Mobile Genetic Elements Associated with Antimicrobial Resistance

Abstract

Strains of bacteria resistant to antibiotics, particularly those that are multiresistant, are an increasing major health care problem around the world. It is now abundantly clear that both Gram-negative and Gram-positive bacteria are able to meet the evolutionary challenge of combating antimicrobial chemotherapy, often by acquiring preexisting resistance determinants from the bacterial gene pool. This is achieved through the concerted activities of mobile genetic elements able to move within or between DNA molecules, which include insertion sequences, transposons, and gene cassettes/integrons, and those that are able to transfer between bacterial cells, such as plasmids and integrative conjugative elements. Together these elements play a central role in facilitating horizontal genetic exchange and therefore promote the acquisition and spread of resistance genes. This review aims to outline the characteristics of the major types of mobile genetic elements involved in acquisition and spread of antibiotic resistance in both Gram-negative and Gram-positive bacteria, focusing on the so-called ESKAPEE group of organisms (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp., and Escherichia coli), which have become the most problematic hospital pathogens.

Keywords: antibiotic resistance; gene cassette; insertion sequence; integrative conjugative element; integron; plasmid; resistance island; transposon.

FIG 1

Examples of mobile genetic elements (MGE) and processes involved in intracellular mobility or intercellular transfer of antibiotic resistance genes. Two cells of different strains or species are represented, with one acting as donor (envelope and chromosome shown in blue; contains two plasmids) and the other as recipient (shown in red). Various MGE are shown, with the functions of the genes they carry color coded as shown in the key. Different resistance genes associated with different MGE are represented by small arrows of various colors. Thin black arrows indicate intracellular processes, with those mediated by a transposase protein labeled Tnp and those mediated by a site-specific recombinase protein labeled Ssr. Thick green arrows represent intercellular (horizontal) transfer. Successive insertions of the same IS on both sides of a resistance gene may allow it to be captured and moved to another DNA molecule (e.g., from the chromosome to a plasmid) as part of a composite Tn (A). A unit Tn carrying a resistance gene may transpose between plasmids (B) or from a plasmid to the chromosome or vice versa. A gene cassette may move between In (a class 1 In/Tn structure is represented here) via a circular intermediate (C). An ICE can be integrated into the chromosome or excised as a circular element that can then conjugate into a recipient cell and integrate (reversibly) into the chromosome at a specific recombination site (D). A plasmid may be able to mediate its own intercellular transfer by conjugation or, if it lacks a conjugation region, be mobilized by another plasmid (or, alternatively, move horizontally by phage transduction or transformation). Tn and/or In and associated resistance genes on an incoming plasmid may move into the chromosome or other plasmid(s) in the recipient cell (E), as illustrated here for class 1 In/Tn, which are known to target unit Tn. See relevant sections of the text for further details.

FIG 2

Insertion sequences and composite transposons. (A) Components of a typical IS. (B) Composite transposon. IS are shown as block arrows, with the pointed end corresponding to IR_R, and a captured resistance gene is shown as a black arrow. The two IS can also be oriented inversely. (C) Outcomes of transposition by IS26. (i) Intermolecular replicative transposition can insert a “translocatable unit” (TU; one copy of IS26 and an adjacent region) into a recipient that lacks IS26, while intermolecular conservative transposition targets an existing copy of IS26 (∼50× higher frequency). Both reactions create the same type of cointegrate, in which a “composite transposon”-like structure is flanked by 8-bp TSD (black lollipops) created during replicative transposition or preserved (if previously present) during conservative transposition. The cointegrate can be resolved by homologous recombination (HR), but not normally by the IS26 transposase. Intermolecular conservative transposition into a region that already contains two copies of IS26 flanking a resistance gene can give an array of TU. (ii) Intramolecular replicative transposition in direct orientation is another way of creating a TU, and in doing so deletes the region between the original IS26 element and the targeted position (white lollipop). Intramolecular replicative transposition in the inverse orientation inverts the region between the original IS26 element and the position targeted, so that TSD on the same strand are now reverse complements of one another (thick and thin lollipops). Diagrams are based on previously reported information (22, 25–28). (D) Transposition units (TPU) mediated by IS1380 family elements. △, 131 bp of IR_R end of ISEnca1. Paired lollipops indicate different TSD sequences. Diagrams were drawn based on sequences from the following INSDC accession numbers: IS1247, AJ971344; ISKpn23, KP689347; ISEnca1, AY939911 (end of TPU found by alignment with Staphylococcus plasmids, e.g., pSTE1 [accession number HE662694]); and ISEcp1, FJ621588. (E) Different structures containing ISApl1 and mcr-1. Deletion of one or both copies of ISApl1 leaves “scars” (asterisks) (41, 45). Diagrams (from top to bottom) were drawn based on sequences from INSDC accession numbers CP016184, KY689635, KP347127, and KX084392. (F) IS1294 has captured part (delimited by dashed lines) of the ISEcp1-bla_CMY-2 TPU plus 159 bp of the adjacent plasmid backbone (see the bottom diagram of panel D), ending with 4 bp matching its ter end (white circle), and targets a related 4-bp sequence (gray circle). The diagram was drawn based on sequences from INSDC accession number HG970648 (55). (G) By analogy with IS1294, ISCR1 captures regions adjacent to its ter end, but these are found adjacent to the ori end after insertion (by homologous recombination) (21, 59) between partial duplications of the 3′-CS of class 1 integrons (see Fig. 4B). The diagram was drawn based on the sequence from INSDC accession number AJ311891. The phenotypes conferred by resistance genes shown in the diagrams are given in Table 1 (panel E), Table 3 (panels D and F), and Table 4 (panel G).

