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Find video protocols related to scientific articles indexed in Pubmed.
Protective role of the capsule and impact of serotype 4 switching on Streptococcus mitis.
Infect. Immun.
PUBLISHED: 06-23-2014
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The polysaccharide capsule surrounding Streptococcus pneumoniae is essential for virulence. Recently, Streptococcus mitis, a human commensal and a close relative of S. pneumoniae, was also shown to have a capsule. In this study, the S. mitis type strain switched capsule by acquisition of the serotype 4 capsule locus of S. pneumoniae TIGR4, following induction of competence for natural transformation. Comparison of the wild type with the capsule-switching mutant and with a capsule deletion mutant showed that the capsule protected S. mitis against phagocytosis by RAW 264.7 macrophages. This effect was enhanced in the S. mitis strain expressing the S. pneumoniae capsule, which showed, in addition, increased resistance against early clearance in a mouse model of lung infection. Expression of both capsules also favored survival in human blood, and the effect was again more pronounced for the capsule-switching mutant. S. mitis survival in horse blood or in a mouse model of bacteremia was not significantly different between the wild type and the mutant strains. In all models, S. pneumoniae TIGR4 showed higher rates of survival than the S. mitis type strain or the capsule-switching mutant, except in the lung model, in which significant differences between S. pneumoniae TIGR4 and the capsule-switching mutant were not observed. Thus, we identified conditions that showed a protective function for the capsule in S. mitis. Under such conditions, S. mitis resistance to clearance could be enhanced by capsule switching to serotype 4, but it was enhanced to levels lower than those for the virulent strain S. pneumoniae TIGR4.
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Novel insight into antimicrobial resistance and sensitivity phenotypes associated to qac and norA genotypes in Staphylococcus aureus.
Microbiol. Res.
PUBLISHED: 06-13-2014
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Staphylococcus aureus strains harboring QacA, QacB, QacC, QacG transporters and norA promoter up-regulating mutations were characterized by phenotype microarray (PM), standard methods for susceptibility testing, and ethidium bromide efflux assays, in order to increase knowledge on phenotypes associated to efflux pumps and their substrates. PM data and standard susceptibility testing lead to the identification of new potential efflux targets, such as guanidine hydrochloride or 8-hydroxyquinoline for QacA and QacC pumps, respectively. The identification of compounds to which the presence of efflux pumps induced increased susceptibility opens new perspectives for potential adjunct anti-resistance treatment (i.e. strains bearing QacB transporters showed increased susceptibility to thioridazine, amitriptyline and orphenadrine). Although the tested isolates were characterized by high degree of heterogeneity, a hallmark of clinical isolates, direct ethidium bromide efflux assays were effective in highlighting differences in efflux efficiency among strains. These data add to characterization of substrate specificity in the different classes of staphylococcal multidrug efflux systems conferring specific substrate profiles and efflux features to each of them.
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Evaluation of epidemiological cut-off values indicates that biocide resistant subpopulations are uncommon in natural isolates of clinically-relevant microorganisms.
PLoS ONE
PUBLISHED: 01-01-2014
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To date there are no clear criteria to determine whether a microbe is susceptible to biocides or not. As a starting point for distinguishing between wild-type and resistant organisms, we set out to determine the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) distributions for four common biocides; triclosan, benzalkonium chloride, chlorhexidine and sodium hypochlorite for 3319 clinical isolates, with a particular focus on Staphylococcus aureus (N?=?1635) and Salmonella spp. (N?=?901) but also including Escherichia coli (N?=?368), Candida albicans (N?=?200), Klebsiella pneumoniae (N?=?60), Enterobacter spp. (N?=?54), Enterococcus faecium (N?=?53), and Enterococcus faecalis (N?=?56). From these data epidemiological cut-off values (ECOFFs) are proposed. As would be expected, MBCs were higher than MICs for all biocides. In most cases both values followed a normal distribution. Bimodal distributions, indicating the existence of biocide resistant subpopulations were observed for Enterobacter chlorhexidine susceptibility (both MICs and MBCs) and the susceptibility to triclosan of Enterobacter (MBC), E. coli (MBC and MIC) and S. aureus (MBC and MIC). There is a concern on the potential selection of antibiotic resistance by biocides. Our results indicate however that resistance to biocides and, hence any potential association with antibiotic resistance, is uncommon in natural populations of clinically relevant microorganisms.
