The Plasmidome of Firmicutes: Impact on the Emergence and the Spread of Resistance to Antimicrobials
- Authors: Val Fernández Lanza1, Ana P. Tedim4, José Luís Martínez7, Fernando Baquero9, Teresa M. Coque12
- Editors: Marcelo Tolmasky15, Juan Carlos Alonso16
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VIEW AFFILIATIONS HIDE AFFILIATIONSAffiliations: 1: Servicio de Microbiología, Hospital Universitario Ramón y Cajal, Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Madrid, Spain; 2: Unidad de Resistencia a Antibióticos y Virulencia Bacteriana (HRYC-CSIC), Madrid, Spain; 3: Centro de Investigación en Red en Epidemiología y Salud Pública (CIBER-ESP), Spain; 4: Servicio de Microbiología, Hospital Universitario Ramón y Cajal, Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Madrid, Spain; 5: Unidad de Resistencia a Antibióticos y Virulencia Bacteriana (HRYC-CSIC), Madrid, Spain; 6: Centro de Investigación en Red en Epidemiología y Salud Pública (CIBER-ESP), Spain; 7: Unidad de Resistencia a Antibióticos y Virulencia Bacteriana (HRYC-CSIC), Madrid, Spain; 8: Centro Nacional de Biotecnología (CNB-CSIC), Madrid, Spain; 9: Servicio de Microbiología, Hospital Universitario Ramón y Cajal, Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Madrid, Spain; 10: Unidad de Resistencia a Antibióticos y Virulencia Bacteriana (HRYC-CSIC), Madrid, Spain; 11: Centro de Investigación en Red en Epidemiología y Salud Pública (CIBER-ESP), Spain; 12: Servicio de Microbiología, Hospital Universitario Ramón y Cajal, Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Madrid, Spain; 13: Unidad de Resistencia a Antibióticos y Virulencia Bacteriana (HRYC-CSIC), Madrid, Spain; 14: Centro de Investigación en Red en Epidemiología y Salud Pública (CIBER-ESP), Spain; 15: California State University, Fullerton, CA; 16: Centro Nacional de Biotecnología, Cantoblanco, Madrid, Spain
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Received 14 January 2015 Accepted 15 January 2015 Published 03 April 2015
- Correspondence: Teresa M. Coque, [email protected]; [email protected]

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Abstract:
The phylum Firmicutes is one of the most abundant groups of prokaryotes in the microbiota of humans and animals and includes genera of outstanding relevance in biomedicine, health care, and industry. Antimicrobial drug resistance is now considered a global health security challenge of the 21st century, and this heterogeneous group of microorganisms represents a significant part of this public health issue.
The presence of the same resistant genes in unrelated bacterial genera indicates a complex history of genetic interactions. Plasmids have largely contributed to the spread of resistance genes among Staphylococcus, Enterococcus, and Streptococcus species, also influencing the selection and ecological variation of specific populations. However, this information is fragmented and often omits species outside these genera. To date, the antimicrobial resistance problem has been analyzed under a “single centric” perspective (“gene tracking” or “vehicle centric” in “single host-single pathogen” systems) that has greatly delayed the understanding of gene and plasmid dynamics and their role in the evolution of bacterial communities.
This work analyzes the dynamics of antimicrobial resistance genes using gene exchange networks; the role of plasmids in the emergence, dissemination, and maintenance of genes encoding resistance to antimicrobials (antibiotics, heavy metals, and biocides); and their influence on the genomic diversity of the main Gram-positive opportunistic pathogens under the light of evolutionary ecology. A revision of the approaches to categorize plasmids in this group of microorganisms is given using the 1,326 fully sequenced plasmids of Gram-positive bacteria available in the GenBank database at the time the article was written.
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Citation: Lanza V, Tedim A, Martínez J, Baquero F, Coque T. 2015. The Plasmidome of Firmicutes: Impact on the Emergence and the Spread of Resistance to Antimicrobials. Microbiol Spectrum 3(2):PLAS-0039-2014. doi:10.1128/microbiolspec.PLAS-0039-2014.




