Exiguobacterium sp. A1b/GX59 isolated from a patient with community-acquired pneumonia and bacteremia: genomic characterization and literature review

The genus Exiguobacterium belongs to the group of coryneform bacteria, firstly described in 1983 by Collins et al. [1]. It is a facultative anaerobic, Gram-positive bacillus. The pathogenic potential of Exiguobacterium spp. seems rather low. To date, only a few cases of bacteremia and skin infection were documented in English literature [2‐6]. However, the clinical infection due to the micro-organism might be probably underdiagnosed or unreported, for it tended to be misidentified by routine commercial methods [2‐6]. As an emerging pathogen, its pathogenesis should be clarified.

Here, we present a case of community-acquired pneumonia (CAP) and bacteremia due to Exiguobacterium sp. strain AT1b/GX59 in a type 2 diabetes mellitus (T2DM) patient. To the best of our knowledge, this is the first fatal case of CAP and bacteremia due to the micro-organism in a healthy male without serious underlying diseases. In order to generate significant insights into pathogenicity of the micro-organism, its genome was sequenced and compared with 3 Exiguobacterium genomes available in NCBI Genbank database.

The clinical characterizations of Exiguobacterium species isolated previously from different infectious samples were listed in Table 3 [2‐6]. As documented, most infections due to Exiguobacterium spp. had underlying diseases, such as liver cirrhosis [2], intravenous drug abuse and multiple myeloma [6]. However, the patient in present study was in a generally healthy condition, though he suffered from T2DM. Although a male patient infected by the micro-organism was previously healthy, different from our patient, he had only an ulcer on a finger with a painful black eschar rather than systematic infection [4]. Bacteria of the genus Exiguobacterium distribute extensively and have been isolated from markedly diverse sources, including water, the rhizosphere of plants, and the environment of food processing plants [8]. The patient in current study was a farmer living on processing plants in humid climate of South China. Considering his early clinical symptoms, inhalation of the micro-organism might be a possible portal of entry of his pneumonia [8].

The literature review demonstrated that Exiguobacterium were rarely isolated as human pathogen. Furthermore, it is difficult to identify Exiguobacterium spp. based on traditional biochemical method. Almost all reported infective strains of this genus were misidentified when the commercial biochemical system was used, including API Coryne kit/VITEK 2 Compact system (Bio-Mérieux, France), and Becton Dickinson Diagnostic Systems [2‐6]. Furthermore, Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) analysis is presently becoming a routinely used tool in many microbiology laboratories, and might be used in identifying the genus Exiguobacterium in future [6], however, in our study, the strain GX59 was identified as Exiguobacterium aurantiacum 290RLT with its score as 1.547 by using MALDI-TOF (Bruker Daltonics, MALDI Biotyper 3.1 software, 2371 species and 5989 entries included).

In current study, the pathogen could not be identified using ANC card of VITEK 2 system, and was confirmed as Exiguobacterium AT1b by 16s rRNA sequencing only in retrospective analysis. Exiguobacterium AT1b was initially isolated from a slightly alkaline, highly carbonate, and hot spring water sample from Yellowstone National Park [9], and had never documented to be isolated clinically. Therefore, the in-depth analysis of the Exiguobacterium sp. AT1b/GX59 isolate in present study might elucidate the pathogenicity of this environmental saprophytic micro-organism. Moreover, the comparison study of the genomes of the strain GX59 and other Exiguobacterium spp. might then shed light on the pathogenicity of the microorganism. The publicly-available genomes of Exiguobacterium spp. were mostly isolated from environment. Through ortholog gene analysis, it was reasonably to speculate that several following genetic characteristics were closely related to the pathogenicity of current GX59 strain. Firstly, secretion systems identified in the strain might be involved its pathogenicity. Desvaux M [10] suggested to use the terms sec, tat, FEA, and FPE in translocation systems across the cytoplasmic membrane of both Gram-positive and -negative bacteria. The above 4 Exiguobacterium strains possessed 37 genes of secretion systems, encoding two translocons, including sec, tat, FEA, and competence-related DNA transformation transporter (Com). In pathogenic Gram-positive bacteria, the vast majority of proteins were exported out of cytosol by conserved general sec system [11] or, by tat system [12]. Other studies discovered Com proteins were required for internalization of extracellular DNA [13, 14]. Taken together, Exiguobacterium had the ability to export toxins to exert its full virulence and to uptake DNA to acquire a variety of virulence and resistance traits. Secondly, a series of unique virulence genes identified in the GX59 strain genome might explain its high virulence. For example, the hemolysin encoded by the gene tlyC was taken as a virulence factor in a variety of Gram-positive infectious bacteria. Carvalho E [15] suggested that the protein tlyC was not directly involved in hemolysis, but contributed to binding of Leptospira to extracellular matrix (ECM) during host infection. The non-hemolytic GX59 strain in present study contained tlyC genes, in consistent with Carvalho E’s finding [15]. Moreover, NprR, as a major transcriptional regulator, was documented to belong to RNPP family of quorum-sensing (QS) receptors, a group of intracellular regulators activated directly by signaling oligopeptides in Gram-positive bacteria [16]. It might control sporulation and necrotrophic properties, ensuring survival and dissemination of the bacteria in clinical infections by feeding on host proteins [16]. Another pathogenetic gene uniquely detected in the GX59 strain was mcp, which got involved in virulence, motility, and biofilm formation of bacteria [17], possibly performing a potential function in invasive infections. Furthermore, Dam mediated the methylation of adenine in the 5′-GATC-3′ sequence shortly after DNA replication, and was implicated as a virulence factor in bacterial pathogenesis. As documented previously, Dam methylation was required for efficient biofilm production in Salmonella enterica serovar enteritidis [18]. Dam was also crucial in modulating the pathogenicity of K. pneumoniae genotype K1 [19]. The Dam gene identified in the GX59 strain might participate in its invasiveness during infection. Finally, apart from the virulence factors unique in the AT1b/GX59 strain, other common virulence factors might be involved in severe infection. For, example, the GX59 strain harboured an extra gene SecDF, which played a role as a chaperone that facilitates the translocation of L. monocytogenes virulence factors during infection [7]. Deletion of secDF resulted in reduced virulence and motility Bacillus cereus ATCC 14579 [20]. Taken together, the severe CAP and following bacteriemia in our patient was possibly explained by the high pathogenicity due to the above-identified virulence genes.

Except for high pathogenic genes, Exiguobacterium spp. also harboured some antimicrobial resistance genes, including tetracycline resistance genes, macrolide resistance genes, aminoglycoside resistance genes, phenicol resistance genes, cationic antimicrobial peptide, multidrug resistance efflux pumps (abcA and bmrA), and vancomycin resistance modules (vanY, vanW). Accordingly, although the strain remained susceptible, it would easily become resistant to many antibiotics. Generally, timely antibiotic therapy often resulted in a favorable outcome in the clinical infections due to the microorganism [2, 3, 5, 6]. However, in current study the isolate was susceptible to the antibiotics used, e.g. meropenem and ciprofloxacin, but the patient died of deteriorated infection. This might be explained by the reasons as follows. The pathogen was not identified timely, the appropriate antibiotic therapy failed to take, and furthermore, the rapidly-deteriorated severe acute type 2 respiratory failure caused his death.

In summary, the Exiguobacterium sp. AT1b/GX59 strain in current study is equipped with a variety of factors that facilitate its adaptation to a pathogenic lifestyle, such as hemolysin, secretion systems, chemotaxis proteins, and antibiotic resistance genes. The Exiguobacterium sp., as a potential pathogen, should attract more attention.