scholarly journals Genetic Markers for Rapid PCR-Based Identification of Gull, Canada Goose, Duck, and Chicken Fecal Contamination in Water

2011 ◽  
Vol 78 (2) ◽  
pp. 503-510 ◽  
Author(s):  
Hyatt C. Green ◽  
Linda K. Dick ◽  
Brent Gilpin ◽  
Mansour Samadpour ◽  
Katharine G. Field
Keyword(s):  
New Zealand ◽  
Fecal Contamination ◽  
The United States ◽  
Rrna Genes ◽  
Most Probable Number ◽  
Rrna Gene ◽  
Probable Number ◽  
Content Type ◽  
16S Rna ◽  
Pcr Assays

ABSTRACTAvian feces contaminate waterways but contribute fewer human pathogens than human sources. Rapid identification and quantification of avian contamination would therefore be useful to prevent overestimation of human health risk. We used subtractive hybridization of PCR-amplified gull fecal 16S RNA genes to identify avian-specific fecal rRNA gene sequences. The subtracters were rRNA genes amplified from human, dog, cat, cow, and pig feces. Recovered sequences were related toEnterobacteriaceae(47%),Helicobacter(26%),Catellicoccus(11%),Fusobacterium(11%), andCampylobacter(5%). Three PCR assays, designated GFB, GFC, and GFD, were based on recovered sequence fragments. Quantitative PCR assays for GFC and GFD were developed using SYBR green. GFC detected down to 0.1 mg gull feces/100 ml (corresponding to 2 gull enterococci most probable number [MPN]/100 ml). GFD detected down to 0.1 mg chicken feces/100 ml (corresponding to 13Escherichia coliMPN/100 ml). GFB and GFC were 97% and 94% specific to gulls, respectively. GFC cross-reacted with 35% of sheep samples but occurred at about 100,000 times lower concentrations in sheep. GFD was 100% avian specific and occurred in gulls, geese, chickens, and ducks. In the United States, Canada, and New Zealand, the three markers differed in their geographic distributions but were found across the range tested. These assays detected four important bird groups contributing to fecal contamination of waterways: gulls, geese, ducks, and chickens. Marker distributions across North America and in New Zealand suggest that they will have broad applicability in other parts of the world as well.

scholarly journals Long-Term Study of Vibrio parahaemolyticus Prevalence and Distribution in New Zealand Shellfish

2015 ◽  
Vol 81 (7) ◽  
pp. 2320-2327 ◽  
Author(s):  
C. D. Cruz ◽  
D. Hedderley ◽  
G. C. Fletcher
Keyword(s):  
New Zealand ◽  
Vibrio Parahaemolyticus ◽  
Most Probable Number ◽  
Pcr Analysis ◽  
Potential Hazard ◽  
Probable Number ◽  
Content Type ◽  
The North ◽  
Long Term Study ◽  
Food Borne

ABSTRACTThe food-borne pathogenVibrio parahaemolyticushas been reported as being present in New Zealand (NZ) seawaters, but there have been no reported outbreaks of food-borne infection from commercially grown NZ seafood. Our study determined the current incidence ofV. parahaemolyticusin NZ oysters and Greenshell mussels and the prevalence ofV. parahaemolyticustdhandtrhstrains. Pacific (235) and dredge (21) oyster samples and mussel samples (55) were obtained from commercial shellfish-growing areas between December 2009 and June 2012. TotalV. parahaemolyticusnumbers and the presence of pathogenic genestdhandtrhwere determined using the FDA most-probable-number (MPN) method and confirmed using PCR analysis. In samples from the North Island of NZ,V. parahaemolyticuswas detected in 81% of Pacific oysters and 34% of mussel samples, while the numbers ofV. parahaemolyticustdhandtrhstrains were low, with just 3/215 Pacific oyster samples carrying thetdhgene.V. parahaemolyticusorganisms carryingtdhandtrhwere not detected in South Island samples, andV. parahaemolyticuswas detected in just 1/21 dredge oyster and 2/16 mussel samples. Numbers ofV. parahaemolyticusorganisms increased when seawater temperatures were high, the season when most commercial shellfish-growing areas are not harvested. The numbers ofV. parahaemolyticusorganisms in samples exceeded 1,000 MPN/g only when the seawater temperatures exceeded 19°C, so this environmental parameter could be used as a trigger warning of potential hazard. There is some evidence that the totalV. parahaemolyticusnumbers increased compared with those reported from a previous 1981 to 1984 study, but the analytical methods differed significantly.

