1. Int J Med Health Sci. July 2015,Vol-4;Issue-3 382
International Journal of Medical and Health Sciences
Journal Home Page: http://www.ijmhs.net ISSN:2277-4505
Isolation of Salmonella enterica ssp. diarizonae in clinical samples: A diagnostic
dilemma
Mohit Bhatia1*
, Archana Thakur2
, Bimbadhar Rath3
, Bibhabati Mishra4
, Vinita Dogra5
1
Senior Resident, 2
Director Professor, 4
Director Professor and Head, 5
Director Professor, Department of Microbiology,
Govind Ballabh Pant Institute of Post Graduate Medical Education and Research, Jawahar Lal Nehru Marg, New
Delhi-110002.
3
Professor and Head, Department of Pediatrics, Saraswathi Institute of Medical Sciences, Hapur Road, Anwarpur,
Uttar Pradesh 245304.
ABSTRACT
A twelve years old boy was admitted in a tertiary care hospital in January, 2015 with history of cough with expectoration, chest
pain and fever respectively. On examination, all his vital parameters were normal except low grade fever. No abnormality was
detected on systemic examination except slightly reduced air entry in right lung fields. His chest X-ray revealed consolidation of
right lung fields with blunting of right costo-phrenic angle. A CT scan of chest was immediately performed which revealed
collection of fluid in right pleural cavity and pleural tapping revealed frank pus. Subsequently, a chest drain was placed for the
drainage of pleural fluid. Pleural fluid was subjected to culture and sensitivity. Two different organisms namely Salmonella
enterica ssp. diarizonae and Escherichia coli were identified by VITEK-2 automated system. Keeping in mind the rare possibility
of human infection caused by Salmonella enterica ssp. diarizonae, this isolate was subjected to conventional biochemical
identification tests and eventually identified as a different strain of Escherichia coli. The patient was successfully treated and
discharged from the hospital after one week. To conclude, we would like to emphasize on the fact that identification by automated
systems of bacterial isolates in clinical samples as Salmonella enterica ssp. diarizonae does not necessarily mean that it is the
causative organism. Such bacterial isolates must be subjected to conventional biochemical identification methods. Laboratories
should consider several possibilities such as specimen contamination, incorrect identification by automated systems and
contamination of sheep blood agar before ascertaining pathogenic status to this organism.
KEYWORDS: Salmonella enterica ssp. diarizonae, Escherichia coli, VITEK-2.
INTRODUCTION
Salmonella spp. are documented to be pathogens that cause
a spectrum of diseases in humans and animals, including
domesticated and wild mammals, reptiles, birds, and
insects. Salmonella spp. infections are caused by
consumption of contaminated food, person-to-person
transmission, waterborne transmission and numerous
environmental and animal exposures [1].
S.enterica ssp. diarizonae is one of the less common
subspecies of Salmonella which like many non-typhoidal
salmonellae is mostly found in animal species (commonly
reptiles) and only occasionally infects humans [2]. It is
becoming increasingly common to keep reptiles as pets and
reports of infection caused by S. enterica ssp. diarizonae are
increasing [3]. Even a pseudo-outbreak of Salmonella
enterica ssp. diarizonae infection has been reported in the
past [4].
We present a case report which highlights the issue of
bacterial isolates obtained from clinical samples being
incorrectly reported as S.enterica ssp. diarizonae by
automated identification systems.
Case Report

2. Int J Med Health Sci. July 2015,Vol-4;Issue-3 383
CASE REPORT
A twelve years old male child was admitted in a tertiary care
hospital in January, 2015 with history of cough and fever
since two months and one month respectively. His cough
was associated with expectoration which was copious in
amount, green in color, not blood tinged and without any
positional and diurnal variation. He also had chest pain on
right side which was aggravated by coughing. He was on
medication (details not available) under the supervision of a
local doctor for more than a month prior to hospitalization
but did not show any symptomatic improvement. On
examination, all his vital parameters were normal except
low grade fever of 99.5˚F. Systemic examination did not
reveal any abnormality except slightly reduced air entry all
over the lung fields on right side. However, the child was
not in respiratory distress. His chest X-ray revealed
consolidation of right lung fields with blunting of right
costo-phrenic angle.
A CT scan of chest was immediately performed which
revealed collection of fluid in right pleural cavity and
pleural tapping revealed frank pus. Subsequently, a chest
drain was placed for the drainage of pleural fluid. The
following investigations were also carried out: hemoglobin-
9.4 g%; total leucocyte count- 11,800; differential leucocyte
count- neutrophils 79% and lymphocytes 15%. Pleural fluid
analysis revealed a cell count of >50,000 cells/cu.mm with
predominance of neutrophils and occasional lymphocytes.
Pleural fluid was also subjected to culture and sensitivity.
Two different types of colonies were obtained on blood and
MacConkey agar (both colonies being lactose fermenting).
These were subjected to identification and antibiotic
susceptibility testing using VITEK-2 automated system
(Biomerieux India Pvt. Ltd.).
Pan drug resistant Salmonella enterica ssp. diarizonae
(resistant to amoxicillin/clavulanate, amikacin,
cotrimoxazole, cefotaxime, cefuroxime, ceftriaxone,
cefepime, ciprofloxacin, ofloxacin, gentamicin, imipenem,
meropenem and piperacillin/tazobactam) and Escherichia
coli (sensitive to imipenem, meropenem, amikacin,
gentamicin and resistant to amoxicillin/clavulanate,
cotrimoxazole, cefotaxime, cefuroxime, ceftriaxone,
cefepime, ciprofloxacin, ofloxacin, imipenem, meropenem
and piperacillin/tazobactam) were identified.
