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Diversity and Global Distribution of IncL/M Plasmids Enabling Horizontal Dissemination of β-Lactam Resistance Genes among the Enterobacteriaceae

Antibiotic resistance determinants are frequently associated with plasmids and other mobile genetic elements, which simplifies their horizontal transmission. Several groups of plasmids (including replicons of the IncL/M incompatibility group) were found to play an important role in the dissemination of resistance genes encoding β-lactamases. The IncL/M plasmids are large, broad host range, and self-transmissible replicons. We have identified and characterized two novel members of this group: pARM26 (isolated from bacteria inhabiting activated sludge from a wastewater treatment plant) and pIGT15 (originating from a clinical strain of Escherichia coli). This instigated a detailed comparative analysis of all available sequences of IncL/M plasmids encoding β-lactamases. The core genome of these plasmids is comprised of 20 genes with conserved synteny. Phylogenetic analyses of these core genes allowed clustering of the plasmids into four separate groups, which reflect their antibiotic resistance profiles. Examination of the biogeography of the IncL/M plasmids revealed that they are most frequently found in bacteria of the family Enterobacteriaceae originating from the Mediterranean region and Western Europe and that they are able to persist in various ecological niches even in the absence of direct antibiotic selection pressure.


Introduction
The discovery of antibiotics initiated a new era in medicine. However, in recent years, the rapidly growing antibiotic resistance of pathogenic bacterial strains has greatly hindered the fight against infectious diseases. This problem is particularly serious in relation to the use of -lactam antibiotics due to the profusion and diversity of -lactamases. The production of these enzymes by Gram-negative bacteria is the most important factor undermining the efficacy of treatment using this group of antibiotics [1].
Two major schemes are used to classify -lactamases: molecular discrimination (proposed by Ambler) based on 2 BioMed Research International proven to be inaccurate as novel enzymes with modified activity profiles are discovered following the introduction of newly developed drugs into clinical practice. For example, a single amino acid alteration in the narrow-spectrumlactamase SHV-1 may lead to the emergence of enzymes that can hydrolyze extended-spectrum cephalosporins or even carbapenems [4].
The dissemination of -lactamase genes and other antibiotic resistance determinants is a consequence of inter-and intraspecies DNA exchange, occurring mainly due to the transfer of bacterial plasmids. Plasmids are extrachromosomal DNA molecules capable of autonomous replication, which are the major players in horizontal gene transfer and are responsible for shaping bacterial resistomes. A harmless environmental strain may be transformed into a pathogen by the acquisition of a multiresistance plasmid [5].
Plasmids promote the horizontal transfer of antibiotic resistance genes among bacteria belonging to various species or genera. The spread of these genes depends on the host range and conjugative properties of plasmids. Thus, conjugative, broad host range replicons have the greatest impact on overall antibiotic resistance transmission [6,7].
Amongst broad host range conjugative plasmids, special attention has been paid to IncL/M replicons, one of the six major resistance plasmid groups identified in clinically relevant Enterobacteriaceae [8,9]. Plasmids representing this incompatibility group have been identified as carriers of various -lactam resistance genes encoding extendedspectrum -lactamases (ESBL), classes A, B, and D carbapenemases, and AmpC -lactamases [10][11][12][13][14][15][16][17][18][19][20][21]. Plasmid pEL60 of Erwinia amylovora was recognized as an archetype for the IncL/M group. It lacks mobile elements and drug resistance genes, typical for other multiresistant IncL/M plasmids [22]. However, a detailed genomic analysis revealed the presence of backbone genes typical for all IncL/M replicons, including replication and stabilization modules, a conjugative transfer system and a mucAB-like mutagenic DNA repair system, which is closely related to the rulAB operon conferring tolerance to UV radiation [22].
This study comprises an analysis of the complete nucleotide sequences of two SHV-5 -lactamase-encoding plasmids (pARM26 and pIGT15) isolated from environmental and clinical samples, plus a detailed meta-analysis of available sequence data describing IncL/M replicons carrying -lactamase genes.

Bacterial Strains and Culture
Conditions. Escherichia coli strains DH5 [23] and CZD1527 [24] were grown in lysogeny broth (LB) medium [25] at 30 ∘ C or 37 ∘ C. Plates of solidified LB medium were prepared by the addition of 1.5% agar. Where necessary, the medium was supplemented with ampicillin (100 g/mL) and/or tetracycline (20 g/mL).

