presence of antibiotic resistant staphylococcus aureus in ... jws-a-14-004.… · 228 a. naquin et...

Presence of Antibiotic Resistant Staphylococcus aureus in Sewage

Treatment Plant

Anthony Naquin, Jacob Clement, Mary Sauce, Richard Grabert, Mingma Sherpa, Raj Boopathy*

Department of Biological Sciences, Nicholls State University, Thibodaux, LA 70310, USA

ABSTRACT

Antibiotic resistance has become very common in the world. After passing through the human or animal body the

antibiotics are entered into the sewage treatment plant, where water is processed and cleaned then returned into the

environment. During the sewage treatment process, antibiotics come into contact with bacteria entering the treatment

process, as well as bacteria used in the treatment process. The bacteria that are exposed to these antibiotics can be-

come resistant during the treatment process and then expose the antibiotic resistance genes (ARGs) to the environ-

ment upon release of treated water from the treatment plant. Because of the contact between bacteria and antibiotics

during the treatment process, sewage treatment plants are considered prime habitat to create antibiotic resistant

bacteria. There are very limited studies on this subject from a small town sewage treatment plant. Therefore, this

study was conducted using raw sewage as well as treated sewage from Thibodaux sewage treatment plant, which

serves 15,000 people in rural southeast Louisiana of USA. Samples were collected monthly from Thibodaux sewage

treatment plant and antibiotic resistance was monitored using Kirby-Bauer assay. Special attention was given to

Methicillin Resistant Staphylococcus aureus (MRSA) in raw and treated sewage samples for the five month of the

study period. Results showed the presence of MRSA consistently in both raw and treated sewage. The presence of

mecA gene responsible for Methicillin resistance was confirmed in isolates of pure culture of S. aureus as well as in

the sewage samples.

Keywords: Sewage treatment; antibiotic resistant; mecA gene, free DNA; Staphylococcus aureus; genetic transfor-

mation

1. INTRODUCTION

Staphylococcus aureus is a Gram positive

human pathogen that is responsible for

dangerous infections in individuals around the

world. S. aureus belongs to a group of bacteria

that normally live on the surface of the skin of

humans. According to Gordon and Lowy

(2008), approximately 30% of individuals are

intermittently colonized with S. aureus, and

approximately 20% of individuals are

persistently colonized. S. aureus is resistant to

penicillin, an antibiotic that disrupts a protein

(penicillin-binding protein) that is essential to

the synthesis of bacterial cell walls. S. aureus

produces a protein called penicillinase that

binds to penicillin and disrupts the antibiotic’s

chemical structure (Morell and Balkin, 2010).

Methicillin, first introduced in 1959, is a

penicillin-like antibiotic that is resistant to the

action of penicillinases. Methicillin was used

as an effective treatment for infections caused

by S. aureus; however, within a year of the

introduction of methicillin, methicillin-

resistant S. aureus (MRSA) were reported

(Gordon and Lowy, 2008). After careful

*Corresponding to: [email protected]

DOI: 10.11912/jws.2014.4.4.227-236

Journal of Water Sustainability, Volume 4, Issue 4, December 2014, 227-236

© University of Technology Sydney & Xi’an University of Architecture and Technology

228 A. Naquin et al. / Journal of Water Sustainability 4 (2014) 227-236

analysis, methicillin resistance was shown to

be conferred by the mecA gene, a gene which

codes for a mutated penicillin-binding protein

called penicillin-binding protein 2a (PBP2a)

(Morell and Balkin, 2010). This protein, unlike

the original penicillin binding proteins, has a

low affinity for beta-lactams like methicillin.

From 1960 until the early 1990s, methicil-

lin-resistant Staphylococcus aureus were

generally associated with hospital settings;

however, in the early 1990s, community-

associated strains of MRSA (CA-MRSA)

emerged (Gordon and Lowy, 2008).

CA-MRSA are resistant to the action of

methicillin and have a gene that codes for a

toxin that hospital-acquired MRSA cannot

produce.

Both hospital-acquired methicillin-resistant

Staphylococcus aureus (HS-MRSA) and

community-associated methicillin-resistant

Staphylococcus aureus (CA-MRSA) pose

significant health risks to human populations.

