Trends in candidemia and antifungal susceptibility in a university hospital in Northern Ireland 2001-2006

Objectives: To describe the species distribution and antifungal susceptibility trends for documented episodes of candidemia at the Royal Hospitals, Belfast, 2001-2006. Methods: Laboratory-based retrospective observational study of all episodes of candidemia. Results: There were 151 episodes of candidemia. The species recovered were: 96 C. albicans; 26 C. glabrata; 18 C. parapsilosis; five C. tropicalis; four C. guilliermondii; one C. famata and one C. dubliniensis. We separated the data into two periods 2001-2003 and 2004-2006; contrary to the findings of other investigators, there was a notable trends toward increasing frequency of C. albicans and decreasing frequency of non-albicans species over time. Although the proportion of C. albicans, C. parapsilosis and C. tropicalis isolates susceptible to fluconazole was unchanged over time, a trend of decreased susceptibility of C. glabrata to fluconazole was noted over the six-year period. Overall, 73% and 7.7% of C. glabrata isolates had susceptible-dose-dependent and resistant phenotypes, respectively. The percentage of C. glabrata isolates susceptible to fluconazole (MIC

Medusins: a new class of antimicrobial peptides from the skin secretions of phyllomedusine frogs

Natural drug discovery represents an area of research with vast potential. The investigation into the use of naturally-occurring peptides as potential therapeutic agents provides a new “chemical space” for the procurement of drug leads. Intensive and systematic studies on the broad-spectrum antimicrobial peptides found in amphibian skin secretions are of particular interest in the quest for new antibiotics to treat multiple drug-resistant bacterial infections. Here we report the molecular cloning of the biosynthetic precursor-encoding cDNAs and respective mature peptides representing a novel group of antimicrobial peptides from the skin secretions of representative species of phyllomedusine leaf frogs: the Central American red-eyed leaf frog (Agalychnis callidryas), the South American orange-legged leaf frog (Phyllomedusa hypochondrialis) and the Giant Mexican leaf frog, (Pachymedusa dacnicolor). Each novel peptide possessed the highly-conserved sequence, LGMIPL/VAISAISA/SLSKLamide, and each exhibited activity against the Gram-positive bacterium, Staphylococcus aureus and the yeast, Candida albicans, but all were devoid of haemolytic effects at concentrations up to and including the MICs for both organisms. The novel peptide group were named medusins, derived from the name of the hylid frog sub-family, Phyllomedusinae, to which all species investigated belong. These data clearly demonstrate that comparative studies of the skin secretions of phyllomedusine frogs can continue to produce novel peptides that have the potential to be leads in the development of new and effective antimicrobials.

Senegalin: a novel antimicrobial/myotropic hexadecapeptide from the skin secretion of the African running frog, Kassina senegalensis

Amphibian skin is a rich and unique source of novel bioactive peptides most of which are endowed with either antimicrobial or pharmacological properties. Here we report the identification and structural characterization of a novel peptide, named senegalin, which possesses both activities. Senegalin is a hexadecapeptide amide (FLPFLIPALTSLISSL-NH2) of unique primary structure found in the skin secretion of the African running frog, Kassina senegalensis. The structure of the biosynthetic precursor of senegalin, deduced from cloned skin cDNA, consists of 76 amino acid residues and displays the typical domain organization of an amphibian skin peptide precursor. Both natural senegalin and its synthetic replicate
displayed antimicrobial and myotropic activities. Senegalin was active against Staphylococcus aureus (MIC 50µM) and Candida albicans (MIC 150µM) but was nonhaemolytic at concentrations up to and including 150µM. In contrast, senegalin induced a dose-dependent contraction of rat urinary bladder smooth muscle (EC50 2.9nM) and a dosedependent relaxation of rat tail artery smooth muscle (EC50 37.7nM). Senegalin thus represents a prototype biologically-active amphibian skin peptide and illustrates the fact thatamphibian skin secretion peptidomes continue to be unique sources of such molecules.

