Broad-range PCR,cloning and sequencing of the full 16S rRNA gene for detection of bacterial DNA in synovial fluid samples of Tunisian patients with reactive and undifferentiated arthritis

Introduction

Broad-range rDNA PCR provides an alternative, cultivation-independent approach for identifying bacterial DNA in reactive and other form of arthritis. The aim of this study was to use broad-range rDNA PCR targeting the 16S rRNA gene in patients with reactive and other forms of arthritis and to screen for the presence of DNA from any given bacterial species in synovial fluid (SF) samples.

Methods

We examined the SF samples from a total of 27 patients consisting of patients with reactive arthritis (ReA) (n = 5), undifferentiated arthritis (UA) (n = 9), rheumatoid arthritis (n = 7), and osteoarthritis (n = 6) of which the latter two were used as controls. Using broad-range bacterial PCR amplifying a 1400 bp fragment from the 16S rRNA gene, we identified and sequenced at least 24 clones from each SF sample. To identify the corresponding bacteria, DNA sequences were compared to the EMBL (European Molecular Biology Laboratory) database.

Results

Bacterial DNA was identified in 20 of the 27 SF samples (74, 10%). Analysis of a large number of sequences revealed the presence of DNA from more than one single bacterial species in the SF of all patients studied. The nearly complete sequences of the 1400 bp were obtained for most of the detected species. DNA of bacterial species including Shigella species, Escherichia species, and other coli-form bacteria as well as opportunistic pathogens such as Stenotrophomonas maltophilia and Achromobacter xylosoxidans were shared in all arthritis patients. Among pathogens described to trigger ReA, DNA from Shigella sonnei was found in ReA and UA patients. We also detected DNA from rarely occurring human pathogens such as Aranicola species and Pantoea ananatis. We also found DNA from bacteria so far not described in human infections such as Bacillus niacini, Paenibacillus humicus, Diaphorobacter species and uncultured bacterium genera incertae sedis OP10.

Conclusions

Broad-range PCR followed by cloning and sequencing the entire 16S rDNA, allowed the identification of the bacterial DNA environment in the SF samples of arthritic patients. We found a wide spectrum of bacteria including those known to be involved in ReA and others not previously associated with arthritis.     

Detection and frequency of Chlamydia trachomatis DNA in synovial samples from Tunisian patients with reactive arthritis and undifferentiated oligoarthritis

We aimed to determine the frequency of Chlamydia trachomatis DNA in the synovial compartment of 34 arthritic patients. Chlamydia trachomatis DNA was detected using a nested PCR targeting the cryptic plasmid, the 16S rRNA gene and the outer membrane protein 1 gene. The presence of serum immunoglobulin (Ig)G and IgA antibodies against C. trachomatis was studied by a microimmunofluorescence assay and by an enzyme-linked immunosorbent assay, respectively. Synovial samples from 20 of 34 (59%) patients [nine with reactive arthritis (ReA), seven with undifferentiated oligoarthritis (UOA), two with rheumatoid arthritis and two with osteoarthritis] were positive for at least one C. trachomatis DNA sequence by nested PCR. The high sensitivity results most likely from the combination of a standardized automated MagNA Pure extraction method, PCR targeting three different C. trachomatis genes and the screening for C. trachomatis in synovial tissue and fluid samples. There was no correlation between the presence of C. trachomatis DNA in the joint and a Chlamydia -specific serologic response. Our data support that PCR is the method of choice to establish the diagnosis of Chlamydia -induced arthritis in patients with ReA. We suggest that this diagnosis might also be considered in C. trachomatis -positive patients previously classified as UOA.     

Optimization of enzymatic hydrolysis and fermentation conditions for improved bioethanol production from potato peel residues

