Ecophysiology and molecular phylogeny of bacteria isolated from alkaline two-phase olive mill wastes

The use of two-phase centrifugal decanters has been widely adopted in the olive oil extraction industry in order to reduce the huge quantities of wastewaters produced during the traditional three-phase extraction process. The resulting sludge-like byproduct, widely known as "alpeorujo", has a pH of 4-6, low water activity (a(w)) and high phytotoxicity. Addition of Ca(OH)(2) to alpeorujo, which is commonly performed at the olive oil mill to handle disposal problems related to acidic pH and odor emissions, creates an alkaline secondary waste (alkaline alpeorujo). Bacteria isolated from alkaline alpeorujo were cultured in order to investigate their physiological and phylogenetic characteristics. The bacterial population at neutral pH was estimated to be 6.0+/-0.4 x 10(7) cells g(-1) dw, while the bacterial population at pH 11 reached 2.1+/-0.3 x 10(5) cells g(-1) dw. Fourteen strains isolated from alkaline pH were halotolerant alkaliphiles, while seven isolates from neutral pH were moderate to extreme halotolerant or/and alkalitolerant bacteria. Based on 16S rRNA gene sequence analysis, four of the halotolerant alkaliphilic isolates showed 98.4-99.2% similarity to known sequences of Bacillus alcalophilus and Nesterenkonia lacusekhoensis, whereas ten isolates demonstrated low percentage similarities (94.4-96.9%) to the genera Idiomarina, Halomonas and Nesterenkonia. As concerns bacteria isolated from neutral pH, four isolates were associated with Corynebacterium, Novosphingobium, Serratia marcescens and Pseudomonas aeruginosa (98.3-99.9% similarities), while three isolates presented 96.5-97.2% sequence similarities to Rhodobacter, Pseudomonas and Ochrobactrum. At least six groups of isolates represent novel phylogenetic linkages among Bacteria.     

Occurrence of crude oil degrading bacteria in gasoline and diesel station soils

Microbial enumeration and identification were carried out on several oil contaminated soil samples collected from gasoline and diesel stations. Bacteria were the most dominant microbiota and were therefore classified to generic level. Eleven main genera were detected and Corynebacterium was the predominant genus in all the samples. Biochemical characterisation and substrate utilisation showed high percentage of lipolytic ability combined with high inorganic nitrogen utilisers. The ability of these cultures to degrade crude oil was tested individually and in mixed bacterial consortium at different temperatures and pH values. Maximum crude oil biodegradation of 78% was achieved using a bacterial consortium containing five cultures (Micrococcus sp. GS2-22, Corynebacterium sp. GS5-66, Flavobacterium sp. DS5-73, Bacillus sp. DS6-86 and Pseudomonas sp. DS10-129) with 1% crude oil at 30 degrees C and pH 7.5. Such a consortium may be useful for bioaugmentation of oil contaminated environments.     

Isolation,characterization and phylogeny of sponge-associated bacteria with antimicrobial activities from Brazil

Bacteria associated with marine sponges represent a rich source of bioactive metabolites. The aim of this study was to isolate and characterize bacteria with antimicrobial activities from Brazilian sponges. A total of 158 colony-forming units were isolated from nine sponge species. Among these, 12 isolates presented antimicrobial activities against pathogenic bacteria. Based on comparative sequence analysis of their 16S rRNA genes, the sponge-associated bacterial strains could be subdivided into three phylogenetically different clusters. Five strains were affiliated with Firmicutes (genera Bacillus and Virgibacillus), three with α-Proteobacteria (Pseudovibrio sp.) and four with γ-Proteobacteria (genera Pseudomonas and Stenotrophomonas). The sponge-associated bacterial strains Pseudomonas fluorescens H40 and H41 and Pseudomonas aeruginosa H51 exhibited antimicrobial activity against both Gram-negative and Gram-positive bacteria, including strains such as vancomycin-resistant Enterococcus faecium and multiresistant Klebsiella pneumoniae. Bacillus pumilus Pc31 and Pc32, Pseudovibrio ascidiaceicola Pm31 and Ca31 and Pseudovibrio denitrificans Mm37 strains were more effective against Gram-positive bacteria. These findings suggest that the identified strains may contribute to the search for new sources of antimicrobial substances, an important strategy for developing alternative therapies to treat infections caused by multidrug-resistant bacteria.     

