SEARCH

SEARCH BY CITATION

Keywords:

  • Bacteraemia;
  • Exiguobacterium aurantacum;
  • human infections;
  • identification;
  • mass spectrum profiles;
  • susceptibility

Abstract

  1. Top of page
  2. Abstract
  3. Acknowledgements
  4. References

Exiguobacterium spp. are alkaliphilic, halotolerant, non-spore-forming Gram-positive bacilli, hitherto uncharacterised from human infections. Six isolates of Exiguobacterium aurantiacum were obtained from patients with bacteraemia, three of whom had myeloma. All isolates formed orange–yellow pigmented colonies on blood agar, were catalase- and DNase-positive, and grew on nutrient agar at pH 10 and in the presence of NaCl 6% w/v. The six isolates were susceptible to all antimicrobial agents tested and were uniform in their fatty acid and mass spectrum profiles.

Coryneform bacteria include a diverse range of bacterial genera grouped together as aerobic non-spore-forming Gram-positive bacilli. Of these, the genus Exiguobacterium was first isolated from potato-processing effluent in 1983 [1] in the form of a single species, Exiguobacterium aurantiacum. Eight other alkaliphilic and halotolerant species have been described subsequently, namely Exiguobacterium acetylicum, Exiguobacterium antarticum, Exiguobacterium undae, Exiguobacterium oxidotolerans, Exiguobacterium aestuarii, Exiguobacterium marinum, Exiguobacterium mexicanum and Exiguobacterium artemiae[2,3]. A report in 2003 used 16S rDNA sequence homology to indicate the possible presence of E. aurantiacum in a patient with periodontitis [4], and a novel Exiguobacterium sp. was identified from blood culture in 2006 using the same technology [5]. The present study reports the identification of a further six isolates of E. aurantiacum from blood cultures over a 10-year period. The first case was a male aged 27 years with a history of intravenous drug abuse. The patient presented to hospital with a swollen and tender left leg, with cellulitis of the left groin where he had injected himself 2 days previously. On the following day, blood samples were taken, from which Gram-positive yellow-pigmented colonies were isolated. Three further isolates were from adult males, two of whom had multiple myeloma and the third of whom had suspected infective endocarditis. The fifth case was a neonate. All of these patients responded to therapy and the organism was not recovered from subsequent specimens.

Full clinical information was available for the sixth patient, who was a Caucasian male aged 55 years, diagnosed with IgG kappa multiple myeloma 4 years previously. This patient received local radiotherapy, corticosteroids and six courses of infusional chemotherapy via an indwelling central venous line. During the final course of chemotherapy, the patient became febrile (38.2°C) and experienced rigors after the indwelling central line was flushed. The patient was otherwise well and not neutropenic, and was therefore treated empirically with intravenous ceftazidime and teicoplanin according to the protocol for fever. The aerobic and anaerobic blood culture bottles drawn through the central line yielded Gram-positive bacilli (identified subsequently as E. aurantiacum) and coagulase-negative staphylococci. Despite treatment with intravenous ceftazidime and teicoplanin for the following 3 days, the fever persisted. Peripheral blood cultures, taken simultaneously, were negative. Only when the central line was removed on day 4 did the fever resolve. The isolation of E. aurantiacum from central, but not peripheral, blood cultures, together with the temporal association between line flushing and the onset of symptoms, supports the diagnosis of an E. aurantiacum long-line infection. The fact that the patient's fever resolved only when the line was removed is also consistent with this diagnosis. Unfortunately, specific details of the line tip culture were no longer available, and a report of ‘normal skin flora’ was issued.

All isolates grew aerobically on Columbia blood agar and formed orange–yellow pigmented colonies 1–1.5 mm in diameter after incubation for 24–48 h at 37°C. All six isolates produced catalase and DNase, and grew on nutrient agar at pH 10 and in the presence of NaCl 6% w/v. Reactions in the API CORYNE kit (bioMérieux, Marcy l'Etoile, France) and in additional biochemical tests indicated a strong similarity to Cellulomonas/Microbacterium spp. (Table 1). All isolates were susceptible to ampicillin, cefotaxime, chloramphenicol, ciprofloxacin, clindamycin, erythromycin, gentamicin, penicillin, rifampicin, teicoplanin, tetracycline and trimethoprim according to Etests (AB Biodisk, Solna Sweden).

Table 1.   Biochemical test results for clinical isolates of Exiguobacterium aurantiacum
  1. +, positive; –, negative; v, variable.

