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 ~  Abstract
 ~ Introduction
 ~ Case Report
 ~ Discussion
 ~ Acknowledgement
 ~  References
 ~  Article Figures

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  Table of Contents  
CASE REPORT
Year : 2015  |  Volume : 33  |  Issue : 1  |  Page : 172-175
 

Shewanella algae in acute gastroenteritis


1 Department of Microbiology & Molecular Biology, Regional Medical Research Centre (Indian Council of Medical Research, Department of Health Research, Govt. of India), Nehru Nagar, Belgaum; KLE University, Nehru Nagar, Belgaum, Karnataka, India
2 Department of Microbiology & Molecular Biology, Regional Medical Research Centre (Indian Council of Medical Research, Department of Health Research, Govt. of India), Nehru Nagar, Belgaum, India
3 Karnataka Institute of Medical Sciences, Government Medical College, Hubli, Karnataka, India

Date of Submission16-Sep-2013
Date of Acceptance17-Feb-2014
Date of Web Publication5-Jan-2015

Correspondence Address:
S Roy
Department of Microbiology & Molecular Biology, Regional Medical Research Centre (Indian Council of Medical Research, Department of Health Research, Govt. of India), Nehru Nagar, Belgaum
India
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Source of Support: The study was supported by the Intramural funds of Indian Council of Medical Research and of Dept. of Medical Education, Govt. of Karnataka, Conflict of Interest: None


DOI: 10.4103/0255-0857.148442

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 ~ Abstract 

Shewanella algae is an emerging bacteria rarely implicated as a human pathogen. Previously reported cases of S. algae have mainly been associated with direct contact with seawater. Here we report the isolation of S. algae as the sole etiological agent from a patient suffering from acute gastroenteritis with bloody diarrhoea. The bacterium was identified by automated identification system and 16S rRNA gene sequence analysis. Our report highlights the importance of looking for the relatively rare aetiological agents in clinical samples that does not yield common pathogens. It also underscores the usefulness of automated systems in identification of rare pathogens.


Keywords: 16S rRNA, bacillary dysentery, diarrhoea, enteric pathogen, gene sequencing


How to cite this article:
Dey S, Bhattacharya D, Roy S, Nadgir S D, Patil A, Kholkute S D. Shewanella algae in acute gastroenteritis. Indian J Med Microbiol 2015;33:172-5

How to cite this URL:
Dey S, Bhattacharya D, Roy S, Nadgir S D, Patil A, Kholkute S D. Shewanella algae in acute gastroenteritis. Indian J Med Microbiol [serial online] 2015 [cited 2019 Apr 19];33:172-5. Available from: http://www.ijmm.org/text.asp?2015/33/1/172/148442



 ~ Introduction Top


Various species of Shewanella are among the many bacterial floras that are known to exist in seawater. Although several pathological conditions like bacteraemia, otitis media, cellulitis, gastroenteritis, abscesses, soft tissue infections and wound infections are attributed to direct exposure to seawater or ingestion of raw or undercooked seafood contaminated with these organisms, [1] reports of infection due to Shewanella has been rare and limited mainly to the species Shewanella algae. [2],[3] Known mostly for their ability of bioremediation/detoxification of heavy/radioactive metals in the environment, infections due to S. algae is being increasingly reported worldwide. [4],[5] However, such reports are scanty from developing countries like India, possibly because of the fact that most of the hospitals and diagnostic laboratories in these countries are not equipped with necessary facilities to identify such rare pathogens. [2] S. algae was isolated from two patients of acute gastroenteritis in North-East India recently in the year 2010, one in pure culture and the other mixed with  Escherichia More Details coli suggesting its role as an aetiological agent. [3] In the present communication we report the isolation of S. algae as the only aetiological agent from a patient suffering from acute gastroenteritis with bloody diarrhoea in Belgaum, South India, confirming its role as a causative agent of gastroenteritis.


