cells/ml) almost all cells are individual spirochetes, but as the concentration increases above 1610 7 cells/ml, cell aggregates start to form. The different stages of aggregate formationof representative cultures of B31 and B31-GTP strains ofBorreliaburgdorferi, cultured in polystyrene tubes with no additional matrix provided, were examined. Figure 1 demonstrates that with an initial cell concentration of 1610 7 cells/ml, aggregates were observed in these cultures under all modes of microscopy used in early (column 1: days 0–2), middle (column 2, days 3–6) and late stages (column 3, days 7–21). With dark field illumination, spirochetes were observed arrayed around the periphery of a semi-spherical core that diffused light strongly and heterogeneously within a day (Figure 1A). In middle-stage samples representing 3–6 days of culturing, the matrix adopted a more filmic aspect and appeared to be pliant, with a relatively low viscosity allowing the film to flatten under the weight of the coverslip (Figure 1B). Moderate densities of spherical elements were visible within the matrix, but dark field illumination did not permit observation of matrix-embedded spirochetes. In late stage structures, representing day 7–21 days (Figure 1C), the matrix exhibited higher rigidity, with hills, valleys and cracks observed in the structures. Using Differential Interference Contrast (DIC) microscopy, the heterogeneity of the film’s composition was more readily visible. The grainy appearance of the film under DIC (Figure 1E, F) indicated the film contained constituents of non-uniform optical density. The late stage sample (Figure 1F) exhibited substantial rigidity, resisting even applied compression of the cover slip. With FITC-band illumination of the GFP-expressing hybrid strain, spirochetes involved in initial formationof the matrix were clearly visible (Figure 1G). Middle-stage colonies were largely composed of now-visible spirochetes (Figure 1H), whereas in late-stage colonies, round bodies predominated (Figure 1I). Figure S1 is a dark field microscopy image showing aggregates developed in a roughly circular manner, with circles apparently converging to form larger biofilm-like structures. Video S1 is a movie file showing two large floating Borreliaburgdorferi aggregates actively growing surrounded by individual spirochetes. Borreliaburgdorferi strain 297 showed similar growth at all culture times (data not shown).
Figure 2. The urokinase receptor (uPAR) is involved in clearance of B. burgdorferi . (A) Urokinase receptor knock-out C57BL/6 mice display higher systemic B. burgdorferi numbers. WT and uPAR 2/2 mice were inoculated with B. burgdorferi and sacrificed two and four weeks post infection. DNA was extracted from the indicated tissues and subjected to quantitative Borrelia flab and mouse b-actin PCR. In sham inoculated mice (2 to 3 per group) we did not detect B. burgdorferi DNA. Six to eight mice per group were used and bars represent the mean6SEM. (B and C) Urokinase receptor knock-out C57BL/6 mice develop more rigorous IgG responses. Sera from C57BL/6 WT and uPAR knock-out mice, 2 and 4 weeks post B. burgdorferi (B burg) or sham inoculation (SHAM) was used for whole cell B. burgdorferi ELISA. Thus, we determined total IgG directed against B. burgdorferi (B) and IgG subclasses, of which only IgG1 (C) is shown. (D) WT and uPAR 2/2 macrophages produce similar levels of pro-inflammatory cytokines when exposed to viable B. burgdorferiinvitro. Peritoneal macrophages were stimulated with control medium (medium) or B. burgdorferi (B burg) for 16 hours. The supernatant was analyzed for cytokine production using a mouse inflammation cytometric bead array. (E and F) Urokinase receptor deficient granulocytes and macrophages are incapable of adequately phagocytosing B. burgdorferi. Whole blood or peritoneal macrophages were incubated with CFSE-labeled viable or heat-killed FITC-labeled B. burgdorferi at 37uC or at 4uC as a control. Phagocytosis was stopped by transferring the tubes to ice and extracellular bacteria were quenched by addition of a quenching dye containing Trypan blue. When whole blood was used erythrocytes were lysed before cells were DAPI stained and subjected to fluorescent microscopy (E) or stained for Gr-1 (granulocytes) and subjected to FACS analysis (F; left panel). Peritoneal macrophages were directly subjected to FACS analysis (F; right panel). Phagocytosis was depicted as the phagocytosis index [64,65]: mean fluorescence intensity (MFI)6percentage (%) positive cells) at 37uC minus (MFI6% positive cells at 4uC). Six to eight mice per group were used, graphs represent the mean6SEM and are representative of three independent experiments. (G) B. burgdorferi binds equally well to WT and uPAR 2/2 macrophages. A similar experiment as described in (F) was performed, albeit at 4uC and without the addition of quenching dye to determine binding of B. burgdorferi to peritoneal macrophages. Binding is expressed as the binding index: % CFSE positive cells6MFI. Four to six mice per group were used and bars represent the mean6SEM. The experiment was repeated twice. A p-value,0.05 was considered statistically significant. * indicating p,0,05; ** p,0,01 and *** p,0,001.
