Detection of linezolid and vancomycin resistant Enterococcus isolates collected from healthy chicken caecum.

AIM: The poultry industry represents an important economic sector in Tunisia. This study aims to determine the antimicrobial resistance phenotypes and genotypes and virulence factors of enterococci collected from chicken caecum in Tunisia. METHODS AND RESULTS: Forty-nine composite chicken caecum samples were recovered in 49 different Tunisian farms (December 2019-March 2020). Each composite sample corresponds to six individual caecum from each farm. Composite samples were plated on Slanetz-Bartley agar both supplemented (SB-Van) and not supplemented (SB) with vancomycin and isolates were identified by matrix-assisted laser desorption/ionization time-of-flight. Antibiotic resistance and virulence genes were tested by Polymerase Chain Reaction (PCR) and sequencing and multilocus-sequence-typing of selected enterococci was performed. One hundred sixty seven enterococci of six different species were recovered. Acquired linezolid resistance was detected in 6 enterococci of 4/49 samples (8.1%): (A) four optrA-carrying Enterococcus faecalis isolates assigned to ST792, ST478, and ST968 lineages; (B) two poxtA-carrying Enterococcus faecium assigned to ST2315 and new ST2330. Plasmid typing highlighted the presence of the rep10, rep14, rep7, rep8, and pLG1 in these strains. One vancomycin-resistant E. faecium isolate (typed as ST1091) with vanA gene (included in Tn1546) was detected in SB-Van plates. The gelE, agg, esp, and hyl virulence genes were found in linezolid- and vancomycin-resistant enterococci. High resistance rates were identified in the enterococci recovered in SB plates: tetracycline [74.8%, tet(M) and tet(L) genes], erythromycin [65.9%, erm(B)], and gentamicin [37.1%, aac(6')-Ie-aph(2″)-Ia]. CONCLUSION: The detection of emerging mechanisms of resistance related to linezolid and vancomycin in the fecal enterococci of poultry farms has public health implications, and further surveillance should be carried out to control their dissemination by the food chain.

Exploring Bacillus amyloliquefaciens strain OM81 for the production of polyhydroxyalkanoate (PHA) bioplastic using olive mill wastewater.

In this study, bacterial strains isolated from olive oil mill wastewater assigned to Bacillus (n = 4) and Klebsiella (n = 1) genera, were evaluated for their ability to accumulate intracellular PHA granules using Sudan Black staining. A maximum PHA production of 0.14 g/L (i.e., 30.2% wt./wt. in dry biomass) was observed in Bacillus amyloliquefaciens strain OM81 after 72 h of incubation in the presence of 2% glucose (synthetic medium). To reduce bioplastic production costs and recover a polluting product, olive mill wastewater was tested as a carbon source. In this context, the maximum growth (1.45 g/L) was observed in the presence of 50% olive mill wastewater. After extracting the biopolymers with chloroform, quantitative and qualitative analyses were conducted using Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). FTIR showed an absorption band at 1730 cm-1 assigned to the elongation of the PHB carbonyl groups. This approach offers a dual benefit of reducing pollution and bioplastic production costs. The Bacillus amyloliquefaciens strain OM81 showed promising results for PHAs production, making it a potential candidate for further investigation. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03808-4.

Efficient Wastewater Treatment and Removal of Bisphenol A and Diclofenac in Mesocosm Flow Constructed Wetlands Using Granulated Cork as Emerged Substrate.

Constructed wetlands (CWs) are considered as low-cost and energy-efficient wastewater treatment systems. Media selection is one of the essential technical keys for their implementation. The purpose of this work was essentially to evaluate the removal efficiency of organic pollution and nitrogen from municipal wastewater (MWW) using different selected media (gravel/gravel amended with granulated cork) in mesocosm horizontal flow constructed wetlands (HFCWs). The results showed that the highest chemical oxygen demand (COD) and ammonium nitrogen removal of 80.53% and 42%, respectively, were recorded in the units filled with gravel amended with cork. The influence of macrophytes (Phragmites australis and Typha angustifolia) was studied and both species showed steeper efficiencies. The system was operated under different hydraulic retention times (HRTs) i.e., 6 h, 24 h, 30 h, and 48 h. The obtained results revealed that the COD removal efficiency was significantly enhanced by up to 38% counter to the ammonium rates when HRT was increased from 6 h to 48 h. Moreover, the removal efficiency of two endocrine-disrupting compounds (EDCs) namely, bisphenol A (BPA) and diclofenac (DCF) was investigated in two selected HFCWs, at 48 h HRT. The achieved results proved the high capacity of cork for BPA and DCF removal with the removal rates of 90.95% and 89.66%, respectively. The results confirmed the role of these engineered systems, especially for EDC removal, which should be further explored.