Growth Inhibitory Activity of Deoxyribonucleic Acid-Containing Factor(s) Isolated from Lepromatous Lesions

Deoxyribonucleic acid-containing factor(s) isolated from Mycobacterium leprae suspensions obtained from lepromas of nine patients showed growth inhibitory activity against Micrococcus and both orange-red-pigmented and coccoid mutants of mycobacteria. No growth inhibition was observed for parent mycobacterial species, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus epidermidis.     

Secretion of phospholipase C by Pseudomonas aeruginosa.

The conditions necessary for the secretion of phospholipase C (phosphatidylcholine cholinephosphohydrolase) by Pseudomonas aeruginosa were studied. Enzyme secretion by washed cell suspensions required a carbon source and ammonium, potassium, and calcium ions. The calcium requirement could be substituted by magnesium and strontium but not by copper, manganese, cobalt, or zinc. During growth in liquid medium, cells secreted phospholipase C during late logarithmic and early stationary phases. Secretion was repressed by the addition of inorganic phosphate but not by organic phosphates, glucose, or sodium succinate. Studies with tetracycline indicated that de novo protein synthesis was necessary for the secretion of phospholipase C and that the exoenzyme was not released from a preformed periplasmic pool. Similarly, extraction of actively secreting cells with 0.2 M MgCl2 at pH 8.4 solubilized large quantities of the periplasmic enzyme alkaline phosphatase but insignificant amounts of phospholipase C. Bacteria continued to secrete enzyme for nearly 45 min after the addition of inorganic phosphate or rifampin.     

Abundance, diversity and antibiotics resistance pattern of Vibrio spp. in coral ecosystem of Kurusadai island

The abundance and species diversity of Vibrio associated with coral reef ecosystem of Kurusadai island, Tamil Nadu, India were evaluated. A total of twelve sampling locations including different live and dead coral surfaces, surrounding water and rock surface (negative control) were selected for the present study. Total viable and TCBS counts were found to be higher in dead coral as compared to that of live coral. Out of total 21 species of Vibrio isolated, 13 were identified up to species level based on biochemical tests and 16S rRNA gene sequence homology, while remaining 8 isolates did not show homology up to species level with any of the sequences available in the NCBI database. Moreover, these unidentified Vibrio spp. exhibited intra-species variation. This study indicated association of hitherto unknown Vibrio species with coral reef ecosystem of Kurusadai island. Assuming that only resistant bacteria can grow in the coral environment, susceptibility against a total of 20 antibiotics was evaluated. All the isolates exhibited resistance towards more than 6 antibiotics. Interestingly, none of the identified bacteria were previously reported to be of coral pathogen reflecting the healthy nature of the ecosystem. However, a continuous monitoring of the region will be prerequisite to envisage the role of these bacteria on the health status of the coral ecosystem.     

A survey of bacterial diversity in ticks,lice and fleas from Australia

We isolated bacteria from ticks, lice and fleas. Partial small subunit rRNA sequences were obtained for each isolate and the closest matches in the FastA database were determined. These bacteria were mostly Gram-positive (Firmicutes), although representatives from the Proteobacteria (alpha, beta, gamma subdivisions) and CFB group were also isolated. Most of the isolates we found were from genera that were present in most of the ectoparasites studied, but a few genera were restricted to one species of ectoparasite. The most commonly isolated genera were Stenotrophomonas, Staphylococcus, Pseudomonas, Acinetobacter and Bacillus. Species of Bacillus and Proteus, which have biopesticide potential, were found in some of these ectoparasites. Overall, the communities of bacteria were similar to those found in other studies of parasitic arthropods.     

