Faecal indicator bacteria enumeration in beach sand: a comparison study of extraction methods in medium to coarse sands

Aims:  The absence of standardized methods for quantifying faecal indicator bacteria (FIB) in sand hinders comparison of results across studies. The purpose of the study was to compare methods for extraction of faecal bacteria from sands and recommend a standardized extraction technique.
Methods and Results:  Twenty-two methods of extracting enterococci and Escherichia coli from sand were evaluated, including multiple permutations of hand shaking, mechanical shaking, blending, sonication, number of rinses, settling time, eluant-to-sand ratio, eluant composition, prefiltration and type of decantation. Tests were performed on sands from California, Florida and Lake Michigan. Most extraction parameters did not significantly affect bacterial enumeration. anova revealed significant effects of eluant composition and blending; with both sodium metaphosphate buffer and blending producing reduced counts.
Conclusions:  The simplest extraction method that produced the highest FIB recoveries consisted of 2 min of hand shaking in phosphate-buffered saline or deionized water, a 30-s settling time, one-rinse step and a 10 : 1 eluant volume to sand weight ratio. This result was consistent across the sand compositions tested in this study but could vary for other sand types.
Significance and Impact of the Study:  Method standardization will improve the understanding of how sands affect surface water quality.     

Iron in Oxidative and Reduction Processes in Marine Sands

The iron content and the ratio of bivalent to total iron in the labile acid-soluble fraction of iron were studied in sublittoral sands of Vostok Bay, Sea of Japan. Iron oxidation and reduction rates in sand sediment were measured in the laboratory. Trivalent iron almost always prevailed in the acid-extractable fraction in the natural environment. The most oxidized state of iron occurred in spring and was associated with a low water temperature and a high seawater oxygen content; the most reduced iron was found in fall during periods of low hydrodynamics and low oxygen concentration. In sand, iron is mainly reduced by bacteria; this process is slow and can be inhibited by adding chloroform. Oxidation of iron is mainly a chemical process and cannot be stopped by chloroform. In sand, the content of redox equivalents such as trivalent iron is much greater than in dissolved oxygen of pore water. It is assumed that labile iron in sands acts as a redox buffer, is oxidized by dissolved pore water oxygen at the periods of advective mixing, and is slowly reduced by benthic bacteria during anaerobic conditions.     

Laboratory contamination over time during low‐biomass sample analysis

Bacteria are not only ubiquitous on earth but can also be incredibly diverse within clean laboratories and reagents. The presence of both living and dead bacteria in laboratory environments and reagents is especially problematic when examining samples with low endogenous content (e.g., skin swabs, tissue biopsies, ice, water, degraded forensic samples or ancient material), where contaminants can outnumber endogenous microorganisms within samples. The contribution of contaminants within high‐throughput studies remains poorly understood because of the relatively low number of contaminant surveys. Here, we examined 144 negative control samples (extraction blank and no‐template amplification controls) collected in both typical molecular laboratories and an ultraclean ancient DNA laboratory over 5 years to characterize long‐term contaminant diversity. We additionally compared the contaminant content within a home‐made silica‐based extraction method, commonly used to analyse low endogenous content samples, with a widely used commercial DNA extraction kit. The contaminant taxonomic profile of the ultraclean ancient DNA laboratory was unique compared to modern molecular biology laboratories, and changed over time according to researcher, month and season. The commercial kit also contained higher microbial diversity and several human‐associated taxa in comparison to the home‐made silica extraction protocol. We recommend a minimum of two strategies to reduce the impacts of laboratory contaminants within low‐biomass metagenomic studies: (a) extraction blank controls should be included and sequenced with every batch of extractions and (b) the contributions of laboratory contamination should be assessed and reported in each high‐throughput metagenomic study.     

