Characterization of <Emphasis Type="Italic">Bacillus</Emphasis> phage-K2 isolated from chungkookjang,a fermented soybean foodstuff

An investigation of a virulent Bacillus phage-K2 (named Bp-K2) isolated from chungkookjang (a fermented soybean foodstuff) was made. Bp-K2 differed in infectivity against a number of Bacillus subtilis strains including starter strains of chungkookjang and natto, being more infectious to Bacillus strains isolated from the chungkookjang, but much less active against a natto strain. Bp-K2 is a small DNA phage whose genome size is about 21 kb. Bp-K2 is a tailed bacteriophage with an isometric icosahedral head (50 nm long on the lateral side, 80 nm wide), a long contractile sheath (85–90 nm × 28 nm), a thin tail fiber (80–85 nm long, 10 nm wide), and a basal plate (29 nm long, 47 nm wide) with a number of spikes, but no collar. The details of the structures of Bp-K2 differ from natto phage ϕBN100 as well as other known Bacillus phages such as SPO1-like or ϕ 29-like viruses. These data suggest that Bp-K2 would be a new member of the Myoviridae family of Bacillus bacteriophages.     

Rhamnolipid-producing thermophilic bacteria of species <Emphasis Type="Italic">Thermus</Emphasis> and <Emphasis Type="Italic">Meiothermus</Emphasis>

Novel rhamnolipid-producing strains of three thermophilic bacteria, Thermus sp., T. aquaticus and Meiothermus ruber were identified that have not been previously described as rhamnolipid producers. Rhamnolipids were extracted from supernatant and further purified by thin-layer chromatography. Mass spectrometry with negative electrospray ionization revealed 77 rhamnolipid homologues varying in chain length and unsaturation. Tandem mass spectrometry identified mono-rhamnolipid and di-rhamnolipid homologues containing one or two 3-hydroxy-fatty acids, saturated, monounsaturated or diunsaturated, even- or odd-chain, up to unusual long chains with 24 carbon atoms. The stereochemistry of rhamnose was L and that of 3-hydroxy-fatty acids was R, the position of double bonds in monoenoic acids was cis ω-9. All three strains produced a rhamnolipid that differs in structure from Pseudomonas aeruginosa rhamnolipids and exhibits excellent surfactant properties. Importantly, in comparison to P. aeruginosa both strains, i.e., Thermus and Meiothermus, are Biosafety level 1 microorganisms and are not pathogenic to humans.     

Characterization of a T7-Like Lytic Bacteriophage of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> B5055: A Potential Therapeutic Agent

Characterization of bacteriophages to be used prophylactically or therapeutically is mandatory, as use of uncharacterized bacteriophages is considered as one of the major reasons of failure of phage therapy in preantibiotic era. In the present study, one lytic bacteriophage, KPO1K2, specific for Klebsiella pneumoniae B5055, with broad host range was selected for characterization. As shown by TEM, morphologically KPO1K2 possessed icosahedral head with pentagonal nature with apex to apex head diameter of about 39 nm. Presence of short noncontractile tail (10 nm) suggested its inclusion into family Podoviridae with a designation of T7-like lytic bacteriophage. The phage growth cycle with a latent period of 15 min and a burst size of approximately 140 plaque forming units per infected cell as well as a genome of 42 kbps and structural protein pattern of this bacteriophage further confirmed its T7-like characteristics. Phage was stable over a wide pH range of 4–11 and demonstrated maximum activity at 37°C. After injection into mice, at 6 h, a high phage titer was seen in blood as well as in kidney and urinary bladder, though titers in kidney and urinary bladder were higher as compared to blood. Phage got cleared completely in 36 h from blood while from kidneys and urinary bladder its clearance was delayed. We propose the use of this characterized phage, KPO1K2, as a prophylactic/therapeutic agent especially for the treatment of catheter associated UTI caused by Klebsiella pneumoniae.     

