Antibacterial potentiality of Argemone mexicana solvent extracts against some pathogenic bacteria

The sensitivity of two Gram positive (Staphylococcus aureus and Bacillus subtilis) and two Gram negative (Escherichia coli and Pseudomonas aeruginosa) pathogenic multi-drug resistant bacteria was tested against the crude extracts (cold aqueous, hot aqueous, and methanol extracts) of leaves and seeds of Argemone mexicana L. (Papaveraceae) by agar well diffusion method. Though all the extracts were found effective, yet the methanol extract showed maximum inhibition against the test microorganisms followed by hot aqueous extract and cold aqueous extract.

Laboratory and field evaluation of an oviposition trap for Culex quinquefasciatus(Diptera: Culicidae)

An ovitrap (BR-OVT) based on physical and chemical stimuli for attracting gravid Culex quinquefasciatus (Diptera: Culicidae) females was developed and evaluated under laboratory and field conditions. Attractants were assayed using alternative chamber bioassays prior to being used in the BR-OVT oviposition trap. A significant preference of gravid females for sites containing conspecific egg rafts was observed, as a response to the natural oviposition pheromone, as well as for sites treated with the synthetic pheromone erythro-6-acetoxy-5-hexadecanolide. Five- to 20-day old grass infusion was strongly attractive to gravid females for laying eggs. On the other hand, entomopathogenic Bacillus sphaericus (Bs) did not influence the choice of an oviposition site when used in combination with grass infusion and can therefore be used as a larvicide in ovitraps. Results from field trials showed that the BR-OVT with grass infusion and with or without Bs works as a preferred oviposition site for Cx. quinquefasciatus. The BR-OVT was more effective for egg collection when placed indoors and comparison with the number of egg rafts laid in cesspits over 40 days indicates that this very simple ovitrap may be a useful tool for monitoring populations of the most important of the vectors of bancroftian filariasis.

First isolation of microorganisms from the gut diverticulum of Aedes aegypti (Diptera: Culicidae): new perspectives for an insect-bacteria association

We show for the first time that the ventral diverticulum of the mosquito gut (impermeable sugar storage organ) harbors microorganisms. The gut diverticulum from newly emerged and non-fed Aedes aegypti was dissected under aseptic conditions, homogenized and plated on BHI medium. Microbial isolates were identified by sequencing of 16S rDNA for bacteria and 28S rDNA for yeast. A direct DNA extraction from Ae. aegypti gut diverticulum was also performed. The bacterial isolates were: Bacillus sp., Bacillus subtilis and Serratia sp. The latter was the predominant bacteria found in our isolations. The yeast species identified was Pichia caribbica.

Antimicrobial activity of Brazilian copaiba oils obtained from different species of the Copaifera genus

The antimicrobial activity of copaiba oils was tested against Gram-positive and Gram-negative bacteria, yeast, and dermatophytes. Oils obtained from Copaifera martii, Copaifera officinalis, and Copaifera reticulata (collected in the state of Acre) were active against Gram-positive species (Staphylococcus aureus, methicillin-resistant S. aureus, Staphylococcus epidermidis, Bacillus subtilis, and Enterococcus faecalis) with minimum inhibitory concentrations ranging from 31.3-62.5 µg/ml. The oils showed bactericidal activity, decreasing the viability of these Gram-positive bacteria within 3 h. Moderate activity was observed against dermatophyte fungi (Trichophyton rubrum and Microsporum canis). The oils showed no activity against Gram-negative bacteria and yeast. Scannning electron microscopy of S. aureus treated with resin oil from C. martii revealed lysis of the bacteria, causing cellular agglomerates. Transmission electron microscopy revealed disruption and damage to the cell wall, resulting in the release of cytoplasmic compounds, alterations in morphology, and a decrease in cell volume, indicating that copaiba oil may affect the cell wall.

