Application of Fourier Transform Infrared Spectroscopy and Chemometrics for Differentiation of Salmonella enterica Serovar Enteritidis Phage Types

Fourier transform infrared (FT-IR) spectroscopy and chemometric techniques were used to discriminate five closely related Salmonella enterica serotype Enteritidis phage types, phage type 1 (PT1), PT1b, PT4b, PT6, and PT6a. Intact cells and outer membrane protein (OMP) extracts from bacterial cell membranes were subjected to FT-IR analysis in transmittance mode. Spectra were collected over a wavenumber range from 4,000 to 600 cm⁻¹. Partial least-squares discriminant analysis (PLS-DA) was used to develop calibration models based on preprocessed FT-IR spectra. The analysis based on OMP extracts provided greater separation between the Salmonella Enteritidis PT1-PT1b, PT4b, and PT6-PT6a groups than the intact cell analysis. When these three phage type groups were considered, the method based on OMP extract FT-IR spectra was 100% accurate. Moreover, complementary local models that considered only the PT1-PT1b and PT6-PT6a groups were developed, and the level of discrimination increased. PT1 and PT1b isolates were differentiated successfully with the local model using the entire OMP extract spectrum (98.3% correct predictions), whereas the accuracy of discrimination between PT6 and PT6a isolates was 86.0%. Isolates belonging to different phage types (PT19, PT20, and PT21) were used with the model to test its robustness. For the first time it was demonstrated that FT-IR analysis of OMP extracts can be used for construction of robust models that allow fast and accurate discrimination of different Salmonella Enteritidis phage types.Over the past 10 years there has been an increase in the incidence of gastrointestinal infections caused by Salmonella enterica serovar Enteritidis, which is now one of the leading S. enterica serotypes worldwide (21, 27). Poultry, poultry products, cattle, and dairy products are the predominant sources of Salmonella-contaminated food products that cause human salmonellosis (28). Large-scale infections continue to occur in developed countries (8). Unrestricted international movement of commercially prepared food and food ingredients and dissimilarities in government and industry food safety controls during the processing, distribution, and marketing of products have surely contributed to the increase in food-borne outbreaks. Salmonella is a tremendous challenge for the agricultural and food processing industries because of its ability to survive under adverse conditions, such as low levels of nutrients and suboptimal temperatures (4, 13).Salmonella Enteritidis isolates can be categorized for epidemiological purposes by using a variety of typing tools (13). These tools include typing techniques such as serological and phage typing (29) and antibiotic resistance patterns (25). These methods are now supplemented by molecular genetics techniques, such as DNA fingerprinting (23), plasmid profiling (16), and pulsed-field gel electrophoresis (26). Phage typing has been used to diagnose Salmonella outbreaks, including S. enterica serovar Typhi and S. enterica serovar Typhimurium outbreaks (29). It is useful to evaluate whether isolates obtained from different sources at different times are similar or distinct in terms of their reactions with a specific collection of bacteriophages used for typing. The correlation between phage type and the source of an epidemic is high (22). Although very effective, existing classification methods are time-consuming, laborious, and expensive, and they often require special training of personnel and expertise, which can prevent a rapid response to the presence of pathogenic bacterial species.Fourier transform infrared (FT-IR) spectroscopy has been successfully used for differentiation and classification of microorganisms at the species and subspecies levels (7, 9, 12, 15, 18, 19, 20). This technique has been shown to have high discriminatory power and allows identification of bacteria at distinct taxonomic levels based on differences in the infrared absorption patterns of microbial cells. FT-IR spectroscopy has been used to differentiate and characterize intact microbial cells based on outer membrane cell components, including lipopolysaccharides (LPS), lipoproteins, and phospholipids (24). Several studies in which S. enterica serotypes have been discriminated using multivariate data analysis and FT-IR spectroscopy have been performed (1, 2, 10, 11). Kim et al. (11) compared the FT-IR spectra of intact cells and the FT-IR spectra of outer membrane protein (OMP) extracts from S. enterica serotypes to discriminate serotypes. Analysis of spectra of OMP extracts in the 1,800- to 1,500-cm⁻¹ region resulted in 100% correct classification of the serotypes investigated.Previously, there have been no reports of differentiation of Salmonella Enteritidis phage types by FT-IR spectroscopy and chemometric methods. To discriminate closely related phage types of Salmonella Enteritidis in this study, intact cells and OMP extracts of bacterial cell membranes were subjected to FT-IR analysis. The isolates analyzed included isolates belonging to five of the phage types of Salmonella Enteritidis found most frequently in Portuguese hospitals in the period from 2004 to 2006, phage type 1 (PT1), PT1b, PT4b, PT6, and PT6a (5, 14). Chemometric models were used to discriminate between phage types based on infrared spectra.     

