scholarly journals Comparison of Rapid Quantitative PCR-Based and Conventional Culture-Based Methods for Enumeration of Enterococcus spp. and Escherichia coli in Recreational Waters

2010 ◽  
Vol 76 (22) ◽  
pp. 7437-7443 ◽  
Author(s):  
Rachel T. Noble ◽  
A. Denene Blackwood ◽  
John F. Griffith ◽  
Charles D. McGee ◽  
Stephen B. Weisberg
Keyword(s):  
Escherichia Coli ◽  
Water Quality ◽  
Quantitative Pcr ◽  
23S Rrna ◽  
Management Decision ◽  
Beach Management ◽  
Recreational Waters ◽  
E Coli ◽  
Conventional Culture ◽  
Pure Cultures

ABSTRACT Recreational water quality is currently monitored using culture-based methods that require 18 to 96 h for results. Quantitative PCR (QPCR) methods that can be completed in less than 2 h have been developed, but they could yield different results than the conventional methods. We present two studies in which samples were processed simultaneously for Enterococcus spp. and Escherichia coli using two culture-based methods (EPA method 1600 and Enterolert/Colilert-18) and QPCR. The proprietary QPCR assays targeted the 23S rRNA (Enterococcus spp.) and uidA (E. coli) genes and were conducted using lyophilized beads containing all reagents. In the first study, the QPCR method developers processed 54 blind samples that were inoculated with sewage or pure cultures or were ambient beach samples. The second study involved 163 samples processed by water quality personnel. The correlation between results of QPCR and EPA 1600 during the first study (r 2) was 0.69 for Enterococcus spp., which was less than that observed between the culture-based methods (r 2, 0.87). During the second study, the correlations were similar. No false positives occurred in either study when QPCR-based assays were used with blank samples. Levels of reproducibility measured through coefficients of variation were similar for results by Enterococcus QPCR and culture-based methods during both studies but were higher for E. coli QPCR results in the first study. Regarding the concentration at which beach management decisions are issued in the State of California, the agreement between results of Enterococcus QPCR and EPA method 1600 was 88%, compared to 94% agreement between EPA method 1600 and Enterolert. The beach management decision agreement between E. coli QPCR and Colilert-18 was 94%. The samples showing disagreement suggested an underestimation bias for QPCR.

Limitations of Immunomagnetic Separation for Detection of the Top Seven Serogroups of Shiga Toxin–Producing Escherichia coli

2017 ◽  
Vol 80 (4) ◽  
pp. 598-603 ◽  
Author(s):  
J. Hallewell ◽  
T. Alexander ◽  
T. Reuter ◽  
K. Stanford
Keyword(s):  
Escherichia Coli ◽  
Quantitative Pcr ◽  
Foodborne Pathogens ◽  
Shiga Toxin ◽  
Immunomagnetic Separation ◽  
Detection Methods ◽  
Conventional Pcr ◽  
E Coli ◽  
Pure Cultures

ABSTRACT Shiga toxin–producing Escherichia coli (STEC) strains are foodborne pathogens that negatively impact human health and compromise food safety. Serogroup O157 is the most frequently isolated and studied STEC serogroup, but six others (O26, O45, O103, O111, O121, and O145) have also been identified as significant sources of human disease and collectively have been referred to as the “top six” pathogenic serogroups. Because detection methods for non-O157 serogroups are not yet refined, the objective of this study was to compare the effectiveness of immunomagnetic separation (IMS) for recovery of serogroup O157 isolates with that for each of the top six E. coli serogroups in pure and mixed cultures of STEC at 103 to 107 CFU/mL. After serogroup-specific IMS, DNA was extracted from cultured isolates to analyze the specificity of each IMS assay using conventional and quantitative PCR. In pure cultures, DNA copy number obtained after IMS was lower for O111 and O157 (P < 0.01) than for other serogroups. Based on quantitative PCR (qPCR) analyses, specificity was reduced for all IMS assays when STEC isolates were mixed at 7 log CFU/mL, although the O157 IMS assays recovered only O157 over a wider range of concentrations than did assays for non-O157 serogroups. At the lowest dilution tested, conventional PCR was specific for all serogroups except O121 and O145. For these two serogroups, no dilution tested recovered only O121 or O145 when evaluated with conventional PCR. Refinements to IMS assays, development of selective media, and determination of optimal enrichment times to reduce background microflora or competition among serogroups would be especially beneficial for recovery of O111, O121, and O145 serogroups to improve STEC detection and isolation.

