The carriage population of Staphylococcus aureus from Mali is composed of a combination of pandemic clones and the divergent Panton-Valentine leukocidin-positive genotype ST152

Staphylococcus aureus is an important human pathogen, but it appears more commonly in asymptomatic colonization of the nasopharynx than in cases of invasive disease. Evidence concerning the global population structure of S. aureus is limited by the overrepresentation in the multilocus sequence testing database of disease isolates recovered from Western Europe, the Americas, Australia, and Japan. We address this by presenting data from the S. aureus carriage population in Mali, the first detailed characterization of asymptomatic carriage from an African population. These data confirm the pandemic spread of many of the common S. aureus clones in the carriage population. We also note the high frequency (approximately 24%) of a single divergent genotype, sequence type 152 (ST152), which has not previously been recovered from nasal carriage isolates but corresponds to a sporadic Panton-Valentine leukocidin (PVL)-positive, community-acquired methicillin-resistant S. aureus clone noted mostly in Central Europe. We show that 100% of the ST152 isolates recovered from nasal carriage samples in Mali are PVL positive and discuss implications relating to the emergence and spread of this virulent genotype.     

Quantification of Key Genes Steering the Microbial Nitrogen Cycle in the Rhizosphere of Sorghum Cultivars in Tropical Agroecosystems

The effect of agricultural management practices on geochemical cycles in moderate ecosystems is by far better understood than in semiarid regions, where fertilizer availability and climatic conditions are less favorable. We studied the impact of different fertilizer regimens in an agricultural long-term observatory in Burkina Faso at three different plant development stages (early leaf development, flowering, and senescence) of sorghum cultivars. Using real-time PCR, we investigated functional microbial communities involved in key processes of the nitrogen cycle (nitrogen fixation, ammonia oxidation, and denitrification) in the rhizosphere. The results indicate that fertilizer treatments and plant development stages combined with environmental factors affected the abundance of the targeted functional genes in the rhizosphere. While nitrogen-fixing populations dominated the investigated communities when organic fertilizers (manure and straw) were applied, their numbers were comparatively reduced in urea-treated plots. In contrast, ammonia-oxidizing bacteria (AOB) increased not only in absolute numbers but also in relation to the other bacterial groups investigated in the urea-amended plots. Ammonia-oxidizing archaea exhibited higher numbers compared to AOB independent of fertilizer application. Similarly, denitrifiers were also more abundant in the urea-treated plots. Our data imply as well that, more than in moderate regions, water availability might shape microbial communities in the rhizosphere, since low gene abundance data were obtained for all tested genes at the flowering stage, when water availability was very limited.Land degradation is one of the most serious threats to food production on the African continent. Soil erosion, nutrient depletion, low organic matter content, and unfavorable pH values are some of the reasons for a deficient soil fertility (30), mainly in Central African countries. Combined with high variability and irregular distribution of rainfall, these factors contribute to negative nutrient balances. For example, 4.4 million tons of nitrogen (N) are lost per year in African soils, but only 0.8 million tons are reapplied by fertilization (12, 34). Since nitrogen is a key nutrient determining the productivity of agroecosystems (7, 11, 43), it is of central importance to optimize the nitrogen balance in these countries, mainly by steering the genetic resources of soil microbes in a way that losses of applied nitrogen are minimized and biological nitrogen fixation is increased. The aim should be to obtain a highly efficient nitrogen turnover, with leaching of nitrate and losses of gaseous products such as nitrous oxide (N₂O) or dinitrogen (N₂) as low as possible.Despite the importance of this issue, not much data are available on microbial community structure and function related to the nitrogen cycle in agroecosystems of Central Africa, and scenarios from moderate climatic regions cannot simply be transferred to tropical agroecosystems. Furthermore, the few studies published thus far only investigated effects of agricultural management on a single process of the nitrogen turnover (18, 20, 32) and ignore the fact that nitrogen turnover is a network of closely interlinked processes.Therefore, we sought to investigate the effects of different fertilizer regimens on multiple transformation processes within the nitrogen cycle in agroecosystems from semiarid areas in Central Africa. We investigated nitrogen dynamics on a full-cycle approach, including the most important steps in well-aerated agricultural soils (nitrification, denitrification, and nitrogen fixation). We hypothesized that each fertilizer regimen results in typical abundance pattern of the functional populations. While in moderate agroecosystems the plant (respectively, the plant development stage and the plant performance) plays an important role in shaping microbial community structure and function in the rhizosphere, we further postulated that nitrogen turnover and the corresponding populations are also influenced by the availability of water in semiarid soils. To test these hypotheses on a molecular basis, we quantified bacterial genes encoding the nitrogenase reductase (nifH), ammonia monooxygenase (amoA), and nitrite reductase (nirK and nirS), as well as archaeal amoA genes, by real-time PCR and linked the data to soil properties and grain yield.     

