Anthracene and Pyrene Biodegradation Performance of Marine Sponge Symbiont Bacteria Consortium.

Every petroleum-processing plant produces sewage sludge containing several types of polycyclic aromatic hydrocarbons (PAHs). The degradation of PAHs via physical, biological, and chemical methods is not yet efficient. Among biological methods, the use of marine sponge symbiont bacteria is considered an alternative and promising approach in the degradation of and reduction in PAHs. This study aimed to explore the potential performance of a consortium of sponge symbiont bacteria in degrading anthracene and pyrene. Three bacterial species (Bacillus pumilus strain GLB197, Pseudomonas stutzeri strain SLG510A3-8, and Acinetobacter calcoaceticus strain SLCDA 976) were mixed to form the consortium. The interaction between the bacterial consortium suspension and PAH components was measured at 5 day intervals for 25 days. The biodegradation performance of bacteria on PAH samples was determined on the basis of five biodegradation parameters. The analysis results showed a decrease in the concentration of anthracene (21.89%) and pyrene (7.71%), equivalent to a ratio of 3:1, followed by a decrease in the abundance of anthracene (60.30%) and pyrene (27.52%), equivalent to a ratio of 2:1. The level of pyrene degradation was lower than that of the anthracene due to fact that pyrene is more toxic and has a more stable molecular structure, which hinders its metabolism by bacterial cells. The products from the biodegradation of the two PAHs are alcohols, aldehydes, carboxylic acids, and a small proportion of aromatic hydrocarbon components.

Ecological role of Acinetobacter calcoaceticus GSN3 in natural biofilm formation and its advantages in bioremediation.

This study assessed the role of a new Acinetobacter calcoaceticus strain, GSN3, with biofilm-forming and phenol-degrading abilities. Three biofilm reactors were spiked with activated sludge (R1), green fluorescent plasmid (GFP) tagged GSN3 (R2), and their combination (R3). More than 99% phenol removal was achieved during four weeks in R3 while this efficiency was reached after two and four further operational weeks in R2 and R1, respectively. Confocal scanning electron microscopy revealed that GSN3-gfp strains appeared mostly in the deeper layers of the biofilm in R3. After four weeks, almost 7.07 × 107 more attached sludge cells were counted per carrier in R3 in comparison to R1. Additionally, the higher numbers of GSN3-gfp in R2 were unable to increase the efficiency as much as measured in R3. The presence of GSN3-gfp in R3 conveyed advantages, including enhancement of cell immobilization, population diversity, metabolic cooperation and ultimately treatment efficiency.

Biofilm Formation and Extended-Spectrum Beta-Lactamase Producer among Acinetobacter Species Isolated in a Tertiary Care Hospital: A Descriptive Cross-sectional Study.

INTRODUCTION: Acinetobacter species are short, stout, gram-negative coccobacilli, generally considered to be a relatively low-grade pathogen. However, its resistance towards multiple classes of antibiotics through an array of resistance mechanisms including its ability to form biofilm has led to its emergence as an important pathogen in hospital settings. This study was done to determine the prevalence of biofilm former and Extended-spectrum Beta-Lactamase producer among Acinetobacter species. METHODS: A descriptive cross-sectional study was done in the clinical microbiology laboratory, Kathmandu Medical College from January to June 2019. Convenient sampling method was used. Ethical approval was taken from the Institutional Review Committee, Ref no. 2812201805. Preliminary identification followed by characterization of Acinetobacter species was done. Antibiotic susceptibility test was done using the Kirby-Bauer method following Clinical and Laboratory Standards Institute guidelines. Extended-spectrum Beta-Lactamase was detected by combined disc method and Biofilm detection was done using congo red agar method. Statistical Package for Social Sciences 16.0 version statistical software package was used for statistical analysis. Point estimate at 95% Confidence Interval was calculated along with frequencyand proportion for binarydata. RESULTS: Among 108 Acinetobacter species, 86 (79.7%) Acinetobacter calcoaceticus-A. baumannii complex was seen. Seventy-eight (72%) of the isolates were multidrug-resistant, 34 (31%) of the isolates were Extended-spectrum Beta-Lactamase producer and only 10 (9.3%) of the isolates, were biofilm producers. CONCLUSIONS: Multidrug-resistant Acinetobacter spp. with the ability to produce Extended-spectrum Beta-Lactamase is prevalent in our hospital settings. Strict compliance with infection control practices is necessary to curb its spread.

