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 Rath

3, Bibhabati Mishra

4, Vinita Dogra

5

1Senior Resident,

2Director Professor,

4Director Professor and Head,

5Director Professor, Department of Microbiology,

Govind Ballabh Pant Institute of Post Graduate Medical Education and Research, Jawahar Lal Nehru Marg, New

Delhi-110002.

3Professor 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

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

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.

REFERENCES

1. Yunsop C, Oh HK, Samuel YL, Ki SC, Toshio S.

Salmonella enterica subspecies diarizonae bacteremia in

an infant with enteritis- A case report. Yonsel Medical

Journal 1991; 32(3): 275-78

2. Habermalz D, Pietzsch O: Identification

of arizona bacteria. A contribution to the problem of

salmonella infections among reptiles and amphibians in

zoological gardens. Zentralbl Bakteriol Orig

A 1973; 225:323-42

3. Bhatt BD, Zuckerman MJ, Foland JA, Polly SM,

Marwah RK: Disseminated Salmonella arizona infection

associated with rattlesnake meat ingestion. Am J

Gastroenterol 1989; 84:433-435

4. J. M. Thiolet, N. J. Da Silva, A. Reggiani, H. De Valk,

B. Coignard, F. X. Weill. Nationwide pseudo-outbreak

of Salmonella enterica ssp. diarizonae,France. Clin

Microbiol Infect 2011; 17: 915–918

5. Aiken AM, Lane C, Adak GK: Risk

of Salmonella infection with exposure to reptiles in

England, 2004-2007. Euro Surveill 2010;15(22):19581

6. Casner PR, Zuckerman MJ: Salmonella arizonae in

patients with AIDS along the U.S.-Mexican border. N

Engl J Med 1990;323:198-199

7. Schneider L, Ehlinger M, Stanchina C, Giacomelli MC,

Gicquel P, Karger C, Clavert JM:Salmonella

enterica subsp. arizonae bone and joints sepsis. A case

report and literature review. Orthop Traumatol Surg

Res 2009; 95:237-242

8. Starakis I, Siagris D, Karatza C, Solomou H, Bassaris

H: Endocarditis due to Salmonella

enterica subsp. arizonae in a patient with sickle cell

disease: a case report and review of the literature.

Cardiovasc Hematol Disord Drug Targets 2007;7:199-

204

9. Editorial team, Bertrand S, Rimhanen-Finne R, Weill

FX, Rabsch W, Thornton L, Perevoscikovs J, van Pelt

W, Heck M: Salmonella infections associated with

reptiles: the current situation in Europe. Euro

Surveill 2008; 13:18902

10. Hoag JB, Sessler CN: A comprehensive review of

disseminated Salmonella Arizona infection with an

illustrative case presentation. South Med

J 2005; 98:1123-1129

11. The Enterobacteriaceae. In: Winn W, Allen SD, Allen S,

Janda W, Koneman EW, Schreckenberger PC, Procop

GW, Woods GL editors. Koneman's Color Atlas and

Textbook of Diagnostic Microbiology. 6th edition.

Baltimore: Lippincott Williams & Wilkins; 2006.

pp.211-300

12. Pamela B.C. Enterobacteriaceae: Escherichia, Klebsiella,

Proteus and other genera In:Coghlan JD. Collee JG,

Fraser AG, Marimon BP, Simmons A, editors. . Mackie

& Mc Cartney Practical Medical Microbiology.14th ed.

New Delhi: Elsevier; 2007 : pp.361-84

13. Funke G, Monnet D, deBernardis C, von Graevenitz

A, Freney J. Evaluation of the VITEK 2 system for rapid

identification of medically relevant gram-negative rods.

J Clin Microbiol. 1998 Jul;36(7):1948-52

______________________________________________

*Corresponding author: Dr. Mohit Bhatia E-Mail: docmb1984@gmail.com