The Pks13/FadD32 Crosstalk for the Biosynthesis of Mycolic Acids in Mycobacterium tuberculosis

The last steps of the biosynthesis of mycolic acids, essential and specific lipids of Mycobacterium tuberculosis and related bacteria, are catalyzed by proteins encoded by the fadD32-pks13-accD4 cluster. Here, we produced and purified an active form of the Pks13 polyketide synthase, with a phosphopantetheinyl (P-pant) arm at both positions Ser-55 and Ser-1266 of its two acyl carrier protein (ACP) domains. Combination of liquid chromatography-tandem mass spectrometry of protein tryptic digests and radiolabeling experiments showed that, in vitro, the enzyme specifically loads long-chain 2-carboxyacyl-CoA substrates onto the P-pant arm of its C-terminal ACP domain via the acyltransferase domain. The acyl-AMPs produced by the FadD32 enzyme are specifically transferred onto the ketosynthase domain after binding to the P-pant moiety of the N-terminal ACP domain of Pks13 (N-ACP_Pks13). Unexpectedly, however, the latter step requires the presence of active FadD32. Thus, the couple FadD32-(N-ACP_Pks13) composes the initiation module of the mycolic condensation system. Pks13 ultimately condenses the two loaded fatty acyl chains to produce α-alkyl β-ketoacids, the precursors of mycolic acids. The developed in vitro assay will constitute a strategic tool for antimycobacterial drug screening.Mycolic acids, α-branched and β-hydroxylated fatty acids of unusual chain length (C30-C90), are the hallmark of the Corynebacterineae suborder that includes the causative agents of tuberculosis (Mycobacterium tuberculosis) and leprosy (Mycobacterium leprae). Members of each genus biosynthesize mycolic acids of specific chain lengths, a feature used in taxonomy. For example, Corynebacterium holds the simplest prototypes (C32-C36), called “corynomycolic acids,” which result from an enzymatic condensation between two regular size fatty acids (C16–C18). In contrast, the longest mycolates (C60-C90) are the products of condensation between a very long meromycolic chain (C40-C60) and a shorter α-chain (C22-C26) (1). These so-called “eumycolic acids” are found in mycobacteria and display various structural features present on the meromycolic chain. Eumycolic acids are major and essential components of the mycobacterial envelope where they contribute to the formation of the outer membrane (2, 3) that plays a crucial role in the permeability of the envelope. They also impact on the pathogenicity of some mycobacterial species (4).The first in vitro mycolate biosynthesis assays have been developed using Corynebacterium cell-wall extracts in the presence of a radioactive precursor (5, 6) and have brought key information about this pathway. Yet, any attempt to fractionate these extracts to identify the proteins involved has ended in failure. Later, enzymes catalyzing the formation of the meromycolic chain and the introduction of functions have been discovered with the help of novel molecular biology tools (for review, see Ref. 1), culminating with the identification of the putative operon fadD32-pks13-accD4 that encodes enzymes implicated in the mycolic condensation step in both corynebacteria and mycobacteria (see Fig. 1) (7–9). AccD4, a putative carboxyltransferase, associates at least with the AccA3 subunit to form an acyl-CoA carboxylase (ACC)³ complex that most likely activates, through a C₂-carboxylation step, the extender unit to be condensed with the meromycolic chain (see Fig. 1). In Corynebacterium glutamicum, the carboxylase would metabolize a C16 substrate (8, 10), whereas in M. tuberculosis the purified complex AccA3-AccD4 was shown to carboxylate C24-C26 acyl-CoAs (11). Furthermore, FadD32, predicted to belong to a new class of long-chain acyl-AMP ligases (FAAL) (12), is most likely required for the activation of the meromycolic chain prior to the condensation reaction. At last, the cmrA gene controls the reduction of the β-keto function to yield the final mycolic motif (13) (see Fig. 1).Open in a separate windowFIGURE 1.Proposed scheme for the biosynthesis of mycolic acids. The asymmetrical carbons of the mycolic motif have a R,R configuration. R₁-CO, meromycolic chain; R₂, branch chain. In mycobacteria, R₁-CO = C40-C60 and R₂ = C20-C24; in corynebacteria, R₁-CO = C16-C18 and R₂ = C14-C16; X₁, unknown acceptor of the mycolic α-alkyl β-ketoacyl chains; X₂, unknown acceptor of the mycolic acyl chains.Although the enzymatic properties of the ACC complex have been well characterized (9, 11), those of Pks13 and FadD32 are poorly or not described. Pks13 is a type I polyketide synthase (PKS) made of a minimal module holding ketosynthase (KS), acyltransferase (AT), and acyl carrier protein (ACP) domains, and additional N-terminal ACP and C-terminal thioesterase domains (Fig. 1). Its ACP domains are naturally activated by the 4′-phosphopantetheinyl (P-pant) transferase PptT (14). The P-pant arm has the general function of carrying the substrate acyl chain via a thioester bond involving its terminal thiol group. In the present article we report the purification of a soluble activated form of the large Pks13 protein. For the first time, the loading mechanisms of both types of substrates on specific domains of the PKS were investigated. We describe a unique catalytic mechanism of the Pks13-FadD32 enzymatic couple and the development of an in vitro condensation assay that generates the formation of α-alkyl β-ketoacids, the precursors of mycolic acids.     