Enhanced biomass production of duckweeds by inoculating a plant growth-promoting bacterium, Acinetobacter calcoaceticus P23, in sterile medium and non-sterile environmental waters.

Duckweed offers the promise of a co-benefit culture combining water purification with biomass production. Acinetobacter calcoaceticus P23 is a plant growth-promoting bacterium isolated from a duckweed, Lemna aequinoctialis. This study quantified its growth-promoting effect on three duckweeds (L. aoukikusa, L. minor, and Spirodela polyrhiza) in sterile Hoagland solution and evaluated its usefulness in duckweed culture under non-sterile conditions. P23 promoted growth of three duckweeds in sterile Hoagland solution at low to high nutrient concentrations (1.25-10 mg NO3-N/L and 0.25-2.0 mg PO4-P/L). It increased the biomass production of L. aequinoctialis 3.8-4.3-fold, of L. minor 2.3-3.3-fold, and of S. polyrhiza 1.4-1.5-fold after 7 days compared with noninoculated controls. P23 also increased the biomass production of L. minor 2.4-fold in pond water and 1.7-fold in secondary effluent of a sewage treatment plant under non-sterile conditions at laboratory-scale experiments. P23 rescued L. minor from growth inhibition caused by microorganisms indigenous to the pond water. The results demonstrate that the use of P23 in duckweed culture can improve the efficiency of duckweed biomass production, and a positive effect of P23 on duckweed-based wastewater treatment can be assumed.

Risk factors and outcomes for patients with bloodstream infection due to Acinetobacter baumannii-calcoaceticus complex.

Identifying patients at risk for bloodstream infection (BSI) due to Acinetobacter baumannii-Acinetobacter calcoaceticus complex (ABC) and providing early appropriate therapy are critical for improving patient outcomes. A retrospective matched case-control study was conducted to investigate the risk factors for BSI due to ABC in patients admitted to the Detroit Medical Center (DMC) between January 2006 and April 2009. The cases were patients with BSI due to ABC; the controls were patients not infected with ABC. Potential risk factors were collected 30 days prior to the ABC-positive culture date for the cases and 30 days prior to admission for the controls. A total of 245 case patients were matched with 245 control patients. Independent risk factors associated with BSI due to ABC included a Charlson's comorbidity score of ≥ 3 (odds ratio [OR], 2.34; P = 0.001), a direct admission from another health care facility (OR, 4.63; P < 0.0001), a prior hospitalization (OR, 3.11; P < 0.0001), the presence of an indwelling central venous line (OR, 2.75; P = 0.011), the receipt of total parenteral nutrition (OR, 21.2; P < 0.0001), the prior receipt of ß-lactams (OR, 3.58; P < 0.0001), the prior receipt of carbapenems (OR, 3.18; P = 0.006), and the prior receipt of chemotherapy (OR, 15.42; P < 0.0001). The median time from the ABC-positive culture date to the initiation of the appropriate antimicrobial therapy was 2 days (interquartile range [IQR], 1 to 3 days). The in-hospital mortality rate was significantly higher among case patients than among control patients (OR, 3.40; P < 0.0001). BSIs due to ABC are more common among critically ill and debilitated institutionalized patients, who are heavily exposed to health care settings and invasive devices.

Plant growth-promoting bacterium Acinetobacter calcoaceticus P23 increases the chlorophyll content of the monocot Lemna minor (duckweed) and the dicot Lactuca sativa (lettuce).

Acinetobacter calcoaceticus P23 is a plant growth-promoting bacterium that was isolated from the surface of duckweed (Lemna aoukikusa). The bacterium was observed to colonize on the plant surfaces and increase the chlorophyll content of not only the monocotyledon Lemna minor but also the dicotyledon Lactuca sativa in a hydroponic culture. This effect on the Lactuca sativa was significant in nutrient-poor (×1/100 dilution of H2 medium) and not nutrient-rich (×1 or ×1/10 dilutions of H2 medium) conditions. Strain P23 has the potential to play a part in the future development of fertilizers and energy-saving hydroponic agricultural technologies.

