Comparative Genomics of blaIMI Plasmids and Enterobacter Lineages

Good to see you again. In the previous lesson, we separated a recurring ST250 label from evidence for a true outbreak clone. The same discipline now applies to plasmids: an IMI allele, a plasmid replicon call, a conserved resistance-gene neighbourhood, and a complete plasmid backbone are related observations—but they are not interchangeable.

This lesson compares the reported vehicles of blaIMI-2 with those of other IMI alleles, then returns to the specific question of ST250 Enterobacter asburiae. By the end, you should be able to state precisely what the current literature supports, what it merely suggests, and what would be required to substantiate an ST250 plasmid-outbreak claim.

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A replicon is not a plasmid lineage

A replicon type is defined by a plasmid’s replication-control system. IncFII(Yp), IncFII(Yb), IncFIB, and IncHI2 are examples of replicon designations. Plasmids with the same incompatibility type may be unable to coexist stably in one host because they use closely related replication systems.

That is useful information, but it is only one layer of description.

A plasmid backbone is broader. It includes:

• the replication module and any additional replicons;
• maintenance functions, such as partitioning and toxin–antitoxin systems;
• transfer machinery, particularly conjugative tra and trb systems;
• accessory regions carrying resistance, metal tolerance, phage-related, or other adaptive genes;
• the order and structural arrangement of these components.

Finally, the IMI genetic module is the local segment containing blaIMI, its regulator imiR where present, and nearby insertion sequences or other mobile elements.

These three statements therefore have very different strengths:

Observation	What it supports	What it does not establish
Two isolates have IncFII(Yp) replicons	Their plasmids share, or contain related, replication modules	Their plasmids are the same throughout
Two plasmids have a similar imiR–blaIMI-2 region	The resistance module may share ancestry or have moved between backbones	The bacterial hosts are clonally related
Two isolates carry highly similar closed plasmids	Strong evidence for plasmid dissemination, if assembly and comparison are adequate	Direct patient-to-patient transmission
Two isolates are ST250	They share an MLST designation	They share the same IMI plasmid or are part of one outbreak

This distinction is essential for hospital plumbing. A sink-associated Enterobacter strain can persist clonally with a chromosomal IMI element. Alternatively, diverse bacteria in a biofilm could acquire related plasmids or a smaller IMI-associated module. Those scenarios demand different control measures and different genomic analyses.

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A high-level catalogue of reported IMI vehicles

The French 2012–2022 collection provides the clearest comparative overview across IMI alleles. Its major result is that IMI-family genes occur in both chromosomal and plasmid-borne forms.

The resulting evidence map is below. It deliberately separates gene location from replicon evidence.

IMI or NMC-A group	Reported principal location in the French collection	Replicon or backbone evidence supported by the resources	Interpretation
NMC-A, IMI-1, IMI-4, IMI-12	Chromosomal	Usually within EcloIMEX-type elements	These are chromosomal mobile-element contexts, not plasmid replicon types. Their persistence may be strongly lineage-associated, although EcloIMEX-like structures can occur across ECC species.
IMI-13	Chromosomal	Distinct context, with nearby insertion sequences; acquisition mechanism unresolved	Do not force every chromosomal IMI allele into the EcloIMEX model.
IMI-2	Plasmid-borne in the French collection	IncFII(Yp) in 8 of 12 producers; no replicase identified in 4	IncFII(Yp) is the most frequently documented replicon association, but it is neither demonstrated in every IMI-2 isolate nor equivalent to one conserved plasmid.
IMI-6	Plasmid-borne	IncFII(Yb), approximately 160–200 kb	This is a distinct reported IncFII subtype association from IMI-2.
IMI-17, IMI-19, IMI-25, IMI-26, IMI-27	Plasmid-borne	The provided evidence establishes plasmid location but does not supply a full replicon catalogue for every allele	It would be inappropriate to assign these alleles an IncFII(Yp) backbone merely by analogy with IMI-2.

The French study also found that the investigated plasmids were conjugative in mating experiments, except for the plasmid carrying IMI-17. That is functional evidence that several IMI-bearing plasmids can transfer under laboratory conditions. It does not, by itself, quantify transfer frequency in drains, establish transfer in a patient, or identify the original donor in an outbreak.

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IMI-2: a recurring IncFII(Yp) association, not one fixed plasmid

The strongest plasmid association for IMI-2 is with IncFII-family plasmids, especially IncFII(Yp). However, a closer look shows substantial variation in size and accessory content.

The Swedish environmental plasmid, pSB89A/IMI-2, illustrates this distinction well. It was a 216 kb circular plasmid recovered from an E. asburiae isolate in a feed-mill environment in Sweden in 2019. It carried three detected replication-related signatures:

• IncFII related to pECLA;
• IncFIB related to pENTAS01;
• a third, IncFII(Yp)-like replicon.