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Contribution of different pneumococcal virulence factors to experimental meningitis in mice.
BMC Infect. Dis.
PUBLISHED: 05-27-2013
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Pneumococcal meningitis (PM) is a life-threatening disease with a high case-fatality rate and elevated risk for serious neurological sequelae. In this study, we investigated the contribution of three major virulence factors of Streptococcus pneumoniae, the capsule, pneumococcal surface protein A (PspA) and C (PspC), to the pathogenesis of experimental PM.
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Evaluation of reduced susceptibility to quaternary ammonium compounds and bisbiguanides in clinical isolates and laboratory-generated mutants of Staphylococcus aureus.
Antimicrob. Agents Chemother.
PUBLISHED: 05-13-2013
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The MICs and minimum bactericidal concentrations (MBCs) for the biocides benzalkonium chloride and chlorhexidine were determined against 1,602 clinical isolates of Staphylococcus aureus. Both compounds showed unimodal MIC and MBC distributions (2 and 4 or 8 mg/liter, respectively) with no apparent subpopulation with reduced susceptibility. To investigate further, all isolates were screened for qac genes, and 39 of these also had the promoter region of the NorA multidrug-resistant (MDR) efflux pump sequenced. The presence of qacA, qacB, qacC, and qacG genes increased the mode MIC, but not MBC, to benzalkonium chloride, while only qacA and qacB increased the chlorhexidine mode MIC. Isolates with a wild-type norA promoter or mutations in the norA promoter had similar biocide MIC distributions; notably, not all clinical isolates with norA mutations were resistant to fluoroquinolones. In vitro efflux mutants could be readily selected with ethidium bromide and acriflavine. Multiple passages were necessary to select mutants with biocides, but these mutants showed phenotypes comparable to those of mutants selected by dyes. All mutants showed changes in the promoter region of norA, but these were distinct from this region of the clinical isolates. Still, none of the in vitro mutants displayed fitness defects in a killing assay in Galleria mellonella larvae. In conclusion, our data provide an in-depth comparative overview on efflux in S. aureus mutants and clinical isolates, showing also that plasmid-encoded efflux pumps did not affect bactericidal activity of biocides. In addition, current in vitro tests appear not to be suitable for predicting levels of resistance that are clinically relevant.
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The use of machine learning methodologies to analyse antibiotic and biocide susceptibility in Staphylococcus aureus.
PLoS ONE
PUBLISHED: 02-19-2013
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The rise of antibiotic resistance in pathogenic bacteria is a significant problem for the treatment of infectious diseases. Resistance is usually selected by the antibiotic itself; however, biocides might also co-select for resistance to antibiotics. Although resistance to biocides is poorly defined, different in vitro studies have shown that mutants presenting low susceptibility to biocides also have reduced susceptibility to antibiotics. However, studies with natural bacterial isolates are more limited and there are no clear conclusions as to whether the use of biocides results in the development of multidrug resistant bacteria.
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The factor H-binding fragment of PspC as a vaccine antigen for the induction of protective humoral immunity against experimental pneumococcal sepsis.
Vaccine
PUBLISHED: 02-21-2011
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Pneumococcal surface protein C (PspC) is a major virulence factor of Streptococcus pneumoniae and interferes with complement activity by binding complement factor H (fH). In this study, protection against experimental sepsis caused by pneumococci carrying different PspC variants was evaluated by immunisation with the fH-binding fragment of PspC. The mechanisms of protection mediated by antibodies to PspC were also studied. Mice were immunised with a PspC fragment (PspC(39-261)) from the type 3 strain HB565 and infected intravenously with either strain HB565 (homologous challenge), or strains D39 and TIGR4 (heterologous challenge). Immunisation with PspC(39-261) elicited high titers (>300,000) of PspC-specific serum IgG and conferred protection from challenge with HB565. In contrast, cross-protection was either limited or absent in vaccinated animals infected with D39 and TIGR4, respectively. To correlate protection with reactivity and function of PspC antibodies, pooled sera from vaccinated mice were tested in IgG binding and complement deposition experiments. IgG antibodies efficiently bound to HB565, while binding was lower with D39 and absent with TIGR4. In the presence of mouse post-immune sera, C3 deposition was increased onto HB565, while no effect was observed with D39 and TIGR4. Antibody cross-reactivity and complement deposition progressively declined with reduced amino acid identity between PspC variants. Antibodies to PspC were also found to interfere with fH binding to HB565. Finally, in vitro and ex vivo phagocytosis assays demonstrated that PspC-specific antibodies promoted opsonophagocytic killing of bacteria.
