Genomic Insights into Drug Resistance Determinants in Cedecea neteri, A Rare Opportunistic Pathogen

Cedecea, a genus in the Enterobacteriaceae family, includes several opportunistic pathogens reported to cause an array of sporadic acute infections, most notably of the lung and bloodstream. One species, Cedecea neteri, is associated with cases of bacteremia in immunocompromised hosts and has documented resistance to different antibiotics, including β-lactams and colistin. Despite the potential to inflict serious infections, knowledge about drug resistance determinants in Cedecea is limited. In this study, we utilized whole-genome sequence data available for three environmental strains (SSMD04, M006, ND14a) of C. neteri and various bioinformatics tools to analyze drug resistance genes in this bacterium. All three genomes harbor multiple chromosome-encoded β-lactamase genes. A deeper analysis of β-lactamase genes in SSMD04 revealed four metallo-β-lactamases, a novel variant, and a CMY/ACT-type AmpC putatively regulated by a divergently transcribed AmpR. Homologs of known resistance-nodulation-cell division (RND)-type multidrug efflux pumps such as OqxB, AcrB, AcrD, and MdtBC were also identified. Genomic island prediction for SSMD04 indicated that tolC, involved in drug and toxin export across the outer membrane of Gram-negative bacteria, was acquired by a transposase-mediated genetic transfer mechanism. Our study provides new insights into drug resistance mechanisms of an environmental microorganism capable of behaving as a clinically relevant opportunistic pathogen.

While Cedecea infections have been reported sporadically in the literature, clinical cases of this emerging opportunistic human pathogen continue to occur worldwide. Clinical strains of Cedecea species exhibit natural resistance to various antimicrobial agents, including ampicillin, ampicillin-sulbactam, cefazolin, cephalothin, cefoxitin, and colistin [3]. The multidrug-resistant phenotype of one C. lapagei isolate was attributed, in part, to the acquisition of the bla NDM-1 gene, which encodes the New Delhi metallo-β-lactamase-1 (NDM-1) [18]. This clinical isolate was resistant to carbapenems, monobactams, and extended-spectrum cephalosporins. Recently, additional bla NDM-1 -harboring Cedecea isolates have been reported [23,24]. The dissemination of NDM-1 is of serious public health concern as this metallo-β-lactamase severely limits the chemotherapeutic options available for treating bacterial infections. Apart from the detection of β-lactamase expression in certain Cedecea strains [18,25], knowledge on the genetic basis of drug resistance phenotypes in this genus is limited. The pathogenic potential of Cedecea species is likely under-recognized due to its low reported frequency of association with human infections and the fact that these organisms are not linked to specific disease states but, instead, cause a wide spectrum of acute infections in compromised hosts.
Opportunistic C. neteri infections typically present as bacteremia [10,12], although recently C. neteri was identified as the etiological agent of a urinary tract infection in a pregnant female with polyhydramnios [26]. One of the rare fatal patient outcomes associated with clinical isolates of Cedecea spp. involved a C. neteri infection in the bloodstream of an individual with systemic lupus erythematosus (SLE) [12]. Given the potentially serious nature of these opportunistic infections, research is needed on identifying the genes implicated in drug resistance phenotypes that impact treatment of Cedecea. In this study, we focused our attention on C. neteri because whole-genome sequence (WGS) information is available for multiple strains of this species. WGS data for three environmental strains of C. neteri and different bioinformatics tools were utilized to provide insight into the predicted drug resistance determinants of this species. Only a draft genome at the unassembled contig level is available for the type strain C. neteri ATCC 33855 [10], isolated from a human foot, so we focused our queries on the completed genome sequence of C. neteri SSMD04 [27] and compared results to the genomes of C. neteri M006 [28] and ND14a. Our findings highlight the value of genome-based explorations in contributing to our understanding of antimicrobial resistance in little studied environmental microorganisms with the capability of behaving as clinically relevant opportunistic pathogens.

Bacterial Genomes
Detailed information about the bacterial genomes analyzed in this study are presented in Table 1. Completely sequenced and annotated genomes for the following strains have been previously deposited in GenBank: C. neteri SSMD04 (GenBank accession no. CP009451.1), C. neteri M006 (GenBank accession no. CP009458.1), C. neteri ND14a (Gen-Bank accession no. CP009459.1), and Klebsiella michiganensis RC10 (GenBank accession no. CP011077.1). These genomes do not contain any extrachromosomal elements. SSMD04 was used as the query strain in sequence homology searches and comparative genomic analyses.

