Yeasts and moulds contaminants of food ice cubes and their survival in different drinks.

AIMS: To evaluate the levels of unicellular and filamentous fungi in ice cubes produced at different levels and to determine their survival in alcoholic beverages and soft drinks. METHODS AND RESULTS: Sixty samples of ice cubes collected from home level (HL) productions, bars and pubs (BP) and industrial manufacturing plants (MP) were investigated for the presence and cell density of yeasts and moulds. Moulds were detected in almost all samples, while yeasts developed from the majority of HL and MP samples. Representative colonies of microfungi were subjected to phenotypic and genotypic characterization. The identification was carried out by restriction fragment length polymorphism (RFLP) analysis of the region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5·8S rRNA gene. The process of yeast identification was concluded by sequencing the D1/D2 region of the 26S rRNA gene. The fungal biodiversity associated with food ice was represented by nine yeast and nine mould species. Strains belonging to Candida parapsilosis and Cryptococcus curvatus, both opportunistic human pathogens, and Penicillium glabrum, an ubiquitous mould in the ice samples analysed, were selected to evaluate the effectiveness of the ice cubes to transfer pathogenic microfungi to consumers, after addition to alcoholic beverages and soft drinks. All strains retained their viability. CONCLUSIONS: The survival test indicated that the most common mode of consumption of ice cubes, through its direct addition to drinks and beverages, did not reduce the viability of microfungi. SIGNIFICANCE AND IMPACT OF THE STUDY: This study evidenced the presence of microfungi in food ice and ascertained their survival in soft drinks and alcoholic beverages.

Substance P expression in the gingival tissue after upper third molar extraction: effect of ketoprofen, a preliminary study.

The aim of this study was to evaluate substance P (SP) levels and the effect of a non-steroidal anti-inflammatory drug (NSAID), ketoprofen, on SP in the pericoronal gingival tissue after extraction of upper third molars. A sample of 20 young non-smoking systemically healthy adults of both sexes, with a healthy upper third molar to extract for orthodontic purposes, was selected. After extraction, a sample of the gingival tissue of the pericoronal region was collected with a sterile scalpel, placed into test tubes and kept frozen at -20°C until the SP determination. SP levels were determined by using a commercially available enzyme immunoassay (ELISA) kit. The subjects were randomly divided into two groups: group 1 received a single dose of ketoprofen 30 minutes prior to the experimental procedure. The subjects of group 2 did not receive any kind of drug administration before extraction. The patients were asked to complete a diary on the postoperative pain. A relevant amount of SP was measured in all the gingival samples. No statistically significant difference could be detected in SP expression between the two groups. In group 1 pain appearance was significantly delayed (6.2±0.13 hours) in comparison with group 2 (3.95±0.2 hours). In this small selected group of subjects and limited study design, preventive administration of ketoprofen did not significantly affect the gingival levels of SP, the clinical recommendation emerging is that of NSAID administration postoperatively but before pain appearance in order to optimize the management of pain of the patient.

Seasonal variations of antimicrobial activity and chemical composition of essential oils extracted from three Citrus limon L. Burm. cultivars.

In order to investigate the seasonal variations of antimicrobial properties and chemical composition of essential oils (EOs), three different cultivars of Citrus limon L. Burm. spp. (Femminello Santa Teresa, Monachello and Femminello Continella) were collected at 6-week intervals, from December 2012 to April 2013, for a total of four harvests. The EOs were extracted from lemon peel by hydro-distillation. The antimicrobial activity, tested by paper disc diffusion method, was evaluated against common food-related pathogenic bacteria (Listeria monocytogenes, Staphylococcus aureus, Salmonella enterica and Enterobacter spp.). EOs were more effective against Gram-positive than Gram-negative bacteria at each collection time, but a strong strain dependence was evidenced. Monachello EOs showed the highest inhibition power. The chemical characterisation of the EOs performed by gas chromatography/mass spectrometry identified from 36 to 42 molecules. The chemical difference registered among samples and seasons may explain the different antimicrobial efficacies recorded.

Persistence of wild Streptococcus thermophilus strains on wooden vat and during the manufacture of a traditional Caciocavallo type cheese.

