Photodynamic and Antibiotic Therapy Impair the Pathogenesis of Enterococcus faecium in a Whole Animal Insect Model

Enterococcus faecium has emerged as one of the most important pathogens in healthcare-associated infections worldwide due to its intrinsic and acquired resistance to many antibiotics, including vancomycin. Antimicrobial photodynamic therapy (aPDT) is an alternative therapeutic platform that is currently under investigation for the control and treatment of infections. PDT is based on the use of photoactive dye molecules, widely known as photosensitizer (PS). PS, upon irradiation with visible light, produces reactive oxygen species that can destroy lipids and proteins causing cell death. We employed Galleria mellonella (the greater wax moth) caterpillar fatally infected with E. faecium to develop an invertebrate host model system that can be used to study the antimicrobial PDT (alone or combined with antibiotics). In the establishment of infection by E. faecium in G. mellonella, we found that the G. mellonella death rate was dependent on the number of bacterial cells injected into the insect hemocoel and all E. faecium strains tested were capable of infecting and killing G. mellonella. Antibiotic treatment with ampicillin, gentamicin or the combination of ampicillin and gentamicin prolonged caterpillar survival infected by E. faecium (P = 0.0003, P = 0.0001 and P = 0.0001, respectively). In the study of antimicrobial PDT, we verified that methylene blue (MB) injected into the insect followed by whole body illumination prolonged the caterpillar survival (P = 0.0192). Interestingly, combination therapy of larvae infected with vancomycin-resistant E. faecium, with antimicrobial PDT followed by vancomycin, significantly prolonged the survival of the caterpillars when compared to either antimicrobial PDT (P = 0.0095) or vancomycin treatment alone (P = 0.0025), suggesting that the aPDT made the vancomycin resistant E. faecium strain more susceptible to vancomycin action. In summary, G. mellonella provides an invertebrate model host to study the antimicrobial PDT and to explore combinatorial aPDT-based treatments.

Assessment of the relationship between entomologic indicators of Aedes aegypti and the epidemic occurrence of dengue virus 3 in a susceptible population, São José do Rio Preto, Sao Paulo, Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Equid herpesvirus type-1 exhibits neurotropism and neurovirulence in a mouse model

Intranasal inoculation of equid herpesvirus type-1 (EHV-1) Brazilian strains A4/72 and A9/92 induced an acute and lethal infection in four different inbred mouse strains. Clinical and neurological signs appeared between the 2nd and 3rd day post inoculation (dpi) and included weight loss, ruffled fur, a hunched posture, crouching in corners, nasal and ocular discharges, dyspnoea, dehydration and increased salivation. These signs were followed by increased reactivity to external stimulation, seizures, recumbency and death. The virus was recovered consistently from the brain and viscera of all mice with neurological signs. Histopathological changes consisted of leptomeningitis, focal haemorrhage, ventriculitis, neuronal degeneration and necrosis, neuronophagia, non-suppurative inflammation, multifocal gliosis and perivascular infiltration of polymorphonuclear and mononuclear cells. Immunohistochemical examination demonstrated that EHV-1 strains A4/72 and A9/92 replicated in neurons of the olfactory bulb, the cortex and the hippocampus. In contrast, mice inoculated with the EHV-1 Brazilian strain A3/97 showed neither weight loss nor apparent clinical or neurological signs; however, the virus was recovered consistently from their lungs at 3 dpi. These three EHV-1 strains showed distinct degrees of virulence and tissue tropism in mice. EHV-1 strains A4/72 and A9/92 exhibited a high degree of central nervous system tropism with neuroinvasion and neurovirulence. EHV-1 strain A3/97 was not neurovirulent despite being detected in the brains of infected BALB/c nude mice. These findings indicate that several inbred mouse strains are susceptible to neuropathogenic EHV-1 strains and should be useful models for studying the pathogenesis and mechanisms contributing to EHV-induced myeloencephalopathy in horses. (C) 2011 Elsevier Ltd. All rights reserved.

