Sphingomonas Paucimobilis Research Articles

Inactivation of environmental and reference strains of heterotrophic bacteria and Escherichia coli O157:H7 by free chlorine and monochloramine

Reference cultures from culture collections are often used in disinfection experiments instead of indigenous environmental bacteria, but they may not accurately represent the organisms found in drinking water distribution systems due to physiological differences. This may explain why disinfectant concentrations and contact times, determined under typical laboratory conditions, are not always sufficient to control microbial growth or survival in distribution systems. The objective of this study was to investigate the effect of chlorine or monochloramine disinfection on Escherichia coli O157:H7 and other heterotrophic bacteria obtained from a culture collection and strains isolated from various environments, including disinfected water. It was hypothesized that previous exposure of the environmental strains to sublethal concentrations of disinfectant may allow them to develop greater resistance. Accordingly, it was observed that environmental strains of E. coli O157:H7 were either equally or less susceptible to chlorine and monochloramine than the reference strain. In contrast however, environmental strains of Brevundimonas vesicularis, Pseudomonas fluorescens, and Sphingomonas paucimobilis were either equally or more susceptible to free chlorine or monochloramine than their corresponding reference strains. This was counterintuitive because the environmental strains were able to survive disinfection in the pipes from which they were isolated. It is hypothesized that upon culturing in the laboratory the environmental strains may have become susceptible to low levels of disinfectant. Other researchers have suggested that changes in culture conditions may impact disinfectant sensitivity by affecting cell permeability, cell composition, or growth rate. This emphasizes the importance of designing laboratory studies to mimic environmental conditions as closely as possible to accurately represent environmental inactivation kinetics, including proper handling of organisms (subculturing, media transfers, etc.) before their use in inactivation studies.

Genetic and Biochemical Investigations on Bacterial Catabolic Pathways for Lignin-Derived Aromatic Compounds

Lignins are the most abundant aromatic compounds in nature, and their decomposition is essential to the terrestrial carbon cycle. White rot fungi secreting phenol oxidases are assumed to be involved in the initial degradation of native lignin, whereas bacteria play a main role in the mineralization of lignin-derived low-molecular-weight compounds in soil. There are a number of reports on the degradation pathways for lignin-derived aromatic compounds, but their catabolism has not been enzymatically or genetically characterized. Sphingomonas paucimobilis SYK-6 is one of the best-characterized lignin-degrading bacteria. It can grow on a wide variety of lignin-related biaryls and monoaryls, including beta-aryl ether, biphenyl, diarylpropane, and phenylpropane. These compounds are degraded via the protocatechuate (PCA) 4,5-cleavage pathway or multiple 3-O-methylgallate (3MGA) catabolic pathways. In this review, the enzyme systems for beta-aryl ether and biphenyl degradation, O demethylation linked with one carbon metabolism, the PCA 4,5-cleavage pathway, and the multiple 3MGA catabolic pathways in SYK-6 are outlined.

VIRULENCE LEVELS OF BIOFILM‐GROWN LISTERIA MONOCYTOGENES LO28 ARE LOWER THAN THOSE OF PLANKTONIC CELLS IN AN ORAL INOCULATION TEST ON MICE

ABSTRACT The aim of this study was to produce Listeria monocytogenes biofilms suitable for virulence assays and to determine whether the released bacteria had the same virulence potential as their planktonic counterparts. Biofilms of Listeria monocytogenes LO28 strain, with or without Sphingomonas paucimobilis CCL10 strain, containing up to 7 log10 cfu/cm2 were produced in polypropylene syringes. The virulence of strain LO28 was analyzed in mice after intravenous, subcutaneous and oral inoculation. Its virulence level in binary cultures was not significantly different from that of monocultures. L. monocytogenes LO28 virulence in biofilms was lower than that of their planktonic counterparts after oral inoculation. Our results suggest that biofilms pose no greater health risk to the consumer than planktonic bacteria.

