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USGS2010

Characterization of Metarhizium species and varieties based on molecular analysis, heat tolerance and cold activity

Aims: The genetic relationships and conidial tolerances to high and low temperatures were determined for isolates of several Metarhizium species and varieties. Methods and Results: Molecular-based techniques [AFLP and rDNA (ITS1, ITS2 and 5??8S) gene sequencing] were used to characterize morphologically identified Metarhizium spp. isolates from a wide range of sources. Conidial suspensions of isolates were exposed to wet heat (45 ?? 0??2??C) and plated on potato dextrose agar plus yeast extract (PDAY) medium. After 8-h exposure, the isolates divided clearly into two groups: (i) all isolates of Metarhizium anisopliae var. anisopliae (Ma-an) and Metarhizium from the flavoviride complex (Mf) had virtually zero conidial relative germination (RG), (ii) Metarhizium anisopliae var. acridum (Ma-ac) isolates demonstrated high heat tolerance (c. 70-100% RG). Conidial suspensions also were plated on PDAY and incubated at 5??C for 15 days, during which time RGs for Ma-an and Ma-ac isolates were virtually zero, whereas the two Mf were highly cold active (100% RG). Conclusions: Heat and cold exposures can be used as rapid tools to tentatively identify some important Metarhizium species and varieties. Significance and Impact of the Study: Identification of Metarhizium spp. currently relies primarily on DNA-based methods; we suggest a simple temperature-based screen to quickly obtain tentative identification of isolates as to species or species complexes. ?? 2009 The Society for Applied Microbiology.

Journal of Applied Microbiology↗

USGS2009

Genetic diversity among brazilian isolates of beauveria bassiana: comparisons with non-brazilian isolates and other beauveria species

Aims: The genetic diversity of Beauveria bassiana was investigated by comparing isolates of this species to each other (49 from different geographical regions of Brazil and 4 from USA) and to other Beauveria spp. Methods and Results: The isolates were examined by multilocus enzyme electrophoresis (MLEE), amplified fragment length polymorphism (AFLP), and rDNA sequencing. MLEE and AFLP revealed considerable genetic variability among B. bassiana isolates. Several isolates from South and Southeast Brazil had high similarity coefficients, providing evidence of at least one population with clonal structure. There were clear genomic differences between most Brazilian and USA B. bassiana isolates. A Mantel test using data generated by AFLP provided evidence that greater geographical distances were associated with higher genetic distances. AFLP and rDNA sequencing demonstrated notable genotypic variation between B. bassiana and other Beauveria spp. Conclusion: Geographical distance between populations apparently is an important factor influencing genotypic variability among B. bassiana populations in Brazil. Significance and Impact of the Study: This study characterized many B. bassiana isolates. The results indicate that certain Brazilian isolates are considerably different from others and possibly should be regarded as separate species from B. bassiana sensu latu. The information on genetic variation among the Brazilian isolates, therefore, will be important to comprehending the population structure of B. bassiana in Brazil. ?? 2009 The Society for Applied Microbiology.

Journal of Applied Microbiology↗

USGS2010

Statistical assessment of DNA extraction reagent lot variability in real-time quantitative PCR

Aims: The aim of this study was to evaluate the variability in lots of a DNA extraction kit using real-time PCR assays for Bacillus anthracis, Francisella tularensis and Vibrio cholerae. Methods and Results: Replicate aliquots of three bacteria were processed in duplicate with three different lots of a commercial DNA extraction kit. This experiment was repeated in triplicate. Results showed that cycle threshold values were statistically different among the different lots. Conclusions: Differences in DNA extraction reagent lots were found to be a significant source of variability for qPCR results. Steps should be taken to ensure the quality and consistency of reagents. Minimally, we propose that standard curves should be constructed for each new lot of extraction reagents, so that lot-to-lot variation is accounted for in data interpretation. Significance and Impact of the Study: This study highlights the importance of evaluating variability in DNA extraction procedures, especially when different reagent lots are used. Consideration of this variability in data interpretation should be an integral part of studies investigating environmental samples with unknown concentrations of organisms. ?? 2010 The Society for Applied Microbiology.

