Untargeted GC-MS Metabolomics Reveals Changes in the Metabolite Dynamics of Industrial Scale Batch Fermentations of Streptoccoccus thermophilus Broth.

An industrial scale biomass production using batch or fed-batch fermentations usually optimized by selection of bacterial strains, tuning fermentation media, feeding strategy, and temperature. However, in-depth investigation of the biomass metabolome during the production may reveal new knowledge for better optimization. In this study, for the first time, the authors investigated seven fermentation batches performed on five Streptoccoccus thermophilus strains during the biomass production at Chr. Hansen (Denmark) in a real life large scale fermentation process. The study is designed to investigate effects of batch fermentation, fermentation time, production line, and yeast extract brands on the biomass metabolome using untargeted GC-MS metabolomics. Processing of the raw GC-MS data using PARAFAC2 revealed a total of 90 metabolites out of which 64 are identified. Partitioning of the data variance according to the experimental design was performed using ASCA and revealed that batch and fermentation time effects and their interaction term were the most significant effects. The yeast extract brand had a smaller impact on the biomass metabolome, while the production line showed no effect. This study shows that in-depth metabolic analysis of fermentation broth provides a new tool for advanced optimization of high-volume-low-cost biomass production by lowering the cost, increase the yield, and augment the product quality.

A super-infection in the cornea caused by Stemphylium, Acremonium, and α-Streptococcus.

BACKGROUND: Polymicrobial keratitis with fungus and bacteria can lead to blindness and is challenging to treat. Here, we introduce a case of fungal keratitis caused by two different strains in addition to definite bacterial super-infection caused by an α-Streptococcus sp., and describe the importance of microscopic examination. CASE PRESENTATION: A 74-year-old woman, who had a past history of infection with leprosy, presented with conjunctival hyperaemia, pain, and corneal opacity in her right eye. Under the presumptive diagnosis of infectious keratitis, corneal scrapings were stained by various reagents and inoculated on several agar plates. Microscopic findings of the scrapings revealed fungi and a small number of Gram-positive cocci. Multiple anti-fungal therapies with levofloxacin ophthalmic solution were administered. Although empiric treatment was initially effective, keratitis recurred 10 days after its initiation. Repeated corneal scraping revealed an abundance of Gram-positive chain cocci and a small amount of fungi, resulting in the switching of an antibiotic medication from levofloxacin to moxifloxacin and cefmenoxime. Keratitis resolved gradually after the conversion. Stemphylium sp., Acremonium sp., and α-Streptococcus sp. were simultaneously isolated from the corneal scrapings. CONCLUSIONS: To the best of our knowledge, this is the first case of fungal keratitis caused by Stemphylium sp., and also the first case of super-infection in the cornea caused by two different fungi and one bacterium. Microscopic examination of the corneal scrapings was beneficial in rapid decision of changing to appropriate drug according to the dominancy of pathogenicity.

Culture-independent detection of microorganisms in traditional Slovakian bryndza cheese.

In order to investigate the microflora of Slovakian bryndza cheese (a cheese containing unpasteurized or pasteurized ewes' milk component) by a culture-independent method, DNA was extracted directly from 7 bryndza samples and analysed by an innovative method. Using the universal prokaryotic and fungal primers, ribosomal DNA internal transcribed spacer (ITS) regions with variable length were amplified. The standard universal reverse primer L1 aligning to bacterial 23s rDNA was found unsuitable for some lactic acid bacteria and other species based on in silico analysis. Therefore, L1 primer was replaced by a combination of novel primers GplusR and GminusR aligning to the adjacent, more conserved DNA region. The amplification profiles were visualised by both standard electrophoresis and by fluorescent capillary gel electrophoresis. From representative samples, major amplicons were excised from the gel, cloned and sequenced. Sequencing revealed that the samples contained Lactobacillus delbrueckii, Lactobacillus brevis, Streptococcus thermophilus, Lactococcus lactis, Lactococcus raffinolactis, Streptococcus macedonicus, Leuconostoc pseudomesenteroides, Debaromyces hansenii, Mucor fragilis, Yarrowia lipolytica and Galactomyces geotrichum. These results represent an extension of the knowledge on the microflora of Slovakian bryndza cheese. The introduced automated ribosomal DNA intergenic spacer analysis of the bacterial and fungal genomes proved to be very effective in the application of studying microflora of cheese.

Quantification of micro-organisms in binary mixed populations by Fourier transform infrared (FT-IR) spectroscopy.

Fourier Transform Infrared (FT-IR) spectroscopy was used for the first time to determine the ratios of different microorganisms in mixtures. Exemplarily, systems composed of two food-associated yeast species (Saccharomyces cerevisiae/Hanseniaspora uvarum) and two yoghurt lactic acid bacteria (Lactobacillus acidophilus/Streptococcus salivarius ssp. thermophilus) were investigated. Determination of the cell number ratio in the lactic acid bacteria system was possible with a minimal prediction accuracy of +/- 16 ratio percentage points while the minimum accuracy of prediction in the yeast two-component system was +/- 4% (both at a 95% confidence level). These results show that FT-IR spectroscopy is potentially a rapid method for the quantification of cell ratios in mixtures of two different microorganisms, provided that the cell ratio does not drop below a certain, system-specific threshold.