DNA Manipulations and the Transformation of Bac-
terial Cells with Plasmids. The isolation of plasmids and common DNA manipulation methods were carried out as described previously [25]. PCR was performed in a Mastercycler (Eppendorf) using Taq DNA polymerase (Qiagen; with the supplied buffer), dNTP mixture, and appropriate primer pairs. Transformation of E. coli strains was performed according to the method of Kushner [28].

2.5.
Bioinformatics. The plasmid nucleotide sequences were analyzed using Clone Manager (Sci-Ed8) and Artemis [29]. Similarity searches were performed using the BLAST programs [30]. Identified insertion sequences were compared with the ISfinder database [31]. For comparative analyses of plasmid sequences, the Easyfig program was used [32]. Phylogenetic analysis of the IncL/M plasmids was performed using the EDGAR tool [33]. To construct the phylogenetic tree, 20 core genes from the 20 IncL/M plasmid genomes were compared. Multiple alignments were created using MUSCLE software [34] and the highly variable regions of these alignments were removed using Gblocks [35]. The remaining sections of the alignments were concatenated into one multiple alignment and used in the construction of a phylogenetic tree with PHYLIP software [36]. The tree was visualized using TreeView version 1.6.6. [37]. Singletons and the core genes, initially defined using the EDGAR tool, were manually verified with BLASTn.  Comparative analyses of the plasmid genomes revealed high levels of synteny and sequence identity. Homologs of all the pIGT15 genes are also present in the pARM26 genome and the aligned nucleotide sequences show 99% identity. Plasmid pARM26 is approximately 12 kb larger than pIGT15 because it possesses two additional DNA regions named region I and region II ( Figure 1). Region I (nucleotide position 18,704-20,614) contains genes encoding (i) a conserved membrane protein (pIGT15 carries a truncated homologous gene encoding only the N-terminal part of this protein), (ii) a putative L-fuculose-phosphate aldolase (EC 4.1.2.17; which can also function as a D-ribulosephosphate aldolase) involved in the utilization of L-fucose and D-arabinose [38], and (iii) a hydroxypyruvate isomerase (EC 5.3.1.22, which catalyzes the reversible isomerization of hydroxypyruvate and tartronate semialdehyde [39]). The region II (nucleotide position 53,618-63,898) carries 17 genes encoding (i) a DNA repair protein RadC, (ii) a HTH XRE transcriptional regulator, (iii) gene expression modulator RmoA, (iv) antirestriction protein KlcA, (v) a mobilization protein C, and (vi) 12 hypothetical proteins of unknown function.

