Comparison of the insecticide susceptibilities of laboratory strains of Aedes aegypti and Aedes albopictus

A susceptible strain of Aedes albopictus derived from the Gainesville strain (Florida, USA) was established in our laboratory. The larvicidal efficacies of the neurotoxic insecticides temephos, permethrin and the pure cis and trans-permethrin isomers and the microbial insecticide Bacillus thuringiensis israelensis (Bti) against Ae. albopictus were estimated and compared to a susceptible strain of Aedes aegypti. The larvicidal effect of insect growth regulator pyriproxyfen was also evaluated in both mosquito strains. The median lethal concentration/median emergency inhibition values for Ae. aegypti and Ae. albopictus, respectively, were: temephos, 3.058 and 6.632 ppb, permethrin, 3.143 and 4.933 ppb, cis-permethrin, 4.457 and 10.068 ppb, trans-permethrin, 1.510 and 3.883 ppb, Bti, 0.655 and 0.880 ppb and pyriproxyfen, 0.00774 and 0.01642 ppb. Ae. albopictus was more tolerant than Ae. aegypti to all six larvicides evaluated. The order of susceptibility for Ae. aegypti was pyriproxyfen > Bti > trans-permethrin > temephos > permethrin > cis-permethrin and for Ae. albopictus was pyriproxyfen > Bti > trans-permethrin > permethrin > temephos > cis-permethrin. Because both species can be found together in common urban, suburban and rural breeding sites, the results of this work provide baseline data on the susceptibility of Ae. albopictus to insecticides commonly used for controlling Ae. aegypti in the field.

Aggregative adherent strains of Corynebacterium pseudodiphtheriticum enter and survive within HEp-2 epithelial cells

Corynebacterium pseudodiphtheriticum is a well-known human pathogen that mainly causes respiratory disease and is associated with high mortality in compromised hosts. Little is known about the virulence factors and pathogenesis of C. pseudodiphtheriticum. In this study, cultured human epithelial (HEp-2) cells were used to analyse the adherence pattern, internalisation and intracellular survival of the ATCC 10700 type strain and two additional clinical isolates. These microorganisms exhibited an aggregative adherence-like pattern to HEp-2 cells characterised by clumps of bacteria with a "stacked-brick" appearance. The differences in the ability of these microorganisms to invade and survive within HEp-2 cells and replicate in the extracellular environment up to 24 h post infection were evaluated. The fluorescent actin staining test demonstrated that actin polymerisation is involved in the internalisation of the C. pseudodiphtheriticum strains. The depolymerisation of microfilaments by cytochalasin E significantly reduced the internalisation of C. pseudodiphtheriticum by HEp-2 cells. Bacterial internalisation and cytoskeletal rearrangement seemed to be partially triggered by the activation of tyrosine kinase activity. Although C. pseudodiphtheriticum strains did not demonstrate an ability to replicate intracellularly, HEp-2 cells were unable to fully clear the pathogen within 24 h. These characteristics may explain how some C. pseudodiphtheriticum strains cause severe infection in human patients.

Terbinafine inhibits Cryptococcus neoformans growth and modulates fungal morphology

Cryptococcus neoformans is an encapsulated fungus that causes cryptococcosis. Central nervous system infection is the most common clinical presentation followed by pulmonary, skin and eye manifestations. Cryptococcosis is primarily treated with amphotericin B (AMB), fluconazole (FLC) and itraconazole (ITC). In the present work, we evaluated the in vitro effect of terbinafine (TRB), an antifungal not commonly used to treat cryptococcosis. We specifically examined the effects of TRB, either alone or in conjunction with AMB, FLC and ITC, on clinical C. neoformans isolates, including some isolates resistant to AMB and ITC. Broth microdilution assays showed that TRB was the most effective drug in vitro. Antifungal combinations demonstrated synergism of TRB with AMB, FLC and ITC. The drug concentrations used for the combination formulations were as much as 32 and 16-fold lower than the minimum inhibitory concentration (MIC) values of FLC and AMB alone, respectively. In addition, calcofluor white staining revealed the presence of true septa in hyphae structures that were generated after drug treatment. Ultrastructural analyses demonstrated several alterations in response to drug treatment, such as cell wall alterations, plasma membrane detachment, presence of several cytoplasmic vacuoles and mitochondrial swelling. Therefore, we believe that the use of TRB alone or in combination with AMB and azoles should be explored as an alternative treatment for cryptococcosis patients who do not respond to standard therapies.

Gene copy number polymorphisms in an arbuscular mycorrhizal fungal population.

