Effects of the spatial repellent metofluthrin on landing rates of outdoor biting anophelines in Cambodia,Southeast Asia

The emergence of artemisinin‐resistant malaria in Southeast Asia is a major problem. The fact that many people become infected with malaria when they are outside has prompted the development of ‘spatial’ rather than topical repellents. The respective effects of one or four slow‐release emanators of metofluthrin, a pyrethroid, were tested in Pailin, Pursat and Koh Kong, Cambodia. Numbers of mosquitoes counted in outdoor landing catches when one or four emanators were suspended close to the collector were compared with control collections. In Pailin, the effects of emanators on catches in Furvela tent traps and Centers for Disease Control (CDC) light traps suspended underneath houses were also investigated. Rate ratios were used to determine differences. A total of 29 255 mosquitoes were collected over 2934 h of landing collections, 87 nights of tent trapping and 81 nights of light trap capture. In Pailin, landing rates were reduced by 48% by a single emanator and by 67% by four emanators (P < 0.001). Similar reductions were observed in the number of mosquitoes collected in tent traps and the number of anophelines only collected in light traps. Results were similar in Pursat, but, for unknown reasons, those in Koh Kong showed no difference between control and metofluthrin collections (P > 0.05). These findings suggest that although the product can produce a significant effect, it requires further improvement.     

Focused Screening and Treatment (FSAT): A PCR-Based Strategy to Detect Malaria Parasite Carriers and Contain Drug Resistant P. falciparum, Pailin, Cambodia

Recent studies have shown that Plasmodium falciparum malaria parasites in Pailin province, along the border between Thailand and Cambodia, have become resistant to artemisinin derivatives. To better define the epidemiology of P. falciparum populations and to assess the risk of the possible spread of these parasites outside Pailin, a new epidemiological tool named “Focused Screening and Treatment” (FSAT), based on active molecular detection of asymptomatic parasite carriers was introduced in 2010. Cross-sectional malariometric surveys using PCR were carried out in 20 out of 109 villages in Pailin province. Individuals detected as P. falciparum carriers were treated with atovaquone-proguanil combination plus a single dose of primaquine if the patient was non-G6PD deficient. Interviews were conducted to elicit history of cross-border travel that might contribute to the spread of artemisinin-resistant parasites. After directly observed treatment, patients were followed up and re-examined on day 7 and day 28. Among 6931 individuals screened, prevalence of P. falciparum carriers was less than 1%, of whom 96% were asymptomatic. Only 1.6% of the individuals had a travel history or plans to go outside Cambodia, with none of those tested being positive for P. falciparum. Retrospective analysis, using 2010 routine surveillance data, showed significant differences in the prevalence of asymptomatic carriers discovered by FSAT between villages classified as “high risk” and “low risk” based on malaria incidence data. All positive individuals treated and followed-up until day 28 were cured. No mutant-type allele related to atovaquone resistance was found. FSAT is a potentially useful tool to detect, treat and track clusters of asymptomatic carriers of P. falciparum along with providing valuable epidemiological information regarding cross-border movements of potential malaria parasite carriers and parasite gene flow.     

Photoreactivating capacity in Clostridium butyricum and Clostridium acetobutylicum

No difference in survival was observed when u.v.-irradiated clostridial cells were subsequently incubated in the dark or exposed to photoreactivating light. This suggests that photoreactivation does not occur in Clostridium butyricum and in Clostridium acetohutylicum.     

Plasmids in Clostridium botulinum and related Clostridium species.

Toxigenic Clostridium botulinum and nontoxigenic C. sporogenes, C. subterminale, and C. botulinum-like organisms from a variety of sources were screened for plasmids. Of the 68 toxigenic C. botulinum isolates, 56% carried one or more plasmids, ranging in mass from 2.1 to 81 megadaltons. Within individual groups (based on the type of neurotoxin produced), many strains showed identical plasmid banding patterns on agarose gels. Of the 15 nontoxigenic strains tested, 40% also carried one or more plasmids ranging from 1.7 to 25.0 megadaltons, with both unique and common banding patterns represented. A total of 67 plasmids from both toxigenic and nontoxigenic strains were detected. At this time, no phenotypic functions have been assessed for these plasmids, and they must therefore be considered cryptic. A variety of lysing and extraction techniques were necessary to detect plasmids in the different C. botulinum groups.     

Glucose metabolism in Clostridium sporogenes and Clostridium sticklandii bacteria

Clostridium sporogenes 272 has a high rate of glucose fermentation. Its cell-free extract contains all glycolytic enzymes catalysing glucose degradation to pyruvate and shows the phosphoroclastic activity. C. sticklandii CSG has a low rate of glucose fermentation. Hence, the activity of the following enzymes is lower in this organism comparing to C. sporogenes: phosphohexoisomerase (EC 5.3.1.9), phosphofructokinase (EC 2.7.1.11), aldolase (EC 4.1.2.13), triosephosphate isomerase (EC 5.3.1.1) and glyceraldehyde phosphate dehydrogenase (EC 1.2.1.12). Moreover, it is possible that the system of glucose transport into the cell is damaged in C. sticklandii.     

