Development of an LB cloning system and its application in expression of fusion genes in Sphingomonas sp. WG / 生物工程学报

In order to overcome the challenges of insufficient restriction enzyme sites, and construct a fusion-expression vector with flexible fusion direction, we designed an LB cloning system based on the type IIS and type IIT restriction enzymes LguⅠ and BbvCⅠ. The LB cloning system is constructed by inserting the LB fragment (GCTCTTCCTCAGC) into the multiple cloning site region of the broad-host plasmid pBBR1MCS-3 using PCR. The LB fragment contains partially overlapped recognition sites of LguⅠ and BbvCⅠ. Therefore, the same non-palindromic sequence will be generated by these two restriction endonucleases digestion. This feature can be used to quickly and flexibly insert multiple genes into the expression vector in a stepwise and directed way. In order to verify the efficacy of the cloning system, two glycosyltransferase genes welB and welK of Sphingomonas sp. WG were consecutively fused to the LB cloning vector, and the recombinant plasmid was transferred into Sphingomonas sp. WG by triparental mating. The results showed that gene fusion expression has little effect on sphingan titer, but enhanced the viscosity of sphingan. The viscosity of the sphingan produced by recombinant strain Sphingomonas sp. WG/pBBR1MCS-3-LB-welKB was 24.7% higher than that of the wild strain after fermentation for 84 h, which would be beneficial for its application. In conclusion, the application of LB cloning system were verified using Sphingomonas sp. WG. The LB cloning system may provide an efficient tool for fusion expression of target genes.

Enzymatic preparation of manno-oligosaccharides from locust bean gum and palm kernel cake, and investigations into its prebiotic activity

BACKGROUND: Manno-oligosaccharides (MOS) is known as a kind of prebiotics. Mannanase plays a key role for the degradation of mannan to produce MOS. In this study, the mannanases of glycoside hydrolase (GH) families 5 Man5HJ14 and GH26 ManAJB13 were employed to prepare MOS from locust bean gum (LBG) and palm kernel cake (PKC). The prebiotic activity and utilization of MOS were assessed in vitro using the probiotic Lactobacillus plantarum strain. RESULTS: Galactomannan from LBG was converted to MOS ranging in size from mannose up to mannoheptose by Man5HJ14 and ManAJB13. Mannoheptose was got from the hydrolysates produced by Man5HJ14, which mannohexaose was obtained from LBG hydrolyzed by ManAJB13. However, the same components of MOS ranging in size from mannose up to mannotetrose were observed between PKC hydrolyzed by the mannanases mentioned above. MOS stability was not affected by high-temperature and high-pressure condition at their natural pH. Based on in vitro growth study, all MOS from LBG and PKC was effective in promoting the growth of L. plantarum CICC 24202, with the strain preferring to use mannose to mannotriose, rather than above mannotetrose. CONCLUSIONS: The effect of mannanases and mannan difference on MOS composition was studied. All of MOS hydrolysates showed the stability in adversity condition and prebiotic activity of L. plantarum, which would have potential application in the biotechnological applications.

Identification and Characterization of Various Pseudomonas Species from Distinct Clinical Specimens: Antibiogram and Resistance Pattern

P. aeruginosa accounts for a significant proportion of nosocomial infections. This study was conducted to assess the prevalence, levels of antimicrobial susceptibility and resistance mechanisms of Pseudomonas from various clinical samples. P. aeruginosa accounts for a significant proportion of nosocomial infections. This study was conducted to assess the prevalence, levels of antimicrobial susceptibility and resistance mechanisms of Pseudomonas from various clinical samples. Methods: The study was conducted in a tertiary care hospital, over a period of 1 year. After identification of genus Pseudomonas, the speciation was done by biochemical tests and by VITEK 2. Antibiotic susceptibility was determined by disc diffusion method. Extended-spectrum β-lactamases (ESBLs) and metallo-β-lactamases (MBLs) production were detected by the combined disc diffusion test. Results: They were predominantly isolated from pus (44.3%), followed by blood (24.05%), body fluids (13.92%) and sputum (12.03%). The highest number of isolates were pseudomonas aeruginosa (64.56%) followed by p. fluroscence 19.62%, p. putida 7.6%, p. stutzeri 1.9%, p. alcaligens 1.9%, burkhelderia cepacia complex (BCC) (previous designation: pseudomonas cepacia) 1.9% and 2.53% isolate of burkhelderia pseudomallei (previous designation: pseudomona spseudomallei). Conclusion: This study examined the prevalence of pseudomonl infections, and its susceptibility patterns to different antibiotics. The presence of antibiotic-resistant P. aeruginosa isolates could be attributed to β-lactamase production and the use of multiple drug resistance efflux pump. It therefore calls for a very judicious, rational treatment regimens prescription by the physicians to limit the further spread of antimicrobial resistance among the P. aeruginosa strains.

