Metabolic adaptability shifts of cell membrane fatty acids of Komagataeibacter hansenii HDM1-3 improve acid stress resistance and survival in acidic environments.

Komagataeibacter hansenii HDM1-3 (K. hansenii HDM1-3) has been widely applied for producing bacterial cellulose (BC). The yield of BC has been frequently limited by the acidification during sugar metabolism, due to the generation of organic acids such as acetic acid. In this study, the acid resistance mechanism of K. hansenii HDM1-3 has been investigated from the aspect of metabolic adaptability of cell membrane fatty acids. Firstly, we observed that the survival rate of K. hansenii HDM1-3 was decreased with lowered pH values (adjusted with acetic acids), accompanied by increased leakage rate. Secondly, the cell membrane adaptability in response to acid stress was evaluated, including the variations of cell membrane fluidity and fatty acid composition. The proportion of unsaturated fatty acids was increased (especially, C18-1w9c and C19-Cyc), unsaturation degree and chain length of fatty acids were also increased. Thirdly, the potential molecular regulation mechanism was further elucidated. Under acid stress, the fatty acid synthesis pathway was involved in the structure and composition variations of fatty acids, which was proved by the activation of both fatty acid dehydrogenase (des) and cyclopropane fatty acid synthase (cfa) genes, as well as the addition of exogenous fatty acids. The fatty acid synthesis of K. hansenii HDM1-3 may be mediated by the activation of two-component sensor signaling pathways in response to the acid stress. The acid resistance mechanism of K. hansenii HDM1-3 adds to our knowledge of the acid stress adaptation, which may facilitate the development of new strategies for improving the industrial performance of this species under acid stress.

Streptophyta and Acetic Acid Bacteria Succession Promoted by Brass in Slow Sand Filter System Schmutzdeckes.

Macro- and microorganism activities are important for the effectiveness of the slow sand filtration (SSF), where native microorganisms remove contaminants mainly by substrate competition, predation, and antagonism. The aim of the present study was to evaluate the addition of the oligodynamic metals iron, copper, and brass, inserted separately into SSF to enhance pollutant removal in water samples. Four laboratory-scale SSFs were built and tested: control, iron, copper, and brass. Water analysis included physicochemical evaluation, total and fecal coliform quantification. An analysis on microbial communities in the SSFs schmutzdecke was achieved by using 16S rRNA amplification, the Illumina MiSeq platform, and the QIIME bioinformatics software. The results demonstrated that inorganic and organic contaminants such as coliforms were removed up to 90%. The addition of metals had no significant effect (p > 0.05) on the other parameters. The microbial community analysis demonstrated different compositions of the SSF with brass-influent, where the eukaryote Streptophyta was predominant (31.4%), followed by the acetic acid bacteria Gluconobacter (24.6%), and Acetobacteraceae (7.7%), these genera were absent in the other SSF treatments. In conclusion, the use of a SSF system can be a low cost alternative to reduce microbial contamination in water and thus reduce gastrointestinal diseases in rural areas.

Characterisation of Roseomonas mucosa isolated from the root canal of an infected tooth.

OBJECTIVE: The genus Roseomonas comprises a group of pink-pigmented, slow-growing, aerobic, non-fermentative Gram-negative bacteria, which have been isolated from environmental sources such as water and soil, but are also associated with human infections. In the study presented here, Roseomonas mucosa was identified for the first time as part of the endodontic microbiota of an infected root canal and characterised in respect to growth, antibiotic susceptibility and biofilm formation. RESULTS: The isolated R. mucosa strain showed strong slime formation and was resistant to most ß-lactam antibiotics, while it was susceptible to aminoglycosides, carbapenemes, fluorochinolones, polymyxines, sulfonamides and tetracyclines. Biofilm formation on artificial surfaces (glass, polystyrene, gutta-percha) and on teeth was tested using colorimetric and fluorescence microscopic assays. While solid biofilms were formed on glass surfaces, on the hydrophobic surface of gutta-percha points, no confluent but localised, spotty biofilms were observed. Furthermore, R. mucosa was able form biofilms on dentin. The data obtained indicate that R. mucosa can support establishment of endodontic biofilms and furthermore, infected root canals might serve as an entrance pathway for blood stream infections by this emerging pathogen.