Gene copy number polymorphism was studied in a population of the arbuscular mycorrhizal fungus Glomus intraradices by using a quantitative PCR approach on four different genomic regions. Variation in gene copy number was found for a pseudogene and for three ribosomal genes, providing conclusive evidence for a widespread occurrence of macromutational events in the population.

Novel Chlamydiales strains isolated from a water treatment plant.

Chlamydiae are obligate intracellular bacteria infecting free-living amoebae, vertebrates and some invertebrates. Novel members are regularly discovered, and there is accumulating evidence supporting a very important diversity of chlamydiae in the environment. In this study, we investigated the presence of chlamydiae in a drinking water treatment plant. Samples were used to inoculate Acanthamoeba monolayers (Acanthamoeba co-culture), and to recover autochthonous amoebae onto non-nutritive agar. Chlamydiae were searched for by a pan-chlamydia 16S rRNA gene PCR from both Acanthamoeba co-cultures and autochthonous amoebae, and phylotypes determined by 16S rRNA gene sequencing. Autochthonous amoebae also were identified by 18S rRNA gene amplification and sequencing. From a total of 79 samples, we recovered eight chlamydial strains by Acanthamoeba co-culture, but only one of 28 amoebae harboured a chlamydia. Sequencing results and phylogenetic analysis showed our strains belonging to four distinct chlamydial lineages. Four strains, including the strain recovered within its natural host, belonged to the Parachlamydiaceae; two closely related strains belonged to the Criblamydiaceae; two distinct strains clustered with Rhabdochlamydia spp.; one strain clustered only with uncultured environmental clones. Our results confirmed the usefulness of amoeba co-culture to recover novel chlamydial strains from complex samples and demonstrated the huge diversity of chlamydiae in the environment, by identifying several new species including one representing the first strain of a new family.

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.

Safety and immunomodulatory effects of three probiotic strains isolated from the feces of breast-fed infants in healthy adults: SETOPROB study.

We previously described the isolation and characterization of three probiotic strains from the feces of exclusively breast-fed newborn infants: Lactobacillus paracasei CNCM I-4034, Bifidobacterium breve CNCM I-4035 and Lactobacillus rhamnosus CNCM I-4036. These strains were shown to adhere to intestinal mucus in vitro, to be sensitive to antibiotics and to resist biliary salts and low pH. In the present study, a multicenter, randomized, double-blind, placebo-controlled trial with 100 healthy volunteers in three Spanish cities was carried out to evaluate the tolerance, safety, gut colonization and immunomodulatory effects of these three probiotics. Volunteers underwent a 15-day washout period, after which they were randomly divided into 5 groups that received daily a placebo, a capsule containing one of the 3 strains or a capsule containing a mixture of two strains for 30 days. The intervention was followed by another 15-day washout period. Patients did not consume fermented milk for the entire duration of the study. Gastrointestinal symptoms, defecation frequency and stool consistency were not altered by probiotic intake. No relevant changes in blood and serum, as well as no adverse events occurred during or after treatment. Probiotic administration slightly modified bacterial populations in the volunteers' feces. Intestinal persistence occurred in volunteers who received L. rhamnosus CNCM I-4036. Administration of B. breve CNCM I-4035 resulted in a significant increase in fecal secretory IgA content. IL-4 and IL-10 increased, whereas IL-12 decreased in the serum of volunteers treated with any of the three strains. These results demonstrate that the consumption of these three bacterial strains was safe and exerted varying degrees of immunomodulatory effects.

Myenteric plexus is differentially affected by infection with distinct Trypanosoma cruzi strains in Beagle dogs

Chagasic megaoesophagus and megacolon are characterised by motor abnormalities related to enteric nervous system lesions and their development seems to be related to geographic distribution of distinct Trypanosoma cruzi subpopulations. Beagle dogs were infected with Y or Berenice-78 (Be-78) T. cruzi strains and necropsied during the acute or chronic phase of experimental disease for post mortem histopathological evaluation of the oesophagus and colon. Both strains infected the oesophagus and colon and caused an inflammatory response during the acute phase. In the chronic phase, inflammatory process was observed exclusively in the Be-78 infected animals, possibly due to a parasitism persistent only in this group. Myenteric denervation occurred during the acute phase of infection for both strains, but persisted chronically only in Be-78 infected animals. Glial cell involvement occurred earlier in animals infected with the Y strain, while animals infected with the Be-78 strain showed reduced glial fibrillary acidic protein immunoreactive area of enteric glial cells in the chronic phase. These results suggest that although both strains cause lesions in the digestive tract, the Y strain is associated with early control of the lesion, while the Be-78 strain results in progressive gut lesions in this model.

