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Indian Journal of Medical Microbiology, Vol. 29, No. 4, October-December, 2011, pp. 422-425 Case Report Isolation of Shewanella algae from rectal swabs of patients with bloody diarrhoea R Nath, L Saikia, G Choudhury, PP Das Department of Microbiology, Assam Medical College and Hospital, Dibrugarh, Assam 786 002, India Date of Submission: 05-Mar-2011 Code Number: mb11102 PMID: 22120808 Abstract Shewanella algae is an emerging bacteria rarely implicated as a human pathogen. It was infrequently recovered from clinical specimens probably because of inadequate processing of non-fermenting oxidase-positive gram-negative bacilli. We report here isolation of S. algae in pure culture and mixed with E. coli from two cases of acute gastroenteritis with bloody mucous containing diarrhea occurring at the same time. As this organism is not a normal flora of the gut, the possible source of infection may be fish contaminated with the organism. Whether this bacterium can be considered an enteric pathogen needs to be evaluated. The cases were clinically diagnosed as acute bacillary dysentery. The bacterium was identified by 16S r-RNA gene sequence analysis. Keywords: Dysentery, Shewanella algae, Shewanella spp Introduction Shewanella is a marine bacteria rarely implicated as a human pathogen. Two species of importance are Shewanella algae and Shewanella putrefaciens. [1] S. algae may cause a variety of clinical symptoms in humans ranging from skin and soft tissue infections to bacteraemia and osteomyelitis. [2],[3],[4],[5],[6],[7] It is an oxidase-positive, hydrogen sulphide producing Gram negative bacilli. It was infrequently recovered from clinical specimens probably because of inadequate processing of non-fermenting oxidase-positive gram-negative bacilli. We report here isolation of S. algae in pure culture and mixed with E. coli from two cases of acute gastroenteritis with bloody mucous containing diarrhoea occurring at the same time. Whether this bacterium can be considered an enteric pathogen needs to be evaluated. The cases were clinically diagnosed as acute bacillary dysentery. Case Reports Case 1 A 70-year-old woman along with another member of her family suffered from bloody mucous containing diarrhoea with a frequency of 5-6 times in a day, which started 12 h after consuming dinner in a remote village of Lakhimpur district, Assam, in September 2010. Both of them consumed dinner with rice and fish. There was no history of fever or vomiting. Rectal swab was collected by health personnel in Cary Blair transport medium before starting empirical therapy for acute bacillary dysentery with ciprofloxacin tablet 500 mg 8 hourly. The other family member was unavailable for taking sample and was also treated empirically for bacillary dysentery. Case 2 We report a case of a 20-year-old man with a history of passing bloody mucous containing stool since last 24 h. There was a history of eating fish and rice for dinner 12 h earlier. No history of fever or vomiting could be found. Both the patients belonged to the same village and the symptoms started at the same time. Other members of his family were not affected. Whether both the patients brought the fish from the same source was not known. There was no history of taking any seafood or visiting any sea side in both the cases. Even though we could not implicate any food directly for lack of microbiological evidence, fish can be suspected as the possible source. In both the families, 7 people consumed fish preparations and 3 persons were affected at the same time with the same symptoms (attack rate 42.8%). Hospitalization was not needed. As the area is endemic for cholera and shigellosis, these rectal swabs were sent as a routine laboratory investigation for culture and sensitivity to the district level microbiology laboratory. Samples of other household members were not sent. Both the persons were otherwise not suffering from any general disease or local lesion. Source of drinking water was tube well. The samples were inoculated into MacConkey agar (BBL: Becton, Dickinson and Company, Sparks, MD, USA), Salmonella-Shigella agar (BBL: Becton, Dickinson and Company, Sparks, MD, USA), nutrient agar and also in alkaline peptone water (all; Hi-Media Laboratories Pvt Ltd, Mumbai, Maharashtra, India) to exclude Vibrionaceae group as these cases came from cholera endemic rural Assam and Vibrio cholerae 01 ogawa was reported in that district recently. Culture plates were incubated at 37°C for 18-24 h, alkaline peptone water was incubated for 6-8 h and subcultured thereafter in MacConkey agar, TCBS (BBL: Becton, Dickinson and Company, Sparks, MD, USA) agar and nutrient agar. In case 1, there was pure growth of non-lactose-fermenting colonies in MacConkey agar (10 4 CFU/plate), yellowish mucoid colonies in nutrient agar [Figure - 1]. Colonies produced haemolysis in sheep blood agar media on subculture. Biochemically it was oxidase positive, catalase positive, nonfermenter in Difco oxidation-fermentation media, produced abundant H 2 S on triple sugar iron agar slant, decarboxylated ornithine, urea was hydrolyzed, glucose and maltose oxidized; lactose, mannitol, and sucrose not oxidized. The organism was presumptively identified as Shewanella spp. For confirmation, 16S r-RNA gene sequence analysis was done. DNA was extracted (QIAamp DNA Purification Kit, Qiagen; Hilden, Germany) and its quality was assessed on 1.2% agarose gel, a single band of high molecular weight DNA was observed. PCR amplification of the 16S rRNA-encoding gene was performed by using the universal primer pair 8F (5′ AGA GTT TGA TCC TGG CTC AG 3′) and 1492R (5′ ACG GCT ACC TTG TTA CGA CTT 3′) commercially obtained from Xcelris Labs Ltd, Ahmedabad, India. A single discrete PCR amplicon band of 1500 bp was observed when resolved on agarose gel. The PCR amplicon was purified to remove contaminants. Forward and reverse DNA sequencing