Cloning, Expression, and Purification of a GDSL-like Lipase/Acylhydrolase from a Native Lipase-Producing Bacterium, Lactobacillus fermentum

Background: Lipase enzymes are of great importance in various industries. Currently, extensive efforts have been focused on exploring new lipase producer microorganism as well as genetic and protein engineering of available lipases to improve their functional features. Methods: For screening lipase-producing lactobacilli, isolated strains were inoculated onto tributyrin agar plates. Molecular identification of lipase-producing Lactobacilli was performed by sequencing the 16Sr DNA gene, and a phylogenetic tree was constructed. The LAF_RS05195 gene, encoding lipase protein in L. fermentum isolates, was identified using specific primers, amplified by PCR (918 bp) and cloned into the pET28a (+) vector. The recombinant proteins were expressed 2, 4, 6, and 12 hours after induction with IPTG and assessed using the SDS polyacrylamide gel electrophoresis (SDS-PAGE). Enzymatic activity of the purified recombinant protein was measured at 410 nm in the presence of ρ-NPA and ρ-NPP. Results: Among five identified native lipase-producing isolates, one isolate showed 98% similarity with Enterococcus species. The other four isolates indicated 98% similarity to L. fermentum. After purification steps with Ni-NTA column, a single protein band of about 34 kDa was detected on SDS-PAGE gel. The enzymatic activity of purified recombinant protein alongside ρ-NPA and ρ-NPP was measured to be 0.6 U/ml and 0.2 U/ml, respectively. Conclusion: In the present research, a novel lipase/esterase from L. fermentum was cloned and expressed. The novel lipase/esterase has the merit to be further studied due to its substrate specificity.

The Inhibitory Effects of Lactobacillus Supernatants and Their Metabolites on the Growth and Biofilm Formation of Klebsiella pneumoniae.

BACKGROUND: Klebsiella pneumoniae is a common cause of hospital-acquired infections, including urinary tract infection (UTI). Biofilm formation makes the K. pneumoniae infection more complicated and carrying extended-spectrum beta-lactamases (ESBLs) genes, it limits antibiotic choices for treatment. Lactobacillus strains are known as natural protective barriers against UTIs. OBJECTIVES: This is a small in-vitro study aimed to determine the effect of probiotic Lactobacillus strains and some types of their metabolites on the growth and biofilm of UTI isolates and reference strain of Klebsiella pneumoniae. METHODS: The efficacy of Lactobacillus supernatants and antibiotics in the prevention and elimination of K. pneumoniae biofilms was determined using a quantitative adherence assay. A rapid colorimetric microplate bioassay was applied for the detection of survived bacterial cells after treatment with antibacterial agents. Biofilm phenotypes were studied by scanning electron microscopy (SEM). RESULTS: The results showed that seven out of eight ESBL producing uropathogenic K. pneumoniae isolates in this study were able to produce biofilm. Lactobacillus supernatants at 1:1 to 1:16 dilutions, had more than 95% biofilm-inhibitory and biofilm-killing properties on a strong biofilm producer isolate. Supra-MIC levels of antibiotics had a much lower anti-biofilm effect than Lactobacillus supernatant and left considerable alive biofilm cells. CONCLUSIONS: Although antibiotic resistance increases in biofilm forms of Klebsiella pneumoniae, Lactobacillus supernatants have strong antibiofilm efficacy even in lower concentrations of MIC. Biofilm formation decreases considerably in the presence of Lactobacillus supernatants. Hydrogen peroxide is an effective product against growth and biofilm formation of Klebsiella pneumoniae.

Antagonistic and Immunomodulant Effects of Two Probiotic Strains of Lactobacillus on Clinical Strains of Helicobacter pylori.

