Two novel Enterobacter species, Enterobacter chinensis sp. nov. and Enterobacter rongchengensis sp. nov., recovered from clinical samples carrying multiple virulence factors

ABSTRACT Two Enterobacter strains 170198T and 170250T were isolated from clinical blood samples from distinct patients in a hospital in Chengdu, China, in 2022. These isolates were subjected to whole-genome sequencing. A phylogenomic tree based on 2,096 concatenated core genes showed that the two strains were clustered within the genus Enterobacter. The average nucleotide identity (ANI) and in silico DNA–DNA hybridization (isDDH) values between each of the two strains and type strains of all currently known Enterobacter species were determined. The two strains belonged to two novel species as the highest ANI and isDDH values with type strains of all currently known Enterobacter species below the cutoff for species demarcation (96% for ANI and 70% for isDDH). Then the physiological and biochemical studies demonstrated that biochemical features and the profile of whole fatty acids of strains 170198T and 170250T were largely consistent with those known Enterobacter species. Nevertheless, the two novel species can be differentiated from all other Enterobacter species by certain biochemical characteristics. In conclusion, 170198T and 170250T represent two novel species of the genus Enterobacter, for which we propose Enterobacter chinensis sp. nov. and Enterobacter rongchengensis sp. nov., as the species names. The type strains of Enterobacter chinensis sp. nov., and Enterobacter rongchengensis sp. nov. are 170198T (=GDMCC 1.3549T=JCM 35826T) and 170250T (=GDMCC 1.3670T=JCM 36189T), respectively. The two novel species have clinical significance with the ability to cause bloodstream infections. IMPORTANCE Enterobacter is a group of bacteria comprising several common opportunistic pathogens and has a complicated taxonomy. Here, we reported two novel Enterobacter species. We demonstrated that the two novel species can be differentiated from other Enterobacter species by certain phenotypic characteristics and therefore provide information for designing tests for identification. We also showed that strains of the two novel species are able to cause human bloodstream infections and carry multiple virulence factors and therefore are of clinical significance. We highlight that the virulence of Enterobacter is less studied and warrants further exploration. We believe that the findings here are valuable for enhancing the appreciation toward Enterobacter, an important pathogen.

under the order Enterobacterales (2).Enterobacter is widely distributed in nature, and some species are common nosocomial opportunistic pathogens causing a wide range of infections (3).Notably, Enterobacter cloacae complex is a common term used clinically and comprises several species sharing ≥60% overall nucleotide similarity of genomes (4)(5)(6).Currently, there are 23 species of the genus Enterobacter (7)(8)(9), and Enterobacter is closely related to the genera of Huaxiibacter, Leclercia, Lelliottia, and Pseudoenterobacter (10)(11)(12).Here, we reported two novel Enterobacter species via characterization of two stains causing bloodstream infection in hospitalized patients at our hospital in 2022.

Strains and in vitro susceptibility
Strains 170198 T and 170250 T were recovered from the blood samples of two hospi talized patients as part of standard care.The strains were preliminarily identified by Vitek II automated system (bioMérieux, Durham, DC, USA) and using matrix-assis ted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF; Bruker, Billerica, MA, USA; MBI compass version 4.1.100).Antimicrobial susceptibility testing was performed using the broth microdilution method except for fosfomycin, which was determined using the agar dilution method, according to guidelines of the Clinical and Laboratory Standards Institute (CLSI) (13).Antimicrobial agents tested were amikacin, ampicillin, ampicillin-sulbactam, amoxicillin-clavulanate, aztreonam, cefazolin, cefepime, cefotaxime, ceftazidime, ceftriaxone, cefuroxime, cephalothin, ciprofloxacin, colistin, doripenem, ertapenem, fosfomycin, gentamicin, imipenem, levofloxacin, meropenem, moxifloxacin, piperacillin, piperacillin-tazobactam, tetracycline, ticarcillin, tigecycline, tobramycin, and trimethoprim/sulfamethoxazole.The susceptibility results were also interpreted based on CLSI except those of moxifloxacin, ticarcillin, and tigecycline, which were based on European Committee on Antimicrobial Susceptibil ity Testing (http://www.eucast.org/),considering the absence of CLSI breakpoints and categories for these agents.

