Probable Number Procedure Research Articles

Levels of Salmonella enterica and Listeria monocytogenes in Alternative Irrigation Water Vary Based on Water Source on the Eastern Shore of Maryland.

ABSTRACTIrrigation water sources have been shown to harbor foodborne pathogens and could contribute to the outbreak of foodborne illness related to consumption of contaminated produce. Determining the probability of and the degree to which these irrigation water sources contain these pathogens is paramount. The purpose of this study was to determine the prevalence of Salmonella enterica and Listeria monocytogenes in alternative irrigation water sources. Water samples (n = 188) were collected over 2 years (2016 to 2018) from 2 reclaimed water plants, 3 nontidal freshwater rivers, and 1 tidal brackish river on Maryland’s Eastern Shore (ESM). Samples were collected by filtration using modified Moore swabs (MMS) and analyzed by culture methods. Pathogen levels were quantified using a modified most probable number (MPN) procedure with three different volumes (10 liters, 1 liter, and 0.1 liter). Overall, 65% (122/188) and 40% (76/188) of water samples were positive for S. enterica and L. monocytogenes, respectively. For both pathogens, MPN values ranged from 0.015 to 11 MPN/liter. Pathogen levels (MPN/liter) were significantly (P < 0.05) greater for the nontidal freshwater river sites and the tidal brackish river site than the reclaimed water sites. L. monocytogenes levels in water varied based on season. Detection of S. enterica was more likely with 10-liter filtration compared to 0.1-liter filtration. The physicochemical factors measured attributed only 6.4% of the constrained variance to the levels of both pathogens. This study shows clear variations in S. enterica and L. monocytogenes levels in irrigation water sources on ESM.IMPORTANCE In the last several decades, Maryland’s Eastern Shore has seen significant declines in groundwater levels. While this area is not currently experiencing drought conditions or water scarcity, this research represents a proactive approach. Efforts, to investigate the levels of pathogenic bacteria and the microbial quality of alternative irrigation water are important for sustainable irrigation practices into the future. This research will be used to determine the suitability of alternative irrigation water sources for use in fresh produce irrigation to conserve groundwater.

Escherichia coli O157:H7 in Drin Prevalence of Escherichia coli O157:H7 in Drink from Different Food Premises in Kota Samarahan Sarawak

Contamination of drinks with E. coli O157:H7 served in food premises such as restaurants can cause haemorrhagic colitis and haemolytic uremic syndrome to humans. The presence or absence of faecal pathogen was demonstrated using coliform group as indicator microorganisms. Therefore, this study was conducted to detect the presence of E. coli O157:H7 in drinking water from food restaurant premise in Kota Samarahan and Kuching to ensure safe and potable drinking water is served to the consumer. A total of thirty (n=30) drink samples including six types of each of the samples are cold plain water, iced tea, iced milo, syrup and iced milk tea. Most Probable Number (MPN) procedure was used in this study to enumerate the MPN values of coliform bacteria in each drink collected. A total of 53.33% (16/30) of the drink samples showed positive E. coli detection. Then, the PCR assay showed 6.25% (one out of 16 isolates) samples were positive and carried stx1 gene produced by E. coli O157:H7 in iced milo sample types. This study showed the drinks collected from food premises was contaminated with faecal contamination, which was not safe to drink by the consumer. Therefore, preventive actions should be taken to prevent foodborne illness outbreak in future

Escherichia coli detection and identification in shellfish from southeastern Brazil

The pathogenic Escherichia coli strain may be responsible for foodborne infections in humans. This study was carried out to evaluate the presence of Escherichia coli and verify the occurrence of virulence genes in mussels (Mytella falcata) and oysters (Crassostrea brasiliana) collected from natural banks of the Estuary Lagunar Complex of Cananéia in the State of São Paulo, southeastern Brazil. A total of 150 shellfish samples (75 mussels and 75 oysters) were collected from June 2016 to February 2017. Total coliforms and Escherichia coli were determined using the most probable number (MPN) procedure. Colonies of Escherichia coli were biochemically characterized. The positive samples were submitted to molecular identification of species and posterior genetic sequencing for identifying Escherichia coli pathotypes. Escherichia coli was detected in 48 samples (32%) in concentrations ranging from <3 to <927 MPN/g. From 240 colonies tested biochemically, ninety-three were positive and submitted to molecular analyses. Fourty-eight colonies were confirmed as Escherichia coli by presenting PhoA gene and six (12.5%) were identified as enteropathogenic Escherichia coli (EPEC) by presenting eaeA gene and by sequencing of positive amplicons. Phylogenetic analyses revealed that the Brazilian samples were grouped in clades along with other EPEC isolates. These results are the first to indicate the presence of EPEC in shellfish in Brazil.

