BHI broth in a shaker for 8 h, collected by centrifugation at 5,000 x g for 15 min and then washed twice with distilled water. The cells were resuspended in 100 µl of TSL solu- tion (Tris-HCl 50 mM, pH 8,0, sucrose 20% and lysozime 2mg/ml) and incubated at 0ºC for 10 min. Then, 50 ml of EDTA 0,25 M were added. The suspension obtained, was diluted with 1,3 ml of Tris-HCl 50 mM pH 8,0 containing 9% of sucrose. Spheroplasts were collected by centrifuga- tion (3,000 x g for 15 min) and resuspended in 8 ml of TSM (Tris-HCl 10 mM pH 7,6, sucrose 0,75 M and mag- nesium sulfate 20 mM). The spheroplast efficiency was de- termined to be greater than 90% on BHI broth without os- motic stabilizers for the three bacterial species. The sphero- plast were treated with different bacteriocin PsVP-10 concentration and lysis was followed by turbidimetry (O.D. 620 nm).
In this study, the ability of three pathogenic species belonging to different environments to adhere to electrospun PCL microfiber meshes and form biofilms was analyzed and compared using different assays. Two of the most important Gram-negative nosocomial pathogens, P. aeruginosa and Acinetobacter baumannii, with the capacity to acquire and develop multiple resistance mechanisms 29, 30 were used in this study. In addition, the ability to form biofilm in these microfibers was studied in L. monocytogenes, one of the most important foodborne pathogens. 31 The attachment, the structure and the viability of the biofilms formed on these meshes were studied using crystal violet (CV) staining, scanning electron microscope (SEM) and colony forming unit (CFU) quantification. Also, since P. aeruginosa and A. baumannii are frequently involved in wound infections, 32 bacterial transfer experiments using PCL meshes in direct contact with bacteria previously grown in agar plates were performed to simulate a wound environment. Furthermore, CFU count assays were carried out to study the influence of the PCL mesh microstructure on bacterial attachment, and to perform comparative experiments with borosilicate glass and electrospun polylactic acid (PLA) fibers, materials commonly used in hospital environments and biomedicine applications respectively.
E. coli DH5α was able to grow on diesel fuel medium after elec- troporation mediated transformation with the 11Kbp plasmids. P. luteola and the transformed E. coli DH5α, growing on diesel fuel media, expressed a 70KDa polypeptide, detected by denatur- ing polyacrylamide gel electrophoresis, which was absent in the non-transformed E. coli DH5α strain grown in Luria broth. The data suggest that the 70KDa polypeptide, probably encoded by the 11Kbp plasmid, is involved in the uptake of diesel fuel type molecules from the extracellular medium to the internal periplas- mic bacterial space. This kind of experimental strategies might constitute biotechnological tools for improving the degradative capabilities of bacterialstrains present in natural environments.
Abstract: Infectious diseases especially those caused by bacterial and viral pathogens are serious loss factors in shrimp farming. In this study, bacteria were isolated from the gut and hepatopancreas of stressed shrimps obtained from a commercial farm. The isolates were screened on Thiosulfate citrate bile salt sucrose (TCBS) agar plates for the selection of Vibrio species. Presumptive vibrios were characterized through tests for hemolytic and enzymatic activity, hydrophobicity, growth and molecular identification. Three experimental infections were conducted in order to confirm the pathogenicity of selected bacterialstrains VHPC18, VHPC23, VHPC24 and VIC30. In the third experimental challenge the LD 50 was obtained, it lasted 10 days with 10 shrimp, weighing 6.9±1.1g, per tank. The treatments in triplicate were: (1) saline solution (control group); (2) 2×10 5 CFU/shrimp;
Open cast coal mining operations involve the use of the explosive ANFO (AN-ammonium nitrate, FO-Fuel Oil), composed of ammonium nitrate and diesel fuel for the detonation processes. Degradation of the second component of ANFO, which is diesel, by four bacterialstrains of Pseudomonas sp and Pseudomonas stutzeri was previously determined by gas chromatography with a flame ionization detector. Analysis for the presence and expression of the alkane monooxygenase gene (alkB) and the alkS regulator gene involved in the degradation of diesel by four strains was estimated in this study. Bacteria were grown on a minimal salts medium with diesel as the only carbon source. PCR was performed to evaluate the presence of alkB and alkS genes, RNA extraction and retro transcription was implemented to evidence the expression of the alkB and alkS genes. In addition an enzymatic assay for the activity of alkB was undertaken. Results for the presence and expression of the alkB gene showed band size products between 500- 600 bp. This suggests that the alkB gene is involved in the degradation of alkanes and that it is expressed under diesel pressure conditions.
