Superficieibacter electus gen. nov., sp. nov., an Extended-Spectrum β-Lactamase Possessing Member of the Enterobacteriaceae Family, Isolated From Intensive Care Unit Surfaces

Two Gram-negative bacilli strains, designated BP-1(T) and BP-2, were recovered from two different Intensive Care Unit surfaces during a longitudinal survey in Pakistan. Both strains were unidentified using the bioMerieux VITEK MS IVD v2.3.3 and Bruker BioTyper MALDI-TOF mass spectrometry platforms. To more precisely determine the taxonomic identity of BP-1(T) and BP-2, we employed a biochemical and phylogenomic approach. The 16S rRNA gene sequence of strain BP-1(T) had the highest identity to Citrobacter farmeri CDC 2991-81(T) (98.63%) Citrobacter amalonaticus CECT 863(T) (98.56%), Citrobacter sedlakii NBRC 105722(T) (97.74%) and Citrobacter rodentium NBRC 105723(T) (97.74%). The biochemical utilization scheme of BP-1(T) using the Analytic Profile Index for Enterobacteriaceae (API20E) indicated its enzymatic functions are unique within the Enterobacteriaceae but most closely resemble Kluyvera spp., Enterobacter cloacae and Citrobacter koseri/farmeri. Phylogenomic analysis of the shared genes between BP-1(T), BP-2 and type strains from Kluyvera, Citrobacter, Escherichia, Salmonella, Kosakonia, Siccibacter and Shigella indicate that BP-1(T) and BP-2 isolates form a distinct branch from these genera. Average Nucleotide Identity analysis indicates that BP-1(T) and BP-2 are the same species. The biochemical and phylogenomic analysis indicate strains BP-1(T) and BP-2 represent a novel species from a new genus within the Enterobacteriaceae family, for which the name Superficieibacter electus gen. nov., sp. nov., is proposed. The type strain is BP-1(T) (= ATCC BAA-2937, = NBRC 113412).


INTRODUCTION
The hospital built environment is a key source of nosocomial pathogens, which can cause high mortality infections in vulnerable patient populations (Oberauner et al., 2013). The danger from these infections is exacerbated by the higher levels of antibiotic resistance harbored by nosocomial pathogens compared to their community-associated relatives (Horcajada et al., 2013). As many pathogens can persist on surfaces for extended periods of time, microbiological and molecular surveillance of high-contact surfaces in health-care settings is an important feature of effective infection control and prevention strategies (Russotto et al., 2015). The isolates reported herein were recovered as part of such a surveillance program of intensive care unit (ICU) surfaces at a tertiary care hospital in Pakistan in 2016.
The Enterobacteriaceae is a diverse family of Gammaproteobacteria that includes many common human pathogens (Paradis et al., 2005;Potter et al., 2016). Enterobacteriaceae taxonomy is complicated, as demonstrated by the use of multilocus sequence analysis to reclassify several Enterobacter species into the Lelliottia, Pluralibacter and Kosakonia genera (Brady et al., 2013). Next-generation sequencing has significantly improved our understanding of Enterobacteriaceae taxonomy by identifying similarities between species of different genera (e.g., Salmonella subterranean and Escherichia hermannii into Atlantibacter subterranea and Atlantibacter hermannii, respectively) or resolving closely related species within the same genus (e.g., Klebsiella pneumoniae and Klebsiella variicola) (Holt et al., 2015;Hata et al., 2016). Many Enterobacteriaceae strains are medically relevant not only as etiological agents of disease but also as reservoirs for transferable antibiotic resistance genes (Sirot et al., 1988). Thus, documenting the appearance of novel Enterobacteriaceae species in hospital settings is an important component in surveilling and preventing emerging infectious diseases (Iredell et al., 2016).