Antibacterial and cytotoxic activity of Kenyan medicinal plants

Seven medicinal plant extracts traditionally used in Kenya, mainly for management of infectious conditions, were chosen and screened for their antibacterial activity against Gram-negative (Pseudomonas aeruginosa and Escherichia coli) and Gram-positive (Bacillus cereus and Staphylococcus aureus) bacteria. Antibacterial activity was tested using the broth dilution method. Harrisonia abyssinica and Terminalia kilimandscharica extracts showed significant activity against Gram+ and Gram- bacteria. The methanolic extracts of T. kilimandscharica bark and H. abyssinica bark and leaves showed minimum inhibitory activity against all tested bacteria, with minimal inhibitory concentrations ranging from 25-150 mg/mL. Ajuga remota and Amaranthus hybridus, which are lethal to brine shrimp nauplii, showed significantly lower antibacterial activity than those that were relatively non-toxic.

Multiplex-PCR serotyping of Listeria monocytogenes isolated from human clinical specimens

The genus Listeria is composed of six species of which Listeria monocytogenes is considered the single pathogenic species that causes listeriosis in humans. Of the 13 serovars of L. monocytogenes, 1/2a, 1/2b and 4b are responsible for the majority of clinical cases. The aim of this work was to detect L. monocytogenes in the cerebrospinal fluid sample of premature newborns and to characterize this sample using biotyping, serotyping and molecular typing. The results indicated the presence of L. monocytogenesin the clinical sample studied. Moreover, the isolate was identified as the 4b serovar that was characterized by the presence of a unique 691 bp band after analysis using the Multiplex-PCR technique. The results of repeated Multiplex-PCR and sequencing have indicated that the L. monocytogenes isolate was an atypical 4b serovar, which is the first time this finding has been reported.

Antibacterial and antifungal activity of extracts and exudates of the Amazonian medicinal tree Himatanthus articulatus (Vahl) Woodson (common name: sucuba)

Himatanthus articulatus (Vahl) Woodson is a tree found in the northern Amazon savannahs (common name: sucuba) that is used in local Amerindian medicine. Leaf, bark and branch wood methanol extracts, sequentially obtained hexane, ethyl acetate and methanol extracts and latex were evaluated for antifungal and antibacterial activities against American Type Culture Collection (ATCC) and local clinical strains using the disc diffusion method. Methanol extracts and latex inhibited Candida albicans, leaf methanol extracts inhibited Staphylococcus aureus and Bacillus subtilis and bark methanol extracts inhibited B. subtilis. Active extracts inhibited the ATCC and clinical strains. Polar antifungal and antibacterial principles in latex and extracts are thought to be responsible for the inhibition.

Survey of Leptospira spp in pampas deer (Ozotoceros bezoarticus) in the Pantanal wetlands of the state of Mato Grosso do Sul, Brazil by serology and polymerase chain reaction

This work reports a survey of Leptospira spp in pampas deer (Ozotoceros bezoarticus) in the Pantanal wetlands of the state of Mato Grosso do Sul, Brazil by serology and polymerase chain reaction (PCR). Seventy pampas deer were captured in the dry season and surveyed using PCR, microscopic agglutination test (MAT) (n = 51) and by both techniques (n = 47). PCR detected infections in two pampas deer and MAT detected infections in three. Through sequencing and phylogenetic analyses, the PCR-amplified fragment detected in deer was identified as Leptospira interrogans. Serovars Pomona and Butembo were detected using MAT and the highest titre was 200 for serovar Pomona. Epidemiological aspects of the findings are discussed.

TLR5-dependent immunogenicity of a recombinant fusion protein containing an immunodominant epitope of malarial circumsporozoite protein and the FliC flagellin of Salmonella Typhimurium

Recently, we described the improved immunogenicity of new malaria vaccine candidates based on the expression of fusion proteins containing immunodominant epitopes of merozoites and Salmonella enterica serovar Typhimurium flagellin (FliC) protein as an innate immune agonist. Here, we tested whether a similar strategy, based on an immunodominant B-cell epitope from malaria sporozoites, could also generate immunogenic fusion polypeptides. A recombinant His6-tagged FliC protein containing the C-terminal repeat regions of the VK210 variant of Plasmodium vivax circumsporozoite (CS) protein was constructed. This recombinant protein was successfully expressed in Escherichia coli as soluble protein and was purified by affinity to Ni-agarose beads followed by ion exchange chromatography. A monoclonal antibody specific for the CS protein of P. vivax sporozoites (VK210) was able to recognise the purified protein. C57BL/6 mice subcutaneously immunised with the recombinant fusion protein in the absence of any conventional adjuvant developed protein-specific systemic antibody responses. However, in mice genetically deficient in expression of TLR5, this immune response was extremely low. These results extend our previous observations concerning the immunogenicity of these recombinant fusion proteins and provide evidence that the main mechanism responsible for this immune activation involves interactions with TLR5, which has not previously been demonstrated for any recombinant FliC fusion protein.