Letters in Applied Microbiology↗

USGS2009

Comparison of traditional and molecular analytical methods for detecting biological agents in raw and drinking water following ultrafiltration

Aims: To compare the performance of traditional methods to quantitative polymerase chain reaction (qPCR) for detecting five biological agents in large-volume drinking-water samples concentrated by ultrafiltration (UF). Methods and Results: Drinking-water samples (100 l) were seeded with Bacillus anthracis, Cryptospordium parvum, Francisella tularensis, Salmonella Typhi, and Vibrio cholerae and concentrated by UF. Recoveries by traditional methods were variable between samples and between some replicates; recoveries were not determined by qPCR. Francisella tularensis and V. cholerae were detected in all 14 samples after UF, B. anthracis was detected in 13, and C. parvum was detected in 9 out of 14 samples. Numbers found by qPCR after UF were significantly or nearly related to those found by traditional methods for all organisms except for C. parvum. A qPCR assay for S. Typhi was not available. Conclusions: qPCR can be used to rapidly detect biological agents after UF as well as traditional methods, but additional work is needed to improve qPCR assays for several biological agents, determine recoveries by qPCR, and expand the study to other areas. Significance and Impact of the Study: To our knowledge, this is the first study to compare the use of traditional and qPCR methods to detect biological agents in large-volume drinking-water samples. ?? 2009 The Society for Applied Microbiology.

Journal of Applied Microbiology↗

USGS2009

Pilot- and bench-scale testing of faecal indicator bacteria survival in marine beach sand near point sources

Aim: Factors affecting faecal indicator bacteria (FIB) and pathogen survival/persistence in sand remain largely unstudied. This work elucidates how biological and physical factors affect die-off in beach sand following sewage spills. Methods and Results: Solar disinfection with mechanical mixing was pilot-tested as a disinfection procedure after a large sewage spill in Los Angeles. Effects of solar exposure, mechanical mixing, predation and/or competition, season, and moisture were tested at bench scale. First-order decay constants for Escherichia coli ranged between -0??23 and -1??02 per day, and for enterococci between -0??5 and -1??0 per day. Desiccation was a dominant factor for E. coli but not enterococci inactivation. Effects of season were investigated through a comparison of experimental results from winter, spring, and fall. Conclusions: Moisture was the dominant factor controlling E. coli inactivation kinetics. Initial microbial community and sand temperature were also important factors. Mechanical mixing, common in beach grooming, did not consistently reduce bacterial levels. Significance and Impact of the Study: Inactivation rates are mainly dependent on moisture and high sand temperature. Chlorination was an effective disinfection treatment in sand microcosms inoculated with raw influent. ?? 2009 The Society for Applied Microbiology.

Journal of Applied Microbiology↗

USGS2009

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. ?? 2009 The Society for Applied Microbiology.

Journal of Applied Microbiology↗

USGS2006

Population structure, persistence, and seasonality of autochthonous Escherichia coli in temperate, coastal forest soil from a Great Lakes watershed

The common occurrence of Escherichia coli in temperate soils has previously been reported, however, there are few studies to date to characterize its source, distribution, persistent capability and genetic diversity. In this study, undisturbed, forest soils within six randomly selected 0.5 m2 exclosure plots (covered by netting of 2.3 mm2 mesh size) were monitored from March to October 2003 for E. coli in order to describe its numerical and population characteristics. Culturable E. coli occurred in 88% of the samples collected, with overall mean counts of 16 MPN g-1, ranging from <1 to 1657 (n = 66). Escherichia coli counts did not correlate with substrate moisture content, air, or soil temperatures, suggesting that seasonality were not a strong factor in population density control. Mean E. coli counts in soil samples (n = 60) were significantly higher inside than immediately outside the exclosures; E. coli distribution within the exclosures was patchy. Repetitive extragenic palindromic polymerase chain reaction (Rep-PCR) demonstrated genetic heterogeneity of E. coli within and among exclosure sites, and the soil strains were genetically distinct from animal (E. coli) strains tested (i.e. gulls, terns, deer and most geese). These results suggest that E. coli can occur and persist for extended periods in undisturbed temperate forest soils independent of recent allochthonous input and season, and that the soil E. coli populations formed a cohesive phylogenetic group in comparison to the set of fecal strains with which they were compared. Thus, in assessing E. coli sources within a stream, it is important to differentiate background soil loadings from inputs derived from animal and human fecal contamination. ?? 2005 Society for Applied Microbiology and Blackwell Publishing Ltd.

