Genomic Characterization of an Emerging Enterobacteriaceae Species for the First Case of Human Infection in Cooperation with a Typical Pathogen

Opportunistic pathogens are important for clinical practice as they often cause antibiotic resistant infections. However, little is documented for many emerging opportunistic pathogens and their biological characteristics. Here, we isolated a novel species of extended-spectrum β-lactamase-producing Enterobacteriaceae from a patient of biliary tract infection. The isolate grows very slowly but confers strong protection for the co-infected cephalosporin-sensitive Klebsiella pneumonia . As the initial laboratory testing failed to identify the taxonomy of the strain, great perplexity was caused in the etiological diagnosis, and anti-infection treatment for the patient. Rigorous sequencing efforts achieved the complete genome sequence of the isolate which we designated as AF18. AF18 is phylogenetically close to a few strains respectively isolated from soil, clinical sewage, and patients, forming a novel species together, while the taxonomic nomenclature of which is still under discussion. And this is the first report of human infection of this novel species. As its relatives, AF18 harbors many genes related to cell mobility, various genes adaptive to both wild environment and animal host, tens of genetic mobile elements, and a plasmid bearing bla CTX-M-3 gene, indicating its ability to disseminate antimicrobial resistant genes from wild to patients. Transcriptome sequencing identified two sRNAs that critically regulate the growth rate of AF18 which could serve as targets for novel antimicrobial strategies. These findings imply that AF18 and its species are not only infection-relevant but also potential disseminators in transferring antibiotic determinants, which highlights the need for continuous monitoring for this novel species and efforts to develop controlling strategies.

. These species often have other animal hosts, or they can be found in the wider environment, such as soil and sewage, or both (Mather et al., 2013).
Enterobacteriaceae species (including E. coli, Klebsiella, and Enterobacter) are also famous for their antibiotic resistance and regarded as one of the most dangerous pathogens that they can efficiently acquire various ARGs through efficient plasmid transmission (Iredell et al., 2016). The ability of these species in transferring between habitats and transferring ARGs potentiates them to be important mediators in the eco-evolutionary feedback loops that disperse ARGs back to clinical settings from wild environment. The taxonomy of Enterobacteriaceae is complex that contains 28 genera and over 75 species (Adeolu et al., 2016), while novel species are continuously discovered. To fully recognize and characterize Enterobacteriaceae species, especially those of emerging opportunistic pathogens, is critical for understanding the dynamics of the evolution of AMR.
Here, from a patient of biliary infection, we isolated a novel strain of unknown taxonomy accompanying an infectious Klebsiella pneumonia strain. The isolate grew slowly but provided drugresistance to its companion by carrying a bla CTX−M−3 resistant gene. The co-infection brought perplexity in both diagnosis and treatment of the patients. Complete genome sequencing based on both SMRT (single-molecular real-time sequencing) and Illumina platform achieved a high-quality genome of this resistant strain, which suggested it as a strain in Enterobacteriaceae but of an undefined novel species. Together with a transcriptome sequencing, we are able to take a deep insight into the genomic characteristics of the rare pathogen and regulation mechanisms of how it adapts to multiple habitats and associates to ARG transfer.

Materials And Methods
Biological Characterization of Strain AF18 Colony of AF18 was applied to assay using the VITEK-II automated bacterial identification system and the API20E Enterobacter biochemical identification system (Biomerieux, France). The TOF-MS -based identification was conducted with a MicroFlex LT mass spectrometer (Bruker Daltonik) to obtain the protein profile of AF18, which was further analyzed using MALDI Biotyper software (Bruker Daltonik).
The morphology of the bacteria was observed under a Hitachi S-3400N scanning electron microscope with a standard procedure. Briefly, a colony of AF18 was fixed with 2.5% glutaraldehyde followed by sequenced with the short-reads Hiseq 2000 platform from Illumina (100bp × 2) for 3G raw reads. The obtained raw SMRT reads were analyzed and de novo assembled using SMRT Analysis 2.3.0 software.
Error correction of tentative complete circular sequences was performed using Pilon version 1.18 with

Phylogenetic Analysis
We used Mash (Ondov et al., 2016) to compare the AF18 genome sequence to genome assembly database from NCBI, and picked 33 non-redundant species with identity score > 75%. Then, we used kSNP3 to identity core genome SNPs of each pair of the 34 genome sequences with an optimal k-mer size of 21 (determined by Kchooser) (Gardner et al., 2015). These core SNPs were used to build a maximum likelihood tree by FastTreeML (Price et al., 2010), and iTOL was used to exhibit the phylogenetic tree (Letunic and Bork, 2016). We used fastANI to calculated the pairwise ANI of the 34 genome sequences (Jain et al., 2018).

