Properties of Antibiotic-Resistant Bacteria Isolated from Onsite Wastewater Treatment Plant in Relation to Biofilm Formation.

The aim of the present study was to determine some properties of antibiotic-resistant bacterial strains isolated from onsite wastewater technology in relation to biofilm formation, e.g., autoaggregation and motility. Additionally, biosurfactant production by the isolates was also evaluated. The ability of selected strains to develop a biofilm was assessed by using the crystal violet method, which allows to indirectly quantify the attached bacterial biomass (live, dead cells, and polysaccharides as well). Obtained results showed that 19 of the analyzed strains were able to produce biofilm after 72 h of incubation. The low values of surface tension in the range between 28 and 36 mN/m were observed in the bacteria, which are not able to produce biofilm or be classified as weak biofilm producers. Among biofilm-forming strains the highest autoaggregation index was observed for Mycobacterium brumae and Bacillus alcalophilus. Noteworthy, that some strains capable of biofilm formation showed no aggregation abilities or were characterized by low autoaggregative properties. The results of visual autoaggregation assay showed no visible flocs after given time of incubation. The results from motility test demonstrated that most of the analyzed strains were motile. Noteworthy, that up to now literature data about physiology, biofilm formation, and autoaggregative capabilities of bacteria isolated from onsite wastewater technology are very limited and this paper gives the information on the antibiotic-resistant bacteria with ability to form biofilm. Thus, the present study points to develop novel bioinocula in antibiotic degradation and to reach novel biofilm-dispersing agents produced by various bacteria that can be used as disinfectants or surface-coating agents to prevent microbial surface colonization and biofilm development.

Structural identification of lipopeptide biosurfactants produced by Bacillus subtilis strains grown on the media obtained from renewable natural resources.

The aim of the study was to identify and characterize lipopeptide (LP) biosurfactants produced by two Bacillus subtilis strains (KP7 and I'-1a) grown on various media prepared from renewable natural resources: two different brewery wastewaters (BW#4 and BW#6), 2% beet molasses (M), apple peels extract (APE) supplemented with 0.25% of yeast extract (YE) or 0.25% peptone (P), and similarly supplemented carrot peels extract (CPE). In all used media both strains retained their individual LP production signature characterized by surfactin and iturin overproduction exhibited by KP7 and I'-1a strain, respectively. The production level and the structural diversity of synthesized LPs were dependent on the medium composition. In the CPE+YE medium it was higher than the yield obtained in Luria-Bertani (140.6 and 100.3 mg L-1, respectively). Surfactins were produced by both strains as a mixture of four homologues (C13-C16) with the domination of variant C14. All other broths prepared from renewable resources strongly stimulated the iturin production by I'-1a strain with the exception of BW media. The highest iturin concentration (428.7 mg L-1) obtained in the CPE+P culture of I'-1a strain was about seven-fold higher than in LB. In all cultures only iturin A was identified. Among four iturin homologues (C13-16) produced by I'-1a strain, the highest relative contents of C16 variant (70-80%) were calculated for samples obtained from APE+P and CPE+P media. The obtained data indicate that the waste composition has an influence on both the types and amounts of biosurfactants produced by studied B. subtilis strains.

Using phenotype microarrays in the assessment of the antibiotic susceptibility profile of bacteria isolated from wastewater in on-site treatment facilities.

The scope of the study was to apply Phenotype Biolog MicroArray (PM) technology to test the antibiotic sensitivity of the bacterial strains isolated from on-site wastewater treatment facilities. In the first step of the study, the percentage values of resistant bacteria from total heterotrophic bacteria growing on solid media supplemented with various antibiotics were determined. In the untreated wastewater, the average shares of kanamycin-, streptomycin-, and tetracycline-resistant bacteria were 53, 56, and 42%, respectively. Meanwhile, the shares of kanamycin-, streptomycin-, and tetracycline-resistant bacteria in the treated wastewater were 39, 33, and 29%, respectively. To evaluate the antibiotic susceptibility of the bacteria present in the wastewater, using the phenotype microarrays (PMs), the most common isolates from the treated wastewater were chosen: Serratia marcescens ss marcescens, Pseudomonas fluorescens, Stenotrophomonas maltophilia, Stenotrophomonas rhizophila, Microbacterium flavescens, Alcaligenes faecalis ss faecalis, Flavobacterium hydatis, Variovorax paradoxus, Acinetobacter johnsonii, and Aeromonas bestiarum. The strains were classified as multi-antibiotic-resistant bacteria. Most of them were resistant to more than 30 antibiotics from various chemical classes. Phenotype microarrays could be successfully used as an additional tool for evaluation of the multi-antibiotic resistance of environmental bacteria and in preliminary determination of the range of inhibition concentration.