Environmental Microbiology↗

USGS2007

Molecular analysis of fungal communities and laccase genes in decomposing litter reveals differences among forest types but no impact of nitrogen deposition

The fungal community of the forest floor was examined as the cause of previously reported increases in soil organic matter due to experimental N deposition in ecosystems producing predominantly high-lignin litter, and the opposite response in ecosystems producing low-lignin litter. The mechanism proposed to explain this phenomenon was that white-rot basidiomycetes are more important in the degradation of high-lignin litter than of low-lignin litter, and that their activity is suppressed by N deposition. We found that forest floor mass in the low-lignin sugar-maple dominated system decreased in October due to experimental N deposition, whereas forest floor mass of high-lignin oak-dominated ecosystems was unaffected by N deposition. Increased relative abundance of basidiomycetes in high-lignin forest floor was confirmed by denaturing gradient gel electrophoresis (DGGE) and sequencing. Abundance of basidiomycete laccase genes, encoding an enzyme used by white-rot basidiomycetes in the degradation of lignin, was 5-10 times greater in high-lignin forest floor than in low-lignin forest floor. While the differences between the fungal communities in different ecosystems were consistent with the proposed mechanism, no significant effects of N deposition were detected on DGGE profiles, laccase gene abundance, laccase length heterogeneity profiles, or phenol oxidase activity. Our observations indicate that the previously detected accumulation of soil organic matter in the high-lignin system may be driven by effects of N deposition on organisms in the mineral soil, rather than on organisms residing in the forest floor. However, studies of in situ gene expression and temporal and spatial variability within forest floor communities will be necessary to further relate the ecosystem dynamics of organic carbon to microbial communities and atmospheric N deposition. ?? 2007 The Authors; Journal compilation ?? 2007 Society for Applied Microbiology and Blackwell Publishing Ltd.

Environmental Microbiology↗

USGS1966

Uptake of 2,4-dichlorophenoxyacetic acid by Pseudomonas fluorescens

Factors influencing the uptake of the sodium salt of 2,4-dichlorophenoxyacetic acid (2,4-D), under conditions in which no net metabolism occurred, were investigated in an effort to determine both the significance of “non-metabolic” uptake as a potential agent in reducing pesticide levels and the mechanisms involved. Uptake of 2,4-D was affected by p H, temperature, and the presence of other organic and inorganic compounds. Uptake was more pronounced at p H values less than 6, which implies that there may be some interaction between charged groups on the cell and the ionized carboxyl group of 2,4-D. Active transport, carrier-mediated diffusion, passive diffusion, and adsorption were considered as possible mechanisms. Though uptake was inhibited by glucose, sodium azide, and fluorodinitrobenzene (but not by uranyl ion), 2,4-D was not accumulated against a concentration gradient, a necessary consequence of an active transport system, nor was isotope counterflow found to occur. Thus, carrier-mediated diffusion was finally precluded, implying that uptake probably occurs by a two-step process: sorption onto the cell wall followed by passive diffusion into the cytoplasm.

