| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Alcon Research Ltd, 6201 South Freeway, R0-17, Fort Worth, TX 76134-2099, USA
Correspondence
David W. Stroman
David.Stroman{at}alconlabs.com
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of MCC10330T is AY953147.
Tables detailing the biochemical and phenotypic characteristics of individual novel strains and the cellular fatty acid composition of the novel strains and related Pseudomonas species are available as supplementary material in IJSEM Online.
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
and since then members of the genus have been isolated from humans, plants and various environmental habitats, such as soil (Roland & Stroman, 2002; Munsch et al., 2002; Ivanova et al., 2002; Sikorski et al., 2001). The genus Pseudomonas was formerly described by Palleroni (1984) as consisting of more than 100 species of phylogenetically unrelated groups of Proteobacteria. Palleroni et al. (1973), using genetic analysis, specifically rRNA–DNA hybridization, subdivided the genus into five rRNA groups (I–V). Kersters et al. (1996) have summarized the changes in the reclassification of the former Pseudomonas species, which are now classified into several genera belonging to the Alphaproteobacteria, Betaproteobacteria or Gammaproteobacteria. Species belonging to the genus Pseudomonas sensu stricto are now placed in rRNA group I (Palleroni, 1984), which belongs to the Gammaproteobacteria (Kersters et al., 1996; Achouak et al., 2000). In the past two decades, polyphasic taxonomic studies, especially those using genetic and molecular techniques, have played a critical role in improving the classification of the pseudomonads (Anzai et al., 2000). Many of the organisms originally described as species of the genus Pseudomonas have been reclassified to other genera.
Novel pseudomonads were recovered from the ears of patients enrolled in anti-infective clinical studies. In total, 41 novel pseudomonads were recovered from ears of patients diagnosed with acute otitis externa (n=34), otitis media with otorrhoea drainage through a tympanostomy tube (n=6) and chronic suppurative otitis media (n=1). A brief description of the source of 11 representative isolates is listed in Table 1. At clinical examination, a swab was used to sample each patient's ears, and the swabs were then immediately placed into transport media. Swabs were removed from transport media and cultured onto various media to obtain viable colonies and then subcultured to obtain a pure culture. Isolated colonies were transferred to trypticase soy broth supplemented with 10 % glycerol and stored in liquid nitrogen. Bacteria were thawed and cultured onto blood agar prior to genetic or phenotypic analyses. DNA–DNA hybridization, comparative 16S rRNA gene sequencing and phenotypic analysis identified these organisms as members of a novel Pseudomonas species. The novel pseudomonads were compared with the type strains of Pseudomonas alcaligenes, P. aeruginosa, P. citronellolis, P. multiresinovorans, P. nitroreducens, P. resinovorans and P. stutzeri, which were acquired from the ATCC.
|
. Strain-level discrimination of the new isolates is shown in Fig. 2. The ribotype pattern of the PstI digests revealed a band at 6·9 kbp that was present for every isolate, and the majority of isolates had double bands at approximately 6·5–7·5 kbp. Other bands were present, but no discernible pattern was noted. The ribotype pattern of PvuII digests showed a distinct band at 3·4 kbp that was present for all the isolates. Ribotype patterns of PvuII digestion also produced numerous bands that did not demonstrate any obvious patterns or similarity between isolates.
|
|
, a phylogenetic tree obtained by using the neighbour-joining method, shows the phylogenetic placement of MCC10330T branching with P. aeruginosa (98·6 % 16S rRNA gene sequence similarity). The other species most closely related (percentage sequence similarity) to MCC10330T were P. alcaligenes (98·2 %), P. stutzeri (98·0 %), P. resinovorans (97·5 %), Pseudomonas pseudoalcaligenes (97·1 %) and P. citronellolis (96·9 %).
|
. DNA–DNA hybridization was carried out as described by De Ley et al. (1970) with the modifications suggested by Huß et al. (1983) using a Cary 100 Bio UV/Vis spectrophotometer equipped with a Peltier-thermostatted 6x6 multicell changer and a temperature controller with in-situ temperature probe (Varian). DNA–DNA hybridizations (performed in duplicate) between the new taxon and other related pseudomonads had the following binding values (percentage relatedness): P. alcaligenes (55 %), P. citronellolis (44 %), P. aeruginosa (40 %), P. nitroreducens (41 %), P. resinovorans (19 %) and P. stutzeri (12 %). Given a threshold level of 70 % DNA–DNA relatedness used to delineate a bacterial species, as recommended by Wayne et al. (1987), the results indicated that MCC10330T did not belong to any of the Pseudomonas species tested. The DNA G+C content of MCC10330T was 67·9 mol%.