Bacterial Isolation
We isolated strain BP-1(T) (bedside rail) and BP-2 (nurse call button) from an intensive care room at a tertiary care hospital in Pakistan with the Eswab collection device and transport system (Becton Dickenson & Company, Franklin Lakes, NJ) in June 2016. Surfaces were swabbed in triplicate during an ongoing yearlong longitudinal sampling study. Swabs were sent to the US at room temperature and arrived in the US within ∼3 days of sampling. Samples from Pakistan were received by the Burnham lab under approval of the Washington University IBC Protocol number 6,572. Swabs were cultured within 24 h of arrival in the US laboratory site. The isolates were recovered on MacConkey agar with 5 µg/ml cefotaxime (Hardy Diagnostics, Santa Maria, CA) and re-streaked on blood agar using the four-quadrant method to recover distinct single colonies. The isolates were unidentified by the MALDI-TOF VITEK MS IVD v2.3.3 (bioMerieux,Durham,NC) and Bruker BioTyper (Bruker Daltonics, Billerica, MA) mass spectrometry systems. For subsequent genomic analysis, frozen stocks of each isolate were made by creating a dense suspension of the isolate in Tryptic Soy Broth (Sigma Aldrich, St. Louis, MO) with 15% Glycerol (Hardy Diagnostics, Santa Maria, CA).

Transmission Electron Microscopy
Glycerol stocks of strain BP-1(T) and BP-2 were plated onto MacConkey agar with ceftriaxone (5 µg/ml) overnight. A single colony of each isolate was separately inoculated into 5 ml of LB broth and grown until log phase at 37 • C with orbital shaking at 220 rpm. For analysis of whole bacteria by negative staining, the isolates absorbed onto formvar/carbon-coated copper grids for 2 min. Grids were washed in dH 2 O and stained with 1% aqueous uranyl acetate (Ted Pella Inc., Redding CA) for 1 min. Excess liquid was gently wicked off and grids were air dried. Bacteria viewed on a JEOL 1200 EX transmission electron microscope (JEOL USA Inc., Peabody, MA) equipped with an AMT 8megapixel digital camera (Advanced Microscopy Techniques, Woburn, MA).
For ultrastructural analysis of cross-sections of bacteria, the isolates were fixed in 2% paraformaldehyde/2.5% glutaraldehyde in 100 mM cacodylate buffer, pH 7.2 for 1 h at room temperature. Samples were washed in cacodylate buffer and postfixed in 1% osmium tetroxide (Polysciences Inc.) for 1 h. Samples were then rinsed extensively in dH 2 0 prior to en bloc staining with 1% aqueous uranyl acetate (Ted Pella Inc., Redding, CA) for 1 h. Following several rinses in dH 2 0, samples were dehydrated in a graded series of ethanol and embedded in Eponate 12 resin (Ted Pella Inc.). Sections of 95 nm were cut with a Leica Ultracut UCT ultramicrotome (Leica Microsystems Inc., Bannockburn, IL), stained with uranyl acetate and lead citrate and viewed on the transmission electron microscope.

16S rRNA Similarity
16S rRNA sequences from the BP-1(T) file were identified using Barrnap with the command: "barrnap -quiet <contigs.fasta>" (https://github.com/tseemann/barrnap) and manually retrieved. The sequence is on contig_69 from base pairs 22 to 1559. The complete 16S rRNA sequence was submitted to the EzBioCloud identify service on 08/31/17 (Kim et al., 2014;Yoon et al., 2017). The 16S rRNA sequences for the top 10 most similar taxa were retrieved and aligned using MUSCLE (Edgar, 2004) using the command "muscle -in <input_multifasta> -out <output_aligned_multifasta>." The aligned 16S rRNA multifasta was made into a phylogenetic tree with 1,000 bootstraps using raxML with the command: "raxmlHPC -s <input_aligned_multifasta> -n output -m GTRGAMMA -p 100 -f a -N 1000 -x 12345." The resulting newick tree was visualized in FigTree with bootstrap values depicted as node label. The aligned multifasta sequences were uploaded into Jalview v2.0 and colored by nucleotide (Waterhouse et al., 2009). The 16S rRNA sequence has been submitted to Genbank under accession number MG866003.