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The encapsulated strain TIGR4 of Streptococcus pneumoniae is phagocytosed but is resistant to intracellular killing by mouse microglia.
Microbes Infect.
PUBLISHED: 02-11-2010
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The polysaccharide capsule is a major virulence factor of Streptococcus pneumoniae as it confers resistance to phagocytosis. The encapsulated serotype 4 TIGR4 strain was shown to be efficiently phagocytosed by the mouse microglial cell line BV2, whereas the type 3 HB565 strain resisted phagocytosis. Comparing survival after uptake of TIGR4 or its unencapsulated derivative FP23 in gentamicin protection and phagolysosome maturation assays, it was shown that TIGR4 was protected from intracellular killing. Pneumococcal capsular genes were up-regulated in intracellular TIGR4 bacteria recovered from microglial cells. Actual presence of bacteria inside BV2 cells was confirmed by transmission electron microscopy (TEM) for both TIGR4 and FP23 strains, but typical phagosomes/phagolysosomes were detected only in cells infected with the unencapsulated strain. In a mouse model of meningitis based on intracranic inoculation of pneumococci, TIGR4 caused lethal meningitis with an LD(50) of 2 × 10² CFU, whereas the LD(50) for the unencapsulated FP23 was greater than 10? CFU. Phagocytosis of TIGR4 by microglia was also demonstrated by TEM and immunohistochemistry on brain samples from infected mice. The results indicate that encapsulation does not protect the TIGR4 strain from phagocytosis by microglia, while it affords resistance to intracellular killing.
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Characteristics of drug-resistant tuberculosis in Abkhazia (Georgia), a high-prevalence area in Eastern Europe.
Tuberculosis (Edinb)
PUBLISHED: 04-13-2009
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Although multidrug-resistant (MDR) tuberculosis (TB) is a major public health problem in Eastern Europe, the factors contributing to emergence, spread and containment of MDR-TB are not well defined. Here, we analysed the characteristics of drug-resistant TB in a cross-sectional study in Abkhazia (Georgia) between 2003 and 2005, where standard short-course chemotherapy is supplemented with individualized drug-resistance therapy. Drug susceptibility testing (DST) and molecular typing were carried out for Mycobacterium tuberculosis complex strains from consecutive smear-positive TB patients. Out of 366 patients, 60.4% were resistant to any first-line drugs and 21% had MDR-TB. Overall, 25% of all strains belong to the Beijing genotype, which was found to be strongly associated with the risk of MDR-TB (OR 25.9, 95% CI 10.2-66.0) and transmission (OR 2.8, 95% CI 1.6-5.0). One dominant MDR Beijing clone represents 23% of all MDR-TB cases. The level of MDR-TB did not decline during the study period, coinciding with increasing levels of MDR Beijing strains among previously treated cases. Standard chemotherapy plus individualized drug-resistance therapy, guided by conventional DST, might be not sufficient to control MDR-TB in Eastern Europe in light of the spread of "highly transmissible" MDR Beijing strains circulating in the community.
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Functional analysis of pneumococcal drug efflux pumps associates the MATE DinF transporter with quinolone susceptibility.
Antimicrob. Agents Chemother.
