Empyema Due to Gemella Morbillorum Is Diagnosed by 16S Ribosomal RNA Gene Sequencing and a Phylogenetic Tree Analysis: a Case Report and Literature Review

□ CASE REPORT □

Empyema due to Gemella morbillorum Is Diagnosed by 16S Ribosomal RNA Gene Sequencing and a Phylogenetic Tree Analysis: A Case Report and Literature Review

Hideaki Yamakawa 1, Masahiro Hayashi 2, Kaori Tanaka 2 and Kazuyoshi Kuwano 3

Abstract

We report a case of empyema due to Gemella morbillorum. In this case, an analysis of the aspirate from the pleural effusion revealed empyema and evidence of a Gram-positive coccal bacteria. A biochemical identification system labelled the bacteria as ‘unclassified’, although we initially suspected the bacterium to be- long to the Streptococcus species. 16S ribosomal RNA (16S rRNA) gene sequencing and a phylogenetic tree analysis of the isolated strain confirmed the presence of Gemella morbillorum. To ascertain the true incidence of Gemella species in empyema, 16S rRNA gene sequencing should be used when the standard conventional biochemical methods fail to identify the organism or it identifies it with a low degree of reliability.

Key words: Gemella morbillorum, empyema, 16S ribosomal RNA gene sequencing

(Intern Med 54: 2231-2234, 2015) (DOI: 10.2169/internalmedicine.54.4950)

Introduction Case Report

Gemella morbillorum, formerly known as Streptococcus The patient was a 77-year-old man who was an ex- morbillorum, is a catalase-negative, facultative anaerobic, smoker and a social drinker. He had been diagnosed with and gram-positive coccus that is present in the normal flora essential thrombocythaemia for which he received hydroxy- of the oral cavity, upper respiratory tract, gastrointestinal carbamide and aspirin for eight years. He presented symp- tract, and genitourinary system. Isolates of G. morbillorum toms of sudden fever, dyspnoea, left-sided pleuritic pain, have been found in cases of soft-tissue abscesses, meningi- and shivering. He was then admitted to our hospital and a tis, endocarditis, septic shock, and arthritis (1). Although vi- chest computed tomography (CT) scan revealed a left pleu- ridans streptococci with similar microbiological properties ral effusion without pulmonary lesions (Fig. 1). His vital have been found to be involved in the pathogenesis of pleu- signs upon admission were as follows: temperature, 38.6℃; ral empyema and lung abscess, the presence of G. morbillo- blood pressure, 106/62 mmHg; pulse, 104 beats/min with rum in this setting has seldom been reported (2). In this case regular rhythm; and oxygen saturation on room air, 88% on of empyema that was caused by G. morbillorum, a diagnosis pulse oximetry sensor. Decreased breath sounds in the left was made by DNA amplification and sequence analyses of lung were detected on auscultation, and heart sounds indi- the 16S ribosomal RNA (16S rRNA) and phylogenetic tree cated a regular rhythm without a heart murmur. His oral analysis. condition was poor, and he had some decayed teeth. The patient’s C-reactive protein (CRP) level was elevated at 8.4 mg/dL. No blast cells were detected in the peripheral blood. The patient’s HbA1c (NGSP: National Glycohemoglobin

１Division of Respiratory Medicine, Department of Internal Medicine, Jikei University School of Medicine, Kashiwa Hospital, Japan, ２Division of Anaerobe Research, Life Science Research Center, Gifu University, Japan and ３Division of Respiratory Medicine, Department of Internal Medicine, Jikei University School of Medicine, Japan Received for publication January 13, 2015; Accepted for publication March 16, 2015 Correspondence to Dr. Hideaki Yamakawa, [email protected]

2231 Intern Med 54: 2231-2234, 2015 DOI: 10.2169/internalmedicine.54.4950

Figure 2. A gram stain of the isolated strain in the pleural Figure 1. Chest computed tomography on admission re- aspirate showed a Gram-positive diplococci with a heteroge- vealed a pleural effusion in the left lung with no swelling of the neous form. hilar or mediastinal lymph nodes.

