pDGO100, a type 1 IncC plasmid from 1981 carrying ARI-A and a Tn1696-like transposon in a novel integrating element

    pDGO100, a type 1 IncC plasmid from 1981 carrying ARI-A and a Tn1696like transposon in a novel integrating element Christopher J. Harmer, Sally R. Partridge, Ruth M. Hall PII: DOI: Reference:

S0147-619X(16)30022-1 doi: 10.1016/j.plasmid.2016.06.002 YPLAS 2297

To appear in: Received date: Revised date: Accepted date:

12 May 2016 13 June 2016 14 June 2016

Please cite this article as: Harmer, Christopher J., Partridge, Sally R., Hall, Ruth M., pDGO100, a type 1 IncC plasmid from 1981 carrying ARI-A and a Tn1696-like transposon in a novel integrating element, (2016), doi: 10.1016/j.plasmid.2016.06.002

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT pDGO100, a type 1 IncC plasmid from 1981 carrying ARI-A and a Tn1696-like

1

SC RI

Christopher J. Harmer1,*, Sally R. Partridge2, Ruth M. Hall1

School of Molecular Bioscience, The University of Sydney, New South Wales,

Australia.

Centre for Infectious Diseases and Microbiology, The Westmead Institute for Medical

NU

2

PT

transposon in a novel integrating element

MA

Research, The University of Sydney, Westmead Hospital, New South Wales, Australia.

PT

ED

Keywords: IncC, A/C2, antibiotic resistance, integrating element, evolution in situ.

CE

*Corresponding author.

AC

Mailing address: School of Molecular Bioscience, Molecular Bioscience Building G08, The University of Sydney, NSW 2006, Australia Phone: 61-2-9351-6028 Fax: 61-2-9351-5858 E-mail: [email protected]

ACCEPTED MANUSCRIPT Abstract Most A/C plasmids sequenced to date were recovered in the last two decades. To gain

PT

insight into the evolution of this group, the IncC plasmid pDGO100, found in a multiply antibiotic resistant Escherichia coli strain isolated in 1981, was sequenced. pDGO100

SC RI

belongs to the type 1 lineage and carries an ARI-A antibiotic resistance island but not an ARI-B island. The A/C2 backbone of pDGO100 has a deletion in the rhs1 gene previously found in pRMH760 and differs by only six single base-pair substitutions

NU

from pRMH760, recovered at the same hospital 16 years later. This confirms that the

MA

separation of type 1 and type 2 IncC plasmids is long standing. The ARI-A islands are also closely related, but pRMH760 contains Tn4352B in tniA of Tn402, while in

ED

pDGO100 Tn4352 has inserted into merA of pDUmer. pDGO100 also carries an additional 46 kb insertion that includes a Tn1696-like transposon with the dfrB3 gene

PT

cassette. This insertion was identified as a novel integrating element, with an int gene at one end, and also includes the fec iron uptake operon that has been acquired from the E.

CE

coli chromosome. Related integrating elements carrying the same int gene were found in

AC

A/C2, IncHI1 and IncHI2 plasmids, and in the chromosomes of Enterobacter cloacae, Klebsiella oxytoca and Cronobacter sakazakii isolates. In the Enterobacteriaceae chromosomes, these integrating elements appear to target a gene encoding a radical SAM superfamily protein. In the A/C2, IncHI1 and IncHI2 plasmids, genes encoding a phosphoadenosine phosphosulfate reductase were interrupted. The extremities of the integrating element are highly conserved, whilst the internal gene content varies. The detection of integrative elements in plasmids demonstrates an increased range of locations into which this type of mobile element can integrate and insertion in plasmids is likely to assist their spread.

ACCEPTED MANUSCRIPT 1. Introduction Plasmids belonging to incompatibility group C were first identified over four

PT

decades ago in Pseudomonas aeruginosa and Klebsiella pneumoniae from Paris hospitals7,26 and are among the earliest plasmids to be associated with antibiotic

SC RI

resistance in Gram-negative bacteria. The IncA group, which only includes one sequenced plasmid, RA1, was later combined with the IncC group as A/C. A/C plasmids have now been found in many Gram-negative species, highlighting their broad host

NU

range.14 A/C2 plasmids, likely corresponding to the IncC group, are divided into two

MA

distinct lineages, type 1 and type 2, defined by backbone features and a characteristic resistance island in type 1.13 All but one sequenced type 1 A/C2 plasmid carries the ARI-

ED

A antibiotic resistance island in precisely the same location, and the majority of sequenced type 1 plasmids also include an ISEcp1-associated blaCMY-2 or blaCMY-2

PT

variant. The ARI-B island, containing sul2, is found in both type 1 and type 2 plasmids.13,14 The type 2 plasmid R55 (GenBank accession no. JQ010984), isolated in

CE

1969,7 is currently the oldest sequenced A/C2 plasmid.8 The oldest sequenced type 1

