Physicochemical and Microbiological Water Quality Assessment of a Northwestern Algerian Dam: Detection of Ichtyopathogenic Bacteria.

Freshwater fish are often exposed to threats from anthropogenic or natural origins, such as pathogenic or opportunistic microorganisms responsible for a broad range of severe infections. In this study, we aimed to assess this microbiological threat to fish in an Algerian northwestern dam Sekkak (Tlemcen) by evaluating the diversity of ichtyopathogenic bacteria. In order to determine the water quality, physicochemical analyses of the dam water were carried out in situ. Ichtyopathogenic bacteria were isolated on selective media and identified by API galleries and molecular techniques (PCR and sequencing of the 16S rRNA gene). Besides, the antibiograms were constructed for all the isolates. The physicochemical and bacteriological analyses allowed us to classify the dam water as moderately polluted to polluted. Furthermore, an important diversity of ichtyopathogenic bacterial species was observed as Aeromonas hydrophila, Providencia rettgeri, and Pseudomonas aeruginosa were retrieved. The antibiogram test revealed notable resistance. The antibiotic family for which most resistances were found was the ß-lactam family, followed by aminoglycosides and macrolides. These results indicate that aquatic environments can shelter multidrug-resistant pathogenic bacteria representing a threat to the endemic fauna. Therefore, it is important to closely monitor these waters in order to improve the fish's living environment and ensure healthier production.

Quorum quenching and anti-biofilm activities of halotolerant Bacillus strains isolated in different environments in Algeria.

AIMS: The current study aimed to screen Bacillus strains with wide-spectrum quorum quenching (QQ) activity against N-acyl-l-homoserine lactones (AHLs), helpful in controlling virulence traits in Gram-negatives, including biofilm formation and also with anti-biofilm activity against Gram-positives. METHODS AND RESULTS: A total of 94 halotolerant strains of Bacillus isolated from soil and salt-lake sediment samples in Algeria were examined for the presence of QQ activity against AHLs, the presence of the aiiA gene, encoding an AHL lactonase enzyme typical of Bacillus spp., antimicrobial and anti-biofilm activities against Pseudomonas aeruginosa and Streptococcus mutans. Of all strains of Bacillus spp. isolated, 48.9% showed antibacterial activity. In addition, 40% of these isolates showed a positive QQ activity against long-chain AHLs, of which seven strains presented the aiiA gene. Among the species with broad-spectrum QQ activity, the cell extract of Bacillus thuringiensis DZ16 showed antibiofilm activity against P. aeruginosa PAO1, reducing 60% using the Amsterdam active attachment (AAA) biofilm cultivation model. In addition, the cell extract of B. subtilis DZ17, also presenting a broad-spectrum QQ activity, significantly reduced Strep. mutans ATCC 25175 biofilm formations by 63% and 53% in the xCELLigence and the AAA model, respectively, without affecting growth. Strain DZ17 is of particular interest due to its explicit halophilic nature because it can thrive at salinities in the range of 6%-30%. CONCLUSIONS: B. thuringiensis DZ16 and B. subtilis DZ17 strains have interesting antibacterial, QQ, and anti-biofilm activities. The high range of salinities accepted by these strains increases their biotechnological potential. This may open up their use as probiotics, the treatment and prevention of conventional and emerging infectious diseases. SIGNIFICANCE AND IMPACT OF STUDY: The use of safe, economical and effective probiotics is limited to control the infections related to multi-resistant bacteria. In our study, we provide two promising agents with QQ, anti-biofilm and antibacterial activities.

Evaluation of the Toxicity of Supernatant Cultures and Spore-Crystal Mixtures of Bacillus thuringiensis Strains Isolated from Algeria.