Lipid composition in a strain of Bacillus subtilis, a producer of iturin A lipopeptides that are active against uropathogenic bacteria.

Urinary tract infections are a common disease in humans. Therefore, new methods are needed to destroy biofilms that are formed by uropathogens. Iturin A lipopeptides (LPs) C14 and C15 are potent biosurfactants synthetized by the Bacillus subtilis I'1a strain. The biological activity of extracted LPs was confirmed by examining extracts from I'1a cultures against uropathogenic bacteria that had been isolated from biofilms on urinary catheters. Compared with cultures of DSM 3257, which produce surfactin at a relatively low level, the extract obtained from strain I'1a exhibited a greater inhibitory effect against both planktonic and sessile forms of Escherichia coli, Serratia marcescens, Enterobacter cloacae, Proteus mirabilis, Citrobacter freundii and Enterococcus faecalis. Moreover, cyclic LP biosurfactants may disturb the integrity of cytoplasmic membranes; therefore, we investigated the effects of synthetized LPs on fatty acids and phospholipids of B. subtilis. LPs and lipids were analyzed using GC-MS, LC-MS/MS and MALDI-TOF/TOF techniques. Compared with B. subtilis DSM 3257, membranes of the I'1a strain were characterized by an increased amount of anteiso fatty acids and a ten-fold higher ratio of phosphatidylglycerol (PG)-to-phosphatidylethanolamine (PE). Interestingly, in cultures of B. subtilis DSM 3257 supplemented with LP extracts of the I'1a strain, the PG-to-PE ratio was fourfold higher, and the amount of anteiso fatty acids was also increased.

Synthesis of silver nanoparticles by Bacillus subtilis T-1 growing on agro-industrial wastes and producing biosurfactant.

In this study, culture supernatnats of Bacillus subtilis T-1 growing on brewery effluents and molasses was used for silver nanoparticles (Ag-NPs) synthesis. The biosurfactant production of B. subtilis T-1 was confirmed by the detection of genes in the genome and by the identification of the product in the supernatants. The genes for synthesis of surfactin (sfp, srfAA) and iturin (ituC) were noted by PCR reactions. Also, in examined culture supernatants the presence of C13, C14 and C15 surfactin homologues with the sodiated molecules [M + Na](+) at m/z 1030, 1044 and 1058 was confirmed using LC/MS/MS analysis. The formation of NPs in the culture supernatants was confirmed by UV-vis spectroscopy. The dynamic light scattering measurements and transmission electron microscopy images showed the nanometric sizes of the biosynthesised Ag-NPs which ranged from several nm to several tens of nm depending on the used culture supernatant. Biological properties of Ag-NPs were evaluated by binding of Ag-NPs with DNA isolated from the Escherichia coli ATCC 25922 and B. subtilis ATCC 6633. Biogenic Ag-NPs were actively bound to DNA in increased concentration which could be the one important mode of antibacterial action of the Ag-NPs.

Microbial Community Profiles in Wastewaters from Onsite Wastewater Treatment Systems Technology.

The aim of the study was to determine the potential of community-level physiological profiles (CLPPs) methodology as an assay for characterization of the metabolic diversity of wastewater samples and to link the metabolic diversity patterns to efficiency of select onsite biological wastewater facilities. Metabolic fingerprints obtained from the selected samples were used to understand functional diversity implied by the carbon substrate shifts. Three different biological facilities of onsite wastewater treatment were evaluated: fixed bed reactor (technology A), trickling filter/biofilter system (technology B), and aerated filter system (the fluidized bed reactor, technology C). High similarities of the microbial community functional structures were found among the samples from the three onsite wastewater treatment plants (WWTPs), as shown by the diversity indices. Principal components analysis (PCA) showed that the diversity and CLPPs of microbial communities depended on the working efficiency of the wastewater treatment technologies. This study provided an overall picture of microbial community functional structures of investigated samples in WWTPs and discerned the linkages between microbial communities and technologies of onsite WWTPs used. The results obtained confirmed that metabolic profiles could be used to monitor treatment processes as valuable biological indicators of onsite wastewater treatment technologies efficiency. This is the first step toward understanding relations of technology types with microbial community patterns in raw and treated wastewaters.

