- Email: [email protected]
First Australian description of Eggerthella lenta bacteraemia identified by 16S rRNA gene sequencing
Pathology (June 2008) 40(4), pp. 409–439
CORRESPONDENCE
First Australian description of Eggerthella lenta bacteraemia identified by 16S rRNA gene sequencing Sir, We wish to report the first Australian case of a patient with Eggerthella lenta infection, demonstrating the usefulness of 16S rRNA gene sequencing technology in a clinical diagnostic microbiology laboratory. A 68-year-old woman was admitted to hospital with a two-day history of malaise, anorexia, nausea and colicky abdominal pain. She had no fever or sweats, no change in bowel habit, no diarrhoea or vomiting and no melaena. She had a history of thyroidectomy, ectopic pregnancy and hypertension. On examination, the temperature was 37.48C, the blood pressure 98/58 mmHg and the pulse rate 86 beats per minute. She had generalised abdominal tenderness, but the rest of the examination was unremarkable. Initial radiological investigations showed no pneumoperitoneum, air-fluid levels or distended loops in the bowel. Ultrasound suggested the presence of free fluid in Morrison’s pouch. At laparotomy, purulent peritoneal fluid was seen and foulsmelling pus found around the uterus. The rest of the abdominal organs were macroscopically normal. There was evidence of previous appendicectomy. The patient underwent total abdominal hysterectomy, left salpingo-oophorectomy and irrigation of the peritoneum. The provisional diagnosis was a tubo-ovarian abscess. Intravenous ampicillin, gentamicin and metronidazole were commenced. Gram positive bacilli were isolated in pure growth from the blood cultures performed on admission and from a swab of the peritoneum taken at surgery. With initial improvement, the antibiotics were stopped. A week after admission, the patient deteriorated. CT scans demonstrated a pelvic collection with retroperitoneal extension. CT guided drainage of the collection yielded faecal fluid. She underwent resection of the sigmoid colon and Hartmann’s procedure. The antibiotics were recommenced. She developed a methicillin resistant Staphylococcus aureus (MRSA) bacteraemia. Her antibiotics were changed to vancomycin, timentin and metronidazole; however, she remained unwell. Three weeks after admission, she had further CT scans which showed a perisplenic collection, which yielded MRSA when drained. She gradually improved hereafter and was eventually discharged home 5 weeks after admission. Histological examination of the specimens obtained from the first surgical intervention confirmed a pyometrium with involvement of the fallopian tube and ovary. Examination of the specimens obtained from the sigmoid resection demonstrated diverticulosis of the sigmoid colon and localised peritonitis. Subculture of the Gram positive bacillus seen in the anaerobic blood culture bottle (BacT/ALERT 3D; bioMerieux, France) yielded small, low-convex, grey colonies on plates incubated anaerobically after 3 days. No growth was obtained on plates incubated aerobically. However,
phenotypic identification did not proceed because the isolate was non-viable hereafter. The organism was subjected to 16S rRNA gene sequencing (MicroSeq500; Applied Biosystems, USA) and identified as Eggerthella lenta (98% identity over 478 bp to GenBank sequence AF292375). The isolate obtained from anaerobic cultures of the peritoneal swab was also non-viable on subculture. However, the primary plates had been discarded and thus were not available for further investigation. Eggerthella lenta is a member of the family Coriobacteriaceae. The differentiation and reclassification from other members of the genus Eubacterium was reported in 19991,2 on the basis of GþC content and 16S rRNA gene sequencing. Eubacterium lentum has a high GþC content (62 mol%) compared with the type species, Eubacterium limosum. Eggerthella lentum is found primarily in human faeces and is bile resistant, whereas Eubacterium exiguum is found predominantly in the mouth, in the context of periodontitis and periapical infections and is bile sensitive. 