A whole-cell hypersensitive biosensor for beta-lactams based on the AmpR-AmpC regulatory circuit from the Antarctic Pseudomonas sp. IB20.

Detecting antibiotic residues is vital to minimize their impact. Yet, existing methods are complex and costly. Biosensors offer an alternative. While many biosensors detect various antibiotics, specific ones for beta-lactams are lacking. To address this gap, a biosensor based on the AmpC beta-lactamase regulation system (ampR-ampC) from Pseudomonas sp. IB20, an Antarctic isolate, was developed in this study. The AmpR-AmpC system is well-conserved in the genus Pseudomonas and has been extensively studied for its involvement in peptidoglycan recycling and beta-lactam resistance. To create the biosensor, the ampC coding sequence was replaced with the mCherry fluorescent protein as a reporter, resulting in a transcriptional fusion. This construct was then inserted into Escherichia coli SN0301, a beta-lactam hypersensitive strain, generating a whole-cell biosensor. The biosensor demonstrated dose-dependent detection of penicillins, cephalosporins and carbapenems. However, the most interesting aspect of this work is the high sensitivity presented by the biosensor in the detection of carbapenems, as it was able to detect 8 pg/mL of meropenem and 40 pg/mL of imipenem and reach levels of 1-10 ng/mL for penicillins and cephalosporins. This makes the biosensor a powerful tool for the detection of beta-lactam antibiotics, specifically carbapenems, in different matrices.

Genetic characterization of clinically relevant class 1 integrons carried by multidrug resistant bacteria (MDRB) isolated from the gut microbiota of highly antibiotic treated Salmo salar.

OBJECTIVES: The main objective of this study was the genetic characterization of clinically relevant class 1 integrons carried by multidrug resistant bacteria isolated from the intestinal microbiota of aquaculture salmon treated with high concentrations of antibiotics. METHODS: In 82 multidrug resistant bacterial isolates, the prevalence of both the conserved elements of the integrons, qacEΔ1 and sul1 genes, and the variable region (VR) was determined. Further, whole genome sequencing and complete genetic analysis was performed in VR-positive isolates. RESULTS: Despite the fact that 100% of the bacterial isolates presented the intI1 gene, only 12.3% carried the qacEΔ1 and sul1 genes and only two (2.4%) presented a VR with gene cassettes. In the Pseudomonas baetica 25P2F9 isolate, a VR carrying aac(6')31, qacH, and blaOXA-2 gene cassettes was described, whereas the VR of Aeromonas salmonicida 30PB8 isolate showed a dfrA14 gene cassette. The array of gene cassettes found in the Pseudomonas isolate appears with high frequency in clinically relevant pathogens such as Pseudomonas aeruginosa or Escherichia coli. Additionally, it was possible to determine that these integrons are contained in plasmids and coul be easily transferred. Resistome analysis demonstrated that both isolates carried a great diversity of antibiotic resistance genes, including many ß-lactamases. Even in the Aeromonas isolate a new oxacillin-hydrolyzing beta-lactamase gene was described (blaOXA-956). CONCLUSION: The presence of multidrug resistant bacteria and clinically relevant genetic elements in the salmon intestinal microbiota make the aquaculture a hotspot in the phenomenon of antibiotic resistance; therefore, the control of antibiotics used in this activity is a key point to avoid its escalation.

Phylogenetic MLSA and phenotypic analysis identification of three probable novel Pseudomonas species isolated on King George Island, South Shetland, Antarctica

ABSTRACT Antarctica harbors a great diversity of microorganisms, including bacteria, archaea, microalgae and yeasts. The Pseudomonas genus is one of the most diverse and successful bacterial groups described to date, but only eight species isolated from Antarctica have been characterized. Here, we present three potentially novel species isolated on King George Island. The most abundant isolates from four different environments, were genotypically and phenotypically characterized. Multilocus sequence analysis and 16S rRNA gene analysis of a sequence concatenate for six genes (16S, aroE, glnS, gyrB, ileS and rpoD), determined one of the isolates to be a new Pseudomonas mandelii strain, while the other three are good candidates for new Pseudomonas species. Additionally, genotype analyses showed the three candidates to be part of a new subgroup within the Pseudomonas fluorescens complex, together with the Antarctic species Pseudomonas antarctica and Pseudomonas extremaustralis. We propose terming this new subgroup P. antarctica. Likewise, phenotypic analyses using API 20 NE and BIOLOG® corroborated the genotyping results, confirming that all presented isolates form part of the P. fluorescens complex. Pseudomonas genus research on the Antarctic continent is in its infancy. To understand these microorganisms' role in this extreme environment, the characterization and description of new species is vital.

