Pharmaceutical Journal o f Sri Lanka 2017 Volume 7 Issue 1

http://doi.org/10.4038/pjsl.v7i0.16

Research Article

Risk of Prophylactic Antibiotics in Livestock and Poultry Farms; A

Growing Problem for Human and Animal Health

Liyanage G.Y and Pathmalal M*

Centre for Water Quality and Algae Research, Department of Zoology, University of Sri
J ayewardenepura.

^Corresponding author: [email protected]

Abstract range of 60 to 720ppm concentrations of the

antibiotics by agar dilution method.
Purpose: Veterinary antibiotics are Results: Oxytetracycline (55%), and
physiologically highly active substances tetracycline (38%) were recorded as most
which are being used on a large scale as frequent antibiotics in samples and having
therapeutic drugs and as feed additives in concentrations of 0.005 ppm and 0.004 ppm
modem agricultural practice. Major purposes respectively. The highest concentration of
of use of antibiotics are prevention and AMX was recorded as 0.003±0.004 ppm.
treatments of bacterial infections and for the However, AMP was not detected during the
improvements of growth rates of farm study period. Oxytetracycline and
animals. Therefore, the present study was tetracycline resistance bacteria were
focused on quantification of some selected identified as Enterococcus sp., E. faecium,
groups of antibiotics; tetracycline E.coli and Clostridium sp. while, A.
[Tetracycline (TET), Oxytetracycline baumanii, E. clocae, A. Iwoffii and H.pylori
(OTC)] and penicillin [Amoxicillin (AMX), were identified as AMPr and AMXr bacteria
Ampicillin (AMP)] in wastewater discharge by 16S rRNA sequencing. The MIC values
drains in large-scale livestock and poultry of tetracycline (TET, OTC) resistant bacteria
farms and to isolate antibiotic resistant ranged from 360 ppm to 720 ppm whereas
bacteria. for penicillin (AMX, AMP) from 360 to 760
Methods: Twenty wastewater discharge ppm.
drains in livestock and poultry farms were Conclusion: The contamination of
selected to collect samples for the study. antibiotics leads to develop antibiotic
Solid-Phase Extraction (SPE) was employed resistance in environmental bacteria. Thus,
and antibiotic quantification was done by the results of the study indicate that presence
High Performance Liquid Chromatography of antibiotic resistant bacteria may limit the
(HPLC). Isolation of resistance (r) bacteria effectiveness of antibiotics in treating animal
was done by standard pour plate method illness, thereby causing a potential risk to the
where Minimum Inhibitory Concentration productivity of livestock and poultry farms.
(MIC) of bacteria was determined using the
Keywords: Tetracycline, Oxytetracycline,
Amoxicillin, Ampicillin, Resistance
I This is an open-access article distributed under the terms of the creative Commons Attribution License, which permits unrestricted use,
distribution, and reproduction in any medium, provided the original author and source are credited.

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Pharmaceutical Journal o f Sri Lanka 2017 Volume 7, Issue 1

Introduction animal wastewater and effluent water from

wastewater treatment. Besides, the
Antibiotics are the most important group of application of manure from livestock farm
antimicrobial drugs, widely prescribed for onto agriculture land contributes to introduce
human, and animal diseases. According to antibiotics in to the aquatic
the World Health Organization (WHO), environment.(3,4) The introduction of these
about half of the antibiotics are consumed for residual compounds into the environment
non-human applications.(1,2) Livestock through different sources will lead to serious
farming is frequently referred to as a environmental problems including
reservoir for potentially pathogenic and environmental pollution and risk of wide
antimicrobial resistant bacteria and as a spread transmission of antibiotic resistant
reservoir of antibiotic resistance genes as pathogenic bacteria to humans.(10)
well.(3,4) Antimicrobial drugs are widely Afterwards, transfer of resistance genes from
applied in animal husbandry to increase antibiotic resistance microorganisms to
production, treatment of infectious diseases indigenous environmental bacteria occurs
and as growth promoters.(5) under selection pressure of antibiotics driven
by the mechanism of horizontal gene transfer
Tetracycline, P-lactams and macrolides are (HGT).(11) In soil, transfer of resistance
the most common antibiotic groups which determinants to pathogens may reduce the
are being used for veterinary purposes, efficiency of antibiotics on both human and
human therapy and in agriculture sector as animals. (12,13) Thus recently, antibiotic
feed additives.(6) Owing to their lower cost resistance genes (ARG) and antibiotic
and their higher antimicrobial activity, resistance bacteria were considered as new
tetracycline antibiotics are widely used as emerging contaminants.(10,14)
veterinary drugs for the prevention and
treatment of several infectious diseases such At present, large amounts of antibiotics are
as leptospirosis, mycoplasma.(7) used to intensively manage animal
Tetracycline antibiotics are used as feed husbandry in Sri Lanka. This has resulted in
additives to promote the growth of the high contamination levels of veterinary
animals.(8) Several P-lactams are commonly antibiotics in wastewater from livestock
used for infections such as; colibacillosis, farms.(5) Thus, the present study was
fowl cholera, respiratory infections due to planned to determine antibiotic
Ornithobacterium rhinotracheale, septicemia contamination status and screening of
due to Riemerella antipestifer (ampicillin, antibiotic resistance bacteria in effluent
amoxicillin), dysbacteriosis (benzyl waters in livestock farms in Sri Lanka.
penicillin), and erysipelas (penicillin) in
poultry.(3, 4) Methods