Sequence Database Search and Bioinformatics Tools
Initial searches based on gene and protein annotation were performed against the KEGG (Kyoto Encyclopedia of Gene and Genomes) integrated database resource (https: //www.genome.jp/kegg/, accessed on 14 August 2021; [29]) using "beta-lactamase", "transporter", "efflux pump", "antibiotic resistance", "drug resistance", and "multidrug resistance" as search terms. Similarity of intra-and interspecies protein coding sequences (CDSs) was computed using BLASTP [30] with a default E-value threshold of 10.0 and the BLOSUM62 amino-acid scoring matrix. For all BLASTP analyses, the C. neteri SSMD04 CDS represented the query sequence. ClustalW [31] was used to construct multiple alignments of protein sequences. Sequence signatures or motifs specific for the different Ambler classes of β-lactamases were identified by a combination of manual scrutiny and multiple sequence alignment. A phylogram (midpoint-rooted tree) was generated using the phylogenetic analysis pipeline of the Environment for Tree Exploration, version 3.1.1 (ETE3) [32] and FastTree v2.1.8 [33] with default parameters.

Multiple Metallo-β-Lactamase-Encoding Genes in C. neteri
Six open-reading frames (ORFs) in the C. neteri SSMD04 genome (Cnt) are annotated as unclassified β-lactamases based on a previous KEGG database search [36], compared with 9 ORFs in strain M006 and 11 ORFs in strain ND14a. As environmental Cedecea species exhibit a natural phenotype of resistance to certain clinically important β-lactam antibiotics [3], we further analyzed these six putative β-lactamase-encoding ORFs in more detail. The deduced primary sequences of four of these genes have the metallo-beta-lactamase (MBL) protein fold (αβ/αβ) distinctive of the metallo-hydrolase/oxidoreductase superfamily based on the presence of Pfam domains (Table 2). Bacterial MBLs are clinically relevant because these enzymes hydrolyze and inactivate a broad spectrum of therapeutically important β-lactam antibiotics, including carbapenems, and are refractory to inhibition by clavulanate, sulbactam, and tazobactam [37]. MBLs require a divalent metal ion, usually zinc, as a cofactor for β-lactam hydrolysis (reviewed in [38]). While Class B metallo-βlactamases exhibit substantial divergence in terms of molecular structure and function, sequence analysis revealed that three (Cnt00700, Cnt16535, and Cnt22070) of the four putative C. neteri SSMD04 MBLs contained the strictly conserved metal-binding motif, H-X-H-X-D-H, which is involved in specific Zn 2+ interactions and functions as the MBL active-site center (Table 2; [39,40]). The other predicted metallo-β-lactamase, Cnt03975, lacked the conserved group B-specific motif involved in zinc ion coordination, but harbored conserved histidine residues at positions 113, 186, and 220, and two aspartic acid residues at positions 85 and 87 which may contribute to activity.  Family-specific motifs can serve as structural 'fingerprints' for the assignment of new members to β-lactamase families, and the three subclasses of MBLs (B1, B2, and B3) possess distinct conserved sets of amino acid residues [41]. We searched the amino acid sequences of the four putative C. neteri MBLs for the presence of molecular fingerprints and identified the sequence 198 P-G-H-T-P-G 203 in a fingerprint (HFMPGHTPGS) previously identified as characterizing subclass B3 MBLs [41]. Of the four SSMD04 ORFs with a predicted MBL protein fold, the PGHTPG motif was only identified in Cnt00700, suggesting that this MBL may belong to subclass B3 ( Table 2). The motif 195 V-P-L-P-G-H-T-P-G-H 204 is conserved in predicted Class B β-lactamase sequences from C. neteri strains (SSMD04, M006, ND14a), C. lapagei NCTC11466, and Klebsiella michiganensis RC10. Interestingly, subclass B3 MBLs have been identified in the genus Chryseobacterium, whose members inhabit environmental niches and exhibit the capacity to behave as opportunistic pathogens [42]. Cnt00700 and the subclass B3-metallo-β-lactamase (CPS-1) from Chryseobacterium piscium share the conserved sequence motif PGHTxG, which was not found in the other putative MBLs encoded in the SSMD04 genome. CPS-1 displayed broad-spectrum hydrolyzing activity against penicillins, cephalothin, some oxyimino-cephalosporins (cefuroxime, ceftriaxone, cefotaxime), cefoxitin, and carbapenems (imipenem, meropenem, doripenem) [43].