Inoculation of phosphate solubilizing bacteria for the improvement of lead accumulation by Brassica juncea.

Two phosphate-solubilizing bacterial strains were isolated and identified as Acinetobacter calcoaceticus YC-5a and Enterobacter agglomerans KMC-7 based on the 16S rRNA gene sequence analysis. A. calcoaceticus YC-5a is less well known as a phosphate-solubilizing plant-associated bacterium. The plant growth-promoting properties of the phosphate-solubilizing bacteria (PSB) were characterized in vitro, including their phosphate-solubilizing activities and their capabilities for producing indole-3-acetic acid and siderophores. A pot experiment was conducted to elucidate the effects of inoculating both strains on the growth and Pb uptake of Brassica juncea grown in different concentrations of Pb-contaminated soils. Inoculation with both PSB not only stimulated the growth of B. juncea, but it also influenced the accumulation of Pb in the shoots and roots of the host plant. The present study demonstrates that PSB are a valuable microbial resource that can be exploited to improve the efficiency of phytoextraction.

Acinetobacter calcoaceticus-baumannii complex strains induce caspase-dependent and caspase-independent death of human epithelial cells.

We investigated interactions of human isolates of Acinetobacter calcoaceticus-baumannii complex strains with epithelial cells. The results showed that bacterial contact with the cells as well as adhesion and invasion were required for induction of cytotoxicity. The infected cells revealed hallmarks of apoptosis characterized by cell shrinking, condensed chromatin, and internucleosomal fragmentation of nuclear DNA. The highest apoptotic index was observed for 4 of 10 A. calcoaceticus and 4 of 7 A. baumannii strains. Moreover, we observed oncotic changes: cellular swelling and blebbing, noncondensed chromatin, and the absence of DNA fragmentation. The highest oncotic index was observed in cells infected with 6 A. calcoaceticus isolates. Cell-contact cytotoxicity and cell death were not inhibited by the pan-caspase inhibitor z-VAD-fmk. Induction of oncosis was correlated with increased invasive ability of the strains. We demonstrated that the mitochondria of infected cells undergo structural and functional alterations which can lead to cell death. Infected apoptotic and oncotic cells exhibited loss of mitochondrial transmembrane potential (ΔΨ(m)). Bacterial infection caused generation of nitric oxide and reactive oxygen species. This study indicated that Acinetobacter spp. induced strain-dependent distinct types of epithelial cell death that may contribute to the pathogenesis of bacterial infection.

Biofilm formation and microbial activity in a biofilter system in the presence of MTBE, ETBE and TAME.

Emerging water contaminants derived from unleaded gasoline such as methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE) and tert-amyl methyl ether (TAME), are in need of effective bioremediation technologies for restoring water resources. In order to design the conditions of a future groundwater bioremediating biofilter, this work assesses the potential use of Acinetobacter calcoaceticus M10, Rhodococcus ruber E10 and Gordonia amicalis T3 for the removal of MTBE, ETBE and TAME in consortia or as individual strains. Biofilm formation on an inert polyethylene support material was assessed with scanning electron microscopy, and consortia were also analysed with fluorescent in situ hybridisation to examine the relation between the strains. A. calcoaceticus M10 was the best coloniser, followed by G. amicalis T3, however, biofilm formation of pair consortia favoured consortium M10-E10 both in formation and activity. However, degradation batch studies determined that neither consortium exhibited higher degradation than individual strain degradation. The physiological state of the three strains was also determined through flow cytometry using propidium iodide and 3'-dihexylocarbocyanine iodide thus gathering information on their viability and activity with the three oxygenates since previous microbial counts revealed slow growth. Strain E10 was observed to have the highest physiological activity in the presence of MTBE, and strain M10 activity with TAME was only maintained for 24 h, thus we believe that biotransformation of MTBE occurs within the active periods established by the cytometry analyses. Viable cell counts and oxygenate removal were determined in the presence of the metabolites tert-butyl alcohol (TBA) and tert-amyl alcohol (TAA), resulting in TBA biotransformation by M10 and E10, and TAA by M10. Our results show that A. calcoaceticus M10 and the consortium M10-E10 could be adequate inocula in MTBE and TAME bioremediating technologies.

