Identification of Raoultella terrigena as a Rare Causative Agent of Subungual Abscess Based on 16S rRNA and Housekeeping Gene Sequencing

A 63-year-old-man was admitted to our hospital with severe subungual abscess. Bacteria were isolated from pus samples, and an inconsistent identification was shown by VITEK 2 system and MALDI-TOF mass spectrometry as Raoultella planticola and Raoultella terrigena, respectively. Molecular identification by 16S rRNA sequencing suggested that the isolate is R. terrigena, and this was further demonstrated by sequencing three housekeeping genes (rpoB, gyrA, and parC) with phylogenetic analysis. To our knowledge, this is the first report of subungual abscess caused by R. terrigena, a rare case of human infection due to soil bacterium. Our study highlights the technique importance on this pathogen identification.


Introduction
Raoultella terrigena is a Gram-negative, oxidase-negative, aerobic, nonmotile, capsulated, non-spore-forming bacterium. It was primarily recovered from soil and considered as a nonpathogenic species, although it harbours numerous virulence factors found in Klebsiella pneumoniae [1]. Very few reports of clinical infections caused by this species are available so far, and its clinical significance is still unclear [1]. A previous study showed that the carriage rate of R. terrigena in healthy people was 0.9% by analyzing 5,377 different stool specimens [2]. The incidence of R. terrigena among clinical Klebsiella isolates was 0.4%, and most of these isolates were obtained from respiratory secretions [2]. We here reported a case of a 63-year-old man who developed a subungual abscess caused by R. terrigena. We further performed a literature review on R. terrigena infections.

Case History
In March 2015, a 63-year-old man was admitted to the surgery department of a university hospital with the complaint of a painful abscess on his right thumb lasting for 3 days. The patient is a farmer and had no prior medical and surgical history. Eight days before his admission, the patient experienced an accidental injury on his thumb during working in the field and later washed the wound by river water. As shown by physical examination, the abscess was at the lateral nail edge, and finger pad was tense and pus presented. No other skin lesions were detected. The patient was consequently diagnosed as the subungual abscess. A longitudinal incision and drainage were thus performed with expression of purulent fluid, and a culture of the fluid was performed. The relief of pain immediately after this treatment was reported by the patient. 500 mg of imipenem twice a day for 10 days was prescribed to the patient, and he recovered after this treatment.
The pus from the incision was inoculated onto sheep blood agar plates and incubated at 37 ∘ C in a CO 2 -enriched (5%) atmosphere for 24 h. Incubation yielded single colonies of Gram-negative rod-shaped bacterium identified by Gram staining (Figure 1(a)). Colonies of isolate Z38 were circular, smooth, glistening, light yellow, and nonhemolysis after 48 h of incubation on sheep blood agar plates at 37 ∘ C (Figure 1(b)).
a MICs were determined using the Vitek 2 GN and AST cards, following the manufacturer's instructions. The susceptibility breakpoints of MICs followed those recommend by the Clinical and Laboratory Standards Institute.
The samples were analyzed by MALDI-TOF mass spectrometry (Autoflex III, Bruker, Germany) and were identified as Raoultella terrigena with a similarity rate of 99.9%. As Raoultella terrigena is rarely identified in clinical samples, identification was subsequently redone by VITEK 2 system with panel AST-GN-13 (BioMérieux, France). The isolate was identified as Raoultella planticola by the VITEK 2 system. Antimicrobial susceptibility testing was also performed by VITEK 2 system with panel AST-GN-13, and Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were as controls. The result interpretation was performed as described in CLSI guidelines. The isolate was susceptible to all antibiotics tested except ampicillin (Table 1).
To get a reliable identification, 16S rRNA and the housekeeping gene sequencing were further used to accurately identify and discriminate. PCR amplification of the 16S rRNA was performed with the universal bacterial primers 27f (5 -AGAGTTTGATCCTGGCTCAG-3 ) and 1492r (5 -TACCTTGTTACGACTT-3 ). The 1.4 kb PCR product was sequenced and blasted in GenBank to identify the species. Alignments of 16S rRNA sequences of the isolate Z38 and type strains of close related species collected from EzTaxon-e database were generated by ClustalW program. A neighborjoining phylogenetic tree based on the Tamura-Nei model was constructed by MAGA6.06. The phylogeny indicated the strain Z38 (KT276326) sharing 99% identity with the type strain Raoultella terrigena NBRC 12941 T (Y17658) ( Figure  S1A in Supplementary Material available online at http://dx .doi.org/10.1155/2016/3879635).
Housekeeping genes rpoB, gyrA, and parC were sequenced to demonstrate the taxonomic identification as described previously [3]. The sequence similarity of gyrA, parC, and rpoB between pus isolate Z38 and the most closely related type strain (R. terrigena NBRC 12941 T ) was 99%, 99%, 98%, respectively (Table 2 and Figures S2, S3, and S4). The phylogenetic tree constructed by concatenating sequences of the 3 housekeeping genes showed that strain Z38 formed a distinct lineage with R. terrigena NBRC 12941 T ( Figure S1B). Together with the typing result of 16S rRNA, the pus isolate Z38 was identified as R. terrigena.