Results and Discussion
Comparison with the GenBank database revealed that pARM26 and pIGT15 are most closely related to plasmid pACM1 of Klebsiella oxytoca [10]. As shown in Figure 1, the three plasmids share gene synteny and show high levels of sequence conservation. Plasmid pACM1, the largest of the replicons, has an additional unique DNA region containing a class I integron carrying two antibiotic resistance gene cassettes ( Figure 1). Each of the plasmids also contains several transposable elements (TEs), including (i) the insertion sequences IS26 (2 copies; some in different genomic locations) and IS6100 (1 copy), both classified within the IS6 family [31] and (ii) a large Tn1721-like noncomposite Tn3 family transposon (of 28,815 bp in pARM26, 26,985 bp in pIGT15, and 31,842 bp in pACM1), which is absent in other members of the IncL/M group. These three closely related plasmids are self-transmissible replicons conferring resistance to multiple antibiotics, including aminoglycosides (aacA4, aacC1, and aadA1), trimethoprim (dfrA1, only in pACM1), sulfonamides (sul1), and tetracycline (tetA). They also encode an extendedspectrum -lactamase (ESBL) belonging to the SHV family, SHV-5 ( Figure 1). Interestingly, all of the resistance determinants are embedded within the aforementioned Tn3 family transposon ( Figure 1).
It is important to mention that the bacterial host strains carrying these plasmids originated from different environments (i.e., nosocomial and nonnosocomial) and were isolated at different times. The plasmid pACM1 was isolated in 1993 from a cephalosporin-resistant K. oxytoca strain from the USA [40], pIGT15 in 2005 from a Polish clinical strain of E. coli, and pARM26 in 2011 from bacteria inhabiting a wastewater treatment plant, also in Poland. Comparative genomic analyses of other IncL/M replicons may shed light on the phylogeny and evolution of this group of antibiotic  (Table 1).
With the addition of the two replicons described in this study (pARM26 and pIGT15) the sequences of 20 plasmids from this group were available for comparative genomic analyses. Comparative analyses revealed that the pan-genome of the IncL/M plasmids consists of 248 genes. This is a relatively small number considering the fact that each of these large plasmids (of 60-90 kb in size; Table 1) carries about 100 genes. This finding indicates that the IncL/M plasmids are highly conserved, sharing the majority of their genes. It is also in accordance with the small number of unique genes (56) identified in the plasmid genomes. Only half of the analyzed IncL/M plasmids contain singletons ( Table 2) and their number ranges from 0 to 15 per replicon. The highest number of singleton genes was found in plasmid R830b of Serratia marcescens, which does not carry any AR determinants. Several of these genes may play an important role in adaptation of the host strain to changing environmental conditions. Some appear to confer resistance to toxic heavy metals (mercury, cobalt, zinc, and cadmium), while others are probably engaged in formaldehyde detoxification (Table 2). Heavy metal resistance genes (an arsenic resistance gene cluster) were also identified in pKOI-34, another member of the IncL/M group (   (Table 2). This finding is in agreement with previous observations suggesting that the majority of AR genes of the IncL/M plasmids have been acquired within TEs (or mobile integron gene cassettes) inserted at "hotspot" sites in the highly conserved plasmid backbone [10].
The core genome of the IncL/M plasmids consists of 20 genes, conserved in all the analyzed replicons. Functional classification of these genes revealed that they encode proteins involved in plasmid replication (repA, repC), stable plasmid maintenance (resD, parA), conjugal transfer  In an effort to understand the evolution of these plasmids, a phylogenetic analysis was performed based on the amino acid sequences of the proteins encoded by the core genes ( Figure 2). Within the constructed phylogenetic tree, four main clusters are evident, grouping closely related replicons. The first cluster (cluster I) is comprised of the plasmids pARM26 and pIGT15, identified in this work, and also the closely related pACM1. All three are multiresistance replicons carrying 7 (or 8 in the case of pACM1 [10]) AR genes, including a tetA gene conferring resistance to tetracycline, which is not present in other IncL/M plasmids (Figure 2). The second cluster (cluster II) contains two multiresistance replicons (pFOX-7a, pNE1280) [15,20] and plasmid pEL60 of the plant pathogen E. amylovora, which does not contain any AR genes [22] (Figure 2). The third and largest cluster (cluster III) is divided into two subclusters. The IIIA branch groups closely related plasmids pKPN-068, pENT-e56, and pENT-d0d, carrying five common, identical AR genes ( TEM-1 , SHV-12 , aadB, qnrB19, and cat) [26], as well as the R830b replicon (containing no AR genes), which forms an outgroup within this subcluster (Figure 2). The IIIB branch gathers four similar plasmids (pOXA-48a, pJEG011, pKPoxa-48N1, and pE71T) with an embedded OXA-48 gene [13,16,17,21]. Interestingly, within this subcluster there is only one multiresistance plasmid, pJEG011 [16] (Figure 2). The fourth cluster (cluster IV) is also divided into two smaller subclusters: IVA, consisting of two plasmids, pNDM-HK and pNDM-OM, with a high level of genetic synteny [18,19], and IVB, grouping closely related plasmids pCTX-M3, pCTX-M360, and pEl1573 [11,12,14] (Figure 2).
The results of this phylogenetic analysis confirmed previous assumptions that there were probably several lineages of the IncL/M plasmids before they began acquiring diverse exogenous segments of DNA [10]. A more detailed comparative analysis showed that the insertions occurred within several "hotspots," which are usually located between conserved core genes in the plasmid backbone. Insertions at other locations were observed in only a few cases, for example, in the plasmids pOXA-48a, pJEG011, pE71T, and pKPoxa-48N1 (all grouped in the subcluster IIIB), in which the acquired DNA interrupted the tir gene encoding a transfer inhibition protein.