Characterization of halophiles isolated from solar salterns in Baja California, Mexico

Solar salterns are extreme hypersaline environments that are five to ten times saltier than seawater (150–300 g L⁻¹ salt concentration) and typically contain high numbers of halophiles adapted to tolerate such extreme hypersalinity. Thirty-five halophile cultures of both Bacteria and Archaea were isolated from the Exportadora de Sal saltworks in Guerrero Negro, Baja California, Mexico. 16S rRNA sequence analysis showed that these cultured isolates included members belonging to the Halorubrum, Haloarcula, Halomonas, Halovibrio, Salicola, and Salinibacter genera and what may represent a new archaeal genus. For the first time, metabolic substrate usage of halophile isolates was evaluated using the non-colorimetric BIOLOG Phenotype MicroArray™ plates. Unique carbon substrate usage profiles were observed, even for closely related Halorubrum species, with bacterial isolates using more substrates than archaeal cultures. Characterization of these isolates also included morphology and pigmentation analyses, as well as salinity tolerance over a range of 50–300 g L⁻¹ salt concentration. Salinity optima varied between 50 and 250 g L⁻¹ and doubling times varied between 1 and 12 h. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Safety and Immunogenicity of the Malaria Vaccine Candidate MSP3 Long Synthetic Peptide in 12–24 Months-Old Burkinabe Children

Background

A Phase Ia trial in European volunteers of the candidate vaccine merozoite surface protein 3 (MSP3), a Plasmodium falciparum blood stage membrane, showed that it induces biologically active antibodies able to achieve parasite killing in vitro, while a phase Ib trial in semi-immune adult volunteers in Burkina Faso confirmed that the vaccine was safe.The aim of this study was to assess the safety and immunogenicity of this vaccine candidate in children aged 12–24 months living in malaria endemic area of Burkina Faso.