Thus, pSB89A/IMI-2 is not simply “an IncFII(Yp) plasmid.” It is a large, multi-replicon IncF-family plasmid with transfer genes, partitioning and stability systems, and accessory genes including an arsenic transport system.

This is a useful reminder for bioinformatic interpretation: a replicon finder reports recognizable replication sequences. It does not automatically tell you whether a plasmid is a simple single-replicon unit, a hybrid plasmid assembled through recombination, or one part of a mixed assembly.

Reported IMI-2 plasmids span multiple hosts and settings

The study’s comparison table places related IncFII(Yp)-associated IMI-2 plasmids in a much wider ecological landscape than one Enterobacter species or one hospital.

Plasmid	Reported host	Source and geography	Size	Replicon finding
pSB89A/IMI-2	E. asburiae	Feed-mill environment, Sweden, 2019	216 kb	IncFII, IncFIB, IncFII(Yp)-group
pEn542	E. mori	Human clinical isolate, Sweden, 2018	181 kb	IncFII(Yp)
pEn701	E. ludwigii	Human clinical isolate, Sweden, 2020	167 kb	IncFII(Yp)
pEn718	E. mori	Human clinical isolate, Sweden, 2020	137 kb	IncFII(Yp)
pN151247-1	Klebsiella aerogenes	Shrimp imported from Bangladesh, sequenced in Canada	60 kb	IncFII(Yp)-group
p3442-IMI-2	Reported as E. cloacae complex	Imported white shrimp, Netherlands, 2017	78 kb	IncFII(Yp)
pJF-787	Klebsiella variicola	Human clinical isolate, Wales, 2011	78 kb	IncFII(Yp)

Several conclusions follow.

First, IMI-2 has been found in environmental, food-associated, and clinical settings. This makes a simple “hospital-only” or “environment-only” explanation untenable.

Second, IncFII(Yp) recurs across these reports, including in Enterobacter and non-Enterobacter hosts. That supports a durable association between IMI-2 and an IncFII(Yp)-related plasmid family or module.

Third, sizes from roughly 60 kb to 216 kb are too different to justify calling these isolates carriers of one unchanged plasmid. Shared replication and local resistance modules can coexist with extensive divergence elsewhere in the plasmid.

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The conserved local module and the variable backbone

The local region around blaIMI-2 is more informative than a replicon call alone, but it still must not be mistaken for an entire plasmid.

Across several IMI-2 plasmids, the study found recurring components near the gene:

• the LysR-like regulator imiR;
• upstream remnants or copies of ISEae1 and ISSba14;
• an ISEae2-related element downstream of imiR in several plasmids;
• small IS1-family fragments near blaIMI-2;
• toxin–antitoxin systems, particularly relBE/stbDE, in the examined IMI-2 plasmids.

The key pattern is local conservation with global rearrangement. pSB89A/IMI-2 and pEn701 had particularly similar blaIMI-2–imiR regions, including an identical ISEae2 element, yet they differed elsewhere in structure and transfer systems. Some IMI-2 regions were also reported within putative prophage-associated segments.

The figure makes an important analytic point: shaded similarity around a resistance locus is evidence for related segments, not necessarily evidence that the complete plasmids are the same. In an isolate collection, this distinction separates a plausible mobile IMI module from confirmed dissemination of one plasmid backbone.

A second genomic map extends this idea to other IMI contexts.

The transposon comparison is especially useful conceptually. A resistance gene may be carried within a nested structure: a local gene module inside an insertion-sequence-rich region, within a transposon-like element, on a plasmid, in a bacterial chromosome. Two isolates can therefore share the same transposon-like structure while carrying different plasmids, or share a plasmid backbone while having acquired distinct resistance regions.

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How IMI-2 differs from other IMI alleles

The literature summarized here supports two broad genomic modes.

Chromosomal IMI/NMC-A mode

NMC-A and several IMI alleles, notably IMI-1, IMI-4, and IMI-12, were found in chromosomal EcloIMEX-type elements in the French collection. These elements were inserted between setB and yieP and had a conserved region but variable overall forms.

This means a chromosomal IMI result should not be casually described as “non-mobile.” The recurrence of related EcloIMEX elements in different ECC species suggests possible movement of the larger chromosomal element. But its epidemiology differs from that of a self-transmissible plasmid: clonal expansion and chromosomal-element movement need to be evaluated separately.

Plasmid-associated IMI mode

IMI-2 and IMI-6 are the best-supported examples in the supplied literature.

• IMI-2: repeatedly associated with IncFII(Yp)-related plasmids, yet found on plasmids with strikingly different sizes and gene contents.
• IMI-6: associated in the French collection with IncFII(Yb) plasmids of approximately 160–200 kb.

