Hoeflea prorocentri sp. nov., isolated from a culture of the marine dinoflagellate Prorocentrum mexicanum PM01.

A Gram-stain negative, aerobic, rod-shaped, non-motile, yellow-pigmented and non-spore-forming bacterial strain, designated PM5-8T, was isolated from a culture of a marine toxigenic dinoflagellate Prorocentrum mexicanum PM01. Strain PM5-8T grew at 15-35 °C (optimum, 25-30 °C) and pH 6-11 (optimum, 7.5-8). Cells required at least 1.5% (w/v) NaCl for growth, and can tolerate up to 7.0% with the optimum of 4%. Phylogenetic analysis based on 16S rRNA gene sequence revealed that the strain PM5-8T is closely related to members of the genus Hoeflea, with high sequence similarities with Hoeflea halophila JG120-1T (97.06%) and Hoeflea alexandrii AM1V30T (97.01%). DNA-DNA hybridization values between the isolate and other type strains of recognized species of the genus Hoeflea were between 11.8 and 25.2%, which is far below the value of 70% threshold for species delineation. The DNA G + C content was 50.3 mol%. The predominant cellular fatty acids of the strain were identified as summed feature 8 (C16:1 ω7c and/or C16:1 ω6c; 51.5%), C18:1 ω7c 11-methyl (20.7%), C16:0 (17.2%) and C18:0 (5.7%). The major respiratory quinone was Q-10. Polar lipids profiles contained phosphatidylcholine, phosphatidylglycerol, sulfoquinovosyl diacylglycerol, phosphatidylmono- methylethanolamine, phosphatidylethanolamine and four unidentified lipids. On the basis of the polyphasic taxonomic data presented, strain PM5-8T (= CCTCC AB 2016294T = KCTC 62490T) represents a novel species of the genus Hoeflea, for which the name Hoeflea prorocentri sp. nov. is proposed.

Sepsis neonatal tardía nosocomial en una unidad de terapia intensiva: agentes etiológicos y localización más frecuente / Late onset neonatal sepsis in an intensive care neonatal unit: etiological agents and most frequent location

Resumen Introducción: La sepsis neonatal nosocomial (SNN) es una entidad frecuente en las unidades de cuidados intensivos, donde causa una gran morbimortalidad. La ubicación más frecuente es bacteriemia, seguido de neumonía asociada a ventilador mecánico y vía urinaria. Objetivo: Conocer la etiología y localización más frecuente de la infección en el SNN. Población, Material y Métodos: Estudio retrospectivo, de prevalencias de enero a diciembre de 2015, realizado en la Unidad de Cuidados Intensivos Neonatal de un hospital de alta complejidad. Fueron incluidos todos los neonatos. Resultados: Se incluyeron 70 pacientes, se analizaron 88 episodios de SNN. La localización más frecuente fue sangre 40% de los casos, seguido de orina y aspirado traqueal en 25% respectivamente. Los microorganismos más frecuentemente aislados fueron Staphylococcus de diferentes tipos, seguido de Acinetobacter baumannii multi-resistente. La afectación del SNC fue de 32%. La mortalidad fue de 34%, elevándose a 50% ante un segundo episodio de SNN. La terapia empírica de elección fue vancomicina y carbapenem, ajustándose a antibiograma. Conclusiones: La infección más frecuente fue la bacteremia, principalmente por Staphylococcus resistentes a meticilina. La afectación del SNC fue elevada, lo mismo que la mortalidad.

Introduction: Nosocomial neonatal sepsis (NNS) is a frequent entity in intensive care units, causing great morbidity and mortality. The most frequent site is blood, followed by lungs and urine. Objective: To know the etiology and most frequent localization of infection in the NNS. Population, Material and Methods: Cross sectional study, from January to December 2015, performed in a teaching hospital. All newborns infants were included. Results: 70 patients were included, 88 episodes of NNS were analyzed. The most frequent localization was bacteremia in 40% of cases, followed by urinary tract infection and VAP in 25% respectively. The bacteria most frequently isolated were staphylococci of different types, followed by multiresistant Acinetobacter. The CNS involvement was 32%. Mortality was 34%, rising up to 50% with a second episode of NNS. The empirical therapy of choice was vancomycin and carbapenem, adjusting to antibiogram. Conclusions: The most frequent infection was bacteremia, mainly by staphylococci resistant to methicillin. CNS involvement was elevated, as well as mortality.

