Diversity and Antimicrobial Activities of Actinobacteria Isolated from Mining Soils in Midelt Region, Morocco.

Multidrug-resistant bacteria have emerged as a serious global health threat that requires, more than ever before, an urgent need for novel and more effective drugs. In this regard, the present study sheds light on the diversity and antimicrobial potential of Actinobacteria isolates in mining ecosystems. We have indeed investigated the production of bioactive molecules by the Actinobacteria isolated from abandoned mining areas in Midelt, Morocco, where average contents of lead (Pb) and cadmium (Cd) are higher than normal world levels. One hundred and forty-five Actinobacteria isolates were isolated and characterized based on morphological, chemotaxonomical, biochemical, and molecular data. Most of the 145 isolates were identified as Streptomyces. Isolates affiliated to the genera Amycolatopsis, Lentzea, Actinopolymorpha, and Pseudonocardia were also found. Antimicrobial producing potentials of Actinobacteria isolates were assessed against eight test microorganisms Gram+ and Gram- bacteria and yeast. Out of 145 isolates, 51 showed antimicrobial activities against at least one test microorganism. 31 isolates inhibited only bacteria, 7 showed activity against bacteria and Candida albicans, and 13 displayed activity against C. albicans solely. Our findings suggest that Actinobacteria isolated from natural heavy metal ecosystems may be a valuable source of novel secondary metabolites and therefore of new biotechnologically promising antimicrobial compounds.

Protective Effects of Two Halophilic Crude Extracts from Pseudomonas zhaodongensis and Bacillus stratosphericus against Memory Deficits and Anxiety- and Depression-Like Behaviors in Methionine-Induced Schizophrenia in Mice Focusing on Oxidative Stress Status.

Recently, the implication of oxidative stress in behavioral-like disorders has received a lot of attention. Many studies were interested in searching for new natural compounds with protective effects on behavioral-like disorders by focusing on oxidative stress as the main causal factor. Here, we assess the potential effect of cell-free extracts from halophilic bacteria on memory, anxiety, and depression-related behaviors in mice, as well as on cognitive deficits, negative symptoms, and some oxidative stress biomarkers in methionine-induced mice models of schizophrenia. Firstly, crude extracts of bacteria isolated from the Dead Sea were screened for their effects on memory and anxiety- and depression-like behaviors through Y-maze, elevated plus maze, and forced swimming test, respectively, using two doses 60 mg/kg and 120 mg/kg. Then, 120 mg/kg of two bacterial crude extracts, from two strains designated SL22 and BM20 and identified as Bacillus stratosphericus and Pseudomonas zhaodongensis, respectively, with significant contents of phenolic and flavonoid-like compounds, were selected for the assessment of cognitive and negative symptom improvement, as well as for their effects on oxidative stress status in methionine-induced mice models of schizophrenia using six groups (controls, methionine, crude extracts solely, and combinations of crude extracts and methionine). Results showed that the administration of the crude extracts caused a significant increase in the spontaneous alternations in the Y-maze task, the time spent in open arms of the elevated plus maze, and a decrease in immobility time in the forced swimming test in comparison with the control group. Furthermore, the administration of bacterial extracts seemed to diminish disorders related to cognitive and negative symptoms of schizophrenia and to improve the oxidative state in the temporal lobes, in comparison with the methionine group. Our findings suggest substantial antioxidant and anti-neuropsychiatric effects of the crude extracts prepared from Pseudomonas zhaodongensis strain BM20 and Bacillus stratosphericus strain SL22 and that further studies are needed to purify and to determine the active fraction from the extracts.

Occurrence, molecular and antimicrobial resistance of Enterococcus spp. isolated from raw cow's milk trade by street trading in Meknes city, Morocco.

