Computing Conservative Consensus Core- and Pan-Genomes

High stringency homologous gene clusters were computed with the GET_HOMOLOGUES (Contreras-Moreira and Vinuesa, 2013) software package by imposing a minimum of 90% query coverage on the all-against-all BLASTP results and performing a PFAM-domain scanning on each sequence to ensure that all homologous gene clusters contain the same domain composition and order. Clustering was performed with the BDBH (-e -D), COGtriangles (-G -t 0 -D) and OMCL (-M -t 0 -D) algorithms implemented in GET_HOMOLOGUES (Contreras-Moreira and Vinuesa, 2013) and the indicated parameters. A consensus core-genome was computed with the aid of the compare_clusters.pl script (-t 34) from the clusters generated by each of the tree algorithms, as detailed elsewhere (Vinuesa and Contreras-Moreira, 2015). Similarly, a consensus pan-genome was computed from the COGtriangles and OMCL clusters using the ‘-m -t 0’ parameters, to generate the pan-genome matrix reporting clusters of all sizes.

Estimating a Robust Maximum-Likelihood Core-Genome Phylogeny for the Genus Serratia

A core-genome phylogeny was estimated under the maximum-likelihood (ML) optimality criterion using the consensus core-genome clusters computed by GET_HOMOLOGUES (Contreras-Moreira and Vinuesa, 2013) as described in the previous section and passing them to the GET_PHYLOMARKERS (Vinuesa et al., 2018, in revision) software suite, which was run in default mode (-R 1 -t DNA). The latter is freely available on GitHub¹. Briefly, the GET_PHYLOMARKERS pipeline was used to select core-genome loci with optimal phylogenetic attributes, namely those passing the phi recombination test (Bruen et al., 2006), producing tree topologies and branch-lengths not significantly deviating (kdetrees test) from the expected distribution of these parameters under the multispecies coalescent (Weyenberg et al., 2014) and displaying average branch support values >0.7 (see Vinuesa et al., 2018 for the details). The clustal–omega (Sievers et al., 2011) codon alignments passing these filters were concatenated and a ML phylogeny estimated with IQ-TREE 1.6.1 (Nguyen et al., 2015) using the best fitting model and selecting the phylogeny with the highest likelihood score from those found among independent searches.

Computing Pairwise Core-Genome Average Nucleotide Identity Values From OMCL Clusters (cgANIb-OMCL)

Pairwise cgANIb values were computed from the BLASTN alignments identified by OMCL as belonging to the core-genome with the aid of the get_homologues.pl script, run with the ‘-A -a CDS’ parameters. The resulting cgANIb-OMCL matrix was then displayed as an ordered, bi-dimensional heatmap with the aid of the plot_matrix_heatmap.sh script, distributed with the GET_HOMOLOGUES package.

Phylogeny of Serratia spp. Based on Its Pan-Genome

A parsimony pan-genome phylogeny for the 32 Serratia genomes was estimated from the consensus pan-genome matrix of presence/absence data for homologous clusters created with the aid of the compare_clusters.pl script from the GET_HOMOLOGUES (Contreras-Moreira and Vinuesa, 2013) package, run with the -m -t 0 -T parameters, which calls PARS from the PHYLIP suite. The total number of resistance determinants and the number of acquired and intrinsic resistance genes, including the number of efflux pump genes identified in each genome, are indicated on the tree.

Identification of Antimicrobial Resistance Genes in the Serratia Genus

The whole genome for all strains was analyzed using BLASTN to identify ARGs (Camacho et al., 2009). Searches were also made against locally maintained versions of the CARD (a rigorously curated collection of known resistance determinants) (McArthur et al., 2013) and ResFinder (Zankari et al., 2012) databases. Mutations of genes associated with resistance were searched with BioEdit 7.2.5 (Hall, 1999) and MEGA7 softwares (Kumar et al., 2008). We aligned the 32 Serratia whole genomes using Mauve 2.4.0 (Darling et al., 2010) in order to enable identification of the chromosomal genes associated with intrinsic antibiotic resistance by sequence homology. Mutations of genes gyrA and gyrB (resistance to quinolones), murA (resistance to fosfomycin) and folP (resistance to sulfamethoxazole) were assessed for acquired resistance using BioEdit 7.2.5. In addition, each genome was screened for additional and strain-exclusive ARGs. In order to identify acquired genes on plasmids or integrons, we used BLASTN (Altschul et al., 1990), Mauve 2.4.0. and ISfinder (Siguier et al., 2006).