Black Currant (Ribes nigrum L.) and Bilberry (Vaccinium myrtillus L.) Fruit Juices Inhibit Adhesion of Asaia spp.

The aim of the study was to evaluate the activity of high-polyphenolic black currant (Ribes nigrum L.) and bilberry (Vaccinium myrtillus L.) juices against bacterial strains Asaia lannensis and Asaia bogorensis isolated as spoilage of commercial soft drinks. The composition of fruit juices was evaluated using chromatographic techniques HPLC and LC-MS. The adhesion to glass, polystyrene, and polyethylene terephthalate in two different culture media was evaluated by luminometry and the plate count method. The major anthocyanins in the V. myrtillus were petunidin-3-glucoside, malvidin-3-glucoside, cyanidin-3-glucoside, and delphinidin-3-glucoside, while in R. nigrum delphinidin-3-rutinoside and cyanidin-3-rutinoside were detected. The LC-MS analysis showed presence of anthocyanins (delphinidin, cyanidin, petunidin, and malvidin derivatives), phenolic acids (chlorogenic and neochlorogenic acids), flavonols (quercetin-3-glucoside, quercetin-3-rutinoside), and flavanols (procyanidin B2 and procyanidin type A2). Additionally, in the bilberry juice A type procyanidin trimer was detected. The adhesion of Asaia spp. cells depended on the type of medium, carbon sources, and the type of abiotic surfaces. We noted that the adhesion was significantly stronger in minimal medium containing sucrose. The addition of bilberry and black currant juices notably reduced bacterial growth as well as cell adhesion to polyethylene terephthalate surfaces.

Asaia lannensis bacteremia in a 'needle freak' patient.

The genus Asaia has gained much interest lately owing to constant new species discoveries and its role as a potential opportunistic pathogen to humans. Here we describe a transient bacteremia due to Asaia lannensis in a patient with a psychiatric disorder (compulsive self-injection of different substances). Common phenotypic methods of identification failed to identify this organism, and only restriction fragment lenght polymorphism of PCR-amplified 16S rRNA gene allowed for proper identification. The isolate was highly resistant to most antibiotics. The paper also discusses the currently available medical literature, acknowledges the potential problems linked to the isolation of these strains and proposes an approach to species identification that can be applied in a clinical microbiology laboratory.

Native microbiome impedes vertical transmission of Wolbachia in Anopheles mosquitoes.

Over evolutionary time, Wolbachia has been repeatedly transferred between host species contributing to the widespread distribution of the symbiont in arthropods. For novel infections to be maintained, Wolbachia must infect the female germ line after being acquired by horizontal transfer. Although mechanistic examples of horizontal transfer exist, there is a poor understanding of factors that lead to successful vertical maintenance of the acquired infection. Using Anopheles mosquitoes (which are naturally uninfected by Wolbachia) we demonstrate that the native mosquito microbiota is a major barrier to vertical transmission of a horizontally acquired Wolbachia infection. After injection into adult Anopheles gambiae, some strains of Wolbachia invade the germ line, but are poorly transmitted to the next generation. In Anopheles stephensi, Wolbachia infection elicited massive blood meal-induced mortality, preventing development of progeny. Manipulation of the mosquito microbiota by antibiotic treatment resulted in perfect maternal transmission at significantly elevated titers of the wAlbB Wolbachia strain in A. gambiae, and alleviated blood meal-induced mortality in A. stephensi enabling production of Wolbachia-infected offspring. Microbiome analysis using high-throughput sequencing identified that the bacterium Asaia was significantly reduced by antibiotic treatment in both mosquito species. Supplementation of an antibiotic-resistant mutant of Asaia to antibiotic-treated mosquitoes completely inhibited Wolbachia transmission and partly contributed to blood meal-induced mortality. These data suggest that the components of the native mosquito microbiota can impede Wolbachia transmission in Anopheles. Incompatibility between the microbiota and Wolbachia may in part explain why some hosts are uninfected by this endosymbiont in nature.

Delayed larval development in Anopheles mosquitoes deprived of Asaia bacterial symbionts.