IncL/M Plasmids: A Reservoir of -Lactamase Genes.
Among the 20 completely sequenced IncL/M plasmids, 18 encode at least one -lactam resistance gene. In total, these plasmids carry 28 genes encoding 8 families of -lactam hydrolyzing enzymes, representing all of the classes distinguished by Ambler: TEM, CTX-M, SHV, and KPC of Ambler class A; IMP and NDM of class B; FOX of class C; and OXA of class D [41,42].
The most common is the TEM-1 gene (encoding the TEM-1 type enzyme), which is present in 9 plasmids: (i-iv) pFOX-7a, pKPN-068, pENT-e56, and pENT-d0d, representing clusters II and III in the phylogenetic tree (Figure 2), and (v-ix) pNDM-OM, pNDM-HK, pCTX-M3, pCTX-M360, and pEl1573, all belonging to cluster IV ( Figure 2). Interestingly, all of the aforementioned plasmids also encode another -lactamase inactivating a wider range of -lactams. The presence of this second -lactamase gene reflects the relatively narrow substrate specificity of the TEM-1 type enzyme, which is capable of hydrolyzing only penicillins and narrow-spectrum cephalosporins (such as cephalothin or cefazolin), but not more recently developed highergeneration cephalosporins with an oxyimino side chain (oxyimino-cephalosporins) [2]. Thus, the resistance conferred by the TEM-1 enzyme (probably the most common among enteric bacteria [43]) is in fact currently ineffective and has been "bolstered" by the acquisition of other genes encoding -lactamases with a broader specificity in the course of IncL/M plasmid evolution.
The IncL/M plasmids also encode enzymes representing the CTX-M family of ESBLs. These enzymes are less closely related to TEM or SHV -lactamases but show high similarity to the -lactamases of various Kluyvera species [46]. Three of the IncL/M plasmids encode the CTX-M family -lactamases: (i-ii) pCTX-M3 and pCTX-M360 ( CTX-M3 ) and (iii) pJEG011 ( CTX-M14 ) ( Figure 2).
Another class of -lactamases encoded by the IncL/M plasmids is represented by KPC-4, a carbapenemase of the KPC family. The gene encoding this enzyme is located within the transposon Tn4401f embedded in the plasmid pNE1280 [20].
An Ambler class C -lactamase (AmpC-type, CBL) is encoded by only one IncL/M plasmid, pFOX-7a, which was isolated from strains of Klebsiella pneumoniae associated with an outbreak in a neonatal intensive care unit in central Italy [15]. The FOX-7 enzyme can hydrolyze extended-spectrum cephalosporins (e.g., cephamycins) and is resistant to various combinations of -lactamase inhibitors [15]. The  gene is unique in the current pan-genome of the IncL/M plasmids ( Figure 2, Table 2).
Epidemiological surveys have revealed that IncL/M plasmids play an important role in the global dissemination of -lactam resistance genes (e.g., [49]). These replicons are not simply carriers of these resistance genes; they may also be considered "donors" of bla genes to other replicons coresiding in a particular host.
Close inspection of the IncL/M plasmid genomes revealed that the majority of their -lactamase genes are associated with transposable elements (e.g., (i) TEM-1 is present within Tn2 of pCTX-M360, pCTX-M3, pNDM-HK, pNDM-OM, and pEl1573, (ii) CTX-M3 was mobilized by ISEcp1 into pCTX-M3 and pCTX-M360, and (iii) OXA-48 occurs in Tn1999 of pOXA-48a) or they persist in the form of integron gene cassettes ( IMP-4 of pEl1573 and IMP-34 of pKOI-34). This localization significantly enhances the "mobility" of the resistance genes, which can be readily transferred from the IncL/M plasmids into bacterial chromosomes, as shown for the OXA-48 gene associated with the IS1R-based composite transposon Tn6237 [50]. However, an even greater epidemiological threat is posed by the transfer of bla genes into other plasmids coresiding in a bacterial cell. This may result in the generation of novel resistance plasmids with a host range that is different from that of the parental IncL/M plasmids. Therefore, plasmidmediated gene transfer can play a fundamental role in the crossing of taxonomic boundaries and may significantly influence the prevalence of -lactam resistance.
It is notable that 13 of the analyzed IncL/M plasmids also contain other AR genes, which confer resistance to antibiotics representing 8 drug classes: aminoglycosides, macrolides, quinolones, trimethoprim, sulfonamides, bleomycin, chloramphenicol, and tetracycline ( Figure 2). Interestingly, this phenomenon of multiresistance has also been observed in several other types of plasmids encoding extended-spectrum -lactamases (e.g., [51]). In consequence, the choice of appropriate antibiotics to eliminate -lactamase-producing pathogenic bacterial strains is becoming more and more limited [52].

