Pfcrt genotypes and related microsatellite DNA polymorphisms on Plasmodium falciparum differed among populations in the Greater Mekong Subregion

Malaria morbidity and mortality have decreased gradually in the Greater Mekong Subregion (GMS). Presently, WHO sets a goal to eliminate malaria by 2030 in the GMS. However, drug-resistant malaria has been reported from several endemic areas. To achieve the goal of elimination, the status of the emergence and spread of drug resistance should be monitored. In this study, the genotype of the Plasmodium falciparum chloroquine (CQ) resistance transporter gene (pfcrt) and 6 microsatellite DNA loci flanking the gene were examined. P. falciparum isolates (n?=?136) was collected from malaria patients in Thailand (n?=?50, 2002–2005), Vietnam (n?=?39, 2004), Laos (n?=?15, 2007) and Cambodia (n?=?32, 2009). Amino acid sequences at codons 72–76 on the gene were determined. All of the isolates from Thailand were CQ-resistant (CVIET), as were all of the isolates from Cambodia (CVIET, CVIDT). Thirteen of the 15 isolates (87%) from Laos were CQ-resistant (CVIET, CVIDT), whereas the other 2 (13%) were CQ-susceptible (CVMNK). In contrast, 27 of the 39 isolates (69%) from Vietnam were CQ-susceptible (CVMNK), whereas the other 12 (31%) were CQ-resistant (CVIET, CVIDT, CVMDT) or mixed (CVMNK/CVIDT). The mean of expected heterozygosity of the microsatellite loci was 0.444 in the Thai population, 0.482 in the Cambodian population, and 0.734 in the Vietnamese population. Genetic diversity in the Thai population was significantly lower than that in the Vietnamese population. These results suggested that chloroquine selective pressure on P. falciparum populations is heterogeneous in the GMS. Therefore, further examination to understand the mechanisms behind the emergence and spread of drug-resistant malaria are needed.     

Antimicrobial properties of endophytic actinomycetes isolated from <Emphasis Type="Italic">Combretum latifolium</Emphasis> Blume,a medicinal shrub from Western Ghats of India

Endophytic actinomycetes were isolated from Combretum latifolium Blume (Combretaceae),Western Ghats of Southern India and identified by its characteristic culture morphology and molecular analysis of 16S rRNA gene sequences. In this survey of endophytic actinomycetes, a total of 117 isolates representing 9 different genera of endophytic actinomycetes were obtained using four different isolation media and several of them seemed to be novel taxa. Streptomyces genera (35%) was the most frequently isolated strains, followed by Nocordiopsis (17%) and Micromonospora (13%). ISP-4 medium recovered more isolates (47%) when compared to rest of the media used. Preliminary antibacterial activity of the isolates was carried out by confrontation test. Ethyl acetate fraction of selected isolates in disc diffusion assay exhibited broad spectrum antimicrobial activity against test human pathogens. All Streptomyces spp. strains displayed significant antimicrobial activity against test pathogens. Strain CLA-66 and CLA-68 which are Nocordipsis spp. inhibited both bacterial and fungal pathogens where as other isolates inhibited atleast three test human pathogens in disc diffusion assay. Antimicrobial screening of endophytic actinomycetes from this host may represent a unique potential niche for antimicrobial compounds of industrial and pharmaceutical applications. This work is the first comprehensive report on incidence of potential endophytic actinomycetes inhabiting C. latifolium Blume.     

Diversity and antimicrobial activity of Pseudovibrio spp. from Irish marine sponges

