Application of molecular methods for the differentiation of acetic acid bacteria in a red wine fermentation.

AIMS: To apply rapid and reliable molecular techniques for typing acetic acid bacteria and studying their population dynamics during wine-making processes. METHODS AND RESULTS: We tested the usefulness of the Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR) and Repetitive Extragenic Palindromic-PCR (REP-PCR) techniques with reference strains of most of the species of acetic acid bacteria. We obtained exclusive patterns for each strain with the ERIC-PCR technique, proving the utility for characterizing below species level. REP-PCR technique was not as adequate for this purpose because some strains yielded identical fingerprint. One hundred twenty isolates from a commercial red wine fermentation were fingerprinted using both techniques. We detected a high degree of strain diversity in the first stage of fermentation that decreased throughout the process. However, several strains and species were dominant in the alcoholic fermentation phases. The identification of different strains or genotypes at the species level was carried out by restriction analysis of the 16S ribosomal DNA gene. Gluconobacter oxydans dominated the fresh must, while Acetobacter aceti was the only isolated species at the end of the process. Gluconacetobacter hansenii and G. liquefaciens were also isolated in significant numbers at the beginning of fermentation. CONCLUSIONS: ERIC-PCR and REP-PCR techniques proved useful for characterizing strains of acetic acid bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The availability of molecular techniques for a fast and reliable genotypic characterization should increase our knowledge of the ecology of acetic acid bacteria and determine more accurately their growth behaviour during various stages of vinification.

Anti-inflammatory activity of Debaryomyces hansenii Cu,Zn-SOD.

BACKGROUND: Cu,Zn-superoxide-dismutase, Cu,Zn-SOD, can be obtained from different sources with different anti-inflammatory activities. In this study we compared the anti-inflammatory capacity of the marine yeast Debaryomyces hanseii Cu,Zn-SOD (Dh-SOD) with that of bovine erythrocytes (Be-SOD) in a preventive and a therapeutic fashion. METHODS: Edema was induced by carrageenan injection into the rat hind paw and was evaluated using a mercury plethysmograph. Development of the inflammatory process was followed by volume displacement at time 0 (carrageenan injection), 1, 2, 3, 4, 5, 6, 9, 12, and 24 h thereafter. Three different SOD doses were used in preliminary experiments to prevent edema: 10, 100, and 1,000 U/kg. RESULTS: The results indicate that, at the lowest dose (10 U/kg), both SOD samples are effective in reducing inflammation in both the prostaglandin and amplification phases (-24.8% and -17.5% in the case of Be-SOD, and 11.8% and -18.7% in the case of Dh-SOD, respectively) (p < 0.05). At 100 U/kg, Be-SOD also shows good anti-inflammatory activity in all edema phase (-27.1% in the serotonin phase; -19.4% in the prostaglandin phase; and -20% in the amplification phase) (p < 0.05), but Dh-SOD was less effective (-10.9%, -9.1%, and -5.7%). At the highest dose tested (1000 U/kg), Dh-SOD was, again, more effective than Be-SOD in all three edema phases (-33.1% and -1.5%; -17.9% and -2.6%; and -13.8% and 6.7%, respectively) (p < 0.05). When evaluated as a therapeutic alternative, single doses of Dh-SOD at 1,000 U/kg, and Be-SOD at 100 U/kg, both showed good anti-inflammatory activities (-31.7% and -23.5%, respectively) (p < 0.05). CONCLUSION: For therapy purposes alone, Dh-SOD appears to be a better anti-inflammatory agent than Be-SOD in carrageenan-induced edema.

Serological reactivity to a synthetic analog of phenolic glycolipid I and early detection of leprosy in an area of low endemicity.

A total of 23,863 individuals living in an area of low endemicity for leprosy were tested by enzyme-linked immunosorbent assay with a semisynthetic analogue of the phenolic glycolipid I antigen of Mycobacterium leprae. The proportion found positive was 3.86% which was significantly higher than that in a sample of a population known to be free of leprosy. Clinical examinations as well as Mitsuda and skin smear tests were organized for those defined as seropositive. The proportion of individuals with lepromin reactions of less than 3 mm increased 18.9% per serological interval as antibodies rose though it was not statistically significant. As a result of the clinical and bacteriological examinations, 2 cases with clinical signs and heavy bacillary load were found, whereas acid-fast bacilli were demonstrated in 2 other individuals without clinical manifestations of leprosy. The usefulness of the system for control purposes is discussed.

Survey for secondary dapsone and rifampicin resistance in Cuba.

