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1 Departamento de Biotecnología, Universidad Autónoma Metropolitana-Iztapalapa, Avenida Michoacán y la Purísima s/n, Col. Vicentina, 09340 México DF, Mexico
2 Institut de Recherche pour le Développement (IRD), Cicerón 609, Col. Los Morales, 11530 México DF, Mexico
3 Laboratoire de Microbiologie IRD, IFR-BAIM, Universités de Provence et de la Méditerranée, ESIL case 925, 163 avenue de Luminy, 13288 Marseille cedex 9, France
4 Central Research Laboratories, Ajinomoto Co., Inc., 1-1, Suzuki-Cho, Kawasaki-ku, Kawasaki-shi, 210-8681, Japan
5 Institut für Klinische Mikrobiologie, Immunologie und Hygiene, Wasserturmstr. 3, 91054 Erlangen, Germany
6 LAMIB-CRSBAN, Département de Biochimie-Microbiologie, Unité de Formation et de Recherches en Sciences de la Vie et de la Terre, Université de Ouagadougou, 03 BP 7021, Ouagadougou 03, Burkina Faso
7 Leukaemia Foundation Research Unit, Queensland Institute of Medical Research, 300 Herston Rd, Herston QLD-4000, Australia
Correspondence
Hervé Macarie
herve.macarie{at}esil.univ-mrs.fr
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Published online ahead of print on 15 June 2004 as DOI 10.1099/ijs.0.02810-0.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains AMX 26BT, UR374_02 and 12-3T are respectively AF273082, AY124375 and AB008507.
Transmission electron micrographs, graphs showing anoxic growth in the presence of nitrite and a dendrogram comparing CFA profiles are available as supplementary material in IJSEM Online.
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TOPABSTRACTMAIN TEXTREFERENCES |
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; Finkmann et al., 2000; Assih et al., 2002). More recently, the isolation of two novel N2O-producing strains from hot springs led to the description of a second species of the genus, Pseudoxanthomonas taiwanensis, which differs from P. broegbernensis mainly by the fact that it is not motile and that its optimum growth temperature is 50 °C instead of 30 °C (Chen et al., 2002). The two species also exhibit differences in the spectrum of substrates used and in their cellular fatty acid (CFA) profiles. Within the framework of a study on the role of aerobic bacteria in anaerobic digesters, a strain identified as AMX 26BT was isolated in Mexico. This strain could not be assigned to any known species on the base of classical biochemical tests (conventional and API 20NE, Biotype 100) and CFA profiles. Analysis of its 16S rRNA gene sequence revealed that it was closely related to a strain (12-3T) previously isolated in Japan (Iizuka et al., 1998) and also to another strain (UR374_02) isolated in Germany and that it was moderately related to P. broegbernensis. In order to clarify their taxonomic position, all strains were subjected to more detailed study. In this paper, we report the characterization of two novel species of Pseudoxanthomonas, Pseudoxanthomonas mexicana sp. nov., including strains AMX 26BT and UR374_02, and Pseudoxanthomonas japonensis sp. nov., including strain 12-3T.
Isolation and cultivation
Unless otherwise indicated, isolation and culture of micro-organisms to perform phenotypic tests were done under aerobic conditions at 35 °C, pH 7. All analyses were performed at least in duplicate. AMX 26BT was isolated from a laboratory-scale upflow anaerobic sludge blanket (UASB) reactor treating the wastewater of a cheese factory. Isolation was performed by serial dilution of grounded reactor sludge in a buffered salt solution (g l–1: MgCl2.6H2O, 0·405; KH2PO4, 0·042; pH 7·2) and surface inoculation of Petri dishes containing medium R2A with 0·1 ml of the highest dilutions (10–5–10–10). Purification was later obtained by streaking single colonies on Petri dishes filled with the same medium. Strain UR374_02 was isolated from the urine of a 10-year-old boy presenting bladder extrophy, a congenital birth defect that requires long-term catheterization and may occasionally cause urinary tract infections. As a routine follow up of the patient, 1 µl urine was plated on trypticase soy agar (TSA) supplemented with 5 % (v/v) sheep blood and incubated at 37 °C. Strain 12-3T was obtained from a sample of polluted urban soil, collected at the riverside of the Tamagawa river in the Tokyo metropolitan area, Japan, after a procedure (direct plating following filtration through a 0·45 µm membrane without enrichment) specially designed to select for extremely small, free-living bacteria. A detailed description of the isolation and purification procedures for this strain is given by Iizuka et al. (1998).
