Therapeutic Effect of an Antibody-Derived Peptide in a Galleria mellonella Model of Systemic Candidiasis.

The synthetic peptide T11F (TCRVDHRGLTF), with sequence identical to a fragment of the constant region of human IgM, and most of its alanine-substituted derivatives proved to possess a significant candidacidal activity in vitro. In this study, the therapeutic efficacy of T11F, D5A, the derivative most active in vitro, and F11A, characterized by a different conformation, was investigated in Galleria mellonella larvae infected with Candida albicans. A single injection of F11A and D5A derivatives, in contrast with T11F, led to a significant increase in survival of larvae injected with a lethal inoculum of C. albicans cells, in comparison with infected animals treated with saline. Peptide modulation of host immunity upon C. albicans infection was determined by hemocyte analysis and larval histology, highlighting a different immune stimulation by the studied peptides. F11A, particularly, was the most active in eliciting nodule formation, melanization and fat body activation, leading to a better control of yeast infection. Overall, the obtained data suggest a double role for F11A, able to simultaneously target the fungus and the host immune system, resulting in a more efficient pathogen clearance.

Proteobacteria Overgrowth and Butyrate-Producing Taxa Depletion in the Gut Microbiota of Glycogen Storage Disease Type 1 Patients.

A life-long dietary intervention can affect the substrates' availability for gut fermentation in metabolic diseases such as the glycogen-storage diseases (GSD). Besides drug consumption, the main treatment of types GSD-Ia and Ib to prevent metabolic complications is a specific diet with definite nutrient intakes. In order to evaluate how deeply this dietary treatment affects gut bacteria, we compared the gut microbiota of nine GSD-I subjects and 12 healthy controls (HC) through 16S rRNA gene sequencing; we assessed their dietary intake and nutrients, their microbial short chain fatty acids (SCFAs) via gas chromatography and their hematic values. Both alpha-diversity and phylogenetic analysis revealed a significant biodiversity reduction in the GSD group compared to the HC group, and highlighted profound differences of their gut microbiota. GSD subjects were characterized by an increase in the relative abundance of Enterobacteriaceae and Veillonellaceae families, while the beneficial genera Faecalibacterium and Oscillospira were significantly reduced. SCFA quantification revealed a significant increase of fecal acetate and propionate in GSD subjects, but with a beneficial role probably reduced due to unbalanced bacterial interactions; nutritional values correlated to bacterial genera were significantly different between experimental groups, with nearly opposite cohort trends.

Phenylketonuria Diet Promotes Shifts in Firmicutes Populations.

Low-phenylalanine diet, the mainstay of treatment for phenylketonuria (PKU), has been shown to increase glycemic index and glycemic load, affecting the availability of substrates for microbial fermentation. Indeed, changes in the PKU gut microbiota compared with healthy controls have been previously reported. In this study we compared the gut microbial communities of children with PKU and with mild hyperphenylalaninemia (MHP, unrestricted diet). For each group, we enrolled 21 children (4-18 years old), for a total dataset of 42 subjects. We assessed dietary intake and performed gut microbiota analysis by sequencing the V3-V4 hypervariable regions of the 16S rRNA gene. Short chain fatty acids (SCFAs) were quantified by gas chromatographic analysis. While alpha-diversity analysis showed no significant differences between PKU and MHP groups, microbial community analysis highlighted a significant separation of the gut microbiota according to both unweighted (p = 0.008) and weighted Unifrac distances (p = 0.033). Major differences were seen within the Firmicutes phylum. Indeed, PKU children were depleted in Faecalibacterium spp. and enriched in Blautia spp. and Clostridium spp (family Lachnospiraceae). We found a divergent response of members of the Firmicutes phylum with respect to daily glycemic index, higher in PKU children. Faecalibacterium prausnitzii, unclassified Ruminococcaceae and, to a lesser extent Roseburia spp. negatively correlated with glycemic index, whereas unclassified Lachnospiraceae were positively associated. Indicator species analysis suggested F. prausnitzii be related to MHP status and Ruminococcus bromii to be associated with PKU. Despite PKU children having a higher vegetable and fiber intake, resembling a vegan diet, their gut microbial profile is different from the microbiota reported in the literature for individuals consuming a high-fiber/low-protein diet. Indeed, beneficial microorganisms, such as F. prausnitzii, considered a biomarker for a healthy status and one of the main butyrate producers, are depleted in PKU gut microbiota. We suggest that both the quality and quantity of carbohydrates ingested participate in determining the observed Firmicutes shifts on the PKU population.

