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HUANG, FANG

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GUSMAN, VERA P.

MEDIĆ, DEANA D.

TRUDIĆ, ANIKA DJ

BANOVIĆ, PAVLE Z.

NIKOLIĆ, NATAŠA M.

Polish Journal of Microbiology
2021, Vol. 70, No 3, 405–407
https://doi.org/10.33073/pjm-2021-037
SHORT COMMUNICATION
* Corresponding author: V.P. Gusman, University of Novi Sad, Faculty of Medicine, Department of Microbiology, Novi Sad, Serbia; 
Institute of Public Health of Vojvodina, Novi Sad, Serbia; e-mail: vera.gusman@mf.uns.ac.rs
© 2021 Vera P. Gusman et al.
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License (https://creativecommons.
org/licenses/by-nc-nd/4.0/).
Exiguobacterium aurantiacum belongs to the genus 
Exiguobacterium, initially isolated from potato-proces-
sing effluent in 1983 by Collins et al. (1983), as the first 
species of this genus. Later, members of this genus have 
been isolated from various environments, including gla-
ciers in Greenland and Siberia and hot springs in Yellow- 
 stone, today, counting 17 species (Vishnivetskaya et al. 
2009; Ramesh and Pandey 2017; Strahsburger et al. 2018).
E. aurantiacum are aerobic, motile, non-spore-form-
ing Gram-positive short coryneform bacilli, catalase- 
and DNase-positive, oxidase-negative, and alkaliphilic. 
These bacilli grow on nutrient agar at pH 10 and in the 
presence of NaCl 6% w/v (halotolerant), reduce nitrate 
to nitrite, and metabolize glucose fermentatively. Bio-
chemically very active, they produce acid from glucose, 
galactose, glycerol, maltose, mannitol, and sucrose 
but not from L-arabinose, dulcitol, lactose, melezitose, 
raffinose, rhamnose, sorbitol, or xylose. Sometimes 
these bacteria produce acid from fructose and sali- 
cin, and hydrolyse starch, casein and gelatin. They 
form orange-yellow pigmented colonies on blood agar 
(Takemura et al. 2009). E. aurantiacum has been very 
rarely reported to cause infections in humans world-
wide, such as bacteremia in the UK, corneal ulcers in 
India, and pneumonia in China (Pitt et al. 2007; Chen 
et al. 2017; Jain and Kamble 2018), however, in most 
works, the origin of the infection is still unclear.
So far, E. aurantiacum, was unidentified from human 
infections in Serbia. Here, we report the first isolation 
of E. aurantiacum in Serbia, still causing bacteremia in 
a patient after surgery.
In December 2020, two samples, distilled water and 
patients’ blood sample, from the Department of Uro-
logy, Clinical Center of Vojvodina, Novi Sad, Serbia, 
First Isolation of Exiguobacterium aurantiacum in Serbia
VERA P. GUSMAN1, 2*  , DEANA D. MEDIĆ1, 2, ANIKA DJ TRUDIĆ1, 3,
PAVLE Z. BANOVIĆ1, 4 and NATAŠA M. NIKOLIĆ1, 2
1 Department of Microbiology, Faculty of Medicine, University of Novi Sad, Novi Sad, Serbia
2 Institute of Public Health of Vojvodina, Novi Sad, Serbia
3 Institute for Pulmonary Diseases of Vojvodina, Sremska Kamenica, Serbia
4 Pasteur Institute of Novi Sad, Novi Sad, Serbia
Submitted 16 June 2021, revised 16 July 2021, accepted 28 July 2021
A b s t r a c t
K e y w o r d s: Exiguobacterium aurantiacum, identification, MALDI-TOF
Exiguobacterium aurantiacum is isolated from a variety of environ-
mental samples but rarely from patients. The aim of the study was 
to represent isolation of unusual bacterial strains that could cause 
infection in patients. Final identification was performed using 
matrix-assisted description/ionization time-of-flight mass spec-
trometry (MALDI-TOF). Two isolates strains of E. aurantiacum 
were isolated, one isolate from distilled water used during surgical 
treatment and the second one from a patient with bacteremia after 
radical prostatectomy, both sensitive to all tested antimicrobials. 
Environmental strains could cause infection, especially in immu-
nocompromised patients; therefore, rare bacteria testing is required, 
in which identification special assistance is provided by an auto-