Sulfitobacter pontiacus subsp. fungiae subsp. nov., Isolated from Coral Fungia seychellensis from Andaman Sea, and Description of Sulfitobacter pontiacus subsp. pontiacus subsp. nov.

Two closely related aerobic, Gram reaction-negative rod-shaped bacteria (S7-75T and S7-80) were isolated from mucus of coral Fungia seychellensis from Andaman Sea, India. Heterotrophic growth on marine agar was observed at 4-35 °C and pH 6.5-10.5; optimum growth occurred at 25-30 °C and pH 7-8. 16 S rRNA sequence analysis confirmed the strains belonged to the genus Sulfitobacter and the two isolates shared more than 99.28% pairwise sequence similarity. DNA-DNA similarity between two isolates S7-75T and S7-80 was above 96%. Strain S7-75T showed maximum 16S rRNA similarity of 99.64% with Sulfitobacter pontiacus LMG 19752T. However, DNA-DNA relatedness between strain S7-75T and S. pontiacus LMG 19752T confirmed the placement of strain S7-75T as subspecies under the species S. pontiacus. Further, pulsed-field gel electrophoresis (PFGE), REP-PCR, ERIC-PCR fingerprint patterns and lipid profiles also differentiated strain S7-75T from the reference strain of S. pontiacus LMG 19752T. The DNA G+C content was 59.8 mol%. Q10 was the major respiratory quinone. Based on polyphasic analysis, the isolate S7-75T represents a subspecies of S. pontiacus for which the name S. pontiacus subsp. fungiae subsp. nov. is proposed with S7-75T (=JCM 31094T = LMG 29158T) as type strain.

Clinical impact of Gram-negative nonfermenters on adults with community-onset bacteremia in the emergency department.

BACKGROUND: To determine clinical predictors and impact of Gram-negative nonfermenters (GNNFs) infections among adults with community-onset bacteremia in the emergency department (ED). METHODS: Adults with bacteremia visiting the ED from January 2007 to June 2008 were identified retrospectively. Demographic characteristics, underlying illnesses, clinical conditions, bacteremic pathogens, antimicrobial agents, and outcome, were retrieved from chart records. RESULTS: After the exclusion of 261 patients with contamination of blood cultures and 24 patients referred from other hospitals, 518 adults with community-onset bacteremia were eligible; their mean age was 65.1 years, with slight predominance of female (262 patients, 50.6%). Of a total of 565 bacteremic isolates, Escherichia coli (228 isolates, 40.4%) and Klebsiella pneumoniae (100, 17.7%) were the major microorganisms. GNNFs caused bacteremia in 31 (6.0%) patients. A higher proportion of inappropriate antibiotic therapy in the ED (87.1% vs. 26.5%, p < 0.001) and higher 28-day crude mortality rate (19.4% vs. 8.4%, p = 0.05) were observed in bacteremic patients caused by GNNFs than those not caused by GNNFs. In further analysis of Kaplan-Meier survival curve, patients with GNNF bacteremia had a worse outcome than those due to other pathogens (p = 0.04). Multivariate analysis revealed that the independent predictors related to GNNF bacteremia included surgery during previous 4 weeks prior to ED arrival [odds ratio (OR), 10.79; 95% confidence interval (CI), 1.84-63.24; p = 0.01], residents in long-term healthcare facilities (OR, 4.62; 95% CI, 2.08-10.29; p < 0.001), and malignancy (OR, 2.24; 95% CI, 1.10-5.40; p = 0.02). CONCLUSION: For adults with bacteremia visiting the ED, GNNF is associated with a higher mortality rate and more inappropriate empirical antibiotic therapy in the ED. To allow early administration of empirical antibiotics, several clinical predictors of GNNF infections were identified.

Acute prostatitis caused by Raoultella planticola in a renal transplant recipient: a novel case.

We present a unique case of acute bacterial prostatitis caused by a very rare human pathogen, Raoultella planticola, in a renal allograft recipient 3.5 months post transplantation. Only a few cases of human infection by this pathogen have been reported worldwide. The present study reports the case of a 67-year-old man who was admitted to our transplant unit 3.5 months post transplantation with fever, dysuria, suprapubic pain, symptoms and signs of acute prostatitis, and elevated markers of inflammation and prostate-specific antigen. R. planticola was isolated in the urine culture. The patient was treated with ciprofloxacin (based on the antibiogram) and had a full recovery, with satisfactory renal function. To the best of our knowledge, this is not only the first reported case of R. planticola prostatitis, but also the first report of such an infection in a solid organ transplant recipient or in a patient on immunosuppressive medication.

