Effects of Different Wastewater Treatment Processes on Occurrence and Prevalence of Antibiotic Resistant Bacteria and Their Resistance Genes

Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 2, Pages: 744-749

J. Environ. Treat. Tech.

ISSN: 2309-1185

Journal web link: http://www.jett.dormaj.com

Effects of Different Wastewater Treatment

Processes on Occurrence and Prevalence of

Antibiotic Resistant Bacteria and Their Resistance

Genes

Mahdi Asadi-Ghalhari , Rahim Aali *, Mohammad Aghanejad , Reza Fouladi Fard , Hassan

Izanloo , Ali Shahryari , Hamed Mirhossaini , Mohsen Mehdipour Rabori , Reza Ghanbari

Research Center for Environmental pollutants, Department of Environmental Health Engineering, Faculty of Health, Qom University of Medical

Sciences, Qom, Iran

Research Center for Environmental pollutants, Department of Environmental Health Engineering, Faculty of Health, Qom University of Medical

Sciences, Qom, Iran

Department of Environmental Health Engineering, Khoy University of Medical Sciences, Khoy, Iran. Email: mamamaxy01@yahoo.com

Research Center for Environmental pollutants, Department of Environmental Health Engineering, Faculty of Health, Qom University of Medical

Sciences, Qom, Iran

Research Center for Environmental pollutants, Department of Environmental Health Engineering, Faculty of Health, Qom University of Medical

Sciences, Qom, Iran

Department of Environmental Health Engineering, Gorgan University of Medical Sciences, Gorgan, Iran

Department of Environmental Health Engineering, Arak University of Medical Sciences, Arak, Iran

Department of Environmental Health Engineering, Kerman University of Medical Sciences. Kerman, Iran

Department of Environmental Health Engineering, Qazvin University of Medical Sciences, Qazvin, Iran

Received: 14/01/2020

Accepted: 13/04/2020

Published: 20/05/2020

Abstract

This study aimed to explore the difference between hospital and municipal wastewater treatment processes regarding the reduction

of antibiotic-resistant bacteria (ARB) and antibiotic resistant genes (ARGs). Samples were collected from raw and final effluent of four

different wastewater treatment plants (WWTPs). ARB were evaluated by modified HPC method. Extraction and purification of DNA

from the samples were conducted by Freeze-Thaw and DNA extraction kit. Real-time PCR (qPCR) was utilized to obtain the quantity

of Sul1 and ErmB genes in the samples. For standard control in qPCR, was used plasmid containing each gene sequence. The average

ARB concentration in the raw wastewater and effluent was 1.03×10 -6.63×10 CFU/100mL. Quantitative range of the Sul1 and ErmB

genes were obtained as 0-8.3×10 Copies/100 mL and 9.29×10 - 9.64×10 Copies/100 mL, respectively. The results show that urban

wastewaters play a more significant role than hospital wastewaters in the emission of sulfonamides and erythromycin-resistant bacteria

and genes to the environment. Findings revealed that conventional wastewater treatment plants cannot be regarded as reliable barriers

for the control of these agents.

Keywords: Antibiotic-resistant bacteria, ARGs,Hospital wastewater,Urban wastewater, Real-time PCR, Sul1, ErmB

and hospital wastewaters are the most important sources that

Introduction¹

release these contaminants to the environment (4). Wastewater

treatment plants are one of the most important and recent

obstacles in the emission of resistant bacteria and genetic

elements to the environment (11). Researchers have not

reached a consensus regarding the effects of WWTPs yet. Some

have reported the reducing effect of these plants. Others,

however, have mentioned the increasing effects of treatment

plants on the emission of agents that can develop antibiotic

resistance. A number of studies have regarded the effect of

urban WWTPs, and other researchers believe the hospital

WWTPs to be more efficient. Research shows that the destiny

of various antibiotic resistance factors in the environment is

contingent upon different factors, including the type of

treatment processes, procedure of operation, wastewater

Increasing concerns have been reported about the negative

impacts of antibiotic residuals on the environment (1-3). The

major representation of this problem is the development of

antibiotic resistance (4-6). Antibiotic resistance has been

reported all around the world. The WHO has mentioned

antibiotic resistance as one of the three major problems of the

1 century (4, 7). This resistance has been observed across a

wide variety of environments such as water, soil, air, and

wastewater (8, 9). Antibiotic resistance can be developed by

different ways, including the direct entrance of resistant

bacteria from therapeutic settings or the antibiotic residual

pressure in environmental resources (10). The developed

resistance can cause changes in the natural ecosystems. Urban

Corresponding author: Rahim Aali, Research Center for Environmental pollutants, Department of Environmental Health Engineering,

Faculty of Health, Qom University of Medical Sciences, Qom, Iran. E mail: r.aali@qum.ac.ir.

Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 2, Pages: 744-749

organic load, the variety of wastewater microorganisms, the

amount of discharged antibiotics, conditions of hospitals,

consumption patterns of antibiotics and the economic and

cultural situation of a society. In Iran, the consumption of

antibiotics has not become standardized in therapeutic settings

and the society. Iran is among countries with the highest

antibiotic consumption. Antibiotic resistance has been reported

in almost all clinical, agricultural, and animal husbandry studies

and against all antibiotic groups (12-16). Limited research has

been conducted on antibiotic resistance in the Iranian

environment (17). In this research, two antibiotic groups, i.e.

sulfonamides (STX) and erythromycins (ER), were

investigated because of their wide application in therapeutic,

agricultural, and animal husbandry environments, as well as

their informal use by the people. The common coding genes

2.3 Quantitate PCR

Plasmid DNA was used as the standard control in real-time

PCR (qPCR). Fresh PCR products of ermB and sul1 were

separated and excised from the agarose gel. The gel fragments

were purified with a gel extraction kit (Gel purification kit, Cat

NO: K-3035-1, Bioneer, USA) and ligated into the PTZ 57R

vector. DNA was transformed into Escherichia coli Top 10

using CaCl and heat-shock. Clones containing the correct

insert were confirmed by PCR amplification with m13

universal primer [F-m13 (5-ttgtaaaacgacggccagt-3) and R-m13

(5-acaggaaacagctatgaccatg-3)] and sequencing (14). Plasmids

were purified and plasmid concentration was then determined

by spectroscopy (Nano drop ND1000). The copy number of

each plasmid was calculated using the molecular weight of

nucleic acid and length (in base pairs) of the cloned plasmid.

To generate the standard curve, the threshold cycle (Ct) value

was used for each concentration. The qPCR was used to

quantify the concentrations of ermB and sul1 genes. Prior work

had shown that ermB and sul1 genes were present in substantial

concentrations in wastewater. The qPCR was carried out on the

applied bio systems (Applied Bio systems, USA) using the

SYBER green method. All qPCR reactions were performed in

(

Sul1, ErmB) in relation to these two antibiotic groups were

also studied quantitatively.

Materials and Methods

.2 Sampling and enumeration of antibiotic resistant bacteria

Samples were collected from wastewater treatment plants

in Isfahan province, Iran. Four sampling sites were selected to

study antibiotic-resistant bacteria (ARB) and antibiotic

0 µL of reaction mixture (23). T-tests were run to compare

quantity averages in influents and effluents. McNamara

statistical test was used to compare the examination results

from just before and after the WWTPs. ANOVA was employed

to compare the variation results from different sites. Finally,

Pearson’s correlation coefficients were used to determine the

correlations between HPC (heterotrophic plate count),

incidence of ARB, and ARG genes (SPSS 16 for Windows,

SPSS Inc., Chicago, IL).

1 2

resistant genes (ARGs). The first (MW ), second (MW ), and

third (MW ) sampling sites were the influent and effluent of

municipal WWTPs with different processes, conventional

activated sludge (two-step), conventional activated sludge, and

stabilization pond, respectively. The capacity of these WWTPs

3 -1

were 25×10 , 13×10 and 9×10 m d , respectively. The

disinfection process of all WWTPs were chlorination. The

fourth sampling site (HW ) was the influent and effluent of

extended aeration supported with high-speed sand filter. Its

-1

capacity was 890 m d , and its disinfection process was

chlorination. To determine ARB concentration, samples were

3 3 Results and Discussion

3.1 ARB and ARGs in wastewater sources

The average concentrations of ARB and ARGs in influent

diluted and 0.1 mL of each dilution was spread on R

amended individually with erythromycin (15µg mL ),

A (Difco),

-1

and effluent were obtained to be 5.39×10 -4.22×10 and

sulfamethoxazole (50 µg mL ), and additional antifungal

nystatin (18, 19). Plates were incubated for 48h at 37˚C. ARB

results were derived by comparing heterotrophic and ARB

cultivable concentrations (20, 21). All assays were performed

in duplicate. Positive samples were rechecked.