According to Klein et al. (2007), approxi-

mately 5,500 people die every year from

MRSA-related infections, and MRSA is the

leading cause of lower respiratory tract

infections and surgical site infections. One

reason that MRSA have become a major source

of dangerous infections is that all S. aureus are

hardy organisms that are capable of living in a

wide variety of environments. S. aureus

colonization is not limited to humans; S.

aureus has been isolated from cats, dogs, pigs,

and cows. The number of outbreaks of

CA-MRSA has steadily increased over recent

years among individuals who share close

contact with others (Goldstein et al., 2012).

Although HA-MRSA strains have traditionally

been associated with hospital settings and

CA-MRSA strains are usually found in

community settings, the two populations of

MRSA are beginning to intermingle (Gordon

and Lowy, 2008). For these reasons, reducing

the amount of contact individuals have with

any MRSA strains is of utmost importance if

the MRSA is to be controlled.

The focus of this study is to find the

prevalence of S. aureus in a small scale rural

sewage treatment plant. Sewage treatment

typically involves three major treatments,

including a primary treatment to remove

suspended solids, a secondary treatment to

remove organic matter, and a tertiary treatment

to disinfect pathogens (Cowan and Talaro,

2006). In the primary treatment, larger floating

materials are skimmed off of the top of the raw

sewage (Cowan and Talaro, 2006). Sedimen-

tation tanks are used to remove suspended

particulate matter (Robens, 1996). In the

secondary treatment, organic matter is digested

by a community of microbes through a process

known as activated sludge. Biodegradation

typically produces solid wastes, known as

sludge, which collect at the bottom of the tank

and break down slowly (Cowan and Talaro,

2006). In order to break down the sludge more

quickly, the sludge is activated through the

injection of air, mechanically stirred, and

recirculated (Cowan and Talaro, 2006). In the

tertiary treatment, the wastewater is disinfected

and discharged (Cowan and Talaro, 2006).

The City of Thibodaux in the state of

Louisiana, USA is a rural community with a

population of 15,000. The sewage treatment in

Thibodaux employs a traditional three-stage

sewage treatment system as mentioned above

along with an anaerobic digester to treat the

sludge. The treated wastewater is pumped to a

nearby wetland to be further purified by the

environment (Goldstein et al., 2012). Prior to

discharge to the wetland, the wastewater passes

through an ultraviolet disinfection system to

eliminate any microbes present in the water

samples.

Sewage treatment plant is an ideal habitat

for the development of antibiotic resistant

bacteria as large numbers of bacteria come in

close contact along with many types of

antibiotics discharged in the wastewater.

Elimination of the bulk of bacterial load occurs

A. Naquin et al. / Journal of Water Sustainability 4 (2014) 227-236 229

in the disinfection step of the sewage treatment

process; however, many problems exist with

current methods of disinfection. Many sewage

treatment facilities employ a chlorination

system, but chlorination systems are expensive

(Robens, 1996). As a result some sewage

treatment facilities have switched to an

ultraviolet disinfection system. Ultraviolet

light destroys the DNA of microbes, thus,

preventing microbes from reproducing.

However, ultraviolet light may induce the

production of heat shock proteins, proteins

which repair DNA damage and guards against

apoptotic processes (Behzadi and Behzadi,

2012). Inefficient treatment processes,

including incomplete disinfection, are a major

method of microbial introduction to the

environment (Hendricks and Pool, 2012).

The purpose of this study was to test samples

from the Thibodaux sewage treatment facility

for the presence of Methicillin-Resistant

Staphylococcus aureus (MRSA). The specific

objectives of this study were to survey MRSA

populations in raw and treated sewage, and to

confirm the presence of MRSA gene (mecA)

using molecular techniques.

2. METHODS

2.1 Sample collection

Monthly samples were collected from the

Thibodaux sewage treatment plant, Thibodaux,

Louisiana, USA for a period of five months

from October 2013 to February 2014. Samples

were collected from raw sewage that come into

the plant and the treated sewage that is

discharged to the wetland. The collected

samples were brought to the lab in a cooler and

processed immediately.

2.2 Quantification of staphylococcus

aureus in the sample

The S. aureus in the raw sewage and treated

sewage were quantified every month using the

Manitol salt agar (MSA) media, which is

selective and differential for the presence of S.

aureus (Leboffe and Pierce, 2010) and it

produces yellow color colonies in MSA. The

samples were serially diluted and the dilutions

from 101 - 105 were plated in Petri dishes using

pour plate technique with MSA to obtain

countable colony forming units (CFU). The

plates were incubated at 37˚C for 24 hours and

the yellow colonies were counted and reported

as CFU/ml of sample.