Repurposing Pilocarpine Hydrochloride for Treatment of Candida albicans Infections.

Acetylcholine modulates the virulence of Candidaalbicans and regulates an appropriate immune response to infection in a Galleria mellonella infection model. Indeed, the evidence suggests that C. albicans possesses a functional cholinergic receptor that can regulate filamentous growth and biofilm formation. Furthermore, G. mellonella immune cell subsets possess repertories of cholinergic receptors which regulate an effective and appropriate cellular immune response to C. albicans infection. This study aimed to investigate the cholinergic receptor subtype involved in regulation of filamentous growth and biofilm formation by C. albicans and determine the roles of cholinergic receptors in modulation of G. mellonella immune cell subsets. The general muscarinic receptor agonist, pilocarpine hydrochloride, inhibited C. albicans biofilm formation and pathogenicity, a phenomenon that could be reversed using the general muscarinic receptor antagonist, scopolamine. Pilocarpine hydrochloride protected G. mellonella larvae from C. albicans infection via inhibition of C. albicans filamentation and appropriate regulation of cellular immunity. However, scopolamine abrogated the capacity of pilocarpine hydrochloride to protect G. mellonella larvae from C. albicans infection. Furthermore, acetylcholine and pilocarpine hydrochloride exhibited differential modulatory capabilities on Galleria mellonella hemocyte responses to C. albicans The data in this article demonstrate that a muscarinic receptor modulates C. albicans filamentation and biofilm formation. Furthermore, the results suggest that G. mellonella hemocyte subsets possess unique repertoires of cholinergic receptors that regulate their differentiation, activation, and function in contrasting manners. Therefore, targeting cholinergic receptors by repurposing currently licensed cholinergic drugs may offer novel therapeutic solutions for the prevention or treatment of fungal infections.IMPORTANCECandida albicans is the most common human fungal pathogen with an estimated crude mortality rate of 40%. The ability of the organism to switch from the yeast to hyphal form and produce biofilms are important virulence factors. C. albicans infections are combatted by the host immune system. However, Candida triggers a strong inflammatory response that, if not appropriately regulated, can damage host tissues. Therefore, it is important that the host immune response eliminates the fungus but limits tissue damage. This study provides evidence that targeting cholinergic receptors cannot only curb the virulence of C. albicans by inhibiting filamentous growth and biofilm formation but can also appropriately regulate the host immune response to induce rapid clearance with limited damage to vital tissues. This article provides evidence that repurposing licensed drugs that target cholinergic receptors may offer novel therapeutic solutions for the prevention or treatment of fungal infections.

Antenatal Microbial Colonization of Mammalian Gut.

The widely accepted dogma of intrauterine sterility and initial colonization of the newborn during birth has been blurred by recent observations of microbial presence in meconium, placenta, and amniotic fluid. Given the importance of a maternal-derived in utero infant seeding, it is crucial to exclude potential environmental or procedural contaminations and to assess fetal colonization before parturition. To this end, we analyzed sterilely collected intestinal tissues, placenta, and amniotic fluid from rodent fetuses and tissues from autoptic human fetuses. Total bacterial DNA was extracted from collected samples and analyzed by Next Generation Sequencing (NGS) techniques using hypervariable 16S ribosomal RNA (rRNA) regions (V3-V4). Colonizing microbes were visualized in situ, using labeled probes targeting 16S ribosomal DNA by fluorescent in situ hybridization. The NGS analysis showed the presence of pioneer microbes in both rat and human intestines as well as in rodent placentas and amniotic fluids. Microbial communities showed fetus- and dam-dependent clustering, confirming the high interindividual variability of commensal microbiota even in the antenatal period. Fluorescent in situ hybridization analysis confirmed the microbes' presence in the lumen of the developing gut. These findings suggest a possible antenatal colonization of the developing mammalian gut.

