Survival strategies of Escherichia coli and Vibrio spp.: contribution of the viable but nonculturable phenotype to their stress-resistance and persistence in adverse environments.

In their natural ecosystems, bacteria are continuously exposed to changing environmental factors including physicochemical parameters (e.g. temperature, pH, etc.), availability of nutrients as well as interaction(s) with other organisms. To increase their tolerance and survival under adverse conditions, bacteria trigger a number of adaptation mechanisms. One of the well-known adaptation responses of the non-spore-forming bacteria is the acquisition of the viable but non-culturable (VBNC) state. This phenotype is induced by different stress factors (e.g. low temperature) and is characterized by the temporal loss of culturability, which can potentially be restored. Moreover, this response can be combined with the bust and boom strategy, which implies the death of the main population of the stressed cells (or their entry into the VBNC state) upon stress, thus enabling the remaining cells (i.e. residual culturable population) to subsist at the expense of the dead or/and VBNC cells. In this review, we discuss the characteristics of the VBNC state, its biological significance and contribution to bacterial survival.

The long-term survival of Acinetobacter baumannii ATCC 19606(T) under nutrient-deprived conditions does not require the entry into the viable but non-culturable state.

Acinetobacter baumannii possesses a tremendous potential to thrive under hostile conditions. To learn more about its survival strategy and capacity to persist in the environment, we studied the effect of temperature, nutrient deprivation and dryness on the long-term survival of two A. baumannii strains (ATCC 19606(T) and a clinical isolate). Our results revealed that both strains show a great persistence under stress that appears to involve a bust-and-boom strategy. Bacterial survival was differentially affected by temperature and physical environment: Desiccation favored cell resistance to stress at 20 and 37 °C, while survival in aqueous environments was temperature dependent and led to changes in several cellular characteristics. In addition, we tested the ability of the A. baumannii ATCC 19606(T) strain to form biofilms by monitoring the expression of adhesion-/biofilm-related genes (ompA, bfmR and csuAB). The observed downregulation of these genes suggests that the potential difficulties to adhere to solid surfaces and form biofilms likely limit the capacity of starved cells to spread and colonize abiotic surfaces.

Forensic evaluation of the AmpFℓSTR Identifiler kit in nine Mexican native populations from the pre-Columbian Mesoamerican region.

Allele frequency distribution and forensic parameters of the AmpFℓSTR Identifiler kit was determined in nine Mexican Amerindian populations based on 1,040 unrelated individuals from the pre-Columbian region known as Mesoamerica. Hardy-Weinberg equilibrium was demonstrated for most of the short tandem repeats (STRs) in all nine populations. The power of discrimination and exclusion were higher than 0.99999 and 0.997942, respectively. In addition, a brief overview of the genetic relatedness and structure (F st = 2.62 %; p = 0.00000) between these populations is presented.

Preeclampsia and risk of maternal pulmonary hypertension at high altitude in Bolivia.

Women with a history of preeclampsia (PE) have a greater risk of pulmonary arterial hypertension (PAH). In turn, pregnancy at high altitude is a risk factor for PE. However, whether women who develop PE during highland pregnancy are at risk of PAH before and after birth has not been investigated. We tested the hypothesis that during highland pregnancy, women who develop PE are at greater risk of PAH compared to women undergoing healthy highland pregnancies. The study was on 140 women in La Paz, Bolivia (3640m). Women undergoing healthy highland pregnancy were controls (C, n = 70; 29 ± 3.3 years old, mean±SD). Women diagnosed with PE were the experimental group (PE, n = 70, 31 ± 2 years old). Conventional (B- and M-mode, PW Doppler) and modern (pulsed wave tissue Doppler imaging) ultrasound were applied for cardiovascular íííassessment. Spirometry determined maternal lung function. Assessments occurred at 35 ± 4 weeks of pregnancy and 6 ± 0.3 weeks after birth. Relative to highland controls, highland PE women had enlarged right ventricular (RV) and right atrial chamber sizes, greater pulmonary artery dimensions and increased estimated RV contractility, pulmonary artery pressure and pulmonary vascular resistance. Highland PE women had lower values for peripheral oxygen saturation, forced expiratory flow and the bronchial permeability index. Differences remained 6 weeks after birth. Therefore, women who develop PE at high altitude are at greater risk of PAH before and long after birth. Hence, women with a history of PE at high altitude have an increased cardiovascular risk that transcends the systemic circulation to include the pulmonary vascular bed.