1.38×10 -9.29×10 Copies/100 mL, respectively (Fig 1, 2 and

3). Results in all of figures were shown by standard deviation.

Raw wastewater

Final effluent

1.00E+08

.00E+06

1.00E+04

.2 DNA extraction and qualified PCR

DNA was extracted from original samples. Fifty mL of the

original samples was prepared (centrifuged at 6000 rpm for 15

min) and the pellet was resuspended in 300 µL of distilled

water. The pellets were frozen in liquid nitrogen and boiling

water for three times (22). The DNA was extracted and purified

by DNA extraction kit (promega wizard genomic DNA

purification kit, Madison, WI) according to the manufacturer’s

manual. Primer pairs were used to amplify sul1 and ermB

genes, as taken from Munir et al (19). The total volume of the

reaction mixture (25µL) contained 0.5 µL of each primer, 1.5

.00E+02

.00E+00

HPC

ARB

HPC

ARB

Municipal

Hospital

Sites

Figure 1: Compare of raw and effluent in municipal and hospital

wastewater

µL MgCl

, 0.5 mM dNTP, 2.5 µL PCR buffer, 1 µL of template

These values are larger than the means of ARGs and ARB

achieved in other studies (19, 24, 25). This can be due to

insufficient management of WWTPs in Iran and the nature of

the produced wastewater. The generated wastewater in Iran has

a larger organic load compared with Western countries because

of different nutritional patterns. For example, Kim et al (2006a)

reported a strong relationship between organic load and the

ARB growth rate. Previous studies have reported a high organic

load in the studied urban WWTPs in Isfahan province, Iran

(26). In addition, high quantitative values of resistant genes in

wastewater can be due to high population and horizontal gene

transfer (19, 27).

DNA and 5 units of Taq DNA polymerase (22). All PCR assays

contained a positive and a negative control. PCR amplification

was performed using a thermal cycler (Corbett, Australia). The

PCR profile included initial denaturation at 94˚C for 10 min,

denaturation at 94 ˚C for 45 s, annealing (varied) for 30 s, and

extension at 72˚C for 45s for 30 cycles, followed by a final

extension at 72˚C for 10 min. PCR products (6 µL) were mixed

with 2 µL of DNA safe stain and loaded on 1.5% agarose gel.

Gels were viewed on a UV trans illuminator, and DNA

fragment sizes were compared with the 100-bp ladder (14).

Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 2, Pages: 744-749

.00E+08

.50E+08

.00E+08

.50E+08

.00E+08

.00E+07

.00E+00

2.50E+08

2.00E+08

1.50E+08

1.00E+08

5.00E+07

0.00E+00

Raw wastewater

Final effluent

Raw wastewater

Final effluent

HPC

STX

HPC

STX

MW2

MW1

(

(a)

.50E+08

.00E+08

.50E+08

.00E+08

.00E+07

.00E+00

3.00E+08

2.50E+08

2.00E+08

1.50E+08

1.00E+08

Raw wastewater Final effluent

Raw wastewater

Final effluent

5.00E+07

.00E+00

HPC

STX

HPC

STX

HW3

MW3

(c)

(d)

Figure 2: Compare of ABR and HPC in municipal (a,b and c) and hospital (d) wastewater treatment plants

The results of this study revealed a moderate reduction in

ARB and ARGs in the WWTPs. The findings of other studies

are in agreement with the results of this study (19, 28). The rate

of ARB and ARGs discharge to the environment through

not agree with the reports of Huang et al. (2012) and Rodriquez-

Mozaz et al. (2015) regarding the great effect of treatment on

the reduction of antibiotic resistance (31). The investigation of

the resistance developed by individual ARB and ARGs

revealed that the resistance patterns in the raw wastewater and

effluent were not necessarily similar (Fig 2 and 4).

