ANTIBIOTIC-RESISTANT AND PATHOGENIC BACTERIA

IN KITCHEN SPONGES

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By

JAIME M. HERNANDEZ

B.S. Syracuse University, 2011

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A THESIS

Presented to the College of Arts and Sciences and Quinnipiac University

in partial fulfillment of the requirements for the degree of

Master of Molecular and Cell Biology

Spring 2014
 UMI Number: 1527035

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 ABSTRACT

ANTIBIOTIC-RESISTANT AND PATHOGENIC BACTERIA

IN KITCHEN SPONGES

Jaime Hernandez

Master of Molecular and Cell Biology

College of Arts and Sciences

Quinnipiac University

Spring 2014

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Kitchen sponges are favorable environments for bacterial contamination and

propagation. In this investigation, 500 bacterial colonies were isolated from 20

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sponges acquired from college students living in houses. Antibiotic-resistance

patterns revealed that every sponge contained at least one antibiotic-resistant

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isolate. Furthermore, 134 isolates (26.8%) were tetracycline-resistant and 54

(10.8%) were multidrug resistant. According to both biochemical and 16S rRNA
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results, 55 of 90 sequenced isolates (61%) were potentially pathogenic, with 16

of those 55 (29.1%) resistant to antibiotics. Furthermore, bacterial load was

compared to previously published data regarding sponges from dormitories.

House sponges exhibited a lower initial bacterial load relative to dormitory

sponges. For both populations, there was a decrease in bacterial load after

microwaving the sponges for 60 seconds. These results will help to alert the

public in understanding the benefits of disinfecting their sponges in order to

prevent a pathogenic outbreak and the proliferation of antibiotic-resistant

bacteria.
 ANTIBIOTIC-RESISTANT AND PATHOGENIC BACTERIA

IN KITCHEN SPONGES

This thesis is approved as creditable and independent investigation by a

candidate for the degree of Master of Molecular and Cell Biology, and is
acceptable as meeting the thesis requirements for this degree, but without
implying that the conclusions reached by the candidate are necessarily the
conclusions of the major department.

Thesis Advisor____________________________________________________

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Lisa A. Cuchara, Ph. D.
Professor, Biomedical Sciences IE
Quinnipiac University
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Quinnipiac University Thesis Committee Member_________________________
Christian H. Eggers, Ph. D.
Assistant Professor, Biomedical Sciences
Quinnipiac University
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Quinnipiac University Thesis Committee Member_________________________

Charlotte I. Hammond, Ph. D.
Professor, Biology
Quinnipiac University

Director, Molecular and Cell Biology Graduate Program____________________

Michelle M. Geremia, Ph.D.
Chair of Biological Sciences
Quinnipiac University
 ACKNOWLEDGEMENTS

I want to express my appreciation to the following individuals for their help and

assistance in the completion of this projectB

• Professor Linda Post

• Dr. Sarah Berke

• Dean Allan Smits

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• Meaghan Donnelly

• Patrick McInnis

• Andrew Kirlis
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• Lindsay Musgrove
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• Alexa Rubino

• Felix Cusano
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• Lauren Aber

• Mahdi Alali

• Frank Rizzo

• Rick Gadzinski

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 TABLE OF CONTENTS

Page

INTRODUCTION BBBBBBBBBBBBBBBBBBBBBBBBBBB.1

MATERIALS AND METHODS..............................................................................9

RESULTSBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB18

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DISCUSSIONBBBBBBBBBBBBBBBBBBBBBBBBBBBB..32
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REFERENCESBBBBBBBBBBBBBBBBBBBBBBBBBBBB41
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APPENDIX I: Raw Data TablesBBBBBBB.BBBBBBBBBBBBB..46

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APPENDIX II: Sequencing Data

16S rRNA Sequencing Data from House IsolatesBBBBB..BBB.89

16S rRNA Sequencing Data from Dormitory IsolatesBBBBB..B134

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 LIST OF TABLES

Table 1: Kirby-Bauer antibiotic resistance standards (CLSI, 2007)BBBBB..14

Table 2: Questionnaire responses pertaining to sponge use at homeBBBB.46

Table 3: Colony morphologies of all house sponge isolatesBBBBBB.....B.47

Table 4: Bacterial load (CFU/g) in sponges collected from houses

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before and after microwave treatmentBBBBBBBBBBBBBBBBBB59

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Table 5: Bacterial load (CFU/g) in sponges collected from dormitories

before and after microwave treatmentBBBBBBBBBBBBBBBBBB60

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Table 6: Triple Sugar Iron test resultsBBBB.BBBBBBBB...BBBBB61