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Abstract:
The phylum Firmicutes is one of the most abundant groups of prokaryotes in the microbiota of humans and animals and includes genera of outstanding relevance in biomedicine, health care, and industry. Antimicrobial drug resistance is now considered a global health security challenge of the 21st century, and this heterogeneous group of microorganisms represents a significant part of this public health issue.
The presence of the same resistant genes in unrelated bacterial genera indicates a complex history of genetic interactions. Plasmids have largely contributed to the spread of resistance genes among Staphylococcus, Enterococcus, and Streptococcus species, also influencing the selection and ecological variation of specific populations. However, this information is fragmented and often omits species outside these genera. To date, the antimicrobial resistance problem has been analyzed under a “single centric” perspective (“gene tracking” or “vehicle centric” in “single host-single pathogen” systems) that has greatly delayed the understanding of gene and plasmid dynamics and their role in the evolution of bacterial communities.
This work analyzes the dynamics of antimicrobial resistance genes using gene exchange networks; the role of plasmids in the emergence, dissemination, and maintenance of genes encoding resistance to antimicrobials (antibiotics, heavy metals, and biocides); and their influence on the genomic diversity of the main Gram-positive opportunistic pathogens under the light of evolutionary ecology. A revision of the approaches to categorize plasmids in this group of microorganisms is given using the 1,326 fully sequenced plasmids of Gram-positive bacteria available in the GenBank database at the time the article was written.

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Figures

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FIGURE 1
Protein content network (PCN) of AbR proteins found in plasmids and chromosomes of Firmicutes and Actinobacteria. To determine the AbR protein catalog of Gram-positive strains (chromosomes and plasmids), a Blastp search was performed of all their proteomes against the ARG-ANNOT database (http://en.mediterranee-infection.com/article.php?laref=283&titre=arg-annot) using a cut-off of 1e-30 and 85% of identity. The presence of the Gram-positive AbR proteins identified above in all bacterial species (only complete sequences, not partial) was determined using a similar Blast search (blastp, 1e-30 E-value and 85% identity) against the NCBI GenBank database. The nodes correspond to bacterial species (circular nodes; each color indicates one genus) and AbR proteins (square nodes). Nodes were connected by an edge when a positive hit between AbR proteins and one or more strains of a given species were identified. Edges further indicate the location of the AbR genes associated with each AbR protein of the Gram-positive catalog. Solid lines represent chromosomal location, and dotted lines represent plasmid location. When an AbR gene was located in both chromosomes and plasmids, both lines were plotted.

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FIGURE 2
PCN of metal-biocide (MetR/BcR) proteins found in plasmids and chromosomes of Firmicutes and Actinobacteria. To determine the MetR/BcR protein catalog of Gram-positive strains (chromosomes and plasmids), a Blastp search was performed of all their proteomes against the BacMet database (http://bacmet.biomedicine.gu.se/) using a cut-off of 1e-30 and 85% of identity. The presence of the Gram-positive MetR/BcR proteins identified above in all bacterial species (only complete sequences, not partial) was determined using a similar Blastp search (blastp, 1e-30 evalue and 85% identity) against the NCBI GenBank database. The nodes correspond to bacterial species (circular nodes) and MetR/BcR proteins (triangular nodes). Nodes were connected by an edge when a positive hit between MetR/BcR proteins on one or more strains of a given species was identified. Edges further indicate the location of the MetR/BcR genes associated with each MetR/BcR protein of the Gram-positive catalog. Solid lines represent chromosomal location, and dotted lines represent plasmid location. When a MetR/BcR gene was located in both chromosomes and plasmids, both lines were plotted.

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FIGURE 3
Plasmid homology network. The genomic homology network was performed using “All-versus-All” genomic Megablast ( 238 ) of 1,326 fully sequenced plasmids from low G+C bacterial species (Firmicutes and Actinobacteria phyla) available at public gene databases. The nodes correspond to bacterial plasmids (circular nodes; different colors representing different genera). Two nodes are connected by an edge if they share homologous DNA.