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The pneumococcal chromosome encodes about 140 transporters, many of which are predicted to be involved in efflux. In order to critically evaluate pneumococcal efflux, a series of transporter mutants were constructed, and their phenotypes were assayed by disk diffusion, microdilution drug susceptibility testing (MIC testing), growth of cultures at sub-MIC concentrations, and phenotype microarray analysis. Mutants with mutations in seven ATP binding cassette (ABC) transporters, three multiantimicrobial extrusion (MATE) family efflux pumps, and one major facilitator superfamily (MFS) transporter were obtained in Streptococcus pneumoniae strain DP1004. The susceptibility of these 11 mutants to over 250 different substances was compared to that of the parent strain. Of the tested transporters, only the ABC transporter PatAB (SP2073-5) presented a clear multidrug resistance (MDR) profile, as the mutant showed significantly increased susceptibility to ethidium bromide, acriflavine, and berberine. Among the other transporters analyzed, the mutants devoid of the MATE efflux pump SP2065 exhibited reduced susceptibility to novobiocin, and those with mutations of the MATE family DinF transport system (SP1939) exhibited increased susceptibility to moxifloxacin, ciprofloxacin, and levofloxacin. This change in quinolone MIC was found to be independent from the competence-mediated effect of quinolones on the cinA-recA-dinF operon. Furthermore, the dinF mutant, in contrast to the parental strain, allowed selection for quinolone-resistant mutants when exposed to moxifloxacin. These data confirm the clear MDR profile of the PatAB ABC transporter and suggest for the MATE DinF a phenotype associated with quinolone susceptibility, particularly for moxifloxacin.
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A novel resistance mechanism to triclosan that suggests horizontal gene transfer and demonstrates a potential selective pressure for reduced biocide susceptibility in clinical strains of Staphylococcus aureus.
Int. J. Antimicrob. Agents
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The widely used biocide triclosan selectively targets FabI, the NADH-dependent trans-2-enoyl-acyl carrier protein reductase, which is an important target for narrow-spectrum antimicrobial drug development. In relation to the growing concern about biocide resistance, we compared in vitro mutants and clinical isolates of Staphylococcus aureus with reduced triclosan susceptibility. Clinical isolates of S. aureus as well as laboratory-generated mutants were assayed for minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) phenotypes and genotypes related to reduced triclosan susceptibility. A potential epidemiological cut-off (ECOFF) MBC of >4 mg/L was observed for triclosan in clinical isolates of S. aureus. These showed significantly lower MICs and higher MBCs than laboratory mutants. These groups of strains also had few similarities in the triclosan resistance mechanism. Molecular analysis identified novel resistance mechanisms linked to the presence of an additional sh-fabI allele derived from Staphylococcus haemolyticus. The lack of predictive value of in-vitro-selected mutations for clinical isolates indicates that laboratory tests in the present form appear to be of limited value. More importantly, detection of sh-fabI as a novel resistance mechanism with high potential for horizontal gene transfer demonstrates for the first time that a biocide could exert a selective pressure able to drive the spread of a resistance determinant in a human pathogen.
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A functional genomics approach to establish the complement of carbohydrate transporters in Streptococcus pneumoniae.
PLoS ONE
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The aerotolerant anaerobe Streptococcus pneumoniae is part of the normal nasopharyngeal microbiota of humans and one of the most important invasive pathogens. A genomic survey allowed establishing the occurrence of twenty-one phosphotransferase systems, seven carbohydrate uptake ABC transporters, one sodium:solute symporter and a permease, underlining an exceptionally high capacity for uptake of carbohydrate substrates. Despite high genomic variability, combined phenotypic and genomic analysis of twenty sequenced strains did assign the substrate specificity only to two uptake systems. Systematic analysis of mutants for most carbohydrate transporters enabled us to assign a phenotype and substrate specificity to twenty-three transport systems. For five putative transporters for galactose, pentoses, ribonucleosides and sulphated glycans activity was inferred, but not experimentally confirmed and only one transport system remains with an unknown substrate and lack of any functional annotation. Using a metabolic approach, 80% of the thirty-two fermentable carbon substrates were assigned to the corresponding transporter. The complexity and robustness of sugar uptake is underlined by the finding that many transporters have multiple substrates, and many sugars are transported by more than one system. The present work permits to draw a functional map of the complete arsenal of carbohydrate utilisation proteins of pneumococci, allows re-annotation of genomic data and might serve as a reference for related species. These data provide tools for specific investigation of the roles of the different carbon substrates on pneumococcal physiology in the host during carriage and invasive infection.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.