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COMMENTARY MobileGeneticElements2:1,55–58;January/February2012;G2012LandesBioscience IncA/C plasmids An emerging threat to human and animal health? Timothy J. Johnson* and Kevin S. Lang DepartmentofVeterinaryandBiomedicalSciences;UniversityofMinnesota;SaintPaul,MNUSA Incompatibility groupIncA/Cplasmids turtle farms. This provided the first are large, low copy, theta-replicating evidencethatIncA/Cplasmidsmighthave plasmids that have been described in the emerged in response to antibiotic use in literature for over 40 years. However, animal agriculture. they have only recently been intensively In the 1990s, the plasmid-encoded © 2012 Lastudiend on dthe geneomicslevel beBcause oif oβ-lacstamasecgenie belaCMYn-2 wascidenteified . their associations with the emergence of among Klebsiella pneumoniae,4 and multidrug resistance in enteric pathogens several reports in the early 2000s identi- of humans and animals. These plasmids fied bla on a conjugative element in CMY-2 are unique among other enterobacterial Escherichia coli and Salmonella enterica plasmidsinmanyaspects,includingtheir from food-producing animals.5-10 This modular structure and gene content. was followed by the identification of Do not distribute. WhiletheIncA/Cplasmidgenomestruc- bla in clinical S. enterica and CMY-2 ture has now been well defined, many K. pneumoniae of humans.11,12 The questionsremainpertainingtotheirbasic applicationofplasmidgenomesequencing biological mechanisms of dissemination andreplicontypingrevealedthatbla CMY-2 and regulation. Here, we discuss the was often localized to IncA/C plasmids, history of IncA/C plasmids in light of and that these plasmids also encoded for our recent understanding of their popu- resistance to numerous other antimicro- lation distribution, genomics, and effects bials.13-16 Studies by the US. Food and on host bacteria. Drug Administration examining clinical isolates from 1940–2000 showed that IncA/C plasmids and their multidrug Since the discovery of the first multidrug- resistance phenotypes emerged in S. enter- resistantShigella strains in 1955, plasmids ica serovar Newport isolates after 1980.17 encoding multidrug resistance have been Thus, the literature would suggest an extensively studied and appreciated for emergence of this plasmid type in food their diversity and plasticity.1 Among the animal and human enteric bacteria origin- large number of defined plasmid incom- ating from an environmental bacterial patibility (Inc.) groups is IncA/C, which reservoir,followedbytherapidacquisition have been described in the literature for of resistance gene modules associated with over 40 years but until recently were not antibiotics used in animal agriculture and extensively studied. IncA/C plasmids were human therapy. Circulation of IncA/C first identified among multidrug resistant plasmids in Gram-negative pathogens is Keywords: IncA/C, plasmid, emergence, Aeromonas hydrophila and Vibrio spp now common, and these plasmids bring resistance, ICE, regulation causing disease in cultured fish in the with them the ability to encode resistance 1970s, where it was hypothesized that to broad arrays of antimicrobial agents. Submitted: 01/02/12 such plasmids emerged in response to the Precisely how IncA/C plasmids have Accepted: 02/06/12 common practice of therapeutic antibiotic emerged among enteric bacteria is a diffi- http://dx.doi.org/10.4161/mge.19626 treatment in response to disease.2,3 Their cult question to address, but it is certainly appearance coincided with the use of not surprising. These plasmids apparently *Correspondenceto:TimothyJ.Johnson; Email:[email protected] antibioticsbyculturedfishandsoft-shelled have an extremely broad host range,18 www.landesbioscience.com MobileGeneticElements 55 coupled with an ability to spread via the hotspot regions of this plasmid have (Fig.1).13-16,23,24 Remarkably, the core conjugative transfer within bacterial com- apparentlyrapidlyevolved.Acaseinpoint backbone of the numerous IncA/C plas- munities. While the conjugal transfer of istherecentfinding that IncA/C plasmids mid genomes described in the literature some IncA/C plasmid variants between acquiring the bla gene encoding the thus far shares 99% nucleotide sequence NDM-1 and among E. coli and Salmonella in vitro New Delhi metallo-β-lactamase have suc- similarity,withsubstantialdifferencesonly is a subject of debate,19 there are docu- cessfully disseminated among clinical K. occurring within the resistance-associated mented cases of the short-term ability of pneumoniae and E. coli originating in genes of their hotspot regions. With the these plasmids to disseminate in hospital India.21,22 Therefore, it seems likely that growing number of unpublished genome settings. For example, a 2-year outbreak both horizontal gene transfer and clonal sequences, however, this level of conserva- study in a Tunisian hospital involving disseminationmustplayanimportantrole tion is not always observed. For example, multidrug-resistant Providencia stuartii in the success of IncA/C plasmids, recent IncA/C plasmid sequences have identified IncA/C plasmid variants that although the abundance of these plasmids beendepositedfromAeromonashydrophila had circulated among three bacterial in the environment and their full range of (FJ705807), Xenorhabdus nematophila clones over this period.20 bacterial hosts is not yet appreciated. (FN667743), and Aeromonas salmonicida Comparative IncA/C plasmid sequenc- There are a number of interesting (CP000645), and these plasmids share ing has revealed that they possess at least traits of IncA/C plasmids that make them only 72–94% nucleotide sequence simila- three hotspots for the integration of unique among known enterobacterial rity in their core backbones with the mobile genetic elements.13-16 IncA/C plas- plasmid types. First, their genome struc- previously mentioned plasmids. It can be mids now have been sequenced from ture is remarkable. These plasmids have hypothesized, then, that