2.3 Isolation of staphylococcus aureus

To isolate Staphylococcus aureus, sterile

cotton swabs were used to transfer bacteria

from the water sample collection vessels to

tryptic soy broth (TSB) tubes (MP, Solon, OH)

according to the procedure described by

Leboffe and Pierce (2010). The TSB tubes

were incubated at 37˚C for 24 hours. From the

TSB cultures, a small amount of sample was

transferred to mannitol salt agar (MSA) plates

(Remel, Lenexa, KA) using sterile cotton

swabs. An inoculation loop was used to isolate

pure culture using the quadrant streak method

(Leboffe and Pierce, 2010). The MSA plates

were incubated at 37˚C for 24 hours. After

twenty-four hours, one yellow colony (yellow

colony in MSA medium indicates the presence

of S. aureus) from each of the MSA plates was

transferred to TSB. The TSB tubes were

incubated at 37˚C for 24 hours. These TSB

tubes were treated as pure cultures for the

duration of the experiment. For long-term

storage of isolated bacteria, bacteria from the

pure TSB cultures were transferred to TSA

slants, and the TSA slants were incubated at

37˚C for 24 hours and after incubation were

stored at 4˚C.

2.4 Kirby-baur disk diffusion assay

The isolated pure culture was tested every

month for antibiotic resistant using Methicillin.


A small amount of the pure culture was

pipetted into a sterile test tube and the turbidity

of culture was adjusted using sterile saline to

match the turbidity of the McFarland standard

(Leboffe and Pierce, 2010). Using a sterile

cotton swab, a bacterial lawn was created on a

MSA plate and Methicillin antibiotic disc was

dispensed onto the surface of the MSA plate.

The plate was incubated at 37˚C for 24 hours.

After the incubation time, the zone size was

measured in millimeter using a manual caliper.

The zone size was compared to interpretive

standards to examine the antibiotic suscepti-

bility of the isolated bacteria.

2.5 Molecular analysis of mecA gene

The presence of mecA gene in the raw and

treated sewage as well as from the isolated S.

aureus was analyzed using the mecA primer,

5’ATGCGCTATAGATTGAAAGGAT-3’ as

demonstrated by Suzuki et al. (1992). The

primer was obtained from Sigma Aldrich Co.,

(St. Louis, USA). Raw sewage and post UV

treated sewage samples were centrifuged at

13,000 rpm for 15 minutes. The pellet was

transferred to 1.5 ml microfuge tube and a

FAST ID DNA extraction kit was used to

extract the DNA according to the instructions

provided by the manufacturer. Similar

procedure was followed for the pure cultures of

S. aureus. After the DNA was extracted,

polymerase chain reaction (PCR) technique

was used to amplify the DNA with 5 µL DNA

extract, 32.5 µL DNA free water, 1 µL of

forward primer, 1 µL reverse primer, 5 µL

deoxynucleoside triphosphates, 5 µL buffer,

and 0.5 µL taq polymerase. The tubes were

then placed in the PCR machine (Life

Technologies, Applied Biosystems, Grand

Island, NY; model number 2720) and allowed

the cycle to completion. The PCR cycle was

similar to the one described by Everage et al.

(2014). A 2% agarose gel with ethidium

bromide was prepared and used to visualize the

PCR samples. 10 µL PCR sample was mixed

with 2 µL 6x loading dye and injected into each

well. The gel was run at 100 V for an hour. The

gel was visualized using FluorChem FC2

imaging system.

2.6 Statistical analysis

All data were subjected to an analysis of

variance (ANOVA) test (p≤ 0.05) followed by

a tukey “post hoc” analysis.

3. RESULTS AND DISCUSSION

3.1 Presence of S. aureus in raw and

treated sewage

S. aureus was consistently present in both raw

and treated samples in all the sampling

periods. Fig. 1 shows the bacterial load during

the various sampling periods. As expected the

population of S. aureus was high in raw

sewage compared to treated sewage and it was

statistically significant (p< 0.05). The

Thibodaux sewage treatment uses UV light as

a disinfecting agent before the treated sewage

was discharged into the nearby wetlands.