Bacillus thuringiensis (Bt) is the most used technology for biological control of insect pathogens worldwide. In order to select new Bt candidates challenging the emergence of insect's resistance, a mass bioassay and molecular screening was performed on an autochthonous collection. Toxicity assays against neonate larvae of three lepidopteran species (Mamestra brassicae, Grapholita molesta, and Spodoptera exigua) were conducted using spore-crystal mixtures and supernatant cultures of 49 Bt isolates harboring at least one gene coding for a lepidopteran-specific insecticidal protein. A threshold of 30% of "functional mortality" was used to discriminate between "nontoxic" and "toxic" isolates. The toxicity of many Bt isolates competed with that of Btk-HD1. However, only three of them (Bl4NA, Bl5NA, and Bl9NA) showed high toxicity in both spore-crystal mixtures and supernatant cultures against the three lepidopteran species. The Bt isolates Bl4NA and Bl9NA express a protein of 130 kDa whereas the Bt isolate Bl5NA expresses a protein of 65-70 kDa. The LC-MS/MS results indicate that the major peptides in the 130 kDa band of Bl9NA were Cry1Da, Cry1Ca, Cry1Ab, and Cry1Aa, and those in the 70 kDa band of Bl5NA were Cry1Aa and Cry1Ca. The evaluation of the protein content of the supernatants by comparison to Btk-HD1 indicates the overproduction of Vip3 proteins in these strains (most likely Vip3Aa in Bl4NA and Bl9NA and Vip3Ca in Bl5NA). In addition, these three Bt strains do not produce ß-exotoxins. Based on our results, the three selected strains could be considered promising candidates to be used in insect pest control.

Multidrug-resistant Opportunistic and Pathogenic Bacteria Contaminate Algerian Banknotes Currency.

Currency is one of the most exchanged items in human communities as it is used daily in exchange for goods and services. It is handled by persons with different hygiene standards and can transit in different environments. Hence, money can constitute a reservoir for different types of human pathogens. This study aimed to evaluate the potential of Algerian banknotes to shelter opportunistic pathogenic and multiresistant bacteria. To that end, 200 circulating notes of four different denominations were collected from various places and analyzed for their bacterial loads and contents. Besides, predominant strains were identified and characterized by biochemical and molecular methods, and their resistance profiles against 34 antibiotics were determined. Our results indicated that 100% of the studied banknotes were contaminated with bacteria. The total bacterial concentrations were relatively high, and different bacterial groups were grown, showing important diversity. In total, 48 predominant strains were identified as belonging to 17 genera. Staphylococcus and Micrococcus were the most prevalent genera, followed by Bacillus, Pseudomonas, and Acinetobacter. Antibiotic susceptibility testing showed that all the isolates harbored resistance to at least two molecules, and worrying resistance levels were observed. These findings prove that Algerian currency harbors opportunistic multiresistant bacteria and could potentially act as a vehicle for the spread of bacterial diseases and as a reservoir for antibiotic resistance genes among the community. Therefore, no cash payment systems should be developed and generalized to minimize cash handling and subsequent potential health risks.

In Vitro Cytotoxicity of Parasporins from Native Algerian Bacillus thuringiensis Strains Against Laryngeal and Alveolar Cancers.

Parasporins (PS), a class of non-insecticidal and non-hemolytic crystal proteins of Bacillus thuringiensis (Bt), are being explored as promising anti-cancer agents due to their specific toxicity to cancer cells. This work is considered as a first initiative aiming at investigating Algerian soil Bt isolates' activity and cytotoxic potential against cancer cells. A total of 48 Bacillus spp. were isolated from different sites in Algeria. Phenotypic and biochemical tests, 16S rDNA molecular identification, and microscopic observation of crystal have confirmed the identification of Bt for ten strains. A screening for non-hemolytic crystalline proteins was performed. Extraction, purification, and activation of non-hemolytic proteins by chromatographic analysis yielded several polypeptides of different molecular weights. A purified PS1, with pro-protein of 81 kDa and several peptides with different molecular weights (18-58 kDa) after activation by trypsin, has been identified from the strain BDzG. The NH2-terminal sequence deciphered in BLAST analysis showed homology to a Bt PS1 protein. Moreover, the screening of parasporin-1 (PS1) gene has also been performed. Cytocidal activity against human epithelial type 2 (HEp2) cells, considered to originate from a human laryngeal carcinoma, was observed with an IC50 equal to 2.33 µg/ml, while moderate cytotoxicity against adenocarcinomic human alveolar basal epithelial (A549) cells has been shown with IC50 equal to 18.54 µg/ml. No cytotoxicity against normal cells was noted. Fluorescence microscopy revealed a condensed or fragmented chromatin indicating the apoptotic death of HEp2 cells. Thus, Bt PS-producer isolated from Algerian soil might have a potential to join the arsenal of natural anti-cancer drugs with high therapeutic potential.