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FIGURE 4
PCN of AbR and MetR/BcR proteins located on plasmids of Firmicutes and Actinobacteria. PCN of AbR and MetR proteins found in Gram-positive plasmids. We formed the PCN by representing plasmids as circular nodes, AbR as square nodes, and MetR/BcR as triangular nodes, connecting two nodes (plasmid and AbR or MetR/BcR) if one plasmid has this AbR or MetR/BcR. The presence of the MetR/BcR gene was determined by blasp of all plasmid proteins against the BacMet database. The presence of the AbR gene was determined by blasp of all plasmid proteins against the ARG-ANNOT database. The colors for the genus are the same as those in Fig. 3 .

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FIGURE 5
Plasmids from Staphylococcus spp. The presence of an orange border in the RIP family indicates that the corresponding RIP is truncated. aPriCT_1; *One of these plasmids (GenBank accession number NC_013381) has a truncated rep_pKH21 (rep_1) gene, and no other known RIPs were identified. **Two of these plasmids (GenBank accession number NC_016054 and NC_019144) appear to have two copies of the MOBV gene. +One of these plasmids (GenBank accession number NC_008354) has two copies of the lnuA gene. §One plasmid (GenBank accession number NC_001393) has a truncated copy of the tetK gene. ¥One plasmid (GenBank accession number NC_010419) has a truncated copy of the blaZ gene. £The plasmid (GenBank accession number NC_005076) appears to have two copies of the MOBV gene. $One plasmid (GenBank accession number NC_018959) has a truncated copy of the blaZ gene. & Two plasmids (GenBank accession numbers NC_007931 and NC_016942) have two copies of the arsB and arsC genes. ¢This plasmid (GenBank accession number NC_013320) appears to have two copies of the MOBV gene. ΠThis plasmid (GenBank accession number NC_005004) has a truncated copy of the blaZ gene. ϙThree plasmids (GenBank accession numbers NC_013321, NC_019007, and NC_018976) have a truncated copy of the blaZ gene. ϪEleven of these plasmids have a truncated copy of the cadD gene (GenBank accession numbers NC_020531, NC_020538, NC_013337, NC_020534, NC_020565, NC_020567, NC_020530, NC_020539, NC_017352, NC_013323, and NC_022610). ϬOne plasmid (GenBank accession number NC_013334) has a truncated copy of the cadX gene. ϻThis plasmid (GenBank accession number NC_020237) appears to have two copies of the MOBV gene. ЂThis plasmid (GenBank accession number NC_022598) appears to have two copies of the MOBV gene. Abbreviations: MRIP, Multi-RIP; S, Staphylococcus spp; Sar, Staphylococcus arlettae; Sa, S. aureus; Sc, Staphylococcus chromogenes; Se, Staphylococcus epidermidis; Sha, Shaemolyticus haemolyticus; Shy, Staphylococcus hyicus; Sle, Staphylococcus lentus; Slu, Staphylococcus lugdunensis; Sp, Staphylococcus pasteuri; Ssa, Staphylococcus saprophyticus; Ssc, Staphylococcus sciuri; Ssi, Staphylococcus simulans; Sw, Staphylococcus warneri.

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FIGURE 6
Hierarchical clustering dendrogram of plasmids from enterococci. The matrix distance used for building the UPGMA dendrogram is based on the Raup-Crick distance of the orthologous protein profile of each plasmid. For each plasmid, a presence/absence protein profile was made using cut-off values of 80% identity and 80% coverage. Protein clustering was made by using CD-HIT ( 239 ). Different background colors are used to emphasize branches of related plasmids and are the same as those defined in Fig. 5 . Names to the left of the dendrogram indicate the RIP family. Background colors were used to point out plasmid groups frequently involved in mobility of AbR genes and E. faecalis pheromone-responsive plasmids. Circles indicate RIPs identified in each plasmid according to data shown in Fig. 7 .

