A follow-up typing scheme for repBIncFIB genes
To perform a scheme for classifying repBIncFIB genes, a maximum likelihood phylogenetic tree (Figure S1 and Table S2) was constructed using repBIncFIB genes presented in our previous study [5] together with additional three ones from plasmids in GenBank (last accessed February 26, 2021). These three ones were clustered to one phylogenetic clade, which was newly designed as a primary IncFIB-8 type. Subsequently, based on the criterion of 95% nucleotide identity for repBIncFIB sequences, an IncFIB-8.1 subtype was further identified (Figure S2). The repBIncFIB sequences in IncFIB-8.1 subtype displayed ≥ 95% nucleotide identity with each other, but showed no significant sequence similarity with those in other IncFIB subtypes.
A Group Of The 20 Analyzed Plasmids Sharing The Inchi3 Core Backbone
Herein, we determined the complete genome sequences of eight plasmids containing the IncHI3 core backbone. Based on the replication gene profiles, the eight plasmids could be divided into three groups: i) four IncHI3 plasmids including three harboring repHI3B and repBIncFIB−6.1 together with one harboring repHI3B, repBIncFIB−6.1, and repBIncFIB−8.1; ii) one IncFIB-6.1 plasmid carrying repBIncFIB−6.1; iii) three IncFIB-6.1:8.1 plasmids containing repBIncFIB−6.1 and repBIncFIB−8.1.
A genomic comparison was applied to a total of 20 plasmids (Table 1, last accessed August 10th 2021), including eight ones sequenced in the study and 12 ones from GenBank. According to the replication gene profiles, the 12 plasmids could be segmented into three groups: i) seven IncHI3 plasmids possessing repHI3B and/or repBIncFIB−6.1; ii) two IncFIB-6.1:8.1 plasmids containing repBIncFIB−6.1 and repBIncFIB−8.1; iii) three IncFIB-8.1 plasmids carrying repBIncFIB−8.1. The first sequenced IncHI3 plasmid (pNDM-MAR) served as the IncHI3 reference plasmid [33], and six more IncHI3 plasmids were selected because they contained a wealth of drug resistance genes. Among the two IncFIB-6.1:8.1 plasmids, one had the most complete IncHI3 core backbone with the least insertions and deletion, and the other with multiple drug resistance genes. The rest three were the only ones harboring the sole repHI3B in GenBank.
Table 1
General features of the 20 analyzed plasmids
Group | Plasmid | Accession number | Host bacterium | Total length (bp) | Total number of ORFs | Mean G + C content, % | Length of backbone (bp) | Mean G + C content of backbone (%) | Reference |
IncHI3 | pNDM-MAR | JN420336 | Klebsiella pneumoniae | 267,242 | 304 | 46.7% | 196,715 | 44.4% | [1] |
pW09308-HI3 | MW013145 | K. pneumoniae | 231,347 | 245 | 45.8% | 201,830 | 44.5% | This study |
p362713-HI3 | MK413719 | K. pneumoniae | 194,664 | 218 | 46.8% | 151,663 | 44.9% | This study |
pA1718-HI3 | MW013142 | K. pneumoniae | 290,842 | 337 | 47.4% | 191,490 | 44.7% | This study |
p721005-HI3 | CP030293 | K. pneumoniae | 269,204 | 310 | 46.5% | 191,484 | 44.7% | This study |
pKPM502 | CP031736 | K. pneumoniae | 250,351 | 279 | 46.4% | 146,568 | 43.9% | Not applicable |
pCP020068 | CP020068 | K. pneumoniae | 276,460 | 317 | 46.6% | 203,134 | 44.5% | Not applicable |
pCP041935 | CP041935 | K. pneumoniae | 287,790 | 330 | 47.3% | 203,486 | 44.6% | Not applicable |
pKOX3-P1 | KY913897 | K. oxytoca | 239,374 | 260 | 47.5% | 155,879 | 44.5% | [2] |
pCP050156 | CP050156 | K. pneumoniae | 259,659 | 294 | 46.4% | 184,229 | 44.5% | Not applicable |
pKX029332 | KX029332 | K. pneumoniae | 209,419 | 219 | 47.3% | 137,271 | 44.3% | Not applicable |