Aims: To evaluate the diversity and antimicrobial activity present among Pseudovibrio spp. isolated from marine sponges. Methods and Results: Seventy‐three bacterial isolates from the marine sponges Polymastia boletiformis, Axinella dissimilis and Haliclona simulans were identified as Pseudovibrio spp. using phylogenetic analysis of 16S rRNA gene sequences. Genetic diversity among these isolates was estimated using random amplification of polymorphic DNA (RAPD), and 33 RAPD types were identified among the 73 Pseudovibrio isolates. These Pseudovibrio spp. were assayed for the production of compounds with antimicrobial activity against various clinically relevant pathogens. Sixty‐two (85%) of the isolates showed activity against at least one of the pathogens tested, including Escherichia coli, Salmonella enterica serotype Typhimurium, methicillin‐resistant Staphylococcus aureus (MRSA), and Clostridium difficile. PCR screens of the Pseudovibrio isolates also revealed the presence of potential antibiotic‐producing polyketide synthase genes. Conclusions: Marine sponges harbour a diverse population of Pseudovibrio spp., the majority of which demonstrate antimicrobial activity. The identification of several different antimicrobial activity spectra suggests that the Pseudovibrio isolates may produce a suite of antimicrobial compounds. Significance and Impact of the Study: This is the first study in which an extended population of Pseudovibrio isolates from marine sponges has been analysed and establishes the little‐studied Pseudovibrio as a potentially important genus in the search for antimicrobial compounds of clinical relevance.     

Molecular Markers Reveal Only Two Mud Crab Species of Genus Scylla (Brachyura: Portunidae) in Indian Coastal Waters

The taxonomic ambiguity of the Indian mud crab (genus Scylla de Hann 1833) is still a cause of concern as several papers have been published with misleading identification. This is the first attempt to resolve the taxonomic uncertainty of the mud crab commonly available in Indian coastal waters using molecular genetic markers (ITS-1 and sequencing of COI gene) combined with traditional morphometry. Additionally, we developed a PCR method by which Indian mud crab species can be identified rapidly and effectively. The results clearly indicate that the green morph of the Indian mud crab is Scylla serrata and the brown morph is S. olivacea. The S. serrata commonly mentioned in the literature from India is S. olivacea; the S. tranquebarica noted by many Indian researchers should belong to S. serrata. Caution should be taken when interpreting or implementing the biological, molecular, and aquaculture data in the literature.     

A Conserved Hydrolase Responsible for the Cleavage of Aminoacylphosphatidylglycerol in the Membrane of Enterococcus faecium

Aminoacylphosphatidylglycerol synthases (aaPGSs) are enzymes that transfer amino acids from aminoacyl-tRNAs (aa-tRNAs) to phosphatidylglycerol (PG) to form aa-PG in the cytoplasmic membrane of bacteria. aa-PGs provide bacteria with resistance to a range of antimicrobial compounds and stress conditions. Enterococcus faecium encodes a triple-specific aaPGS (RakPGS) that utilizes arginine, alanine, and lysine as substrates. Here we identify a novel hydrolase (AhyD), encoded immediately adjacent to rakPGS in E. faecium, which is responsible for the hydrolysis of aa-PG. The genetic synteny of aaPGS and ahyD is conserved in >60 different bacterial species. Deletion of ahyD in E. faecium resulted in increased formation of Ala-PG and Lys-PG and increased sensitivity to bacitracin. Our results suggest that AhyD and RakPGS act together to maintain optimal levels of aa-PG in the bacterial membrane to confer resistance to certain antimicrobial compounds and stress conditions.     

Antimicrobial activity of bacteria from marine sponge Suberea mollis and bioactive metabolites of Vibrio sp. EA348

Discovery of potential bioactive metabolites from sponge-associated bacteria have gained attraction in recent years. The current study explores the potential of sponge (Suberea mollis) associated bacteria against bacterial and fungal pathogens. Sponge samples were collected from Red sea in Obhur region, Jeddah, Saudi Arabia. Of 29 isolated bacteria belong to four different classes i.e. Firmicutes (62%), γ-Proteobacteria (21%), α-Proteobacteria (10%) and Actinobacteria (7%). Among them nineteen (65%) bacterial strains showed antagonistic activity against oomycetes and only 3 (10%) bacterial strains were active against human pathogenic bacteria tested. Most bioactive genera include Bacillus (55%), Pseudovibrio (13%) and Ruegeria (10%). Enzyme production (protease, lipase, amylase, cellualse) was identified in 12 (41%) bacterial strains where potential strains belonging to γ-Proteobacteria and Firmicutes groups. Production of antimicrobial metabolites and hydrolysates in these bacteria suggest their potential role in sponge against pathogens. Further bioactive metabolites from selected strain of Vibrio sp. EA348 were identified using LC-MS and GC–MS analyses. We identified many active metabolites including antibiotics such as Amifloxacin and fosfomycin. Plant growth hormones including Indoleacetic acid and Gibberellin A3 and volatile organic compound such as methyl jasmonate were also detected in this strain. Our results highlighted the importance of marine bacteria inhabiting sponges as potential source of antimicrobial compounds and plant growth hormones of pharmaceutical and agricultural significance.     

