DETERMINATION OF ANTIBIOTIC RESISTANCE IN THE BACTERIAL COMMUNITY OF BOILING ...

107 Pages·2012·1.69 MB·English

DETERMINATION OF ANTIBIOTIC RESISTANCE IN THE BACTERIAL
COMMUNITY OF BOILING SPRINGS LAKE IN LASSEN VOLCANIC NATIONAL
PARK
By

Carrie Lynn Hudson

A Thesis Presented to
The Faculty of Humboldt State University
In Partial Fulfillment of the Requirements for the Degree
Master of Science in Biology

Committee Membership
Dr. Patricia Siering, Professor, Committee Chair
Dr. Jacob Varkey, Professor
Dr. Mark Wilson, Associate Professor
Dr. Jianmin Zhong, Associate Professor
Dr. Michael Mesler, Professor, Graduate Coordinator

December 2012
ABSTRACT
Determination of Antibiotic Resistance in the Bacterial Community of Boiling Springs
Lake in Lassen Volcanic National Park

Carrie Hudson

Boiling Springs Lake is a pristine, oligotrophic, acidic hot spring in Lassen
Volcanic National Park. In 2008, 25 isolates from BSL were tested for resistance to 14
different antimicrobial compounds or a sub-set of the 14 antimicrobial agents. All isolates
were resistant to at least two of the antimicrobials and some were resistant to six or more.
This thesis project expanded upon the preliminary research to achieve the following
goals: (1) determine the antimicrobial resistance profiles of 50 BSL isolates; (2)
determine the minimum inhibitory concentrations for select multi-drug resistant isolates;
(3) analyze microarray data obtained from Geochip 3.0 to assess the levels of antibiotic-
and metal-resistance genes present and their phylogenetic origins; and (4) use PCR to
screen selected BSL isolates for the presence of multi-drug resistant efflux genes. All 50
isolates that were screened were resistant to at least one antimicrobial compound with
74% being resistant to four or more. One hundred and twenty-four antibiotic-resistance
genes and 221 metal-resistance genes were detected in BSL. The emrB/qacA efflux gene
amplicon sequence was obtained from 20 of the BSL isolates and was found to be 99-
ii
100% identical. This identity spanned species, genus, and phylum levels of taxonomic
hierarchy. These results support the findings of other studies that multi-drug resistance is
common in pristine habitats, and that pristine habitats are reservoirs of antimicrobial
resistance genes. These results also suggest that the emrB/qacA gene has been
horizontally transferred between the BSL isolates.

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ACKNOWLEDGEMENTS
A special thank you to my advisor Dr. Patricia Siering for all of her guidance and support
throughout this project, my committee members Dr. Mark Wilson, Dr. Jacob Varkey, and
Dr. Jianmin Zhong, the Humboldt Extreme Acidiphile Team (HEAT) lab, Clayton Carey
for all of his help during the first year of this project, and the National Science
Foundation and the Howard Hughes Medical Institute for funding this project.

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TABLE OF CONTENTS

ABSTRACT ........................................................................................................................ ii
ACKNOWLEDGEMENTS ............................................................................................... iv
CHAPTER 1: SITE DESCRIPTION AND PROJECT GOALS ........................................ 1
CHAPTER 2: LITERATURE REVIEW ............................................................................ 4
INTRODUCTION………………………………………………………………...4
POSSIBLE FUNCTIONS OF ANTIBIOTICS IN THE ENVIRONMENT…...…5
ORIGINS OF ANTIBIOTIC RESISTANCE DETERMINENTS AND THEIR
SELECTION IN NATURAL ECOSYSTEMS……………………………………...……8

CHAPTER 3: ANTIBIOTIC RESISTANCE SCREENINGS ......................................... 16
INTRODUCTION……………………………………………………………….16
MATERIALS AND METHODS………………………………………………...19
Media and antibiotic preparations………………………………………..19
Isolation and identification of isolates………………………………...…22
Antibiotic resistance screenings…………………………………………24
Minimum inhibitory concentrations (MIC)……………………………..25
Antibiotic stability………………………………………………………26
RESULTS…………………………………………………………………….…27
Identity of isolates……………………………………………………….27
Antibiotic resistance screenings………………………………………...30
Minimum inhibitory concentrations (MIC)…………………………..…36
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DISCUSSION……………………………………………………………………38
Identity of isolates………………………………………………………..38
Antibiotic resistance screenings………………………………………….39
Minimum inhibitory concentrations……………………………………..46
Antibiotic stability……………………………………………………….47
CHAPTER 4: GENETIC ANALYSIS…………………………………………………..52
INTRODUCTION……………………………………………………………….52
MATERIALS AND METHODS………………………………………………..54
Geochip 3.0 microarray analysis………………………………………...54
Multi-drug resistance efflux genes………………………………………55
RESULTS………………………………………………………………………..52
Geochip 3.0 microarray analysis……………………………………...…56
Antibiotic resistance genes………………………………………56
Metal resistance genes………………..………………………….62
Multi-drug resistance efflux genes………………………...…….70
DISCUSSION……………………………………………………………………73
Geochip 3.0 microarray analysis…………………………………………73
Multi-drug resistance efflux genes……………………………………….76
CHAPTER 5: SUMMARY............................................................................................... 78
REFERENCES…………………………………………………………………………..79