Identification of atypical Aeromonas salmonicida: inter-laboratory evaluation and harmonization of methods

The atypical isolates of Aeromonas salmonicida are becoming increasingly important as the frequency of isolation of bacteria belonging to this group continues to rise. The primary object of this study was to compare and evaluate the results obtained in various laboratories concerning the biochemical identification of atypical Aer. salmonicida before and after standardization of media and methods. Five laboratories examined 25 isolates of Aer. salmonicida from diverse fish species and geographical locations including the reference strains of Aer. salmonicida subsp. salmonicida (NCMB 1102) and Aer. salmonicida subsp. achromogenes (NCMB 1110). Without standardization of the methods, 100% agreement was obtained only for two tests: motility and ornithine decarboxylase. The main reason for the discrepancies found was the variation of the incubation time prior to reading the biochemical reactions. After standardization, improvement was obtained with the identification; however, disagreement was still observed between the different laboratories. These findings demonstrate the difficulties involved in a proper identification of atypical Aer. salmonicida and also that data presented in the literature on various strains of Aer. salmonicida are not readily comparable. This paper seems to be the first on standardization of microbiological tests for identification of fish pathogens and the results obtained show the need for standardization of methods both within and between laboratories.     

Diversity and Transport of Microorganisms in Intertidal Sands of the California Coast

Forced by tides and waves, large volumes of seawater are flushed through the beach daily. Organic material and nutrients in seawater are remineralized and cycled as they pass through the beach. Microorganisms are responsible for most of the biogeochemical cycling in the beach; however, few studies have characterized their diversity in intertidal sands, and little work has characterized the extent to which microbes are transported between different compartments of the beach. The present study uses next-generation massively parallel sequencing to characterize the microbial community present at 49 beaches along the coast of California. In addition, we characterize the transport of microorganisms within intertidal sands using laboratory column experiments. We identified extensive diversity in the beach sands. Nearly 1,000 unique taxa were identified in sands from 10 or more unique beaches, suggesting the existence of a group of “cosmopolitan” sand microorganisms. A biogeographical analysis identified a taxon-distance relationship among the beaches. In addition, sands with similar grain size, organic carbon content, exposed to a similar wave climate, and having the same degree of anthropogenic influence tended to have similar microbial communities. Column experiments identified microbes readily mobilized by seawater infiltrating through unsaturated intertidal sands. The ease with which microbes were mobilized suggests that intertidal sands may represent a reservoir of bacteria that seed the beach aquifer where they may partake in biogeochemical cycling.     

News & Notes: Comparison of Methods to Enumerate Bacteria in Dental Unit Water Lines

Millipore HPC samplers are simple, self-contained test devices that can be used by personnel in dental offices who do not have microbiologic training to easily and economically monitor dental unit water quality without laboratory support. This study evaluated the correlation of HPC samplers to R2A agar for enumerating planktonic bacteria in dental unit treatment water. Eight different dental units were sampled. Five replicates were performed for each media at each dilution. The Pearson correlation coefficient between the R2A agar and HPC sampler is 0.89. These data suggest HPC samplers correlate with conventional laboratory-based R2A culture techniques for determining dental unit water line contamination. Received: 16 June 1998 / Accepted: 31 August 1998     

Bacteria-Induced Cementation in Sandy Soils

Bacteria-induced calcite precipitation (BICP) is a promising technique that utilizes bacteria to form calcite precipitates throughout the soil matrix, leading to an increase in soil strength and stiffness. This research investigated BICP in two types of sands under sterile and nonsterile conditions. Bacteria formation and BICP in the sterilized sand specimens are higher than those in the nonsterilized sand specimens. The development of calcite with time is initially greater for the sand specimens containing calcite. Scanning electron microscope imaging allowed the detection of cementation from calcite precipitation on the surface and pores of the sand matrix. The effects of injecting nutrient mediums and bacteria into the specimens, as well as pH of soil samples on BICP were investigated. The bearing capacity of biologically treated vs. untreated sand specimens were determined especially by laboratory foundation loading tests.     