Exploration of the parameter space in an agent-based model of tuberculosis spread: emergence of drug resistance in developing vs developed countries

In this work we present an agent-based model for the spread of tuberculosis where the individuals can be infected with either drug-susceptible or drug-resistant strains and can also receive a treatment. The dynamics of the model and the role of each one of the parameters are explained. The whole set of parameters is explored to check their importance in the numerical simulation results. The model captures the beneficial impact of the adequate treatment on the prevalence of tuberculosis. Nevertheless, depending on the treatment parameters range, it also captures the emergence of drug resistance. Drug resistance emergence is particularly likely to occur for parameter values corresponding to less efficacious treatment, as usually found in developing countries.

BEM modeling of saturated porous media susceptible to damage

This paper deals with the numerical analysis of saturated porous media, taking into account the damage phenomena on the solid skeleton. The porous media is taken into poro-elastic framework, in full-saturated condition, based on Biot's Theory. A scalar damage model is assumed for this analysis. An implicit boundary element method (BEM) formulation, based on time-independent fundamental solutions, is developed and implemented to couple the fluid flow and two-dimensional elastostatic problems. The integration over boundary elements is evaluated using a numerical Gauss procedure. A semi-analytical scheme for the case of triangular domain cells is followed to carry out the relevant domain integrals. The non-linear problem is solved by a Newton-Raphson procedure. Numerical examples are presented, in order to validate the implemented formulation and to illustrate its efficacy. (C) 2011 Elsevier Ltd. All rights reserved.

A stochastic spatially structured epidemic model with diffusive processes

We developed a stochastic lattice model to describe the vector-borne disease (like yellow fever or dengue). The model is spatially structured and its dynamical rules take into account the diffusion of vectors. We consider a bipartite lattice, forming a sub-lattice of human and another occupied by mosquitoes. At each site of lattice we associate a stochastic variable that describes the occupation and the health state of a single individual (mosquito or human). The process of disease transmission in the human population follows a similar dynamic of the Susceptible-Infected-Recovered model (SIR), while the disease transmission in the mosquito population has an analogous dynamic of the Susceptible-Infected-Susceptible model (SIS) with mosquitos diffusion. The occurrence of an epidemic is directly related to the conditional probability of occurrence of infected mosquitoes (human) in the presence of susceptible human (mosquitoes) on neighborhood. The probability of diffusion of mosquitoes can facilitate the formation of pairs Susceptible-Infected enabling an increase in the size of the epidemic. Using an asynchronous dynamic update, we study the disease transmission in a population initially formed by susceptible individuals due to the introduction of a single mosquito (human) infected. We find that this model exhibits a continuous phase transition related to the existence or non-existence of an epidemic. By means of mean field approximations and Monte Carlo simulations we investigate the epidemic threshold and the phase diagram in terms of the diffusion probability and the infection probability.

Pair approximation for a model of vertical and horizontal transmission of parasites.

We apply Stochastic Dynamics method for a differential equations model, proposed by Marc Lipsitch and collaborators (Proc. R. Soc. Lond. B 260, 321, 1995), for which the transmission dynamics of parasites occurs from a parent to its offspring (vertical transmission), and by contact with infected host (horizontal transmission). Herpes, Hepatitis and AIDS are examples of diseases for which both horizontal and vertical transmission occur simultaneously during the virus spreading. Understanding the role of each type of transmission in the infection prevalence on a susceptible host population may provide some information about the factors that contribute for the eradication and/or control of those diseases. We present a pair mean-field approximation obtained from the master equation of the model. The pair approximation is formed by the differential equations of the susceptible and infected population densities and the differential equations of pairs that contribute to the former ones. In terms of the model parameters, we obtain the conditions that lead to the disease eradication, and set up the phase diagram based on the local stability analysis of fixed points. We also perform Monte Carlo simulations of the model on complete graphs and Erdös-Rényi graphs in order to investigate the influence of population size and neighborhood on the previous mean-field results; by this way, we also expect to evaluate the contribution of vertical and horizontal transmission on the elimination of parasite. Pair Approximation for a Model of Vertical and Horizontal Transmission of Parasites.

