Molecular identification of Giardia duodenalis in Ecuador by polymerase chain reaction-restriction fragment length polymorphism

The aim of this study was to determine the genetic diversity of Giardia duodenalis present in a human population living in a northern Ecuadorian rain forest. All Giardia positive samples (based on an ELISA assay) were analysed using a semi-nested polymerase chain reaction-restriction fragment length polymorphism assay that targets the glutamate dehydrogenase (gdh) gene; those amplified were subsequently genotyped using NlaIV and RsaI enzymes. The gdh gene was successfully amplified in 74 of 154 ELISA positive samples; 69 of the 74 samples were subsequently genotyped. Of these 69 samples, 42 (61%) were classified as assemblage B (26 as BIII and 16 as BIV), 22 (32%) as assemblage A (3 as AI and 19 as AII) and five (7%) as mixed AII and BIII types. In this study site we observe similar diversity in genotypes to other regions in Latin America, though in contrast to some previous studies, we found similar levels of diarrheal symptoms in those individuals infected with assemblage B compared with those infected with assemblage A.     

Phlebotomus (Legeromyia) multihamatus subg. nov., sp. nov. from Gabon (Diptera: Psychodidae)

During a research project aimed at the study of the Culicinae fauna of Gabon and carried out in the National Park of La Lopé, we captured an unknown sandfly male specimen (genus Phlebotomus) by CDC miniature light trap belonging to a new species for Science. Furthermore, the originality of his genitalia does not allow us to include this species in one of the existing subgenus, thus in this paper we propose the creation of a new subgenus, as Phlebotomus (Legeromyia) multihamatus sp. nov., subg. nov. described from the National Park of La Lopé, through one male captured with CDC miniature light trap. A new species and a new subgenus of sandfly is characterised by a short style with three spines, a paramere wearing a basal hook as well as a basal pouch and the absence of basal lobe on the coxite. The originality of the genitalia of the male gives way to discussion about potential primary homologies between P. multihamatus sp. nov. and Phlebotomus (Abonnencius) fortunatarum, Phlebotomus (Anaphlebotomus) stantoni and Phlebotomus (Euphlebotomus) argentipes, which should be verified for future studies. The discovery of this new species in Gabon must encourage the study of sandflies in this country.     

Monitoring resistance to Bacillus thuringiensis subsp. israelensis in the field by performing bioassays with each Cry toxin separately

Bacillus thuringiensis subsp. israelensis (Bti) is increasingly used worldwide for mosquito control and is the only larvicide used in the French Rhône-Alpes region since decades. The artificial selection of mosquitoes with field-persistent Bti collected in breeding sites from this region led to a moderate level of resistance to Bti, but to relatively high levels of resistance to individual Bti Cry toxins. Based on this observation, we developed a bioassay procedure using each Bti Cry toxin separately to detect cryptic Bti-resistance evolving in field mosquito populations. Although no resistance to Bti was detected in none of the three mosquito species tested (Aedes rusticus, Aedes sticticus and Aedes vexans), an increased tolerance to Cry4Aa (3.5-fold) and Cry11Aa toxins (8-fold) was found in one Ae. sticticus population compared to other populations of the same species, suggesting that resistance to Bti may be arising in this population. This study confirms previous works showing a lack of Bti resistance in field mosquito populations treated for decades with this bioinsecticide. It also provides a first panorama of their susceptibility status to individual Bti Cry toxins. In combination with bioassays with Bti, bioassays with separate Cry toxins allow a more sensitive monitoring of Bti-resistance in the field.     

Use of sodC versus ctrA for real-time polymerase chain reaction-based detection of Neisseria meningitidis in sterile body fluids

We evaluated the use of a newly described sodC-based real-time-polymerase chain reaction (RT-PCR) assay for detecting Neisseria meningitidis in normally sterile sites, such as cerebrospinal fluid and serum. The sodC-based RT-PCR assay has an advantage over ctrA for detecting nongroupable N. meningitidis isolates, which are commonly present in asymptomatic pharyngeal carriage. However, in our study, sodC-based RT-PCR was 7.5% less sensitive than ctrA. Given the public health impact of possible false-negative results due to the use of the sodC target gene alone, sodC-based RT-PCR for the diagnosis of meningococcal meningitis should be used with caution.     