16S rRNA gene sequencing favoured the creation of a new genus, Eggerthella, to accommodate Eubacterium lentum, now renamed Eggerthella lenta gen. nov., comb. nov. The genus is named after Eggerth, the first person to isolate the organism. The cells are 0.2–0.4 6 0.2–2.0 mm, occur singly, in pairs and short chains and are Gram positive. Colonies are 0.5–2.0 mm, circular, raised to low-convex, translucent to semi-opaque, dull to shiny and smooth. The organism is an obligate anaerobe without spores or flagella. Glucose is fermented to produce acetate, lactate and succinate, phenotypically differentiating it from Propionibacterium (propionate), Lactobacillus (lactate), Actinomyces (succinate) and Bifidobacterium (acetate and lactate). Hydrogen is not produced. It is catalase negative, does not hydrolyse gelatin or aesculin but reduces nitrate. It is indole negative. In agar dilution studies3 performed according to National Committee of Clinical and Laboratory Standards (NCCLS) guidelines, around 40–50% of tested strains of Eggerthella lenta are inhibited by concentrations of 0.06 mg/mL or less, of amoxycillin, amoxycillin-clavulanate and clindamycin. Fewer strains still are inhibited at these concentrations of imipenem, metronidazole and moxifloxacin. The original strains of Eggerthella lenta were obtained from human faeces. Together with other anaerobes, it has also been isolated with a frequency of 50% from human appendices,4 whether non-inflamed, inflamed or gangrenous, as well as from the aortic walls5 from patients with abdominal aortic aneurysms. In both these conditions, it is unclear whether this association is pathogenic. The paucity of reported pathological associations is likely due to difficulty in cultivation and difficulty of differentiating these organisms from other non-sporulating anaerobic Gram positive bacilli. More recent studies, however, have begun to suggest both strong causal associations as well as importance in terms of frequencies. A group in Hong Kong6,7 found that Eggerthella species caused 18% of clinically significant bacteraemia due to anaerobic Gram positive bacilli. Half of these were due to E. lenta. There was a strong association with gastrointestinal disease with an
Print ISSN 0031-3025/Online ISSN 1465-3931 # 2008 Royal College of Pathologists of Australasia
410
CORRESPONDENCE
attributable mortality of 31%. Interestingly, this group from Hong Kong has isolated two novel Eggerthella species, E. hongkongensis sp. nov. and E. sinensis sp. nov. These authors found their strains to be susceptible to metronidazole. This case has been instructive. It is likely that the Gram positive bacillus isolated from the peritoneal swab was also E. lenta, although this could not be confirmed. The importance of identifying organisms isolated from a sterile site such as blood or from an abdominal surgical specimen is reinforced by this case. A finding of a Gram positive bacillus in the context of abdominal sepsis should prompt further clinical and laboratory investigation. This case also clearly demonstrates the utility of 16S rRNA gene sequencing as an extension of traditional phenotypic methods. Its use here enabled the identification of a newly described clinicopathological correlation which might otherwise have been missed.
Raymond C. Chan*{ Joanne Mercer* Departments of Microbiology and Infectious Diseases, Sydney South West Pathology Service, *(SWAPS) and {(RPAH), Sydney, New South Wales, Australia Contact Dr R. C. Chan. E-mail: [email protected]
1. Wade WG, Downes J, Dymock D, et al. The family Coriobacteriaceae: reclassification of Eubacterium exiguum (Poco et al. 1996) and Peptostreptococcus heliotrinreducens (Lanigan 1976) as Slakia exigua gen. nov., comb. nov. and Slakia heliotrinireducens gen. nov., comb. nov., and Eubacterium lentum (Prevot 1938) as Eggerthella lenta gen. nov., comb. nov. Int J Syst Bacteriol 1999; 49: 595–600. 2. Kageyama A, Benno Y, Nakase T. Phylogenetic evidence for the transfer of Eubacterium lentum to the genus Eggerthella as Eggerthella lenta gen. nov., comb. nov. Int J Syst Bacteriol 1999; 49: 1725–32. 3. Liebetrau A, Rodloff AC, Behra-Miellet J, Dubreuil L. In vitro activities of a new des-fluoro(6) quinolone, garenoxacin, against clinical anaerobic bacteria. Antimicrob Agents Chemother 2003; 47: 3667–71. 4. Rautio M, Saxen H, Siitonen A, Nikku R, Jousimies-somer H. Bacteriology of histopathologically defined appendicitis in children. Paed Infect Dis J 2000; 19: 1078–83. 5. Da Silva RM, Lingaas PS, Geiran O, Trionstad L, Olsen I. Multiple bacteria in aortic aneurysms. J Vasc Surg 2003; 38: 1384–9. 6. Lau SKP, Woo PCY, Fung AMY, Chan K, Woo GKS, Yuen K, Anaerobic, non-sporulating Gram-positive bacilli bacteraemia characterized by 16 S rRNA gene sequencing. J Med Microbiol 2004; 53: 1247–53. 7. Lau SKP, Woo PCY, Woo GKS, et al. Eggerthella hongkongensis sp. nov. and Eggerthella sinensis sp. nov., two novel Eggerthella species, account for half of the cases of Eggerthella bacteremia. Diagn Microbiol Infect Dis 2004; 49: 255–63.