Core Genome Phylogeny
Fifty-six genomes from type strains in species of Escherichia, Shigella, Citrobacter, Salmonella, Kluyvera, Klebsiella, Kosakonia, Siccibacter and Pasteurella (Table S1) were obtained from NCBI Genomes on 09/01/17. The protein coding sequences of these genomes and strains BP-1(T) and BP-2 were annotated using Prokka with the previously described command (Seemann, 2014). The core genome, representing genes shared by 100% of all isolates in the cohort was constructed and aligned using the PRANK module within roary with the following command: "roary -e -group_limit 700000 -p 8 -i <percent identity> * .gff " (Page et al., 2015). Eighty percent of gene identity was used for core genome alignment of Enterobacteriaceae genomes plus the P. multocida outgroup and for core genome alignment of only the Enterobacteriaceae genomes.

Average Nucleotide Identity
Whole genome sequences of strains BP-1(T) and BP-2 were compared against each other using the mummer method of average nucleotide identity via the jspecies webserver (http://jspecies.ribohost.com/jspeciesws/) on 09/18/17 (Richter et al., 2016). The contig file for strains BP-1(T), BP-2 and the set of Enterobacteriaceae genomes (Table S1) were compared using the mummer method of average nucleotide identity via the standalone pyANI tool (https://github.com/widdowquinn/pyani) with command "python3 average_nucleotide_identity.py The Hadamard matrix representing the multiplication of the percentage identity and alignment length was converted into a dendrogram using scipy.cluster.hierarchy.dendrogram. within python3 using "euclidian" metric and "weighted" method. The dendrogram was viewed in matplotlib.

Phenotypic Antibiotic Susceptibility Testing
Antimicrobial susceptibility testing was performed on the isolates using Kirby Bauer disk diffusion on Mueller Hinton Agar (Hardy Diagnostics, Santa Maria, CA) according to CLSI standards (CLSI, 2013). Results were interpreted using CLSI M100 criteria for Enterobacteriacace (CLSI, 2013).

Colony Appearance and Ultrastructural Analysis
Both strains BP-1(T) and BP-2 appeared identical on agar plates. BP-1(T) formed circular, shiny, mucoid, gray colonies and were non-hemolytic on blood agar plates ( Figure 1A). The colonies were small, blue and rigid on HardyCHROM TM ESBL Agar ( Figure 1B). BP-1(T) colonies were bright pink on MacConkey agar (i.e., lactose fermenting) with an opaque zone surrounding the edge of the colony ( Figure 1C). Coral colored colonies were produced on HE agar ( Figure 1D).
Analysis by negative staining and transmission electron microscopy revealed that BP-1(T) and BP-2 are rod shaped and ∼2.7 µm in length (Figures 2A,B). Cross sections from resin embedding also show rod shaped bacteria (Figures 2C,D) with two electron dense lipid bilayers of the outer membrane and cytoplasmic membrane ( Figure 2E). No evidence of flagella was observed, but pili-like structures were present between a group of isolates (Figures 2F,G).

Biochemical and Temperature Studies
Strains BP-1(T) and BP-2 had identical results from phenotypic assays (Table 1). Aerobically, BP-1(T) showed no growth at 4 • C, scant growth at 25 and 42 • C, but robust growth at 35 • C ( Table 1). BP-1(T) and BP-2 both showed robust growth anaerobically at 35 • C using an anaerobic bag ( Table 1). The isolates were negative for motility, oxidase, indole, catalase and urease. Both strains BP-1(T) and BP-2 were positive for gas production in TSI and LIA reactions ( Table 1). The strains grew on bile esculin agar and can ferment glucose ( Table 1).
As evaluated using the API 20E, BP-1(T) and BP-2 were positive for arabinose, amygdalin, melibiose, sacharose, rhamnose, sorbitol and mannitol fermentation, but negative for inositol fermentation ( Table 2). BP-1(T) and BP-2 can utilize citrate and decarboxylate ornithine for metabolism in addition to utilizing disaccharides (melibiose) ( Table 2). These biochemical test results were unable to assign a high confidence genus identification to strains BP-1(T) and BP-2. The analytic profile index (API) algorithm indicates that strains BP-1(T) and BP-2 most resembles Kluyvera spp. and to a lesser extent E. cloacae and Citrobacter koseri/farmeri ( Table 3). Analysis of the biochemical utilization profile from these related taxa indicate that strains BP-1(T)/BP-2 and Kluyvera cryocrescens ATCC 33435 are both negative for arginine dihydrolase activity but E. cloacae and C. koseri are positive for it. Additionally, strains BP-1(T)/BP-2, E. cloacae and C. koseri can ferment D-sorbitol but K. cryocrescens ATCC 33435 cannot. Both BP-1(T) and BP-2 were tested as non-motile but K. cryocrescens ATCC 33435, E. cloacae and C. koseri are positive. Accordingly, BP-1(T)/BP-2 can be uniquely distinguished from its closest neighbors based on being negative for arginine hydrolase activity, positive for D-sorbitol fermentation and non-motile.