Differentiation of acetic acid bacteria based on sequence analysis of 16S-23S rRNA gene internal transcribed spacer sequences.

The 16S-23S gene internal transcribed spacer sequence of sixty-four strains belonging to different acetic acid bacteria genera were analyzed, and phylogenetic trees were generated for each genera. The topologies of the different trees were in accordance with the 16S rRNA gene trees, although the similarity percentages obtained between the species was shown to be much lower. These values suggest the usefulness of including the 16S-23S gene internal transcribed spacer region as a part of the polyphasic approach required for the further classification of acetic acid bacteria. Furthermore, the region could be a good target for primer and probe design. It has also been validated for use in the identification of unknown samples of this bacterial group from wine vinegar and fruit condiments.

Fourier transform infrared spectroscopy for rapid identification of nonfermenting gram-negative bacteria isolated from sputum samples from cystic fibrosis patients.

The accurate and rapid identification of bacteria isolated from the respiratory tract of patients with cystic fibrosis (CF) is critical in epidemiological studies, during intrahospital outbreaks, for patient treatment, and for determination of therapeutic options. While the most common organisms isolated from sputum samples are Pseudomonas aeruginosa, Staphylococcus aureus, and Haemophilus influenzae, in recent decades an increasing fraction of CF patients has been colonized by other nonfermenting (NF) gram-negative rods, such as Burkholderia cepacia complex (BCC) bacteria, Stenotrophomonas maltophilia, Ralstonia pickettii, Acinetobacter spp., and Achromobacter spp. In the present study, we developed a novel strategy for the rapid identification of NF rods based on Fourier transform infrared spectroscopy (FTIR) in combination with artificial neural networks (ANNs). A total of 15 reference strains and 169 clinical isolates of NF gram-negative bacteria recovered from sputum samples from 150 CF patients were used in this study. The clinical isolates were identified according to the guidelines for clinical microbiology practices for respiratory tract specimens from CF patients; and particularly, BCC bacteria were further identified by recA-based PCR followed by restriction fragment length polymorphism analysis with HaeIII, and their identities were confirmed by recA species-specific PCR. In addition, some strains belonging to genera different from BCC were identified by 16S rRNA gene sequencing. A standardized experimental protocol was established, and an FTIR spectral database containing more than 2,000 infrared spectra was created. The ANN identification system consisted of two hierarchical levels. The top-level network allowed the identification of P. aeruginosa, S. maltophilia, Achromobacter xylosoxidans, Acinetobacter spp., R. pickettii, and BCC bacteria with an identification success rate of 98.1%. The second-level network was developed to differentiate the four most clinically relevant species of BCC, B. cepacia, B. multivorans, B. cenocepacia, and B. stabilis (genomovars I to IV, respectively), with a correct identification rate of 93.8%. Our results demonstrate the high degree of reliability and strong potential of ANN-based FTIR spectrum analysis for the rapid identification of NF rods suitable for use in routine clinical microbiology laboratories.

[Vitamin B12-independent strains of Methylophaga marina isolated from Red Sea algae].

Two strains (KM3 and KM5) of halophilic methylobacteria isolated from Red Sea algae do not require vitamin B12 for growth and can use methanol, methylamine, dimethylamine, trimethylamine, dimethyl sulfide, and fructose as sources of carbon and energy. The cells of these strains are gram-negative motile monotrichous (strain KM3) or peritrichous (strain KM5) rods. The strains are strictly aerobic and require Na+ ions but not growth factors for growth. They are oxidase- and catalase-positive and reduce nitrates to nitrites. Both strains can grow in a temperature range of 4 to 37 degrees C (with optimal growth at 29-34 degrees C), at pH between 5.5 and 8.5 (with optimal growth at pH 7.5-8.0), and in a range of salt concentrations between 0.5 and 15% NaCl (with optimal growth at 5-9% NaCl). The phospholipids of these strains are dominated by phosphatidylethanolamine and phosphatidylglycerol and also include phosphatidylcholine, phosphatidylserine, and cardiolipin. The dominant fatty acids are C(16:1omega7c) and C(16:0). The major ubiquinone is Q8. The cells accumulate ectoin, glutamate, and sucrose as intracellular osmoprotectants. The strains implement the 2-keto-3-deoxy-6-phosphogluconate-dependent variant of the ribulose monophosphate pathway. The G+C content of the DNA is 44.4-44.7 mol %. Analysis of the 16S rRNA genes showed that both strains belong to Gammaproteobacteria and have a high degree of homology (99.4%) to Methylophaga marina ATCC 35842T . Based on the data of polyphasic taxonomy, strains KM3 and KM5 are identified as new strains M. marina KM3 (VKM B-2386) and M. marina KM5 (VKM B-2387). The ability of these strains to produce auxins (indole-3-acetic acid) suggests their metabolic association with marine algae.