FIG 3

Tn3 family transposons. The extents and orientations of various genes are shown by arrows, with thick arrows used to indicate antibiotic resistance genes (apart from those in gene cassettes). Terminal IR are indicated by black bars and putative ancestral IR relics by gray bars. res sites are shown as black boxes. (A) Tn3 family. Gene cassettes in Tn1331 are shown as narrower boxes. (B) Tn21 subfamily. For Tn21, the insertion site for class 1 In/Tn (see Fig. 4) and the 5-bp TSD are shown. Different integron structures and different cassettes may be present. IS4321 or IS5075 may be found inserted into IR_L and/or IR_R, in the indicated orientations. (C) Tn4401. The approximate position of deletions that lead to different promoter variants is indicated. Diagrams were drawn based on sequences from the following INSDC accession numbers: Tn2, AY123253; Tn1331, AF479774; Tn5393, AF262622; Tn1546, M97297; Tn21, AF071413; Tn1696, U12338; Tn6452; KY807920; Tn1721, X61367; and Tn4401, EU176011. The resistance genes shown confer resistance to the following antibiotics: bla_TEM-1, broad-spectrum β-lactams; bla_OXA-9, oxacillin; aadA1, streptomycin and spectinomycin; strAB, streptomycin; vanXAH, vancomycin/teicoplanin; mcr-5, colistin; tet(A), tetracycline; and bla_KPC, carbapenems.

FIG 4

Tn7-like transposons. Most features are shown as described in the legend to Fig. 3. (A) Tn7. The asterisk indicates the position of a common stop codon in intI2. The diagram was drawn based on the sequence from INSDC accession number AP002527. (B) Evolution of class 1 In/Tn. The diagrams show capture of intI1/attI1/Pc and gene cassettes, with qacE in the last position, by a Tn5053-like transposon. Subsequent deletion of parts of the final qacE cassette and tni region and insertion of sul1 create the 3′-CS, giving a typical “clinical” class 1 In/Tn which is not self-transposable, e.g., In2. Diagrams are based on information in reference and sequences from INSDC accession numbers U67194 and AF071413. Different extents of tni and different IS may be present beyond the 3′-CS (see Fig. 5 in reference for further details). (C) Tn552. Gene names shown in parentheses indicate relationships to those in Tn7/Tn5053 elements. The diagram was drawn based on the sequence from INSDC accession number X52734. (D) Transposons making up resistance islands in A. baumannii. The top diagram represents Tn6022; differences in minor variants Tn6021 (a short region with only 90% identity matches Tn6172) and Tn6022Δ are shown. The main part of the Tn6174 diagram corresponds to the hypothetical, ancestral Tn6173, which is also related to Tn6022 (percentage identities in different regions shown above) but has the ars/feo region replacing uspA and sup. Tn6174 itself has the two insertions shown above the diagram. Tn6172 was generated from Tn6174 by addition of Tn5393 (Fig. 3) and an internal deletion. In AbGRI1-0, a region flanked by Tn6022 and Tn6172 is inserted into the chromosomal comM gene. The backbone Tn6019 of AbaR3-like islands is related to Tn6022 (percent identities in different regions are shown) but contains an additional segment, shown above the relevant diagram. Various regions containing different antibiotic resistance genes are found between the two copies of Tn6018 (designated RR). Diagrams are based on previously published information (109, 110) and on sequences from the following INSDC accession numbers: Tn6022, CP012952; Tn6021 and Tn6164, CP012005; Tn6022Δ, JN247441; Tn6172, KU744946; and Tn6019, FJ172370. (E) GIsul2 (15.460 kb [188] rather than the initially reported 15.456 kb [61], apparently due to errors in the S. flexneri sequence). ars, arenite/arsenate resistance gene; TA, toxin-antitoxin system; alpA, regulation gene. The diagram is based on information in reference and the sequence from INSDC accession number KX709966. The resistance genes shown confer resistance to the following antibiotics: aadA1, streptomycin and spectinomycin; sat2, streptothricin; dfrA1 and dfrB3, trimethoprim; qacE, quaternary ammonium compounds; sul1 and sul2, sulfonamides; blaZ, penicillins; and strAB, streptomycin.