Up-Regulation of Antimicrobial Peptides Gallerimycin and Galiomicin in Galleria mellonella Infected with Candida Yeasts Displaying Different Virulence Traits.

Galleria mellonella has been described as a cheap and an easy-to-reproduce model for the study of fungal infections. We hypothesized that yeasts with higher virulence potential decrease survival and significantly trigger an immune response in G. mellonella through the regulation of innate immunity-related genes encoding antimicrobial peptides (AMPs) such as gallerimycin and galiomicin. Candida albicans SC5314 and Candida dubliniensis CBS 7987, selected because of their different virulence potential, were used for a killing assay followed by the determination of gene expression using qPCR. In vivo results confirmed a significantly (p = 0.0321) lower pathogenicity for C. dubliniensis than for C. albicans. Accordingly, the induction of C. dubliniensis AMPs was lower at all the selected time points post-infection (1 h, 24 h, 48 h). Moreover, we observed an extremely high regulation of the galiomicin gene compared to the gallerimycin one, suggesting a different role of the tested AMPs in protecting G. mellonella from candidiasis.

Body Mass Index and Sex Affect Diverse Microbial Niches within the Gut.

Gut microbiota is considered a separate organ with endocrine capabilities, actively contributing to tissue homeostasis. It consists of at least two separate microbial populations, the lumen-associated (LAM) and the mucosa-associated microbiota (MAM). In the present study, we compared LAM and MAM, by collecting stools and sigmoid brush samples of forty adults without large-bowel symptoms, and through a 16S rRNA gene next-generation sequencing (NGS) approach. MAM sample analysis revealed enrichment in aerotolerant Proteobacteria, probably selected by a gradient of oxygen that decreases from tissue to lumen, and in Streptococcus and Clostridium spp., highly fermenting bacteria. On the other hand, LAM microbiota showed an increased abundance in Bacteroides, Prevotella, and Oscillospira, genera able to digest and to degrade biopolymers in the large intestine. Predicted metagenomic analysis showed LAM to be enriched in genes encoding enzymes mostly involved in energy extraction from carbohydrates and lipids, whereas MAM in amino acid and vitamin metabolism. Moreover, LAM and MAM communities seemed to be influenced by different host factors, such as diet and sex. LAM is affected by body mass index (BMI) status. Indeed, BMI negatively correlates with Faecalibacterium prausnitzii and Flavonifractor plautii abundance, putative biomarkers of healthy status. In contrast, MAM microbial population showed a significant grouping according to sex. Female MAM was enriched in Actinobacteria (with an increased trend of the genus Bifidobacterium), and a significant depletion in Veillonellaceae. Interestingly, we found the species Gemmiger formicilis to be associated with male and Bifidobacterium adolescentis, with female MAM samples. In conclusion, our results suggest that gut harbors microbial niches that differ in both composition and host factor susceptibility, and their richness and diversity may be overlooked evaluating only fecal samples.

Anti-biofilm activity of antibody directed against surface antigen complement receptor 3-related protein-comparison of Candida albicans and Candida dubliniensis.

Candida albicans and C. dubliniensis are related yeasts that differ in the expression of virulence-associated proteins involved in adherence and biofilm development. CR3-RP (complement receptor 3-related protein) is one of the surface antigens expressed by Candida species. The main objective of this research was to elucidate the effect of the polyclonal anti-CR3-RP antibody (Ab) on adherence and the biofilm formed by C. albicans SC5314 and C. dubliniensis CBS 7987 and two clinical isolates in vitro, ex vivo and in vivo. A comparison of species, and of treated vs. non-treated with the anti-CR3-RP Ab showed a reduction in adherence (22%-41%) that was dependent on the time point of evaluation (60, 90 or 120 min), but did not prove to be species-dependent. Confocal microscopy revealed a decreased thickness in biofilms formed by both species after pre-treatment with the anti-CR3-RP Ab. This observation was confirmed ex vivo by immunohistochemistry analysis of biofilms formed on mouse tongues. Moreover, anti-CR3-RP Ab administration, 1 h post-infection, has been shown to promote larval survival compared to the control group in a Galleria mellonella infection model. Our data suggest a potential activity of the anti-CR3-RP Ab relevant to immunotherapy or vaccine development against biofilm-associated Candida infections.