Sphingomonas Paucimobilis: A Rare Infectious Agent Found in Cerebrospinal Fluid

Sphingomonas paucimobilis (S. paucimobilis) is a gram negative bacillus. It has existed in soil, drinking water and plants. It has been isolated from distilled water tanks, respirators, and hemodialysis devices at the hospital setting. Patients with chronic disorders or immune suppression may be susceptible to infections with it. This microorganism has also been reported to infect healthy persons. Both nosocomial and community-acquired infections have been reported. So far, a variety of infections have been reported, including sepsis, septic pulmonary embolism, septic arthritis, peritonitis, and endophthalmitis. Only 2 cases of meningitis have been reported so far in the literature. So far, no previous reports of culture proliferation have been reported in patients with external ventricular drains, as was the case in our patient. Therefore, our case is the first to have S. paucimobilis proliferation in cerebrospinal fluid culture during intensive care unit stay for an external ventricular drain.

Sphingomonas from petroleum-contaminated soils in Shenfu, China and their PAHs degradation abilities

Abstract Members of the Sphingomonas genus are often isolated from petroleum-contaminated soils due to their unique abilities to degrade polycyclic aromatic hydrocarbons (PAHs), which are important for in situ bioremediation. In this study, a combined phenotypic and genotypic approach using streptomycin-containing medium and Sphingomonas -specific PCR was developed to isolate and identify culturable Sphingomonas strains present in petroleum-contaminated soils in the Shenfu wastewater irrigation zone. Of the 15 soil samples examined, 12 soils yielded yellow streptomycin-resistant colonies. The largest number of yellow colony-forming units (CFUs) could reach 105 CFUs g-1 soil. The number of yellow CFUs had a significant positive correlation (p < 0.05) with the ratio of PAHs to total petroleum hydrocarbons (TPH), indicating that Sphingomonas may play a key role in degrading the PAH fraction of the petroleum contaminants at this site. Sixty yellow colonies were selected randomly and analyzed by colony PCR using Sphingomonas -specific primers, out of which 48 isolates had PCR-positive signals. The 48 positive amplicons generated 8 distinct restriction fragment length polymorphism (RFLP) patterns, and 7 out of 8 phylotypes were identified as Sphingomonas by 16S rRNA gene sequencing of the representative strains. Within these 7 Sphingomonas strains, 6 strains were capable of using fluorene as the sole carbon source, while 2 strains were phenanthrene-degrading Sphingomonas. To the best of our knowledge, this is the first report to evaluate the relationship between PAHs contamination levels and culturable Sphingomonas in environmental samples.

Sphingomonas paucimobilis septicemia in a neonate: A rare case report.

Sphingomonas paucimobilis, a yellow‑pigmented, aerobic, glucose nonfermenting, Gram‑negative bacilli is a rare cause of human infection. It was first discovered as an infective agent in humans in 1977 and named Pseudomonas paucimobilis. It was renamed as S. paucimobilis in 1990 in accordance with phylogenetic data. S. paucimobilis is an aerobic bacterium found in soil and water; it is a rare cause of healthcare associated infections. S. paucimobilis can cause infections in healthy as well as immunocompromised individuals. At first, its colony looks like Gram‑positive bacilli colony, so by mistake it is discarded as contaminants. S. paucimobilis is an emerging pathogen and it should not be discarded as contaminants. Here, we report a case of S. paucimobilis bacteremia in a neonate who presented with respiratory distress.

Sphingomonas paucimobilis bacteraemia and shock in a patient with rheumatic carditis.