Restriction endonuclease activity in Clostridium thermocellum and Clostridium thermosaccharolyticum

 Clostridium thermocellum cell extracts exhibit specific endonuclease activity with very little non-specific exonuclease activity at 55°C. The Dam methylation system of Escherichia coli offers complete protection from digestion by C. thermocellum ATCC 27405 cell extracts for all DNA tested (totaling >100 kb, insuring that most potential restriction sequences have been exposed). Based on both the Dam recognition sequence and the similarity of cell extract and MboI DNA digests, the C. thermocellum restriction enzyme recognition sequence appears to be 5′ GATC 3′. Cell extracts made from a second thermophile, C. thermosaccharolyticum ATCC 31960 do not exhibit specific endonuclease activity under the conditions tested. Genomic DNA from C. thermocellum exhibits a Dam⁺ phenotype while genomic DNA from C. thermosaccharolyticum exhibits a Dam^- phenotype. Received: 10 March 1995/Received revision: 4 September 1995/Accepted: 13 September 1995     

Carbon Monoxide Oxidation by Clostridium thermoaceticum and Clostridium formicoaceticum

Cultures of Clostridium formicoaceticum and C. thermoaceticum growing on fructose and glucose, respectively, were shown to rapidly oxidize CO to CO₂. Rates up to 0.4 μmol min⁻¹ mg of wet cells⁻¹ were observed. Carbon monoxide oxidation by cell suspensions was found (i) to be dependent on pyruvate, (ii) to be inhibited by alkyl halides and arsenate, and (iii) to stimulate CO₂ reduction to acetate. Cell extracts catalyzed the oxidation of carbon monoxide with methyl viologen at specific rates up to 10 μmol min⁻¹ mg of protein⁻¹ (35°C, pH 7.2). Nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate and ferredoxin from C. pasteurianum were ineffective as electron acceptors. The catalytic mechanism of carbon monoxide oxidation was “ping-pong,” indicating that the enzyme catalyzing carbon monoxide oxidation can be present in an oxidized and a reduced form. The oxidized form was shown to react reversibly with cyanide, and the reduced form was shown to react reversibly with alkyl halides: cyanide inactivated the enzyme only in the absence of carbon monoxide, and alkyl halides inactivated it only in the presence of carbon monoxide. Extracts inactivated by alkyl halides were reactivated by photolysis. The findings are interpreted to indicate that carbon monoxide oxidation in the two bacteria is catalyzed by a corrinoid enzyme and that in vivo the reaction is coupled with the reduction of CO₂ to acetate. Cultures of C. acidi-urici and C. cylindrosporum growing on hypoxanthine were found not to oxidize CO, indicating that clostridia mediating a corrinoid-independent total synthesis of acetate from CO₂ do not possess a CO-oxidizing system.     

New materials of Cisuralian (early Permian) radiolarians from western Cambodia: Paleobiogeographic implications in the Paleotethys

Radiolarians, including the new species Entactinia pailinensis Ito n. sp., were obtained from the Pailin area, western Cambodia. The radiolarians indicated an age around the Asselian–Sakmarian of the Cisuralian (early Permian). The new species is characterized by an evenly-sized-porous exosphere composed of a polygonal outer porous plate and circular inner porous plate with some radial outer spines. A previous study concluded that Entactinia Foreman rapidly expanded in the early–middle Cisuralian. Our discovery of this new species adds further occurrence data of Entactinia in the early–middle Cisuralian. In addition, another previous study highlighted the uneven distribution of Pseudotormentus De Wever and Caridroit compared with the wider distribution of Quadriremis Nazarov and Ormiston through the Permian, in particular the Guadalupian (middle Permian). Our results combined with those of previous studies indicate that the uneven distribution might extend back to the Sakmarian of the Cisuralian at least.     

Taxonomic relationships among Clostridium novyi Types A and B, Clostridium haemolyticum and Clostridium botulinum type C

The present study was undertaken to examine the genetic relationships among the closely related species, Clostridium novyi types A and B, C. haemolyticum and C. botulinum type C. These species were tested for DNA-DNA homology and thermostability of DNA duplexes and sorted into three genetically related groups: I, C. novyi type A; II, C. novyi type B, C. haemolyticum and one C. botulinum type C strain (Stockholm); III, the remaining C. botulinum type C strains. A few biochemical criteria corresponding to the genetic differences were recommended to differentiate each group. These studies imply that C. haemolyticum might be considered as C. novyi type D and that there are two genetically different groups in C. botulinum type C.     

Presence of Cytochrome and Menaquinone in Clostridium formicoaceticum and Clostridium thermoaceticum

Cytochrome b and menaquinone have been demonstrated in the homoacetate-fermenting Clostridium formicoaceticum and Clostridium thermoaceticum.     