Therefore, “IMI plasmid” is too vague for interpretation. At minimum, report the allele and whether the evidence supports chromosomal EcloIMEX, an IncFII(Yp)-related plasmid, an IncFII(Yb)-related plasmid, or an unresolved locus.

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What does this establish about ST250 E. asburiae?

The answer is deliberately narrow:

The provided evidence does not document an ST250 E. asburiae outbreak driven by an IMI-2 plasmid.

The Swedish study provides a particularly relevant E. asburiae observation: an environmental E. asburiae isolate carried pSB89A/IMI-2. But the reported material identifies the source as a feed-mill environment and does not identify this isolate as ST250, link it to a hospital, or link it to an outbreak.

Likewise, the French collection shows that ST250 recurred among IMI/NMC-A-positive ECC isolates, as discussed in the preceding lesson. Yet the plasmid evidence provided here does not show that those ST250 isolates:

• were all E. asburiae by genome-based species assignment;
• carried IMI-2 rather than another IMI allele;
• carried IncFII(Yp) plasmids;
• carried pSB89A-like plasmids;
• were closely related at the core-genome level;
• came from one institution, ward, sink, shower, or clinical cluster.

The correct conclusion is not that an ST250 outbreak is impossible. It is that the required connecting evidence has not been demonstrated in these sources.

Evidence required for an ST250 plasmid-outbreak claim

A defensible claim would need an isolate-resolved evidence chain:

1. Species assignment: each isolate identified as E. asburiae using a genome-based method, rather than a broad ECC label.
2. Lineage assignment: ST250 reported with the MLST scheme and allele profile.
3. Allele assignment: the exact IMI allele, such as IMI-2, reported for each isolate.
4. Locus placement: adequate long-read or hybrid evidence placing blaIMI-2 on a plasmid rather than on a short unresolved contig.
5. Plasmid comparison: nucleotide coverage, sequence identity, synteny, replicons, transfer region, and resistance-region structure compared across isolates.
6. Strain comparison: recombination-aware core-genome analysis assessing whether the bacterial hosts form a recent cluster.
7. Epidemiological coherence: dates, wards, patient movement, sample types, and plumbing locations consistent with a shared transmission setting.

Without these layers, the strongest permissible wording is typically: “ST250 and IMI were detected in the same surveillance collection” or “an environmental E. asburiae isolate carried an IMI-2 IncF-family plasmid.” Neither statement establishes a clonal hospital outbreak.

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A practical reporting template for your own genomes

For each IMI-positive Enterobacter isolate, record the following before drawing an epidemiological conclusion:

Field	Example of a sufficiently specific result
Species	Enterobacter asburiae, ANI-supported
Lineage	ST250, with stated MLST scheme
IMI allele	blaIMI-2, full-length sequence and identity reported
Locus evidence	Circular 181-kb hybrid-assembled plasmid
Replicon profile	IncFII(Yp), or multi-replicon IncFII plus IncFIB
Local context	imiR, flanking ISEae1/ISEae2 status, other nearby IS elements
Backbone comparison	Percentage coverage and identity against relevant reference plasmids, plus synteny assessment
Transfer potential	Complete or interrupted conjugation machinery; experimental evidence if available
Epidemiological context	Patient, drain, shower, or water source; dates and location

This format prevents the common overstatement: “The isolate has an IncFII(Yp) replicon, therefore it carries the Swedish IMI-2 plasmid.” The appropriate statement would be: “The isolate contains an IncFII(Yp)-related replicon and blaIMI-2; complete plasmid identity remains to be assessed.”

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Key takeaways

• IMI-family carbapenemase genes occupy both chromosomal and plasmid-borne contexts in Enterobacter.
• IMI-2 is most often reported on IncFII(Yp)-related plasmids, but the reported plasmids vary substantially in size, replicon composition, transfer systems, and accessory content.
• IMI-6 has a distinct reported association with IncFII(Yb) plasmids in the French collection.
• A conserved imiR–blaIMI-2 region and recurrent insertion sequences can indicate a related mobile module, but do not prove that whole plasmids are identical.
• The environmental Swedish E. asburiae plasmid pSB89A/IMI-2 is important evidence that IMI-2 plasmids occur outside clinical settings, but it is not documented as ST250 or as part of a hospital outbreak.
• No provided evidence substantiates an ST250 E. asburiae IMI-2 plasmid outbreak. Such a conclusion requires linked species, ST, allele, complete-plasmid, core-genome, and epidemiological data.

Next, the course turns to hospital-water ecology: the physiological and ecological reasons Enterobacter can persist in nutrient-limited plumbing environments before considering how biofilms enable long-term resistance-gene maintenance and transfer.