Native Valve Endocarditis Due to a Novel Strain of Legionella ^▿

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Of the 54 validly named Legionella species, 21 have been isolated from human infections (14, 24). Legionella pneumophila represents 90% of the cases, with L. longbeachae and L. bozemanae being the next most common, causing 4% and 2% of cases, respectively (25). The primary site for Legionella infection is the lung, but in rare cases, Legionella is found in extrapulmonary sites, either as a consequence of dissemination from the lung or as isolated primary infections (13). Here, we describe an unusual case of Legionella endocarditis in a native heart valve. Genotypic and phenotypic analyses indicate that the bacterial isolate is unlike previously described strains of Legionella.

A 68-year-old woman with Goodpasture's syndrome who underwent living related kidney transplantation in 2004 presented to the hospital in August 2009 complaining of chest pain and dyspnea on exertion. Her history was notable for cytomegalovirus viremia in 2004 and an episode of organ rejection in 2008 requiring methylprednisolone and thymoglobulin therapy. Her maintenance immune suppression was mycophenolic acid and tacrolimus. At presentation, she reported a 1-day history of chest pain associated with nausea, diaphoresis, and anxiety. She was afebrile, with a heart rate of 100 bpm, blood pressure 113/42 mm Hg, and pulse oximetry of 96% on 2 liters of oxygen. A grade II holosystolic murmur was present. Her leukocyte count was 13,500/mm³ (normal range, 4.1 to 10.9/mm³), creatinine level was 3.5 mg/dl (normal range, 0.4 to 1.0 mg/dl), potassium level was 5.7 meq/liter (normal range, 3.4 to 5.1 meq/liter), and troponin level was 0.2 ng/ml (normal range, ≤0.1 ng/ml). A chest radiograph demonstrated an infiltrate in the right lower lobe, which was confirmed by computed tomography (CT) scan of the chest.

Due to a history of penicillin allergy, vancomycin and ciprofloxacin were initiated for suspected health care-associated pneumonia (she had been hospitalized 3 weeks earlier for “gastroenteritis”). L. pneumophila urine antigen and urine pneumococcal antigen were undetectable, and blood cultures revealed no growth. Her troponin level continued to rise and peaked at 2.18 ng/ml. A transesophageal echocardiogram showed a 1.09-cm freely mobile mass on the left coronary cusp of the aortic valve, consistent with vegetation.

Vancomycin and ciprofloxacin were continued, and gentamicin was added for empirical coverage of culture-negative endocarditis. Her clinical status deteriorated, and she underwent emergency aortic valve replacement with a bovine bioprosthesis on the 6th day of hospitalization. Intraoperative findings included a trileaflet aortic valve with a large vegetation on the left coronary cusp. No aortic annulus abscess was found. Gram stain of the resected aortic valve revealed lightly staining Gram-negative bacilli; however, routine tissue culture showed no growth. Broad-range 16S rRNA PCR and sequencing were performed on the resected aortic valve tissue (17, 22). The 16S rRNA genes were PCR amplified with broad-range bacterial primers, using HotStar Taq master mix (Qiagen, Inc., Valencia, CA) (3). Bidirectional sequencing was performed using the BigDye terminator kit (v 3.0) on the ABI 3130xl gene analyzer (Applied Biosciences, Foster City, CA). The DNA sequences were aligned in DNAStar SeqMan Pro v7.2 software (DNAStar, Inc., Madison, WI) and compared to reference sequences in GenBank (3). The 16S rRNA sequencing identified the organism as a Legionella species (see below). The organism subsequently grew after plating on buffered charcoal-yeast extract (BCYE) agar.

The patient's antibiotics were changed to moxifloxacin, and she completed a 6-week course of therapy. Her clinical condition improved initially, but she subsequently restarted dialysis due to worsening renal failure. Serial transthoracic echocardiograms were unchanged until April 2010, when she was readmitted to the hospital with volume overload. A transthoracic echocardiogram showed evidence of a perivalvular abscess with disruption of the valve. Blood cultures remained sterile. She was restarted on therapy with vancomycin, azithromycin, and rifampin. Despite treatment, her clinical status deteriorated, and she became hypotensive, requiring vasopressors to maintain her blood pressure. A repeat valve replacement and debridement of infected tissue were recommended. However, the patient refused repeat surgery and expired 2 weeks following admission. A request for an autopsy was declined.