Even though the UV light removed most of

the S. aureus, the sample contained on an

average around 1000 CFU/ml. S. aureus in the

sample was positively identified by using the

selective and differential medium of MSA.

MSA is selective and differential for S. aureus

as it contains 7.5% salt which will eliminate

most of the bacteria and the substrate manitol

in the medium is only metabolized by S.

aureus, which will produce fatty acid from

manitol. The fatty acid turns the medium to

acidic and the pH indicator present in the

medium will turn yellow color under acidic

condition (Duguid, 1989). The presence of

yellow color colony positively identifies the

presence of S. aureus in the sample.


Figure 1 S. aureus population in raw and treated sewage for each month of sampling during

2013-2014 (Data represent average of two samples per sampling event. S.D was less than 5%)

Figure 2 Average resistance of S. aureus to Methicillin by each month during 2013-2014

sampling period in raw and treated sewage (Data represent average of three isolates for each

month. The S.D was less than 5%).

1.0E+00

1.0E+01

1.0E+02

1.0E+03

1.0E+04

1.0E+05

1.0E+06

1.0E+07

1.0E+08

October November December January February

Bacte

ria

l L

oa

d (

CF

U/m

L)

Month Sampled

Raw Sewage

Treated Sewage

0

2

4

6

8

10

12

14

October November December January February

Aver

age Z

on

e D

iam

ete

r (m

m)

Month Sampled

Raw Sewage

Treated Sewage


Figure 3 MecA assay for February 2014 sample. The lanes are, lane 1 raw sewage, lane 2

treated sewage, lane 3 negative control with deionized water, lane 4 positive control with mecA

primer, and lane 5 negative control with DNA free water

Figure 4 MecA gene in five different isolates of S. aureus isolated from sewage sample during

the study period. Lanes 1 to 5 represent five isolates, lane 6 is the positive control of mecA gene

primer, and lane 7 is negative control with deionized water

3.2 Methicillin resistance

The isolated S. aureus from the sewage sample

was tested for the antibiotic resistance,

specifically to the antibiotic, Methicillin to see

whether MRSA is prevalent in sewage. The

result indicated MRSA is commonly present in

both raw and treated sewage (Fig. 2). The

antibiotic resistance was significantly higher

with a p value of < 0.05 in treated sewage than

sewage. This implies that the bacteria are

picking up antibiotic resistant gene during the

treatment time when the sewage was held for a

hydraulic retention time (HRT) of five days.

During this HRT, the activated sludge is an

ideal place with bacteria habituating in close

1 2 3 4 5


quarters to go through bacterial gene transfer in

one or several methods such as genetic

transformation, conjugation, and transduction

(Everage et al., 2014). When different species

of bacteria grow in close proximity to each

other, the growing bacteria produce com-

pounds not normally produced when they grow

alone (Traxler et al., 2013). Some bacteria can

produce extracellular enzymes to deactivate

the antibiotic, allowing sensitive bacteria the

chance to grow in the environment (Salyers

and Whitt, 2001). Sensitive bacteria can also

coexist with resistant bacteria in the presence

of antibiotics by covering the sensitive bacteria

with a layer of resistant bacteria (Narisawa et

al., 2008). In this study, the possible mecha-

nism for antibiotic resistance is the genetic

transformation as mecA gene was present in the

treated sewage as a free DNA, which might

have played a key role in conferring antibiotic

resistance to bacteria that are sensitive to

antibiotics as described by Everage et al.

(2014).

3.3 Presence of mecA gene

To positively confirm the presence of mecA

gene, which is the gene that confers the

resistance to Methicillin, molecular analysis

was done in raw and treated sewage samples.

The mecA gene was consistently observed in

both raw as well as treated sewage. Fig. 3

shows the result of mecA assay for the month

of February 2014 as an example. Lane 1 in the

gel is the raw sewage sample, lane 2 represents

treated sewage, lane 3 is the negative control

with deionized water, and lane 4 is the positive

control with the primer of mecA gene. From

Fig. 3, we can positively identify the presence

of mecA gene in both raw and treated sewage

sample. This was consistent for all sampling

periods. Moreover, the isolated colonies also

showed the presence of mecA gene as

indicated in Fig. 4. This figure shows various S.

aureus isolates for five months and all the

isolates showed positive band for the mecA

gene. This result confirmed S. aureus isolated

from the sewage sample as MRSA. Suzuki et al.