Gene expression analysis in ‘Candidatus Phytoplasma mali’-resistant and -susceptible Malus genotypes

Apple proliferation (AP) disease is the most important graft-transmissible and vector-borne disease of apple in Europe. ‘Candidatus Phytoplasma mali’ (Ca. P. mali) is the causal agent of AP. Apple (Malus x domestica) and other Malus species are the only known woody hosts. In European apple orchards, the cultivars are mainly grafted on one rootstock, M. x domestica cv. M9. M9 like all other M. x domestica cultivars is susceptible to ‘Ca. P. mali’. Resistance to AP was found in the wild genotype Malus sieboldii (MS) and in MS-derived hybrids but they were characterised by poor agronomic value. The breeding of a new rootstock carrying the resistant and the agronomic traits was the major aim of a project of which this work is a part. The objective was to shed light into the unknown resistance mechanism. The plant-phytoplasma interaction was studied by analysing differences between the ‘Ca. P. mali’-resistant and -susceptible genotypes related to constitutively expressed genes or to induced genes during infection. The cDNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) technique was employed in both approaches. Differences related to constitutively expressed genes were identified between two ‘Ca. P. mali’-resistant hybrid genotypes (4551 and H0909) and the ‘Ca. P. mali’-susceptible M9. 232 cDNA-AFLP bands present in the two resistant genotypes but absent in the susceptible one were isolated but several different products associated to each band were found. Therefore, two different macroarray hybridisation experiments were performed with the cDNA-AFLP fragments yielding 40 sequences encoding for genes of unknown function or a wide array of functions including plant defence. In the second approach, individuation and analysis of the induced genes was carried out exploiting an in vitro system in which healthy and ‘Ca. P. mali’-infected micropropagated plants were maintained under controlled conditions. Infection trials using in vitro grafting of ‘Ca. P. mali’ showed that the resistance phenotype could be reproduced in this system. In addition, ex vitro plants were generated as an independent control of the genes differentially expressed in the in vitro plants. The cDNA-AFLP analysis in in vitro plants yielded 63 bands characterised by over-expression in the infected state of both the H0909 and MS genotypes. The major part (37 %) of the associated sequences showed homology with products of unknown function. The other genes were involved in plant defence, energy transport/oxidative stress response, protein metabolism and cellular growth. Real-time qPCR analysis was employed to validate the differential expression of the genes individuated in the cDNA-AFLP analysis. Since no internal controls were available for the study of the gene expression in Malus, an analysis on housekeeping genes was performed. The most stably expressed genes were the elongation factor-1 α (EF1) and the eukaryotic translation initiation factor 4-A (eIF4A). Twelve out of 20 genes investigated through qPCR were significantly differentially expressed in at least one genotype either in in vitro plants or in ex vitro plants. Overall, about 20% of the genes confirmed their cDNA-AFLP expression pattern in M. sieboldii or H0909. On the contrary, 30 % of the genes showed down-regulation or were not differentially expressed. For the remaining 50 % of the genes a contrasting behaviour was observed. The qPCR data could be interpreted as follows: the phytoplasma infection unbalance photosynthetic activity and photorespiration down-regulating genes involved in photosynthesis and in the electron transfer chain. As result, and in contrast to M. x domestica genotypes, an up-regulation of genes of the general response against pathogens was found in MS. These genes involved the pathway of H2O2 and the production of secondary metabolites leading to the hypothesis that a response based on the accumulation of H2O2 in MS would be at the base of its resistance. This resembles a phenomenon known as “recovery” where the spontaneous remission of the symptoms is observed in old susceptible plants but occurring in a stochastic way while the resistance in MS is an inducible but stable feature. As additional product of this work three cDNA-AFLP-derived markers were developed which showed independent distribution among the seedlings of two breeding progenies and were associated to a genomic region characteristic of MS. These markers will contribute to the development of molecular markers for the resistance as well as to map the resistance on the Malus genome.