16S rRNA gene sequence analyses, DNA–DNA hybridization and G+C content
DNA extraction, 16S rRNA gene amplification, purification and sequencing were done as described previously by Assih et al. (2002) for strain AMX 26BT, Iizuka et al. (1998) for strain 12-3T and Relman et al. (1992) for strain UR374_02. A non-redundant BLASTN search of full sequences through GenBank (Altschul et al., 1990; Benson et al., 1999) identified the closest relatives. Sequences used in the phylogenetic analysis were obtained from the RDP II (Maidak et al., 2001) and GenBank (Benson et al., 1999). The alignment was produced by importing sequences into BioEdit version 5.0.9 (Hall, 1999) and using the RDP II Sequence Aligner program (Maidak et al., 2001). Positions of sequence and alignment ambiguity were omitted and a masked dataset of 1410 unambiguous nucleotides was produced. Pairwise evolutionary distances were calculated using the method of Jukes & Cantor (1969). Dendrograms were constructed using the neighbour-joining method (Saitou & Nei, 1987) as implemented in the TreeCon for Windows package (Van de Peer & De Wachter, 1994). Confidence in the tree topology was determined by bootstrapping 100 replicates (Felsenstein, 1985) and expressed as a percentage near the branching point. Determination of G+C content and DNA renaturation studies were performed by the DSMZ using HPLC and spectrophotometric methods, respectively, as described by Assih et al. (2002). Hybridization percentages obtained by the spectrophotometric technique presented a standard deviation of less than 2·5 %. In any case, hybridizations between 60 and 75 % were repeated in order to confirm the results.
16S rRNA gene sequences of 1464, 1466 and 1540 nucleotides, respectively, were determined for strains 12-3T, UR374_02 and AMX 26BT. Phylogenetic analysis revealed that the three isolates clustered within the Xanthomonas–Stenotrophomonas–Pseudoxanthomonas branch of the Proteobacteria (Fig. 1). The sequences of isolates AMX 26BT and UR374_02 were 100 % identical and they both shared 99·6 % similarity with that of strain 12-3T. The three strains were related almost equidistantly to Xanthomonas (95·4–96·6 %) and Stenotrophomonas (95·3–96·1 %) species. P. broegbernensis DSM 12573T, which shared 97·5 % sequence similarity with strains AMX 26BT and UR374_02 and 97·1 % with strain 12-3T, appeared to be their closest described relative. Within the genus Pseudoxanthomonas, the strains were only moderately related to P. taiwanensis ATCC BAA-4040T (95·3–95·4 % similarity), which was itself only moderately related to P. broegbernensis DSM 12573T (95·8 % similarity). In order to clarify definitively the phylogenetic position of the three isolates, DNA–DNA hybridization experiments were performed between them and with P. broegbernensis DSM 12573T. Strain AMX 26BT hybridized at 80·4 % with UR374_02 and 58·7–61·3 % with 12-3T. It showed a much lower level of hybridization with P. broegbernensis DSM 12573T (33·7 %), similar to that observed between this species and strain 12-3T (37 %). The DNA G+C content of the isolates is given in the species description and was similar to that of the two Pseudoxanthomonas species described to date.
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. Cell morphology and motility were deduced from direct observations of fresh cultures using a Nikon phase-contrast microscope. The presence, number and position of flagella were determined by flagellar staining (Kodaka et al., 1982; Mayfield & Inniss, 1977) and/or transmission electron microscopy after negative staining. To detect the presence of spores, slant cultures of the strains were prepared on yeast-peptone medium and one drop of the condensation water at the bottom of the slants was observed by optical microscopy. Gram staining was performed according to Murray et al. (1994). The capacity of the strains to hydrolyse Tween 80 and ONPG as well as the presence of arginine dihydrolase and lysine and ornithine decarboxylases were determined using the traditional techniques outlined by Marchal et al. (1987). All other classical biochemical phenotypic tests were performed with conventional procedures in Petri plates or tubes as described by Smibert & Krieg (1994). The ranges of temperature and NaCl concentration that allowed growth of the strains were examined on solid media. TSA, which contains 5 g NaCl l–1, was used for the temperature measurement, while nutrient agar was used for the salt concentration assay. For the range of pH, a liquid medium (nutrient broth with no NaCl added) was utilized instead of a solid one. The profile of substrates used by the strains was obtained by inoculating API 20NE (12 carbon sources) and Biotype 100 (98 carbon sources) strips according to the manufacturer's instructions (bioMérieux). The strips were incubated at 30 °C and the readings were done after 1–4 days. The API 20NE strips also allowed cross-checking of some of the classical phenotypic tests and presented 11 common substrates with Biotype 100. A good correlation was found between these different tests, since a discrepancy was only observed for gelatin hydrolysis in the case of strain 12-3T.
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). Strain UR374_02 appeared to be the least versatile and the most fastidious. It could use only 9–11 substrates when the Biotype test was performed with Biotype medium 1 and could use 21–22 substrates in the presence of medium Biotype 2, which contains more growth factors (37 instead of 16, including vitamins and amino acids). Despite the poor carbon source utilization by the strains, several sugars, amino acids and organic acids supported growth (Table 1). The test performed on MEVAG medium indicated that glucose was metabolized by oxidation and that none of the strains, including P. broegbernensis DSM 12573T, could ferment it.