Background: Enterococcus spp. belongs to a group of pathogens which are responsible for serious infections. This study aims at highlighting the raw milk microbiological contamination and at providing data for prevalence and antimicrobial resistance of Enterococcus spp. isolated from raw cow's milk marketed (without any pasteurization) by street traders. Methods: During the period of May 2015 through April 2016, 150 cow's raw milk samples were collected from street traders in Meknes city. They were examined for the identification of Enterococcus spp. using biochemical tests and 16S rRNA gene sequencing. The antimicrobial susceptibility of the isolates was determined. Results: The results showed that 11.3% (17/150) of samples were positive for the presence of Enterococcus spp., of which 64.7% were identified as Enterococcus faecalis, 17.6% as Enterococcus faecium, 11.8% as Enterococcus durans and 5.9% as Enterococcus hirae. The antimicrobial susceptibility showed that all Enterococcus spp. were resistant to ampicillin. The species E. faecalis, E. faecium, E. durans and E. hirae were resistant to streptomycin, with percentages of 52.9% (9/17), 11.8% (2/17), 11.8% (2/17), and 5.9% (1/17) respectively. All isolated strains of E. faecalis and E. faecium were resistant to tetracycline. The multiple antibiotic resistance index was elevated in the majority of Enterococcus spp., reaching values higher than 0.5, indicating a risk for public health. Conclusion: This study shows that the raw milk consumed by the population is contaminated with strains of Enterococcus resistant to antibiotics used in breeding for prophylactic purposes. This requires raising the awareness of those involved in the production and marketing of milk, so as to take measures to apply good hygienic practices and rationalize the use of zootechnical antibiotics.

A comparative study of the antioxidant scavenging activity of green tea, black tea and coffee extracts: a kinetic approach.

The antioxidant activities of three beverages, coffee, black tea and green tea, along with their major components, were investigated in terms of their reaction with the stable radical 2,2'-diphenyl-2-picrylhydrazyl (DPPH). We used a kinetic approach in parallel with quantification methods based on a fixed end-point to determine the scavenging efficiency of compounds abundant in these beverages during their reaction with DPPH using a stopped-flow spectrophotometer-based method. Ascorbic acid, (+)-catechin, (-)-epigallocatechin, tannic acid, and caffeic acid were selected as model antioxidants to study in coffee, black tea and green tea. We applied a second-order model to demonstrate similarities in the kinetics behavior of beverages and related compounds. Our findings showed the slopes k2(')((mol/L)(-1)s(-1)) and k2max(')((mol/L)(1)s(-1)) exhibited similar and correlated values; we suggest the variation in k2(') as a function of time is more informative about antioxidant properties than reaction with DPPH alone. We also used IC100 to test the reliability of the relative stoichiometry using a new comparative parameter "n", which was calculated as: n=c0DPPHIC100 (mol/L(mol/L)(-1), (mol/L)mlmg(-1) or molg(-1)).

Lipoamide dehydrogenase mediates retention of coronin-1 on BCG vacuoles, leading to arrest in phagosome maturation.

Mycobacterium tuberculosis evades the innate antimicrobial defenses of macrophages by inhibiting the maturation of its phagosome to a bactericidal phagolysosome. Despite intense studies of the mycobacterial phagosome, the mechanism of mycobacterial persistence dependent on prolonged phagosomal retention of the coat protein coronin-1 is still unclear. The present study demonstrated that several mycobacterial proteins traffic intracellularly in M. bovis BCG-infected cells and that one of them, with an apparent subunit size of M(r) 50,000, actively retains coronin-1 on the phagosomal membrane. This protein was initially termed coronin-interacting protein (CIP)50 and was shown to be also expressed by M. tuberculosis but not by the non-pathogenic species M. smegmatis. Cell-free system experiments using a GST-coronin-1 construct showed that binding of CIP50 to coronin-1 required cholesterol. Thereafter, mass spectrometry sequencing identified mycobacterial lipoamide dehydrogenase C (LpdC) as a coronin-1 binding protein. M. smegmatis over-expressing Mtb LpdC protein acquired the capacity to maintain coronin-1 on the phagosomal membrane and this prolonged its survival within the macrophage. Importantly, IFNgamma-induced phagolysosome fusion in cells infected with BCG resulted in the dissociation of the LpdC-coronin-1 complex by a mechanism dependent, at least in part, on IFNgamma-induced LRG-47 expression. These findings provide further support for the relevance of the LpdC-coronin-1 interaction in phagosome maturation arrest.