Applied Microbiology↗

USGS2007

Bacterial Cycling of Methyl Halides

This chapter focuses on the monohalogenated methanes methyl chloride (MeCl) and methyl bromide (MeBr), their natural and anthropogenic sources, and their degradation by microorganisms, specifically by aerobic bacteria that can use MeBr and MeCl as sole source of carbon and energy. The biogeochemical cycle of methyl halides and the microbiology, biochemistry, genetics, and biotechnological potential of methyl halide-degrading microorganisms are discussed in the chapter. Methyl halides are the dominant halocarbons in the atmosphere. They play an important role in regulating stratospheric ozone concentrations and global warming as well, two factors governing planetary habitability. The monohalomethanes—methyl chloride (MeCl), methyl bromide (MeBr), and methyl iodide (MeI)—are trace gases in the atmosphere with average tropospheric-mixing ratios of 600, 10, and 2 parts per trillion (ppt), respectively. However, methyl halides are radiatively active and hence contribute to global warming by absorbing radiation in the infrared region. This is evident in their elevated global warming potential (GWP), a value calculated to quantify each compound's warming effect (on a mass basis) relative to the same mass of CO 2 . Compounds with high GWP including those like methyl halides with low concentrations may have considerable impact on atmospheric warming when compared with other “greenhouse” gases with low GWP.

Advances in Applied Microbiology↗

USGS2005

Microbial biogeochemistry of uranium mill tailings

Uranium mill tailings (UMT) are the crushed ore residues from the extraction of uranium (U) from ores. Among the radioactive wastes associated with the nuclear fuel cycle, UMT are unique in terms of their volume and their limited isolation from the surficial environment. For this latter reason, their management and long-term fate has many interfaces with environmental microbial communities and processes. The interactions of microorganisms with UMT have been shown to be diverse and with significant consequences for radionuclide mobility and bioremediation. These radionuclides are associated with the U-decay series. The addition of organic carbon and phosphate is required to initiate the reduction of the U present in the groundwater down gradient of the mills. Investigations on sediment and water from the U-contaminated aquifer, indicates that the addition of a carbon source stimulates the rate of U removal by microbial reduction. Moreover, most attention with respect to passive or engineered removal of U from groundwaters focuses on iron-reducing and sulfate-reducing bacteria.

Advances in Applied Microbiology↗

USGS2003

Abundance and characteristics of the recreational water quality indicator bacteria Escherichia coli and enterococci in gull faeces

Aims: To evaluate the numbers and selected phenotypic and genotypic characteristics of the faecal indicator bacteria Escherichia coli and enterococci in gull faeces at representative Great Lakes swimming beaches in the United States. Methods and Results: E. coli and enterococci were enumerated in gull faeces by membrane filtration. E. coli genotypes (rep‐PCR genomic profiles) and E. coli (Vitek ® GNI+) and enterococci (API ® rapid ID 32 Strep and resistance to streptomycin, gentamicin, vancomycin, tetracycline and ampicillin) phenotypes were determined for isolates obtained from gull faeces both early and late in the swimming season. Identical E. coli genotypes were obtained only from single gull faecal samples but most faecal samples yielded more than one genotype (median of eight genotypes for samples with 10 isolates). E. coli isolates from the same site that clustered at ≥85% similarity were from the same sampling date and shared phenotypic characteristics, and at this similarity level there was population overlap between the two geographically isolated beach sites. Enterococcus API ® profiles varied with sampling date. Gull enterococci displayed wide variation in antibiotic resistance patterns, and high‐level resistance to some antibiotics. Conclusions: Gull faeces could be a major contributor of E. coli (10 5 –10 9 CFU g −1 ) and enterococci (10 4 –10 8 CFU g −1 ) to Great Lakes recreational waters. E. coli and enterococci in gull faeces are highly variable with respect to their genotypic and phenotypic characteristics and may exhibit temporal or geographic trends in these features. Significance and Impact of the Study: The high degree of variation in genotypic or phenotypic characteristics of E. coli or enterococci populations within gull hosts will require extensive sampling for adequate characterization, and will influence methods that use these characteristics to determine faecal contamination sources for recreational waters.