BACKGROUND: The present study aimed to evaluate the in vitro and in situ antagonistic effects of Lactobacillus probiotic strains on clinical strains of Helicobacter pylori. Also to investigate their immunomodulation effects on a macrophage cell model. MATERIALS AND METHODS: Anti-microbial effects of probiotic lactobacilli against H. pylori was assessed using the well and disk diffusion methods. Effects of lactobacilli probiotics strains, as well as their cell-free supernatant on adhesion of H. pylori to MKN-45 gastric epithelial cells, were examined in their presence and absence. Immunomodulation effects of probiotic lactobacilli were performed using the U937 macrophage cell model. Incubation of host cells with probiotics and their cell-free supernatants with cultured host cells was performed in different optimized conditions. The supernatant of host cells cultured in their presence and absence was used for cytokines measurement. RESULTS: Two probiotics,Lactobacillus acidophilus ATCC4356, and Lactobacillus rhamnosus PTCC1607, could inhibit the growth of clinical H. pylori in vitro. They could also inhibit attachment of H. pylori to MKN-45 cells. Cell-free supernatant of L. acidophilus had a stimulating effect on the production of Interferon-gamma (IFN-γ) by U937 cells. CONCLUSION: The present study demonstrates that, L. acidophilus ATCC4356 and L. rhamnosus PTCC1607 probiotic strains can inhibit the growth of clinical H. pylori in vitro. Treatment of U937 with alive H. pylori plus cell-free supernatant of L. acidophilus, have a significantly higher capacity to stimulate IFN-γ production than H. pylori alone. So, the metabolite (s) of this probiotic may have an immunomodulatory effect in immune response versus H. pylori.

Microencapsulation of Omeprazole by Lactobacillus acidophilus ATCC 4356 Surface Layer Protein and Evaluation of its Stability in Acidic Condition.

The present study introduces a novel method for encapsulation of the acid-labile drug called Omeprazole using Lactobacillus acidophilus (L. acidophilus) ATCC 4356 S-layer protein. Before preparing the Omeprazole suspension, a series of preliminary studies were performed on the Omeprazole powder. For this purpose, some parameters such as melting point, IR spectrum, UV spectrum, and the particle size of Omeprazole powder were investigated. The size reduction process was done in order to achieve an ideal formulation. Ultimately, the resulting powder had an average particle size of 35.516 µm and it was almost uniform. After calculating the amount of S-layer protein required for complete covering of drug particles, the effect of different factors on the drug coating process was investigated with one factor at a time method. Then stability of coated Omeprazole was evaluated in acetate buffer (pH 5). Finally, the maximum coat of drug particles was determined using S- layer protein of Lactobacillus acidophilus ATCC 4356 at 25 °C for 2 h, shaking rate of 100 rpm and ratio of 2:1 for S-layer protein amount/Omeprazole Surface in Tris hydrochloride buffer medium (50 mM, pH 8). The coating of Omeprazole by the S-layer protein decreased the drug decomposition rate up to 2.223.

Antibacterial activity of lactobacilli probiotics on clinical strains of Helicobacter pylori.

OBJECTIVES: Treatment of Helicobacter pylori infection by common drugs may be associated with several problems such as antimicrobial resistance to commonly used antibiotics and side effects of employed drugs. Therefore, exploration of non-chemical compounds which are safer than chemical ones is becoming important as an alternative therapy. The purpose of this study was to evaluate the effects of lactic acid bacteria (LAB) against clinical strains of H. pylori. MATERIALS AND METHODS: Study of antibacterial effects of LAB against H. pylori strains included: evaluation of LAB effects as well as its cell-free supernatant (CFS) to reduce the number of H. pylori, and to examine the effects of CFS to inhibit the urease activity of H. pylori. The anti-adhesion effect of LAB on adherence of H. pylori to epithelial cell line was also evaluated. RESULTS: Evaluation of the anti H. pylori effect of LAB depended on the strain of H. pylori and Lactobacillus. However, CFS of LAB reduced significantly the growth of all H. pylori strains. Also, urease activity of H. pylori strains was inhibited by CFS of LAB demonstrating that their organic acid may have a role in this inhibition. The significant anti-adhesion effect of LAB on adherence of H. pylori was also observed. CONCLUSION: Presence of LAB and/or their CFS can reduce the count of H. pylori, inhibit the urease activity of H. pylori, and reduce adhesion of H. pylori to epithelial cell line. This may be important for the impact of H. pylori colonization in the host stomach.

The role of probiotic Lactobacillus acidophilus ATCC 4356 bacteriocin on effect of HBsu on planktonic cells and biofilm formation of Bacillus subtilis.