ABSTRACT: The extracts of plants from Brazilian savanna are currently used in popular medicine. This study evaluated the inhibitory activity of the alcoholic and aqueous extracts from savanna plants on periodontal bacteria. The minimal inhibitory concentrations were evaluated by the agar dilution method, using Wilkins-Chalgren agar. Antimicrobial activity of plants extracts on microbial biofilms was determined in microplates. Psidium cattleianum and Myracrodruon urundeuva extracts demonstrated significant inhibitory activity on all bacterialstrains tested; alcoholic and aqueous extracts showed similar results. The extracts from these two species were able to inhibit both planktonic cells and microbial biofilm.
Ludwing et al. 1993, Olsen et al. 1994, Wallington and Lund 1994, Woese 1994) have greatly contributed to discerning the evolutionary relatíonshíps between bacteria, and therefore have substantially helped to identify different bacterial groups. Unfortunately, the same properties that make these molecules suítable for phylogenetic classification at a certain level, make them poor tools for defining bacterial species within close sister dades. For instance, 1 agree with the concept that there are genes (such as those cited aboye) which can approach the phylogeny of an organism; however, 1 also understand that at the species level they do not have enough resolution for defining bacterial species as separate entities (Stackebrandt and Goebel 1994). Furthermore, sorne of these bacterial genes are not single units but rather are duplicated in the same chromosome (Dryden and Kaplan 1990, Viale ei al. 1994) or repeated in a second (different) chromosome of sorne bacteria (Allardet-Servent et al. 1988, Dryden and Kaplan 1990) and are therefore subjected to different positional constraints that may promote diversity within the same group. It can be argued that, given a gene (or the entire genome), one can always discern between close bacterial relatives and resol ve them into species. The main problem with this is the absence of limits in the prediction of a terminal taxa: if one tries hard enough, sequence differences can be found to díagnose virtually any bacterial populatíon. The "pendular movement machíne" operated by the "lumpers" and the "splitters" of bacterial taxonomy depends on the data provided by them, generating from one to an ample number of species.
RESUMEN. Recientemente han sido desarrollados conjugados este- roide-antibiótico como agentes potenciales terapéuticos para enfer- medades infecciosas. En este trabajo fue evaluada la actividad anti- bacteriana de dos derivados de pregnenolona sobre S. aureus, K. pneumoniae y E. coli usando el método de dilución y la concentra- ción mínima inhibitoria (CMI). Los resultados obtenidos indican que el crecimiento bacterial de S. aureus fue inhibido con cefotaxi- ma (MIC = 0.25 mg/ml), gentamicina (MIC = 0.0125 mg/ml), he- misuccinato-pregnenolona (MIC = 1 mg/ml), etilenediamina-hemi- succinato-pregnenenolona (MIC = 0.25 mg/ml) y la mezcla de los dos derivados de pregnenolona (MIC = 0.5 mg/ml). Otros resulta- dos mostraron que el crecimiento bacterial de E. coli fue también inhibido con cefotaxima (MIC = 0.25 mg/ml), gentamicina (MIC = 0.00625 mg/ml), hemisuccinato-pregnenolona (MIC = 1 mg/ml), etilenediamina-hemisuccinato-pregnenenolona (MIC = 0.5 mg/ml) y la mezcla de los dos derivados de pregnenolona (MIC = 0.5 mg/ ml). Experimentos alternativos, mostraron que el crecimiento bacte- rial de K. pneumoniae fue inhibido con cefotaxima (MIC = 0.125 mg/ml), gentamicina (MIC = 0.0.125 mg/ml), hemisuccinato-preg- nenolona (MIC = 1 mg/ml), etilenediamina-hemisuccinato-pregne- nenolona (MIC = 0.5 mg/ml) y la mezcla de los dos derivados de pregnenolona (MIC = 0.5 mg/ml). Nuestros resultados sugieren que el efecto inducido por los derivados de pregnenolona podría ser por la interacción con algunos factores de la membrana bacterial que son específicos para la resistencia bacterial. En este sentido, la actividad antibacterial de los derivados de pregnenolona puede de- pender de la naturaleza de los grupos funcionales involucrados en su estructura química que parece ser la llave requerida para la acti- vidad antibacterial.