Molecular typing and antimicrobial susceptibility testing to six antimicrobials of <Emphasis Type="Italic">Clostridium difficile</Emphasis> isolates from three Czech hospitals in Eastern Bohemia in 2011–2012

In 2011–2012, a survey was performed in three regional hospitals in the Czech Republic to determine the incidence of Clostridium difficile infections (CDIs) and to characterize bacterial isolates. C. difficile isolates were characterized by PCR ribotyping, toxin genes detection, multiple-locus variable-number tandem-repeat analysis (MLVA), and antimicrobial susceptibility testing to fidaxomicin, vancomycin, metronidazole, clindamycin, LFF571, and moxifloxacin using agar dilution method. The incidence of CDI in three studied hospitals was 145, 146, and 24 cases per 100,000 inhabitants in 2011 and 177, 258, and 67 cases per 100,000 inhabitants in 2012. A total of 64 isolates of C. difficile was available for molecular typing and antimicrobial susceptibility testing. 60.9% of the isolates were classified as ribotype 176. All 41 isolates of ribotypes 176 and 078 were positive for the presence of binary toxin genes. Ribotype 176 also carried 18-bp deletion in the regulatory gene tcdC. Tested isolates of C. difficile were fully susceptible to vancomycin and metronidazole, whereas 65.1% of the isolates were resistant to moxifloxacin. MLVA results indicated that isolates from three different hospitals were genetically related, suggesting transmission between healthcare facilities.     

Heterotrophic bacteria associated with the green alga <Emphasis Type="Italic">Ulva rigida</Emphasis>: identification and antimicrobial potential

Heterotrophic bacteria associated with the green alga Ulva rigida, collected from the coast of Tunisia, were isolated and subsequently identified by their 16S rRNA gene sequences and by phylogenetic analysis. The 71 isolates belong to four phyla: Proteobacteria (Alpha-and Gamma- subclasses), Bacteroidetes, Firmicutes, and Actinobacteria. Most of the isolates belong to Proteobacteria. The Gram-positive Firmicutes and especially the genus Bacillus were well-represented at the surface of U. rigida, collected from the coast as well as from the lagoon, while Actinobacteria were represented only at the surface of algae collected from the coast of Cap Zebib. Bacteroidetes were more represented at the surface of algae collected from the Ghar El Melh lagoon. The bacterial community of the water surrounding the algae was different from that associated with the surface of the algae. Moreover, the abundance of bacteria in the surrounding water was much lower compared to the density of bacteria associated with the surface of the algae. Bacteria isolated from the algal surface were tested for their antimicrobial potential. The results show that ~?36% of the algae-associated bacterial isolates possess antibacterial activity whereas free-living bacteria, isolated from the surrounding water, did not show such activity. The surface of U. rigida was colonized by a high diversity of culturable and possibly novel epiphytic bacteria that may be an important source of antimicrobial compounds and are therefore of biotechnological interest.     

Diversity of antagonistic bacteria isolated from medicinal plant Peganum harmala L.

The antimicrobial activity of plant extract of Peganum harmala, a medicinal plant has been studied already. However, knowledge about bacterial diversity associated with different parts of host plant antagonistic to different human pathogenic bacteria is limited. In this study, bacteria were isolated from root, leaf and fruit of plant. Among 188 bacterial isolates isolated from different parts of the plant only 24 were found to be active against different pathogenic bacteria i.e. Escherichia coli, Methicillin-resistant Staphylococcus aureus (MRSA), Enterococcus faecium, Enterococcus faecalis and Pseudomonas aeruginosa. These active bacterial isolates were identified on the basis of 16S rRNA gene analysis. Total population of bacteria isolated from plant was high in root, following leaf and fruit. Antagonistic bacteria were also more abundant in root as compared to leaf and fruit. Two isolates (EA5 and EA18) exhibited antagonistic activity against most of the targeted pathogenic bacteria mentioned above. Some isolates showed strong inhibition for one targeted pathogenic bacterium while weak or no inhibition for others. Most of the antagonistic isolates were active against MRSA, following E. faecium, P. aeruginosa, E. coli and E. faecalis. Taken together, our results show that medicinal plants are good source of antagonistic bacteria having inhibitory effect against clinical bacterial pathogens.     

