Detection and characterization of antibiotic resistance plasmids in Cheney biosolids

91 Pages·2014·1.15 MB·English

by  Gaayathiri Paramasivam

Eastern Washington University
EWU Digital Commons
EWU Masters Thesis Collection Student Research and Creative Works
2013
Detection and characterization of antibiotic
resistance plasmids in Cheney biosolids
Gaayathiri Paramasivam
Eastern Washington University
Follow this and additional works at:http://dc.ewu.edu/theses
Recommended Citation
Paramasivam, Gaayathiri, "Detection and characterization of antibiotic resistance plasmids in Cheney biosolids" (2013).EWU Masters
Thesis Collection. 241.
http://dc.ewu.edu/theses/241
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DETECTION AND CHARACTERIZATION OF ANTIBIOTIC RESISTANCE PLASMIDS IN
CHENEY BIOSOLIDS

A Thesis
Presented To
Eastern Washington University
Cheney, Washington

In Partial Fulfillment of the Requirements
For the Degree
Biology (Master of Science)

By
Gaayathiri Paramasivam
Winter 2013
THESIS OF GAAYATHIRI PARAMASIVAM APPROVED BY

DATE
DR. PRAKASH BHUTA, GRADUATE STUDY COMMITTEE

DATE
DR. ANDREA CASTILLO, GRADUATE STUDY COMMITTEE

DATE
DR. LAVONA REEVES, GRADUATE STUDY COMMITTEE

ii
MASTER’S THESIS

In presenting this thesis in partial fulfillment of the requirements for a master’s degree
at Eastern Washington University, I agree that the JFK Library shall make copies freely
available for inspection. I further agree that copying of this project in whole or in part is
allowable only for scholarly purposes. It is understood, however, that any copying or
publication of this thesis for commercial purposes, or for financial gain, shall not be
allowed without my written permission.

Signature

Date

iii
ABSTRACT

Biosolids are organic matter produced at the end of sewage treatment process. It has
been shown that during sewage treatment, resistant bacteria are selected because of
the presence of antibiotics and their byproducts. These resistant bacteria are more likely
to transfer resistance genes to other bacteria. In the current study, we examined
Cheney biosolids for the presence of drug resistant bacteria and the role of these
bacteria in transfer of resistance genes to others. We screened 100 bacteria for drug
resistance and found that 68% of the isolates were resistant to two or more drugs
tested. Plasmids were separated from the resistant bacteria and 13.2% of them showed
the presence of plasmids. These resistance plasmids were introduced into E. coli MM294
to screen for the presence of antibiotic resistance genes. Plasmids were isolated from
the transformants and 77.7% of the transformants showed the presence of plasmids
with similar size and mobility on an agarose gel. The plasmids extracted from the
transformants were digested with a restriction enzyme, EcoRI to verify the presence of
multiple plasmids in the samples. The resistant bacteria (13.2%) that showed the
presence of plasmid were tested whether they were conjugative or mobilizable type.
Unfortunately, none of the isolates were conjugative or mobilizable plasmid. In short,
Cheney biosolids do contain drug resistant bacteria, so there is a chance that these
resistant bacteria will transfer their resistance genes to other bacteria present in
biosolids.

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ACKNOWLEDGEMENTS
I would like to thank Mike Lambert at the Cheney wastewater plant for his help in
collecting biosolid samples. I would like to thank Dr. Steve Moseley at University of
Washington for coliphage samples and bacterial cultures. I would also like to thank Dr.
Nicholas Burgis at EWU Chemistry Department for bacterial cultures. I would like to
thank Dr. Prakash Bhuta, Dr. Andrea Castillo and Dr. LaVona Reeves for their help and
guidance. Finally, I would like to thank my friends and family for their support and much
more.

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TABLE OF CONTENTS
ABSTRACT…………………………………………………………………………………………………………iv

ACKNOWLEDGEMENTS…………………………………………………………………………………....v

1.0 INTRODUCTION……………………………………………………………………………………...…1

1.1 Significance of Antibiotics………………………………………………………...1
1.2 Issues with Drug Resistant Bacteria…………………………………………..4

1.3 Development of Antibiotic Resistance…………………………………...…6
1.4 Plasmids and Antibiotic Resistance……………………………………….….10
1.5 Accumulation of Antibiotics in the Environment…………………..….12

1.6 Production of Biosolids…………………………………………………………....13
1.7 Regulations for Biosolids………………………….…………………………..….16
1.8 Potential Hazards of Biosolids………………………………………………....17

1.9 Significance of Bacteria in Enterobacteriaceae Family………………18
1.10 Plasmid Extraction and Transformation……………………………….19

1.11 Phage Sensitivity Assay………………………………………………………..21
1.12 Mechanism of Conjugation…………………………………………….……24
1.13 Objectives…………………………………………………………………….……..28