Isolation and characterization of a novel uronic acid-containing acidic glycosphingolipid from the ascidian Halocynthia roretzi

A novel uronic acid-containing glycosphingolipid (UGL-1) was isolated from the ascidian Halocynthia roretzi. UGL-1 was prepared from chloroform-methanol extracts and purified by the use of successive column chromatography on DEAE-Sephadex, Florisil, and Iatrobeads. Chemical structural analysis was performed using methylation analysis, gas chromatography, gas chromatography-mass spectrometry, matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry, and 1H-NMR spectra. The chemical structure of UGL-1 was determined to be a glucuronic acid-containing glycosphingolipid, Galbeta1-4(Fucalpha1-3)GlcAbeta1-1Cer. The ceramide component was composed of C16:0 and C18:0 acids and C16-, C17-, and C18-phytosphingosines as major components.

A novel pathway for the biodegradation of ?-hexachlorocyclohexane by a Xanthomonas sp. strain ICH12

To isolate gamma-hexachlorocyclohexane (HCH)-degrading bacteria from contaminated soil and characterize the metabolites formed and the genes involved in the degradation pathway. A bacterial strain Xanthomonas sp. ICH12, capable of biodegrading gamma- HCH was isolated from HCH-contaminated soil. DNA-colony hybridization method was employed to detect bacterial populations containing specific gene sequences of the gamma-HCH degradation pathway. linA (dehydrodehalogenase), linB (hydrolytic dehalogenase) and linC (dehydrogenase) from a Sphingomonas paucimobilis UT26, reportedly possessing gamma-HCH degradation activity, were used as gene probes against isolated colonies. The isolate was found to grow and utilize gamma-HCH as the sole carbon and energy source. The 16S ribosomal RNA gene sequence of the isolate resulted in its identification as a Xanthomonas species, and we designated it as strain ICH12. During the degradation of gamma-HCH by ICH12, formation of two intermediates, gamma-2,3,4,5,6-pentachlorocyclohexene (gamma-PCCH), and 2,5-dichlorobenzoquinone (2,5-DCBQ), were identified by gas chromatography-mass spectrometric (GC-MS) analysis. While gamma-PCCH was reported previously, 2,5-dichlorohydroquinone was a novel metabolite from HCH degradation. A Xanthomonas sp. for gamma-HCH degradation from a contaminated soil was isolated. gamma-HCH was utilized as sole source of carbon and energy, and the degradation proceeds by successive dechlorination. Two degradation products gamma-PCCH and 2,5-DCBQ were characterized, and the latter metabolite was not known in contrasts with the previous studies. The present work, for the first time, demonstrates the potential of a Xanthomonas species to degrade a recalcitrant and widespread pollutant like gamma-HCH. This study demonstrates the isolation and characterization of a novel HCH-degrading bacterium. Further results provide an insight into the novel degradation pathway which may exist in diverse HCH-degrading bacteria in contaminated soils leading to bioremediation of gamma-HCH.

Improvement in Production and Quality of Gellan Gum by Sphingomonas paucimobilis Under High Dissolved Oxygen Tension Levels

The effect of agitation rate and dissolved oxygen tension (DOT) on growth and gellan production by Sphingomonas paucimobilis was studied. Higher cell growth of 5.4 g l(-1) was obtained at 700 rpm but maximum gellan (15 g l(-1)) was produced at 500 rpm. DOT levels above 20% had no effect on cell growth but gellan yield was increased to 23 g l(-1 )with increase in DOT level to 100%. Higher DOT levels improved the viscosity and molecular weight of the polymer with change in acetate and glycerate content of the polymer.

Microflora en semillas de frijol (Phaseolus vulgaris L.).

