Characterization and Technological Features of Autochthonous Coagulase-Negative Staphylococci as Potential Starters for Portuguese Dry Fermented Sausages.

The manufacture of dry fermented sausages is an important part of the meat industry in Southern European countries. These products are usually produced in small shops from a mixture of pork, fat, salt, and condiments and are stuffed into natural casings. Meat sausages are slowly cured through spontaneous fermentation by autochthonous microbiota present in the raw materials or introduced during manufacturing. The aim of this work was to evaluate the technological and safety features of coagulase-negative staphylococci (CNS) isolated from Portuguese dry fermented meat sausages in order to select autochthonous starters. Isolates (n = 104) obtained from 2 small manufacturers were identified as Staphylococcus xylosus, Staphylococcus equorum, Staphylococcus saprophyticus, and Staphylococcus carnosus. Genomically diverse isolates (n = 82) were selected for further analysis to determine the ability to produce enzymes (for example, nitrate-reductases, proteases, lipases) and antibiotic susceptibility. Autochthonous CNS producing a wide range of enzymes and showing low antibioresistance were selected as potential starters for future use in the production of dry fermented meat sausages.

Characterization and optimization of the haemozoin-like crystal (HLC) assay to determine Hz inhibiting effects of anti-malarial compounds.

BACKGROUND: The haem-haemozoin biocrystallization pathway is an attractive target where several efficacious and safe anti-malarial drugs act. Consequently, in vitro haemozoin (Hz) inhibition assays have been developed to identify novel compounds. However, results may differ between assays and often require complex methods or sophisticated infrastructure. The recently reported growth of haemozoin-like crystals (HLC) appears to be a simple alternative although the endproduct is structurally different to Hz. This study set out to characterize this assay in depth, optimize it, and assess its performance. METHODS: The HLC assay was used as previously described but a range of different growth conditions were examined. Obtained HLCs were investigated and compared to synthetic (sHz) and natural haemozoin (nHz) using scanning electron microscopy, powder X-ray diffraction (PXRD), Fourier Transform Infrared spectroscopy (FTIR) and Raman spectroscopy (RS). Interactions of HLC with quinolines was analysed using RS. Inhibitory effects of currently used anti-malarial drugs under four final growth conditions were established. RESULTS: HLC growth requires Mycoplasma Broth Base, Tween 80, pancreatin, and lysed blood or haemin. HLCs are similar to nHz and sHz in terms of solubility, macroscopic and microscopic appearance although PXRD, FTIR and RS confirm that the haem aggregates of HLCs are structurally different. RS reveals that CQ seems to interact with HLCs in similar ways as with Hz. Inhibition of quinoline drugs ranged from 62.5 µM (chloroquine, amodiaquine, piperaquine) to 500 µM in mefloquine. CONCLUSIONS: The HLC assay provides data on inhibiting properties of compounds. Even if the end-product is not structurally identical to Hz, the inhibitory effects appear consistent with those obtained with sHz assays, as illustrated by the results obtained for quinolines. The assay is simple, inexpensive, robust, reproducible and can be performed under basic laboratory conditions with a simple visual positive/negative read-out.

Assessing anti-malarial drug effects ex vivo using the haemozoin detection assay.

BACKGROUND: In vitro sensitivity assays are crucial to detect and monitor drug resistance. Plasmodium falciparum has developed resistance to almost all anti-malarial drugs. Although different in vitro drug assays are available, some of their inherent characteristics limit their application, especially in the field. A recently developed approach based on the flow cytometric detection of haemozoin (Hz) allowed reagent-free monitoring of parasite maturation and detection of drug effects in culture-adapted parasites. In this study, the set-up, performance and usefulness of this novel assay were investigated under field conditions in Gabon. METHODS: An existing flow cytometer (Cyflow Blue) was modified on site to detect light depolarization caused by Hz. Blood from malaria patients was incubated for 72 hrs with increasing concentrations of chloroquine, artesunate and artemisinin. The percentage of depolarizing red blood cells (RBC) was used as maturation indicator and measured at 24, 48 and 72 hrs of incubation to determine parasite growth and drug effects. RESULTS: The flow cytometer was easily adapted on site to detect light depolarization caused by Hz. Analysis of ex vivo cultures of parasites, obtained from blood samples of malaria patients, showed four different growth profiles. In 39/46 samples, 50% inhibitory concentrations (IC50) were successfully determined. IC50 values for chloroquine were higher than 200 nM in 70% of the samples, indicating the presence of chloroquine-resistant parasites. For artesunate and artemisinin, IC50 values ranged from 0.9 to 60 nM and from 2.2 nM to 124 nM, respectively, indicating fully sensitive parasites. CONCLUSION: Flow cytometric detection of Hz allowed the detection of drug effects in blood samples from malaria patients, without using additional reagents or complex protocols. Adjustment of the initial parasitaemia was not required, which greatly simplifies the protocol, although it may lead to different IC50 values. Further investigation of set-up conditions of the Hz assay, as well as future studies in various settings should be performed to further determine the usefulness of this assay as a tool for rapid resistance testing in malaria-endemic countries.

Light depolarization measurements in malaria: A new job for an old friend.

The use of flow cytometry in malaria research has increased over the last decade. Most approaches use nucleic acid stains to detect parasite DNA and RNA and require complex multi-color, multi-parameter analysis to reliably detect infected red blood cells (iRBCs). We recently described a novel and simpler approach to parasite detection based on flow cytometric measurement of scattered light depolarization caused by hemozoin (Hz), a pigment formed by parasite digestion of hemoglobin in iRBCs. Depolarization measurement by flow cytometry was described in 1987; however, patent issues restricted its use to a single manufacturer's hematology analyzers until 2009. Although we recently demonstrated that depolarization measurement of Hz, easily implemented on a bench top flow cytometer (Cyflow), provided useful information for malaria work, doubts regarding its application and utility remain in both the flow cytometry and malaria communities, at least in part because instrument manufacturers do not offer the option of measuring depolarized scatter. Under such circumstances, providing other researchers with guidance as to how to do this seemed to offer the most expeditious way to resolve the issue. We accordingly examined how several commercially available flow cytometers (CyFlow SL, MoFLo, Attune and Accuri C6) could be modified to detect depolarization due to the presence of free Hz on solution, or of Hz in leukocytes or erythrocytes from rodent or human blood. All were readily adapted, with substantially equivalent results obtained with lasers emitting over a wide wavelength range. Other instruments now available may also be modifiable for Hz measurement. Cytometric detection of Hz using depolarization is useful to study different aspects of malaria. Adding additional parameters, such as DNA content and base composition and RNA content, can demonstrably provide improved accuracy and sensitivity of parasite detection and characterization, allowing malaria researchers and eventually clinicians to benefit from cytometric technology.