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  • Open access
  • 54 Reads
Characterisation and enzymatic profiling of a halotolerant Penicillium strain

Salt adapted fungi acquire great importance economically and biotechnologically due to their ability to grow and resist to saline conditions as well as other harsh ones like ultraviolet radiations, extremes pH and temperature beside their production’s capabilities of salt-resistant metabolites like compatible solutes, biopolymers, proteins, hemolysis, antibacterial, antioxidants and enzymes. These latter display a combination of other stresses like alkaline pH, high temperature, ultraviolet radiation and organic solvents alongside the salt resistance. The currently work aims to explore the physiologic and enzymatic characteristics of a halotolerant Penicillium strain isolated from an Algerian saline soil. For this purpose Penicillium sp GS15 was isolated on PDA supplemented with 20% NaCl then subjected to morphologic and B-tubulin molecular identification to confirm its affiliation to Penicillium chrysogenum strains. The physiologic characterisation on solid media displays that P.chrysogenum GS15 presents an optimal growth at 25° C, pH 5, dark and aerobic conditions with no significance effect of agitation while on liquid medium, the salinity test showed that it is highly halotolerant microorganism because of its ability to grow between 0% and 21% NaCl (w/v) with most favourable growth at 15 %. In addition, a secreted enzyme profile was also detected with agar plate method. In fact P.chrysogenum exhibits diverse extracellular enzymes such as laccases, amylases, caseinases, tannases, esterases, and lipases while no production of pectinase, lactase, cellulase, gelatinase and albuminase is revealed. On account of the capability of P. chrysogenum GS15 to tolerate salinity and to grow on non-exigent conditions beside its ability to produce various enzymes this fungus may be an interesting organism for several industrial processes.

  • Open access
  • 117 Reads
Unlocking rhizospheric bacteria secondary metabolism: genome analysis for the discovery of novel antimicrobial compounds

The emergence of antimicrobial resistance in pathogenic agents has raised awareness among society and nowadays is a recognized threat to public health. This problem is aggravated due to the misuse of current antibiotics and the lack of novel antimicrobial compounds1. Soil microorganisms are a potential source of new antibiotics and, thanks to the study of their genomes, we can guide the search for undescribed antimicrobial compounds2,3. We have isolated two bacterial strains from a rhizospheric soil, belonging to the genera Brevibacillus and Streptomyces, which were revealed as antimicrobial agents, inhibiting the growth of bacteria and fungi with different profiles of antimicrobial resistance. We sequenced the genome of these strains using the Illumina MiSeq platform. The gene calling and genome annotation were done through the RAST tool (v2.0)4. antiSMASH (v5.1)5 was used to annotate in depth those genes related to the secondary metabolism of both strains. Genome analyses showed diverse antimicrobial potential encoded within these 2 genomes. In sum, 61 biosynthetic gene clusters (BGCs) related with the secondary metabolism were annotated, of which 16 correspond to the Brevibacillus strain and 45 to the Streptomyces strain. The most abundant BGCs were non-ribosomal peptide synthetase (NRPS), terpenes and siderophores. Interestingly, some of these BGCs showed no similarity to any of the already described ones involved in the production of antimicrobial compounds. Therefore, the genetic machinery encoded in both genomes might provide us the basis for the discovery of novel antibiotics against multidrug resistance pathogens.
1. Arias CA, Murray BE. Enterococcus species, Streptococcus gallolyticus group, and Leuconostoc species. In: Bennett JE, Dolin R, Blaser MJ, editors. Mandell, Douglas, and Bennett’s; Principles and Practice of Infectious Diseases. 8th ed. Philadelphia: Elsevier Saunders; 2015, p. 2328-2339
2. Ziemert N, Alanjary M, Weber T. The evolution of genome mining in microbes – a review. RSC. 2016; 33: 988–1005
3. Adamek M, Alanjary M, Ziemert N. Applied evolution: phylogeny-based approaches in natural products research. RSC. 2019; 36: 1295–1312
4. Aziz RK, Bartels D, Best AA. et al. The RAST Server: Rapid Annotations using Subsystems Technology. BMC Genomics. 2008; 9: 75
5. Blin K, Shaw S, Steinke K, Villebro R, Ziemert N, Lee SY, Medema MH, Weber T. antiSMASH 5.0: updates to the secondary metabolite genome mining pipeline. Nucleic Acids Research. 2019; 47: 81–87

  • Open access
  • 30 Reads
Analysis of Beneficial Bacterial Populations from Chinese Longbeans

Chinese Longbean (Vigna unguiculate (L.) Walp. Ssp. Sesquipedalis (L.) Verdc.)

