The microbial ecology of a typical Italian salami during its natural fermentation.

We have investigated the bacteria and yeast ecology of the typical Italian Ciauscolo salami that is produced in Central Italy using a polyphasic approach based on culture-dependent and -independent methods. The physico-chemical analyses showed a progressive drop in pH and water activity (aw) during ripening. The viable counts revealed a dominance of lactic acid bacteria (LAB) over coagulase negative cocci (CNC) and yeasts. From the molecular identification of the isolates, the prevalence of Lactobacillus curvatus, Lb. plantarum and Staphylococcus xylosus was shown among the bacteria, while Debaryomyces hansenii was the prevalent species among the yeasts, and it was isolated throughout the whole ripening process. Minority species, namely Rhodotorula mucillaginosa and Trichosporon brassicae, were also recovered from the meat batter. The total microbial community was profiled without cultivation by analyzing the DNA that was directly extracted from the salami samples. Moreover, the cultivable community was profiled by analyzing the DNA recovered from bulk cells that were obtained by harvesting the colonies from serial-dilution agar plates. The 16S rRNA gene V1 and V3 regions were used as targets in the denaturing gradient gel electrophoresis (DGGE) profiling of the LAB and CNC communities, respectively, while the diversity and dynamics of the yeast population were assessed by analyzing a portion of the 28S rRNA gene. Our findings suggest that the microbial diversity of fermented meat products can be successfully investigated by this polyphasic approach that is based on the assessment of both the total and the cultivable community diversity.

Microbial ecology of the soppressata of Vallo di Diano, a traditional dry fermented sausage from southern Italy, and in vitro and in situ selection of autochthonous starter cultures.

The microbial ecology of "soppressata of Vallo di Diano," a traditional dry fermented sausage from southern Italy, was studied by using both culture-dependent and culture-independent approaches. The ripened fermented sausages were characterized by high microbial loads of both staphylococci and lactobacilli. Using PCR-denaturing gradient gel electrophoresis (PCR-DGGE) targeting the variable V3 and V1 regions of the 16S rRNA gene and direct DNA sequencing, it was possible to identify Staphylococcus xylosus, S. succinus, and S. equorum among the staphylococci and Lactobacillus sakei and L. curvatus within the lactobacilli. Moreover, Debaryomyces hansenii was the main yeast species found by targeting the yeast 26S rRNA gene by PCR-DGGE. Selected strains of S. xylosus, L. sakei, and L. curvatus were characterized for their technological properties in the ripening conditions of the fermented sausages so as to select an autochthonous starter formulation. The selection included the determination of nitrate reductase, lipolytic, and antioxidant activity and proteolysis with myofibrillar and sarcoplasmic protein fractions. Such properties were evaluated in both in vitro and in situ assays; the latter were performed by using each strain as a starter in the laboratory-scale manufacture of soppressata of Vallo di Diano and by monitoring the microbiological and chemical changes at the end of ripening. The results show differences between the in vitro and in situ selection results and indicate that in situ evaluation of the technological performance of specific strains is better suited to selecting autochthonous starter cultures for fermented-meat products than in vitro evaluation.

Sources of the adventitious microflora of a smear-ripened cheese.

AIMS: To determine the relationships between the major organisms from the cheese-making personnel and environment and the surface of a smear cheese. METHODS AND RESULTS: 360 yeast and 593 bacteria from the cheese surface, the dairy environment and the hands and arms of personnel were collected. Pulsed-field gel electrophoresis, repetitive sequence-based polymerase chain reaction and 16S rDNA sequencing were used for typing and identifying the bacteria, and mitochondrial DNA restriction fragment length polymorphism and Fourier-transform infrared spectroscopy for typing and identifying the yeast. The three most dominant bacteria were Corynebacterium casei, Corynebacterium variabile and Staphylococcus saprophyticus, which were divided into three, five and seven clusters, respectively, by macrorestriction analysis. The same clones from these organisms were isolated on the cheese surface, the dairy environment and the skin of the cheese personnel. Debaryomyces hansenii was the most dominant yeast. CONCLUSIONS: A 'house' microflora exists in the cheese plant. Although the original source of the micro-organisms was not identified, the brines were an important source of S. saprophyticus and D. hansenii and, additionally, the arms and hands of the workers the sources of C. casei and C. variabile. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time that the major contribution of the house microflora to the ripening of a smear-ripened cheese has been demonstrated.

Staphylococcus equorum subsp. linens, subsp. nov., a starter culture component for surface ripened semi-hard cheeses.