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Table 7: Methyl Red & Vogues-Proskauer test resultsBBBBB..BB..BBB69

Table 8: Kirby-Bauer test resultsBBBBBBBBBBBBBBBBBBBB.72

Table 9: 16S rRNA sequencing results from house isolatesBBBBBBBB..84

Table 10: 16S rRNA sequencing results from dormitory isolatesBBBBBB..86

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 LIST OF FIGURES

Figure 1: Antibiotic-resistance patterns among house bacterial isolatesBBB.19

Figure 2: Percentage of house isolates exhibiting multi-drug resistanceBBB.20

Figure 3: Disinfection patterns among sponges collected from

Quinnipiac University students living in houses (n=20)BBBBBBBBBBB22

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Figure 4: Disinfection patterns among sponges collected from

Quinnipiac University students living in dormitories (n=44)BBBBBBBBB.22

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Figure 5: Percent reduction of bacteria from house sponges after
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microwave treatmentBBBBBBBBBBBBBBBBBBBBBBBBB..25
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Figure 6: Percent reduction of bacteria from dormitory sponges after

microwave treatmentBBBBBBBBBBBBBBBBBBBBBBBBB..26

Figure 7: Gram stain data from sequenced bacterial isolates from

house spongesBBBBBBBBBBBBBBBBBBBBBBBBBBB...27

Figure 8: Gram stain data from sequenced bacterial isolates from

dormitory spongesBBBBBBBBBBBBBBBBBBBBBBBBBB..28

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 LIST OF FIGURES (continued)

Figure 9: 16S rRNA sequencing results for the house sponge isolates.BBB..29

Figure 10: Potential pathogenicity of the sequenced house sponge

isolates.................................................................................................................29

Figure 11: 16S rRNA sequencing results for the dormitory sponge

isolates.BBBBBBBBBBBBBBBBBBBBBBBBBBBBB.......30

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Figure 12: Potential pathogenicity of the sequenced dormitory sponge

isolatesBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB.31
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INTRODUCTION:

Food poisoning, defined as illnesses caused by bacteria in food, occurs

frequently around the world; approximately 48 million cases of food poisoning are

reported in the United States every year at an estimated cost of 78 billion dollars

(Scharff, 2012). The most frequent cases of food poisoning occur at home, with

the kitchen being the area with the most contamination (Rusin et al., 1998). A

study showed that fecal coliforms, which are known to cause gastroenteritis,

have the ability to survive on countertop surfaces for up to 24 hours (Scott and

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Bloomfield, 1990). In kitchens, the floor contains the least amount of bacteria

compared to the sponge, which harbors the most (Rusin et al., 1998). Since
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sponges retain moisture, they provide a suitable environment for microbial

growth. In addition to moisture, food particle remnants influence bacterial

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propagation in sponges. Foods that originate from raw meat, poultry, fruits and

vegetables can potentially become vehicles of transmission for numerous

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infectious diseases caused by microorganisms (Erdogrul and Erbilir, 2005; Park

et al., 2006). Some of these pathogenic microorganisms include Salmonella,

Escherichia coli, and Campylobacter (Josephson et al., 1997; Mattick et al.,

2002). These pathogenic microorganisms have also been known to contribute to

food poisoning, specifically gastroenteritis.

Numerous studies pertaining to microorganisms in kitchen sponges have

been performed. In one study, researchers investigated the efficacy of using

liquid dishwashing detergents to control microbial growth in kitchen sponges

(Nielsen et al., 2002). Specifically, store-bought sponges were inoculated with a

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variety of microorganisms. In order to simulate used sponges, some of the

inoculated sponges were soiled with nonfat dry milk. All sponges were then

treated with a dishwashing detergent and incubated. After 24 hours, both the

clean and soiled sponges showed a significant decrease in bacterial load, with

the clean sponges exhibiting a higher decrease. The researchers concluded that

detergents are effective in reducing bacterial load in kitchen sponges (Nielsen et

al., 2002).

Another study investigated the potential for foodborne-pathogen survival

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during plate washing and subsequent cross-contamination onto sponges and

kitchen surfaces (Mattick et al., 2002). Since Salmonella, E. coli, and

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Campylobacter are common foodborne pathogens that can potentially

contaminate the kitchen, the researchers used these, along with grease soil in
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order to simulate soiled dishes (Josephson et al., 1997; Mattick et al., 2002).