Discrepancies were found between some of the characteristics reported in the original description of P. broegbernensis (Lipski et al., 1992; Finkmann et al., 2000) and observations in the present study: hydrolysis of aesculin (positive instead of negative), growth at 37 °C and the use of L-arabinose, D-fructose and L-proline as carbon sources. Except for aesculin hydrolysis, however, our results are in line with those of Chen et al. (2002), who also re-evaluated the phenotype of this strain. Considering that P. broegbernensis DSM 12573T has repeatedly given a positive result in the aesculin hydrolysis assay performed in our different laboratories when using API 20NE and Biotype 100 strips but also in conventional tests in tubes or Petri plates, it seems that the negative result reported by Finkmann et al. (2000) is in fact restricted to the microtitration plate technique (Lipski et al., 1992) that they used to determine this parameter and the criteria that they selected to consider that the response to the test was positive or negative. Beyond the specific scope of this paper, this suggests that, in order to avoid evaluation-dependent phenotypes and so to improve stability in the description of strains, the use of widely available commercial kits or conventional techniques easily reproducible everywhere should be recommended for analysis of physiological properties.
Nitrate and nitrite reduction
The ability of the strains to reduce nitrate and nitrite was first investigated by the standard technique with the Griess reagent (Smibert & Krieg, 1994) and Escherichia coli W3110 (=ATCC 27325), a bacterium able to reduce nitrate to nitrite but not nitrite, as a control. Since no clear and reproducible result could be obtained from the previous experiments between our different laboratories, despite the fact that the control worked well (the same situation was observed with the API 20NE 
test), a new assay was prepared. This time, the strains were cultivated in trypticase soy broth (TSB), a medium of choice to study denitrification (Tiedje, 1988). The medium was pre-reduced by boiling and distributed under a nitrogen atmosphere in hermetically closed Hungate tubes. NaNO3 or NaNO2 was added to the tubes (three per strain and electron acceptor) immediately prior to inoculation (10 % v/v). Five concentrations were tested for 
(1·47, 5, 10, 15, 20 mM) and one for 
(10 mM). One tube per strain was inoculated but not amended with either nitrate or nitrite. These tubes served as negative controls. Another series of tubes prepared with unreduced TSB under an air atmosphere was also inoculated to check the viability of the strains. Growth was followed daily or every 2–3 days by measuring OD580. The capacity of the strains to grow anoxically in presence of 
or 
but not in their absence was considered to indicate their ability to use them as electron acceptors. If no growth occurred after 46 days, the test was considered negative. Tubes showing growth were subcultured at least once in the same conditions in order to confirm the results. The reduction of 
and 
was later validated by measuring their final concentrations in the tubes using Quantofix nitrate/nitrite paper test sticks (Macherey-Nagel). The presence of N2O in the tube gas phase was also investigated qualitatively by GC with a thermal conductivity detector.
The three isolates showed the capacity to grow under anoxic conditions in the presence of nitrite at all the concentrations tested but not in its absence (Supplementary Fig. B; results for only some concentrations are shown) or in the presence of 10 mM nitrate (data not shown). Less than 0·43 mM 
was detected in the tubes initially amended with 5 and 10 mM 
after 46 days of incubation for the primary cultures and 25–30 days of incubation for the subcultures. At higher initial concentrations (15 and 20 mM), the amount of nitrite remaining in the tubes after the same length of time was above the upper concentration detectable (1·74 mM) by the Quantofix test sticks. The presence of N2O in the gas phase of all the tubes containing nitrite definitively established the ability of the strains to reduce it. However, the conditions of the test, performed with a nitrogen atmosphere and a complex medium containing organic nitrogen that may liberate 
upon degradation, did not allow us to determine whether nitrous oxide was the sole and final product of nitrite reduction. The growth efficiency of the three isolates under anoxic conditions with nitrite as the electron acceptor was much lower than under aerobic conditions. Indeed, the increase in OD was limited to 1·2–1·7 times that of the inoculated control without added nitrite, while the maximal OD achieved represented only one-fifth to one-third of that reached in the aerobic viability controls. All the strains also presented a lag phase ranging from 4 to 12 days before starting to grow when transferred from an aerobic to an anoxic environment. This lag phase decreased or even disappeared upon subculturing anoxically with nitrite. P. broegbernensis DSM 12573T, used as a positive control for 
reduction and absence of 
reduction, behaved similarly to the novel isolates. Nevertheless, its growth in the presence of 
appeared to be much more efficient, since it was characterized by shorter lag phases, at least in the case of the primary cultures, and it attained higher OD for the same nitrite concentrations. In the case of P. broegbernensis DSM 12573T, the maximal OD reached by the cultures increased with the concentration of nitrite (Supplementary Fig. B). This indicates that, in contrast to what was originally hypothesized (Finkmann et al., 2000), the reduction of nitrite to N2O by this strain is not only a detoxification process but is also coupled to energy production, since it influences the biomass yield. The positive correlation between OD and nitrite concentration was less evident for strains AMX 26BT, UR374_02 and 12-3T (data not shown). Nevertheless, the fact that they could grow anoxically only in presence of this electron acceptor shows that they can also get energy from its reduction.