Microbial colonizers in leprosy skin ulcers and intensity of inflammation.

The microflora of 55 patients with leprosy skin ulcers was studied and related to a weighted inflammatory score (IS). The control group consisted of 18 ulcers with different underlying pathology. Leprosy ulcers were characterized by the exclusive presence of two types of branching gram-positive rods; a particular interesting proposal is that Mycobacterium leprae share common antigens with these unusual "leprosy ulcer associated" organisms and group G beta-hemolytic streptococci. In the leprosy group, corynebacteria and branching rods accounted for 97% of gram-positive bacilli and Bacillus species constituted only 3%. In the control group, B. species formed 50% of gram-positive rods; the rest were corynebacteria (p = 0.03). In the leprosy group, one third of the gram-positive bacteria were branching rods; none of them was acid fast. Ten of them were identified as Arcanobacterium haemolyticum, and the remaining 7 could not be identified. The IS of leprosy patients was lower than in the control group. The presence of more than two species of facultative or aerobic gram-negative rods or single species of pyogenic gram-positive cocci correlated with a high IS. The presence of two or more different pyogenic cocci resulted in a lower IS. Further studies into the nature of leprosyunique organisms as well as the inflammation inhibition factors in mixed infections are warranted. It is recommended that management of ulcers should consist of the application of local disinfection and early treatment of episodes of inflammation with a combination of fluoroquinolone and penicillin.

Human T cell lymphotropic virus (HTLV-1): cutaneous manifestations

Skin manifestations are a common feature of HTLV-1 associated disorders and of HTLV-1 infection itself. These include the lymphomatous skin infiltrates in adult T-cell lymphoma/leukaemia, most commonly manifesting as persistent, generalised papules, nodules and plaques with later ulceration, acquired ichthyosis and xeroderma in HAM/TSP, infective dermatitis of children, dermatomyositis, crusted (Norwegian) scabies, psoriasiform rashes which may precede one of the more serious disease associations, and possibly also seborrhoeic dermatitis. Disorders typically associated with immunosuppression such as disseminated herpes zoster, and ulcerative non-healing herpes simplex may also be seen occasionally both in ATK as well as in other wise asymptomatic HTLV-1 infection (AU)

Phylogenetic analysis of Ruminococcus flavefaciens, the type species of the genus Ruminococcus, does not support the reclassification of Streptococcus hansenii and Peptostreptococcus productus as ruminococci.

The 16S rRNA gene sequence of the type strain of Ruminococcus flavefaciens, the type species of the genus Ruminococcus, was determined by PCR direct sequencing. A comparative sequence analysis showed that R. flavefaciens is phylogenetically related to a small cluster (cluster IV of Collins et al. [M. D. Collins, P. A. Lawson, A. Willems, J. J. Cordoba, J. Fernandez-Garayzabal, P. Garcia, J. Cai, H. Hippe, and J. A. E. Farrow, Int. J. Syst. Bacteriol. 44:812-826, 1994]) of organisms which includes several Clostridium and Eubacterium species. R. flavefaciens was found to be phylogenetically only remotely related to Ruminococcus gnavus, Ruminococcus torques, Peptostreptococcus productus, and Streptococcus hansenii. These findings demonstrate that the genus Ruminococcus is not a monophyletic group, and the proposed transfer of P. productus and S. hansenii to this genus (T. Ezaki, N. Li, Y. Hashimoto, H. Miura, and H. Yamamoto, Int. J. Syst. Bacteriol. 44:130-136, 1994) is not supported.

Importance of group G streptococci in human pyogenic infections.

Streptococci that belong to groups other than A are involved in a variety of human infections. In the present study, we report the pyogenic infections associated with group G streptococci (GGS), observed at the Christian Medical College Hospital, Vellore, Tamil Nadu, India. Of 2314 beta-haemolytic streptococci isolated from 31,857 pus specimens processed during the period 1975-1986, GGS accounted for 338 (14.6%). Twelve of these were grown in pure culture. The clinical conditions associated with them included pyogenic arthritis, osteomyelitis, paravertebral sinus and an abdominal burst. Clinical analysis of 99 of 125 patients with GGS, grown in mixture with another organism or skin contaminants, showed that 59% of them were from surgical wound infections, diabetic lesions or wound infections in leprosy patients. Surgical management and antibiotic treatment resulted in full recovery of these clinical conditions.

Plantar ulcers with osteomyelitis underneath. A bacteriological study.

39 consecutive cases of plantar ulcers with underlying chronic osteomyelitis admitted in the Sacred Heart Hospital during 1975/1976 were studied for the infecting organisms and their sensitivity to easily available antibiotics. Single organism was iasolated in only 10 cases, the infection in the rest being a mixed one. The commonest organisms were Staphylococcus, Streptococcus and Proteus mirabilis. In a few cases Pseudomonas and E-Coli were also isolated. Chloramphenicol was the most effective antibiotic in general and Streptomycin the least. 70% of the staphylococcus strains isolated were found to be resistant to Penicillin. Empirical use of antibiotics especially Penicillin and Streptomycin is hence deprecated.

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.