Atmospheric Growth Requirements for Alloiococcus Species and

attracted public attention but also to better describe diseases considered commonplace in our society, such as ear infections. A unique bacterial organism, ...
Télécharger le PDF
181KB taille 2 téléchargements 347 vues
commentaire
Report
JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1996, p. 1027–1028 0095-1137/96/$04.0010 Copyright q 1996, American Society for Microbiology

Vol. 34, No. 4

Atmospheric Growth Requirements for Alloiococcus Species and Related Gram-Positive Cocci P. H. MILLER,* R. R. FACKLAM,

AND

J. M. MILLER

Centers for Disease Control and Prevention, Atlanta, Georgia 30333 Received 4 October 1995/Returned for modification 28 November 1995/Accepted 23 January 1996

The growth of Alloiococcus otitis under different atmospheres and nutritional conditions was studied. The growth rates of 25 strains of gram-positive cocci representing five genera on heart infusion agar plates containing 5% rabbit blood and on brucella agar plates with and without sheep blood under aerobic, increased CO2, and anaerobic atmospheres were compared. Eight strains of alloiococci plated on heart infusion agar with rabbit blood and on brucella sheep blood agar grew under aerobic and candle jar atmospheres. Two of these strains showed poor anaerobic growth after 7 days. Strains of Aerococcus viridans, Aerococcus urinae, Helcococci kunzi, Dolosigranulum pigrum, Gemella haemolysans, and Gemella morbillorum grew well under all three atmospheres and on the three types of media and in thioglycolate broth. These results confirm the aerobic atmospheric requirements for Alloiococcus strains and show that aerobic growth characteristics help distinguish the alloiococci from the other gram-positive cocci that are facultatively anaerobic. 39-95). Aerococcus viridans ATCC 11563 and ATCC 29723 and Gemella morbillorum ATCC 27824 and Gemella haemolysans ATCC 10379 were obtained from the American Type Culture Collection. Four A. urinae strains were taken from the Centers for Disease Control culture collection (1515-85, 2653-87, 998-93, and 1891-94), and A. urinae NCTC 12142 was obtained from the National Collection of Type Cultures, London, England. H. kunzii 0007, 0014, and 0022 (CDC SS-1333, SS-1334, and SS-1335, respectively) were received from K. Ruoff, Boston, Mass., and Dolosigranulum pigrum NCFB 2975 was obtained from the National Collection of Food Bacteria, Reading, England. Plates of heart infusion agar supplemented with 5% rabbit blood (HIA-RB), brucella agar with and without 5% sheep blood, and fluid thioglyocolate broth were obtained from Becton-Dickinson Microbiology Systems, Cockeysville, Md. Colonies from the pure isolates were suspended in Lombard-Dowell broth to a McFarland standard of 1, and 0.001 ml (1 ml) of each organism was inoculated onto three HIA-RB plates, three brucella agar plates with sheep blood, and three brucella agar plates without blood and into thioglycolate broth. One plate was incubated under normal atmospheric conditions at 378C, one plate was placed in a candle extinction jar with the candle lighted and the candle extinction jar was placed in an incubator at 378C, and the third plate was placed in an anaerobic glove box under strict anaerobic conditions at 378C. The thioglycolate broth was also placed in the glove box and incubated at 378C. All cultures were inspected visually daily for 3 days and after 7 days. The thioglycolate broth was read for turbidity at 24 and 48 h and was subcultured aerobically for 3 days to test for growth and survival. Criteria for recording the degree of growth on the blood agar plates were as follows: abundant, .300 colonies; moderate, 30 to 300 colonies; and sparse, ,30 colonies. Colony diameters were also noted as follows: large, $5 mm; small, 1 to 3 mm; and tiny, ,1 mm. The growth of A. otitis under aerobic and candle jar atmospheres was better on HIA-RB than on brucella agar, on which some strains did not show growth until 72 h (Table 1). At 72 h, there was no difference in the growth rates under aerobic atmospheres on HIA-RB and brucella agar. Under candle jar atmospheres at 72 h, all strains grew, but they were judged to grow moderately, with tiny colonies, on brucella agar (value of 4). Most strains were judged to grow abundantly on HIA-RB