wastewaters was very high (the output range of ND-7.7×10

CFU/100mL and ND-8.29×10 Copies/100 mL). The findings

of this research generally show that the identified values are

much larger than the values of ARB and ARGs available in the

receiver sources such as water resources (19, 29). Therefore,

abundant release of these agents causes a high pressure on

water resources and natural ecosystems (30). The results of the

effluent indicated that although ARB and ARGs were slightly

affected during the treatment stages, conventional wastewater

treatment facilities could not have a significant effect on the

removal of ARB and ARGs. Thus, the findings of this study do

The pattern obtained based on quantitative results of genes

in raw wastewater and effluent indicated that the Sul1 gene had

the highest value in the input and output (Fig 4). In a study

conducted by Munir et al. (2011), the quantitative value of sul1

gene identified in the WWTPs was the highest (19). These

results are also in accordance with those of the investigation of

Rodriquez-Mozaz et al. (2015). The high frequency of this gene

can be due to the high frequency of genetic elements in the

wastewater.

.00E+10

.50E+10

.00E+10

.50E+10

.00E+10

.00E+09

.00E+00

2.50E+08

.00E+08

.50E+08

.00E+08

Raw wastewater

Final effluent

5.00E+07

.00E+00

raw

final effluent

raw

final effluent

HPC

STX

wastewater

MW2

Municipal

Hospital

(

Total

(

Figure 3: Compare of ARGs in hospital and municipal wastewater treatment plants

Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 2, Pages: 744-749

.2 ARB and ARGs in municipal and hospital wastewater

The results of this study indicate that the average ARB and

ARGs are larger in urban wastewaters (UWW) than in hospital

wastewaters (HWW) (Fig 1 and 3). This can be due to the

higher microbial load of UWW, presence of organic and

nutrient materials to bacterial growth, and easy transmission of

resistance among bacterial population because of receiving

wastewater from a wider spectrum of sources (32, 33).

Excessive use of antibiotics, especially oral antibiotics by

people and the subsequent release of antibiotics from body to

the UWW are not negligible. Geli et al. (2012) and Udekwe et

al. (2009) have suggested that the increased resistance to

vancomycin in the US in 1980s could have been a result of the

over-consumption of oral vancomycin to control C. difficile

1-1.5 log. This plant has a strong management system. From

among the studied treatment plants, MW had the lowest

reduction percentage, possessing a dual active sludge process.

This plant has no significant effect on ARB removal, despite

benefiting from a dual process. It also suffers from a poor

management system. The pattern of ARB reduction indicates

that in addition to the effect of the process type, the operation

procedure also affects the efficiency of ARB removal. The

MW process is a stabilization pond. Some researchers attribute

the reduction of ARB and ARGs in this process to solar

radiation. The present study showed that the level of log

reduction for STX and ER was equal to 1.43. This reduction is

1.44 for ARGs (ermB and sul1).

Investigating the effects of sunlight on resistant bacteria,

Mezrioui and Echab (1995) observed that these ponds can

cause an increase in population of these bacteria. Rizzo et al.

(2013) attribute the increased number of resistant strains of

E.coli in aeration lagoons to the support of the process type

from the mentioned strains (32).

(34). This trend can also be applied to other antibiotics. Overall,

many researchers consider UWW as a key source of ARB and

ARGs’ emission into the environment (19) The lower levels of

ARB and ARGs by hospitals in comparison with the UWW

may be attributed to the vast application of disinfection and

acidic compounds, as well as the transmission of these

compounds into the wastewater. This is not in congruence with

the findings suggesting that HWW are richer in ARB and ARGs

Conclusions

Municipal wastewater plays a more significant role in

(24, 35). These studies attribute higher ARB and ARG levels to

discharging ARB and ARGs into the environment. This study

proves that although the processes of UWW treatment can be

effective to some extent in removing genes, they are not

successful to sustainable and reliable controlling of the

bacterial resistant genes to the environment. Performance of

treatment plants based on their process demonstrated that the

process of active sludge with a widespread aeration mechanism

supported by a dual disinfection system together with sand

filter is the most efficient method. It seems that multiple

disinfection systems complemented by a sand filter are

effective and efficient in the removal of bacteria and genetic-

the high concentration of antibiotics in the HWW and in turn,

the increased chance of contact between bacteria and antibiotics

and resistant strains (24). Some studies consider the wide

application of antibiotics in hospital as a selective advantage

for ARB. In an investigation of the biofilm of urban and

hospital wastewaters, Shwarz et al. (2003) demonstrated that

ARB was higher in UWW and ARGs in HWW. In addition,

some researchers argue that the mechanisms of resistance

development in the HWW and UWW are different (36). The

incidence of antibiotic resistance in the treatment plants

effluent is not necessarily similar in different antibiotics and

microbial groups.