Mycobacterium bovis BCG attenuates surface expression of mature class II molecules through IL-10-dependent inhibition of cathepsin S.

We have previously shown that macrophage infection with Mycobacterium tuberculosis and M. bovis bacillus Calmette-Guérin (BCG) partially inhibits MHC class II surface expression in response to IFN-gamma. The present study examined the nature of class II molecules that do in fact reach the surface of infected cells. Immunostaining with specific Abs that discriminate between mature and immature class II populations showed a predominance of invariant chain (Ii)-associated class II molecules at the surface of BCG-infected cells suggesting that mycobacteria specifically block the surface export of peptide-loaded class II molecules. This phenotype was due to inhibition of IFN-gamma-induced cathepsin S (Cat S) expression in infected cells and the subsequent intracellular accumulation of alphabeta class II dimers associated with the Cat S substrate Ii p10 fragment. In contrast, infection with BCG was shown to induce secretion of IL-10, and addition of blocking anti-IL-10 Abs to cell cultures restored both expression of active Cat S and export of mature class II molecules to the surface of infected cells. Consistent with these findings, expression of mature class II molecules was also restored in cells infected with BCG and transfected with active recombinant Cat S. Thus, M. bovis BCG exploits IL-10 induction to inhibit Cat S-dependent processing of Ii in human macrophages. This effect results in inhibition of peptide loading of class II molecules and in reduced presentation of mycobacterial peptides to CD4(+) T cells. This ability may represent an effective mycobacterial strategy for eluding immune surveillance and persisting in the host.

Cross-talk between CD14 and complement receptor 3 promotes phagocytosis of mycobacteria: regulation by phosphatidylinositol 3-kinase and cytohesin-1.

The glycosylphosphatidyl anchored molecule CD14 to the monocyte membrane plays a prominent role in innate immunity, and the paradigms for CD14 selective signaling are beginning to be elucidated. In this study, transfected human monocytic cell line THP-1 and Chinese hamster ovary (CHO) fibroblastic cells were used to examine phagocytosis of Mycobacterium bovis bacillus Calmette-Guerin (BCG). Flow cytometry was combined with molecular and biochemical approaches to demonstrate a dual mechanism for BCG internalization involving either CD14 alone or a CD14-regulated complement receptor (CR)3-dependent pathway. Phagocytosis by CD14-positive THP-1 cells was attenuated by phosphatidylinositol-3 inhibitors LY294002 and wortmannin and experiments using transfected CHO cells showed substantial accumulation of phosphatidylinositol-3,4,5-trisphosphate at the BCG attachment site in CHO cells expressing CD14 and TLR2 suggesting that bacteria bind to CD14 and use TLR2 to initiate a PI3K signaling pathway. Additional experiments using blocking Abs showed that anti-TLR2 Abs inhibit phagocytosis of BCG by THP-1 cells. Furthermore, knockdown of cytohesin-1, a PI3K-regulated adaptor molecule for beta(2) integrin activation, specifically abrogated CD14-regulated CR3 ingestion of BCG consistent with the observation of physical association between CR3 and cytohesin-1 in cells stimulated with mycobacterial surface components. These findings reveal that mycobacteria promote their uptake through a process of "inside-out" signaling involving CD14, TLR2, PI3K, and cytohesin-1. This converts low avidity CR3 into an active receptor leading to increased bacterial internalization.

Mycobacterium bovis BCG urease attenuates major histocompatibility complex class II trafficking to the macrophage cell surface.