Antiviral activity of Inonotusin A an active compound isolated from Boletus bellinii and Boletus subtomentosus.

Mushrooms produce various classes of secondary metabolites that could be used as antivirals in the future. The aim of this study was to determine the antiviral activity of methanolic extracts obtained from two edible mushrooms, Boletus bellinii (B. bellinii) and Boletus subtomentosus (B. subtomentosus), collected from the north forests of Tunisia, against Herpes Simplex Virus type 2 and Coxsackie Virus B type 3. In vitro micro-inhibition assays and cytotoxicity screening were performed on Vero cells. The tested Boletus methanolic extracts were found to be non-cytotoxic at high doses (50% cytotoxic concentration - CC50 > 1 mg/mL) and exhibited relevant viral inhibition with 50% inhibitory concentration, i.e., IC50 of 3.60 ± 0.66 µg/mL and 35.70 ± 7.42 µg/mL for B. bellinii, and 5.67 ± 1.02 µg/mL and 56.88 ± 9.56 µg/mL for B. subtomentosus, against HSV-2 and CVB-3, respectively. Interestingly, Boletus methanolic extracts showed high selectivity index (SI) values against both viruses, with the highest values against HSV-2 (SI > 800). Both viral strains were inhibited when treated with extracts during the early stages of virus replication. Inonotusin A was isolated and identified as the compound responsible for these activities. The latter is a novel antiviral agent that may have clinical utility or serve as a lead compound for further development. This study is the first attempt to investigate the antiviral activity of inonotusin A, isolated from the genus Boletus. The information from the present work should be a valuable reference for future studies on the antiviral activity of inonotusin A.

Treatment of olive mill wastewater through employing sequencing batch reactor: performance and microbial diversity assessment.

This work describes the performance of a sequencing batch reactor (SBR) and the involvement of a novel reconstituted bacterial consortium in olive mill wastewater (OMW) treatment. The organic loading rate applied to the SBR was serially increased in terms of initial COD from 10 to 75 g L-1 to allow gradual acclimatization of activated sludge to high concentrations of toxic compounds in OMW. After the acclimatization period, up to 60% of the total COD content were effectively biodegraded from OMW at 75 g L-1 COD within 30 day hydraulic retention time. The diversity and community composition of cultivable bacteria participating in the aerobic process of treating OMW were further assessed. A total of 91 bacterial strains were isolated from the reactor and analyzed by amplification of the 16S-23S rRNA internal transcribed spacer (ITS) region and by 16S rRNA gene sequencing. The most abundant phylum was Firmicutes (57.1%) followed by Proteobacteria (35.2%) and Actinobacteria (7.7%). The use of the Biolog® Phenotype Microarray system to evaluate the ability of isolated strains to utilize OMW phenolic compounds is reported in this work for the first time. Interestingly, results showed that all species tested were able to utilize phenolics as sole carbon and energy sources. The removals of COD and phenolics from undiluted OMW by the reconstituted bacterial consortium were almost similar to those obtained by the acclimatized activated sludge, which suggest that cultivable bacteria play the major role in OMW biodegradation. Phytotoxicity assays using tomato seeds showed a significant improvement of seed germination values for treated OMW. Our overall results suggest that the novel developed bacterial consortium could be considered as a good prospect for phenolics-rich wastewaters bioremediation applications.

A sustainable use of low-cost raw substrates for biodiesel production by the oleaginous yeast Wickerhamomyces anomalus.

Over the past decade, the increasing demand of vegetable oils for biodiesel production has highlighted the need for alternative oil feedstocks that do not compete with food production. In this context, the combined use of agro-industrial wastes and oleaginous microorganisms could be a promising strategy for sustainable biodiesel production. The present investigation involves the performance of the oleaginous yeast Wickerhamomyces anomalus strain EC28 to produce lipids from different agro-industrial wastewaters (i.e., deproteinized cheese whey, olive mill wastewater, and wastewaters from confectionary industries) and waste frying oils (i.e., waste oil from frying fish, waste oil from frying potato and waste oil from frying meat). Results indicated that this strain can adequately grow on agro-industrial wastewater-based media and produce substantial amounts of lipids [up to 24%, wt/wt in deproteinized cheese whey-based medium and olive mill wastewater-based medium (75%, v/v in water)] of similar fatty acid composition to that of the most commonly used vegetable oils in the biodiesel industry. However, the addition of frying oils to the culture media resulted in a significant decrease in total lipid content, probably due to excess of available nitrogen released from meat, fish, and potato into the frying oil. The estimated properties of the resulting biodiesels, such as SV (190.69-203.13), IV (61.77-88.32), CN (53.45-59.32), and CFPP (-0.54 to 10.4), are reported, for the first time, for W. anomalus and correlate well with specified standards. In conclusion, W. anomalus strain EC28, for which there is very limited amount of available information, might be regarded as a promising candidate for biodiesel production and additional efforts for process improvement should be envisaged.

