Dear Editor,
Ochrobactrum pseudogrignonense is a gram-negative, non-spore-forming, aerobic bacillus, rarely encountered in clinical specimens. It was originally isolated in Scandinavia from the blood of a 28-year-old man in 1992 and the ear of a newborn in 2000 [1]. Since then, O. pseudogrignonense has been isolated from environmental sources [2,3,4]; however, its pathogenicity in humans has not been explored extensively. We report the first case of O. pseudogrignonense bacteremia in Korea—and only the third in the world [1], to our knowledge—and species identification was verified by 16S rRNA and recA gene sequencing. This study was approved by the Institutional Review Board (IRB) of Yonsei University of Medicine, Seoul, Korea (2019-1369). This is a retrospective report and hence IRB approval was obtained without consent forms.
A 44-year-old man with hypertension, diabetes mellitus, and dilated cardiomyopathy presented with aggravated dyspnea and falling blood pressure and was admitted to a hospital in March 2018. After three days, the patient received extracorporeal membrane oxygenation (ECMO) and was referred to a tertiary-care hospital in Seoul for heart transplantation. After transfer, the patient's vital signs were as follows: blood pressure, 110/87 mm Hg; pulse rate, 120/minutes; respiration rate, 16/minutes; and body temperature, 36.5℃. The initial laboratory results were as follows: hemoglobin, 175 g/L; white blood cell (WBC) count, 28.5×109/L; and platelet count, 118×109/L. The patient was initially placed on a vancomycin and piperacillin-tazobactam regimen. Although the patient had no fever one day after admission, his serum C-reactive protein level was 514.3 nmol/L, WBC count was 42.6×109/L, and delta neutrophil index was 12.4%, suggesting an increased immature granulocyte fraction [5]. As fever may not be apparent in ECMO patients with bacteremia [6] and considering the deteriorated laboratory results, the antibiotic regimen was changed from piperacillin-tazobactam to meropenem.
One set of aerobic and anaerobic blood cultures from the central venous line and two sets from different peripheral venipunctures were collected before initiation of antibiotic therapy and incubated for 36 hours in the BACT/ALERT 3D blood culture system (bioMérieux, Durham, NC, USA). All three aerobic vials demonstrated bacterial growth; they formed beige, non-hemolytic, distinct small circular colonies on blood and MacConkey agar plates within 24 hours of incubation at 35℃ (Fig. 1A & B). Gram-staining indicated gram-negative, rod-shaped bacteria without spores (Fig. 1C). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS; Bruker Daltonics, Bremen, Germany) of the isolate revealed signals characteristic of O. gallinifaecis with a score of 1.81; however, both the VITEK MS and VITEK2 systems (bioMérieux, Marcy l'Etoile, France) failed to identify the isolate.
Fig. 1. Colony and microscopic morphology of the O. pseudogrignonense isolate. (A) Colonies on blood agar (10×) and (B) MacConkey agar (10×) plates. (C) Gram staining of the isolate (1,000×).
To characterize the bacterium, molecular identification was performed by DNA amplification and sequencing of 16S rRNA [7]. Based on the EzTaxon database (http://www.ezbiocloud.net), both 373 and 1,374 bp 16S rRNA sequences of the isolate shared 100% sequence identity with those of O. pseudogrignonense (GenBank accession number AM422371) and 99.3% with those of Ochrobactrum thiophenivorans (GenBank number AM490617). Based on the Ribosomal Database Project (https://rdp.cme.msu.edu/), the isolate had a sequence match score of 1.0 for O. pseudogrignonense and 0.96 for O. thiophenivorans.
As 16S rRNA did not provide the resolution to distinguish the species [7], alternative analysis using the recA gene was performed [8,9]. We constructed primer pairs for partial sequencing O. pseudogrignonense (GenBank number AM422877.1) and O. thiophenivorans (GenBank number KF866345) recA: recA-O.pseudo-f (5′-TTCGGGTAAAACCACTCTCG-3′)/recA-O.pseudo-r (5′-ATGTCGAACTCGACCTGCTT-3′) and recA-O.thio-f (5′-GCAAGGGCTCAATCATGC-3′)/recA-O.thio-r (5′-AATCACCCATTTCACCTTCG-3′). The isolate was amplified successfully only with the O. pseudogrignonense recA primer pair (510 bp). This PCR product shared 100% sequence identity with the recA gene of O. pseudogrignonense, as determined using the GenBank alignment search tool (BLAST; https://blast.ncbi.nlm.nih.gov/Blast.cgi).
We performed antimicrobial susceptibility testing (AST) of the isolate using the VITEK2 system with the AST-N225 card, which was interpreted based on the breakpoints for other non-Enterobacteriaceae [10]. The results showed that the isolate was susceptible to meropenem and resistant to piperacillin-tazobactam (Table 1). Consistent with the AST results, two days after meropenem treatment, the patient's WBC count decreased to 16.0 × 109/L, and blood culture results were negative for five days. The patient recovered without any evident sequelae.
