Gene Transfer in Wastewater Treatment Plants
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From: "Edo McGowan" <edo_mcgowan@hotmail.com>
To: "Helane Shields" <hshields@worldpath.net>
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Subject: RE: to EDo - EPA says no gene transfer in POTWs - needs rebuttal MDRB
There were three little monkeys that saw no, heard no, and said no gene transfer.
"We are great. We are free. We are wonderful. We are the most
wonderful people in all Washington, said the monkey people! We all say so, and so it must be true," they shouted.
"Now as you are a new listener and can carry our words back to the Jungle People so that they may notice us in
future, we will tell you all about our most excellent selves."
Mowgli made no objection, and the monkeys gathered by hundreds and hundreds on the on the steps of EPA to
listen to their own speakers singing the praises of the Bandar-log, and whenever a speaker stopped for want of
breath they would all shout together: "This is true; we all say so."
Enterococcus faecalis Gene Transfer under Natural Conditions in Municipal Sewage Water Treatment Plants
Herbert Marcinek, Reinhard Wirth,* Albrecht Muscholl-Silberhorn, and Matthias Gauer
Microbiology-NWFIII, University of Regensburg, D-93053 Regensburg, Germany
Received 2 October 1997/Accepted 12 November 1997
The ability of Enterococcus faecalis to transfer various genetic elements under natural conditions was tested in two
municipal sewage water treatment plants. Experiments in activated sludge basins of the plants were performed in a
microcosm which allowed us to work under sterile conditions; experiments in anoxic sludge digestors were
performed in dialysis bags. We used the following naturally occurring genetic elements: pAD1 and pIP1017 (two so-
called sex pheromone plasmids with restricted host ranges, which are transferred at high rates under laboratory
conditions); pIP501 (a resistance plasmid possessing a broad host range for gram-positive bacteria, which is
transferred at low rates under laboratory conditions); and Tn916 (a conjugative transposon which is transferred
under laboratory conditions at low rates to gram-positive bacteria and at very low rates to gram-negative bacteria).
The transfer rate between different strains of E. faecalis under natural conditions was, compared to that under
laboratory conditions, at least 105-fold lower for the sex pheromone plasmids, at least 100-fold lower for pIP501,
and at least 10-fold lower for Tn916. In no case was transfer from E. faecalis to another bacterial species detected.
By determining the dependence of transfer rates for pIP1017 on bacterial concentration and extrapolating to actual
concentrations in the sewage water treatment plant, we calculated that the maximum number of transfer events for
the sex pheromone plasmids between different strains of E. faecalis in the municipal sewage water treatment plant
of the city of Regensburg ranged from 10/5th to 10/8th events per 4 h, indicating that gene transfer should take
place under natural conditions.
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* Corresponding author. Mailing address: Mikrobiologie-NWFIII, University of Regensburg, Universitätsstrasse 31, D-
93053 Regensburg, Germany. Phone: (49) 941 943 1825. Fax: (49) 941 943 1824. E-mail: Reinhard.
Wirth@biologie.uni-regensburg.de.
This paper is dedicated to Herbert Marcinek, who died in a tragic accident.
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Behavior of drug resistant fecal coliforms and R plasmids in a wastewater treatment plant][Article in Japanese]
Nakamura S, Shirota H.
Department of Food and Nutrition, Ube College.
Fecal coliforms were isolated from the inlet, the primary sedimentation tank, the activated sludge digestion tank, the
final settling tank, the outlet and the return activated sludge drain at the municipal wastewater plant in Ube City, and
examined for drug resistance and presence of R plasmids. Drug concentrations employed to distinguish resistant
isolates from sensitive isolates were 25 micrograms/ml for tetracycline, kanamycin, chloramphenicol and
streptomycin, 50 micrograms/ml for ampicillin, nalidixic acid and rifampicin, and 200 micrograms/ml for sulfisoxazole,
respectively. Of a total of 900 isolates, 45.7% were drug resistant and 51.1% of them carried R plasmids. The
further along that wastewater had progressed through the treatment process the greater the tendency was for
appearance of the multiresistant isolates. These isolates also were shown to simultaneously carry transferable R
plasmids. Observed resistant patterns of R plasmids were mainly multiple and encoded to resistance to tetracycline,
chloramphenicol, streptomycin and sulfisoxazole. It became clear that multiplication of R plasmids took place in the
activated sludge digestion tank. This study show that drug resistance transfer mediated by these R plasmids may
occur in actual wastewater treatment plants.
