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 |
AF18 | K. pneumuniae strain |
MIC (µg/ml) | Phenotype | MIC (µg/ml) | Phenotype |
Ampicillin | ≥ 32 | R | 16 | I |
Ampicillin/sulbactam | ≥ 32 | R | 4 | S |
Piperacillin | ≥ 128 | R | ≤ 4 | S |
Piperacillin/tazobactam | ≥ 128 | R | ≤ 4 | S |
Cefazolin | ≥ 64 | R | ≤ 4 | S |
Cefuroxime | ≥ 64 | R | ≤ 1 | S |
Cefuroxime axetil | ≥ 64 | R | ≤ 1 | S |
Cefotetan | ≤ 4 | S | ≤ 4 | S |
Ceftazidime | 16 | R | ≤ 1 | S |
Ceftriaxone | ≥ 64 | R | ≤ 1 | S |
Cefepime | ≥ 64 | R | ≤ 1 | S |
Aztreonam | ≥ 64 | R | ≤ 1 | S |
Imipenem | ≤ 1 | S | ≤ 1 | S |
Meropenem | ≤ 0.25 | S | ≤ 0.25 | S |
Amikacin | ≤ 2 | S | ≤ 2 | S |
Gentamicin | ≤ 1 | S | ≤ 1 | S |
Tobramycin | 2 | S | ≤ 1 | S |
Ciprofloxacin | 2 | I | ≤ 0.25 | S |
Levofloxacin | 1 | S | ≤ 0.25 | S |
Nitrofurantoin | 256 | R | ≤ 16 | S |
Trimethoprim/sulfamt | ≤ 20 | S | ≤ 20 | S |
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).
Table 2
Overview of genome information for AF18
Replicon | Nucleotide length (bp) | Coding Genes | GC% | Inc type | Antimicrobial resistance genes | GenBank ID |
Chromosome | 5,676,372 | 5651 | 53.06 | NA | ksgA | CP025982 |
pAF18_1 | 140,420 | 181 | 51.14 | IncFII | tetC | CP025983 |
pAF18_2 | 42,923 | 53 | 51.28 | IncN | qnrS, blaCTX−M−3 and dfrA | CP025984 |
By using Mash (Ondov et al., 2016) to search the publicly available bacterial genomes and drafts with a cutoff of mutation distance < 0.25, we identified 33 non-redundant close relatives of AF18, all of which were in the Enterobacteriaceae family. The average nucleotide identity (ANI) matrix of the 34 strains (Fig. 2A) shows that the closest five with identity > 98.5% (i.e. regarded as strains of the same species) are nominated as [Kluyvera] intestini, Matakosakonia sp., Enterobacter sp. (two strains), and just Enterobacteriaceae bacterium, respectively, indicating that the nomenclature of this novel species is still under discussion due to very limited documentation (Alnajar and Gupta, 2017). The first report of the novel species was in 2016 when [Kluyvera] intestini str. GT-16 was isolated from the stomach of a patient with gastric cancer (Tetz and Tetz, 2016), and in the following years, strains of this species were emergingly discovered (Sekizuka et al., 2018; Weingarten et al., 2018). Of note, AF18 is the first clear report of human infection of this novel species, as [Kluyvera] intestine GT-16 and Matakosakonia sp. MRY16_398 were more likely a common resident in the gastrointestinal tract or a by-stander of the diverticulitis. Although the first strain of [Kluyvera] intestini str. GT-16 had been assigned to the genus Kluyvera, the ANI of strains in this novel species to typical Kluyvera spp are less than 80.8%, even farer than the distance to other genus, such as Kasokonia (ANI, 82.3%), and typical Enterobacter spp. (ANI, 81%), suggesting that AF18 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 blaCTX−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 blaCTX−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 antibiotic-resistance 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 co-cultivated 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.