A critical assessment of four most probable number procedures for routine enumeration of cheese-damaging clostridia in milk

In cheese production, knowledge of clostridial spore levels in milk is crucial to avoid economic losses due to late-blowing, a characteristic quality defect observed especially in hard cheeses. However, no international standard method is available to quantify clostridial spores in milk. Since performance data on the currently applied enumeration methods have not been systematically investigated, in this study we assessed four routine media and most probable number (MPN) methods for the enumeration of cheese-damaging clostridia in milk. Among the four evaluated methods, statistically significant differences became evident, while moderate to optimal correlations were observed. Bacterial isolates from positive reactions were identified using molecular methods. All four methods assessed showed a severe lack of selectivity due to the incorporation of non-clostridial spore-formers in the counts produced. The results illustrate major drawbacks of currently used methods and stress the necessity for improving method selectivity, sensitivity and reliability before standardisation can be envisaged.

Isolation, Screening, and Characterization of Naphthalene-Degrading Bacteria from Zarand Mine, Iran

ABSTRACTPAHs are aromatic hydrocarbons with two or more fused benzene rings. They are formed during the thermal breakdown of organic molecules and their succeeding recombination. Naphthalene is the simplest (PAHs) that is carcinogenic. Bioremediation method is considered as an economical and safe approach for the elimination of aromatic compounds from environment. The bacteria were capable to grow on various hydrocarbons like naphthalene. The aim of this research is to isolate and identify naphthalene-degrading bacteria from the coal mine of Zarand. Four samples of water and sludge from various sites of the mine were collected. These sites include the following: Main coal vacate site (MC), Inoculum Sump site (IB), Sludge aggregate site (SA), and Near sludge aggregate site (NF). In this study, 12 bacterial strains that utilize naphthalene at initial concentration 200 mg/L (ppm) as carbon and energy sources for growth were isolated from the Zarand mine in Iran. In addition, bacterial cell density was assayed by measuring the OD600. In addition, total naphthalene-degrading bacteria were quantified with the most probable number (MPN) procedure using microtiter plates and the colony-forming unit (CFU) method. The results had shown that most of the naphthalene degrader bacteria aggregated in (SA) site. Six bacteria, isolated from wastewater and oil-contaminated soil showed great potential as naphthalene degraders up to 400 (ppm) and selected for biochemical characteristics. Naphthalene tolerance of the strains in various concentrations of naphthalene indicates that the strain 38 N can grow best at 600 (ppm) naphthalene. This strain was identified based on 16S rRNA gene analysis that showed belonging to Sphingobacterium multivorum AHB38N.

Bioremediation of hydrocarbon degradation in a petroleum-contaminated soil and microbial population and activity determination

Bioremediation of hydrocarbon degradation in petroleum-polluted soil is carried out by various microorganisms. However, little information is available for the relationships between hydrocarbon degradation rates in petroleum-contaminated soil and microbial population and activity in laboratory assay. In a microcosm study, degradation rate and efficiency of total petroleum hydrocarbons (TPH), alkanes, and polycyclic aromatic hydrocarbons (PAH) in a petroleum-contaminated soil were determined using an infrared photometer oil content analyzer and a gas chromatography mass spectrometry (GC-MS). Also, the populations of TPH, alkane, and PAH degraders were enumerated by a modified most probable number (MPN) procedure, and the hydrocarbon degrading activities of these degraders were determined by the Biolog (MT2) MicroPlates assay. Results showed linear correlations between the TPH and alkane degradation rates and the population and activity increases of TPH and alkane degraders, but no correlation was observed between the PAH degradation rates and the PAH population and activity increases. Petroleum hydrocarbon degrading microbial population measured by MPN was significantly correlated with metabolic activity in the Biolog assay. The results suggest that the MPN procedure and the Biolog assay are efficient methods for assessing the rates of TPH and alkane, but not PAH, bioremediation in oil-contaminated soil in laboratory.