16S rRNA gene sequence analysis
The nearly complete 16S rRNA gene sequence (1,465 bp) of the strains were obtained using PCR with primers 27F and 1492R (14) and subsequent Sanger sequencing.The 16S rRNA gene sequences were compared with those of type strains of all known species belonging to the genera of Enterobacter, Huaxiibacter, Leclercia, Lelliottia, and Pseudoen terobacter using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

Average nucleotide identity (ANI) and in silico DNA-DNA hybridization (isDDH) analysis
Pairwise average nucleotide identity (ANI) and in silico DNA-DNA hybridization (isDDH) values were determined between each of the strains and the type or reference strains of all known species within the genus Enterobacter and the closely related genera Huaxiibacter, Leclercia, Lelliottia, and Pseudoenterobacter for species demarcation.ANI and isDDH values were calculated using FastANI v1.33 (21) and Genome-to-Genome Distance Calculator (formula 2) (22), respectively.Values of ≥96% ANI (23,24) and ≥70% isDDH (25) were used as the cutoff to define species.

Morphological observation and biochemical assays
A set of morphological and phenotypic experiments were performed to characterize the two strains.The cell morphology and motility of the strains were observed using a CX21FS1 light microscope (Olympus, Tokyo, Japan) and HT7800 transmission electron microscopy (Hitachi, Tokyo, Japan).Gram staining was performed as described previously (28).Bacterial growth was determined at different temperatures (4°C, 15°C, 20°C, 25°C, 30°C, 37°C, 40°C, 42°C, 45°C, and 50°C), at different pH values (3.0-12.0, at intervals of 1.0 pH), and at multiple concentrations of NaCl (0%-10%, wt/vol, at intervals of 1%) after a 2-day incubation in 15-mL test tubes containing 3 mL of tryptic soy broth (TSB; Hopebio, Qingdao, China) placed in a thermostatically controlled water bath as described previously (29).Anaerobic growth was examined using an anaerobic bag (bioMérieux) by incubating the isolates on nutrient agar (Sangon Biotech, Shanghai, China) for 7 days.Catalase activity was tested by dropping 3% (vol/vol) H 2 O 2 on culture grown for 24 h on nutrient agar.Oxidase activity was determined using oxidase strips (Hopebio).Hemolysis was observed on blood agar plates.The physiological and biochemical properties were evaluated using the API 20E and API 50CH kits (bioMérieux) according to the manufacturer's instructions.Escherichia coli strain ATCC 25922 was used as the control.

Whole-cell fatty acid profiling
Whole-cell fatty acid profiles were determined by Guangdong Institute of Microbiology (Guangzhou, China) as described previously (30).

