Genetic characterization of a novel Salinicola salarius isolate applied for the bioconversion of agro-industrial wastes into polyhydroxybutyrate.

BACKGROUND: Polyhydroxybutyrate (PHB) has emerged as a promising eco-friendly alternative to traditional petrochemical-based plastics. In the present study, we isolated and characterized a new strain of Salinicola salarius, a halophilic bacterium, from the New Suez Canal in Egypt and characterized exclusively as a potential PHB producer. Further genome analysis of the isolated strain, ES021, was conducted to identify and elucidate the genes involved in PHB production. RESULTS: Different PHB-producing marine bacteria were isolated from the New Suez Canal and characterized as PHB producers. Among the 17 bacterial isolates, Salinicola salarius ES021 strain showed the capability to accumulate the highest amount of PHB. Whole genome analysis was implemented to identify the PHB-related genes in Salinicola salarius ES021 strain. Putative genes were identified that can function as phaCAB genes to produce PHB in this strain. These genes include fadA, fabG, and P3W43_16340 (encoding acyl-CoA thioesterase II) for PHB production from glucose. Additionally, phaJ and fadB were identified as key genes involved in PHB production from fatty acids. Optimization of environmental factors such as shaking rate and incubation temperature, resulted in the highest PHB productivity when growing Salinicola salarius ES021 strain at 30°C on a shaker incubator (110 rpm) for 48 h. To maximize PHB production economically, different raw materials i.e., salted whey and sugarcane molasses were examined as cost-effective carbon sources. The PHB productivity increased two-fold (13.34 g/L) when using molasses (5% sucrose) as a fermentation media. This molasses medium was used to upscale PHB production in a 20 L stirred-tank bioreactor yielding a biomass of 25.12 g/L, and PHB of 12.88 g/L. Furthermore, the produced polymer was confirmed as PHB using Fourier-transform infrared spectroscopy (FTIR), gas chromatography-mass spectroscopy (GC-MS), and nuclear magnetic resonance spectroscopy (NMR) analyses. CONCLUSIONS: Herein, Salinicola salarius ES021 strain was demonstrated as a robust natural producer of PHB from agro-industrial wastes. The detailed genome characterization of the ES021 strain presented in this study identifies potential PHB-related genes. However, further metabolic engineering is warranted to confirm the gene networks required for PHB production in this strain. Overall, this study contributes to the development of sustainable and cost-effective PHB production strategies.

Probiotic potentials of lactic acid bacteria isolated from Egyptian fermented food.

Lactic acid bacteria (LAB) are of major concern due to their health benefits. Fermented food products comprise variable LAB demonstrating probiotic properties. Discovering and evaluating new probiotics in fermented food products poses a global economic and health importance. Therefore, the present work aimed to investigate and evaluate the probiotic potentials of LAB strains isolated from Egyptian fermented food. In this study, we isolated and functionally characterized 100 bacterial strains isolated from different Egyptian fermented food sources as probiotics. Only four LAB strains amongst the isolated LAB showed probiotic attributes and are considered to be safe for their implementation as feed or dietary supplements. Additionally, they were shown to exert antimicrobial activities against pathogenic bacteria and anticancer effects against the colon cancer cell line Caco-2. The Enterococcus massiliensis IS06 strain was exclusively reported in this study as a probiotic strain with high antimicrobial, antioxidant, and anti-colon cancer activity. Hitherto, few studies have focused on elucidating the impact of probiotic supplementation in vivo. Therefore, in the current study, the safety of the four strains was tested in vivo through the supplementation of rats with potential probiotic strains for 21 days. The results revealed that probiotic bacterial supplementation in rats did not adversely affect the general health of rats. The Lactiplantibacillus plantarum IS07 strain significantly increased the growth performance of rats. Furthermore, the four strains exhibited increased levels of antioxidants such as superoxide dismutase and glutathione in vivo. Consistently, all strains also showed high antioxidant activity of the superoxide dismutase enzyme in vitro. Overall, these findings demonstrated that these isolated potential probiotics harbor desirable characteristics and can be applied widely as feed additives for animals or as dietary supplements for humans to exert their health benefits and combat serious diseases.

The evolutionary trajectories of P. aeruginosa in biofilm and planktonic growth modes exposed to ciprofloxacin: beyond selection of antibiotic resistance.

Ciprofloxacin (CIP) is used to treat Pseudomonas aeruginosa biofilm infections. We showed that the pathways of CIP-resistance development during exposure of biofilms and planktonic P. aeruginosa populations to subinhibitory levels of CIP depend on the mode of growth. In the present study, we analyzed CIP-resistant isolates obtained from previous evolution experiments, and we report a variety of evolved phenotypic and genotypic changes that occurred in parallel with the evolution of CIP-resistance. Cross-resistance to beta-lactam antibiotics was associated with mutations in genes involved in cell-wall recycling (ftsZ, murG); and could also be explained by mutations in the TCA cycle (sdhA) genes and in genes involved in arginine catabolism. We found that CIP-exposed isolates that lacked mutations in quorum-sensing genes and acquired mutations in type IV pili genes maintained swarming motility and lost twitching motility, respectively. Evolved CIP-resistant isolates showed high fitness cost in planktonic competition experiments, yet persisted in the biofilm under control conditions, compared with ancestor isolates and had an advantage when exposed to CIP. Their persistence in biofilm competition experiments in spite of their fitness cost in planktonic growth could be explained by their prolonged lag-phase. Interestingly, the set of mutated genes that we identified in these in vitro-evolved CIP-resistant colonies, overlap with a large number of patho-adaptive genes previously reported in P. aeruginosa isolates from cystic fibrosis (CF) patients. This suggests that the antibiotic stress is contributing to the bacterial evolution in vivo, and that adaptive laboratory evolution can be used to predict the in vivo evolutionary trajectories.

