- Volume 61, Issue 12, 2012

Staphylococcus aureus can invade epithelial cells, and the host-cell receptor α5β1 integrin is thought to mediate this process. The aim of this study was to investigate S. aureus invasion of epithelial cell lines derived from oral (H357), skin (UP) and nasopharyngeal (Detroit 562) sites and to determine whether any differences were due to the levels of α5β1 integrin expressed. While the adhesion and invasion of two S. aureus strains were similar in both oral and skin-derived keratinocytes, this was markedly reduced in the nasopharyngeal cell line, despite it expressing similar levels of α5β1. While this might be explainable on the basis of availability of cell receptor, adhesion to and invasion of H357 and UP cells by S. aureus were enhanced when the epithelial cells were in suspension rather than on a surface, and levels of α5 integrin subunit mRNA were also increased. Detroit 562 cells exhibited a similar α5 gene upregulation, but this did not result in enhanced adhesion and invasion of S. aureus. The Detroit 562 cells also showed reduced adhesion to fibronectin compared with the other cell types. This, and the low S. aureus invasion, may result from reduced α5β1 integrin activity or from variation in an as-yet-unidentified additional receptor or accessory molecule. These studies shed further light on the mechanisms of S. aureus invasion of human cells.

A multiplex PCR was developed to detect 15 serotypes of Streptococcus suis. This multiplex PCR was separated into two reaction sets. The first set identified nine serotypes (serotypes 1/2, 1, 2, 3, 4, 7, 9, 14 and 16) and the second set identified six serotypes (serotypes 5, 8, 10, 19, 23 and 25). This assay correctly detected serotypes 2, 5 and 14 in human isolates, and serotypes 1, 2, 1/2, 3, 4, 5, 7, 9, 14, 16 and 19 in pig isolates from Thailand. No cross-reaction was observed with other streptococcal species. This assay may be useful for the serotype surveillance of human and pig isolates of S. suis.

The genetic diversity between major meticillin-resistant Staphylococcus aureus (MRSA) lineages was probed using fluorescent amplified fragment length polymorphism (FAFLP) as a random genome sampling tool. Genomic DNA was digested with endonucleases BglII and Csp6I and a subset of the restricted fragments were amplified using the primer pair BglII+A and Csp6I+0. Sixty-seven FAFLP profiles consisting of 46–68 amplified fragments ranging in size from 50 to 600 bp were exhibited amongst the 71 isolates analysed. Cluster analysis of FAFLP data revealed concordance with spa typing and MLST clonal complexes (CC), with isolates of each CC grouping in the same FAFLP cluster. Furthermore, FAFLP could differentiate subtypes within the homogeneous CC22 isolates and also between MLST sequence types 8 and 239. The discriminatory power of FAFLP was 0.998 compared to values of 0.975 and 0.909 for spa typing and MLST, respectively. Thus, FAFLP analysis proved to be a rapid, reproducible and high-resolution tool that displayed the microheterogeneity within MRSA lineages. Using FAFLP data, lineage-specific fragments were identified and sequenced; these encoded toxins, antibiotic resistance determinants and bacteriophage resistance factors. Lineage-specific sequence variations were observed, which may provide insights into the evolution and fitness of successful lineages. This will also aid in the development of rapid and high-throughput diagnostic PCR-based assays for the identification of MRSA lineages in resource-poor settings.

Leptospirosis is a zoonotic disease that is highly prevalent in tropical countries; uveitis is one of the manifestations of leptospirosis. The leptospiral aetiology of uveitis is difficult to predict because of overlapping clinical symptoms with uveitis due to other causes. The objective of this study was to evaluate the leptospiral haemin-binding protein HbpA as a diagnostic antigen for the serodiagnosis of leptospiral uveitis. Serum samples from patients, clinically diagnosed with leptospiral uveitis, were tested by ELISA for anti-HbpA antibodies and compared against the ‘gold standard’ microscopic agglutination test (MAT). Non-leptospiral uveitis and normal healthy individuals were used as controls. A total of 60 serum samples from patients suffering from leptospiral uveitis were studied, obtained from Aravind Eye Hospital, Madurai. Anti-HbpA IgG antibodies were detected in 92 % of patients clinically diagnosed with leptospiral uveitis, indicating that it is more sensitive than MAT, which had a seropositivity of only 50 %, and better than the commercially available Pan Bio IgM ELISA (81 %). The mean anti-HbpA antibody titre was significantly higher in leptospiral uveitis patients compared with controls (P<0.05). The antigen showed negligible cross-reactivity with non-leptospiral uveitis samples and cataract controls. We conclude that HbpA IgG ELISA identified cases of uveitis with leptospirosis aetiology and proved to be useful in differentiating them from other forms of uveitis.

