Spatial distribution of triatomines in domiciles of an urban area of the Brazilian Southeast Region

Reports of triatomine infestation in urban areas have increased. We analysed the spatial distribution of infestation by triatomines in the urban area of Diamantina, in the state of Minas Gerais, Brazil. Triatomines were obtained by community-based entomological surveillance. Spatial patterns of infestation were analysed by Ripley’s K function and Kernel density estimator. Normalised difference vegetation index (NDVI) and land cover derived from satellite imagery were compared between infested and uninfested areas. A total of 140 adults of four species were captured (100 Triatoma vitticeps, 25Panstrongylus geniculatus, 8 Panstrongylus megistus, and 7 Triatoma arthurneivai specimens). In total, 87.9% were captured within domiciles. Infection by trypanosomes was observed in 19.6% of 107 examined insects. The spatial distributions ofT. vitticeps, P. geniculatus, T. arthurneivai, and trypanosome-positive triatomines were clustered, occurring mainly in peripheral areas. NDVI values were statistically higher in areas infested by T. vitticeps and P. geniculatus. Buildings infested by these species were located closer to open fields, whereas infestations of P. megistus andT. arthurneivai were closer to bare soil. Human occupation and modification of natural areas may be involved in triatomine invasion, exposing the population to these vectors.     

Analysis of the structure of bacteria communities and detection of resistance genes of quinolones from pharmaceutical wastewater

The aim of the present study was to investigate the distribution of bacteria and detect the presence of quinolone resistance gene (qnrA) and integrons (intI1, intI2) in a habitat polluted by pharmaceutical sewage. The bacteria were isolated by nutrient agar and nutrient broth from waste water and sludge collected from the sewage outfall of a pharmaceutical factory. The bacteria were identified by Gram staining and biochemical tests, and the bacterial community diversity was analyzed by Shannon–Wiener diversity index (H), Pielou evenness index (J) and Simpson’s diversity index (D). The occurrence of qnrA and integrons (intI1, intI2) were detected by Real-time PCR assays. The results showed that 90 strains were isolated from water samples and sludge samples including 22 genera and 26 species. Types of bacteria in water samples contained 18 genera and 20 species, while 13 genera and 14 species were detected in sludge samples. Fifty-five Enterobacteriaceae isolates (61.11 %, 55 of 90) were the predominant bacteria in water and sludge samples. Bacterial species richness and evenness in water samples were higher than in sludge samples. The resistance genes of qnrA and integrons (intI1, intI2) with the total DNA and single isolate plasmid DNA were detected. There were a variety of bacterial species and the presence of qnrA and integrons (intI1, intI2) genes in pharmaceutical wastewater habitats, in which Enterobacteriaceae strains were the dominant bacteria. These results suggested that pharmaceutical wastewater had potential risks to public health.     

Differential Rates of Digestion of Bacteria by Freshwater and Marine Phagotrophic Protozoa

Differential decreases over time of two bacterial species, Escherichia coli and Enterococcus faecalis, in a freshwater and a marine ecosystem were observed and explained by a differential rate of digestion of these bacteria by phagotrophic flagellates and ciliates. For this purpose, fluorescence-labeled bacteria (FLB) were used and prepared from the two species cited above. The number of FLB was observed for 5 days in fresh and marine waters in the presence or absence (0.2-μm-pore-size-filtered water) of natural microbiota. These experiments showed a longer persistence of Enterococcus faecalis FLB as opposed to Escherichia coli FLB in the presence of natural microbiota. Removal of FLB was due to protozoan grazing because no decrease of FLB number was observed in the absence of natural microbiota. In short-term (about 40 min) ingestion experiments, we found similar clearance rates of Escherichia coli and Enterococcus faecalis FLB by assemblages of flagellates from the freshwater and the marine ecosystem and by cultured assemblages of ciliates from the marine ecosystem. Clearance rates of Enterococcus faecalis FLB were greater than those of Escherichia coli FLB for assemblages of ciliates from the freshwater ecosystem. Comparison of rates of ingestion and digestion of FLB by protozoa showed that Escherichia coli FLB were digested and ingested at similar rates. However, Enterococcus faecalis FLB were digested slower than they were ingested. These results suggest that a longer persistence of Enterococcus faecalis as opposed to Escherichia coli can be explained by a differential digestion by flagellates and ciliates in aquatic ecosystems. Moreover, rates of ingestion and digestion were strongly correlated for both FLB types.     