According to the available literature (4, 9) Standards and reagents

antibiotics can enter into the aquatic Two tetracyclines including oxytetracycline
environment via the direct discharge of (OTC) (97%), tetracyclines (TET) (97%),

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Pharmaceutical Journal o f Sri Lanka 2017 Volume 7, Issue 1

two penicillin including amoxicillin (AMX) including 10 livestock farms (Ambewala -

(96%), ampicilline (AMP) (97%) and all the New Zealand, Ambewala - Highland,
HPLC grade chemicals were purchased from Bopaththalawa, Weerawila, Jaffna,
Sigma Aldrich, USA. Batticaloa, Dayagama, Ridiyagama,
Nikwaratiya) and 10 poultry farms
Sample collection (Karandagolla, Katunayake, Jaffna, Bairaha,
Triplicate effluent water samples were Gampaha) following standard methods
collected from 20 sampling locations: (Figure 1).(15)

Figurel: Effluent water sampling locations of the present study

Antibiotic analysis of 100 ppm and subjected to SPE.(16,17) In

One liter of wastewater sample was adjusted this procedure, C18 cartridge was
to pH 3 and the sample was filtered through preconditioned with 5 ml 100% methanol
0.22 p,m nuclearpore filter. Samples were and then 5ml double-deionized water. Pre­
spiked with antibiotic at final concentration prepared samples were then passed through
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Pharmaceutical Journal o f Sri Lanka 2017 Volume 7, Issue 1

the C18 cartridges at flow rate of antibiotic (OTC, TET, AMP, AMX) at 0-100
approximately 1-2 ml/min and then rinsed ppm were prepared. The linearity of the
with 5 ml of deionized water. The analytes method was evaluated by plotting peak area
were eluted with 5ml of 100% methanol as a function of the concentration of each
followed by 2ml of 100% isopropanol. The analyte.
extract was purged by 99.9% nitrogen stream
and re-dissolved with 1ml of mobile Enrichment and isolation of antibiotic
phase.(18,19) The target antibiotics were resistance bacteria
quantified by using Agilent 1200 series 50 ml of water from each sampling sites
HPLC equipped with diode array and were enriched by spiking, antibiotic at final
fluorescence detector. Antibiotics in effluent concentration of 60 ppm in 100 ml
water were analyzed according to the erlenmeyer flasks and the final volume was
Fernandez et al. (2010) with a topped up to 100 ml with sterile water and
modification.(20) 0.1% glacial acetic acid then the flasks were incubated at 28 0C ± 1
(polar protic solvent) was used as mobile 0C in 100 rpm for 14 days in a shaking
phase and injected volume was 20 pl and incubator. After 14 days of incubation, 1 ml
chromatography was performed at 30 0C. of sample aliquot was taken from each flask.
The mobile phase of the mixture of 0.1% Isolation and enumeration of bacteria was
glacial acetic acid in water (Component A): carried out by standard pour plate
0.1% glacial acetic acid in acetonitrile method.(10) LB medium containing 60 ppm
(Component B), 99:1 (v/v) was pumped of antibiotics (TET, OTC, AMP, AMX) was
beginning at a flow rate of 0.7 ml/min for used to isolate TET, OTC, AMP and AMX
OTC, TC, AMP and AMX. Then followed resistant (r) bacteria.(15,17,21)
linear elution gradient from 99% to 70% A
in 25 min for antibiotics.(15,17,19) Table 1 After three days of incubation at 28 0C,
shows conditions and retention time bacteria colonies with different
employed for each antibiotic analysis. morphological characters were picked up and
re-suspended in sterilized liquid LB medium.
Table 1: Wave length and retention time for Subsequently pure bacterial cultures were
different antibiotic tested in the present study sub cultured and stored in agar slants at -20
X SD 0C in LB-glycerol media for further studies
Retention
Analyte absorption (min) and identification purposes.(22)
time (min)
(nm) (n=3)
Antibiotic susceptibility test
OTC 280 17.34 0.002
The LB broth culture was prepared and a
TET 280 16.56 0.001 loop of isolated bacteria strain was
AMP 230 13.08 0.001 inoculated and incubated at 28 0C shaking
AMX 230 11.23 0.006 under 100 rpm overnight. The cell density of
SD, Standard deviation
the bacteria suspension was equalized using
McFarland No 0.5.(15,21) MIC was
Calibration plots
determined by using an agar dilution method
Individual calibration standards of each
following the CLSI guidelines.(23)