The present work was undertaken to evaluate the influence of the wooden dairy plant equipment on the microbiological characteristics of curd to be transformed into Caciocavallo Palermitano cheese. Traditional raw milk productions were performed concomitantly with standard cheese making trials carried out in stainless steel vat inoculated with a commercial starter. Milk from two different farms (A and B) was separately processed. The wooden vat was found to be a reservoir of lactic acid bacteria (LAB), while unwanted (spoilage and/or pathogenic) microorganisms were not hosted or were present at very low levels. All microbial groups were numerically different in bulk milks, showing higher levels for the farm B. LAB, especially thermophilic cocci, dominated the whole cheese making process of all productions. Undesired microorganisms decreased in number or disappeared during transformation, particularly after curd stretching. LAB were isolated from the wooden vat surface and from all dairy samples, subjected to phenotypic and genetic characterization and identification. Streptococcus thermophilus was the species found at the highest concentration in all samples analyzed and it also dominated the microbial community of the wooden vat. Fourteen other LAB species belonging to six genera (Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Streptococcus and Weissella) were also detected. All S. thermophilus isolates were genetically differentiated and a consortium of four strains persisted during the whole traditional production process. As confirmed by pH and the total acidity after the acidification step, indigenous S. thermophilus strains acted as a mixed starter culture.

Chemical composition and antibacterial potential of Artemisia arborescens L. essential oil.

This study was undertaken to characterize the essential oil (EO) of Artemisia arborescens growing wild in Sicily. EO, extracted by steam distillation, was examined for its chemical composition and for its capability to inhibit some food-borne pathogen bacteria. A total of 43 compounds (13 monoterpene hydrocarbons, 14 oxygenated monoterpenes, 10 sesquiterpene hydrocarbons, three oxygenated sesquiterpenes and less amount of other three compounds), which account 93.73% of the total oil, were identified by gas chromatography and gas chromatography-mass spectrometry. Oxygenated monoterpenes (57.32%) constituted the main fraction, with ß-thujone as the main compound (45.04%), followed by the sesquiterpene hydrocarbon chamazulene (22.71%). Undiluted EO showed a large inhibition spectrum against strains of Listeria monocytogenes (34 out of 44), whilst it was ineffective against enterobacteria and salmonellas. The minimum inhibition concentration (MIC) was evaluated for the two most sensitive strains (L. monocytogenes 186 and 7BO) at two cellular concentrations (10(6) and 10(7) CFU ml(-1)). The lowest MIC (0.625 µl ml(-1), dilution of oil with acetone) was found for strain L. monocytogenes 186 at 10(6) CFU ml(-1).

Could halophilic archaea improve the traditional salted anchovies (Engraulis encrasicholus L.) safety and quality?

AIMS: The positive influence of two selected extremely halophilic archaea strains in the production of salted anchovies (Engraulis encrasicolus, L., 1758) was highlighted. METHODS AND RESULTS: Anchovies produced with salt artificially contaminated with halophiles exhibited lower loads of staphylococci, Enterobacteriaceae and lactic acid bacteria, and a reduced content of histamine as well as an improved organoleptic acceptance. CONCLUSIONS: The findings of this survey are expected to enhance the safety of salted anchovies, with regard to the histamine formation during ripening, and to improve the sensory attributes of this product. SIGNIFICANCE AND IMPACT OF THE STUDY: This study represents the first report on the positive influence of halophilic archaea in traditional salted anchovies production, thus suggesting new perspectives about a conscious employment of properly selected haloarchaea strains in this traditional manufacture.

Heterotrophic microorganisms in deteriorated medieval wall paintings in southern Italian churches.