A Novel β-Lactamase Gene in the SSMD04 Genome
Most of the genes with predicted functions in β-lactamase-mediated antibiotic resistance in C. neteri SSMD04 were also conserved in the genomes of C. neteri strains M006 and ND14a. The exception was cnt22350 (ORF JT31_22350), which exhibited 93% amino acid identity to a putative 6-aminohexanoate-dimer hydrolase (NylB) from C. lapagei NCTC11466 but had no orthologs in C. neteri strains M006 and ND14a and in K. michiganensis ( Table 2). The protein product of cnt22350 displayed 87% amino acid identity with an unclassified β-lactamase from Hafnia alvei, a member of the Enterobacteriaceae family associated with infrequent diarrheal cases in humans, as well as nosocomial systemic infections [44,45]. BLASTP analysis of Cnt22350 against the KEGG database demonstrated notable amino acid sequence identity to AmpC from Kluyvera ascorbata and Enterobacter cloacae subsp. cloacae ATCC 13047 (66 and 65%, respectively). Multiple sequence alignment further revealed that Cnt22350 lacked most of the known conserved structural elements found in Ambler Class A, C, and D β-lactamases. The conserved 70 S-X-X-K 73 motif in Class A and D families was replaced by S-Y-E-G, and the 64 S-X-X-K 67 sequence element characterizing Class C β-lactamases was replaced by 65 69 . The sequence 144 S-V-G-K 147 was identified within a larger element 142 S-R-S-V-G-K-S-V-V-S-T-L-V-G 155 that was highly conserved among homologs and may constitute a novel serine-containing active-site signature. We suggest that Cnt22350 represents a variant β-lactamase that may possess some novel functional features.

CMY/ACT-Type AmpC β-Lactamase
Previously, we proposed that cnt10470 (ORF JT31_10470) in the C. neteri SSMD04 genome encodes an Ambler Class C (AmpC) β-lactamase (cephalosporinase) based on the presence of signature motifs characteristic of Class C active-site serine β-lactamases and a functionally verified Cedecea davisae AmpC [25,36]. Various families have been described for AmpC β-lactamases according to polymorphisms in amino acid sequence and include CMY (currently the largest family), ACC, ACT, FOX, LAT, MIR, MOX, and DHA, as well as others (reviewed in [46,47]). AmpC-type variants with expanded-spectrum activity toward imipenem also have been described in Pseudomonas aeruginosa and are collectively designated as PDC [48]. To investigate the evolutionary relationship between Cnt10470 and various AmpC clones, a phylogram was constructed using the amino acid sequences from plasmid and chromosomal AmpCs belonging to the families CMY, ACT, LAT, DHA, and PDC based on the top BLASTP hits ( Figure 1). The predicted C. neteri AmpC is more related to polymorphic CMY-and ACT-type AmpCs compared with DHA-and PDCtype variants. Further sequence analysis indicated that the chromosomal-borne SSMD04 AmpC likely belongs to the CMY/ACT family based on the presence of the sequence fingerprint RYWR(v)G(s)MYQ [41] at amino acid positions 279 to 288 (Table 2). This motif was conserved among Cnt10470 and various CMY and ACT clones, except at positions 283 and 285; however, the same sequence motif was absent in DHA and PDC representatives, which diverged from a less recent common ancestor. The phylogenetic results suggest that the SSMD04 AmpC represents yet another variant in the highly polymorphic CMY/ACT families.
Recent preliminary kinetic studies conducted by this research group demonstrated that purified polyhistidine-tagged versions of the predicted C. neteri AmpC from the SSMD04 environmental strain and a clinical isolate, ATCC 33855, readily hydrolyzed chromogenic cephalosporin substrates CENTA [49] and nitrocefin, demonstrating that both recombinant AmpC proteins are functional (data not shown). While CMY variants can exhibit functional differences, the hydrolysis activity profile of CMY-type AmpC enzymes includes penicillins, cephalosporins, cephamycins (e.g., cefoxitin, cefotetan), oxyimino-cephalosporins (e.g., ceftazidime, cefotaxime, ceftriaxone), and monobactams such as aztreonam. In-depth biochemical characterization of the recombinant AmpCs from C. neteri SSMD04 and C. neteri ATCC 33855 is ongoing.

