Cytotoxicity, Intracellular Replication, and Contact-Dependent Pore Formation of Genotyped Environmental Legionella pneumophila Isolates from HospitalWater Systems in the West Bank, Palestine

Legionella pneumophila is the causative agent of Legionnaires’ disease. Due to the hot climate and intermittent water supply, the West Bank, Palestine, can be considered a high-risk area for this often fatal atypical pneumonia. L. pneumophila occurs in biofilms of natural and man-made freshwater environments, where it infects and replicates intracellularly within protozoa. To correlate the genetic diversity of the bacteria in the environment with their virulence properties for protozoan and mammalian host cells, 60 genotyped isolates from hospital water systems in the West Bank were analyzed. The L. pneumophila isolates were previously genotyped by high resolution Multi Locus Variable Number of Tandem Repeat Analysis (MLVA-8(12)) and sorted according to their relationship in clonal complexes (VACC). Strains of relevant genotypes and VACCs were compared according to their capacity to infect Acanthamoeba castellanii and THP-1 macrophages, and to mediate poreforming cytotoxicity in sheep red blood cells (sRBCs). Based on a previous detailed analysis of the biogeographic distribution and abundance of the MLVA-8(12)-genotypes, the focus of the study was on the most abundant L. pneumophila- genotypes Gt4(17), Gt6 (18) and Gt10(93) and the four relevant clonal complexes [VACC1, VACC2, VACC5 and VACC11]. The highly abundant genotypes Gt4(17) and Gt6(18) are affiliated with VACC1 and sequence type (ST)1 (comprising L. pneumophila str. Paris), and displayed seroroup (Sg)1. Isolates of these two genotypes exhibited significantly higher virulence potentials compared to other genotypes and clonal complexes in theWest Bank. Endemic for theWest Bank was the clonal complex VACC11 (affiliated with ST461) represented by three relevant genotypes that all displayed Sg6. These genotypes unique for the West Bank showed a lower infectivity and cytotoxicity compared to all other clonal complexes and their affiliated genotypes. Interestingly, the L. pneumophila serotypes ST1 and ST461 were previously identified by in situ-sequence based typing (SBT) as main causative agents of Legionnaires’ disease (LD) in the West Bank at a comparable level. Overall, this study demonstrates the site-specific regional diversity of L. pneumophila genotypes in theWest Bank and suggests that a combination of MLVA, cellular infection assays and hierarchical agglomerative cluster analysis allows an improved genotype-based risk assessment

Microevolution of Pandemic Vibrio parahaemolyticus Assessed by the Number of Repeat Units in Short Sequence Tandem Repeat Regions

The emergence of the pandemic strain Vibrio parahaemolyticus O3:K6 in 1996 caused a large increase of diarrhea outbreaks related to seafood consumption in Southeast Asia, and later worldwide. Isolates of this strain constitutes a clonal complex, and their effectual differentiation is possible by comparison of their variable number tandem repeats (VNTRs). The differentiation of the isolates by the differences in VNTRs will allow inferring the population dynamics and microevolution of this strain but this requires knowing the rate and mechanism of VNTRs' variation. Our study of mutants obtained after serial cultivation of clones showed that mutation rates of the six VNTRs examined are on the order of 10−4 mutant per generation and that difference increases by stepwise addition of single mutations. The single stepwise mutation (SSM) was deduced because mutants with 1, 2, 3, or more repeat unit deletions or insertions follow a geometric distribution. Plausible phylogenetic trees are obtained when, according to SSM, the genetic distance between clusters with different number of repeats is assessed by the absolute differences in repeats. Using this approach, mutants originated from different isolates of pandemic V. parahaemolyticus after serial cultivation are clustered with their parental isolates. Additionally, isolates of pandemic V. parahaemolyticus from Southeast Asia, Tokyo, and northern and southern Chile are clustered according their geographical origin. The deepest split in these four populations is observed between the Tokyo and southern Chile populations. We conclude that proper phylogenetic relations and successful tracing of pandemic V. parahaemolyticus requires measuring the differences between isolates by the absolute number of repeats in the VNTRs considered

Pretubulysin: From Hypothetical Biosynthetic Intermediate to Potential Lead in Tumor Therapy

Pretubulysin is a natural product that is found in strains of myxobacteria in only minute amounts. It represents the first enzyme-free intermediate in the biosynthesis of tubulysins and undergoes post-assembly acylation and oxidation reactions. Pretubulysin inhibits the growth of cultured mammalian cells, as do tubulysins, which are already in advanced preclinical development as anticancer and antiangiogenic agents. The mechanism of action of this highly potent compound class involves the depolymerization of microtubules, thereby inducing mitotic arrest. Supply issues with naturally occurring derivatives can now be circumvented by the total synthesis of pretubulysin, which, in contrast to tubulysin, is synthetically accessible in gram-scale quantities. We show that the simplified precursor is nearly equally potent to the parent compound. Pretubulysin induces apoptosis and inhibits cancer cell migration and tubulin assembly in vitro. Consequently, pretubulysin appears to be an ideal candidate for future development in preclinical trials and is a very promising early lead structure in cancer therapy