Comparative Analysis Of Paf18_2
We blast pAF18_2 against the nr/nt database, and picked top 10 non-redundant plasmids according to the query coverage and alignment score (Johnson et al., 2008). We then performed BLASTN for pAF18_2 against the other 10 plasmids with E-value < e-50 (Camacho et al., 2009), and generated the comparative map using CGView (Stothard and Wishart, 2005).

Drug-resistant Plasmid Elimination Test
A single colony of AF18 was inoculated into LB medium without antibiotics and cultured for 24 hours at 37 °C. Then the culture was 1:1000 diluted and re-inoculated in LB medium for another 24 hours.
The procedure was repeated while an aliquot was collected and spread on LB agar for each round of re-inoculation. Colonies grown on the LB agar were randomly selected and tested for the presence of

Differential Gene Expression Analysis
The read count data of each transcript was first normalized using DEseq (Anders and Huber, 2012).
According to the binomial distribution model, hypothesis testing was performed on each transcript between the AF18 strain and the AF18-NC strain, and confirmed by multiple hypothesis tests.

Functions Of Differentially Expressed Genes
Functions of the differentially expressed genes between AF18 and AF18-NC were annotated with GO database (Harris et al., 2004), and the probability of enrichment for each cluster was calculated by using the Goseq algorithm based on the Wallenius non-central hyper-geometric distribution (Young et al., 2012). Clusters with corrected p value < 0.05 was regarded as significantly enriched.

Biological Identification of the Strain AF18
A patient of obstructive jaundice who suffered an infection two days after the percutaneous transhepatic cholangial drainage (PTCD) surgery was admitted to our hospital. From the bile sample of the patient, two types of colonies were isolated after serial dilutions and isolations on MacConkey agar plates. One type was mucous, entirely pink, and of 4-5 mm in diameter, which was finally identified as a K. pneumonia clone sensitive to common antibiotics (Table 1); the other type was small colonies of red center, clear and transparent edge, and of 2-3 mm in diameter (Fig. 1A). The bacteria of the small colonies seems prone to adhere to the cells of K.
pneumonia and were not able to be isolated until extensive dilutions. The taxonomy of the small colonies was not immediately identified by the microbiological laboratory in the hospital and we designated it as strain AF18. AF18 exhibited remarkably resistance to most β-lactam antibiotics in antimicrobial susceptibility testing ( Table 1). As the infection was rather intractable and finally cured by intravenous amikacin, the final diagnosis for the patient was a co-infection caused by a sensitive K. pneumonia strain and a multidrug resistant strain of unknown species. Table 1 The antibiotic resistance profile of AF18 and K. pneumuniae isolate.

Drug
Antibiotic susceptibility Microscope observation showed that AF18 was a Gram-negative bacillus (Fig. 1B), and its cells were surrounded by flagella under transmission electron microscope (Fig. 1C). Scanning electron microscope confirmed the tubular shape of AF18 and a smooth surface with no polysaccharide particle (Fig. 1D), in line with the mucus-free characteristics of its colony. VITEK-II in hospital laboratory did not result in any bacterial species identical to the biochemical properties of AF18 (Table S1), whereas API20E biochemical identification system suggested AF18 as Pantoea sp. but with low reliability. The mass spectrometry which scans the protein profile of samples did not identify the species of AF18 either.