The bacterial microflora present and its relationship with Xanthomonas campestris pv. phaseoli (Xcp) were studied in bean seeds, on 118 genotypes coming from VIDAC - 98, INTA - Nicaragua, TARS - USDA and Isabela - P.R. Five isolation methods were used: seed decontaminated with sodium hypochlorite, seed treated with refrigerated nutrient broth for one hour, dispersion of 0.1 ml of suspension of seeds between solid, liquid sowing of 1 ml suspension and seed in nutritious, agitated broth and refrigerated by 24 hours. One hundred and four yellow colonies from 41 genotypes were isolated. Thirty-six colonies were positive KOH (Gram negative), 68 negative (Gram positive) and 34 were starch hydrolyzers. The yellow colonies were nonpathogenic under greenhouse conditions. These were identified with the BIOLOG system as: Pantoea agglomerans (25), Xanthomonas campestris (2), Enterobacter agglomerans (2), Sphingomonas paucimobilis (2), Pseudomonas fluorescens and Flavimonas oryzihabitans. In addition, the genotypes carried colonies with other pigmentations. Antagonistic colonies were identified with deoxyribonuclease activity and antibiosis to Xcp. Of these, 15 colonies inhibited Xcp significantly. The fungi were identified as: Rhizoctonia solani, Penicillium spp., Fusarium spp., Aspergillus flavus, Rhizopus nigricans and Macrophomina phaseolina in 52.9% of the total evaluated genotypes.

Postoperative endophthalmitis caused by Sphingomonas paucimobilis

We present a case in which a new organism, Sphingomonas paucimobilis, caused endophthalmitis after phacoemulsification in a 73-year-old woman. The case shows a recurrent acute endophthalmitis with complete resolution only after vitrectomy. This organism has not been described as a cause of endophthalmitis and was resistant to initial medical management. We also describe an interaction between this organism and a co-infective organism that may account for the unusual clinical course.

Enhanced Degradation of a Mixture of Polycyclic Aromatic Hydrocarbons by a Defined Microbial Consortium in a Two-Phase Partitioning Bioreactor

Biological treatment methods are effective at destroying polycyclic aromatic hydrocarbons (PAHs), and some of the highest rates of PAH degradation have been achieved using two-phase-partitioning bioreactors (TPPBs). TPPBs consist of a cell-containing aqueous phase and a biocompatible and immiscible organic phase that partitions toxic and/or recalcitrant substrates to the cells based on their metabolic demand and on maintaining the thermodynamic equilibrium of the system. In this study, the degradation of a 5-component mixture of high and low molecular weight PAHs by a defined microbial consortium of Sphingomonas aromaticivorans B0695 and Sphingomonas paucimobilis EPA505 in a TPPB was examined. The extremely low aqueous solubilities of the high molecular weight (HMW) PAHs significantly reduce their bioavailability to cells, not only in the environment, but in TPPBs as well. That is, in the two-phase system, the originally selected solvent, dodecane, was found to sequester the HMW PAHs from the cells in the aqueous phase due to the inherent high solubility of the hydrophobic compounds in this solvent. To circumvent this limitation, the initial PAH concentrations in dodecane were increased to sufficient levels in the aqueous phase to support degradation: LMW PAHs (naphthalene, phenanthrene) and fluoranthene were degraded completely in 8 h, while the HMW PAHs, pyrene and benzo[a]pyrene, were degraded by 64% and 11%, at rates of 42.9 mg l(-1) d(-1) and 7.5 mg l(-1) d(-1), respectively. Silicone oil has superior PAH partitioning abilities compared to dodecane for the HMW PAHs, and was used to improve the extent of degradation for the PAH mixture. Although silicone oil increased the bioavailability of the HMW PAHs and greater extents of biodegradation were observed, the rates of degradation were lower than that obtained in the TPPB employing dodecane.