is a crop plant that originated in the southern Asia region from domestication of Cowpea. Longbeans are now grown in Asia, Europe, and North America as a food source and for ornamental applications. Longbean pods, leaves, and stems are edible and the violet-blue flowers with draping pods are a useful ornamental. Despite their popularity, little information is known about the beneficial bacteria associated with this productive alternative food crop. The objectives of this project were to detect, enumerate, and identify beneficial Methylobacterium spp. on the leaves and symbiotic rhizobia spp. in the root nodules. The long-term goal is to develop natural agricultural biologicals that support the growth of crop plants. For this study, longbean plants were divided into two groups and cultivated in a garden setting (summer 2018) and a greenhouse setting (fall 2018). For Methylobacterium detection, macerated leaves were plated on selective agar containing methanol and incubated for seven days at 25ºC. Pink colonies typical for Methylobacterium spp. were counted and identified using cultural, microscopic, and molecular identification. Leaf samples were also plated on Plate Count Agar to determine total bacterial count. For rhizobia detection, root nodules were surface sterilized, crushed, plated on Congo Red Yeast Extract Mannitol Agar (CRYMA), and incubated for fourteen days at 25ºC. Suspected rhizobia colonies were counted and identified using cultural, microscopic, and molecular identification. Interestingly, Root-nodule rhizobia were identified as Bradyrhizobium spp. Two types of Bradyrhizobium spp. were isolated from nodules but only one sp. type per nodule. The typical Bradyrhizobium sp. (elkanii) was found in a majority of nodules. The atypical Bradyrhizobium sp. (japonicum) was found in an occasional nodule. Heterotrophic bacterial counts for greenhouse longbeans were slightly higher (43 CFU /g leaf x 106) versus the garden setting (36 CFU /g leaf x 106). The Methylobacterium counts for greenhouse-grown longbeans (8.7 CFU /g leaf x 104) were much lower versus the garden longbean plants 1230 CFU /g leaf x 104). The root-nodule rhizobia bacterial count for greenhouse longbeans was slightly lower (9.8 CFU / nodule x 106) than the garden longbeans plants (16.0 CFU/ nodule x 106). Although none of the bacterial count comparisons were statistically significant due to variability, there was clearly a trend that growth conditions impact Methylobacterium counts on longbeans. This is the first report on detection and identification of beneficial Methylobacterium and Bradyrhizobium spp. associated with the productive alternative food crop Chinese Longbeans. It is also unusual to find two different rhizobia species form a symbiosis with the same crop plant. The beneficial bacterial species isolated from productive longbeans can be used to develop natural microbial inoculants that support the growth of other crop plants.

  • Open access
  • 87 Reads
Growth Assessment of Bifidobacterium on Glucansucrase-Derived Oligosaccharides

Prebiotics are dietary supplements used to selectively stimulate the growth of beneficial intestinal bacteria such as Bifidobacterium in humans and animals. Maintenance of beneficial intestinal bacteria may provide ecological-based, health-promoting factors such as disease suppression and improved metabolism. A promising source of potential prebiotic supplements is through enzymatic synthesis of oligosaccharides using microbial glucansucrase enzymes. Glucansucrases can be used to prepare a seemingly endless variety of potential prebiotic oligosaccharides containing various linkage structures and monosaccharide components. In this study, six oligosaccharide products synthesized by bacterial glucansucrase were assessed for ability to support bacterial growth (growth rate, µ h-1) and biomass (cell dry weight, mg ml-1) formation using two Bifidobacterium species commonly associated with the gastrointestinal tract of animals. Each growth assessment was performed in triplicate and reported as the mean. Overall, oligosaccharide products Sucromalt (Cargill, Inc) and isomelezitose supported the highest growth rates for B. adolescentis (0.60, 0.69 µ h-1) and B. pseudocatenulatum (0.66, 0.52 µ h-1) when compared to glucose (0.51, 0.37 µ h-1). The raffinose-derived and gentiobiose-derived oligosaccharide products supported the lowest growth rates for the listed species. In addition to growth rate, isomelezitose also supported the highest biomass formation for both Bifidobacterium species (497, 464 mg ml-1) when compared to glucose and the other oligosaccharides. The raffinose-derived product, however, supported the second highest biomass formation for both species (404, 416 mg ml-1) even though the growth rates were lowest indicating a slow but steady metabolism of this supplement by Bifidobacterium. In summary, the Sucromalt, isomelezitose, and raffinose products supported significant growth of intestinal Bifidobacterium and are good candidates for further studies of their prebiotic potential.

  • Open access
  • 88 Reads
Clonal diversity and antimicrobial resistance of Staphylococcus pseudintermedius isolated from canine pyoderma

Staphylococcus pseudintermedius is a predominant cause of skin infections in dogs and the most common causative agent of pyoderma. Methicillin-resistant S. pseudintermedius (MRSP) have been identified in increasing frequencies in canine pyoderma. MRSP strains are usually resistant to several classes of antibiotics which leads to therapeutic failure and, potentially, zoonotic problems. This study aimed to characterize the antimicrobial resistance and genetic lineages of S. pseudintermedius isolated from canine pyoderma.