Detection and quantitation of colored deposit-forming <Emphasis Type="Italic">Meiothermus</Emphasis> spp. in paper industry processes and end products

Colored biofilms cause problems in paper industry. In this work we used real-time PCR to detect and to quantitate members of the genus Meiothermus from the process samples and end products from 24 machines manufacturing pulp, paper and board in four countries. The results obtained from 200 samples showed the importance of members of the genus Meiothermus as ubiquitous biofoulers in paper machines. This genus was the dominant biofouler in some mills. From ≤10⁴ to 10¹¹ copies of Meiothermus 16S rRNA genes were found per gram of process deposit (wet weight). Meiothermus spp. were found in paper and board products with colored defects and connection between deposit-forming microbes and end-product spots was shown. 16S rRNA gene sequences of 29 biofilm producing bacterial isolates from different mills were determined. Based on sequence data, 25 of the isolates were assigned to the genus Meiothermus, with Meiothermus silvanus and M. ruber as the most frequent species.     

BVPaP-3, a T7-Like Lytic Phage of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>: Its Isolation and Characterisation

The increasing emergence of antibiotic-resistant bacteria has produced a growing interest among scientists in bacteriophages as alternative antimicrobial agents. This article reports a lytic phage against an antibiotic-resistant strain of Pseudomonas aeruginosa. Phage BVPaP-3 is a member of the Podoviridae family and morphologically similar to the T7-like phage gh-1. The phage has a hexagonal head of 58–59 nm in diameter and a short tail of 10 × 8 nm. It is stable at a wide range of pH (6–10) and temperatures (4–40°C). Its optimal growth temperature is 37°C and the adsorption rate constant is 1.19 × 10⁻⁹. Latent and eclipse periods are 20 and 15 min, respectively, and the burst size is 44 after 35 min at 37°C. The phage has a DNA size of 41.31 kb and a proteome of 11 proteins. The major protein is 33 kDa in size.     

Occurrence of Vibrio parahaemolyticus and Its Specific Phages from Shrimp Ponds in East Coast of India

Vibrio parahaemolyticus is a natural microflora of marine and coastal water bodies and associated with mortality of larval shrimp in penaeid shrimp in ponds. Bacteriophages occur virtually in all places where their hosts exist. In this study, total distribution of V. parahaemolyticus and its phages were examined in shrimp ponds, seawater, estuary, animal surface, and tissues. Total vibrio count in sediments of two ponds was found to be 2.6 × 10³ and 5.6 × 10³ cfu/g. Incidence of V. parahaemolyticus in the ponds was close, while it was markedly higher in the animal surface and tissue samples. Biochemically identified eight strains of V. parahaemolyticus (V1, V3–V6, V9, V11, and V12) were taken for further infection studies with bacteriophage. Totally five bacteriophages capable of infecting V. parahaemolyticus MTCC-451 strain were isolated from all the samples. One of the isolated bacteriophage Vp1 from estuary was able to lyse all the isolated V. parahaemolyticus strains we used. Therefore, the morphology of Vp1 was estimated in TEM. Vp1 phage head measuring approximately about 50–60 nm diameter with icosahedral outline and a contractile tails of diameter 7 nm and length 100 nm and it was identified as Myoviridae. Therefore, the phages have the potential application in destroying bacterial pathogens.     

Batch and fed-batch bioreactor cultivations of a Thermus species with thermophilic BTEX-degrading activity

The thermophilic bacterium, Thermus species ATCC 27978, which is capable of aerobically degrading benzene, toluene, ethylbenzene, and the xylenes (BTEX), was cultured in 5-1 fermentors on a Castenholz salts-tryptone medium. This bacterium can be cultivated more conveniently at 45 °C, a temperature substantially lower than its optimal growth temperature (approx. 60 °C). Yet, the washed harvested cells from such cultures display the same initial BTEX-degrading activity as those when Thermus sp. is grown at its higher optimal temperature. Two bioreactor cultivation modes, batch and fed batch, were investigated. More biomass and more BTEX-degrading activity (assayed at 60 °C) were generated in fed-batch cultures than in the growth-limited batch cultures. The former yielded a biomass concentration of 2.5 g dry cell weight (DCW) l⁻¹ and whole-cell degrading specific activities of 7.6 ± 1.3, 10.1 ± 1.9, 9.8 ± 2.1, 2.3 ± 0.5, and 4.6 ± 0.9 nmol degraded (mg DCW)⁻¹ min⁻¹ for benzene, toluene, ethylbenzene, m-xylene, and the o- plus p-xylenes (unresolved mixture), respectively. Although the formation of cellular BTEX-degrading activity is growth-associated, a slow to moderate specific growth rate of 0.02–0.07 h⁻¹ favors the production of BTEX-degrading activity, while a high growth rate, of the order of 0.16 h⁻¹, is detrimental to its production. The washed harvested Thermus sp. cells were capable of degrading BTEX over a broad range of thermophilic incubation temperatures, 45–77 °C. Received: 28 June 1996 / Received revision: 31 December 1996 / Accepted: 31 January 1997     