prepared Hsp70 IBs (and latter FGF-2 IBs, Figure 2), which have recently been observed as having an anti-apoptotic effect when administered as Nanopills (32) . For a comparative analysis of Hsp70 administered as Nanopills (top-down) or as a scaffold (bottom-up), we first determined the efficiency of the surface coating with Hsp70 Nanopills. An almost linear dependence between the amounts of added IB protein and the final amount of IB protein retained after the final washing steps was observed with approx. 60% of the initial protein retained in the IB protein range tested (Figure 3 a). This relation was considered for further comparison. The anti-apoptotic effect of Hsp70 on the model HL- 60 cells challenged with cisplatinum was determined when administered as both Nanopills or topographically as a biofunctional scaffold. It was seen (Figure 3 b) that Hsp70 Nanopills indeed rescued cell viability as expected (32) , thus providing control to our experiment. In a new observation, it was also seen that the topographical Hsp70 scaffold also rescued cells from apoptosis. Cell viability on the Hsp70 nanotopography was significantly higher than in absence of IBs (C bars), or when the surface had been functionalized with non-functional IB controls (made from VP1LAC, denoted as IR, irrelevant). These data confirmed the concept of functional scaffolds based on bacterial IBs and formed by a releasable, bioavailable form of protein drugs.
sistant IR strains were used. The combined application of insecticide-susceptible and insecticide-resistant strains should, in principle, detect somatic cell recombinagens in the Drosophila melanogaster in vivo w r w q assay. The IS strain was more susceptible to toxicity induced by the test chemicals than the IR stocks. Its performance in the biotransformation of the chemicals tested was rather poor. TU was inactive in all strains. With the active compounds, spot frequencies increased approximately linearly with dose for each spot type. SDEH gave a strong positive result in all three female genotypes
a) Pathogenicity test on leaves: The leaves were washed with running water, they were disinfected with sodium hypochlorite solution 2% for 2 min and two rinses with sterile distilled water were performed. In each leaf, four fragments of mycelium (~5mm diameter) grown on PDA medium for 10 days incubation at 25 ± 1 ° C were placed. Two fragments were placedin areas where incisions of 0.5 cm in length were made and two in zones without incisions. The same scheme was used in leafs where free PDA fragments of Fusarium were placed. Two leafs were used for each strain. The samples were placed in humid chambers retaining 100% relative humidity at 25 ± 1° C for 12 days. It was monitored daily for the initial stages in which the strains were able to generate
Previous research at Centro de Investigaciones Microbiológicas (CIMIC), showed in vitro antibiotic resistance transfer between two L. sphaericus strains, and identification of genes involved in plasmid transference, these findings could show plasmid presence in both strains (Rojas, Cárdenas & Dussán, s.f.). To confirm this assumption, two plasmid isolation techniques were tested. A total of 21 isolates were obtained and visualized by agarose gel electrophoresis, those possible-plasmids bands were validated by PCR of three genes: V3-V5 Region of 16S ARNr, traG involved in conjugation and the gene coding for erythromycin resistance. The size of the possible plasmids was determined by enzymatic digestion, finally a first approach of plasmid organization is proposed.