Two staphylococcal strains, RP29T and RP33, were isolated from the main microflora of a surface ripened Swiss mountain cheese made from raw milk. These two strains were differentiated from the most closely related species Staphylococcus equorum on the basis of DNA-DNA hybridisation and phenotypic characteristics and are proposed as Staphylococcus equorum subsp. linens subsp. nov. They could be distinguished phenotypically from S. equorum by their sensitivity to all 14 tested antibiotics, especially to novobiocin, their incapability to ferment alpha-D-lactose, maltose, sucrose, D-trehalose, D-xylose, L-arabinose, salicin, D-ribose, D-raffinose, D-mannitol, and D-alanine. The GenBank accession numbers for the reference sequences of the 16S rDNA and the hsp60 gene used in this study are AF527483 and AF527484, respectively. 30 tons of a semi-hard Swiss cheese were produced with Staphylococcus equorum subsp. linens DSM 15097T as starter culture component in addition to Debaryomyces hansenii, Geotrichum candidum, Brevibacterium linens, Corynebacterium casei for surface ripened cheeses. The products were sensorically and hygienically perfect. Therefore, Staphylococcus equorum subsp. linens DSM 15097T can be proposed as starter culture component for surface ripened cheeses without any detected antibiotic resistances. The type strain of Staphylococcus equorum subsp. linens is DSM 15097T (CIP 107656T).

Characterization of microbial flora of leprous ulcers infested with maggots.

Swabs from 64 maggot infested leprosy ulcers before and after treatment for maggots and 100 non-infested leprosy ulcers were studied for the bacterial flora. From maggot infested ulcers (before treatment), the cultures usually showed mixed growth. Among the Gram positive isolates, Staphylococcus aureus (37%), S. albus (18%) and Streptococcus pyogenes (36%) were frequently isolated. Gram negative bacteria isolated were Proteus spp. (21%) and Escherichia coli (7%). Anaerobic bacteria isolated were Micrococcus (3%) and Bacteroides (4%). After treatment of maggot infested ulcers, S. aureus (36%) continued to be isolated with almost the same frequency. The isolates of other Gram negative organisms were slightly reduced. Among the Gram negatives the Proteus spp. (10%) were also less in number. In few cases Neisseria (3%) was found. Anaerobic isolates were M. luteus (2%) and B. necrophorus (3%). From the cases without maggot infestation, a single organism was isolated from 16 cases and 84 mixed cultures were obtained. Isolates included the aerobic Gram positives S. aureus (46%), S. albus (21%) and S. pyogenes (38%), and the Gram negative Proteus spp. (19%) and E. coli (7%). The anaerobic isolate was M. luteus (3%). From this study no apparent association between the type of bacterial flora and maggot infestation could be observed.

Bacteriological study of trophic ulcers in leprosy patients (a preliminary study).

Swabs from trophic ulcers from 108 cases were studied by culture. 37 cases yielded single organism (Ps. aeruginosa, 18, Proteus species 11, Staph. pyogenes 4, Others 4). 71 cases yielded mixed growth with two or more organisms. Ps. aeruginosa, Proteus species and Diphtheroids were the predominant organisms in these cultures. Ps. aeruginosa was sensitive to Gentamycin (96.6%), Streptomycin (62.7%) and Chloramphenicol (33.9%). Other organisms although comparatively more sensitive showed a similar pattern.

Variably acid-fast bacteria in a case of systemic sarcoidosis and hypodermitis sclerodermiformis

The histologic finding of variably acid-fast coccoid forms in all the available biopsy material (skin, lymph nodes, and lung) from a case of coexisting scleroderma-like cutaneous disease (hypodermitis sclerodermiformis) and systemic sarcoidosis is reported. The morphologic size, shape, and staining characteristics of these microbes, along with the presence of the lung of 'large bodies', suggest that these microbes are cell wall deficient L forms of mycobacteria. Culture of the skin of the scleroderma-like lesion yielded Staphylococcus epidermidis, and the relationship of this isolate to the histologic findings of bacteria is discussed, as well as the possible pathogenic role played by L forms of mycobacteria in collagen disease and systemic sarcoidosis.

Bacteria isolated from plantar ulcers of Ethiopian leprosy patients, with the antibacterial drug sensitivities of the isolates

Bacteriological sampling of grossly infected or chronic plantar ulcers was perfomed in 39 untreated patients with leprosy and in 22 patients who had received antibiotic treatment. Samples were cultured aerobically and anaerobically, and films of pus were stained by Gram´s method. Stained films gave little indication of the type of infecting pathogen, except when Gram-positive cocci alone were seen. From the ulcers of patients untreated with antibiotics anaerobic streptococci were isolated more frequently than any other organism, and and this may be an original observation. Of the 8 Staphylococcus aureus isolates 5 were penicillin sensitive. A renge of Gram-negative bacteria, but no Clostridia, were isolated. From the ulcers of patients who had received antibiotics penicillin-resistant Staph. aureus was most frequently isolated. Some Gram-positive bacteria resistant to tetracycline were sensitive to doxycycline.