resistant elements. Furthermore, the poor performance of MW

plant benefiting from a dual system of active sludge highlights

the fact that in addition to the process type, management can

play a major role in this regard

.3 The effect of WWTPs on ARB and ARGs

As can be seen in Fig 2, the reduction of ARB in the HW

treatment plant had the highest percentage (over 2 logs) among

the studied treatment plants. This hospital treatment plant

possesses an extended aeration process supported by a dual

disinfection system (CL+UV) and rapid sand filter. Although

STX and ER were not detected in the effluent, the results of

quantitative ARGs indicated that ermB was identified and

Aknowledgment

We would like to thank the staff of hospitals and municipal

WWTPs in Isfahan and Research Center for Environmental

Pollutants in Qom University of Medical Sciences.

measured in the effluent (9.64×10 copies/100 mL). The lack of

Ethical issue

identification of resistant bacteria in the effluent is possibly

related to the dual system of disinfection and filtration. On the

other hand, positive results of molecular experiments indicated

that genetic parts (genome DNA, plasmid, etc.) related to ARB

existed in the wastewater. This shows that cultivation-based

tests on their own are not reliable for investigation of the status

of ARB and ARGs in environmental sources. Thus, the

application of cultivation-based and molecular tests can present

reliable results for the estimation of the level of activity and the

genetic makeup of resistant agents (37). Our results

demonstrated that the reduction of ARB and ARGs had taken

place desirably. Rizzo et al. (2013) believe that rapid passage

over surfaces of infiltration systems is not suitable for the

process of conjugation of microorganisms and can cause sexual

cell damage in them (32). It seems that more studies are

required based on the applied advanced systems for the removal

of contributing factors to the development of resistance in

Authors are aware of, and comply with, best practice in

publication ethics specifically with regard to authorship

(avoidance of guest authorship), dual submission, manipulation

of figures, competing interests and compliance with policies on

research ethics. Authors adhere to publication requirements

that submitted work is original and has not been published

elsewhere in any language.

Competing interests

The authors declare that there is no conflict of interest that

would prejudice the impartiality of this scientific work.

Authors’ contribution

All authors of this study have a complete contribution for

data collection, data analyses and manuscript writing.

WWTPs (36). According to the results of the study, after HW ,

the MW plant had the second highest reduction of ARB with

Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 2, Pages: 744-749

.50E+10

.00E+10

.50E+10

.00E+10

.50E+10

.00E+10

.50E+10

.00E+10

.00E+09

.00E+00

2.50E+10

.00E+10

1.50E+10

.00E+10

5.00E+09

.00E+00

Raw wastewater Final effluent

Raw wastewater

Final effluent

Sul1

ErmB

Sul1

ErmB

MW2

MW1

(

.00E+11

.00E+10

.00E+00

4.00E+10

.00E+10

.00E+00

Raw wastewater

Final effluent

Raw wastewater

ErmB

Sul1

ErmB

HW3

MW3

(

Figure 4: Quantities average of Sul1 and ermB genes in different WWTPs

0. Vaz-Moreira I, Nunes OC, Manaia CM. Bacterial diversity and

antibiotic resistance in water habitats: searching the links with the

References

Barancheshme F, Munir M. Strategies to Combat Antibiotic

Resistance in the Wastewater Treatment Plants. Frontiers in

microbiology. 2018;8:2603.

human

microbiome.

FEMS

microbiology

reviews.

014;38(4):761-78.

1. Zhang S, Han B, Gu J, Wang C, Wang P, Ma Y, et al. Fate of

antibiotic resistant cultivable heterotrophic bacteria and antibiotic

resistance genes in wastewater treatment processes. Chemosphere.

Mostafaloo R, Mahmoudian MH, Asadi-Ghalhari M.

BiFeO /Magnetic Nanocomposites for the Photocatalytic

Degradation of Ceﬁxime From Aqueous Solutions under Visible

Light. Journal of Photochemistry and Photobiology A: Chemistry.

015;135:138-45.