For construction of the TEF1-BCR1 overexpression plasmid pCJN491, PCR was done using primers OE723-ATG (59-ATGTCAGG GACATCACAAGTACTTCA-39) and OE723-908 (59-AATAA TAGTTTCCCAATTGAAAAAAGAGAGGAC-39) to generate a 2,723-bp fragment beginning from the ATG of the BCR1 ORF (orf19.8342) to 500 bp downstream of the stop codon. This fragment was inserted into the pGEMT-Easy vector (Promega, Madison, Wisconsin, United States) and then digested with EcoRI and SpeI (releasing a 1,650-bp fragment containing the larger portion of the BCR1 ORF including the start codon and 1,650 bp downstream of the start codon), and cloned into an EcoRI- and SpeI-linearized vector pTEF1 , to yield plasmid pCJN491 in the correct orientation. pTEF1  is a vector that harbors the constitutively active TEF1 promoter that is derived from pDDB78, a HIS1 vector . A unique SbfI site lying within the 1,650-bp portion of BCR1 was used to direct integration of the plasmid to the natural BCR1 locus via SbfI digestion. The TEF1-BCR1 overexpression C. albicans strains CJN1011, CJN1035, and CJN1039 were constructed by transforming CJN459 (a His bcr1/bcr1 insertion mutant), CJN308 (a His tec1/tec1 insertion mutant), and DAY286 (a His reference strain), respectively, with SbfI-linearized pCJN491 to generate Hisþ strains overexpressing BCR1. The TEF1 vector alone C. albicans strains CJN1060, CJN1052, and CJN1015 were constructed by transforming CJN459, CJN308, and DAY286, respectively, with NruI-linearized pTEF1 to generate Hisþ strains with the vector alone.
After culturing for 6 hours, Co-Cr-Mo had a significantly lower BCR than Ti-6Al-4V, cp-Ti and stainless steel ( P,0.05). However, the total biofilm mass determined by CV staining and the viable cell counts did not differ significantly between the materials ( P.0.05). Boks et al reported that bond strengthening for four strains of S. epidermidis on a hydrophobic surface was limited to a minor increase . Tang et al showed that more bacteria adhered to a hydrophilic surface than a hydrophobic surface . As water molecules adjacent to a hydrophobic surface are not able to form hydrogen bonds with that surface (hydrophobic effect), bacterial adhesion to a hydrophobic surface is brought about by an entropically favorable release of water molecules. With regards to surface free energy, numerous studies in the dental field agree that surfaces with high surface free energy foster microbial adherence invitro and in vivo [31–33,54–56]. Glantz et al reported that when analyzed gravimetrically, there was less dental plaque on low surface free energy hydrophobic substrata than on hydrophilic substrata due to the effect of interfacial thermodynamics . On the other hand, Van Pelt et al suggested that surface free energy is presumably more directly related to the binding force rather than to the number of bacteria on the surface area . Therefore, it can be speculated that bacteria on the relatively hydrophobic Co-Cr-Mo surface, which has the lowest surface free energy, binds cell-to-cell more tightly with polysaccharides than to a cell-to-material surface (bacter- iophobic effect), and that it is difficult for bacteria to develop a biofilm on the horizontal plane on the Co-Cr-Mo surface. However, the ability of bacteria to adhere and form a biofilm, as described by Cerca et al, varies to a wide degree depending on the strain of S. epidermidis . Schildhauer et al also reported that S epidermidis varied in its adherence to various metallic implants and there was no significant difference between them . Thus, the literature does not agree on how the physical characteristics of a biomaterial influence early biofilmformation. It is also possible that additional physico-chemical characteristics, such as released metal ions and chemical structure, may have some influence that inhibits or delays biofilm development. Poortinga et al showed that the change in substratum potential as a function of the number of adhering bacteria is a measure of the amount of electric charge transferred between the substratum and the bacteria during adhesion . Thus, early biofilmformation is a multi-factorial process that is unlikely to be explained by a single