After washing the soiled dishes with sponges and dishwasher detergent, the
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sponges were then used to wipe a kitchen surface.

To assess pathogen survival after cleaning, the researchers examined

both a sample of sponge rinse and the “cleaned” kitchen surface for bacterial

analysis. The results of the analyses demonstrated that both E. coli and

Salmonella transferred onto the sponges from the soiled plates, but only E. coli

was present on the kitchen surface after wiping with the same sponge.

Campylobacter was not present in either the sponge or on the kitchen surface

most likely because it is significantly more sensitive to both desiccation and

dishwasher detergent than the other two microorganisms (Mattick et al., 2002).

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 Based on these results, the researchers recommended washing dishes at

the maximum temperature to ensure that most microorganisms would become

inactivated. Furthermore, the researchers recommend that sponges used in the

kitchen should be periodically replaced or decontaminated, due to the amount of

microorganisms present in them and the potential for these microbes to spread

elsewhere (Mattick et al., 2002) A sponge containing a significant amount of

bacteria still poses a risk of bacterial transfer to other kitchen items that come

into contact with the sponge.

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Another investigation evaluated the effects of subjecting bacteria-laden

sponges with and without food particles to dishwashing detergent in a laboratory

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setting (Erdogrul and Erbilir, 2005). In terms of household sponge use after ten

days, daily use of detergent had no effect in the amount of yeast/molds,

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Pseudomonas, or E. coli present in the sponge, but significantly reduced the

amount of Salmonella and Staphylococcus aureus. Additionally, the presence of

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food particles on the sponge hindered the ability of the detergent to reduce

bacterial load (Erdogrul and Erbilir, 2005).

Park and colleagues sought to elucidate the efficacy of microbial

inactivation through the use of microwave radiation. They determined that, at the

highest power level, complete inactivation of E. coli occurred after only 30

seconds of microwaving the sponge, but that inactivation of Bacillus cereus, a

spore-forming microorganism, occurred after 2 minutes (Park et al., 2006). The

researchers concluded that all microorganisms in a sponge were completely

inactivated by microwave radiation within 1 to 2 minutes, therefore, microwave

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radiation is a practical, safe, and cost-effective method of decontaminating

household sponges.

Supporting the investigation of microbial inactivation through the use of

microwave radiation, an additional investigation sought out effective methods to

disinfect kitchen sponges. Different soiled sponges were disinfected using

chemical treatments including bleach, lemon juice, and water, while other soiled

sponges were disinfected through using either a microwave or a dishwasher

(Sharma et al., 2009). The amount of bacteria post-decontamination with these

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methods was compared to the amount of bacteria residing in an untreated

sponge. The results of this investigation indicated that after treatment with
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bleach, lemon juice, or water, a significant amount of bacteria still remained.

Decontamination through dishwashing eliminated 99.9998% of bacteria, and

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disinfection through microwave radiation proved to be the most effective method

for disinfecting sponges, eliminating 99.99999% of bacteria (Sharma et al.,

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2009). The researchers concluded that disinfecting sponges through the use of a

microwave or dishwasher is fast and effective, while using chemical treatments to

disinfect the sponges proved far less effective.

Using microwave radiation to inactivate microorganisms is nothing new; in

order to reduce the spread of pathogenic microorganisms, the food industry has

used microwave radiation as a means to pasteurize and sterilize foods

(Rosenberg and Bogl, 1987). On the molecular level, microwaving bacteria

causes many effects on bacterial structure. Some effects include incomplete cell

lysis, which contributes to the leakage of nucleic acids and proteins from the cell

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(Woo et al., 2000). Additionally, microwave radiation causes the membranes of

Gram-negative bacteria, especially E. coli, to become damaged, rough, and

swollen; on the other hand, no apparent damage was observed in the

membranes of Bacillus species (Gram-positive) (Woo et al., 2000). Internally,

microwave radiation causes proteins to denature and form aggregate clusters as

well as heat shock protein activation for both Gram-positive and Gram-negative

bacteria (Xiong et al., 1997).

Recently there has been an increase in public awareness regarding

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antibiotic-resistant microorganisms. As a result, antibacterial dishwashing

detergents are prevalent in many households. Many of these antibacterial

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products contain chemicals such as triclosan, tricolocarban, pine oils, or

quarternary ammonium compounds. When used, the antibacterial chemicals

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leave residues that may affect the microenvironment of the application site; the

remaining microorganisms that are exposed to these residues acquire resistance

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to the antibacterial chemical, which may also induce cross-resistance to

antibiotics (Levy, 2001; Moken et al., 1997).