Identification of a New Serine Alkaline Peptidase from the Moderately Halophilic Virgibacillus natechei sp. nov., Strain FarDT and its Application as Bioadditive for Peptide Synthesis and Laundry Detergent Formulations.

A new peptidase designated as SAPV produced from a moderately halophilic Virgibacillus natechei sp. nov., strain FarDT was investigated by purification to homogeneity followed by biochemical and molecular characterization purposes. Through optimization, it was determined that the optimum peptidase activity was 16,000 U/mL. It was achieved after 36 h incubation at 35°C in the optimized enzyme liquid medium (ELM) at pH 7.4 that contains only white shrimp shell by-product (60 g/L) as sole energy and carbon sources. The SAPV enzyme is a monomer protein with a molecular mass of 31 kDa as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance liquid chromatography (HPLC) gel filtration chromatography. The sequence of its NH2-terminal amino-acid residues showed homology with those of Bacillus peptidases S8/S53 superfamily. The SAPV showed optimal activity at pH 9 and 60°C. Irreversible inhibition of enzyme activity by diiodopropyl fluorophosphates (DFP) and phenylmethanesulfonyl fluoride (PMSF) confirmed its belonging to the serine peptidases. Considering its interesting biochemical characterization, the sapV gene was cloned, sequenced, and heterologously overexpressed in the extracellular fraction of E. coli BL21(DE3)pLysS. The biochemical properties of the recombinant peptidase (rSAPV) were similar to those of the native one. The highest sequence identity value (97.66%) of SAPV was obtained with peptidase S8 from Virgibacillus massiliensis DSM 28587, with 9 amino-acid residues of difference. Interestingly, rSAPV showed an outstanding and high resistance to several organic solvents than SPVP from Aeribacillus pallidus VP3 and Thermolysin type X. Furthermore, rSAPV exhibited an excellent detergent stability and compatibility than Alcalase 2.4 L FG and Bioprotease N100L. Considering all these remarkable properties, rSAPV has attracted the interest of industrialists.

Insights into the draft genome sequence of bioactives-producing Bacillus thuringiensis DNG9 isolated from Algerian soil-oil slough.

Bacillus thuringiensis is widely used as a bioinsecticide due to its ability to form parasporal crystals containing proteinaceous toxins. It is a member of the Bacillus cereus sensu lato, a group with low genetic diversity but produces several promising antimicrobial compounds. B. thuringiensis DNG9, isolated from an oil-contaminated slough in Algeria, has strong antibacterial, antifungal and biosurfactant properties. Here, we report the 6.06 Mbp draft genome sequence of B. thuringiensis DNG9. The genome encodes several gene inventories for the biosynthesis of bioactive compounds such as zwittermycin A, petrobactin, insecticidal toxins, polyhydroxyalkanoates and multiple bacteriocins. We expect the genome information of strain DNG9 will provide another model system to study pathogenicity against insect pests, plant diseases, and antimicrobial compound mining and comparative phylogenesis among the Bacillus cereus sensu lato group.

Biodegradation of anionic surfactants by Alcaligenes faecalis, Enterobacter cloacae and Serratia marcescens strains isolated from industrial wastewater.

Pseudo-persistent organic pollutants, such as anionic surfactants (AS), are nowadays among the more complex problems that threaten the aquatic environments and other environmental compartments. The present work describes the identification and efficiency of a consortium, isolated from Algerian industrial wastewater, to remove three anionic surfactants (i.e., sodium dodecylbenzenesulfonate (SDBS), sodium dodecyl sulfate (SDS) and sodium lauryl ether sulfate (SLES)). The genetic analysis of 16S rRNA indicated that these strains are Alcaligenes faecalis, Enterobacter cloacae and Serratia marcescens. Under aerobic conditions, pH 7.0 and optimum temperature of 30 °C, the mixed consortium allowed to degrade 85.1% of initial SDBS amount after 144 h of incubation with half-life of 20.8 h. While E. cloacae and S. marcescens pure strains eliminated 46% and 41% less SDBS respectively. Evenly, SDS was degraded at only 23.71% by A. faecalis strain. However, the degradation capacity of SDS by the consortium was very high (94.2%) with a half-life of 9.8 h. The SLES anionic surfactant showed a lower biodegradation by the consortium (47.53%) due to the presence of ether oxide units in the chemical structure of SLES which induced toxicity to the medium. The investigation of the biodegradation of this type of organic pollutants by microorganisms has recently become a key issue for the environmental protection area.