UTI is the major public health problem in the developing countries and it is one of the most commonly encountered clinical conditions. Present study showed E. coli was the most frequently isolated pathogen followed by Klebsiella pneumoniae. Both are known to be responsible for high percentage of UTI and causes symptomatic UTI [1, 4, 10]. These bacteria have multiple virulence factors including biofilmformation to establish them in the urinary tract. Biofilmformation helps the organisms to survive in adverse conditions even in the presence of antibacterial agents.
Inflammation has long been implicated as a contributor to pathogenesis in many CNS illnesses, including Lyme neuroborreliosis. Borreliaburgdorferi is the spirochete that causes Lyme disease and it is known to potently induce the production of inflammatory mediators in a variety of cells. In experiments where B. burgdorferi was co-cultured invitro with primary microglia, we observed robust expression and release of IL-6 and IL-8, CCL2 (MCP-1), CCL3 (MIP-1a), CCL4 (MIP-1b) and CCL5 (RANTES), but we detected no induction of microglial apoptosis. In contrast, SH-SY5Y (SY) neuroblastoma cells co- cultured with B. burgdorferi expressed negligible amounts of inflammatory mediators and also remained resistant to apoptosis. When SY cells were co-cultured with microglia and B. burgdorferi, significant neuronal apoptosis consistently occurred. Confocal microscopy imaging of these cell cultures stained for apoptosis and with cell type-specific markers confirmed that it was predominantly the SY cells that were dying. Microarray analysis demonstrated an intense microglia- mediated inflammatory response to B. burgdorferi including up-regulation in gene transcripts for TLR-2 and NFkb. Surprisingly, a pathway that exhibited profound changes in regard to inflammatory signaling was triggering receptor expressed on myeloid cells-1 (TREM1). Significant transcript alterations in essential p53 pathway genes also occurred in SY cells cultured in the presence of microglia and B. burgdorferi, which indicated a shift from cell survival to preparation for apoptosis when compared to SY cells cultured in the presence of B. burgdorferi alone. Taken together, these findings indicate that B. burgdorferi is not directly toxic to SY cells; rather, these cells become distressed and die in the inflammatory surroundings generated by microglia through a bystander effect. If, as we hypothesized, neuronal apoptosis is the key pathogenic event in Lyme neuroborreliosis, then targeting microglial responses may be a significant therapeutic approach for the treatment of this form of Lyme disease.
PCD facilitates the removal of aged, damaged, infected, problematic or unnecessary cells, and is a vital process in the development and homeostasis of eukaryotic multicellular organ- isms, including animals, plants and fungi [4–6]. In bacteria, PCD is counterproductive for an individual cell; however, it might be advantageous for a whole population . For example, mazEF- mediated death can act as a defense mechanism that prevents the spread of phages in E. coli . Bacterial cell death mediated by mazEF may have several other roles; the mazEF system might act as a guardian of the bacterial chromosome [50,51]. When, for example, DNA repair systems fail to overcome excess damage of the chromosome, mazEF-mediated cell death might be activated. Thus, by eliminating cells that carry genomic defects and mutations, the mazEF system might contribute to the maintenance of genomic stability of the whole population. Cell death mediated by mazEF may also be important in the response of bacteria to severe nutritional stress . The second PCD program is found in B. subtilis, in which the skf and sdp operons mediate the death of a subpopulation of sporulating cells [8,9]. PCD is also important for biofilm development in S. aureus . Following cell death, a sub-population of the dead bacteria lyse and release genomic Figure 6. Genomic DNA remains intact in induced cell death.