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 Pharmaceutical Journal o f Sri Lanka 2017 Volume 7, Issue 1

Results and Discussion administered antibiotics are excreted without

undergoing metabolism.(25)
Antibiotics are commonly used at sub
therapeutic levels in livestock to prevent Based upon the WHO estimates,
diseases and promote growth.(24) Despite considerable differences were recorded in
the long history of antibiotic usage, antibiotic usage among different animal
information regarding antibiotic production species (swine vs. poultry). Therefore, the
and usage patterns in Sri Lanka is severely types of antibiotic compounds that are likely
limited due to the lack of research to be found in effluent water of livestock
information, comprehensive monitoring and farms will strongly depend upon the types of
documenting efforts. The large amounts of livestock operations within the
antibiotic usage may result in their presence environment.(7)
in environment as up to 90 percent of the

0.01

Sampling locations

Figure 2: Antibiotic contamination levels in livestock farms (n=3); ^ OTC,H TET, 0A M X ,

■ AMP

In the present study antibiotic concentrations 0.021 ppm) was detected in effluent water at
in effluent water of livestock farms ranged a poultry farm in Katunayake.
between 0.001 ppm-0.004 ppm for OTC,
0.001 ppm - 0.005 ppm for TET, 0.001 ppm- Among the selected antibiotics,
0.003 ppm for AMX respectively (Figure 2). approximately 45% from the TET, 35% from
However, AMP was not detected during the the OTC and 20% of AMX contributed to
study period. The maximum concentrations environment pollution through the effluent
of AMX (0.003 ppm) and OTC (0.004 ppm) water.
were detected in samples collected from
livestock farms in Jaffna (Figure 2). The The WHO has recommended less than 0.001
considerable concentration of TET (0.005± ppm as guideline value for antibiotic
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Pharmaceutical Journal o f Sri Lanka 2017 Volume 7, Issue 1

residues in the aquatic environment. the present study too, effluent water of
(26,27,28) Thus, the results of the present livestock farms were recorded as a
study revealed that the antibiotic significant source of antibiotic
contamination level in effluent water had contamination. In the present study OTC
exceeded the values given by the WHO.(26) (0.001-0.004 ppm) and TET (0.001-0.006
ppm) were detected at high concentrations
The high detection frequency and while AMX (0.001-0.00 2ppm) was recorded
concentration of tetracyclines are likely due in low concentrations.
to the large amount of OTC and TET usage
in livestock and poultry as feed additives and These antibiotic residues promote the
therapeutic drugs for diseases in farm sites. evolution or the development of antibiotic
The results of the present study are resistant microorganisms, which can induce
comparable to the findings of Brown et al., adverse effect to human health when present
(2010), who reported concentrations of OTC in drinking water or irrigation water.(21,29)
(0.001-0.008 ppm), TET (0.001-0.005 ppm), These antibiotic resistant bacteria may
AMX (not detected) and AMP (not disturb the microflora of the human
detected) in livestock effluents in New intestines and increase the risk of certain
Mexico. Similar to previous studies(21,29) infectious diseases as well.(3,26)
conducted in different parts of the world, in

