Mobile Insertion Cassette Research Articles

Characterization of FosL1, a Plasmid-Encoded Fosfomycin Resistance Protein Identified in Escherichia coli.

Fosfomycin is gaining renewed interest for treating urinary tract infections. Monitoring fosfomycin resistance is therefore important in order to detect the emergence of novel resistance mechanisms. Here, we used the Rapid Fosfomycin NP test to screen a collection of extended-spectrum-β-lactamase-producing Escherichia coli isolates from Switzerland and found a fosfomycin-resistant isolate in which a novel plasmid-mediated fosfomycin resistance gene, named fosL1, was identified. The FosL1 protein is a putative glutathione S-transferase enzyme conferring high-level resistance to fosfomycin and sharing between 57% to 63% amino acid identity with other FosA-like family members. Genetic analyses showed that the fosL1 gene was embedded in a mobile insertion cassette and had likely been acquired by transposition through a Tn7-related mechanism. In silico analysis over GenBank databases identified the FosL1-encoding gene in addition to another variant (fosL1 and fosL2, respectively) in two Salmonella enterica isolates from the United States. Our study further highlights the necessity of monitoring fosfomycin resistance in Enterobacteriaceae to identify the emergence of novel mechanisms of resistance.

Unique Features of Aeromonas Plasmid pAC3 and Expression of the Plasmid-Mediated Quinolone Resistance Genes.

A highly fluoroquinolone-resistant isolate of Aeromonas species was isolated from a wastewater treatment plant and found to possess multiple resistance mechanisms, including mutations in gyrA and parC, efflux pumps, and plasmid-mediated quinolone resistance (PMQR) genes. Complete sequencing of the IncU-type plasmid, pAC3, present in the strain revealed a circular plasmid DNA 15,872bp long containing two PMQR genes [qnrS2 and aac(6')-Ib-cr]. A mobile insertion cassette element containing the qnrS2 gene and a typical miniature inverted-repeat transposable element (MITE) structure was identified in the plasmid. The present study revealed that this MITE sequence appears in other Aeromonas species plasmids and chromosomes. Plasmid pAC3 was introduced into Escherichia coli, and its PMQR genes were expressed, resulting in the acquisition of resistance. Proteome analysis of the recipient E.coli strain harboring the plasmid revealed that aac(6')-Ib-cr expression was constitutive and that qnrS2 expression was dependent upon fluoroquinolone stress through regulation by regulator of sigma D (Rsd). To the best of our knowledge, this is the first report to characterize a novel MITE sequence upstream of the PMQR gene within a mobile insertion cassette, as well as the regulation of qnrS2 expression. Our results suggest that this mobile element may play an important role in qnrS2 dissemination. IMPORTANCE In the present study, plasmid pAC3 isolated from a highly fluoroquinolone-resistant isolate of Aeromonas species was sequenced and found to contain two fluoroquinolone resistance genes, aac(6')-Ib-cr and qnrS2. Comparative analyses of plasmid pAC3 and other Aeromonas sp. IncU-type plasmids revealed a mobile insertion cassette element with a unique structure containing a qnrS2 gene and a typical miniature inverted-repeat transposable element (MITE) structure. This study also revealed that this MITE sequence appears in other Aeromonas species plasmids and chromosomes. Our results also demonstrate that the fluoroquinolone-dependent expression of qnrS2 is associated with rsd in E.coli DH5α harboring plasmid pAC3. Our findings suggest that the mobile element may play an important role in qnrS2 dissemination and that Aeromonas species constitute an important reservoir of fluoroquinolone resistance determinants in the environment.

High Prevalence of Plasmid-Mediated Quinolone Resistance and IncQ Plasmids Carrying qnrS2 Gene in Bacteria from Rivers near Hospitals and Aquaculture in China.