Microbiota in anorexia nervosa: The triangle between bacterial species, metabolites and psychological tests.

Anorexia nervosa (AN) is a psychiatric disease with devastating physical consequences, with a pathophysiological mechanism still to be elucidated. Metagenomic studies on anorexia nervosa have revealed profound gut microbiome perturbations as a possible environmental factor involved in the disease. In this study we performed a comprehensive analysis integrating data on gut microbiota with clinical, anthropometric and psychological traits to gain new insight in the pathophysiology of AN. Fifteen AN women were compared with fifteen age-, sex- and ethnicity-matched healthy controls. AN diet was characterized by a significant lower energy intake, but macronutrient analysis highlighted a restriction only in fats and carbohydrates consumption. Next generation sequencing showed that AN intestinal microbiota was significantly affected at every taxonomic level, showing a significant increase of Enterobacteriaceae, and of the archeon Methanobrevibacter smithii compared with healthy controls. On the contrary, the genera Roseburia, Ruminococcus and Clostridium, were depleted, in line with the observed reduction in AN of total short chain fatty acids, butyrate, and propionate. Butyrate concentrations inversely correlated with anxiety levels, whereas propionate directly correlated with insulin levels and with the relative abundance of Roseburia inulinivorans, a known propionate producer. BMI represented the best predictive value for gut dysbiosis and metabolic alterations, showing a negative correlation with Bacteroides uniformis (microbiota), with alanine aminotransferase (liver function), and with psychopathological scores (obsession-compulsion, anxiety, and depression), and a positive correlation with white blood cells count. In conclusion, our findings corroborate the hypothesis that the gut dysbiosis could take part in the AN neurobiology, in particular in sustaining the persistence of alterations that eventually result in relapses after renourishment and psychological therapy, but causality still needs to be proven.

A rare case of subcutaneous fusariosis in an immunocompetent patient.

Fusarium species can cause diseases in immunocompromised patients, whereas have rarely been reported as pathogens in immunocompetent individuals. Fusarium oxysporum and F. solani species complex are the most frequent pathogenic species. Primary subcutaneous hyalohyphomycosis due to these filamentous fungi is an extremely rare pathology, especially in immunocompetent patients. Only 37 cases of deep Fusarium infection in healthy patient have been reported in international literature. We report a case of subcutaneous infection due to F. solani species complex in a healthy 45-year-old man presenting with painful nodular lesion on the left ankle, appeared seven years earlier.

Relative Abundance in Bacterial and Fungal Gut Microbes in Obese Children: A Case Control Study.

BACKGROUND: Differences in relative proportions of gut microbial communities in adults have been correlated with intestinal diseases and obesity. In this study we evaluated the gut microbiota biodiversity, both bacterial and fungal, in obese and normal-weight school-aged children. METHODS: We studied 28 obese (mean age 10.03 ± 0.68) and 33 age- and sex-matched normal-weight children. BMI z-scores were calculated, and the obesity condition was defined according to the WHO criteria. Fecal samples were analyzed by 16S rRNA amplification followed by denaturing gradient gel electrophoresis (DGGE) analysis and sequencing. Real-time polymerase chain reaction (PCR) was performed to quantify the most representative microbial species and genera. RESULTS: DGGE profiles showed high bacterial biodiversity without significant correlations with BMI z-score groups. Compared to bacterial profiles, we observed lower richness in yeast species. Sequence of the most representative bands gave back Eubacterium rectale, Saccharomyces cerevisiae, Candida albicans, and C. glabrata as present in all samples. Debaryomyces hansenii was present only in two obese children. Obese children revealed a significantly lower abundance in Akkermansia muciniphyla, Faecalibacterium prausnitzii, Bacteroides/Prevotella group, Candida spp., and Saccharomyces spp. (P = 0.031, P = 0.044, P = 0.003, P = 0.047, and P = 0.034, respectively). CONCLUSION: Taking into account the complexity of obesity, our data suggest that differences in relative abundance of some core microbial species, preexisting or diet driven, could actively be part of its etiology. This study improved our knowledge about the fungal population in the pediatric school-age population and highlighted the need to consider the influence of cross-kingdom relationships.