BACKGROUND: In recent years, acetic acid bacteria have been shown to be frequently associated with insects, but knowledge on their biological role in the arthropod host is limited. The discovery that acetic acid bacteria of the genus Asaia are a main component of the microbiota of Anopheles stephensi makes this mosquito a useful model for studies on this novel group of symbionts. Here we present experimental results that provide a first evidence for a beneficial role of Asaia in An. stephensi. RESULTS: Larvae of An. stephensi at different stages were treated with rifampicin, an antibiotic effective on wild-type Asaia spp., and the effects on the larval development were evaluated. Larvae treated with the antibiotic showed a delay in the development and an asynchrony in the appearance of later instars. In larvae treated with rifampicin, but supplemented with a rifampicin-resistant mutant strain of Asaia, larval development was comparable to that of control larvae not exposed to the antibiotic. Analysis of the bacterial diversity of the three mosquito populations confirmed that the level of Asaia was strongly decreased in the antibiotic-treated larvae, since the symbiont was not detectable by PCR-DGGE (denaturing gradient gel electrophoresis), while Asaia was consistently found in insects supplemented with rifampicin plus the antibiotic-resistant mutant in the diet, and in those not exposed to the antibiotic. CONCLUSIONS: The results here reported indicate that Asaia symbionts play a beneficial role in the normal development of An. stephensi larvae.

Recurrent Granulibacter bethesdensis infections and chronic granulomatous disease.

Chronic granulomatous disease (CGD) is characterized by frequent infections, most of which are curable. Granulibacter bethesdensis is an emerging pathogen in patients with CGD that causes fever and necrotizing lymphadenitis. However, unlike typical CGD organisms, this organism can cause relapse after clinical quiescence. To better define whether infections were newly acquired or recrudesced, we use comparative bacterial genomic hybridization to characterize 11 isolates obtained from 5 patients with CGD from North and Central America. Genomic typing showed that 3 patients had recurrent infection months to years after apparent clinical cure. Two patients were infected with the same strain as previously isolated, and 1 was infected with a genetically distinct strain. This organism is multidrug resistant, and therapy required surgery and combination antimicrobial drugs, including long-term ceftriaxone. G. bethesdensis causes necrotizing lymphadenitis in CGD, which may recur or relapse.

Acidocella aluminiidurans sp. nov., an aluminium-tolerant bacterium isolated from Panicum repens grown in a highly acidic swamp in actual acid sulfate soil area of Vietnam.

An aluminium-tolerant bacterium, strain AL46(T), was isolated from a waterweed, Panicum repens, grown in a highly acidic swamp (pH 3) at an actual acid sulfate soil area of Vietnam. Cells were Gram-negative, aerobic, non-spore-forming, non-motile rods (0.3 microm wide and 1.2-1.6 microm long). 16S rRNA gene sequence analysis indicated that strain AL46(T) belongs to the genus Acidocella, class Alphaproteobacteria. Strain AL46(T) was related most closely to the type strains of Acidocella facilis and Acidocella aminolytica (99.4 and 97.8 % 16S rRNA gene sequence similarity, respectively). Levels of DNA-DNA relatedness between strain AL46(T) and the above type strains were 40 %. The results of physiological and biochemical tests allowed the novel strain to be differentiated phenotypically from the two recognized Acidocella species. Data for predominant cellular fatty acids (cyclopropyl C(19 : 0) and C(18 : 1)), major isoprenoid quinone (Q-10) and DNA G+C content (65.6 mol%) were in accordance with those reported for the genus Acidocella. Therefore, strain AL46(T) is considered to represent a novel species of the genus Acidocella, for which the name Acidocella aluminiidurans sp. nov. is proposed. The type strain is AL46(T) (=NBRC 104303(T) =VTCC-D9-1(T)).

Naphthalene-utilizing and mercury-resistant bacteria isolated from an acidic environment.

Soil samples were taken from areas of low pH (2.5-3.5) surrounding an outdoor coal storage pile. These samples were added to medium with naphthalene as the sole carbon source to enrich for organisms capable of degrading polycyclic aromatic hydrocarbons (PAH) at low pH. Five such bacterial strains were isolated. Sequencing of the 16S rDNA showed them to be members of the genera Clavibacter, Arthrobacter and Acidocella. These organisms were all capable of growth with naphthalene as a sole carbon source at low pH. The genes nahAc, nahAd, phnAc, nahH, xylE or GST, which are known to be associated with PAH degradation were not detected. Isolate 10, the Acidocella strain, tolerated high levels of mercury. PCR amplification and sequencing of genes from the mer operon from isolate 10 DNA suggested that mercury is transported into the bacterial cell and subsequently detoxified since the enzymes encoded by genes in this operon are involved in these processes.