The effect of parasitism by the mermithid Romanomermis culicivorax on the dry weight and hemolymph soluble protein content of three species of mosquitoes

The dry weight, hemolymph soluble protein composition, and content of three species of mosquitoes, Culex pipiens, Aedes taeniorhynchus, and Anopheles quadrimaculatus were examined to determine the effects of parasitism by the mermithid nematode Romanomermis culicivorax. The dry weights of infected fourth-stage larvae of all three species were significantly lower than controls. The differences in weight found between infected early and late C. pipiens and A. quadrimaculatus larvae were attributed to the weight of the parasite itself. This difference was not noticeable in A. taeniorhynchus larvae. Hemolymph proteins were severely depleted in all three mosquito species during parasitism by R. culicivorax. Analysis of protein composition by PAGE showed that these depletions were accompanied by a reduction in the number of proteins. Differences between protein composition concentrations were evident between early and late fourthstage control larvae of C. pipiens and A. quadrimaculatus. The concentration of some low-molecular-weight proteins (below 68,000) remained constant between infected and control samples of all three mosquito species.     

Detection of plasmid-mediated quinolone resistance in clinical isolates of Enterobacteriaceae strains in Hamadan,West of Iran

Plasmid mediated quinolone resistance (PMQR) determinants have arisen as a significant concern in recent years. The aim of this study was screening of resistant-clinical isolates to fluoroquinolone antibiotics and detection of qnr and aac(6′)-Ib-cr genes.For this purpose we collected 100 fluoroquinolone-resistant Enterobacteriaceae which were from 3 hospitals in Hamadan, west provinces of Iran, between October 2012 and June 2013. The all samples were identified by biochemical tests and confirmed by PCR method. Antimicrobial susceptibility to 14 antimicrobial agents including levofloxacin and ciprofloxacin were determined by disk diffusion methods and ciprofloxacin MIC was obtained by broth microdilution method as Clinical Laboratory Standards Institute (CLSI) recommendations. The isolates were screened for the presence of qnrA, qnrB, qnrS and aac(6′)-Ib-cr genes using PCR assay. Among the screened isolates, 64 strains (64%) of Escherichia coli, 23 strains (23%) of Klebsiella pneumoniae, 13 strains (13%) of Proteus mirabilis were collected as quinolone-resistant isolates. out of 100 isolates, two (2%) were positive for qnrS, seventeen (17%) isolates were positive for qnrB and we did not find qnrA gene in any of the isolates. There were also 32 positive isolates for aac(6′)-Ib-cr determinant. We described the prevalence of qnr and aac(6′)-Ib-cr genes in fluoroquinolone-resistant Enterobacteriaceae in Hamadan city. The carriage rate of multidrug-resistant Enterobacteriaceae in healthy people in Hamadan City is extremely high. Moreover, genes encoding transferable quinolones, in particular aac(6′)-Ib-cr, are highly prevalent in these strains.     

Taxonomic significance of the distribution of constituents of leaf cuticular waxes of Croton species (Euphorbiaceae)

Foliar epicuticular waxes of specimens of 13 Croton species native in Brazil were extracted. The fractions containing alkanes and primary alcohols were isolated by preparative thin layer chromatography. Derivatized n-primary alcohols were identified by gas chromatography (GC) coupled with mass spectrometry and n-alkanes by GC and comparison with known standards. Relative abundances were estimated by GC coupled with flame ionization detector. The distribution of constituents of both classes was analyzed by cluster analysis, using the UPGMA method and Euclidean distances. The chemical affinities among species were compared with published data of molecular phylogenetic relationships. The distribution of n-alkanes and primary alcohols were shown to be useful markers of Croton species. Primary alcohols were more consistent than n-alkanes for species fingerprinting.     

