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Bacteraemia caused by Catabacter hongkongensis
Anaerobe 18 (2012) 366e368
Contents lists available at SciVerse ScienceDirect
Anaerobe journal homepage: www.elsevier.com/locate/anaerobe
Note
Bacteraemia caused by Catabacter hongkongensis Kevin Smith a, Sushil K. Pandey b,1, James E. Ussher a, * a b
Clinical Microbiology Laboratory, North Shore Hospital, Private Bag 93-503, Takapuna, Auckland 0740, New Zealand Department of Microbiology, LabPlus, PO Box 110031, Auckland City Hospital, Auckland 1148, New Zealand
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 January 2012 Received in revised form 24 March 2012 Accepted 27 March 2012 Available online 4 April 2012
Catabacter hongkongensis is a recently recognised anaerobic gram positive bacillus. We report the isolation of a C. hongkongensis strain that does not utilise mannose, a novel phenotype, from the blood cultures of a patient with a perforated appendix. The isolate was identified by 16s rRNA gene sequencing. This case highlights the difficulties with phenotypic identification of rare anaerobic isolates. It also illustrates the association of C. hongkongensis bacteraemia with a gastrointestinal source and intestinal perforation. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Catabacter hongkongensis Bacteraemia
A 47 year old New Zealand man was admitted to hospital with a one day history of abdominal pain. He had no past medical history except for a recent admission for the drainage of a perineal abscess from which Streptococcus pyogenes was isolated. This had been treated with gentamicin, amoxicillin and metronidazole followed by amoxicillin-clavulanate, which he was still taking when readmitted. On examination he was febrile, tachycardic and tender with guarding in the right iliac fossa. One set of blood cultures was taken before the commencement of intravenous cefuroxime and metronidazole. A CT scan was consistent with appendicitis and a localised perforation. The appendix was removed laparoscopically and the right iliac fossa and pelvis washed out. Histopathological examination showed extensive mucosal ulceration, full-thickness neutrophil infiltration, and areas of full-thickness necrosis, consistent with perforation. He received intravenous cefuroxime and metronidazole for 4 days and oral amoxicillin-clavulanate for a further 12 days and made an uneventful recovery. A non-sporing gram positive coccobacillus was isolated from the BACTEC FX Anaerobic/F lytic bottle (Becton Dickinson, MD) after 4 days incubation. The aerobic bottle and a subsequent set of blood cultures taken after the initiation of antibiotics failed to grow. No other specimens were submitted for culture. Non-haemolytic * Corresponding author. Present address: Peter Medawar Building for Pathogen Research, South Parks Road, Oxford, OX1 3SY, United Kingdom. Tel.: þ44 1865 281232 (office), þ44 7407 242410 (mobile); fax: þ44 1865 281531. E-mail addresses: [email protected] (K. Smith), sushil_ [email protected] (S.K. Pandey), [email protected] (J.E. Ussher). 1 Present address: Mycobacterium Reference Laboratory, Pathology Queensland, Sir Raphael Cilento Building, Herston Hospitals Complex, Herston, Queensland 4029, Australia. 1075-9964/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.anaerobe.2012.03.006
pinpoint colonies were detected on fastidious anaerobic agar (Fort Richard Laboratories) after 48 h anaerobic incubation. There was no growth on Columbia sheep blood agar (Fort Richard Laboratories) or supplemented chocolate agar (Fort Richard Laboratories) after 4 days incubation at 37 C in 5% CO2. A Gram stain from the plate confirmed the isolate to be a non-sporulating gram positive coccobacillus. The isolate was catalase positive, had a rapid ID32A (bioMérieux SA) profile of 0010000010 (positive for b-galactosidase and glutamic acid decarboxylase activity only); notably it failed to utilise D-mannose. It was nitrate negative (BBL Taxo Differentiation Discs Nitrate, USA) and motile (BBL Motility Media, USA). It did not grow after 48 h in 5% CO2, however when these plates were re-incubated anaerobically