Population structure and paternal admixture landscape on present-day Mexican-Mestizos revealed by Y-STR haplotypes.

Mestizos currently represent most of the Mexican population (>90%); they are defined as individuals born in the country having a Spanish-derived last name, with family antecedents of Mexican ancestors back at least to the third generation. Mestizos are result of 500 years of admixture mainly among Spaniards, Amerindians, and African slaves. Consequently, a complex genetic pattern has been generated throughout the country that has been scarcely studied from the paternal point of view. This fact is important, taking into account that gene flow toward the New World comprised largely males. We analyzed the population structure and paternal admixture of present-day Mexican-Mestizo populations based on Y-STRs. We genotyped at least 12 Y-STRs in DNA samples of 986 males from five states: Aguascalientes (n = 293); Jalisco (n = 185); Guanajuato (n = 168); Chiapas (n = 170); and Yucatán (n = 170). AmpFlSTR Y-filer and Powerplex-Y(R) kits were used. Inclusion of North and Central Y-STR databases in the analyses allowed obtaining a Y-STR variability landscape from Mexico. Results confirmed the population differentiation gradient previously noted in Mestizos with SNPs and autosomal STRs throughout the Mexican territory: European ancestry increments to the Northwest and, correspondingly, Amerindian ancestry increments to the Center and Southeast. In addition, SAMOVA test and Autocorrelation Index for DNA Analysis autocorrelogram plot suggested preferential gene flow of males with neighboring populations in agreement with the isolation-by-distance model. Results are important for disease-risk studies (principally male-related) and for human identification purposes, because Y-STR databases are not available on the majority of Mexican-Mestizo populations.

Origin and genetic differentiation of three Native Mexican groups (Purépechas, Triquis and Mayas): contribution of CODIS-STRs to the history of human populations of Mesoamerica.

BACKGROUND: CODIS-STRs in Native Mexican groups have rarely been analysed for human identification and anthropological purposes. AIM: To analyse the genetic relationships and population structure among three Native Mexican groups from Mesoamerica. SUBJECTS AND METHODS: 531 unrelated Native individuals from Mexico were PCR-typed for 15 and 9 autosomal STRs (Identifiler™ and Profiler™ kits, respectively), including five population samples: Purépechas (Mountain, Valley and Lake), Triquis and Yucatec Mayas. Previously published STR data were included in the analyses. RESULTS: Allele frequencies and statistical parameters of forensic importance were estimated by population. The majority of Native groups were not differentiated pairwise, excepting Triquis and Purépechas, which was attributable to their relative geographic and cultural isolation. Although Mayas, Triquis and Purépechas-Mountain presented the highest number of private alleles, suggesting recurrent gene flow, the elevated differentiation of Triquis indicates a different origin of this gene flow. Interestingly, Huastecos and Mayas were not differentiated, which is in agreement with the archaeological hypothesis that Huastecos represent an ancestral Maya group. Interpopulation variability was greater in Natives than in Mestizos, both significant. CONCLUSION: Although results suggest that European admixture has increased the similarity between Native Mexican groups, the differentiation and inconsistent clustering by language or geography stresses the importance of serial founder effect and/or genetic drift in showing their present genetic relationships.

Analysis of Acinetobacter baumannii survival in liquid media and on solid matrices as well as effect of disinfectants.