1991.9

AC

A/C2 plasmid, p199061_160 (GenBank accession no. HQ023863), was isolated in

pDGO100 is a conjugative plasmid that was first isolated from a multiresistant

Escherichia coli isolate (VA292) at Royal North Shore Hospital, Sydney, Australia in 1981. It was classified as IncC using traditional incompatibility testing.11 It confers resistance to gentamicin, kanamycin, tobramycin, neomycin, trimethoprim, sulfamethoxazole, ampicillin and chloramphenicol.11 VA292 was part of an outbreak of gentamicin and tobramycin resistant Gram-negative bacteria at Royal North Shore hospital that began in 1976.1

ACCEPTED MANUSCRIPT pDGO100 is of particular historical importance in the study of antibiotic resistance, as it was one of the first plasmids shown to possess a class 1 integron.21 The

PT

only gene cassette carried by In7 in pDGO100 is aadB, conferring resistance to gentamicin, kanamycin and tobramycin4 (GenBank accession no. L06418). pDGO100

SC RI

was one of the plasmids used to define a 2,154 bp region that is common to the In6 and In7 integrons, now known as common region 1 (CR1 or ISCR1).17,23 In6 and In7 also contain a partial duplication of the 3′-conserved segment (3′-CS), resulting in a

NU

duplication of the sul1 sulphonamide resistance gene. In pDGO100 a 2.8 kb segment

MA

between the two 3′-CS contains CR1 and the trimethoprim resistance gene dfrA10.15 An identical integron is found in pRMH760, a type 1 A/C2 plasmid recovered 16 years later

ED

(1997) at the same Sydney hospital.13,17 This illustrates how complex structures containing antibiotic resistance genes can persist in a local environment over an

PT

extended period of time.

Subsequent analysis of pDGO100 suggested that it possessed a second integron,

CE

reported to carry the dfrA7 trimethoprim resistance gene cassette, and this was the first

AC

time that two integrons with different gene cassettes had been found in the same naturally occurring plasmid.2 However, later analysis yielded the dfrB3 gene cassette in the second integron (GenBank accession no. AY123252). The study reported here was stimulated by the historical importance of pDGO100 and to determine where it fits into the evolutionary history of A/C2 plasmids. Sequence data for historical A/C2 plasmids is limited, as most A/C2 plasmids sequenced to date were recovered in the last two decades (see compilations in references13 and14). Here, we have determined the pDGO100 sequence and compared it to other sequenced A/C2 plasmids. We report that pDGO100 is an A/C2 plasmid, and hence that A/C2 corresponds to IncC.

ACCEPTED MANUSCRIPT 2. Materials and methods 2.1 Bacterial strains and conjugation

PT

Plasmid pDGO100 was transferred from E. coli VA292 to nalidixic acid resistant E. coli DH5α (supE44 ΔlacU169 [ϕ80 lacZ ΔM15] hsdR17 recA1 endA1 gyrA96 thi-1

SC RI

relA1) by mixing equal amounts of stationary phase cultures on Mueller-Hinton agar and growing overnight at 37oC. Cells were resuspended in 0.9% (w/v) sterile saline, serially diluted, and transconjugants recovered on plates containing nalidixic acid to select

NU

against the donor and for the recipient, and sulfamethoxazole, trimethoprim and

MA

kanamycin to select for transfer of pDGO100. The resulting transconjugants were screened using PCR-based replicon typing5 and their resistance phenotypes determined by patching onto plates containing chloramphenicol (25 μg/ml), gentamicin (8 μg/ml),

ED

kanamycin (20 μg/ml), neomycin (50 μg/ml), sulfamethoxazole (100 μg/ml), tobramycin

PT

(12.5 μg/ml) or trimethoprim (25 μg/ml). To calculate the transfer efficiency of pDGO100, DH5α cells containing pDGO100 were mated with a rifampicin (Rif)

CE

resistant derivative of E. coli UB1637 as described above. Conjugation frequencies were

AC

calculated as the number of kanamycin and rifampicin resistant transconjugants per kanamycin resistant donor from three independent determinations.

2.2 Polymerase chain reaction Polymerase chain reactions (PCR) to confirm the presence of the A/C replicon in the transconjugants were performed on approximately 20 ng of diluted plasmid DNA. All PCR reagents were sourced from New England Biolabs. PCR conditions were as described previously.27 Published primers were used to detect the A/C replicon.5 Amplicons were resolved by electrophoresis on 1% (w/v) agarose gels with molecular

ACCEPTED MANUSCRIPT weight standards, stained with ethidium bromide, and visualized using a GelDoc1000

PT

image analysis station (BioRad).