The Campania region in southern Italy is noted for its large number of churches that harbour invaluable frescoes, dated from the beginnings of the 4th up to the 13th century. The wall paintings represent an integral part of the monuments, and their deterioration constitutes a potentially significant loss for the world's cultural heritage. Heterotrophic microorganisms such as bacteria and mould can grow on the surface of paintings that contain a wide range of organic and inorganic constituents, and provide different ecological niches that are exploited by a large variety of microbial species. We isolated and identified the heterotrophic microorganisms found in the biodegraded medieval wall paintings of seven historical churches in Campania. The paintings showed different levels of microbial contamination. Microbiological analysis of different paintings gave an overview of the different heterotrophic microorganisms. Bacteria and moulds were isolated from 77% of the sampling points analysed, in which the most common type of alteration was discolouration often associated with detachment of the paint layer. Bacterial strains were identified by 16S rRNA partial sequence analysis. The Bacillus genus was isolated in all churches, even though the type of species was variable, whereas all actinomycetes strains, isolated in five of the seven churches analysed, could be referred to the Streptomyces genus. The similarity of the sequences analysed of the 42 Bacillus spp., 2 Paenibacillus spp. and reference strains of different species showed that these bacteria differentiated in 14 groups. The most frequently occurring taxa were most closely related to Bacillus cereus/thurigiensis/anthracis and Bacillus pumilus groups. Thirteen Streptomyces spp. were differentiated in seven groups on the basis of neighbor-joining analysis of 16S rRNA. Fungi belonging to the genera Penicillium, Aspergillus, Fusarium and Alternaria were also isolated from deteriorated wall paintings.

Fennel waste-based films suitable for protecting cultivations.

Biodegradable, flexible, and moisture-resistant films were obtained by recycling fennel waste and adding to fennel homogenates the bean protein phaseolin that was modified or not modified by the enzyme transglutaminase. All films were analyzed for their morphology, mechanical properties, water vapor permeability, and susceptibility to biodegradation under soil-like conditions. Our experiments showed that transglutaminase treatment of the phaseolin-containing fennel waste homogenates allowed us to obtain films comparable in their mechanical properties and water vapor permeability to the commercial films Ecoflex and Mater-Bi. Furthermore, biodegradability tests demonstrated that the presence of the enzyme in the film-casting sample significantly influences the integrity of such a product that lasts longer than films obtained either with fennel waste alone or with a mixture of fennel waste and phaseolin. These findings indicate the fennel-phaseolin film prepared in the presence of transglutaminase to be a promising candidate for a new environmentally friendly mulching bioplastic.

Presence and characterisation of verotoxin producing E. coli in fresh Italian pork sausages, and preparation and use of an antibiotic-resistant strain for challenge studies.

One hundred and twenty six samples of fresh pork sausages were analysed for the presence of verocytotoxigenic Escherichia coli (VTEC). Selective enrichment followed by DNA extraction and PCR amplification of the stx1 and stx2 genes highlighted the occurrence of the above mentioned genes in 20 out of 126 samples screened. From the stx positive enriched cultures, isolation was performed on CT-SMAC agar plates after immuno-magnetic separation of E. coli O157. Fifty three non-sorbitol fermenting isolates were obtained and further characterised, along with the reference strain E. coli ATCC 35150(T). All the isolates were characterised by PCR assays, assessing the presence of stx1, stx2, rfbE(O157:H7), eae and hlyA genes. The overall prevalence of VTEC was found to be 16%. VTEC strains were also characterised by plasmid profiling and REA-PFGE analysis, which allowed strain clustering into 5 and 8 groups, respectively. In addition, an antibiotic resistant E. coli O157:H7 strain was selected and used in challenge tests of raw pork at 4°C. This strain could be selectively counted in the presence of a normal background microflora and it was shown that it could survive for 1week at 4°C in the raw food studied.

PCR-DGGE fingerprints of microbial succession during a manufacture of traditional water buffalo mozzarella cheese.

AIMS: To monitor the process and the starter effectiveness recording a series of fingerprints of the microbial diversity occurring at different steps of mozzarella cheese manufacture and to investigate the involvement of the natural starter to the achievement of the final product. METHODS AND RESULTS: Samples of raw milk, natural whey culture (NWC) used as starter, curd after ripening and final product were collected during a mozzarella cheese manufacture. Total microbial DNA was directly extracted from the dairy samples as well as bulk colonies collected from the plates of appropriate culture media generally used for viable counts of mesophilic and thermophilic lactic acid bacteria (LAB) and used in polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) experiments. The analysis of the DGGE profiles showed a strong influence of the microflora of the NWC on the whole process because after the starter addition, the profile of all the dairy samples was identical to the one shown by the NWC. Simple indexes were calculated for the DGGE profiles to have an objective estimation of biodiversity and of technological importance of specific groups of organisms. LAB grown on Man Rogosa Sharp (MRS) and Rogosa agar at 30 degrees C showed high viable counts and the highest diversity in species indicating their importance in the cheese making, which had not been considered so far. Moreover, the NWC profiles were shown to be the most similar to the curd profile suggesting to be effective in manufacture. CONCLUSIONS: The PCR-DGGE analysis showed that in premium quality manufacture the NWC used as starter had a strong influence on the microflora responsible for process development. SIGNIFICANCE AND IMPACT OF THE STUDY: The molecular approach appeared to be valid as a tool to control process development, starter effectiveness and product identity as well as to rank cheese quality.