Phylogenetic Structure of Serratia spp. Pan-Genome and the ARGs Number

The parsimony pan-genome tree was derived from a consensus presence/absence matrix of homologous genes based on the clustering algorithms OMCL and COGtriangles. The Venn diagram shows the number of gene clusters of the 32 Serratia spp. pan-genome (12,347 clusters) (Supplementary Figure S3) that were used to generate the pan-genome matrix and the phylogeny.

The pan-genome tree shows five main groups (Figure ​Figure22). The results of the phylogenetic analysis placed all the S. marcescens strains in group A, together with the misclassified S. ureilytica, an environmental strain from a geothermal spring and S. nematodiphila DSM 21420, a nematode symbiont and an insect pathogen and 3 uncharacterized strains: Serratia sp. YD25, Serratia sp. FS14 and Serratia sp. SCB1. All these strains should be re-classified as S. marcescens, based on the evidence gained from the core-genome phylogeny (Figure ​Figure11) and the cgANIb data (Supplementary Figure S2). Most of the acquired ARGs and efflux pump genes are found in strains of this group. Group B consists of two sub-groups: one with the strains belonging to all S. plymuthica strains; and the other with the S. liquefaciens and S. proteamaculans strains, which are all environmental strains, with the exception of S. liquefaciens HUMV-21 and S. liquefaciens FDAARGOS 125, which are both nosocomial strains. Group C comprises only two strains, Serratia sp. FGI94, a fungus symbiont and S. rubidaea 1122 isolated from a patient in China. The former has a cgANIb value of 99.2 when compared to the latter, and hence strain FGI94 should be classified as S. rubidea. S. fonticola GS2 and S. fonticola DSM4576 are placed in group D, both environmental strains. Finally, group E, holds 3 distantly related strains: Chania multitudinisentens isolated from the soil of an ex-landfill site; Serratia sp. ATCC 39006 isolated from water; and the co-obligate aphid symbiont, S. symbiotica ‘Cinara Cedri’. The genomes of these 3 strains harbor the fewest ARGs on their chromosomes. Their cgANIb values are <85% with respect to the other species in the main Serratia spp. cluster, and therefore their classification as members of the former genus is questionable.

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FIGURE 2

Parsimony pan-genome tree for 32 Serratia genomes derived from presence/absence of homolog genes in a consensus pan-genome matrix computed by the COGtriangles y OrthoMCL clustering algorithms. This phylogeny was the most parsimonious tree found in a tree search made with PARS from the PHYLIP suite. The tree has a total length of 20,453 steps. Capital letters A-E indicate the phylogenetic groups.

Phylogenetically, S. marcescens SmUNAM836 and S. marcescens SM39 are closely related with both genomes sharing the same intrinsic ARGs and efflux pump genes (Iguchi et al., 2014) but different acquired ARGs.

Identification of Antimicrobial Resistance Genes in the 32 Serratia spp. Genomes

Using the CARD database, we looked for ARGs reported to be associated with enterobacteria and more specifically with Serratia spp., which were identified on the genomes of the Serratia spp. strains using BLASTN. We then aligned the 32 genomes using Mauve 2.4.0 and performed BLASTN to look for the percentage of identity and coverage between the different ARGs. The sequence alignments showed that those genes that appeared to be homologous in Mauve, shared more than 70% of identity between them when performing BLASTN; this, together with the use of global multiple sequence alignments, makes our identification of ARGs more robust (Pearson, 2013). Strain specific ARGs and efflux pump components were detected running a blast search against the CARD database using the following parameters: coverage = 95/90% and identity = 70/50%, respectively.