2.3 Plasmid recovery and sequencing

SC RI

pDGO100 plasmid DNA was extracted from a DH5α transconjugant using an alkaline lysis method and sequenced on an Ion Torrent platform (Life Technologies) as previously described.13 The sequence reads (37-fold coverage) were assembled de novo

NU

into contigs using Geneious version 6.1.6 (Biomatters). PCR and sequencing was used

MA

to join contigs that ended with the outer ends of repeated sequences, namely IS4321, IS26, and sul1. Overlapping contigs were assembled with Sequencher version 5.1 (Gene

ED

Codes). The final assembly was confirmed by comparing the fragments obtained from in silico BamHI or EcoRI digests to fragments obtained when pDGO100 was digested with

PT

BamHI or EcoRI (New England Biolabs). Single residues missing from the majority of

and added.

CE

reads but present in some were assumed to be due to the use of the Ion Torrent platform,

AC

Putative coding regions in pDGO100 were identified and annotated based on the available annotations for the A/C2 plasmids pRMH760 (GenBank accession no. KF976462), pR148 (JX141473) and/or pKEC-39c (CP008824), and as described in a recent review.14

2.4 Sequence analysis The backbone of pDGO100 was derived by removing ARI-A and a novel integrating element, circularizing and reopening at the same location as several other sequenced A/C2 plasmids, 1,139  bp upstream of repA. Pairwise comparisons to the backbones of pR148 (GenBank accession no. JX141473) and pRMH760 (KF976462)

ACCEPTED MANUSCRIPT were performed using the BLAST paired alignment facility

PT

(http://blast.ncbi.nlm.nih.gov) and were visualized using the Artemis Comparison Tool.6

2.5 Nucleotide sequence accession number

SC RI

The nucleotide sequence of pDGO100 has been submitted to GenBank under accession no. KU997026.

NU

3. Results

MA

3.1 pDGO100 is a large type 1 A/C2 plasmid carrying a novel insertion pDGO100 was originally recovered from a clinical E. coli isolate by conjugation.

ED

Here, the efficiency of transfer of pDGO100 (E. coli to E. coli) was found to be 3 x 10-3 transconjugants per donor (average of three independent determinations). This is 20-fold

PT

lower than the transfer frequency of the closely related plasmid pRMH760 tested under the same conditions (6.4 x 10-2).

CE

The complete pDGO100 sequence determined here (GenBank accession no.

AC

KU997026) is 217,306 bp and the sequence of the repA gene revealed that pDGO100 is an A/C2 plasmid. Hence, A/C2 corresponds to IncC. The presence of rhs1 and orf1832 revealed that pDGO100 is a type 1 IncC (A/C2) plasmid. pDGO100 contains two resistance regions, ARI-A and a Tn1696-like transposon. ARI-A in pDGO100 is in the same location as in other type 1 A/C2 plasmids (see reference14 for boundaries). However, the Tn1696-like transposon is contained within a larger (46.4 kb) region that has inserted into orf102 of the A/C2 backbone (Fig. 1). Like pRMH760, pDGO100 does not contain either the ISEcp1-blaCMY-2 island or the sul2containing ARI-B island seen in many type 1 A/C2 plasmids.

ACCEPTED MANUSCRIPT Removal of the two large insertions generated a backbone of 125,445 bp (Fig. 1) that includes a replication initiation gene (repA), genes for conjugative transfer (tra),

PT

partitioning (par), a variety of other genes and open reading frames with no known function that have been described elsewhere.14 However, pDGO100 has a 2,358 bp

SC RI

deletion within the 5,571 bp rhs1 gene that was first identified in pRMH760,13 which was isolated in the same hospital as pDGO100. A detailed comparison of pDGO100 and pRMH760 revealed that the backbones are the same size and differ by only six single

NU

nucleotide polymorphisms (SNP). Two of the SNPs fall within intergenic regions, two

MA

within hypothetical proteins with no known function and one each within the traC and traL genes (Table 1). One SNP results in replacement of glycine at amino acid 34 in

ED

TraL of pDGO100 with arginine, and the other a valine-669-glycine substitution in TraC. One or more of these substitutions may account for the 20-fold difference in the transfer

PT

frequencies of pDGO100 and pRMH760 (3 x 10-3 vs. 6 x 10-2 transconjugants per

CE

donor), but this remains to be proven. Until recently, the rhs1 deletion had not been seen in any other sequenced A/C2

AC

plasmid. However, comparison to the backbones of other sequenced A/C2 plasmids showed that pKPC_CAV1344 (GenBank accession no. CP011622), recovered from a clinical K. pneumoniae strain in the US in 2010 and sequenced in 2015, also shares the characteristic 2,358 bp deletion within rhs1 and has a backbone almost identical to pDGO100 and pRMH760 (6 SNPs compared to pDGO100 and 2 SNPs compared to pRMH760, Table 1). This suggests a common ancestor for these three plasmids. The geographic distribution between Australia and the US suggests this lineage of type 1 may be globally disseminated.