Effluents from hospital and aquaculture are considered important sources of quinolone resistance. However, little information is available on the impact of this effluent on nearby rivers. In this study, 188 ciprofloxacin-resistant bacterial isolates obtained from rivers near hospitals and aquaculture were screened for plasmid-mediated quinolone resistance (PMQR) genes. Species identification, antibiotic susceptibility testing, and PMQR gene transferability assessment were conducted for PMQR-positive bacteria. Representative qnrS2-encoding plasmids were subsequently sequenced using a primer-walking approach. In total, 44 isolates (23.4%) were positive for qnr genes (16 qnrB2, 3 qnrS1, and 25 qnrS2) and 32 isolates (17.0%) were positive for aac(6′)-Ib-cr. Other PMQR genes were not detected. The qnrB2 and aac(6′)-Ib-cr genes had a higher prevalence in aquaculture samples than in hospital samples, and were significantly associated with Enterobacteriaceae (p < 0.05). In contrast, the prevalence of qnrS2 was not site-related, but was significantly associated with Aeromonas spp. (p < 0.05). All PMQR isolates were resistant to three or more classes of antibiotics. Eleven qnrS2-harboring plasmids from Aeromonas spp., including a novel conjugative plasmid pHP18, were selected for sequencing. These plasmids were small in size (6,388–16,197 bp) and belonged to the IncQ or IncU plasmid family, with qnrS2 being part of a mobile insertion cassette. Taken together, our findings suggest that aquaculture is a possible source for aac(6′)-Ib-cr and qnrB2 dissemination, and demonstrate the ubiquity of qnrS2 in aquatic environments. Finally, Aeromonas spp. served as vectors for qnrS2 with the help of IncQ-type plasmids.

Complete Sequences of IncU Plasmids Harboring Quinolone Resistance Genes qnrS2 and aac(6')-Ib-cr in Aeromonas spp. from Ornamental Fish.

The nucleotide sequences of three IncU plasmids from Aeromonas spp. isolated from ornamental fish are described. They had a typical IncU backbone for plasmid replication and maintenance functions, but conjugative transfer modules were disrupted. The gene qnrS2 was inserted into mpR as a mobile insertion cassette. Novel Tn3 family transposons carrying putative toxin-antitoxin and plasmid stability genes were identified. The study demonstrates high plasticity of IncU plasmids from aquatic environments.

Complete sequence and detailed analysis of the first indigenous plasmid from Xanthomonas oryzae pv. oryzicola.

BackgroundBacterial plasmids have a major impact on metabolic function and adaptation of their hosts. An indigenous plasmid was identified in a Chinese isolate (GX01) of the invasive phytopathogen Xanthomonas oryzae pv. oryzicola (Xoc), the causal agent of rice bacterial leaf streak (BLS). To elucidate the biological functions of the plasmid, we have sequenced and comprehensively annotated the plasmid.MethodsThe plasmid DNA was extracted from Xoc strain GX01 by alkaline lysis and digested with restriction enzymes. The cloned and subcloned DNA fragments in pUC19 were sequenced by Sanger sequencing. Sequences were assembled by using Sequencher software. Gaps were closed by primer walking and sequencing, and multi-PCRs were conducted through the whole plasmid sequence for verification. BLAST, phylogenetic analysis and dinucleotide calculation were performed for gene annotation and DNA structure analysis. Transformation, transconjugation and stress tolerance tests were carried out for plasmid function assays.ResultsThe indigenous plasmid from Xoc strain GX01, designated pXOCgx01, is 53,206-bp long and has been annotated to possess 64 open reading frames (ORFs), including genes encoding type IV secretion system, heavy metal exporter, plasmid stability factors, and DNA mobile factors, i.e., the Tn3-like transposon. Bioinformatics analysis showed that pXOCgx01 has a mosaic structure containing different genome contexts with distinct genomic heterogeneities. Phylogenetic analysis indicated that the closest relative of pXOCgx01 is pXAC64 from Xanthomonas axonopodis pv. citri str. 306. It was estimated that there are four copies of pXOCgx01 per cell of Xoc GX01 by PCR assay and the calculation of whole genome shotgun sequencing data. We demonstrate that pXOCgx01 is a self-transmissible plasmid and can replicate in some Xanthomonas spp. strains, but not in Escherichia coli DH5α. It could significantly enhance the tolerance of Xanthomonas oryzae pv. oryzae PXO99A to the stresses of heavy metal ions. The plasmid survey indicated that nine out of 257 Xoc Chinese isolates contain plasmids.ConclusionspXOCgx01 is the first report of indigenous plasmid from Xanthomonas oryzae pv. oryzicola, and the first completely sequenced plasmid from Xanthomonas oryzae species. It is a self-transmissible plasmid and has a mosaic structure, containing genes for macromolecule secretion, heavy metal exportation, and DNA mobile factors, especially the Tn3-like transposon which may provide transposition function for mobile insertion cassette and play a major role in the spread of pathogenicity determinants. The results will be helpful to elucidate the biological significance of this cryptic plasmid and the adaptive evolution of Xoc.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0562-x) contains supplementary material, which is available to authorized users.