reaction of the PCR amplicon was carried out with 8F and 1492R primers using Big Dye Terminator v3.1 Cycle sequencing kit on ABI 3730xl Genetic analyzer (Applied Biosystems Inc, California, USA). Consensus sequence of 1287 bp 16S rDNA gene was generated from forward and reverse sequence data using aligner software. The 16S rDNA gene sequence was used to carry out BLAST with the nr database of NCBI GenBank database. Based on maximum identity score the first 10 sequences were selected and aligned using multiple alignment software programme Clustal W. Distance matrix was generated using RDP database and the phylogenetic tree [Figure - 2] was constructed using MEGA 4. The organism was found to have 100% similarity to S. alga (GenBank Accession Number: SA U91544.1). Phylogenetic tree (constructed by the maximum likelihood method). The strain from our patient, which is called C33, was compared with other strains of Shewanella spp. already described in the literature. In case 2, Escherichia coli colonies mixed with non-lactose-fermenting, mucoid, yellow, oxidase-positive colonies were detected, which were biochemically same as that of case 1. E. coli colonies were tested for the presence of enterotoxigenic pili by serotyping with K88, K99, and 987p and O157: H7 anti-sera (Denka Seikan, Tokyo, Japan), which were found to be negative. Other tests for detection of shiga-like toxin in E. coli isolate were not done. This non-lactose-fermenting bacterium was presumptively identified as S. algae as it was biochemically exactly same as that of case 1 and reported from the same area. Clonality of the strains was not tested. Antibiotic susceptibility was tested by Kirby-Bauer disc diffusion method (Clinical Laboratory Standards Institute), [8] using Mόller-Hinton agar and antibiotic discs from Hi-Media Laboratories Pvt Ltd. The strains were found to be sensitive to gentamicin, cotrimoxazole, amoxicillin-clavulanic acid, ciprofloxacin, norfloxacin, chloramphenicol, azithromycin, ofloxacin, and resistant to nalidixic acid, ceftazidime, and cefotaxime. The MICs to different antibiotics as determined by Hi-Comb (Hi-Media) were azithromycin 0.1 mcg/mL, ciprofloxacin 0.5 mcg/mL, chloramphenicol 0.1 mcg/mL, norfloxacin 2 mcg/mL, ofloxacin 2 mcg/mL, cefotaxime 30 mcg/mL, nalidixic acid ≥240 mcg/mL, and ceftazidime ≥240 mcg/mL. E coli 25922 was used as the control strain. Both the cases received ciprofloxacin tablet 500 mg 8 hourly as empirical therapy of acute bacillary dysentery after collection of the samples. The symptoms stopped after ciprofloxacin therapy within 24 h in both the cases. Repeat sample after 15 days did not yield the bacteria. Samples of water as well as samples of rectal swabs sent for culture from other patients of the same area did not yield the organism. Discussion Shewanella algae is a recently described marine bacterial species, which plays a role in the environment in the turnover of Fe (III) and other metal ions. [9],[10] Originally it was named as S. alga and later on it was changed to S. algae. [11],[12] It can play a role in in situ bioremediation. [13] S. algae may cause a variety of clinical symptoms in humans. [2],[3],[4],[5],[6],[7] Most of the infections are reported from a warm climate. It is not found as a normal flora of the gut and no other sample in the hospital yielded Shewanella spp., excluding laboratory contamination as a possible cause. In case 2, as it was isolated in mixed culture with E coli, the significance of the isolate remained doubtful. But in case 1, as it was in pure culture, the significance of the isolate remains to be seen. We searched for available literature and found only one case of acute gastroenteritis reported from India where the etiological evidence was said to be doubtful as it was mixed with E. coli and the patient later developed haemolytic uremic syndrome. [14] In both our cases though we could not implicate any food directly for lack of microbiological evidence, fish can be suspected as the possible source. Sea water is known to be the most common source though. Dominguez et al. in 1996 reported the presence of this organism in stagnant water. [10] Both our patients did not have any history of travelling to any seaside in the recent past. Shewanella spp., especially Shewanella putrefaciens is known to cause spoilage of marine fish, especially finfish. As it is also isolated from stagnant waters, it can cause spoilage of fish at warm temperature as this organism is mesophilic. S. algae is not found as a normal flora of gut; therefore, probable source of this organism in these two cases of dysentery could have been the contaminated fish. Most of the S. algae infections in man are skin and soft tissue infections. [4],[5] It has also been reported from a case of osteomyelitis. [7] Bacteraemia is also reported to occur with S. algae. [3] The pathogenic potential of the bacteria is said to be conferred by the haemolytic activity of S. algae. [1] No other case of gastroenteritis caused due to this organism could be found in the literature. Even though S. algae was isolated in pure culture in the first case, the possibility of other causes could not be ruled out because of lack of environmental and microbiological evidence of the organism in food material and water. Further environmental and clinical studies will be necessary to know the significance of the organism as an enteric pathogen. This case report emphasizes the need to further characterize the oxidase-positive, H 2 S-producing non-fermenter gram-negative bacilli to know the prevalence of this organism even in areas not exposed to sea in different clinical specimens, including stool and rectal swab samples from diarrhoea and dysentery cases. References
Copyright 2011 - Indian Journal of Medical Microbiology The following images related to this document are available:Photo images[mb11102f1.jpg] [mb11102f2.jpg] |
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