Specific detection of toxigenic vibrio cholerae based on in situ PCR in combination with flow cytometry.

OBJECTIVE: To develop an in situ PCR in combination with flow cytometry (ISPCR-FCM) for monitoring cholera toxin positive Vibrio cholerae. METHODS: In running this method, 4% paraformaldehyde was used to fix the Vibrio cholerae cells and 1 mg/mL lysozyme for 20 min to permeabilize the cells. Before the PCR thermal cycling, 2.5% glycerol was added into the PCR reaction mixture in order to protect the integrality of the cells. RESULTS: A length of 1037bp DNA sequence was amplified, which is specific for the cholera toxin gene (ctxAB gene). Cells subjected to ISPCR showed the presences of ctxAB gene both in epifluorescence microscopy and in flow cytometric analysis. The specificity and sensitivity of the method were investigated. The sensitivity was relatively low (10(5) cells/mL), while the specificity was high. CONCLUSION: We have successfully developed a new technique for detection of toxigenic Vibrio cholerae strains. Further study is needed to enhance its sensitivities. ISPCR-FCM shows a great promise in monitoring specific bacteria and their physiological states in environmental samples.

[The recent research progress on Bdellovibrio bacteriourus].

The recent research progress on taxonomy and physiological of B. bacteriouorus were briefly touched. The bacteriouorus HD100 and the system for genetic recombination and repair in B. genome characteristics of B. bacteriouorus were summarized in great detail. Prey cells invading mechanism(s) and biochemical characteristics of B. bacteriouorus, including respiration and synthesis and nutrients transportation and absorption were introduced in detail too. Recent practical applications of B. bacteriouorus were reviewed and the potential problems in applications were also pointed out.

[Study of bacteria-lysis abilities and growth conditions of 4 Bdellovibrio].

Bdellovibrio can lyse pathogenic bacteria and clean up waters. 4 strains of Bdellovibrio sp., designated Bh04-4, Bh04-41a, Bh04-A + and Bh04-1f, were isolated from seawaters used Bh04 as host bacterium. After confirmation to be Bdellovibrio sp. by electron microscopy and specific PCR method, their growth conditions and lytic ability on 61 bacteria from various sources were performed. Results showed that all four Bdellovibrio grew in salinity in the range of between 1% and 3%, with 3% salinity being the most suitable one. They grew in the range of temperature from 15 to 30 degrees C, with 20 - 25 degrees C as their best growth temperature. These four Bdellovibrio only grew on live host bacteria rather than the dead ones. When 61 strains of bacteria were used as hosts, Bh04-4 lysed 21 strains, corresponding to 34.4% of lysis ability (21/61), Bh04-41a lysed 24 strains (39.3% lysis ability), Bh04-A + lysed 40 strains (65.6% lysis ability) and Bh04-1f 43 strains (70.5% lysis ability). Taken all four Bdellovibrio together, they lysed 55 out of 61 strains, amounting to 90.2% lysis ability. Results fully demonstrate the potential application of Bdellovibrio in lysing pathogenic bacteria from the marine environments.

[Studies on extracellular virulent factors produced by potential pathogenic bacteria isolated from abalone postlarvae and biofilms].

To find out the potential pathogen(s) that caused massive death of abalone postlarvae( Haliotis diversicolor supertesta ) in Southern China, 105 bacterial strains were isolated from the water, whitened postlarvae and their biofilms of an abalone farm in Guangdong Province. Extra-cellular protease, gelatinase, lipase as well as haemolysis tests were performed on them. Lysophospholipase (Tlh)-targeted PCR was also carried out in order to reveal if the haemolysis caused by bacterial strains were related to Tlh. Results showed that 35 out of 105 strains possessed extra-cellular enzymatic abilities and strains 1, 2, 3, 5, 9 and 16 were the most powerful ones of all. Among these 35 strains, 85.6% strains (30/35) possessed haemolytic activities on goat blood agar and specific PCR revealed that 16 strains were PCR positive, indicating that they contained Tlh genes. In order to identify these strains, API 20 E strips were employed and results indicated that 50% were vibrios, among them Vibrio alginolyticus composed 70% of the vibrio population. In conclusion, compared to other strains, 6 Vibrio alginolyticus (strain 1, 2, 3, 5, 13, and 16 ) and 2 Vibrio parahaemolyticus (strain 9 and 21) which all were isolated from whitened abalone postlarvae, warrant further studies due to their better abilities in extra-cellular enzyme(s) production and/or haemolytic activities.