RESULTS
Strains 170198 T and 170250 T were recovered from the blood samples of two hospitalized patients with bloodstream infections.Given that no other pathogens were detected from the blood of the two patients during the disease duration, the two strains were determined as the pathogen causing bloodstream infection.The two patients were in an immunocompromised status, with neutropenia due to cancer chemotherapy or receiving immunosuppressants after a renal transplantation.Both patients had fever but did not develop septic shock and recovered uneventfully after antimicrobial chemother apy, meropenem in one patient and levofloxacin in another.The two clinical strains were identified as E. cloacae complex via conventional identification using the Vitek II and as Enterobacter bugandensis using MALDI-TOF.To determine the exact species of the isolates, we obtained the nearly complete sequence of the 16S rRNA gene of the two strains and found that the 16S rRNA sequence of 170198 T and 170250 T shared the maximum identity with Enterobacter kobei (99.92%) and Huaxiibacter chinensis (98.11%), respectively.Considering the low resolution of 16S rRNA gene analysis for species identification (31), we performed whole-genome sequencing for the two strains and phylogenomic analysis based on core genes.
Whole-genome sequencing generated a total of 10,047,104 [1.51 gigabases (Gb)] and 12,662,322 (1.90 Gb) reads for strain 170198 T and 170250 T , respectively, with a 200× coverage.The draft genome of strain 170198 T was 4.9 Mb containing 28 contigs (≥200 bp; N50, 702,505 bp) with a 55.12 mol% DNA G + C content.The 4.9 Mb draft genome of strain 170250 T contains 89 contigs (≥200 bp; N50, 199,762 bp; 56.18 mol% G + C content).The GTDB tree indicated strains 170198 T and 170250 T were located within the Enterobacter cluster (Fig. S1 in the Supplementary material).However, this phylogenomic analysis only utilized 120 core genes that reflect species characteristics with a relatively lower resolution (32).We therefore inferred another phylogenomic tree based on 2,096 core genes, which also demonstrated that strains 170198 T and 170250 T were located within the genus Enterobacter, most closely to Enterobacter sichuanensis and Enterobacter chengduensis, respectively (Fig. 1).Strain 170250 T had the highest ANI (92.93%) and isDDH (49.9%) values with the type strain of E. chengduensis (accession no.GCA_001984825) (Table 1).Strain 170198 T had the highest ANI (95.58%) and isDDH (64.4%) values with the type strain of Enterobacter asburiae (accession no.GCA_016027695) (Table 1).
Biochemical characteristics of strains 170198 T and 170250 T are shown in Table S1 in the Supplementary material and were compared with those of type strains of species of the genera Enterobacter, Huaxiibacter, Leclercia, Lelliottia, and Pseudoenterobacter (3, 7, 8, 10, 12, 29, 33-39) (Table S2 in the Supplementary material).The two species can be distinguished from each other and all other Enterobacter species by certain characteris tics, which are described in the following species description sections.The major cellular fatty acids of 170198 T and 170250 T are C 16:0 , C 17:0 cyclo, C 18:1 ω7c, and C 16:1 ω7c/ C16:1 ω6c, which is consistent with other Enterobacter species (Table S3 in the Supplementary material).
Four antimicrobial resistant genes, AmpC β-lactamase-encoding bla ACT , fosfomycinresistance gene fosA, and oqxA-oqxB associated with reduced susceptibility to quino lones, all of which are intrinsic, chromosomally located to Enterobacter, were identified from the genome of strains 170198 T and 170250 T .Notably, 170250 T has bla ACT-48 , while 170198 T has a variant of bla ACT encoding a novel ACT of a highest amino acid iden tity (94.23%) with ACT-6.The differences of ACT β-lactamases in the two strains may provide an explanation for the discrepancy of their susceptibility to certain β-lactams.A variety of virulence factors were identified in the genome of both strains 170198 T and 170250 T including aerobactin siderophore encoding the gene iucABCD-iutA, enterobac tin encoding the ent gene cluster, type 1 fimbriae encoding fim operon, and type three fimbriae encoding the gene mrkABCDF, hemorrhagic E. coli pilus (HCP) encoding the gene hcp, curli fiber encoding the gene csg, and components encoding the genes of type VI secretion system.In addition, strain 170198 T harbored genes for endotoxin synthesis and a capsular polysaccharide (CPS) locus (40), while strain 170250 T had genes encoding multiple toxins including alpha-hemolysin, cytolethal distending toxin, and Shiga-like toxin (Table S5 in the Supplementary material).All of the virulence factors are presumably located on the chromosome except for type 3 fimbriae encoding the gene mrkABCDF, which is likely on the plasmid.