IncFIB-6.1:8.1 | pKp_Goe_414-1 | CP018339 | K. pneumoniae | 204,862 | 236 | 44.8% | 192,370 | 44.4% | Not applicable |
pE17KP0019-1 | CP052242 | K. pneumoniae | 266,878 | 295 | 46.6% | 198,024 | 44.7% | Not applicable |
pBJ20-1FIB | MW013146 | K. pneumoniae | 286,220 | 331 | 46.8% | 190,130 | 44.5% | This study |
pE20-1FIB | MG288682 | K. aerogenes | 239,779 | 277 | 45.3% | 199,074 | 44.3% | This study |
pKPN43G-1FIB | MW575452 | K. pneumoniae | 220,325 | 261 | 45.6% | 179,723 | 44.3% | This study |
IncFIB-6.1 | pK5880C-1FIB | MW307890 | K. pneumoniae | 259,711 | 300 | 46.2% | 197,006 | 44.6% | This study |
IncFIB-8.1 | pLR890543 | LR890543 | K. pneumoniae | 268,509 | 292 | 47.6% | 173,911 | 44.5% | Not applicable |
pRHB26-C08_2 | CP057460 | K. pneumoniae | 256,547 | 290 | 47.4% | 142,757 | 44.4% | Not applicable |
pCP069904 | CP069904 | K. pneumoniae | 358,127 | 392 | 47.4% | 171,133 | 44.6% | Not applicable |
References |
1. Villa L, Poirel L, Nordmann P, Carta C, Carattoli A: Complete sequencing of an IncH plasmid carrying the blaNDM-1, blaCTX-M-15 and qnrB1 genes. J Antimicrob Chemother 2012, 67(7):1645–1650. |
2. Wang J, Yuan M, Chen H, Chen X, Jia Y, Zhu X, Bai L, Bai X, Fanning S, Lu J et al: First Report of Klebsiella oxytoca Strain Simultaneously Producing NDM-1, IMP-4, and KPC-2 Carbapenemases. Antimicrob Agents Chemother 2017, 61(9). |
The modular structure (Figure S3 and Table 2) of each plasmid was divided into the conserved backbone and the accessory modules that were defined as acquired DNA regions associated/bordered with mobile elements. A pairwise sequence comparison using BLASTN showed that the 20 analyzed plasmids displayed considerable nucleotide identity (each ≥ 96%) across their backbone sequences (each ≥ 61%) (Table S3). They shared the IncHI3 core backbone markers, including tra1 and tra2, and parABC, but displayed different profiles of replication genes. Within the whole genomes of these plasmids, at least 51 drug resistance genes were identified and involved in resistance to 19 different categories of antibiotics and heavy metal (Fig. 1 and Table S4).
Table 2
Accessory modules in the 20 analyzed plasmids
Group (n = 3) | Plasmid (n = 20) | Accessory resistance modules | Accessory non-resistance modules |
Tn1696-related regions (n = 14) | MDR regions (n = 3) | The others | Metabolism-related | The others |
iuc regions (n = 3) | lac regions (n = 2) | moa regions (n = 2) |
IncHI3 | pNDM-MAR | 22.8-kb Tn7067 | 25.8-kb MDR region | | | | | Tn6754, Se.ma.I1 intron, 2.1-kb stbDE region, ΔIS10R–ISKpn34–ISKpn26, ISKpn8–ISKpn28, ISKpn34, ISKpn21, IS3000, and ISKpn50 |
pW09308-HI3 | | | 13.8-kb blaSHV−12 region | | | | Se.ma.I1 intron, ISKpn21:ISKpn38, ISEc33, IS1R, and ΔISKpn41 |
p362713-HI3 | 21.3-kb Tn7066 | | | | | | Se.ma.I1 intron, 5.7-kb relEB region, 4.4-kb stbDE region, IS903B, IS5, ISKpn8, ISKox3, ISEc52, and IS2 |
pA1718-HI3 | 25.8-kb T1696R region | | | 54.6-kb iuc region | | | Se.ma.I1 intron, 5.7-kb relEB region, 4.4-kb stbDE region, ISEc33, ISKpn26, and ISEc52 |
p721005-HI3 | 4.0-kb T1696R region | | | 54.6-kb iuc region | | | Se.ma.I1 intron, 5.7-kb relEB region, 4.4-kb stbDE region, ISKpn26, IS5075, and ISEc52 |
pKPM502 | 42.8-kb Tn7068 | 45.0-kb MDR region | | | | | Se.ma.I1 intron, 9.8-kb stbDE region, IS200/IS605 family IS element-tnpA mutated, and ISKpn21 |
pCP020068 | 62.1-kb Tn7070 | | | | | | Se.ma.I1 intron, 4.4-kb stbDE region, ΔIS26–IS26, ISSen4, and ISEc23 |
pCP041935 | 31.7-kb Tn7073 | | | | 39.8-kb lac region | | 4.4-kb stbDE region, Interrupted ISEc52, ISKpn28, and ISKpn21 |