Extended antibiotic treatment in salmon farms select multiresistant gut bacteria with a high prevalence of antibiotic resistance genes.

The high use of antibiotics for the treatment of bacterial diseases is one of the main problems in the mass production of animal protein. Salmon farming in Chile is a clear example of the above statement, where more than 5,500 tonnes of antibiotics have been used over the last 10 years. This has caused a great impact both at the production level and on the environment; however, there are still few works in relation to it. In order to demonstrate the impact of the high use of antibiotics on fish gut microbiota, we have selected four salmon farms presenting a similar amount of fish of the Atlantic salmon species (Salmo salar), ranging from 4,500 to 6,000 tonnes. All of these farms used treatments with high doses of antibiotics. Thus, 15 healthy fish were selected and euthanised in order to isolate the bacteria resistant to the antibiotics oxytetracycline and florfenicol from the gut microbiota. In total, 47 bacterial isolates resistant to florfenicol and 44 resistant to oxytetracycline were isolated, among which isolates with Minimum Inhibitory Concentrations (MIC) exceeding 2048 µg/mL for florfenicol and 1024 µg/mL for oxytetracycline were found. In addition, another six different antibiotics were tested in order to demonstrate the multiresistance phenomenon. In this regard, six isolates of 91 showed elevated resistance values for the eight tested antibiotics, including florfenicol and oxytetracycline, were found. These bacteria were called "super-resistant" bacteria. This phenotypic resistance was verified at a genotypic level since most isolates showed antibiotic resistance genes (ARGs) to florfenicol and oxytetracycline. Specifically, 77% of antibiotic resistant bacteria showed at least one gene resistant to florfenicol and 89% showed at least one gene resistant to oxytetracycline. In the present study, it was demonstrated that the high use of the antibiotics florfenicol and oxytetracycline has, as a consequence, the selection of multiresistant bacteria in the gut microbiota of farmed fish of the Salmo salar species at the seawater stage. Also, the phenotypic resistance of these bacteria can be correlated with the presence of antibiotic resistance genes.

Phylogenetic MLSA and phenotypic analysis identification of three probable novel Pseudomonas species isolated on King George Island, South Shetland, Antarctica.

Antarctica harbors a great diversity of microorganisms, including bacteria, archaea, microalgae and yeasts. The Pseudomonas genus is one of the most diverse and successful bacterial groups described to date, but only eight species isolated from Antarctica have been characterized. Here, we present three potentially novel species isolated on King George Island. The most abundant isolates from four different environments, were genotypically and phenotypically characterized. Multilocus sequence analysis and 16S rRNA gene analysis of a sequence concatenate for six genes (16S, aroE, glnS, gyrB, ileS and rpoD), determined one of the isolates to be a new Pseudomonas mandelii strain, while the other three are good candidates for new Pseudomonas species. Additionally, genotype analyses showed the three candidates to be part of a new subgroup within the Pseudomonas fluorescens complex, together with the Antarctic species Pseudomonas antarctica and Pseudomonas extremaustralis. We propose terming this new subgroup P. antarctica. Likewise, phenotypic analyses using API 20 NE and BIOLOG® corroborated the genotyping results, confirming that all presented isolates form part of the P. fluorescens complex. Pseudomonas genus research on the Antarctic continent is in its infancy. To understand these microorganisms' role in this extreme environment, the characterization and description of new species is vital.

Alginate overproduction and biofilm formation by psychrotolerant Pseudomonas mandelii depend on temperature in Antarctic marine sediments

Background: In recent years, Antarctica has become a key source of biotechnological resources. Native microorganisms have developed a wide range of survival strategies to adapt to the harsh Antarctic environment, including the formation of biofilms. Alginate is the principal component of the exopolysaccharide matrix in biofilms produced by Pseudomonas, and this component is highly demanded for the production of a wide variety of commercial products. There is a constant search for efficient alginate-producing organisms. Results: In this study, a novel strain of Pseudomonas mandelii isolated from Antarctica was characterized and found to overproduce alginate compared with other good alginate producers such as Pseudomonas aeruginosa and Pseudomonas fluorescens. Alginate production and expression levels of the alginate operon were highest at 4°C. It is probable that this alginate-overproducing phenotype was the result of downregulated MucA, an anti-sigma factor of AlgU. Conclusion: Because biofilm formation is an efficient bacterial strategy to overcome stressful conditions, alginate overproduction might represent the best solution for the successful adaptation of P. mandelii to the extreme temperatures of the Antarctic. Through additional research, it is possible that this novel P. mandelii strain could become an additional source for biotechnological alginate production.