We have previously shown that Mycobacterium tuberculosis attenuates cell surface expression of major histocompatibility complex class II molecules in response to gamma interferon (IFN-gamma) by a mechanism dependent on intracellular sequestration of alpha,beta dimers. In this study we examined whether intracellular alkalinization due to mycobacterial urease could account for the defect in intracellular trafficking of class II molecules. Phagocytosis of wild-type Mycobacterium bovis BCG was associated with secretion of ammonia intracellularly, which increased substantially upon addition of exogenous urea to the culture medium. Increased intracellular ammonia, due to urea degradation by the bacterium, correlated with inhibition of class II surface expression. Conversely, no ammonia was detected in cells infected with a urease-negative mutant strain of M. bovis BCG, which also displayed a reduced effect on surface expression of class II molecules. A direct cause-effect relationship between urease and class II molecule trafficking was established with experiments where cells ingesting beads coated with purified urease showed an increased ammonia level and decreased surface expression of class II in response to IFN-gamma. In contrast to BCG, infection of macrophages with Mycobacterium smegmatis, which expresses relatively greater urease activity in cell-free culture, had a marginal effect on both the intracellular level of ammonia and class II expression. The limited effect of M. smegmatis was consistent with a failure to resist intracellular killing, suggesting that urease alone is not sufficient to resist macrophage microbicidal mechanisms and that this is required for a more distal effect on cell regulation. Our results demonstrate that alkalinization of critical intracellular organelles by pathogenic mycobacteria expressing urease contributes significantly to the intracellular retention of class II dimers.

Quantitative analysis of phagolysosome fusion in intact cells: inhibition by mycobacterial lipoarabinomannan and rescue by an 1alpha,25-dihydroxyvitamin D3-phosphoinositide 3-kinase pathway.

Macrophage cell membranes were labeled with PKH26 and subsequently incubated with latex beads to generate phagosomes surrounded by a red-fluorescent membrane suitable for flow cytometry. Following cell disruption and partial purification of phagosomes, these vesicles were readily distinguished from both cell debris and free beads released from disrupted vacuoles. Flow cytometry analysis of phagosomes stained with specific mAbs and FITC-labeled secondary antibodies showed progressive acquisition of both Rab7 and LAMP-1 consistent with movement along the endocytic pathway. Alternatively, macrophages were preloaded with the lysosomal tracer FITC-dextran before membrane labeling with PKH and incubation with latex beads. Phagosome-lysosome fusion was then quantified on the basis of the colocalization of red and green signals. Using these flow cytometry-based systems, we showed that co-internalization of beads with lysates of Mycobacterium tuberculosis, but not lysates from the nonpathogenic organism Mycobacterium smegmatis, markedly decreased phagosome acquisition of Rab7 and LAMP-1 and vesicle fusion with FITC-dextran-loaded lysosomes. Inhibition of phagolysosome fusion could be attributed, at least in part, to the mycobacterial cell wall glycolipid lipoarabinomannan, and further analysis showed complete rescue of phagosome maturation when cells were pretreated with vitamin D3 before exposure to lipoarabinomannan. Moreover, the ability of vitamin D3 to reverse the phenotype of phagosomes in the presence of the glycolipid was completely abrogated by LY-294002, suggesting that vitamin D3 promotes phagolysosome fusion via a phosphoinositide 3-kinase signaling pathway. These findings establish a robust platform technology based on labeling of phagocyte cell membranes and flow cytometry capable of supporting broad-based screens to identify microbial and other bioactive compounds that influence phagosome biology.

Biodegradation of polyphenols with immobilized Candida tropicalis under metabolic induction.

During olive oil production, large quantities of olive mill wastewater (OMW) are produced. This wastewater material, containing a high level of phenolic compounds, poses a serious environmental problem in almost all Mediterranean countries. Candida tropicalis YMEC14 was used as an extremophile strain to design an aerobic biotreatment process to detoxify OMW and reduce its polluting organic load. The process was enhanced by directing yeast metabolism towards biodegradation pathways using hexadecane as co-metabolite and by immobilizing yeast cells in calcium alginate beads. Under immobilization conditions, C. tropicalis YMEC14 grown at 40 degrees C in OMW supplemented with hexadecane resulted in 69.7%, 69.2% and 55.3% reduction of chemical oxygen demand, monophenols and polyphenols, respectively, after a 24-h fermentation cycle.