for a further 48 h growth was noted. Antimicrobial susceptibility testing was performed by the gradient diffusion method on fastidious anaerobic agar inoculated with a 1.0 McFarland suspension and incubated anaerobically for 48 h; CLSI interpretative criteria for anaerobes were used [Clinical and Laboratory Standards Institute, [1]]. The MICs of metronidazole, vancomycin, penicillin, amoxicillinclavulanate, imipenim (all M.I.C.Evaluator, Oxoid, UK), and ceftriaxone (Etest, AB bioMérieux, France) were <0.016 mg ml1, 2 mg ml1, 4 mg ml1, 2 mg ml1, 0.12 mg ml1, and 8 mg ml1 respectively. The isolate was b-lactamase negative (BBL Cefinase paper disc, BD, USA). The identity of the isolate was confirmed by 16s rRNA gene sequencing [2]. A BLAST search of the sequence revealed 100% sequence homology with Catabacter hongkongensis (GenBank ID: AY574991 [3]). C. hongkongensis is an anaerobic non-sporulating gram positive coccobacillus which has been recently identified from the blood cultures of four patients from Hong Kong and Canada [3]. Two
K. Smith et al. / Anaerobe 18 (2012) 366e368
367
Table 1 Summary of reported cases of Catabacter hongkongensis bacteraemia. Demographics
Location
Diagnosis
Past medical history
Source (day of culture isolated)
Treatment
Outcome
Reference
48 yo, male
Hong Kong
Partial small bowel obstruction with secondary sepsis
Blood (ANO2, day 3)
Cefuroxime þ metronidazole
Recovered
Lau et al., 2007
39 yo, male
Hong Kong
Acute appendicitis with perforation
ESRF e haemodialysis Tuberculous peritonitis Recurrent bowel obstruction Nil
Blood (ANO2, day 3)
Recovered
Lau et al., 2007
74 yo, male
Canada
Fever post exchange of biliary stent
Blood (NS)
Surgery Cefuroxime þ metronidazole / oral antibiotics Ciprofloxacin
Recovered
Lau et al., 2007
66 yo, female
Canada
NS 52 yo, male
North Carolina, USA France
Fever, sepsis syndrome NS
47 yo, male
New Zealand
Intestinal perforation, peritonitis, septic shock Acute appendicitis with perforation
Plasmacytoma, Secondary biliary obstruction with multiple stents Metastatic carcinoma of the lung (palliative) NS
Blood (ANO2, day 5)
Cefuroxime þ ciprofloxacin
Died
Lau et al., 2007
Blood (NS)
NS
NS
Hypertension
Blood (ANO2, day 3)
Died
Perineal abscess
Blood (ANO2, day 4)
Surgery Amoxicillin-clavulanate þ gentamicin Surgery Cefuroxime þ metronidazole / amoxicillin-clavulanate
Simmon et al., 2008 Elsendoorn et al., 2011
Recovered
This report
NS, not stated; yo, years old; ANO2, anaerobic bottle; ESRF, end stage renal failure.
further clinical isolates have been reported: one by 16s rRNA sequencing of 316 anaerobic isolates from bloodstream infections from over 5 years in North Carolina, USA [4], and the other from the blood cultures of a patient from France [5]. Our isolate was highly similar to the phenotypic description of the type strain, HKU16, except that it was mannose negative as assessed by the Rapid ID32A. All previous isolates have utilised mannose in both the Rapid ID32A and API 20A systems [3,5]. The identity of our isolate was confirmed by 16s rRNA sequencing. Therefore in addition to the previously reported variable utilisation of glycerol and rhamnose and production of leucine arylamidase [3,5], C. hongkongensis is also variable in its ability to utilise mannose. The following profiles have been reported in the Rapid ID32A system: 0012000010 [3], 0002000010 [5], and 0010000010. Of the seven reported clinical isolates of C. hongkongensis all were from blood cultures, five from patients with a gastrointestinal source, one from a patient with metastatic lung carcinoma and a sepsis syndrome but no identified source, and one in which no clinical details were provided (Table 1). Three patients had intestinal perforation. The median time to detection was 3 days (range 3e5 days); while this is similar to the common contaminant Propionibacterium acnes, C. hongkongensis can be differentiated by its gram stain appearance and the lack of indole production [3]. While our patient made a rapid recovery with surgery and antimicrobial therapy, in 2 of the 6 reported cases of C. honkongensis bacteraemia the patient died. This is similar to the mortality