Iprodione degradation by isolated soil microorganisms

Three bacterial strains were isolated from soils adapted to iprodione and identified as Pseudomonas fluorescens, Pseudomonas sp. and Pseudomonas paucimobilis. The first two strains transformed iprodione to N-(3,5-dichlorophenyl)-2,4-dioxoimidazolidine (II) and under restrictive conditions to 3,5-dichlorophenylurea acetic acid (III); the latter subsequently degraded II to III and III to 3,5-dichloroaniline (3,5-D). We constructed bacterial combinations consisting of Pseudomonas paucimobilis plus one of the iprodione degraders and showed that these combinations transformed iprodione into 3,5-D. It is known that 3,5-D was the major metabolite found in adapted soils, suggesting that such a bacterial combination might be responsible for degrading iprodione into 3,5-D in adapted soils. Plasmids could only be isolated in Pseudomonas fluorescens but we did not investigate if one of these was involved in the ability to degrade iprodione.

Distribution of γ-hexachlorocyclohexane-degrading genes on three replicons inSphingobium japonicumUT26

Sphingobium japonicum (formerly Sphingomonas paucimobilis) UT26 utilizes the important insecticide gamma-hexachlorocyclohexane as a sole source of carbon and energy. In previous studies, we isolated and characterized six structural genes (linA to linF) and one regulatory gene (linR) of UT26 for the degradation of gamma-hexachlorocyclohexane to beta-ketoadipate. Our analysis in this study indicated that the UT26 genome consists of three large circular replicons of 3.6 Mb, 670 kb, and 185 kb. The 3.6 Mb and the 670 kb replicons had one and two copies, respectively, of the 16S ribosomal RNA gene, and these replicons were designated as chromosomes (Chr) I and II, respectively. Chr I was indicated to be a main chromosome carrying the dnaA gene. The first three lin genes, linA to linC, for conversion of gamma-hexachlorocyclohexane to 2,5-dichlorohydroquinone, were dispersed on Chr I. The 185 kb plasmid, pCHQ1, carried the linRED operon for the conversion of 2,5-dichlorohydroquinone to maleylacetate and was conjugatively transferred to another sphingomonad strain. The linF gene encoding maleylacetate reductase was located on Chr II. These results indicated that the genes for the complete gamma-hexachlorocyclohexane degradation are dispersed on the three large replicons of UT26.

Gram-negative bacteria from patients seeking medical advice in Stockholm after the tsunami catastrophe

Microbiological cultures from 229 patients seeking medical advice in Stockholm after the tsunami catastrophe of December 2004 were analysed at the Clinical Microbiology Laboratory, Karolinska University Hospital, Stockholm, Sweden. Gram-negative bacilli were the most common findings from wound cultures. Common human pathogens such as Escherichia coli, Proteus species, Klebsiella spp., and Pseudomonas aeruginosa were isolated. More rare species of Gram-negative bacilli, e.g. Myroides odoratus, Sphingomonas paucimobilis and Bergeyella zoohelcum were also isolated. Resistance towards ordinary antibiotics was more extensive compared to our Swedish reference material for Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis and Acinetobacter spp., but not for Pseudomonas aeruginosa, probably reflecting that the resistant isolates were nosocomially acquired in Asia.

Microbial Contamination of Nebulization Solution and Its Measures

We evaluated the microbial contamination of nebulization solutions in medication cups from a total of 76 ultrasonic nebulizers in use in 10 hospitals. In addition, an interview survey was given to nurses to evaluate the disinfection methods of these ultrasonic nebulizers. Of a total of 76 nebulization solution samples, 11 (14.5%) were contaminated with 10-10(2) colony-forming units (CFU)/ml and 9 (11.8%) with 10(3)-10(5) CFU/ml. The major contaminants were glucose non-fermentative bacilli such as Burkholderia cepacia, CDC gr.IV C-2, and Sphingomonas paucimobilis. Comparison of microbial contamination between the frequencies of disinfection showed a significantly lower number of contaminated samples when the cups were disinfected once daily than when disinfected once at intervals of 2-7 d (p=0.00037). In addition, comparison between the presence and absence of preservatives contained in the nebulization solution showed a significantly lower number of contaminated samples in the presence, rather than in the absence, of preservatives (p=0.00001). These results show that disinfection of ultrasonic nebulizers at 24-h intervals is desirable. In particular, when nebulization solutions not containing preservatives are used, disinfection at 24-h intervals is indispensable.