Coagulase-negative staphylococci strains resistant to oxacillin isolated from neonatal blood cultures

Coagulase-negative staphylococci (CoNS) are the microorganisms most frequently isolated from clinical samples and are commonly found in neonatal blood cultures. Oxacillin is an alternative treatment of choice for CoNS infections; however, resistance to oxacillin can have a substantial impact on healthcare by adversely affecting morbidity and mortality. The objective of this study was to detect and characterise oxacillin-resistant CoNS strains in blood cultures of newborns hospitalised at the neonatal ward of the University Hospital of the Faculty of Medicine of Botucatu. One hundred CoNS strains were isolated and the mecA gene was detected in 69 of the CoNS strains, including 73.2% of Staphylococcus epidermidis strains, 85.7% of Staphylococcus haemolyticus strains, 28.6% of Staphylococcus hominis strains and 50% of Staphylococcus lugdunensis strains. Among these oxacillin-resistant CoNS strains, staphylococcal cassette chromosome mec (SCCmec) type I was identified in 24.6%, type II in 4.3%, type III in 56.5% and type IV in 14.5% of the strains. The data revealed an increase in the percentage of CoNS strains isolated from blood cultures from 1991-2009. Furthermore, a predominant SCCmec profile of the oxacillin-resistant CoNS strains isolated from neonatal intensive care units was identified with a prevalence of SCCmec types found in hospital-acquired strains.     

Cloning and Expression of Genes Encoding F107-C and K88-1NT Fimbrial Proteins of Enterotoxigenic Escherichia coli from Piglets

We cloned two genes coding F107-C and K88-1NT fimbrial subunits from strains E. coli C and 1NT isolated from Thua Thien Hue province, Vietnam. The mature peptide of faeG gene from strain E. coli 1NT (called faeG-1NT) is 100 % similarity with faeG gene, while the CDS of fedA gene from strain C (called fedA-C) has a similarity of 97 % with the fedA gene. Expression of the faeG-1NT and fedA-C genes in E. coli BL21 Star™ (DE3) produced proteins of ~31 and 22 kDa, respectively. The effect of IPTG concentration on the K88-1NT and F107-C fimbriae production was investigated. The results showed that 0.5 mM IPTG is suitable for higher expression of K88-1NT subunit, while 0.75 mM IPTG strongly stimulated expression of F107-C subunit. The optimal induction time for expression was also examined. Generally, highest expression of K88-1NT subunit occurred after 6 h of induction, while that of F107-C subunit is after 14 h.     

Shotgun Analysis of the Secretome of Fusarium graminearum

Fusarium head blight, caused predominately by Fusarium graminearum, is one of the most destructive diseases of wheat (Triticum aestivum L.) worldwide. To characterize the profile of proteins secreted by F. graminearum, the extracellular proteins were collectively obtained from F. graminearum culture supernatants and evaluated using one-dimensional SDS-PAGE and liquid chromatography-tandem mass spectrometry. A total of 87 proteins have been identified, of which 63 were predicted as secretory proteins including those with known functions. Meanwhile, 20 proteins that are not homologous to genomic sequences with known functions have also been detected. Some of the identified proteins are possible virulence factors and may play extracellular roles during F. graminearum infection. This study provides a valuable dataset of F. graminearum extracellular proteins, and a better understanding of the virulence mechanisms of the pathogen.     

A Method for Molecular Analysis of Catalase Gene Diversity in Seawater

Catalase plays an important role in the metabolism of marine bacteria and has potential impact on the marine environment. Four PCR primers were designed to amplify the catalase gene fragments in marine bacteria by applying metagenomic DNA from Yellow Sea surface water as the template. Of the four reproducible target PCR products, the longest one with 900 bp were chosen for catalase gene library construction by the T-vector and the white Escherichia coli colonies in the library was screened through restriction-digesting the reamplified insert fragments by the selected restriction endonuclease MboI, and then the bands of the resulting products were displayed in the agarose gel by electrophoresis. The unique restriction fragment length polymorphism (RFLP) pattern was selected and the corresponding catalase gene fragments were sequenced, which verified that every unique RFLP pattern represented one type of catalase. This PCR–RFLP method above was established to investigate the bacterial catalase diversity in seawater.     

丙肝病毒核心基因靶向性M1GS 核酶的体外抗病毒活性

[目的]丙型肝炎病毒(Hepatitis C Virus,HCV)是引起病毒性肝炎的重要病原.目前临床HCV感染多采用干扰素联合病毒唑进行治疗,但其应答率低且感染易反复,探索新型抗HCV治疗策略与药物具有紧迫的现实意义.[方法]基于大肠埃希菌来源的核糖核酸酶P(RNase P),针对HCV核心基因的序列,设计一小段与之互补的外部引导序列(Guide Sequence,GS),通过PCR将其共价连接至大肠埃希菌RNase P催化性亚基(M1 RNA)的3'末端,从而构建一种靶向性的核酶——M1GS.[结果]构建的M1 GS-HCV/C52核酶在体外可对靶RNA片段产生特异性切割;在HCV感染的Huh7.5.1细胞,该人工核酶也能够显著抑制HCV核心基因的表达,并使培养上清中HCV RNA的拷贝数减少约1500倍.[结论]本研究构建的M1GS-HCV/C52核酶具有显著的体外抗病毒活性,从而为HCV的治疗研究提供了一条潜在途径.