An Improved Membrane Filtration Method for Enumeration of Faecal Coliforms and E. Coli by a Fluorogenic β-Glucuronidase Assay

1993 ◽  
Vol 27 (3-4) ◽  
pp. 267-270 ◽  
Author(s):  
M. T. Augoustinos ◽  
N. A. Grabow ◽  
B. Genthe ◽  
R. Kfir
Keyword(s):  
Escherichia Coli ◽  
Water Samples ◽  
Membrane Filtration ◽  
Faecal Coliforms ◽  
Environmental Waters ◽  
Filtration Method ◽  
E Coli ◽  
Wide Range ◽  
Pure Cultures ◽  
Glucuronidase Assay

A fluorogenic β-glucuronidase assay comprising membrane filtration followed by selective enumeration on m-FC agar at 44.5°C and further confirmation using tlie 4-metliylumbelliferyl-β-D-glucuronide (MUG) containing medium was evaluated for the detection of Escherichia coli in water. A total of 200 typical blue and non-typical blue colonies were isolated from sea and fresh water samples using initial selective enumeration on m-FC agar. Pure cultures of the selected colonies were further tested using the MUG assay and identified using the API 20E method. Of the colonies tested which were shown to be positive using the MUG assay 99.4% were Escherichia coli. The results of this study indicate the combination of the m-FC method followed by the MUG assay to be highly efficient for the selection and confirmation of E. coli from a wide range of environmental waters.

scholarly journals Binding Site of the Bridged Macrolides in the Escherichia coli Ribosome

2005 ◽  
Vol 49 (1) ◽  
pp. 281-288 ◽  
Author(s):  
Liqun Xiong ◽  
Yakov Korkhin ◽  
Alexander S. Mankin
Keyword(s):  
Escherichia Coli ◽  
Tight Binding ◽  
Dimethyl Sulfate ◽  
23S Rrna ◽  
Side Chain ◽  
Macrolide Antibiotics ◽  
Side Chains ◽  
Alkyl Aryl ◽  
E Coli ◽  
Exit Tunnel

ABSTRACT Ketolides represent the latest group of macrolide antibiotics. Tight binding of ketolides to the ribosome appears to correlate with the presence of an extended alkyl-aryl side chain. Recently developed 6,11-bridged bicyclic ketolides extend the spectrum of platforms used to generate new potent macrolides with extended alkyl-aryl side chains. The purpose of the present study was to characterize the site of binding and the action of bridged macrolides in the ribosomes of Escherichia coli. All the bridged macrolides investigated efficiently protected A2058 and A2059 in domain V of 23S rRNA from modification by dimethyl sulfate and U2609 from modification by carbodiimide. In addition, bridged macrolides that carry extended alkyl-aryl side chains protruding from the 6,11 bridge protected A752 in helix 35 of domain II of 23S rRNA from modification by dimethyl sulfate. Bridged macrolides efficiently displaced erythromycin from the ribosome in a competition binding assay. The A2058G mutation in 23S rRNA conferred resistance to the bridged macrolides. The U2609C mutation, which renders E. coli resistant to the previously studied ketolides telithromycin and cethromycin, barely affected cell susceptibility to the bridged macrolides used in this study. The results of the biochemical and genetic studies indicate that in the E. coli ribosome, bridged macrolides bind in the nascent peptide exit tunnel at the site previously described for other macrolide antibiotics. The presence of the side chain promotes the formation of specific interactions with the helix 35 of 23S rRNA.

scholarly journals In vitro reconstitution of the GTPase-associated centre of the archaebacterial ribosome: the functional features observed in a hybrid form with Escherichia coli 50S subunits

2006 ◽  
Vol 396 (3) ◽  
pp. 565-571 ◽  
Author(s):  
Takaomi Nomura ◽  
Kohji Nakano ◽  
Yasushi Maki ◽  
Takao Naganuma ◽  
Takashi Nakashima ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ribosomal Proteins ◽  
Synthetic Activity ◽  
23S Rrna ◽  
Elongation Factors ◽  
Hybrid Form ◽  
Pyrococcus Horikoshii ◽  
E Coli ◽  
Functional Features