Discussion
R. terrigena is a soil microorganism with only two reports of clinical infection. Goegele et al. described the first case of human infection in a liver transplant recipient who developed fatal endocarditis due to R. terrigena in 2007 [4]. The association between R. terrigena and sepsis was reported in 2011, which represents the second case of human infection [5]. We speculated that the pathogen in our case may originate from environment, since the patient injured in the farmland and had the risk of infection by soil bacterium. Accurate identification of Raoultella spp. is a challenge by routine work as this genus shares highly similar phenotypes with other genera of Enterobacteriaceae, especially Klebsiella spp. [5]. In 2001, Raoultella spp. as a novel species was separated from Klebsiella spp. on the basis of 16S rDNA and rpoB typing results [6]. Phylogenetic analysis of gyrB sequences showed that K. terrigena (R. terrigena) and K. pneumoniae belong to two separate lineages [7]. Difficulties in correct identification of Raoultella spp. may have led to an underestimation of its incidence and uncertainty on its pathogenic role [8]. Currently, the identification of Raoultella species from clinical specimens mainly relied on phenotypic methods. A study has validated that MALDI-TOF MS increases the accuracy of Raoultella identifications to the genus level [9]. However, the three species of Raoultella genus shared very close genetic relationship and can only be distinguished by a very few biochemical characters. In our study, a discrepancy was caused by MALDI-TOF mass spectrometry and VITEK 2 identification. Thus, molecular techniques are highly recommended to discriminate the three species [5].
However, no molecular markers are available for this purpose from the literature due to the rarity of the pathogen. A study showed that rpoB was demonstrated on the correct identifications of all Raoultella isolates [8]. Furthermore, three housekeeping genes gyrA (383 bp), rpoB (512 bp), and parC (319 bp) were used for species-level identification of members of the Klebsiella/Raoultella complex [3]. Therefore, in our study three housekeeping genes (gyrA, rpoB, and parC) were chosen for the identification of R. terrigena. The phylogenetic analysis by concatenating the 3 housekeeping genes showed that strain Z38 was clustered with R. terrigena. This result is consistent with the results of MALDI-TOF and 16S rRNA sequencing ( Figure S1). We therefore concluded that VITEK 2 system is not suitable for R. terrigena identification. Our study further implies that human infections by R. terrigena may be underestimated due to the difficulties in correctly identifying this bacterium during routine diagnosis in clinical microbiology laboratories.
The clinical features and outcomes of human infections caused by R. terrigena have been rarely reported. The first R. terrigena infection case was reported in 2007, and a 45year-old patient developed endocarditis due to R. terrigena after liver transplant [4]. Another infection case occurred in a patient who underwent pancreatic resection for pancreatic cancer [5]. However, the pathogenic role of Raoultella spp. in human infection is still difficult to elucidate. Notably, some species of Raoultella spp., for example, Raoultella ornithinolytica, have been suggested as opportunistic pathogens that mainly infect elderly patients with immunosuppression or comorbidities, particularly solid tumours [8].
In conclusion, this report describes the first case of subungual abscess caused by R. terrigena infection. Our finding highlights that the soil microflora enables causing severe infections. Additionally, molecular identification enables us to accurately identify fastidious organisms from clinical specimens when phenotypic identification fails.