Pulmonary Granuloma Caused by Pseudomonas andersonii sp nov

Anatomic Pathology / PULMONARY GRANULOMA CAUSED BY PSEUDOMONAS ANDERSONII SP NOV

Pulmonary Granuloma Caused by Pseudomonas andersonii sp nov Xiang Y. Han, MD, PhD,1 Audrey S. Pham, PhD,1 Kim U. Nguyen, MT(ASCP),1 W. Roy Smythe, MD,2 Nelson G. Ordonez, MD,3 Kalen L. Jacobson, MD,4 and Jeffrey J. Tarrand, MD1 Key Words: Pulmonary granuloma; Pseudomonas; Gram-negative bacterium

Abstract Pulmonary granuloma is a common lesion for which gram-negative bacteria are rarely implicated as a cause. Hence, most physicians are unaware of this etiology. We isolated a gram-negative bacterium from a surgically resected pulmonary granuloma in a 42-yearold, nonimmunocompromised woman. Within the necrotizing granuloma, numerous organisms also were demonstrated by Gram stain, suggesting a causedisease relationship. Characterization of the bacterium by sequence analysis of the 16S ribosomal gene, cellular fatty acid profiling, and microbiologic studies revealed a novel bacterium with a close relationship to Pseudomonas. We propose a new species for the bacterium, Pseudomonas andersonii. These results suggest that the differential diagnosis of a lung granuloma also should include this gram-negative bacterium as a potential causative agent, in addition to the more common infections caused by acid-fast bacilli and fungi. This bacterium was shown to be susceptible to most antibiotics that are active against gramnegative bacteria.

© American Society of Clinical Pathologists

Pulmonary granulomas can be of both infectious and noninfectious causes. They frequently manifest as solitary or multiple nodular lesions in the lung and can be confused diagnostically with other nodular lesions such as primary or secondary tumors, particularly in the absence of systemic manifestations. The treatment options for lung nodules differ drastically depending on the cause. Generally, most granulomatous nodules are treated medically, while cancerous nodules are surgically resected if possible. Mycobacteria (acid-fast bacilli [AFB]) and fungi are the most frequent infectious agents causing pulmonary granulomas, and we commonly include these in the differential diagnosis. On histologic diagnosis of such a lesion (commonly by frozen sections), efforts usually are made to identify the responsible organism by special stains, culture isolation, or both. In our experience, however, it is not uncommon that a microbe cannot be identified. Infections by several gram-negative bacteria, such as Brucella,1,2 Bartonella,3 Afipia,4 and Salmonella1,2 species, can lead to granulomatous lesions. Most of these granulomas, however, involve lymph nodes and/or reticuloendothelial organs, with the exception of lung involvement by Brucella suis. A few such cases of lung involvement were reported decades ago, in which B suis was isolated from well-formed lung granulomas.5-8 Since then, to our knowledge, there have been no other reports of lung granulomas due to gram-negative bacteria. Therefore, gram-negative bacteria are rarely considered in the differential diagnosis. We report isolation and characterization of a novel gramnegative bacterium from a surgically resected lung granuloma of a 42-year-old woman who was not clinically immunocompromised. Numerous organisms were demonstrated histologically within the lesion, providing strong evidence for a causal relationship. Am J Clin Pathol 2001;116:347-353

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Materials and Methods Case Report A 42-year-old woman sought care at the M.D. Anderson Cancer Center, Houston, TX, in January 2000 for evaluation and treatment of a lung nodule that was discovered at another institution in September 1999 on a chest radiograph and computed tomography (CT) scan before elective cholecystectomy. A high-resolution chest CT scan revealed an 8mm, well-circumscribed nodule in the posterolateral right lower lobe, without accompanying hilar lesions or adenopathy, which was interpreted as most likely representing a noncalcified granuloma ❚Image 1❚. The patient also had a 2-week history of cough productive of clear sputum, which she attributed to “a recent cold.” The patient had a long history of gallstones for which she underwent a laparoscopic cholecystectomy in October 1999. The patient also related that 2.5 years earlier, following several episodes of swimming in a stagnating river in Texas, she developed severe unilateral left tinnitus (without fever) resulting in deafness. Otologic examination at that time failed to find ear fluid or infection; the cause thus remained unknown. Since then, she also experienced daily headache with some intermittent blurred vision. The patient had a 20-year history of smoking (20-40 cigarettes per day) and consumed 60 to 80 g of alcohol daily (marked hepatic steatosis revealed on a recent biopsy). She worked in an office environment in Houston, owned a few cats, and traveled frequently to the United States–Mexican border region. She had no known exposure to tuberculosis.