Draft Genome Sequence of Bacillus paralicheniformis F47, Isolated from an Algerian Salty Lake.

Bacillus paralicheniformis F47 was isolated from a salty lake in Ain Baida-Ouargla, southern Algeria. The genome contains genes for the production of several bioactive secondary metabolites, including the siderophore bacillibactin, the lipopeptides fengycin, surfactin, and lichenysin, the antibiotics bacitracin and kanosamine, and a putative circular bacteriocin.

Bacillus amyloliquefaciens ssp. plantarum F11 isolated from Algerian salty lake as a source of biosurfactants and bioactive lipopeptides.

In this study, we identified a new Bacillus strain isolated from an Algerian salty lake that produces metabolites that are active against Gram-positive and Gram-negative bacteria, as well as fungal pathogens. The draft genome sequence of the strain is presented herein. Genome sequence analysis identified the strain to be B. amyloliquefaciens subspecies plantarum F11, and showed that the strain carries the gene clusters for the production of a number of bioactive and surface-active compounds. These include the lipopeptides surfactin and fengycin, antibacterial polyketides macrolactin and bacillaene, and a putative novel lanthipeptide, among others. Through an activity-guided purification method using hydrophobic interaction chromatographic techniques, we confirmed the ability of the strain to produce fengycin lipopeptides. The identities of the isolated fengycin homologs were ascertained through tandem mass spectrometry.

Multiresistant opportunistic pathogenic bacteria isolated from polluted rivers and first detection of nontuberculous mycobacteria in the Algerian aquatic environment.

Opportunistic infections constitute a major challenge for modern medicine mainly because the involved bacteria are usually multiresistant to antibiotics. Most of these bacteria possess remarkable ability to adapt to various ecosystems, including those exposed to anthropogenic activities. This study isolated and identified 21 multiresistant opportunistic bacteria from two polluted rivers, located in Algiers. Cadmium, lead, and copper concentrations were determined for both water samples to evaluate heavy metal pollution. High prevalence of Enterobacteria and non-fermentative Gram-negative rods was found and a nontuberculous Mycobacterium (NTM) strain was isolated. To the best of our knowledge, this is the first detection of NTM in the Algerian environment. The strains were tested for their resistance against 34 antibiotics and 8 heavy metals. Multiple antibiotics and heavy metals resistance was observed in all isolates. The two most resistant strains, identified as Acinetobacter sp. and Citrobacter freundii, were submitted to plasmid curing to determine if resistance genes were plasmid or chromosome encoded. Citrobacter freundii strain P18 showed a high molecular weight plasmid which seems to code for resistance to zinc, lead, and tetracycline, at the same time. These findings strongly suggest that anthropized environments constitute a reservoir for multiresistant opportunistic bacteria and for circulating resistance genes.

Draft Genome Sequences of Bacillus cereus E41 and Bacillus anthracis F34 Isolated from Algerian Salt Lakes.

Two strains of Bacillus, B. cereus E41 and B. anthracis F34, were isolated from a salt lake in Aïn M'lila-Oum El Bouaghi, eastern Algeria, and Ain Baida-Ouargla, southern Algeria, respectively. Their genomes display genes for the production of several bioactive secondary metabolites, including polyhydroxyalkanoate, iron siderophores, lipopeptides, and bacteriocins.

Assessment of the Antimicrobial Activity and the Entomocidal Potential of Bacillus thuringiensis Isolates from Algeria.