Sixty-one S. pseudintermedius were isolated from dogs with pyoderma in a veterinary hospital. The presence of mecA gene was detected by PCR. Antimicrobial susceptibility testing was performed by the Kirby-Bauer disk diffusion method against 17 antimicrobial agents. Multilocus‐sequence‐typing (MLST) was performed in all MRSP isolates as previously described (

From the 61 isolates, 31 harbored the mecA gene and were therefore classified as MRSP. The majority of S. pseudintermedius isolates showed resistance to penicillin (88,5%), erythromycin (60,7%), clindamycin (60,7%), tetracycline (65,6%) and trimethoprim-sulfamethoxazole (55,7%). Most MRSP also showed resistance to aminoglycosides. MSRP isolates were ascribed to 9 previously described sequence types (ST): ST123, ST727, ST339, ST537, ST45, ST1029, ST118, ST1468, ST71; and to 5 ST described for the first time in this study: ST2024, ST2025, ST2026, ST2027 and ST2028.

These results show that more than half of S. pseudintermedius isolated from pyoderma were resistant to methicillin. There was a difference in the antimicrobial susceptibility pattern between methicillin-resistant and -sensible S. pseudintermedius, in particular for aminoglycosides. Furthermore, there was a high diversity of genetic lineages among MSRP causing pyoderma.

  • Open access
  • 151 Reads
Antimicrobial and Antioxidant Activity of Phenolic Compounds Extracted from Wine Industry By-Products of Tinto Cão Variety

In Portugal, wine production is one of the most important and relevant socio-economic activities. Winery by-products are rich in phenolic compounds which exhibit a wide range of physiological properties. Therefore, the aim of this study was to extract phenolic compounds from grape skin, seeds, stems, shoats and leaves from the Tinto Cão variety and evaluate their antioxidant activity and antibacterial properties against antibiotic resistant bacterial strains.

The polyphenolic constituents were extracted using water/ethanol (20:80) mixture. The extracts were dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 100 μg/mL. Antimicrobial susceptibility assay was performed using Kirby-Bauer disc diffusion method and was tested against ten different bacteria: Listeria, Pseudomonas aeruginosa, Enterecoccus faecium, Klebsiella pneumoniae, Staphylococcus aureus, Salmonella enteriditis, Enterococcus faecalis, Bacillus cereus, Staphylococcus epidermidis e Escherichia coli. The antioxidant activity of the extracts was determined by DPPH, FRAP and CuPRAC methods.

The higher antioxidant activity was found in the seed extracts, followed by the leaves extracts. The shoats extracts showed the highest antimicrobial properties with minimum inhibitory concentration (MIC) of 25mg/mL, except for B. Cereus, followed by the stems extracts. None of the extracts showed antibacterial activity against E. faecium, S. enteriditis, E.faecalis and E.coli.

These results show that, natural products, such as polyphenols, may represent a promising mean to fight microbial resistance. However, they were not effective against all bacteria which shows that polyphenols, alone, might not substitute antibiotics. Thus, the phenolic compounds may be used in food and pharmaceutical industries as natural food preservers and antibiotic adjuvants.

  • Open access
  • 85 Reads
Antimicrobial Activity of Phenolic Compounds Extracted from Platanus hybrida: Exploring Alternative Therapies for a Post-Antibiotic Era

Multidrug-resistant bacteria are a significant threat to public health and new classes of antibiotics and approaches to treatment are needed. Several studies showed that natural plant-derived compounds could be a promising mean to fight microbial resistance but only a few were conducted with antibiotic resistant bacteria. Therefore, the aim of this study was to extract phenolic compounds from the leaves, fruits and tree trunk of Platanus hybrida and evaluate their antibacterial activity against antibiotic resistant bacterial strains.

The polyphenolic constituents were extracted using a water/ethanol (20:80) mixture. Two grams of powder of each sample was extracted with 100 mL of solvent by stirring for 2h. The extracts were redissolved in dimethyl sulfoxide (DMSO) to a final concentration of 100 μg/mL. Antimicrobial susceptibility assay was performed using Kirby-Bauer disc diffusion method and was tested against ten different bacteria: Listeria, Pseudomonas aeruginosa, Enterecoccus faecium, Klebsiella pneumoniae, Staphylococcus aureus, Salmonella enteriditis, Enterococcus faecalis, Bacillus cereus, Staphylococcus epidermidis e Escherichia coli.

The fruits had the highest antibacterial activity showing a minimum inhibitory concentration (MIC) of 10mg/mL, contrary to the tree trunk that showed the lowest antibacterial activity. None of the extracts showed antimicrobial properties against E. faecium, S. enteriditis and E. faecalis.