Prevalence and behavior of multidrug-resistant shiga toxin-producing Escherichia coli, enteropathogenic E. coli and enterotoxigenic E. coli on coriander

The prevalence and behavior of multidrug-resistant diarrheagenic Escherichia coli pathotypes on coriander was determined. One hundred coriander samples were collected from markets. Generic E. coli were determined using the most probable number procedure. Diarrheagenic E. coli pathotypes (DEPs) were identified using two multiplex polymerase chain reaction procedures. Susceptibility to sixteen antibiotics was tested for the isolated DEPs strains by standard test. The behavior of multidrug-resistant DEPs isolated from coriander was determined on coriander leaves and chopped coriander at 25°± 2 °C and 3°± 2 °C. Generic E. coli and DEPs were identified, respectively, in 43 and 7% of samples. Nine DEPs strains were isolated from positive coriander samples. The identified DEPs included Shiga toxin-producing E. coli (STEC, 4%) enterotoxigenic E. coli (ETEC, 2%) and enteropathogenic E. coli (EPEC, 1%). All isolated DEPs strains exhibited multi-resistance to antibiotics. On inoculated coriander leaves stored at 25°± 2 °C or 3°± 2 °C, no growth was observed for multidrug-resistant DEPs strains. However, multidrug-resistant DEPs strains grew in chopped coriander: after 24 h at 25° ± 2 °C, DEPs strains had grown to approximately 3 log CFU/g. However, at 3°± 2 °C the bacterial growth was inhibited. To the best of our knowledge, this is the first report of the presence and behavior of multidrug-resistant STEC, ETEC and EPEC on coriander and chopped coriander.

Presence of Multidrug-Resistant Shiga Toxin-Producing Escherichia coli, Enteropathogenic E. coli and Enterotoxigenic E. coli, on Raw Nopalitos (Opuntia ficus-indica L.) and in Nopalitos Salads from Local Retail Markets in Mexico.

The presence of multidrug-resistant pathogenic bacteria in food is a significant public health concern. Diarrheagenic Escherichia coli pathotypes (DEPs) are foodborne bacteria. In Mexico, DEPs have been associated with diarrheal illness. There is no information about the presence of multidrug-resistant DEPs on fresh vegetables and in cooked vegetable salads in Mexico. "Nopalitos" (Opuntia ficus-indica L.) is a Cactacea extensively used as a fresh green vegetable throughout Mexico. The presence of generic E. coli and multidrug-resistant DEPs on raw whole and cut nopalitos and in nopalitos salad samples was determined. One hundred raw whole nopalitos (without prickles) samples, 100 raw nopalitos cut into small square samples, and 100 cooked nopalitos salad samples were collected from markets. Generic E. coli was determined using the most probable number procedures. DEPs were identified using two multiplex polymerase chain reaction procedures. Susceptibility to 16 antibiotics was tested for the isolated DEP strains by standard test. Of the 100 whole nopalitos samples, 100 cut nopalitos samples, and 100 nopalitos salad samples, generic E. coli and DEPs were identified, respectively, in 80% and 10%, 74% and 10%, and 64% and 8%. Eighty-two DEP strains were isolated from positive nopalitos samples. The identified DEPs included Shiga toxin-producing E. coli (STEC), enteropathogenic E. coli (EPEC), and enterotoxigenic E. coli (ETEC). All isolated strains exhibited resistance to at least six antibiotics. To the best of our knowledge, this is the first report of the presence of multidrug-resistant and antibiotic resistance profiles of STEC, ETEC, and EPEC on raw nopalitos and in nopalitos salads in Mexico.