Chemotaxonomic analyses
The quinone systems of strains AMX 26BT and 12-3T were extracted with an organic solvent mixture, evaporated, purified by TLC and separated by reverse-phase TLC (Hiraishi et al., 1984). Both strains were characterized by the absence of menaquinones and the presence of a ubiquinone with eight isoprenoid units as sole ubiquinone. The accuracy of this result was checked by extracting and analysing in parallel the quinones of P. broegbernensis DSM 12573T, known to contain exclusively Q-8 (Lipski et al., 1992; Finkmann et al., 2000), as well as those of E. coli W3110 (=ATCC 27325), which contain MK-8 and Q-8.
The CFA composition was determined by Microbial ID using the fully automated GC Sherlock Microbial Identification System (MIDI) and MIDI standard procedures (Sasser, 1990) for strain cultivation (24 h at 28 °C in TSB supplemented with 15 g agar l–1) as well as CFA extraction and analysis. Some 24–29 different CFA were detected in the novel isolates (Table 2). Nevertheless, eight to ten of them appeared only in very small amounts (0·07–0·32 %) and represented altogether less than 1·6 % of the total CFA content. Saturated, hydroxy and unsaturated linear fatty acids were almost absent, while methyl-branched fatty acids corresponded to 66–71 % of the total CFA. In order of decreasing abundance of this class were C15 : 0 iso, C16 : 0 iso, C11 : 0 iso, C16 : 1 iso H and C17 : 0 iso. The branched, unsaturated fatty acid C17 : 1 iso cis7 and the branched hydroxy fatty acid C11 : 0 iso 3-OH were also found in large amounts, particularly the first, which represented 18–20 % of the total fatty acid content. The branched saturated and branched hydroxy fatty acid patterns obtained for the three strains were similar to those of Xanthomonas and Stenotrophomonas species, but they lacked C13 : 0 iso 3-OH. Qualitatively and quantitatively, the CFA profiles of the three strains were almost indistinguishable to the eye. Their comparison by unweighted arithmetic average clustering together with the CFA profiles of closely related species from the genera Pseudoxanthomonas, Stenotrophomonas and Xanthomonas showed that strains AMX 26BT and UR374_02 had the most similar profiles, since they linked at a Euclidean distance of 3·9, and that they linked together at a distance of 6·8 with strain 12-3T (Supplementary Fig. C). This last value nevertheless remained well below the usual cut-off limit of 10 found by MIDI between species. In the same analysis, P. broegbernensis DSM 12573T linked with them at a Euclidean distance of over 25. Qualitatively, P. broegbernensis DSM 12573T can be distinguished from the novel isolates by the fact that it possesses unsaturated (C16 : 1 cis7, C17 : 1 cis9) and hydroxy (C10 : 0 3-OH, C12 : 0 3-OH) fatty acids and that, within the methyl-branched fatty acids, it contains C11 : 0 anteiso but not C16 : 1 iso H or C18 : 1 iso H. Quantitative differences can also be found. For instance, C16 : 0 iso and C17 : 1 iso cis7 are respectively 3–5 and 1·7–1·9 times more abundant in strains AMX 26BT, UR374_02 and 12-3T than in P. broegbernensis DSM 12573T, while the latter strain contains 15–22 times more C16 : 0 and 2·4–3·75 times more C16 : 1 cis9/C15 : 0 iso 2-OH mixture than the others (Table 2). In addition, the ratio of C15 : 0 iso/C15 : 0 anteiso is completely different between P. broegbernensis DSM 12573T and the three strains (1·89 against 8·5–19).
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) on Mueller–Hinton solid medium (bioMérieux) with E. coli CIP 76.24 and Pseudomonas aeruginosa CIP 76.110 as controls. Inhibition diameters were recorded after 18–24 h incubation at 37 °C. MICs were extrapolated from the inhibition diameters using adapted software. The isolates showed a similar response and were found to be highly susceptible (low MIC values) to most of the antibiotics tested except those characterized by a mechanism of action based on the inhibition of protein synthesis and pipemidic acid, a quinolone, acting on the synthesis of nucleic acids (Table 3). Within the antibiotics that affect protein synthesis, the strains seemed to be particularly resistant (very high MIC) to the aminoglycosides but remained sensitive to doxycycline (MIC=0·01 mg l–1). Despite its medical origin, strain UR374_02 appeared to be the most susceptible of the three isolates. For instance, it presented low MICs for gentamicin, fusidic acid and erythromycin, in contrast to the others. Strain AMX 26BT had the lowest MIC for penicillin G. P. broegbernensis DSM 12573T produced a susceptibility pattern completely different from those of the new isolates (Table 3). It was the only one to show low MICs to the aminoglycosides. Nevertheless, it must be noted that, among this antibiotic class, it was reported as resistant to gentamicin and kanamycin in the original published description (Finkmann et al., 2000). In the same study, it was also reported as resistant to penicillin G, while we found it susceptible. Since Finkmann et al. (2000) did not report MIC values or inhibition zone diameters for the antibiotics that they tested, and these parameters cannot be estimated from the given resistant and susceptible categories, their results are not in fact comparable to those obtained here. The antibiotic susceptibility profile presented in Table 3 for P. broegbernensis DSM 12573T therefore corresponds in reality to the first antibiogram defined on an adequate basis for this strain and which can serve as a reference for future comparisons.