Genetic Environment of AmpC Gene in C. neteri SSMD04
As shown in Figure 2, the genomic organization of the C. neteri ampC (cnt10470) locus closely resembles the structure of plasmid-mediated E. coli CMY-13-type ampC, which was originally derived from Citrobacter freundii via a transposition-mediated genetic transfer mechanism [50]. The C. neteri AmpC shares 73% amino acid identity to CMY-13. Like the E. coli CMY-13 gene, C. neteri ampC is linked to a divergently oriented gene annotated as a LysR family transcriptional regulator (Cnt10465), which we previously proposed is AmpR [36]. The Cnt10465 gene product shares high amino acid identity to AmpR from Cedecea lapagei (94%), Enterobacter cloacae ATCC 13047 (82%), Kluyvera intermedia (81%), Enterobacter ludwigii (80%), and Pseudomonas aeruginosa (64%). The N-terminal helix-turnhelix (HTH) motif in the predicted SSMD04 AmpR contains conserved residues S-38 and K-42 which have been shown to be critical for the DNA-binding activity of AmpR in P. aeruginosa [51]. The predicted C. neteri AmpR also contains conserved residues G-102 and D-135, which play important structural roles in P. aeruginosa AmpR. Microorganisms 2021, 9, x FOR PEER REVIEW 8 of 17 The E. coli and C. neteri ampR-ampC loci are flanked by fumarate reductase genes and sugE, which encodes an efflux pump of the small multidrug resistance family (SMR) that confers resistance to quaternary ammonium compounds ( Figure 2; [50,52]). A similar genetic organization was identified in the genomes of C. neteri M006 and ND14a. The fumarate reductase cluster frdABCD is located immediately downstream of SSMD04 ampR, and an outer membrane lipoprotein gene, cnt10475, is immediately downstream of ampC. The deduced amino acid sequence of Cnt10475 is 76% identical to Blc (apolipoprotein D/lipocalin family protein) from Salmonella enterica and E. coli.
The presence of a divergently transcribed AmpR homolog suggests that C. neteri exhibits an inducible AmpC phenotype in the presence of β-lactam antibiotics. This chromosomal β-lactamase induction mechanism has previously been shown to involve three additional major gene products connected to the peptidoglycan recycling pathway: AmpD (a cytosolic N-acetyl-anhydromuramyl-L-alanine amidase), AmpE (specific function unknown), and AmpG (inner membrane permease responsible for muropeptide transport) [53; reviewed in 54]. The SSMD04 chromosome harbors the following genes comprising the specific transcriptional regulatory system for ampC expression: ampD (cnt08695, amidase), ampE (cnt08690, regulatory protein), and ampG (cnt07450, muropeptide transporter). The putative ampD and ampE loci are closely linked on the SSMD04 chromosome and may constitute an ampDE operon, the same gene organization that has been reported in other enterobacteria [55,56].

Predicted Multidrug Efflux Pumps in C. neteri
A ubiquitous microbial mechanism of resistance is drug extrusion via integral membrane transporters, which reduces exposure of the bacterial target to active concentrations of the drug. Five different transporter families confer clinical resistance to various antibiotic classes [57]: ATP-binding cassette (ABC), multidrug and toxic compound extrusion (MATE), major facilitator superfamily (MFS), resistance-nodulationdivision (RND), and small multidrug resistance (SMR). Database searches revealed the presence of at least 34, 32, and 33 annotated efflux pump or transporter genes in C. neteri strains SSMD04, M006, and ND14a, respectively. Classifications of these predicted efflux pump/transporter genes were distributed across the five different families (ABC, MATE, MFS, RND, and SMR). Efflux pumps of the RND family represent some of the most clinically significant transporter proteins in Gram-negative bacteria because of their broad substrate specificity and association with multidrug resistance (MDR) [58]. Genomes for all three C. neteri strains encoded several RND MDR transporters generally annotated as efflux pumps. The E. coli and C. neteri ampR-ampC loci are flanked by fumarate reductase genes and sugE, which encodes an efflux pump of the small multidrug resistance family (SMR) that confers resistance to quaternary ammonium compounds ( Figure 2; [50,52]). A similar genetic organization was identified in the genomes of C. neteri M006 and ND14a. The fumarate reductase cluster frdABCD is located immediately downstream of SSMD04 ampR, and an outer membrane lipoprotein gene, cnt10475, is immediately downstream of ampC. The deduced amino acid sequence of Cnt10475 is 76% identical to Blc (apolipoprotein D/lipocalin family protein) from Salmonella enterica and E. coli.
The presence of a divergently transcribed AmpR homolog suggests that C. neteri exhibits an inducible AmpC phenotype in the presence of β-lactam antibiotics. This chromosomal β-lactamase induction mechanism has previously been shown to involve three additional major gene products connected to the peptidoglycan recycling pathway: AmpD (a cytosolic N-acetyl-anhydromuramyl-L-alanine amidase), AmpE (specific function unknown), and AmpG (inner membrane permease responsible for muropeptide transport) ( [53]; reviewed in [54]). The SSMD04 chromosome harbors the following genes comprising the specific transcriptional regulatory system for ampC expression: ampD (cnt08695, amidase), ampE (cnt08690, regulatory protein), and ampG (cnt07450, muropeptide transporter). The putative ampD and ampE loci are closely linked on the SSMD04 chromosome and may constitute an ampDE operon, the same gene organization that has been reported in other enterobacteria [55,56].