The persistence of symptoms in Lyme disease patients following antibiotic therapy, and their causes, continue to be a matter of intense controversy. The studies presented here explore antibiotic efficacy using nonhuman primates. Rhesus macaques were infected with B. burgdorferi and a portion received aggressive antibiotic therapy 4–6 months later. Multiple methods were utilized for detection of residual organisms, including the feeding of lab-reared ticks on monkeys (xenodiagnosis), culture, immunofluorescence and PCR. Antibody responses to the B. burgdorferi-specific C6 diagnostic peptide were measured longitudinally and declined in all treated animals. B. burgdorferi antigen, DNA and RNA were detected in the tissues of treated animals. Finally, small numbers of intact spirochetes were recovered by xenodiagnosis from treated monkeys. These results demonstrate that B. burgdorferi can withstand antibiotic treatment, administered post- dissemination, in a primate host. Though B. burgdorferi is not known to possess resistance mechanisms and is susceptible to the standard antibiotics (doxycycline, ceftriaxone) invitro, it appears to become tolerant post-dissemination in the primate host. This finding raises important questions about the pathogenicity of antibiotic-tolerant persisters and whether or not they can contribute to symptoms post-treatment.
The Lyme diseases and babesiosis are emerging zoonosis with socio-economic importance to the country, and it is necessary the laboratorial and epidemiological characterization. This study has the aim of recognizing asymptomatics individuals through the research of anti- Borreliaburgdorferi antibodies using the method of ELISA and Western blotting and anti- Babesia bovis by ELISA in the serum of one hundred and fifteen blood donors of Hemosul in Campo Grande-MS. It has been used as antigen, whole sonicated of B. burgdorferi sensu stricto (american strain G39/40) and from B. bovis. Positivity was observed for the antibodies to Borreliaburgdorferiin 7,8% of the samples analyzed by ELISA. All the positive results identified by this technique were confirmed by Western blotting. For anti-Babesia bovis antibodies a prevalence of 10,4% was verified. Concomitance of the two antibodies was observed in two (1,73%) of the one hundred and fifteen donors researched. The results observed in this study indicate the widespread distribution of these agents in our region and the possibility of existing human cases without symptoms. This way, we stand out the importance of carrying out more clinico-labotarial and epidemiological studies in our State, which will make possible a better understanding of the interaction between these two zoonosis in our environment.
Saraiva et al, 2013 evaluated the synergism of the S. brasiliensis leaf at a concentration of 25 and 50 μg / mL, which were associated with antibiotics (tetracycline and oxacillin), the results showed additive and synergic actions for the concentration of 5 0 μg / mL, although not enough for MIC to reach values below 2 and 4 μg / mL, necessary to be classified as sensitive strains CLSI (2005) to oxacillin and tetracycline, respectively. Therefore, it was concluded that in the concentration of 50 μg / mL of the leaves of S. brasiliensis presented antimicrobial potential against the multiresistant strains of S. aureus MRSA and that the associations of the fractions with the antibiotics tested did not present benefits, not justifying the concomitant use. This was the only study in the literature found to perform the association with the S. brasiliensis extract.
Regarding interspecific bacterial interactions impact on biofilmformation, bacterial supernatants from several soil borne strains were used. The bacteria were chosen due to their importance as PGPR and wide range of biological activity in the rhizosphere. Moreover, P. putida X236, P. fluorescens 20130311XA1 were selected due to Pseudomonads biocontrol capabilities (51, 52, 53, 54). Not only, but also species of Bacillus have those proprieties (55, 56, 57, 58, 59). Thus, strains of B. pumilus, B. subtilis, B. licheniformis and B. megaterium were selected. In addition, due to their nitrogen fixation strains of B. japonicum, A. chroococcum and M. loti (85, 86, 87) were picked too. Considering the supernatants of the selected bacteria, P. putida X236 had a positive and significant effect on A. brasilense. After examining the results, there was no link with the bacteria phylogeny. As consequence of the screening and posterior supernatant influence inbiofilmformation, was provided further focus on P. putida X236, which showed more potential. Therefore, P. putida X236 had a direct effect in A. brasilense biofilmformation.