A combined approach of VNTR and MLST analysis: improving molecular typing of Argentinean isolates of Leptospira interrogans

Leptospirosis is an emerging infectious disease that has been identified as both a human and animal health problem worldwide. Regular outbreaks associated with specific risk factors have been reported in Argentina. However, there are no available data concerning the genetic population level for this pathogen. Therefore, the aim of this work was to describe the genetic diversity of Leptospira interrogans through the application of two molecular typing strategies: variable number of tandem repeats (VNTR) and multilocus sequence typing (MLST). For this purpose, seven reference strains and 18 non-epidemiologically related isolates from diverse hosts and Argentinean regions were analysed. Among them, nine genotypes and seven sequence types (STs), including three unreported STs, were described using VNTR and MLST, respectively. eBURST analysis demonstrated that ST37 was the most frequent and founder genotype of a clonal complex (CCs) containing STN1 and STN3, suggesting the importance of studying the serovars belonging to this CC in Argentina. The data from maximum parsimony analysis, which combined both techniques, achieved intra-serovar discrimination, surmounted microscopic agglutination test discrepancies and increased the discriminatory power of each technique applied separately. This study is the first to combine both strategies for L. interrogans typing to generate a more comprehensive molecular genotyping of isolates from Argentina in a global context.

Increased hepcidin expression in multibacillary leprosy

Iron is essential for all organisms and its availability can control the growth of microorganisms; therefore, we examined the role of iron metabolism in multibacillary (MB) leprosy, focusing on the involvement of hepcidin. Erythrograms, iron metabolism parameters, pro-inflammatory cytokines and urinary hepcidin levels were evaluated in patients with MB and matched control subjects. Hepcidin expression in MB lesions was evaluated by quantitative polymerase chain reaction. The expression of ferroportin and hepcidin was evaluated by immunofluorescence in paucibacillary and MB lesions. Analysis of hepcidin protein levels in urine and of hepcidin mRNA and protein levels in leprosy lesions and skin biopsies from healthy control subjects showed elevated hepcidin levels in MB patients. Decreases in haematologic parameters and total iron binding capacity were observed in patients with MB leprosy. Moreover, interleukin-1 beta, ferritin, soluble transferrin receptor and soluble transferrin receptor/log ferritin index values were increased in leprosy patients. Hepcidin was elevated in lepromatous lesions, whereas ferroportin was more abundant in tuberculoid lesions. In addition, hepcidin and ferroportin were not colocalised in the biopsies from leprosy lesions. Anaemia was not commonly observed in patients with MB; however, the observed changes in haematologic parameters indicating altered iron metabolism appeared to result from a mixture of anaemia of inflammation and iron deficiency. Thus, iron sequestration inside host cells might play a role in leprosy by providing an optimal environment for the bacillus.

Mycobacterium leprae in six-banded (Euphractus sexcinctus) and nine-banded armadillos (Dasypus novemcinctus) in Northeast Brazil

Human beings are the main reservoir of the causative agent of leprosy, Mycobacterium leprae. In the Americas, nine-banded armadillos (Dasypus novemcinctus) also act as a reservoir for the bacillus. In the state of Ceará (CE), which is located in Northeast Brazil and is an endemic area of leprosy, there are several species of armadillos, including D. novemcinctus and Euphractus sexcinctus (six-banded armadillo). Contact between humans and armadillos occur mainly through hunting, cleaning, preparing, cooking and eating. This study identified M. leprae DNA in the two main species of armadillos found in Northeast Brazil. A total of 29 wild armadillos (27 D. novemcinctus and 2 E. sexcinctus) were captured in different environments of CE countryside. Samples from the ear, nose, liver and spleen from each of these animals were tested by a nested M. leprae-specific repetitive element polymerase chain reaction assay. The samples that tested positive were confirmed by DNA sequencing. M. leprae was detected in 21% (6/29) of the animals, including five D. novemcinctus and one E. sexcinctus. This is the first Brazilian study to identify the presence of a biomarker of M. leprae in wild armadillos (D. novemcinctus and E. sexcinctus) in a leprosy hyperendemic area where there is continuous contact between humans and armadillos.