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). Moreover, their 16S rRNA gene sequences show a difference of 4·6–4·7 % from that of P. taiwanensis ATCC BAA-4040T, which is greater than the 3 % difference recognized as sufficient to separate bacteria in distinct species (Stackebrandt & Goebel, 1994). The same criteria would indicate that all isolates could be members of P. broegbernensis, since their 16S rRNA gene sequences have only 2·5–2·9 % difference from that of P. broegbernensis DSM 12573T. This conclusion is strengthened by the fact that the type strain of this species shares numerous phenotypic traits with the novel strains (see legend to Table 1), especially that of being mesophilic and motile by means of a single polar flagellum. Nevertheless, beside these similarities, strains AMX 26BT, UR374_02 and 12-3T present more phenotypic differences from P. broegbernensis DSM 12573T at the level of substrate utilization (at least 13 differences, when each strain uses only 17–24 carbon sources of the 99 tested), CFA composition and antibiotic susceptibility. These differences, combined with the fact that the DNA of strains AMX 26BT and 12-3T hybridized with that of P. broegbernensis DSM 12573T at a level significantly below the value of 70 % proposed for species delineation (Wayne et al., 1987; Stackebrandt et al., 2002), support definitively the conclusion that these novel isolates are not members of this species. DNA–DNA hybridization also revealed that the three strains could be divided in two genomic groups. The first was composed of AMX 26BT and UR374_02 and the second of 12-3T. The two genomic groups could not be easily distinguished by their morphology, antibiotic susceptibility or CFA profiles. Notwithstanding this observation, and despite the heterogeneous biochemical profile presented by the members of the first group (Table 1), strain 12-3T could be clearly distinguished from it by other phenotypic properties such as a catalase-negative reaction, the inability to grow in the presence of 40 g NaCl l–1 and the capacity to hydrolyse ONPG/PNPG and to use D-galactose, D-glucosamine, lactulose, D-xylose, fumarate and L-malate as carbon sources but not L-proline (Table 1). Taking into account the phenotypic and phylogenetic characteristics described above, we propose that strains AMX 26BT and UR374_02 represent a novel species within the genus Pseudoxanthomonas, Pseudoxanthomonas mexicana sp. nov., with AMX 26BT as the type strain, and that strain 12-3T represents another novel species of the genus, Pseudoxanthomonas japonensis sp. nov. The genus Pseudoxanthomonas now contains four species. Three of them, P. broegbernensis, P. japonensis and P. mexicana, form a phenotypically and phylogenetically homogeneous group, since they are all mesophilic and motile by means of a single polar flagellum and share 97·1–99·6 % 16S rRNA gene sequence similarity. In this scheme, the thermophilic and non-motile P. taiwanensis appears isolated. The phylogenetic distance (>4·5 % difference in 16S rRNA gene sequence) that separates it from the others indicates that a new genus could be set up to accommodate it. Nevertheless, this splitting appears premature, since only one thermophilic Pseudoxanthomonas species has been described so far. As a consequence, it seems judicious to wait for the description of more thermophilic and mesophilic Pseudoxanthomonas species, in order to determine whether they clearly form two independent homogeneous groups. In any case, the additional information brought by the description of P. japonensis and P. mexicana and also the re-evaluation of the phenotype of P. broegbernensis DSM 12573T in this work indicate the necessity of emending the descriptions of the genus Pseudoxanthomonas and P. broegbernensis. The new data brought by the recent description of P. taiwanensis will be also incorporated in the emended description of the genus.