Microbial communities associated with the root system of wild olives (Olea europaea L. subsp. europaea var. sylvestris) are good reservoirs of bacteria with antagonistic potential against Verticillium dahliae

Wild olive trees, namely oleaster, are considered the ancestor of cultivated olive and a unexplored source of genetic variability that might contain important traits of agronomic and biotechnological interest. The longevity and genetic diversity of oleasters may have favoured selection of specific and well adapted rhizosphere microbial populations that can constitute unique reservoirs of microbial antagonists of Verticillium dahliae, the main soilborne fungal pathogen of olive worldwide. The objective of this present study was to determine the structure and diversity of bacterial communities in the rhizosphere and endosphere of oleaster from 11 havens in Cádiz and Córdoba provinces of Andalusia, southern Spain. To carry out the study we used a multiphasic approach. First, the occurrence and diversity of rhizosphere bacteria was monitored by a cultivation-independent-approach, using fluorescent terminal restriction fragment length polymorphism (FT-RFLP) analyses of amplified 16S rDNA sequences. FT-RFLP patterns revealed a high heterogeneity in the composition of the sampled rhizosphere bacterial communities and suggested the existence of plant genotype-site-specific communities, with each oleaster haven being a unique reservoir of bacterial diversity. Secondly, to investigate the antagonistic potential of these root-associated bacterial populations, a total of 675 bacterial isolates obtained from oleaster rhizosphere and endosphere were screened by dual testing for inhibition of in vitro growth of the highly virulent, olive defoliating pathotype of V. dahliae. Out of 675 tested bacterial isolates, 94 (14%) showed a strong antagonistic activity against a defoliating V. dahliae pathotype. Of the antagonistic bacteria, a slightly lower proportion (12.9% of total bacteria) were inhabitant of the oleaster rhizosphere compared to that in the endosphere (16.5%). The biotechnological potential of those isolates was assessed by in vitro production of different hydrolytic enzymes, indole-1.3-acetic acid (IAA), siderophores, and antimicrobial compounds. Overall, most of bacterial antagonists (58.5 to 78.3%) showed proteolytic, lipolytic, and chitinolytic activity, and produced IAA and siderophores. Finally, analysis of the 16S rDNA gene sequence indicated that most of the 94 bacterial antagonists belong to genera Bacillus (56.4%), Pseudomonas (27.7%), and Paenibacillus (7.4%). Overall, the rhizosphere and endosphere of wild olives were proved as a good reservoir of bacteria antagonists against V. dahliae. Several of those bacteria showing high and broad antagonism potential may therefore be considered for further analyses as promising biocontrol agents against V. dahliae in olive.     

Isolation and Selection of Potential Probiotic Bacteria from the Pig Gastrointestinal Tract

The present study aimed to isolate bacterial strains from the pig gastrointestinal tract that have antagonistic activity against potential pathogens and are able to produce antimicrobial compounds. That ability would be a first requirement for the strains’ possible use as probiotics. Samples obtained from pig intestinal mucosa and contents were screened for the presence of antagonistic activity against pathogenic indicator strains of Escherichia coli, Salmonella, and Listeria by means of the double-layer technique. Samples displaying the largest inhibitory halos were further studied for the production of inhibitory substances using the agar diffusion and microtitration methods. The three most promising isolates were identified by sequencing of the 16S rRNA gene and showed highest affiliation to Lactobacillus salivarius. Optimal growth conditions and bacteriocin production were recorded in de Man, Rogosa, and Sharpe broth under anaerobic conditions at 37 °C. The antimicrobial substances were found to be sensitive to proteolytic enzymes but showed good stability at pH values below 6. Our findings suggest that these three intestinal strains are able to produce antimicrobial substances capable of inhibiting the growth of potential enteric pathogens and might have potential as probiotic feed additives for the prevention of gastrointestinal diseases.     