Growth Inhibition by Metabolites of γ-Hexachlorocyclohexane inSphingobium japonicumUT26

The growth of a gamma-hexachlorocyclohexane (gamma-HCH)-degrading bacterium Sphingobium japonicum (formerly Sphingomonas paucimobilis) UT26 in rich medium was inhibited by gamma-HCH. This growth inhibition was not observed in a mutant that lacked the initial or second step enzymatic activity for gamma-HCH degradation, suggesting that metabolites of gamma-HCH are toxic to UT26. Two metabolites of gamma-HCH, 2,5-dichlorophenol (2,5-DCP) and 2,5-dichlorohydroquinone (2,5-DCHQ), showed a direct toxic effect on UT26 and other sphingomonad strains. Because only 2,5-DCP accumulated during gamma-HCH degradation, 2,5-DCP is thought to be a main compound for growth inhibition.

Suspected Ventilator-Associated Pneumonia in Cardiac Patients Admitted to the Coronary Care Unit

To determine the incidence, risk factors, associated pathogens, and outcome of ventilator-associated pneumonia (VAP) in patients admitted to a coronary care unit (CCU). This retrospective cohort study was performed in the CCU of a single tertiary medical center. Patients who were admitted to the CCU between March 23, 2002, and May 25, 2003, and who required invasive mechanical ventilation for more than 48 hours were included. Of the 92 patients who met the study criteria, 17 (18.5%; 95% confidence interval, 11.9%-27.6%) developed VAP. The incidence of VAP was 36.3 (95% confidence interval, 21.1-58.1) per 1000 days of mechanical ventilation. There were no statistically significant differences in demographics, presence of chronic obstructive pulmonary disease, or use of continuous intravenous sedatives or neuromuscular blockers between patients with and without VAP. The most commonly isolated organisms were methicillin-sensitive Staphylococcus aureus, Sphingomonas paucimobilis, and Stenotrophomonas maltophilia. The median length of stay in the CCU for patients with VAP was 10 days compared to 6 days for patients without VAP (P < .01). Eight (47%) of the 17 patients with VAP died compared to 29 (39%) of 75 patients without VAP (P = .52). The incidence of VAP in the CCU is similar to or higher than that reported in other intensive care units. The development of VAP in CCU patients is associated with a prolonged CCU stay but not with an increased hospital mortality.

P14.16 Large Outbreak with Sphingomonas Paucimobilis Due to the Ventilation System at a Traumatologic Intensiv Care Unit in Austria

P14.16 Large Outbreak with Sphingomonas Paucimobilis Due to the Ventilation System at a Traumatologic Intensiv Care Unit in Austria

Efficient production of 2-pyrone 4,6-dicarboxylic acid as a novel polymer-based material from protocatechuate by microbial function

Sphingomonas paucimobilis SYK-6, which can degrade various low molecular weight compounds derived from plant polyphenols such as lignin, lignan, and tannin, metabolizes these substances via 2-pyrone-4,6-dicarboxylic acid (PDC). We focused on this metabolic intermediate as a potential raw material for novel, bio-based polymers. We cloned the ligAB and ligC genes of SYK-6, which respectively encode protocatechuate 4,5-dioxygenase and 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase, into a broad host range plasmid vector, pKT230MC. The resulting plasmid, pDVABC, was introduced into the PpY1100 strain of Pseudomonas putida, and we found that PDC could be stably produced from protocatechuate and accumulated. In addition, we examined the efficiency of production of PDC from protocatechuate on a 5-L scale in a Luria-Bertani medium containing 100 mM glucose and determined that PDC was stably produced from protocatechuate to yield 10 g/L or more.

Enantioselective transformation of alpha-hexachlorocyclohexane by the dehydrochlorinases LinA1 and LinA2 from the soil bacterium Sphingomonas paucimobilis B90A.