Endophytic bacteria in Coffea arabica L

Eighty-seven culturable endophytic bacterial isolates in 19 genera were obtained from coffee plants collected in Colombia (n = 67), Hawaii (n = 17), and Mexico (n = 3). Both Gram positive and Gram negative bacteria were isolated, with a greater percentage (68%) being Gram negative. Tissues yielding bacterial endophytes included adult plant leaves, various parts of the berry (e.g., crown, pulp, peduncle and seed), and leaves, stems, and roots of seedlings. Some of the bacteria also occurred as epiphytes. The highest number of bacteria among the berry tissues sampled was isolated from the seed, and includes Bacillus , Burkholderia , Clavibacter , Curtobacterium , Escherichia , Micrococcus , Pantoea , Pseudomonas , Serratia , and Stenotrophomonas . This is the first survey of the endophytic bacteria diversity in various coffee tissues, and the first study reporting endophytic bacteria in coffee seeds. The possible role for these bacteria in the biology of the coffee plant remains unknown.     

Role of bacteria in mediating the oviposition responses of Aedes albopictus (Diptera: Culicidae)

The responses of Aedes albopictus to sources of oviposition attractants and stimulants were evaluated with a behavioral bioassay in which females attracted to odorants emanating from water were trapped on screens coated with an adhesive. Gravid mosquitoes were attracted to volatiles from larval-rearing water and soil-contaminated cotton towels. Bacteria were isolated from these substrates and from an organic infusion made with oak leaves. Through fatty acid-methyl ester analyses, six bacterial isolates from larval-rearing water, two isolates from soil-contaminated cotton towels, and three isolates from oak leaf infusion were identified to species. The response of gravid mosquitoes to these isolates was also evaluated in behavioral bioassays. Water containing Psychrobacter immobilis (from larval-rearing water), Sphingobacterium multivorum (from soil-contaminated cotton towels), and an undetermined Bacillus species (from oak leaf infusion) elicited significantly higher oviposition than control water without bacteria. Only volatiles collected from larval rearing water elicited significant electroantennogram responses in females.     

Polyphosphate accumulation and cell-surface properties by autochthonous bacteria from Argentinian Patagonia

Bacteria persisting in environments contaminated with polycyclic aromatic hydrocarbons (PAHs) have developed physiological mechanisms to counteract environmental stress. Inorganic polyphosphate accumulation represents one of these possible mechanisms. Likewise, properties such as cell-surface hydrophobicity, auto-aggregation, biofilm formation and bioemulsifying activity could facilitate interaction of microorganisms with hydrophobic organic compounds. In this work, these physiological properties were compared in indigenous bacteria from polluted sediments from Argentinian Patagonia, which were cultivated in two culture media (LBm and JPP) as a way to improve in the next future the PAHs removal. The highest hydrophobicity values were obtained in Rhodococcus strains, while Bacillus sp. B18 showed the highest auto-aggregation percentage and emulsion index. The highest numerical values of biofilm formation were determined in Rhodococcus sp. F27, Pseudomonas sp. P26, and Gordonia sp. H19 either on hydrophilic or on hydrophobic support. The qualitative and quantitative polyP determinations confirmed the presence of this biopolymer in the strains evaluated. The highest intracellular phosphate mean values were obtained in Bacillus sp. B18 in LBm and Rhodococcus erythropolis 20 in JPP. The bacteria evaluated belonging to different genera showed significant differences in their cell-surface characteristics, bioemulsifying activity and polyP accumulation. The low-cost JPP culture medium was selected for future contaminant removal studies.     

Characterization of Antarctic psychrotrophic bacteria with antibacterial activities against terrestrial microorganisms

Five-hundreds and eighty bacterial strains, isolated from various Antarctic marine sources and locations, were screened for antimicrobial activity against terrestrial microorganisms. Twenty-two Antarctic isolates (3.8%), mainly retrieved from the water column at Terra Nova Bay (Ross Sea), expressed antagonistic activity against one to three indicator organisms. Escherichia coli and Proteus mirabilis resulted as the more susceptible, followed by Micrococcus luteus and Bacillus subtilis. None of the isolates inhibited the growth of Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella enterica and the eukaryotic fungus Candida albicans.Active Antarctic isolates, identified by 16S rDNA sequencing and phenotypically characterized by classical methods, were phylogenetically affiliated to the Actinobacteria (16 strains) and the gamma-Proteobacteria (6 strains). Inhibition patterns, as well as phenotypic characteristics, highly vary for different isolates, even though they were affiliated to the same genus or closely related to the identical microorganism retrieved from the database, suggesting that these features were more likely strain-rather than species-specific.Results obtained from the present study confirm previous observations and highlight the potentiality of Antarctic marine bacteria as novel source of antibacterial substances.     