Pefloxacin therapy for experimental endocarditis caused by methicillin-susceptible or methicillin-resistant strains of Staphylococcus aureus

The therapeutic efficacy of pefloxacin in experimental endocarditis caused by methicillin-susceptible or methicillin-resistant Staphylococcus aureus was evaluated. In rabbits infected with a methicillin-susceptible strain, 4 days of pefloxacin therapy significantly reduced both the number of bacteria per gram of vegetation and the mortality rate compared with untreated controls, and pefloxacin was equivalent to cephalothin. Pefloxacin was also as effective as vancomycin in reducing vegetation titers and mortality rate in animals with endocarditis caused by a methicillin-resistant strain. These results suggest that pefloxacin may be an effective agent in the therapy of serious infections caused by either methicillin-susceptible or -resistant strains of S. aureus.

Toll-like receptor 2-deficient mice are highly susceptible to Streptococcus pneumoniae meningitis because of reduced bacterial clearing and enhanced inflammation

Toll-like receptor-2 (TLR2) mediates host responses to gram-positive bacterial wall components. TLR2 function was investigated in a murine Streptococcus pneumoniae meningitis model in wild-type (wt) and TLR2-deficient (TLR2(-/-)) mice. TLR2(-/-) mice showed earlier time of death than wt mice (P<.02). Plasma interleukin-6 levels and bacterial numbers in blood and peripheral organs were similar for both strains. With ceftriaxone therapy, none of the wt but 27% of the TLR2(-/-) mice died (P<.04). Beyond 3 hours after infection, TLR2(-/-) mice had higher bacterial loads in brain than did wt mice, as assessed with luciferase-tagged S. pneumoniae by means of a Xenogen-CCD (charge-coupled device) camera. After 24 h, tumor necrosis factor activity was higher in cerebrospinal fluid of TLR2(-/-) than wt mice (P<.05) and was related to increased blood-brain barrier permeability (Evans blue staining, P<.02). In conclusion, the lack of TLR2 was associated with earlier death from meningitis, which was not due to sepsis but to reduced brain bacterial clearing, followed by increased intrathecal inflammation.

Tuberculosis in Cape Town: An age-structured transmission model.

BACKGROUND Tuberculosis (TB) is the leading cause of death in South Africa. The burden of disease varies by age, with peaks in TB notification rates in the HIV-negative population at ages 0-5, 20-24, and 45-49 years. There is little variation between age groups in the rates in the HIV-positive population. The drivers of this age pattern remain unknown. METHODS We developed an age-structured simulation model of Mycobacterium tuberculosis (Mtb) transmission in Cape Town, South Africa. We considered five states of TB progression: susceptible, infected (latent TB), active TB, treated TB, and treatment default. Latently infected individuals could be re-infected; a previous Mtb infection slowed progression to active disease. We further considered three states of HIV progression: HIV negative, HIV positive, on antiretroviral therapy. To parameterize the model, we analysed treatment outcomes from the Cape Town electronic TB register, social mixing patterns from a Cape Town community and used literature estimates for other parameters. To investigate the main drivers behind the age patterns, we conducted sensitivity analyses on all parameters related to the age structure. RESULTS The model replicated the age patterns in HIV-negative TB notification rates of Cape Town in 2009. Simulated TB notification rate in HIV-negative patients was 1000/100,000 person-years (pyrs) in children aged <5 years and decreased to 51/100,000 in children 5-15 years. The peak in early adulthood occurred at 25-29 years (463/100,000 pyrs). After a subsequent decline, simulated TB notification rates gradually increased from the age of 30 years. Sensitivity analyses showed that the dip after the early adult peak was due to the protective effect of latent TB and that retreatment TB was mainly responsible for the rise in TB notification rates from the age of 30 years. CONCLUSION The protective effect of a first latent infection on subsequent infections and the faster progression in previously treated patients are the key determinants of the age-structure of TB notification rates in Cape Town.

The crucial role of polymorphonuclear leukocytes in resistance to Salmonella dublin infections in genetically susceptible and resistant mice