Seasonal Dynamics of Bacterial Population Degrading Dimethylarsenic Acid in Lake Kahokugata

The degradation processes of organoarsenic compounds in aquatic environments would depend on the bioactivities of microorganisms and significantly influence the cycles of the arsenic species. The concentrations of arsenic species were determined in Lake Kahokugata. During the all season, the inorganic arsenic was detected, while the dimethylarsinic acid (DMAA) appeared in only winter seasons, and MMAA was not detected. Moreover, when DMAA was added to the water samples of Lake Kahokugata collected at every month from June in 2005 to November in 2006, the DMAA in the sample water of all seasons decreased and was converted to inorganic arsenic until the 28th day of incubation. According to the Most Probable Number (MPN) procedure, the DMAA-degrading bacteria were detected at cell densities ranged from 120 cells/mL to 2100 cells/mL during the all season. These facts suggested that the lake water of all season in Lake Kahokugata possessed the microbial activities for DMAA decomposition. To determine the composition of DMAA-degrading bacteria, the total 352 isolates obtained as dominated bacterial species were analyzed by the restriction-fragment-length polymorphism (RFLP) analysis of 16S rDNA genes. As a result, total 352 isolates were classified into 10 types of which the rates of total isolate numbers indicated seasonal changes. The diverse compositions of DMAA-degrading bacteria would seasonally change and control the organoarsenic degradation contributing to the seasonal arsenic cycles in Kahokugata.

Distribution, serological and molecular characterization of Vibrio parahaemolyticus from shellfish in the eastern coast of China

To investigate the prevalence of Vibrio parahaemolyticus in shellfish, a total of 288 samples from retail markets in four coastal provinces of eastern China were collected and analyzed monthly from December 2008 to November 2009. Altogether 172 isolates were isolated and identified, of which 2 isolates were tdh + and 5 were trh +. The levels of V. parahaemolyticus were estimated by most probable number procedure and results suggested that the distribution of V. parahaemolyticus was disparity in season. Serotyping was performed among all isolates, 157 isolates were grouped into 9 O-groups and 42 isolates were determined by specific K-typing. Two tdh + isolates were identified as O3:K6 and O4:K68 serovars. Random amplified polymorphic DNA (RAPD) was performed to assess the genetic diversity of all isolates. The results showed that there were 73 different patterns, which were clustered into 18 groups except 6 miscellaneous patterns. The two tdh + isolates and two clinical isolates were grouped into the same cluster. This study demonstrated that V. parahaemolyticus from shellfishes were of high antigenic and genetic diversity. Comparison with serological method, RAPD might be a more efficient vehicle for epidemiology and risk assessment of V. parahaemolyticus.

Occurrence and distribution of Vibrio parahaemolyticus in retail oysters in Sao Paulo State, Brazil

Vibrio parahaemolyticus is a potentially pathogenic bacterium that occurs naturally in estuarine environments worldwide, and is often associated with gastroenteritis in humans following consumption of raw bivalve mollusks, especially raw oysters. The occurrence of total and pathogenic V. parahaemolyticus in 74 samples of raw oysters collected in restaurants, supermarkets, groceries and beach huts in Sao Paulo State, was monitored between February 2006 and January 2007. Enumeration of V. parahaemolyticus was performed according to the most probable number (MPN) procedure. Five to ten typical colonies were selected from thiosulfate-citrate-bile salts-sucrose (TCBS) agar plates for confirmation by the presence of the species-specific gene tlh and the virulence genes tdh and trh by multiplex PCR. V. parahaemolyticus was detected in 100% of samples. The densities of total V. parahaemolyticus varied from 1.78 to 6.04 log 10 (MPN/g), with higher densities being detected in fall and summer, and lower densities in winter ( P < 0.05). There was no statistical difference among densities of V parahaemolyticus regarding the site of collection. None of the 1943 V. parahaemolyticus isolates contained tdh and/or trh. These data provide information for the assessment of exposure to V. parahaemolyticus in oysters consumed in Sao Paulo, State, Brazil.

