INTRODUCTION

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Lautropia mirabilis, a recently characterized motile gram-negative coccus, has been isolated from oral and pulmonary sites (4). Its pathogenicity is unknown, as it has been recovered from both ill and healthy persons. This report describes the isolation of L. mirabilis from the oral cavities of human immunodeficiency virus (HIV)-infected children and its absence in noninfected children. This association suggests a possible link between immunocompromise and oral colonization with L. mirabilis.

L. mirabilis was described in 1994 by P. Gerner-Smidt et al. (4) and subsequently was given species status (6). The organism is gram negative, facultatively anaerobic, and oxidase positive, with cell morphology varying from coccobacilli 1 µm in diameter to spheroblast-like forms more than 10 µm in diameter. The colonies are pleomorphic. This organism appears to be identical to Sarcina mirabilis, described by Orskov in 1930 (7). Both Orskov and Gerner-Smidt et al. isolated the organism from oral or upper respiratory sites. Subsequently, it has been isolated as the predominant microorganism from the sputum of one Australian patient with cystic fibrosis (1).

	MATERIALS AND METHODS

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Population. The study population consisted of 60 children, ranging in age from newborn to 12 years, who had been infected with HIV vertically, i.e., who were born to infected mothers. HIV culture and/or PCR determined the infection status of the children. The control population consisted of 25 uninfected children born to HIV-infected mothers. This prospective study was designed to evaluate the scope and possible causes of oral lesions in HIV-infected children, as oral disease is a cause of significant morbidity in HIV-infected children and adults.

Clinical evaluation. Each child received a complete oral examination by a pediatric dentist at entry into the study and every 6 months subsequently, or at the time of any illness. During the examination, aerobic and anaerobic cultures were initiated.

Microbiology. The gingivae or alveolar mucosae of patients and controls were swabbed, and the swabs were transported to the laboratory in an S/P Culturette (Baxter Diagnostics, Deerfield, Ill.). The specimens were inoculated immediately upon receipt at the Texas Children's Hospital Microbiology Laboratory onto a variety of media chosen to identify common oral organisms and were subsequently processed in a dedicated portion of the laboratory.

Biochemical identification. Standard biochemical methods were used. The production of urease was determined by using a heavy inoculum on Christensen's urea agar slant (Becton Dickinson, Cockeysville, Md.), with incubation at 35°C for 24 to 72 h. Urease was also tested for with Christensen's urea broth (rapid urea broth; Becton Dickinson) and with the API 20E system (bioMérieux Vitek, Hazelwood, Mo.). Catalase was tested for with 10 and 3% hydrogen peroxide.

Electron microscopy. Colonies of L. mirabilis were obtained from MacConkey's II agar (Becton Dickinson) and fixed in 3% glutaraldehyde for 24 h. Postfixation staining was carried out for 90 min in 1% osmium tetroxide, followed by 2% uranyl acetate. The colonies were dehydrated and embedded in plastic, and thin sections were produced for ultrastructural examination.

Statistical analysis. The statistical significance of associations was tested with the chi-square test, or Fisher's exact test when the expected cell count was <5. Mean values were compared by the t test.

	RESULTS

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L. mirabilis was initially isolated from MacConkey's II agar at 35°C in 6% CO₂. The organism was visible in 48 to 72 h. On primary isolation on this agar the colony was circular, less than 1 mm in diameter, and dark purplish; it had a rough surface and was extremely pitted in the agar. With further incubation, the colony increased to 1 to 2 mm in diameter and became irregular in shape, lighter purple, and less pitted in the agar (Fig. 1A). The organism was successfully reisolated on Sabouraud dextrose agar (Emmons modification) (Becton Dickinson), chocolate II agar with bacitracin (Becton Dickinson), and TSA II-5% sheep blood agar (Becton Dickinson). CO₂ was not required for growth, and the organism grew at room temperature as well as at 35°C. Subsequent work has shown that growth is best on Regan-Lowe charcoal agar (Becton Dickinson). L. mirabilis was not recovered from specimens processed anaerobically.

View larger version (54K): [in this window] [in a new window]   FIG. 1.   (A) L. mirabilis colonies on MacConkey's agar. (B) Very small forms (1 to 2 µm in diameter; arrows) of L. mirabilis with larger forms (up to 10 µm in diameter; arrowheads). The staining was with lactophenol cotton blue. Magnification, ×100 (bar = 10 µm). (C) Large aggregates of L. mirabilis organisms, with individual organisms at the periphery. The staining was with lactophenol cotton blue. Magnification, ×100 (bar = 10 µm).

Wet mounts of L. mirabilis demonstrated the morphological forms that have previously been described (4). Very small (approximately 1 µm in diameter) round forms frequently showed a rapid, circular motility. Individual cells of larger size, up to 10 µm in diameter, often with a vaguely perceptible internal structure (Fig. 1B), and large aggregates (>10 µm in diameter) of nonmotile cells of various sizes were also seen (Fig. 1C). All forms were gram negative. The cells were also well visualized by lactophenol cotton blue staining; this stain is generally used in mycology and yields results comparable to those with methylene blue.