G-CSF alone vs cyclophosphamide plus G-CSF in PBPC mobilization of patients with lymphoma: results depend on degree of previous pretreatment.

We performed a randomized study to compare 'G-CSF alone' (administered at dose of 10 mcg/kg/day) and 'cyclophosphamide plus G-CSF' (cyclophosphamide at dose of 4 g/m(2) and G-CSF at dose of 10 microg/kg/day), as PBPC mobilization schedules in 52 patients with NHL or HD. Randomization was stratified according to the amount of previous chemotherapy (< or =2 and >2 lines of previous chemotherapy). Mean CD34+ cell peak in P.B., mean 'Total CD34+ cells' harvested and percentage of patients successfully mobilized, in the group mobilized with 'G-CSF alone' vs the group mobilized with 'cyclophosphamide plus G-CSF', were: 35.3 x 10(6) vs 45.8 x 10(6)/l (P=0.3), 5.4 x 10(6) vs 6.8 x 10(6)/kg (P>0.9) and 50 vs 61% (P=0.4). No differences were observed in the stratum of less pretreated patients. However, in the stratum of patients who had previously received more than two lines of chemotherapy, CD34+cell peak (P=0.05) and percentage of successful mobilization (P=0.01) were higher when 'cyclophosphamide plus G-CSF' was used. Using logistic regression, both age and mobilization with 'G-CSF alone' were significantly associated with a low CD34+ cell peak in P.B. However, in the stratum of less pretreated patients, only age was significantly associated with this risk.

Characterization of lactic acid bacteria strains on the basis of neutral volatile compounds produced in whey.

AIMS: Seventy-eight strains of lactic acid bacteria belonging to five genera and showing six different phenotype combinations of Lac (lactose fermentation), Prt (proteolytic activity) and Cit (citrate degradation) characters were investigated for their main flavouring properties with the aim to detect variability among and within the groups. METHODS AND RESULTS: High resolution gas chromatography-mass spectrometry analysis of neutral volatile compounds produced in whey showed that, considering both neo-formation compounds and substances quantified in the whey cultures at different concentrations in comparison to the extract from sterile whey, the groups of lactococci, enterococci, thermophilic streptococci and mesophilic lactobacilli produced a higher number of volatiles than thermophilic lactobacilli and leuconostocs. Applying principal component analysis (PCA) to the results, enterococci, mesophilic lactobacilli and thermophilic streptococci showed a broad diversity, while lactococci included rather similar strains as well as strains with special flavouring properties. Applying PCA to thermophilic streptococci and enterococci, to lactococci and enterococci, to lactococci and thermophilic streptococci, or to mesophilic and thermophilic lactobacilli, the strains gathered consistently with their systematic position. CONCLUSION: The study evidenced strains producing some volatile compounds responsible for food flavouring. Flavouring properties were variable among the systematic groups and in some cases different within the same bacterial group. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential of the findings is discussed with reference to the development of flavouring adjuncts for the dairy industry.

Comparison of statistical methods for identification of Streptococcus thermophilus, Enterococcus faecalis, and Enterococcus faecium from randomly amplified polymorphic DNA patterns.