Biogeography of the IncL/M Plasmids.
Although IncL/M broad host range, self-transferable plasmids play an important role in the horizontal transmission of multiresistance phenotypes among Enterobacteriaceae, still very little is known about the diversity and distribution of these replicons in different environments. As mentioned above, the genetic structure of only 20 replicons of this group has been revealed to date. However, database searches (performed at the NCBI website) produced some information about four other members of the IncL/M group that have been  partially sequenced and subjected to molecular analyses. These are R446b of Morganella morganii [53], R471a of Serratia marcescens [54], pMU407.1 of Klebsiella pneumoniae [55], and p61/9 of Salmonella enterica serovar Typhimurium [56]. Moreover, in depth analysis of sequence accessions in the NCBI database yielded further information concerning IncL/M plasmids (usually identified by PCR-based replicon typing) in samples originating from various environments. Most of the detected IncL/M plasmids were carried by pathogenic strains of Enterobacteriaceae isolated from hospitalized patients. These strains originated from 27 countries: Albania [57], Algeria [58], Argentina [59], Australia [60], Belgium [61], Brazil [62], Bulgaria [63], Canada [64], China [65], Croatia [66], Denmark [67], Egypt [21], France [68], Greece [69], Kenya [70], Korea [71], Lebanon [21], Libya [72], Morocco [21], Philippines [69], Portugal [73], Russia [74], Spain [75], Switzerland [76], Tunisia [77], Turkey [21], and USA [78]. IncL/M plasmids were also found in enterobacteria isolated from companion animals in Italy [79] and from an urban wastewater treatment plant in Algeria [80]. Thus, IncL/M plasmids have so far been identified in 31 countries on 6 continents, which points to their global distribution ( Figure 3). Notably, the majority of strains carrying the IncL/M plasmids were identified in countries located near the Mediterranean Sea and in Western Europe (Figure 3).
Majority of the IncL/M plasmids were isolated from pathogenic strains of Enterobacteriaceae in the course of epidemiological studies. The unique genes found within these plasmids are usually involved in antibiotic resistance ( Table 2) and may be linked with the specific medical treatment in the particular health care unit. This exemplifies the role of mobile genetic elements in transfer of genes, enabling bacteria to improve their fitness under local stress conditions [81]. Such phenomenon is common for environmental bacterial strains, which are able to survive under various harsh conditions (e.g., [82][83][84]). This study illustrates that the IncL/M plasmids are not exclusively associated with pathogenic enterobacteria but are also found in nonnosocomial environments, including communal wastes. Wide dissemination of these multiresistance plasmids and their possible transfer into pathogenic bacteria make them an important and considerable epidemiological threat.

Conclusions
The plasmids of the IncL/M incompatibility group are broad host range, multiresistance, self-transmissible replicons, which actively participate in the horizontal transfer of antibiotic resistance genes. These replicons have been associated with several outbreaks of disease caused by pathogenic strains of Enterobacteriaceae all over the world. They are also involved in the dissemination of -lactam resistance, since they carry genes encoding enzymes representing all four Ambler classes of -lactamases: Detailed analysis of the IncL/M plasmid genomes revealed that they have highly conserved backbones and share significant gene synteny. Moreover, phylogenetic analysis showed that these replicons may be grouped into four clusters, which also reflect the distribution of the antibiotic resistance determinants.
This study is the first to present available data indicating the global dissemination of bacteria carrying IncL/M plasmids. These multiresistance replicons not only are maintained by pathogenic strains of Enterobacteriaceae but also have been detected in bacteria inhabiting different nonnosocomial environments. A good example is the -lactamaseencoding plasmid pARM26 characterized in this study, which was isolated from a wastewater treatment plant in Poland. Its identification strongly suggests that IncL/M plasmids have the ability to persist in various ecological niches even in the absence of direct antibiotic selection. Therefore, these plasmids constitute a rich reservoir of AR genes that may be disseminated among diverse bacteria (including pathogenic strains) in the natural environment.