Discovery of a new source of rifamycin antibiotics in marine sponge actinobacteria by phylogenetic prediction

Phylogenetic analysis of the ketosynthase (KS) gene sequences of marine sponge-derived Salinispora strains of actinobacteria indicated that the polyketide synthase (PKS) gene sequence most closely related to that of Salinispora was the rifamycin B synthase of Amycolatopsis mediterranei. This result was not expected from taxonomic species tree phylogenetics using 16S rRNA sequences. From the PKS sequence data generated from our sponge-derived Salinispora strains, we predicted that such strains might synthesize rifamycin-like compounds. Liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis was applied to one sponge-derived Salinispora strain to test the hypothesis of rifamycin synthesis. The analysis reported here demonstrates that this Salinispora isolate does produce compounds of the rifamycin class, including rifamycin B and rifamycin SV. A rifamycin-specific KS primer set was designed, and that primer set increased the number of rifamycin-positive strains detected by PCR screening relative to the number detectable using a conserved KS-specific set. Thus, the Salinispora group of actinobacteria represents a potential new source of rifamycins outside the genus Amycolatopsis and the first recorded source of rifamycins from marine bacteria.     

Gut bacterium of Dendrobaena veneta (Annelida: Oligochaeta) possesses antimycobacterial activity

The new bacterial strain with antimycobacterial activity has been isolated from the midgut of Dendrobaena veneta (Annelida). Biochemical and molecular characterization of isolates from 18 individuals identified all as Raoultella ornithinolytica genus with 99% similarity. The bacterium is a possible symbiont of the earthworm D. veneta. The isolated microorganism has shown the activity against four strains of fast-growing mycobacteria: Mycobacterium butiricum, Mycobacterium jucho, Mycobacterium smegmatis and Mycobacterium phlei. The multiplication of the gut bacterium on plates with Sauton medium containing mycobacteria has caused a lytic effect. After the incubation of the cell free extract prepared from the gut bacterium with four strains of mycobacteria in liquid Sauton medium, the cells of all tested strains were deformed and divided to small oval forms and sometimes created long filaments. The effect was observed by the use of light, transmission and scanning microscopy. Viability of all examined species of mycobacteria was significantly decreased. The antimycobacterial effect was probably the result of the antibiotic action produced by the gut bacterium of the earthworm. The application of ultrafiltration procedure allowed to demonstrate that antimicrobial substance with strong antimycobacterial activity from bacterial culture supernatant, is a protein with the molecular mass above 100 kDa.     

Isolation and identification of mycobacteria from soils at an illegal dumping site and landfills in Japan

In order to study the diversity and community of genus Mycobacterium in polluted soils, we tried to isolate mycobacteria from 11 soil samples collected from an illegal dumping site and 3 landfills in Japan. Using culture methods with or without Acanthamoeba culbertsoni, a total of 19 isolates of mycobacteria were obtained from 5 soil samples and 3 of them were isolated only by the co-culture method with the amoeba. Conventional biochemical tests and sequencing of the hsp65, rpoB, and 16S rRNA genes were performed for species identification of 17 of the 19 isolates. Among the 17 isolates, there was one isolate each of Mycobacterium vanbaalenii, Mycobacterium mageritense, Mycobacterium frederiksbergense, M. vanbaalenii or Mycobacterium austroafricanum, and Mycobacterium chubuense or Mycobacterium chlorophenolicum. The remaining 12 isolates could not be precisely identified at the species level. A phylogenic tree based on the hsp65 sequences indicated that 2 of the 12 isolates were novel species. In addition, 4 isolates were phylogenically close to species that degrade polycyclic aromatic hydrocarbons, which induce some cancers in humans. These results demonstrated that there were many hitherto-unreported mycobacteria in the polluted soils, and suggested that some mycobacteria might play roles in the natural attenuation and engineered bioremediation of contaminated sites with other microorganisms.     