Stochastic exits from dormancy give rise to heavy‐tailed distributions of descendants in bacterial populations

Variance in reproductive success is a major determinant of the degree of genetic drift in a population. While many plants and animals exhibit high variance in their number of progeny, far less is known about these distributions for microorganisms. Here, we used a strain barcoding approach to quantify variability in offspring number among replicate bacterial populations and developed a Bayesian method to infer the distribution of descendants from this variability. We applied our approach to measure the offspring distributions for five strains of bacteria from the genus Streptomyces after germination and growth in a homogenous laboratory environment. The distributions of descendants were heavy‐tailed, with a few cells effectively ‘winning the jackpot’ to become a disproportionately large fraction of the population. This extreme variability in reproductive success largely traced back to initial populations of spores stochastically exiting dormancy, which provided early‐germinating spores with an exponential advantage. In simulations with multiple dormancy cycles, heavy‐tailed distributions of descendants decreased the effective population size by many orders of magnitude and led to allele dynamics differing substantially from classical population genetics models with matching effective population size. Collectively, these results demonstrate that extreme variability in reproductive success can occur even in growth conditions that are far more homogeneous than the natural environment. Thus, extreme variability in reproductive success might be an important factor shaping microbial population dynamics with implications for predicting the fate of beneficial mutations, interpreting sequence variability within populations and explaining variability in infection outcomes across patients.     

Variation in foraging success among predators and its implications for population dynamics

The effects of the expected predation rate on population dynamics have been studied intensively, but little is known about the effects of predation rate variability (i.e., predator individuals having variable foraging success) on population dynamics. In this study, variation in foraging success among predators was quantified by observing the predation of the wolf spider Pardosa pseudoannulata on the cricket Gryllus bimaculatus in the laboratory. A population model was then developed, and the effect of foraging variability on predator–prey dynamics was examined by incorporating levels of variation comparable to those quantified in the experiment. The variability in the foraging success among spiders was greater than would be expected by chance (i.e., the random allocation of prey to predators). The foraging variation was density‐dependent; it became higher as the predator density increased. A population model that incorporates foraging variation shows that the variation influences population dynamics by affecting the numerical response of predators. In particular, the variation induces negative density‐dependent effects among predators and stabilizes predator–prey dynamics.     

Water supply and not nitrate concentration determines primary root growth in Arabidopsis

Understanding how root system architecture (RSA) adapts to changing nitrogen and water availability is important for improving acquisition. A sand rhizotron system was developed to study RSA in a porous substrate under tightly regulated nutrient supply. The RSA of Arabidopsis seedlings under differing nitrate (NO₃^‐) and water supplies in agar and sand was described. The hydraulic conductivity of the root environment was manipulated by using altered sand particle size and matric potentials. Ion‐selective microelectrodes were used to quantify NO₃^‐ at the surface of growing primary roots in sands of different particle sizes. Differences in RSA were observed between seedlings grown on agar and sand, and the influence of NO₃^‐ (0.1–10.0 mm ) and water on RSA was determined. Primary root length (PRL) was a function of water flux and independent of NO₃^‐. The percentage of roots with laterals correlated with water flux, whereas NO₃^‐ supply was important for basal root (BR) growth. In agar and sand, the NO₃^‐ activities at the root surface were higher than those supplied in the nutrient solution. The sand rhizotron system is a useful tool for the study of RSA, providing a porous growth environment that can be used to simulate the effects of hydraulic conductivity on growth.     

Multiple Laboratory Comparison of the Doubly Labeled Water Technique*

A double-blind study was conducted to determine between-laboratory variability in the doubly labeled water method for measurement of total energy expenditure in humans, and to compare the accuracy and precision of three widely-used procedures for calculating rates of carbon dioxide production from the original isotope data. Eighteen laboratories from five countries participated in the study. All laboratories were provided with five water standards containing varying amounts of ²H and ¹⁸O, and in addition 11 laboratories were provided with urine and dose specimens from one (six laboratories) or two (five laboratories) healthy elderly subjects of normal height and weight undergoing a calorimetric validation of the doubly labeled water method. The data from the five water standards were analyzed to predict between-laboratory variability in the doubly labeled water technique in all laboratories. In addition, data from the subjects were analyzed using the “slope-intercept”, “2-point” and “modified” methods of calculation. The results confirm that the doubly labeled water method can be an accurate technique for the measurement of energy expenditure in adult human subjects in some laboratories. However, there was substantial between-laboratory variability in the results and some laboratories returned physiologically impossible results. There was no significant effect of calculation procedure on the accuracy of the technique in this limited comparison, although the slope-intercept procedure appeared to be more susceptible to analytical error than the other procedures. The isotope standards analyzed by participants in this study will be made available to other investigators on request.     