Mobile Insertion Cassette Elements Found in Small Non-Transmissible Plasmids in Proteeae May Explain qnrD Mobilization

qnrD is a plasmid mediated quinolone resistance gene from unknown origin, recently described in Enterobacteriaceae. It encodes a pentapeptide repeat protein 36–60% different from the other Qnr (A, B, C, S and VC). Since most qnrD-positive strains were described as strains belonging to Proteus or Providencia genera, we hypothesized that qnrD originated in Proteeae before disseminating to other enterobacterial species. We screened 317 strains of Proteeae for qnrD and its genetic support by PCR. For all the seven qnrD-positive strains (4 Proteus mirabilis, 1 Proteus vulgaris and 2 Providencia rettgeri) the gene was carried onto a small non-transmissible plasmid, contrarily to other qnr genes that are usually carried onto large multi-resistant plasmids. Nucleotide sequences of the qnrD-bearing plasmids were 96% identical. Plasmids contained 3 ORFs apart from qnrD and belonged to an undescribed incompatibility group. Only one plasmid, in P. vulgaris, was slightly different with a 1,568-bp insertion between qnrD and its promoter, leading to absence of quinolone resistance. We sought for similar plasmids in 15 reference strains of Proteeae, but which were tested negative for qnrD, and found a 48% identical plasmid (pVERM) in Providencia vermicola. In order to explain how qnrD could have been inserted into such native plasmid, we sought for gene mobilization structures. qnrD was found to be located within a mobile insertion cassette (mic) element which sequences are similar to one mic also found in pVERM. Our conclusions are that (i) the small non-transmissible qnrD-plasmids described here may result from the recombination between an as-yet-unknown progenitor of qnrD and pVERM, (ii) these plasmids are maintained in Proteeae being a qnrD reservoir (iii) the mic element may explain qnrD mobilization from non-transmissible plasmids to mobilizable or conjugative plasmids from other Enterobacteriaceae, (iv) they can recombined with larger multiresistant plasmids conjugated in Proteeae.

Modular Genetic Architecture of the Toxigenic Plasmid pIS56-63 Harboring cry1Ab21 in Bacillus thuringiensis subsp. thuringiensis strain IS5056