Since the beginning of the HIV epidemic, there has been a significant increase in the number of histoplasmosis cases in Ceará, a state in north-east Brazil. The lack of epidemiological data on the genotypes circulating in the north-east region shows the importance of more detailed studies on the molecular epidemiology of Histoplasma capsulatum var. capsulatum in this region. Different molecular techniques have been used to better characterize the genetic profile of H. capsulatum var. capsulatum strains. The aim of this study was to analyse the genetic diversity of H. capsulatum var. capsulatum isolates in Fortaleza, the capital of Ceará, through the sequencing of the internal transcribed spacer (ITS)1-5.8S-ITS2 region, and establish the molecular profile of these isolates, along with strains from south-east Brazil, by RAPD analysis, featuring the different clusters in those regions. The isolates were grouped into two clusters. Cluster 1 included strains from the south-east and north-east regions with separation of isolates into three distinct subgroups (subgroups 1a, 1b and 1c). Cluster 2 included only samples from north-east Brazil. Sequencing of the ITS1–5.8S–ITS2 region allowed the detection of two major clades, which showed geographical correlation between them and their subgroups. Therefore, it can be concluded that the H. capsulatum var. capsulatum isolates from Ceará have a high degree of genetic polymorphism. The molecular data also confirm that populations of this fungus are composed of different genotypes in Brazil and worldwide.

The aim of the present study was to evaluate the in vitro activity of baicalein, the flavone constituent of Scutellaria baicalensis, and synergism of the combination of baicalein and fluconazole against Candida albicans, Candida tropicalis and Candida parapsilosis. The MIC50 (lowest concentration at which there was 50 % inhibition of growth) of baicalein alone against six Candida strains ranged from 13 to 104 µg ml−1. For the three species tested, exposure to baicalein at the MIC50 concentrations obtained for each strain resulted in a high loss of viability. The fluconazole plus baicalein combination markedly reduced the MICs of both drugs for all three strains analysed. In addition, a synergistic effect between baicalein and fluconazole was observed for C. parapsilosis in terms of MIC50 (fractional inhibitory concentration index = 0.207). Scanning electron microscopy analysis revealed that yeast cells exposed to baicalein at MIC50 produced a profusely flocculent extracellular material, resembling a biofilm-like structure. In conclusion, these results showed the antifungal capability of baicalein against Candida species and highlight a promising role of baicalein when used in combination with fluconazole against Candida infections.

Leishmaniasis is one of the major tropical parasitic diseases, and the condition ranges in severity from self-healing cutaneous lesions to fatal visceral manifestations. There is no vaccine available against visceral leishmaniasis (VL) (also known as kala-azar in India), and current antileishmanial drugs face major drawbacks, including drug resistance, variable efficacy, toxicity and parenteral administration. We report here that n-hexane fractions of Artemisia annua leaves (AAL) and seeds (AAS) possess significant antileishmanial activity against Leishmania donovani promastigotes, with GI50 of 14.4 and 14.6 µg ml−1, respectively, and the IC50 against intracellular amastigotes was found to be 6.6 and 5.05 µg ml−1, respectively. Changes in the morphology of promastigotes and growth reversibility analysis following treatment confirmed the leishmanicidal effect of the active fractions, which presented no cytotoxic effect on mammalian cells. The antileishmanial activity was mediated via apoptosis, as evidenced by externalization of phosphatidylserine, in situ labelling of DNA fragments by terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling (TUNEL) and cell-cycle arrest at the sub-G0/G1 phase. High-performance thin-layer chromatography (HPTLC) fingerprinting showed that the content of artemisinin in crude bioactive extracts (~1.4 µg per 100 µg n-hexane fraction) was too low to account for the observed antileishmanial activity. Characterization of the active constituents by GC-MS showed that α-amyrinyl acetate, β-amyrine and derivatives of artemisinin were the major constituents in AAL and cetin, EINECS 211-126-2 and artemisinin derivatives in AAS. Our findings indicate the presence of antileishmanial compounds besides artemisinin in the n-hexane fractions of A. annua leaves and seeds.

Silver nanoparticles (nano-Ags), which have well-known antimicrobial properties, are used extensively in various medical and general applications. In this study, the combination effects between nano-Ags and the conventional antibiotics ampicillin, chloramphenicol and kanamycin against various pathogenic bacteria were investigated. The MIC and fractional inhibitory concentration index (FICI) were determined to confirm antibacterial susceptibility and synergistic effects. The results showed that nano-Ags possessed antibacterial effects and synergistic activities. The antibiofilm activities of nano-Ags alone or in combination with antibiotics were also investigated. Formation of biofilm is associated with resistance to antimicrobial agents and chronic bacterial infections. The results indicated that nano-Ags also had antibiofilm activities. To understand these effects of nano-Ags, an ATPase inhibitor assay, permeability assay and hydroxyl radical assay were conducted. The antibacterial activity of nano-Ags was influenced by ATP-associated metabolism rather than by the permeability of the outer membrane. Additionally, nano-Ags generated hydroxyl radicals, a highly reactive oxygen species induced by bactericidal agents. It was concluded that nano-Ags have potential as a combination therapeutic agent for the treatment of infectious diseases by bacteria.