Genetic diversity and antimicrobial resistance in Streptococcus agalactiae strains recovered from female carriers in the Bucharest area

For the first time, we used multilocus sequence typing (MLST) to understand how Romanian group B streptococcus (GBS) strains fit into the global GBS population structure. Colonising isolates recovered from adult human females were tested for antibiotic resistance, were molecularly serotyped based on the capsular polysaccharide synthesis (cps) gene cluster and further characterised using a set of molecular markers (surface protein genes, pilus-encoded islands and mobile genetic elements inserted in the scpB-lmb intergenic region). Pulsed-field gel electrophoresis was used to complement the MLST clonal distribution pattern of selected strains. Among the 55 strains assigned to six cps types (Ia, Ib, II-V), 18 sequence types (STs) were identified by MLST. Five STs represented new entries to the MLST database. The prevalent STs were ST-1, ST-17, ST-19 and ST-28. Twenty molecular marker profiles were identified. The most common profiles (rib+GBSi1+PI-1, rib+GBSi1+PI-1, PI-2b and alp2/3+PI-1, PI-2a) were associated with the cps III/ST-17 and cps V/ST-1 strains. A cluster of fluoroquinolone-resistant strains was detected among the cps V/ST-19 members; these strains shared alp1 and IS1548 and carried PI-1, PI-2a or both. Our results support the usefulness of implementing an integrated genotyping system at the reference laboratory level to obtain the reliable data required to make comparisons between countries.

Virulence variation among epidemic and non-epidemic strains of Saint Louis encephalitis virus circulating in Argentina

Saint Louis encephalitis virus caused an outbreak of febrile illness and encephalitis cases in Córdoba, Argentina, in 2005. During this outbreak, the strain CbaAr-4005 was isolated from Culex quinquefasciatus mosquitoes. We hypothesised that this epidemic variant would be more virulent in a mouse model than two other non-epidemic strains (78V-6507 and CorAn-9275) isolated under different epidemiological conditions. To test this hypothesis, we performed a biological characterisation in a murine model, including mortality, morbidity and infection percentages and lethal infection indices using the three strains. Mice were separated into age groups (7, 10 and 21-day-old mice) and analysed after infection. The strain CbaAr-4005 was the most infective and lethal of the three variants, whereas the other two strains exhibited a decreasing mortality percentage with increasing animal age. The strain CbaAr-4005 produced the highest morbidity percentages and no significant differences among age groups were observed. The epidemic strain caused signs of illness in all inoculated animals and showed narrower ranges from the onset of symptoms than the other strains. CbaAr-4005 was the most virulent for Swiss albino mice. Our results highlight the importance of performing biological characterisations of arbovirus strains likely to be responsible for emerging or reemerging human diseases.

Next-generation sequencing of experimental mouse strains.

Since the turn of the century the complete genome sequence of just one mouse strain, C57BL/6J, has been available. Knowing the sequence of this strain has enabled large-scale forward genetic screens to be performed, the creation of an almost complete set of embryonic stem (ES) cell lines with targeted alleles for protein-coding genes, and the generation of a rich catalog of mouse genomic variation. However, many experiments that use other common laboratory mouse strains have been hindered by a lack of whole-genome sequence data for these strains. The last 5 years has witnessed a revolution in DNA sequencing technologies. Recently, these technologies have been used to expand the repertoire of fully sequenced mouse genomes. In this article we review the main findings of these studies and discuss how the sequence of mouse genomes is helping pave the way from sequence to phenotype. Finally, we discuss the prospects for using de novo assembly techniques to obtain high-quality assembled genome sequences of these laboratory mouse strains, and what advances in sequencing technologies may be required to achieve this goal.

Molecular epidemiology of Candida albicans and Candida glabrata strains isolated from intensive care unit patients in Poland

Over the last decades, Candida spp have been responsible for an increasing number of infections, especially in patients requiring intensive care. Knowledge of local epidemiology and analysis of the spread of these pathogens is important in understanding and controlling their transmission. The aim of this study was to evaluate the genetic diversity of 31 Candida albicans and 17 Candida glabrata isolates recovered from intensive care unit patients from the tertiary hospital in Krakow between 2011-2012. The strains were typed by random amplified polymorphic DNA (RAPD) polymerase chain reaction using five primers (CD16AS, HP1247, ERIC-2, OPE-3 and OPE-18). The results of the present investigation revealed a high degree of genetic diversity among the isolates. No clonal relationship was found among the C. albicans strains, whereas two C. glabrata isolates were identical. The source of Candida infection appeared to be mostly endogenous; however, the presence of two clonal C. glabrata strains suggested the possibility of cross-transmission of these pathogens. Our study confirmed the high discriminatory power of the RAPD technique in the molecular typing of Candida clinical isolates. This method may be applied to the evaluation of transmission routes of pathogenic fungi on a local level.