We cloned the genes encoding the ribosomal proteins Ph (Pyrococcus horikoshii)-P0, Ph-L12 and Ph-L11, which constitute the GTPase-associated centre of the archaebacterium Pyrococcus horikoshii. These proteins are homologues of the eukaryotic P0, P1/P2 and eL12 proteins, and correspond to Escherichia coli L10, L7/L12 and L11 proteins respectively. The proteins and the truncation mutants of Ph-P0 were overexpressed in E. coli cells and used for in vitro assembly on to the conserved domain around position 1070 of 23S rRNA (E. coli numbering). Ph-L12 tightly associated as a homodimer and bound to the C-terminal half of Ph-P0. The Ph-P0·Ph-L12 complex and Ph-L11 bound to the 1070 rRNA fragments from the three biological kingdoms in the same manner as the equivalent proteins of eukaryotic and eubacterial ribosomes. The Ph-P0·Ph-L12 complex and Ph-L11 could replace L10·L7/L12 and L11 respectively, on the E. coli 50S subunit in vitro. The resultant hybrid ribosome was accessible for eukaryotic, as well as archaebacterial elongation factors, but not for prokaryotic elongation factors. The GTPase and polyphenylalanine-synthetic activity that is dependent on eukaryotic elongation factors was comparable with that of the hybrid ribosomes carrying the eukaryotic ribosomal proteins. The results suggest that the archaebacterial proteins, including the Ph-L12 homodimer, are functionally accessible to eukaryotic translation factors.

In Vitro Inhibition of the Growth of Salmonella typhimurium and Escherichia coli 0157:H7 by Bacteria Isolated from the Cecal Contents of Adult Chickens

1991 ◽  
Vol 54 (7) ◽  
pp. 496-501 ◽  
Author(s):  
ARTHUR HINTON ◽  
GEORGE E. SPATES ◽  
DONALD E. CORRIER ◽  
MICHAEL E. HUME ◽  
JOHN R. DELOACH ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Lactic Acid ◽  
Salmonella Typhimurium ◽  
Mixed Cultures ◽  
Enterococcus Durans ◽  
E Coli ◽  
Pure Cultures ◽  
In Vitro Inhibition ◽  
Lactic Acids

A Veillonella species and Enterococcus durans were isolated from the cecal contents of adult broilers. Mixed cultures of Veillonella and E. durans inhibited the growth of Salmonella typhimurium and Escherichia coli 0157:H7 on media containing 2.5% lactose (w/v). The growth of S. typhimurium or E. coli 0157:H7 was not inhibited by mixed cultures containing Veillonella and E. durans on media containing only 0.25% lactose or by pure cultures of Veillonella or E. durans on media containing either 0.25% or 2.5% lactose. The mixed cultures of Veillonella and E. durans produced significantly (P<0.05) more acetic, propionic, and lactic acids in media containing 2.5% lactose than in media containing 0.25% lactose. The inhibition of the enteropathogens was related to the production of lactic acid from lactose by the E. durans and the production of acetic and propionic acids from lactic acid by the Veillonella.

Multiplex Quantitative PCR Assays for the Detection and Quantification of the Six Major Non-O157 Escherichia coli Serogroups in Cattle Feces†

2016 ◽  
Vol 79 (1) ◽  
pp. 66-74 ◽  
Author(s):  
P. B. SHRIDHAR ◽  
L. W. NOLL ◽  
X. SHI ◽  
B. AN ◽  
N. CERNICCHIARO ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Quantitative Pcr ◽  
Foodborne Pathogens ◽  
Virulence Genes ◽  
Target Genes ◽  
Culture Methods ◽  
Fecal Samples ◽  
E Coli ◽  
Cattle Feces ◽  
Before And After