Frequency of ARGs According to Each Class of Antibiotic in the Serratia spp. Resistome

The general frequency of ARGs found in the 32 Serratia spp. genomes (efflux pumps not included in this analysis) according to the class of antibiotic was: 0.13% for macrolides; 0.54% for sulfonamides; 3.69% for chloramphenicol; 3.83% for phosphonic antimicrobials; 3.96% for quinolones; 4.1% for aminocoumarin antibiotics (novobiocin); 7.11% associated with aminoglycoside resistance; 20.38% for β-lactam, 56.22% for antimicrobial polypeptides (Figure ​Figure33). It is not surprising that the highest frequency of ARGs corresponded to the genes that confer resistance to polypeptide antibiotics since S. marcescens and the whole genus has a natural resistance to polymyxin B (Olaitan et al., 2014). The second highest frequency in our sample corresponded to β-lactam resistance genes. This fact was expected due to the high number of β-lactamases acquired through HGT in plasmids or transposons found in this genus. The importance of β-lactam resistance genes is reinforced with the fact that up to 1,000 genes related to β-lactam resistance have been described and classified in recent years, among them many new alleles and new genes found in remote geographical niches (Allen et al., 2009; Bush and Jacoby, 2010). In our analysis, the fact that the β-lactamases acquired on plasmids were found exclusively in nosocomial bacteria, except for bla_ACT-28, which was identified in an environmental strain associated with plants, is a noteworthy observation (Supplementary Table S3). HDT has been shown to play a major role in the transmission and evolution of β-lactam resistance genes in pathogenic and environmental enteric bacteria (Davies and Davies, 2010; Iredell et al., 2016).

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FIGURE 3

Frequency of ARGs according to the antibiotic class in Serratia spp. resistome.

Total Antibiotic Resistance Genes

A total of 123 different classes of genes associated with intrinsic and acquired antibiotic resistance were found among the 32 Serratia species (detailed information is shown in Supplementary Table S2). These ARGs include genes that encode modifying enzymes, antibiotic hydrolysis enzymes, efflux pumps, porines, regulatory proteins, genes with mutations that confer antibiotic resistance, and alleles of some genes and efflux pumps. From the 123 total resistance genes: 33 are intrinsic resistance genes; 16 are acquired resistance genes [13 on plasmids (Supplementary Table S3), 2 on transposons, and the gene gyrA with the Ser83 mutation that confers quinolone resistance] (Figure ​Figure44); and 74 are genes associated with efflux systems, from which 3 were localized on plasmids (Figure ​Figure55).

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FIGURE 4

Number of intrinsic and acquired ARGs identified in the 32 Serratia genomes (efflux pump genes not included). Intrinsic ARGs in orange color, acquired in red, and acquired by mutation in green.

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FIGURE 5

Box plot showing the number of genes associated with the five families of efflux pumps identified in the 32 Serratia genomes: multidrug and toxic-compound extrusion family (MATE), the small multidrug resistance family (SMR), the major facilitate superfamily (MFS), the resistance nodulation division family (RND), and the ATP binding cassette superfamily (ABC).

Intrinsic Resistance Genes

Intrinsic resistance comprises all of the inherent properties provided by the characteristics of a particular microorganism that limit the action of antimicrobials (Fernández and Hancock, 2012). Most of the genes related to antimicrobial resistance are encoded on the chromosome of the Serratia genus. In most genomes, we identified the Ambler class C β-lactamase gene ampC, which confers resistance to all penicillins, as well as to 3rd generation cephalosporins and aztreonam when overexpressed (Paterson and Bonomo, 2005), in addition, we found all its regulatory genes: ampD, ampE, ampG, and ampR (MacDougall, 2011). A class A β-lactamase gene bla_CTX-M and class C β-lactamase gene bla_ACT-29 were found only in the two S. fonticola genomes. Of the 14 ARGs associated with β-lactam resistance, 10 were found in S. marcescens CAV1492, a nosocomial strain (Supplementary Table S2).

Three aminoglycoside resistance genes, the aminoglycoside phosphotransferase gene aph(3″), which confers resistance to streptomycin, and the aminoglycoside acetyltransferase gene aac(6′)-Ic to amikacin and tobramycin (Shaw et al., 1992) were present on most of the chromosomes studied. We found the aminoglycoside nucleotidyltransferase gene aadA16 in only 4 genomes from environmental strains. S. symbiotica, and C. multitudinisentens RB-25 did not present any of these genes.