tococcus species was the most likely pathogen responsible for the empyema because the catalase test for the strain in our patient was negative, and Streptococcus species is the most common aetiologic agent responsible for empyema (3). We inserted a chest tube for natural drainage and treated the patient with an antibiotic therapy of ampicillin/sulbactam (ABPC/SBT) (9.0 g/day). Thereafter, an additional biochemical investigation of this isolated strain was performed with the API rapid ID 32 Strep system (bioMérieux sa, Marcy l’Etoile, France), which identified the organisms G. haemolysans and G. morbillorum with degrees of reliability of 85.7% and 14.1%, respectively, indicating that the strain could not be identified precisely. Routine antimicrobial sus- Figure 3. Neighbour-joining phylogenetic tree of the genus ceptibility testing with a disc diffusion on Mueller-Hinton Gemella morbillorum based on the 16S rRNA (1,500 bp) gene agar that contained 5% sheep’s blood showed a good sus- sequences. ceptibility of the strain to various drugs, including ABPC/ SBT. Subsequently, we asked the Division of Anaerobe Re- search, Life Science Research Center of Gifu University to Standardization Program) level was 5.6%. Anti-HIV- perform an additional analysis of this isolated strain using antibodies were negative. An arterial blood gas analysis on molecular biology techniques. We performed molecular room air revealed hypoxemia with a partial pressure of oxy- identification by polymerase chain reaction (PCR) amplifica- gen in arterial blood (PaO2) of 53.4 Torr. Contrast-enhanced tion and sequence analysis of the 16S rRNA gene using whole-body CT showed no other lesions, and transthoracic DNA extracted from the isolated strain. The universal prim- echocardiography showed no evidence of infective endo- ers 8UA (5'-AGAGTTTGATCMTGGCTCAG-3') and 1485B carditis. An analysis of the pleural aspirate prior to antibiotic (5'-ACGGG CGGTGTGTRC-3') were used for amplification therapy showed a pH of 7.2, an elevated lactate dehydroge- and sequencing. We performed a sequence analysis using the nase level of 3,063 IU/L, and decreased glucose of 39 mg/ basic local alignment search tool (BLAST) search and dL. A gram stain of this specimen revealed a predominance CLUSTAL X (neighbor-joining method) phylogenetic tools. of neutrophils with Gram-positive cocci. This sample was The phylogenetic tree was drawn by TreeView and shown in cultured aerobically using chocolate and sheep’s blood agar/ Fig. 3. The sequence of the 16S rRNA gene showed 98.8 % bromothymol blue (BTB) lactose agar, and anaerobically concordance (1,434 bp over the entire 1,451 bp fragment) cultured using Brucella HK agar. After 48 hours of incuba- with a type strain of G. morbillorum (ATCC 27824; Se- tion, α-haemolytic, catalase-negative colonies were obtained quence accession no. L14327). Based on this result, we on the sheep’s blood agar medium. A gram stain of this iso- identified the isolate as G. morbillorum. After insertion of lated strain showed a Gram-positive diplococci with a het- the chest tube, he was maintained on antibiotics (ABPC/ erogeneous form (Fig. 2). Sputum cultures were negative for SBT), and irrigation of the thoracic cavity with saline solu- clinically significant bacteria, fungi, and acid-fast bacilli. tion was started twice a day during hospitalization. The pa- Blood cultures were also negative. We believed that a Strep- tient’s condition improved gradually after local and systemic

