mode of IS26 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 IS26 such that the ISs are in direct orientation. Repeating this process would lead to arrays of resistance genes with directly oriented copies of IS26 at each end and between each unique segment. This model requires that IS26 recognizes another IS26 as a target, and in transpo- sition experiments, the frequency of cointegrate formation was 60-fold higher when the target plasmid contained IS26. This re- action was conservative, with no additional IS26 or target site duplication generated, and orientation specific as the IS26s in the cointegrates were always in the same orientation. Consequently, the cointegrates were identical to those formed via the known mode of IS26 movement when a target IS26 was not present. Intact transposase genes in both IS26s were required for high- frequency cointegrate formation as inactivation of either one reduced the frequency 30-fold. However, the IS26 target specificity was retained. Conversion of each residue in the DDE motif of the Tnp26 transposase also reduced the cointegration frequency

Aquariums as Reservoirs for Multidrug-resistant Salmonella Paratyphi B

Multidrug-resistant Salmonella enterica serovar Paratyphi B dT+ isolates from patients with gastroenteritis were identical with isolates from their home aquariums. Matched isolates had identical phage types, XbaI and IS200 profiles, and Salmonella genomic island 1 (SGI1). Ornamental fish tanks are reservoirs for SGI1-containing S. Paratyphi B dT+