❚Image 1❚ High-resolution computed tomography scan of the lung nodule (arrow) in a 42-year-old woman (film courtesy of Robert T. Brown, MD).

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Physical examination on admission to M.D. Anderson was unremarkable except for some erythema in the left ear canal and dull tympanic membranes with diffuse light reflex in both ears. No palpable lymph nodes or abnormal auscultation of the heart or lungs was noted particularly. Results of blood chemistry tests were normal. The WBC count was 6,700/µL (6.7 × 109/L); hemoglobin, 14.0 g/dL (140 g/L); mean corpuscular volume, 108 µm3 (108 fL); mean corpuscular hemoglobin, 37.5 pg; distribution width, 18.8%; and platelet count, 277 × 103/µL (277 × 109/L). The patient underwent video-assisted thoracoscopy and wedge resection of the lung nodule (specimen, 5.0 × 3.5 × 1.3 cm; nodule, 0.8 cm). Approximately 1 g of the resected tissue was submitted for microbiologic cultures. Inoculations included blood and chocolate agars (incubated at 35°C with 5% carbon dioxide) and thioglycolate broth (35°C) for bacteria, Sabouraud dextrose agar for fungi (30°C without carbon dioxide), and Löwenstein-Jensen agar and Middlebrook 7H10 agar for AFB (35°C with 8% carbon dioxide). Pathologic examinations revealed a necrotizing granuloma with occasional giant cells ❚Image 2A❚ and ❚Image 2B❚ and unremarkable surrounding lung parenchyma. No AFB or fungi were identified on Ziehl-Neelsen or Gomori methenamine silver stains. A Gram stain (Brown-Hopps method), however, demonstrated the presence of numerous gram-negative coccobacilli within the necrotic center of the lesion ❚Image 3A❚, some of which were located within giant cells ❚Image 3B❚. The organism was isolated 6 days later from the thioglycolate broth only, but it could not be identified by routine microbiologic tests. No AFB or fungi were isolated. The patient’s immediate postoperative course was uneventful, and she was discharged on day 3. By postoperative day 6, however, she developed a new and worsening right pleural effusion at the thoracotomy site and was readmitted to the hospital. Bilateral pulmonary infiltrates with fever and leukocytosis followed during the next 3 days, and despite broad-spectrum intravenous antibiotics, she developed respiratory insufficiency necessitating endotracheal intubation. Her fever (temperature up to 39°C), leukocytosis (WBC count up to 19,000/µL [19 × 109/L]), and hypoxia persisted for 2 weeks. An extensive evaluation for the cause of this pneumonia continued, in addition to attempts at identification of the bacterial isolate from the resected lung nodule, although the significance of this bacterium was considered doubtful for this event in view of its antibiotic susceptibility pattern (see “Results”). Numerous cultures of blood, pleural fluid, and lower respiratory specimens (bronchoalveolar lavage and sputum specimens and tracheal aspirates) were performed without recovery of bacteria, viruses, or mycobacteria except a few yeasts in the sputum specimens (probably contaminants). Serologic studies for antibodies against human immunodeficiency virus 1 and 2, © American Society of Clinical Pathologists

Anatomic Pathology / ORIGINAL ARTICLE

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B

❚Image 2❚ Histologic examination of the resected lung nodule (A, H&E, ×10; B, H&E ×100). A, Bar equals 1 mm. B, Arrows indicate giant cells.

Brucella (IgM and IgG), Histoplasma, Blastomyces, and Coccidioides were negative, as were screens for cryptococcal antigens, antinuclear antibody, and rheumatoid factor. The cause of the pneumonia thus remained unclear, although a viral infection was most likely by exclusion. The patient’s condition gradually improved, and she was extubated and eventually discharged without evidence of infection 4 weeks later. Microbiologic Studies A routine workup pathway for a gram-negative bacterium was used to study the culture and biochemical

characteristics of the organism, including conventional tube media and/or commercial kits API 20E and 20NE (BioMerieux, Marcy-l’Etoile, France) and Vitek (BioMerieux, Hazelwood, MO). Scanning electron microscopy was performed using a 4-day culture of the bacterium grown in thioglycolate broth after fixation in a buffered glutaraldehyde solution. Antibiotic susceptibility was performed using the disk diffusion method (KirbyBauer),9 E-test (AB Bio-disk, Solna, Sweden), and microdilution method.10 The disk diffusion method was performed on blood Mueller-Hinton agar with 48-hour incubation at 35°C and 5% carbon dioxide owing to slow growth and,

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❚Image 3❚ Gram-negative short bacilli to coccobacilli in the necrotic center of the lesion (A, Gram, ×1,000) and intracellularly (arrows) (B, Gram, ×1,000).