Thermophilic streptococci play an important role in the manufacture of many European cheeses, and a rapid and reliable method for their identification is needed. Randomly amplified polymorphic DNA (RAPD) PCR (RAPD-PCR) with two different primers coupled to hierarchical cluster analysis has proven to be a powerful tool for the classification and typing of Streptococcus thermophilus, Enterococcus faecium, and Enterococcus faecalis (G. Moschetti, G. Blaiotta, M. Aponte, P. Catzeddu, F. Villani, P. Deiana, and S. Coppola, J. Appl. Microbiol. 85:25-36, 1998). In order to develop a fast and inexpensive method for the identification of thermophilic streptococci, RAPD-PCR patterns were generated with a single primer (XD9), and the results were analyzed using artificial neural networks (Multilayer Perceptron, Radial Basis Function network, and Bayesian network) and multivariate statistical techniques (cluster analysis, linear discriminant analysis, and classification trees). Cluster analysis allowed the identification of S. thermophilus but not of enterococci. A Bayesian network proved to be more effective than a Multilayer Perceptron or a Radial Basis Function network for the identification of S. thermophilus, E. faecium, and E. faecalis using simplified RAPD-PCR patterns (obtained by summing the bands in selected areas of the patterns). The Bayesian network also significantly outperformed two multivariate statistical techniques (linear discriminant analysis and classification trees) and proved to be less sensitive to the size of the training set and more robust in the response to patterns belonging to unknown species.

Molecular evaluation of microbial diversity occurring in different types of Mozzarella cheese.

AIMS: The microbial community of different types of unripened Pasta Filata cheese was investigated by culture-independent methods with the aim of rapidly achieving knowledge about cheese microbiota and discriminating traditional and industrial cheeses. METHODS AND RESULTS: The microbial DNA extracted directly from the samples was used as a template in PCR experiments to amplify the 16S-23S rDNA spacer region and the V3 region of the 16S rDNA. Conventional electrophoresis of the amplified spacers allowed known classes of these DNA fragments belonging to genera and species of lactic acid bacteria to be distinguished. Denaturing gradient gel electrophoresis analysis of V3 amplicons was supported by reference cultures of LAB used as markers. CONCLUSION: Both molecular approaches furnished the expected information about microbial diversity and were quite valid for discriminating industrial, semi-artisanal or traditional cheeses, characterized by increasingly complex DNA profiles. SIGNIFICANCE AND IMPACT OF THE STUDY: Both methods could be used for legal purposes when products obtained through prescribed manufacturing regulations are to be analysed.

Detection and characterization of a bacteriocin, garviecin L1-5, produced by Lactococcus garvieae isolated from raw cow's milk.

AIMS: The identification of a bacteriocin-producing lactococcal strain isolated from raw cow's milk is reported, along with production conditions, physical and chemical properties, and mode of action of the bacteriocin. METHODS AND RESULTS: On the basis of resistance to clindamycin, species-specific PCR and amplification of the 16S-23S rDNA spacer region, the strain was identified as Lactococcus garvieae. Its bacteriocin, designated garviecin L1-5, was bactericidal against closely related species and strains of species from different genera, including Listeria monocytogenes and Clostridium spp. Garviecin L1-5 was shown to be proteinaceous by protease inactivation and was unaffected by heat treatments, also at low pH values. When amplifying known lactococcal bacteriocin genes using DNA from strain L1-5 as template, no amplification products were observed on the agarose gel. The molecular weight of garviecin L1-5 was about 2.5 kDa. As far as is known, no bacteriocins have been detected from Lactococcus garvieae. CONCLUSION: The general properties of garviecin L1-5 are characteristic of the low-molecular-weight bactericidal peptide group. SIGNIFICANCE AND IMPACT OF THE STUDY: The survey of micro-organisms for novel antimicrobial substances provided valuable information on their physiology, ecology and practical application.

Behavior of variable V3 region from 16S rDNA of lactic acid bacteria in denaturing gradient gel electrophoresis.

Separation of amplified V3 region from 16S rDNA by denaturing gradient gel electrophoresis (DGGE) was tested as a tool for differentiation of lactic acid bacteria commonly isolated from food. Variable V3 regions of 21 reference strains and 34 wild strains referred to species belonging to the genera Pediococcus, Enterococcus, Lactococcus, Lactobacillus, Leuconostoc, Weissella, and Streptococcus were analyzed. DGGE profiles obtained were species-specific for most of the cultures tested. Moreover, it was possible to group the remaining LAB reference strains according to the migration of their 16S V3 region in the denaturing gel. The results are discussed with reference to their potential in the analysis of LAB communities in food, besides shedding light on taxonomic aspects.

Nisin-producing organisms during traditional 'Fior di latte' cheese-making monitored by multiplex-PCR and PFGE analyses.