rate previously reported for clinically significant anaerobic bacteraemia [6]. Our isolate was susceptible to metronidazole, vancomycin, amoxicillin-clavulanate, imipenem, and ceftriaxone but had reduced susceptibility to penicillin (MIC 4 mg ml1). The isolate was b-lactamase negative. Lau et al. reported a similar susceptibility pattern with variable susceptibility to penicillin (MICs of 0.5e4 mg ml1) [3]. The recent isolate from France also showed reduced susceptibility to penicillin (MIC 2 mg ml1) but in contrast to the report by Lau et al. was resistant to kanamycin and susceptible to colistin [5]. In addition to clinical specimens, C. hongkongensis has also been detected by molecular methods in environmental samples. The detection of C. hongkongensis DNA in four of twenty nine polluted water samples from Spain (three from a waste water inlet and one from a river in an urban area) [7] and in sediment from an Egyptian
lake polluted with human waste [8] may either reflect a high level of faecal contamination or an alternative environmental reservoir. A Catabacter sp. was also identified in multiple urban air samples from San Antonio and Austin, Texas, by a 16s rRNA DNA microarray [9], although this may not be C. hongkongensis but a related species. Therefore while an environmental reservoir for C. hongkongensis cannot be excluded, it is likely that it is at least a transient part of the gastrointestinal microflora and has a global distribution. Indeed, the detection by 16s rRNA PCR of C. hongkonensis in the faeces of a captive Dugong dugong from Japan suggests that it may also be part of the gastrointestinal microflora in other mammals [10]. In conclusion, isolation of C. hongkongensis from blood cultures should be considered suggestive of a gastrointestinal source, with intestinal perforation common. In addition to variable production of leucine arylamidase and utilisation of glycerol and rhamnose, C. hongkongensis is also variable in its utilisation of mannose. The difficulty accurately identifying rare anaerobic isolates such as C. hongkongensis by standard laboratory phenotypic methods highlights the utility of 16s rRNA gene sequencing [4]. Acknowledgements We would like to thank Dr Dragana Drinkovic (North Shore Hospital) and Dr Susan Taylor (Middlemore Hospital) for their helpful comments on the manuscript. References [1] Clinical and Laboratory Standards Institute. Methods for antimicrobial susceptibility testing of anaerobic bacteria. Approved standard M11-A7. 7th ed. Wayne, PA: Clinical and Laboratory Standards Institute; 2007. [2] Lane DJ. 16s/23s rRNA sequencing. In: Stackebrant E, Goodfellow M, editors. Nucleic acid techniques in bacterial systematics. London: John Wiley & Sons Ltd; 1991. p. 115e75. [3] Lau SKP, McNabb A, Woo GKS, Hoang L, Fung AMY, Chung LMW, et al. Catabacter hongkongensis gen. nov., sp. nov., isolated from blood cultures of patients from Hong Kong and Canada. J Clin Microbiol 2007;45:395e401. [4] Simmon KE, Mirrett S, Reller LB, Petti CA. Genotypic diversity of anaerobic isolates from bloodstream infections. J Clin Microbiol 2008;46:1596e601. [5] Elsendoorn A, Robert R, Culos A, Roblot F, Burucoa C. Catabacter hongkongensis bacteremia with fatal septic shock. Emerg Infect Dis 2011;17:1330e1. [6] Salonen JH, Eerola E, Meurman O. Clinical significance and outcome of anaerobic bacteremia. Clin Infect Dis 1998;26:1413e7.
368
K. Smith et al. / Anaerobe 18 (2012) 366e368
[7] Codony F, Adrados B, Pérez LM, Fittipaldi M, Morató J. Detection of Catabacter hongkongensis in polluted European water samples. J Zhejiang Univ Sci B 2009;10:867e9. [8] El Saied HE. Molecular genetic monitoring of bacterial communities in Manzala lake, Egypt, based on 16s rRNA gene analysis. Egypt J Aquatic Res 2007; 33:179e94.
[9] Brodie EL, DeSantis TZ, Parker JPM, Zubietta IX, Piceno YM, Andersen GL. Urban aerosols harbor diverse and dynamic bacterial populations. Proc Natl Acad Sci U S A 2007;104:299e304. [10] Tsukinowa E, Karita S, Asano S, Wakai Y, Oka Y, Furuta M, et al. Fecal microbiota of a dugong (Dugong dugong) in captivity at Toba Aquarium. J Gen Appl Microbiol 2008;54:25e38.