Diversity of fungal isolates from three Hawaiian marine sponges

Sponges harbor diverse prokaryotic and eukaryotic microbes. However, the nature of sponge-fungal association and diversity of sponge-derived fungi have barely been addressed. In this study, the cultivation-dependent approach was applied to study fungal diversity in the Hawaiian sponges Gelliodes fibrosa, Haliclona caerulea, and Mycale armata. The cultivated fungal isolates were representatives of 8 taxonomic orders, belonging to at least 25 genera of Ascomycota and 1 of Basidiomycota. A portion of these isolates (n=15, 17%) were closely affiliated with fungal isolates isolated from other marine habitats; the rest of the isolates had affiliation with terrestrial fungal strains. Cultivated fungal isolates were classified into 3 groups: 'sponge-generalists'-found in all sponge species, 'sponge-associates'-found in more than one sponge species, and 'sponge-specialists'-found only in one sponge species. Individuals of G. fibrosa collected at two different locations shared the same group of 'sponge-specialists'. Also, representatives of 15 genera were identified for the first time in marine sponges. Large-scale phylogenetic analysis of sponge-derived fungi may provide critical information to distinguish between 'resident fungi' and 'transient fungi' in sponges as it has been done in other marine microbial groups. This is the first report of the host specificity analysis of culturable fungal communities in marine sponges.     

Culture-dependent and culture-independent diversity within the obligate marine actinomycete genus Salinispora

Salinispora is the first obligate marine genus within the order Actinomycetales and a productive source of biologically active secondary metabolites. Despite a worldwide, tropical or subtropical distribution in marine sediments, only two Salinispora species have thus far been cultivated, suggesting limited species-level diversity. To further explore Salinispora diversity and distributions, the phylogenetic diversity of more than 350 strains isolated from sediments collected around the Bahamas was examined, including strains cultured using new enrichment methods. A culture-independent method, using a Salinispora-specific seminested PCR technique, was used to detect Salinispora from environmental DNA and estimate diversity. Overall, the 16S rRNA gene sequence diversity of cultured strains agreed well with that detected in the environmental clone libraries. Despite extensive effort, no new species level diversity was detected, and 97% of the 105 strains examined by restriction fragment length polymorphism belonged to one phylotype (S. arenicola). New intraspecific diversity was detected in the libraries, including an abundant new phylotype that has yet to be cultured, and a new depth record of 1,100 m was established for the genus. PCR-introduced error, primarily from Taq polymerase, significantly increased clone library sequence diversity and, if not masked from the analyses, would have led to an overestimation of total diversity. An environmental DNA extraction method specific for vegetative cells provided evidence for active actinomycete growth in marine sediments while indicating that a majority of sediment samples contained predominantly Salinispora spores at concentrations that could not be detected in environmental clone libraries. Challenges involved with the direct sequence-based detection of spore-forming microorganisms in environmental samples are discussed.     

Diversity of culturable actinobacteria isolated from marine sponge <Emphasis Type="Italic">Haliclona</Emphasis> sp.

This study describes actinobacteria isolated from the marine sponge Haliclona sp. collected in shallow water of the South China Sea. A total of 54 actinobacteria were isolated using media selective for actinobacteria. Species diversity and natural product diversity of isolates from marine sponge Haliclona sp. were analysed. Twenty-four isolates were selected on the basis of their morphology on different media and assigned to the phylum Actinobacteria by a combination of 16S rRNA gene based restriction enzymes digestion and 16S rRNA gene sequence analysis. The 16S rRNA genes of 24 isolates were digested by restriction enzymes TaqI and MspI and assigned to different groups according to their restriction enzyme pattern. The phylogenetic analysis based on 16S rRNA gene sequencing showed that the isolates belonged to the genera Streptomyces, Nocardiopsis, Micromonospora and Verrucosispora; one other isolate was recovered that does not belong to known genera based on its unique 16S rRNA gene sequence. To our knowledge, this is the first report of a bacterium classified as Verrucosispora sp. that has been isolated from a marine sponge. The majority of the strains tested belong to the genus Streptomyces and three isolates may be new species. All of the 24 isolates were screened for genes encoding polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS). PKS and NRPS sequences were detected in more than half of the isolates and the different "PKS-I-PKS-II-NRPS" combinations in different isolates belonging to the same species are indicators of their potential natural product diversity and divergent genetic evolution.     