scholarly journals Repeated Anaerobic Microbial Redox Cycling of Iron

2011 ◽  
Vol 77 (17) ◽  
pp. 6036-6042 ◽  
Author(s):  
Aaron J. Coby ◽  
Flynn Picardal ◽  
Evgenya Shelobolina ◽  
Huifang Xu ◽  
Eric E. Roden
Keyword(s):  
Microbial Communities ◽  
Microscopic Analysis ◽  
Redox Cycling ◽  
Most Probable Number ◽  
Rrna Gene ◽  
Electron Microscopic ◽  
16S Rrna Gene Analysis ◽  
Probable Number ◽  
Content Type ◽  
Floodplain Sediments

ABSTRACTSome nitrate- and Fe(III)-reducing microorganisms are capable of oxidizing Fe(II) with nitrate as the electron acceptor. This enzymatic pathway may facilitate the development of anaerobic microbial communities that take advantage of the energy available during Fe-N redox oscillations. We examined this phenomenon in synthetic Fe(III) oxide (nanocrystalline goethite) suspensions inoculated with microflora from freshwater river floodplain sediments. Nitrate and acetate were added at alternate intervals in order to induce repeated cycles of microbial Fe(III) reduction and nitrate-dependent Fe(II) oxidation. Addition of nitrate to reduced, acetate-depleted suspensions resulted in rapid Fe(II) oxidation and accumulation of ammonium. High-resolution transmission electron microscopic analysis of material from Fe redox cycling reactors showed amorphous coatings on the goethite nanocrystals that were not observed in reactors operated under strictly nitrate- or Fe(III)-reducing conditions. Microbial communities associated with N and Fe redox metabolism were assessed using a combination of most-probable-number enumerations and 16S rRNA gene analysis. The nitrate-reducing and Fe(III)-reducing cultures were dominated by denitrifyingBetaproteobacteria(e.g.,Dechloromonas) and Fe(III)-reducingDeltaproteobacteria(Geobacter), respectively; these same taxa were dominant in the Fe cycling cultures. The combined chemical and microbiological data suggest that bothGeobacterand variousBetaproteobacteriaparticipated in nitrate-dependent Fe(II) oxidation in the cycling cultures. Microbially driven Fe-N redox cycling may have important consequences for both the fate of N and the abundance and reactivity of Fe(III) oxides in sediments.

Characterization and Identification of Numerically Abundant Culturable Bacteria from the Anoxic Bulk Soil of Rice Paddy Microcosms

1999 ◽  
Vol 65 (11) ◽  
pp. 5042-5049 ◽  
Author(s):  
Kuk-Jeong Chin ◽  
Dittmar Hahn ◽  
Ulf Hengstmann ◽  
Werner Liesack ◽  
Peter H. Janssen
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Dry Weight ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Most Probable Number ◽  
Rrna Gene ◽  
16S Rrna Gene Sequences ◽  
Probable Number ◽  
Clostridial Cluster

