Extended-spectrum cephalosporin-resistant Gram-negative organisms in livestock: an emerging problem for human health?
Bacteria carry extrachromosomal, self-replicating genetic elements called plasmids. A plasmid is defined as a doublestranded, circular DNA molecule capable of autonomous replication. By definition, plasmids do not carry genes essential for the growth of host cells under nonstressed conditions (109). Plasmids have systems which guarantee their autonomous replication but also have mechanisms controlling their copy number and ensuring stable inheritance during cell division. They can promote lateral transfer among bacteria of different genera and kingdoms through the conjugation process. Many plasmids encode addiction systems generally based on toxin-antitoxin factors, which are able to kill daughter cells that do not inherit the plasmid during cell division (46). These systems efficiently promote plasmid maintenance in the bacterial population, regardless of other selective pressure, and do not provide any apparent benefit to the bacterium hosting the plasmid. However, most of the plasmids confer positively selectable phenotypes with the presence of antimicrobial resistance genes. We consider plasmids to be living organisms in spite of their simple structure, since they are unit elements of a continuous lineage with individual evolutionary history. Conjugative plasmids resemble lambdoid phages, which are capable of theta replication during vegetative growth and rolling-circle replication during “packaging” of the DNA into a recipient cell (62). The ability to recognize and categorize plasmids in homogeneous groups on the basis of their phylogenetic relatedness is helpful to analyze their distribution in nature and their relationship to host cells and to discover their evolutionary origins (34). Identification and classification of plasmids should be based on genetic traits that are present and constant. These criteria are best met by traits concerned with plasmid maintenance, especially replication controls (28). In 1971, Hedges and Datta proposed a plasmid classification scheme based on the stability of plasmids during conjugation, a phenomenon called plasmid incompatibility (27, 47). Incompatibility is a manifestation of the relatedness of plasmids that share common replication controls (27, 78). Incompatibility was defined as the inability of two related plasmids to be propagated stably in the same cell line; thus, only compatible plasmids can be rescued in transconjugants. The first incompatibility (Inc) groups were defined as follows: IncI, plasmids producing type I pili susceptible to phage Ifl; IncN, N3-related plasmids susceptible to phage IKe; IncF, plasmids producing type F pili susceptible to phage Ff; and IncP, RP4-related plasmids susceptible to the PRR1 phage (27, 47). Currently, 27 Inc groups are recognized in Enterobacteriaceae by the Plasmid Section of the National Collection of Type Cultures (London, United Kingdom), including six IncF (FII to VII) and three IncI (I1, I , I2) variants. In 1988, Couturier and colleagues proposed a genetic plasmid typing scheme based on Southern blot hybridization, using cloned replication regions (replicons) as probes (26). This approach successfully provided classification for both conjugative and nonconjugative plasmids, but the low specificity of the hybridization method underestimated plasmid diversity because of the cross-hybridization reaction among highly related replicons (repI, repB/O, repFII, repFIC). Since 2005, a PCRbased replicon typing (PBRT) scheme has been available, targeting the replicons of the major plasmid families occurring in Enterobacteriaceae (HI2, HI1, I1, X, L/M, N, FIA, FIB, FIC, W, Y, P, A/C, T, K, B/O) and also including PCR assays (FrepB and FIIAs PCRs), detecting the FII, FIII, FIV, and FIV variants and the FII replicon of the Salmonella virulence plasmids, respectively (12). However, the PBRT scheme still has several limitations, since the classification is currently based on plasmids belonging to the classic Inc groups and can fail to identify divergent or novel replicons. The most accurate method to characterize a plasmid is based on the determination of the full-length DNA sequence, and to date, more than 800 plasmids from Gammaproteobacteria have been fully sequenced (http://www.ncbi.nlm.nih.gov/genome/), contributing to the identification of novel plasmid families. Furthermore, more than 1,000 resistance plasmids have been typed and assigned to specific plasmid families by PBRT and hybridization/ conjugation methods. This review aims to provide an overview of the major plasmid families that are currently emerging in multidrug-resistant Enterobacteriaceae strains isolated worldwide among those conferring resistance to clinically relevant antibiotics, such as extended-spectrum cephalosporins, fluoroquinolones, and aminoglycosides (Table 1).