Characterization of <Emphasis Type="Italic">Deinococcus sahariens</Emphasis> sp. nov., a radiation-resistant bacterium isolated from a Saharan hot spring

An ultraviolet-radiation-resistant, Gram-positive, orange-pigmented, thermophilic and strictly aerobic cocci was isolated from Saharan water hot spring in Tunisia. The newly isolated bacterium, designated HAN-23^T, was identified based on polyphasic taxonomy including genotypic, phenotypic and chemotaxonomic characterization. Phylogenetic analysis based on 16S rRNA gene sequences placed this strain within Deinococcus genus. However, strain HAN-23^T is different from recognized species of the genus Deinococcus, showing less than 94.0% similarity values to its closest relatives. The predominant cellular fatty acids determined by gas chromatography were iso-C_15:0, iso-C_17:0 and iso C_17:1 ω9c. The major respiratory quinone was MK-8. The DNA G + C content was 66.9 mol%. DNA–DNA hybridization measurements revealed low DNA relatedness (6%) between the novel isolate and its closest neighbor, the type strain Deinococcus geothermalis DSM 11300. On the basis of the phenotypic, chemotaxonomic and phylogenetic data, strain HAN-23^T represents a novel species of the genus Deinococcus, for which the name Deinococcus sahariens sp. nov. is proposed, the type strain being HAN-23^T (=DSM 18496^T; LMG 23756^T).     

A novel thermophilic lysozyme from bacteriophage phiIN93

The lysozyme of bacteriophage φIN93 was purified to apparent homogeneity with Carboxymethyl Sepharose and Hydroxyapatie columns from lysates of the phage grown on Thermus aquaticus TZ2. The enzyme is a single polypeptide chain with a molecular weight of 33,000. From the determined N-terminal amio acids of the enzyme, the locus of the gene was specified on a φIN93 genome. The enzyme was not similar to egg white lysozyme, T4 phage lysozyme, or lambda phage lysozyme. The enzyme, φIN93 lysozyme, was found to be a novel type of thermophilic lysozyme, which lyses specifically Thermus sp. cells, and exhibited conspicuous thermal stability at 95 °C for 1 h in the presence of β-mercaptoethanol.     

A new anchovy, <Emphasis Type="Italic">Stolephorus teguhi</Emphasis> (Clupeiformes: Engraulidae), from North Sulawesi,Indonesia

Stolephorus teguhi sp. nov. is described from the holotype and 14 paratypes, 49–77 mm in standard length, collected from North Sulawesi, Indonesia. The species is characterized by having numerous gill rakers (31–35 + 41–46 = 72–82) and a short upper jaw, its posterior tip reaching to or extending slightly beyond the anterior margin of the preopercle. Stolephorus pacificus and S. multibranchus also have relatively numerous gill rakers for species of this genus (21–27 + 29–36 = 53–61 and 21–28 + 30–35 = 54–60, respectively), but counts for S. teguhi exceed those for the two species. Although S. advenus also has a short upper jaw similar to that of S. teguhi, the former has far fewer gill rakers (19 + 24 = 43) than the latter.     