The aim of this work was the optimization of the enzyme hydrolysis of potato peel residues (PPR) for bioethanol production. The process included a pretreatment step followed by an enzyme hydrolysis using crude enzyme system composed of cellulase, amylase and hemicellulase, produced by a mixed culture of Aspergillus niger and Trichoderma reesei. Hydrothermal, alkali and acid pretreatments were considered with regards to the enhancement of enzyme hydrolysis of potato peel residues. The obtained results showed that hydrothermal pretreatment lead to a higher enzyme hydrolysis yield compared to both acid and alkali pretreatments. Enzyme hydrolysis was also optimized for parameters such as temperature, pH, substrate loading and surfactant loading using a response surface methodology. Under optimized conditions, 77 g L^?1 of reducing sugars were obtained. Yeast fermentation of the released reducing sugars led to an ethanol titer of 30 g L^?1 after supplementation of the culture medium with ammonium sulfate. Moreover, a comparative study between acid and enzyme hydrolysis of potato peel residues was investigated. Results showed that enzyme hydrolysis offers higher yield of bioethanol production than acid hydrolysis. These results highlight the potential of second generation bioethanol production from potato peel residues treated with onsite produced hydrolytic enzymes. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:397–406, 2017     

Analysis of bacterial DNA in synovial tissue of Tunisian patients with reactive and undifferentiated arthritis by broad-range PCR, cloning and sequencing

Introduction

Bacteria and/or their antigens have been implicated in the pathogenesis of reactive arthritis (ReA). Several studies have reported the presence of bacterial antigens and nucleic acids of bacteria other than those specified by diagnostic criteria for ReA in joint specimens from patients with ReA and various arthritides. The present study was conducted to detect any bacterial DNA and identify bacterial species that are present in the synovial tissue of Tunisian patients with reactive arthritis and undifferentiated arthritis (UA) using PCR, cloning and sequencing.

Methods

We examined synovial tissue samples from 28 patients: six patients with ReA and nine with UA, and a control group consisting of seven patients with rheumatoid arthritis and six with osteoarthritis (OA). Using broad-range bacterial PCR producing a 1,400-base-pair fragment from the 16S rRNA gene, at least 24 clones were sequenced for each synovial tissue sample. To identify the corresponding bacteria, DNA sequences were compared with sequences from the EMBL (European Molecular Biology Laboratory) database.

Results

Bacterial DNA was detected in 75% of the 28 synovial tissue samples. DNA from 68 various bacterial species were found in ReA and UA samples, whereas DNA from 12 bacteria were detected in control group samples. Most of the bacterial DNAs detected were from skin or intestinal bacteria. DNA from bacteria known to trigger ReA, such as Shigella flexneri and Shigella sonnei, were detected in ReA and UA samples of synovial tissue and not in control samples. DNA from various bacterial species detected in this study have not previously been found in synovial samples.

Conclusion

This study is the first to use broad-range PCR targeting the full 16S rRNA gene for detection of bacterial DNA in synovial tissue. We detected DNA from a wide spectrum of bacterial species, including those known to be involved in ReA and others not previously associated with ReA or related arthritis. The pathogenic significance of some of these intrasynovial bacterial DNAs remains unclear.     

The Attractive Serratia plymuthica BMA1 Strain With High Rock Phosphate-Solubilizing Activity and Its Effect on the Growth and Phosphorus Uptake by Vicia faba L. Plants

Abstract

The present work aims to investigate the attractive ability of the newly isolated bacterium Serratia plymuthica BMA1, to release phosphorus (P) from rock phosphate (RP) and also to assess its beneficial effect in promoting the growth of Vicia faba. This strain exhibited the highest RP-solubilization activity ever reported, with 450?mg l^?1 of soluble P at 30?°C. At 10 and 20?°C, its RP-solubilization was estimated at 100 and 200?mg l^?1, respectively. HPLC analysis revealed that RP-solubilizing activity was associated with the release of gluconic acid. The hydroxyapatite (HA) solubilization activity concomitantly occurred with the secretion of gluconic acid and lactic acid. Under greenhouse conditions, application of BMA1 strain as an inoculant in presence of RP as the sole P source, considerably increased phosphorus uptake by V. faba L. and upgraded its overall growth in terms of dry weight and length by 76% and 39%, respectively. This growth promoting effect was accompanied by a substantial increase in chlorophyll contents. Additionally, phosphorus levels within roots and shoots of S. plymuthica BMA1-treated plants were approximately three times greater, compared to the non-inoculated control plants. When HA was used instead of RP, bacterization with BMA1 strain also enhanced the plant growth parameters and P contents, but significantly less than RP. These findings revealed that S. plymuthica BMA1 could be a potential candidate to improve the agronomic effectiveness of RP, toward a clean P-nutrition through the formulation of bio-phosphate fertilizers for plant growth promotion.