In the present study, five intergenic spacers were selected from the alignment of B. crocidurae, B. duttonii and B. recurrentis reference genomes, representing approximately ,0.2% of the total genome length. The spacers were scattered across the chromosome thus representative of the whole genome. Such a multi-target approach offers distinct advantages over the one single locus methods previously used, such as the 16S–23S IGS for typing that may be less representative of the whole genome. Based on this spacer sequencing, a total of 61 RF strains could be separated into 12 STs. Interestingly, we observed that isolates grouped into three clades corresponding to the three Borrelia organisms under study. Indeed, MST yielded no overlap between B. duttonii and B. recurrentis organisms contrary to that observed when using IGS typing [1,7]. We observed that sequencing MST7 spacer alone accurately discriminated between B. duttonii and B. recurrentis with 3% sequence divergence, a result not previously achieved. Therefore, sequencing MST7 spacer alone could be used for the molecular identification of RF Borreliain Africa at the species level, but not for genotyping which requires sequencing the four other spacers in addition to MST7.
crônica são os mais relacionados com a BL crônica, sendo referidos como fadiga profunda e extremamente debilitante. A causa da BL crônica ainda não está totalmente elucidada, embora vários estudos apontem como principal fator a infecção persistente por B. burgdorferi, a despeito do tratamento correto da doença. Alguns autores postulam que essas manifestações seriam decorrentes de fenômenos inflamatórios e autoimunes, desencadeados pela persistência da infecção, não pela presença em si do agente infeccioso, de modo semelhante ao que ocorre na sífilis. Isso justifica o fato de muitos pacientes com BL crônica não responderem a esquemas prolongados com antibióticos. 53
Lyme borreliosis (LB) is a tick-borne disease caused by genospecies of the Borreliaburgdorferi sensu lato (s.l.) complex (Steere, 1997). The genospecies causing LB vary according to the geographic region: B. andersonii, is mainly found in North America, B. afzelli and B. garinii in Europe, B. japonica in Japan, and B. burgdorferi sensu stricto (s.s.) has been detected on several continents (Qiu et al., 2008; Rudenko et al., 2011). Migratory birds cause the dissemination ofBorrelia spp. between continents, and the establishment and maintenance of these spirochetes in a new environment depends on the presence of their reservoir hosts (tick species) and host-vector interactions (Hasle, 2013; Norte et al., 2013).
Studies of the Brazilian lyme-Like disease, or Bag- gio-Yoshinari syndrome, have revealed differences in epi- demiological, clinical and laboratorial characteristics compared with those reported in affected individuals in the northern hemisphere, suggesting the existence of differing etiological agents in the two locations (Yoshinari et al., 2010). In Brazil, despite the wide geographical distribution of both invertebrate and vertebrate hosts for Borrelia spp., there are few descriptions of these spirochetes. Thus, fur- ther serological and molecular studies are needed in hu- mans, different species of domestic and wild animals, and ticks, in particular, to better understand the epidemiology ofBorrelia spp.
microorganisms that can be associated with tick-borne. It was found a higher frequency of antibodies against borrelia and babesia in patients in relation to the controls. Although small, this difference was statistically significant. This finding does not certify that the clinical features of patients is caused by species ofborrelia or babesia, but raised the possibility of involvement of some infectious agent that could be related to these micro- organisms. The studies should continue in order to improve the delineation of this syndrome and to try the isolation of the etiologic agent in our State.
monitorized by counting the total number of spirochetes in 0.1ml of medium in a dark field microscope, using a 10x30 mm cover slip. For the first 12 days, counting was done each 24 hours, and afterwards once a week during 14 weeks. There occurred growth of B. burgdorferiin all tested media, with the best performance of three of them: BSK with rabbit serum, BSK swine serum + 5 fluorouracil, and CTB medium. Growth of B. burgdorferi was seen from the 4th week on, reaching its maximum within 8-12 weeks, depleting the culture medium after this time. Cystic forms of B. burgdorferi were observed with all tested media.