IQ-motif peptides as novel anti-microbial agents

The IQ-motif is an amphipathic, often positively charged, a-helical, calmodulin binding sequence found in a number of eukaryote signalling, transport and cytoskeletal proteins. They share common biophysical characteristics with established, cationic a-helical antimicrobial peptides, such as the human cathelicidin LL-37. Therefore, we tested eight peptides encoding the sequences of IQ-motifs derived from the human cytoskeletal scaffolding proteins IQGAP2 and IQGAP3. Some of these peptides were able to inhibit the growth of Escherichia coli and Staphylococcus aureus with minimal inhibitory concentrations (MIC) comparable to LL-37. In addition some IQ-motifs had activity against the fungus Candida albicans. This antimicrobial activity is combined with low haemolytic activity (comparable to, or lower than, that of LL-37). Those IQ-motifs with anti-microbial activity tended to be able to bind to lipopolysaccharide. Some of these were also able to permeabilise the cell membranes of both Gram positive and Gram negative bacteria. These results demonstrate that IQ-motifs are viable lead sequences for the identification and optimisation of novel anti-microbial peptides. Thus, further investigation of the anti-microbial properties of this diverse group of sequences is merited.

Arbuscular mycorrhizal fungi and collembola non-additively increase soil aggregation

Soil aggregation is a principal ecosystem process mediated by soil biota. Collembola and arbuscular mycorrhizal (AM) fungi are important groups in the soil, and can interact in various ways. Few studies have examined collembola effects on soil aggregation, while many have quantified AM effects. Here, we asked if collembola have any effect on soil aggregation, and if they alter AM fungi-mediated effects on soil aggregation.

We carried out a factorial greenhouse study, manipulating the presence of both collembola and AM fungi, using two different plant species, Sorghum vulgare and Daucus carota. We measured root length and biomass, AMF (and non-AMF) soil hyphal length, root colonization, and collembolan populations, and quantified water stable soil aggregates (WSA) in four size classes.

Soil exposed to growth of AMF hyphae and collembola individually had higher WSA than control treatments. Moreover, the interaction effects between AMF and collembola were significant, with nonadditive increases in the combined application compared to the single treatments.

Our findings show that collembola can play a crucial role in maintaining ecological sustainability through promoting soil aggregation, and point to the importance of considering organism interactions in understanding formation of soil structure. (C) 2011 Elsevier Ltd. All rights reserved.

Two peptides, TsAP-1 and TsAP-2, from the venom of the Brazilian yellow scorpion, Tityus serrulatus: evaluation of their antimicrobial and anticancer activities

Here we report two novel 17-mer amidated linear peptides (TsAP-1 and TsAP-2) whose structures were deduced from cDNAs cloned from a venom-derived cDNA library of the Brazilian yellow scorpion, Tityus serrulatus. Both mature peptides were structurally-characterised following their location in chromatographic fractions of venom and synthetic replicates of each were subjected to a range of biological assays. The peptides were each active against model test micro-organisms but with different potencies. TsAP-1 was of low potency against all three test organisms (MICs 120-160µM), whereas TsAP-2 was of high potency against the Gram-positive bacterium, Staphylococcus aureus (MIC 5µM) and the yeast, Candida albicans (10µM). Haemolytic activity of TsAP-1 was low (4% at 160µM) and in contrast, that of TsAP-2 was considerably higher (18% at 20µM). Substitution of four neutral amino acid residues with Lys residues in each peptide had dramatic effects on their antimicrobial potencies and haemolytic activities, particularly those of TsAP-1. The MICs of the enhanced cationic analogue (TsAP-S1) were 2.5µM for S.aureus/C.albicans and 5µM for E.coli but with an associated large increase in haemolytic activity (30% at 5µM). The same Lys residue substitutions in TsAP-2 produced a dramatic effect on its MIC for E.coli lowering this from >320µM to 5µM. TsAP-1 was ineffective against three of the five human cancer cell lines tested while TsAP-2 inhibited the growth of all five. Lys residue substitution of both peptides enhanced their potency against all five cell lines with TsAp-S2 being the most potent with IC50 values ranging between 0.83 and 2.0 µM. TsAP-1 and TsAP-2 are novel scorpion venom peptides with broad spectrum antimicrobial and anticancer cell activities the potencies of which can be significantly enhanced by increasing their cationicity.