BACKGROUND: Acinetobacter baumannii is a cause of healthcare-associated infections and has considerable potential to survive on inanimate hospital surfaces under hostile conditions (e.g. disinfection or desiccation). AIM: To learn more about its survival strategy and capacity to persist in liquid media and on surfaces mimicking hospital environments. METHODS: The effect of temperature, nutrient deprivation, permanence on inanimate surfaces, and exposure to disinfectants on the survival of four A. baumannii strains (ATCC 19606T and three clinical isolates) was studied by monitoring the number of total and viable cells using fluorescent microscopy and of culturable cells by standard cultures. FINDINGS: Bacterial survival was differentially affected by temperature (cells maintained at 20°C remained culturable at least within 30 days) and physical environment (desiccation favoured cell resistance to stress at 37°C). Moreover, persistence was associated with two adaptation patterns: one linked to entry into the viable but non-culturable state, whereas the other apparently followed a bust-and-boom model. During a study on the effect of disinfectant (commercial bleach and quaternary ammonium compounds), it was found that treatment with these antibacterial compounds did not eliminate A. baumannii populations and provoked the reduction of culturable populations, although a fraction of cells remained culturable. CONCLUSION: The ability to persist for long periods on different surfaces, mimicking those usually found in hospitals, along with A. baumannii's capacity to survive after a disinfection process may account for the recurrent outbreaks in intensive care units.

Y-linked haplotypes in Amerindian chromosomes from Mexican populations: genetic evidence to the dual origin of the Huichol tribe.

We studied six Y-linked short tandem repeats (Y-STRs) to describe the internal diversity of the Amerindian haplogroup Q-M3 in 129 males from eight Mexican populations. The low gene diversity in the Huichol tribe demonstrated the effects of genetic drift, attributable to geographic isolation and founder effect. The presence of two principal paternal lineages supported the historical and anthropometric records, which indicate that Huichols were formed by the fusion of two ancestral Mexican tribes. Moreover, genetic distances and close relationships of haplotypes between Huichols and Tarahumaras were in agreement with their linguistic affiliation. The high genetic diversity of the Purépechas and wide distribution of haplotypes along the constructed network-joining tree suggest that the present genetic composition was influenced by Purépecha dominance in western Mesoamerica. The Y-haplotypes shared between populations suggest that, among the Amerindian tribes studied herein, the paternal genetic pool of Nahuas could have contributed more importantly to the European-admixed population, the Mexican-Mestizos.

Autosomal dominant inheritance in Cantú syndrome (congenital hypertrichosis, osteochondrodysplasia, and cardiomegaly).

Cantú syndrome (CS) is characterized by congenital hypertrichosis, osteochondrodysplasia, cardiomegaly, and coarse facial appearance; autosomal recessive inheritance has been postulated. We report on a Mexican family with CS; the affected members are the 44-year-old father and his two children (a male and female), aged 14 and 4 years, respectively; each shows the classic characteristics, but the father and the brother also have a previously unreported feature, namely, a thick calvarium. This is the first reported instance of male-to-male transmission of CS. With the paternal age effect found in the reported sporadic cases and the segregation analysis [Robertson et al., 1999], autosomal dominant inheritance is more likely than autosomal recessive inheritance. The cases of affected sibs reported by Cantú et al. [1982] could be explained by parental gonadal mosaicism.

Humic Materials Offer Photoprotective Effect to Escherichia coli Exposed to Damaging Luminous Radiation.

The behavior of Escherichia coli immersed in aqueous systems amended with humic acids, under PAR, UV-A, UV-B, and simulated solar radiation was examined. Culturability, ability to elongate, functioning of the electron transport systems, and glucose uptake were assessed. Humic substances in the range from 1 to 50 mg L-1 protected cells from photoinactivation. Decrease in culturability and cellular activities was significantly (p <0.05) less in the presence of humic material. However, humic acids were not used as nutrients. Neither irradiated nor nonirradiated humic solutions (50 mg L-1) supported the growth of 105 cells ml-1. However, humic acids dissolved in 0.9% NaC1 efficiently absorbed light over wavelengths from 270 to 500 nm. Also, a photoprotective effect against simulated sunlight was observed when humic acids were not in contact with but rather enveloped the cellular suspensions in double-wall microcosms. The protection afforded by humic acids against luminous radiation likely derives from their ability to absorb these radiations and hence reduces the amount of energy reaching the cells.