Bacterial diversity in five Icelandic geothermal waters: temperature and sinter growth rate effects

The microbial ecology associated with siliceous sinters was studied in five geochemically diverse Icelandic geothermal systems. Bacterial 16S rRNA clone libraries were constructed from water-saturated precipitates from each site resulting in a total of 342 bacterial clone sequences and 43 species level phylotypes. In near-neutral, saline (2.6–4.7% salinity) geothermal waters where sinter growth varied between 10 and ~300 kg year⁻¹ m⁻², 16S rRNA gene analyses revealed very low (no OTUs could be detected) to medium (9 OTUs) microbial activity. The most dominant phylotypes found in these waters belong to marine genera of the Proteobacteria. In contrast, in alkaline (pH = 9–10), meteoric geothermal waters with temperature = 66–96°C and <1–20 kg year⁻¹m⁻² sinter growth, extensive biofilms (a total of 34 OTUs) were observed within the waters and these were dominated by members of the class Aquificae (mostly related to Thermocrinis), Deinococci (Thermus species) as well as Proteobacteria. The observed phylogenetic diversity (i.e., number and composition of detected OTUs) is argued to be related to the physico-chemical regime prevalent in the studied geothermal waters; alkaliphilic thermophilic microbial communities with phylotypes related to heterotrophic and autotrophic microorganisms developed in alkaline high temperature waters, whereas halophilic mesophilic communities dominated coastal geothermal waters.     

Life cycle of tortoise tick <Emphasis Type="Italic">Hyalomma aegyptium</Emphasis> under laboratory conditions

The tortoise tick Hyalomma aegyptium has a typical three-host life-cycle. Whereas its larvae and nymphs are less host-specific feeding on a variety of tetrapods, tortoises of the genus Testudo are principal hosts of adults. Ticks retained this trait also in our study under laboratory conditions, while adults were reluctant to feed on mammalian hosts. Combination of feeding larvae and nymphs on guinea pigs and feeding of adults on Testudo marginata tortoises provided the best results. Feeding period of females was on average 25 days (range 17–44), whereas males remain after female engorgement on tortoise host. Female pre-oviposition period was 14 days (3–31), followed by 24 days of oviposition (18–29). Pre-eclosion and eclosion, both together, takes 31 days (21–43). Larvae fed 5 days (3–9), then molted to nymphs after 17 days (12–23). Feeding period of nymphs lasted 7 days (5–10), engorged nymphs molted to adults after 24 days (19–26). Sex ratio of laboratory hatched H. aegyptium was nearly equal (1:1.09). The average weight of engorged female was 0.95 (0.72–1.12) g. The average number of laid eggs was 6,900 (6,524–7,532) per female, it was significantly correlated with weight of engorged female. Only 2.8% of engorged larvae and 1.8% of engorged nymphs remained un-molted and died. Despite the use of natural host species, feeding success of females reached only 45%. The whole life-cycle was completed within 147 days (98–215).     

Characterization of MspNI (G/GWCC) and MspNII (R/GATCY), novel thermostable Type II restriction endonucleases from <Emphasis Type="Italic">Meiothermus</Emphasis> sp., isoschizomers of AvaII and BstYI

MspNI and MspNII, isoschizomers of prototype Type II restriction endonucleases AvaII and BstYI, were extracted from an extreme thermophile bacterium belonging to the genus Meiothermus, isolated from the hot sulphur springs in north Himalayan region of India where temperature and pH ranged from 60 to 80°C and 7.5 to 8.5, respectively. The two enzymes were purified to homogeneity using Cibacron-Blue 3GA Agarose, Q-Sepharose and SP-Sepharose chromatography and were homodimers with subunit molecular weights of 27 and 45 kDa, respectively. Restriction mapping and run-off sequencing of MspNI and MspNII cleaved pBR322 DNA showed that they recognized and cleaved 5′-G/GWCC-3′ and 5′-R/GATCY-3′ sites, respectively. MspNI and MspNII worked optimally at 60 and 70°C, 6 and 5 mM MgCl₂, respectively and showed no star activity in organic solvents. Both were resistant to sequence methylation and were stable up to 25 PCR cycles.     