 ~ Case Report Top


A 45-years-old male patient was admitted to the Karnataka Institute of Medical Sciences (KIMS), Hubli, located in the North-West part of the South Indian State of Karnataka, with complaint of gastroenteritis and acute diarrhoea with blood and mucous on 30 th July, 2012. The patient had mild dehydration and did not have any history of fever or vomiting. The patient was put on rehydration therapy and stool sample was collected for laboratory investigations before administration of any antibiotic. He was empirically treated for bacillary dysentery with oral ciprofloxacin, 500 mg tablet thrice daily.

After routine microscopy, the collected stool sample was processed for isolation of common bacterial enteric pathogens like Shigella,  Salmonella More Details, diarrhoeagenic E. coli, Vibrio, etc., following WHO (1987) [6] by enrichment in Selenite F broth, peptone water and alkaline peptone water for 6-8 hr before plating differential and selective media viz., McConkey's Agar, Hektoen Enteric agar (HEA), Thiosulphate and Citrate Bile Salt Sucrose (TCBS) Agar (Becton, Dickinson and Company, USA). Rapid lysate of the culture concentrate grown in peptone water was used as template in Polymerase Chain Reaction for detection of various diarrhoeagenic E. coli.[7] The stool samples were also tested for Group A Rotavirus by ELISA.

Pure growth of non-lactose-fermenting colonies was observed on MacConkey agar while yellowish mucoid colonies were noted in nutrient agar plates [Figure 1]. TCBS agar plates showed transparent colonies while there was no growth on HEA. The organisms were found to be oxidase positive. Although morphologically colony appearance was not typical of V. cholerae on TCBS agar, serology was performed with O1 and O139 Ogawa and Inaba antisera (Denka Seiken, Japan) and was found negative. Further biochemical tests revealed that these organisms were catalase positive, lactose non-fermenter in Difco oxidation-fermentation media, produced abundant H 2 S on TSI slant, urea was hydrolysed, glucose and maltose oxidised; lactose, mannitol and sucrose were not oxidised. The organism was presumptively identified as Shewanella sp. Automated identification system, Vitec2 Compact (bioMerieux, France) was subsequently used to confirm the identity of this organism as Shewanella algae.
Figure 1: Yellowish mucoid colonies of S. algae (M981) in nutrient agar plate

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For further confirmation, 16S rRNA gene sequence analysis was carried out. DNA was isolated by CTAB/NaCl method [8] and subjected to PCR amplification of the 16S rRNA-encoding gene using the universal primer 8F (5' AGA GTT TAG TCC TGG CTC AG 3') and 1492R (5' ACG GCT ACC TTG TTA CGA ATT 3') (Xcelris Labs Ltd, Ahmedabad, India). A single discrete PCR amplicon band of 1500 bp was obtained when resolved on agarose gel [Figure 2]. The PCR amplicon was purified to remove contaminants and cycle sequenced using BigDye chemistry (Applied Biosystems, Foster City, USA). Sequence data obtained was aligned and analyzed using Seqscape v2.7 (Applied Biosystems, USA) and BLAST5. [9] The 16S rRNA gene sequence of the present isolate was compared with other sequences submitted in the NCBI gene bank to understand its genetic relationship with other S. algae isolated from elsewhere by Neighbor Joining phylogenetic analysis. Based on maximum identity score the first 10 sequence were selected and aligned using multiple alignment software programme. The organism was found to have 100% similarity to S. algae isolated from different parts of the world [Figure 3] confirming the microbiological identifications.
Figure 2: Agarose gel picture of the 16s rRNA PCR product; Lane 1- Ladder, Lane 2 - S. algae isolate (M981), Lane 3 - E. coli (Positive control), Lane 4 - Negative control

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Figure 3: Phlylogenetic analysis of the 16S rRNA gene sequence of the S.algae isolate (M981) in comparison to other isolates belonging to other species and genus, respectively