Pediatric obesity is associated with an altered gut microbiota and discordant shifts in Firmicutes populations.

An altered gut microbiota has been linked to obesity in adulthood, although little is known about childhood obesity. The aim of this study was to characterize the composition of the gut microbiota in obese (n = 42) and normal-weight (n = 36) children aged 6 to 16. Using 16S rRNA gene-targeted sequencing, we evaluated taxa with differential abundance according to age- and sex-normalized body mass index (BMI z-score). Obesity was associated with an altered gut microbiota characterized by elevated levels of Firmicutes and depleted levels of Bacteroidetes. Correlation network analysis revealed that the gut microbiota of obese children also had increased correlation density and clustering of operational taxonomic units (OTUs). Members of the Bacteroidetes were generally better predictors of BMI z-score and obesity than Firmicutes, which was likely due to discordant responses of Firmicutes OTUs. In accordance with these observations, the main metabolites produced by gut bacteria, short chain fatty acids (SCFAs), were higher in obese children, suggesting elevated substrate utilisation. Multiple taxa were correlated with SCFA levels, reinforcing the tight link between the microbiota, SCFAs and obesity. Our results suggest that gut microbiota dysbiosis and elevated fermentation activity may be involved in the etiology of childhood obesity.

A Naturally Occurring Antibody Fragment Neutralizes Infectivity of Diverse Infectious Agents.

A phosphorylated peptide, named K40H, derived from the constant region of IgMs was detected in human serum by liquid chromatography coupled to high-resolution mass spectrometry. Synthetic K40H proved to exert a potent in vitro activity against fungal pathogens, and to inhibit HIV-1 replication in vitro and ex vivo. It also showed a therapeutic effect against an experimental infection by Candida albicans in the invertebrate model Galleria mellonella. K40H represents the proof of concept of the innate role that naturally occurring antibody fragments may exert against infectious agents, shedding a new light upon the posthumous role of antibodies and opening a new scenario on the multifaceted functionality of humoral immunity.

Fungal Biofilms: Update on Resistance.

Over the past decade, the emergence of biofilm-related invasive fungal diseases has been the subject of numerous studies focused on antifungal resistance and its impact on antifungal therapy in severely ill patients. The majority of the studies investigated the molecular mechanisms involved in antifungal resistance and pathogenicity of biofilm production by Candida albicans and Aspergillus fumigatus, the most common etiologic agents of yeast and mold invasive infections. The main mechanism characterizing biofilm-related antifungal resistance is the production of extracellular matrix, a physical barrier preventing the drugs from entering and expressing their activity. However, over-expression of efflux pumps, genetic changes of drug targets, persister cells, biofilm-host immune system interaction, proteins leading to filamentation, all together contribute to the onset of biofilm antifungal resistance. Some of these mechanisms are shared with planktonic cells and are often related to developmental phases of biofilm formation. All physical and genetic factors leading to biofilm-related antifungal resistance have been briefly discussed.

Molecular Identification and Echinocandin Susceptibility of Candida parapsilosis Complex Bloodstream Isolates in Italy, 2007-2014.