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thus, was considered nonstandard according to the National Committee for Clinical Laboratory Standards (24-hour incubation).9 Analysis of 16S Ribosomal Gene Genomic DNA of the bacterium, used as a template for amplification of the 16S ribosomal gene (rDNA) by a polymerase chain reaction (PCR), was purified using a method previously described.11 Briefly, the bacterium in a 3-day culture in thioglycolate broth was pelleted by centrifugation and homogenized in STET (sodium chloride, Tris [tris(hydroxymethyl)aminomethane], EDTA, and Triton X100 [Sigma, St Louis, MO]) buffer. Following lysis of the bacterium by lysozyme treatment and boiling (10 minutes), the DNA was extracted using phenol in a mixture of 25:24:1 phenol/chloroform/amyl alcohol, re-extracted with chloroform, and washed with 70% ethanol. PCR was performed using a gradient thermocycler (Eppendorf, Westbury, NY) in a 50-µL reaction volume containing a 10-mmol/L concentration of potassium chloride, a 0.4-mmol/L concentration of the deoxynucleoside triphosphates, 50 pmol each of the primers, a 2.5-mmol/L concentration of magnesium chloride, PCR enhancer, and 1.5 U of Taq polymerase (Eppendorf). Thermal cycles were 95°C for 5 minutes, 39 cycles of 95°C for 30 seconds, 63°C for 30 seconds, 72°C for 60 seconds, and a last cycle of 63°C for 2 minutes and 72°C for 5 minutes. A set of conserved primers for bacterial 16S rDNA,12 forward 5'-AACTGGAGGAAGGTGGGGA-3' (1155-1173) and reverse 5'-TGCGGTTGGATCACCTCCT-3' (1507-1525), was used initially to amplify a 371-base-pair (bp) fragment. Upon sequence analysis of this amplicon indicating homology to Pseudomonas, a forward primer conserved among Pseudomonas species, 5'-AGCGGCGGACGGGTGAGTAAT-3' (86-106), was designed, and in conjunction with the above reverse primer, a 1,440-bp fragment was amplified.

This fragment was sequenced following treatment of the PCR product with a shrimp alkaline phosphatase/exonuclease I (US Biochemicals, Cleveland, OH). The sequencing analysis was performed using the method of dye terminator cycle (ABI 377 automated sequencer, Applied Biosystems, Foster City, CA). Additional internal primers were designed to complete the entire sequence analysis of the 1,440-bp fragment. Fatty Acid Analysis Fatty acid analysis was performed at the Texas Department of Health (Austin) using a MIDI System (Newark, DE) with a method similar to that described by Cartwright.13 Briefly, the bacterial cells were grown to a stationary phase and then lysed by saponification. The total cellular fatty acids were esterified in 10% BCl3CH3OH to the volatile derivatives and then extracted. Fatty acids were identified by gas-liquid chromatography.

Results The bacterium was initially isolated after being enriched in thioglycolate broth for 6 days. Subsequent culture further suggested fastidiousness in view of slow growth in chocolate agar (2 days) and sheep blood agar (3 days). Culture characteristics and results of biochemical reactions are summarized in ❚Table 1❚ . On the basis of these characteristics, this bacterium could not be identified. By scanning electron microscopy, as well as negative staining, this bacterium appeared to be 0.5 × 0.9-1.9 µm, short bacillary, and singly flagellated ❚Image 4❚ . Antibiotic susceptibility tests suggested that the bacterium was susceptible to extendedspectrum penicillins, fluoroquinolones, aminoglycosides, and third- and fourth-generation cephalosporins ❚Table 2❚. In an attempt to identify this bacterium, analysis of 16S rDNA was performed. A 1440-bp rDNA fragment was

❚Table 1❚ Culture and Biochemical Characteristics of Pseudomonas andersonii* Characteristic Growth temperatures Preferred growth agar Additional growth agar Colony morphologic features on 3-d sheep blood agar Gram stain Biochemical reactions Positive Negative

*

25°C, 35°C Chocolate agar and sheep blood agar (2-3 d) MacConkey agar, Brucella agar, trypticase soy agar, and buffered charcoal yeast extract agar (4-5 d) Small, smooth, opaque, nearly entire, nonhemolytic, light brown pigmented Gram-negative, short rods Oxidase, catalase, nitrite (but not nitrate) reduction, hydrolysis of urea, motility, acetoin Glucose fermentation; utilization of maltose, lactose, arabinose, inositol, sorbitol, rhamnose, sucrose, melibiose, and amygdalin; indole formation; esculin hydrolysis; beta-galactosidase; H2S production; citrate utilization; tryptophan deaminase; hydrolysis of gelatin; arginine dihydrolase; lysine decarboxylase; ornithine decarboxylase

A combination of tube media and commercial test kits were used. The codes of commercial test results were 0200054 for API 20NE (BioMerieux, Marcy-l’Etoile, France) and 2272401 for Vitek (BioMerieux, Hazelwood, MO).