Genotypes of pathogenic Leptospira spp isolated from rodents in Argentina

Leptospirosis is the most widespread zoonosis in the world and significant efforts have been made to determine and classify pathogenic Leptospira strains. This zoonosis is maintained in nature through chronic renal infections of carrier animals, with rodents and other small mammals serving as the most important reservoirs. Additionally, domestic animals, such as livestock and dogs, are significant sources of human infection. In this study, a multiple-locus variable-number tandem repeat analysis (MLVA) was applied to genotype 22 pathogenic Leptospira strains isolated from urban and periurban rodent populations from different regions of Argentina. Three MLVA profiles were identified in strains belonging to the species Leptospira interrogans (serovars Icterohaemorrhagiae and Canicola); one profile was observed in serovar Icterohaemorrhagiae and two MLVA profiles were observed in isolates of serovars Canicola and Portlandvere. All strains belonging to Leptospira borgpetersenii serovar Castellonis exhibited the same MLVA profile. Four different genotypes were isolated from urban populations of rodents, including both mice and rats and two different genotypes were isolated from periurban populations.

Molecular characterisation and disease severity of leptospirosis in Sri Lanka

Leptospirosis is a re-emerging zoonotic disease all over the world, important in tropical and subtropical areas. A majority of leptospirosis infected patients present as subclinical or mild disease while 5-10% may develop severe infection requiring hospitalisation and critical care. It is possible that several factors, such as the infecting serovar, level of leptospiraemia, host genetic factors and host immune response, may be important in predisposition towards severe disease. Different Leptospirastrains circulate in different geographical regions contributing to variable disease severity. Therefore, it is important to investigate the circulating strains at geographical locations during each outbreak for epidemiological studies and to support the clinical management of the patients. In this study immunochromatography, microscopic agglutination test and polymerase chain reaction were used to diagnose leptospirosis. Further restriction fragment length polymorphism and DNA sequencing methods were used to identify the circulating strains in two selected geographical regions of Sri Lanka.Leptospira interrogans, Leptospira borgpeterseniiandLeptospira kirschneristrains were identified to be circulating in western and southern provinces. L. interroganswas the predominant species circulating in western and southern provinces in 2013 and its presence was mainly associated with renal failure.

Widespread nasal carriage of Mycobacterium lepraeamong a healthy population in a hyperendemic region of northeastern Brazil

A case-control study was conducted to determine the presence ofMycobacterium leprae DNA in nasal secretions of leprosy cases and nonleprosy individuals in Fortaleza, Brazil. It included 185 cases identified by physicians at the Dona Libânia National Reference Centre for Sanitary Dermatology (CDERM). A control group (Co) (n = 136) was identified among individuals from CDERM not diagnosed as leprosy cases. To augment the spatial analysis of M. leprae specific repetitive element (RLEP) positive prevalence, an external group (EG) (n = 121), a convenience sample of healthy students, were included. Polymerase chain reaction for the RLEP sequence was conducted for all participants. Prevalence of RLEP positivity for cases and Co were 69.2% and 66.9%, respectively, significantly higher than for EG (28.1%), and reported elsewhere. Male sex, belonging to a lower socioeconomic status (D/E), history of a previous contact with a case and being older, were associated with being a leprosy case. Our geographical analysis demonstrated that the bacillus is widespread among the healthy population, with clusters of RLEP positive multibacillary cases concentrated in distinct areas of the city. Our results suggest that in endemic areas, as in Fortaleza, surveillance for both nonhousehold leprosy contacts and members of the general population living in cluster areas should be implemented.