Screening of Probiotic Activities of Forty-Seven Strains of Lactobacillus spp. by In Vitro Techniques and Evaluation of the Colonization Ability of Five Selected Strains in Humans

The probiotic potential of 47 selected strains of Lactobacillus spp. was investigated. The strains were examined for resistance to pH 2.5 and 0.3% oxgall, adhesion to Caco-2 cells, and antimicrobial activities against enteric pathogenic bacteria in model systems. From the results obtained in vitro, five strains, Lactobacillus rhamnosus 19070-2, L. reuteri DSM 12246, L. rhamnosus LGG, L. delbrueckii subsp. lactis CHCC 2329, and L. casei subsp. alactus CHCC 3137, were selected for in vivo studies. The daily consumption by 12 healthy volunteers of two doses of 10¹⁰ freeze-dried bacteria of the selected strains for 18 days was followed by a washout period of 17 days. Fecal samples were taken at days 0 and 18 and during the washout period at days 5 and 11. Lactobacillus isolates were initially identified by API 50CHL and internal transcribed spacer PCR, and their identities were confirmed by restriction enzyme analysis in combination with pulsed-field gel electrophoresis. Among the tested strains, L. rhamnosus 19070-2, L. reuteri DSM 12246, and L. rhamnosus LGG were identified most frequently in fecal samples; they were found in 10, 8, and 7 of the 12 samples tested during the intervention period, respectively, whereas reisolations were less frequent in the washout period. The bacteria were reisolated in concentrations from 10⁵ to 10⁸ cells/g of feces. Survival and reisolation of the bacteria in vivo appeared to be linked to pH tolerance, adhesion, and antimicrobial properties in vitro.     

rpoD Gene Pyrosequencing for the Assessment of Pseudomonas Diversity in a Water Sample from the Woluwe River

A water sample from a noncontaminated site at the source of the Woluwe River (Belgium) was analyzed by culture-dependent and -independent methods. Pseudomonas isolates were identified by sequencing and analysis of the rpoD gene. Culture-independent methods consisted of cloning and pyrosequencing of a Pseudomonas rpoD amplicon from total DNA extracted from the same sample and amplified with selective rpoD gene primers. Among a total of 14,540 reads, 6,228 corresponded to Pseudomonas rpoD gene sequences by a BLAST analysis in the NCBI database. The selection criteria for the reads were sequences longer than 400 bp, an average Q₄₀ value greater than 25, and >85% identity with a Pseudomonas species. Of the 6,228 Pseudomonas rpoD sequences, 5,345 sequences met the established criteria for selection. Sequences were clustered by phylogenetic analysis and by use of the QIIME software package. Representative sequences of each cluster were assigned by BLAST analysis to a known Pseudomonas species when the identity with the type strain was greater than or equal to 96%. Twenty-six species distributed among 12 phylogenetic groups or subgroups within the genus were detected by pyrosequencing. Pseudomonas stutzeri, P. moraviensis, and P. simiae were the only cultured species not detected by pyrosequencing. The predominant phylogenetic group within the Pseudomonas genus was the P. fluorescens group, as determined by culture-dependent and -independent analyses. In all analyses, a high number of putative novel phylospecies was found: 10 were identified in the cultured strains and 246 were detected by pyrosequencing, indicating that the diversity of Pseudomonas species has not been fully described.     

Effects of Meloidogyne spp. and Rhizoctonia solani on the Growth of Grapevine Rootings

A disease complex involving Meloidogyne incognita and Rhizoctonia solani was associated with stunting of grapevines in a field nursery. Nematode reproduction was occurring on both susceptible and resistant cultivars, and pot experiments were conducted to determine the virulence of this M. incognita population, and of M. javanica and M. hapla populations, to V. vinifera cv. Colombard (susceptible) and to V. champinii cv. Ramsey (regarded locally as highly resistant). The virulence of R. solani isolates obtained from roots of diseased grapevines also was determined both alone and in combination with M. incognita. Ramsey was susceptible to M. incognita (reproduction ratio 9.8 to 18.4 in a shadehouse and heated glasshouse, respectively) but was resistant to M. javanica and M. hapla. Colombard was susceptible to M. incognita (reproduction ratio 24.3 and 41.3, respectively) and M. javanica. Shoot growth was suppressed (by 35%) by M. incognita and, to a lesser extent, by M. hapla. Colombard roots were more severely galled than Ramsey roots by all three species, and nematode reproduction was higher on Colombard. Isolates of R. solani assigned to putative anastomosis groups 2-1 and 4, and an unidentified isolate, colonized and induced rotting of grapevine roots. Ramsey was more susceptible to root rotting than Colombard. Shoot growth was inhibited by up to 15% by several AG 4 isolates and by 20% by the AG 2-1 isolate. AG 4 isolates varied in their virulence. Root rotting was higher when grapevines were inoculated with both M. incognita and R. solani and was highest when nematode inoculation preceded the fungus. Shoot weights were lower when vines were inoculated with the nematode 13 days before the fungus compared with inoculation with both the nematode and the fungus on the same day. It was concluded that both the M. incognita population and some R. solani isolates were virulent against both Colombard and Ramsey, and that measures to prevent spread in nursery stock were therefore important.     