Sustainable biodegradation of phenol by Acinetobacter calcoaceticus P23 isolated from the rhizosphere of duckweed Lemna aoukikusa.

Phenol-degrading bacteria were isolated from the rhizosphere of duckweed (Lemna aoukikusa) using an enrichment culture method. One of the isolates, P23, exhibited an excellent ability to degrade phenol and attach to a solid surface under laboratory conditions. Phylogenetic analysis revealed that P23 belongs to the genera Acinetobacter and has the highest similarity to Acinetobacter calcoaceticus. P23 rapidly colonized on the surface of sterilized duckweed roots and formed biofilms, indicating that the conditions provided by the root system of duckweed are favorable to P23. A long-term performance test (160 h) showed that continuous removal of phenol can be attributed to the beneficial symbiotic interaction between duckweed and P23. P23 is the first growth-promoting bacterium identified from Lemna aoukikusa. The results in this study suggest the potential usefulness of dominating a particular bacterium in the rhizosphere of duckweeds to achieve efficient and sustainable bioremediation of polluted water.

Adhesion and biofilm formation on polystyrene by drinking water-isolated bacteria.

This study was performed in order to characterize the relationship between adhesion and biofilm formation abilities of drinking water-isolated bacteria (Acinetobacter calcoaceticus, Burkholderia cepacia, Methylobacterium sp., Mycobacterium mucogenicum, Sphingomonas capsulata and Staphylococcus sp.). Adhesion was assessed by two distinct methods: thermodynamic prediction of adhesion potential by quantifying hydrophobicity and the free energy of adhesion; and by microtiter plate assays. Biofilms were developed in microtiter plates for 24, 48 and 72 h. Polystyrene (PS) was used as adhesion substratum. The tested bacteria had negative surface charge and were hydrophilic. PS had negative surface charge and was hydrophobic. The free energy of adhesion between the bacteria and PS was > 0 mJ/m(2) (thermodynamic unfavorable adhesion). The thermodynamic approach was inappropriate for modelling adhesion of the tested drinking water bacteria, underestimating adhesion to PS. Only three (B. cepacia, Sph. capsulata and Staphylococcus sp.) of the six bacteria were non-adherent to PS. A. calcoaceticus, Methylobacterium sp. and M. mucogenicum were weakly adherent. This adhesion ability was correlated with the biofilm formation ability when comparing with the results of 24 h aged biofilms. Methylobacterium sp. and M. mucogenicum formed large biofilm amounts, regardless the biofilm age. Given time, all the bacteria formed biofilms; even those non-adherents produced large amounts of matured (72 h aged) biofilms. The overall results indicate that initial adhesion did not predict the ability of the tested drinking water-isolated bacteria to form a mature biofilm, suggesting that other events such as phenotypic and genetic switching during biofilm development and the production of extracellular polymeric substances (EPS), may play a significant role on biofilm formation and differentiation. This understanding of the relationship between adhesion and biofilm formation is important for the development of control strategies efficient in the early stages of biofilm development.

Benzoate catabolite repression of the phenol degradation in Acinetobacter calcoaceticus PHEA-2.

Acinetobacter calcoaceticus PHEA-2 exhibited a delayed utilization of phenol in the presence of benzoate. Benzoate supplementation completely inhibited phenol degradation in a benzoate 1,2-dioxygenase knockout mutant. The mphR encoding the transcriptional activator and mphN encoding the largest subunit of multi-component phenol hydroxylase in the benA mutant were significantly downregulated (about 7- and 70-fold) on the basis of mRNA levels when benzoate was added to the medium. The co-transformant assay of E. coli JM109 with mphK::lacZ fusion and the plasmid pETR carrying mphR gene showed that MphR did not activate the mph promoter in the presence of benzoate. These results suggest that catabolite repression of phenol degradation by benzoate in A. calcoaceticus PHEA-2 is mediated by the inhibition of the activator protein MphR.