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Antibiotic susceptibility tests were carried out by Kirby-Bauer disc diffusion method, [10] using 22 antibiotic impregnated discs (Hi-Media Laboratories Pvt Ltd., India) that included ampicillin (AMP, 10 μg), tetracycline (TET, 30 μg), co-trimoxazole (CoT, 20 μg), nalidixic acid (NAL, 30 μg), ciprofloxacin (CIP, 30 μg), gentamicin (GEN, 10 μg), norfloxacin (NOR, 10 μg), nitrofurantoin (NIT, 300 μg), ofloxacin (NIT, 5 μg), gatifloxacin (GAT, 5 μg), amikacin (AMK, 30 μg), azithromycin (AZM, 30 μg), imipenem (IMP, 30 μg), chloramphenicol (CHL, 30 μg), carbenicillin (CAR, 100 μg), cefixime (CFM, 30 μg), cefuroxime (CXM, 5 μg), cephalothin (CEF, 30 μg), ceftriaxone (CRO, 30 μg), cefotaxime (CTX, 30 μg), ceftazidime (CAZ, 30 μg) and augmentin/amoxicillin-clavulanic acid combination (AMC, 30 μg). Some of the drugs such as nitrofurantoin, imipenem, azithromycin and aminoglycosides were included though these are not recommended for treatment of bacterial diarrhoea, resistance to these could be used as a phenotypic characteristic to study the evolution of the pathogen over a period of time. Quality control strains Staphyllococcus aureus ATCC 25923 and E. coli ATCC 25922 were included in each test. The S. algae isolate was found to be resistant to cephalothin, and amoxicillin-clavulanic acid while it showed intermediate resistance to ampicillin. The isolate was sensitive to all the other antibiotics tested.

There was no other pathogenic bacterium isolated from the stool sample. Microscopic examination also did not reveal the presence of any parasitic infection. The patient responded to ciprofloxacin treatment and all symptoms disappeared within 48 hours and he was discharged soon after.


 ~ Discussion Top


S. algae, an otherwise very important environment-friendly bacterium, extensively studied and used in detoxification of metals and radioactive wastes, [4] is increasing being reported to be associated with a variety of clinical manifestations in human. [1],[5],[11] Among the 50 odd species of the genus S. algae, appears to be gradually being established as the predominant Shewanella species isolated from clinical specimens. [12] Mouse pathogenicity studies also indicated S. algae as the more virulent species of the genus with haemolytic activity which has been speculated to be an important virulence factor. [13]

Although reports of S. algae causing gastroenteritis has been rare and is only recently being reported, [5] isolation of S. algae had been reported from a couple of cases of diarrhoea, one in association with E. coli and the other in pure culture by [3] from North-East India recently. The role of S. algae as the sole aetiological agent is still being speculated. In our case, we did not isolate any bacterial species other than S. algae that can be attributed to have caused the disease. Interestingly the symptoms presented by the patient, i.e., acute diarrhoea with blood and mucous without fever and vomiting identical to the earlier observation [3] and this information will be useful for clinicians and microbiologists to keep this aetiology in mind while dealing with patients presenting such symptoms. The presentation of blood may be due to the haemolytic activity identified as a virulence factor of S. algae.[13] Although qnrA, the gene responsible for plasmid-mediated quinolone resistance has been shown to originate in the chromosome of S. algae, [14] the isolated strain was sensitive to most of the antibiotics including ciprofloxacin that was used to treat the patient although on wrong presumption of bacillary dysentery, and the patient responded to the treatment. Due to non-availability of the patient after discharge from hospital, further follow-up was not possible and we could not trace the source of his infection.

In a particular study from India, Sharma and Kalawat has documented that some of the organisms isolated from clinical cases including that of gastroenteritis that were identified as Pseudomonas species were in fact Shewanella species. [2] Because of the rarity of isolation of S. algae, lack of adequate infrastructure required for identification of such rare species and lack of awareness about the possibility of this organism causing infections, cases of S. algae infection is likely to be largely under-reported. In our case, the availability of automated microbial identification system (Vitec2 Compact) helped in correct identification of the microbiologically suspected S. algae isolate which was later confirmed by 16S rRNA gene sequencing.