mated system MALDI-TOF.
Gusman V.P. et al. 3406
were submitted to the Department of Sanitary Bac-
teriology of the Institute of Public Health of Vojvodina, 
Novi Sad, Serbia, for microbiological analysis.
First, distilled water was brought on routine micro-
biological examination according to European Pharma-
co poeia 9th edition. 100 ml of distilled water was asepti-
cally filtrated through the membrane filter system using 
sterile mixed cellulose esters membrane filters 0.45 μm 
pore size (Sartorius, Goettingen, Germany) after which 
membrane filter paper was placed on R2A agar (Oxoid, 
Basingstoke, Hampshire, UK), and incubated for seven 
days in 22°C. The colonies were then sub-cultured on 
blood agar (Oxoid, Basingstoke, Hampshire, UK), and 
further examination and confirmation were perfor- 
med using matrix-assisted description/ionization time- 
of-flight mass spectrometry spectrometry (MALDI-
TOF-MS) (Brucker, Carteret, NJ, USA). 
The following day, a blood sample was taken from an 
adult male, aged 65 years, who became febrile (38.4°C) 
after a radical prostatectomy. The samples were incu-
bated in aerobic and anaerobic blood culture bottles 
(BioMerieux, Marcy l’Etoile, France). Gram-positive 
bacilli have been found in both bottles and were sub-
sequently identified as E. aurantiacum using MALDI-
TOF-MS. The aerobic and anaerobic blood culture bot-
tles were inoculated on nutrient agar (HiMedia, India), 
incubated for 24 h at 35 ± 1°C. The colonies were trans-
ferred to a highly polished stainless steel plate to be 
analyzed in vacuum. In this particular application of the 
MALDI-TOF-MS method, the surface-associated mol-
ecules of the cell yield a species-specific mass spectral 
profile that can be aligned with profiles in an existing 
database (Keys et al. 2004). Both isolates matched the 
E. aurantiacum profile, with characteristic mass ions at 
772 and 1,379 Da reinforcing this identification.
Antimicrobial susceptibility testing was perfor- 
med according to EUCAST Broth Microdilution in 
accordance to ISO 20776-1, where the following anti-
microbial drugs were tested: penicillin, meropenem, 
genta micin, ciprofloxacin, rifampicin, vancomycin, 
tetra cycline, erythromycin, clindamycin according to 
EUCAST 2020, Clinical Breakpoint v.10.0 for nonspe-
cies related MIC breakpoint.
Two isolates of E. aurantiacum were obtained, the 
first one from distilled water used during surgery in the 
Department of Urology, Clinical Center of Vojvodina, 
Novi Sad, Serbia, and the second from the patient with 
bacteremia after radical prostatectomy. 
Antimicrobial susceptibility testing was performed 
by using the microdilution method, and the MIC 
results were as follows: penicillin (≤ 0.064), mero-
penem (≤ 0.064), gentamicin (≤ 0.125), ciprofloxacin 
(≤ 0.125), rifampicin (≤ 0.064), vancomycin (≤ 0.125), 
tetracycline (≤ 0.125), erythromycin (≤ 0.125), clinda-
mycin (≤ 0.125).
During the identification of bacteria empirical ther-
apy, ceftazidime, according to the protocol for fever, 
was applied. As bacterial strain was susceptible to all 
tested antimicrobial drugs, the patient responded well 
to administrated therapy, and microorganism was not 
recovered from subsequent blood culture specimens. 
Nevertheless, the mentioned bacterium was again iso-
lated from a repeated sample of distilled water, indi-
cating inadequate preparation of such water, which 
became bacterial contamination originating from the 
environment.
E. aurantiacum, a microorganism widely distrib-
uted in nature, is recognized as a rare human patho-
gen affecting mostly sensitive population groups, espe-
cially with an impaired immune system. It is difficult 
to be identified based on the traditional biochemical 
method due to a substantial similarity to Cellulomonas/
Microbacterium spp. (Pitt et al. 2007). Also, it can be 
misidentified as Oerskovia xanthineolytica when API 