2232 Intern Med 54: 2231-2234, 2015 DOI: 10.2169/internalmedicine.54.4950 therapy, and his CRP level reverted to normal values. The pathogen of empyema because of the negative catalase test chest tube was removed on the 24th hospital day, and antibi- for this strain. This abnormality has also been reported for otic therapy with ABPC/SBT was continued for 30 days. He other organisms and may be responsible for the frequent was discharged from our hospital on the 34th hospital day. misidentification of Gemella, and possibly, the fact that so few cases of Gemella infection are reported. On the other Discussion hand, Kawanami et al. reported that some cases showed dis- crepancies between the results from the cultivation method Microorganisms of the Gemella species are opportunistic and the clone library method in the analysis of bacterial pathogens that are Gram-positive, with a cocci morphology, pleurisy. These inconsistencies suggest that the cultivation and are facultative anaerobic. These pathogens have the abil- method alone may not fully express precise bacterial infor- ity to cause serious local and systemic infections, mainly in mation of the infected foci. They concluded that the clone immunocompromised patients. The species G. haemolysans library analysis using the 16S rRNA gene of pleural fluid and G. morbillorum are the most important pathogens. G. showed a higher incidence of anaerobic bacteria in infec- haemolysans can cause endocarditis and meningitis, whereas tious pleurisy than previously expected, and also provided G. morbillorum causes endocarditis, especially of native additional bacterial information of cultivation method (12). valves, septic arthritis, and meningitis (4). Respiratory tract Therefore, clinicians should consider the possibility of a infections caused by Gemella species have rarely been re- mixed infection in the pleuropulmonary infections. Although ported, but they can cause lung abscesses, necrotizing pneu- the pathogen that was detected in our case was G. morbillo- monia, and pleural empyema. However, there are a few pub- rum, the possibility of a mixed infection that would include lications referring to pleural involvement, and these describe anaerobic bacteria cannot be discounted. We believe that a isolated cases by conventional biochemical identification (5). biochemical and molecular approach is important in order to In the literature, there are 14 cases of empyema due to G. detect the precise pathogens in the pleuropulmonary infec- morbillorum. Ten patients (71.4%) were men and 4 (28.6%) tions. were women, with a median age of 66 (range: 26-80) years, We report the first case, to the best of our knowledge, of which indicates that middle-aged men are predominantly af- empyema due to G. morbillorum that was diagnosed by 16S fected. All patients had predisposing factors such as poor rRNA gene sequencing and a phylogenetic tree analysis. oral hygiene, smoking, chronic cardiovascular or respiratory Further studies using 16S rRNA gene sequencing need to be disease, drug abuse, alcohol abuse or malignancies, indicat- conducted to ascertain the true incidence of empyema that is ing that these patients were frequently associated with con- caused by Gemella species because this species might be ditions that favoured aspiration (4-10). Our patient’s oral frequently misidentified as a species of Streptococcus. condition was poor and he had some decayed teeth. These conditions might result in silent aspiration. Moreover, he The authors state that they have no Conflict of Interest (COI). had a haematological disorder, which might be an additional risk factor. Prognosis of G. morbillorum-associated pleuro- References pulmonary infections is generally good with early and ade- quate therapy, and species isolated from clinical specimens 1. Young Ann J, Kwon JC, Eun Song J, et al. Sternal osteomyelitis in the past were highly susceptible to penicillin (6, 9). with a mediastinal abscess caused by Gemella morbillorum fol- The identification of infrequently encountered bacterial lowing blunt force trauma. Intern Med 52: 511-514, 2013. 2. Meyer CN, Rosenlund S, Nielsen J, Friis-Møller A. Bacteriologi- species in clinical microbiology laboratories has always been cal aetiology and antimicrobial treatment of pleural empyema. a problem. Since the number of reference strains used to Scand J Infect Dis 43: 165-169, 2011. build up databases in commercial kits is usually small for 3. Lisboa T, Waterer GW, Lee YC. Pleural infection: changing bacte- rare bacteria, it is not uncommon to encounter clinical iso- riology and its implications. Respirology 16: 598-603, 2011. lates of these rare bacterial species with ambiguous bio- 4. Senent C, Sancho JN, Chiner E, Signes-Costa J, Camarasa A, Andreu AL. Pleural empyema caused by Gemella species: a rare chemical profiles. Furthermore, even if they are ‘successful- condition. Arch Bronconeumol 44: 574-577, 2008. ly’ identified by the commercial kits, the low prevalence 5. Valipour A, Koller H, Setinek U, Burghuber OC. Pleural em- rate would imply a low positive predictive value. Using 16S pyema associated with Gemella morbillorum: report of a case and rRNA gene sequencing, Woo et al. reported that Gemella review of the literature. Scand J Infect Dis 37: 378-381, 2005. species accounted for 0.7% of bacteraemias caused by α- 6. da Costa CT, Porter C, Parry K, Morris A, Quoraishi AH. Em- pyema thoracis and lung abscess due to Gemella morbillorum.Eur haemolytic streptococci, with the exception of Streptococcus J Clin Microbiol Infect Dis 15: 75-77, 1996. pneumonia. Because commercially available phenotypic sys- 7. Canet JJ, Hernández R, Almagro P, Garau J. Pleural empyema due tems frequently misidentified the organism, they recom- to Gemella morbillorum. Enferm Infecc Microbiol Clin 19: 189, mended identification of Gemella and Gemella-like isolates 2001. by 16S rRNA gene analysis (11). In fact, biochemical iden- 8. Hayashi Y, Ito G. A case of bacterial empyema caused by Gemella tification with the API Rapid ID 32 Strep system labelled morbillorum. Kansenshogaku Zasshi (The Journal of the Japanese Association for Infectious Diseases) 70: 259-263, 1996 (in Japa- the strain in our patient as ‘unclassified’, although we in- nese, Abstract in English). itially suspected a Streptococcus species as the most likely

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9. Marcos Sánchez F, Celdrán Gil J, Arbol Linde F, Med Chil 140: 1544-1547, 2012 (in Spanish, Abstract in English). Caballero Sánchez-Robles L. Pleural empyema due to Gemella 11. Woo PC, Lau SK, Fung AM, Chiu SK, Yung RW, Yuen KY. Ge- morbillorum with a favorable outcome. An Med Interna 17: 112- mella bacteraemia characterised by 16S ribosomal RNA gene se- 113, 2000 (in Spanish). quencing. J Clin Pathol 56: 690-693, 2003. 10. Aibar-Arregui MÁ, De Escalante-Yangüela B, Garrido-Buenache 12. Kawanami T, Fukuda K, Yatera K, Kido M, Mukae H, Taniguchi A, Navarro-Aguilar ME, Montoya-Arenas J, Rodero-Roldán Mdel H. A higher significance of anaerobes: the clone library analysis M. Pleural empyema due to Gemella spp: report of 12 cases. Rev of bacterial pleurisy. Chest 139: 600-608, 2011.

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