Macrophages are considered to be the mediators of resistance to extra-intestinal Salmonella infections. Nevertheless, the initial cellular response to Salmonella infections consists primarily of polymorphonuclear leukocytes (PMN). To determine whether PMN serve an important function for the infected host, we made mice neutropenic with the rat mAb to RB6–8C5 and infected them i.v. with ≈103 Salmonella dublin or an isogenic derivative that lacks the virulence plasmid (LD842). We infected BALB/c mice, which have a point mutation in the macrophage-expressed gene Nramp1 that makes them susceptible to Salmonella, and BALB/c.D2 congenic mice, which have the wild-type Nramp1 gene that makes them resistant to Salmonella. Both mouse strains were resistant to LD842, and neutropenia made only the BALB/c strain susceptible to this infection. Neutropenic congenic mice, however, were susceptible only to wild-type S. dublin (plasmid+). These results show a complex interplay between plasmid-virulence genes in Salmonella, host macrophages, and PMN. Mice with normal macrophages need PMN to defend against nontyphoid Salmonella that carry a virulence plasmid but not against Salmonella without virulence plasmids. Mice with a mutant Nramp1 gene need PMN to defend against all Salmonella, even those that lack virulence plasmids. These results, plus the evidence that PMN kill Salmonella efficiently in vitro, suggest that Salmonella have adapted to grow inside macrophages where they are relatively sheltered from PMN. The adaptations that allow Salmonella to survive in macrophages do not protect them from PMN.

Human mesothelial cells are unusually susceptible to simian virus 40-mediated transformation and asbestos cocarcinogenicity

Mesothelioma, a malignancy associated with asbestos, has been recently linked to simian virus 40 (SV40). We found that infection of human mesothelial cells by SV40 is very different from the semipermissive infection thought to be characteristic of human cells. Mesothelial cells are uniformly infected but not lysed by SV40, a mechanism related to p53, and undergo cell transformation at an extremely high rate. Exposure of mesothelial cells to asbestos complemented SV40 mutants in transformation. Our data provide a mechanistic explanation for the ability of SV40 to transform mesothelial cells preferentially and indicate that asbestos and SV40 may be cocarcinogens.

Older plasma lipoproteins are more susceptible to oxidation: a linking mechanism for the lipid and oxidation theories of atherosclerotic cardiovascular disease.

Increases in plasma cholesterol are associated with progressive increases in the risk of atherosclerotic cardiovascular disease. In humans plasma cholesterol is contained primarily in apolipoprotein B-based low density lipoprotein (LDL). Cells stop making the high-affinity receptor responsible for LDL removal as they become cholesterol replete; this slows removal of LDL from plasma and elevates plasma LDL. As a result of this delayed uptake, hypercholesterolemic individuals not only have more LDL but have significantly older LDL. Oxidative modification of LDL enhances their atherogenicity. This study sought to determine whether increased time spent in circulation, or aging, by lipoprotein particles altered their susceptibility to oxidative modification. Controlled synchronous production of distinctive apolipoprotein B lipoproteins (yolk-specific very low density lipoproteins; VLDLy) with a single estrogen injection into young turkeys was used to model LDL aging in vivo. VLDLy remained in circulation for at least 10 days. Susceptibility to oxidation in vitro was highly dependent on lipoprotein age in vivo. Oxidation, measured as hexanal release from n-6 fatty acids in VLDLy, increased from 13.3 +/- 5.5 nmol of 2-day-old VLDLy per ml, to 108 +/- 17 nmol of 7-day-old VLDLy per ml. Oxidative instability was not due to tocopherol depletion or conversion to a more unsaturated fatty acid composition. These findings establish mathematically describable linkages between the variables of LDL concentration and LDL oxidation. The proposed mathematical models suggest a unified investigative approach to determine the mechanisms for acceleration of atherosclerotic cardiovascular disease risk as plasma cholesterol rises.

On the time to reach a critical number of infections in epidemic models with infective and susceptible immigrants.

In this paper we examine the time T to reach a critical number K0 of infections during an outbreak in an epidemic model with infective and susceptible immigrants. The underlying process X, which was first introduced by Ridler-Rowe (1967), is related to recurrent diseases and it appears to be analytically intractable. We present an approximating model inspired from the use of extreme values, and we derive formulae for the Laplace-Stieltjes transform of T and its moments, which are evaluated by using an iterative procedure. Numerical examples are presented to illustrate the effects of the contact and removal rates on the expected values of T and the threshold K0, when the initial time instant corresponds to an invasion time. We also study the exact reproduction number Rexact,0 and the population transmission number Rp, which are random versions of the basic reproduction number R0.