Biodiversity of chloramphenicol-resistant mesophilic heterotrophs from Southeast Asian aquaculture environments

In the present study, samples of pond water, sediment and farmed species were collected at 12 fish and shrimp farms in Malaysia, Thailand and Vietnam to determine the biodiversity and environmental distribution of chloramphenicol-resistant (CmR) mesophilic heterotrophs in Southeast Asian aquaculture sites. Following isolation on Iso-Sensitest agar supplemented with 35mug ml(-1) Cm and dereplication using (GTG)(5)-PCR fingerprinting, 557 genotypically unique CmR strains were subjected to polyphasic identification. The 557 mesophilic heterotrophic CmR isolates represented 18 different genera largely dominated by the genera Escherichia (40.2%), Pseudomonas (11.7%), Acinetobacter (11.1%), Klebsiella (7.5%) and Bacillus (5.9%). A total of 439 CmR isolates were further assigned to 31 described species or species groups, mainly including organisms that have been associated with various human opportunistic infections such as Escherichia coli (n=219), Pseudomonas putida (n=47), Klebsiella pneumoniae (n=38) and Acinetobacter baumannii (n=23). Strains of Escherichia, and most notably, of E. coli, were the only common group of CmR heterotrophs irrespective of country, sample type or farm type. Together with other predominant but less widespread groups such as acinetobacters and pseudomonads, the results of this biodiversity study suggest that E. coli can be regarded as a potential indicator of Cm resistance in Southeast Asian aquaculture environments.     

Bacterial flora of captive houbara (Chlamydotis undulata), kori (Ardeotis kori) and rufous-crested (Eupodotis ruficrista) bustards

A survey was carried out to investigate the aerobic bacterial flora of captive houbara (Chlamydotis undulata), kori (Ardeotis kori) and rufous-crested (Eupodotis ruficrista) bustards maintained at the National Avian Research Center, Abu Dhabi, United Arab Emirates. None of the houbara or rufous-crested bustards sampled were bred in captivity by the Center but four of the kori bustards sampled were captive-bred. Faecal, cloacal, oropharyngeal and skin samples (from under the wing) from clinically normal bustards were taken during routine handling of the birds. Samples were also taken from clinical cases, carcasses and failed eggs. Culture for fungi was also carried out. Bacteria belonging to several different genera were isolated from the three species, they included Staphylococcus aurais and Escherichia coli and also species of Aeromonas, Bacillus, Citrobacter, Enterobacter, Klebsiella, Micrococcus, Proteus, Pseu-domonas, Salmonella, Serratia, Sphingobacterium, Staphylococcus, Streptococcus and Vibrio. Fungal cultures yielded species of Aspergillus and Candida albicans. This appears to be the first bacterial survey carried out on bustards and shows that they are colonized by a wide range of bacteria, some of which were also isolated from disease conditions.     

Antimicrobial and Free Radical Scavenging Activities of Five Palestinian Medicinal Plants

Extracts from five indigenous Palestinian medicinal plants including Rosmarinus officinalis, Pisidium guajava, Punica granatum peel, grape seeds and Teucrium polium were investigated for antimicrobial and free radical scavenging activities against eight microorganisms, using well diffusion method. The microorganisms included six bacterial isolates (i.e. Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginos, Klebsiella pneumonia, Bacillus subtilis and Micrococcus luteus) and two fungal isolates (i.e. Candida albicans and Aspergillus niger). A standard antioxidant assay was performed on the plant extracts to assess their capability in scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH). Of the five tested plant extract, only Rosmarinus offcinalis extract contained significant antimicrobial activity against all eight microbial isolates including Pseudomonas aeruginosa. Extracts from other four plants exhibited a variable antimicrobial activity against all microorganisms, except Pseudomonas aeruginosa. Significant antioxidant activity was detected in all plant extracts. However, extracts from Pisidium guajava leaves contained significantly higher antioxidant activity compared to the other extracts tested. The antimicrobial and scavenging activities detected in this in vitro study in extracts from the five Palestinian medicinal plants suggest that further study is needed to identify active compounds to target diseases caused by a wide-spectrum pathogens.     