PMID: 2131972 [PubMed - indexed for MEDLINE]
Related Links[The occurrence of antibiotic resistant coliforms in the waste water of a water treatment plant]
[Zentralbl Mikrobiol. 1988]Occurrence of multiple antibiotic resistance and R plasmids in Enterobacteriaceae
isolated from children in the Sudan. [Epidemiol Infect. 1988]Effect of UV light disinfection on antibiotic-resistant
coliforms in wastewater effluents. [Appl Environ Microbiol. 1982]Transferable resistance to gentamicin and other
antibiotics in Enterobacteriaceae isolates from municipal wastewater. [J Hyg Epidemiol Microbiol Immunol. 1984]
[Isolation and characterization of gentamicin resistance plasmids of coliform bacteria from the waste water of a
water treatment plant] [Zentralbl Mikrobiol. 1988]See all Related Articles...
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Biodiversity and horizontal gene transfer in culturable bacteria isolated from activated sludge enriched in
nonylphenol ethoxylates.
Barberio C, Pagliai L, Cavalieri D, Fani R.
Dipartimento di Biologia Animale e Genetica, Florence, Italy. cbarberio@dbag.unifi.it
One hundred and twenty bacterial isolates, from activated sludge of a treatment plant collecting wastes enriched in
ethoxylated nonylphenols, were studied. Sixty isolates were selected on rich medium and 60 on mineral medium
containing two nonylphenol ethoxylates as the sole carbon source. Analysis of biodiversity at the species level was
performed by comparing the AluI restriction patterns of the 16S ribosomal DNA amplified by PCR from 120 isolates.
The rDNA restriction analysis enabled us to cluster the isolates into 15 groups, five of which represented nearly
77% of the community. Phylogenetic analysis of five strains belonging to these main groups made it possible to
assign four of them to the genera Acinetobacter, Aeromonas and Shewanella and one to the Proteus group. The
analysis of plasmid content showed a high variability and suggested that horizontal gene transfer had taken place
at the intraspecific, interspecific and intergeneric levels.
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Combined Molecular and Conventional Analyses of Nitrifying Bacterium Diversity in Activated Sludge: Nitrosococcus
mobilis and Nitrospira-Like Bacteria as Dominant Populations
Stefan Juretschko,1 Gabriele Timmermann,2 Markus Schmid,1 Karl-Heinz Schleifer,1 Andreas Pommerening-Röser,
3 Hans-Peter Koops,3 and Michael Wagner1,*
Lehrstuhl für Mikrobiologie, Technische Universität München, D-80290 Munich,1 Max-Planck-Institut für Limnologie,
D-24302 Plön,2 and Institut für Allgemeine Botanik, Abteilung Mikrobiologie, Universität Hamburg, D-22609
Hamburg,3 Germany
Received 5 February 1998/Accepted 27 May 1998
The ammonia-oxidizing and nitrite-oxidizing bacterial populations occurring in the nitrifying activated sludge of an
industrial wastewater treatment plant receiving sewage with high ammonia concentrations were studied by use of a
polyphasic approach. In situ hybridization with a set of hierarchical 16S rRNA-targeted probes for ammonia-oxidizing
bacteria revealed the dominance of Nitrosococcus mobilis-like bacteria. The phylogenetic affiliation suggested by
fluorescent in situ hybridization (FISH) was confirmed by isolation of N. mobilis as the numerically dominant
ammonia oxidizer and subsequent comparative 16S rRNA gene (rDNA) sequence and DNA-DNA hybridization
analyses. For molecular fine-scale analysis of the ammonia-oxidizing population, a partial stretch of the gene
encoding the active-site polypeptide of ammonia monooxygenase (amoA) was amplified from total DNA extracted
from ammonia oxidizer isolates and from activated sludge. However, comparative sequence analysis of 13 amoA
clone sequences from activated sludge demonstrated that these sequences were highly similar to each other and
to the corresponding amoA gene fragments of Nitrosomonas europaea Nm50 and the N. mobilis isolate. The
unexpected high sequence similarity between the amoA gene fragments of the N. mobilis isolate and N. europaea
indicates a possible lateral gene transfer event. Although a Nitrobacter strain was isolated, members of the nitrite-
oxidizing genus Nitrobacter were not detectable in the activated sludge by in situ hybridization. Therefore, we used
the rRNA approach to investigate the abundance of other well-known nitrite-oxidizing bacterial genera. Three
different methods were used for DNA extraction from the activated sludge. For each DNA preparation, almost full-
length genes encoding small-subunit rRNA were separately amplified and used to generate three 16S rDNA
libraries. By comparative sequence analysis, 2 of 60 randomly selected clones could be assigned to the nitrite-
oxidizing bacteria of the genus Nitrospira. Based on these
clone sequences, a specific 16S rRNA-targeted probe was developed. FISH of the activated sludge with this probe
demonstrated that Nitrospira-like bacteria were present in significant numbers (9% of the total bacterial counts) and
frequently occurred in coaggregated microcolonies with N. mobilis.