Figure 3 : Phylogenetic affiliations for OTCr, TETr, AMXr and AMPr bacteria with their
phylogeny and Minimum Inhibition Concentration (MIC) values, The phylogeny was
constructed by neighbor-joining method based on Mega 6/ Cluster W from alignment of 16s
rRNA gene sequence comparison of antibiotic resistance bacteria.
N am e o f isolate A ccession No M IC (ppm )
B a cillu ssu b tilis (A M X 4) HF 586509 480
B acillus sp. (TET 3) HF586507 640
B acillus sp. (TET 4) HF657890 720
B acillus sp (AM P 4) KU567678 360
B acillus thurengiensis (AMP 3) NR. 0423 87 400
E nterobacter sp. (OTC 5) EU. 159482 420
E nterobacter cloacae (A M X 7) KL 234156 680
1__ E nterobacter cloacae (A M X 1) K M 435657 360
E scherichia coli (A M X 3) K N 514297 700
— E scherichia coli (A M X 11) F J 982654 480
E scherichia coli (TET 2) F J 922659 660
i__ E scherichia coli (AM P 1) F J 982657 720
E scherichia coli (O TC 3) □ 1186353 360
E nterobacterfaecium (TET 1) KM 234561 380
E nterobacter cloacae (A M X 5) K X 495499 400
k Streptococcus eq u i (A M X 9) HF 947328 540
L actobacillus sp. (AM P 7) E U 321541 400
E nterococcus sp. (OTC 2) A Y 499112 360
E nterococcus sp. (TET 4) K X 458221 480
i H elicobacter p y lo r i(AM P 80
C itrobacterfreundii (A M X 2)
FS 464023
E U 321541
380
680
C itrobacterfreundii (A M X 8) A Y 4 9 9 112 720
A cinetobacter baum anii (AM P 2) K X 458221 360
A cinetobacterlw ojjii (AMP 6) FS 464023 580
Streptom yces rim osus (A M X 5) K U 690365 620
C lostridium perjringens (A M X 10) K U 691844 360
Clostridium sp. (OTC 1) KS 678976 380
Streptom yces sp , (A M X 6) F J 982654 460

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Pharmaceutical Journal o f Sri Lanka 2017 Volume 7, Issue 1

In the present study different antibiotic followed by Bacillus sp.(4) These results
resistance bacteria were isolated based on may reflect regional differences in species
MIC test. The isolates were subjected to the composition of the bacterial population.
16S rRNA analysis to identify the isolated
OTCr, TETr, AMPr and AMXr. Species In the present study, most of OTCr and TETr
composition results for OTC, TET, AMP, bacteria were isolated from livestock farms
and AMX resistant bacteria in environmental and those bacteria mainly caused nonscomial
samples from different sampling sites and infections (Enterococcus sp., E. faecium,
their MIC values are summarized in Figure Enterococcus sp.) and intestinal infectious
3. diseases (E.coli, Clostridium sp.). However,
Enterobacter sp. and Bacillus sp. were
Based on the 16S rRNA sequences, most of recorded as non pathogenic bacteria. Most
the resistance bacteria belonged to genera of AMXr and AMPr bacteria which were
Bacillus, Enterobacter and Escherichia. The isolated from livestock farms were recorded
majority of tetracycline resistant bacteria as causative agents for intestinal diseases and
belonged to the genera Bacillus, opportunistic infections, whereas only few
Enterococcus, Enterobacter, Clostridium, non-pathogenic bacteria were detected.
Escherichia whereas penicillin (AMX, (8,29)
AMP) resistant bacteria belonged to
Escherichia, Enterobacter, Streptomyces, There is a further concern that antibiotic-
Streptococcus, Lactobacillus, Bacillus, resistant bacteria might develop from long­
Helicobacter, Acinetobacter and term environmental exposure to low
Clostridium. concentrations of antibiotic (ng/L-^g/L),
present in wastewater and surface waters.
The MIC values of OTCr bacteria in (30) It was highlighted that continuous
livestock farms ranged between 360 ppm to exposure to a particular antibiotic residues in
720 ppm whereas for TETr bacteria were the environment, multidrug resistant
from 360 to 700 ppm (Figure 2). MIC values pathogens can make drugs ineffective and
of AMXr bacteria ranged between 360- 720 pose a serious risk to the global
ppm; the highest MIC was recorded for pharmaceutical and healthcare industry.(10,
Citrobacter freundii where the lowest was 31)
for Bacillus sp. MIC of AMPr bacteria
varied from 360 ppm to 720 ppm whereas Thus, greater attention must be given to the
the highest MIC was recorded for E.coli and overall pattern of antibiotics usage in the
the lowest for Bacillus sp. respectively. farms, including environmental risk
assessment and research on the mitigation of
The results of the present study were antibiotic contaminants. Therefore the
different from the study conducted in baseline data collected in the present study
Vietnam, where Acinetobacter was the most will provide first-hand information to
common tetracycline resistant bacterium.(5) authorities to open eyes to see the
However, another study in a Vietnamese contamination status and develop national
poultry farm found that Vibrio sp. was the strategic plan to reduce antibiotic
commonest tetracycline-resistant bacteria, contamination from anthropological
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Pharmaceutical Journal o f Sri Lanka 2017 Volume 7, Issue 1