3.2 ARI-A in pDGO100

ACCEPTED MANUSCRIPT The ARI-A resistance island in pDGO100 (Fig. 2A) is 45,453 bp and is closely related to ARI-A in pRMH760. The In7 complex class 1 integron described previously,

PT

conferring resistance to gentamicin, kanamycin, tobramycin, trimethoprim and sulphamethoxazole,12,22 is contained within ARI-A. ARI-A of pDGO100 (Fig. 2A) is a

SC RI

complex mosaic of transposons, containing a copy of blaTEM-1b (ampicillin resistance) within Tn2, catA1 (chloramphenicol resistance) within a Tn9-like-derived region, and aphA1a in Tn4352 (IS26-aphA1a-IS26) conferring resistance to kanamycin and

NU

neomycin. It is very closely related to ARI-A in pRMH760, with some minor variations

MA

that are highlighted in Fig. 2A. Whereas pRMH760 contains a copy of Tn4352B in tniA of Tn402, only a single IS26 is found at this position in pDGO100. The aphA1a gene in

ED

pDGO100 is in Tn4352, which has inserted into the merA gene of pDUmer. The remainder of tniA and part of the mer region of Tn21, found between Tn4352B and Tn2

PT

in pRMH760, has been replaced by a part of Tn5393 and a 1,125 bp segment that shares 96% nucleotide identity with a segment from the Enterobacter cloacae IncL/M plasmid

CE

pNE1280 (GenBank accession no. JQ83726), but is not related to any other sequence in

AC

GenBank. This segment includes a single open reading frame with no known function. Compared to pRMH760 and Tn2670, a 17 bp deletion has also occurred between IS1 and IRmer of Tn21, immediately adjacent to IS1 (indicated by an asterisk in Fig. 2A). A characteristic of the type 1 A/C2 ARI-A island is the presence of an insertion sequence, IS4321, IS5075 or a close relative, in each IRtnp and IRmer.18 IS4321 and related IS target the 38 bp terminal inverted repeats of Tn21-family transposons, and prevent further movement.18 In the pDGO100 ARI-A, IS4321 is found in the same inverted repeats as in the pRMH760 ARI-A, except IRtnp of Tn21 in pDGO100, which has not yet acquired IS4321 or a close relative.

ACCEPTED MANUSCRIPT 3.3 The second resistance region in pDGO100 The second insertion in the pDGO100 backbone is 46,403 bp. This region has

PT

inserted within a 2,454 bp open reading frame in the A/C2 backbone (orf102, Fig. 1) predicted to encode a protein sharing 30% amino acid identity with a phosphoadenosine

SC RI

phosphosulfate reductase family protein (Pfam PF01507),14 though the function of this gene has not been experimentally verified. There is no flanking duplication or loss of backbone compared with other A/C2 plasmids. This insertion includes a 14,791 bp

NU

Tn1696-like transposon containing an In4-type class 1 integron. The Tn1696-like

MA

transposon is not flanked by a 5 bp duplication of the target sequence and IRtnp is interrupted by IS4321. The 5′- and 3′-CS are both complete, and the integron contains a

ED

single gene cassette, dfrB3 (trimethoprim resistance) replacing the aacC1, aadA2, orfE and cmlA1 gene cassettes of In4. This is consistent with a previous analysis

PT

demonstrating that pDGO100 possessed a second integron containing dfrB3 (GenBank

CE

accession no. AY123252).

AC

3.4 The novel integrating element in pDGO100 A detailed analysis of the open reading frames within the 46.4 kb insert revealed

a gene at the left end (Fig. 3) that encodes an integrase (Pfam PF00589), suggesting that this insert is an integrating element (IE). In addition to the Tn1696-like transposon, the IE contains four complete IS (ISEc36, ISSen3, IS1-like and IS903) and two partial IS (ISEhe3 and ISEc21). It also includes a copy of the complete fecABCDE operon encoding the citrate-dependent iron (III) transport system,20 and the fecI and fecR twocomponent regulator of the operon.24 The fec module plays an important role in the utilization of insoluble Fe3+,20 and originates from the E. coli chromosome (>97.7% nucleotide identity with E. coli K-12).

ACCEPTED MANUSCRIPT A BLAST search performed using the int sequence revealed that a relative of this IE that does not include the Tn1696-like transposon is also found in the same position in

PT

another A/C2 plasmid, pKEC-39c (GenBank accession no. CP008824), from E. cloacae ECNIH2 isolated in 2012. However, pKEC-39c is a type 2 A/C2 plasmid, whereas

SC RI

pDGO100 comes from the type 1 lineage. The backbones of type 1 and type 2 plasmids differ by approximately 1 bp difference/100 bp. The 1 kb of plasmid backbone sequence on either side of the IE in pDGO100 and pKEC-39c corresponds to the type 1 and type 2

NU

backbones, respectively, indicating that this element has been independently acquired by

MA

the two lineages. Hence, it appears that this element has been introduced on two separate occasions into the same site in A/C2 plasmids. A nearly identical IE, which only differs

ED

from that in pKEC-39c by 9 single base pair deletions and one single base insertion, is also present in the ECNIH2 chromosome (GenBank accession no. CP008823),

PT

suggesting that pKEC-39c may have acquired the element from the chromosome, or vice

CE

versa.