FIG 5

Representative diagrams of the backbone organization of major plasmid types associated with antibiotic resistance in Enterobacteriaceae. Plasmid types are indicated on the left. Diagrams are approximately to scale, with those in boxes at a different scale (see scale bars). Selected genes/gene regions involved in various functions are shown by the following colors: red, replication/oriV; blue, conjugation; green, maintenance; brown, entry exclusion; and purple, TA. Additional features may be shown for different plasmid types, with most explained further in the text, except for the following: ssb, single-stranded DNA binding protein gene; pri/sog, primase gene; resD/resP, resolvase gene; stb, stability/partitioning gene; psiAB, plasmid SOS inhibition gene; impABC/mucAB, mutagenic DNA polymerase gene; ardA, antirestriction gene; korAB, kill override gene (involved in regulation of tra); ccr, central control region; LDR, long direct repeats. Origins of transfer (oriT) are indicated by “T,” if they have been defined. Insertion points for resistance regions common to plasmids of the same type are also indicated, in some cases, by labeled vertical arrows. C backbones are represented by a single line, with differences (presence/absence of ARI-A, orf1832 versus orf1847, rhs1 versus rhs2, and presence/absence of i1 and i2) shown above (C₁) and below (C₂). L and M backbones are also represented by a single line, with different insertions in common plasmids shown above (L) and below (M) (modified versions of Tn2 with additional resistance genes are also found at the site indicated for Tn2). These two plasmid types differ mainly in traY/excA (entry exclusion) and traX (relaxase), with differences in inc distinguishing the M1 and M2 types. For HI1 plasmids, the type 1 backbone is shown, with insertions found in type 2 plasmids indicated above (A to E; region D from reference was recognized as a transposon, TnD, in reference 208). Insertions that give resistance to various heavy metals are indicated as follows; Te, tellurite; Ag, silver; Cu, copper; and As, arsenic. Targets for pMLST schemes are underlined (for C plasmids, repA, parA, parB, and 053; for I1 plasmids, repA, ardA, trbA, sogS, and pilL; for N plasmids, repA, korA, and traJ; for HI1 plasmids, repA [HCM1.64] as well as HCM1.99, HCM1.116, HCM1.178ac, HCM1.259, and HCM143 [abbreviated “99,” etc.]; and for HI2 plasmids, 0199 and 0018). Shufflons in I1 plasmid R64 (above) and I2 plasmid R721 (below) are shown in a separate box. Segment A contains partial open reading frames A and A′, etc. sfx repeats are represented by flags. Diagrams are based on information in previous publications and/or sequences from INSDC accession numbers for prototype plasmids, as follows: C₁ and C₂, references and ; FII, accession number AP000342; I1, references and ; I2, reference and accession number KP347127; I1 and I2 shufflons, reference ; L/M, references , , and ; N, reference and accession number AY046276; P, reference and accession number U67194; W, reference and accession number BR000038; X, reference ; HI1, references , , and and accession numbers AF250878 and AL513383; HI2, references and and accession number BX664015; Q-1, reference ; and ColE1, reference and accession number J01566.

FIG 6

S. aureus multiresistance plasmids. Representative multiresistance plasmids (pI258, pSK1, and pUSA300-HOU-MR) and pSK41-, pWBG749-, and pWBG4-family conjugative multiresistance plasmids (pLW1043, pBRZ01, and pWBG4, respectively) are shown (15, 104, 359, 384, 390, 398, 505, 516). IS, transposons, cointegrated plasmids, and resistance genes are shown, with resistances conferred by the latter listed in Table 2 or as follows: arsBC, arsenic resistance; bcrAB, bacitracin resistance; cadA and cadD, cadmium resistance; merAB, mercury resistance; msrA and mphC, macrolide resistance; and qacA, antiseptic/disinfectant resistance. The following plasmid maintenance genes/systems are also shown: par, novel partitioning system; parAB, type I partitioning system; parMR, type II partitioning system; rep, initiation of replication; res and sin, multimer resolution; TA, Fts-like toxin-antitoxin system. The conjugation-associated genes of pLW1043, pBRZ01, and pWBG4 are denoted tra, smp, and det, respectively.

FIG 7

Enterococcal multiresistance plasmids. Representatives of the Inc18 and RepA_N families are shown (, , , , , ; see the text for additional references). IS, transposons, and resistance genes are shown, with resistances conferred by the latter listed in Table 2 or as follows: cat (chloramphenicol resistance) and fexB (chloramphenicol/florfenicol resistance). The following plasmid maintenance genes/systems are also shown: cop, copy number control; parAB, type I partitioning system; rep, repA, and repB, initiation of replication; res, multimer resolution; txe-axe, toxin-antitoxin system. Note that pS177 is a pRUM-like plasmid.

FIG 8

Representative SCCmec elements (, ; see the text for additional references). IS, transposons, cointegrated plasmids, and resistance genes are shown, with resistances conferred by the latter listed in Table 2 or as follows: arsBC, arsenic resistance; cadA, cadmium resistance; fusC (previously known as far), fusidic acid resistance (517); and mecA/mecC, β-lactam resistance. Cassette recombinase genes (ccrA1 to -4, ccrB1 to -4, and ccrC1 and -2), mecA/C regulatory genes (mecI and mecR1), and an arginine catabolic mobile element (ACME) (441) are also shown; mec classes and ccr types are denoted by colored shading. Note that cch genes, polA, and SAUGI are not shown.