Table 1. Antimicrobial susceptibilities of the O. pseudogrignonense isolate.
| Antimicrobial agents | Susceptibility | MIC (µg/mL) |
|---|---|---|
| Amikacin | S | 8 |
| Ampicillin/Sulbactam | S | 8/4 |
| Aztreonam | R | ≥ 64 |
| Ceftazidime | S | 8 |
| Ciprofloxacin | S | ≤ 0.25 |
| Colistin | S | ≤ 0.5 |
| Cefepime | S | ≤1 |
| Cefotaxime | S | 4 |
| Gentamicin | S | ≤1 |
| Imipenem | S | 0.5 |
| Meropenem | S | 1 |
| Minocycline | S | ≤1 |
| Piperacillin | R | ≥ 128 |
| Piperacillin-Tazobactam | R | ≥ 128/4 |
| Cotrimoxazole | S | ≤ 20 |
| Tigecycline | S | ≤ 0.5 |
Abbreviations: MIC, minimum inhibitory concentration; S, susceptible; R, resistant.
Ochrobactrum species are emerging pathogens with low virulence that can cause severe systemic infections not only in patients with underlying diseases but also in immunocompetent patients [8,9]. This is the first case of human infection with O. pseudogrignonense in Korea. As the isolate showed resistance to piperacillin-tazobactam, accurate identification and AST were critical for timely and appropriate treatment; however, it was difficult to identify the isolate using commercial bacterial identification kits and MALDI-TOF MS. Owing to the high similarity of 16S rRNA sequences among Ochrobactrum species, as well as Brucella species [8,9], additional sequencing, such as of the recA gene, is needed for accurate species-level identification.
ACKNOWLEDGEMENTS
We are grateful to Myungsook Kim, Hyeon Ju Oh and the medical laboratory technologists from the department of clinical microbiology for the excellent technical assistance.
Footnotes
AUTHOR CONTRIBUTIONS: HWC and DY conceptualized and designed the study; searched the scientific literature; collected, analyzed, and interpreted the data; and wrote and revised the report. JHB, DK, HL, and KWL designed the study and revised the report. DY and KWL supervised the study and gave administrative, technical, and material support and secured the funding.
CONFLICTS OF INTEREST: No potential conflicts of interest relevant to this article are reported.
RESEARCH FUNDING: This work was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI14C1324).
References
- 1.Kampfer P, Scholz HC, Huber B, Falsen E, Busse HJ. Ochrobactrum haematophilum sp. nov. and Ochrobactrum pseudogrignonense sp. nov., isolated from human clinical specimens. Int J Syst Evol Microbiol. 2007;57:2513–2518. doi: 10.1099/ijs.0.65066-0. [DOI] [PubMed] [Google Scholar]
- 2.Yang Y, Yu X, Zhang R. Draft genome sequence of Ochrobactrum pseudogrignonense strain CDB2, a highly efficient arsenate-resistant soil bacterium from arsenic-contaminated cattle dip sites. Genome Announc. 2013;1:e00173-13. doi: 10.1128/genomeA.00173-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Zhao G, Ma F, Wei L, Chua H, Chang CC, Zhang XJ. Electricity generation from cattle dung using microbial fuel cell technology during anaerobic acidogenesis and the development of microbial populations. Waste Manag. 2012;32:1651–1658. doi: 10.1016/j.wasman.2012.04.013. [DOI] [PubMed] [Google Scholar]
- 4.Wu Z, Peng W, He X, Wang B, Gan B, Zhang X. Mushroom tumor: a new disease on Flammulina velutipes caused by Ochrobactrum pseudogrignonense. FEMS Microbiol Lett. 2016;363:fnv226. doi: 10.1093/femsle/fnv226. [DOI] [PubMed] [Google Scholar]
- 5.Seok Y, Choi JR, Kim J, Kim YK, Lee J, Song J, et al. Delta neutrophil index: a promising diagnostic and prognostic marker for sepsis. Shock. 2012;37:242–246. doi: 10.1097/SHK.0b013e3182454acf. [DOI] [PubMed] [Google Scholar]
- 6.Burket JS, Bartlett RH, Vander Hyde K, Chenoweth CE. Nosocomial infections in adult patients undergoing extracorporeal membrane oxygenation. Clin Infect Dis. 1999;28:828–833. doi: 10.1086/515200. [DOI] [PubMed] [Google Scholar]
- 7.CLSI. MM18. 2nd ed. Wayne, PA: Clinical and Laboratory Standards Institute; 2018. Interpretive criteria for identification of bacteria and fungi by targeted DNA sequencing. [Google Scholar]
- 8.Scholz HC, Al Dahouk S, Tomaso H, Neubauer H, Witte A, Schloter M, et al. Genetic diversity and phylogenetic relationships of bacteria belonging to the Ochrobactrum-Brucella group by recA and 16S rRNA gene-based comparative sequence analysis. Syst Appl Microbiol. 2008;31:1–16. doi: 10.1016/j.syapm.2007.10.004. [DOI] [PubMed] [Google Scholar]
- 9.Scholz HC, Tomaso H, Al Dahouk S, Witte A, Schloter M, Kampfer P, et al. Genotyping of Ochrobactrum anthropi by recA-based comparative sequence, PCR-RFLP, and 16S rRNA gene analysis. FEMS Microbiol Lett. 2006;257:7–16. doi: 10.1111/j.1574-6968.2006.00153.x. [DOI] [PubMed] [Google Scholar]
- 10.CLSI. M100. 28th ed. Wayne, PA: Clinical and Laboratory Standards Institute; 2018. Performance standards for antimicrobial susceptibility testing. [Google Scholar]