DOI: 10.1080/00313020802036772
A case of acute necrotising eosinophilic myocarditis with an associated raised myoplasma serology: an incidental finding or a causal association? Sir, Myocarditis is an inflammatory disease of the myocardium with a wide range of clinical presentations, ranging from
Pathology (2008), 40(4), June
asymptomatic to rapidly progressive heart failure and sudden death. The diagnosis is usually based on clinical history and examination, together with ancillary tests like 2Dechocardiography and electrocardiography (ECG). A definite diagnosis would require an endomyocardial biopsy or autopsy. Histologically, myocarditis is classified into four main categories, mainly lymphocytic, neutrophilic, eosinophilic and giant cell myocarditis. We report a case of acute necrotising eosinophilic myocarditis (ANEM) which represents the most severe form of eosinophilic myocarditis. The deceased was a 15-year-old Chinese male, with no significant medical history or history of any allergies. He visited a general practitioner (GP), complaining of fever associated with abdominal bloatedness and three episodes of vomiting within 2 days. He had been to a 3-day field camp on an offshore island near Singapore 3 weeks before the illness. The GP noted that he was well hydrated but febrile (398C). His abdomen was soft. No rash was present. A clinical diagnosis of viral gastritis was made. The GP prescribed metoclopropamide, omeprazole and voltaren suppository. The teenager revisited the GP the next day, complaining of persistent fever with new onset of mild cough and shortness of breath. The abdominal bloatedness and vomiting had resolved. Physical examination then revealed no fever or rash, and was otherwise unremarkable. A diagnosis of upper respiratory tract infection was made. The GP prescribed ciprofloxacin and cough mixture. About 2 hours after the second visit, the patient was brought back to the clinic with symptoms of cyanosis, breathless and foaming around the mouth. Clinical examination revealed features of acute pulmonary oedema, characterised by bilateral crepitations and poor air entry. The patient collapsed and resuscitation was attempted. The patient was conveyed to the emergency department where he was pronounced dead on arrival. A coronial autopsy was performed 20 hours after death. External examination showed a well nourished Chinese adolescent, measuring 169 cm tall and weighing 59 kg with no rash or jaundice. Internal examination revealed an enlarged oedematous heart, weighing 420 g. Serial sections of the heart showed patchy and confluent serpiginous yellowish areas of discolouration extensively involving the myocardium. The lungs weighed 1410 g in total and were markedly oedematous. There was no other diagnostic macroscopic pathology. Blood was collected from the femoral veins for toxicology and serology. Brain, heart, lung and intestinal tissues were sampled for virological studies. Heart and lung tissues were sampled for detection of mycoplasma. Microscopic sections of the heart showed a dense and diffuse infiltrate rich in eosinophils, admixed with lymphocytes and histiocytes, associated with necrosis of the myocardial fibres (Fig. 1A,B). There were no discrete granulomas. No fungus or acid fast bacillus were identified. Immunohistochemistry confirmed abundant CD3 positive and CD8 positive (CD3þ/CD8þ) lymphocytes within the inflammatory infiltrate (Fig. 2A,B). There were scattered CD20 positive lymphocytes. There was no cytoplasmic or membrane staining for CD4. Microscopic examination of the lungs, kidneys, liver, spleen and brain confirmed macroscopic observations and revealed no additional pathological features. Toxicology of the post-mortem blood sample showed the presence of codeine, ephedrine, methorphan,
CORRESPONDENCE