activities in order to safe guard human and 4. Davies J, Davies D. Origins and
animal health. evolution of antibiotic resistance.
Microbiol. Mol. Biol. Rev. 2010;
Conclusion 74(3):417-33.
5. Bien TLT, Sato-Takabe Y, Ogo M, Usui
In conclusion, the present study M, Suzuki S. Persistence of Multi-Drug
demonstrates that livestock and poultry Resistance Plasmids in Sterile Water
farms have been a reservoir of antibiotics under Very Low Concentrations of
and antibiotic-resistant bacteria. The TETr, Tetracycline. Microbes and
OTCr, AMPr and AMXr bacteria; especially Environments. 2015; 30(4): 339-44.
the opportunistic pathogens were isolated 6. Li W, Shi Y, Gao L, Liu J, Cai
from farm wastewater and the Y.vOccurrence of antibiotics in water,
contaminations of TET, OTC, AMP and sediments, aquatic plants, and animals
AMX implies an urgent need for from Baiyangdian Lake in North
constructing a monitoring system for China. Chemosphere. 2012; 89(11):
antibiotic usage in livestock and poultry 1307-15.
farms. Because isolated antibiotic resistance 7. Chenia HY. Vietze C. Tetracycline
bacteria in farm wastewater may lead to resistance determinants of heterotrophic
problems in the efficiency of antibiotics bacteria isolated from a South African
which used to treat animal diseases. Further tilapia aquaculture system. Afr. J.
research is particularly necessary to better Microbiol. Res. 2012; 6(39): 6761-68.
understand the route and mechanisms of 8. Giorgia ML, Davide C, Paolo G, Sara C,
ARGs proliferation and to thus mitigate the Giuseppe C, Roberto F. Preliminary
risks to public health. investigation on the environmental
occurrence and effects of antibiotics
used in aquaculture in Italy.
References
Chemosphere. 2004; 54: 661-68.
9. Chopra I. Roberts M. Tetracycline
1. Tello A, Austin B, Telfer T. Selective
Antibiotics: Mode of Action,
pressure of antibiotic pollution on
Epidemiology of Bacterial Resistance
bacteria of importance to public health.
Applications, Molecular Biology, and
.2012 Environment health perspective
Epidemiology of Bacterial Resistance.
120(8) 1100-06.
Microbiol. Mol. Biol. Rev. 2001;
2. WHO. Tuberculosis MDR-TB & XDR-
65(2):232-60.
TB 2011 Progress Report. 2011 [cited
10. Manage PM, Zen ichiro K, Shin ichi N.
2016 Oct 14]. Available from:
Algicidal effect of the bacterium
http://www.who.int/tb/challenges/mdr/fa
Alcaligenes denitrification on
ctsheet_mdr_progress_march2011.pdf
Microcystis spp. Aquatic Microbial
3. Costanzo SDJ, Murby, Bates J.
Ecology. 2009; 22: 111-17.
Ecosystem response to antibiotics
11. Perez-Burgos R, Grzelak EM, Gokce G,
entering the aquatic environment.
Saurina J, Barbosa J, Barron D.
Marine Pollution Bulletin. 2005; 51:
Quechers methodologies as an
218-23.
alternative to solid phase extraction
20
Pharmaceutical Journal o f Sri Lanka 2017 Volume 7, Issue 1