Acute rheumatic fever (ARF) carditis is treated with steroids, which can cause changes in the cellular immune response, especially decreased CD3 (+) T cells. Nosocomial infections due to steroid use for treatment of ARF carditis or secondary to the changes in the cellular immune response have not been reported in the literature. Sphingomonas paucimobilis is a Gram‑negative bacillus causing community‑ and hospital‑acquired infections. It has been reported as causing bacteraemia/sepsis, pneumonia or peritonitis in patients with malignancies, immunosuppression or diabetes. We present a case with S. paucimobilis bacteraemia/sepsis and shock after administration of steroids for treatment of ARF carditis. We suggest early identification of the causative agent and appropriate adjustments of the treatment plan to avoid shock and possible mortality. This is the first reported case of S. paucimobilis bacteraemia/sepsis in the setting of steroid use for ARF carditis.

Endoftalmitis poscirugía de catarata por Sphingomonas paucimobilis / Sphingomonas paucimobilis-induced endophthalmitis after cataract surgery

Se presenta la caracterización y manejo terapéutico de un caso de endoftalmitis bacteriana posoperatoria causada por el germen Sphingomonas paucimobilis. La endoftalmitis es la inflamación de los tejidos intraoculares, considerada como la más devastadora de las complicaciones posoperatorias; posee pronóstico visual muy reservado y un elevado riesgo de secuela. Las Sphingomonas paucimobilis son bacterias gramnegativas con forma de bacilo, quimioheterótrofa y estrictamente aerobias que causan enfermedades en los seres humanos, principalmente infecciones hospitalarias que típicamente son tratadas fácilmente con antibióticos. Por sus capacidades biodegradantes y biosintéticas, son pocos los reportes hallados de infección intraocular por este germen. El pronóstico visual es favorable con un diagnóstico precoz y la aplicación del tratamiento adecuado. En este artículo se presentan un caso de endoftalmitis poscirugía de catarata por Sphingomonas paucimobilis reportado en Cuba en el mes de septiembre de 2009.

The characterization and therapeutic management of a case of postsurgery bacterial endophthalmitis caused by Shingomonas paucimobilis was presented. Endophthalmitis is the inflammation of the intraocular tissues and considered as the most devastating postoperative complication. The visual prognosis of this disease is very reserved judgement and high risk of sequelae. Sphingomonas paucimobilis are bacillus-shaped, chemoheterotrophic and strictly aerobic Gramnegative bacteria that cause diseases in the human being, mainly nosocomial infections that are typically treated with antibiotics. Based on its biodegrading and biosynthetic capacities, there are few reports on intraocular infections caused by this germ. The visual prognosis is favourable when the disease is early diagnosed and adequately managed. A case of endophthalmitis following a cataract surgery and caused by Shingomonas paucimobilis in Cuba in September 2009 was presented in this article.

Microbial community in a full-scale drinking water biosand filter.

To remove turbidity and minimize microbiological risks, rapid sand filtration is one of main drinking water treatment processes in the world. However, after a long-term operation, sand particles will be colonized by microorganisms which can remove biodegradable organic matters and nitrogen compounds. In this study, 16S rRNA gene clone library analysis was applied to characterize the microbial community in a full-scale biosand filter used for drinking water treatment. The results indicate that phylum Nitrospirae and class Alphaproteobacteria were the dominant bacterial groups in the biosand sample collected from the upper filter layer. The dominance of Sphingomonas species might pose a microbiological risk. This work could provide some new insights into microbial community in drinking water biofilter.

A case of peritoneal dialysis-associated peritonitis caused by Sphingomonas paucimobilis

Sphingomonas paucimobilis is an aerobic Gram-negative bacillus found in soil and water. Knowledge regarding the role of this infectious agent is limited because it is rarely isolated from human material. Furthermore, it is an unusual pathogen in cases of peritoneal dialysis (PD)-associated peritonitis. The clinical courses and outcomes of peritonitis caused by S. paucimobilis are variable. Whereas some patients were cured with appropriate antibiotic therapy, others required catheter removal. Cases of PD-associated peritonitis caused by S. paucimobilis have been reported worldwide, and there was a case report of coinfection with S. paucimobilis and Chryseobacterium indologenes in Korea. However, there has been no case caused by S. paucimobilis as a single pathogen. We report a case of PD-associated peritonitis due to S. paucimobilis in which the patient recovered after catheter removal.

Outbreak of Sphingomonas paucimobilis Septicemia in a Neonatal Intensive Care Unit.