First Australian description of Eggerthella lenta bacteraemia identified by 16S rRNA gene sequencing Sir, We wish to report the first Australian case of a patient with Eggerthella lenta infection, demonstrating the usefulness of 16S rRNA gene sequencing technology in a clinical diagnostic microbiology laboratory. A 68-year-old woman was admitted to hospital with a two-day history of malaise, anorexia, nausea and colicky abdominal pain. She had no fever or sweats, no change in bowel habit, no diarrhoea or vomiting and no melaena. She had a history of thyroidectomy, ectopic pregnancy and hypertension. On examination, the temperature was 37.48C, the blood pressure 98/58 mmHg and the pulse rate 86 beats per minute. She had generalised abdominal tenderness, but the rest of the examination was unremarkable. Initial radiological investigations showed no pneumoperitoneum, air-fluid levels or distended loops in the bowel. Ultrasound suggested the presence of free fluid in Morrison’s pouch. At laparotomy, purulent peritoneal fluid was seen and foulsmelling pus found around the uterus. The rest of the abdominal organs were macroscopically normal. There was evidence of previous appendicectomy. The patient underwent total abdominal hysterectomy, left salpingo-oophorectomy and irrigation of the peritoneum. The provisional diagnosis was a tubo-ovarian abscess. Intravenous ampicillin, gentamicin and metronidazole were commenced. Gram positive bacilli were isolated in pure growth from the blood cultures performed on admission and from a swab of the peritoneum taken at surgery. With initial improvement, the antibiotics were stopped. A week after admission, the patient deteriorated. CT scans demonstrated a pelvic collection with retroperitoneal extension. CT guided drainage of the collection yielded faecal fluid. She underwent resection of the sigmoid colon and Hartmann’s procedure. The antibiotics were recommenced. She developed a methicillin resistant Staphylococcus aureus (MRSA) bacteraemia. Her antibiotics were changed to vancomycin, timentin and metronidazole; however, she remained unwell. Three weeks after admission, she had further CT scans which showed a perisplenic collection, which yielded MRSA when drained. She gradually improved hereafter and was eventually discharged home 5 weeks after admission. Histological examination of the specimens obtained from the first surgical intervention confirmed a pyometrium with involvement of the fallopian tube and ovary. Examination of the specimens obtained from the sigmoid resection demonstrated diverticulosis of the sigmoid colon and localised peritonitis. Subculture of the Gram positive bacillus seen in the anaerobic blood culture bottle (BacT/ALERT 3D; bioMerieux, France) yielded small, low-convex, grey colonies on plates incubated anaerobically after 3 days. No growth was obtained on plates incubated aerobically. However,
phenotypic identification did not proceed because the isolate was non-viable hereafter. The organism was subjected to 16S rRNA gene sequencing (MicroSeq500; Applied Biosystems, USA) and identified as Eggerthella lenta (98% identity over 478 bp to GenBank sequence AF292375). The isolate obtained from anaerobic cultures of the peritoneal swab was also non-viable on subculture. However, the primary plates had been discarded and thus were not available for further investigation. Eggerthella lenta is a member of the family Coriobacteriaceae. The differentiation and reclassification from other members of the genus Eubacterium was reported in 19991,2 on the basis of GþC content and 16S rRNA gene sequencing. Eubacterium lentum has a high GþC content (62 mol%) compared with the type species, Eubacterium limosum. Eggerthella lentum is found primarily in human faeces and is bile resistant, whereas Eubacterium exiguum is found predominantly in the mouth, in the context of periodontitis and periapical infections and is bile sensitive. 16S rRNA gene sequencing favoured the creation of a new genus, Eggerthella, to accommodate Eubacterium lentum, now renamed Eggerthella lenta gen. nov., comb. nov. The genus is named after Eggerth, the first person to isolate the organism. The cells are 0.2–0.4 6 0.2–2.0 mm, occur singly, in pairs and short chains and are Gram positive. Colonies are 0.5–2.0 mm, circular, raised to low-convex, translucent to semi-opaque, dull to shiny and smooth. The organism is an obligate anaerobe without spores or flagella. Glucose is fermented to produce acetate, lactate and succinate, phenotypically differentiating it from Propionibacterium (propionate), Lactobacillus (lactate), Actinomyces (succinate) and Bifidobacterium (acetate and lactate). Hydrogen is not produced. It is catalase negative, does