Bacillus thuringiensis subsp. thuringiensis IS5056, a strain highly toxic to Trichoplusia ni larvae, produces the newly described Cry1Ab21 delta-endotoxin encoded by a gene located in the 63.8 kb pIS56-63 plasmid. In this report we present the structure and functional similarity of this plasmid to other B. thuringiensis large toxigenic plasmids with particular interest focused on its modular architecture. The 61 open reading frames (ORFs) of the plasmid made four functional modules: (i) M1-mic, the mobile insertion cassette harboring cry1Ab21; (ii) M2-tra, the putative conjugative element; (iii) M3-reg, regulation sequence; and (iv) M4-rep, the ori44 replicon. These modules display similarity to corresponding sequences in distinct B. thuringiensis plasmids, but, in general, not to plasmid of other Bacillus cereus sensu lato. The nucleotide sequence and organization of genes in pIS56-63 were highly similar (80-100%) to those in pHT73 of B. thuringiensis HT73, and in p03 of B. thuringiensis HD771, particularly within the M3-reg and M4-rep modules, and slightly less in M2-tra, the latter of which is composed of two segments exhibiting homology to sequences in pBMB28, pAH187_45, pCT83, and pIS56-85 or to pCT72, pBMB67, p04, and pIS56-68. The tetrapartite structure of the toxigenic pIS56-63 plasmid strongly suggests that its hybrid nature is a result of recombination of various genetic elements originating from different extrachromosomal and chromosomal sources in B. thuringiensis. The presence of cry1Ab21 in the mobile cassette suggests that its occurrence on pIS56-63 resulted from recombination and transposition events during the evolution of the plasmid.

First description of ColE-type plasmid in Aeromonas spp. carrying quinolone resistance (qnrS2) gene

Isolation and full sequence analysis of ColE-type plasmid, which carries the qnrS2 gene. Quinolone resistance (qnrS2) gene-carrying plasmids were isolated from Aeromonas sobria and Aeromonas hydrophila strains, and plasmid sequencing was achieved by a primer-walking approach. The total sizes of these plasmids (pAQ2-1 and pAQ2-2) were 6900 bp and 6903 bp, respectively, and they were 99·1% identical to each other. The genes (oriV and repA) for plasmid replication were organized similar to the corresponding genes in the ColE2-type plasmids, pAsa3 and pAsa1, isolated from Aeromonas salmonicida subsp. salmonicida, but the gene (mobA) for mobilization was homologue to ColE1-type plasmid (pAsa2) from Aer. salmonicida subsp. salmonicida. Additionally, the qnrS2 gene was part of a mobile insertion cassette element in the plasmid. Two plasmids were assumed to be the same plasmid, and this identification of a plasmid-mediated qnrS2 gene from the two different strains underlines a possible diffusion of these resistance determinants in an aquaculture system. This is the first finding of the ColE-type plasmid carrying the qnrS2 gene.

Plasmid-mediated quinolone resistance in Aeromonas allosaccharophila recovered from a Swiss lake

To search for plasmid-mediated qnr genes among waterborne environmental Aeromonas spp. recovered from Switzerland. Isolates presenting MICs of nalidixic acid or ciprofloxacin > or = 1 mg/L were screened for qnr genes by a multiplex PCR approach followed by sequencing. Plasmids were transferred by transformation, and further analysis of the genetic structures surrounding the qnrS2 gene was carried out by PCR and sequencing. A qnrS2 gene was identified from a single Aeromonas allosaccharophila isolate (Lugano lake, Lugano), as part of a mobile insertion cassette located on a broad host range IncU-type plasmid. This plasmid co-harboured a class 1 integron containing the aac(6')-Ib-cr, bla(OXA-1), catB3 and arr-3 gene cassettes. These findings strengthen further the role of Aeromonas spp. as a reservoir of antimicrobial resistance determinants in the environment.

Unexpected Occurrence of Plasmid-Mediated Quinolone Resistance Determinants in EnvironmentalAeromonasspp.

We searched for plasmid-mediated quinolone resistance determinants of the Qnr type in several water samples collected at diverse locations from the Seine River (Paris, France). The qnrS2 genes were identified from Aeromonas punctata subsp. punctata and A. media. The qnrS2 gene was located on IncU-type plasmids in both isolates, which resulted in increased MIC values of quinolones and fluoroquinolones, once they were transferred into Escherichia coli. The qnrS2 gene identified in A. punctata was part of novel genetic structure corresponding to a mobile insertion cassette element. This identification of plasmid-mediated qnr genes outside Enterobacteriaceae underlines a possible diffusion of those resistance determinants within gram-negative rods.