Methods

The study was a double-blind, randomized, controlled, dose escalation phase Ib trial, designed to assess the safety, reactogenicity and immunogenicity of three doses of either 15 or 30 µg of MSP3-LSP adsorbed on aluminum hydroxide in 45 children 12 to 24 months of age randomized into three equal groups. Each group received 3 vaccine doses (on days 0, 28 and 56) of either 15 µg of MSP3-LSP, 30 µg of MSP3-LSP or of the Engerix B hepatitis B vaccine. Children were visited at home daily for the 6 days following each vaccination to solicit symptoms which might be related to vaccination. Serious adverse events occurring during the study period (1 year) were recorded. Antibody responses to MSP3-LSP were measured on days 0, 28, 56 and 84.

Results

All 45 enrolled children received three MSP3 vaccine doses. No serious adverse events were reported. Most of the adverse events reported were mild to moderate in severity. The only reported local symptoms with grade 3 severity were swelling and induration, with an apparently dose related response. All grade 3 adverse events resolved without any sequelae. Both MSP3 doses regimens were able to elicit high levels of anti-MSP3 specific IgG1 and IgG3 antibodies in the volunteers with very little or no increase in IgG2, IgG4 and IgM classes: i.e. vaccination induced predominantly the isotypes involved in the monocyte-dependent mechanism of P. falciparum parasite-killing.

Conclusion

Our results support the promise of MSP3-LSP as a malaria vaccine candidate, both in terms of tolerability and of immunogenicity. Further assessment of the efficacy of this vaccine is recommended.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00452088","term_id":"NCT00452088"}}NCT00452088     

Prevalence and Spatial Distribution of Entamoeba histolytica/dispar and Giardia lamblia among Schoolchildren in Agboville Area (C?te d'Ivoire)

Background

New efforts are being made to improve understanding of the epidemiology of the helminths and intensifying the control efforts against these parasites. In contrast, relatively few studies are being carried out in this direction for the intestinal protozoa. To contribute to a better comprehension of the epidemiology of the intestinal protozoa, prevalence, and spatial distribution of Entamoeba histolytica/dispar and Giardia lamblia, and their association with drinking water supplies, were determined in the Agboville department in southeast Côte d''Ivoire.

Methods/Findings

Stool samples were taken from more than 1,300 schoolchildren in the third year of primary education (CE1) from 30 primary schools and preserved in SAF (sodium acetate-acetic acid-formalin). The samples were analyzed by formalin-ether concentration. Then, a survey questionnaire addressed to schoolchildren and school directors was used to collect data on water supplies. Prevalence of E. histolytica/dispar and G. lamblia were, respectively, 18.8% and 13.9%. No particular focus zone was observed in the spatial distribution of the two species. Significant negative association was observed between use of tap water and high prevalence of E. histolytica/dispar infection (OR = 0.83, p = 0.01). High prevalence of G. lamblia infection was positively associated with use of ponds as the source of drinking water (OR = 1.28, p = 0.009).

Conclusion

These two species of pathogenic protozoa are present with substantial prevalence in this area of Côte d''Ivoire. Although their spatial distribution is not focused in any one place, determination of the population segments with the highest levels of infection will help to target the chemotherapeutic fight. To reinforce treatment with chemotherapeutic agents, tap water should be made available in all the localities of this area.     

Where Do We Go from Here? Prevalence of Trachoma Three Years after Stopping Mass Distribution of Antibiotics in the Regions of Kayes and Koulikoro,Mali

Objectives

A national survey in 1997 demonstrated that trachoma was endemic in Mali. Interventions to control trachoma including mass drug administration (MDA) with azithromycin were launched in the regions of Kayes and Koulikoro in 2003. MDA was discontinued after three annual rounds in 2006, and an impact survey conducted. We resurveyed all districts in Kayes and Koulikoro in 2009 to reassess trachoma prevalence and determine intervention objectives for the future. In this paper we present findings from both the 2006 and 2009 surveys.