Hydrogel-forming microneedle arrays exhibit antimicrobial properties:potential for enhanced patient safety

We describe, for the first time, the microbial characterisation of hydrogel-forming polymeric microneedle arrays and the potential for passage of microorganisms into skin following microneedle penetration. Uniquely, we also present insights into the storage stability of these hydroscopic formulations, from physical and microbiological viewpoints, and examine clinical performance and safety in human volunteers. Experiments employing excised porcine skin and radiolabelled microorganisms showed that microorganisms can penetrate skin beyond the stratum corneum following microneedle puncture. Indeed, the numbers of microorganisms crossing the stratum corneum following microneedle puncture were greater than 105 cfu in each case. However, no microorganisms crossed the epidermal skin. When using a 21G hypodermic needle, more than 104 microorganisms penetrated into the viable tissue and 106 cfu of Candida albicans and Staphylococcus epidermidis completely crossed the epidermal skin in 24 h. The hydrogel-forming materials contained no microorganisms following de-moulding and exhibited no microbial growth during storage, while also maintaining their mechanical strength, apart from when stored at relative humidities of 86%. No microbial penetration through the swelling microneedles was detectable, while human volunteer studies confirmed that skin or systemic infection is highly unlikely when polymeric microneedles are used for transdermal drug delivery. Since no pharmacopoeial standards currently exist for microneedle-based products, the exact requirements for a proprietary product based on hydrogel-forming microneedles are at present unclear. However, we are currently working towards a comprehensive specification set for this microneedle system that may inform future developments in this regard.

Dietary exposure to aflatoxin and fumonisin among Tanzanian children as determined using biomarkers of exposure

SCOPE: The study aims to evaluate the status of dietary exposure to aflatoxin and fumonisin in young Tanzanian children, using previously validated biomarkers of exposure. METHODS AND RESULTS: A total of 148 children aged 12-22 months, were recruited from three geographically distant villages in Tanzania; Nyabula, Kigwa, and Kikelelwa. Plasma aflatoxin-albumin adducts (AF-alb) and urinary fumonisin B1 (UFB1) were measured by ELISA and LC-MS, respectively. AF-alb was detectable in 84% of children, was highest in fully weaned children (p <0.01) with higher levels being associated with higher maize intake (p <0.05). AF-alb geometric mean (95% CI) was 43.2 (28.7-65.0), 19.9 (13.5-29.2), and 3.6 (2.8-4.7) pg/mg albumin in children from Kigwa, Nyabula, and Kikelelwa, respectively. UFB1 was detectable in 96% of children and the level was highest in children who had been fully weaned (p <0.01). The geometric UFB1 mean (95% CI) was 327.2 (217.1-493.0), 211.7 (161.1-278.1), and 82.8 (58.3-117.7) pg/mL in Kigwa, Nyabula, and Kikelelwa, respectively. About 82% of all the children were exposed to both mycotoxins. CONCLUSION: Young children in Tanzania are chronically exposed to both aflatoxin and fumonisin through contaminated diet, although the level of exposure varies markedly between the three villages studied.

Hylaranins: prototypes of a new class of amphibian antimicrobial peptide from the skin secretion of the oriental broad-folded frog, Hylarana latouchii

Amphibian skin secretions contain a broad spectrum of biologically active compounds, particularly antimicrobial peptides, which are considered to constitute a first line of defence against bacterial infection. Here we describe the identification of two prototype peptides representing a novel structural class of antimicrobial peptide from the skin secretion of the oriental broad-folded frog, Hylarana latouchii. Named hylaranin-L1 (GVLSAFKNALPGIMKIIVamide) and hylaranin-L2 (GVLSVIKNALPGIMRFIAamide), both peptides consist of 18 amino acid residues, are C-terminally amidated and are of unique primary structures. Their primary structures were initially deduced by MS/MS fragmentation sequencing from reverse-phase HPLC fractions of skin secretion that demonstrated antimicrobial activity. Subsequently, their precursor-encoding cDNAs were cloned from a skin secretion-derived cDNA library and their primary structures were confirmed unequivocally. Synthetic replicates of both peptides exhibited broad-spectrum antimicrobial activity with mean inhibitory concentrations (MICs) of 34 µM against Gram-negative Escherichia coli, 4.3 µM against Gram-positive Staphylococcus aureus and 4–9 µM against the yeast, Candida albicans. Both peptides exhibited little haemolytic activity (<6 %) at the MICs for S. aureus and C. albicans. Amphibian skin secretions thus continue to provide novel antimicrobial peptide structures that may prove to be lead compounds in the design of new classes of anti-infection therapeutics.