(SPE) for the determination and 18. Liyanage GY, Manage PM. Occurrence,
characterization of residues of fate and ecological risk of antibiotics in
cephalosporins in beef muscle using hospital effluent water and sediments
LC-MS/MS. Journal of in Sri Lanka, International Journal of
Chromatography B. 2012; 899: 57-65. Agriculture and Environmental
12. Butler MS, Buss AD. Natural Research. 2016; 4: 909-35.
products--the future scaffolds for novel 19. Nikokar I, Tishayar A, Flakiyan Z,
antibiotics? Biochem Pharmacol. 2006; Alijani, K, Rehana-Banisaeed S,
71(7): 919-29. Hossinpou M. et al. Antibiotic
13. Shah SQ, Cabello FC, LAbee-Lund resistance and frequency of class 1
TM, Tomova A, Godfrey HP. integrons among Pseudomonas
Buschmann AH. et al. Antimicrobial aeruginosa, isolated from burn patients
resistance and antimicrobial resistance in Guilan, Iran. Iranian journal of
genes in marine bacteria from salmon microbiology. 2013; 5(1): 36-41.
aquaculture and non-aquaculture 20. Fernandez-Torres MO, Consentino MA,
sites. Environmental microbiology. Bello Lopez, Larsen, JL. Simultaneous
2014; 16(5): 1310-20. determination of 11 antibiotics and their
14. Pham DK., Chu J, Do NT, Brose F, main metabolites from four Different
Degand G, Delahaut P. et al. Monitoring groups by reversed-phase high
antibiotic use and residue in freshwater performance liquid chromatography-
aquaculture for domestic use in diode array -fluorescence (HPLC-DAD-
vietnam. EcoHealth. 2015; 12(3): 480­ FLD) in human urine sample, Talanta.
89. 2010; 81: 871-80.
15. Liyanage GY, Manage PM. 21. Kim SJ, Ogo M, Oh MJ, Suzuki S.
Quantification of Oxytetracycline and Occurrence of tetracycline resistant
Amphicillin in two waste water bacteria and tet (M) gene in seawater
discharging points in Colombo, Sri from Korean coast. Interdisciplinary
Lanka, Journal of Environment and studies on environmental chemistry—
Natural Resources. 2014;129-30. Environmental pollution and
16. Baquero F, Martinez JL, Canton R. ecotoxicology. TERRAPUB, Tokyo.
“Antibiotics and antibiotic resistance in 2012; 367-75.
water Environments,” Current Opinion 22. Manage PM, Zen’ichiro K., Shin-ichi N.
Biotechnology. 2008; 18: 123-34. Algicidal effect of the bacterium
17. Liyanage GY, Manage PM. Presence of Alcaligenes denitrification on
Tetracycline and Oxytetracycline Microcystis spp. Aquatic Microbial
Resistant Bacteria and Resistant Genes Ecology.2000; 22: 111-17.
in Effluent Water of Zoological Garden, 23. CLSI guide lines. 2015. [cited 2016 Nov
Sri Lanka. Proceeding of 11th 03]. Available from:http://clsi.org/wp-
International Academic Conference on content/uploads/sites/14/2013/07/CLSI-
Development in Science and 2015-Catalog.pdf
Technology (IACDST-2015). 2015; 11­
14.

21
Pharmaceutical Journal o f Sri Lanka 2017 Volume 7, Issue 1

24. Fedorova G, Nebesky V, Randak T, 28. Marti E, Variatza E, Balcazar JL. The
Grabic R. Simultaneous determination role of aquatic ecosystems as reservoirs
of 32 antibiotics in aquaculture products of antibiotic resistance. Trends in
using LC-MS/MS. Chemical Papers. microbiology. 2014; 22(1): 36-41.
2014; 68(1): 29-3 29. Gueimonde M, Sanchez B, De los
25. Pruden A, Larsson DJ, Amezquita A, Reyes-Gavilan CG, Margolles A.
Collignon P, Brandt KK., Graham DW. Antibiotic resistance in probiotic
et al. Management options for reducing bacteria. Front Microbiol, 2013; 4(202):
the release of antibiotics and antibiotic 1-6.
resistance genes to the 30. Suga N, Ogo M, Suzuki S. Risk
environment.Environmental Health assessment of oxytetracycline in water
Perspectives. 2013; 121(8): 878- 94. phase to major sediment bacterial
26. Connor SO, Aga DS. Analysis of community: a water-sediment
tetracycline antibiotics in soil; advances microcosm study. Environmental
in Extraction, clean-up, and toxicology and pharmacology.
quantification. TrAC Trends in 2013; 36(1): 142-48.
Analytical chemistry. 2007; 26: 456-65. 31. Foley CA. Pennsylvania Veterinarian
27. WHO. Tuberculosis MDR-TB & XDR- Perspectives of Antibiotic Use and
TB 2011 Progress Report. 2013 [cited Antibiotic Resistance; The Pennsylvania
2016 Oct 24]. Available from: State University: 2014.
http://www.who.int/tb/challenges/mdr/fa
ctsheet_mdr_progress_march2011.pdf.
Accessed 12 May 2016

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