Triclosan is a common antibacterial agent found in dishwashing

detergents. It works as an antibacterial by inhibiting fatty acid synthesis in

bacteria (McMurry et al., 1998). Inhibition of fatty acid synthesis prevents the

bacterial cell from undergoing membrane biogenesis, thus making triclosan a

bacteriostatic agent when used at low concentrations (Escalada et al., 2005). In

terms of resistance, research has shown that resistance to triclosan is restricted

to certain types of enteric bacterial species including E. coli (Ledder et al., 2006).

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 Marshall and her colleagues sought to determine the frequency of

antibiotic-resistant bacteria in households that use differing amounts of these

antibacterial agents (Marshall et al., 2012). Households involved were either

categorized as users of antibacterial chemicals or non-users. Additionally,

samples were taken from each home to characterize the types of

microorganisms. Overall, there was no apparent difference in the amount of

antibiotic-resistant microorganisms in user and non-user households.

The majority of studies pertaining to bacteria in kitchen sponges have

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been performed in a simulated laboratory setting; in other words, sponges were

inoculated with known bacterial samples and were purposely soiled using agents
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like dry milk. Furthermore, there are very few studies that compare the number

and types of bacteria between two subject populations. Moreover, no studies

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have elucidated a possible connection between microwaving a sponge and the

reduction of antibiotic-resistant microorganisms afterwards.

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During the 1980s, it was determined that almost all bacteria can be

identified by conserved genes (Woese, 2000). These conserved genes among

bacteria included ones coding for ribosomal subunits 5S, 16S, and 23S; the 16S

rRNA gene is commonly used to identify bacteria (Clarridge, 2004). The gene

coding for 16S rRNA is used because of its adequate size (1500 bp), its

occurrence in all types of bacteria, and the conservation of the gene over time

(Patel, 2001; Clarridge, 2004). Furthermore, the 16S rRNA gene of bacteria can

be compared to the 16S rRNA gene of archaebacteria and the 18S rRNA gene of

eukaryotes (Clarridge, 2004). Additionally, Dubnau and his colleagues

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discovered that the 16S rRNA gene from different Bacillus species were highly

conserved (1965). It was later postulated that this genetic conservation was

attributed to 16S rRNA having a critical role in bacterial function (Woese, 1987).

For these reasons, the 16S rRNA gene from bacteria is adequate in order to

elucidate the identity of individual bacterial isolates.

The primary purpose of the proposed thesis was to characterize bacteria

that were found on sponges collected from college students living in houses.

Specifically, the Kirby-Bauer antibiotic resistance test was used to assess

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isolates that were resistant to different classes of broad-spectrum antibiotics that

included cephalosporins, fluoroquinolones, and tetracyclines. In order to further

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characterize the bacteria, 16S rRNA sequence analysis was performed on the

isolates. With these sequence results, along with results from several
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biochemical tests, the genera of the sequenced isolates were determined.

The secondary purpose of this thesis was to compare the bacteria isolated
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from house sponges to bacteria found on sponges collected from college

students living in dormitories. The dormitory house sponge data was previously

collected and analyzed by Patrick McInnis, Meaghan Donnelly and their

colleagues in Dr. Lisa Cuchara’s research laboratory at Quinnipiac University in

2013 (McInnis et al., 2013). Comparisons between both populations were made

regarding disinfection patterns, initial bacterial loads, effectiveness of

microwaving, and 16S rRNA sequencing results.

In the proposed thesis, 20 sponges were collected from college students

living in houses. The average amount of bacterial colonies from each sponge

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was determined using quantitative plate counts. This data was compared to

previous data pertaining to dormitory sponges. In addition to comparing colony

counts, bacterial isolates were identified using selective and differential media,

biochemical tests, and 16S rRNA analysis.

The results from this investigation will hopefully assist the public in

becoming conscious toward disinfecting their sponges in order to prevent a

potential outbreak caused by the microorganisms in kitchen sponges, especially

the ones resistant to antibiotics.

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MATERIALS AND METHODS:

1. Acquisition and preparation of sponge samples

20 household sponges were acquired from college-aged individuals (20-

28 yrs) living in houses. When collecting the sponges, the individuals were

asked the amount of time the sponge has been in use; the amount of people

that use the sponge; the method of decontamination if disinfected and the

frequency of decontamination. The sponges were placed in a Ziploc bag in

order to retain moisture.