抗肿瘤海绵放线菌的分离及菌株HA01184的鉴定

目的：对采自海南、湛江等海域的海绵样品进行放线菌选择性分离,采用其发酵液进行抗肿瘤活性筛选,并对活性较好的菌株进行鉴定。方法：用含50μg／mL重铬酸钾为抑制剂的海水高氏一号合成培养基分离培养海绵放线菌;以MTT法进行菌株的抗肿瘤活性筛选;通过培养特征、形态特征、生理生化特征、16S rDNA序列测定及系统发育分析,对菌株HA01184进行鉴定。结果与结论：海水高氏一号合成培养基用于海绵放线菌分离培养具有很好的选择性,从海绵样品中共分离得到放线菌165株,细胞毒活性达80％以上的阳性菌株有10株,其中菌株HA01184的发酵液细胞毒活性为90％。结合形态观察、生理生化特征和16S rDNA序列比对分析,将HA01184归于链霉菌属,可能是来自海洋环境的一个潜在新种。     

Evolution of secondary metabolite genes in three closely related marine actinomycete species

The marine actinomycete genus Salinispora is composed of three closely related species. These bacteria are a rich source of secondary metabolites, which are produced in species-specific patterns. This study examines the distribution and phylogenetic relationships of genes involved in the biosynthesis of secondary metabolites in the salinosporamide and staurosporine classes, which have been reported for S. tropica and S. arenicola, respectively. The focus is on "Salinispora pacifica," the most recently discovered and phylogenetically diverse member of the genus. Of 61 S. pacifica strains examined, 15 tested positive for a ketosynthase (KS) domain linked to the biosynthesis of salinosporamide K, a new compound in the salinosporamide series. Compound production was confirmed in two strains, and the domain phylogeny supports vertical inheritance from a common ancestor shared with S. tropica, which produces related compounds in the salinosporamide series. There was no evidence for interspecies recombination among salA KS sequences, providing further support for the geographic isolation of these two salinosporamide-producing lineages. In addition, staurosporine production is reported for the first time for S. pacifica, with 24 of 61 strains testing positive for staD, a key gene involved in the biosynthesis of this compound. High levels of recombination were observed between staD alleles in S. pacifica and the cooccurring yet more distantly related S. arenicola, which produces a similar series of staurosporines. The distributions and phylogenies of the biosynthetic genes examined provide insight into the complex processes driving the evolution of secondary metabolism among closely related bacterial species.     

Isolation and automated ribotyping of Mycobacterium lentiflavum from drinking water distribution system and clinical specimens

Automated ribotyping as a tool for identifying of nontuberculous mycobacteria was evaluated. We created a database comprising of riboprints of 60 strains, representing 32 species of nontuberculous mycobacteria. It was shown that combined ribopatterns generated after digestion with EcoRI and PvuII were distinguishable between species of both slow-growing and rapid-growing mycobacteria. The findings were in good agreement with the 16S rRNA gene sequencing results, allowing correct identification of Mycobacterium lentiflavum isolated from clinical specimens and from biofilms growing in public water distribution system. The automated ribotyping was powerful in discriminating between M. lentiflavum and closely related species M. simiae and M. palustre. Mycobacterium lentiflavum strains from drinking water biofilms were resistant to two to four antimycobacterial drugs. The drinking water distribution system may, thus, be a source of nontuberculous mycobacteria resistant to multiple drugs.     