Predicted Multidrug Efflux Pumps in C. neteri
A ubiquitous microbial mechanism of resistance is drug extrusion via integral membrane transporters, which reduces exposure of the bacterial target to active concentrations of the drug. Five different transporter families confer clinical resistance to various antibiotic classes [57]: ATP-binding cassette (ABC), multidrug and toxic compound extrusion (MATE), major facilitator superfamily (MFS), resistance-nodulation-division (RND), and small multidrug resistance (SMR). Database searches revealed the presence of at least 34, 32, and 33 annotated efflux pump or transporter genes in C. neteri strains SSMD04, M006, and ND14a, respectively. Classifications of these predicted efflux pump/transporter genes were distributed across the five different families (ABC, MATE, MFS, RND, and SMR). Efflux pumps of the RND family represent some of the most clinically significant transporter proteins in Gram-negative bacteria because of their broad substrate specificity and association with multidrug resistance (MDR) [58]. Genomes for all three C. neteri strains encoded several RND MDR transporters generally annotated as efflux pumps.
By performing in-depth homology searches against the KEGG database, we found homologs of the RND MDR efflux systems AcrAB-TolC, AcrD, OqxAB, and MdtABCD in the SSMD04 genome ( Figure 3). BLAST searches indicated that one predicted RND efflux system resembled both OqxAB and AcrAB. Cnt17100 (ORF JT31_17100) has 95% identity to OqxB in Enterobacter cloacae and 84% identity to AcrB in Xanthomonas citri at the amino acid level. Both OqxB and AcrB are RND transporters that share a consistent transmembrane helical structure [59]. Immediately upstream of JT31_17100 is a gene with 88% amino acid identity to AcrA in E. cloacae and 85% identity to OqxA in Klebsiella variicola. OqxA and AcrA function as membrane fusion proteins (MFPs) in the RND MDR efflux system. Upstream of the oqxAB/acrAB gene cluster is a gene (JT31_17110) encoding an AraC family transcriptional regulator that is transcribed divergently. The deduced gene product shares 84% amino acid identity to the transcriptional activator MarA in Enterobacter ludwigii and 78% identity to RarA in Klebsiella pneumoniae. A second putative regulatory gene (JT31_17095), encoding a Rrf2-type regulator, is located immediately downstream of the oqxAB/acrAB gene cluster and may be involved in repressing transcription of the putative RND MDR efflux system based on its homology to OqxR. Substrates of the AcrAB efflux pump include cationic dyes (acriflavine), detergents, and antibiotics such as penicillins, cephalosporins, fluoroquinolones, macrolides, chloramphenicol, and tetracycline [60]. OqxAB confers resistance to multiple antimicrobial agents (quinoxalines, quinolones, tigecycline, nitrofurantoin, and chloramphenicol), as well as detergents and disinfectants [59]. The function of the E. coli AcrB transporter is dependent on TolC, a multifunctional outer membrane channel [61,62], and a homologous gene with 75% amino acid sequence identity to TolC in Salmonella enterica was identified upstream of the C. neteri acrB/oqxB gene (JT31_17100), suggesting a similar tripartite system in this species ( Figure  3). Additionally, the small adaptor protein, AcrZ, which interacts with the AcrAB efflux pump, was identified some distance downstream of the acrB gene in a similar genetic organization as the E. coli cluster [63]. The AcrZ protein was shown to aid in the binding and export of chloramphenicol and tetracycline by the AcrAB efflux pump, thus enhancing the drug resistance phenotype of E. coli [64]. By performing in-depth homology searches against the KEGG database, we found homologs of the RND MDR efflux systems AcrAB-TolC, AcrD, OqxAB, and MdtABCD in the SSMD04 genome (Figure 3). BLAST searches indicated that one predicted RND efflux system resembled both OqxAB and AcrAB. Cnt17100 (ORF JT31_17100) has 95% identity to OqxB in Enterobacter cloacae and 84% identity to AcrB in Xanthomonas citri at the amino acid level. Both OqxB and AcrB are RND transporters that share a consistent transmembrane helical structure [59]. Immediately upstream of JT31_17100 is a gene with 88% amino acid identity to AcrA in E. cloacae and 85% identity to OqxA in Klebsiella variicola. OqxA and AcrA function as membrane fusion proteins (MFPs) in the RND MDR efflux system. Upstream of the oqxAB/acrAB gene cluster is a gene (JT31_17110) encoding an AraC family transcriptional regulator that is transcribed divergently. The deduced gene product shares 84% amino acid identity to the transcriptional activator MarA in Enterobacter ludwigii and 78% identity to RarA in Klebsiella pneumoniae. A second putative regulatory gene (JT31_17095), encoding a Rrf2-type regulator, is located immediately downstream of the oqxAB/acrAB gene cluster and may be involved in repressing transcription of the putative RND MDR efflux system