ABSTRACT Most-probable-number (liquid serial dilution culture) counts were obtained for polysaccharolytic and saccharolytic fermenting bacteria in the anoxic bulk soil of flooded microcosms containing rice plants. The highest viable counts (up to 2.5 × 108 cells per g [dry weight] of soil) were obtained by using xylan, pectin, or a mixture of seven mono- and disaccharides as the growth substrate. The total cell count for the soil, as determined by using 4′,6-diamidino-2-phenylindole staining, was 4.8 × 108cells per g (dry weight) of soil. The nine strains isolated from the terminal positive tubes in counting experiments which yielded culturable populations that were equivalent to about 5% or more of the total microscopic count population belonged to the divisionVerrucomicrobia, theCytophaga-Flavobacterium-Bacteroides division, clostridial cluster XIVa, clostridial cluster IX, Bacillus spp., and the class Actinobacteria. Isolates originating from the terminal positive tubes of liquid dilution series can be expected to be representatives of species whose populations in the soil are large. None of the isolates had 16S rRNA gene sequences identical to 16S rRNA gene sequences of previously described species for which data are available. Eight of the nine strains isolated fermented sugars to acetate and propionate (and some also fermented sugars to succinate). The closest relatives of these strains (except for the two strains of actinobacteria) were as-yet-uncultivated bacteria detected in the same soil sample by cloning PCR-amplified 16S rRNA genes (U. Hengstmann, K.-J. Chin, P. H. Janssen, and W. Liesack, Appl. Environ. Microbiol. 65:5050–5058, 1999). Twelve other isolates, which originated from most-probable-number counting series indicating that the culturable populations were smaller, were less closely related to cloned 16S rRNA genes.

scholarly journals A Pilot Study of Chicago Waterways as Reservoirs of Multidrug-Resistant Enterobacteriaceae (MDR-Ent) in a High-Risk Region for Community-Acquired MDR-Ent Infection in Children

2020 ◽  
Vol 64 (4) ◽  
Author(s):  
Latania K. Logan ◽  
Liqing Zhang ◽  
Stefan J. Green ◽  
Samuel Dorevitch ◽  
Gustavo A. Arango-Argoty ◽  
...  
Keyword(s):  
Microbial Community ◽  
Pilot Study ◽  
Microbial Community Composition ◽  
Antibiotic Resistance Genes ◽  
The United States ◽  
Multidrug Resistant ◽  
Most Probable Number ◽  
Metagenomic Sequencing ◽  
Probable Number ◽  
Content Type

ABSTRACT Community-acquired multidrug resistant Enterobacteriaceae (MDR-Ent) infections continue to increase in the United States. In prior studies, we identified neighboring regions in Chicago, Illinois, where children have 5 to 6 times greater odds of MDR-Ent infections. To prevent community spread of MDR-Ent, we need to identify the MDR-Ent reservoirs. A pilot study of 4 Chicago waterways for MDR-Ent and associated antibiotic resistance genes (ARGs) was conducted. Three waterways (A1 to A3) are labeled safe for “incidental contact recreation” (e.g., kayaking), and A4 is a nonrecreational waterway that carries nondisinfected water. Surface water samples were collected and processed for standard bacterial culture and shotgun metagenomic sequencing. Generally, A3 and A4 (neighboring waterways which are not hydraulically connected) were strikingly similar in bacterial taxa, ARG profiles, and abundances of corresponding clades and genera within the Enterobacteriaceae. Additionally, total ARG abundances recovered from the full microbial community were strongly correlated between A3 and A4 (R2 = 0.97). Escherichia coli numbers (per 100 ml water) were highest in A4 (783 most probable number [MPN]) and A3 (200 MPN) relative to A2 (84 MPN) and A1 (32 MPN). We found concerning ARGs in Enterobacteriaceae such as MCR-1 (colistin), Qnr and OqxA/B (quinolones), CTX-M, OXA and ACT/MIR (beta-lactams), and AAC (aminoglycosides). We found significant correlations in microbial community composition between nearby waterways that are not hydraulically connected, suggesting cross-seeding and the potential for mobility of ARGs. Enterobacteriaceae and ARG profiles support the hypothesized concerns that recreational waterways are a potential source of community-acquired MDR-Ent.

scholarly journals Spatial and Temporal Patterns in the Microbial Diversity of a Meromictic Soda Lake in Washington State

2008 ◽  
Vol 74 (15) ◽  
pp. 4877-4888 ◽  
Author(s):  
Pedro A. Dimitriu ◽  
Holly C. Pinkart ◽  
Brent M. Peyton ◽  
Melanie R. Mormile
Keyword(s):  
16S Rrna ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Bacterial Community Composition ◽  
Soda Lake ◽  
Community Diversity ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Most Probable Number ◽  
Rrna Gene ◽  
Probable Number