Application of optical coherence tomography enhances reproducibility of arthroscopic evaluation of equine joints

Background

Staphylococcus aureus is an important cause of clinical mastitis in dairy cows worldwide. The cure rate after antimicrobial treatment of clinical S. aureus mastitis is very variable due to both cow and bacterial factors. Studies have shown that bacterial genotype might affect short-term bacteriological and clinical cure, but the long-term outcome has been less studied. The objectives of this study were to investigate associations between bacterial genotype and long-term outcome of veterinary-treated clinical mastitis (VTCM) caused by S. aureus during a follow-up period of 120 days and to study genotype variation among Swedish S. aureus isolates. S. aureus isolates from cases of VTCM were genotyped by pulsed-field gel electrophoresis. Long-term outcome measurements used were somatic cell count (SCC), additional diagnoses of VTCM, milk yield and culling. Isolates were classified into clusters (>80% similarity) and pulsotypes (100% similarity). Clusters and pulsotypes were grouped according to occurrence. Multivariable mixed-effect linear regression models including cow and bacterial factors with possible influence on SCC or milk yield were used to calculate differences in SCC or milk yield between groups. Additional outcome measures were calculated using a test of proportions.

Results

The isolates (n?=?185) were divided into 18 clusters and 29 pulsotypes. Two pulsotypes were classified as common, and were found in 64% of the cases of VTCM. Remaining isolates were classified as less common or rare pulsotypes. The distribution was similar at cluster level. Outcome was calculated from follow-up data on 111 cows. Significantly lower SCC during the follow-up period was found in cows infected with common clusters compared to in cows infected with less common/rare clusters. The proportion of cows with SCC <200 000 cells/ml during the whole follow-up period was significantly higher in the group common clusters than in the group less common/rare clusters. Bacterial genotype did not influence the other outcome parameters.

Conclusions

In Sweden, two S. aureus pulsotypes, identified in about 64% of clinical S. aureus cases, were widespread. Cows infected with the common genotypes had significantly lower SCC during 120 days after treatment compared to cows infected with less common or rare genotypes.     

Synthesis and in vitro antimicrobial activity of new 4-phenyl-5-methyl-4H-1,2,4-triazole-3-thione derivatives

This study presents the synthesis, spectral analysis and antimicrobial evaluation of a new series of substituted 1,2,4-triazole (5a–i) and 1,3,4-thiadiazole derivatives (9a, c, g, h). New compounds were obtained by cyclization reaction of acyl thiosemicarbazide derivatives in the presence of alkaline and acidic media. All synthesized compounds were screened for their in vitro antimicrobial activities. Nine of the compounds had potential activity against Gram-positive bacteria (MIC?=?3.91–500 µg/mL). Some compounds showed good activity especially against: Micrococcus luteus ATCC 10240 (MIC?=?3.91?31.25 µg/mL), Bacillus subtilis ATCC 6633 (MIC?=?15.63? 62.5 µg/mL), and Staphylococcus aureus ATCC 25923 (MIC?=?15.63?125 µg/mL).     

Amphibian Symbiotic Bacteria Do Not Show a Universal Ability To Inhibit Growth of the Global Panzootic Lineage of Batrachochytrium dendrobatidis

Microbiomes associated with multicellular organisms influence the disease susceptibility of hosts. The potential exists for such bacteria to protect wildlife from infectious diseases, particularly in the case of the globally distributed and highly virulent fungal pathogen Batrachochytrium dendrobatidis of the global panzootic lineage (B. dendrobatidis GPL), responsible for mass extinctions and population declines of amphibians. B. dendrobatidis GPL exhibits wide genotypic and virulence variation, and the ability of candidate probiotics to restrict growth across B. dendrobatidis isolates has not previously been considered. Here we show that only a small proportion of candidate probiotics exhibited broad-spectrum inhibition across B. dendrobatidis GPL isolates. Moreover, some bacterial genera showed significantly greater inhibition than others, but overall, genus and species were not particularly reliable predictors of inhibitory capabilities. These findings indicate that bacterial consortia are likely to offer a more stable and effective approach to probiotics, particularly if related bacteria are selected from genera with greater antimicrobial capabilities. Together these results highlight a complex interaction between pathogens and host-associated symbiotic bacteria that will require consideration in the development of bacterial probiotics for wildlife conservation. Future efforts to construct protective microbiomes should incorporate bacteria that exhibit broad-spectrum inhibition of B. dendrobatidis GPL isolates.     