Sponge-specific clusters revisited: a comprehensive phylogeny of sponge-associated microorganisms

Marine sponges often contain diverse and abundant communities of microorganisms including bacteria, archaea and eukaryotic microbes. Numerous 16S rRNA-based studies have identified putative 'sponge-specific' microbes that are apparently absent from seawater and other (non-sponge) marine habitats. With more than 7500 sponge-derived rRNA sequences (from clone, isolate and denaturing gradient gel electrophoresis data) now publicly available, we sought to determine whether the current notion of sponge-specific sequence clusters remains valid. Comprehensive phylogenetic analyses were performed on the 7546 sponge-derived 16S and 18S rRNA sequences that were publicly available in early 2010. Overall, 27% of all sequences fell into monophyletic, sponge-specific sequence clusters. Such clusters were particularly well represented among the Chloroflexi, Cyanobacteria, 'Poribacteria', Betaproteobacteria and Acidobacteria, and in total were identified in at least 14 bacterial phyla, as well as the Archaea and fungi. The largest sponge-specific cluster, representing the cyanobacterium 'Synechococcus spongiarum', contained 245 sequences from 40 sponge species. These results strongly support the existence of sponge-specific microbes and provide a suitable framework for future studies of rare and abundant sponge symbionts, both of which can now be studied using next-generation sequencing technologies.     

Comparison of NaOH-N-acetyl cysteine and sulfuric acid decontamination methods for recovery of mycobacteria from clinical specimens

We compared the NaOH-N-acetyl cysteine (NaOH-NALC) and the sulfuric acid decontamination procedure in the detection of mycobacteria using the Mycobacteria Growth Indicator Tube (MGIT). In total 219 sputum specimens were collected from 142 Zambian patients and subjected to mycobacterial culture. One half of the specimen was decontaminated with NaOH-NALC and the other half was decontaminated with sulfuric acid. From the 438 samples a total of 261 (60%) cultures yielded growth of mycobacteria, consisting of 22 different species. The sulfuric acid method was more successful than the NaOH-NALC method in recovering mycobacteria in MGITs (146 versus 115 respectively, p = 0.001). Of the 146 positive mycobacterial cultures recovered after sulfuric acid decontamination 28 were Mycobacterium tuberculosis, 84 nontuberculous mycobacteria (NTM) and 34 acid fast bacterial isolates which could not be identified to the species level. The 115 mycobacteria recovered by the NaOH-NALC method consisted of 34 M. tuberculosis strains, 55 NTM and 26 acid fast bacteria that could not be identified. The most frequently isolated NTM were Mycobacterium lentiflavum and Mycobacterium intracellulare. Comparing the two decontamination methods the recovery of NTM in the sulfuric acid group was significant higher than in the NaOH-NALC group (p = 0.001). In contrast, no significant difference was found for the recovery of M. tuberculosis. These results show that the decontamination method used affects the recovery of nontuberculous mycobacteria in particular.     

Multiple antibiotic resistances of <Emphasis Type="Italic">Enterococcus</Emphasis> isolates from raw or sand-filtered sewage

Fifty antibiotic-resistant Enterococcus strains were isolated from raw sewage of a wastewater treatment plant and from the same sewage after trickling through a 25-cm sand column, which retained >99% of the initial population. All 50 Enterococcus isolates were resistant against triple sulfa and trimethoprim/sulfamethoxazole and none were resistant against vancomycin. Most of the isolates from raw sewage were resistant to more antibiotics than the isolates from sand column effluent. One Enterococcus isolate from raw sewage (no. 61) and one Enterococcus isolate from sand column effluent (no. 95) had ten antibiotic resistances each. Isolate no. 95 maintained its resistances in the absence of antibiotics during the whole study. It was compared with isolate no. 70, which was one of the isolates, being resistant only against the two sulfonamides. Phenotypically and biochemically, the two organisms were strains of Enterococcus faecalis. Sequence analysis of partical 16S rDNA allowed alignment of isolate no. 95 as a strain of Enterococcus faecium and of isolate no. 70 as a strain of E. faecalis. E. faecium strain no. 95 carried at least six different plasmids, whereas for E. faecalis strain no. 70, no discrete plasmid band was seen on the gels.     