Long-Term Changes in Chemostat Cultures of Cytophaga johnsonae

Long-term studies with a gliding, heterotrophic bacterium, Cytophaga johnsonae, were conducted in a glucose-limited chemostat at a high and a low dilution rate. To test the stability of the steady state during long-term experiments the following parameters were monitored: optical density, glucose concentration, glucose uptake potential, ATP content of the cells, and plate counts on two different agar media. Biomass remained relatively constant, although the observed changes could have been possible in both directions. During all steady states, glucose uptake showed a stepwise increase and the glucose concentration showed a corresponding decrease. Glucose uptake potential and glucose concentration in the chemostat were inversely proportional. The ATP content of the cells varied up to 33% during the steady state, but did not show a general trend. After long cultivation in all chemostats, plate counts on both agars dropped to values less than 20% of the original steady-state level. These decreases were due to an inability of the cells to grow on agar plates, not to a lack of vitality of the cells in the chemostat. This study showed that even during shorter chemostat runs, e.g., 1 week, changes in important parameters with the steady state must be expected, especially in the uptake potential and the concentration of the limiting substrate

Changes in Bacterioplankton Community Structure and Activity with Depth in a Eutrophic Lake as Revealed by 5S rRNA Analysis

The community structure of bacterioplankton was studied at different depths (0 to 25 m) of a temperate eutrophic lake (Lake Plußsee in northern Germany) by using comparative 5S rRNA analysis. The relative amounts of taxonomic groups were estimated from 5S rRNA bands separated by high-resolution electrophoresis. Comparison of partial 5S rRNA sequences enabled detection of changes in single taxa over space and during seasons. Overall, the bacterioplankton community was dominated by 3 to 14 abundant (>4% of the total 5S rRNA) taxa. In general, the number of 5S rRNA bands (i.e., the number of bacterial taxa) decreased with depth. In the fall, when thermal stratification and chemical stratification were much more pronounced than they were in the spring, the correlation between the depth layers and the community structure was more pronounced. Therefore, in the fall each layer had its own community structure; i.e., there were different community structures in the epilimnion, the metalimnion, and the hypolimnion. Only three 5S rRNA bands were detected in the hypolimnion during the fall, and one band accounted for about 70% of the total 5S rRNA. The sequences of individual 5S rRNA bands from the spring and fall were different for all size classes analyzed except two bands, one of which was identified as Comamonas acidivorans. In the overall analysis of the depth profiles, the diversity in the epilimnion contrasted with the reduced diversity of the bacterioplankton communities in the hypolimnion, and large differences occurred in the composition of the epilimnion at different seasons except for generalists like C. acidivorans

Distribution and Life Strategies of Two Bacterial Populations in a Eutrophic Lake

Monoclonal antibodies and epifluorescence microscopy were used to determine the depth distribution of two indigenous bacterial populations in the stratified Lake Plußsee and characterize their life strategies. Populations of Comamonas acidovorans PX54 showed a depth distribution with maximum abundances in the oxic epilimnion, whereas Aeromonas hydrophila PU7718 showed a depth distribution with maximum abundances in the anoxic thermocline layer (metalimnion), i.e., in the water layer with the highest microbial activity. Resistance of PX54 to protist grazing and high metabolic versatility and growth rate of PU7718 were the most important life strategy traits for explaining the depth distribution of the two bacterial populations. Maximum abundance of PX54 was 16,000 cells per ml, and maximum abundance of PU7718 was 20,000 cells per ml. Determination of bacterial productivity in dilution cultures with different-size fractions of dissolved organic matter (DOM) from lake water indicates that low-molecular-weight (LMW) DOM is less bioreactive than total DOM (TDOM). The abundance and growth rate of PU7718 were highest in the TDOM fractions, whereas those of PX54 were highest in the LMW DOM fraction, demonstrating that PX54 can grow well on the less bioreactive DOM fraction. We estimated that 13 to 24% of the entire bacterial community and 14% of PU7718 were removed by viral lysis, whereas no significant effect of viral lysis on PX54 could be detected. Growth rates of PX54 (0.11 to 0.13 h(−1)) were higher than those of the entire bacterial community (0.04 to 0.08 h(−1)) but lower than those of PU7718 (0.26 to 0.31 h(−1)). In undiluted cultures, the growth rates were significantly lower, pointing to density effects such as resource limitation or antibiosis, and the effects were stronger for PU7718 and the entire bacterial community than for PX54. Life strategy characterizations based on data from literature and this study revealed that the fast-growing and metabolically versatile A. hydrophila PU7718 is an r-strategist or opportunistic population in Lake Plußsee, whereas the grazing-resistant C. acidovorans PX54 is rather a K-strategist or equilibrium population