pKOX3-P1 | 47.9-kb Tn7071 | | | | | | Se.ma.I1 intron, 12.3-kb relEB region, 2.9-kb stbDE region, ISKox3–to–orf555, ISEc36, ISEc52, IS903B, ISSpu2, and IS26 |
pCP050156 | 51.4-kb Tn7069 | | | | | | Tn6754, Se.ma.I1 intron, 4.4-kb stbDE region, ISKpn34–ΔIS10R, ISKpn8–ISKpn28, Se.ma.I1–ISKpn21:IS903B–ISKpn21, and ISKpn14 |
pKX029332 | | | 54.2-kb MDR region | | | | Se.ma.I1 intron, 4.1-kb stbDE region, ISKpn21–ISKpn84, ISKpn26, ISKpn20, IS26, IS903, IS903B, and IS5 |
IncFIB-6.1:8.1 | pKp_Goe_414-1 | | | | | | | Se.ma.I1 intron, 1.1-kb inserted regionorf471, and ISEc23 |
pE17KP0019-1 | | | 35.9-kb MDR region | | 9.7-kb truncated lac region | | Se.ma.I1 intron, 5.5-kb stbDE region, ISKpn21:ISKpn38, IS5, and ISEc33 |
pBJ20-1FIB | 18.4-kb Tn7072 | | | 57.4-kb iuc region | | | Se.ma.I1 intron, 4.4-kb stbDE region, IS903B, IS4321, IS1F, and ISKpn26 |
pE20-1FIB | 19.7-kb T1696R-1 region, and 6.8-kb T1696R-2 region | | | | | | 4.4-kb stbDE region, IS1R, and ISKpn21 |
pKPN43G-1FIB | 19.7-kb T1696R-1 region, and 5.2-kb T1696R-2 region | | | | | | Se.ma.I1 intron, 4.4-kb stbDE region, IS1R, and ISKpn21 |
IncFIB-6.1 | pK5880C-1FIB | | 40.4-kb MDR region | | | | | Se.ma.I1 intron, 6.8-kb stbDE region, 1.1-kb inserted regionorf471, ΔISKpn8–ISKpn28, ISKpn21–ISKpn75, ISKpn78, IS102, and ΔISKpn41 |
IncFIB-8.1 | pLR890543 | | | Tn6968, and 15.5-kb relEB region | | | | Se.ma.I1 intron, 4.4-kb stbDE region, ISSen4, IS903B, ISKpn26, IS4321R, and IS903 |
pRHB26-C08_2 | | | | | | 58.4-kb/52.9-kb moa region | 1.5-kb inserted regionorf1272, and ISKpn14 |
pCP069904 | | | 38.4-kb lac region | | | | Se.ma.I1 intron, 61.2-kb relEB region, 9.9-kb stbDE region, 7.7-kb inserted regionumuC, IS200/IS605 family IS element-tnpA mutated, IS903B–to–ISKpn38, ISKpn26–to–ISSpu2, IS4321R, ISKpn24, ISKpn8, ISEc52, ISSen4, IS102, and IS903B |
Massive Gene Acquisition And Loss Across The 20 Analyzed Plasmids Genomes
At least 39 regions/sites were recognized to display major modular diversity across the whole genomes of the 20 analyzed plasmids (Figure S4 and Table S5). Firstly, a collection of 189 events of gene acquisition occurred at 37 regions/sites within the 20 analyzed plasmids, and each of these plasmids acquired different profiles of accessory modules, including IS elements, transposons, MDR regions, drug resistance/metabolism/toxin-antitoxin regions, introns, and so-called “inserted regions”. These insertion events of foreign genetic contents often led to deletion and/or inversion of surrounding backbone regions. Secondly, a total of 49 deletion events of backbone regions, 35 of which were caused by the acquisition of exogenous DNA regions, occurred at 35 regions/sites in 17 plasmids. There were 12 plasmids each with a > 5 kb deletion of backbone regions. The deletion of tra region (covering tra1 and/or tra2) was observed in five plasmids, where one plasmid had a 14.7-kb deletion of tra2 region (24.5 kb in length) due to unknown reasons, while the rest four plasmids underwent completely or partially deletion of tra region due to the insertions of exogenous modules at a site within or upstream of tra region. All the above insertions and deletions of tra region might impair self-transferability of the corresponding plasmids. Thirdly, four of the analyzed 20 plasmids underwent 23.8-kb or 36.6-kb inversion of backbone regions, all of which were caused by exogenous insertions. Finally, A total of 41 substitution events of the backbone regions occurred at ten regions/sites in 12 plasmids. Taken together, the large genomes of the analyzed 20 plasmids exhibited a high level of modular diversity that mainly manifested as massive gene acquisition and loss.