based on its homology to OqxR. Substrates of the AcrAB efflux pump include cationic dyes (acriflavine), detergents, and antibiotics such as penicillins, cephalosporins, fluoroquinolones, macrolides, chloramphenicol, and tetracycline [60]. OqxAB confers resistance to multiple antimicrobial agents (quinoxalines, quinolones, tigecycline, nitrofurantoin, and chloramphenicol), as well as detergents and disinfectants [59]. The function of the E. coli AcrB transporter is dependent on TolC, a multifunctional outer membrane channel [61,62], and a homologous gene with 75% amino acid sequence identity to TolC in Salmonella enterica was identified upstream of the C. neteri acrB/oqxB gene (JT31_17100), suggesting a similar tripartite system in this species (Figure 3). Additionally, the small adaptor protein, AcrZ, which interacts with the AcrAB efflux pump, was identified some distance downstream of the acrB gene in a similar genetic organization as the E. coli cluster [63]. The AcrZ protein was shown to aid in the binding and export of chloramphenicol and tetracycline by the AcrAB efflux pump, thus enhancing the drug resistance phenotype of E. coli [64]. In our analysis, a BLAST search suggested that C. neteri JT31_18485 is a homolog of E. coli AcrD, an aminoglycoside efflux pump from the RND family [65]. The JT31_18485 In our analysis, a BLAST search suggested that C. neteri JT31_18485 is a homolog of E. coli AcrD, an aminoglycoside efflux pump from the RND family [65]. The JT31_18485 gene product showed 87% identity in amino acid sequence to AcrD from E. coli and Shigella sonnei. In addition, homologous genes encoding the MdtABCD efflux pump system were identified on the C. neteri SSMD04 chromosome (Figure 3). The C. neteri mdtABCD locus encodes a putative membrane fusion protein (mdtA), two RND-type transporters (mdtB and mdtC), and an MFS-type transporter (mdtD). The predicted MdtB and MdtC transmembrane exporter subunits in C. neteri exhibited high amino acid sequence identity (89% and 90%, respectively) to their counterparts in E. cloacae and E. coli. MdtABCD has previously been shown to comprise a multidrug efflux system that confers resistance to novobiocin and deoxycholate [66,67]. The genes baeS and baeR, which encode a two-component signal transduction system, were identified immediately downstream of the C. neteri mdtABCD locus in a similar genetic organization as described for E. coli. The BaeSR two-component system positively regulates drug resistance in E. coli via the MdtABCD multidrug efflux system [67,68], suggesting that the predicted mdtABCD locus in C. neteri may be under the transcriptional control of the BaeR response regulator. These putative RND efflux pump systems likely contribute to the intrinsic antimicrobial drug resistance reported for clinical isolates of C. neteri. However, the functional role of these homologs of RND efflux pumps and the identity of the specific drug substrates of each pump remain to be established.
A KEGG database search also revealed two pairs of linked genes annotated as emrA-like and emrB-like MDR transporters of the MFS family on the chromosomes of all three C. neteri strains. In C. neteri SSMD04, these emrA/emrB-like gene pairs are JT31_07770/JT31_07775 and JT31_16920/JT31_16915. Multiple sequence alignment showed that the JT31_16920/JT31_16915 pair had the highest sequence identity with the known EmrAB counterpart in Klebsiella pneumoniae, Salmonella enterica, and E. coli. The emrAB locus in E. coli encodes a MDR pump involved in the extrusion of chemically unrelated antimicrobial agents, including the antibiotics nalidixic acid and thiolactomycin [69]. The putative SSMD04 emrB, which encodes a MFS-type efflux pump, shared 93% amino acid sequence identity with K. pneumoniae EmrB, 92% identity with E. coli EmrB, and 90% identity with S. enterica EmrB. The putative emrA, which encodes a membrane fusion protein, shared high amino acid sequence identity (82%-86%) with EmrA in K. pneumoniae, E. coli, and S. enterica. In addition, a gene (JT31_16925) annotated as mprA is located adjacent to emrA. Previous research demonstrated that mprA (renamed emrR) is part of the emrAB operon and functions to repress transcription of emrAB [70]. A recent study showed that the EmrAB pump system contributes to colistin resistance in the nosocomial pathogen Acinetobacter baumannii [71]. Colistin resistance has been noted as one of the defining properties characterizing Cedecea species and is a trait shared by established opportunistic pathogens in the genus Serratia [1,10], but the specific mechanism conferring this resistance in Cedecea is not known. The possibility that the predicted MFS-type EmrAB efflux pump system may be responsible, at least in part, for the colistin resistance phenotype in Cedecea should be explored further, particularly since these species are gaining increased recognition as opportunistic pathogens in the clinical setting.