Antibacterial and biofilm removal activity of a podoviridae Staphylococcus aureus bacteriophage SAP-2 and a derived recombinant cell-wall-degrading enzyme

Antibacterial and biofilm removal activity of a new podoviridae Staphylococcus aureus bacteriophage (SAP-2), which belongs to the φ29-like phage genus of the Podoviridae family, and a cell-wall-degrading enzyme (SAL-2), which is derived from bacteriophage SAP-2, have been characterized. The cell-wall-degrading enzyme SAL-2 was expressed in Escherichia coli in a soluble form using a low-temperature culture. The cell-wall-degrading enzyme SAL-2 had specific lytic activity against S. aureus, including methicillin-resistant strains, and showed a minimum inhibitory concentration of about 1 μg/ml. In addition, this enzyme showed a broader spectrum of activity within the Staphylococcus genus compared with bacteriophage SAP-2 in its ability to remove the S. aureus biofilms. Thus, the cell-wall-degrading enzyme SAL-2 can be used to prevent and treat biofilm-associated S. aureus infections either on its own or in combination with other cell-wall-degrading enzymes with anti-S. aureus activity.     

Amoxicillin and specific bacteriophage can be used together for eradication of biofilm of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> B5055

Despite the efficacy of antibiotics as well as bacteriophages in treatment of bacterial infections, their role in treatment of biofilm associated infections is still under consideration especially in case of older biofilms. Here, efficacy of bacteriophage alone or in combination with amoxicillin, for eradication of biofilm of Klebsiella pneumoniae B5055 has been assessed. Planktonic cells as well as biofilm of K. pneumoniae B5055 grown in 96-well microtiter plates were exposed to bacteriophage and amoxicillin at various Multiplicity of Infections (MoIs) as well as at three different antibiotic concentrations (512, 256 and 128 μg/ml), respectively. After exposure to 256 μg/ml (MIC) of amoxicillin, bacterial load of planktonic culture as well as 1-day-old biofilm was reduced by a log factor of 4.1 ± 0.31 (P = 0.008) and 1.24 ± 0.27 (P < 0.05), respectively but reduction in the bacterial load of mature biofilm (8-day-old) was insignificant (P = 0.23). When 8-day-old biofilm was exposed to higher antibiotic concentration (512 μg/ml) or phage alone (MoI = 0.01) a log reduction of 2.97 ± 0.11 (P = 0.182) and 3.51 ± 0.19 (P = 0.073), respectively was observed. While on exposing to a combination of both the amoxicillin and phage, a significant reduction (P < 0.01) in bacterial load of the biofilm was seen. Hence, when antibiotic was used in combination with specific bacteriophage a greater destruction of the biofilm structure suggested that the phages could be used successfully along with antibiotic therapy. An added advantage of the combination therapy would be its ability to check formation of resistant mutants that otherwise develop easily upon using phage or antibiotic alone.     

Thermoanaerobacterium thermostercus sp. nov., a new anaerobic thermophilic hydrogen-producing bacterium from buffalo-dung

A novel thermophilic, anaerobic, rod-shaped bacterium strain, designated Buff, was isolated from buffalo-dung samples collected from a buffalo-farm located in Caserta (Campania, south of Italy). Strain Buff was Gram-positive, motile and no spore-forming. The growth temperature range was 40–65°C with an optimum at 60°C, while pH growth range at 60°C was 5.5–8.0 with an optimum at about pH 6.5. NaCl growth concentration ranged from 0 to 2.0% with an optimum at 0.5% (w/v); no growth was observed with the presence of NaCl 3.0% (w/v). The strain produced ethanol, acetate, lactate, H₂, H₂S and CO₂ by glucose fermentation. The DNA G + C content was 34.4 mol%. As determined by 16S rRNA sequence analysis, this organism belonged to the genus Thermoanaerobacterium. On the basis of the physiological and molecular properties, we propose for strain Buff the new species designation Thermoanaerobacterium thermostercus sp. nov. This novel organism represents the first species of the genus Thermoanaerobacterium isolated from buffalo-dung. The type strain is Buff (=DSM 22141 = ATCC BAA-1776).     

