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Detection by metagenomic functional analysis and improvement by experimental evolution of β-lactams resistance genes present in oil contaminated soils.

ABSTRACT: The spread of antibiotic resistance genes has become a global health concern identified by the World Health Organization as one of the greatest threats to health. Many of antimicrobial resistance determinants found in bacterial pathogens originate from environmental bacteria, so identifying the genes that confer resistance to antibiotics in different habitats is mandatory to better understand resistance mechanisms. Soil is one of the most diverse environments considered reservoir of antimicrobial resistance genes. The aim of this work is to study the presence of genes that provide resistance to antibiotics used in clinical settings in two oil contaminated soils by metagenomic functional analysis. Using fosmid vectors that efficiently transcribe metagenomic DNA, we have selected 12 fosmids coding for two class A β-lactamases, two subclass B1 and two subclass B3 metallo-β-lactamases, one class D β-lactamase and three efflux pumps that confer resistance to cefexime, ceftriaxone, meropenem and/or imipenem. In some of them, detection of the resistance required heterologous expression from the fosmid promoter. Although initially, these environmental genes only provide resistance to low concentrations of antibiotics, we have obtained, by experimental evolution, fosmid derivatives containing β-lactamase ORFs with a single base substitution, which substantially increase their β-lactamase activity and resistance level. None of the mutations affect β-lactamase coding sequences and are all located upstream of them. These results demonstrate the presence of enzymes that confer resistance to relevant β-lactams in these soils and their capacity to rapidly adapt to provide higher resistance levels.

SUBMITTER: Alvarez-Marin MT

PROVIDER: S-EPMC9243250 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:In this study, we compared the culturability of aerobic bacteria isolated from long-term oil-contaminated soils via enrichment and direct-plating methods; bacteria were cultured at 30°C or ambient temperatures. Two soil samples were collected from two sites in the Shengli oilfield located in Dongying, China. One sample (S0) was close to the outlet of an oil-production water treatment plant, and the other sample (S1) was located 500 m downstream of the outlet. In total, 595 bacterial isolates belonging to 56 genera were isolated, distributed in Actinobacteria, Firmicutes, Bacterioidetes, and Proteobacteria. It was interesting that Actinobacteria and Firmicutes were not detected from the 16S rRNA gene clone library. The results suggested the activation of rare species during culture. Using the enrichment method, 239 isolates (31 genera) and 96 (22 genera) isolates were obtained at ambient temperatures and 30°C, respectively, from S0 soil. Using the direct-plating method, 97 isolates (15 genera) and 163 isolates (20 genera) were obtained at ambient temperatures and 30°C, respectively, from two soils. Of the 595 isolates, 244 isolates (41.7% of total isolates) could degrade n-hexadecane. A greater number of alkane-degraders was isolated at ambient temperatures using the enrichment method, suggesting that this method could significantly improve bacterial culturability. Interestingly, the proportion of alkane degrading isolates was lower in the isolates obtained using enrichment method than that obtained using direct-plating methods. Considering the greater species diversity of isolates obtained via the enrichment method, this technique could be used to increase the diversity of the microbial consortia. Furthermore, phenol hydroxylase genes (pheN), medium-chain alkane monooxygenases genes (alkB and CYP153A), and long-chain alkane monooxygenase gene (almA) were detected in 60 isolates (11 genotypes), 91 isolates (27 genotypes) and 93 isolates (24 genotypes), and 34 isolates (14 genotypes), respectively. This study could provide new insights into microbial resources from oil fields or other environments, and this information will be beneficial for bioremediation of petroleum contamination and for other industrial applications.

Project description:Soil contamination by heavy metals, particularly mercury (Hg), is a problem that can seriously affect the environment, animals, and human health. Hg has the capacity to biomagnify in the food chain. That fact can lead to pathologies, of those which affect the central nervous system being the most severe. It is convenient to know the biological environmental indicators that alert of the effects of Hg contamination as well as the biological mechanisms that can help in its remediation. To contribute to this knowledge, this study conducted comparative analysis by the use of Shotgun metagenomics of the microbial communities in rhizospheric soils and bulk soil of the mining region of Almadén (Ciudad Real, Spain), one of the most affected areas by Hg in the world The sequences obtained was analyzed with MetaPhlAn2 tool and SUPER-FOCUS. The most abundant taxa in the taxonomic analysis in bulk soil were those of Actinobateria and Alphaproteobacteria. On the contrary, in the rhizospheric soil microorganisms belonging to the phylum Proteobacteria were abundant, evidencing that roots have a selective effect on the rhizospheric communities. In order to analyze possible indicators of biological contamination, a functional potential analysis was performed. The results point to a co-selection of the mechanisms of resistance to Hg and the mechanisms of resistance to antibiotics or other toxic compounds in environments contaminated by Hg. Likewise, the finding of antibiotic resistance mechanisms typical of the human clinic, such as resistance to beta-lactams and glycopeptics (vancomycin), suggests that these environments can behave as reservoirs. The sequences involved in Hg resistance (operon mer and efflux pumps) have a similar abundance in both soil types. However, the response to abiotic stress (salinity, desiccation, and contaminants) is more prevalent in rhizospheric soil. Finally, sequences involved in nitrogen fixation and metabolism and plant growth promotion (PGP genes) were identified, with higher relative abundances in rhizospheric soils. These findings can be the starting point for the targeted search for microorganisms suitable for further use in bioremediation processes in Hg-contaminated environments.

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Detection by metagenomic functional analysis and improvement by experimental evolution of β-lactams resistance genes present in oil contaminated soils.

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