On several media, L. mirabilis had two colony morphologies: a smooth, mucoid form and a drier, rough form. These morphologies corresponded very approximately to the microscopic morphology, as the small, motile forms were more likely to come from the mucoid colonies. The best long-term viability was noted in Trypticase soy broth with 20% glycerol (Becton Dickinson) at 70°C. The growth of L. mirabilis was sometimes poor, and the organism was resistant to multiple reisolations. Confirmation of the species was provided for three of the isolates by Brita Bruun of the Statens Serum Institut in Copenhagen, Denmark (2).

Biochemical identification. L. mirabilis was positive for oxidase, urease, catalase, nitrate reduction, esculin, acid formation from glucose, mannitol, sucrose, and maltose. L. mirabilis was negative for o-nitrophenyl--D-galactopyranoside, ornithine decarboxylase, citrate utilization, tryptophan deaminase, indole, H₂S production, acid formation from inositol, rhamnose, melibiose, amygdalin, arabinose, lactose, inulin, and raffinose. The urease test was positive on Christensen's urea agar slants or rapid urea broth but was often negative with the API 20E system. The catalase test was weakly positive with 10% hydrogen peroxide, and catalase was undetectable with 3% H₂O₂. Variable reactions were observed for arginine dihydrolase, lysine decarboxylase, Voges-Proskauer test, gelatin at 35°C, and sorbitol.

Electron microscopy. An ultrastructural examination of organisms obtained from the colonies showed large groups of irregular cells surrounded by a thin surface membrane which in many locations was either lost or disrupted (Fig. 2). These large groups of cells measured approximately 2 to 3.5 µm in diameter in aggregate, with individual cells having maximum dimensions varying from 0.5 to 1.2 µm. Septa of intermediate electron density divided the cells. The internal regions of the cells were composed of fine, granular, homogenous, electron-dense material with only occasional myeloid-like figures and fine punctate dense granules. The cells appeared to divide by binary fission. The cell walls were generally characteristic of those described for gram-negative organisms. No flagella or fimbria were identified in the specimens examined, although these have been previously reported (4).

View larger version (132K): [in this window] [in a new window]   FIG. 2.   Electron microscopic appearance of L. mirabilis colonies at two magnifications. Bars, 2.0 µm (A) and 0.5 µm (B).

Culture. Of 85 subjects evaluated in this study, 35 (41.4%) harbored cultivable L. mirabilis organisms at some time during the study. L. mirabilis was recovered on two separate occasions from nine of these individuals. No clinical manifestations of this colonization were found. Specifically, those infected showed no increase in the number of oral lesions.

	DISCUSSION

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L. mirabilis is an unusual organism in many respects. Both its colonial and microscopic morphologies are striking. Optimal growth conditions remain uncertain; other workers have not successfully isolated this organism on MacConkey's media (1, 4). The range of biochemical reactions included within strains of this species has also not been fully defined.

Clinically, L. mirabilis has generally been considered a saprophyte (4), although its isolation has occurred in several patients with respiratory disease (1, 4). In our study it has been isolated from a large number of children; this series encompasses the largest number of isolations of L. mirabilis reported to date. No specific disease manifestation has been identified with this unusual bacterium. However, a strong association between the presence of cultivable L. mirabilis and HIV infection in these children is seen.

Recent work on the transmission of oral pathogens has shown a close identity at the molecular level between strains found in children and those found in their parents (5). No direct evidence for the presence of L. mirabilis in the parents of the children in this study is available, as specimens from the parents were not cultured.

In an attempt to elucidate the basis and mechanism of the strong association between HIV and L. mirabilis, several other differences between the infected and uninfected children were considered. Sex did not appear to be an important variable in the acquisition of Lautropia. The ages of the HIV-positive subjects were generally greater than those of the HIV-negative controls; this is a natural effect of the success of current therapy in reducing HIV transmission from HIV-infected pregnant women to their children. Within the HIV-infected group, age was not a significant variable. Data from this study do not suggest that the acquisition of L. mirabilis is simply an age-related phenomenon. However, the effect of age cannot be clearly determined from these limited data.

The lack of correlation between the presence of L. mirabilis and immunological or clinical status could be due to medications, as many children with more advanced disease were receiving antibacterial or antifungal treatment, either for clinical disease or as prophylaxis. Such drugs might affect the usual oral flora, resulting, for example, in increased growth of L. mirabilis by suppression of more numerous and commonly found organisms. An initial examination of the oral flora of these children did not reveal major differences between HIV-infected and uninfected children which might account for the difference in L. mirabilis isolation (8). The HIV-infected children in this study took a wide variety of anti-infective drugs. Of special interest is the possible effect of topical oral agents on the growth of L. mirabilis. The organism has been seen to be sensitive to a wide variety of antimicrobial agents (1, 4). Such an effect would not, of course, account for the lack of L. mirabilis in the noninfected population, as these children would not be receiving this medication. However, this or similar topical agents may play a role in the reduced incidence of L. mirabilis seen in the most severely immunocompromised and most severely clinically affected groups.

The incidence of oral and periodontal disease in HIV-infected individuals is striking, and the possible role of this organism remains to be investigated. Further work is also needed to clarify the role of topical oral agents such as nystatin in the presence of this organism. The association between HIV and L. mirabilis is fascinating, suggesting a possible role for immune function in the acquisition of this little-understood organism.