Evaluating spatial within plot crop variability for different management practices with an optical sensor?

Subsoil constraints to root growth exacerbate frequent water and nutrient limitations to crop yields in Mediterranean-type environments. Amelioration of subsoil constraints can relieve these limitations by opening root-access to subsoil water and nutrients. However, decisions in subsoil amelioration are hampered by seasonally variable yield responses in these environments. We used the APSIM model to analyse the impact of subsoil constraints on yield and yield variability. The simulated yield data were used to calculate the financial benefits of subsoil amelioration across several scenarios. There was a strong yield-dependence on accessible soil water governed by root depth. Root depth development was limited to a minimum of either the effect of subsoil constraints or the weather-dependent depth of the soil wetting front. Insufficient rainfall in dry years or in a drier region often resulted in shallow soil wetting fronts and correspondingly shallow roots even in the absence of subsoil compaction. In these situations, there is little response to subsoil amelioration. Positive yield responses and positive financial returns to subsoil amelioration are therefore greater in good rainfall years and are more likely in a wetter region. A yield response to amelioration is also greater in coarser textured sand than finer textured sandy loam in an average rainfall season because the same amount of rainfall results in a deeper wetting front in sand than in sandy loam. Hence, roots in a sand are required to grow deeper compared to a sandy loam to access the same amount of water and therefore benefited more from subsoil amelioration in an average rainfall year. In wet years, sands leach more nitrate than sandy loam, which decreases yields and the response to subsoil amelioration in sands is more than in the sandy loam. Environmental threats occur along with yield loss when roots cannot access subsoil water. These include increased nitrate leaching and deep drainage due to unused water remaining in the soil profile. By allowing roots to access deep soil water, ameliorating subsoil is expected to yield financial gains in average to good rainfall seasons and decrease the environmental risk of drainage and leaching loss. The financial gains are expected to offset potential financial losses in dry and dry finish seasons especially in coarser textured soils and wetter environment. Responsible Editor: Jan Vos.     

An evaluation of the repeatability and reproducibility of a surface test for the activity of disinfectants

A collaborative study was carried out to determine the precision of a disinfectant surface test method which is currently under consideration for development as a harmonized European standard surface test. Results indicate that significant variation in microbicidal effect occurs both within and between test laboratories despite careful standardization of test conditions, but that the variability may be less than that associated with suspension tests. Indications are that much of this variability derives from random variations in the resistance of the test strains from day to day and, most particularly, from test period to test period both within as well as between laboratories. It is concluded that although the test may be sufficiently reliable to be used as a standard method, adequate replication must be specified to distinguish borderline pass from borderline fail concentrations.     

Sorption and desorption of arsenic from sandy soils: Column studies

Rate‐limited sorption/desorption can have a profound effect upon the transport of sorbing contaminants. Numerical and analytical models used to predict chemical movement through the subsurface rarely incorporate the effects of nonlinear sorption and desorption kinetics, resulting in potentially large overestimates of mass extractability. Mass transfer characteristics of arsenic‐contaminated soils at the site of a former arsenical herbicide manufacturer in Houston, Texas, were examined in the laboratory using soil columns. Unaffected soils comprised of silty sands to coarse sands were collected from the uppermost aquifer. Two soil columns were loaded with a known mass of mixed organic and inorganic forms of arsenic resident in site ground water. A third control column was prepared with dry 20 × 30 mesh ASTM silica sand. Leachate samples were collected from each void volume until arsenic breakthrough was achieved. The dynamic test applied a continuing head of water, operating in an upflow mode through 4‐in. diameter by 12‐in. long soil columns repacked to in situ density. A flow‐through velocity of one void volume per day was chosen for arsenic loading to the columns and 0.08 void volume per day during the desorption phase of the test. Uncontaminated ground water was then passed through the columns, and the tests were restarted in the desorption mode. Analysis of the leachate and resulting arsenic concentrations in the test columns allowed for the calculation of distribution coefficients that describe arsenic behavior. Measured distribution coefficients during desorption ranged from 0.26 after one void volume to 3.3 after six void volumes had been passed through the column.     