the diversity of ©isolate s o2f E. 0coli, S1. ente2rica, V ibLrio aan itneron-codntainieng thseta r eplBicon, ait obactesrial scpeciesihaerborinng InccA/C eplas- . cholerae, Yersinia pestis, Photobacterium least three integrative hotspots capable of mids is vastly underappreciated, as is the damselae subsp piscicida, Yersinia ruckeri, acquiring a variety of mobile genetic genetic diversity within this plasmid and Klebsiella pneumonia. Multiple line- elements, a putative transfer region group. This previously unrecognized agesofIncA/Cplasmidshavesubsequently resembling that of an integrative conjuga- diversity also suggests that the aforemen- been identified, with each containing tive element, a number of putative tioned plasmids sharing high conservation unique arrays of mobile genetic elements transcriptional regulators belonging to in their backbone regions have a relatively Do not distribute. within their hotspot regions. Since the various classes, and an even largernumber recent ancestry, supporting the notion entrance of IncA/C plasmids into human of hypothetical genes that appear to be that IncA/C plasmids of enteric bacteria and animal enterobacterial populations, derived from a variety of different sources originatefromacommonancestralplasmid Figure1.BackbonestructureofIncA/Cplasmids.Integrationhotspotsaredepictedwithblackarrows,andinsertedmodulesofsequencedIncA/C plasmidsaresummarizednexttothearrows.Thereplicon(Rep),transcriptionalregulators(HU-b-like,GntR,andH-NS),andplasmidstabilityregions (Toxin-antitoxinandPar)oftheIncA/Cbackbonearedepictedinthecircularmap.Regionswithsimilaritytointegrativeconjugativeelement(ICE) SXT/R391andputativetransferregionsarecoloredaccordingtothelegend. 56 MobileGeneticElements Volume2Issue1 type. These findings further support the encode H-NS-like proteins and other IncA/Cplasmidstheopportunitytoreside rapid dissemination of these plasmids transcriptional regulators.26,28,29 Some of in a microbial community in the absence between enteric bacteria and a rapid these proteins have been shown to exhibit of benefit to an enteric host, persist, and evolution via recombinational events. silencing effects on the bacterial chro- subsequently emerge in pathogens when- IncA/C plasmids have been shown to mosome, suggesting that this might be a ever conditions require. modulate changes to their bacterial host key activity in the successful dissemina- Many questions remain related to chromosomes. For example, IncA/C plas- tion of these plasmids to a broad range IncA/C plasmid biology. What is the mids confer the mobilization in trans of of bacterial hosts. However, the high nature of the IncA/C plasmid-bacteria chromosomal mobile elements, as demon- number of putative regulatory genes that relationship, and how does this relation- strated in Salmonella enterica using chro- IncA/C plasmids possess suggests that ship differ in enteric bacteria vs. the mosomal island SGI1.25 They are also strict regulation of both the plasmid and assumed long-term hosts of these plas- capable of integration into the bacterial naïve host chromosome is critical for their mids? Notably, all studies examining chromosome.26 On the transcriptional broad-host dissemination. Interestingly, IncA/C plasmid biology thus far have level, we have demonstrated effects on the closest database matches to these utilized a limited number of bacterial the E. coli chromosome after the acquisi- protein sequences are from non-enteric species and strains. It is evident, though, tion of an IncA/C plasmid, the most bacteria such as Vibrio spp, Aeromonas that additional hosts should be con- notable effect being the upregulation of spp, etc., indicating that the most recent sidered when studying its mechanisms of the multiple antimicrobial resistance long-term hosts of these plasmids may transfer and stability. Another lingering (mar)locusinvolvedinlow-levelresistance indeedhavebeensoilandwaterbacteria.16 question is how frequently, and to what ©to mu ltip2le anti0microb1ial age2nts.27 ThLus, aIt is tnherefodre temeptingsto spe cuBlate thait oextenst,doecsrecoimbeinationnoccucrontehese . the ability of these plasmids to interact these plasmids are well suited to persist plasmids? Is their dissemination due with the bacterial host using multiple in certain subsets of reservoir bacteria, primarily to the emergence of highly regulatory mechanisms and to mobilize in which they provide a lower imposed successful clones or frequent horizontal other genetic elements suggests that the fitness cost to their bacterial host and gene transfer events? Given their appar- broad host range of IncA/C plasmids may are better able to persist in a microbial ently recent introduction into enteric be enabled not only by its basic replicon community. It has been demonstrated bacteria of humans and animals, are we Do not distribute. butalsothroughtranscriptional regulation that IncA/C plasmids in hosts such as in the beginning stages of a long battle and extensive genetic recombination. E. coli and Salmonella might require with these plasmids in efforts to circum- IncA/C plasmids possess a number of selective pressure for their long-term vent the dissemination of antimicrobial putative transcriptional regulators with persistence.30 However, if other reservoir resistance? Furthermore, what led to this sequencesimilarity(albeitlow)toproteins bacteria are available as better hosts for scenarioanddoesitrepresent anepidemic of the H-NS, HU-β, Xre, LysR, GntR IncA/C plasmids when resistance pheno- threat to the treatment of Gram-negative andLuxRfamilies.16Thepresenceofsuch typesarenotrequired,adauntingscenario bacterial infection? In theworld ofmobile proteins on large, low copy plasmids is emerges where many suitable reservoirs genetic elements, IncA/C plasmids are not unexpected. Other large plasmids, for this plasmid might exist in any given both fascinating and concerning and there such as Inc.P, Inc.H and Inc.X, also ecological niche. 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