Recognition of greater diversity of Bacillus species and related bacteria in human faeces

In a study looking at culturable aerobic Actinobacteria associated with the human gastrointestinal tract, the vast majority of isolates obtained from dried human faeces belonged to the genus Bacillus and related bacteria. A total of 124 isolates were recovered from the faeces of 10 healthy adult donors. 16S rRNA gene sequence analyses showed the majority belonged to the families Bacillaceae (n=81) and Paenibacillaceae (n=3), with Bacillus species isolated from all donors. Isolates tentatively identified as Bacillus clausii (n=32) and Bacillus licheniformis (n=28) were recovered most frequently, with the genera Lysinibacillus, Ureibacillus, Oceanobacillus, Ornithinibacillus and Virgibacillus represented in some donors. Phenotypic data confirmed the identities of isolates belonging to well-characterized species. Representatives of the phylum Actinobacteria were recovered in much lower numbers (n=11). Many of the bacilli exhibited antimicrobial activity against one or more strains of Clostridium difficile, Clostridium perfringens, Listeria monocytogenes and Staphylococcus aureus, with some (n=12) found to have no detectable cytopathic effect on HEp-2 cells. This study has revealed greater diversity within gut-associated aerobic spore-formers than previous studies, and suggests that bacilli with potential as probiotics could be isolated from the human gut.     

Genetic diversity of endophytic bacteria which could be find in the apoplastic sap of the medullary parenchym of the stem of healthy sugarcane plants

The genetic diversity of 29 endophytic bacterial strains isolated from apoplastic sap of the medullary parenchym of the stem of healthy sugarcane plants grown in Cuba was analysed by Two Primers-Ramdom Amplified Polymorphic DNA fingerprinting (TP-RAPD) and 16S rRNA gene sequencing. The strains were distributed into 17 groups on the basis of their TP-RAPD patterns, and a representative strain from each group was subjected to 16S rRNA gene sequencing. Analysis of these sequences showed that the isolates belong to a wide variety of phylogenetic groups being closely related to species of genera Bacillus and Staphylococcus from Firmicutes, Microbacterium, Micrococcus and Kokuria from Actinobacteria, Rhizobium and Gluconacetobacter from alpha -Proteobacteria, Comamonas and Xanthomonas from beta-Proteobacteria, and Acinetobacter and Pantoea from gamma-Proteobacteria. These results show the complexity of the bacterial populations present in inner tissues of sugarcane, and indicate the interest and relevance of the studies on microbial diversity to improve our knowledge on the plant endophytic bacterial communities.     

Diversity and phylogeny of plant growth-promoting bacilli from moderately acidic soil

The molecular diversity of aerobic endospore-forming bacteria, typically Bacillus and its derived genera, has been investigated in various environments. However, there have been few investigations concerning Bacillus in acidic soils. In this study, the genotypic diversity and phylogenetic relationships among plant growth-promoting (PGP) bacilli isolated from the rice rhizosphere growing in acidic soils of Kerala (pH varying from 6.3 to 6.8) were investigated. For assessing their biocontrol potential and PGP attributes, 115 isolates were randomly selected and 49 isolates that were positive for multiple traits were selected. Metabolic characterization of representative strains, using the Biolog GP2 (Gram Positive) MicroPlate(TM) , revealed a large versatility with respect to carbohydrate utilization. Amplified ribosomal DNA restriction analysis revealed 13 clusters at 65% similarity level, which consisted of 1-21 strains. 16S rDNA partial sequencing assigned all the isolates, except for one, to the Bacillus genus, with close relatedness to Bacillus humi, B. megaterium, B. drentensis, B. pocheonensis, B. aestuarii, B. arbutinivorans, B. niacini, and Brevibacterium casei. The Bacillus species with different metabolic capabilities, PGP abilities, and genetic diversity found in this study are likely to have ecological relevance.     