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At the Quinnipiac University Microbiology Laboratory, the sponge was

sprayed with sterile H2O, and two 1 g pieces were cut from the sponge; one of
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the pieces was placed in a tube containing 9 mL of 1.25% Phosphate

Buffered Saline (PBS) solution while the other was placed in the center of the
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microwave (1100 W) on high for 1 min, then placed in another PBS containing

tube. Using a 1000 µL micropipette with sterile disposable tips, serial dilutions
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ranging from 10-1 to 10-6 were made from the PBS solutions containing the

treated and untreated sponge.

2. Preparation of media

In order to culture bacterial colonies from the sponge, different types of

media needed to be prepared. These media types include Xylose Lysine

Desoxycholate Agar (XLD), Trypticase Soy Agar (TSA), and MacConkey

Agar.

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 XLD is a selective media that differentiates between Salmonella and

Shigella species (Pollock and Dahlgren, 1974). Salmonella growth results in

red colonies, sometimes with black centers. Additionally, Shigella growth

results in red colonies and E. coli growth results in yellow colonies. TSA is a

nonselective, general growth medium that is able to grow both aerobic and

anaerobic bacteria (Leavitt et al., 1955). MacConkey is a Gram-negative-

selective media for the differentiation of coliform and enteric bacteria

(MacFaddin, 1985). Coliform growth results in pink to purple colonies, while

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enteric growth results in colorless colonies.

In order to perform antibiotic resistance testing, Mueller-Hinton Agar was

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also prepared. Other media that needed to be prepared were Trypticase Soy

Broth (TSB; grow bacterial isolates), Triple Sugar Iron slants (TSI;
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biochemical test) and Methyl Red – Vogues Proskauer broth (MR-VP;

biochemical test). All media types were prepared per the manufacturers’
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instructions.

3. Culturing bacterial colonies

Using aseptic techniques, 100 µL of the final three dilutions (10-4 to 10-6)

from the untreated sponge, and 100 µL of dilutions 10-2 to 10-4 from the

microwaved sponge were plated onto XLD, TSA, and MacConkey agars and

spread using a glass rod. Lower dilutions were plated for the microwaved

sponge because at higher dilutions, little to no bacterial growth occurred.

Once inoculated, plates were incubated at 37oC for 24 h.

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4. Colony count and colony isolation

After incubation, the bacteria present on plates containing 30-300 colonies

were counted. Having a plate with less than 30 colonies is unacceptable

because there are too few colonies to represent the sample. On the other

hand, having more than 300 colonies is also unacceptable because colonies

will be too close to each other, complicating the number of distinct colony-

forming units (CFUs). Later, the morphologies of 5 distinct colonies from

each different agar plate were recorded (Appendix, Table 2). Each set of the

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5 distinct colonies were then inoculated into TSB tubes using aseptic

techniques and incubated at 37oC for 24 h.

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5. Freezing bacterial isolates and subsequent handling
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When isolates had grown in TSB for 24 h, 850 µL of the isolate, along with

150 µL of 99% glycerol was placed in a freeze tube. After all isolates were
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placed in freeze tubes, the tubes were labeled and placed in a -70oC freezer.

When the isolates were needed for subsequent experiments, precautions

were taken to prevent thawing the samples; the freeze tubes were removed

from the freezer and placed in an ice bath. Using a sterile loop, a small

amount of the frozen isolate was scraped and then transferred into a TSB

tube and incubated for 24 h at 37oC. Afterwards, the freeze tubes were then

placed back in the freezer.

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6. Biochemical analysis: Triple Sugar Iron test

The TSI test is a differential analysis used to identify carbohydrate

fermentation, gas (CO2 and O2) production, and H2S production (Hajna,

1945). This test is used for the identification of Gram-negative bacteria. For

the TSI test, slants were stabbed and streaked with isolates from TSA and

MacConkey plates. The slants were then incubated at 37oC for 24 h.

7. Biochemical analysis: Methyl Red & Vogues-Proskauer test

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The MR-VP test is used to identify E. coli and Enterobacter bacteria (Clark

and Lubs, 1915). Samples that tested positive for coliform bacteria (yellow
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slant/yellow butt, gas production) from the TSI test (Appendix, Table 5) were

chosen for the MR-VP test. For the test, isolates from TSB after 24 h
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incubation were inoculated via loop into the MR-VP broth and incubated at

37oC for 24-48 h. After incubation, the broth was divided into two tubes, one
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tube for the MR test and the other for the VP test.

For the MR test, 5 drops of methyl red were added to the broth. If the

broth turned red, then E. coli was most likely present. If no color change

occurred, Enterobacter bacteria were most likely present.