We describe an outbreak of Sphingomonas paucimobilis in 13 newborn infants with septicemia and septic shock. Fifteen available isolates were obtained from patients and distilled water. Pulsed-field gel electrophoresis showed that there was a cross-transmission of S. paucimobilis in eleven patients but these types were not the same the isolate obtained from the distilled water. The outbreak was terminated by taking appropriate control measures for infection and change of source of distilled water.

Efecto de la inoculación de la cepa Sphingomonas paucimobilis 20006FA sobre la composición de un consorcio bacteriano degradador de fenantreno / Effect of the inoculant strain Sphingomonas paucimobilis 20006FA on the bacterial composition of a phenanthrene-degrading consortium

Se estudió el efecto de la inoculación con la cepa Sphingomonas paucimobilis 20006FA sobre la composición bacteriana de un consorcio degradador de fenantreno en cultivos discontinuos (batch) con 8 repiques sucesivos. El consorcio original se obtuvo a partir de un suelo prístino. A los fines del estudio, se obtuvieron y mantuvieron dos consorcios: uno inoculado (F200+I) y otro sin inocular (F200). Se estudió la diversidad bacteriana de los consorcios mediante el análisis de microorganismos cultivables (por caracterización fenotípica y genotípica) y totales (por PCR-DGGE). A lo largo de los repiques sucesivos pudo observarse en ambos consorcios una tendencia a la pérdida de la capacidad degradadora de fenantreno, acompañada por una disminución de la diversidad bacteriana. Si bien la inoculación no produjo cambios significativos en la capacidad degradadora de fenantreno de los consorcios (29,9% para F200 y 27,6% para F200+I hacia el tercer repique), sí produjo cambios en la composición bacteriana, ya que los perfiles de DGGE revelaron una dinámica estructural diferente en el consorcio inoculado. En ambos consorcios se pudo observar la presencia de una banda intensa posicionada a la misma altura que el ADN del inóculo en el gel de DGGE; sin embargo, los cultivos aislados de los consorcios que presentaban idéntica posición de banda en el perfil PCR-DGGE que la cepa S. paucimobilis 20006FA mostraron baja similitud con la cepa inoculada mediante la técnica de RAPD.

The effect of the inoculant strain Sphingomonas paucimobilis 20006FA on the bacterial composition of a phenanthrene-degrading consortium obtained from a pristine soil in sequencing batch cultures was studied. Inoculated (F200+I) and non-inoculated (F200) phenanthrene-degrading consortia, were obtained. Bacterial diversity of consortia was studied at cultivable (phenotype and genotype characterization) and non-cultivable (PCR-DGGE) levels. During the successive cultures, a loss in the phenanthrene-degrading capacity and a decrease in the bacterial diversity were observed in both consortia. Although inoculation did not produce any significant changes in the consortia phenanthrene-degrading capacity (29.9% F200 and 27.6% F200+I), it did produce changes in the bacterial composition, showing a differential structural dynamics in the DGGE profiles of the inoculated consortium. In both consortia, a dominant band placed at the same position as that of the DNA of the inoculant strain in the DGGE gel could be observed. However, isolated cultures from the consortia which had an identical band position to that of S. paucimobilis 20006FA in the PCR-DGGE profile showed low similarity with respect to the inoculant strain (RAPD).

A Case of Postoperative Sphingomonas paucimobilis Endophthalmitis After Cataract Extraction

PURPOSE: To report a case of an acute onset of delayed postoperative endophthalmitis that was caused by Sphingomonas paucimobilis. METHODS: This case demonstrates an acute onset of delayed postoperative endophthalmitis at 3 months after uneventful cataract extraction and posterior chamber intraocular lens implantation. We performed vitrectomy, intraocular lens and capsular bag removal, and intravitreal antibiotics injection. On the smear stains from the aspirated vitreous humor, gram-negative bacilli were detected and S. paucimobilis was found in culture. RESULTS: At three months after vitrectomy, the best corrected visual acuity was 20/300. Fundus examination showed mild pale color of optic disc and macular degeneration. CONCLUSIONS: Vitrectomy with intravitreal ceftazidime injection had contributed to the favorable result in case of an acute onset of delayed postoperatire endophthalmitis caused by S. paucimobilis.