Generation of novel plasmids in Escherichia coli S17-1(pSUP106).

When the highly metal-resistant acidophilic heterotrophic strain, Acidiphilium symbioticum KM2, was incubated with two Escherichia coli strains, viz. S17-1 (pSUP106) and K12, on a medium that supported growth of these two divergent species of different habitats, E. coli transconjugants were isolated that contained novel plasmids and were resistant to Zn(2+) (48 m M), Cu(2+) (12 m M), Ni(2+) (12 m M), chloramphenicol (50 microg/ml), and tetracycline (25 microg/ml). The transconjugant plasmids did not hybridize with any of the A. symbioticum KM2 plasmids. After curing of the plasmids, the transconjugants became sensitive to 12 m M Zn(2+), 12 m M Cu(2+), and 12 m M Ni(2+), but remained chloramphenicol and tetracycline resistant-the phenotypic markers that were originally present in pSUP106. That a part of pSUP106 was integrated into the chromosome of the transconjugants was evident from the hybridization of pSUP106 with chromosomal DNA of the cured derivatives of the transconjugants. Further, the transconjugant plasmids hybridized only with the chromosomal DNA of E. coli S17-1 and not with the chromosomal DNA of A. symbioticum KM2 or E. coli K12, suggesting their host chromosomal origin. Thus, the present study describes a unique event of genetic rearrangements in the E. coli strain S17-1 (pSUP106), resulting in the formation of novel plasmids conferring metal-resistance phenotypes in the cell.

Influence of residual ethanol concentration on the growth of Gluconacetobacter xylinus I 2281.

The influence of residual ethanol on metabolism of food grade Gluconacetobacter xylinus I 2281 was investigated during controlled cultivations on 35 g/l glucose and 5 g/l ethanol. Bacterial growth was strongly reduced in the presence of ethanol, which is unusual for acetic acid bacteria. Biomass accumulated only after complete oxidation of ethanol to acetate and carbon dioxide. In contrast, bacterial growth initiated without delay on 35 g/l glucose and 5 g/l acetate. It was found that acetyl CoA was activated by the acetyl coenzyme A synthetase (Acs) pathway in parallel with the phosphotransacetylase (Pta)-acetate kinase (Ack) pathway. The presence of ethanol in the culture medium strongly reduced Pta activity while Acs and Ack remained active. A carbon balance calculation showed that the overall catabolism could be divided into two independent parts: upper glycolysis linked to glucose catabolism and lower glycolysis liked to ethanol catabolism. This calculation showed that the carbon flux through the tricarboxylic cycle is lower on ethanol than on acetate. This corroborated the diminution of carbon flux through the Pta-Ack pathway due to the inhibition of Pta activity on ethanol.

Inducible aluminum resistance of Acidiphilium cryptum and aluminum tolerance of other acidophilic bacteria.

Aluminum ions are highly soluble in acidic environments. Toxicity of aluminum ions for heterotrophic, facultatively and obligately chemolithoautotrophic acidophilic bacteria was examined. Acidiphilium cryptum grew in glucose-mineral medium, pH 3, containing 300 mM aluminum sulfate [Al(2)(SO(4))(3)] after a lag phase of about 120 h with a doubling time of 7.6 h, as compared to 5.2 h of growth without aluminum. Precultivation with 1 mM Al(2)(SO(4))(3) and transfer to a medium with 300 mM Al(2)(SO(4))(3) reduced the lag phase from 120 to 60 h, and immediate growth was observed when A. cryptum was precultivated with 50 mM Al(2)(SO(4))(3), suggesting an aluminum-induced resistance. Aluminum resistance was not induced by Fe(3+) ions and divalent cations. Upon exposure of A. cryptum to 300 mM Al(2)(SO(4))(3), the protein profile changed significantly as determined by SDS-PAGE. When other acidophiles were cultivated with 50-200 mM aluminum sulfate, no lag phase was observed while the growth rates and the cellular yields were significantly reduced. This growth response was observed with Acidobacterium capsulatum, Acidiphilium acidophilum, Acidithiobacillus ferrooxidans, and Acidithiobacillus thiooxidans. Precultivation of these strains with aluminum ions did not alter the growth response caused by aluminum. The content of A. cryptum cultivated with 300 mM Al(2)(SO(4))(3)was 0.44 microg Al/mg cell dry weight, while that of the other strains cultivated with 50 mM Al(2)(SO(4))(3) ranged from 0.30 to 3.47 microg Al/mg cell dry weight.