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LIST OF TABLES

Table Page
1 Summary of antimicrobials utilized for this project 18
2 Antibiotic disc preparation 21
3 Identity of isolates 29
4 Screening results for pH 2.9, low nutrient isolates 31
5 Screening results for pH 2.9, medium nutrient isolates 32
6 Screening results for pH 2.9, high nutrient isolates 33
7 Screening results for pH 5.0 and 7.0 isolates 34
8 Minimum inhibitory concentration results 37
9 Summary of antibiotic resistance genes (ABR) detected in BSL and 61
averaged normalized signal intensities (NSI) for detected ABR genes
on hybridization of 2004 and 2009 samples

10 Summary metal resistance genes (MR) detected in BSL and 69
averaged normalized signal intensities (NSI) for detected MR genes
on hybridization of 2004 and 2009 samples

11 Distance matrix for emrB/qacA genes and 16S rRNA genes 72
12 Most probable number results 98

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LIST OF FIGURES
Figure Page
1 Percent of BSL strains resistant to each antimicrobial 35
2 Predicted structure of BceB signal transduction system and ABC 44
transporter translocator

3 Structure of ciproflaxin with arrow indicating N 49
1

4 Antibiotic resistance genes detected in BSL 59

5 Phylogenetic origins of detected antibiotic resistance genes 60

6 Metal resistance genes detected in BSL 67

7 Phylogenetic origins of detected metal resistance genes 68

APPENDICES

Page

1 Antibiotics as a single carbon source 93
2 Most probable number (MPN) quantification of antibiotic resistant 96
bacteria from BSL

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1

CHAPTER 1: SITE DESCRIPTION AND PROJECT GOALS

Lassen Volcanic National Park is located in northeastern California at the
southern end of the Cascade mountain range and harbors many hydrothermal features
within its boundaries. One of these features is Boiling Springs Lake (BSL), an
approximately 18,000 m2 hot spring where the average temperature of the surface water
during the summer months (June-August) is approximately 52°C +/- 3°C, and the average
pH value is 2 +/- 0.4 (116,118). The southern end of the lake contains continuously active
bubbling springs and mud pots and is the hottest and most heterogeneous region of the
lake with an average summer temperature of 65-95°C (118). Research since the mid-
1970’s has determined that BSL is a stable feature in terms of pH, temperature and area
of coverage, although this is based almost exclusively on summer sampling. Recent
remote sensing during the winter months (October-May, 2008-2010) has revealed a
seasonal depression of temperature by 5-10° C along with transient and further
temperature depressions during snow-fall followed by slow recovery (118). These results
suggest that although BSL is stable over the long-term, seasonal variation does occur.
Unlike many other low pH environments being studied in Yellowstone National
Park and other regions, the water chemistry at BSL is one that is low in metals, with all
metals except SiO and Al being present at very low concentrations (97,98). BSL is an
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oligotrophic habitat with measurements of dissolved organic carbon (DOC) ranging from
2

0.3-1.2 ppm in 2000-2010 (97). NH + was found to be 1.43 ppm or ~44 μM; the
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concentration of NO - was measured at less than 0.02 ppm, and PO 3- was undetectable at
3 4
less than 0.05 ppm (97,98). Past and current research has revealed BSL contains an
exclusively microbial community, with novel species of Archaea and Bacteria being the
most abundant phylotypes in clone libraries derived from sediment samples (116,118).
The planktonic community consists primarily of well-characterized bacteria in the
Actinobacteria and Aquificales phyla (118). Both the planktonic and sediment
communities are low in species richness and evenness as assessed by the clone library
analysis: e.g. only 22 phylotypes were detected in 3 libraries prepared from BSL water,
and 2 of the phylotypes represented 69% of the total clones (n=235) (116, 118).
In addition to the assessment of diversity and the chemistry of BSL, our
laboratory began investigating issues associated with organismal and gene biogeography,
physiology and evolution in extreme habitats including intra-population diversity and
ecotype differentiation. In the summer of 2008, 25 isolates from BSL were tested for
resistance to 14 different antibiotics and bacteriocins or a sub-set of the 14 antimicrobial
agents (75): ampicillin, penicillin-G, vancomycin, bacitracin, nisin, kanamycin,
streptomycin, gentamycin, tetracycline, trimethoprim, rifampim, sulfisoxazole,
ciproflaxin, and chloramphenicol. All of the isolates were found to be resistant to at least
2 of the antimicrobial compounds with some resistant to 6 or more, and surprisingly,
most of the strains were resistant to the completely synthetic antibiotic ciproflaxin.
Since BSL is a pristine habitat devoid of agricultural and municipal run-off, we
attributed the abundance of resistant phenotypes to be a secondary effect of living in the