Five years of nosocomial Gram-negative bacteremia in a general intensive care unit: epidemiology, antimicrobial susceptibility patterns, and outcomes.

OBJECTIVES: Nosocomial Gram-negative bacteremia in the critically ill is associated with significant morbidity and mortality. This study provides epidemiological and antimicrobial susceptibility data for nosocomial Gram-negative bacteremia in a general intensive care unit (ICU) over a five-year period. METHODS: Positive blood cultures from January 1, 1999 to December 31, 2003 were reviewed for microbial etiology and susceptibilities. Patient charts were reviewed to determine the source of infection and outcome. RESULTS: Forty-five nosocomial Gram-negative bacteremias occurred in 44 patients. Infection rates of 6.9/1000 admissions and 11.3/10,000 patient days remained stable. Admitting diagnoses included respiratory failure, solid organ transplant, post-surgery, and multi-trauma. Seven bacterial species were identified; Pseudomonas aeruginosa and Enterobacter spp were most common. Sources of bacteremia included pneumonia (48.9%), and central venous catheterization (22.2%). Antimicrobial susceptibilities were highest for imipenem, gentamicin, tobramycin, ceftazidime, and piperacillin-tazobactam. Ciprofloxacin susceptibility was inferior to imipenem, gentamicin, and tobramycin (p < 0.05). Mortality rates were 53.3% in the ICU, and 60% for overall hospitalization. Average length of ICU stay was 50.5 days compared to 6.13 days for all-comers. CONCLUSIONS: Nosocomial Gram-negative bacteremia is associated with marked morbidity and mortality in critically ill patients. Significant resistance to ciprofloxacin was demonstrated. Empiric treatment regimens should be based on unit-specific data.

In vitro antimicrobial activity of gatifloxacin compared with other quinolones against clinical isolates from cancer patients.

Owing to the predominance of gram-positive pathogens in neutropenic cancer patients, newer generation quinolones with an expanded gram-positive spectrum and enhanced potency, may have a role to play for prophylaxis and/or empiric therapy in such patients. The in vitro activity of gatifloxacin was compared with that of ciprofloxacin, levofloxacin and trovafloxacin against 848 recent clinical isolates from cancer patients. Against gram-positive organisms, gatifloxacin was the most active agent tested inhibiting all Aerococcus, Listeria monocytogens, Micrococcus, Stomatococcus mucilaginous, Bacillus, and Rhodococcus equi strains at < or =2 mg/l, its designated susceptibility breakpoint. It was also very active against methicillin-susceptible staphylococci and Streptococcus spp. (including penicillin nonsusceptible Streptococcus pneumoniae and viridans streptococci). It had moderate activity against methicillin-resistant staphylococci and Enterococcus faecalis, inhibiting 68-80% of these strains at < or =2 mg/l. Gatifloxacin also had good activity against the Enterobacteriaceae (although ciprofloxacin was more potent) inhibiting >95% of isolates at < or =1 mg/l. Nonfermentative gram-negative organisms were less susceptible to all 4 agents. Gatifloxacin was very active against Acinetobacter lwoffi (MIC100 0.12 mg/l) and had moderate activity against Acinetobacter baumanii, Chryseobacterium spp., Stenotrophomonas maltophilia, Pseudomonas aeruginosa and other Pseudomonas species. Alcaligenes xylosoxidans strains were relatively resistant to all 4 agents.

Bacteriologic characterization of 36 strains of Roseomonas species and proposal of Roseomonas mucosa sp nov and Roseomonas gilardii subsp rosea subsp nov.