ABSTRACT The microbial community diversity and composition of meromictic Soap Lake were studied using culture-dependent and culture-independent approaches. The water column and sediments were sampled monthly for a year. Denaturing gradient gel electrophoresis of bacterial and archaeal 16S rRNA genes showed an increase in diversity with depth for both groups. Late-summer samples harbored the highest prokaryotic diversity, and the bacteria exhibited less seasonal variability than the archaea. Most-probable-number assays targeting anaerobic microbial guilds were performed to compare summer and fall samples. In both seasons, the anoxic samples appeared to be dominated by lactate-oxidizing sulfate-reducing prokaryotes. High numbers of lactate- and acetate-oxidizing iron-reducing bacteria, as well as fermentative microorganisms, were also found, whereas the numbers of methanogens were low or methanogens were undetectable. The bacterial community composition of summer and fall samples was also assessed by constructing 16S rRNA gene clone libraries. A total of 508 sequences represented an estimated >1,100 unique operational taxonomic units, most of which were from the monimolimnion, and the summer samples were more diverse than the fall samples (Chao1 = 530 and Chao1 = 295, respectively). For both seasons, the mixolimnion sequences were dominated by Gammaproteobacteria, and the chemocline and monimolimnion libraries were dominated by members of the low-G+C-content group, followed by the Cytophaga-Flexibacter-Bacteroides (CFB) group; the mixolimnion sediments contained sequences related to uncultured members of the Chloroflexi and the CFB group. Community overlap and phylogenetic analyses, however, not only demonstrated that there was a high degree of spatial turnover but also suggested that there was a degree of temporal variability due to differences in the members and structures of the communities.

scholarly journals Anaerobic Mineralization of Quaternary Carbon Atoms: Isolation of Denitrifying Bacteria on Dimethylmalonate

1999 ◽  
Vol 65 (8) ◽  
pp. 3319-3324 ◽  
Author(s):  
Olaf Kniemeyer ◽  
Christina Probian ◽  
Ramon Rosselló-Mora ◽  
Jens Harder
Keyword(s):  
Sewage Sludge ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Most Probable Number ◽  
Rrna Gene ◽  
Quaternary Carbon ◽  
Probable Number ◽  
Gene Similarity

ABSTRACT The microbial capacity to degrade simple organic compounds with quaternary carbon atoms was demonstrated by enrichment and isolation of five denitrifying strains on dimethylmalonate as the sole electron donor and carbon source. Quantitative growth experiments showed a complete mineralization of dimethylmalonate. According to phylogenetic analysis of the complete 16S rRNA genes, two strains isolated from activated sewage sludge were related to the genusParacoccus within the α-Proteobacteria (98.0 and 98.2% 16S rRNA gene similarity to Paracoccus denitrificans T), and three strains isolated from freshwater ditches were affiliated with the β-Proteobacteria (97.4 and 98.3% 16S rRNA gene similarity to Herbaspirillum seropedicae T andAcidovorax facilis T, respectively). Most-probable-number determinations for denitrifying populations in sewage sludge yielded 4.6 × 104dimethylmalonate-utilizing cells ml−1, representing up to 0.4% of the total culturable nitrate-reducing population.

scholarly journals Detection of Verrucomicrobia in a Pasture Soil by PCR-Mediated Amplification of 16S rRNA Genes

1999 ◽  
Vol 65 (9) ◽  
pp. 4280-4284 ◽  
Author(s):  
Katrina A. O’Farrell ◽  
Peter H. Janssen
Keyword(s):  
16S Rrna ◽  
Restriction Analysis ◽  
Comparative Sequence Analysis ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Most Probable Number ◽  
Rrna Gene ◽  
Soil Dna ◽  
Probable Number ◽  
Pasture Soil