Ecological aspects
The isolation of strains AMX 26BT, UR374_02 and 12-3T broadens the number of biotopes known to be occupied by Pseudoxanthomonas species and indicates that they are widely distributed over the world, since they have been found in three continents (Europe, Asia and America). The case of P. mexicana also shows that each species may colonize different environments. The presence of this species in an anaerobic digester seems on first inspection to be casual. Indeed, its growth under anoxic conditions is only possible with nitrite as the electron acceptor, a compound unlikely to be found in sufficient concentrations in the cheese factory effluent used to feed the reactor. This suggests that P. mexicana was probably introduced in this biotope as a transient micro-organism through the wastewater. Nevertheless, it is surprising to see that an as-yet uncultured bacterium (clone SH-39) phylogenetically very close to P. mexicana (99·7 % 16S rRNA gene sequence similarity; Fig. 1) was also detected in an anaerobic reactor (Schlötelburg, 2001) and that other respiratory denitrifiers have been found to survive for very long periods of time (even years) in such nitrate-/nitrite-free anaerobic environments (Jørgensen & Tiedje, 1993). The question then remains open as to whether this species can or can not be an autochthonous member of the microflora of this biotope, as was previously observed in the case of another denitrifying member of the family Xanthomonadaceae, Stenotrophomonas acidaminiphila (Assih et al., 2002). The presence of P. mexicana UR374_02 in the urine of a child at a titre compatible with urinary tract infections (105 c.f.u. ml–1) also raises the question of its potential role (and that of other Pseudoxanthomonas species) as an opportunistic pathogen. The fact that it was obtained in mixed culture with environmental or saprophytic bacterial species (Enterococcus sp., Acinetobacter sp., coagulase-negative staphylococci and Corynebacterium sp.) and the absence of infective signs at the moment of isolation suggest that it came from contamination (i.e. saprophytic skin flora) and propagated in the bag used to collect the catheter urine. P. mexicana therefore appears not to have clinical significance. In any case, it seems to be highly susceptible to treatment by antibiotics, as are the other mesophilic Pseudoxanthomonas species. It is also worth noting that, similarly to P. mexicana, uncultured bacteria (clones KL-15-2-16 and KL-59-7-1) phylogenetically close to P. japonensis (99·2–99·3 % 16S rRNA gene sequence similarity; Fig. 1) have been found in a completely different biotope (spacecraft assembly facilities). This suggests that novel strains belonging to the known species should soon be isolated or that novel species of the genus should be described.
Emended description of the genus Pseudoxanthomonas Finkmann et al. 2000
Members of the genus are non-spore-forming rods, usually 0·4–0·8x0·9–1·5 µm, which stain Gram-negative. They have a strictly respiratory type of metabolism with O2 as preferential terminal electron acceptor and can reduce nitrite but not nitrate. N2O is always a main product of nitrite reduction if not the only one. Most species are mesophilic (optimum temperature 30–37 °C), slightly alkalophilic (optimum pH over 7 and preferentially around 8) and motile by means of a single polar flagellum; however, one species is thermophilic (optimum temperature 50 °C) and non-motile. Colonies on solid media are generally yellow to pale yellow or beige. Oxidase-positive and heterotrophic. They use a limited range of carbon sources, including some sugars, organic and amino acids. The cellular fatty acids are of the iso/anteiso type with 15 : 0 iso normally predominating and 13 : 0 iso 3-OH always absent. Quinones are generally of the Q8 type. DNA G+C content is 65–70 mol%. Members of the genus are widely distributed in nature. The type species is Pseudoxanthomonas broegbernensis.
Emended description of Pseudoxanthomonas broegbernensis Finkmann et al. 2000
Exhibits all of the characteristics of the mesophilic members of the genus. Tests for catalase and hydrolysis of ONPG/PNPG, aesculin, DNA and Tween 80 are positive, while those for gelatin, starch and urea hydrolysis, indole production, lysine and ornithine decarboxylase but also arginine dihydrolase are negative. Substrates utilized and susceptibility to antibiotics are detailed in Tables 1 and 3. Can be distinguished from the other mesophilic species by its ability to use L-arabinose, lactose, maltitol, D-melibiose, palatinose, sucrose, D-turanose and citrate. Predominant fatty acids are, in decreasing abundance, 15 : 0 iso, 15 : 0 anteiso, 16 : 1 cis9, 17 : 1 iso cis7 and 16 : 0. The fatty acid 17 : 0 cyclo is absent. Reduces nitrite to nitrous oxide as sole end product. Growth is possible with 0–40 g NaCl l–1, at 10–37 °C and pH 6·5–9·75 and is optimal at 30 °C, pH 8 and without NaCl. The DNA G+C content is 66·5±0·8 mol%.
The type strain, B1616/1T (=ATCC BAA-10T=CCUG 46890T=CIP 107227T=DSM 12573T), was isolated from an experimental biofilter supplied with the waste gas of an animal-rendering plant.
Description of Pseudoxanthomonas mexicana sp. nov.
Pseudoxanthomonas mexicana (me.xi.ca'na. N.L. fem. adj. mexicana pertaining to Mexico, where the type strain was isolated).
Exhibits all of the characteristics of the mesophilic members of the genus. Tests for catalase and hydrolysis of aesculin, gelatin and DNA are positive, while those for ONPG/PNPG and urea hydrolysis, indole production, lysine and ornithine decarboxylase as well as arginine dihydrolase are negative. Tween 80 and starch hydrolysis may give a positive or negative result depending on the strain. The spectrum of substrates utilized is listed in Table 1; it may fluctuate from strain to strain. Members of this species can be distinguished from the other mesophilic species by their inability to use D-galactose, D-glucosamine, lactulose and D-xylose. Susceptible to most classes of antibiotics except aminoglycosides. Predominant fatty acids are, in decreasing abundance, 15 : 0 iso, 17 : 1 iso cis7, 16 : 0 iso, 11 : 0 iso 3-OH and 11 : 0 iso. Growth is possible with 0–40 g NaCl l–1, at 10–37 °C and pH 5·8–9·75 and is optimal at 30–37 °C, pH 7–8 and without NaCl. The DNA G+C content is 67·8±2 mol%.