We used a polyphasic approach (sequencing analysis of the 16S ribosomal RNA gene and phenotypic analyses) to characterize 36 strains of Roseomonas species isolated from blood. Five strains, represented by strain MDA5176 (M.D. Anderson Cancer Center), were identified as Roseomonas gilardii. One strain belonged to Roseomonas genomospecies 4. The 22 strains represented by strain MDA5527 showed significant differences genotypically and phenotypically with R gilardii and other Roseomonas species and represented a new Roseomonas species; Roseomonas mucosa sp nov was proposed to denote its prominent mucoid, almost runny colonies. Eight strains, represented by strain MDA5605, had minor differences with R gilardii and displayed obvious pink to red colonies; Roseomonas gilardii subsp rosea subsp nov was proposed. For subspecies differentiation, R gilardii was proposed to be R gilardii subsp gilardii subsp nov. Unique patterns of biochemical reactions were established for these Roseomonas species, which may assist routine identification of these organisms. All 36 strains and 2 American Type Culture Collection strains were susceptible to amikacin and ciprofloxacin but resistant to cefepime and ceftazidime. They also were frequently susceptible to imipenem and ticarcillin-clavulanate but far less susceptible to ceftriaxone, trimethoprim-sulfamethoxazole, and ampicillin. R mucosa strains were most resistant, whereas R gilardii subsp gilardii strains were most susceptible.

Polyphasic identification of Bacillus and Brevibacillus strains from clinical, dairy and industrial specimens and proposal of Brevibacillus invocatus sp. nov..

Thirty-three clinical, dairy and industrial isolates of aerobic endospore-forming bacteria which were unreactive in routine identification tests were characterized genotypically by using amplified rDNA restriction analysis (ARDRA), 16S rDNA sequencing and DNA-DNA reassociation, and phenotypically by using fatty acid methyl ester (FAME) analysis, SDS-PAGE of whole-cell proteins, API Biotype 100 assimilation tests and 16 other routine phenotypic tests. Three isolates were identified as strains of Bacillus badius, 12 as Brevibacillus agri, including 3 strains associated with an outbreak of waterborne illness, 4 as Brevibacillus centrosporus and 2 as Brevibacillus parabrevis; 12 strains contaminating an antibiotic production plant were recognized as members of a new species, for which the name Brevibacillus invocatus is proposed, with the type strain LMG 18962T (= B2156T = CIP 106911T = NCIMB 13772T).

Polyphasic evidence for the reclassification of Rhodothermus obamensis Sako et al. 1996 as a member of the species Rhodothermus marinus Alfredsson et al. 1988.

DNA-DNA reassociation studies, 16S rRNA gene sequence comparisons and fatty acid analysis were used to reassess the taxonomic status of the type strain of Rhodothermus obamensis and several strains of the genus Rhodothermus isolated from widely distributed shallow marine hot springs. The results show that the type strain of R. obamensis, JCM 9785T, has a DNA-DNA reassociation value of 78% with the type strain of R. marinus, DSM 4252T. The other strains examined had DNA-DNA reassociation values that varied between about 68 and 94% with R. marinus. The 165 rRNA gene sequence was determined for the type strain of R. obamensis and found to share 99.5% similarity with the type strain of R. marinus. The fatty acid composition of R. obamensis was slightly different from that of the other strains examined, but indicated that this strain is very closely related to the other strains examined in this study. On the basis of DNA-DNA reassociation values, 16S rRNA gene sequence comparison and fatty acid profiles, it was concluded that R. obamensis and R. marinus represent the same species and that the name Rhodothermus obamensis should be regarded as a junior synonym of Rhodothermus marinus.

Taxonomy of Antarctic Flavobacterium species: description of Flavobacterium gillisiae sp. nov., Flavobacterium tegetincola sp. nov., and Flavobacterium xanthum sp. nov., nom. rev. and reclassification of [Flavobacterium] salegens as Salegentibacter salegens gen. nov., comb. nov.