Acinetobacter strains IH9 and OCI1, two rhizospheric phosphate solubilizing isolates able to promote plant growth, constitute a new genomovar of Acinetobacter calcoaceticus.

During a screening of phosphate solubilizing bacteria (PSB) in agricultural soils, two strains, IH9 and OCI1, were isolated from the rhizosphere of grasses in Spain, and they showed a high ability to solubilize phosphate in vitro. Inoculation experiments in chickpea and barley were conducted with both strains and the results demonstrated their ability to promote plant growth. The 16S rRNA gene sequences of these strains were nearly identical to each other and to those of Acinetobacter calcoaceticus DSM 30006(T), as well as the strain CIP 70.29 representing genomospecies 3. Their phenotypic characteristics also coincided with those of strains forming the A. calcoaceticus-baumannii complex. They differed from A. calcoaceticus in the utilization of l-tartrate as a carbon source and from genomospecies 3 in the use of d-asparagine as a carbon source. The 16S-23S intergenic spacer (ITS) sequences of the two isolates showed nearly 98% identities to those of A. calcoaceticus, confirming that they belong to this phylogenetic group. However, the isolates appeared as a separate branch from the A. calcoaceticus sequences, indicating their molecular separation from other A. calcoaceticus strains. The analysis of three housekeeping genes, recA, rpoD and gyrB, confirmed that IH9 and OCI1 form a distinct lineage within A. calcoaceticus. These results were congruent with those from DNA-DNA hybridization, indicating that strains IH9 and OCI1 constitute a new genomovar for which we propose the name A. calcoaceticus genomovar rhizosphaerae.

Gibberellin production and phosphate solubilization by newly isolated strain of Acinetobacter calcoaceticus and its effect on plant growth.

Plant growth-promoting rhizobacteria with gibberellins (GA)-producing potential were isolated from soil and screened for plant growth promotion. A new strain, Acinetobacter calcoaceticus SE370, produced extracellular GA and also had phosphate solubilising potential. It produced 10 different gibberellins, including the bioactive GA(1), GA(3) and GA(4) which were at, respectively, 0.45, 6.2 and 2.8 ng/100 ml. The isolate solubilised tricalcium phosphate and lowered pH of the medium during the process. Culture filtrates of the organism after growth on broth promoted growth of cucumber, Chinese cabbage and crown daisy.

Intergeneric coaggregation of strains isolated from phenol-degrading aerobic granules.

This work aims at exploring the intergeneric coaggregation of the pairs of strains, Acinetobacter calcoaceticus I6 and Bacillus thuringiensis I2 or Candida tropicalis I9 (with GenBank accession numbers EU250016, EU036759, and DQ515822) isolated from phenol-degrading aerobic granules. The I2 and I6 are functionally similar stains, while the I6 and I9 are functionally dissimilar strains. The lectin-saccharide interaction controlled the coaggregation of both the I2+I6 and I6+I9 pairs, with the protein adhesin being associated with the strain I6, and the complementary galactosamine-like or fucose-like sugar receptor with the strain I2 or I9, respectively. The rod-like I2 cells bridged the clusters of I2 or I6 cells to form aggregates, while the small I6 cells attached on and modified the surface of I9 to form aggregates.

Biofilm formation and interactions of bacterial strains found in wastewater treatment systems.

Biofilm formation and adherence properties of 13 bacterial strains commonly found in wastewater treatment systems were studied in pure and mixed cultures using a crystal violet microtiter plate assay. Four different culture media were used, wastewater, acetate medium, glucose medium and diluted nutrient broth. The medium composition strongly affected biofilm formation. All strains were able to form pure culture biofilms within 24 h in at least one of the tested culture media and three strains were able to form biofilm in all four culture media, namely Acinetobacter calcoaceticus ATCC 23055, Comamonas denitrificans 123 and Pseudomonas aeruginosa MBL 0199. The adherence properties assessed were initial adherence, cell surface hydrophobicity, and production of amyloid fibers and extracellular polymeric substances. The growth of dual-strain biofilms showed that five organisms formed biofilm with all 13 strains while seven formed no or only weak biofilm when cocultured. In dual-strain cultures, strains with different properties were able to complement each other, giving synergistic effects. Strongest biofilm formation was observed when a mixture of all 13 bacteria were grown together. These results on attachment and biofilm formation can serve as a tool for the design of tailored systems for the degradation of municipal and industrial wastewater.