tion in sterile distilled water was prepared. Fifty µ l of each dilution were sown in lawn over Mueller-Hinton agar and incubated at 37ºC for 24 h in order to obtain isolated colo- nies. Later on, the dishes were covered with a thin layer of 5 ml of fresh BHI medium containing 0.7% agar (soft agar), mixed with 2 ml of a log phase culture of the indica- tor E. coli strain and the dishes were incubated at 37ºC for 5 h in order to observe the inhibitory zones. From these colonies, ten bacteriocinogenic S. flexneri strains were se- lected that showed different sizes of inhibitory zones, in- cluding some with or without any inhibitory zone,. These colonies were separately grown in 5 ml of BHI broth, incu- bated overnight at 37º C and then the cultures were centri- fuged at 6,000 x g for 10 minutes. One ml of the each of the supernatants was serially diluted in sterile distilled wa- ter (in two-fold decrements) and 5 µ l of each dilution was assayed to detect bacteriocin activity by the method de- scribed before. One arbitrary unit ml -1 (AU/ml) of bacterio-
The low mortality observed in the con- trols of the coarse bioassays indicates that any increase in mortality above ten percent, is due to the toxic effect of Bt crystal proteins or to a synergistic effect of spore and crystal proteins. However, the latter should be examined in fine bioassays using purified crystals. It is important to mention that, in order to eliminate any puta- tive toxic effect of the β-exotoxin (as it may be present in some of these strains) in coarse bioassays, spore and crystal suspensions were thoroughly washed by centrifugation before incorporating them into the diet. This report shows that the strains 40-X-m and HD1, that contain cry1Aa, cryIAb, cryIAc and cryIAd genes (Mora and Espinoza 2004) showed mor- talities above 80 %.
QS-disrupting activity exhibited by the extracts of these endophytic filamentous fungi was unrelated to toxic effects on the reporter strain Chromobacterium violaceum CVO26. LC-HRMS profiles of these extracts revealed the presence of a broad diversity of fungal metabolites (Figure 5), some of them of recent identification in marine-derived strains [62–71]. Interestingly, all these fungal extracts contained compounds that might represent new natural products whose identity and biological activity will be determined in further experiments. The identity of the compounds tentatively identified by HRMS and UV analysis and whether the presence of the abovementioned metabolites can explain the QS-inhibitory activity observed in the extracts or it is due to the presence of minor components, or even to the synergistic effect of combinations of these molecules are issues that remain to be determined. To the best of our knowledge, this is also the first report of QS inhibition in endophytic fungi isolated from marine organisms.
The functionality of these tfdA gene related sequences is also suggested by other kind of evidence. The func- tional structure of the soil bacterial community, i.e. the number of tfdA gene related sequences (functional rich- ness), and their relative abundance, is significantly affected by the specific herbicide compound used during enrichment (Fig. 4). The shifts in the taxonomic structure of the soil bacterial community, based on tracking of the 16S rRNA gene sequences, parallel those of the func- tional structure, indicating that the bacterial groups that possess the tfdA gene related sequences are also those that react to the presence of the herbicide. Indeed, more direct approaches tracking the expression of particular tfdA gene related sequences are required to evaluate the functionality of these sequences, as it has been demon- strated for tfdA gene from C. necator during degradation of MCPA in an agricultural soil (Nicolaisen et al., 2008). Changes in the relative abundance of variants of the tfdA gene sequences have been reported in soils exposed to MCPA herbicide using PCR primer pairs targeting the class III proteobacterial tfdA genes (Bælum et al., 2006). More recently, the use of primer pairs that distinguish among subgroups of proteobacterial TfdA protein sequences allowed establishment of some differences in the contribution of specific tfdA gene sequences to deg- radation of MCPA or 2,4-D in soils (Bælum et al., 2008; Bælum and Jacobsen, 2009).
For each plant species, we determined the number of OTUs (relative abundance >0.03%) shared across all three compartments, which constituted what we called the plant core microbiome (Fig. S1). We observed that around 56% of all OTUs (64% in CAL, 45.4% in NAS, 57% in JAR, and 52.3% in PYC) present in the three com- partments constituted the core microbiome. A substantial number of OTUs was also shared between the bulk soil and the RSS, and also between the RSS and the rhizosphere (Fig. S1). Thus, the RSS bacterial community appeared to be a subset of the bulk soil and, in turn, the rhizobacterial community a subset of the RSS. Actually, each compartment only exhibited an average of 3.7% of exclusive or non-core OTUs, that is, OTUs not found in any other compartment. Besides, the rhizosphere presented the highest percentage of non-core OTUs, with an average of 6%.