Sphingomonas paucimobilis B90A contains two variants, LinA1 and LinA2, of a dehydrochlorinase that catalyzes the first and second steps in the metabolism of hexachlorocyclohexanes (R. Kumari, S. Subudhi, M. Suar, G. Dhingra, V. Raina, C. Dogra, S. Lal, J. R. van der Meer, C. Holliger, and R. Lal, Appl. Environ. Microbiol. 68:6021-6028, 2002). On the amino acid level, LinA1 and LinA2 were 88% identical to each other, and LinA2 was 100% identical to LinA of S. paucimobilis UT26. Incubation of chiral alpha-hexachlorocyclohexane (alpha-HCH) with Escherichia coli BL21 expressing functional LinA1 and LinA2 S-glutathione transferase fusion proteins showed that LinA1 preferentially converted the (+) enantiomer, whereas LinA2 preferred the (-) enantiomer. Concurrent formation and subsequent dissipation of beta-pentachlorocyclohexene enantiomers was also observed in these experiments, indicating that there was enantioselective formation and/or dissipation of these enantiomers. LinA1 preferentially formed (3S,4S,5R,6R)-1,3,4,5,6-pentachlorocyclohexene, and LinA2 preferentially formed (3R,4R,5S,6S)-1,3,4,5,6-pentachlorocyclohexene. Because enantioselectivity was not observed in incubations with whole cells of S. paucimobilis B90A, we concluded that LinA1 and LinA2 are equally active in this organism. The enantioselective transformation of chiral alpha-HCH by LinA1 and LinA2 provides the first evidence of the molecular basis for the changed enantiomer composition of alpha-HCH in many natural environments. Enantioselective degradation may be one of the key processes determining enantiomer composition, especially when strains that contain only one of the linA genes, such as S. paucimobilis UT26, prevail.

Characterization of the novel HCH-degrading strain, Microbacterium sp. ITRC1

A gram-positive Microbacterium sp. strain, ITRC1, that was able to degrade the persistent and toxic hexachlorocyclohexane (HCH) isomers was isolated and characterized. The ITRC1 strain has the capacity to degrade all four major isomers of HCH present in both liquid cultures and aged contaminated soil. DNA fragments corresponding to the two initial genes involved in gamma-HCH degradative pathway, encoding enzymes for gamma-pentachlorocyclohexene hydrolytic dehalogenase (linB) and a 2,5-dichloro-2,5-cyclohexadiene-1,4-diol dehydrogenase (linC), were amplified by PCR and sequenced. Their presence in the ITRC1 genomic DNA was also confirmed by Southern hybridization. Sequencing of the amplified DNA fragment revealed that the two genes present in the ITRC1 strain were homologous to those present in Sphingomonas paucimobilis UT26. Both 16S rRNA sequencing and phylogenetic analysis resulted in the identification of the bacteria as a Microbacterium sp. We assume that these HCH-degrading bacteria evolved independently but possessed genes similar to S. paucimobilis UT26. The reported results indicate that catabolic genes for gamma-HCH degradation are highly conserved in diverse genera of bacteria, including the gram-positive groups, occurring in various environmental conditions.

Isolation and characterization of phenanthrene-degrading Sphingomonas paucimobilis strain ZX4

Phenanthrene-degrading bacterium strain ZX4 was isolated from an oil-contaminated soil, and identified as Sphingomonas paucimobilis based on 16S rDNA sequence, cellular fatty acid composition, mol% G + C and Biolog-GN tests. Besides phenanthrene, strain ZX4 could also utilize naphthalene, fluorene and other aromatic compounds. The growth on salicylic acid and catechol showed that the strain degraded phenanthrene via salicylate pathway, while the assay of catechol 2,3-dioxygenase revealed catechol could be metabolized through meta-cleavage pathway. Three genes, including two of meta-cleavage operon genes and one of GST encoding gene were obtained. The order of genes arrangement was similar to S-type metapathway operons. The phylogenetic trees based on 16S rDNA sequence and meta-pathway gene both revealed that strain ZX4 is clustered with strains from genus Sphingomonas.