Bacillus subtilis is a Gram positive, aerobic and motile bacterium. Biofilm formation is an important feature of this bacterium which confers resistance to antimicrobial agents. The use of new antimicrobial reagents which eliminate biofilms are important and necessary. In this study, the effect of secondary metabolites (bacteriocin) from Lactobacillus acidophilus ATCC 4356 on Bacillus subtilis BM19 in the presence and absence of HBsu which is involved in the growth of planktonic cells and biofilm formation, is reported. HBsu nucleoprotein plays several roles in different processes of Bacillus subtilis cells such as replication, transcription, cell division, recombination and repair. In this study, for the first time, the effect of HBsu on biofilm formation is presented. RESULTS: In the absence of HBsu, purified bacteriocin from L. acidophilus ATCC 4356 was more effective in inhibiting growth of B. subtilis BM19 planktonic cells as well as biofilm formation. The presence of HBsu on the other hand led to increased biofilm formation.

Evaluation of Synergistic Interactions Between Cell-Free Supernatant of Lactobacillus Strains and Amikacin and Genetamicin Against Pseudomonas aeruginosa.

BACKGROUND: The indiscriminate use of antibiotics in the treatment of infectious diseases can increase the development of antibiotic resistance. Therefore, there is a big demand for new sources of antimicrobial agents and alternative treatments for reduction of antibiotic dosage required to decrease the associated side effects. OBJECTIVES: In this study, the synergistic action of aminoglycoside antibiotics and cell-free supernatant (CFS) of probiotic (Lactobacillus rahmnosus and L. casei) against Pseudomonas aeruginosa PTCC 1430 was evaluated. MATERIALS AND METHODS: A growth medium for culturing of probiotic bacteria was separated by centrifugation. The antimicrobial effects of CFS of probiotic bacteria were evaluated using the agar well diffusion assay. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were evaluated using the micro dilution method. Finally, an interaction between CFS and amikacin or gentamicin against P. aeruginosa PTCC 1430 was examined through the checkerboard method and fractional inhibitory concentration (FIC). Furthermore, CFSs from Lactobacillus strains were analyzed by reversed phase HPLC (RP-HPLC) for antimicrobial compounds. RESULTS: The results showed a significant effect of CFS on the growth of P. aeruginosa. The MIC and MBC of CFS from L. casei were 62.5 µL/mL while the MIC and MBC of CFS from L. rhamnosus were 62.5 µL/mL and 125 µL/mL, respectively. Using the FIC indices, synergistic interactions were observed in combination of CFS and antibiotics. Fractional Inhibitory Concentration indices of CFS from L. casei and aminoglycoside antibiotics were 0.124 and 0.312 while FIC indices of CFS from L. rhamnosus and aminoglycoside antibiotics were 0.124 and 0.56, respectively showing a synergism effect. The results of RP-HPLC showed that CFS of Lactobacillus strains contained acetic acid, lactic acid and hydrogen peroxide (H2O2). CONCLUSIONS: Our findings indicate that probiotic bacterial strains of Lactobacillus have a significant inhibitory effect on the growth of P. aeruginosa PTCC 1430. The antimicrobial potency of this combination can be useful for designing and developing alternative therapeutic strategies against P. aeruginosa infections.

The inhibitory effect of a Lactobacillus acidophilus derived biosurfactant on biofilm producer Serratia marcescens.

OBJECTIVES: Serratia marcescens is one of the nosocomial pathogen with the ability to form biofilm which is an important feature in the pathogenesis of S. marcescens. The aim of this study was to determine the anti-adhesive properties of a biosurfactant isolated from Lactobacillus acidophilus ATCC 4356, on S. marcescens strains. MATERIALS AND METHODS: Lactobacillus acidophilus ATCC 4356 was selected as a probiotic strain for biosurfactant production. Anti-adhesive activities was determined by pre-coating and co- incubating methods in 96-well culture plates. RESULTS: The FTIR analysis of derived biosurfactant revealed the composition as protein component. Due to the release of such biosurfactants, L. acidophilus was able to interfere with the adhesion and biofilm formation of the S. marcescens strains. In co-incubation method, this biosurfactant in 2.5 mg/ml concentration showed anti-adhesive activity against all tested strains of S. marcescens (P<0.05). CONCLUSION: Our results show that the anti-adhesive properties of L. acidophilus biosurfactant has the potential to be used against microorganisms responsible for infections in the urinary, vaginal and gastrointestinal tracts, as well as skin, making it a suitable alternative to conventional antibiotics.