not hydrolyse gelatin or aesculin but reduces nitrate. It is indole negative. In agar dilution studies3 performed according to National Committee of Clinical and Laboratory Standards (NCCLS) guidelines, around 40–50% of tested strains of Eggerthella lenta are inhibited by concentrations of 0.06 mg/mL or less, of amoxycillin, amoxycillin-clavulanate and clindamycin. Fewer strains still are inhibited at these concentrations of imipenem, metronidazole and moxifloxacin. The original strains of Eggerthella lenta were obtained from human faeces. Together with other anaerobes, it has also been isolated with a frequency of 50% from human appendices,4 whether non-inflamed, inflamed or gangrenous, as well as from the aortic walls5 from patients with abdominal aortic aneurysms. In both these conditions, it is unclear whether this association is pathogenic. The paucity of reported pathological associations is likely due to difficulty in cultivation and difficulty of differentiating these organisms from other non-sporulating anaerobic Gram positive bacilli. More recent studies, however, have begun to suggest both strong causal associations as well as importance in terms of frequencies. A group in Hong Kong6,7 found that Eggerthella species caused 18% of clinically significant bacteraemia due to anaerobic Gram positive bacilli. Half of these were due to E. lenta. There was a strong association with gastrointestinal disease with an
Print ISSN 0031-3025/Online ISSN 1465-3931 # 2008 Royal College of Pathologists of Australasia
410
CORRESPONDENCE
attributable mortality of 31%. Interestingly, this group from Hong Kong has isolated two novel Eggerthella species, E. hongkongensis sp. nov. and E. sinensis sp. nov. These authors found their strains to be susceptible to metronidazole. This case has been instructive. It is likely that the Gram positive bacillus isolated from the peritoneal swab was also E. lenta, although this could not be confirmed. The importance of identifying organisms isolated from a sterile site such as blood or from an abdominal surgical specimen is reinforced by this case. A finding of a Gram positive bacillus in the context of abdominal sepsis should prompt further clinical and laboratory investigation. This case also clearly demonstrates the utility of 16S rRNA gene sequencing as an extension of traditional phenotypic methods. Its use here enabled the identification of a newly described clinicopathological correlation which might otherwise have been missed.
Raymond C. Chan*{ Joanne Mercer* Departments of Microbiology and Infectious Diseases, Sydney South West Pathology Service, *(SWAPS) and {(RPAH), Sydney, New South Wales, Australia Contact Dr R. C. Chan. E-mail: [email protected]
1. Wade WG, Downes J, Dymock D, et al. The family Coriobacteriaceae: reclassification of Eubacterium exiguum (Poco et al. 1996) and Peptostreptococcus heliotrinreducens (Lanigan 1976) as Slakia exigua gen. nov., comb. nov. and Slakia heliotrinireducens gen. nov., comb. nov., and Eubacterium lentum (Prevot 1938) as Eggerthella lenta gen. nov., comb. nov. Int J Syst Bacteriol 1999; 49: 595–600. 2. Kageyama A, Benno Y, Nakase T. Phylogenetic evidence for the transfer of Eubacterium lentum to the genus Eggerthella as Eggerthella lenta gen. nov., comb. nov. Int J Syst Bacteriol 1999; 49: 1725–32. 3. Liebetrau A, Rodloff AC, Behra-Miellet J, Dubreuil L. In vitro activities of a new des-fluoro(6) quinolone, garenoxacin, against clinical anaerobic bacteria. Antimicrob Agents Chemother 2003; 47: 3667–71. 4. Rautio M, Saxen H, Siitonen A, Nikku R, Jousimies-somer H. Bacteriology of histopathologically defined appendicitis in children. Paed Infect Dis J 2000; 19: 1078–83. 5. Da Silva RM, Lingaas PS, Geiran O, Trionstad L, Olsen I. Multiple bacteria in aortic aneurysms. J Vasc Surg 2003; 38: 1384–9. 6. Lau SKP, Woo PCY, Fung AMY, Chan K, Woo GKS, Yuen K, Anaerobic, non-sporulating Gram-positive bacilli bacteraemia characterized by 16 S rRNA gene sequencing. J Med Microbiol 2004; 53: 1247–53. 7. Lau SKP, Woo PCY, Woo GKS, et al. Eggerthella hongkongensis sp. nov. and Eggerthella sinensis sp. nov., two novel Eggerthella species, account for half of the cases of Eggerthella bacteremia. Diagn Microbiol Infect Dis 2004; 49: 255–63.