Evaluation of emission of greenhouse gases from soils amended with sewage sludge

Increase in concentrations of various greenhouse gases and their possible contributions to the global warming are becoming a serious concern. Anthropogenic activities such as cultivation of flooded rice and application of waste materials, such as sewage sludge which are rich in C and N, as soil amendments could contribute to the increase in emission of greenhouse gases such as methane (CH4) and nitrous oxide (N2O) into the atmosphere. Therefore, evaluation of flux of various greenhouse gases from soils amended with sewage sludge is essential to quantify their release into the atmosphere. Two soils with contrasting properties (Candler fine sand [CFS] from Florida, and Ogeechee loamy sand [OLS] from Savannah, GA) were amended with varying rates (0, 24.7, 49.4, 98.8, and 148.3 Mg ha− 1) of 2 types of sewage sludge (industrial [ISS] and domestic [DSS] origin. The amended soil samples were incubated in anaerobic condition at field capacity soil water content in static chamber (Qopak bottles). Gas samples were extracted immediately after amending soils and subsequently on a daily basis to evaluate the emission of CH4, CO2 and N2O. The results showed that emission rates and cumulative emission of all three gases increased with increasing rates of amendments. Cumulative emission of gases during 25-d incubation of soils amended with different types of sewage sludge decreased in the order: CO2 > N2O > CH4. The emission of gases was greater from the soils amended with DSS as compared to that with ISS. This may indicate the presence of either low C and N content or possible harmful chemicals in the ISS. The emission of gases was greater from the CFS as compared to that from the OLS. Furthermore, the results clearly depicted the inhibitory effect of acetylene in both soils by producing more N2O and CH4 emission compared to the soils that did not receive acetylene at the rate of 1 mL g− 1 soil. Enumeration of microbial population by fluorescein diacetate (FDA) and most probable number (MPN) procedure at the end of 25-d incubation demonstrated a clear relationship between microbial activity and the emission of gases. The results of this study emphasize the need to consider the emission of greenhouse gases from soils amended with organic soil amendments such as sewage sludge, especially at high rates, and their potential contribution to global warming.

Response of nematode-trapping fungi to organic substrates in a coastal grassland soil

To understand why Arthrobotrys oligospora and other nematode-trapping fungi are common and sometimes abundant in the coastal grassland soils of the Bodega Marine Reserve (BMR, Sonoma County, CA), we examined how resident trapping fungi responded to the addition of eight organic substrates (lupine leaves, grass leaves, dead isopods, dead moth larvae, isopod faeces, deer faeces, shrimp shells, and powdered chitin). We were especially interested in the effects of dead isopods because isopods are abundant at BMR and because previous studies had documented strong responses of A. oligospora to other arthropods (dead moth larvae). Soil from BMR was packed into vials (40 g dry mass equivalent per vial with water potential at −230 kPa and bulk density at 0.9 g cm −3), and one substrate or no substrate was added to the soil surface. After 30 d at 20 °C, trapping fungi were quantified by dilution plating and most probable number procedures. The response of A. oligospora was inversely related to substrate carbon:nitrogen (C:N) ratio: substrates with low C:N ratios (dead isopods, lupine leaves, dead moth larvae) usually caused large increases in A. oligospora whereas those with higher C:N ratios (isopod faeces, deer faeces, grass leaves) did not. An exception was chitin powder, which had a low C:N ratio, but which did not cause A. oligospora to proliferate. Responses of A. oligospora were directly related to the quantity of nitrogen added with each substrate, and those substrates that caused large increases in resident nematodes usually caused large increases in A. oligospora. Other trapping fungi did not respond as strongly as A. oligospora.

Two Rapid Methods for Detection of Escherichia coli Exceeding 104/g Action Levels: Precollaborative Study

The current AOAC Method 966.24 for enumeration of Escherichia coli in foods uses a most probable number (MPN) procedure with extensive confirmation steps. Two new methods based on membrane filtration (MF) were compared to the MPN reference method for detection of high levels of E. coli in 5 food types, some of which represent categories for which the U.S. Food and Drug Administration (FDA) mandates additional testing if an action level of 10(4)/g E. coli is exceeded. Ground beef, which is not FDA regulated, was also tested. The 5 food types were all inoculated at 3 levels: 10(2)/g, > or = 10(4)/g, and > or = 10(5)/g E. coli. An MF protocol using either m-ColiBlue24 (CB) or lauryl sulfate tryptose plus BCIG (LST/BCIG) was an effective potential alternative to the reference method. Sensitivity and specificity for both CB and LST/BCIG were 98 and 100%, respectively. Agreement between MPN and both CB and LST/BCIG was 98%. The 2 proposed methods allow completion of both presumptive and confirmatory steps in 1-3 days, whereas the reference method requires as many as 11 days. Exclusivity testing with 50 non-E. coli strains indicated 100% were correctly ruled out by the proposed protocols. Inclusivity testing was used to determine whether typical results were obtained after incubation of E. coli cultures on CB or LST/BCIG for 24 h. Of 50 E. coli strains tested, 100% yielded typical results after incubation on CB, and 98% yielded typical results after incubation on LST/BCIG.