We identified the qnr gene that confers quinolone resistance in the chromosome of 25 strains. Six different alleles of this gene were identified: allele qnrB15 in the S. phymutica group, allele qnrB23 in the S. marcescens and S. nematodiphila group, qnrB31 also in the S. marcescens group, qnrB32 in the S. liquefaciens and S. proteamaculans group, qnrB37 in the S. marcescens and S. ureilytica (reclassified as S. marcescens) group, and qnrB57 in the rest of the S. phymuthica strains. Due to the fact that this gene is unique to the Serratia spp. chromosome, it could be the possible source of Qnr determinants for the plasmid-encoded Qnr in other species of bacteria (Velasco et al., 2009).

Serratia spp. is intrinsically resistant to polymyxin B. Polymyxin B resistance in this genus can be explained by the presence of the arnBCADTEF operon, the two-component systems pmrAB/phoPQ and the regulatory gene mgrB in all strains, except for S. symbiotica ‘Cinara Cedri’. Activation of the two-component systems is set off by environmental stimuli that result in an overexpression of LPS-modifying genes (Lin et al., 2014; Olaitan et al., 2014) and leading to polymyxin B and colistin resistance.

Besides the above mentioned antibiotics, the genus Serratia is intrinsically resistant to other classes of antibiotics: 31 out of 32 strains have the bacA gene on their chromosomes, conferring resistance to bacitracin, another polypeptide antibiotic. In addition, 30 strains harbor the gene hslJ and 28 strains the gene fosA, which confer novobiocin and fosfomycin resistance, respectively. The chloramphenicol acetyltransferase cat gene, which confers resistance to chloramphenicol is also present in 27 strains (Potrykus and Wegrzyn, 2001; Schwarz et al., 2004). Only 12 strains, mostly environmental, possess the vancomycin resistance gene vanX on its chromosome (detailed information is shown in Supplementary Table S2).

Intrinsic Resistance Conferred by Efflux Pumps

Active drug extrusion outside of the bacterial cell is one of the most common mechanisms associated with resistance. We identified 74 different genes related to the 5 efflux pump families: the multidrug and toxic-compound extrusion family (MATE), the small multidrug resistance family (SMR), the major facilitator superfamily (MFS), the resistance nodulation division family (RND), and the ATP binding cassette superfamily (ABC) (Supplementary Table S2).

Some of these efflux pumps have been described previously in the Serratia genus and have homolog in other bacteria: the SsmE efflux pump of the SMR family (conferring resistance to ethidium bromide) is homologous to the E. coli EmrE (Minato et al., 2008); SmfY of the MFS family (conferring resistance to norfloxacin, acriflavine and ethidium bromide) homologous to QacA of Staphylococcus aureus (Shahcheraghi et al., 2007); SdeXY (conferring resistance to erythromycin, tetracycline, norfloxacin, benzalkonium chloride, ethidium bromide, acriflavine, and rhodamine); SdeAB (conferring resistance to fluoroquinolones) and SdeCDE (conferring resistance to novobiocin) all belonging to the RND family and having a high degree of homology with the AcrAB-TolC, OqxAB and MdtABC efflux pumps of E. coli respectively (Chen et al., 2003; Kumar and Worobec, 2005; Begic and Worobec, 2008); and finally, SmdAB (conferring resistance to norfloxacin, tetracycline, and tetraphenylphosphonium chloride) of the ABC superfamily, an homolog of MdlAB of E. coli (Matsuo et al., 2008).

Other homologous genes of efflux systems from different bacteria that confer resistance to several kinds of antibiotics and antiseptics were identified on the 32 genomes: 2 alleles of the aminoglycoside efflux pump AcrD from the RND family (Rosenberg et al., 2000); 2 quinolone efflux pumps, DinF and MdtK, from the MATE family (Brown et al., 2007); SugE, a SMR family efflux pump which confers resistance to quaternary ammonium compounds (Chung and Saier, 2002); 9 different MSF family efflux pumps, among them two alleles of EmrAB conferring resistance to thiolactomycin (Furukawa et al., 1993), Bcr to bacitracin (Bernard et al., 2005), Fsr, an homolog of E. coli RosA, which confers fosmidomycin resistance, and MdfA, which confers resistance to chloramphenicol (Heng et al., 2015). The efflux pump MacAB from the ABC superfamily, which confers resistance to macrolides (Lu and Zgurskaya, 2012; Fitzpatrick et al., 2017) was also identified on the chromosomes of the Serratia spp. genomes, and the efflux pump TetA from the MFS family conferring resistance to tetracycline (Aldema et al., 1996). In addition, 16 genes belonging to 8 efflux systems had alleles on different chromosomal loci, including copies on genomic islands, for example, the aminoglycoside efflux protein AcrD, a transporter belonging to the resistance-nodulation-division family (RND family), which has three different alleles (acrD, acrD′, and acrD″) distributed among the 32 Serratia genomes. The nucleotidic identity percentage between acrD and acrD′ is 67.2%, while it is 60.9% between acrD and acrD″ and 62.6% between acrD′ and acrD″. Two of the nosocomial strains, SmUNAM836 and Sm39, were the only strains that presented all three alleles.