2. Noorbakhsh Sabet N, Japoni A, Mehrabani D, Japoni S. Multi-drug

resistance bacteria in Qom hospitals, Central Iran. Iran Red

Crescent Med J. 2010;12:501-3.

3. Dallal MMS, Doyle MP, Rezadehbashi M, Dabiri H, Sanaei M,

Modarresi S, et al. Prevalence and antimicrobial resistance profiles

of Salmonella serotypes, Campylobacter and Yersinia spp. isolated

from retail chicken and beef, Tehran, Iran. Food Control.

019:111926.

Mostafaloo, R., Asadi-Ghalhari, M., Izanloo, H., Zayadi, A.

Photocatalytic degradation of ciprofloxacin antibiotic from

aqueous solution by BiFeO nanocomposites using response

surface methodology. Global Journal of Environmental Science

and Management, 2020; 6(2): 191-202.

Rodriguez-Mozaz S, Chamorro S, Marti E, Huerta B, Gros M,

Sànchez-Melsió A, et al. Occurrence of antibiotics and antibiotic

resistance genes in hospital and urban wastewaters and their impact

on the receiving river. Water Research. 2015;69:234-42.

Kamani H, Bazrafshan E, Ashrafi SD, Sancholi F. Efficiency of

sono-nano-catalytic process of TiO2 nano-particle in removal of

erythromycin and metronidazole from aqueous solution. Journal of

Mazandaran University of Medical Sciences. 2017;27(151):140-

010;21(4):388-92.

4. Fallahi G-H, Maleknejad S. Helicobacter pylori culture and

antimicrobial resistance in Iran. The Indian Journal of Pediatrics.

007;74(2):127-30.

5. Askarian M, Zeinalzadeh A, Japoni A, Alborzi A, Memish ZA.

Prevalence of nasal carriage of methicillin-resistant

Staphylococcus aureus and its antibiotic susceptibility pattern in

healthcare workers at Namazi Hospital, Shiraz, Iran. International

Journal of Infectious Diseases. 2009;13(5):e241-e7.

6. Aali R, Ghanbari R. Antibiotic Resistance in Environment and its

Public Health Risks in Iran. Journal of Environmental Health and

Sustainable Development. 2017;2(4):371-3.

7. Aali R, Nikaeen M, Khanahmad H, Hejazi Z, Kazemi M,

Hassanzadeh A. Occurrence of tetracycline resistant bacteria and

resistance gene(tetW) in hospital and municipal wastewaters.

Fresenius Environmental Bulletin. 2014;23(10a):2560-6.

8. Gao P, Munir M, Xagoraraki I. Correlation of tetracycline and

sulfonamide antibiotics with corresponding resistance genes and

resistant bacteria in a conventional municipal wastewater treatment

plant. Science of The Total Environment. 2012;421–422(0):173-

Al-Musawi TJ, Kamani H, Bazrafshan E, Panahi AH, Silva MF,

Abi G. Optimization the effects of physicochemical parameters on

the degradation of cephalexin in sono-Fenton reactor by using box-

Behnken response surface methodology. Catalysis Letters.

019;149(5):1186-96.

Organization WH. Antimicrobial resistance global report on

surveillance: 2014 summary. 2014.

Hernando MD, Mezcua M, Fernández-Alba AR, Barceló D.

Environmental risk assessment of pharmaceutical residues in

wastewater effluents, surface waters and sediments. Talanta.

006;69(2):334-42.

Ghanbari R, Shahryari A, Asgari E, Hosseinpoor S, Yeganeh J,

Salighehdar Iran N, et al. Environmental cycle of antibiotic

resistance encoded genes: A systematic review. The Journal of

Qazvin University of Medical Sciences. 2017;21(5):71-55.

9. Munir M, Wong K, Xagoraraki I. Release of antibiotic resistant

bacteria and genes in the effluent and biosolids of five wastewater

utilities in Michigan. Water Research. 2011;45(2):681-93.

Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 2, Pages: 744-749

0. J.P.brooks SLm, C.Rensing, C.P. Gerba, and I.L.Pepper.

Occurrence of antibiotic-resistant bacteria and endotoxin

associated with the land application of biosolids. Can J Microbial.

007;53:616-22.

1. Yeganeh J, Mehrdadi N, Baghdadi M, Aali RJJoMUoMS. Effect

of Zero Valent Iron Nanoparticles on Removal of Antibiotic

Resistant Genes of Heterotrophic Bacteria in Urban Wastewater.