High Prevalence of Multidrug-Tolerant Bacteria and Associated Antimicrobial Resistance Genes Isolated from Ornamental Fish and Their Carriage Water

Background

Antimicrobials are used to directly control bacterial infections in pet (ornamental) fish and are routinely added to the water these fish are shipped in to suppress the growth of potential pathogens during transport.

Methodology/Principal Findings

To assess the potential effects of this sustained selection pressure, 127 Aeromonas spp. isolated from warm and cold water ornamental fish species were screened for tolerance to 34 antimicrobials. Representative isolates were also examined for the presence of 54 resistance genes by a combination of miniaturized microarray and conventional PCR. Forty-seven of 94 Aeromonas spp. isolates recovered from tropical ornamental fish and their carriage water were tolerant to ≥15 antibiotics, representing seven or more different classes of antimicrobial. The quinolone and fluoroquinolone resistance gene, qnrS2, was detected at high frequency (37% tested recent isolates were positive by PCR). Class 1 integrons, IncA/C broad host range plasmids and a range of other antibiotic resistance genes, including floR, bla _TEM−1, tet(A), tet(D), tet(E), qacE2, sul1, and a number of different dihydrofolate reductase and aminoglycoside transferase coding genes were also detected in carriage water samples and bacterial isolates.

Conclusions

These data suggest that ornamental fish and their carriage water act as a reservoir for both multi-resistant bacteria and resistance genes.     

Diversity of cultivable fungi associated with Antarctic marine sponges and screening for their antimicrobial, antitumoral and antioxidant potential

The diversity of sponge-associated fungi has been poorly investigated in remote geographical areas like Antarctica. In this study, 101 phenotypically different fungal isolates were obtained from 11 sponge samples collected in King George Island, Antarctica. The analysis of ITS sequences revealed that they belong to the phylum Ascomycota. Sixty-five isolates belong to the genera Geomyces, Penicillium, Epicoccum, Pseudeurotium, Thelebolus, Cladosporium, Aspergillus, Aureobasidium, Phoma, and Trichocladium but 36 isolates could not be identified at genus level. In order to estimate the potential of these isolates as producers of interesting bioactivities, antimicrobial, antitumoral and antioxidant activities of fungal culture extracts were assayed. Around 51 % of the extracts, mainly from the genus Geomyces and non identified relatives, showed antimicrobial activity against some of the bacteria tested. On the other hand, around 42 % of the extracts showed potent antitumoral activity, Geomyces sp. having the best performance. Finally, the potential of the isolated fungi as producers of antioxidant activity seems to be moderate. Our results suggest that fungi associated with Antarctic sponges, particularly Geomyces, would be valuable sources of antimicrobial and antitumoral compounds. To our knowledge, this is the first report describing the biodiversity and the metabolic potential of fungi associated with Antarctic marine sponges.     

Antimicrobial resistance,virulence genes and PFGE-profiling of Escherichia coli isolates from South Korean cattle farms