Complete Genome of Enterobacteriaceae bacterium AF18
To determine the taxonomy and genetic features of AF18, we performed whole genome sequencing on both platforms of short-reads Illumina Hiseq and long-reads PacBio sequencer and achieved a high-quality completed genome sequence of AF18 which possesses circulated chromosome and two plasmids ( Table 2, Fig. S1).  and its species is not a typical Kluyvera species or should not be included in this genus. Phylogenetic relationship of these relatives was further inferred with core genome SNPs (Fig. 2B) which confirmed the relationships inferred from the ANI matrix and indicated the novel species including AF18 possibly stands for another genus than Kluyvera. Herein, we temporarily nominated our stain as Enterobacteriaceae bacterium AF18 as the nomenclature of its species even genus name is still undefined.
The chromosome of AF18 possesses 5651 protein-coding genes which functions facilitate the survival and adaptation of AF18 in various habits (Table S2). For example, motility-related genes, including a complete flagellar gene cluster that encodes all components of flagellar, csg gene cluster that encodes curli assembly proteins to mediate adhesion, and other genes of ompA, pilRT, ibeB, icaA, htpB and fimB, together confer the ability of adhesion, invasion, chemotaxis, and escape to the host strain. Genes of the hcp-clp and mprAB system are powerful in implementing persistence status which endows resistance to many environmental stresses including all kinds of antibiotics. Efflux pump genes which confer resistance to macrolides, quinolones and aminoglycosides were also identified. Meanwhile, the AF18 genome possesses 20 genomic islands, 11 prophages and five CRISPR sequences (Table S3), indicating active transfer of stress-adaptive genes by these genetic mobile elements and bacteriophages of this species. More importantly, markers of bacteria inhabited in soil, including a complete nitrogen fixation gene cluster and ksgA--a pesticide-resistant gene, were found in AF18 genome which suggests that AF18 is adaptive to dwell in nature environment or a wider range of habits. And the mobility of this strain potentiates it to shuttle between various habits.
Analysis of conserved genes in plasmids shows that most of the antibiotic-resistant genes of AF18, including qnrS, dfrA and bla CTX−M−3 , are carried by the smaller plasmid pAF18_2 (Fig. 3) which is responsible for the antibiotic resistance profile of AF18. Sequence alignment shows that pAF18_2 are similar to many plasmids from host of other Enterobacteriaceae species, such as E. coli, K. pneumonia, and C. freundii, and they contain identical replication origin, replication and transcription system, plasmid partition system, and a partial gene cluster responsible for plasmid conjugation, which indicates that the plasmid might be compatible with all these Enterobacteriaceae host species. Besides, these plasmids share a common anti-restriction system that ensures they would not be destroyed by the restriction-modified system in other host strains. Specifically, the pAF18_2 contains an active transposase system with complete IS elements which had acquired the bla CTX−M−3 gene and an arsenical resistant system. Many other DNA manipulating enzymes such as integrase and DNA invertase were also identified in the plasmid, all of which facilitate the plasmid in efficiently acquiring and transferring antibioticresistance genes and other stress-adaptive genes among Enterobacteriaceae strains.

Growth of AF18 in Co-cultures and Its Transcriptional Regulation
To disentangle the respective contribution of AF18 and the sensitive K. pneumonia in the co-infection, we cocultivated the two strain in various concentration of ceftriaxone, and found that addition of 1% AF18 was able to elevated the MIC from 0.125 µg/ml of pure K. pneumonia culture to 64 µg/ml. Furthermore, when spread the co-culture onto the MacConkey agar containing ceftriaxone, the sensitive K. pneumonia colonies were able to withstand 8 µg/ml ceftriaxone (Fig. 4A), indicating a strong protective effect of AF18 to the co-infected K. pneumonia.
Although important in the co-infection for antibiotic-resistance, AF18 only took less than 1% in the initial sample.
Even when equally input, the proportion of AF18 decreased to 1% of the co-culture if without antibiotic pressure (Fig. 4B). It seems that AF18 may be less aggressive and its growth rate is much slower than the co-inhabited K.
pneumonia. It has been reported that bearing plasmid may slow down growth rate due to the cellular cost caused by the addition of plasmid (Bouma and Lenski, 1988), and thus we generate a new strain-AF18-NC by deleting the resistant plasmid of AF18. Then we measured the independent growth curve of the three strains-K.
pneumonia, AF18, and AF18-NC, respectively (Fig. 4C). As expected, AF18-NC did grow faster than its mother strain AF18 as relieved from the plasmid-caused cellular cost. However, the growth rate of AF18-NC was still much slower than that of K. pneumonia, suggesting that slow growth is an inherent property of the novel species.
Next, we analyzed the genes involved in regulation of growth rate by a comparison between the transcriptomes of AF18 and AF18-NC. A total of 3,309 genes of chromosomal coding genes were differentially expressed in significance, with 1675 upregulated and 1634 downregulated in AF18 (Fig. 4D). Functional cluster analysis with GO (Gene Ontology) database showed that most of differentially expressed genes were in the categories of transcriptional regulation, biosynthesis regulation, metabolic process regulation, signal transduction, DNA binding, and signal sensing. Analysis of the non-coding sRNA expression profile identified a total of 15 sRNAs differentially expressed between AF18 and AF18-NC. Interestingly, two of the down regulated sRNAs in AF18, sRNA00063 and sRNA00291 (Fig. S2) shared 98% of their predicted target genes which took up 56% of the above-mentioned differentially expressed coding genes, suggesting their key roles in promoting growth. This result indicated the importance of the two sRNAs in globe regulation of growth rate, and consequently the contribution and competition of the host AF18 in co-infections.