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FIGURE 7
Plasmids from Enterococcus spp. The presence of an orange border in the RIP family indicates that the corresponding RIP is truncated. aRep_2; *One of these plasmids (GenBank accession number NC_015849) has a truncated rep_AUS0004_p2 (Rep_1) gene, and no other known replication initiator proteins were found. **This plasmid (GenBank accession number NC_017962) has two copies of Tn401; in one of them the add(6) gene is not truncated; this plasmid also appears to have two copies of the MOBP1 gene. +These two plasmids (GenBank accession numbers NC_008768 and NC_008821) have a truncated copy of the str gene. Abbreviations: MRIP, multi-RIP; Efm, E. faecium; Efc, E. faecalis; Emu, E. mundtii; Edu, E. durans; Ehi, E. hirae.

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FIGURE 8
Plasmids from Streptococcus spp. aRep_trans; §This plasmid (GenBank accession number NC_015219) has two similar replication genes belonging to the Rep_3 family. ¥This plasmid (GenBank accession number NC_006979) has two similar replication genes belonging to the PriCT_1 family. Abbreviations: MRIP, Multi-RIP; Sag, S. agalactiae; Sdy, Streptococcus dysgalactiae; Sga, Streptococcus gallolyticus; Siu, Streptococcus infantarius; Sin, Streptococcus infantis; Sma, Streptococcus macedonicus; Smu, Streptococcus mutans; Spa, Streptococcus parasanguinis; Spn, S. pneumoniae; Sps, Streptococcus pseudopneumoniae; Spy, S. pyogenes; Ssu, Streptococcus suis; Sth, Streptococcus thermophilus.

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FIGURE 9
Plasmids from Listeria spp. arep_3. bOne plasmid (GenBank NC_013767) has two copies of the mco gene, one of which is truncated. cOne plasmid (GenBank NC_018888) has a truncated copy of the mco gene. dThis plasmid (GenBank NC_022045) has two copies of the cadC-cadA operon. Abbreviations: MRIP, multi-RIP; Lm, L. monocytogenes; Lg, Listeria grayi; Li, Listeria innocua.

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FIGURE 10
Plasmids from LAB. aRep_1. bRep_trans. cPriCT_1. *This plasmid (GenBank accession number NC_010540) has two copies of the ermB gene. **One of these plasmids (GenBank accession number NC_010603) has a truncated copy of the arsC gene. §One of these plasmids (GenBank accession number NC_014133) appears to have three copies of the MOBV gene. ¥One of these plasmids (GenBank accession number NC_022123) appears to have two copies of the MOBP1 gene. Abbreviations: MRip, multi-RIP; Lb, Lactobacillus spp; Lca, Lactobacillus acidophilus; Lam, Lactobacillus amylovorus; Lbr, Lactobacillus brevis; Lbu, Lactobacillus bucheneri; Lca, Lactobacillus casei; Lfe Lactobacillus fermentum; Lpa, Lactobacillus paracasei; Lpl, Lactobacillus plantarum; Lre, Lactobacillus reuteri; Lsa, Lactobacillus sakei; Lga, Lactococcus garvieae; Lla, Lactococcus lactis; Lcm, Leuconostoc carnosum; Lci, Leuconostoc citreum; Lki, Leuconostoc kimchii; Lme, Leuconostoc mesenteroides.

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FIGURE 11
Similarity of rep-like sequences encoding RIPs of the Rep_1 family. A neighbor-joining tree of gene sequences coding for RIPs of the Rep_1 family was built using MEGA 6.06. A cut-off equal to or higher than 80% and a bootstrap analysis based on 1,000 permutations were applied to the analysis. Alignment of nucleotide sequences was performed using ClustalW2 (http://www.ebi.ac.uk/Tools/phylogeny/clustalw2_phylogeny/), and sequences showing an identity equal to or higher than 80% were clustered in groups that were highlighted by different backgrounds colors. Black dots indicate the RIP of the plasmid used for further comparison in Figs. 5 to 10 .