This work represents the first initiative to analyze the distribution of B. thuringiensis in Algeria and to evaluate the biological potential of the isolates. A total of 157 isolates were recovered, with at least one isolate in 94.4% of the samples. The highest Bt index was found in samples from rhizospheric soil (0.48) and from the Mediterranean area (0.44). Most isolates showed antifungal activity (98.5%), in contrast to the few that had antibacterial activity (29.9%). A high genetic diversity was made evident by the finding of many different crystal shapes and various combinations of shapes within a single isolate (in 58.4% of the isolates). Also, over 50% of the isolates harbored cry1, cry2, or cry9 genes, and 69.3% contained a vip3 gene. A good correlation between the presence of chitinase genes and antifungal activity was observed. More than half of the isolates with a broad spectrum of antifungal activity harbored both endochitinase and exochitinase genes. Interestingly, 15 isolates contained the two chitinase genes and all of the above cry family genes, with some of them harboring a vip3 gene as well. The combination of this large number of genes coding for entomopathogenic proteins suggests a putative wide range of entomotoxic activity.

Virgibacillus ainsalahensis sp. nov., a Moderately Halophilic Bacterium Isolated from Sediment of a Saline Lake in South of Algeria.

A Gram-positive, moderately halophilic, endospore-forming bacterium, designated MerVT, was isolated from a sediment sample of a saline lake located in Ain Salah, south of Algeria. The cells were rod shaped and motile. Isolate MerVT grew at salinity interval of 0.5-25% NaCl (optimum, 5-10%), pH 6.0-12.0 (optimum, 8.0), and temperature between 10 and 40 °C (optimum, 30 °C).The polar lipids comprised diphosphatidylglycerol, phosphatidylglycerol, a glycolipid, a phospholipid, and two lipids, and MK-7 is the predominant menaquinone. The predominant cellular fatty acids were anteiso C15:0 and anteiso C17:0. The DNA G+C content was 45.3 mol%. Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that strain MerVT was most closely related to Virgibacillus halodenitrificans (gene sequence similarity of 97.0%). On the basis of phenotypic, chemotaxonomic properties, and phylogenetic analyses, strain MerVT (=DSM = 28944T) should be placed in the genus Virgibacillus as a novel species, for which the name Virgibacillus ainsalahensis is proposed.

Mycobacterium icosiumassiliensis sp. nov., a New Member in the Mycobacterium terrae Complex Isolated from Surface Water in Algeria.

An acid-fast, rapidly growing, rod-shaped microorganism designated 8WA6 was isolated from a lake in Algiers, Algeria. The lake water was characterized by a temperature of 18 °C, a pH of 7.82, a copper concentration of 8.6 µg/L, and a cadmium concentration of 0.6 µg/L. First-line molecular identification confirmed the 8WA6 isolate to be a member of the Mycobacterium terrae complex, sharing 99.4 % 16S rRNA gene sequence similarity with M. arupense AR-30097, 98.2 % partial hsp65 gene sequence similarity with M. terrae 28K766, and 97.1 % partial rpoB gene sequence similarity with Mycobacterium sp. FI-05396. Its 4.89-Mb genome exhibits a 66.8 GC % and an average nucleotide identity of 64.5 % with M. tuberculosis, 70.5 % with M. arupense, and 75 % with M. asiaticum. In the M. terrae complex, Mycobacterium 8WA6 was unique in exhibiting growth at 42 °C, negative reaction for nitrate reduction, urease activity and Tween 80 hydrolysis, and a positive reaction for α-glucosidase and ß-glucosidase. Its protein profile determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry revealed a unique spectrum similar to M. arupense and M. terrae, exhibiting eleven specific peaks at 3787.791, 4578.019, 6349.630, 6855.638, 7202.310, 8149.608, 8775.257, 10,224.588, 10,484.116, 12,226.379, and 12,636.871 m/z. Minimal inhibitory concentrations (MIC) for antibiotics, determined by microdilution, indicated a broad spectrum resistance, except for rifabutin (MIC, 0.5 g/L) and cefoxitin (MIC, 16 g/L). We concluded that the 8WA6 isolate is a representative isolate of a previously undescribed species in the M. terrae complex, which was named M. icosiumassiliensis sp. nov. with strain 8WA6 (Collection de Souches de l'Unité des Rickettsies, CSUR P1561, Deutsche Sammlung von Mikroorganismen und Zellkulturen, DSM 100711) as the type strain.

Screening for genes coding for putative antitumor compounds, antimicrobial and enzymatic activities from haloalkalitolerant and haloalkaliphilic bacteria strains of Algerian Sahara Soils.