For the VP test, 12 drops of VP-A were added to the broth, followed by 4

drops of VP-B. Once both reagents were added, the broth was shaken to mix.

If the broth turned red, then Enterobacter bacteria was most likely present. If

no color change occurred, E. coli was most likely present.

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8. Kirby-Bauer antibiotic resistance test

For the Kirby-Bauer antibiotic resistance test, the mature isolates grown in

TSB were placed into a tube containing only TSB until a McFarland turbidity

standard of 0.5 was reached. The turbidity of the solution was measured

using the Grant-Bio DEN1 densitometer. Once the standard was reached, the

contents of the tube were streaked onto Mueller-Hinton agar plates using a

sterile cotton swab. Afterwards, antibiotic disks (ceftriaxone: 30 µg,

ciprofloxacin: 5 µg, penicillin: 10 units, tetracycline: 30 µg) were placed on the

agar and the plates were incubated at 37oC for 24 h. After 24 h, if there was

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an apparent zone of inhibition, the diameter of the zone was measured in
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mm. These values were then analyzed to establish if the bacterial isolate was

susceptible, intermediate, or resistant to the antibiotic. This was determined

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using Kirby-Bauer antibiotic-resistance standards (Table 1).

Ceftriaxone, a 3rd generation cephalosporin, inhibits bacterial cell wall

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synthesis by interfering with peptidoglycan production (Medscape, 2013a).

Since it is a broad-spectrum antibiotic, it is effective against both Gram-

positive and Gram-negative bacteria.

Ciprofloxacin, a 2nd generation fluroquinolone, promotes DNA stress by

interfering with DNA gyrase, thus causing the DNA to break (Medscape,

2013b). Resistance to ciprofloxacin has become prevalent today because it

has since become one of the most commonly prescribed antibiotics to adults

(Linder et al., 2005). This obviously contradicts its intended use as a last-

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resort antibiotic (Medscape, 2013b). Since it is a broad-spectrum antibiotic, it

is effective against both Gram-positive and Gram-negative bacteria.

Penicillin is a β-lactam antibiotic that inhibits the formation of

peptidoglycan cross-linkages that occur in the cell wall of bacteria (Medscape,

2013c). Penicillin was used as a control due to its narrow-spectrum properties

against Gram-positive bacteria. Furthermore, it is widely recognized that a

significant amount of bacteria have evolved resistance for this antibiotic.

Tetracycline is a broad-spectrum antibiotic that inhibits protein synthesis

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through binding to the 30S subunit of the ribosome (Medscape, 2013d). The

effectiveness of tetracycline has decreased due to the rise of antibiotic-

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resistant bacteria (Speer et al., 1992).
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Table 1: Kirby-Bauer antibiotic resistance standards (CLSI, 2007)

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Zone Diameter Interperative

Standards (mm)
Resistant Intermediate Sensitive

Ceftriaxone ≤ 13 14 - 20 ≥ 21

Ciprofloxacin ≤ 15 16 - 20 ≥ 21

Penicillin ≤ 28 ≥ 29

Tetracycline ≤ 14 15 - 18 ≥ 19

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9. Extracting DNA from isolates

Three sponges (90 isolates, 30 from each sponge) were chosen for DNA

extraction and analysis. After incubating isolates in TSB for 48 h at 37oC,

DNA from the isolates was extracted using the UltraClean® Microbial DNA

Isolation Kit (cat #12224-250, available from MOBIO Laboratories, Inc.).

To begin extraction, the isolate was concentrated into a pellet through

centrifugation. The pellet, along with a mixture of buffer, salts, and lysis

solution was added to a microbead-containing tube and placed on a vortex,

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causing bacterial cell lysis through both chemical and mechanical methods.

After, a reagent that precipitates non-DNA matter was added to the tube and
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centrifuged. Later, the supernatant was transferred to another tube, where a

high-salt solution was added. This solution facilitated the DNA to readily bind
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to a filter. The mixture was then added to a filter tube and then placed in a

centrifuge, causing the DNA to adhere to the filter. After discarding the flow-
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through, an ethanol-based solution was added to the filter tube in order to

further purify the bound DNA. After placing the filter tube in the centrifuge and

discarding the flow-through, an elution buffer was added to the filter tube in

order to release the bound DNA from the filter.

10. 16S rRNA sequencing and analysis

Each DNA isolate (90 isolates as described above) was placed in a 96-

well plate and was shipped to Functional Biosciences, Inc. (Madison, WI)

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