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Detection of antibiotic-resistant bacteria and their resistance genes in wastewater, surface water, and drinking
water biofilms
Thomas Schwartza,aForschungszentrum Karlsruhe GmbH, Department of Environmental Microbiology, ITC-WGT, P.
O. Box 3640, D-76021 Karlsruhe, Germany**Corresponding author. Tel.: +49 (7247) 826 802; Fax: +49 (7247) 826
858. E-mail address: thomas.schwartz@itc-wgt.fzk.de, Wolfgang KohnenbbUniversity of Mainz, Department of
Hygiene and Environmental Medicine, Hochhaus am Augustusplatz, D-55131 Mainz, Germany, Bernd
JansenbbUniversity of Mainz, Department of Hygiene and Environmental Medicine, Hochhaus am Augustusplatz, D-
55131 Mainz, Germany, Ursula ObstaaForschungszentrum Karlsruhe GmbH, Department of Environmental
Microbiology, ITC-WGT, P.O. Box 3640, D-76021 Karlsruhe, GermanyaForschungszentrum Karlsruhe GmbH,
Department of Environmental Microbiology, ITC-WGT, P.O. Box 3640, D-76021 Karlsruhe, GermanybUniversity of
Mainz, Department of Hygiene and Environmental Medicine, Hochhaus am Augustusplatz, D-55131 Mainz, Germany
*Corresponding author. Tel.: +49 (7247) 826 802; Fax: +49 (7247) 826 858. E-mail address: thomas.schwartz@itc-
wgt.fzk.de
Abstract
In view of the increasing interest in the possible role played by hospital and municipal wastewater systems in the
selection of antibiotic-resistant bacteria, biofilms were investigated using enterococci, staphylococci,
Enterobacteriaceae, and heterotrophic bacteria as indicator organisms. In addition to wastewater, biofilms were also
investigated in drinking water from river bank filtrate to estimate the occurrence of resistant bacteria and their
resistance genes, thus indicating possible transfer from wastewater and surface water to the drinking water
distribution network. Vancomycin-resistant enterococci were characterized by antibiograms, and the vanA
resistance gene was detected by molecular biology methods, including PCR. The vanA gene was found not only in
wastewater biofilms but also in drinking water biofilms in the absence of enterococci, indicating possible gene
transfer to autochthonous drinking water bacteria. The mecA gene encoding methicillin resistance in staphylococci
was detected in hospital wastewater biofilms but not in any other compartment. Enterobacterial ampC resistance
genes encoding β-lactamase activities were amplified by PCR from wastewater, surface water and drinking water
biofilms.
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Vancomycin-resistant enterococci from nosocomial, community, and animal sources in the United States
TM Coque, JF Tomayko, SC Ricke, PC Okhyusen and BE Murray
Center for the Study of Emerging and Reemerging Pathogens, University of Texas Medical School, Houston 77030,
USA.
The presence of vancomycin-resistant enterococci (VRE) was looked for in fecal samples from 104 healthy
volunteers (3 with hospital exposure), 100 selected hospitalized patients, and various environmental sources (44
commercial chickens, 5 farm-raised chickens, 3 turkeys, and 2 chicken farm lagoon slurries). Five probiotic
preparations were also studied. No VRE with vanA or vanB genes were isolated from the healthy volunteers without
hospital exposure, environmental sources, or probiotic preparations. VRE with vanB were found in the stools of 16%
of the high-risk hospitalized patients and in one volunteer with hospital contact. All VRE examined could be
classified into one of two clones by pulsed-field gel electrophoresis. VRE from 11 of the colonized patients were
quantified and ranged from 10(3) to 10(6) CFU/g of stool. This study, in contrast to findings in Europe, failed to find
evidence of VanA- or VanB-type VRE in the community or environmental sources in Houston, Texas, and suggests
that these settings are not a likely source of VRE in hospitals in this geographic area.