Changes in DNA Content and Cellular Death during a Starvation-Survival Process of Escherichia coli in River Water.

Abstract Four nucleoid staining procedures were compared during the starvation-survival process of Escherichia coli in river water. Only the method performed as a modification of the standard acridine orange direct procedure allowed us to visualize nucleoids during the 95 days of experimentation. Moreover, with this method the total number of cells and nucleoid-containing cells can be simultaneously enumerated. The decrease of the chromosomal DNA content of population and of the nucleoid-containing cells indicates that ghosts form and cellular death occurs throughout the starvation-survival process. A long time (<30 days) is needed for non-nucleoid-containing cells to appear in river water; plasmid DNA is also negatively affected by environmental stress. After 4 days of storage in river water, the need to increase the volume of lysed cells used for the plasmid band visualization as well as the decrease in the plasmid band intensity would indicate a decrease in the plasmid DNA content during the starvation-survival process. According to our results, both chromosomal and plasmid DNA content decrease during the starvation-survival process of E. coli in river water.

The Effect of Simulated Solar Radiation on Escherichia coli: The Relative Roles of UV-B, UV-A, and Photosynthetically Active Radiation.

The relative role of components of solar radiation (UV-B, UV-A, and photosynthetically active radiation) as well as the effect of simulated sunlight upon the physiological state of Escherichia coli in fresh water were evaluated. Simulated solar radiation had a sublethal effect on E. coli populations in a short-time exposure by provoking loss of culturability and the formation of viable but nonculturable cells. Prolonged exposure increased the damage to cells but cellular integrity was never affected. However, important differences between the way the sunlight components acted were detected. After photosynthetically active radiation (PAR) exposure, cells remained metabolically active but only 10% of the cells were culturable. When cells were exposed to UV-A, the culturable fraction was similar to the one obtained after PAR irradiation, although formation of viable but nonculturable cells was not observed. For UV-B radiation short-time exposures (6 h) were enough to provoke loss of culturability and a reduction in activity similar to that of simulated sunlight exposed cells. The effect of simulated solar radiation on E. coli cells was mainly attributable to shorter wavelengths, but a synergistic interaction of the UV-B, UV-A and PAR components was detected.

A process-oriented reasoner about physiology.

This paper presents the RAP system: a reasoner about physiology. RAP performs two tasks: (1) it infers the behaviour of a complex physiological process using the behaviours of its subprocesses and the relationships between them; (2) it reasons about the effect of introducing a fault into the model. In order to reason about the behaviour of a complex process, RAP uses a mechanism which: (i) represents how subprocesses behave; (ii) establishes how these subprocesses affect each others behaviors; (iii) 'aggregates' these behaviors together to obtain the behavior of the top level process; (iv) gives that process a temporal context in which to act. RAP uses limited common sense knowledge about faults to reason about their effect in terms of the generation of new processes and the misbehavior of existing ones. The effects are then propagated throughout the model to obtain the overall effect of the fault.

[Benthic macrofauna associated to Thalassia testudinum in Bahía de Mochima, Sucre, Venezuela]. / Macrofauna béntica asociada con Thalassia testudinum en Bahía de Mochima, Sucre, Venezuela.

Diversity and abundance of benthic macrofauna associated to Thalassia testudinum were studied at Ensenada de Reyes, Mochima Bay, in the northeastern coast of Venezuela. Samples were taken monthly in six stations, three at 1 m in depth and three at 6 m, between December 1992 and February 1994, using a quadrat of 0.25 m2 for collecting plants and sediment; each sample was washed with seawater through a 1 mm sieve. The specimens were fixed in 6% formaldehyde. A total of 1722 organisms (6 888 ind x m2) and 127 species of macroinvertebrates were collected. Mollusks dominated with 53 species, followed by polychaetes (40), crustaceans (18) and echinoderms (8). Remaining groups were represented by 1-2 species. The highest abundance was in October (214 specimens), and the lowest in December 1993 (79 specimens). Specific richness was between 47 species in October and 18 in May 1993. Mean species diversity was 2.79-1.36 bits/ind. There were differences (ANOVA p<0.01) in number of specimens at the two depths but not throughout the 15 month study period (p>0.05). There were more specimens and species at the lowest depth and in stations with higher Thalassia testudinum biomass.