Biofilm was higher in M9 medium as compared to LB medium. It seems that biofilm development protected the cells at elevated stress of nutrients and salt with increasing time. Decrease in planktonic and loosely bound cells with a subsequent increase in tightly bound cells was observed. It may be speculated that bacterial cells fight back with stress to stay alive in the form of micro colonies (25). Environmental stress like nutrients and osmotic stress poses increased bacterial competition for available nutrients, and bacteria revert from the planktonic stage to sessile assemblages at various biotic and abiotic surfaces to protect them in the rhizosphere (19, 20). Moreover, increased production of exopolysaccharide against higher salt stress also favors biofilmformation and protects this mini assembly by retaining a water layer around the cells (9, 15, 20, 23). This is also in line with the previous findings that EPS of Staphylococcus was visible extending in various directions from the cells and help them to adhere to surfaces (18). Sticky nature of EPS depends on its composition, i.e., sugars, proteins and lipids (12). Our results are in line with previous studies where exopolysaccharide production reduced in the medium with the addition of salt (29). It may be speculated from previous reports that variable exopolysaccharide production in different bacterial genera Halomonas variabilis (HT1) and Planococcus rifietoensis (RT4) in response to salt stress may
Detection ofbiofilmformation was measured at a lower scale compared to similar studies by Pan et al. (2010). The main difference between the present study and Pan et al. (2010) was that, in the last one, the colonies were incubated with tryptic soy broth as nutrient and NaCl as ad- ditional nutrients. The formationofbiofilm was enhanced as a synergistic effect on the combination of glucose, NaCl and TSB (Pan et al., 2010). In this study, emphasis was placed on the cellular stress by NaCl instead of NaCl as ad- ditional nutrient to the cells. Nonetheless, it has been noted that addition of NaCl to TSB enhances the adherence of the cells and increases the invasiveness of the strains (Jensen et al., 2007). According Norwood and Gilmour (2001), the adherence capability of L. monocytogenes FM876 (sero- type 1/2c) was found to be greater than L. monocytogenes Scott A, as seen at 4 °C, 18 °C and 30 °C. Formationofbiofilmby L. monocytogenes can be affected depending on the existence as monoculture or multispecies inbiofilm ma- trix, such as Staphylococcus biofilm may produce extra- cellular polysaccharides that may prevent the adhesion to stainless steel surfaces (Leriche and Carpentier, 2000). Changes in the cell surface can also be caused by the changes in regulation of virulence and environmental genes as a result of temperature changes (Leimeister-Wachter et al., 1992; Liu et al., 2002) and subsequently affects the at- tachments of cells such as properties of surfaces being hy- drophobic or hydrophilic.
spirochete proteins, reduced with SDS, was electrophoresed (Mini- Protean II System, Bio Rad) on a 10% acrylamide gel. After running, gel proteins were transferred to nitrocellulose paper over night (Mini- Trans Blot System, Bio Rad), and the paper cut in strips, which were washed in distilled water. One strip containing Borreliaburgdorferi antigens and other with molecular weight markers were separated and stained with a solution 1:1 of colloidal gold (Bio Rad). The others strips were blocked with 0.1% TBS Tween 20 and 5% skimmed milk for 1 hour at room temperature and then washed five times with 0.1% TBS Tween 20. The sera samples to be tested (positive and negative controls, and test sera), were diluted 1:100 in blocking solution and added to incubate with strips for 1 hour at room temperature. After washing, the strips were incubated for 1 hour with alkaline phosphatase-conjugated rabbit serum anti-dog IgG (Sigma Chemical) diluted at 1/1000 with blocking solution. Washing was repeated and substrate consisting of NBT/BCIP diluted in bicarbonate buffer pH 9.8 was added. The reaction was blocked when positive control developed color.