The type strain, AMX 26BT (=ATCC 700999T=CIP 106674T=JCM 11524T), was isolated from the sludge of an anaerobic reactor treating the wastewater of a cheese factory. Strain UR374_02 (=DSM 15133) was isolated from the urine of a child.
Description of Pseudoxanthomonas japonensis sp. nov.
Pseudoxanthomonas japonensis (ja.po.nen'sis. N.L. fem. adj. japonensis pertaining to Japan, where the type strain was isolated).
Exhibits all of the characteristics of the mesophilic members of the genus. Tests for the hydrolysis of ONPG/PNPG, aesculin, gelatin, DNA and Tween 80 are positive, while those for starch and urea hydrolysis, catalase, indole production, lysine and ornithine decarboxylase but also arginine dihydrolase are negative. The spectrum of substrates used is listed in Table 1. Can be distinguished from the other mesophilic species by its ability to use fumarate and L-malate but not L-proline. Susceptible to most classes of antibiotics except aminoglycosides, fusidic acid and pipemidic acid. Predominant fatty acids are, in decreasing abundance, 15 : 0 iso, 17 : 1 iso cis7, 16 : 0 iso and 11 : 0 iso 3-OH. Growth is possible with 0–30 g NaCl l–1, at 10–37 °C and pH 6·2–9·5 and is optimal at 30–37 °C, pH 8 and without NaCl. The DNA G+C content is 65·2±1 mol%.
The type strain, 12-3T (=CCUG 48231T=CIP 107388T=JCM 11525T), was isolated from polluted urban soil.
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|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Assih, E. A., Ouattara, A. S., Thierry, S., Cayol, J.-L., Labat, M. & Macarie, H. (2002). Stenotrophomonas acidaminiphila sp. nov., a strictly aerobic bacterium isolated from an upflow anaerobic sludge blanket (UASB) reactor. Int J Syst Evol Microbiol 52, 559–568.[Abstract]
Bauer, A. W., Kirby, W. M. M., Sherris, J. C. & Turk, M. (1966). Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol 45, 493–496.[Medline]
Benson, D. A., Boguski, M. S., Lipman, D. J., Ostell, J., Ouellette, B. F., Rapp, B. A. & Wheeler, D. L. (1999). GenBank. Nucleic Acids Res 27, 12–17.
CASFM (2002). Communiqué 2002. Special number SFM, pp. 1–47. Paris: Comité de l'antibiogramme de la Société Française de Microbiologie (in French). http://www.sfm.asso.fr
Chen, M.-Y., Tsay, S.-S., Chen, K.-Y., Shi, Y.-C., Lin, Y.-T. & Lin, G.-H. (2002). Pseudoxanthomonas taiwanensis sp. nov., a novel thermophilic, N2O-producing species isolated from hot springs. Int J Syst Evol Microbiol 52, 2155–2161.[Abstract]
Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791.[CrossRef]
Finkmann, W., Altendorf, K., Stackebrandt, E. & Lipski, A. (2000). Characterization of N2O-producing Xanthomonas-like isolates from biofilters as Stenotrophomonas nitritireducens sp. nov., Luteimonas mephitis gen. nov., sp. nov. and Pseudoxanthomonas broegbernensis gen. nov., sp. nov. Int J Syst Evol Microbiol 50, 273–282.[Abstract]
Hall, T. A. (1999). BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 48, 95–98.
Hiraishi, A., Hoshino, Y. & Kitamura, H. (1984). Isoprenoid quinone composition in the classification of Rhodospirillaceae. J Gen Appl Microbiol 30, 197–210.
Iizuka, T., Yamanaka, S., Nishiyama, T. & Hiraishi, A. (1998). Isolation and phylogenetic analysis of aerobic copiotrophic ultramicrobacteria from urban soil. J Gen Appl Microbiol 44, 75–84.
Jørgensen, K. S. & Tiedje, J. M. (1993). Survival of denitrifiers in nitrate-free, anaerobic environments. Appl Environ Microbiol 59, 3297–3305.
Jukes, T. H. & Cantor, C. R. (1969). Evolution of protein molecules. In Mammalian Protein Metabolism, vol. 3, pp. 21–132. Edited by H. N. Munro. New York: Academic Press.
Kodaka, H., Armfield, A. Y., Lombard, G. L. & Dowell, V. R. (1982). Practical procedure for demonstrating bacterial flagella. J Clin Microbiol 16, 948–952.
Lipski, A., Klatte, S., Bendinger, B. & Altendorf, K. (1992). Differentiation of Gram-negative, non fermentative bacteria isolated from biofilters on the basis of fatty acid composition, quinone system, and physiological reaction profile. Appl Environ Microbiol 58, 2053–2065.