DOI: 10.1080/00313020802036772
A case of acute necrotising eosinophilic myocarditis with an associated raised myoplasma serology: an incidental finding or a causal association? Sir, Myocarditis is an inflammatory disease of the myocardium with a wide range of clinical presentations, ranging from
Pathology (2008), 40(4), June
asymptomatic to rapidly progressive heart failure and sudden death. The diagnosis is usually based on clinical history and examination, together with ancillary tests like 2Dechocardiography and electrocardiography (ECG). A definite diagnosis would require an endomyocardial biopsy or autopsy. Histologically, myocarditis is classified into four main categories, mainly lymphocytic, neutrophilic, eosinophilic and giant cell myocarditis. We report a case of acute necrotising eosinophilic myocarditis (ANEM) which represents the most severe form of eosinophilic myocarditis. The deceased was a 15-year-old Chinese male, with no significant medical history or history of any allergies. He visited a general practitioner (GP), complaining of fever associated with abdominal bloatedness and three episodes of vomiting within 2 days. He had been to a 3-day field camp on an offshore island near Singapore 3 weeks before the illness. The GP noted that he was well hydrated but febrile (398C). His abdomen was soft. No rash was present. A clinical diagnosis of viral gastritis was made. The GP prescribed metoclopropamide, omeprazole and voltaren suppository. The teenager revisited the GP the next day, complaining of persistent fever with new onset of mild cough and shortness of breath. The abdominal bloatedness and vomiting had resolved. Physical examination then revealed no fever or rash, and was otherwise unremarkable. A diagnosis of upper respiratory tract infection was made. The GP prescribed ciprofloxacin and cough mixture. About 2 hours after the second visit, the patient was brought back to the clinic with symptoms of cyanosis, breathless and foaming around the mouth. Clinical examination revealed features of acute pulmonary oedema, characterised by bilateral crepitations and poor air entry. The patient collapsed and resuscitation was attempted. The patient was conveyed to the emergency department where he was pronounced dead on arrival. A coronial autopsy was performed 20 hours after death. External examination showed a well nourished Chinese adolescent, measuring 169 cm tall and weighing 59 kg with no rash or jaundice. Internal examination revealed an enlarged oedematous heart, weighing 420 g. Serial sections of the heart showed patchy and confluent serpiginous yellowish areas of discolouration extensively involving the myocardium. The lungs weighed 1410 g in total and were markedly oedematous. There was no other diagnostic macroscopic pathology. Blood was collected from the femoral veins for toxicology and serology. Brain, heart, lung and intestinal tissues were sampled for virological studies. Heart and lung tissues were sampled for detection of mycoplasma. Microscopic sections of the heart showed a dense and diffuse infiltrate rich in eosinophils, admixed with lymphocytes and histiocytes, associated with necrosis of the myocardial fibres (Fig. 1A,B). There were no discrete granulomas. No fungus or acid fast bacillus were identified. Immunohistochemistry confirmed abundant CD3 positive and CD8 positive (CD3þ/CD8þ) lymphocytes within the inflammatory infiltrate (Fig. 2A,B). There were scattered CD20 positive lymphocytes. There was no cytoplasmic or membrane staining for CD4. Microscopic examination of the lungs, kidneys, liver, spleen and brain confirmed macroscopic observations and revealed no additional pathological features. Toxicology of the post-mortem blood sample showed the presence of codeine, ephedrine, methorphan,