ABSTRACT Oligonucleotide primers were designed and used to amplify, by PCR, partial 16S rRNA genes of members of the bacterial divisionVerrucomicrobia in DNA extracted from a pasture soil. By applying most-probable-number theory to the assay, verrucomicrobia appeared to contribute some 0.2% of the soil DNA. Amplified ribosomal DNA restriction analysis of 53 cloned PCR-amplified partial 16S rRNA gene fragments and comparative sequence analysis of 21 nonchimeric partial 16S rRNA genes showed that these primers amplified only 16S rRNA genes of members of the Verrucomicrobia in DNA extracted from the soil.

scholarly journals Effects of Intertidal Harvest Practices on Levels of Vibrio parahaemolyticus and Vibrio vulnificus Bacteria in Oysters

2016 ◽  
Vol 82 (15) ◽  
pp. 4517-4522 ◽  
Author(s):  
J. L. Jones ◽  
T. P. Kinsey ◽  
L. W. Johnson ◽  
R. Porso ◽  
B. Friedman ◽  
...  
Keyword(s):  
New Jersey ◽  
Vibrio Parahaemolyticus ◽  
Vibrio Vulnificus ◽  
Ambient Air ◽  
The United States ◽  
Washington State ◽  
Most Probable Number ◽  
Probable Number ◽  
Content Type ◽  
Handling Practices

ABSTRACTVibrio parahaemolyticusandVibrio vulnificuscan grow rapidly in shellfish subjected to ambient air conditions, such as during intertidal exposure. In this study, levels of total and pathogenic (tdh+and/ortrh+)V. parahaemolyticusand totalV. vulnificuswere determined in oysters collected from two study locations where intertidal harvest practices are common. Samples were collected directly off intertidal flats, after exposure (ambient air [Washington State] or refrigerated [New Jersey]), and after reimmersion by natural tidal cycles. Samples were processed using a most-probable-number (MPN) real-time PCR method for total and pathogenicV. parahaemolyticusorV. vulnificus. In Washington State, the mean levels ofV. parahaemolyticusincreased 1.38 log MPN/g following intertidal exposure and dropped 1.41 log MPN/g after reimmersion for 1 day, but the levels were dependent upon the container type utilized. PathogenicV. parahaemolyticuslevels followed a similar trend. However,V. vulnificuslevels increased 0.10 log MPN/g during intertidal exposure in Washington but decreased by >1 log MPN/g after reimmersion. In New Jersey, initial levels of all vibrios studied were not significantly altered during the refrigerated sorting and containerizing process. However, there was an increase in levels after the first day of reimmersion by 0.79, 0.72, 0.92, and 0.71 log MPN/g for total,tdh+andtrh+V. parahaemolyticus, andV. vulnificus, respectively. The levels of all targets decreased to those similar to background after a second day of reimmersion. These data indicate that the intertidal harvest and handling practices for oysters that were studied in Washington and New Jersey do not increase the risk of illness fromV. parahaemolyticusorV. vulnificus.IMPORTANCEVibrio parahaemolyticusandVibrio vulnificusare the leading causes of seafood-associated infectious morbidity and mortality in the United States.Vibriospp. can grow rapidly in shellfish subjected to ambient air conditions, such as during periods of intertidal exposure. When oysters are submersed with the incoming tide, the vibrios can be purged. However, data on the rates of increase and purging during intertidal harvest are scarce, which limits the accuracy of risk assessments. The objective of this study was to help fill these data gaps by determining the levels of total and pathogenic (tdh+and/ortrh+)V. parahaemolyticusandV. vulnificusin oysters from two locations where intertidal harvest practices are common, using the current industry practices. The data generated provide insight into the responses ofVibriospp. to relevant practices of the industry and public health, which can be incorporated into risk management decisions.

scholarly journals Abundance of Vibrio cholerae, V. vulnificus, and V. parahaemolyticus in Oysters (Crassostrea virginica) and Clams (Mercenaria mercenaria) from Long Island Sound