AC

3.5 Related integrating elements IE with identical outer ends were also found in a total of 19 E. cloacae,

Klebsiella oxytoca and Cronobacter sakazakii chromosomal sequences, in three IncHI1 plasmids and one IncHI2 plasmid (Table 2). The internal composition of the IE appears to be highly variable, with 17 different variants identified to date (Table 2), but in all instances the int (left) end, as defined in Fig. 3, is the same. In 24 of the 25 sequenced examples the right hand (RH) end is also the same. In the remaining two, one in the IncHI2 plasmid and one in an E. cloacae chromosome, a deletion originating from within the IE has removed the RH end and some of the adjacent DNA. The internal

ACCEPTED MANUSCRIPT variation of the element appears to have been driven by the acquisition of various IS, including IS903, ISEc36, ISSen3, ISEc21, and many others.

PT

Analysis of the position of the IE in the E. cloacae, K. oxytoca and C. sakazakii chromosomes showed that in each instance it had integrated in precisely the same

SC RI

location (Fig. 4A), within closely related genes that encode proteins from the radical SAM superfamily (Pfam PF04055). These proteins are predicted to catalyze diverse reactions, including methylations and sulphur insertion. Unlike other integrating

NU

elements that insert into the 3′-end of a gene and maintain the reading frame, this novel

MA

IE inserts in the middle of its target genes and may inactivate them. The gene in the A/C2 backbone in which the IE has inserted does not appear to share any identity with

ED

the genes found in the chromosomes listed above. However, the sequence surrounding the element in the IncHI1 and IncHI2 plasmids (gene R0070 in GenBank accession no.

PT

AF250878) also encode a putative phosphoadenosine phosphosulfate reductase family protein (Pfam PF01507). The IncHI1 and IncHI2 genes share 52.6% and 52.7%

CE

nucleotide identity, respectively, with the corresponding gene from the A/C2 backbone.

AC

An alignment of the sequences surrounding the IE revealed that the IE had integrated in precisely the same location in each of these genes (Fig. 4B), suggesting it is recognized by the integrase. To examine a potential integrase binding site, the circular form of the integrating element was aligned with the chromosomal and plasmid sequences (Fig. 4A and Fig. 4B). Only short regions of homology were detected (6/8 bp with the chromosomal sequences and 8/9 bp with the plasmid sequences).

4. Discussion Here, the complete sequence of pDGO100 has revealed the structure of the two resistance regions it contains. ARI-A is typical of type 1 A/C2 plasmids though not all of

ACCEPTED MANUSCRIPT them contain it, and the other is a Tn1696-like transposon contained within a novel IE. The presence of only six SNPs between the backbones of pRMH760 and pDGO100,

PT

recovered from the same hospital some 16 years apart, highlights how closely related these plasmids are. The deletion within the rhs1 gene that they share with

SC RI

pKEC_CAV1344 (isolated in 2010), along with the small number of SNPs in their backbone, suggests that these plasmids may define a sub-lineage of type 1 A/C2 plasmids circulating in hospitals on the east coast of Australia and in the United States.

NU

The backbone of the ARI-A island is likely to have been acquired once by an

MA

ancestor of the type 1 A/C2 plasmids, and then evolved over at least the last four decades. The ARI-A island in pDGO100 is very similar to that in pRMH760. The minor

ED

variations in the internal content of the ARI-A island in pDGO100 and pRMH760 highlight the continual evolution in situ that is occurring in these plasmids as they

PT

circulate. The ARI-A island in pKPC_CAV1344 contains the aadB cassette as seen in pRMH760 and pDGO100, though the remainder of the island differs by insertions,

CE

deletions and inversions. It has also acquired the Tn4401 transposon containing blaKPC-2,

AC

conferring resistance to carbapenems. The first type 1 A/C2 plasmid to be sequenced, pSN254 (isolated in 2000),25 is often included in comparisons of A/C2 plasmids.3,9,10,25,28 We have included ARI-A of this plasmid in Fig. 2B to reveal how it relates to the ARI-A in pDGO100 and pRMH760. The extremities (Tn1696tnp and pDUmer) of ARI-A in pSN254 are conserved, but much of the internal content of the island has been lost when compared to pDGO100 and pRMH760 (Fig. 2B). In addition to building on the evolutionary history of A/C2 plasmids, the pDGO100 sequence has also uncovered the existence of a novel integrating element. Whilst the internal gene content is highly variable, with 17 types identified amongst 25 sequenced examples available in GenBank, the extremities are highly conserved. The

ACCEPTED MANUSCRIPT integrase appears to recognize a particular sequence within A/C2, IncHI1 and IncHI2 plasmid backbones, and another sequence within the chromosomes of

PT

Enterobacteriaceae. A search of the Whole Genome Shotgun (WGS) database revealed that variants of the IE are present in over one hundred different draft whole genome

SC RI

sequences. The prevalence of this element and the role that it plays in shaping bacterial evolution is yet to be fully elucidated. However, the carriage of the fec operon in the IE could be important in mobilizing a potential pathogenicity determinant that would assist

NU

pathogenic bacteria to acquire iron and overcome host nutritional immunity.19

MA

Interestingly, the presence of a compete lac operon in the IE of pKEC-39c (Fig. 3B) could potentially complicate the identification of any organism carrying it.