16S rRNA phylogenetic analysis of a number of yellow- and orange-pigmented strains isolated from a variety of Antarctic habitats including sea ice, lakewater and cyanobacterial mats indicated a close relationship to the genus Flavobacterium but distinct from known Flavobacterium species. Phenotypic properties, DNA G+C content and whole-cell fatty acid profiles of the Antarctic strains were consistent with those of the genus Flavobacterium. DNA-DNA hybridization analysis indicated the presence of two distinct and novel genospecies each isolated from a different Antarctic habitat. From polyphasic taxonomic data it is proposed that the two groups represent new species with the following proposed names: Flavobacterium gillisiae (ACAM 601T) and Flavobacterium tegetincola (ACAM 602T). In addition polyphasic analysis of the species '[Cytophaga] xantha' (Inoue and Komagata 1976), isolated from Antarctic mud, indicated it was a distinct member of the genus Flavobacterium and was thus revived as Flavobacterium xanthum. Phylogenetic and fatty acid analyses also indicate that the species [Flavobacterium] salegens (Dobson et al. 1993), from Organic Lake, Antarctica, is misclassified at the genus level. It is proposed that this species belongs to a new genus, Salegentibacter salegens gen. nov., comb. nov.

Pseudoalteromonas peptidolytica sp. nov., a novel marine mussel-thread-degrading bacterium isolated from the Sea of Japan.

A new bacterial species belonging to the genus Pseudoalteromonas is described on the basis of phenotypic characterization, and sequence analysis of its 16S rRNA-coding and gyrase B (gyrB) genes. Ten strains, isolated from sea water of Yamato Island, Sea of Japan, were Gram-negative, yellow, motile, polarly flagellated, aerobic, rod-shaped eubacteria and had a G + C content of 42 mol%. Analysis of the 16S rDNA sequence revealed a clear affiliation between these strains and members of the gamma-Proteobacteria. High similarity values were found with members of the genus Pseudoalteromonas and this was supported by fatty acid profiles. The 16S rDNA sequence similarity between strain F12-50-A1T and Pseudoalteromonas piscicida was very high (99.1%). However, molecular characterizations employing small subunit 16S rDNA sequences were at the limits of resolution for the differentiation of species in this genus. As a result, DNA-DNA hybridization and sequence analyses of a more rapidly evolving gyrB gene were performed. Our assertion that this strain represents a distinct bacterial species within the genus Pseudoalteromonas was supported by both of these molecular analyses. Species-specific PCR probes were designed for the gyrB gene and used for the rapid screening of F12-50-A1T-like strains, thereby confirming the species. As these strains cleave complex protein compounds of the Mytilus edulis foot by secreting proteases, the name Pseudoalteromonas peptidolytica sp. nov. is proposed, with strain F12-50-A1T (= MBICC F1250A1T) as the type strain.

Phylogenetic characterization of a novel radiation-resistant bacterium from irradiated pork: description of Hymenobacter actinosclerus sp. nov.

A phylogenetic analysis was performed on a red-pigmented, radiation-resistant, Gram-negative, rod-shaped organism originating from irradiated pork. Comparative 16S rRNA gene sequencing showed the bacterium was a member of the Cytophaga-Flavobacterium-Bacteroides line of descent and represents a new subline within the genus Hymenobacter. A new species, Hymenobacter actinosclerus, is described for this novel radiation-resistant bacterium. The type strain of Hymenobacter actinosclerus is CCUG 39621T.

Idiomarina gen. nov., comprising novel indigenous deep-sea bacteria from the Pacific Ocean, including descriptions of two species, Idiomarina abyssalis sp. nov. and Idiomarina zobellii sp. nov.

Two bacterial strains, KMM 227T and 231T, were isolated from seawater samples collected from the north-western Pacific Ocean at a depth of 4000-5000 m and were characterized using polyphasic taxonomy. Both were Gram-negative, psychrotolerant, heterotrophic, aerobic and required NaCl for growth (0.6-15.0%). The temperature for growth was 4-30 degrees C. Both strains were rod-shaped, with a single flagellum. However, strain KMM 231T revealed a single long fimbrium. Cellular fatty acids detected in the isolates were predominantly odd-numbered and iso-branched, with 15 and 17 carbons (ca. 70%). Also present were saturated and monounsaturated straight-chain fatty acids. Results of phylogenetic analyses, employing three tree-making methods, strongly indicated that the two strains formed a distinct lineage within a clade containing the genera Alteromonas, Colwellia and Pseudoalteromonas, in the gamma-Proteobacteria. The two strains shared 16S rDNA sequence similarity of 96.9% and genomic DNA relatedness of 27%; the latter was determined by dot-blot hybridization. The strains were differentiated by the presence of fimbria, production of chitinase, ability to grow on 15% NaCl and BIOLOG profiles. Given the polyphasic evidence accumulated in this study, it is proposed that the two deep-sea isolates be classified in the genus Idiomarina gen. nov., as Idiomarina abyssalis sp. nov. (type strain is KMM 227T) and Idiomarina zobellii sp. nov. (type strain is KMM 231T).