Contents lists available at SciVerse ScienceDirect
Anaerobe journal homepage: www.elsevier.com/locate/anaerobe
Note
Bacteraemia caused by Catabacter hongkongensis Kevin Smith a, Sushil K. Pandey b,1, James E. Ussher a, * a b
Clinical Microbiology Laboratory, North Shore Hospital, Private Bag 93-503, Takapuna, Auckland 0740, New Zealand Department of Microbiology, LabPlus, PO Box 110031, Auckland City Hospital, Auckland 1148, New Zealand
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 January 2012 Received in revised form 24 March 2012 Accepted 27 March 2012 Available online 4 April 2012
Catabacter hongkongensis is a recently recognised anaerobic gram positive bacillus. We report the isolation of a C. hongkongensis strain that does not utilise mannose, a novel phenotype, from the blood cultures of a patient with a perforated appendix. The isolate was identified by 16s rRNA gene sequencing. This case highlights the difficulties with phenotypic identification of rare anaerobic isolates. It also illustrates the association of C. hongkongensis bacteraemia with a gastrointestinal source and intestinal perforation. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Catabacter hongkongensis Bacteraemia
A 47 year old New Zealand man was admitted to hospital with a one day history of abdominal pain. He had no past medical history except for a recent admission for the drainage of a perineal abscess from which Streptococcus pyogenes was isolated. This had been treated with gentamicin, amoxicillin and metronidazole followed by amoxicillin-clavulanate, which he was still taking when readmitted. On examination he was febrile, tachycardic and tender with guarding in the right iliac fossa. One set of blood cultures was taken before the commencement of intravenous cefuroxime and metronidazole. A CT scan was consistent with appendicitis and a localised perforation. The appendix was removed laparoscopically and the right iliac fossa and pelvis washed out. Histopathological examination showed extensive mucosal ulceration, full-thickness neutrophil infiltration, and areas of full-thickness necrosis, consistent with perforation. He received intravenous cefuroxime and metronidazole for 4 days and oral amoxicillin-clavulanate for a further 12 days and made an uneventful recovery. A non-sporing gram positive coccobacillus was isolated from the BACTEC FX Anaerobic/F lytic bottle (Becton Dickinson, MD) after 4 days incubation. The aerobic bottle and a subsequent set of blood cultures taken after the initiation of antibiotics failed to grow. No other specimens were submitted for culture. Non-haemolytic * Corresponding author. Present address: Peter Medawar Building for Pathogen Research, South Parks Road, Oxford, OX1 3SY, United Kingdom. Tel.: þ44 1865 281232 (office), þ44 7407 242410 (mobile); fax: þ44 1865 281531. E-mail addresses: [email protected] (K. Smith), sushil_ [email protected] (S.K. Pandey), [email protected] (J.E. Ussher). 1 Present address: Mycobacterium Reference Laboratory, Pathology Queensland, Sir Raphael Cilento Building, Herston Hospitals Complex, Herston, Queensland 4029, Australia. 1075-9964/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.anaerobe.2012.03.006
pinpoint colonies were detected on fastidious anaerobic agar (Fort Richard Laboratories) after 48 h anaerobic incubation. There was no growth on Columbia sheep blood agar (Fort Richard Laboratories) or supplemented chocolate agar (Fort Richard Laboratories) after 4 days incubation at 37 C in 5% CO2. A Gram stain from the plate confirmed the isolate to be a non-sporulating gram positive coccobacillus. The isolate was catalase positive, had a rapid ID32A (bioMérieux SA) profile of 0010000010 (positive for b-galactosidase and glutamic acid decarboxylase activity only); notably it failed to utilise D-mannose. It was nitrate negative (BBL Taxo Differentiation Discs Nitrate, USA) and motile (BBL Motility Media, USA). It did not grow after 48 h in 5% CO2, however when these plates were re-incubated anaerobically for a further 48 h growth was noted. Antimicrobial susceptibility testing was performed