ED

5. Acknowledgements

AC

CE

PT

CJH was supported by NHMRC Project Grant 1032465.

ACCEPTED MANUSCRIPT 6. References 1.

Ackerman V.P., Groot Obbink D.J., and Sivertsen T. 1983. Transferable

PT

antibiotic resistance in a general hospital: a two year survey. Aust J Exp Biol Med Sci 61 (Pt 2): 251-62.

Burnside J.M., and Groot Obbink D.J. 1996. Plasmid pDGO100 contains a

SC RI

2.

second integron with the trimethoprim resistance gene dfrA7 as the inserted cassette. Plasmid 35: 67-70.

Call D.R., Singer R.S., Meng D., Broschat S.L., Orfe L.H., Anderson J.M.,

NU

3.

2-positive

MA

Herndon D.R., Kappmeyer L.S., Daniels J.B., and Besser T.E. 2010. blaCMYIncA/C plasmids from Escherichia coli and Salmonella enterica are

ED

a distinct component of a larger lineage of plasmids. Antimicrob Agents Chemother 54: 590-6.

Cameron F.H., Groot Obbink D.J., Ackerman V.P., and Hall R.M. 1986.

PT

4.

CE

Nucleotide sequence of the AAD(2'') aminoglycoside adenylyltransferase determinant aadB. Evolutionary relationship of this region with those

AC

surrounding aadA in R538-1 and dhfrII in R388. Nucleic Acids Res 14: 862535.

5.

Carattoli A., Bertini A., Villa L., Falbo V., Hopkins K.L., and Threlfall E.J. 2005. Identification of plasmids by PCR-based replicon typing. J Microbiol Methods 63: 219-28.

6.

Carver T.J., Rutherford K.M., Berriman M., Rajandream M.A., Barrell B.G., and Parkhill J. 2005. ACT: the Artemis Comparison Tool. Bioinformatics 21: 3422-3.

ACCEPTED MANUSCRIPT 7.

Chabbert Y.A., Scavizzi M.R., Witchitz J.L., Gerbaud G.R., and Bouanchaud D.H. 1972. Incompatibility groups and the classification of fi -

8.

PT

resistance factors. J Bacteriol 112: 666-75. Doublet B., Boyd D., Douard G., Praud K., Cloeckaert A., and Mulvey M.R.

SC RI

2012. Complete nucleotide sequence of the multidrug resistance IncA/C plasmid pR55 from Klebsiella pneumoniae isolated in 1969. J Antimicrob Chemother 67: 2354-60.

Fernandez-Alarcon C., Singer R.S., and Johnson T.J. 2011. Comparative

NU

9.

MA

genomics of multidrug resistance-encoding IncA/C plasmids from commensal and pathogenic Escherichia coli from multiple animal sources.

10.

ED

PLoS One 6: e23415.

Fricke W.F., Welch T.J., McDermott P.F., Mammel M.K., LeClerc J.E.,

PT

White D.G., Cebula T.A., and Ravel J. 2009. Comparative genomics of the

11.

CE

IncA/C multidrug resistance plasmid family. J Bacteriol 191: 4750-7. Groot Obbink D.J., Ritchie L.J., Cameron F.H., Mattick J.S., and Ackerman

AC

V.P. 1985. Construction of a gentamicin resistance gene probe for epidemiological studies. Antimicrob Agents Chemother 28: 96-102.

12.

Hall R.M., and Stokes H.W. 1990. The structure of a partial duplication in the integron of plasmid pDGO100. Plasmid 23: 76-9.

13.

Harmer C.J., and Hall R.M. 2014. pRMH760, a precursor of A/C2 plasmids carrying blaCMY and blaNDM genes. Microb Drug Resist 20: 416-23.

14.

Harmer C.J., and Hall R.M. 2015. The A to Z of A/C plasmids. Plasmid 80: 63-82.

ACCEPTED MANUSCRIPT 15.

Parsons Y., Hall R.M., and Stokes H.W. 1991. A new trimethoprim resistance gene, dhfrX, in the In7 integron of plasmid pDGO100. Antimicrob

16.

PT

Agents Chemother 35: 2436-9. Partridge S.R. 2011. Analysis of antibiotic resistance regions in Gram-

17.

SC RI

negative bacteria. FEMS Microbiol Rev 35: 820-55.