Lack of the Major Multifunctional Catalase KatA in Pseudomonas aeruginosa Accelerates Evolution of Antibiotic Resistance in Ciprofloxacin-Treated Biofilms.

During chronic biofilm infections, Pseudomonas aeruginosa bacteria are exposed to increased oxidative stress as a result of the inflammatory response. As reactive oxygen species (ROS) are mutagenic, the evolution of resistance to ciprofloxacin (CIP) in biofilms under oxidative stress conditions was investigated. We experimentally evolved six replicate populations of P. aeruginosa lacking the major catalase KatA in colony biofilms and stationary-phase cultures for seven passages in the presence of subinhibitory levels (0.1 mg/liter) of CIP or without CIP (eight replicate lineages for controls) under aerobic conditions. In CIP-evolved biofilms, a larger CIP-resistant subpopulation was isolated in the ΔkatA strain than in the wild-type (WT) PAO1 population, suggesting oxidative stress as a promoter of the development of antibiotic resistance. A higher number of mutations identified by population sequencing were observed in evolved ΔkatA biofilm populations (CIP and control) than in WT PAO1 populations evolved under the same conditions. Genes involved in iron assimilation were found to be exclusively mutated in CIP-evolved ΔkatA biofilm populations, probably as a defense mechanism against ROS formation resulting from Fenton reactions. Furthermore, a hypermutable lineage due to mutL inactivation developed in one CIP-evolved ΔkatA biofilm lineage. In CIP-evolved biofilms of both the ΔkatA strain and WT PAO1, mutations in nfxB, the negative regulator of the MexCD-OprJ efflux pump, were observed while in CIP-evolved planktonic cultures of both the ΔkatA strain and WT PAO1, mutations in mexR and nalD, regulators of the MexAB-OprM efflux pump, were repeatedly found. In conclusion, these results emphasize the role of oxidative stress as an environmental factor that might increase the development of antibiotic resistance in in vivo biofilms.

Evolution of Antibiotic Resistance in Biofilm and Planktonic Pseudomonas aeruginosa Populations Exposed to Subinhibitory Levels of Ciprofloxacin.

The opportunistic Gram-negative pathogen Pseudomonas aeruginosa, known for its intrinsic and acquired antibiotic resistance, has a notorious ability to form biofilms, which often facilitate chronic infections. The evolutionary paths to antibiotic resistance have mainly been investigated in planktonic cultures and are less studied in biofilms. We experimentally evolved P. aeruginosa PAO1 colony biofilms and stationary-phase planktonic cultures for seven passages in the presence of subinhibitory levels (0.1 mg/liter) of ciprofloxacin (CIP) and performed a genotypic (whole-bacterial population sequencing) and phenotypic assessment of the populations. We observed a higher proportion of CIP resistance in the CIP-evolved biofilm populations than in planktonic populations exposed to the same drug concentrations. However, the MICs of ciprofloxacin were lower in CIP-resistant isolates selected from the biofilm population than the MICs of CIP-resistant isolates from the planktonic cultures. We found common evolutionary trajectories between the different lineages, with mutations in known CIP resistance determinants as well as growth condition-dependent adaptations. We observed a general trend toward a reduction in type IV-pilus-dependent motility (twitching) in CIP-evolved populations and a loss of virulence-associated traits in the populations evolved in the absence of antibiotic. In conclusion, our data indicate that biofilms facilitate the development of low-level mutational resistance, probably due to the lower effective drug exposure than in planktonic cultures. These results provide a framework for the selection process of resistant variants and the evolutionary mechanisms involved under the two different growth conditions.

Inhibition and Dispersal of Pseudomonas aeruginosa Biofilms by Combination Treatment with Escapin Intermediate Products and Hydrogen Peroxide.

Escapin is an l-amino acid oxidase that acts on lysine to produce hydrogen peroxide (H2O2), ammonia, and equilibrium mixtures of several organic acids collectively called escapin intermediate products (EIP). Previous work showed that the combination of synthetic EIP and H2O2 functions synergistically as an antimicrobial toward diverse planktonic bacteria. We initiated the present study to investigate how the combination of EIP and H2O2 affected bacterial biofilms, using Pseudomonas aeruginosa as a model. Specifically, we examined concentrations of EIP and H2O2 that inhibited biofilm formation or fostered disruption of established biofilms. High-throughput assays of biofilm formation using microtiter plates and crystal violet staining showed a significant effect from pairing EIP and H2O2, resulting in inhibition of biofilm formation relative to biofilm formation in untreated controls or with EIP or H2O2 alone. Similarly, flow cell analysis and confocal laser scanning microscopy revealed that the EIP and H2O2 combination reduced the biomass of established biofilms relative to that of the controls. Area layer analysis of biofilms posttreatment indicated that disruption of biomass occurs down to the substratum. Only nanomolar to micromolar concentrations of EIP and H2O2 were required to impact biofilm formation or disruption, and these concentrations are significantly lower than those causing bactericidal effects on planktonic bacteria. Micromolar concentrations of EIP and H2O2 combined enhanced P. aeruginosa swimming motility compared to the effect of either EIP or H2O2 alone. Collectively, our results suggest that the combination of EIP and H2O2 may affect biofilms by interfering with bacterial attachment and destabilizing the biofilm matrix.