Community Composition of a Hypersaline Endoevaporitic Microbial Mat

A hypersaline, endoevaporitic microbial community in Eilat, Israel, was studied by microscopy and by PCR amplification of genes for 16S rRNA from different layers. In terms of biomass, the oxygenic layers of the community were dominated by Cyanobacteria of the Halothece, Spirulina, and Phormidium types, but cell counts (based on 4′,6′-diamidino-2-phenylindole staining) and molecular surveys (clone libraries of PCR-amplified genes for 16S rRNA) showed that oxygenic phototrophs were outnumbered by the other constituents of the community, including chemotrophs and anoxygenic phototrophs. Bacterial clone libraries were dominated by phylotypes affiliated with the Bacteroidetes group and both photo- and chemotrophic groups of α-proteobacteria. Green filaments related to the Chloroflexi were less abundant than reported from hypersaline microbial mats growing at lower salinities and were only detected in the deepest part of the anoxygenic phototrophic zone. Also detected were nonphototrophic γ- and δ-proteobacteria, Planctomycetes, the TM6 group, Firmicutes, and Spirochetes. Several of the phylotypes showed a distinct vertical distribution in the crust, suggesting specific adaptations to the presence or absence of oxygen and light. Archaea were less abundant than Bacteria, their diversity was lower, and the community was less stratified. Detected archaeal groups included organisms affiliated with the Methanosarcinales, the Halobacteriales, and uncultured groups of Euryarchaeota.     

Microbial Diversity of Bovine Mastitic Milk as Described by Pyrosequencing of Metagenomic 16s rDNA

Dairy cow mastitis is an important disease in the dairy industry. Different microbial species have been identified as causative agents in mastitis, and are traditionally diagnosed by bacterial culture. The objective of this study was to use metagenomic pyrosequencing of bacterial 16S rRNA genes to investigate bacterial DNA diversity in milk samples of mastitic and healthy dairy cows and compare the results with those obtained by classical bacterial culture. One hundred and thirty-six milk samples were collected from cows showing signs of mastitis and used for microbiological culture. Additionally, 20 milk samples were collected from healthy quarters. Bacterial DNA was isolated from the same milk samples and the 16S rRNA genes were individually amplified and pyrosequenced. Discriminant analysis showed that the groups of samples that were most clearly different from the rest and thus easily discriminated were the normal milk samples from healthy cows and those characterised by culture as Trueperella pyogenes and Streptococcus spp. The mastitis pathogens identified by culture were generally among the most frequent organisms detected by pyrosequencing, and in some cases (Escherichia coli, Klebsiella spp. and Streptococcus uberis mastitis) the single most prevalent microorganism. Trueperella pyogenes sequences were the second most prevalent sequences in mastitis cases diagnosed as Trueperella pyogenes by culture, Streptococcus dysgalactiae sequences were the second most prevalent sequences in mastitis cases diagnosed as Streptococcus dysgalactiae by culture, and Staphyloccocus aureus sequences were the third most prevalent in mastitis cases diagnosed as Staphylococcus aureus by culture. In samples that were aerobic culture negative, pyrosequencing identified DNA of bacteria that are known to cause mastitis, DNA of bacteria that are known pathogens but have so far not been associated with mastitis, and DNA of bacteria that are currently not known to be pathogens. A possible role of anaerobic pathogens in bovine mastitis is also suggested.     