Mercury biomethylation assessment in the estuary of Bilbao (North of Spain).

The relationship between the microbial methylation of mercury and the microbial activities in sediments and water collected from the estuary of Bilbao (North of Spain) was studied in three different sampling points and in two different seasons. Three different cultures were prepared with a sediment slurry to distinguish between biotic and abiotic methylation pathways and the variations of the methylmercury concentration and the variations of the population of total number of bacteria (TDC), anaerobic heterotrophic bacteria (AHB), sulphate-reducing bacteria (SRB) and Desulfovibrio were measured. From this work, it can be concluded that the variation of MeHg concentrations is a result of the methylation/demethylation processes in the sediments, and that the abiotic processes have a negligible contribution to those processes. According to the statistical analysis of the results (partial least squares analysis) a significant statistical correlation was established between methylmercury and the SRB counts.

Development and validation of a computer program to design and calculate ROPS.

In Spain, there are more than 250,000 tractors built before 1980, when it became mandatory for all new tractors to be equipped with a rollover protective structure (ROPS). A similar situation is found in the European Union, but the situation is worse in the U.S. and in developing countries. Directive 2003/37/EEC establishes that tractors over 800 kg weight can be homologated by using the OECD standard code for the official testing of protective structures on agricultural and forestry tractors (static test), called Code 4. A ROPS attachable to the rear axle of different tractor models has been designed, and a computer program for the calculation of the ROPS design has been developed. The program, named ESTREMA, is available at: www.cfnavarra.es/insl. Using this program, it has been possible to design a ROPS for the Massey Ferguson model 178 tractor, one of the most common tractor models without a ROPS in Spain. After the tractor was equipped with the designed ROPS, it was tested at the Spanish Authorized Station for testing ROPS and passed the homologation test (OECD Code 4), the main results being a maximum distortion of 21.3 cm when the absorbed energy was 5437 N and the maximum force applied was 34 kN during loading from the side. The ROPS was improved, redesigned, and remounted on the tractor, the tractor was tested in a real overturn, and no part of the structure intruded on the driver's clearance zone during the test. In conclusion, the ESTREMA program worked correctly, and the designed ROPS was able to pass the authorized test and provide adequate protection to the operator during a real overturn.

Influence of light and natural microbiota of the Butrón river on E. coli survival.

The survival of an E. coli strain in water samples from the Butrón river has been studied. The input of E. coli cells in the aquatic system breaks down the established balance among the components of the natural microbiota: E. coli becomes the object of the active protozoal predation whereas the autochtonous heterotrophic community become alternative preys. As a result of this new situation, the natural microbiota increases but returns to the initial values once the E. coli cells have been removed from the system. The effect of the temperature of incubation on the survival is exerted through the effect of this parameter on the predatory activity of the protozoa. Light has a lethal and direct action on the E. coli cells, the effect of this parameter is even superior to that of predation.

Chlorination and ozonation of waste-water: comparative analysis of efficacy through the effect on Escherichia coli membranes.

The effect of chlorine and ozone on Escherichia coli cells resuspended in waste-water was compared. Selected chlorination and ozonation conditions produced a similar decrease in culturability (2-2.5 log). Under these conditions, differences in membrane permeability and cell surface hydrophobicity, depending on the disinfectant tested, were detected. After ozonation, while no changes in cell surface hydrophobicity were observed, approximately 95.5% of cells showed altered membrane permeability. The effect of chlorine was not linked to changes in membrane permeability. After chlorination, E. coli cells showed a tendancy to aggregate. The possibility that aggregation of cells could interfere with conventional colony counts is discussed. The degree of toxicity (Microtox assay) was unrelated to the effect on cellular activity.