Methods

Population-based cluster surveys were conducted in each of the nine districts in Koulikoro in 2006 and 2009, whilst in Kayes, four of seven districts in 2006 and all seven districts in 2009 were surveyed. Household members present were examined for clinical signs of trachoma.

Results

Overall, 29,179 persons from 2,528 compounds, in 260 clusters were examined in 2006 and 32,918 from 7,533 households in 320 clusters in 2009. The prevalence of TF in children aged 1–9 years in Kayes and Koulikoro was 3.9% (95%CI 2.9–5.0%, range by district 1.2–5.4%) and 2.7% (95%CI 2.3–3.1%, range by district 0.1–5.0%) respectively in 2006. In 2009 TF prevalence was 7.26% (95%CI 6.2–8.2%, range by district 2.5–15.4%) in Kayes and 8.19% (95%CI 7.3–9.1%, range by district 1.7–17.2%) in Koulikoro among children of the same age group. TT in adults 15 years of age and older was 2.37% (95%CI 1.66–3.07%, range by district 0.30–3.54%) in 2006 and 1.37% (95%CI 1.02–1.72%, range by district 0.37–1.87%) in 2009 in Kayes and 1.75% (95%CI 1.31–2.23%, range by district 1.06–2.49%) in 2006 and 1.08% (95%CI 0.86–1.30%, range by district 0.34–1.78%) in 2009 in Koulikoro.

Conclusions

Using WHO guidelines for decision making, four districts, Bafoulabe in Kayes Region; and Banamba, Kolokani and Koulikoro in Koulikoro Region, still meet criteria for district-wide implementation of the full SAFE strategy as TF in children exceeds 10%. A community-by-community approach to trachoma control may now be required in the other twelve districts. Trichiasis surgery provision remains a need in all districts and should be enhanced in six districts in Kayes and five in Koulikoro where the prevalence exceeded 1.0% in adults. Since 1997 great progress has been observed in the fight against blinding trachoma; however, greater effort is required to meet the elimination target of 2015.     

Ectomycorrhizal fungi are shared between seedlings and adults in a monodominant Gilbertiodendron dewevrei rain forest in Cameroon

Ectomycorrhizal networks may facilitate the establishment and survival of seedlings regenerating under the canopies of tropical forests and are often invoked as a potential contributor to monodominance. We identified ectomycorrhizal fungi in a monodominant Gilbertiodendron dewevrei (Fabaceae) rain forest in Cameroon, using sporocarps and ectomycorrhizae of three age categories (seedlings, intermediate trees, and large trees) and tentatively revealed nutrient transfer through ectomycorrhizal networks by measuring spontaneous isotopic (¹³C and ¹⁵N) abundances in seedlings. Sporocarp surveys revealed fewer ectomycorrhizal fungal taxa (59 species from 1030 sporocarps) than molecular barcoding of ectomycorrhizal roots (75 operational taxonomic units from 828 ectomycorrhizae). Our observations suggest that ectomycorrhizal fungal diversity is similar to that in other mixed tropical forests and provide the first report of the Tuber‐Helvella lineage in a tropical forest. Despite some differences, all age categories of G. dewevrei had overlapping ectomycorrhizal fungal communities, with families belonging to Thelephoraceae, Russulaceae, Sebacinaceae, Boletaceae, and Clavulinaceae. Of the 49 operational taxonomic units shared by the three age categories (65.3% of the ectomycorrhizal fungal community), 19 were the most abundant on root tips of all categories (38.7% of the shared taxa), supporting the likelihood of ectomycorrhizal networks. However, we obtained no evidence for nutrient transfer from trees to seedlings. We discuss the composition of the ectomycorrhizal fungal community among the G. dewevrei age categories and the possible role of common ectomycorrhizal networks in this rain forest.