Vibrio cholerae O1 biotype El Tor (ET), causing the seventh cholera pandemic, was recently replaced in Bangladesh by an altered ET possessing ctxB of the Classical (CL) biotype, which caused the first six cholera pandemics. In the present study, V. cholerae O1 strains associated with endemic cholera in Dhaka between 2006 and 2011 were analysed for major phenotypic and genetic characteristics. Of 54 representative V. cholerae isolates tested, all were phenotypically ET and showed uniform resistance to trimethoprim/sulfamethoxazole (SXT) and furazolidone (FR). Resistance to tetracycline (TE) and erythromycin (E) showed temporal fluctuation, varying from year to year, while all isolates were susceptible to gentamicin (CN) and ciprofloxacin (CIP). Year-wise data revealed erythromycin resistance to be 33.3 % in 2006 and 11 % in 2011, while tetracycline resistance accounted for 33, 78, 0, 100 and 27 % in 2006, 2007, 2008, 2009 and 2010, respectively; interestingly, all isolates tested were sensitive to TE in 2011, as observed in 2008. All V. cholerae isolates tested possessed genetic elements such as SXT, ctxAB, tcpA ET, rstR ET and rtxC; none had IntlI (Integron I). Double mismatch amplification mutation assay (DMAMA)-PCR followed by DNA sequencing and analysis of the ctxB gene revealed a point mutation at position 58 (C→A), which has resulted in an amino acid substitution from histidine (H) to asparagine (N) at position 20 (genotype 7) since 2008. Although the multi-resistant strains having tetracycline resistance showed minor genetic divergence, V. cholerae strains were clonal, as determined by a PFGE (NotI)-based dendrogram. This study shows 2008–2010 to be the time of transition from ctxB genotype 1 to genotype 7 in V. cholerae ET causing endemic cholera in Dhaka, Bangladesh.

Trichosporon asahii is the causative agent of both superficial and deep-seated infections of increasing morbidity and mortality. Urinary tract infections (UTIs) due to T. asahii, frequently associated with indwelling medical devices, have been reported over the years. However, few studies have specifically focused on the genotypic diversity of T. asahii isolates from urine specimens from intensive care units (ICUs), let alone potential virulence factors and antifungal susceptibility testing. In the present study, 23 T. asahii isolates were collected from UTI patients in ICUs between January 2008 and January 2012. Three genotypes (I, III, IV) were determined based on the combination of internal transcribed spacer and intergenic spacer locus PCR. Protease, phospholipase and haemolysin production was assessed by halo formation on corresponding agar plates. Only haemolytic activity was observed to varying degrees. Neither protease nor phospholipase was detectable. Biofilm formation on polystyrene surfaces was detected through a formazan salt reduction assay. All clinical isolates had the ability to form biofilm. In contrast to the susceptibility of planktonic T. asahii cells to clinically used amphotericin B, 5-flucytosine, fluconazole, itraconazole and voriconazole, a remarkable rise in the MICs of these for biofilm T. asahii cells was observed. Our results suggested that genotype IV was the most prevalent genotype among T. asahii isolates from ICUs in China. Haemolysin and biofilm might contribute to the pathogenicity and recurrence of T. asahii-related UTIs. Although triazoles, especially voriconazole, were effective against planktonic T. asahii cells, they failed to treat preformed biofilms.

Zygomycosis is characterized by tissue invasion with broad, non-septate hyphae of species such as Rhizopus, Rhizomucor, Lichtheimia (Absidia) and Basidiobolus. Basidiobolus ranarum usually causes subcutaneous infection, and gastrointestinal manifestations in immunocompetent patients have rarely been reported. It is difficult to diagnose gastrointestinal basidiobolomycosis because of the non-specific clinical presentation and the absence of a definite risk factor. This study identified 14 cases of gastrointestinal basidiobolomycosis, all of which were diagnosed after surgery by characteristic histopathological findings. Diagnosis of this disease requires a high index of suspicion in patients presenting with abdominal symptoms, fever, gastrointestinal mass and eosinophilia accompanied by a high erythrocyte sedimentation rate.

Coxiella burnetii is the causative bacterium of Q fever, a vaccine-preventable infection. C. burnetii is an unusual cause of culture-negative endocarditis. Here, we present a case of Q fever native valve endocarditis that developed in a young man despite prior vaccination. Definitive diagnosis was difficult and required C. burnetii-specific PCR testing.