Hydrogenophaga intermedia sp. nov., a 4-aminobenzenesulfonate degrading organism.

The taxonomic status of a gram-negative, oxidase positive rod (strain S1) able to degrade 4-aminobenzenesulfonate was studied using a polyphasic approach. Chemotaxonomic investigations of quinones and polar lipids established the allocation of this strain to the beta-subclass of the Proteobacteria and revealed similarities to Hydrogenophaga palleronii. 16S rRNA sequence comparisons demonstrated that this strain clusters phylogenetically with H. palleronii and H. taeniospiralis, but clearly represents a new species. The fatty acid patterns and substrate utilization profile displayed similarity to the characteristics of the four validly published species of Hydrogenophaga, although clear differentiating characters were also observed. No close similarities between the type strains of H. palleronii and H. taeniospiralis were detected in hybridization experiments with the genomic DNAs. On basis of these results, the new species Hydrogenophaga intermedia sp. nov. is proposed, with the type strain S1T (= DSM 5680).

Biodegradation of methyl t-butyl ether by pure bacterial cultures.

Three pure bacterial cultures degrading methyl t-butyl ether (MTBE) were isolated from activated sludge and fruit of the Gingko tree. They have been classified as belonging to the genuses Methylobacterium, Rhodococcus, and Arthrobacter. These cultures degraded 60 ppm MTBE in 1-2 weeks of incubation at 23-25 degrees C. The growth of the isolates on MTBE as sole carbon source is very slow compared with growth on nutrient-rich medium. Uniformly-labeled [14C]MTBE was used to determine 14CO2 evolution. Within 7 days of incubation, about 8% of the initial radioactivity was evolved as 14CO2. These strains also grow on t-butanol, butyl formate, isopropanol, acetone and pyruvate as carbon sources. The presence of these compounds in combination with MTBE decreased the degradation of MTBE. The cultures pregrown on pyruvate resulted in a reduction in 14CO2 evolution from [14C]MTBE. The availability of pure cultures will allow the determination of the pathway intermediates and the rate-limiting steps in the degradation of MTBE.

Involvement of an ATP-dependent carboxylase in a CO2-dependent pathway of acetone metabolism by Xanthobacter strain Py2.

The metabolism of acetone by the aerobic bacterium Xanthobacter strain Py2 was investigated. Cell suspensions of Xanthobacter strain Py2 grown with propylene or glucose as carbon sources were unable to metabolize acetone. The addition of acetone to cultures grown with propylene or glucose resulted in a time-dependent increase in acetone-degrading activity. The degradation of acetone by these cultures was prevented by the addition of rifampin and chloramphenicol, demonstrating that new protein synthesis was required for the induction of acetone-degrading activity. In vivo and in vitro studies of acetone-grown Xanthobacter strain Py2 revealed a CO2-dependent pathway of acetone metabolism for this bacterium. The depletion of CO2 from cultures grown with acetone, but not glucose or n-propanol, prevented bacterial growth. The degradation of acetone by whole-cell suspensions of acetone-grown cells was stimulated by the addition of CO2 and was prevented by the depletion of CO2. The degradation of acetone by acetone-grown cell suspensions supported the fixation of 14CO2 into acid-stable products, while the degradation of glucose or beta-hydroxybutyrate did not. Cultures grown with acetone in a nitrogen-deficient medium supplemented with NaH13CO3 specifically incorporated 13C-label into the C-1 (major labeled position) and C-3 (minor labeled position) carbon atoms of the endogenous storage compound poly-beta-hydroxybutyrate. Cell extracts prepared from acetone-grown cells catalyzed the CO2- and ATP-dependent carboxylation of acetone to form acetoacetate as a stoichiometric product. ADP or AMP were incapable of supporting acetone carboxylation in cell extracts. The sustained carboxylation of acetone in cell extracts required the addition of an ATP-regenerating system consisting of phosphocreatine and creatine kinase, suggesting that the carboxylation of acetone is coupled to ATP hydrolysis. Together, these studies provide the first demonstration of a CO2-dependent pathway of acetone metabolism for a strictly aerobic bacterium and provide direct evidence for the involvement of an ATP-dependent carboxylase in bacterial acetone metabolism.