by the gradient diffusion method on fastidious anaerobic agar inoculated with a 1.0 McFarland suspension and incubated anaerobically for 48 h; CLSI interpretative criteria for anaerobes were used [Clinical and Laboratory Standards Institute, [1]]. The MICs of metronidazole, vancomycin, penicillin, amoxicillinclavulanate, imipenim (all M.I.C.Evaluator, Oxoid, UK), and ceftriaxone (Etest, AB bioMérieux, France) were <0.016 mg ml1, 2 mg ml1, 4 mg ml1, 2 mg ml1, 0.12 mg ml1, and 8 mg ml1 respectively. The isolate was b-lactamase negative (BBL Cefinase paper disc, BD, USA). The identity of the isolate was confirmed by 16s rRNA gene sequencing [2]. A BLAST search of the sequence revealed 100% sequence homology with Catabacter hongkongensis (GenBank ID: AY574991 [3]). C. hongkongensis is an anaerobic non-sporulating gram positive coccobacillus which has been recently identified from the blood cultures of four patients from Hong Kong and Canada [3]. Two
K. Smith et al. / Anaerobe 18 (2012) 366e368
367
Table 1 Summary of reported cases of Catabacter hongkongensis bacteraemia. Demographics
Location
Diagnosis
Past medical history
Source (day of culture isolated)
Treatment
Outcome
Reference
48 yo, male
Hong Kong
Partial small bowel obstruction with secondary sepsis
Blood (ANO2, day 3)
Cefuroxime þ metronidazole
Recovered
Lau et al., 2007
39 yo, male
Hong Kong
Acute appendicitis with perforation
ESRF e haemodialysis Tuberculous peritonitis Recurrent bowel obstruction Nil
Blood (ANO2, day 3)
Recovered
Lau et al., 2007
74 yo, male
Canada
Fever post exchange of biliary stent
Blood (NS)
Surgery Cefuroxime þ metronidazole / oral antibiotics Ciprofloxacin
Recovered
Lau et al., 2007
66 yo, female
Canada
NS 52 yo, male
North Carolina, USA France
Fever, sepsis syndrome NS
47 yo, male
New Zealand
Intestinal perforation, peritonitis, septic shock Acute appendicitis with perforation
Plasmacytoma, Secondary biliary obstruction with multiple stents Metastatic carcinoma of the lung (palliative) NS
Blood (ANO2, day 5)
Cefuroxime þ ciprofloxacin
Died
Lau et al., 2007
Blood (NS)
NS
NS
Hypertension
Blood (ANO2, day 3)
Died
Perineal abscess
Blood (ANO2, day 4)
Surgery Amoxicillin-clavulanate þ gentamicin Surgery Cefuroxime þ metronidazole / amoxicillin-clavulanate
Simmon et al., 2008 Elsendoorn et al., 2011
Recovered
This report
NS, not stated; yo, years old; ANO2, anaerobic bottle; ESRF, end stage renal failure.
further clinical isolates have been reported: one by 16s rRNA sequencing of 316 anaerobic isolates from bloodstream infections from over 5 years in North Carolina, USA [4], and the other from the blood cultures of a patient from France [5]. Our isolate was highly similar to the phenotypic description of the type strain, HKU16, except that it was mannose negative as assessed by the Rapid ID32A. All previous isolates have utilised mannose in both the Rapid ID32A and API 20A systems [3,5]. The identity of our isolate was confirmed by 16s rRNA sequencing. Therefore in addition to the previously reported variable utilisation of glycerol and rhamnose and production of leucine arylamidase [3,5], C. hongkongensis is also variable in its ability to utilise mannose. The following profiles have been reported in the Rapid ID32A system: 0012000010 [3], 0002000010 [5], and 0010000010. Of the seven reported clinical isolates of C. hongkongensis all were from blood cultures, five from patients with a gastrointestinal source, one from a patient with metastatic lung carcinoma and a sepsis syndrome but no identified source, and one in which no clinical details were provided (Table 1). Three patients had intestinal perforation. The median time to detection was 3 days (range 3e5 days); while this is similar to the common contaminant Propionibacterium acnes, C. hongkongensis can be differentiated by its gram stain appearance and the lack of indole production [3]. While our patient made a rapid recovery with surgery and antimicrobial therapy, in 2 of the 6 reported cases of C. honkongensis bacteraemia the patient died. This is similar to the mortality rate previously reported for clinically