Comparison of Predicted Genomic Islands in C. neteri and K. michiganensis
Comparative sequence analysis of chromosome-encoded β-lactamase genes in C. neteri SSMD04 revealed a high degree of amino acid sequence identity (89-96%) to orthologous genes in K. michiganensis RC10 (Table 2). K. michiganensis is an emerging human pathogen that was recently identified as the causative agent of bacteremia in a neutropenic patient with acute myeloid leukemia [72]. Clinical cases of C. neteri are markedly similar to the reported K. michiganensis case in terms of clinical presentation and the opportunistic nature of infection. Therefore, we performed a whole-genome comparison of C. neteri SSMD04 and K. michiganensis RC10 to further explore the genetic similarity of these two organisms. The SSMD04 and RC10 genomes share a high average pairwise amino acid identity of 95%, whereas the average pairwise nucleotide identity of 88.8% indicates greater interspecies genetic divergence at the nucleotide level.
IslandViewer 4 [35] was utilized to predict GIs present in the genomes of C. neteri and K. michiganensis, as well as homologs of antimicrobial resistance genes. GIs represent gene regions that originated through probable horizontal gene transfer mechanisms and disproportionately encode for phenotypes that confer enhanced adaptability or competitiveness to microorganisms [35]. Acquired phenotypes include antimicrobial resistance, metal resistance, pathogenicity, as well as phenotypes that contribute to intraspecies diversity and ecological adaptations. In our analysis, two predicted GIs (denoted as GI-1 in Figure 4), one in the C. neteri genome and the other in the K. michiganensis genome, were strikingly similar in terms of the annotations of clustered genes (Table 3), indicating a probable horizontal transfer event between these bacteria or with a common source. These GIs have not been characterized previously. Notably, several annotated genes clustering on both GI-1s are associated with drug resistance and virulence ( Table 3). The first gene, hlyD, encodes a membrane fusion protein that is a component of the type 1 secretion machinery responsible for secretion of hemolytic toxin HlyA in uropathogenic E. coli strains [73]. The second gene encodes a type I secretion system (T1SS) permease/ATPase and is involved in the secretion of various factors to the extracellular space for biofilm formation and host invasion [74]. Both GI-1s also shared a putative tolC, which encodes a multifunctional outer membrane channel. E. coli TolC interacts with various inner membrane transporters (e.g., AcrAB, AcrD, and MdtABC) and forms part of the HlyA T1SS complex (reviewed in [75]), thereby playing critical mechanistic roles in the excretion of a wide range of molecules, including antibiotics [76], bile salts [77,78], antimicrobial peptides such as colicin V [79], and the toxin, α-hemolysin [80,81]. Inactivation of tolC has been shown to result in bacterial susceptibility to various antibiotics [82]. The C. neteri and K. michiganensis GI-associated tolC shared 96% sequence identity at the amino acid level. Both GI-1s harbored genes encoding transposases and integrases, suggesting that tolC was acquired by horizontal means involving genetic transposition.  , and IslandPick (green) methods using the webserver IslandViewer 4 [35]. Selected genomic islands are labeled GI-1 through GI-4. Predicted AMR genes (pink circles) are also shown.  Solid rectangles denote GIs predicted by IslandPath-DIMOB (blue), SIGI-HMM (orange), and IslandPick (green) methods using the webserver IslandViewer 4 [35]. Selected genomic islands are labeled GI-1 through GI-4. Predicted AMR genes (pink circles) are also shown. Interestingly, the tolC-harboring GI-1s in the C. neteri and K. michiganensis chromosomes reside in the same genomic region and in close proximity to genes encoding the RND efflux pump system AcrAB/OqxAB and the multidrug resistance efflux complex, EmrAB, both of which have been described previously in this report. Like the AcrAB efflux pump, the E. coli EmrAB pump forms a tripartite efflux system with the outer membrane channel TolC, with the membrane fusion EmrA protein directly interacting with TolC to form an extended periplasmic canal [83]. A gene encoding EmrR, a MarR family-type transcriptional repressor, was also found adjacent to the emrAB operon in the SSMD04 and RC10 genomes. In general, homologous genes encoding previously characterized antimicrobial resistance (AMR) genes were not found within predicted GIs, suggesting that known AMR genes in C. neteri SSMD04 and K. michiganensis RC10 comprise part of the intrinsic drug resistance mechanisms in these organisms. Putative AMR genes were distributed across the entire SSMD04 and RC10 genomes (Figure 4).