Journal of Applied Microbiology↗

USGS2002

A rapid and efficient assay for extracting DNA from fungi

Aims: A method for the rapid extraction of fungal DNA from small quantities of tissue in a batch-processing format was investigated. Methods and Results: Tissue (< 3.0 mg) was scraped from freshly-grown fungal isolates. The tissue was suspended in buffer AP1 and subjected to seven rounds of freeze/thaw using a crushed dry ice/ethanol bath and a boiling water bath. After a 30 min boiling step, the tissue was quickly ground against the wall of the microfuge tube using a sterile pipette tip. The Qiagen DNeasy Plant Tissue Kit protocol was then used to purify the DNA for PCR/ sequencing applications. Conclusions: The method allowed batch DNA extraction from multiple fungal isolates using a simple yet rapid and reliable assay. Significance and Impact of the Study: Use of this assay will allow researchers to obtain DNA from fungi quickly for use in molecular assays that previously required specialized instrumentation, was time-consuming or was not conducive to batch processing.

Letters in Applied Microbiology↗

USGS2009

Rapid detection of Escherichia coli and enterococci in recreational water using an immunomagnetic separation/adenosine triphosphate technique

Aims: The aim of this study was to examine a rapid method for detecting Escherichia coli and enterococci in recreational water. Methods and Results: Water samples were assayed for E. coli and enterococci by traditional and immunomagnetic separation/adenosine triphosphate (IMS/ATP) methods. Three sample treatments were evaluated for the IMS/ATP method: double filtration, single filtration, and direct analysis. Pearson's correlation analysis showed strong, significant, linear relations between IMS/ATP and traditional methods for all sample treatments; strongest linear correlations were with the direct analysis (r = 0.62 and 0.77 for E. coli and enterococci, respectively). Additionally, simple linear regression was used to estimate bacteria concentrations as a function of IMS/ATP results. The correct classification of water-quality criteria was 67% for E. coli and 80% for enterococci. Conclusions: The IMS/ATP method is a viable alternative to traditional methods for faecal-indicator bacteria. Significance and Impact of the Study: The IMS/ATP method addresses critical public health needs for the rapid detection of faecal-indicator contamination and has potential for satisfying US legislative mandates requiring methods to detect bathing water contamination in 2 h or less. Moreover, IMS/ATP equipment is considerably less costly and more portable than that for molecular methods, making the method suitable for field applications. ?? 2009 The Authors.

Journal of Applied Microbiology↗

USGS2011

Microbial water quality before and after the repair of a failing onsite wastewater treatment system adjacent to coastal waters

Aims: The objective was to assess the impacts of repairing a failing onsite wastewater treatment system (OWTS, i.e., septic system) as related to coastal microbial water quality. Methods and Results: Wastewater, groundwater and surface water were monitored for environmental parameters, faecal indicator bacteria (total coliforms, Escherichia coli, enterococci) and the viral tracer MS2 before and after repairing a failing OWTS. MS2 results using plaque enumeration and quantitative reverse transcriptase polymerase chain reaction (qRT‐PCR) often agreed, but inhibition limited the qRT‐PCR assay sensitivity. Prerepair, MS2 persisted in groundwater and was detected in the nearby creek; postrepair, it was not detected. In groundwater, total coliform concentrations were lower and E. coli was not detected, while enterococci concentrations were similar to prerepair levels. E. coli and enterococci surface water concentrations were elevated both before and after the repair. Conclusions: Repairing the failing OWTS improved groundwater microbial water quality, although persistence of bacteria in surface water suggests that the OWTS was not the singular faecal contributor to adjacent coastal waters. A suite of tracers is needed to fully assess OWTS performance in treating microbial contaminants and related impacts on receiving waters. Molecular methods like qRT‐PCR have potential but require optimization. Significance and Impact of Study: This is the first before and after study of a failing OWTS and provides guidance on selection of microbial tracers and methods.