Screening of rifamycin producing marine sponge bacteria by LC-MS-MS

HPLC-MS-MS has been used for the identification and characterisation of rifamycin B and rifamycin SV in various strains of the marine sponge-derived bacterium Salinispora. Gradient elution using acetonitrile/water/ammonium acetate was used to separate the rifamycins from the matrix and negative ion-electrospray mass spectrometry was used for detection and confirmation. The presence of rifamycin in bacterial extracts was confirmed by matching retention times, parent ion spectra and the fragmentated parent ion spectra of the standard compounds and the bacterial extracts. All strains of the marine sponge bacterium Salinispora tested were found to contain rifamycin thus an alternate actinobacterial source of rifamycin was established.     

Isolation and phylogenetic analysis of bacteria with antimicrobial activities from the Mediterranean sponges Aplysina aerophoba and Aplysina cavernicola

The aim of this study was to isolate bacteria with antimicrobial activities from the marine sponges Aplysina aerophoba and Aplysina cavernicola. The obtained 27 isolates could be subdivided into eight phylogenetically different clusters based on comparative sequence analysis of their 16S rDNA genes. The sponge isolates were affiliated with the low (Bacillus) and high G+C Gram-positive bacteria (Arthobacter, Micrococcus), as well as the alpha-Proteobacteria (unknown isolate) and gamma-Proteobacteria (Vibrio, Pseudoalteromonas). One novel Bacillus species was identified and two species were closely related to previously uncharacterized strains. Isolates with antimicrobial activity were numerically most abundant in the genera Pseudoalteromonas and the alpha-Proteobacteria. The sponge isolates show antimicrobial activities against Gram-positive and Gram-negative reference strains but not against the fungus Candida albicans. A general pattern was observed in that Gram-positive bacteria inhibited Gram-positive strains while Gram-negative bacteria inhibited Gram-negative isolates. Antimicrobial activities were also found against clinical isolates, i.e. multi-resistant Staphylococcus aureus and Staphylococcus epidermidis strains isolated from hospital patients. The high recovery of strains with antimicrobial activity suggests that marine sponges represent an ecological niche which harbors a hitherto largely uncharacterized microbial diversity and, concomitantly, a yet untapped metabolic potential.     

Imatinib-sensitive tyrosine kinases regulate mycobacterial pathogenesis and represent therapeutic targets against tuberculosis

The lengthy course of treatment with currently used antimycobacterial drugs and the resulting emergence of drug-resistant strains have intensified the need for alternative therapies against Mycobacterium tuberculosis (Mtb), the etiologic agent of tuberculosis. We show that Mtb and Mycobacterium marinum use ABL and related tyrosine kinases for entry and intracellular survival in macrophages. In mice, the ABL family tyrosine kinase inhibitor, imatinib (Gleevec), when administered prophylactically or therapeutically, reduced both the number of granulomatous lesions and bacterial load in infected organs and was also effective against a rifampicin-resistant strain. Further, when coadministered with current first-line drugs, rifampicin or rifabutin, imatinib acted synergistically. These data implicate host tyrosine kinases in entry and intracellular survival of mycobacteria and suggest that imatinib may have therapeutic efficacy against Mtb. Because imatinib targets host, it is less likely to engender resistance compared to conventional antibiotics and may decrease the development of resistance against coadministered drugs.     

