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Made available in DSpace on 2019-10-04T12:42:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-08-20 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) A series of experiments were conducted to characterize a novel restorative material. We explored the effect on biological, physical and chemical properties of glass ionomer cement (GIC) adding-the naturally occurring tt-farnesol (900 mM). Two groups were accomplished for all assays: GIC+tt-farnesol and GIC (control). Biological assays: 1) agar diffusion against some cariogenic bacteria; 2) S. mutans biofilm formation and confocal laser scanning microscopy-CLSM. 3) gtfB, gtfC, gtfD, gbpB, vicR, and covR expression; 4) MTT and microscopic morphology. Physical properties assays: 1) roughness; 2) hardness; 3) compressive strength and 4) diametral tensile strength. Chemical assay: Raman spectroscopy. The adding of tt-farnesol to GIC led to larger zones of inhibition (p0.05). Polysaccharides levels increased over time, similarly over groups (p>0.05). Viable and non-viable S. mutans were seen on the specimens' surface by CLSM but their virulence was not modulated by tt-farnesol. The tt-farnesol increased the HaCaT cell viability without impact on compressive and diametral tensile strength and roughness although the hardness was positively affected (p

The phenol equivalence assay is the current industry-adopted test used to quantify the antibacterial activity of honeys in Australia and New Zealand. Activity is measured based on the diffusion of honey through agar and resulting zone of growth inhibition. Due to differences in the aqueous solubilities of antibacterial compounds found in honeys, this method may not be optimal for quantifying activity. Therefore, a new method was developed based on the existing broth microdilution assay that is widely used for determining minimum inhibitory concentrations (MICs). It utilises the four organisms Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853, and an optical density endpoint to quantify bacterial growth. Decreases in bacterial growth in the presence of honey, relative to the positive growth control, are then used to derive a single value to represent the overall antibacterial activity of each honey. Antibacterial activity was quantified for a total of 77 honeys using the new method, the phenol equivalence assay and the standard broth microdilution assay. This included 69 honeys with undisclosed floral sources and the comparators Manuka, Jarrah (Eucalyptus marginata), Marri (Corymbia calophylla), artificial and multifloral honey. For the 69 honey samples, phenol equivalence values ranged from 0–48.5 with a mean of 34 (% w/v phenol). Mean MICs, determined as the average of the MICs obtained for each of the four organisms for each honey ranged from 7–24% (w/v honey). Using the new assay, values for the 69 honeys ranged from 368 to 669 activity units, with a mean of 596. These new antibacterial activity values correlated closely with mean MICs (R2 = 0.949) whereas the relationship with phenol equivalence values was weaker (R2 = 0.649). Limit of detection, limit of quantitation, measuring interval, limit of reporting, sensitivity, selectivity, repeatability, reproducibility, and ruggedness were also investigated and showed that the new assay was both robust and reproducible.

The survival of Listeria (L.) monocytogenes in foods and food production environments (FPE) is dependent on several genes that increase tolerance to stressors; this includes competing with intrinsic bacteria. We aimed to uncover genes that are differentially expressed (DE) in L. monocytogenes sequence type (ST) 121 strain 6179 when co-cultured with cheese rind bacteria. L. monocytogenes was cultivated in broth or on plates with either a Psychrobacter or Brevibacterium isolate from cheese rinds. RNA was extracted from co-cultures in broth after two or 12 hours and from plates after 24 and 72 hours. Broth co-cultivations with Brevibacterium or Psychrobacter yielded up to 392 and 601 DE genes, while plate co-cultivations significantly affected the expression of up to 190 and 485 L. monocytogenes genes, respectively. Notably, the transcription of virulence genes encoding the Listeria adhesion protein and Listeriolysin O were induced during plate and broth co-cultivations. The expression of several systems under the control of the global stress gene regulator, σB, increased during co-cultivation. A cobalamin-dependent gene cluster, responsible for the catabolism of ethanolamine and 1,2-propanediol, was upregulated in both broth and plate co-cultures conditions. Finally, a small non-coding (nc)RNA, Rli47, was induced after 72 hours of co-cultivation on plates and accounted for 50-90% of the total reads mapped to L. monocytogenes. A recent study has shown that Rli47 may contribute to L. monocytogenes stress survival by slowing growth during stress conditions through the suppression of branch-chained amino acid biosynthesis. We hypothesize that Rli47 may have an impactful role in the response of L. monocytogenes to co-cultivation by regulating a complex network of metabolic and virulence mechanisms.