Application of the BIOLOG system for characterization of Serratia marcescens ss marcescens isolated from onsite wastewater technology (OSWT).

The scope of this study was to apply the Biolog system to identify and characterize a Serratia strain isolated from the surface of black plastic pieces which constitute the fluidized bed filter (onsite wastewater technology, OSWT). The preliminary isolation of the strain was done in the medium with tetracycline at a 16 mg/l concentration. To characterize the isolated strain, the following Biolog methods were applied: (1) EcoPlates microplates for evaluation of physiological profiling, (2) GEN III OmniLog® ID System for identification of the isolate, and (3) phenotypic microarrays (PM) technology for evaluation of sensitivity to antibiotics (PM11 and PM12). Results were recorded using the original OmniLog® software. The Serratia strain was identified as Serratia marcescens ss marcescens with similarity index 0.569. The same identification was obtained by the 16S rDNA analysis. PM analysis showed an enhancement of phenotype (resistance or growth) of this strain to 35 antibiotics. The loss of phenotype (sensitivity or non-growth) was observed only for 5 antibiotics: lomefloxacin (0.4 µg/ml), enoxacin (0.9 µg/ml), nalidixic acid (18.0 µg/ml), paromomycin (25.0 µg/ml) and novobiocin (1100 µg/ml). This study acknowledges that the methods proposed by the Biolog system allow correct and complete identification and characterization of the microbes isolated from different environments. Phenotypic microarrays could be successfully used as a new tool for identification of the multi-antibiotic resistance of bacteria and for determination of the minimal inhibition concentrations (MIC).

Strong and Nonspecific Synergistic Antibacterial Efficiency of Antibiotics Combined with Silver Nanoparticles at Very Low Concentrations Showing No Cytotoxic Effect.

The resistance of bacteria towards traditional antibiotics currently constitutes one of the most important health care issues with serious negative impacts in practice. Overcoming this issue can be achieved by using antibacterial agents with multimode antibacterial action. Silver nano-particles (AgNPs) are one of the well-known antibacterial substances showing such multimode antibacterial action. Therefore, AgNPs are suitable candidates for use in combinations with traditional antibiotics in order to improve their antibacterial action. In this work, a systematic study quantifying the synergistic effects of antibiotics with different modes of action and different chemical structures in combination with AgNPs against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus was performed. Employing the microdilution method as more suitable and reliable than the disc diffusion method, strong synergistic effects were shown for all tested antibiotics combined with AgNPs at very low concentrations of both antibiotics and AgNPs. No trends were observed for synergistic effects of antibiotics with different modes of action and different chemical structures in combination with AgNPs, indicating non-specific synergistic effects. Moreover, a very low amount of silver is needed for effective antibacterial action of the antibiotics, which represents an important finding for potential medical applications due to the negligible cytotoxic effect of AgNPs towards human cells at these concentration levels.

Biosurfactant mediated biosynthesis of selected metallic nanoparticles.

Developing a reliable experimental protocol for the synthesis of nanomaterials is one of the challenging topics in current nanotechnology particularly in the context of the recent drive to promote green technologies in their synthesis. The increasing need to develop clean, nontoxic and environmentally safe production processes for nanoparticles to reduce environmental impact, minimize waste and increase energy efficiency has become essential in this field. Consequently, recent studies on the use of microorganisms in the synthesis of selected nanoparticles are gaining increased interest as they represent an exciting area of research with considerable development potential. Microorganisms are known to be capable of synthesizing inorganic molecules that are deposited either intra- or extracellularly. This review presents a brief overview of current research on the use of biosurfactants in the biosynthesis of selected metallic nanoparticles and their potential importance.