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Detection of clinically relevant antibiotic-resistance genes in municipal wastewater using real-time PCR (TaqMan)
Holger Volkmann, Thomas Schwartz, , Petra Bischoff, Silke Kirchen and Ursula Obst
Forschungszentrum Karlsruhe GmbH, Institute for Technical Chemistry, Watertechnology and Geotechnology
Division, Department of Environmental Microbiology, P.O. Box 3640, D-76021, Karlsruhe, Germany
Received 2 October 2003; revised 29 October 2003; accepted 29 October 2003. ; Available online 2 December
2003.
Abstract
Real-time PCR assays were developed for the quantifiable detection of the antibiotic-resistance genes vanA of
enterococci, ampC of Enterobacteriaceae, and mecA of staphylococci in different municipal wastewater samples.
Primer and probe designs for these resistance genes were constructed and optimised for application in
standardised TaqMan PCR assays. Using reference strains, the linear measurement ranges of the assays were
defined and covered concentration ranges of five to seven exponential values. Wastewater isolates of vancomycin-
resistant enterococci (VRE) and β-lactam-resistant Enterobacteriaceae were cultivated from municipal wastewaters
in order to verify the specificity and sensitivity of the primer–probe systems. Additionally, clinical strains of
staphylococci resistant to methicillin (MRSA) confirmed the applicability of the mecA-specific detection system. Total
DNAs were extracted from five different wastewater treatment plants and used for direct TaqMan PCR detection of
the resistance genes without prior cultivation. In municipal wastewater, the resistance gene vanA was detected in
21% of the samples, and ampC in 78%. The gene mecA was not found in municipal wastewater, but in two clinical
wastewater samples.
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Items 1 - 7 of 7 One page.
1:
Schluter A, Szczepanowski R, Puhler A, Top EM.
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Genomics of IncP-1 antibiotic resistance plasmids isolated from wastewater treatment plants provides evidence for
a widely accessible drug resistance gene pool.
FEMS Microbiol Rev. 2007 Jul;31(4):449-77. Epub 2007 Jun 6. Review.
PMID: 17553065 [PubMed - indexed for MEDLINE]
2:
Szczepanowski R, Krahn I, Bohn N, Puhler A, Schluter A.
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Novel macrolide resistance module carried by the IncP-1beta resistance plasmid pRSB111, isolated from a
wastewater treatment plant.
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PMID: 17101677 [PubMed - indexed for MEDLINE]
3:
Gallert C, Fund K, Winter J.
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Antibiotic resistance of bacteria in raw and biologically treated sewage and in groundwater below leaking sewers.
Appl Microbiol Biotechnol. 2005 Nov;69(1):106-12. Epub 2005 Oct 20.
PMID: 16001254 [PubMed - indexed for MEDLINE]
4:
Szczepanowski R, Braun S, Riedel V, Schneiker S, Krahn I, Puhler A, Schluter A.
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The 120 592 bp IncF plasmid pRSB107 isolated from a sewage-treatment plant encodes nine different antibiotic-
resistance determinants, two iron-acquisition systems and other putative virulence-associated functions.
Microbiology. 2005 Apr;151(Pt 4):1095-111.
PMID: 15817778 [PubMed - indexed for MEDLINE]
5:
Szczepanowski R, Krahn I, Linke B, Goesmann A, Puhler A, Schluter A.
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Antibiotic multiresistance plasmid pRSB101 isolated from a wastewater treatment plant is related to plasmids
residing in phytopathogenic bacteria and carries eight different resistance determinants including a multidrug
transport system.
Microbiology. 2004 Nov;150(Pt 11):3613-30.
PMID: 15528650 [PubMed - indexed for MEDLINE]
6:
Mancini P, Fertels S, Nave D, Gealt MA.
Related Articles, Links
Mobilization of plasmid pHSV106 from Escherichia coli HB101 in a laboratory-scale waste treatment facility.
Appl Environ Microbiol. 1987 Apr;53(4):665-71.
PMID: 3555335 [PubMed - indexed for MEDLINE]
7:
Kralikova K, Krcmery V, Krcmery V Jr.
Related Articles, Links
Transferable resistance to gentamicin and other antibiotics in Enterobacteriaceae isolates from municipal
wastewater.
J Hyg Epidemiol Microbiol Immunol. 1984;28(2):161-6.
PMID: 6470479 [PubMed - indexed for MEDLINE]