Newly isolated yeasts from Tunisian microhabitats: Lipid accumulation and fatty acid composition.

Newly isolated yeasts from different Tunisian microhabitats, such as soil, milk, olive brine, vinegar, and from olive mill wastewater-contaminated biotopes were extensively studied for their biochemical arsenal and morphological features, i.e. cell, ascospore, and lipid body morphology. All strains were classified into the Ascomycota phylum. However, they showed great functional diversity, including different morphological and biochemical features, lipid production ability, and fatty acid profiles. Accordingly, the strains were placed in three different groups: Group I, which includes Candida species; Group II (Pichia and related); and Group III (Kluyveromyces marxianus strain CC1). Group I and II were characterized by a high percentage of oleic acid (41.6-65.3% of total lipids) while in Group III, linoleic acid was the major fatty acid (37.2%). Members of Group I and II were further grouped into subgroups according to their fatty acid composition. Among the newly isolated strains, Pichia etchellsii BM1 was able to accumulate around 25% wt/wt lipid per dry cell mass and thus characterized as oleaginous. Some other strains, such as Candida metapsilosis strain EL2, C. parapsilosis strain LV2, C. pararugosa strain BM24, and K. marxianus strain CC1, which are able to produce extracellular lipases, may be of interest for specific environmental applications and/or for the production of novel lipases.

Fatty acid biosynthesis during the life cycle of Debaryomyces etchellsii.

Fatty acid biosynthesis during the life cycle of the ascomycetous yeast Debaryomyces etchellsii cultivated on a non-fermentable substrate, i.e. glycerol, in nitrogen rich media (NRM) and nitrogen limited media (NLM) has been studied. Although considerable activities of key lipogenic enzymes, such as ATP citrate lyase (ACL) and malic enzyme (ME), were detected in vegetative cells during asexual proliferation (which occurred in the first growth stages in both NRM and NLM), lipid accumulation was restricted due to the high activities of NAD+-isocitrate dehydrogenase (NAD+-ICDH). A similar enzymatic profile has been found in ascii and free ascospores produced in NRM; thus lipid accumulation was low. On the contrary, very high activities of both ACL and ME and low activities of NAD+-ICDH were detected in ascii and free ascospores produced in NLM resulting in lipid accumulation. Neutral lipids (NL) were the predominant fraction of cellular lipids produced in vegetative cells and ascospores in both NRM and NLM. On the other hand, phospholipids (P) were the major polar lipids while glycolipids (G) were synthesized in low proportions. During transition from asexual to sexual phase, the percentage of NL increased with a significant decrease of P and, to a lesser extent, of G. High quantities of linoleic acid were found esterified in polar lipids, especially in P, during the vegetative stage of growth, while, with a few exceptions, during transition from asexual to sexual stage, linoleic acid concentration decreased markedly, mainly in P, while oleic acid concentration increased.

Biosynthesis of single-cell biomass from olive mill wastewater by newly isolated yeasts.

The aim of this study was to assess the potential of newly isolated yeast strains Schwanniomyces etchellsii M2 and Candida pararugosa BM24 to produce yeast biomass on olive mill wastewater (OMW). Maximum biomass yield was obtained at 75% (v/v) OMW, after 96 h of incubation at 30 °C and 5% (v/v) inoculum size. The optimal carbon/nitrogen (C/N) ratio was in the range of 8:1 to 10:1, and ammonium chloride was selected as the most suitable nitrogen source. Under these conditions, a maximum biomass production of 15.11 and 21.68 g L(-1) was achieved for Schwanniomyces etchellsii M2 and Candida pararugosa BM24, respectively. Proteins were the major constituents of yeast cells (35.9-39.4% dry weight), lipids were 2.8-5% dry weight, and ash ranged from 4.8 to 9.5 % dry weight. Besides biomass production, yeast strains were also able to reduce toxicity and polluting parameter levels of the spent OMW-based medium. The practical results presented show that pH rose from initial value of 5.5 to 7.24-7.45 after fermentation. Approximately 23.1-41.4% of the chemical oxygen demand (COD) and 15.4-19.2% of the phenolic compounds were removed. The removal of phenolic compounds was associated with their biodegradation and their partial adsorption on yeast cells.