A total of 1211 Cuban multibacillary leprosy patients treated for at least 5 years were clinically and bacteriologically examined. They were being treated according to a 2-phase monotherapy regimen with RMP first and DADDS afterwards. On skin-smear examination 50 patients were found positive, of which 9 showed a BI of 3+ or higher at any site. With regard to the clinical status the only cases found with clinical signs of relapse were 5 out of 7 long-standing patients with BI of 4+ and 5+. A 6th patient of this high BI group who showed a good clinical condition, except for a heavy infiltration of both earlobes, was receiving a second RMP course when examined and biopsied for this research. These 9 patients were biopsied and susceptibility tests to RMP and DDS performed. The results showed that in 1 case the Mycobacterium leprae were resistant to both drugs; the organisms from 2 other patients were susceptible to RMP but low-grade resistant to DDS. Those from another patient were susceptible to RMP and fully resistant to DDS. In 3 other cases the bacilli did not multiply in any of the mice but 1 of these strains was from the patient taking a second RMP course, therefore this strain might also be susceptible to RMP and resistant to DDS. In the last 2 cases multiplication was only observed in 2 of the controls and in 1 of the 0.0001% DDS treated mice; therefore, these experiments were not conclusive, and the AFB recovered were inoculated into fresh mice to repeat the tests but these failed to multiply.

Functional interactions between putative intramembrane charged residues in the lactose permease of Escherichia coli.

Using a lactose permease mutant devoid of Cys residue ("C-less permease"), we systematically replaced putative intramembrane charged residues with Cys. Individual replacements for Asp-237, Asp-240, Glu-269, Arg-302, Lys-319, His-322, Glu-325, or Lys-358 abolish active lactose transport. When Asp-237 and Lys-358 are simultaneously replaced with Cys and/or Ala, however, high activity is observed. Therefore, when either Asp-237 or Lys-358 is replaced with a neutral residue, leaving an unpaired charge, the permease is inactivated, but neutral replacement of both residues yields active permease [King, S. C., Hansen, C. L. & Wilson, T. H. (1991) Biochim. Biophys. Acta 1062, 177-186]. Remarkably, moreover, when Asp-237 is interchanged with Lys-358, high activity is observed. The observations provide a strong indication that Asp-237 and Lys-358 interact to form a salt bridge. In addition, the data demonstrate that neither residue nor the salt bridge plays an important role in the transport mechanism. Thirteen additional double mutants were constructed in which a negative and a positively charged residue were replaced with Cys. Only Asp-240-->Cys/Lys-319-->Cys exhibits significant activity, accumulating lactose to 25-30% of the steady state observed with C-less permease. Replacing either Asp-240 or Lys-319 individually with Ala also inactivates the permease, but double mutants with neutral substitutions (Cys and/or Ala) at both positions exhibit essentially the same activity as Asp-240-->Cys/Lys-319-->Cys. In marked contrast to Asp-237 and Lys-358, interchanging Asp-240 and Lys-319 abolishes active lactose transport. The results demonstrate that Asp-240 and Lys-319, like Asp-237 and Lys-358, interact functionally and may form a salt bridge. However, the interaction between Asp-240 and Lys-319 is clearly more complex than the interaction between Asp-237 and Lys-358. In any event, the findings suggest that putative transmembrane helix VII lies next to helices X and XI in the tertiary structure of lactose permease.

Serodiagnosis of leprosy in patients' contacts by enzyme-linked immunosorbent assay.

Serum samples from 3336 contacts of leprosy patients were tested for antiphenolic glycolipid I antibodies by enzyme-linked immunosorbent assay with the albumin coupled synthetic disaccharide antigen. The overall positivity rate was 9.3%. No significant differences were seen between a group of household contacts of lepromatous patients and those of the other types of the disease. The proportion of ELISA positives was slightly higher in the relatives as compared to workplace contacts and neighbours but significantly different only between the two former (p less than 0.05). Among those contacts with absorbance values higher than 0.100, 5 new leprosy patients were diagnosed, 2 of them with positive skin smears. A sixth contact was detected with a very high absorbance value in whom no single skin lesion was found but whose lepromin reaction was 0 mm and the skin smear showed a bacteriological index of 3+.

[Monitoring of the treatment effectiveness in patients with leprosy using solid-phase immunoenzyme assay]. / Monitoreo de la eficacia del tratamiento en pacientes con lepra mediante el ensayo inmunoenzimático sobre fase sólida.

Serum samples from 184 lepers are studied by means of solid phase ELISA with the semisynthetic antigen disaccharide-bovine albumin analogous of phenolic glycolipid I of Mycobacterium leprae. Patients are grouped according to the clinical presentation of the disease and course of time elapsed since the institution of therapy. Bacilloscopic examinations were made also in 116 patients who were positive at diagnosis. For serologic testing, absorbance values over 0,160 were considered positive. Results in multibacillary patients showed a gradual and significant decrease both of mean absorbance values and in the seropositive ratio in connection with the length of treatment. It was also found that phenolic anti glycolipid antibodies increased with the value of the bacteriologic index. This system is useful in monitoring the effectiveness of chemotherapy in multibacillary leprosy.