A goal of the emerging infectious disease plan from the Centers for Disease Control and Prevention (2) is not only to enhance surveillance of emerging diseases that have recently attracted public attention but also to better describe diseases considered commonplace in our society, such as ear infections. A unique bacterial organism, Alloiococcus otitis, has been recovered in cases of otitis media with effusion. There is limited information about the atmospheric requirements for the growth of A. otitis. In their original description of the isolation of the organism from ear fluids, Faden and Dryja stated that the organism failed to grow under anaerobic conditions (4). The details of how growth was tested under various atmospheric conditions were not described. Aguirre and Collins (1) restated that the organisms failed to grow in an anaerobic environment. It was not clear if the organisms were actually tested or if the authors were restating the report of Faden and Dryja. The alloiococci are gram-positive cocci arranged in pairs and clusters and are weakly catalase positive. These characteristics seem to indicate that the organisms are more closely related to the micrococci or staphylococci than to the aerococci or streptococci. However, the guanine-plus-cytosine (GC) percentage of the alloiococci is more like that of the streptococci and enterococci (44 to 45%). The aerococci, enterococci, streptococci, and other gram-positive cocci with GC ratios of 32 to 44% are all facultatively anaerobic bacteria. This study compares the atmospheric requirements for the growth of alloiococci with those for other recently described gram-positive cocci, such as Helcocccus kunzi and Aerococcus urinae. Three A. otitis strains (7760, 890, and 492 [the type strain]) were received from Faden and Dryja, three strains (1992–94, 1993–94, and 1995–94) were received from M. Baquero, Madrid, Spain, and two strains were taken from the Centers for Disease Control and Prevention culture collection (1984-93 and 277-88). Five additional Alloiococcus-like strains were taken from the Centers for Disease Control and Prevention culture collection (106-91, 810-92, 811-92, 3295-94, and * Corresponding author. Mailing address: Centers for Disease Control and Prevention, 1600 Clifton Rd. NE, Mail Stop C-01, Atlanta, GA 30333. Phone: (404) 639-3475. Fax: (404) 639-3822. Electronic mail address: [email protected]. 1027

1028

NOTES

J. CLIN. MICROBIOL. TABLE 1. Growth of selected strains under three atmospheres and on two media Growth on HIA-RB/brucella blood agarb

Strain

7760 890 1984-93 492 277-88 1992-94 1993-94 1995-94 106-91 810-92 811-92 3295-94 39-95 SS-1333 SS-1334 SS-1335 29723 11563 12142 2653-87 1515-85 998-93 984-94 1871-94 27824 10379 2975

Sourcea

Ear Ear Ear Ear Ear Ear (Spain) Ear (Spain) Ear (Spain) Non-ear Non-ear Non-ear Non-ear Non-ear ATCC Ear Ear ATCC ATCC NCTC Urine Urine Urine Urine Urine ATCC ATCC NCFB

Organism

A. otitis A. otitis A. otitis A. otitis A. otitis A. otitis A. otitis A. otitis ALO ALO ALO ALO ALO H. kunzii H. kunzii H. kunzii A. viridans A. viridans A. urinae A. urinae A. urinae A. urinae A. urinae A. urinae G. morbillorum G. haemolysan D. pigrum

Aerobic atmosphere

Candle jar atmosphere

Anaerobic atmosphere

24 h

48 h

72 h

24 h

48 h

72 h

24 h

48 h

72 h

0/1 4/1 4/0 0/0 0/0 4/0 4/0 4/0 0/0 4/0 7/4 7/0 0/0 7/7 7/7 7/7 7/7 7/7 7/7 7/7 7/7 7/7 7/ND 7/7 0/4 7/7 4/0

7/4 7/4 7/0 8/4 4/4 7/4 7/4 7/4 0/4 7/4 7/7 8/0 4/0 7/7 7/7 7/7 7/7 7/7 7/7 7/7 7/7 7/7 7/ND 7/7 0/4 7/7 7/4

7/7 7/7 7/7 8/7 7/7 7/7 7/7 7/7 7/7 7/7 7/7 8/4 7/4 7/7 7/7 7/7 7/7 7/7 7/7 7/7 7/7 7/7 7/ND 7/7 4/7 8/8 7/7

4/0 4/0 4/0 0/0 0/0 4/0 4/0 4/0 0/0 7/4 7/4 7/0 7/0 7/7 7/7 7/7 7/7 7/7 7/7 7/7 7/7 7/7 7/ND 7/7 0/4 7/7 4/0

4/4 7/4 7/0 8/4 4/4 7/1 7/4 7/0 4/4 7/7 7/7 8/0 8/0 7/7 7/7 7/7 7/7 7/7 7/7 7/7 7/7 7/7 7/ND 7/7 4/7 8/7 7/4

4/4 7/4 7/4 8/4 7/4 7/4 7/4 7/4 7/4 7/7 7/7 8/4 8/4 7/7 7/7 7/7 7/7 7/7 7/7 7/7 7/7 7/7 7/ND 7/7 7/7 8/8 7/7

0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 4/4 4/4 4/4 7/4 7/7 7/7 7/7 7/0 7/7 7/0 7/7 7/7 7/7 7/ND 7/7 4/7 4/7 4/0