Phosphatase activity is a constant feature of all isolates of all major species of the family Enterobacteriaceae.

In this study we evaluated phosphatase activity in members of the family Enterobacteriaceae by conventional methods and by a novel method. The novel method is based on the formation of bright-green-strained colonies by phosphatase-positive, but not phosphatase-negative, strains in the presence of a phosphate substrate, such as phenolphthalein monophosphate or 6-benzoylnaphthyl phosphate (6-BNP), and methyl green. A total of 1,055 strains belonging to 65 different species of Enterobacteriaceae were tested for green staining of the colonies in the presence of methyl green and either phenolphthalein monophosphate or 6-BNP and for phosphatase activity by three different conventional methods. With the sole exception of one Leminorella richardii type strain, all isolates of all of the species formed green-stained colonies in the presence of the substrate 6-BNP. All strains were phosphatase positive by all of the conventional methods.     

Phosphatase activity on the cell wall of Fonsecaea pedrosoi.

The activity of a phosphatase was characterized in intact mycelial forms of Fonsecaea pedrosoi, a pathogenic fungus that causes chromoblastomycosis. At pH 5.5, this fungus hydrolyzed p-nitrophenylphosphate (p-NPP) to p-nitrophenol (p-NP) at a rate of 12.78 +/- 0.53 nmol p-NP per h per mg hyphal dry weight. The values of Vmax and apparent Km for p-NPP hydrolyses were measured as 17.89 +/- 0.92 nmol p-NP per h per mg hyphal dry weight and 1.57 +/- 0.26 mmol/l, respectively. This activity was inhibited at increased pH, a finding compatible with an acid phosphatase. The enzymatic activity was strongly inhibited by classical inhibitors of acid phosphatases such as sodium orthovanadate (Ki = 4.23 micromol/l), sodium molybdate (Ki = 7.53 micromol/l) and sodium fluoride (Ki = 126.78 micromol/l) in a dose-dependent manner. Levamizole (1 mmol/l) and sodium tartrate (10 mmol/l), had no effect on the enzyme activity. Cytochemical localization of the acid phosphatase showed electrondense cerium phosphate deposits on the cell wall, as visualized by transmission electron microscopy. Phosphatase activity in F. pedrosoi seems to be associated with parasitism, as sclerotic cells, which are the fungal forms mainly detected in chromoblastomycosis lesions, showed much higher activities than conidia and mycelia did. A strain of F. pedrosoi recently isolated from a human case of chromoblastomycosis also showed increased enzyme activity, suggesting that the expression of surface phosphatases may be stimulated by interaction with the host.     

Occurrence of antimicrobial activities of bacteria from soybean leaf spots

Bacteria were isolated from leaf spots of field grown soybeans during two growing seasons. The leaf spots yielded up to 4 different species and a total population size of about 10⁷–10⁸ bacteria/cm². The majority of the 192 isolates belonged to the species Pseudomonas syringae pv. glycinea (55%), causing leaf spots of bacterial blight on soybeans, and Erwinia herbicola (22%). The remaining isolates included bacteria from other genera, but occurred occasionally. The determination of biological activity of the isolates demonstrated that a high percentage of strains from the group Erwinia/Enterobacter produced biological active substances against Escherichia coli (69%) and against Chlorella pyrenoidosa (88%). The majority of P. syringae pv. glycinea strains failed to do so. None of the isolates affected the growth of Geotrichum candidum. The E. herbicola strains showed clear antagonistic properties against a wide range of isolated bacteria. Four E. herbicola strains inhibited the growth of nearly all other E. herbicola isolates and 6 other strains were active against most of the P. syringae pv. glycinea isolates. However, antagonistic interactions among strains isolated from a distinct leaf spot were very rarely.