Seasonal Dynamics of Dimethylarsenic Acid Degrading Bacteria Dominated in Lake Kibagata

Degradation processes of organoarsenic compounds significantly influence arsenic cycles in aquatic environments and would depend on the bacterial activities. The bacterial population involving dimethylarsinic acid (DMAA) degradation was investigated in Lake Kibagata from April to December in 2003. During the experimental period, the methylated arsenic was not detected, although the inorganic arsenic concentration ranged from 3.4 nM to 9.2 nM. Moreover, in the sample water of Lake Kibagata to which DMAA added, DMAA decreased while inorganic arsenic increased for 25 days. These facts suggested that the bacteria remineralized methylate arsenic species to inorganic arsenic. In fact, monitoring the use of Most Probable Number (MPN) procedure demonstrated that the DMAA-degrading bacteria exist at cell densities ranged from 41 cells/ml to 510 cells/ml. To determine the composition of DMAA-degrading bacteria, the total 110 isolates obtained as dominated bacterial species were analyzed by the restriction-fragment-length polymorphism (RFLP) analysis of 16S rDNA. As a result, total 110 isolates were classified into 12 types, of which 4 types dominated during the spring and/or fall seasons, and the rest 8 types dominated during summer season. DMAA degrading activities of the 110 isolates ranged at various degrees. Especially, the some isolates of fall season tend to show high degradation activities. The phylogenetic analysis using 16S rDNA revealed that the representative isolates formed several clusters in the gram-positive bacterial group and the proteobacteria subdivision. The diverse compositions of DMAA-degrading bacteria would seasonally change to control the rates of organoarsenic degradation in Kibagata.

Isolation and characterization of Pseudoalteromonas strains with divergent polycyclic aromatic hydrocarbon catabolic properties

Fifteen strains of polycyclic aromatic hydrocarbon (PAH)-catabolizing bacteria, identified as Pseudoalteromonas spp. were isolated from Eagle Harbor, Puget Sound, USA, using a most probable number procedure in which naphthalene or phenanthrene was the sole carbon and energy source. Despite having identical 16S rDNA sequences, some catabolized many PAHs, whereas others oxidized only naphthalenes. A putative naphthalene 1,2-dioxygenase gene fragment was polymerase chain reaction-amplified from the naphthalene-degrading strains and shown to be almost identical to a gene present in Neptunomonas naphthovorans, suggesting horizontal transfer.

Selective enrichment of a pyrene degrader population and enhanced pyrene degradation in Bermuda grass rhizosphere

Rhizosphere soil has a more diverse and active microbial community compared to nonvegetated soil. Consequently, the rhizosphere pyrene degrader population (PDP) and pyrene degradation may be enhanced compared to nonvegetated bulk soil (NVB). The objectives of this growth chamber study were to compare (1) Bermuda grass (Cynodon dactylon cv. Guymon) growth in pyrene-contaminated and noncontaminated soils and (2) pyrene degradation and PDP among NVB, Bermuda grass bulk (BB), and Bermuda grass rhizosphere soil (BR). Soils were amended with pyrene at 0 and 500 mg kg−1, seeded with Bermuda grass, and thinned to two plants per pot 14 days after planting (DAP). Pyrene degradation was evaluated over 63 days. The PDP was enumerated via a most probable number (MPN) procedure at 63 DAP. Bermuda grass root growth was more sensitive to pyrene contamination than shoot growth. Pyrene degradation followed first-order kinetics. Pyrene degradation was significantly greater in BR compared to BB and NVB with rate constants of 0.082, 0.050, and 0.052 day−1, respectively. The PDPs were 8.01, 7.30, and 6.83 log10 MPN g−1 dry soil for BR, BB, and NVB, respectively. The largest PDP was in soil with the most rapid pyrene degradation. These results indicate that Bermuda grass can grow in pyrene-contaminated soil and enhance pyrene degradation through a rhizosphere effect.