The majority of the genes associated with efflux pumps of the 32 genomes belong to the RND efflux family (Figure ​Figure55). Most of these genes are shared by all the strains, except for S. symbiotica ‘Cinara Cedri’ that only contains 5 of the reported 74 efflux pumps genes. It is not surprising that this strain harbors very few ARGs and efflux pumps because of its reductive genome evolution related to its lifestyle inside the stable and protected niche of the host cell of the aphid Cinara cedri (Lamelas et al., 2011; Dutta and Paul, 2012). The highest number of efflux pump genes (57) is present in the Mexican nosocomial strain S. marcescens SmUNAM836 sequenced by our group (Sandner-Miranda et al., 2016). Pathogenic strains, including nosocomial strains, show an average number of 50.1 efflux-associated genes, whereas the environmental strains show an average of 44.1 efflux-associated genes and the symbiotic strains only 33.5 efflux-associated genes (Supplementary Table S2).

Acquired Resistance Genes on Plasmids, Transposons, and Integrons

In order to identify genes associated with acquired antibiotic resistance, we screened all the plasmids of the 32 Serratia spp. strains. A total of 16 plasmids are reported in the NCBI database for this strain collection (Supplementary Table S3). We found 13 plasmid-borne ARGs, 7 β-lactam resistance genes, 3 aminoglycoside resistance genes encoding modifying enzymes, one macrolide resistance gene, one sulfonamide resistance gene, one trimethoprim resistance gene and additionally 3 efflux pump genes associated with resistance to quaternary ammonium compounds, disinfectants and macrolides also on plasmids.

The β-lactam resistance genes class A bla_TEM-1, bla_KPC-2 and bla_{SV H-30,} class B bla_IMP-1, class C bla_ACT-28 and bla_{CMY -8,} and class D bla_OXA-9 were identified in plasmids of nosocomial strains, with the exception of bla_ACT-28, a class C β-lactamase found on plasmid pSF001 of S. fonticola GS2, a strain that is associated with a plant. Looking at the nosocomial strains S. marcescens SmUNAM836 and S. marcescens CAV1429, its plasmids (pSmUNAM836 and pKPC_CAV1492 respectively) harbor class A bla_TEM-1 β-lactamase, which is classified as a broad spectrum β-lactamase (Bush and Jacoby, 2010) and confers resistance to all penicillins and β-lactamase inhibitors. The strain S. marcescens CAV1429 also carries two copies of the class A β-lactamase, namely bla_KPC-2, a carbapenemase (Wang et al., 2014), and the bla_OXA-9 gene that encodes an oxacillinase-carbenicillinase (Bojorquez et al., 1998) on plasmid pKPC_CAV1492. The bla_{SV H-30} gene, which encodes an ESBL that confers resistance to penicillins and cephalosporines (Bradford, 2001), was located on plasmid pCAV1492-73 of strain S. marcescens CAV1492. The imipenem metallo-β-lactamase gene, bla_IMP-1, and the cephalosporin resistance gene, bla_CMY-8, were only found on the plasmid pSMC1 of the nosocomial strain S. marcescens Sm39.