Heterodera avenae, as an obligate endoparasite, causes severe yield loss in wheat (Triticum aestivum). Investigation on the mechanisms how H. avenae perceives wheat roots is limited. Here, the attractiveness of root exudates from eight plant genotypes to H. avenae were evaluated on agar plates. Results showed that the attraction of H. avenae to the root exudates from the non-host Brachypodium distachyon variety Bd21-3 was the highest, approximately 50 infective second-stage juveniles (J2s) per plate, followed by that from three H. avenae-susceptible wheat varieties, Zhengmai9023, Yanmai84 and Xiangmai25, as well as the resistant one of Xinyuan958, whereas the lowest attractive activity was observed in the two H. avenae-resistant wheat varieties, Xianmai20 (approximately 12 J2s/plate) and Liangxing66 (approximately 11 J2s/plate). Then Bd21-3, Zhengmai9023 and Heng4399 were selected for further assays as their different attractiveness and resistance to H. avenae, and attractants for H. avenae in their root exudates were characterized to be heat-labile and low-molecular compounds (LM) by behavioral bioassay. Based on these properties of the attractants, a principle of identifying attractants for H. avenae was set up. Then LM of six root exudates from the three plants with and without heating were separated and analyzed by HPLC-MS. Finally, dihydroxyacetone (DHA), methylprednisolone succinate, embelin and diethylpropionin in the root exudates were identified to be putative attractants for H. avenae according to the principle, and the attraction of DHA to H. avenae was validated by behavioral bioassay on agar. Our study enhances the recognition to the orientation mechanism of H. avenae towards wheat roots.

Biofilm-impaired tissue is a significant factor in chronic wounds such as diabetic foot ulcers. Most, if not all, anti-biotics in clinical use have been optimized against planktonic phenotypes. In this study, an in vitro assessment was performed to determine the potential efficacy of a unique first-in-class series of antibiofilm antibiotics and compare outcomes to current clinical standards of care. The agent, CZ-01179, was formulated into a hydrogel and tested against mature biofilms of a clinical isolate of methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa ATCC 27853 using two separate methods. In the first method, biofilms were grown on cellulose discs on an agar surface. Topical agents were spread on gauze and placed over the biofilms for 24 h. Biofilms were quantified and imaged with confocal and scanning electron microscopy. In the second method, biofilms were grown on bioabsorbable collagen coupons in a modified CDC biofilm reactor. Coupons were immersed in treatment for 24 h. The first method was limited in its ability to assess efficacy. Efficacy profiles against biofilms grown on collagen were more definitive, with CZ-01179 gel eradicating well-established biofilms to a greater degree compared to clinical standards. In conclusion, CZ-01179 may be a promising topical agent that targets the biofilm phenotype. Pre-clinical work is currently being performed to determine the translatable potential of CZ-01179 gel.

ObjectivesThe aim of this study was to compare the Candida bromcresol green (BCG) medium with the chromogenic (CHROM) Brilliance Candida agar and Sabouraud dextrose agar (SDA) media in regard to their capability of detecting Candida isolates from mono-or dual-species cultures.MethodsWe prepared Candida isolates’ suspensions to obtain mono-species (n = 18) or dual-species (n = 153) culture plates per each medium, and three readers independently observed 513 plates at 24-h, 48-h and 72-h incubation time. We scored reading results as correct, over or under detection compared to the expected species number(s).ResultsBCG showed significantly higher correct-detection and lower under-detection rates for all Candida species when observed by at least one reader. At 24-h reading, 12 mono-species cultures had correct (or over) detections in all media, whereas 106, 60 and 78 dual-species cultures had correct (or over) detections in BCG, CHROM or SDA, respectively.ConclusionBCG provides the basis for an accurate laboratory diagnosis of Candida infections.