Apart from confirming the role of Shewanella algae in acute gastroenteritis, our report highlights the importance of looking for the relatively rare aetiological agents in clinical samples that does not yield common pathogens. It also underscores the usefulness of automated systems in identification of rare pathogens.

Nucleotide sequence accession numbers

The 16S rRNA sequence of the S. algae has been deposited in the GenBank database under accession number KF499100.


 ~ Acknowledgement Top


The authors are thankful to Ms. Swapnali Kadam and Ms. Shanta Kalal for their technical assistance in the laboratory work. The authors are also thankful to the Biomedical Informatics Center (BIC), Belgaum for technical assistance in phylogenetic analysis. The authors are also thankful to the Indian Council of Medical Research for providing financial grants for the study.

 
 ~ References Top

1.
Myung DS, Jung YS, Kang SJ, Song YA, Park KH, Jung SI, et al. Primary Shewanella algae bacteremia mimicking Vibrio septicemia. J Korean Med Sci 2009;24:1192-4.  Back to cited text no. 1
    
2.
Sharma KK, Kalawat U. Emerging infections: Shewanella-A series of five cases. J Lab Physicians 2010;2:61-5.  Back to cited text no. 2
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3.
Nath R, Saikia L, Choudhury G, Das PP. Isolation of Shewanella algae from rectal swabs of patients with bloody diarrhea. Indian J Med Microbiol 2011;29:422-5.  Back to cited text no. 3
[PUBMED]  Medknow Journal  
4.
Janda JM, Abbott SL. The genus Shewanella: From the briny depths below to human pathogen. Crit Rev Microbiol 2014;40:293-312.  Back to cited text no. 4
    
5.
Vignier N, Barreau M, Olive C, Baubion E, Théodose R, Hochedez P, et al. Human infection with Shewanella putrefaciens and S. algae: Report of 16 cases in Martinique and review of the literature. Am J Trop Med Hyg 2013;89:151-6.  Back to cited text no. 5
    
6.
World Health Organization. Program for control of diarrheal diseases (CDD/83.3 REv. 1). In: Manual for laboratory investigations of acute enteric infections. Geneva: WHO, 1987. p. 5.27.  Back to cited text no. 6
    
7.
Orlandi PP, Magalhães GF, Matos NB, Silva T, Penatti M, Nogueira PA, et al. Etiology of diarrheal infections in children of Porto Velho (Rondonia, Western Amazon region, Brazil). Braz J Med Biol Res 2006;39:507-17.  Back to cited text no. 7
    
8.
In: Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, et al., editors Current Protocols in Molecular Biology. New York: John Wiley and Sons, Inc; 1999.  Back to cited text no. 8
    
9.
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 2011;28:2731-9.  Back to cited text no. 9
    
10.
Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; Twenty Second Informational Supplement. M100-S22. Wayne: CLSI; 2012.  Back to cited text no. 10
    
11.
Holt HM, Sogaard P, Gahrn-Hansen B. Ear infections with Shewanella algae: A bacteriologic, clinical and epidemiologic study of 67 cases. Clin Microbiol Infect 1997;3:329-34.  Back to cited text no. 11
    
12.
Nozue H, Hayashi T, Hashimoto Y, Ezaki T, Hamasaki K, Ohwada K, et al. Isolation and characterization of Shewanella alga from human clinical specimens and emendation of the description of S. alga Simidu et al., 1990, 335. Int J Syst Bacteriol 1992;42:628-34.  Back to cited text no. 12
    
13.
Khashe S, Janda JM. Biochemical and pathogenic properties of Shewanella alage and Shewanella putrefaciens. J Clin Microbiol 1998;36;783-7.  Back to cited text no. 13
    
14.
Poirel L, Rodriguez-Martinez JM, Mammeri H, Liard A, Nordmann P. Origin of plasmid-mediated quinolone resistance determinant QnrA. Antimicrob Agents Chemother 2005;49:3523-5.  Back to cited text no. 14
    


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  [Figure 1], [Figure 2], [Figure 3]



 

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