Identification, distribution pattern of IS 231 elements in Bacillus thuringiensis and their phylogenetic analysis

In order to better understand the fundamental biology of Bacillus thuringiensis, a single oligonucleotide primer (5′-CATSSCCATCAASYTAAVR-3′) was used to investigate the distribution pattern of IS 231 elements in B. thuringiensis by PCR. The results indicated that IS 231 elements appeared in 20 standard strains and 107 of 111 China isolates. Three novel IS 231, IS 231J, IS 231O and IS 231Q, five variants and a mobile insertion cassette MICBth 4 were cloned from eight standard strains of B. thuringiensis, respectively. Interestingly, BLAST analysis revealed that the 5′ end of novel IS 231J shared 99% identity in 495-bp with a DNA segment adjacent to the 3′ end of B. thuringiensis vip1Ac gene (GenBank Accession No. AY245547). Two phylogenetic trees of IS 231 elements were constructed and analyzed by neighbor-joining and UPGMA methods from PHYLIP 3.6b program, respectively.

IS231-MIC231 elements from Bacillus cereus sensu lato are modular.

Summary IS231A was originally discovered in Bacillus thuringiensis as a typical 1.6 kb insertion sequence (IS) displaying 20 bp inverted repeats (IR) flanking a transposase gene. A first major variation of this canonical organization was found in MIC231A1. This mobile insertion cassette (MIC), delineated by IS231A-related extremities, contained an active d-stereospecific endopeptidase (adp) gene instead of a transposase. Interestingly, it was shown that MIC231A1 can be mobilized in trans by the IS231A transposase. In this paper, we show that this family of IS231-MIC231 elements can be extended to a broad range of related entities displaying higher levels of structural complexity. Several IS231A-like elements contained, upstream of their transposase gene, passenger genes coding for putative antibiotic resistances or regulatory factors. Furthermore, the diversity of the MIC231 elements ranged from empty cassettes to structures carrying up to three passenger genes. Among these, MIC231V carried, in addition to an adp gene, an active fosfomycin resistance determinant. In vivo transposition assays showed that MIC231V is also trans-activated by the IS231A transposase. These results lend further support to the potential contribution of these modular mobile elements to the genome plasticity of the Bacillus cereus/B. thuringiensis group.

MIC231, a naturally occurring mobile insertion cassette from Bacillus cereus.

Recent dissection of numerous plasmids and transposable elements has given more credence to the modular organization of these genetic and genomic entities. Although many variations on each theme exist, the number of basic functional cassettes is thought to be relatively limited. In this paper, a novel type of mobile cassette is described. A naturally occurring assemblage consisting of two left IS231 ends flanking a D-stereospecific endopeptidase (adp) gene was found in several natural isolates of Bacillus cereus. This 1.9 kb genetic entity was shown to transpose in the presence of IS231A transposase, not only in Escherichia coli but also in Bacillus. The acronym MIC231 is proposed for this mobile insertion cassette trans-activated (teletransposed) by IS231. Using (D-Phe)4 tetrapeptide as substrate, the endopeptidase activity of the MIC231 adp gene could be demonstrated in E. coli and B. subtilis. Interestingly, this D-stereospecific endopeptidase activity was not limited to the original B. cereus isolates but was also detected in all but one of the 69 B. cereus sensu lato strains tested, indicating its important, yet dispensable, biological function. However, inactivation of the MIC231 adp gene in two B. cereus strains did not result in any detectable variation of their activity on (D-Phe)4, suggesting the presence of other distantly related adp gene(s). Thus, although the exact role of MIC231 adp remains elusive, its presence inside a mobile cassette represents the archetype of a novel insertion sequence modular organization.