The role of whole-genome sequencing in species discrimination of Elizabethkingia genus
The traditional DDH was used to be one of the most important criterion for discrimination of bacterial species. Recently, in silico DDH has been deemed as a more accurate substitution for traditional DDH [18]. In our study, genome-to-genome distance calculator (GGDC) plus in silico DDH was used to reveal the delineation of Elizabethkingia species. The 20 clinical strains were initially identified as E. meningoseptica, however, they were found to be E. anophelis (14 strains), E. meningoseptica (5 strains) and E. miricola (1 strain). E. anophelis turned out to be the majority species of the Elizabethkingia genus (70%, 14/20), consistent with previous research [12, 16, 19].
Comparative genome analysis of antibiotic resistance genes in Elizabethkingia genus
E. meningoseptica differed from E. anophelis or E. miricola in many aspects as our comparative genome analysis demonstrated. For instance, genomes of E. meningoseptica possessed more GC content than those of E. anophelis and E. miricola. As to antibiotic resistance genes, blaGOB−16 and blaB−12 carbapenemase-encoding genes were more in E. meningoseptica, while blaCME−1 was more in E. anophelis. blaGOB−9 and blaGOB−10 also existed in E. anophelis (Fig. 3). blaGOB−13 and blaB−6 were identified in E. miricola. Extended-spectrum serine-beta-lactamase carboxymethyl ether (CME) (class D) and two unrelated wide-spectrum metallo-beta-lactamases (MBLs), BlaB (subclass B1) and GOB (subclass B3) belong to the family of beta-lactamases. Due to the divergence of CME and MBLs among Elizabethkingia species, it might be a potential evidence for species discrimination to analyze the homology of different phylogenetic cluster.
The relationship of antibiotic resistance genes, antimicrobial resistance-associated proteins and antimicrobial phenotype
Elizabethkingia isolates have been reported to be resistant to varieties of antimicrobial agents, not only most beta-lactams or beta-lactams/beta-lactamase inhibitors, but also aminoglycosides, macrolides, tetracycline, vancomycin, and carbapenems. While Elizabethkingia were susceptible to piperacillin, piperacillin-tazobactam, minocycline, fluoroquinolones, tigecycline, and trimethoprim-sulfamethoxazole [2–5]. Studies revealed that antibiotic susceptibility patterns of Elizabethkingia were closely related to species and geographical locations [20].
According to our previous study, our clinical strains of Elizabethkingia genus possessed high level of multi-drug resistance [17]. The present study demonstrated that proteins resistant to beta-lactamases, vancomycin, tetracyclines, quinolones, macrolides, and multidrug resistance efflux pumps were identified in our clinical strains, while proteins resistant to aminoglycosides and sulfonamides were absent. The coincidence rate between antimicrobial resistance genes and phenotype of beta-lactamases was 100%. Although antimicrobial resistance genes of sulfonamides, macrolides, tetracyclines and aminoglycosides were absent, these strains of Elizabethkingia possessed resistance phenotype to them. Interestingly, TetA, a kind of protein in charge of tetracycline efflux, was found in our clinical Elizabethkingia strains, but these strains were 100% susceptible to minocycline (Table S4). It has been reported that vancomycin was used to treat neonatal meningitis with E. meningoseptica successfully [21], however, all of the 20 clinical Elizabethkingia strains presented minimum inhibitory concentration (MIC) of vancomycin more than 8 µg/ml [22], the similar results were also discovered in other studies [19, 20]. vanW, a vanB-type glycopeptide resistance gene, was identified in all the three Elizabethkingia species. The exact function of vanW still remained uncertain, but an involvement of its mutations has been observed in the regulation of resistance to teicoplanin [23]. Hence, the use of glycopeptide should be cautious in treating infection by Elizabethkingia [19, 20].
Comparative genome analysis of virulence factors predicted among Elizabethkingia species
Bacterial virulence factors are essential for pathogenesis, in our study, exopolysaccharide, heme biosynthesis, urease, and Mg2+ transport were predicted in three species of 20 Elizabethkingia strains (Table 4 and Fig. 3), while they existed only in E. miricola GTC 862T by Liang et al. [16]. The adeG gene existed in 18 strains of Elizabethkingia (Table 4), it was related to biofilm which could cause bacteria to adhere to the medical devices and resist against disinfectant [24–26].
Virulence factors about lipid and fatty acid metabolism, serum resistance and immune evasion and phospholipase D were found mostly in E. meningoseptica, that might be the reason why E. meningoseptica tends to cause more neonatal meningitis and sepsis. It was not clear whether O-antigen involved in immune evasion played a role in outbreaks largely triggered by E. anophelis. There were less categories of virulence factors in E. miricola, which could explain why E. miricola caused occasional clinical infection case reports [27].
COGs and KEGG analysis of Elizabethkingia genus
COGs, clusters of orthologous groups, that involve species-related genes evolving from a common gene, remain the original function during the evolution process. Detection of COGs and prediction of their functions are of fundamental importance in many fields, particularly in pathogenic analysis with new sequence and function of intracellular survival related to COGs with “information storage and processing” [28, 29]. Both COGs and KEGG analysis of Elizabethkingia genus indicated that function of metabolism occupied the largest part, most of which were linked to carbohydrate metabolism.
Pan genome proposed by Tettelin et al. was introduced to discriminate genomes, to investigate the core (conserved), accessory (dispensable), and unique (strain-specific) genes, to trace horizontal gene-flux among strains, and to acquire information about species evolution [30]. The only strain of E. miricola possessed most unique genomes among these 20 clinical Elizabethkingia strains, indicating a higher degree of species evolution than other species. Further study on more strains are needed to explore the complicated species evolution of Elizabethkingia genus.
Clinical characteristics of Elizabethkingia infections
Chronic underlying illnesses, such as cardiovascular disease, hypertension, diabetes mellitus, malignancy, and liver cirrhosis were common in most patients with Elizabethkingia infections [3–5, 31, 32]. Although mortality rate of E. anophelis and E. meningoseptica was 24–34% and 30% reported by Lin et al. (2018) and Lin et al. (2019) respectively [5, 19], the death rate of patients in our study was lower than that. We also found that among mortality-affecting factors, cerebral infarction was an independent risk factor (Table S5). Therefore, we suggest that more attention should be paid on patients with cerebral infarction and Elizabethkingia infection in order to reduce mortality.