A Case of Postoperative Sphingomonas paucimobilis Endophthalmitis After Cataract Extraction

PURPOSE: To report a case of an acute onset of delayed postoperative endophthalmitis that was caused by Sphingomonas paucimobilis. METHODS: This case demonstrates an acute onset of delayed postoperative endophthalmitis at 3 months after uneventful cataract extraction and posterior chamber intraocular lens implantation. We performed vitrectomy, intraocular lens and capsular bag removal, and intravitreal antibiotics injection. On the smear stains from the aspirated vitreous humor, gram-negative bacilli were detected and S. paucimobilis was found in culture. RESULTS: At three months after vitrectomy, the best corrected visual acuity was 20/300. Fundus examination showed mild pale color of optic disc and macular degeneration. CONCLUSIONS: Vitrectomy with intravitreal ceftazidime injection had contributed to the favorable result in case of an acute onset of delayed postoperatire endophthalmitis caused by S. paucimobilis.

Pyogenic Sacroiliitis with Psoas Abscess: A case report

Pyogenic sacroiliitis is a rare infection that may accompany psoas muscle abscess with vague clinical presentation, which present a diagnostic challenge requiring a high index of suspicion. We report a 48-year-old male, diagnosed pyogenic sacroiliitis with psoas abscess caused by Streptococcus intermedius and Sphingomonas paucimobilis. Magnetic resonance imaging showed multiloculated rim-enhancing lesion in right psoas muscle and joint effusion with osteomyelitis around sacroiliac joint. Intravenous antibiotics were administered after ultrasonographically guided abscess aspiration. Surgical drainage was done and his fever and symptom gradually subsided.

Rizoremediação de pentaclorofenol em um solo argiloso por sphingomonas chlorophenolica ATCC 39723 / Pentachlorophenol rhizoremediation in a loamy soil by sphingomonas chlorophenolica ATCC 39723

O principal objetivo deste trabalho foi estudar a degradação de PCP por Sphingomonas chlorophenolicaem solo argiloso na presença e ausência de trigo. As concentrações de PCP foram determinadas através de Análises de Alta Performance de Cromatografia Líquida. Os efeitos tóxicos de PCP foram estudados através do monitoramento do crescimento das plantas. A biodegradação de PCP por S. chlorophenolica foi acompanhada por testes de bioluminescência de Escherichia coli HB101 pUCD607 e contagens bacterianas no solo e nas raízes. A degradação de PCP ocorreu de forma mais rápida no solo plantado e inoculado quando comparada ao solo sem plantas. Houve um aumento significativo nas populações dos organismos testados nas raízes quando comparadas com as populações presentes no solo. O monitoramento do crescimento da planta mostrou o papel protetor exercido pela S.chlorophenolica contra a toxicidade do PCP.

The main objective of this study was study the PCP degradation by Sphingomonas chlorophenolica in a loamy soil in the presence and absence of plants (Winter wheat). Measurements of PCP concentrations were carried out in a laboratory basis using High performance liquid chromatography analysis (HPLC). The toxic effect of PCP on plants was studied through the monitoring of the plant growth. The biodegradation of PCP by S. chlorophenolica in soil was assessed with a bioluminescence assay of Escherichia coli HB101 pUCD607 and bacterial analyses in roots and soil. The planted and inoculated soil showed a faster degradation when compared to the inoculated soil without plants. There was a significative increase in the populations of the organisms tested in the roots when compared to the soil. The monitoring of the plant growth showed a protective role of S. chlorophenolica against the toxicity of PCP in the loamy soil.

Peritonitis by Chryseobacterium indologenes and Sphingomonas paucimobilis in a Patient Undergoing Continuous Ambulatory Peritoneal Dialysis (CAPD) / 대한신장학회지

Bacterial peritonitis is a well-recognized complication of continuous ambulatory peritoneal dialysis (CAPD) in patients with end-stage renal failure. Chryseobacterium indologenes is a non-fermentative Gram-negative bacillus that is a rare pathogen in humans and Sphinomomas paucimobilis has rarely been reported as an opportunistic human pathogen. We present a case of peritonitis due to unusual pathogens, C. indologenes and S. paucimobilis, unresponsive to the standard antibiotics therapy. A 51-year-old diabetic man undergoing CAPD for 45 days developed the first peritonitis due to C. indolegenes. Although he had received intraperitoneal antibiotics with good in vitro activity against organism, the signs of peritonitis persisted. S. paucimobilis was isolated from dialysate sample. The Tenckhoff catheter was finally removed on 19th day of hospitalization and the fever and abdominal pain subsided.