significant anaerobic bacteraemia [6]. Our isolate was susceptible to metronidazole, vancomycin, amoxicillin-clavulanate, imipenem, and ceftriaxone but had reduced susceptibility to penicillin (MIC 4 mg ml1). The isolate was b-lactamase negative. Lau et al. reported a similar susceptibility pattern with variable susceptibility to penicillin (MICs of 0.5e4 mg ml1) [3]. The recent isolate from France also showed reduced susceptibility to penicillin (MIC 2 mg ml1) but in contrast to the report by Lau et al. was resistant to kanamycin and susceptible to colistin [5]. In addition to clinical specimens, C. hongkongensis has also been detected by molecular methods in environmental samples. The detection of C. hongkongensis DNA in four of twenty nine polluted water samples from Spain (three from a waste water inlet and one from a river in an urban area) [7] and in sediment from an Egyptian
lake polluted with human waste [8] may either reflect a high level of faecal contamination or an alternative environmental reservoir. A Catabacter sp. was also identified in multiple urban air samples from San Antonio and Austin, Texas, by a 16s rRNA DNA microarray [9], although this may not be C. hongkongensis but a related species. Therefore while an environmental reservoir for C. hongkongensis cannot be excluded, it is likely that it is at least a transient part of the gastrointestinal microflora and has a global distribution. Indeed, the detection by 16s rRNA PCR of C. hongkonensis in the faeces of a captive Dugong dugong from Japan suggests that it may also be part of the gastrointestinal microflora in other mammals [10]. In conclusion, isolation of C. hongkongensis from blood cultures should be considered suggestive of a gastrointestinal source, with intestinal perforation common. In addition to variable production of leucine arylamidase and utilisation of glycerol and rhamnose, C. hongkongensis is also variable in its utilisation of mannose. The difficulty accurately identifying rare anaerobic isolates such as C. hongkongensis by standard laboratory phenotypic methods highlights the utility of 16s rRNA gene sequencing [4]. Acknowledgements We would like to thank Dr Dragana Drinkovic (North Shore Hospital) and Dr Susan Taylor (Middlemore Hospital) for their helpful comments on the manuscript. References [1] Clinical and Laboratory Standards Institute. Methods for antimicrobial susceptibility testing of anaerobic bacteria. Approved standard M11-A7. 7th ed. Wayne, PA: Clinical and Laboratory Standards Institute; 2007. [2] Lane DJ. 16s/23s rRNA sequencing. In: Stackebrant E, Goodfellow M, editors. Nucleic acid techniques in bacterial systematics. London: John Wiley & Sons Ltd; 1991. p. 115e75. [3] Lau SKP, McNabb A, Woo GKS, Hoang L, Fung AMY, Chung LMW, et al. Catabacter hongkongensis gen. nov., sp. nov., isolated from blood cultures of patients from Hong Kong and Canada. J Clin Microbiol 2007;45:395e401. [4] Simmon KE, Mirrett S, Reller LB, Petti CA. Genotypic diversity of anaerobic isolates from bloodstream infections. J Clin Microbiol 2008;46:1596e601. [5] Elsendoorn A, Robert R, Culos A, Roblot F, Burucoa C. Catabacter hongkongensis bacteremia with fatal septic shock. Emerg Infect Dis 2011;17:1330e1. [6] Salonen JH, Eerola E, Meurman O. Clinical significance and outcome of anaerobic bacteremia. Clin Infect Dis 1998;26:1413e7.
368
K. Smith et al. / Anaerobe 18 (2012) 366e368
[7] Codony F, Adrados B, Pérez LM, Fittipaldi M, Morató J. Detection of Catabacter hongkongensis in polluted European water samples. J Zhejiang Univ Sci B 2009;10:867e9. [8] El Saied HE. Molecular genetic monitoring of bacterial communities in Manzala lake, Egypt, based on 16s rRNA gene analysis. Egypt J Aquatic Res 2007; 33:179e94.
[9] Brodie EL, DeSantis TZ, Parker JPM, Zubietta IX, Piceno YM, Andersen GL. Urban aerosols harbor diverse and dynamic bacterial populations. Proc Natl Acad Sci U S A 2007;104:299e304. [10] Tsukinowa E, Karita S, Asano S, Wakai Y, Oka Y, Furuta M, et al. Fecal microbiota of a dugong (Dugong dugong) in captivity at Toba Aquarium. J Gen Appl Microbiol 2008;54:25e38.