Nitratev
Pyrazinamidase+
Pyrrolidonyl-arylamidase
Alkaline phosphatasev
β-Glucuronidase
β-Galactosidasev
α-Glucosidase+
β-n-Acetyl-glucosaminidasev
Aesculin+
Urease
Gelatinv
Acid from:
 Glucose+
 Ribose+
 Xylosev
 Mannitolv
 Maltose+
 Lactose
 Sucrose+
 Glycogen+
Catalase+
Oxidase
Casein hydrolysis+
Tween-80 hydrolysis
Tyrosine (growth)+
Tyrosine (hydrolysis)
Tyrosine (pigment)
DNase+

Cultures were grown on Trypticase Soya Broth Agar (BBL, Cockeysville, MD, USA) at 30°C for 48 h, after which their long-chain cellular fatty acids were extracted and analysed by gas chromatography (MIDI Sherlock, Newark, NJ, USA). As shown in Table 2, the major fatty acid peaks were isoC13:0, anteisoC13:0, C16:0,isoC17:0, and C18:0. This profile gave a score that was unacceptably low for identification of E. acetylicum, but E. aurantiacum was not included in the database. Of note, the n-alcohol C16:0 values for the six isolates ranged between 1.09 and 8.62. The type strain gave a value of 1.98, which could explain the previous failure of Lopez-Cortez et al. [3] to detect this fatty acid. Universal primers for 16S rDNA genes were used to amplify a near complete fragment using PCR, with subsequent sequencing revealing high sequence homology (99.2%) with E. aurantiacum (GenBank).

Table 2.   Whole-cell fatty acids of clinical isolates of Exiguobacterium aurantiacum
Fatty acidE. aurantiacum
isoC11:01.05–1.60
isoC12:01.05–2.98
C12:00.2.20–5.93
isoC13:010.39–20.65
anteisoC13:05.21–10.95
isoC14:00–0.65
C14:02.06–6.07
isoC15:01.38–6.34
anteisoC15:01.17–1.51
n-Alcohol C16:01.09–8.62
isoC16:00.93–1.62
C16:1 w11c2.38–15.87
C16:015.45–32.65
isoC17:1 w10c0–0.91
isoC17:03.40–11.52
anteisoC17:01.23–1.65
C17:00.69–0.77
C18:3 w6c (6,9,12)0–2.46
C16:0 2OH0.35–0.82
C18:1 w9c1.08–3.58
C18:03.45–11.91

The patient isolates and the type strain of E. aurantiacum (NCIMB 11798) were analysed further by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS). In this particular application of the method, the surface-associated molecules of the cell yield a species-specific mass spectral profile that can be aligned with profiles in an existing database [6]. All of the isolates matched the E. aurantiacum profile, with characteristic mass ions at 772 and 1379 Da reinforcing this identification (Fig. 1).

image

Figure 1.  Mass spectral profiles of two isolates of Exiguobacterium aurantiacum, showing characteristic peaks at 772 and 1397 Da.

Download figure to PowerPoint

In recent years, an increasing number of environmental saprophytic bacterial species have emerged that have the capacity to breach environmental barriers and cause infection in immunocompromised hosts and individuals at extremes of age. Although E. aurantiacum has been recovered from clinical specimens in the past, it has not hitherto been reported as a cause of bacteraemia. Three of the six cases described in the present study had myeloma, which might indicate that individuals with immunosuppression are predisposed to infection by E. aurantiacum. The species is characteristically extremely tolerant of alkaline conditions; thus, growth at pH 10 would appear to be a useful screen to presumptively classify yellow-pigmented coryneforms as E. aurantiacum. Other supportive biochemical characteristics are catalase production, fermentation of glucose, maltose and sucrose, aesculin hydrolysis, motility and salt tolerance.

In summary, this report describes the characterisation of an hitherto unsuspected bacterial species from cases of bacteraemia, and underlines the need to examine clinically relevant isolates of obscure species using a polyphasic taxonomic approach.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Acknowledgements
  4. References

We are grateful to the hospital laboratories that submitted isolates for species identification, and to T. Rogers for permission to publish the clinical details of case 6.

References

  1. Top of page
  2. Abstract
  3. Acknowledgements
  4. References
  • 1
    Collins MD, Lund BM, Farrow JAE, Schleifer KH. Chemotaxonomic study of an alkalophilic bacterium Exiguobacterium aurantiacum gen. nov. sp. nov. J Gen Microbiol 1983; 129: 20372042.
  • 2
    Kim I-G, Lee M-H, Jung S-Y, Song JJ, Oh T-K, Yoon J-H. Exiguobacterium aestuarii sp. nov. and Exiguobacterium marinum sp. nov., isolated from a tidal flat of the Yellow Sea in Korea. Int J Syst Evol Microbiol 2005; 55: 885889.
  • 3
    Lopez-Cortes A, Schumann P, Pukall R, Stackenbrandt E. Exiguobacterium mexicanum sp. nov. and Exiguobacterium artemiae sp. nov., isolated from the brine shrimp Artemia franciscana. Syst Appl Microbiol 2006; 29: 183190.
  • 4
    Zijnge V, Harmsen HJM, Kleinfelder JW, Van Der Rest ME, Degener JE, Welling GW. Denaturing gradient gel electrophoresis analysis to study bacterial community structure in pockets of periodontitis patients. Oral Microbiol Immunol 2003; 18: 5965.
  • 5
    Millar BC, Kenny F, Xu J, Moore JE, McClurg RB. Potential misidentification of a new Exiguobacterium sp. as Oerskovia xanthineolytica isolated from blood culture. Br J Biomed Sci 2006; 63: 86.
  • 6
    Keys CJ, Dare DJ, Sutton H et al. Compilation of a MALDI-TOF mass spectral database for the rapid screening and characterization of bacteria implicated in human infectious diseases. Infect Genet Evol 2004; 4: 221242.