Many microbial phenotypes are differentially or exclusively expressed on agar surfaces, including biofilms, motility, and sociality. However, agar-based assays are limited by their low throughput, which increases costs, lab waste, space requirements, and the time required to conduct experiments. Here, we demonstrate the use of wax-printed microfluidic paper-based analytical devices (μPADs) to measure linear growth rate of microbes on an agar growth media as a means of circumventing the aforementioned limitations. The main production materials of the proposed μPAD design are a wax printer, filter paper, and empty pipet boxes. A single wax-printed μPAD allowing 8 independent, agar-grown colonies costs $0.07, compared to $0.20 and $9.37 for the same number of replicates on traditional microtiter/spectrophotometry and Petri dish assays, respectively. We optimized the μPAD design for channel width (3 mm), agar volume (780 μL/channel), and microbe inoculation method (razor-blade). Comparative analyses of the traditional and proposed μPAD methods for measuring growth rate of nonmotile (Saccharomyces cerevisiae) and motile (flagellated Escherichia coli) microorganisms suggested the μPAD assays conferred a comparable degree of accuracy and reliability to growth rate measurements as their traditional counterparts. We substantiated this claim with strong, positive correlations between the traditional and μPAD assay, a significant nonzero slope in the model relating the two assays, a nonsignificant difference between the relative standard errors of the two techniques, and an analysis of inter-device reliability. Therefore, μPAD designs merit consideration for the development of enhanced-throughput, low-cost microbial growth and motility assays.

Background Isolation of the soil bacterium Burkholderia pseudomallei from tropical environments is important to generate a global risk map for man and animals to acquire the infectious disease melioidosis. There is increasing evidence, that the currently recommended soil culture protocol using threonine-basal salt solution with colistin (TBSS-C50) for enrichment of B. pseudomallei and Ashdown agar for subsequent subculture lacks sensitivity. We therefore investigated, if the otherwise rarely encountered erythritol catabolism of B. pseudomallei might be exploited to improve isolation of this bacterium from soil. Methodology/Principal findings Based on TBSS-C50, we designed a new colistin-containing medium with erythritol as the single carbon source (EM). This medium was validated in various culture protocols by analyzing 80 soil samples from 16 different rice fields in Vietnam. B. pseudomallei enrichment was determined in all culture supernatants by a specific quantitative PCR (qPCR) targeting the type three secretion system 1. 51 out of 80 (63.8%) soil samples gave a positive qPCR signal in at least one of the culture conditions. We observed a significantly higher enrichment shown by lower median cycle threshold values for B. pseudomallei in a two-step culture with TBSS-C50 for 48 h followed by EM for 96h compared to single cultures in TBSS-C50 for either 48h or 144h (p, Author summary Melioidosis is a vastly underdiagnosed infectious disease caused by the bacterial soil pathogen B. pseudomallei. Global environmental suitability for B. pseudomallei has recently been predicted to cover huge areas with three billion people potentially at risk to acquire infection. Although sensitive as well as specific molecular methods are available, cultural isolation of B. pseudomallei strains from the environment will be indispensable to verify this prediction in potential endemic, low-income countries and is a prerequisite to assess the virulence potential and genetic diversity of strains. The currently applied cultural detection lacks sensitivity, which is at least partly due to a lack in selectivity allowing other highly abundant soil microorganisms to outcompete B. pseudomallei. We therefore aimed to develop a more selective medium by taking advantage of B. pseudomallei erythritol catabolism, a metabolic pathway, which is not commonly encountered in other related bacteria. Validation of our new medium with erythritol as the single carbon source was performed by molecular and cultural detection using soil samples from rice paddies in Vietnam. We observed an enhanced enrichment and selectivity resulting in the isolation of B. pseudomallei strains from soil samples, which were otherwise culture-negative when tested by the standard method.