Microdiversity and evidence for high dispersal rates in the marine actinomycete 'Salinispora pacifica'

In July of 2006 and January of 2008, a total of 671 marine sediment samples were collected at depths from 5 to 2012?m throughout the Fijian islands and selectively processed for the cultivation of marine actinomycetes belonging to the genus Salinispora. The primary objectives were to assess the diversity, distribution and phylogeny of 'S. pacifica', the least well studied of the three species in the genus. Employing a sequential screening method based on antibiotic sensitivity, RFLP patterns, and 16S rRNA and ITS sequence analyses, 42 of 750 isolates with Salinispora-like features were identified as 'S. pacifica'. These strains represent the first report of 'S. pacifica' from Fiji and include 15 representatives of 4 new 'S. pacifica' 16S rRNA sequence types. Among the 'S. pacifica' strains isolated, little evidence for geographical isolation emerged based on 16S, ITS or secondary metabolite biosynthetic gene fingerprinting. The inclusion of isolates from additional collection sites and other Salinispora spp. revealed a high degree of dispersal among 'S. pacifica' populations and phylogenetic support for the delineation of this lineage as a third species.     

Mycobacterial ecology of the Rio Grande

This is the first study to characterize the environmental conditions which contribute to the presence and proliferation of environmental mycobacteria in a major freshwater river. Over 20 different species of environmental mycobacteria were isolated, including the pathogenic M. avium and M. kansasii. Species of the rapidly growing M. fortuitum complex were the most commonly isolated mycobacteria, and one-third of all isolates were not identified at the species level, even by 16S sequencing. PCR restriction analysis of the hsp65 gene was more accurate and rapid than biochemical tests and as accurate as yet less expensive than 16S sequencing, showing great promise as a new tool for species identification of environmentally isolated mycobacteria. Total environmental mycobacteria counts positively correlated with coliform and Escherichia coli counts and negatively correlated with chemical toxicity and water temperature. Environmental mycobacteria can survive in the alkaline conditions of the river despite previous reports that especially acidic conditions favor their presence. A representative river isolate (M. fortuitum) survived better than E. coli O157:H7 at pHs below 7 and above 8 in nutrient broth. The river strain also retained viability at 8 ppm of free chlorine, while E. coli was eliminated at 2 ppm and above. Thus, in vitro studies support environmental observations that a variety of extreme conditions favor the hardy environmental mycobacteria.     

Effect of mucoid property on antibiotic susceptibility ofPseudomonas aeruginosa

Clinical isolates ofPseudomonas aeruginosa from patients with cystic fibrosis (CF) were examined for susceptibility to the antibiotics carbenicillin, ticarcillin, tobramycin, gentamicin, and tetracycline. Minimal inhibitory concentrations of the antibiotics were determined for mucoid and nonmucoid isolates from the same patient by a single-colony replica plating method. This method allows the rapid determination of antibiotic susceptibility of a single cell’s progeny and the individual screening of each colony against all antibiotics. Twenty of 34 (58%) cystic fibrosis patients had a mucoid isolate which was more susceptible to antibiotics than their nonmucoid isolate of the same serotype. Nonmucoid revertant segregants of mucoid strains isolated from 50% of the patients demonstrated greater resistance to at least one antibiotic than the original mucoid strain. Multiple isolates from 25 patients were serotyped by Difco (Liu) or Homma antiserum; only 2 patients harbored multiple strains with no common serotyping antigens. Serotypes of the nonmucoid revertants were the same as the original mucoid isolate even if the susceptibilities of the two strains were not similar.     

In vitro activity of SPR719 against Mycobacterium ulcerans,Mycobacterium marinum and Mycobacterium chimaera

Nontuberculosis mycobacterial (NTM) infections are increasing in prevalence across the world. In many cases, treatment options for these infections are limited. However, there has been progress in recent years in the development of new antimycobacterial drugs. Here, we investigate the in vitro activity of SPR719, a novel aminobenzimidazole antibiotic and the active form of the clinical-stage compound, SPR720, against several isolates of Mycobacterium ulcerans, Mycobacterium marinum and Mycobacterium chimaera. We show that SPR719 is active against these NTM species with a MIC range of 0.125–4 μg/ml and that this compares favorably with the commonly utilized antimycobacterial antibiotics, rifampicin and clarithromycin. Our findings suggest that SPR720 should be further evaluated for the treatment of NTM infections.