To estimate the prevalence of Escherichia coli with potential pathogenicity in cattle farm in South Korea, a total of 290 E. coli isolates were isolated from cattle farms over a period of 2 years in South Korea. These were examined for phenotypic and genotypic characteristics including antimicrobial susceptibility, serotype, and gene profiles of virulence and antimicrobial resistance. The most dominant virulence gene was f17 (26.2%), followed by stx2 (15.9%), ehxA (11.0%), stx1 (8.3%), eae (5.2%), and sta (4.1%). Some shiga-toxin producing E. coli isolates possessed eae (15.9%). All isolates except for one showed resistance to one or more antimicrobials, with 152 isolates exhibiting multidrug-resistance. The most prevalent resistance phenotype detected was streptomycin (63.1%), followed by tetracycline (54.5%), neomycin (40.3%), cephalothin (32.8%), amoxicillin (30.0%), ampicillin (29.7%), and sulphamethoxazole/trimethoprim (16.6%). The associated resistance determinants detected were strA-strB (39.0%), tet(E) (80.0%), tet(A) (27.6%), aac(3)-IV (33.1%), aphA1 (21.4%), bla _TEM (23.8%), and sul2 (22.1%). When investigated by O serotyping and PFGE molecular subtyping, the high degree of diversity was exhibited in E. coli isolates. These results suggest that E. coli isolates from South Korean cattle farms are significantly diverse in terms of virulence and antimicrobial resistance. In conclusion, the gastroinstestinal flora of cattle could be a significant reservoir of diverse virulence and antimicrobial resistance determinants, which is potentially hazardous to public health.     

Bioremediation of volatile oil hydrocarbons by epiphytic bacteria associated with American grass (Cynodon sp.) and broad bean (Vicia faba) leaves

Fresh leaves of American grass and broad beans grown in pristine soil were naturally colonized with cultivable volatile oil hydrocarbon-utilizing bacteria, whose numbers increased significantly in plants grown in oily soil. According to their 16S rRNA gene sequences those bacteria were affiliated to various species of the genera Rhodococcus and Pseudomonas. Qualitative growth studies revealed that pure cultures of these phyllospheric bacteria could grow successfully on a solid mineral medium containing individual alkanes with chain lengths of C₉ through C₄₀ and the aromatics phenanthrene, naphthalene, and biphenyl as sole sources of carbon and energy. Quantitative measurements showed that the individual pure bacterial isolates degraded between about 20 and 30% of crude oil, n-hexadecane, or phenanthrene in batch culture after a one-week incubation. These results reflect the high hydrocarbon degradation potential of those bacteria. The isolates were diazotrophic (nitrogen fixers), meaning that they were self-dependent in covering their nitrogen requirements. Incubating fresh leaves in closed microcosms containing volatile oil hydrocarbons resulted in up to more than 80% attenuation of these compounds after two weeks. Experimental evidence was provided that the leaf tissues did not contribute to this attenuation, which was exclusively due to the bacterial activity.     

Identification,Characterization, and Efficacy Evaluation of Bacillus velezensis for Shot-Hole Disease Biocontrol in Flowering Cherry

Though information exists regarding the pathogenesis of the shot-hole disease (SH) in flowering cherry (FC), there has been a lack of research focusing on SH management. Therefore, here, we investigated the inhibitory activities of antagonistic bacteria against SH pathogens both in vitro and in vivo as well as their biochemical characteristics and bioactive compounds. Two biosurfactant-producing bacterial antagonists, identified as Bacillus velezensis strains JCK-1618 and JCK-1696, exhibited the best effects against the growth of both bacterial and fungal SH pathogens in vitro through their cell-free culture filtrates (CFCFs). These two strains also strongly inhibited the growth of the pathogens via the action of their antimicrobial diffusible compounds and antimicrobial volatile organic compounds (VOCs). Crude enzymes, solvent extracts, and biosurfactants of the two strains exhibited antimicrobial activities. Liquid chromatography/electrospray ionization time-of-flight mass spectrometric analysis of the partially purified active fractions revealed that the two antagonists produced three cyclic lipopeptides, including iturin A, fengycin A, and surfactin, and a polyketide, oxydifficidin. In a detached leaf assay, pre-treatment and co-treatment of FC leaves with the CFCFs led to a large reduction in the severity of the leaf spots caused by Epicoccum tobaicum and Bukholderia contaminans, respectively. In addition, the two antagonists produced indole-3-acetic acid, siderophore, and a series of hydrolytic enzymes, along with the formation of a substantial biofilm. To our knowledge, this is the first report of the antimicrobial activities of the diffusible compounds and VOCs of B. velezensis against the SH pathogens and their efficiency in the biocontrol of SH.