Extreme environments may often contain unusual bacterial groups whose physiology is distinct from those of normal environments. To satisfy the need for new bioactive pharmaceuticals compounds and enzymes, we report here the isolation of novel bacteria from an extreme environment. Thirteen selected haloalkalitolerant and haloalkaliphilic bacteria were isolated from Algerian Sahara Desert soils. These isolates were screened for the presence of genes coding for putative antitumor compounds using PCR based methods. Enzymatic, antibacterial, and antifungal activities were determined by using cultural dependant methods. Several of these isolates are typical of desert and alkaline saline soils, but, in addition, we report for the first time the presence of a potential new member of the genus Nocardia with particular activity against the yeast Saccharomyces cerevisiae. In addition to their haloalkali character, the presence of genes coding for putative antitumor compounds, combined with the antimicrobial activity against a broad range of indicator strains and their enzymatic potential, makes them suitable for biotechnology applications.

The world bacterial biogeography and biodiversity through databases: a case study of NCBI Nucleotide Database and GBIF Database.

Databases are an essential tool and resource within the field of bioinformatics. The primary aim of this study was to generate an overview of global bacterial biodiversity and biogeography using available data from the two largest public online databases, NCBI Nucleotide and GBIF. The secondary aim was to highlight the contribution each geographic area has to each database. The basis for data analysis of this study was the metadata provided by both databases, mainly, the taxonomy and the geographical area origin of isolation of the microorganism (record). These were directly obtained from GBIF through the online interface, while E-utilities and Python were used in combination with a programmatic web service access to obtain data from the NCBI Nucleotide Database. Results indicate that the American continent, and more specifically the USA, is the top contributor, while Africa and Antarctica are less well represented. This highlights the imbalance of exploration within these areas rather than any reduction in biodiversity. This study describes a novel approach to generating global scale patterns of bacterial biodiversity and biogeography and indicates that the Proteobacteria are the most abundant and widely distributed phylum within both databases.

Bioinformatics and molecular approaches to detect NRPS genes involved in the biosynthesis of kurstakin from Bacillus thuringiensis.

Degenerated primers designed for the detection by polymerase chain reaction of nonribosomal peptide synthetases (NRPS) genes involved in the biosynthesis of lipopeptides were used on genomic DNA from a new isolate of Bacillus thuringiensis CIP 110220. Primers dedicated to surfactin and bacillomycin detection amplified sequences corresponding respectively to the surfactin synthetase operon and to a gene belonging to a new NRPS operon identified in the genome of B. thuringiensis serovar pondicheriensis BSCG 4BA1. A bioinformatics analysis of this operon led to the prediction of an NRPS constituted of seven modules beginning with a condensation starter domain and which could be involved in the biosynthesis of a heptalipopeptide similar to kurstakin. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF-MS) performed on whole cells of B. thuringiensis CIP 110220 confirmed the production of kurstakin by this strain. The kurstakin operon was thus used to design a new set of degenerated primers specifically to detect kurstakin genes. These primers were used to screen kurstakin producers in a collection of nine B. thuringiensis strains isolated from different areas in Algeria and two from the Pasteur Institute collection. For eight among the 11 tested strains, the amplified fragment matched with an operon similar to the kurstakin operon and found in the newly sequenced genome of Bacillus cereus or B. thuringiensis serovar pulsiensis, kurstaki, and thuringiensis. Kurstakin production was detected by MALDI-ToF-MS on whole cells for six strains. This production was compared with the spreading of the strains and their antimicrobial activity. Only the spreading can be correlated with the kurstakin production.

Application of the resazurin microtitre assay for detection of multidrug resistance in Mycobacterium tuberculosis in Algiers.

This study assessed the performance of a rapid, low-cost, colorimetric method, the resazurin microtitre assay (REMA) plate method, for the detection of resistance to isoniazid and rifampicin in 136 clinical isolates of Mycobacterium tuberculosis from two hospitals in Algiers. MICs were determined and the results were compared with those obtained with the conventional proportion method on Löwenstein-Jensen medium. Excellent results were obtained for the REMA plate method, with a sensitivity of 100 % for both isoniazid and rifampicin and a specificity of 98.3 and 99.2 %, respectively. The REMA plate method appears to be a reliable method for the rapid determination of multidrug-resistant tuberculosis and is a good alternative for use in resource-limited countries such as Algeria.