Phenotypic and genetic characterization of a lipolytic Ralstonia mannitolilytica isolate from petroleum-contaminated soil in Iraq.

BACKGROUND: Lipases play a crucial role in various industrial applications, and microbial lipases, particularly those from bacteria, possess significant properties. With increasing concerns about the environmental and health impacts of hydrocarbons from pipelines and refineries, there is a growing need to mitigate the risks associated with these compounds. METHODS: In this study, 40 bacterial isolates were recovered from contaminated soil samples collected from multiple refineries across Iraq. Using the Vitek system, bacterial isolates were identified up to the species level, revealing that only 12 isolates exhibited lipase-producing capabilities. RESULTS: Among the lipase-producing isolates, Ralstonia mannitolilytica demonstrated the highest extracellular lipase activity, as determined by an olive oil plate assay supplemented with rhodamine B. Confirmation of the species identity was achieved through 16S rRNA gene sequencing, with the obtained sequence deposited under accession number LC772176.1. Further sequence analysis revealed single nucleotide polymorphisms (SNPs) in the genome of Ralstonia mannitolilytica strain H230303-10_N19_7x_R2 (CP011257.1, positions 1,311,102 and 1,311,457). Additionally, the presence of the lipase gene was confirmed through amplification and sequencing using a thermocycler PCR. Sequence analysis of the gene, aligned using Geneious Prime software, identified SNPs (CP010799, CP049132, AY364601, CP011257, and CP023537), and a phylogenetic tree was constructed based on genetic characterization. CONCLUSION: Our findings highlight the potential of Ralstonia mannitolilytica as a promising candidate for lipase production and contribute to our understanding of its genetic diversity and biotechnological applications in hydrocarbon degradation and industrial processes.

New Phage cocktail against infantile Sepsis bacteria.

A total of 54 positive blood aerobic cultures of suspected blood sepsis in neonates were purified, diagnosed and tested for antibiotic susceptibility. Six bacterial pathogens of Escherichia coli (10 isolates), Klebsiella pneumoniae (10 isolates), Haemophilus influenzae (4 isolates), Pseudomonas aeruginosa (3 isolates), Citrobacter fruendii (1 isolate) and Moraxella catarrhalis (1 isolate) were selected for preparation of phages active against all isolates of each species. Virulent phages towards bacterial isolates were isolated from sewage water by spot lysis method, and a total of 29 phages active towards selected bacterial pathogens were isolated and purified. Phage(s) active against each bacterial species showed activity spectrum within each species of 30-80%, 50-80%, 50%, 50%, 100% and 100% for isolates of Escherichia coli, Klebsiella spp., Haemophilus influenzae, Pseudomonas aeruginosa, Citrobacter fruendii and Moraxella catarrhalis, respectively. A total of 29 phages were formulated in one cocktail in a concentration of 106 PFU/ml in SM buffer for each of a total of 29 phages in SM buffer and showed activity spectrum of 100% against all their bacterial hosts in vitro.

An experimental mouse model for phage therapy of bacterial pathogens causing bacteremia.

A minimum lethal dose on mice for five bacterial pathogens were identified with PCR of neonatal septicemia of Escherichia coli, Klebsiella pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa, Citrobacter freundii and Moraxella catarrhalis were studied. Bacteriophage preparations active against 10 isolates of E. coli, 10 isolates of K. pneumoniae, 4 isolates of H. influenzae, 3 isolates of P. aeruginosa, 1 isolate of C. freundii and 1 isolate of M. catarrhalis were tested for their efficacy in treatment of experimental mouse bacteremia of those isolates through dose-ranging and delayed-treatment studies. Efficacy of polyvalent phage preparations of different phages belonging to same species against pathogens of the same species was studied. Results showed that monovalent and polyvalent phage preparations were active in treatment of mice bacteremia after 45 min of injection of bacterial pathogens and on intervals of 5 h, 8 h, 14 h, 18 h and 24 h after bacterial pathogen injection.