The 11 representative novel strains were evaluated for phenotypic characteristics and compared with the type strains of P. aeruginosa, P. citronellolis and P. stutzeri (Table 2; results for the individual novel strains are given in Supplementary Table S1 in IJSEM Online). Carbon substrate utilization was determined using the Biolog GN MicroPlate System and VITEK32 (bioMérieux). The presence of oxidase, catalase and urease activity, production of fluorescent pigments, hydrolysis of Tween 80, aesculin, starch and casein, motility in semisolid media, tolerance of NaCl and minimal and maximal temperature range were all assayed according to standard procedures. Cells of the novel strains were motile, Gram-negative, non-spore-forming rods occurring as single cells. Growth occurred at temperatures of 7–45 °C, but no growth was observed at 4 or 47 °C. Growth occurred on solid media supplemented with 0–4 % NaCl (w/v), but not at higher salinity. Growth was observed in media only at pH 5–10. The majority of the novel strains (10 of 11) failed to produce pyocyanin or fluorescein, whereas both P. aeruginosa and P. citronellolis produced these pigments. Each strain had positive reactions for oxidase and catalase, but all were negative for urease. The novel strains were relatively homogeneous in their metabolic characteristics. They differed from the reference strains in utilization of glycerol, D-gluconic acid, itaconic acid, D-fructose, D-mannitol, acetic acid, cytosine, 
-phenylethylamine and sebacic acid.
|
7c (32·9 %), C16 : 0 (26·9 %), C12 : 0 (9·1 %), C12 : 0 3-OH (4·0 %) and C10 : 0 3-OH (2·8 %). C12 : 0 3-OH is consistently found in members of the genus Pseudomonas sensu stricto (Oyauzu & Komagata, 1983). The fatty acid compositions of the reference pseudomonads assayed were qualitatively similar to but varied in concentration from those of the novel strains.
Minimal inhibitory concentrations (MIC) were determined using the National Committee for Clinical Laboratory Standards broth microdilution method (NCCLS, 2003). Microdilution plates (96 wells) contained specific concentrations of antibiotic incorporated into the wells (PML Microbiologicals). Microdilution plates were inoculated with approximately 5x105 c.f.u. bacteria ml–1 and incubated overnight at 35 °C. The MIC was defined as the lowest concentration of antibiotic that inhibits growth of an organism as determined by lack of turbidity. The concentrations of antibiotic that inhibited the growth of 50 % (MIC50) or 90 % (MIC90) of the number of novel strains tested were determined. Antibiotic plates were analysed using a 96-well microplate reader (Dynatech Laboratories) at a wavelength of 590 nm and further analysed by visual inspection to determine turbidity. The novel strains exhibited similar antibiotic susceptibility profiles (Table 3). The isolates were susceptible to aminoglycosides (tobramycin, neomycin, gentamicin), fluoroquinolones (ciprofloxacin, ofloxacin, moxifloxacin) and polymyxin B. However, piperacillin, erythromycin, tetracycline, chloramphenicol, trimethoprim and sulfamethoxazole had minimal ability to inhibit growth of the organisms.
|
Cells are Gram-negative, motile rods. Colonies on trypticase soy agar are circular, concave and non-pigmented. Colonies grown on trypticase soy agar supplemented with 5 % sheep blood are not haemolytic. Growth occurs at 7–45 °C, pH 5–10 and in the presence of up to 4 % NaCl. Colonies typically do not produce fluorescent pigment, and all strains are positive for oxidase and catalase, but negative for urease. Tween 80 and gelatin are hydrolysed, but casein and aesculin are not. Utilizes glycogen, 
-D-glucose, acetic acid, citric acid, formic acid, lactic acid, propionic acid, succinic acid, D-alanine, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-glutamic acid, L-histidine, L-leucine, L-ornithine, L-proline, L-serine, 
-aminobutyric acid, phenylethylamine and putrescine. None of the strains utilizes N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, adonitol, D-arabitol, L-arabinose, i-erythritol, myo-inositol, glycerol, D-mannitol, D-sorbitol, xylitol, D-fructose, L-fucose, D-galactose, gentiobiose, -D-lactose, maltose, D-mannose, D-melibiose, D-raffinose, L-rhamnose, sucrose, D-trehalose, turanose, D-xylose, D-galacturonic acid, D-gluconic acid, D-glucuronic acid, itaconic acid, glucuronamide, L-phenylalanine, D-serine, inosine, uridine, thymidine, cytosine or sebacic acid. The predominant cellular fatty acids are C18 : 17c, C16 : 0, C12 : 0, C12 : 0 3-OH and C10 : 0 3-OH. Susceptible to aminoglycosides, fluoroquinolones and polymyxin B, but resistant to piperacillin, erythromycin, tetracycline, chloramphenicol, trimethoprim and sulfamethoxazole. The DNA G+C content of the type strain is 67·9 mol%.