Maidak, B. L., Cole, J. R., Lilburn, T. G. & 7 other authors (2001). The RDP-II (Ribosomal database project). Nucleic Acids Res 29, 173–174.
Marchal, N., Bourdon, J. L. & Richard, C. (1987). Culture Medium for the Isolation and Biochemical Identification of Bacteria, 3rd edn. Paris: Doin Editeurs (in French).
Mayfield, C. I. & Inniss, W. E. (1977). A rapid, simple method for staining bacterial flagella. Can J Microbiol 23, 1311–1313.[Medline]
Murray, R. G. E., Doetsch, R. N. & Robinow, C. F. (1994). Determinative and cytological light microscopy. In Methods for General and Molecular Bacteriology, pp. 21–41. Edited by P. Gerhardt, R. G. E. Murray, W. A. Wood & N. R. Krieg. Washington, DC: American Society for Microbiology.
Relman, D. A., Lepp, P. W., Sadler, K. N. & Schmidt, T. M. (1992). Phylogenetic relationships among the agent of bacillary angiomatosis, Bartonella bacilliformis, and other alpha-proteobacteria. Mol Microbiol 6, 1801–1807.[CrossRef][Medline]
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]
Sasser, M. (1990). Identification of bacteria through fatty acid analysis. In Methods in Phytobacteriology, pp. 119–204. Edited by Z. Klement, K. Rudolph & D. C. Sands. Budapest: Akademiai Kiado.
Schlötelburg, C. (2001). Microbial diversity and dynamic in a 1,2-dichloropropane dechlorinating mixed culture. PhD thesis, Humboldt-University, Berlin, Germany (in German). http://dochost.rz.hu-berlin.de/abstract.php3/dissertationen/schloetelburg
Smibert, R. M. & Krieg, W. R. (1994). Phenotypic characterization. In Methods for General and Molecular Bacteriology, pp. 607–654. Edited by P. Gerhardt, R. G. E. Murray, W. A. Wood & N. R. Krieg. Washington, DC: American Society for Microbiology.
Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44, 846–849.
Stackebrandt, E., Frederiksen, W., Garrity, G. M. & 10 other authors (2002). Report of the ad hoc committee for the re-evaluation of the species definition in bacteriology. Int J Syst Evol Microbiol 52, 1043–1047.[Abstract]
Tiedje, J. M. (1988). Ecology of denitrification and dissimilatory nitrate reduction to ammonium. In Biology of Anaerobic Microorganisms, pp. 179–244. Edited by A. J. M. Zenhder. Chichester: Wiley.
Van de Peer, Y. & De Wachter, R. (1994). TREECON for Windows: a software package for the construction and drawing of evolutionary trees for the Microsoft Windows environment. Comput Appl Biosci 10, 569–570.
Wayne, L. G., Brenner, D. J., Colwell, R. R. & 9 other authors (1987). International Committee on Systematic Bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463–464.
Yang, P., Vauterin, L., Vancanneyt, M., Swings, J. & Kersters, K. (1993). Application of fatty acid methylesters for the taxonomic analysis of the genus Xanthomonas. Syst Appl Microbiol 16, 47–71.
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-D-galactoside; +, positive test or growth occurred; –, negative test or no growth; V, variable growth between duplicates; W, weak response; ND, not done. P. broegbernensis DSM 12573T and P. taiwanensis ATCC BAA-404T shared the following characteristics with the isolates: presence of an aesculin and DNA hydrolase and ability to use as carbon sources D-cellobiose and D-glucose but not D-raffinose, L-rhamnose, glycerol, myo-inositol, D-sorbitol or D-malate. In addition, P. broegbernensis DSM 12573T shared with the isolates the following characteristics not tested for P. taiwanensis ATCC BAA-404T: ability to use as growth substrates N-acetyl-D-glucosamine, gentiobiose and maltotriose and inability to use several alcohols (adonitol, D-arabitol, L-arabitol, dulcitol, i-erythritol, D-mannitol, xylitol), amines (ethanolamine, tryptamine), amino acids (D-alanine, L-alanine, tryptophan), benzenic and heterocyclic aromatic compounds (benzoate, 3-hydroxybenzoate, 4-hydroxybenzoate, m-coumarate, gentisate, phenylacetate, 3-phenylpropionate, protocatechuate, trigonelline), organic acids (cis-aconitate, trans-aconitate, adipate, 5-aminovalerate, caprate, caprylate, D-galacturonate, D-gluconate, 2-ketogluconate, 5-ketogluconate, D-glucuronate, glutarate, DL-glycerate, itaconate, malonate, mucate, quinate, D-saccharate, L-tartrate, D-tartrate, meso-tartrate, tricarballylate) and sugars plus related compounds (L-fucose, hydroxyquinoline 
-galactoside, 3-O-methyl D-glucose, D-ribose, L-sorbose, D-tagatose) tested. Data for P. taiwanensis ATCC BAA-404T were taken from Chen et al. (2002)