Thirteen Tn 1696 derivatives
Tn3-family unit transposon Tn1696, originally found in Pseudomonas aeruginosa plasmid R1003, was generated from a concise class 1 integron In4 with the gene cassette array (GCA) aacC1–gcuE–aadA2–cmlA1 into the resolution (res) site of a core backbone structure consisting of IRL (inverted repeat left)–tnpA (transposase)–tnpR (resolvase)–res–mer (mercury resistance operon)–IRR (inverted repeat right) [34]. A collection of 13 Tn1696-related (T1696R) regions were found in 12 plasmids, and 12 of 13 were inserted upstream of the plasmid backbone gene orf366 (hypothetical protein), while the remaining one were inserted downstream of the plasmid backbone gene orf333 (hypothetical protein).
Eight of 13 T1696R regions (Fig. 2) manifested as intact transposons: Tn7066 (p362713-HI3), Tn7067 (pNDM-MAR), Tn7068 (pKPM502), Tn7069 (pCP050156), Tn7070 (pCP020068), Tn7071 (pKOX3-P1), Tn7072 (pBJ20-1FIB), and Tn7073 (pCP041935). They had the whole Tn1696 backbone (except Tn7070 with absence of gene Δres-3’and urf2) with a paired terminal 38-bp IRL/IRR, and were (except Tn7072) further surrounded by the same 5-bp direct repeats (DRs; target site duplication signals for transposition). In4 in Tn1696 was substituted by a concise class 1 integron or its remnant as well as sole or multiple resistance modules. Three T1696R regions, as observed in Tn7066, Tn7067, and Tn7072, carried a concise class 1 integron In1765 (GCA: fosE–blaOXA−1–catB3), a 1.1-kb In remnant, and a concise class 1 integron In37 (GCA: aacA4cr–blaOXA−1–catB3–arr-3), respectively. And each of them acquired another resistance module: i) blaCTX−M−14-containing ISEcp1-based transposition unit Tn6503a; ii) a 9.3-kb Tn2670-related region (containing catA1; Figure S5); iii) a truncated chrA–orf98 unit. The rest five T1696R regions carried Tn1548-related (T1548R) regions (containing In27 with GCA: dfrA12–gcuF–aadA2) and/or In714 (GCA: gcu50–dfrA1–gcu37–aadA5) plus multiple resistance loci: i) Tn7068, Tn7069, and Tn7070 shared an 18.0-kb T1548R region (see below), a truncated ISAba14–aphA6–ISAba14 unit, and blaNDM−1/bleMBL-carrying Tn125:ISEc33. Besides, Tn7070 was integrated with additional three resistance loci: a truncated aacC2–tmrB region, IS26–blaSHV−11–IS3000 unit, and strAB-containing ΔTn6590; ii) Tn7071 and Tn7073 shared In714, but each of them possessed unique resistance loci: the former harboring a 13.6-kb T1548R region (see below), IS26–catA2–IS26 unit, and strAB-containing ΔTn6590, and the latter harboring a truncated chrA–orf98 unit, a 6.0-kb Tn21 remnant carrying mer locus, and aacC2–tmrB region.