Coryne kit (BioMérieux, Marcy l’Étoile, France) is used 
(Kenny et al. 2006). Inadequate and partial microbial 
diagnosis can lead to an inaccurate conclusion. There-
fore, it is necessary to include modern automated sys-
tems, such as MALDI-TOF, which helps in everyday 
work to identify bacteria to the species level. Identify-
ing environmental isolates to the species level is crucial 
for obtaining a correct coverage of the health impact 
caused by these microorganisms.
Based on literature reviews, E. aurantiacum is isola-
ted occasionally from various human clinical speci-
mens like blood, broncho-alveolar lavage fluid, or cor-
neal ulcers in only a few countries worldwide, such as 
the UK, China, and India. In most cases, the origin of 
the infection remained unclear or unknown, while in 
pneumonia, the inhalation route was confirmed, and 
in corneal ulcer, the connection with contact lenses 
was certified (Pitt et al. 2007; Chen et al. 2017; Jain and 
Kamble 2018). Our research concluded that the source 
of bacteria was distilled water, especially since the same 
pathogen was reisolated from the repeated samples of 
distilled water. Since the ability of biofilm production 
has been proven for Exiguobacterium sp. in several stud-
ies (Chen et al. 2017; Gutiérrez-Preciado et al. 2017), we 
believe that this is the most probable reason why this 
bacterium was reisolated. As a corrective measure, the 
remediation of the water distillation apparatus was pro-
posed. After this action, the distilled water samples were 
correct until micro contamination with potential path-
ogens capable of creating a biofilm occurs again, and 
there are more and more of them in the environment. 
This study is one of the rare examples, which try to elu-
cidate the link between the environment and the clinic.
Pitt et al. (2007) determined susceptibility of E. aura n- 
 tiacum to all tested antimicrobial drugs, as we did in 
our study, while Jain with associates (2018) and Chen 
First isolation of Exiguobacterium aurantiacum3 407
with collaborators (2017) determined the susceptibility 
of the bacteria to penicillin, meropenem, gentamicin, 
ciprofloxacin and rifampicin, and their resistance to tet-
racycline, erythromycin, clindamycin. The susceptibility 
to vancomycin was railed in these two studies; the bac-
teria were sensitive according to Chen et al. (2017) and 
resistant in the publication of Jain and Kamble (2018). It 
has been shown that environmental bacteria are involved 
in the horizontal flow of resistance genes. Especially 
soil microorganisms contribute to or acquire resist-
ance determinants from pathogens (Yang et al. 2014). 
Authors from the United Kingdom have reported 
similar findings of E. aurantiacum isolated from blood 
culture (Pitt et al. 2007), but this is the first report of 
such very rare isolate in Serbia.
Even if E. aurantiacum is unusual cause of human 
infection, this report describes the ability of environ-
mental strains to cause infection, especially in immu-
nocompromised patients, and emphasizes the need for 
testing rare bacteria, in which identification special 
assistance is provided by an automated system such as 
MALDI-TOF.
The isolation of E. aurantiacum from a sample of 
distilled water arouses a big doubt about the hygiene 
of the used device, which can cause a severe infec-
tion in patients who are subjected to the treatment. 
As a potential pathogen, E. aurantiacum should attract 
more attention.
   ORCID
Vera P. Gusman https://orcid.org/0000-0003-4112-239X 
Contribution
Each author contributed equally to the development of the 
manuscript.
Conflict of interest
The authors do not report any financial or personal connections 
with other persons or organizations, which might negatively affect 
the contents of this publication and/or claim authorship rights to 
this publication.
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