Seasonal dynamics of dimethylarsinic-acid-decomposing bacteria dominating in Lake Kahokugata

Decomposition processes of organoarsenic compounds significantly influence arsenic cycles in aquatic environments, and such processes depend on bacterial activity. However, the bacterial characteristics in these environments are obscure. Accordingly, we observed seasonal variations of arsenic species and the bacterial population decomposing dimethylarsinic acid (DMAA) in Lake Kahokugata from April 2002 to January 2003. Monitoring of bacterial biomass involving DMAA decomposition using the most probable number procedure showed that the bacterial cell densities ranged from 36 to 3600 ml -1 . On the other hand, methylated arsenic was not detected during the experimental period, although the inorganic arsenic concentration was over 4 nM. This suggests that bacteria remineralized methylated arsenic species to inorganic arsenic. Furthermore, the composition of bacterial communities involving DMAA decomposition was examined by restriction-fragment-length polymorphism analysis of the 16S rDNA nucleotide. As a result, a total of 49 isolates were classified into 10 type groups, and 32 of these isolates belonged to three dominant type groups. Phylogenetic analysis using 16S rDNA partial sequences (ca 320 bp) suggests that the representative isolates of the dominant type groups are specific to the summer or winter season. Moreover, as a result of the culture experiments to examine DMAA decomposition activity, the representative isolates decomposed 1 μM DMAA at a decomposition percentage of below 80%. In conclusion, some bacterial communities in a specific season can decompose DMAA to varying degrees, contributing to the annual cycle of arsenic species.

Ecology of Vibrio vulnificus and Vibrio parahaemolyticus in brackish environments of the Sada River in Shimane Prefecture, Japan

While there are several studies on the ecology of Vibrio vulnificus and Vibrio parahaemolyticus in estuarine water environments around the world, there is little information on the distribution of both organisms during the cold-weather months. Thus, we conducted a multi-year study on the ecology of both organisms in brackish environments of the Sada River, a drainage canal flowing slowly into the Japan Sea from Lake Shinji in Shimane Prefecture, Japan. Water samples were collected twice a month at five sites from August 2000 to May 2002. Both organisms were enumerated in 10 l water, 100 g sediment and 10 g shellfish by the most probable number (MPN) procedure. Isolates were confirmed as V. vulnificus using hemolysin gene PCR. During the last 7 months (including winter) of the study, water and sediment samples were also analyzed for the presence of both organisms. V. parahaemolyticus was isolated from river mouths and coastal environments of average salinity ≧4.4 ± 2.0 ppt throughout the year at cell concentrations of 10 −3 to 10 1 MPN ml −1. Similar concentrations of V. vulnificus were isolated from coastal environments of average salinity 24.0 ± 5.4 ppt, except for two times when water moved to the upper reaches due to high tide and V. vulnificus was rifted to the upper reaches. These findings suggest that both organisms are continuously distributed in the Sada estuary throughout the year regardless of water temperature.

Correlations Between Most Probable Number and Activity of Nematode-Trapping Fungi

ABSTRACT Soil cages were used to determine whether nematode-trapping fungi population density, as measured by most probable number (MPN) procedures, was correlated with the trapping of nematodes. Fungi studied (and trap type) were Arthrobotrys oligospora (adhesive networks), A. eudermata (adhesive networks), A. dactyloides (constricting rings), Dactylellina ellipsospora (adhesive knobs), and D. haptotyla (adhesive knobs). The fungi were formulated as assimilative hyphae in dried alginate pellets. Pellets were added to field soil, the soil was packed into 80-cm(3) cages (PVC pipe, 3.0 cm long and 3.9 cm in diameter), and the cages were buried in vineyards. After 14 to 61 days, the cages were recovered, and MPN data and trapping activity were determined. For all five fungi, MPN data were correlated with the number of pellets added. Regardless of fungus population density, A. oligospora and A. eudermata trapped few if any nematodes in soil, and consequently, trapping and fungus population density were not correlated. The correlation between population density and trapping was weak for A. dactyloides but relatively strong for D. ellipsospora and D. haptotyla. High levels of trapping by the latter two fungi required more than 10(2) fungus propagules per gram of soil.