The remaining five T1696R regions (Fig. 2) in four plasmids carried only 5’- or 3’-terminal regions (with IRLs or IRRs, respectively) of Tn1696 and, thereby, could not be recognized as intact transposons. Each of the 5.2-kb, 6.8-kb, 4.0-kb, and 25.8-kb T1696R regions, as observed in pKPN43G-1FIB, pE20-1FIB, p721005-HI3, and pA1718-HI3, only carried 3’-terminal regions of Tn1696 together with one or more accessory modules: i) IS26, a truncated ISEc29–mph(E)–IS26 unit, and a truncated chrA–orf98 unit displayed in the first three, respectively; ii) a 7.1-kb Tn21 remnant carrying mer locus, IS26–mph(A)–IS6100 unit, an interrupted chrA–orf98 unit, and aacC2–tmrB region sequentially in the last one. Meanwhile, pE20-1FIB and pKPN43G-1FIB shared additionally a 19.7-kb T1696R-1 region, which was composed of 5’- terminal regions of Tn1696, In37, and an 8.9-kb T1548R region (see below).
Three Tn 1548 derivatives
IS26-composite transposon Tn1548 was initially identified on IncL/M plasmid pIP1204 in K. pneumoniae BM4536, and exhibited three resistance loci: In27, ISCR1–armA unit, and ISEc29–mph(E)–IS26 unit [35]. Each of five T1696R regions (Figure S6) as mentioned above carried the 8.9-kb/18.0-kb/13.6-kb T1548R regions: the 8.9-kb T1548R region in the 19.7-kb T1696R-1 region of pE20-1FIB and pKPN43G-1FIB, the 18.0-kb T1548R region in Tn7068, Tn7069, and Tn7070, and the 13.6-kb T1548R region in Tn7071. All these T1548R regions underwent deletion of 5′-terminal IS26, but each of them displayed unique modular structures: i) ISCR1–armA unit plus a truncated ISEc29–mph(E)–IS26 unit constituted the 8.9-kb T1548R region; ii) ISKpn21 bordered by 5-bp DRs was inserted downstream of mph(E) in the 18.0-kb T1548R region; iii) ISEc29–mph(E)–IS26 unit was truncated as a result of the insertion of IS15DI together with a truncated chrA–orf98 unit, and In27 was moved and inversed to the downstream of ISCR1–armA unit in the 13.6-kb T1548R region.
Five Mdr Regions
The 40.4-kb, 25.8-kb, 45.0-kb, 35.9-kb, and 54.2-kb MDR regions were located at different positions of the backbone of pK5880C-1FIB, pNDM-MAR, pKPM502, pE17KP0019-1, and pKX029332, respectively. The former three (Fig. 3) shared only an In37-like element harboring a truncated GCA aacA4cr–blaOXA−1–ΔcatB3, but each of them integrated one or more additional resistance loci: i) ISEcp1-based transposition unit Tn6502b containing blaCTX−M−15 shared by the 40.4-kb and 25.8-kb MDR regions; ii) a 15.4-kb Tn1721-related region (containing a class A tetracycline resistance module tetAR and a concise class 1 integron In1022 with GCA aacA4cr–arr-3–dfrA27–aadA16), an IS26-composite transposon Tn6415 containing aacC2 and tmrB, and a truncated IS26–blaLAP−2–qnrS1–IS26 unit in the 40.4-kb MDR region; iii) a truncated IS3000–qnrB1–IS26 unit plus blaNDM−1/bleMBL-containing Tn3000 in the 25.8-kb MDR region; and iv) a 20.4-kb sil–cop region in the 45.0-kb MDR region. Each of the last two (Figure S7 and Figure S8) carried a complex class 1 integron: i) In522 (VR1/GCA: dfrA15–cmlA1–aadA2 and VR2: ISCR1–blaCTX−M−59 unit) in the 54.2-kb MDR region of pKX029332; ii) In1891 (VR1/GCA: aacA4–blaOXA−4–aadA2 and VR2: ISCR1–qnrB4–blaDHA−1 unit) in the 35.9-kb MDR region of pE17KP0019-1. Besides, the 54.2-kb MDR region of pKX029332 acquired additional resistance components, namely an interrupted ISCR2–sul2 unit and a 7.3-kb Tn21 remnant containing mer locus.