ABSTRACT Shiga toxin–producing Escherichia coli (STEC) serogroups O26, O45, O103, O111, O121, and O145, called non-O157 STEC, are important foodborne pathogens. Cattle, a major reservoir, harbor the organisms in the hindgut and shed them in the feces. Although limited data exist on fecal shedding, concentrations of non-O157 STEC in feces have not been reported. The objectives of our study were (i) to develop and validate two multiplex quantitative PCR (mqPCR) assays, targeting O-antigen genes of O26, O103, and O111 (mqPCR-1) and O45, O121, and O145 (mqPCR-2); (ii) to utilize the two assays, together with a previously developed four-plex qPCR assay (mqPCR-3) targeting the O157 antigen and three virulence genes (stx1, stx2, and eae), to quantify seven serogroups and three virulence genes in cattle feces; and (iii) to compare the three mqPCR assays to a 10-plex conventional PCR (cPCR) targeting seven serogroups and three virulence genes and culture methods to detect seven E. coli serogroups in cattle feces. The two mqPCR assays (1 and 2) were shown to be specific to the target genes, and the detection limits were 4 and 2 log CFU/g of pure culture–spiked fecal samples, before and after enrichment, respectively. A total of 576 fecal samples collected from a feedlot were enriched in E. coli broth and were subjected to quantification (before enrichment) and detection (after enrichment). Of the 576 fecal samples subjected, before enrichment, to three mqPCR assays for quantification, 175 (30.4%) were quantifiable (≥4 log CFU/g) for at least one of the seven serogroups, with O157 being the most common serogroup. The three mqPCR assays detected higher proportions of postenriched fecal samples (P < 0.01) as positive for one or more serogroups compared with cPCR and culture methods. This is the first study to assess the applicability of qPCR assays to detect and quantify six non-O157 serogroups in cattle feces and to generate data on fecal concentration of the six serogroups.

scholarly journals Colistin-Resistant mcr-1-Positive Escherichia coli on Public Beaches, an Infectious Threat Emerging in Recreational Waters

2017 ◽  
Vol 61 (7) ◽  
Author(s):  
Miriam R. Fernandes ◽  
Fábio P. Sellera ◽  
Fernanda Esposito ◽  
Caetano P. Sabino ◽  
Louise Cerdeira ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recreational Waters ◽  
Content Type ◽  
E Coli ◽  
Rapid Spread ◽  
Environmental Reservoir ◽  
Public Beaches ◽  
Urban Beaches

ABSTRACT The emergence and rapid spread of colistin-resistant Escherichia coli carrying the mcr-1 gene have generated an urgent need to strengthen surveillance. We performed a meticulous investigation of strains of this sort, which resulted in the identification of international clones of E. coli carrying IncX4-plasmid-mediated mcr-1 and bla CTX-M genes in recreational waters of public urban beaches in cities with high tourist turnover, highlighting a new environmental reservoir.

Evidence that tropical soil environment can support the growth of Escherichia coli

1998 ◽  
Vol 38 (12) ◽  
pp. 171-174 ◽  
Author(s):  
M. N Byappanahalli ◽  
R. S. Fujioka
Keyword(s):  
Escherichia Coli ◽  
Water Quality ◽  
Tropical Soil ◽  
Soil Conditions ◽  
Natural Soil ◽  
Minimal Amount ◽  
Soil Environment ◽  
Faecal Coliforms ◽  
E Coli ◽  
Faecal Bacteria

Concentrations of faecal coliforms and Escherichia coli in environmental waters have historically been used to establish recreational water quality standards. When these bacteria are used as indices of water quality, it is assumed that there are no significant environmental sources of these bacteria which are unrelated to direct faecal contamination. However, we have previously reported that in tropical island environments such as in Hawaii, these faecal indicators are consistently found at high concentrations in all streams and the source of these faecal bacteria is the soil. To become so well established in soil we hypothesized that these faecal bacteria must have the ability to multiply in the natural soil environment at ambient temperature (23–25°C). Three lines of evidence support this hypothesis: (1) E. coli was shown to grow on 10% soil extract agar, (2) populations of faecal coliforms and E. coli from sewage were shown to immediately increase by about three logs when simple nutrients (glucose and salts) were added to natural soil and (3) faecal coliforms and E. coli increased by two logs within 24 h when a minimal amount of sewage was added to cobalt-irradiated soil. These results indicate that tropical soil environments provide sufficient means to support the growth of faecal coliforms and E. coli. However, under natural soil conditions, indigenous soil microorganisms are much more efficient in obtaining nutrients and we hypothesize that faecal bacteria grow sporadically in response to available nutrients.

scholarly journals Non-point source pollution: Determination of replication versus persistence of Escherichia coli in surface water and sediments with correlation of levels to readily measurable environmental parameters