AcT-2: A Novel Myotropic and Antimicrobial Type 2 Tryptophyllin from the Skin Secretion of the Central American Red-Eyed Leaf Frog, Agalychnis callidryas

Tryptophyllins are a diverse family of amphibian peptides originally found in extracts of phyllomedusine frog skin by chemical means. Their biological activities remain obscure. Here we describe the isolation and preliminary pharmacological characterization of a novel type 2 tryptophyllin, named AcT-2, from the skin secretion of the red-eyed leaf frog, Agalychnis callidryas. The peptide was initially identified during smooth muscle pharmacological screening of skin secretion HPLC fractions and the unique primary structure—GMRPPWF-NH2—was established by both Edman degradation and electrospray MS/MS fragmentation sequencing. A. cDNA encoding the biosynthetic precursor of AcT-2 was successfully cloned from a skin secretion-derived cDNA library by means of RACE PCR and this contained an open-reading frame consisting of 62 amino acid residues with a single AcT-2 encoding sequence located towards the C-terminus. A synthetic replicate of AcT-2 was found to relax arterial smooth muscle (EC50 = 5.1 nM) and to contract rat urinary bladder smooth muscle (EC50 = 9.3 μM). The peptide could also inhibit the growth of the microorganisms, Staphylococcus aureus, (MIC = 256 mg/L) Escherichia coli (MIC = 512 mg/L), and Candida albicans (128 mg/L). AcT-2 is thus the first amphibian skin tryptophyllin found to possess both myotropic and antimicrobial activities.

Balteatide: A Novel Antimicrobial Decapeptide from the Skin Secretion of the Purple-Sided Leaf Frog, Phyllomedusa baltea

The skin secretions of Neotropical phyllomedusine leaf frogs have proven to be a rich source of biologically-active peptides, including antimicrobials. The major families of antimicrobial peptides (AMPs) reported are the dermaseptins and phylloseptins and the minor families, the dermatoxins, phylloxins, plasticins, distinctins and the medusins. Here, we report a novel AMP of 10 amino acid residues (LRPAILVRIKamide), named balteatide, from the skin secretion of wild Peruvian purple-sided leaf frogs, Phyllomedusa baltea. Balteatide was found to exhibit a 90% sequence identity with sauvatide, a potent myotropic peptide from the skin secretion of Phyllomedusa sauvagei. However, despite both peptides exhibiting only a single amino acid difference (I/T at position 9), sauvatide is devoid of antimicrobial activity and balteatide is devoid of myotropic activity. Balteatide was found to have differential activity against the Gram-positive bacterium, Staphylococcus aureus, the Gram-negative bacterium, Escherichia coli and the yeast, Candida albicans, and unusually for phyllomedusine frog skin AMPs, was most potent (MIC 32 mg/L) against the yeast. Balteatide was also devoid of haemolytic activity up to concentrations of 512 mg/L. Phyllomedusine frog skin secretions thus continue to provide novel AMPs, some of which may provide templates for the rational design of new classes of anti-infective therapeutics.

Feleucin-BO1: A Novel Antimicrobial Non-Apeptide Amide from the Skin Secretion of the Toad, Bombina orientalis, and Design of a Potent Broad-Spectrum Synthetic Analogue, Feleucin-K3

Feleucins-BV1 and -BV2 are recently-described prototypes of a novel antimicrobial nonapeptide (AMP) family identified in the skin secretion of the bombinid toad, Bombina variegata. They are encoded on different precursors that also encode a novel bombinin. Here we describe the identification of feleucin-BO1 (FLGLLGSLLamide) which is co-encoded with a different novel bombinin, named feleucin precursor-associated bombinin (FPA-bombinin-BO), from the skin secretion of Bombina orientalis. Synthetic feleucin-BO1 displayed activity against a reference Gram-positive bacterium. Staphylococcus aureus (MIC 34 μM) but was inactive (> 250 μM) against the Gram-negative bacterium, Escherichia coli, and the yeast, Candida albicans. This pattern of activity was similar to that of the prototypes. Design and synthesis of a cationicity-enhanced analogue, feleucin-K3 (F-K3), in which the amino acid residues at positions 3 (G), 6 (G) and 7 (S) of feleucin-BO1 were substituted with Lys (K) residues, resulted in a peptide with significantly-enhanced potency and spectrum of activity. The MICs of F-K3 against the reference microorganisms were 7 μM (S. aureus), 14 μM (E. coli) and 7 μM (C. albicans). These data indicate that the skin secretions of amphibians can continue to provide novel peptide templates for the rational design of analogues with possible therapeutic utility.