Two iuc regions
The iuc locus (iucABCD-iutA) and rmpA2 were recognized in the 54.6-kb iuc region of pA1718-HI3 and p721005-HI3, and the 57.4-kb iuc region in pBJ20-1FIB, all of which were located upstream of pgpB (phosphatidylglycerol-phosphate phosphatase). These iuc regions (Fig. 4) carried different profiles of accessory modules, which included various intact or truncated IS elements, Tn4651-subfamily unit transposon Tn6752, and Se.ma.I1-like intron. They displayed high similarity in gene organizations, but exhibited two major modular differences: i) Se.ma.I1-like intron and ISKpn21 were displayed in the 54.6-kb iuc region, while IS102 in the 57.4-kb iuc region; ii) fep locus involved in ferric dicitrate uptake was located in the 57.4-kb iuc region.
Eleven Other Key Accessory Regions
A total of 11 other key accessory regions were observed in ten plasmids, which were a combination of various IS elements together with drug resistance and/or metabolism genes.
The 13.8-kb blaSHV−12 region (Figure S9) was found downstream of repHI3B in pW09308-HI3 and contained a truncated IS26–blaSHV−12–IS26 unit, which was likely to be an important vector to disseminate the drug resistance gene blaSHV−12.
Tn6968 (Figure S10), as observed in pLR890543, was inserted within orf802 (bacterial Ig-like domain). Tn6968 displayed as a composite transposon that was flanked by two separate IS elements IS903B and IS102 (two closely related IS elements) and further bracketed by 9-bp DRs at both ends. It contained a 21.2-kb sil–cop region and two kinds of metabolism gene loci: lac (galactoside uptake) and fec (iron uptake).
The 5.7-kb/15.5-kb/62.1-kb/12.3-kb relEB regions (Figure S11) were present in p362713-HI3/pA1718-HI3/p721005-HI3, pLR890543, pCP069904, and pKOX3-P1, respectively, and all of them were located upstream of orf207 (hypothetical protein) and then bordered by 6-bp DRs. These relEB regions had highly similar gene organizations, but some of them were equipped with its unique structure: i) ncr locus (nickel resistance) and chr locus (chromate resistance) in the 15.5-kb relEB region; ii) 52.4-kb vagCD region in the 62.1-kb relEB region; iii) Shg.f.I1 intron in the 12.3-kb relEB region. These two RelEB and VagCD toxin-antitoxin systems could maintain plasmid stability in several stressful conditions [36, 37].
The 39.8-kb, 38.4-kb, and 9.7-kb lac regions (Figure S12), displayed in pCP041935, pCP069904, and pE17KP0019-1, respectively, were located within orf95 (hypothetical protein) and exhibited three major modular differences: i) intact and truncated versions of lac locus, an intact version, and a truncated version in the 39.8-kb, 38.4-kb, and 9.7-kb lac regions, respectively; ii) fec locus in the 39.8-kb and 38.4-kb lac regions; iii) ars locus (arsenic resistance) in the 38.4-kb lac region.
The 58.4-kb and 52.9-kb moa regions (Figure S13) were inserted at different locations of pRHB26-C08_2. They only shared the identical moa locus (molybdopterin biosynthesis) and displayed considerable modular diversity in gene organizations.
Transferability And Antimicrobial Susceptibility
Plasmids pW09308-HI3 (IncHI3) and pE20-1FIB (IncFIB-6.1:8.1) were selected as the representatives for IncHI3 plasmids and their derivatives, respectively. Each of these two plasmids could be successfully transferred from the wild-type isolates into EC600 through conjugation, generating transconjugants EC600/pW09308-HI3 and EC600/pE20-1FIB, respectively. The self-transferable nature was in accordance with the fact that each plasmid possessed the complete conjugal transfer regions (tra1 and tra2). The wild-type isolate W09308 and its transconjugant EC600/pW09308-HI3 were resistant to ceftazidime with minimum inhibitory concentration (MIC) values ≥ 64 µg/mL due to the production of SHV enzyme. And the wild-type isolate E20 and its transconjugant EC600/pE20-1FIB were resistant to amikacin with MIC values ≥ 64 µg/mL owing to the expression of 16S rRNA methyltransferase.
Newly Identified Or Designated Ages
There were totally two newly identified AGEs: integron In1765 and unit transposon Tn7066. Additionally, there were 14 newly designated (first designated in this study but with previously determined sequences): eight unit transposons Tn6752 and Tn7067-7073, IS-based transposition unit Tn6754, composite transposon Tn6869, and IS elements ISKpn69, ISKpn78, ISKpn80, and ISKpn85.