The aminoglycoside resistance genes aac(6′)-1b and aadA1, encoding modifying enzymes tobramycin and amikacin 6′ acetyltransferase (Rather et al., 1992) and a streptomycin 3″-adenylyltransferase were found on plasmid pKPC_CAV1492 of the nosocomial strain S. marcescens CAV1429. Gene aadA2, encoding another streptomycin 3″-adenylyltransferase was found on plasmid pSMC1 of the nosocomial strain S. marcescens Sm39. Strain S. marcescens Sm39 harbors two copies of the sulfonamide resistance gene sul1 on plasmid pSMC1.

Three efflux pump genes, qacE, qacH, and msrE, were found on plasmids of the nosocomial strain S. marcescens CAV1492. Two copies of gene qacE were found on plasmid pSMC1 of S. marcescens SM39. These efflux pumps confer resistance to quaternary ammonium compounds, disinfectants and macrolides, respectively.

As previously reported, it is common to find various ARGs on the same mobile element. Such is the case of the genes sul1, qacE and some β-lactamases, which are found together in the clinical class 1 integron (Wyrsch et al., 2016). In this study we found two plasmids harboring this integron, plasmid pSMC1 of the nosocomial strain S. marcescens Sm39 and plasmid pKPC_CAV1492 of the nosocomial strain S. marcescens CAV1492. The class 1 integron is associated with various families of transposons, the Tn3 transposon present in plasmid pKPC_CAV1492 (Deng et al., 2015) and the Tn91 transposon present in plasmid pSMC1. Other plasmids harboring heavy metal resistance are also present in some of the Serratia spp. genomes (mercury resistance genes in plasmid pCAV1492-73 and pSMC1from the nosocomial strains S. marcescens CAV1492 and S. marcescens Sm39, respectively, and copper resistance genes in plasmid pCAV1492-199 also from strain S. marcescens CAV1492).

The aminoglycoside phosphotransferase gene, aph(3′), was only found on S. rubidaea 1122 and Serratia sp. FGI94 chromosomes, associated with a transposon. Gene vanW, which confers resistance to vancomycin, was found on a transposon of the environmental strain S. fonticola DSM4576. These mobile elements have not been previously classified and were inferred in this analysis on the basis of being unique sequences harboring transposases (Supplementary Table S2).

Many other genes encoding transposases and integrases were found on all Serratia spp. chromosomes, but neither associated with ARGs.

Efflux Pumps Acquired on the Genomic Islands of Serratia spp.

Three efflux pumps were identified on different genomic islands of some of the S. marcescens strains using IslandViewer 4 (Bertelli et al., 2017). These genomic islands have not been characterized before. Two of them, MexGHI (Aendekerk et al., 2005) and MexPQ-OprE (Mima et al., 2005), which confer resistance to norfloxacin and macrolides, respectively, showed 82 and 84% homology to the P. aeruginosa efflux pumps, indicating a probable horizontal transfer event between these bacteria. The third efflux pump was an allele of etsABC that encodes an E. coli efflux pump of a putative ABC transport system contained within the pAPEC-O2-ColV plasmid, that confers resistance to macrolides (Johnson et al., 2006). These efflux pumps are also present in other Serratia species and most probably on genomic islands as well (Supplementary Table S2).

Resistance Acquired by Gene Mutations

The gene gyrA mutation at codon Ser83 was identified in three nosocomial strains, namely S. marcescens SmUNAM836, S. marcescens Sm39, and S. marcescens CAV1492, and also on S. symbiotica ‘Cinara Cedri,’ an aphid symbiont. Resistance may arise due to point mutations that result in amino acid substitutions within the topoisomerase genes (gyrA, gyrB, parC, parE), with or without a decreased expression of outer membrane porins and overexpression of multidrug efflux pumps (Hopkins et al., 2005). S. marcescens SmUNAM836 and S. marcescens CAV1492 had an isoleucine residue instead of the expected serine at position 83 of GyrA while the strain S. marcescens Sm39 had an arginine residue. S. symbiotica, which is a co-obligate symbiont, had a threonine in this position. These mutations are likely to induce a local conformation change of the A subunit of the DNA gyrase, modifying the binding affinity of the quinolones for this enzyme (Yoshida et al., 1990; Weigel et al., 1998). Resistance to one type of quinolone will confer resistance to all (Webber and Piddock, 2001). Neither of the strains had the mutation on gene folP that confers resistance to sulfamethoxazole. The folP single C→T transition resulting in a Pro→Ser substitution at amino acid position 64 is absent in all genomes (Vedantam et al., 1998). Amino acid substitutions (Asp369Asn and Leu370Ile) in MurA are a major factor in fosfomycin resistance, but neither of the strains showed these mutations (Takahata et al., 2010).