Discussion
In this study, we reported a case of co-infection caused by a typical pathogen and a rare opportunistic pathogen with taxonomical nomenclature undefined. In the pathogenic consortium of the co-infection, the dominant K.
pneumonia strain is virulent enough to cause an aggressive infection, while the AF18 strain, although taking a very small proportion, provides strong protection for the entire pathogenic consortium against antibiotic damage.
The co-operation between the K. pneumonia and AF18 makes the infectious situation more complicated and difficult in term of therapies than infections caused by either of them. Meanwhile, as the strain AF18 only took a minor proportion and a close adhesion to co-infected K. pneumonia, it was prone to be concealed by the dominant K. pneumonia and hard to be detected and isolated, which led to inaccurate etiological diagnosis and improper anti-infective treatment at first admission. As AF18 and other strains of the same species are rare opportunistic pathogen with little documentation, and conventional testing for bacterial identification are not always correct for such novel species, as shown in this study, WGS comprised a straightforward approach for accurate taxonomy identification.
Sequencing strategy combining both long-and short-read platforms makes it easy to obtain high-quality complete genome including plasmids, which will be helpful for overall characterization of novel species GT-16 contains many ARGs that even resistant to polymyxin (Tetz et al., 2017). A little more distant relative of AF18-Pytobacter ursingii (previously named Kluyvera intermedia) was found to be KPC positive and carbapenemresistant (Sheppard et al., 2016). As adapted to both wild environment and host habitats and mobility to transfer in-between, this species is able to acquire various ARGs from wild environments and when co-inhabit with their Enterobacteriaceae relatives in gut, such as E. coli and K. pneumonia, is able to share these ARGs through various genetic mobile elements or even in a more efficient manner of conjugating resistant plasmids. Thus, the species of AF18 may prove important in spreading ARGs and function as a mediator in the eco-evolutionary feedback loops of AMR. In this regard, the novel species and many other emerging opportunistic pathogens of Enterobacteriaceae family, such as Kluyvera spp. and Enterobacter spp., deserve more attention in clinical practice and the field of antibiotic resistance control.
In our study, the resistant AF18 does not have to transfer its resistance gene to the co-infected sensitive K.
pneumonia to confer protection. Being antibiotic-resistant by itself, AF18 just upregulated the production of antibiotic-hydrolase to generate a niche of low antibiotic concentration for the sensitive K. pneumonia to hide. Our study provides an empirical evidence for these hypothesis and highlights the importance of mutualistic relationships between co-infected microbes in clinical settings. Transcriptome analysis further identified genes involved in growth regulation and pointed to two novel sRNAs that might be the key regulators of the process. As antibiotics are not always successful especially in treating opportunistic pathogens, the sRNAs that promote the growth of host strains as we had identified may serve as targets of bacteriostatic agents and deserve further investigations.
In conclusion, opportunistic pathogenic strain Enterobacteriaceae bacterium AF18 is in a novel species with little documentation. In-depth genomic analysis indicates the ability of this species in transferring between wild and host habitats and activity in transferring antibiotic resistant genes, which potentiates it to be an important disseminator of antibiotic resistance to clinical pathogens. When co-infected with typical pathogens, the resistant opportunistic strain is able to provide temporary protection for the whole consortium and causes confusions in the etiological diagnosis and antibiotic treatment, although the strain by itself is not a pernicious pathogen for immunocompetent patients. Taken together, Enterobacteriaceae bacterium AF18 and other newly emerging opportunistic pathogens complicate the situation of antibiotic resistance control in clinical practice and deserve in-depth investigation including methods for critical surveillance and controls on them.

Ethics Statement
The study protocol was approved by the ethics committee of Peking university people's hospital (Approval No. 2015PHB037-01, 17/01/2015). Written consent was acquired from the patient.

Funding
The study was funded by National Science and Technology Major Project (2017ZX10103004-006), National Natural Science Foundation of China (81870010, 31970568). Programs of the Chinese Academy of Sciences The funding sources had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.

Author Contributions
Zhancheng Gao and Yu Kang designed and conducted the study. Yusheng Chen collected the samples and

Figure S1
The circular map of AF18 chromosome and plasmids. From the outside to the center: Genes on the forward strand, genes on the reverse strand, the annotation genes in COG database, the annotation genes in GO database, the annotation genes in KEGG database, ncRNA, GC content, GC skew.

Figure S2
The Sequences and the secondary structures of sRNAs Table S1 The results of biochemical testing of AF18 isolate by VITEK II2

Table S2
The genome annotation results of AF18    The circular map of pAF18_2 and comparison to similar plasmids. The outmost slot represents the predicted genes of pAF18_2, whose functions are shown in different color arrows. From outward, slot 2-

Figure 4
The properties and regulation of the growth rate of AF18. (A) Over-night co-culture of AF18 and the co-

Supplementary Files
This is a list of supplementary files associated with this preprint. Click to download. Fig S1.tiff Table S2.xlsx   Table S1.docx