In this work we studied using different molecular methods the population dynamics of nisin-producing organisms and the persistence of such organisms within a complex ecosystem, 'Fior di latte' cheese, a traditional high-moisture pasta filata cheese. Using the primers targeting the eubacterial 16S-23S rRNA spacer region, together with those amplifying the nisA or nisZ gene, we were able to provide a rapid species identification of the isolates. Inhibitors of Lactococcus lactis subsp. lactis DSM 20481T used as indicator occurred during the whole process of cheese manufacture as a significant part of lactic microflora; however, only 12 among 109 isolates of bacteriocin producers were nisin producers. Amplification of the nisA or nisZ gene, using DNA extracted directly from dairy samples as templates, showed that the nisin structural gene was detected during cheese-making from milk samples up to the end of curd ripening but not in the final cheese. In order to monitor nisin-producing strains during cheese manufacturing, the 12 Lactococcus lactis nis+ strains were analysed by low frequency restriction fragment and PFGE. Nine isolates among the 12 nisin-producers exhibited an unique and distinct DNA banding pattern and are considered to be genetically diverse. The other three isolates from curd after ripening showed the same restriction pattern and could be the same strain. In fact, it was also isolated 2 months after the first analysis of cheese-making of 'Fior di latte'.

The potential of a polyphasic PCR-dGGE approach in evaluating microbial diversity of natural whey cultures for water-buffalo Mozzarella cheese production: bias of culture-dependent and culture-independent analyses.

A polyphasic PCR-DGGE approach was used to describe the microbial population occurring in natural whey cultures (NWCs) for water-buffalo Mozzarella cheese production. Total microbial community was assessed without cultivation by analyzing DNA directly extracted from the original samples of NWC. In addition, DNA extracted from bulks of cells formed by harvesting colonies from the serial dilution agar plates of a variety of culture media was used to profile the "cultivable" community. The 16S rDNA V3 region was amplified using DNA from NWC as well as DNA from bulks as templates and the amplicons were separated by DGGE. The microbial entities occurring in NWCs were identified by partial 16S rDNA sequencing of DGGE bands: four lactic acid bacteria (LAB) closest relative of Streptococcus thermophilus, Lactococcus lactis, Lactobacillus delbrueckii and Lactobacillus crispatus were revealed by the analysis of DNA directly extracted from NWC while two other LAB, Lactobacillus fermentum and Enterococcus faecalis, were identified by analyzing DNA from the cultivable community. The developed PCR-DGGE analysis of the "cultivable" community showed good potential in evaluating microbial diversity of a dairy environment: it usefully highlighted the bias introduced by selective amplification when compared to the analysis of the total community from NWC and allowed suitability of media and growth conditions to be evaluated. Moreover, it could be used to complete the culture independent study of microbial diversity to give information on concentration ratios among species occurring in a particular environment and can be proposed for rapid identification of dominant microorganisms in alternative to traditional tools.

Conditions for conjugative transposon transfer in Lactococcus lactis.

Three different techniques for bacterial mating were applied to wild type and culture collection strains of Lactococcus lactis harbouring transposons: direct plate conjugation, filter mating and mating on milk agar. Efficiencies and frequencies of transfer were compared. Transconjugants were characterized by marker properties and molecular assays. Transposon-coded Suc+ Nis+ phenotype as well as Suc+ Bac+ Nis- phenotype were transferred with frequencies ranging between 10-9 and 10-6. Milk agar plate mating was the best technique for obtaining gene transfer events involving wild type lactococci.

Antibiotic resistance of coagulase-negative staphylococci isolated from artisanal Naples-type salami.

In the present paper 42 isolates from Italian salami were specified as Staphylococcus xylosus (30), Staph. capitis (1), Staph. saprophyticus (1), Staph. hominis (1), Staph. simulans (1), Staph. cohnii (1) and as Staph. spp. (7). These strains were coagulase-negative and were examined for resistance/sensitivity against 25 antibiotics including beta-lactams (7), macrolides (3), amynoglicosides (5), glycopeptides, lincosamides (4) and novobiocin, fusidic acid, chloramphenicol, rifampicin, tetracycline, minocycline. More than 64% of the strains were resistant to lincomycin, penicillin G, amoxicillin, fusidic acid and novobiocin. All the strains were multiresistant and displayed at least three resistances. Over 75% had a multiple antibiotic resistance (MAR) index between 0.2 and 0.5.