These results show that, Platanus hybrida’s phenolic compounds act as antibacterial agents which may become useful therapeutic tools and may represent a source for the development of novel antimicrobials. However, they were not effective against all bacteria which shows that polyphenols, alone, might not substitute antibiotics.

  • Open access
  • 59 Reads
Exploring the core microbial commonalities of alcoholics and CRC patients’

Understanding the link between the effect of lifestyle on gut microbiome and health status of an individual is one of the biggest scientific challenges in this decade. Our main goal was to find the commonalities between alcoholism and colorectal cancer (CRC) by evaluating the host gut microbiome. The microbial profiles of CRC patients were compared with the microbial findings of our study to establish alcoholism linked bacterial markers responsible for CRC development. A dataset of 504 colorectal cancer patients and CRC induced mice gut bacterial profiles were compared with the alcoholic volunteers and alcohol treated mice gut bacterial profiles, to find out the bacterial biomarkers specific for CRC. The core microbial profiles were evaluated using the raw fastq files of the previously reported data in both QIIME and MicrobiomeAnalyst pipelines. Interestingly, Bifidobacterium, Ruminococcaceae and Prevotella were found to be depleted (p≤0.01) in both CRC and alcoholic groups, while Clostridiales, Porphyromonadaceae, Bacteroides, Helicobacter, Lactobacillus, Klebsiella were found to be elevated compared to the healthy individuals (p≤0.01). The finding may serve as a key role in both early-stage detection and therapeutic intervention in the pathophysiological effect of alcohol leading to colorectal cancer development.

  • Open access
  • 117 Reads
Diversity of Staphylococcus species isolated from surface waters

Antimicrobial resistant bacteria in aquatic systems represents one the greatest challenges and emerging threats since wateris one of the most relevant vehicles of bacterial dissemination. Monitoring the presence of antimicrobial resistance human pathogens, in particular staphylococci, may lead to a better understanding of the epidemiology of infections caused by these bacteria. Thus, we aimed to investigate the prevalence of Staphylococcus spp. in surface waters in Portugal.

Seventy-seven water samples (30 rivers, 18 streams, 11 irrigation systems, 10 dams, 5 fountains and 3 lakes) were collected from the hydrographic basins of Douro river in Trás-os-Montes and Alto Douro region, Portugal. Samples were filtered through a cellulose nitrate pore membrane filter. The filters were incubated at 37°C for 24 h in BHI broth supplemented with 6.5% of NaCl. After that, the inoculum was seeded on Mannitol Salt agar plates and incubated at 37°C for 24 h. Staphylococci species were identified by MALDI-TOF MS. Antimicrobial susceptibility testing was performed by the Kirby-Bauer disk diffusion method.

Seventy-five staphylococci were isolated from water samples of which 30 were coagulase-positive (CoPS) and 45 were coagulase-negative staphylococci (CoNS). Among the CoPS, 29 were identified as S. aureus and one as S. pseudintermedius. The CoNS isolated included S. sciuri, S. lentus, S. xylosus and S. epidermidis, among others. S. aureuswere susceptible to almost all antibiotics tested, and CoNS were mostly resistant to penicillin, clindamycin and fusidic acid.

This study demonstrated that staphylococci, including S. aureus, is present in surface waters. A high diversity of staphylococci was detected in surface water samples, in particular for CoNS.

  • Open access
  • 77 Reads
The cellulase activity of the fungi that grow on the bio-based thermal insulation composite materials

Bio-based thermal insulation materials are ecological materials made from a hemp, flax, jute,
wood wastes. Such composites also consist the corn starch, the poly-L-lactide or natural lignin as the binders.
Such materials are the environment-friendly, but under their exploitation the biodestructive factors arrise. At
certain conditions like high humidity or the occumulation of moisture, such kind of materials may be
susceptable to the microbial action which can lead to the changes of physical-chemical properties of materials,
their destruction, and also the human health problems.
In this work, we isolated and identified several fungal strains that grow on hemp-shives based
biocomposite boards. Such boards were made of the hemp fibres with the naturally present lignin, and either
the corn starch or the acrylate resin Acrodur 9501 binders. Using sequencing of the ribosomal ITS regions, we
identified the belonging to the Rhizopus oryzae, Sporothrix schenckii, Talaromyces pinophilus, Aspergillus
fumigatus, and Trichoderma spp. genera. Such fungi were expected to use the cellulase as the carbon and
energy source, therefore the initial detection of the cellulase activities was determined by the Gram‘s iodine.
The cellulase activities were subsequently determined for A.fumigatus using the 3,5-dinitrosalicylic acid ( DNS
method). The cellulase activity was detected when the composite boards with A.fumigatus were immersed into
the distilled water compared to the growth of these fungi in Czapek medium or natural corn starch medium