The type strain, MCC10330T (=ATCC BAA-1130T=DSM 17224T), and other strains were isolated from the ears of patients with acute otitis externa, acute otitis media with otorrhoea and chronic suppurative otitis media.
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Anzai, Y., Kim, H., Park, J., Wakabayashi, H. & Oyaizu, H. (2000). Phylogenetic affiliation of the pseudomonads based on 16S rRNA sequence. Int J Syst Evol Microbiol 50, 1563–1589.[Abstract]
Cashion, P., Holder-Franklin, M. A., McCully, J. & Franklin, M. (1977). A rapid method for the base ratio determination of bacterial DNA. Anal Biochem 81, 461–466.[CrossRef][Medline]
De Ley, J., Cattoir, H. & Reynaerts, A. (1970). The quantitative measurement of DNA hybridization from renaturation rates. Eur J Biochem 12, 133–142.[Medline]
Huß, V. A. R., Festl, H. & Schleifer, K. H. (1983). Studies on the spectrophotometric determination of DNA hybridization from renaturation rates. Syst Appl Microbiol 4, 184–192.
Ivanova, E., Gorshkova, N., Sawabe, T. & 8 other authors (2002). Pseudomonas extremorientalis sp. nov., isolated from a drinking water reservoir. Int J Syst Evol Microbiol 52, 2113–2120.[Abstract]
Kersters, K., Ludwig, W., Vancanneyt, M., De Vos, P., Gillis, M. & Schleifer, K. H. (1996). Recent changes in the classification of the pseudomonads: an overview. Syst Appl Microbiol 19, 465–477.
Migula, W. (1894). Ube rein neues System der Bakterien. Arb Bakteriol Inst Karlsruhe 1, 235–238 (in German).
Munsch, P., Alatossava, T., Marttinen, N., Meyer, J., Christen, R. & Gardan, L. (2002). Pseudomonas costantinii sp. nov., another causal agent of brown blotch disease, isolated from cultivated mushroom sporophores in Finland. Int J Syst Evol Microbiol 52, 1973–1983.[Abstract]
NCCLS (2003). Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 6th edn. Approved Standard M7-A6. Wayne, PA: National Committee for Clinical Laboratory Standards.
Oyauzu, H. & Komagata, K. (1983). Grouping of Pseudomonas species on the basis of cellular fatty acid composition and the quinone system with special reference to 3-hydroxy fatty acids. J Gen Appl Microbiol 29, 17–40.
Palleroni, N. J. (1984). Genus I. Pseudomonas Migula 1894. In Bergey's Manual of Systematic Bacteriology, vol. 1, pp. 141–199. Edited by N. R. Krieg & J. G. Holt. Baltimore: Williams & Wilkins.
Palleroni, N. J., Kunisawa, R., Contopoulou, R. & Doudoroff, M. (1973). Nucleic acid homologies in the genus Pseudomonas. Int J Syst Bacteriol 23, 333–339.
Roland, P. & Stroman, D. (2002). Microbiology of acute otitis externa. Laryngoscope 112, 1166–1177.[CrossRef][Medline]
Sikorski, J., Stackebrandt, E. & Wackernagel, W. (2001). Pseudomonas kilonensis sp. nov., a bacterium isolated from agricultural soil. Int J Syst Evol Microbiol 51, 1549–1555.[Abstract]
Wayne, L. G., Brenner, D. J., Colwell, R. R. & 9 other authors (1987). Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463–464.
This article has been cited by other articles:
|
|
 |
|
  L. Tvrzova, P. Schumann, C. Sproer, I. Sedlacek, Z. Pacova, O. Sedo, Z. Zdrahal, M. Steffen, and E. Lang Pseudomonas moraviensis sp. nov. and Pseudomonas vranovensis sp. nov., soil bacteria isolated on nitroaromatic compounds, and emended description of Pseudomonas asplenii. Int J Syst Evol Microbiol, November 1, 2006; 56(Pt 11): 2657 - 2663. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
1500 bp), showing the phylogenetic relationship of Pseudomonas otitidis sp. nov. and other closely related members of the genus Pseudomonas. Numbers at nodes are percentages ofbootstrap support based on neighbour-joining analyses of 1500 resampled datasets. GenBank accession numbers are given in parentheses. The scale indicates percentage sequence divergence.