The Candida parapsilosis group encompasses three species: C. parapsilosis, C. orthopsilosis, and C. metapsilosis. Here, we describe the incidence and echinocandin susceptibility profile of bloodstream isolates of these three species collected from patients admitted to an Italian university hospital from 2007 to 2014. Molecular identification of cryptic species of the C. parapsilosis complex was performed using polymerase chain reaction amplification of the gene encoding secondary alcohol dehydrogenase, followed by digestion with the restriction enzyme BanI. Minimum inhibitory concentrations were determined using the broth microdilution method according to European Committee for Antimicrobial Susceptibility Testing (EUCAST EDef 7.2) and Clinical Laboratory Standards Institute (CLSI M27-A3) guidelines, and the results were compared with those obtained using the E-test and Sensititre methods. Of the 163 C. parapsilosis complex isolates, 136 (83.4%) were identified as C. parapsilosis, and 27 (16.6%) as C. orthopsilosis. The species-specific incidences were 2.9/10,000 admissions for C. parapsilosis and 0.6/10,000 admissions for C. orthopsilosis. No resistance to echinocandins was detected with any of the methods. The percent essential agreement (EA) between the EUCAST and E-test/Sensititre methods for anidulafungin, caspofungin, and micafungin susceptibility was, respectively, as follows: C. parapsilosis, 95.6/97.8, 98.5/88.2, and 93.4/96.3; C. orthopsilosis, 92.6/92.6, 96.3/77.8, and 63.0/66.7. The EA between the CLSI and E-test/Sensititre methods was, respectively, as follows: C. parapsilosis, 99.3/100, 98.5/89.0, and 96.3/98.5; C. orthopsilosis, 96.3/92.6, 100/81.5, and 92.6/88.9. Only minor discrepancies, ranging from 16.9% (C. parapsilosis) to 11.1% (C. orthopsilosis), were observed between the CLSI and E-test/Sensititre methods. In conclusion, this epidemiologic study shows a typical C. parapsilosis complex species distribution, no echinocandin resistance, and it reinforces the relevance of using commercially available microbiological methods to assess antifungal susceptibility. These data improve our knowledge of the national distribution of species of the psilosis group, as there are very few studies of these species in Italy.

Antifungal activity of Myriocin on clinically relevant Aspergillus fumigatus strains producing biofilm.

BACKGROUND: The human pathogenic mold Aspergillus fumigatus is able to form a complex biofilm embedded in extracellular matrix. Biofilms confer antimicrobial resistance and it is well known that aspergillosis is often refractory to the conventional antifungal therapy. The treatment of biofilm-related infections poses a significant clinical challenge on a daily basis, promoting the search for new therapeutic agents. Our aim was to exploit the modulation of sphingolipid mediators as new therapeutic target to overcome antifungal resistance in biofilm-related infections. RESULTS: Antifungal susceptibility testing was performed on 20 clinical isolates of Aspergillus fumigatus and one reference strain (A. fumigatus Af293) according the EUCAST protocol. Sessile MICs were assessed on 24-h preformed-biofilm by means of XTT-reduction assay. Myriocin (0.25-64 mg/L), a commercial sphingolipid synthesis inhibitor, was used. The MEC50 value (mg/L) of Myriocin was 8 (range 4-16) for both planktonic and sessile cells. Drug-induced morphological alterations were analyzed by optical and electron microscopy (TEM) on 24h preformed A. fumigatus Af293 biofilms. An evident hyphal damage, resulting in short, stubby, and highly branched hyphae was observed by optical microscopy. At 24h, TEM studies showed important morphological alterations, such as invaginations of the cell membrane, modification in the vacuolar system and presence of multilamellar bodies, in some cases within vacuoles. CONCLUSIONS: The direct antifungal activity, observed on both planktonic and sessile fungi, suggests that inhibition of sphingolipid synthesis could represent a new target to fight biofilm-related A. fumigatus resistance.

New strategic insights into managing fungal biofilms.

Fungal infections have dramatically increased in the last decades in parallel with an increase of populations with impaired immunity, resulting from medical conditions such as cancer, transplantation, or other chronic diseases. Such opportunistic infections result from a complex relationship between fungi and host, and can range from self-limiting to chronic or life-threatening infections. Modern medicine, characterized by a wide use of biomedical devices, offers new niches for fungi to colonize and form biofilm communities. The capability of fungi to form biofilms is well documented and associated with increased drug tolerance and resistance. In addition, biofilm formation facilitates persistence in the host promoting a persistent inflammatory condition. With a limited availability of antifungals within our arsenal, new therapeutic approaches able to address both host and pathogenic factors that promote fungal disease progression, i.e., chronic inflammation and biofilm formation, could represent an advantage in the clinical setting. In this paper we discuss the antifungal properties of myriocin, fulvic acid, and acetylcholine in light of their already known anti-inflammatory activity and as candidate dual action therapeutics to treat opportunistic fungal infections.