Extrapulmonary manifestations of Legionella infections include myocarditis, pericarditis, and endocarditis. There have been 16 cases of Legionella pericarditis or myocarditis, attributed mostly to L. pneumophila and L. dumoffii, and 15 cases of Legionella endocarditis linked to L. pneumophila, L. micdadei, and L. dumoffii (2, 5, 6, 10, 11, 13, 15, 18, 20). All but one of the endocarditis cases occurred in patients who had received aortic homografts or prosthetic valve replacements, and in most cases, the suspected route of infection was via direct inoculation with contaminated surgical equipment. The single case of native valve endocarditis reported in the literature was due to L. pneumophila (18). In contrast, 16S rRNA PCR and sequencing of the aortic value tissue and culture isolate in our case revealed that the Legionella strain, designated H63, shared the highest similarity with L. brunensis ATCC 43878^T (98.4%), an environmental strain originally isolated from a cooling tower in Czechoslovakia and never implicated in disease (23). Among the next closest species were L. jordanis ATCC 33623^T (96.5%), L. nautarum ATCC 49506^T (96.5%), and L. jamestowniensis ATCC 35298^T (96.0%).

While bacterial strains displaying less than 97% similarity in 16S rRNA sequence are to be considered distinct species, it has recently been suggested, based on retrospective analysis of 16S rRNA data and DNA-DNA hybridization values, that the cutoff value be raised to 98.7% (19). Indeed, Legionella species can share up to 99.4% 16S rRNA similarity but still be defined as distinct based on DNA-DNA hybridization values below 70% (7, 12). The mip gene has been proposed as an additional sequencing target because of its wide distribution among legionellae and increased resolution over the 16S rRNA gene (16). In congruence with the 16S rRNA analysis, mip of H63 shared its highest degree of similarity with mip in L. brunensis ATCC 43878^T (85.6%), followed by L. hackeliae ATCC 35250^T (85.3%), L. jamestowniensis ATCC 35298^T (84.1%), and L. feeleii ATCC 35072^T (84.3%). The 14.5% difference between H63 and L. brunensis is within the range of variation that is typical of Legionella species (3 to 31%) (16). In sum, genomic data suggest that H63 represents either a novel strain of L. brunensis or a new Legionella species.

H63 grew well on BCYE agar and displayed l-cysteine auxotrophy (21). It produced a brown pigment upon growth in buffered yeast extract (BYE) broth, did not autofluoresce under UV light, and was negative for nitrate reduction, glucose fermentation, and urease (1, 21). Regarding the published chemotaxonomic traits that vary among Legionella species, H63 produced catalase, β-lactamase, and gelatinase. As related to a final variable trait, H63 hydrolyzed hippurate, unlike L. brunensis, the nearest neighbor based on 16S rRNA sequence (8, 9). Cellular fatty acids of H63 were determined with the Microbial Identification System (MIDI, Newark, DE) after 72 h of incubation at 35°C on BCYE agar (4). The main fatty acids of H63 were C_15:0 anteiso (29%), C_16:1w6c/w7c (22%), and C_16:0 (21%). Compared to entries in the Sherlock CLIN database, H63 had a fatty acid composition most similar to L. steigerwaltii and L. jamestowniensis, with similarity indices of 0.397 and 0.357, respectively. H63 was further distinguished from L. brunensis by the fact the main fatty acid of that type strain is C_15:0 at 39%, followed by C_17:0 anteiso at 24% and C_16:0 at 12% (23). Taken together, the results from standard phenotypic analysis support the view that H63 represents a novel strain of Legionella.

Strain H63 displays the typical genetic, physiological, and biochemical characteristics of the Legionellaceae family. Based upon 16S rRNA and mip sequencing, the closest Legionella species is L. brunensis; however, there are notable differences between the strains in terms of hippurate hydrolysis and fatty acid makeup. While it has yet to be determined if H63 is a novel strain of L. brunensis or represents a new species of Legionella, this is only the second reported case of native valve endocarditis due to a Legionella species and a reminder that Legionella should be considered in the differential diagnosis of culture-negative endocarditis.