North Carolina↗

USGS2016

Natural inactivation of Escherichia coli in anoxic and reduced groundwater

Aims Inactivation rates of E. coli in groundwater have most often been determined in aerobic and oxidized systems. This study examined E. coli inactivation rates in anaerobic and extremely reduced groundwater systems that have been identified as recharge zones. Methods and Results Groundwater from six artesian wells was diverted to above ground, flow through mesocosms that contained laboratory grown E. coli in diffusion chambers. All groundwater was anaerobic and extremely reduced (ORP < - 300 mV). Cells were plated onto mTEC agar during 21 day incubation periods. All data fit a bi-phasic inactivation model, with > 95% of the E. coli population being inactivated < 11.0 hrs (mean k = 0.488± 0.188 h −1 ). Conclusions The groundwater geochemical conditions enhanced the inactivation of E. coli to rates approximately 21-fold greater than previously published inactivation rate in groundwater (mean k = 0.023 ± 0.030 h −1 ). Also, mTEC agar inhibits E. coli growth following exposure to anaerobic and reduced groundwater. Significance and Impact of the Study Aquifer recharge zones with geochemical characteristics observed in this study complement above ground engineered processes (e.g., filtration, disinfection), while increasing the overall indicator microorganism log-reduction rate of a facility.

Journal of Applied Microbiology↗

USGS2017

Environmental Escherichia coli : Ecology and public health implications - A review

Escherichia coli is classified as a rod-shaped, Gram-negative bacterium in the family Enterobacteriaceae . The bacterium mainly inhabits the lower intestinal tract of warm-blooded animals, including humans, and is often discharged into the environment through feces or wastewater effluent. The presence of E. coli in environmental waters has long been considered as an indicator of recent fecal pollution. However, numerous recent studies have reported that some specific strains of E. coli can survive for long periods of time, and potentially reproduce, in extra-intestinal environments. This indicates that E. coli can be integrated into indigenous microbial communities in the environment. This naturalization phenomenon calls into question the reliability of E. coli as a fecal indicator bacterium (FIB). Recently, many studies reported that E. coli populations in the environment are affected by ambient environmental conditions affecting their long-term survival. Large-scale studies of population genetics provide the diversity and complexity of E. coli strains in various environments, affected by multiple environmental factors. This review examines the current knowledge on the ecology of E. coli strains in various environments in regards to its role as a FIB and as a naturalized member of indigenous microbial communities. Special emphasis is given on the growth of pathogenic E. coli in the environment, and the population genetics of environmental members of the genus Escherichia . The impact of environmental E. coli on water quality and public health is also discussed.

Journal of Applied Microbiology↗

USGS2022

Natural inactivation of MS2, poliovirus type 1 and Cryptosporidium parvum in an anaerobic and reduced aquifer

Aims The study of microbial inactivation rates in aquifer systems has most often been determined in aerobic and oxidized systems. This study examined the inactivation (i.e. loss of infectivity) of MS2, poliovirus type 1 (PV1) and Cryptosporidium parvum in an anaerobic and reduced groundwater system that has been identified as storage zones for aquifer storage and recovery (ASR) facilities. Methods and Results Anaerobic and reduced (ORP < − 250 mV) groundwater from an artesian well was diverted to an above-ground, flow-through mesocosm that contained diffusion chambers filled with MS2, PV1 or Cryptosporidium parvum . The respective infectivity assays were performed on microorganisms recovered from the diffusion chambers during 30- to 58-day experiments. The net reduction in infectivity was 5.73 log 10 over 30 days for MS2, 5.00 log 10 over 58 days for PV1 and 4.07 log 10 over 37 days for C . parvum . The best fit inactivation model for PV1 was the log-linear model and the Weibull model for MS2 and C . parvum , with respective inactivation rates (95% confidence interval) of 0.19 (0.17–0.21) log 10 day −1 , 0.31 (0.19–0.89) log 10 day −1 and 0.20 (0.14–0.37) log 10 day −1 . Conclusions The groundwater geochemical conditions in this aquifer enhanced the inactivation of MS2, PV1, and C . parvum at rates approximately 2.0–5.3-fold, 1.2–17.0-fold, and 4.5–5.6-fold greater, respectively, than those from published studies that used diffusion chambers in aerobic-to-anoxic groundwater systems, with positive redox potentials. Significance and Impact of the Study Geochemical conditions like those in the aquifer zone in this study can naturally and significantly reduce concentrations of microbial indicators and pathogens of human health concern in injected surface water. Appropriate storage times for injected surface water could complement above-ground engineered processes for microorganism removal and inactivation (e.g. filtration, disinfection) by naturally increasing overall microorganism log-inactivation rates of ASR facilities.

Journal of Applied Microbiology↗