Epidemiology and antifungal susceptibility of bloodstream fungal isolates in pediatric patients: a Spanish multicenter prospective survey.

Data on fungemia epidemiology and antifungal susceptibility of isolates from children are scarce, leading frequently to pediatric empirical treatment based on available adult data. The present study was designed to update the epidemiological, mycological, and in vitro susceptibility data on fungal isolates from children with fungemia in Spain. All fungemia episodes were identified prospectively by blood culture over 13 months at 30 hospitals. Tests of susceptibility to amphotericin B, flucytosine, fluconazole, itraconazole, posaconazole, voriconazole, anidulafungin, caspofungin, and micafungin were performed at participant institutions by a microdilution colorimetric method. New species-specific clinical breakpoints for fluconazole, voriconazole, and echinocandins were also applied. A total of 203 episodes of fungemia in 200 children were identified. A higher proportion of fungal isolates was from general wards than intensive care units (ICU). Candida parapsilosis (46.8%), Candida albicans (36.5%), Candida tropicalis (5.9%), Candida glabrata (3.9%), and Candida guilliermondii (2.5%) were the leading species. C. parapsilosis was the predominant species except in neonates. C. albicans was the most frequent in neonatal ICU settings (51.9%). Intravascular catheter (79.3%), surgery (35%), prematurity (30%), and neutropenia (11%) were the most frequent predisposing factors. Most Candida isolates (95.1%) were susceptible to all antifungals. When the new species-specific clinical breakpoints were applied, all C. parapsilosis isolates were susceptible to echinocandins except one, which was micafungin resistant. This is the largest published series of fungemia episodes in the pediatric setting. C. parapsilosis is the most prevalent species in Spain, followed by C. albicans and C. tropicalis. Resistance to azole and echinocandin agents is extremely rare among Candida species. The fluconazole resistance rate in Spain has decreased in the last 10 years.

Arbuscular mycorrhizal fungi (Glomeromycota) harbour ancient fungal tubulin genes that resemble those of the chytrids (Chytridiomycota).

The genes encoding alpha- and beta-tubulins have been widely sampled in most major fungal phyla and they are useful tools for fungal phylogeny. Here, we report the first isolation of alpha-tubulin sequences from arbuscular mycorrhizal fungi (AMF). In parallel, AMF beta-tubulins were sampled and analysed to identify the presence of paralogs of this gene. The AMF alpha-tubulin amino acid phylogeny was congruent with the results previously reported for AMF beta-tubulins and showed that AMF tubulins group together at a basal position in the fungal clade and showed high sequence similarities with members of the Chytridiomycota. This is in contrast with phylogenies for other regions of the AMF genome. The amount and nature of substitutions are consistent with an ancient divergence of both orthologs and paralogs of AMF tubulins. At the amino acid level, however, AMF tubulins have hardly evolved from those of the chytrids. This is remarkable given that these two groups are ancient and the monophyletic Glomeromycota probably diverged from basal fungal ancestors at least 500 million years ago. The specific primers we designed for the AMF tubulins, together with the high molecular variation we found among the AMF species we analysed, make AMF tubulin sequences potentially useful for AMF identification purposes.

Interrogation of related clinical pan-azole-resistant Aspergillus fumigatus strains: G138C, Y431C, and G434C single nucleotide polymorphisms in cyp51A, upregulation of cyp51A, and integration and activation of transposon Atf1 in the cyp51A promoter.

Multiple Aspergillus fumigatus isolates from a patient with two aspergillomas complicating chronic pulmonary aspergillosis were pan-azole resistant. Microsatellite typing was identical for all isolates despite major phenotypic and some growth rate differences. Three different cyp51A mutations were found (G138C, Y431C, and G434C), of which the first two were demonstrated by heterologous expression in a hypersusceptible Saccharomyces cerevisiae strain to be at least partly responsible for elevated MICs. cyp51A and cyp51B gene duplication was excluded, but increased expression of cyp51A was demonstrated in three isolates selected for additional study (7-to 13-fold increases). In the isolate with the greatest cyp51A expression, an Aft1 transposon was found inserted 370 bp upstream of the start codon of the cyp51A gene, an integration location never previously demonstrated in Aspergillus. Two transcription start sites were identified at 49 and 136 bp upstream of the start codon. The role of the Aft1 transposon, if any, in modulating cyp51A expression remains to be established. Increased mRNA expression of the transporters AfuMDR1 and AfuMDR4 also was demonstrated in some isolates, which could contribute to azole resistance or simply represent a stress response. The diversity of confirmed and possible azole resistance mechanisms demonstrated in a single series of isogenic isolates is remarkable, indicating the ability of A. fumigatus to adapt in the clinical setting.