Glaesserella parasuis is the cause of Glӓsser's disease in pigs and is a significant contributor to post-weaning mortality in the swine industry. Prevention of G. parasuis disease relies primarily on bacterin vaccines, which have shown good homologous protection and variable heterologous protection. Bacterin production involves large scale growth of the bacteria and proteins produced during the proliferation phase of production become important antigens that stimulate the immune response. In order to evaluate genes activated during G. parasuis growth on different media substrates, the transcriptome of broth and agar grown G. parasuis strain 29755 were sequenced and compared. The transcription of most purported virulence genes were comparable between broth and agar grown G. parasuis; however, four virulence-associated genes, including ompA and vapD, had elevated expression under agar growth, while six virulence-associate genes had elevated expression during broth growth, including several protease genes. Additionally, there were metabolic shifts toward increased protein and lipid production and increased cellular division in broth grown G. parasuis. The results contribute to the understanding of how growth substrate alters gene transcription and protein expression, which may impact vaccine efficacy if immunogens important to the protective immune response are not produced under specific in vitro conditions. While the results of this work are unable to fully elucidate which growth medium presents a transcriptome more representative of in vivo samples or best suited for bacterin production, it forms a foundation that can be used for future comparisons and provides a better understanding of the metabolic differences in broth and agar grown bacteria.

Vibrio(Listonella)anguillarumis a pathogenic bacterium causing septicaemia in a wide range of marine organisms and inducing severe mortalities, thus it is crucial to conduct its accurate and rapid identification. The aim of this study was to assess MALDI-TOF MS as a method of choice for identification of clinicalV.anguillarumisolates from affected marine fish. Since the method accuracy might be influenced by the type of the medium used, as well as by the incubation conditions, we testedV.anguillarumisolates grown on standard media with and without the addition of NaCl, cultured at three incubation temperatures, and at three incubation periods. The best scores were retrieved forV.anguillarumstrains grown on NaCl-supplemented tryptone soy agar (TSA) at 22°C and incubated for 48h (100% identification to species level; overall score 2.232), followed by incubation at 37°C and 48h (100% to species level; score 2.192). The strains grown on non-supplemented TSA gave the best readings when incubated at 22°C for 72h (100% identification to species level; overall score 2.182), followed by incubation at 15°C for 72h (100% to species level; score 2.160). Unreliable identifications and no-identifications were growing with the incubation duration at 37°C, on both media, amounting to 88.89% for 7d incubation on supplemented TSA, and 92.60% for 7d incubation on non-supplemented TSA. The age of the cultured strains and use of media significantly impacted the mass spectra, demonstrating that for reliable identification, MALDI-TOF MS protein fingerprinting with the on-target extraction should be performed on strains grown on a NaCl-supplemented medium at temperatures between 15 and 22°C, incubated for 48–72 hours.

Some strains of motile bacteria self-organize to form spatial patterns of high and low cell density over length scales that can be observed by eye. One such collective behavior is the formation in semisolid agar media of a high cell density swarm band. We isolated 7 wild strains of the Enterobacter cloacae complex capable of forming this band and found its propagation speed can vary 2.5 fold across strains. To connect such variability in collective motility to strain properties, each strain's single-cell motility and exponential growth rates were measured. The band speed did not significantly correlate with any individual strain property; however, a multilinear analysis revealed that the band speed was set by a combination of the run speed and tumbling frequency. Comparison of variability in closely-related wild isolates has the potential to reveal how changes in single-cell properties influence the collective behavior of populations.

Most patients with chronic lower back pain (CLBP) exhibit degenerative disc disease. Disc specimens obtained during initial therapeutic discectomies are often infected/colonized with Propionibacterium acnes, a Gram-positive commensal of the human skin. Although pain associated with infection is typically ascribed to the body's inflammatory response, the Gram-positive bacterium Staphylococcus aureus was recently observed to directly activate nociceptors by secreting pore-forming α-hemolysins that disrupt neuronal cell membranes. The hemolytic activity of P. acnes in cultured disc specimens obtained during routine therapeutic discectomies was assessed through incubation on sheep-blood agar. The β-hemolysis pattern displayed by P. acnes on sheep-blood agar was variable and phylogroup-dependent. Their molecular phylogroups were correlated with their hemolytic patterns. Our findings raise the possibility that pore-forming proteins contribute to the pathogenesis and/or symptomology of chronic P. acnes disc infections and CLBP, at least in a subset of cases.