Phylogenomic Analysis
We used a phylogenomic approach to better delineate the taxonomic context of strains BP-1(T) and BP-2 within the Enterobacteriaceae. Submission of the 1,512-base pair 16S rRNA locus from strain BP-1(T) to the EzBioCloud taxonomic database indicated that strain BP-1(T) had the highest identity to Citrobacter farmeri CDC 2991-81(T), C. amalonaticus CECT 863(T), C. sedlakii NBRC 105722(T) and C. rodentium NBRC 105723(T) ( Table 4) Figure S1). The greatest amount of variation in 16S rRNA identity occurred at the likely V3 region, around nucleotides 426-450 of the BP-1(T) sequence, using standard E. coli nomenclature (Chakravorty et al., 2007). The 6mer of GAGAAT in the BP-1(T) sequence from sites 426 to 435 is unique in comparison to the other sequences at this position. Although the GAG nucleotides from 426 to 428 are shared by K. sacchari SP1(T)/P. gergoviae JCM 1234(T) and the AT site at 434-435 is shared by C. farmeri CDC 2991-81 T , the A residue at position 429 is discriminatory, as all other taxa contain a thymine at this site ( Figure S2). As 98.7-98.65% is a proposed standard for species delineation via rRNA gene sequencing, we endeavored to gain further insight into the taxonomic identity of BP-1(T) and BP-2 based on their whole genome sequence characteristics.
To determine the relative taxonomic identity of strains BP-1(T) and BP-2 beyond 16S rRNA similarity, we constructed an equal angle neighbor net tree produced from the aligned 750 core genes shared between type strains in Enterobacter,  Siccibacter, Escherichia, Enterobacter, Kosakonia, Kluyvera, Klebsiella, Shigella, Salmonella and Citrobacter at >80% identity (Table S1). Analysis of the network indicates that strains BP-1(T) and BP-2 cluster together and distinctly from these major Enterobacteriaceae taxa (Figure 3A).
A maximum likelihood tree from the 48 genes shared between the same Enterobacteriaceae genomes and a Pasteurella multocida outgroup with >80% identity again placed strains BP-1(T) and BP-2 in a cluster separate from existing genera. The BP-1(T)/BP-2 branch separates the genomes analyzed into two major clades composed of Escherichia [excluding Escherichia vulneris NBRC 192420(T), which other groups have suggested is misclassified (Walk et al., 2009)], Shigella, Citrobacter, Salmonella in one branch and Klebsiella, Kluyvera, Enterobacter, Siccibacter and Kosakonia on the other branch ( Figure 3B).
Pairwise Average Nucleotide Identity (ANI) analysis using MUMer between strains BP-1(T) and BP-2 indicate that they are the same species, as the ANI value of 99.97% is greater than the proposed species cutoff of 95% (Thompson et al., 2013). The dendrogram of a distance matrix representing the product of ANI and percentage of the genome alignment created a clustermap that is similar to the rooted Phylogenetic tree with BP-1(T) and BP-2 separating the Escherichia, Shigella, Citrobacter and Salmonella clade from the Klebsiella, Kluyvera, Enterobacter, Siccibacter and Kosakonia branch (Figure 4). While the ordering of the two Citrobacter clades, Salmonella and Escherichia/Shigella is similar between the ANI dendrogram and the core genome phylogenetic tree, in the ANI dendrogram Kosakonia and not Kluyvera/Klebsiella is the closest genera to BP-1(T)/BP-2.
Preceding it was a tetR response regulator with similarity to a protein from Klebsiella sp. RIT-PI-d (WP_049839935.1).
To gain further insight into the potential for antibiotic resistance gene dissemination from strains BP-1(T) and BP-2, we analyzed the draft genomes of BP-1(T) and BP-2 for contigs that had ARGs co-localized with mobile gene elements. An IS1380 family transposase was found co-localized in both genomes with an erythromycin resistance gene ere(A), a putative rifampin resistance gene that was unidentified by ResFinder or Resfams, a putative aminoglycoside N-acetyltransferase unidentified by Resfinder or Resfams, a hypothetical protein and aac(6 ′ )-IIC ( Figure 5).