Chemical integration of Thorictus myrmecophilous beetles into Cataglyphis ant nests

Thorictus beetles of the Dermestidae are obligate myrmecophiles. To understand how these beetles are integrated into and tolerated by their host colonies, the cuticular hydrocarbon profiles of different species of the Thorictus castaneus group that are generally associated with Cataglyphis were examined. The beetles are characterized by small amounts of cuticular hydrocarbons, which render them partly chemically “insignificant”. They also have the same cuticular hydrocarbon profiles as their hosts and thus likely use chemical mimicry to evade worker hostility but, like slaves in slave-maker species, they maintain some partial chemical identity. Thorictus martinezi from Burkina Faso were immediately adopted by conspecific colonies of their host, Cataglyphis sp. aff. bicolor, but were never adopted by colonies of other species (i.e. Cataglyphis viatica and Formica selysi). Thorictus buigasi from Morocco also mimicked the chemical profile of its host, C. viatica, but, in contrast to T. martinezi, individuals were adopted by colonies of Cataglyphis velox from Spain. This result can be explained by the similarity between the hydrocarbon profiles of C. viatica and C. velox, which may facilitate adoptions. T. buigasi beetles remained in Formica selysi colonies for some time but were ultimately rejected, probably due to their very different hydrocarbon profiles. In contrast, they were sometimes adopted by Camponotus herculeanus colonies and eventually chemically matched their new hosts, probably by passive camouflage. These data suggest that Thorictus of castaneus group myrmecophily is the result of coevolution with Cataglyphis hosts and that the mimicry is plastic, such that beetles can live with different hosts if the hosts show very limited CHC differences.     

Isolation and Mapping of t Gene Mutants of Bacteriophage T4D

A procedure for selective isolation of T4 t mutants is described. At 120 min after infection of Escherichia coli cells with a low multiplicity of T4 bacteriophage, the mixture was sedimented through a linear sucrose gradient, and infected cells that remained intact were collected as the fastest sedimenting fraction. Ten to 50% of the phage released by chloroform treatment of this fraction were t mutants. Collection of a high proportion of t mutants depended on efficient elimination of cells that would survive because of superinfection lysis inhibition. This was accomplished by early addition of anti-T4 serum and heat-killed cells to inactivate progeny wild-type phage released at the normal burst time. Of 85 t mutants that were isolated and mapped, 23 new mutations were found, 14 of which are suppressible by an rII mutation and 9 of which are suppressible by rII or amber suppressors. Two hot-spot sites for spontaneous mutations were found; 14 mutants at one site, represented by a frameshift mutation, and 12 mutants at a second site were obtained from 39 spontaneous mutants independently isolated from different parental plaques. On our map of the t gene, the distance between the farthest t mutations is 6% recombination. A nonreverting triple t mutant, constructed to contain a frameshift mutation between two amber mutations, exhibited the same t mutant phenotype observed with revertible t mutants.     

Volatile constituents of Plus and minus strains of Blakeslea trispora

Identification of the predominant constituents produced by the plus and the minus strains of Blakeslea trispora is described. The occurrence of xylenes in the volatiles produced by the plus strain is reported. Additionally, production of 3-hydroxy-3-methylbutanal by the plus strain and dimethyl allyl alcohol by the minus strains were confirmed. Isoamyl alcohol, 1-octen-3-ol, 3-octanol and β-phenethyl alcohol were identified in volatiles from both strains.     

Genetic resources of primitive upland rice in Laos

Primitive upland cultivars (Oryza sativa L.) were collected in northern Laos. One-hundred-thirty-two cultivars were collected in upland fields at 27 sites. Morphological and physiological traits were recorded. The materials were classified intoindica and japonica types based on isozyme genotypes. We classified 106japonica, 16indica, two intermediate, and eight heterozygous cultivars. Thejaponica cultivars were characterized by glabrous hulls and sticky grains. Only two out of 16indica cultivars were glabrous. The heterozygotes were estimated to be generated by out-crosses betweenjaponica andindica cultivars in upland fields. The intermediate type would be the progeny of such heterozygotes. Higher polymorphism was found at two isozyme loci—Amp1 andEst2—among thejaponica cultivars. Genotypic frequencies differed between populations collected from upland fields along roads and along a branch of the Mekong river. Such differences would be caused by different origins of these two populations. In this report, isozymes were indicated as valuable markers to recognize the cultivars to be of independent stock.     