The Serratia spp. Resistome

We classified the resistome according to the niche or lifestyle of the bacteria. We divided the sample of 32 Serratia spp. strains into three lifestyles: pathogens (nosocomial and animal pathogens), environmental (bacteria associated with plant, soil, water, or food) and symbiotic bacteria (Figure ​Figure66). The diagram shows that pathogenic (brown) and environmental strains (blue) do not show a significant different median number of total ARGs, intrinsic and acquired, but symbiotic bacteria (green) do show the smallest number. Regarding efflux pump genes, pathogenic bacteria harbor more of these genes than environmental and symbiotic bacteria. We can appreciate that the difference among resistance gene content between nosocomial/pathogenic and environmental strains is not significant. We believe that this is due to the environment providing a natural reservoir for ARGs.

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FIGURE 6

Box plot showing the relationship between the number of resistance determinants and the lifestyle of the strains. There are 3 main lifestyle groups, nosocomial/pathogen [P (brown)], environmental/free living [E (blue)] and environmental/symbiotic [S (green)]. P, E, and S represent the total resistance determinants. P.1, E.1, and S.1 are the total efflux pump genes. P.2, E.2, and S.2 represent the acquired resistance genes and P.3, E.3, and S.3 the intrinsic AGRs.

In this comparative genomic study, we report that the Serratia spp. resistome consists of genes shared by the majority of the strains and genes exclusive of niche. With the analysis of 32 Serratia spp. genomes, we report a resistome for the Serratia genus totaling 123 ARGs, 49 intrinsic and acquired ARGs, and 74 efflux pump-associated genes. Of the 123 ARGs, 80 are present in one or more strains from each niche, 7 are shared by pathogens and environmental strains only, 2 are shared by pathogens and symbiotic strains, and one is shared by environmental and symbiotic strains. 18 ARGs were found exclusively in pathogenic strains, 14 in environmental strains and one in symbiotic strains (Figure ​Figure77).

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FIGURE 7

Diagram showing the Serratia spp. resistome based on the bacterial niche. Pathogens (red), environmental (blue), and symbiotic (green).

The polymyxin resistance operon and the genes catA, bacA, fosA and hslJ, which confer resistance to chloramphenicol, bacitracin, fosfomycin and novobiocin, respectively, are among the shared ARGs. In addition, the aminoglycoside acelyltransferase gene, aac(6′)-Ic, the aminoglycoside phosphotransferase gene, aph(3″), and the quinolone resistance gene, qnr, are shared by the majority of the strains, regardless of their lifestyles. The class C β-lactamase gene, ampC, and all the regulatory genes were also found in most of the strain chromosomes. Most of the strains share most of the genes associated with efflux systems.

The 18 ARGs exclusive of pathogens are genes located on plasmids, mainly β-lactamase coding genes, with the exception of the alleles of the efflux pumps EmrAB and AcrD, which are located on the chromosome (Supplementary Table S2). The 14 ARGs exclusive of environmental strains are genes found on the chromosome, all of intrinsic resistance except for the vancomycin resistance gene vanW found on a transposon. Only one ARG was found to be exclusive of a symbiotic strain, an allele of the efflux pump AcrF that confers resistance to ciprofloxacin. Five efflux pumps genes, mexP, mexQ, opmE and triC, from the RND family conferring resistance to macrolides, fluoroquinolones and triclosan, respectively, and the gene mdsC, which is part of the efflux pump MdsAB (absent in this sample), are shared by pathogen and environmental strains. The aminoglycoside phosphotransferase gene aph(3′) and the mutation of gyrA, which confers resistance to quinolones, were located in pathogens and symbiotic strains. Only the streptogramin efflux pump gene, vgaC, is shared by environmental and symbiotic strains and is absent in pathogens.

This work was supported by El Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México (UNAM) of which LS-M is a doctoral student. We are also grateful to Gabriela Delgado for her helpful discussions, to José Luis Méndez for his support in the laboratory essays and to Luis Fernando Espinosa for his technical support.