Isolation and Characterization of a Novel Magnetotactic Bacterium From Iran: Iron Uptake and Producing Magnetic Nanoparticles in Alphaproteobacterium MTB-KTN90.

BACKGROUND: Magnetotactic bacteria (MTB) have the ability to biomineralize unique intracellular magnetic nanosize particles. These bacteria and their magnetosomes are under special attraction because of their great useful potential in nano-biotechnological and biomedical applications. MTB are ubiquitous in aquatic environments, but their isolation and axenic cultivation in pure culture is very difficult and only a limited number of them have been isolated in pure culture. OBJECTIVES: The main goal of this study was screening, isolation and cultivation of a new strain of these fastidious bacteria in pure culture from Iran to use them and their magnetosomes. MATERIALS AND METHODS: Thirty samples were collected from various aquatic habitats. Most important physicochemical environmental factors that are involved in growth of MTB in the microcosms were investigated using inductively coupled plasma atomic emission spectroscopy (ICP-AES), portable dissolved oxygen meter, etc. Capillary racetrack technique and magnetic separation were used to purify and enrich MTB. Various isolation media were simultaneously used for isolation of a new magnetotactic bacterium in pure culture. Two imaging techniques were used to visualize the characterizations and cell division: transmission electron microscopy (TEM) and field-emission scanning electron microscopy (FESEM). Polymerase chain reaction (PCR), ChromasPro software and MEGA5 were applied for sequence analysis of the 16S rRNA gene. RESULTS: The results revealed a correlation of important physicochemical factors such as pH and iron with growth and blooms of these bacteria in the microcosms. New strain MTB-KTN90 was isolated in a modified isolation medium at microaerophilic zone from Anzali lagoon, Iran and cultured in a modified growth medium subsequently. The phylogenetic analysis showed that the strain belongs to Alphaproteobacteria. Growth and iron uptake studies indicated an important role by this bacterium in the iron biogeochemical cycle. For the first time, this paper introduced a cultured magnetotactic Alphaproteobacterium, able to synthesize magnetosomes in the temperatures above 30°C and reduce selenate oxyanion. CONCLUSIONS: This paper may serve as a guide to screening, isolation, and cultivation of more new MTB. The new isolated strain opens up good opportunities for biotechnological applications such as medicine to bioremediation processes due to its unique abilities.

Studying the Stability of S-Layer Protein of Lactobacillus Acidophilus ATCC 4356 in Simulated Gastrointestinal Fluids Using SDS-PAGE and Circular Dichroism.

Crystalline arrays of proteinaceous subunits forming surface layers (S-layers) are now recognized as one of the most common outermost cell envelope components of prokaryotic organisms. The surface layer protein of Lactobacillus acidophilus ATCC4356 is composed of a single species of protein of apparent molecular weight of 43-46 KDa. Considering the Lactobacillus acidophilus ATCC4356 having the S-layer is stable in harsh gastrointestinal (GI) conditions, a protective role against destructive GI factors which has been proposed for these nanostructures. It opens interesting perspectives in the using and development of this S-layer as a protective coat for oral administration of unstable drug nanocarriers. To achieve this goal, it is necessary to study the in-vitro stability of the S-layers in the simulated gastrointestinal fluids (SGIF). This study was planned to evaluate the in-vitro stability of the extracted S-layer protein of Lactobacillus acidophilus ATCC4356 in SGIF using it as a protective coat in oral drug delivery. Sodium dodecyl sulfate gel electrophoresis (SDS-PAGE) and circular dichroism (CD) spectroscopy were used to study the stability of the S-layer protein incubated in SGIF. Both the SDS-PAGE and CD spectra results showed that Lactobacillus acidophilus ATCC4356 S-layer protein is stable in simulated gastric fluid (SGF) with pH = 2 up to 5 min. It is stable in SGF pH = 3.2 and above it, with and without pepsin. It is also stable in all the simulated intestinal fluids. This S-layer is also stable in all of the simulated intestinal fluids.