Nitrogen-fixation genes and nitrogenase activity in Clostridium acetobutylicum and Clostridium beijerinckii

Several solvent-producing clostridia, including Clostridium acetobutylicum and C. beijerinckii, were previously shown to be nitrogen-fixing organisms based on the incorporation of ¹⁵N₂ into cellular material. The key nitrogen-fixation (nif) genes, including nifH, nifD, and nifK for nitrogenase component proteins as well as nifE, nifN, nifB and nifV for synthesis of the iron–molybdenum cofactor (FeMoco) of nitrogenase, have now been identified in C. acetobutylicum or C. beijerinckii or both. The organization of these genes is similar to the distinctive pattern that was first observed in Clostridium pasteurianum, with the nifN and nifB genes fused into the nifN-B gene and with the nifV gene split into the nifVω and nifVα genes. The corresponding nif genes of these three clostridial species are highly related to each other. However, in the two solvent-producing clostridia, the nifH and nifD genes are interspersed by two glnB-like genes, which are absent in the corresponding region in C. pasteurianum. However, the nifN-B and nifVω genes of C. pasteurianum are interspersed by the putative modA and modB genes (for molybdate transport), which are absent in the corresponding region in C. acetobutylicum. C. acetobutylicum and C. beijerinckii grew well under nitrogen-fixing conditions, and the acetylene-reducing activity of nitrogenase was measured in the two species. Acetone, butanol, and isopropanol production occurred in nitrogen-fixing cultures, but the peak of nitrogen-fixing activity preceded the active solventogenic phase. Journal of Industrial Microbiology & Biotechnology (2001) 27, 281–286. Received 02 September 2000/ Accepted in revised form 22 November 2000     

Modified plasmid isolation method for Clostridium perfringens and Clostridium absonum

A rapid plasmid isolation procedure for Clostridium perfringens and C. absonum is described. The ratio of culture volume to lysis buffer volume was found to be crucial for efficient plasmid isolation. The method can be scaled up, without difficulty, for large-scale plasmid preparation.     

Differential amylosaccharide metabolism of Clostridium thermosulfurogenes and Clostridium thermohydrosulfuricum.

Clostridium thermosulfurogenes displayed faster growth on either glucose, maltose, or starch than Clostridium thermohydrosulfuricum. Both species grew faster on glucose than on starch or maltose. The fermentation end product ratios were altered based on higher ethanol and lactate yields on starch than on glucose. In C. thermohydrosulfuricum, glucoamylase, pullulanase, and maltase were mainly responsible for conversion of starch and maltose into glucose, which was accumulated by a putative glucose permease. In C. thermosulfurogenes, beta-amylase was primarily responsible for degradation of starch to maltose, which was accumulated by a putative maltose permease and then hydrolyzed by glucoamylase. Regardless of the growth substrate, the rates of glucose, maltose, and starch transformation were higher in C. thermosulfurogenes than in C. thermohydrosulfuricum. Both species had a functional Embden-Meyerhof glycolytic pathway and displayed the following catabolic activities: ferredoxin-linked pyruvate dehydrogenase, acetate kinase, NAD(P)-ethanol dehydrogenase, NAD(P)-ferredoxin oxidoreductase, hydrogenase, and fructose-1,6-diphosphate-activated lactate dehydrogenase. Ferredoxin-NAD reductase activity was higher in C. thermohydrosulfuricum than NADH-ferredoxin oxidase activity, but the former activity was not detectable in C. thermosulfurogenes. Both NAD- and NADP-linked ethanol dehydrogenases were unidirectional in C. thermosulfurogenes but reversible in C. thermohydrosulfuricum. The ratio of hydrogen-producing hydrogenase to hydrogen-consuming hydrogenase was higher in C. thermosulfurogenes. Two biochemical models are proposed to explain the differential saccharide metabolism on the basis of species enzyme differences in relation to specific growth substrates.     

Differentiation Between Clostridium sordellii and Clostridium bifermentans by Gas Chromatography

Clostridium bifermentans can be differentiated from Clostridium sordellii by gas chromatography on the basis of amines detected after growth for 6 hr in cookedmeat medium. Products detected after exposure of resting cells to amino acids gave evidence for the probable origin of the amines.     

Differentiation of Clostridium difficile,Clostridium bifermentans,Clostridium sordellii,and Clostridium perfringens from Diarrheal Stool by API ZYM and API LRA Oxidase Test

A simple, rapid and reliable outline for identification of clostridia isolates from human infections was developed. It consists of a combination of API ZYM and API LRA Oxidase tests. The enzymatic activities were performed with strains sub-cultured onto carbohydrate-free medium (Columbia blood agar). Fifty-five strains of Clostridium difficile, C. bifermentans, C. sordellii, and C. perfringens from clinical specimens and eight reference standard strains representing different species of the same genus were analyzed. The accuracy of the new method was evaluated by comparison with the results obtained by DNA/DNA analysis.