DISCUSSION
Strains 170198 T and 170250 T were preliminarily assigned to the Enterobacter cloacae complex by Vitek II, and 16S rRNA gene sequencing failed to assign exact species.We therefore performed whole-genome sequencing for the two strains.We inferred a core-genome phylogenomic tree comprising type strains of all species belonging to Enterobacter and its closely related genera Huaxiibacter, Leclercia, Lelliottia, and Pseudoenterobacter.This allowed us to uncover that strains 170198 T and 170250 T were clustered within the genus Enterobacter.Next, we determined the ANI and isDDH values.Strain 170250 T had the highest ANI (92.93%) and isDDH (49.9%) values with the type strain of E. chengduensis, confirming that it is of a novel species.Strain 170198 T had the highest ANI (95.58%) and isDDH (64.4%) value with the type strain of Enterobacter asburiae.The 95.58% ANI falls into the 95%-96% inconclusive zone for species demarca tion (23,24), but the isDDH values are well below the ≥70% cutoff to define the species (25).Notably, Enterobacter dykesii exhibited even a higher ANI (96.37% with the type strain of Enterobacter muelleri, with a 69.5% isDDH) when it was proposed as a new species (7), and its species status has been validated later (41).Therefore, strain 170198 T also represents a novel Enterobacter species.The biochemical profile and the whole fatty acid composition of the two that were isolated were generally consistent with those of other Enterobacter species with the exception of some specific metabolic characteristics.
In addition, the two strains can be differentiated from all known Enterobacter species by intricate difference of biochemical characteristics (see the following species description sections).
The two strains caused bloodstream infection, indicating that the two novel species are clinically relevant.We therefore examined the profile of virulence factors of the two strains.The two strains were equipped with a variety of virulence factors for adherence (type I fimbriae and HCP), biofilm formation (curli fibrils, HCP, and type 3 fimbriae), invasion (HCP), and iron acquisition (iucABCD-iutA and ent genes) (42)(43)(44)(45)(46).In particular, aerobactin siderophore encoding the gene iucABCD-iutA and enterobactin encoding the ent gene cluster, both for iron acquisition, have been found as major virulence factors in Enterobacter (44).In addition, the two strains had their respective important virulence factors.Notably, strain 170198 T encoded CPS that has been identified as a critical virulence factor for promoting the pathogenesis of Enterobacter infections (47).In contrast, strain 170250 T had genes encoding hemolysin, cytolethal distending toxin, and Shiga-like toxin, which have been found related to invasion in Enterobacter (48).The presence of multiple virulence factors of the two strains corresponds to their pathogenic role in human infection, although the two patients had no septic shock and recovered uneventfully after antimicrobial therapy, indicating the relatively low virulence of the two strains.Nevertheless, compared to the two most common clinically encountered species of Enterobacteriaceae, E. coli and Klebsiella pneumoniae, the virulence of Enterobacter is less studied and warrant further exploration.
There are limitations of this study.First, only one strain was obtained for each species.The phenotypic characteristics may vary when more strains are recovered and studied in the future.Second, this study focused on the taxonomy of two strains.Experimental studies of the exact mechanisms for resistance to antimicrobial agents such as fosfomy cin resistance in strain 170198 and discrepant susceptibility to several β-lactams between the two strains are beyond the scope of this study but warrants further studies.Likewise, we did not validate the function of predicted virulence genes.Nevertheless, despite the limitations, the characterization of two novel species that are likely opportunistic pathogens for human of clinical relevance.
In conclusion, genotypic and phenotypic characteristics confirmed that strains 170198 T and 170250 T represent two novel species of the genus Enterobacter, for which we propose the name Enterobacter chinensis and Enterobacter rongchengensis.The type strains is 170198 T and 170250 T , respectively.
The type strain is 170198 T (= GDMCC 1.3549 T = JCM 35826 T ) isolated from a human blood sample at West China Hospital, Chengdu, China.
The type strain is 170250 T (= GDMCC 1.3670 T = JCM 36189 T ), recovered from a human blood sample at West China Hospital, Chengdu, China.

TABLE 1
Average nucleotide identity (ANI) and in silico DNA-DNA hybridization (isDDH) values between strains 170198 T , 170250 T , and type strains of the genus Enterobacter and the closely related genera Huaxiibacter, Leclercia, Lelliottia, and Pseudoenterobacter a,b a The closest matches are highlighted in bold.b -, not applicable.