In vitro studies with mammalian cell lines and gum arabic-coated magnetic nanoparticles

Iron oxide magnetic nanoparticles (MNPs) were synthesized by the chemical co-precipitation method and coated with gum arabic (GA) by physical adsorption and covalent attachment. Cultures of mammalian cell lines (HEK293, CHO and TE671) were grown in the presence of uncoated and GA-coated MNPs. Cellular growth was followed by optical microscopy in order to assess the proportion of cells with particles, alterations in cellular density and the presence of debris. The in vitro assays demonstrated that cells from different origins are affected differently by the presence of the nanoparticles. Also, the methods followed for GA coating of MNPs endow distinct surface characteristics that probably underlie the observed differences when in contact with the cells. In general, the nanoparticles to which the GA was adsorbed had a smaller ability to attach to the cells' surface and to compromise the viability of the cultures.     

Genetic and functional analysis of common MRC1 exon 7 polymorphisms in leprosy susceptibility

The chromosomal region 10p13 has been linked to paucibacillary leprosy in two independent studies. The MRC1 gene, encoding the human mannose receptor (MR), is located in the 10p13 region and non-synonymous SNPs in exon 7 of the gene have been suggested as leprosy susceptibility factors. We determined that G396S is the only non-synonymous exon 7-encoded polymorphism in 396 unrelated Vietnamese subjects. This SNP was genotyped in 490 simplex and 90 multiplex leprosy families comprising 704 patients (47% paucibacillary; 53% multibacillary). We observed significant under-transmission of the serine allele of the G396S polymorphism with leprosy per se (P = 0.036) and multibacillary leprosy (P = 0.034). In a sample of 384 Brazilian leprosy cases (51% paucibacillary; 49% multibacillary) and 399 healthy controls, we observed significant association of the glycine allele of the G396S polymorphism with leprosy per se (P = 0.016) and multibacillary leprosy (P = 0.023). In addition, we observed a significant association of exon 7 encoded amino acid haplotypes with leprosy per se (P = 0.012) and multibacillary leprosy (P = 0.004). Next, we tested HEK293 cells over-expressing MR constructs (293-MR) with three exon 7 haplotypes of MRC1 for their ability to bind and internalize ovalbumin and zymosan, two classical MR ligands. No difference in uptake was measured between the variants. In addition, 293-MR failed to bind and internalize viable Mycobacterium leprae and BCG. We propose that the MR–M. leprae interaction is modulated by an accessory host molecule of unknown identity.     

Bombesin derivative radiolabeled with technetium-99m as agent for tumor identification

A bombesin derivative was successfully radiolabeled in high labeling yield. Biodistribution studies and scintigraphic images in Ehrlich tumor-bearing mice were performed. This compound showed high accumulation in tumor tissue with high tumor-to-muscle and tumor-to-blood ratios. Thus, ^99mTc-HYNIC-Bombesin_(7–14) could be used as an agent for tumor diagnosis.     