018;28(160):28-44.

2. Koike S KI, Oliver HD, Yannarell AC, Chee-Sanford JC, Aminov

RI, Mackie RI. Monitoring and source tracking of tetracycline

resistance genes in lagoons and groundwater adjacent to swine

production facilities over a 3-year period. Appl Environ Microbiol.

007;73(15):4813-23.

3. Pei R, Kim S-C, Carlson KH, Pruden A. Effect of River Landscape

on the sediment concentrations of antibiotics and corresponding

antibiotic resistance genes (ARG). Water Research.

006;40(12):2427-35.

4. Reinthaler FF, Posch J, Feierl G, Wüst G, Haas D, Ruckenbauer

G, et al. Antibiotic resistance of E. coli in sewage and sludge.

Water Research. 2003;37(8):1685-90.

5. Auerbach EA, Seyfried EE, McMahon KD. Tetracycline resistance

genes in activated sludge wastewater treatment plants. Water

Research. 2007;41(5):1143-51.

6. Aali R, Nikaeen M. Detection of enteroviruses in biosolids. Health

system research. 2013;9(4):370-7.

7. Aminov R, Garrigues-Jeanjean N, Mackie R. Molecular ecology

of tetracycline resistance: development and validation of primers

for detection of tetracycline resistance genes encoding ribosomal

protection proteins. Applied and environmental microbiology.

001;67(1):22-32.

8. Rijal G, Zmuda J, Gore R, Abedin Z, Granato T, Kollias L, et al.

Antibiotic resistant bacteria in wastewater processed by the

Metropolitan Water Reclamation District of Greater Chicago

System. Water Science and Technology. 2009;59(12):2297-304.

9. Xi C, Zhang Y, Marrs CF, Ye W, Simon C, Foxman B, et al.

Prevalence of Antibiotic Resistance in Drinking Water Treatment

and Distribution Systems. Applied and Environmental

Microbiology. 2009;75(17):5714-8.

0. Iwane T, Urase T, Yamamoto K. Possible impact of treated

wastewater discharge on incidence of antibiotic resistant bacteria

in river water. Water science and technology: a journal of the

International Association on Water Pollution Research.

001;43(2):91.

1. Huang J-J, Hu H-Y, Lu S-Q, Li Y, Tang F, Lu Y, et al. Monitoring

and evaluation of antibiotic-resistant bacteria at a municipal

wastewater treatment plant in China. Environment international.

012;42:31-6.

2. Rizzo L, Manaia C, Merlin C, Schwartz T, Dagot C, Ploy MC, et

al. Urban wastewater treatment plants as hotspots for antibiotic

resistant bacteria and genes spread into the environment: A review.

Science of The Total Environment. 2013;447:345-60.

3. Schlüter A, Szczepanowski R, Kurz N, Schneiker S, Krahn I,

Pühler A. Erythromycin resistance-conferring plasmid pRSB105,

isolated from a sewage treatment plant, harbors a new macrolide

resistance determinant, an integron-containing Tn402-like

element, and a large region of unknown function. Applied and

environmental microbiology. 2007;73(6):1952-60.

4. Geli P, Laxminarayan R, Dunne M, Smith DL. “One-Size-Fits-

All”? Optimizing Treatment Duration for Bacterial Infections.

PloS one. 2012;7(1):e29838.

5. Hadi M sR, ebrahimzadeh namvar A.M, Karimi M, Solaimany

Aminabad M. antibiotic Resistance of Isolated Bacteria from

Urban and Hospital Wastewaters in Hamedan city. IranJHealth&

environ. 2011;4(1):105-14.

6. Tuméo E, Gbaguidi-Haore H, Patry I, Bertrand X, Thouverez M,

Talon D. Are antibiotic-resistant Pseudomonas aeruginosa isolated

from hospitalised patients recovered in the hospital effluents?

International Journal of Hygiene and Environmental Health.

008;211(1-2):200-4.

7. Menceloglu YZ, Balcioglu IA. Comparison of the effectiveness of

chlorine, ozone, and photocatalytic disinfection in reducing the

risk of antibiotic resistance pollution. Journal of Advanced

Oxidation Technologies. 2011;14(2):196-203.