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polyunsaturated, or cyclopropane acids ❚Table 3❚. Straightchain fatty acids C16:0, C16:1, and C18:1, which are characteristic of Pseudomonas species,14,15 were demonstrated in this bacterium. The relative proportions of these and other fatty acids, however, were unique to this organism (Table 3).

Discussion

❚Image 4❚ Scanning electron microscopy of the bacterium. Bar equals 1 µm (×15,000).

amplified by PCR and sequenced (Genbank accession number AF291818). Homology search through Genbank BLAST revealed no complete matches with any known sequences. However, the closest matches fell in the genus Pseudomonas, with 95% identity with Pseudomonas aeruginosa, the type species of Pseudomonas, and 96% identity with Pseudomonas stutzeri, Pseudomonas mendocina, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas resinovoran, and Pseudomonas pseudoalcaligenes. The next level of matches was 86% with Acinetobacter and Achromatium. The close resemblance of this bacterium to Pseudomonas also was suggested by analysis of cellular fatty acids that mainly consisted of straight-chain saturated,

In this report, we have presented strong evidence that a novel gram-negative bacterium can cause lung granuloma. On the basis of the 16S rDNA analysis and the cellular fatty acid composition, we proposed a new species for the organism, Pseudomonas andersonii. Phenotypically, P andersonii is similar to most Pseudomonas species by its nonfermentative metabolism, aerobic growth, positive reactions of oxidase and catalase, and motility by single polar flagellum.16 However, its fastidious growth was different from that of known Pseudomonas species, which can be readily cultured. Pseudomonas species are widely distributed in nature; some are pathogens for plants, animals, and humans.15 The type species as well as the most pathogenic species of Pseudomonas, P aeruginosa, that causes necrotizing pneumonia and other severe infections, bears 95% sequence identity of the 16S rDNA to our isolate. P stutzeri and P mendocina, also closely related to P andersonii, have been implicated as rare causes of bacteremia, communityacquired pneumonia, and infective endocarditis.17-19 Presumably P andersonii is also an environmental organism, one that has not been recognized before. Whether isolation of this organism in this case is a rare event or whether the disease entity is underrecognized or both remains to be seen. Perhaps its fastidious growth might have contributed to its previous lack of recognition.

❚Table 2❚ Antibiotic Susceptibility of Pseudomonas andersonii Minimal Inhibitory Concentration (µg/mL) Agent Ampicillin Ticarcillin-clavulanate Imipenem Ceftazidime Cefepime Amikacin Ciprofloxacin Trimethoprim-sulfamethoxazole

Disk Diffusion Method*† S S S S S S S R

E-Test‡ 1.0 1.0 0.25 0.75 1.0 0.25 0.125 4 or more

Microdilution Test 4 2.0 0.125 1.0 2.0 0.25 0.03 2.4

Interpretation* S S S S S S S R

R, resistant; S, susceptible. * Interpretation of susceptibility is based on breakpoints set for Pseudomonas aeruginosa and other non-Enterobacteriaceae established by the National Committee for Clinical Laboratory Standards (NCCLS).9,10 † Test was performed with 48-h incubation and, thus, is considered nonstandard according to the NCCLS (24-h incubation). Additional antibiotics also tested by this method included susceptible antibiotics: ampicillin-sulbactam, mezlocillin, piperacillin, piperacillin-tazobactam, aztreonam, meropenem, ceftriaxone, gentamicin, tobramycin, levofloxacin, ofloxacin, and tetracycline; and resistant antibiotics: cefazolin and cefoxitin. ‡ AB Bio-disk, Solna, Sweden.


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❚Table 3❚ Fatty Acid Profile of Pseudomonas andersonii* Fatty Acid 10:0 10:0, 3OH 12:0 12:0, 2OH 12:0, 3OH 14:0 15:0 16:0† 16:1 w7c/ 15 iso 2OH† 17:0 cyclo 18:1 w9c† 18:1 w7c† 18:2 w7c 19:0 19:0 cyclo w8c Total

Percentage in Profile