(1992) showed the presence of mecA gene in S.

aureus and also in S. epidermidis. Genetic

material that confers methicillin resistance

may be passed from one organism to another

through a process known as transformation in

which free DNA from one organism is taken up

by another organism and as a result develop

antibiotic resistance. UV light kills most

bacteria and at the same time may promote the

release of free DNA into the wastewater as

shown in this study. Thus through transfor-

mation process, bacteria may acquire antibiotic

resistance trait.

The purpose of this study was to investigate

the presence of ARGs in the form of mecA

gene and MRSA in raw and treated sewage

sample and the results clearly showed the

presence of not only MRSA, but the presence

of free mecA gene floating in the raw and

treated sewage. This antibiotic resistance could

potentially spread to the native bacteria present

in the wetland where the treated sewage is

discharged. This study demonstrated that the

free DNA from the treated sewage, which

contained the mecA gene is the main source for

antibiotic resistance in the natural environment.

Other studies have also shown that wastewater

treatment plants are a common source of

resistance genes (Everage et al., 2014; LaPara

et al., 2011) to the natural environment.

Antibiotics are among the most commonly

used and successful group of pharmaceuticals

used for human medicine (Bouki et al., 2013).

Therefore, rapid spread in resistance to these

antibiotics has caused medial concerns to both

public and health professionals. 1 in 5 trips to

the emergency room (ER) are due to side

effects of antibiotics. At least 2 million people

become infected with resistant bacteria each

year in the US and at least 23,000 die directly

as a result, resulting in a cost of over $30

billion (CDC, 2014). Resistance is a result of


both the appropriate use of antibiotics, such as

normal exposure due to usage, and inappro-

priate use, such as not finishing a prescription

or over-use of the drugs. Other reasons include

the selective pressure of antibiotic use, as well

as technical changes that enhance the

transmission of resistant organisms. There are

many modes of resistance, such as decreased

permeability, altered target site, or enzyme

inactivation. MRSA is a commonly discussed

resistant bacteria in public health. More people

die annually in the US from MRSA than AIDS

and homicides combined (Spellberg et al.,

2011). The goal is to slow down the rise in

antibiotic resistance genes (ARGs) by imple-

menting better hygiene, preventing infections,

controlling the nosocomial transmission of

organisms, treating the source of the causative

agent, and changing and developing new

treatment methods (Dzidic et al., 2003).

Recent studies show that incomplete

metabolism in humans and improper disposal

of antibiotics to sewage treatment plants has

been a main source of antibiotic release into the

environment (Rizzo et al., 2013). This gives

bacteria enough time and sufficient contact to

shield themselves by altering their genes and

cellular mechanisms, favoring their growth and

reproduction (Galvin et al., 2010). These genes

can go on to infect the wildlife in the estuaries

when the treatment plants discharge their

treated wastewater. Since 2007, over 3 million

hunting and fishing licenses have been sold in

Louisiana (Louisiana Wildlife and Fisheries,

2013). This has the potential to spread to

humans that come into contact and consume

the wildlife here in the wetland, where the

sewage is discharged. More studies are needed

to broaden other antibiotic resistance such as

tetracycline, erythromycin, streptomycin, and

sulfanilamide, which are some of the common

antibiotics prescribed in Thibodaux area

clinics.

CONCLUSIONS

This study clearly demonstrated the prevalence

of S. aureus, MRSA, and free DNA of mecA

gene in raw and treated sewage samples of

Thibodaux treatment plant. Bacterial load was

higher in raw sewage compared to treated

sewage. However, the antibiotic resistance was

higher in treated sewage than raw sewage. This

is due to the ideal condition in the sewage

treatment plant for bacteria to develop

antibiotic resistance. The mecA gene was

present both in raw and treated sewage in all

sampling periods. MRSA was identified in all

the pure culture isolates isolated from sewage

samples. Thibodaux sewage treatment plant is

operating according to its designed purpose,

which is to remove organic carbon. Sewage

treatment plants are not designed to remove

antibiotic resistance genes. This is an emerging

problem and it should be addressed by public

health officials.

ACKNOWLEDGEMENTS

This work was supported by the funds from

Nicholls Research Council and Louisiana

Board of Regents. We thank Dr. Rajkumar

Nathaniel and Dr. Michelle Thiaville for their

help in the molecular analysis.

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