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FIGURE 12
Similarity of rep-like sequences encoding RIPs of the Rep_trans family. A neighbor-joining tree of gene sequences coding for RIPs of the Rep_trans family was built using MEGA 6.06. A cut-off equal to or higher than 80% and a bootstrap analysis based on 1,000 permutations were applied to the analysis. Alignment of nucleotide sequences was performed using ClustalW2 (http://www.ebi.ac.uk/Tools/phylogeny/clustalw2_phylogeny/), and sequences showing an identity equal to or higher than 80% were clustered in groups that were highlighted by different backgrounds colors. Black dots indicate the RIP of the plasmid used for further comparison in Figs. 5 to 10 . *Truncated gene. **Similar to E. faecalis ant6-Ia and aadE. Abbreviations: ND, not determined.

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FIGURE 13
Similarity of rep-like sequences encoding RIPs of the Rep_2 family. A neighbor-joining tree of gene sequences coding for RIPs of the Rep_2 family was built using MEGA 6.06. A cut-off equal to or higher than 80% and a bootstrap analysis based on 1,000 permutations were applied to the analysis. Alignment of nucleotide sequences was performed using ClustalW2 (http://www.ebi.ac.uk/Tools/phylogeny/clustalw2_phylogeny/), and sequences showing an identity equal to or higher than 80% were clustered in groups that were highlighted by different background colors. Black dots indicate the RIP of the plasmid used for further comparison in Figs. 5 to 10 .

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FIGURE 14
Similarity of rep-like sequences encoding RIPs of the Rep_L family. A neighbor-joining tree of gene sequences coding for RIPs of the Rep_L family was built using MEGA 6.06. A cut-off equal to or higher than 80% and a bootstrap analysis based on 1,000 permutations were applied to the analysis. Alignment of nucleotide sequences was performed using ClustalW2 (http://www.ebi.ac.uk/Tools/phylogeny/clustalw2_phylogeny/), and sequences showing an identity equal to or higher than 80% were clustered in groups that were highlighted by different background colors. Black dots indicate the RIP of the plasmid used for further comparison in Figs. 5 to 10 .

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FIGURE 15
Similarity of rep-like sequences encoding RIPs of the Rep_3 family. A neighbor-joining tree of gene sequences coding for RIPs of the Rep_3 family was built using MEGA 6.06. A cut-off equal to or higher than 80% and a bootstrap analysis based on 1,000 permutations were applied to the analysis. Alignment of nucleotide sequences was performed using ClustalW2 (http://www.ebi.ac.uk/Tools/phylogeny/clustalw2_phylogeny/), and sequences showing an identity equal to or higher than 80% were clustered in groups that were highlighted by different background colors. Black dots indicate the RIP of the plasmid used for further comparison in Figs. 5 to 10 . Abbreviations: ND, not determined.

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FIGURE 16
Similarity of rep-like sequences encoding RIPs with the PriCT_1 domain. A neighbor-joining tree of gene sequences coding for RIPs with PriCT_1 domains was built using MEGA 6.06. A cut-off equal to or higher than 80% and a bootstrap analysis based on 1,000 permutations were applied to the analysis. Alignment of nucleotide sequences was performed using ClustalW2 (http://www.ebi.ac.uk/Tools/phylogeny/clustalw2_phylogeny/), and sequences showing an identity equal to or higher than 80% were clustered in groups that were highlighted by different background colors. Black dots indicate the RIP of the plasmid used for further comparison in Figs. 5 to 10 . Abbreviations: ND, not determined.

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FIGURE 17
Similarity of rep-like sequences encoding RIPs of the RepA_N family. A neighbor-joining tree of gene sequences coding for replication initiator proteins of the RepA_N family was built using MEGA 6.06. A cut-off equal to or higher than 80% and a bootstrap analysis based on 1,000 permutations were applied to the analysis. Alignment of nucleotide sequences was performed using ClustalW2 (http://www.ebi.ac.uk/Tools/phylogeny/clustalw2_phylogeny/), and sequences showing an identity equal to or higher than 80% were clustered in groups that were highlighted by different background colors. Black dots indicate the RIP of the plasmid used for further comparison in Figs. 5 to 10 . *Truncated gene. +Similar to E. faecalis ant6-Ia and aadE. Abbreviations: ND, not determined.
Tables

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TABLE 1
Fully characterized plasmids from low G+C bacteria available in GenBank database (updated September 2014)
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