2004 ◽  
Vol 2 (2) ◽  
pp. 103-114 ◽  
Author(s):  
Julie Kinzelman ◽  
Sandra L. McLellan ◽  
Annette D. Daniels ◽  
Susan Cashin ◽  
Ajaib Singh ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Water Quality ◽  
Point Source ◽  
Wave Height ◽  
Surface Waters ◽  
Lake Michigan ◽  
Recreational Water ◽  
Point Source Pollution ◽  
E Coli ◽  
Non Point Source Pollution

Racine, Wisconsin, located on Lake Michigan, experiences frequent recreational water quality advisories in the absence of any identifiable point source of pollution. This research examines the environmental distribution of Escherichia coli in conjunction with the assessment of additional parameters (rainfall, turbidity, wave height, wind direction, wind speed and algal presence) in order to determine the most probable factors that influence E. coli levels in surface waters. Densities of E. coli were highest in core samples taken from foreshore sands, often exceeding an order of magnitude greater than those collected from submerged sands and water. Simple regression and multivariate analyses conducted on supplementary environmental data indicate that the previous day's E. coli concentration in conjunction with wave height is significantly predictive for present-time E. coli concentration. Genetic fingerprinting using repetitive element anchored PCR and cellular fatty acid analysis were employed to assess the presence of clonal isolates which indicate replication from a common parent cell. There were relatively few occurrences of clonal patterns in isolates collected from water, foreshore and submerged sands, suggesting that accumulation of E. coli, rather than environmental replication, was occurring in this system. Non-point source pollution, namely transport of accumulated E. coli from foreshore sands to surface waters via wave action, was found to be a major contributor to poor recreational water quality at the Lake Michigan beaches involved in this study.

scholarly journals A comparison of the unfolding and dissociation of the large ribosome subunits from Rhodopseudomonas·spheroides N.C.I.B. 8253 and Escherichia coli M.R.E. 600

1973 ◽  
Vol 133 (4) ◽  
pp. 739-747 ◽  
Author(s):  
A. Robinson ◽  
J. Sykes
Keyword(s):  
Escherichia Coli ◽  
Protein Interactions ◽  
Ribosomal Proteins ◽  
Ribosomal Subunit ◽  
23S Rrna ◽  
Core Particle ◽  
Protein Loss ◽  
E Coli ◽  
Rrna Species ◽  
Rhodopseudomonas Spheroides

1. The behaviour of the large ribosomal subunit from Rhodopseudomonas spheroides (45S) has been compared with the 50S ribosome from Escherichia coli M.R.E. 600 (and E. coli M.R.E. 162) during unfolding by removal of Mg2+ and detachment of ribosomal proteins by high univalent cation concentrations. The extent to which these processes are reversible with these ribosomes has also been examined. 2. The R. spheroides 45S ribosome unfolds relatively slowly but then gives rise directly to two ribonucleoprotein particles (16.6S and 13.7S); the former contains the intact primary structure of the 16.25S rRNA species and the latter the 15.00S rRNA species of the original ribosome. No detectable protein loss occurs during unfolding. The E. coli ribosome unfolds via a series of discrete intermediates to a single, unfolded ribonucleoprotein unit (19.1S) containing the 23S rRNA and all the protein of the original ribosome. 3. The two unfolded R. spheroides ribonucleoproteins did not recombine when the original conditions were restored but each simply assumed a more compact configuration. Similar treatments reversed the unfolding of the E. coli 50S ribosomes; replacement of Mg2+ caused the refolding of the initial products of unfolding and in the presence of Ni2+ the completely unfolded species (19.1S) again sedimented at the same rate as the original ribosomes (44S). 4. Ribosomal proteins (25%) were dissociated from R. spheroides 45S ribosomes by dialysis against a solution with a Na+/Mg2+ ratio of 250:1. During this process two core particles were formed (21.2S and 14.2S) and the primary structures of the two original rRNA species were conserved. This dissociation was not reversed. With E. coli 50S approximately 15% of the original ribosomal protein was dissociated, a single 37.6S core particle was formed, the 23S rRNA remained intact and the ribosomal proteins would reassociate with the core particle to give a 50S ribosome. 5. The ribonuclease activities in R. spheroides 45S and E. coli M.R.E. 600 and E. coli M.R.E. 162 50S ribosomes are compared. 6. The observations concerning unfolding and dissociation are consistent with previous reports showing the unusual rRNA complement of the mature R. spheroides 45S ribosome and show the dependence of these events upon the rRNA and the importance of protein–protein interactions in the structure of the R. spheroides ribosome.

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