The dual function of barred plumage in birds: camouflage and communication

A commonly held principle in visual ecology is that communication compromises camouflage: while visual signals are often conspicuous, camouflage provides concealment. However, some traits may have evolved for communication and camouflage simultaneously, thereby overcoming this functional compromise. Visual patterns generally provide camouflage, but it was suggested that a particular type of visual pattern – avian barred plumage – could also be a signal of individual quality. Here, we test if the evolution of sexual dimorphism in barred plumage, as well as differences between juvenile and adult plumage, indicate camouflage and/or signalling functions across the class Aves. We found a higher frequency of female- rather than male-biased sexual dimorphism in barred plumage, indicating that camouflage is its most common function. But we also found that, compared to other pigmentation patterns, barred plumage is more frequently biased towards males and its expression more frequently restricted to adulthood, suggesting that barred plumage often evolves or is maintained as a sexual communication signal. This illustrates how visual traits can accommodate the apparently incompatible functions of camouflage and communication, which has implications for our understanding of avian visual ecology and sexual ornamentation.     

Chromosomes of Theridiidae spiders (Entelegynae): Interspecific karyotype diversity in Argyrodes and diploid number intraspecific variability in Nesticodes rufipes

Theridiidae is a derived family within the Araneoidea clade. In contrast to closely related groups, the 2n(male) = 20+X(1) X (2) with acro/telocentric chromosomes is the most widespread karyotype among the theridiid spiders. In this work, the cytogenetic analysis of Argyrodes elevatus revealed original chromosome features different from those previously registered for Theridiidae, including the presence of 2n(male) = 20+X with meta/submetacentric chromosomes. Most individuals of Nesticodes rufipes showed family conserved karyotype characteristics. However, one individual had a 2n(male) = 24 due to the presence of an extra chromosome pair, which exhibited regular behavior and reductional segregation during meiosis. After silver staining, mitotic cells exhibited NORs localized on the terminal regions of the short arms of pairs 2, 3, and 4 of A. elevatus and on the terminal regions of long arms of pair 4 of N. rufipes. The comparative analysis with data from phylogenetically related species allowed the clarification of the origin of the interspecific and intraspecific chromosome variability observed in Argyrodes and in N. rufipes, respectively.     

External morphology of the cycliophoran dwarf male: a comparative study of Symbion pandora and S. americanus

Cycliophora is a recently described phylum to which only two species have been assigned so far, Symbion pandora and S. americanus. The cycliophoran life cycle is complex and alternates between asexual and sexual stages. Although not recognized as an entirely independent free-swimming stage when the phylum was first described, the dwarf male has a remarkably complex bodyplan albeit its very small size (approx. 30–40 μm in length). Aiming to increase the knowledge on the gross morphology of the cycliophoran dwarf male, specimens from S. pandora and S. americanus were analyzed by scanning electron microscopy. In both species, anterior and ventral ciliated fields, as well as paired lateral sensorial organs, were identified, thus confirming previous observations. However, new details are described herein such as the penial pouch that encloses the penis. We compare our findings on both Symbion species with the data currently available on other metazoan dwarf males.     

Evolution of female carotenoid coloration by sexual constraint in <Emphasis Type="Italic">Carduelis</Emphasis> finches

Background  

Females often express the same ornaments as males to a similar or lesser degree. Female ornaments can be adaptive, but little is known regarding their origins and mode of evolution. Current utility does not imply evolutionary causation, and therefore it is possible that female ornamentation evolved due to selection on females, as a correlated response to selection on males (sexual constraint), or a combination of both. We tested these ideas simulating simple models for the evolution of male and female correlated traits, and compared their predictions against the coloration of finches in the genus Carduelis.     

The serendipitous origin of chordate secretin peptide family members

Background  

The secretin family is a pleotropic group of brain-gut peptides with affinity for class 2 G-protein coupled receptors (secretin family GPCRs) proposed to have emerged early in the metazoan radiation via gene or genome duplications. In human, 10 members exist and sequence and functional homologues and ligand-receptor pairs have been characterised in representatives of most vertebrate classes. Secretin-like family GPCR homologues have also been isolated in non-vertebrate genomes however their corresponding ligands have not been convincingly identified and their evolution remains enigmatic.