ObjectivesTo determine the influence of local spread of clonal strains and testing of follow-up isolates on categorical (CA) and essential agreement rates (EA) of common colistin (COL) drug susceptibility testing methods with the broth microdilution (BMD) reference method.MethodsCOL MICs were determined for 178 bacterial isolates (Enterobacteriaceae, n = 97; Pseudomonas aeruginosa, n = 81) collected within one year from 64 patients by BMD according to ISO standard 20776-1 (reference method), the SensiTest BMD panel (ST), agar dilution (AD), the VITEK 2 instrument, and gradient diffusion (GD) using antibiotic strips of two and Muller-Hinton agar plates of three manufacturers. CA and EA with BMD were calculated for all isolates and compared to the subset of 68 unique isolates.ResultsCA ranges were 79.4% to 94.1% for the unique isolateq panel and 89.9% to 96.1% for all tested isolates. EA ranges were 64.7% to 86.8% and 67.4% to 91.0%, respectively. In both panels, EA for all GD assays was lower than 90%. Both lower and higher EA values ranging from-18.3% (MTS on BD agar) to + 6.3% (AD, Vitek 2) were observed in the full one-year sample. Acquisition of colistin resistance under therapy was observed for 3 patients.Conclusionsi) Repeat testing and local spread of clonal strains can positively or negatively affect CA and EA, ii) CA is more robust towards local influences than EA, iii) EA of GD and AD methods for COL with the reference BMD method is insufficient.

The aim of the study was the evaluation of a three-step method for the selection of bacterial strains capable of producing surfactin. The procedure consisted of the following steps: 1.blood agar test, 2. measurement of the surface tension (ST) of the medium using the du Nouy method before and after submerged culture, 3. qualitative and quantitative assessment of surfactin by HPLC. Forty five Bacillus subtilis natto strains producing haemolysis zones (≥3mm) were selected. Nineten of them reduced ST of the medium to ≤ 40 mN/m; in six cases, the reduction was as much as 50%. All indicated strains produced surfactin. Positive correlations (p

Tuberculosis (TB) is an infectious bacterial disease that kills approximately 1.3 million people every year. Despite global efforts to reduce both the incidence and mortality associated with TB, the emergence of drug resistant strains has slowed any progress made towards combating the spread of this deadly disease. The current TB drug regimen is inadequate, takes months to complete and poses significant challenges when administering to patients suffering from drug resistant TB. New treatments that are faster, simpler and more affordable are urgently required. Arguably, a good strategy to discover new drugs is to start with an old drug. Here, we have screened a library of 1200 FDA approved drugs from the Prestwick Chemical library®using a GFP microplate assay. Drugs were screened against GFP expressing strains ofMycobacterium smegmatisandMycobacterium bovisBCG as surrogates forMycobacterium tuberculosis,the causative agent of TB in humans. We identified several classes of drugs that displayed antimycobacterial activity against bothM. smegmatisandM. bovisBCG, however each organism also displayed some selectivity towards certain drug classes. Variant analysis of whole genomes sequenced for resistant mutants raised to florfenicol, vanoxerine and pentamidine highlight new pathways that could be exploited in drug repurposing programmes.