Deoxynivalenol exposure assessment in young children in Tanzania

Scope: This study assessed deoxynivalenol (DON) exposure in children from three geographic locations within Tanzania, over three time points in 1 year, using a urinary biomarker of exposure.

Methods and results: A total of 166 children aged 6-14 months were studied at a maize harvest and followed up twice at 6-month intervals. On two consecutive days, morning urine was collected from each child and urinary DON was measured using an LC-MS method, with and without beta-glucuronidase hydrolysis in order to assess free DON (fDON) and glucuronide DON. Overall, urinary DON increased significantly along with the three visits (geometric mean 1.1, 2.3, and 5.7 ng/mL, at visits 1, 2, and 3, respectively, p <0.01). fDON was 22% of urinary total DON. Urinary DON excretion rate was 74% in village Kikelelwa based on food DON level and food consumption. Assuming 360 mL of urine excreted per day, 10, 19, and 29% of children at visits 1, 2, and 3, respectively, exceeded the provisional maximum tolerable daily intake of 1000 ng/kg b.w./day.

Conclusion: Young children in Tanzania are chronically exposed to DON due to eating contaminated maize, although exposure levels varied markedly by region and season.

Quantitative correlation of aflatoxin biomarker with dietary intake of aflatoxin in Tanzanian children

The association between aflatoxin intake from maize-based weaning food and aflatoxin albumin adducts (AF-alb) was investigated in 148 Tanzanian children aged between 12 and 22 months, at 2 visits 6 months apart. At the first visit (storage season) there was a significant correlation at the individual level between AF-alb (geometric mean 43.2 pg/mg albumin) and aflatoxin intake (geometric mean 81.7 ng/kg b.w./d) through maize-based weaning food (r = 0.51, p < 0.01). Overall, this correlation was r = 0.43 (p < 0.01). The AF-alb level in weaning-age children in Tanzania closely reflects aflatoxin intake from maize in weaning food. Exposure levels suggest children may be at risk from aflatoxin associated health effects.

A Prospective Study of Growth and Biomarkers of Exposure to Aflatoxin and Fumonisin during Early Childhood in Tanzania

BACKGROUND: Aflatoxin and fumonisin are toxic food contaminants. Knowledge about effects of their exposure and co-exposure on child growth is inadequate.

OBJECTIVE: To investigate the association between child growth and aflatoxin and fumonisin exposure in Tanzania.

METHODS: A total of 166 children were recruited at 6 to 14 months of age and studied at recruitment, and at the sixth and twelfth month following recruitment. Blood and urine samples were collected and analysed for plasma aflatoxin albumin adducts (AF-alb) using ELISA, and urinary fumonisin B1 (UFB1) using LC-MS, respectively. Anthropometric measurements were taken and growth index Z-scores were computed.

RESULTS: AF-alb geometric mean concentrations (95% confidence intervals) were 4.7 (3.9, 5.6), 12.9 (9.9, 16.7) and 23.5 (19.9, 27.7) pg/mg albumin at recruitment, six months, and 12 months from recruitment, respectively. At these respective sampling times, geometric mean UFB1 concentrations (95% CI) were 313.9 (257.4, 382.9), 167.3 (135.4, 206.7) and 569.5 (464.5, 698.2) pg/mL urine, and the prevalence of stunted children were 44%, 55% and 56%, respectively. UFB1 concentrations at recruitment were negatively associated with length for age Z-scores (LAZ) at six months (p = 0.016) and at 12 months from recruitment (p = 0.014). The mean UFB1 of the three sampling times (at recruitment, at six and 12 months from recruitment) in each child was negatively associated with LAZ (p < 0.001) and length velocity (p = 0.004) at 12 months from recruitment. The negative association between AF-alb and child growth did not reach statistical significance.

CONCLUSIONS: Exposure to fumonisin alone, or co-exposure with aflatoxins may contribute to child growth impairment.