Antibiotic Susceptibility of Biofilm Cells and Molecular Characterisation of Staphylococcus hominis Isolates from Blood

Objectives

We aimed to characterise the staphylococcal cassette chromosome mec (SCCmec) type, genetic relatedness, biofilm formation and composition, icaADBC genes detection, icaD expression, and antibiotic susceptibility of planktonic and biofilm cells of Staphylococcus hominis isolates from blood.

Methods

The study included 67 S. hominis blood isolates. Methicillin resistance was evaluated with the cefoxitin disk test. mecA gene and SCCmec were detected by multiplex PCR. Genetic relatedness was determined by pulsed-field gel electrophoresis. Biofilm formation and composition were evaluated by staining with crystal violet and by detachment assay, respectively; and the biofilm index (BI) was determined. Detection and expression of icaADBC genes were performed by multiplex PCR and real-time PCR, respectively. Antibiotic susceptibilities of planktonic cells (minimum inhibitory concentration, MIC) and biofilm cells (minimum biofilm eradication concentration, MBEC) were determined by the broth dilution method.

Results

Eighty-five percent (57/67) of isolates were methicillin resistant and mecA positive. Of the mecA-positive isolates, 66.7% (38/57) carried a new putative SCCmec type. Four clones were detected, with two to five isolates each. Among all isolates, 91% (61/67) were categorised as strong biofilm producers. Biofilm biomass composition was heterogeneous (polysaccharides, proteins and DNA). All isolates presented the icaD gene, and 6.66% (1/15) isolates expressed icaD. This isolate presented the five genes of ica operon. Higher BI and MBEC values than the MIC values were observed for amikacin, vancomycin, linezolid, oxacillin, ciprofloxacin, and chloramphenicol.

Conclusions

S. hominis isolates were highly resistant to methicillin and other antimicrobials. Most of the detected SCCmec types were different than those described for S. aureus. Isolates indicated low clonality. The results indicate that S. hominis is a strong biofilm producer with an extracellular matrix with similar composition of proteins, DNA and N-acetylglucosamine; and presents high frequency and low expression of icaD gene. Biofilm production is associated with increased antibiotic resistance.     

Characterization of Bacteria by Gas Chromatography: Comparison of Trimethylsilyl Derivatives of Whole-Cell Hydrolysates

A method for the formation of the trimethylsilyl (TMS) derivatives of the whole-cell hydrolysate of bacteria was developed. The TMS derivatives were examined by gas-liquid chromatography. TMS profiles of various bacteria at the genus and species level were compared. Differences in TMS profiles of Listeria, Neisseria, and Clostridium were significant as were differences between the TMS profiles of C. perfringens and C. sporogenes. Two types of C. perfringens, two serotypes of L. monocytogenes, and one culture of C. sporogenes and N. meningitidis were studied. The possible application of TMS profiles as an aid in differentiating closely related organisms which are troublesome to separate by conventional means is discussed.     

Species Diversity and Seasonal Distribution of Culicoides spp. (Diptera: Ceratopogonidae) in Jeju-do,Republic of Korea

Biting midges belonging to the genus Culicoides (Diptera: Ceratopogonidae) were collected by Mosquito Magnet^® and black light traps at 5 sites on Jeju-do, Republic of Korea (Korea), from May-November 2013 to determine species diversity and seasonal distribution. A total of 4,267 specimens were collected, of which 99.9% were female. The most common species was Culicoides tainanus (91.8%), followed by C. lungchiensis (7.2%) and C. punctatus (0.6%), while the remaining 4 species accounted for <0.5% of all Culicoides spp. that were collected. High numbers of C. tainanus were collected in May, followed by decreasing numbers through August, and then increasing numbers through November when surveillance was terminated. Peak numbers of C. lungchiensis were collected during September, with low numbers collected from May-August and October-November. The presence of C. lungchiensis in Korea was confirmed by morphological and molecular analyses.     

Sequence Analysis of Tn10 Insertion Sites in a Collection of Escherichia coli Strains Used for Genetic Mapping and Strain Construction

The chromosomal insertion sites of Tn10-containing Escherichia coli strains were amplified by inverse PCR, and the nucleotide sequences of the junctions were determined. In 95 strains analyzed, 88 unique Tn10 positions were determined and matched to the E. coli chromosome sequence. Two gaps in insertion site positions were noted, one including the terminus of DNA replication and another bounded by recombination hot spots RhsA and RhsB.