DISCUSSION
The purpose of our investigation was to determine the taxonomic identity of strains BP-1(T) and BP-2, two isolates recovered from selective culturing of swabs of two Pakistani ICU room surfaces which could not be identified using MALDI-ToF mass spectroscopy. The colony morphology of strains BP-1(T) and BP-2 on blood agar is indistinguishable from non-swarming Enterobacteriaceae, but the results of growth on MacConkey plates and ChromID ESBL agar show phenotypic similarities to lactose fermenting and extended spectrum β-lactamase producing species such as E. coli and K. pneumoniae (Grohs et al., 2013). Ultrastructural analysis showed morphological and size similarity to other Enterobacteriaceae species (Feng et al., 2014). Consistent with the observed lack of a flagellum, strains BP-1(T) and BP-2 were non-motile like Yersinia, Klebsiella and Shigella genera of Enterobacteriaceae (Kramer et al., 2006).
Differential growth assays and API results were not able to assign strains BP-1(T) and BP-2 to an existing genus with high confidence. API testing determined that both isolates share a unique suite of phenotypes that distinguish BP-1(T) and BP-2 from other Enterobacteriaceae genera. The closest identity to strains BP-1(T) and BP-2 based on biochemical utilization and API algorithm predictions were Kluyvera species (41.2%), E. cloacae (30.7%) and Citrobacter koseri/farmeri (8.4%). Strains BP-1(T) and BP-2 differed from these most closely related taxa by API in arginine dihydrolase, indole production, acetoin production, sorbitol fermentation and motility.  To further probe the appropriate classification of BP-1(T) and BP-2, we performed Illumina whole genome sequencing on both isolates. As the BP-1(T) 16S rRNA gene sequence is 98.7% identical to the C. farmeri CDC 2991-81 rRNA gene, we speculated that these isolates may represent a novel species. To gain greater taxonomic resolution beyond the limitations of using just the 16S rRNA sequence, we identified and aligned all shared protein coding sequences between strains BP-1(T), BP-2 and type strains from Enterobacter, Klebsiella, Kluyvera, Citrobacter, Kosakonia, Escherichia, Shigella, Salmonella, and Siccibacter. Although the BP-1 T 16S rRNA sequence had the greatest similarity to several Citrobacter species, the unrooted NeighborNet tree and rooted maximum likelihood tree indicate BP-1(T) and BP-2 form a cluster separate from known Enterobacteriaceae species. A rooted phylogenetic tree placed the taxonomic position of BP-1(T) and BP-2 in between two large clades; one clade contains Escherichia, Shigella, Salmonella and Citrobacter and the other contains Klebsiella, Kluyvera, Enterobacter, Siccibacter and Kosakonia. ANI is the accepted in silico version of a DNA-DNA hybridization assay, the gold standard for demarcation of new bacterial species (Richter and Rossello-Mora, 2009). The 99.97% ANI between strains BP-1(T) and BP-2 indicate that they are the same species and possibly clonal isolates. A dendrogram of the ANI output reflected a tree topology like the rooted core genome tree but had Kosakonia and not Kluyvera/Klebsiella as the closest genera to BP-1(T) and BP-2.
Both strains BP-1(T) and BP-2 are multidrug resistant Enterobacteriaceae which harbor extended spectrum βlactamases and are therefore classified as a serious antimicrobial threat by the CDC (CDC, 2013;Iredell et al., 2016). The Class A β-lactamase identified by Resfams had high identity to bla HER family genes, which were previously described in Atlantibacter hermanii but only conferred resistance to penicillins (Beauchef-Havard et al., 2003). As this gene was proximal to a tetR response regulator, it is possible that it may be inducible, like the ampR/ampC system widespread in Enterobacter and Citrobacter (Vadlamani