Paternal alleles enhance female reproductive success in tropical pythons

The conventional view that female reproductive success is unlikely to benefit from multiple mating has come under strong challenge in recent years. In the present study, we demonstrate that the time wild-caught reproductive female water pythons (Liasis fuscus) spent in the laboratory prior to oviposition affected both hatching success and the number of male microsatellite alleles detected in the broods. A negative correlation between time in captivity and number of male alleles observed in the broods suggests that reduced hatching success was most likely not caused by environmental factors such as non-optimal temperatures, but rather by restricted mating opportunities. Maternal nutritional status and mean hatchling mass did not affect brood viability. However, our results revealed a positive correlation between number of male microsatellite alleles observed in the broods and hatching success, suggesting that increased paternal genetic variability enhanced female reproductive success. As microsatellite loci are unlikely to be direct targets of selection, we suggest that variability at these loci may cosegregate with other polymorphic genes directly linked to fitness.     

Particulate matter filtration and seasonal nutrient dynamics in permeable carbonate and silicate sands of the Gulf of Aqaba,Red Sea

This study compares mineralization in permeable silicate and carbonate sands in the shallow shelf of the Gulf of Aqaba. From July 1999 to March 2000, we monitored concentrations of inorganic nutrients in water and pore water at two neighboring sites, one dominated by silicate, the other by carbonate sand. Although the carbonate was coarser than the quartz sand, organic matter, dissolved inorganic nitrogen (DIN), and ortho-phosphate concentrations in the biogenic carbonate sediment always exceeded those in the terrigenic silicate sands (factor 1.5–2.0 for organic matter, 1.7–14.0 for nutrients). Higher nutrient concentrations in the water column during winter months caused increases in pore-water nutrient concentrations in both sediments down to 10 cm depth with no significant delay, emphasizing the effect of advective transport of solutes and particles into permeable sands. An experiment was conducted where sieved clean quartz and carbonate sands of same grain size (250–500 µm) were incubated in-situ. Although exposed to the same water and boundary current conditions, the sieved carbonate sand accumulated more organic matter and developed higher nutrient concentrations than the incubated silicate sediment. We conclude that the mineralogical characteristics of the carbonate sand (higher porosity, sorption capacity and pH buffer capacity) enhance the filtration capacity, and the biocatalytic conversion efficiency relative to the smooth crystalline quartz grains.     

The transport of Mycobacterium avium subsp. paratuberculosis through saturated aquifer materials

Aims:  To investigate the processes controlling the transport of Mycobacterium avium subsp. paratuberculosis ( Map ) through aquifer materials.
Methods and Results:  We measured two important surface characteristics known to affect bacterial attachment to sediment surfaces: surface charge and hydrophobicity. We then measured the transport of Map through laboratory columns packed with aquifer sand with varying ionic strength solutions and sediment surface charge. We found that Map has a strong negative charge and is highly hydrophobic and that the transport of Map through positively charged Fe-coated sands was reduced compared with transport through negatively charged clean quartz sand, although Map transport for all treatments was low compared with the transport behaviour reported in the literature for other bacteria.
Conclusions:  Our results suggest that the potential for groundwater contamination by Map is low; however, the organism may remain bound to the soil near the surface where it can be ingested by grazing animals or be released during run off events.
Significance and Impact of the Study:  This is the first study looking at the surface characteristics and transport behaviour of Map through aquifer materials and therefore provides important information for understanding the movement of Map in the environment.     