Other GIs predicted for C. neteri and K. michiganensis contained phage proteins, transposon-related proteins, insertion sequences, and numerous hypothetical proteins. Genomic islands 2 and 4 in C. neteri SSMD04 contained largely phage-associated proteins and hypothetical proteins (Figure 4). Seven arsenical resistance-related genes were identified on GI-2 in K. michiganensis RC10 and included an arsenical efflux pump system, a transcriptional repressor (ArsD) of the arsenical resistance operon, and arsenate reductase, as well as 23 hypothetical proteins. A similar metal resistance GI was not identified in C. neteri SSMD04. The RC10 GI-2 also contained an unknown toxin and antitoxin, as well as a MFS transporter gene (VW41_RS19820, WP_045783884.1). Furthermore, GI-2 in K. michiganensis was located on the chromosome in close proximity to an unknown MFS transporter gene (VW41_RS19520, WP_045783865.1). GI-3 in the RC10 genome contained genes encoding numerous hypothetical proteins as well as unknown transcriptional regulators, conjugal transfer protein TraG, and multiple integrating conjugative element proteins.
Comparative GI analysis revealed a gene encoding the virulence factor SrfB on GI-3 in C. neteri SSMD04 (locus JT31_RS01505, GenBank accession no. WP_038472517.1) and GI-4 in K. michiganensis RC10 (locus VW41_RS04305, GenBank accession no. WP_045781468.1). The putative srfB (JT31_01560) on SSMD04 GI-3 is immediately flanked by two ORFs annotated as hypothetical proteins. BLAST searches and multiple sequence alignments suggest that the two hypothetical ORFs linked with srfB may represent srfA (JT31_01555) and srfC (JT31_01565). ORF JT31_01555 showed 53% identity at the deduced amino acid level with S. enterica SrfA, and JT31_01565 showed 41% identity with S. enterica SrfC. Initially identified in S. enterica [84], the srfABC operon encodes a tripartite toxin that was shown to exhibit injectable insecticidal activity [85]. Recently, Sun et al. [86] demonstrated that each component of the SrfABC toxin is capable of independently inducing varying degrees of cytotoxicity and apoptosis in human cervical carcinoma cells, while all three proteins are required for full cytotoxicity. The presence of srfB on a predicted GI in C. neteri and K. michiganensis suggests its acquisition via a horizontal gene transfer mechanism and raises the question of whether the product of srfB contributes to the opportunistic pathogenicity of these organisms.

Conclusions
Cedecea species are members of the Enterobacteriaceae family that have been found in a wide range of natural environments, as well as in human clinical specimens. Reported clinical isolates have been associated with a spectrum of acute infections (e.g., pneumonia, bacteremia, oral ulcers, and dialysis-related peritonitis) in primarily immunocompromised hosts, and antibiotic susceptibility testing has indicated varying degrees of drug resistance among documented isolates. As emerging opportunistic pathogens of environmental origin, C. neteri and the closely related species C. davisae and C. lapagei have received little research attention to date. This study exploited whole-genome sequence information for three C. neteri strains (SSMD04, M006, and ND14a) to gain a deeper understanding of the genetic potential for drug resistance in this species and to identify drug-resistance candidate genes for further investigation. We focused our genomic analyses on C. neteri SSMD04, an isolate originating from retailed sashimi, since only a draft genome was available for type strain C. neteri ATCC 33855. Our work reports the presence of multiple β-lactamase-encoding genes in the C. neteri SSMD04 chromosome, including four putative MBLs, a CMY/ACT-type AmpC variant, and a novel β-lactamase gene not described previously. Homologous genes encoding RND-and MFS-type efflux pumps were also identified, along with associated regulatory genes known to be involved in the control of these efflux systems in other bacteria. Comparative analysis of predicted genomic islands suggested the acquisition of some drug resistance determinants and virulence factors by horizontal genetic transfer. The findings of this study advance our currently limited understanding of the molecular basis of antimicrobial resistance in C. neteri. Future research is needed to correlate the genetic data with resistance phenotypes impacting public health management of this opportunistic pathogen.