Resistance to cadmium and zinc in Acidiphilium symbioticum KM2 is plasmid mediated.

The acidophilic heterotroph, Acidiphilium symbioticum KM2, is highly resistant to several metals and harbors three plasmids of 3.8, 7.1, and 56 kb in size. The bacterium becomes extremely sensitive to metals when it is cured of its plasmids. A mini-plasmid library was constructed by ligating the plasmid DNA fragments generated by MboI partial digestion into the BamHI site of pBluescriptII KS+. The Lac(-)Amp(r) transformants of Escherichia coli DH5alpha, isolated after transformation with the library, were counter-selected on Cu(2+), Cd(2+), Ni(2+), and Zn(2+)-containing plates. Only Cd(2+)- and Zn(2+)-resistant colonies were developed, and, after screening, four types of recombinant plasmids designated as pNM201 (7.2 kb), pNM206 (3.4 kb), pNM208 (4.5 kb), and pNM215 (4.9 kb) were obtained. The DNA insert in pNM206 hybridized strongly with the 3.8-kb plasmid and weakly with the 7.1-kb plasmid of Acidiphilium symbioticum KM2. The DNA insert in pNM215 hybridized only with the 7.1-kb plasmid. These results strongly suggested that resistance to cadmium and zinc in A. symbioticum KM2 is mediated by these plasmids. The smallest insert of 422 bp in pNM206 conferring metal resistance in E. coli has no sequence similarity with the reported metal-resistant genes. All the putative ORFs are significantly rich (up to 37%) in basic amino acids, mainly arginine.

Acetate-specific stress response in acetate-resistant bacteria: an analysis of protein patterns.

Many metabolic byproducts have toxic effects on bacteria, and acetic acid is an excellent model for such molecules. The negative effects of acetate, which include decreased growth rates and specific productivities, appear for Escherichia coli at acetate concentrations lower than 5 g/L. Acetic acid bacteria, however, are naturally resistant to the detrimental effects of acetate in their surroundings; they remain active at acetate levels well over 40 g/L. This study investigated the response to acetate challenges by the naturally acetate-resistant bacteria Acetobacter aceti and Gluconobacter suboxydans to learn more about possible mechanisms of tolerance to otherwise toxic low molecular weight metabolites. Growth studies showed that the resistant bacteria grow more slowly in the presence of acetate but are not slowed nearly so much as is E. coli. In addition, two-dimensional gel electrophoresis (2DE) was applied to study the relative protein patterns of acetate-resistant bacteria during growth in the presence and absence of acetate. In each organism, growth in acetate-containing medium led to elevated levels of many stress response proteins. 2DE analysis of heat-shocked cultures was used to determine which were nonspecific. Elimination of those proteins that were also amplified following heat shock left only eight proteins, here designated acetate-specific stress proteins (Asps), which are overexpressed specifically in response to acetate. Three of these, AspA, AspB, and AspC, appear to be analogous in the two bacterial strains studied, based on their apparent pIs and molecular weights.

Influence of constant and oscillating dissolved oxygen concentrations on keto acid production by Gluconobacter oxydans subsps. melanogenum.

Gluconobacter species are known to oxidise glucose via a direct oxidation pathway which is distinct from the pentose phosphate pathway. In the present communication results of an investigation on the influence of different dissolved oxygen concentrations (DO) on the production of 2,5-diketogluconic acid in batch and chemostat cultures are given. DO of 30% relative to air at 1 bar was found as a threshold level for optimum productivity. The positive influence of continuous availability of dissolved oxygen on the process of rapid glucose oxidation was unambiguously shown as the result of induction of membrane bound dehydrogenases involved in direct glucose oxidation. Furthermore data of scale-down experiments in which the organism was cultivated under oscillations of dissolved oxygen, are given. The influences of such oscillations of DO in the region of the established threshold (30% saturation) were found to result in a prolonged lag phase for growth and product formation. The data obtained in this study revealed critical residence times at low DO that could be employed as a criterion for scale up of this aerobic process.