Quorum sensing networks have been identified in over one hundred bacterial species to date. A subset of these networks regulate group behaviors, such as bioluminescence, virulence, and biofilm formation, by sending and receiving small molecules called homoserine lactones (HSLs). Bioengineers have incorporated quorum sensing pathways into genetic circuits to connect logical operations. However, the development of higher-order genetic circuitry is inhibited by crosstalk, in which one quorum sensing network responds to HSLs produced by a different network. Here, we report the construction and characterization of a library of ten synthases including some that are expected to produce HSLs that are incompatible with the Lux pathway, and therefore show no crosstalk. We demonstrated their function in a common lab chassis,Escherichia coliBL21, and in two contexts, liquid and solid agar cultures, using decoupled Sender and Receiver pathways. We observed weak or strong stimulation of a Lux Receiver by longer-chain or shorter-chain HSL-generating Senders, respectively. We also considered the under-investigated risk of unintentional release of incompletely deactivated HSLs in biological waste. We found that HSL-enriched media treated with bleach is still bioactive, while autoclaving deactivates LuxR induction. This work represents the most extensive comparison of quorum sensing synthases to date and greatly expands the bacterial signaling toolkit while recommending practices for disposal based on empirical, quantitative evidence.

Background Bile esculin azide with vancomycin (BEAV) medium is a sensitive, but slightly less specific method for vancomycin-resistant enterococci (VRE) screening. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a rapid method for identification of clinical pathogens. This study aimed to assess the performance of a novel combination screening test for VRE, using BEAV broth combined with MALDI-TOF MS. Materials and methods Clinical specimens were collected from patients at risk of VRE carriage, and tested by the novel combination method, using selective BEAV broth culture method followed by MALDI-TOF MS identification (SBEAVM). The reference method used for comparison was the ChromID VRE agar method. Results A total of 135 specimens were collected from 78 patients, and 63 specimens tested positive for VRE positive using the ChromID VRE method (positive rate 46.7%). The sensitivity, specificity, positive predictive value, and negative predictive value of SBEAVM method after an incubation period of 28 hours were 93.7%, 90.3%, 89.4%, and 94.2%, respectively. The SBEAVM method when compared to the ChromID VRE method had a shorter turnaround time (29 vs. 48–72 hours) and lower laboratory cost ($2.11 vs. $3.23 per test). Conclusions This study demonstrates that SBEAVM is a rapid, inexpensive, and accurate method for use in VRE screening.

Standard methods to detect Escherichia coli contamination in food use the polymerase chain reaction (PCR) and agar culture plates. These methods require multiple incubation steps and take a long time to results. An improved rapid flow-cytometry based detection method was developed, using a fluorescence-labeled oligonucleotide probe specifically binding a16S rRNA sequence. The method positively detected 51 E. coli isolates as well as 4 Shigella species. All 27 non-E. coli strains tested gave negative results. Comparison of the new genetic assay with a total plate count (TPC) assay and agar plate counting indicated similar sensitivity, agreement between cytometry cell and colony counts. This method can detect a small number of E.coli cells in the presence of large numbers of other bacteria. This method can be used for rapid, economical, and stable detection of E. coli and Shigella contamination in the food industry and other contexts.

Recent experiments with the bacteria Paenibacillus vortex reveal a remarkable strategy enabling it to cope with antibiotics by cooperating with a different bacterium-Escherichia coli. While P. vortex is a highly effective swarmer, it is sensitive to the antibiotic ampicillin. On the other hand, E. coli can degrade ampicillin but is non-motile when grown on high agar percentages. The two bacterial species form a shared colony in which E. coli is transported by P. vortex and E. coli detoxifies the ampicillin. The paper presents a simplified model, consisting of coupled reaction-diffusion equations, describing the development of ring patterns in the shared colony. Our results demonstrate some of the possible cooperative movement strategies bacteria utilize in order to survive harsh conditions. In addition, we explore the behavior of mixed colonies under new conditions such as antibiotic gradients, synchronization between colonies and possible dynamics of a 3-species system including P. vortex, E. coli and a carbon producing algae that provides nutrients under illuminated, nutrient poor conditions. The derived model was able to simulate an asymmetric relationship between two or three micro-organisms where cooperation is required for survival. Computationally, in order to avoid numerical artifacts due to symmetries within the discretizing grid, the model was solved using a second order Vectorizable Random Lattices method, which is developed as a finite volume scheme on a random grid.