Methicillin-Resistant Staphylococcus aureus in Raw Milk: Prevalence, SCCmec Typing, Enterotoxin Characterization, and Antimicrobial Resistance Patterns.

Staphylococcus aureus is a known major cause of foodborne illnesses, and raw milk and dairy products are often contaminated by enterotoxigenic and antimicrobial-resistant S. aureus strains. In the present study, 35 S. aureus strains were isolated from 383 raw milk samples collected from various dairy herds in the province of Milan (northern Italy). The isolates were characterized based on their antimicrobial susceptibility patterns and the presence of genes encoding staphylococcal enterotoxins (sea, seb, sec, sed, and see). About half (45.7%) of the strains were enterotoxigenic, and 37.1% were resistant to at least one of the antimicrobial drugs tested. Seven (20%) of 35 isolates were identified as methicillin-resistant S. aureus (MRSA), and SCCmec typing performed with a multiplex PCR assay revealed the presence of gene cassettes IV and V, typical of community-acquired MRSA, and I and II, characteristic of health care-associated MRSA. The MRSA strains were evaluated for the presence of the Panton-Valentine leukocidin gene, but this gene was not found. The results of the present study revealed the presence of toxin-producing S. aureus and MRSA strains in raw milk. MRSA and enterotoxigenic S. aureus in dairy farms are an important risk factor for the spread of staphylococcal infections; therefore, further studies are needed to find strategies for monitoring and controlling the presence of S. aureus, especially MRSA, in dairy products.

Detection of Pneumocystis jirovecii and Aspergillus spp. DNa in bronchoalveolar lavage fluids by commercial real-time PCr assays: comparison with conventional diagnostic tests.

The present study employed two commercial real-time PCR kits, MycAssay� Pneumocystis (PJ-PCR) and MycAssay� Aspergillus (ASP-PCR), for the search of fungal DNA on 44 bronchoalveolar lavage (BAL) fluids from patients at risk of invasive fungal disease. Operationally, on the basis of clinical diagnosis and according to the European Organization for Research and Treatment Cancer/Mycoses Study Group (EORTC/MSG) criteria, patients were clustered in 3 groups: a P. jirovecii pneumonia (PCP) group, an invasive aspergillosis (IA) group and a control (CTRL) group, consisting of 8, 10 and 24 patients, respectively. The results were compared to those obtained with conventional diagnostic assays, including BAL culture, galactomannan-ELISA (GM) and immunofluorescence (IF). The PJ-PCR assay returned a sensitivity and specificity of 100% and 94.4%, respectively. The ASP-PCR assay showed a sensitivity and specificity of 80% and 97.1%. When compared to the culture assay, the ASP-PCR showed enhanced sensitivity, and a good level of agreement (kappa = 0.63) was observed between ASP-PCR and GM assays. Overall, our data emphasize the diagnostic usefulness of the two commercial real-time PCR assays, especially in high-risk patients where timing is critical and a low fungal burden may hamper correct and prompt diagnosis by conventional tests.

How to manage aspergillosis in non-neutropenic intensive care unit patients.

Invasive aspergillosis has been mainly reported among immunocompromised patients during prolonged periods of neutropenia. Recently, however, non-neutropenic patients in the ICU population have shown an increasing risk profile for aspergillosis. Associations with chronic obstructive pulmonary disease and corticosteroid therapy have been frequently documented in this cohort. Difficulties in achieving a timely diagnosis of aspergillosis in non-neutropenic patients is related to the non-specificity of symptoms and to lower yields with microbiological tests compared to neutropenic patients. Since high mortality rates are typical of invasive aspergillosis in critically ill patients, a high level of suspicion and prompt initiation of adequate antifungal treatment are mandatory. Epidemiology, risk factors, diagnostic algorithms, and different approaches in antifungal therapy for invasive aspergillosis in non-neutropenic patients are reviewed.