et al., 2015). However further work comparing the expression levels under basal or induced conditions would be needed to ascertain this. TEM-1 βlactamase genes are widespread in E. coli and K. pneumoniae and can confer resistance to penicillins and first/second generation cephalosporins, but not 3rd generation drugs like ceftazidime (Cantu et al., 1997). bla TEM−1b , the variant found in strains BP-1(T) and BP-2 is not considered an ESBL (Udomsantisuk et al., 2011). Therefore, a combination of blaTEM-1 and bla SHV−12 are therefore likely the factor most critical for cephalosporin resistance (Teshager et al., 2000;Newire et al., 2013). However, efflux and porin activity may augment β-lactamase production to achieve clinical resistance, as seen in other Enterobacteriaceae species (Wozniak et al., 2012;Taherpour and Hashemi, 2013). Several predicted antibiotic resistance genes in these genomes were co-localized with IS1247, an IS1380 family transposase. A previously discovered IS1380 family member, ISEcp1 can transfer bla CTX−M via transposition (Poirel et al., 2005;Toleman et al., 2006).
One limitation of our study is that we have only procured two strains and given the temporal similarity they may be clonally related. Although not ideal from a taxonomic perspective, the Enterobacteriaceae species Klebsiella michiganensis, Enterobacter soli, Enterobacter muelleri and genera Chania and Nissabacter were all established from single strain investigations (Manter et al., 2011;Saha et al., 2013;Kampfer et al., 2015;Ee et al., 2016;Mlaga et al., 2017). This study further demonstrates the utility of whole genome sequencing for pathogen identification in a clinical setting. Similarity, another report found that Vitek2 identified a blood culture derived isolate as E. cloacae but Illumina whole genome sequencing unequivocally determined it to be Kosakonia radicincitans (Bhatti et al., 2017). Further comprehensive work is therefore warranted to investigate clinical outcomes that result from these species misidentification.
Further work using long-read assembly such as PacBio or Oxford Nanopore is warranted to construct a highquality complete genome. These additional efforts may also unambiguously identify plasmid components that replicate independently of the chromosome but could be collapsed into the same fasta file using our draft genome approach. This may additionally inform the presence of specific ARGs as chromosomal or plasmid-borne. Unfortunately, these techniques were not available to us during the course of this investigation.
Description of Superficieibacter gen. nov.
Cells are facultative aerobic Gram-negative bacilli visualized without chains but occasionally in clusters under electron microscopy. Non-motile with no evidence of a flagella. Cells are mesophilic with robust growth at 35 • C, scant growth at 25 and 32 • C and no growth at 4 • C. Cells can grow anaerobically at 35 • C. Cells are catalase positive and can produce gas in the TSI and LIA slants. Cells can ferment arabinose, amygdalin, melibiose, sacharose, rhamnose, sorbitol, lactose, glucose and mannitol. Strains can be differed from related genera by being negative for arginine dihydrolase, indole production, acetoin production and motility but positive for sorbitol fermentation. Members belong to class Gammaproteobacteria, order Enterobacteriales and family Enterobacteriaceae. The type species of the genus is Superficieibacter electus.

AUTHOR CONTRIBUTIONS
RP and AD wrote the manuscript and performed in silico analysis. MW and AS performed culture work, growth assays and API test. DG isolated the bacteria from hospital surfaces. SP extracted genomic DNA and prepared Illumina sequencing libraries. WB performed electron microscopy. JK, SA, C-AB and GD devised the study.