Keeping in mind the rare possibility of human infection
caused by Salmonella enterica ssp. diarizonae, we subjected
this isolate to conventional biochemical identification tests,
the results of which were as follows: Catalase test: Positive;
Indole test: Positive; Triple Sugar Iron test: Acid slant/Acid
butt with gas; Urea hydrolysis test: Negative; Citrate
utilization test: Negative; Malonate utilization test:
Negative; Methyl red test: Positive; Fermentation of glucose
with production of gas, lactose, sucrose and mannitol
respectively. These results indicated that the two
supposedly different bacterial isolates were in fact two
different strains of Escherichia coli with different antibiotic
susceptibility patterns.
Before the final culture and sensitivity report was prepared,
the patient was empirically treated with injection ampicillin-
cloxacillin (ampiclox) which was stopped after two days and
replaced by tablet linezolid, injection gentamicin and tablet
metronidazole for one week. General condition of the
patient improved. Chest drainage gradually decreased in
volume and the drain was subsequently removed.
DISCUSSION
Arizona group organism was first isolated by Caldwell and
Ryerson in 1939. This group has been called by various
names such as Salmonella arizonae, Paracolobacterium
arizonae, Arizona arizonae, Arizona hinshawii etc [1] . S.
enterica ssp. diarizonae is part of the normal reptile
intestinal flora but can cause disease in monotremes,
turkeys, chickens, goats, and rarely in humans [5].
S. enterica ssp. diarizonae enteritis or systemic infections
have been well described in patients resident in the southern
states of the USA and Europe it is much rarer, with only a
few cases reported in the literature [6-9]. Most cases of
invasive S. enterica ssp. diarizonae infection have been
either in younger patients or those with underlying diseases
including collagen vascular diseases, malignancy, organ
transplantation and HIV infection [10].
It can be difficult to identify S. enterica ssp. diarizonae due
to their distinguishing biochemical features such as the
ability to utilize malonate, liquefy gelatin and the inability to
grow in the presence of KCN (potassium cyanide), which
are generally not looked into for routine identification of
bacterial isolates. Isolation of S. enterica ssp. arizonae from
the stools is difficult as some strains ferment lactose within
48 hours (approximately 15%) and they may be routinely
discarded as non-pathogens. However the presence of
hydrogen sulfide is an important diagnostic clue during
routine screening [11].
In our case out of the two organisms (Escherichia coli and
Salmonella enterica ssp. diarizonae) isolated in culture of
pleural fluid obtained from our patient, Salmonella enterica
ssp. diarizonae was pan-drug resistant. However,
Escherichia coli was relatively sensitive to certain
antibiotics including gentamicin which was eventually used
(as one of the drugs) to successfully treat this patient.
Isolation of Salmonella enterica ssp. diarizonae from
pleural fluid probably occurred as a result of
misidentification by VITEK-2 system as this isolate was
later correctly identified as Escherichia coli using
conventional biochemical identification tests [12] Similar
observation was made a month later when an isolate from
sputum sample of another patient admitted in our hospital
was incorrectly identified as Salmonella enterica ssp.
diarizonae. VITEK-2 sytem cannot be completely relied
upon for identification of Gram negative rods. In a study
conducted by Guido Funke et al, 84.7% of the isolates of
Gram negative bacilli (either belonging to the family
Enterobacteriaceae or otherwise) were correctly identified at
the species level [13]
Isolation of unusual bacteria from clinical specimens might
sound an alarm regarding a likely outbreak keeping in mind
the background prevalence and pattern of bacterial isolates
in hospital settings. Thiolet et al had reported pseudo-
outbreak of Salmonella enterica ssp. diarizonae infection in
France in 2008. The authors had successfully demonstrated
that the rare SIIIb serotype 61:k:1,5,7 or its monophasic
variant 61:-:1,5,7 considered to be adapted to sheep, may

3. Int J Med Health Sci. July 2015,Vol-4;Issue-3 384
cause sheep blood agar contamination and lead to false-
positive culture results and to pseudo-outbreaks [4].
Concomitant isolation of Salmonella enterica ssp.
diarizonae from pleural fluid, stool and blood samples
obtained from our patient would have pointed towards this
organism being the most likely pathogen. However, since
stool and blood samples were not sent for culture, therefore,
we cannot correlate that Salmonella enterica ssp. diarizonae
was the causative organism. This isolate was pan-drug
resistant but the patient responded to antibiotic therapy.
Also, the pleural fluid sample was obtained from a drainage
tube. All these findings indicate that Salmonella enterica
ssp. diarizonae was most probably a contaminant.
CONCLUSION
To conclude, we would like to emphasize on the fact that
identification (by automated systems) of bacterial isolates in
clinical samples as Salmonella enterica ssp. diarizonae does
not necessarily mean that it is the causative organism. Such
bacterial isolates must be subjected to conventional
biochemical identification methods. Concomitant isolation
of this organism from different clinical samples obtained
from the same patient and clinical history should be
considered before reaching any conclusion. Laboratories
should consider several possibilities such as specimen
contamination, incorrect identification by automated
systems and contamination of sheep blood agar with
Salmonella enterica ssp. diarizonae in case the
aforementioned criteria of ascertaining the pathogenic status
of this organism are not met.
ACKNOWLEDGEMENTS
Dr. Bimbadhar Rath, Professor and Head, Department of
Pediatrics, Saraswathi Institute of Medical Sciences, Hapur
Road, Anwarpur, Uttar Pradesh 245304.
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______________________________________________
*Corresponding author: Dr. Mohit Bhatia
E-Mail: docmb1984@gmail.com