Partridge S.R., and Hall R.M. 2003. In34, a complex In5 family class 1 integron containing orf513 and dfrA10. Antimicrob Agents Chemother 47:

Partridge S.R., and Hall R.M. 2003. The IS1111 family members IS4321 and

MA

18.

NU

342-9.

IS5075 have subterminal inverted repeats and target the terminal inverted

19.

ED

repeats of Tn21 family transposons. J Bacteriol 185: 6371-84. Porcheron G., Garenaux A., Proulx J., Sabri M., and Dozois C.M. 2013.

PT

Iron, copper, zinc, and manganese transport and regulation in pathogenic

CE

Enterobacteria: correlations between strains, site of infection and the relative importance of the different metal transport systems for virulence.

20.

AC

Front Cell Infect Microbiol 3: 90. Staudenmaier H., Van Hove B., Yaraghi Z., and Braun V. 1989. Nucleotide sequences of the fecBCDE genes and locations of the proteins suggest a periplasmic-binding-protein-dependent transport mechanism for iron(III) dicitrate in Escherichia coli. J Bacteriol 171: 2626-33. 21.

Stokes H.W., and Hall R.M. 1989. A novel family of potentially mobile DNA elements encoding site-specific gene-integration functions: integrons. Mol Microbiol 3: 1669-83.

ACCEPTED MANUSCRIPT 22.

Stokes H.W., Tomaras C., Parsons Y., and Hall R.M. 1993. The partial 3'conserved segment duplications in the integrons In6 from pSa and In7 from

23.

PT

pDGO100 have a common origin. Plasmid 30: 39-50. Toleman M.A., Bennett P.M., and Walsh T.R. 2006. ISCR elements: novel

SC RI

gene-capturing systems of the 21st century? Microbiol Mol Biol Rev 70: 296316. 24.

Van Hove B., Staudenmaier H., and Braun V. 1990. Novel two-component

NU

transmembrane transcription control: regulation of iron dicitrate transport

25.

MA

in Escherichia coli K-12. J Bacteriol 172: 6749-58. Welch T.J., Fricke W.F., McDermott P.F., White D.G., Rosso M.L., Rasko

ED

D.A., Mammel M.K., Eppinger M., Rosovitz M.J., Wagner D. and others. 2007. Multiple antimicrobial resistance in plague: an emerging public health

Witchitz J.L., and Chabbert Y.A. 1971. High level transferable resistance to

CE

26.

PT

risk. PLoS One 2: e309.

gentamicin. J Antibiot (Tokyo) 24: 137-9. Yau S., Liu X., Djordjevic S.P., and Hall R.M. 2010. RSF1010-like plasmids

AC

27.

in Australian Salmonella enterica serovar Typhimurium and origin of their sul2-strA-strB antibiotic resistance gene cluster. Microb Drug Resist 16: 24952.

28.

Ye L., Li R., Lin D., Zhou Y., Fu A., Ding Q., Chan E.W., Yao W., and Chen S. 2016. Characterization of an IncA/C multidrug resistance plasmid in Vibrio alginolyticus. Antimicrob Agents Chemother 60: 3232-3235.

ACCEPTED MANUSCRIPT Figure legends FIG. 1 Genetic organization of pDGO100. The backbone sequence of pDGO100 was

PT

linearized 1,139 bp upstream of repA and is drawn to scale. Horizontal arrows indicate

SC RI

the location, size and orientation of ORFs, and every tenth reading frame is numbered below the arrows. The positions of the integrating element, ARI-A, and a deletion within the rhs gene are indicated by vertical arrows. Genes coding for proteins of known

NU

function are named above and colored according to the key. Figure was generated using Adobe Illustrator CS6.

MA

FIG. 2 (A) ARI-A resistance islands in pDGO100 and pRMH760. The islands are drawn to scale from GenBank accession nos. KF976462 (bases 101004-146166) and

ED

KU997026 (bases 147407-192859). IS and CR1 are shown as open boxes with IS

PT

numbers/names indicated inside and > or < indicating orientation. The aadB gene cassette is shown as an open box inserted within the attI1 site. Genes and ORFs are

CE

shown below the line as named arrows indicating the direction of transcription.

AC

Segments derived from known transposons and integrons are indicated below. Segments unique to pDGO100 are shaded in blue, and those unique to pRMH760 in green. An asterisk in pDGO100 denotes a 17 bp deletion relative to pRMH760. (B) ARI-A resistance island in pSN254. The island is drawn to scale from CP000604 (bases 119747-149647). Segments shared with pDGO100 and pRMH760 are shaded in orange. ARI-A of pSN254 and pRMH760 are also compared in reference 16. FIG. 3 (A) Novel integrating element in pDGO100. The island is drawn to scale from GenBank accession no. KU997026 (bases 93121-139523). IS are shown as open boxes with IS numbers/names indicated inside. Inverted repeats are indicated by vertical lines. Genes and ORFs are shown below the line as named arrows indicating the direction of

ACCEPTED MANUSCRIPT transcription. The extent of the Tn1696-like transposon is indicated below and is shaded orange. Green shading indicates regions shared between the IE in pDGO100 and the IE

PT

in pKEC-39c. (B) Integrating element in pKEC-39c. Drawn to scale from CP008824

SC RI

(bases 138119-185797).