2014 ◽  
Vol 80 (24) ◽  
pp. 7667-7672 ◽  
Author(s):  
Jessica L. Jones ◽  
Catharina H. M. Lüdeke ◽  
John C. Bowers ◽  
Kristin DeRosia-Banick ◽  
David H. Carey ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Vibrio Vulnificus ◽  
Long Island ◽  
Long Island Sound ◽  
The United States ◽  
Mitigation Strategies ◽  
Most Probable Number ◽  
Probable Number ◽  
Content Type ◽  
Island Sound

ABSTRACTVibriosis is a leading cause of seafood-associated morbidity and mortality in the United States. Typically associated with consumption of raw or undercooked oysters, vibriosis associated with clam consumption is increasingly being reported. However, little is known about the prevalence ofVibriospp. in clams. The objective of this study was to compare the levels ofVibrio cholerae,Vibrio vulnificus, andVibrio parahaemolyticusin oysters and clams harvested concurrently from Long Island Sound (LIS). Most probable number (MPN)–real-time PCR methods were used for enumeration of totalV. cholerae,V. vulnificus,V. parahaemolyticus, and pathogenic (tdh+and/ortrh+)V. parahaemolyticus.V. choleraewas detected in 8.8% and 3.3% of oyster (n= 68) and clam (n= 30) samples, with levels up to 1.48 and 0.48 log MPN/g in oysters and clams, respectively.V. vulnificuswas detected in 97% and 90% of oyster and clam samples, with median levels of 0.97 and −0.08 log MPN/g, respectively.V. parahaemolyticuswas detected in all samples, with median levels of 1.88 and 1.07 log MPN/g for oysters and clams, respectively. The differences betweenV. vulnificusand total and pathogenicV. parahaemolyticuslevels in the two shellfish species were statistically significant (P< 0.001). These data indicate thatV. vulnificusand total and pathogenicV. parahaemolyticusare more prevalent and are present at higher levels in oysters than in hard clams. Additionally, the data suggest differences in vibrio populations between shellfish harvested from different growing area waters within LIS. These results can be used to evaluate and refine illness mitigation strategies employed by risk managers and shellfish control authorities.

scholarly journals Development of a Real-Time PCR Assay for Detection and Quantification of Rhizobium leguminosarum Bacteria and Discrimination between Different Biovars in Zinc-Contaminated Soil

2011 ◽  
Vol 77 (13) ◽  
pp. 4626-4633 ◽  
Author(s):  
Catriona A. Macdonald ◽  
Ian M. Clark ◽  
Penny R. Hirsch ◽  
Fang-Jie Zhao ◽  
Steve P. McGrath
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Rhizobium Leguminosarum ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Most Probable Number ◽  
Rrna Gene ◽  
Probable Number ◽  
Content Type ◽  
Gene Copies

ABSTRACTPrimers were designed to target 16S rRNA andnodDgenes ofRhizobium leguminosarumfrom DNA extracted from two different soil types contaminated with Zn applied in sewage sludge. Numbers of rhizobia estimated using 16S rRNA gene copy number showed higher abundance than those estimated by bothnodDand the most-probable-number (MPN) enumeration method using a plant trap host. Both 16S rRNA gene copies and the MPN rhizobia declined with increased levels of Zn contamination, as did the abundance of the functional genenodD, providing compelling evidence of a toxic effect of Zn onR. leguminosarumpopulations in the soil. Regression analysis suggested the total Zn concentration in soil as a better predictor of rhizobial numbers than both NH4NO3-extractable and soil solution Zn.R. leguminosarumbv. viciaenodDgene copies were generally less sensitive to Zn thanR. leguminosarumbv. trifoliinodD.The latter were generally below detection limits at Zn levels of >250 mg kg−1. Although there were differences in the actual numbers estimated by each approach, the response to Zn was broadly similar across all methods. These differences were likely to result from the fact that the molecular approaches assess the potential for nodulation while the MPN approach assesses actual nodulation. The results demonstrate that the use of targeted gene probes for assessing environmental perturbations of indigenous soil rhizobial populations may be more sensitive than the conventional plant bioassay and MPN methods.

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