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

Authors: Yu Wang; Xiawei Jiang; Zemin Xu; Chaoqun Ying; Wei Yu; Yonghong Xiao
Journal: Canadian Journal of Infectious Diseases and Medical Microbiology
 (2016)

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## Abstract

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 asRaoultella planticola andRaoultella terrigena, respectively. Molecular identification by 16S rRNA sequencing suggested that the isolate isR. terrigena, and this was further demonstrated by sequencing three housekeeping genes (rpoB,gyrA, andparC) with phylogenetic analysis. To our knowledge, this is the first report of subungual abscess caused byR. terrigena, a rare case of human infection due to soil bacterium. Our study highlights the technique importance on this pathogen identification.


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## Body

## 1. 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 inKlebsiella 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 ofR. terrigena in healthy people was 0.9% by analyzing 5,377 different stool specimens [2]. The incidence ofR.terrigena among clinicalKlebsiella 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 byR. terrigena. We further performed a literature review onR. terrigena infections.


## 2. 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 CO2-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)). The samples were analyzed by MALDI-TOF mass spectrometry (Autoflex III, Bruker, Germany) and were identified asRaoultella terrigena with a similarity rate of 99.9%.Figure 1
Gram staining and morphology ofR. terrigena Z38 colony. (a) Gram-negative, short-rod-shaped bacterium was observed (magnification, ×100). (b) Shown are circular, smooth, glistening, light yellow, and nonhemolysis colonies after a 48-hour culture on sheep blood agar under aerobic conditions.
(a)
(b)AsRaoultella 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 asRaoultella planticola by the VITEK 2 system. Antimicrobial susceptibility testing was also performed by VITEK 2 system with panel AST-GN-13, andEscherichia coli ATCC 25922 andPseudomonas 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).Table 1
Antibiotic resistance profiles ofRaoultella terrigena Z38.
Antibiotics
MIC (μg/mL)a
Susceptibility
Ampicillin
16
R
Amikacin
≤2
S
Ciprofloxacin
≤0.25
S
Levofloxacin
≤0.25
S
Cefoperazone
≤1
S
Imipenem
≤1
S
Trimethoprim-sulfamethoxazole
≤20
S
Tobramycin
≤1
S
Piperacillin-tazobactam
≤4
S
Ampicillin-sulbactam
≤2
S
Aztreonam
≤1
S
Cefotetan
≤4
S
Cefazolin
≤4
S
Gentamicin
≤1
S
Ceftazidime
≤1
S
Cefepime
≤1
S
Ertapenem
≤0.5
S
aMICs 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.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 neighbor-joining 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 strainRaoultella terrigena NBRC 12941
T (Y17658) (Figure S1A in Supplementary Material available online at http://dx.doi.org/10.1155/2016/3879635).Housekeeping genesrpoB,gyrA, andparC were sequenced to demonstrate the taxonomic identification as described previously [3]. The sequence similarity ofgyrA,parC, andrpoB 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 withR. terrigena NBRC 12941
T (Figure S1B). Together with the typing result of 16S rRNA, the pus isolate Z38 was identified asR. terrigena.Table 2
The list of gene sequences used for phylogenetic tree construction.
Species
16S rRNA
gyrA
rpoB
parC
Raoultella terrigena NBRC 12941T
Y17658
AF303617
AY367362
AF303651
Raoultella planticola NBRC 12939T
AF129443
AF303621
AY367361
AF303655
Raoultella ornithinolytica NBRC 105727T
AF129441
AF303618
AF129447
AF303652
Raoultella electrica 1GBT
AB762091
AB828204
AB828205
AB828206
Klebsiella oxytoca ATCC 13182T
U78183
AF052257
AY367363
—
Klebsiella oxytoca KCTC 1686
NR_102982
CP003218
CP003218
CP003218
Klebsiella michiganensis W14T
JQ070300
JQ990329
JQ269337
—
Klebsiella variicola At-22
NR_074729
NC_013850
NC_013850
CP001891
Klebsiella pneumoniae subsp. pneumonia ATCC 13883T
Y17656
DQ673325
DQ673324
AF303641
Klebsiella pneumoniae subsp. rhinoscleromatis ATCC 13884T
NR_114507
FO203501
ACZD01000183
ACZD01000120
Klebsiella pneumoniae subsp. ozaenae ATCC 11297
AF130982
AF303619
AF129445
AF303653
Serratia liquefaciens ATCC 27592T
NR_121703
CP006252
NC_021742
CP006252


## 3. 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 toR. terrigena in 2007 [4]. The association betweenR. 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 ofRaoultella spp. is a challenge by routine work as this genus shares highly similar phenotypes with other genera of Enterobacteriaceae, especiallyKlebsiella spp. [5]. In 2001,Raoultella spp. as a novel species was separated fromKlebsiella spp. on the basis of 16S rDNA andrpoB typing results [6]. Phylogenetic analysis ofgyrB sequences showed thatK. terrigena (R. terrigena) andK. pneumoniae belong to two separate lineages [7]. Difficulties in correct identification ofRaoultella spp. may have led to an underestimation of its incidence and uncertainty on its pathogenic role [8]. Currently, the identification ofRaoultella species from clinical specimens mainly relied on phenotypic methods. A study has validated that MALDI-TOF MS increases the accuracy ofRaoultella identifications to the genus level [9]. However, the three species ofRaoultella 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 thatrpoB was demonstrated on the correct identifications of allRaoultella isolates [8]. Furthermore, three housekeeping genesgyrA (383 bp),rpoB (512 bp), andparC (319 bp) were used for species-level identification of members of theKlebsiella/Raoultella complex [3]. Therefore, in our study three housekeeping genes (gyrA,rpoB, andparC) were chosen for the identification ofR. terrigena. The phylogenetic analysis by concatenating the 3 housekeeping genes showed that strain Z38 was clustered withR. 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 forR. terrigena identification. Our study further implies that human infections byR. 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 byR. terrigena have been rarely reported. The firstR. terrigena infection case was reported in 2007, and a 45-year-old patient developed endocarditis due toR. terrigena after liver transplant [4]. Another infection case occurred in a patient who underwent pancreatic resection for pancreatic cancer [5]. However, the pathogenic role ofRaoultella spp. in human infection is still difficult to elucidate. Notably, some species ofRaoultellaspp., 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 byR. 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.



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*Source: 3879635-2016-06-09.xml*