2.0 MATERIALS and METHODS…………………………………………….…………………..…..….29

2.1 Bacterial Growth Media…………………………………………………………....29

2.2 Preparation of Antibiotic Stock Solutions………………………………..…29
2.3 Preparation of Buffer and Solutions…………………………………………..30
2.4 Collection of Biosolids and Inoculation onto Selective Media…….31

2.5 Bacterial Strains Used………………………………………………………………..31
2.6 Long Term Storage of Bacterial Isolates……………………………………..33

2.7 Plasmid DNA Extraction……………………………………………………………..33
2.8 Agarose Gel Electrophoresis………………………………………………………34
2.9 Identification of Bacteria……………………………………………………………35

2.10 Preparation of Competent Cells…………………………………………….35
2.11 Transformation……………………………………………………………………..36
2.12 Restriction Digestion……………………………………………………………..37

2.13 Assay of Phage Sensitivity……………………………………………………..37
2.14 Tripartite Mating…………………………………………………………………..38

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TABLE OF CONTENTS
3.0 RESULTS and DISCUSSION………………………………………………………………………….…40
3.1 Collection of Biosolids and Isolation of Resistant Bacteria…………….....40
3.2 Isolation of Plasmids from Resistant Bacteria……………………………………47
3.3 Identification of Resistant Bacteria………………………………………………..….50
3.4 Transformation……………………………………………………………………………..….53
3.5 Restriction Digestion……………………………………………………………..………….59
3.6 Phage Sensitivity Testing…………………………………………………………..………64
3.7 Tripartite Mating………………………………………………………………………..…….66

4.0 CONCLUSIONS……………………………………………………………….………………………….….69
5.0 REFERENCES………………………………………………………………………………………….………71
VITA……………………………………………………………………………………………………….…..………79
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List of Tables and Figures:

Table 1 – Antibiotics: classes, targets and resistance mechanisms
Table 2 – The U.S EPA – Pathogen concentration limits
Table 3 – Clinical syndromes caused by members of the Enterobacteriaceae family
Table 4a – Bacterial strains used
Table 4b – Phages used
Table 5a – Resistance profile of the isolates X1-X36
Table 5b – Resistance profile of the isolates X37-X68
Table 6 – Identity of resistant bacteria and their antibiotic resistance profile
Table 7 – Transformation efficiency of parental plasmids

Figure 1 – Mechanism of horizontal gene transfer (HGT) in bacteria
Figure 2 – Mechanism of antibiotic resistance
Figure 3 – Production of biosolids
Figure 4 – Mechanism of transformation in bacteria
Figure 5 – Infection of RNA phage and cell lysis
Figure 6 – Mechanism of conjugation in bacteria
Figure 7 – Percent of isolates resistant to different antibiotics
Figure 8 – Percent of isolates resistant to number of drugs used
Figure 9 – Antibiotic sensitivity testing of the isolates
Figure 10a and 10b – Agarose gel showing the presence of plasmids
Figure 11 – API-20E strips used for the identification of bacteria
Figure 12a to 12f – A comparison between plasmids isolated from transformants with parental plasmids
Figure 13a to 13g – Restriction digestion of the plasmids extracted from transformants
Figure 14 – Phage sensitivity testing
Figure 15a – Resistance profile of the donor, helper and recipient strains
Figure 15b – Tripartite mating process

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1. Introduction:
1.1 Significance of Antibiotics:
Antibiotics are chemicals that restrict the growth of bacteria. They are divided into
two major categories based on their effect on a target cell: bacteriostatic (growth
inhibitor) and bactericidal (killing agent) (Davies, 2010; Walsh, 2003). Antibiotics affect
bacterial growth by many different mechanisms including: 1. disruption of bacterial cell
wall (e.g., Penicillin), 2. inhibition of protein synthesis (e.g., Aminoglycosides and
Tetracyclines), and 3. inhibition of DNA replication and transcription (e.g., Quinolones
and Rifampin). The most commonly used antibiotics and those used in this study are
listed in Table 1. Antibiotics are used everywhere - in agriculture, aquaculture, livestock,
veterinary and human treatment. From the 1950s, antibiotics such as oxytetracycline
and streptomycin have been used as pesticides when growing fruit, vegetable, and
ornamental plants. Streptomycin is primarily used for control of fire blight in apples,
pears and plants of the Rosaceae family. In the USA, approximately 10,000 tons of
antibiotics are used on plants (McManus et al., 2002). Animal farms and aquaculture are
also using antibiotics to promote growth and prevent diseases (Cabello, 2006). For
example, in swine breeding farms, antibiotics are detected in dust from the air
ventilators (Hamscher et al., 2003). It has been shown that antibiotic misuse or overuse
has resulted in the development of drug resistant bacteria in the environment (Rhodes
et al., 2000). Such resistant bacteria pass
1