FIG. 4 (A) Insertion point of the integrating element in Enterobacteriaceae chromosomes. Alignment of GenBank accession nos. FP929040 (bases 3284486-

NU

3284548), CP011636 (bases 1193326-1193368) and CP000783 (bases 34300003430042). The vertical arrow indicates the insertion point of the integrating element in

MA

the three target sequences, and the point between the last and first base of the circularized form of the integrating element (IE circle). Asterisks indicate bases

ED

conserved between the three target sequences and the circular form of the integrating element. Shading indicates conservation of the base in three or more sequences. (B)

PT

Insertion point of the integrating element in A/C2, IncHI1 and IncHI2 plasmids.

CE

Alignment of KF976462 (bases 93092-93143), NC_002305 (bases 70602-70653) and

AC

NC_005211 (bases 237840-237789).

ACCEPTED MANUSCRIPT Table 1. Single nucleotide polymorphisms between pDGO100, pRMH760 and pKPC_CAV1344

Intergenic traL orf1832 traC orf Intergenic orf

A G T T G G C

G A T G A C G

G S V P H

a

PT

Gene

pRMH760 Nucleotide aaa R S G S Q

SC RI

Position in pDGO100 43665 52247 57092 64782 72400 74242 143428

pDGO100 Nucleotide aaa

pKPC_CAV1344 Nucleotide aaa G G A G A C G

AC

CE

PT

ED

MA

NU

aa, amino acid. – denotes no amino acid, as the nucleotide is within an untranslated intergenic region

G T G S Q

ACCEPTED MANUSCRIPT Table 2. Sequences containing an integrating element sharing the same int gene

a

IE size (bp) Variant Accession No

Australia USA USA USA USA Taiwan

E. coli E. cloacae E. cloacae Pantoea sp. Pantoea sp. K. pneumoniae

46403 47679 21152 160606a 166811a 21385

USA USA USA USA USA USA USA USA USA USA USA China USA USA USA USA S. Korea China USA

E. cloacae E. cloacae E. cloacae E. cloacae E. cloacae E. cloacae E. cloacae E. cloacae E. cloacae C. sakazakii E. cloacae Enterobacter sp. E. cloacae E. cloacae K. oxytoca E. cloacae K. oxytoca C. sakazakii E. cloacae

PT

1 2 3 4 5 6

KU997026 CP008824 CP010380 CP009883 CP009869 EF382672

2 2 2 2 2 2Δb 7 7 7 8 9 10 11 12 13 14 15 16 17

CP011650 CP011584 CP011581 CP011572 CP008823 CP009850 CP009854 CP008905 CP008897 CP011047 CP010384 CP012999 CP010376 CP011591 CP008788 CP012165 CP008841 CP006731 CP010377

SC RI 47679 47679 47679 47679 47671 18623 37081 37080 37080 47912 36684 39175 34577 26172 41193 35707 44262 44812 -c

NU

MA

PT

Species

CE

AC

Chromosome CAV1669 CAV1668 CAV1411 CAV1311 ECNIH2 ECNIH4 ECNIH5 ECR091 ECNIH3 ATCC29544 34399 E20 34977 CAV1043 KONIH1 34978 M1 CMCC45402 34983

Country

ED

Name Plasmids pDGO100 (A/C2) pKEC-39c (A/C2) p34983 (IncHI1) pPSP-a3e (IncHI1) pPSP-75c (IncHI1) pK29 (IncHI2)

Large inversion within the plasmid backbone has split the IE Internal deletion variant c The size of this variant cannot be determined due to complex rearrangements and deletions within and adjacent to the element. b

SC RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT

ED

MA

NU

Fig. 1

Fig. 2

AC

CE

PT

ED

MA

NU

SC RI

PT

ACCEPTED MANUSCRIPT

Fig. 3

AC

CE

PT

ED

MA

NU

SC RI

PT

ACCEPTED MANUSCRIPT

MA

NU

SC RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT

ED

Fig. 4

ACCEPTED MANUSCRIPT

AC

CE

PT

ED

MA

NU

SC RI

PT

Highlights  A 217,306 bp IncC plasmid, pDGO100, was isolated from an Escherichia coli strain isolated in 1981  The backbone of pDGO100 is very closely related to another plasmid, pRMH760, recovered from the same hospital 16 years later.  A 45.5 kb ARI-A island includes six antibiotic resistance genes: aadB, sul1, dfrA10, blaTEM, catA1 and aphA1a.  A second 46 kb insertion includes a Tn1696-like transposon with a dfrB3 gene cassette. This insertion was identified as a novel integrating element.  Related integrating elements were found in other plasmid and in the chromosomes of Enterobacteriaceae.