Characterization of a new Bacillus megaterium bacteriophage,MJ-1, from tropical soil

A new Bacillus megaterium bacteriophage is characterized. It is a tailed phage with regular polyhedral head belonging to Bradley's group B. Head and tail dimensions are 56.4 and 300 nm, respectively. Lysis was restricted to strains of B. megaterium. No antigenic relationship with pumilus phage FP-1 or subtilis phage FS-1 was observed. The phage is sensitive to 60°C and moderately sensitive to chloroform. The nucleic acid is double-stranded linear DNA with a G-C mole % of 38.8 and a mol wt of (53±3)×10⁶.     

<Emphasis Type="Italic">Pseudonocardia serianimatus</Emphasis> sp. nov., a novel actinomycete isolated from the surface-sterilized leaves of <Emphasis Type="Italic">Artemisia annua</Emphasis> L.

An aerobic, non-motile, catalase-positive, Gram-stain positive actinomycete designated YIM 63233^T was isolated from the surface-sterilized leaves of Artemisia annua L. and characterized using a polyphasic taxonomic approach. Optimal growth occurred at 20–28°C, pH 6.0–7.0 and in the presence of 0–3% (w/v) NaCl. 16S rRNA gene sequence-based phylogenetic analysis showed that strain YIM 63233^T clustered with species of the genus Pseudonocardia, displaying ≥1.2% sequence divergence with recognized species of this genus (from 98.8 to 94.0%). Relatively low levels of DNA–DNA relatedness were found between strain YIM 63233^T and Pseudonocardia petroleophila IMSNU 22072^T, which supported the classification of strain YIM 63233^T within a novel species of the genus Pseudonocardia. The G + C content of genomic DNA was 72.0 mol%. Strain YIM 63233^T possessed chemotaxonomic markers that were consistent with classification in the genus Pseudonocardia, i.e. the predominant fatty acids were iso-C16:0 (32.27%), C16:0 10-methyl (8.73%) and C17:1ω8c (8.30%), whilst the predominant menaquinone was MK-8(H₄). The diagnostic diamino acid of the cell-wall peptidoglycan was meso-diaminopimelic acid. The major cell wall sugars were glucose, arabinose, galactose, mannose and rhamnose. The results of physiological and biochemical tests and DNA–DNA hybridization allowed the phenotypic and genotypic differentiation of strain YIM 63233^T from its closest phylogenetic neighbours. Therefore, the new isolate YIM 63233^T represents a novel species of the genus Pseudonocardia, for which the name Pseudonocardia serianimatus sp. nov. is proposed. The type strain is YIM 63233^T (=DSM 45302^T = CCTCC AA 208079^T).     

Phylogenetic,Inter, and Intraspecific Sequence Analysis of <Emphasis Type="Italic">spo0A</Emphasis> Gene of the Genus <Emphasis Type="Italic">Geobacillus</Emphasis>

In this work, the variability of spo0A gene in the genus Geobacillus and applicability of this gene for the taxonomy within this genus were evaluated. The protein Spo0A is the master regulator of the endospore-forming process in the all endospore-forming bacteria. Geobacillus genus-specific primers GEOSPO were designed based on the sequences of Geobacillus spo0A gene available through the public databases. Inter and intraspecific variability of Geobacillus spo0A gene was determined after sequencing of the GEOSPO-PCR products. Geobacillus spo0A sequence analysis showed that three species—Geobacillus thermodenitrificans, G. stearothermophilus, and G. jurassicus—could be easily identified. Similarity between the sequences of these species and the other species were in the range of 83.3%–92.0%. In contrast, intraspecific similarity of G. thermodenitrificans and G. stearothermophilus was high—above 99.0%. Similarity of spo0A sequences of G. subterraneus–G. uzenensis species cluster also matched this interval. Intercluster similarity between G. lituanicus–G. thermoleovorans–G. kaustophilus–G. vulcani and G. thermocatenulatus–G. gargensis–G. caldoxylosilyticus–G. toebii–G. thermoglucosidasius species clusters, as well as interspecific similarity within these two clusters was in the range of the intraspecific similarity determined for G. thermodenitrificans and G. stearothermophilus. It was also determined that spo0A cannot be used as the phylogenetic marker for the genus Geobacillus.