Intergeneric coaggregation among drinking water bacteria: evidence of a role for Acinetobacter calcoaceticus as a bridging bacterium.

Intergeneric coaggregation of drinking water bacteria was tested. Acinetobacter calcoaceticus was found not only to autoaggregate but also to coaggregate with four of the five other isolates (Burkholderia cepacia, Methylobacterium sp., Mycobacterium mucogenicum, Sphingomonas capsulata, and Staphylococcus sp.). In its absence, no coaggregation was found. Interactions were lectin-saccharide mediated. The putative bridging function of A. calcoaceticus was evidenced by multispecies biofilm studies, through a strain exclusion process.

Physiological conditions conducive to high cyanophycin content in biomass of Acinetobacter calcoaceticus strain ADP1.

The effects of the inorganic medium components, the initial pH, the incubation temperature, the oxygen supply, the carbon-to-nitrogen ratio, and chloramphenicol on the synthesis of cyanophycin (CGP) by Acinetobacter calcoaceticus strain ADP1 were studied in a mineral salts medium containing sodium glutamate and ammonium sulfate as carbon and nitrogen sources, respectively. Variation of all these factors resulted in maximum CGP contents of only about 3.5% (wt/wt) of the cell dry matter (CDM), and phosphate depletion triggered CGP accumulation most substantially. However, addition of arginine to the medium as the sole carbon source for growth promoted CGP accumulation most strikingly. This effect was systematically studied, and an optimized phosphate-limited medium containing 75 mM arginine and 10 mM ammonium sulfate yielded a CGP content of 41.4% (wt/wt) of the CDM at 30 degrees C. The CGP content of the cells was further increased to 46.0% (wt/wt) of the CDM by adding 2.5 microg of chloramphenicol per ml of medium in the accumulation phase. These contents are by far the highest CGP contents of bacterial cells ever reported. CGP was easily isolated from the cells by using an acid extraction method, and this CGP contained about equimolar amounts of aspartic acid and arginine and no detectable lysine; the molecular masses ranged from 21 to 29 kDa, and the average molecular mass was about 25 kDa. Transmission electron micrographs of thin sections of cells revealed large CGP granules that frequently had an irregular shape with protuberances at the surface and often severely deformed the cells. A cphI::OmegaKm mutant of strain ADP1 with a disrupted putative cyanophycinase gene accumulated significantly less CGP than the wild type accumulated, although the cells expressed cyanophycin synthetase at about the same high level. It is possible that the intact CphI protein is involved in the release of CGP primer molecules from initially synthesized CGP. The resulting lower concentration of primer molecules could explain the observed low rate of accumulation at similar specific activities.

Response of biofilm bacteria to dissolved organic matter from decomposing maple leaves.

Stream bacteria play an important role in the utilization of dissolved organic matter (DOM) leached from leaves, and in transfer of this DOM to other trophic levels. Leaf leachate is a mixture of labile, recalcitrant, and inhibitory compounds, and bacterial communities vary in their ability to utilize leachate. The purpose of this study was to determine the effects of DOM from sugar maple leaves on bacterial populations in biofilms on decomposing leaf surfaces. Populations of Acinetobacter calcoaceticus, Burkholderia cepacia, and Pseudomonas putida were enumerated on decomposing maple leaves in a northeast Ohio stream using fluorescence in situ hybridization. Additionally, artificial substrata consisting of PVC-end caps filled with agar supplemented with leaf leachate and covered with cellulose filters were used to determine bacterial response to leachate from leaves at different stages of decomposition. Population sizes of bacterial species exhibited different responses. Leachate did not affect A. calcoaceticus. B. cepacia was tolerant of phenolic compounds released from leaves and the population size increased when DOM concentrations were greatest. In contrast, P. putida was inhibited by phenolic components of leachate when total DOM concentrations were greatest. Differences in response of the bacterial species to components of leaf leachate indicate the complexity of microbial population dynamics and interactions with DOM. Differences among species in response to DOM have the potential to influence transport and retention of organic matter in stream ecosystems.