0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 7/7 7/7 7/7 7/7 7/7 7/7 7/7 7/4 7/7 7/4 7/7 7/7 7/7 7/ND 7/7 7/7 7/7 4/4

0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 7/7 7/7 7/7 7/7 7/7 7/7 7/7 7/7 7/7 7/7 7/7 7/7 7/7 7/ND 7/7 7/7 7/7 4/7

a

ATCC, American Type Culture Collection; NCTC, National Collection of Type Cultures; NCFB, National Collection of Food Bacteria. 0, no growth; 1, sparse growth (,30 colonies with diameters of ,1 mm); 4, moderate growth (30 to 300 colonies with diameters of ,1 mm); 7, abundant growth (.300 colonies with diameters of ,1 mm); 8, abundant growth (.300 colonies with diameters of 1 to 3 mm); ND, not done. b

under these conditions. No growth was observed at 72 h on the plated media under anaerobic atmospheres. Two alloiococcal strains grew moderately under an anaerobic atmosphere after 7 days: 1995-94 on HIA-RB and 7760 on brucella blood agar. None of the A. otitis cultures grew on brucella agar without blood under any of the atmospheres. None of the alloiococcal strains showed turbidity in thioglycolate broth, but all the strains remained viable after 72 h, which was shown when viable cells were successfully subcultured on HIA-RB. Five strains of Alloiococcus-like organisms (ALO) did not have the same atmospheric requirements as A. otitis. Four of these strains grew under anaerobic atmospheres on both brucella agar and HIA-RB media. None of the ALO showed growth in thioglycolate broth, and three of the five ALO strains from the broth were not viable upon subculture. The failure of the ALO to grow in thioglycolate broth, even though the same strains grew well in an anaerobic environment on agar plates, limits the usefulness of thioglycolate broth as a measure of atmospheric requirements. These strains have the same phenotypic characteristics as the alloiococci (3). None of these strains were isolated from ear cultures. The only apparent differences between documented strains of A. otitis and ALO are the atmospheric requirements for growth. Additional studies are required to identify the taxonomic positions of these bacteria. One strain each of A. viridans and A. urinae showed no growth under an anaerobic atmosphere at 24 h on brucella agar, but growth was abundant on both plates at 72 h. The same two strains showed abundant growth on HIA-RB at 24 h under anaerobic conditions. All other strains showed abundant growth under all three atmospheres on both brucella agar and HIA-RB after 24 h.

All three strains of H. kunzii grew abundantly under all three atmospheres on both media. The growth of G. morbillorum under an anaerobic atmosphere was better than that under either an aerobic or candle jar atmosphere on both HIA-RB and brucella agar media. G. haemolysans grew equally well under all three atmospheres and on both media. Moderate growth of D. pigrum was observed on HIA-RB at 24 h and on brucella blood agar at 48 h, but at 72 h, there was no significant difference in the growth rates under the three atmospheres and on the two different media. These results confirm the facultatively anaerobic growth requirements of both Aerococcus species, both Gemella species, H. kunzii, and D. pigrum and confirm the aerobic atmospheric requirements of the alloiococci, a characteristic that can help distinguish members of the genus Alloiococcus from other grampositive cocci that are facultatively anaerobic. REFERENCES 1. Aguirre, M., and M. D. Collins. 1992. Phylogenetic analysis of Alloiococcus otitis gen. nov., sp. nov., an organism from human middle ear fluid. Int. J. Syst. Bacteriol. 42:79–83. 2. Centers for Disease Control and Prevention. 1994. Addressing emerging infectious disease threats: a prevention strategy for the United States. Public Health Service, U.S. Department of Health and Human Services, Atlanta. 3. Facklam, R., and J. A. Elliott. 1995. Identification, classification, and clinical relevance of catalase-negative, gram-positive cocci, excluding the streptococci and enterococci. Clin. Microbiol. Rev. 8:479–495. 4. Faden, H., and D. Dryja. 1989. Recovery of a unique bacterial organism in human middle ear fluid and its possible role in chronic otitis media. J. Clin. Microbiol. 27:2488–2491.