Evidence of lethal and sublethal injury in food-borne bacterial pathogens exposed to high-intensity pulsed-plasma gas discharges

AIMS: To apply scanning electron microscopy, image analysis and a fluorescent viability stain to assess lethal and sublethal injury in food-borne bacteria exposed to pulsed-plasma gas discharges (PPGD). METHODS AND RESULTS: The fluorescent redox probe 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) was used for enumerating actively respiring cells of Campylobacter jejuni, Escherichia coli, Listeria monocytogenes, Staphylococcus aureus and Salmonella enterica serovar Typhimurium that were suspended in sterile water at 4 degrees C and exposed to separate PPGD and heat treatments. While there was good agreement between use of respiratory staining (RS) and direct-selective agar plate counting (PC) for enumerating untreated bacteria, there were c. 1 and 3 log-unit differences in surviving cell numbers per millilitre for test organisms subjected to PPGD and heat treatments respectively, when enumerated by these different viability indicators. PPGD-treated bacteria were markedly altered at the cellular level when examined by scanning electron microscopy. CONCLUSIONS: Use of this RS method revealed that substantial subpopulations of test bacteria rendered incapable of forming colonies by separate PPGD and heat treatments may remain metabolically active. SIGNIFICANCE AND IMPACT OF THE STUDY: Use of this RS method offers interesting perspectives on assessing established and novel microbial inactivation methods, and may also provide a better understanding of mechanisms involved in microbial inactivation induced by high-intensity PPGD treatments.     

Referential learning of French and Czech labels in African grey parrots (Psittacus erithacus): Different methods yield contrasting results

Some African grey parrots (Psittacus erithacus), the most famous being Pepperberg's parrot Alex, are able to imitate human speech and produce labels referentially. In this study, the aim was to teach ten African grey parrots from two laboratories to label items. Training three parrots from the first laboratory for several months with the Model/Rival method, developed by Pepperberg, in which two humans interact in front of the subject to demonstrate the use of a label, led to disappointing results. Similarly, seven parrots from the second laboratory, having been trained with several variants of Model/Rival attained little success. After the informal observation of the efficiency of other methods (i.e. learning to imitate labels either spontaneously or with specific learning methods and use of these labels referentially), four different teaching methods were tested with two birds: the Model/Rival; Repetition/Association which consisted of repeating a label and presenting the item only when the parrot produced the label; Intuitive in which the experimenter handled an item and repeated its name in front of the subject; Diffusion in which labels with either variable or flat intonation were played back daily to parrots. One bird learned three labels, one of which was used referentially, with the Repetition/Association method. He learned one label non-referentially with the Model/Rival but no labels were acquired using the other methods. The second bird did not learn any labels. This study demonstrates that different methods can be efficient to teach labels referentially and it suggests that rearing conditions and interindividual variability are important features when assessing learning ability of African grey parrots.     

Persistence, dissipation, and activity of Escherichia coli O157:H7 within sand and seawater environments

Runoff from agricultural land into watercourses may transport and deposit animal-derived waste contaminated with Escherichia coli O157:H7 onto beaches, which may in turn lead to human infection. To simulate contamination, freshwater mixed with cattle slurry containing E. coli O157:H7 was added to sand from three recreational beaches. The sand was then maintained in a dry state (nontidal) or subjected to a repeated seawater tidal simulation. The pathogen could still be recovered from all sands by day 5. Although survival of the pathogen did not statistically vary between sands of different origin under nontidal conditions, significant differences in numbers occurred between sands when subject to tidal simulation. In the tidal simulations, a considerable proportion of the E. coli O157:H7 rapidly dissipated from sand into the seawater. In a separate experiment, the activity of bioluminescent (lux-marked) E. coli O157:H7 cells was monitored in various mixtures of contaminated runoff water and seawater over 5 days. Pathogen activity declined with increasing seawater concentration; however, cells remained viable in all treatments over the 5-day period. The addition of nutrients to water rapidly increased pathogen activity in all treatments. Our findings highlight the resilience of E. coli O157:H7 in aquatic and marine environments.