Bacteria isolation from soil

1. A Project Report
On
“ISOLATION AND CHARACTERIZATION OF
BACTERIA FROM Agron remedies pvt ltd”
A Project Submitted
In Partial Fulfillment of the Requirements for the Degree of
Master of Science
By
Student name Suneel kumar
Under the Supervision of
Mrs Purnima chuahan,
Assistant Professor,
IFTMU,
Moradabad.
To the
Department of Biotechnology
IFTM University, Moradabad
Session 2022-2023

2. CERTIFICATE
Certified that Suneel kumar has carried out the project work presented in this thesis entitled
“ISOLATION AND CHARACTERIZATION OF BACTERIA FROM SIOL IN AGRON
REMEDIES PVT LTD” for the award of Master of Science from IFTM University, Moradabad under
my/our (print only that is applicable)supervision.The thesis embodies results of original work and studies
are carried out by the student himself/herself(print only that is applicable)and the contents of the thesis
do not form the basis for the award of any other degree to the candidate any body else from this or any
other University/Institution.
Mrs. Purnima Chauhan
Assistant Professor,
IFTM University,
Moradabad,

3. CONTENT
S. NO. TITLE Page no.
1 Introduction
2 Aim and Objectives
3 Review of literature
4 Materials and methods
5 Results and Discussion
6 Conclusion
7 References

4. ACKNOWLEDGEMENT
I am using this opportunity to express my gratitude to everyone who supported me throughout the
course of this Msc (microbiology) project on “Isolation and characterization of bacteria from soil.”
I am thankful for inspiring guidance, invaluably constructive criticism and friendly advice during
the project work. I would like to thank my project supervisor Mrs. Purnima Chauhan and all
the people who provide me with the facilities being required and conductive condition for my
project. I am sincerely grateful to them for sharing their truthful and illuminating views on the
number of issue related to the project.
Thank you
Suneel kumar 20183029

5. CHAPTER -1
INTRODUCTION
INTRODUCTION
Bacteria are the smallest and most numerous of the free living microorganism in
soil.Bacteria are the most abundant microbes in the soil. They are single celled organisms, and
there can be billions of bacteria in a single gram of soil. Populations of bacteria can boom or bust

6. in the space of a few days in response to changes in soil moisture, soil temperature or carbon
substrate. Some bacteria species are very fragile and may be killed by slight changes in the soil
environment. Others are extremely tough, able to withstand severe heat, cold or drying. Some
bacteria are dependent on specific plant species [1]. Therefore, antibiotic resistance is not only
found in pathogenic bacteria but also in environmental organisms inhabiting terrestrial and aquatic
habitats. Higher numbers of resistant bacteria occur in polluted habitats compared with unpolluted
habitats, indicating that humans have contributed substantially to the increased proportion of
resistant bacteria occurring in the environment [2]. Antibiotics exert a selection in favor of resistant
bacteria by killing or inhibiting growth of susceptible bacteria, resistant bacteria can adapt to
environmental conditions and serve as vectors for the spread of antibiotic resistance [3]. The
evolution and spread of antibiotic resistance in pathogenic bacteria is one of the most urgent
challenges in public health today [4]. However, most antibiotics used in medicine today are derived
from biomolecules and secondary metabolites produced by soil-dwelling microorganisms [5].
While the biosynthesis and the role of antibiotics in microbial ecosystems is a matter of active
investigation [6]. Antibiotic resistance (ABR) is a major global public health problem, which has
attracted considerable attention and research efforts in the recent past. The factors associated with
the global rise in ABR are multi-factorial and thus require multi-pronged strategies to prevent
further development and spread of resistance [7].The realization of the multi-factorial nature of
causes governing antibiotic use and ABR has brought into focus the need for an integrated
approach to study the problem in its entirety and points to the necessity to devise comprehensive
interventions addressing multi-factorial issues the ‘One health’ approach. ‘One health’ is defined
as “the collaborative multi-disciplinary team-working locally, nationally, and globally — to attain
optimal health for- people, animals and the environment[8]. Indeed, genomic and phylogenetic
analyses of β-lactamases, a group of enzymes that degrade penicillin and other β-lactam
antibiotics, predict that precursors of the enzymes originated and diversified in bacteria millions
of years ago [9]. Under thick layers of ice and soil, bacteria found in permafrost have been
unaffected by physical and biological factors experienced at the surface for thousands of years
[10]. Given current coring and sampling methods, it is now possible to extract from such ancient
milieus culturable cells or DNA free from surface contaminants [11]. For example, bacterial efflux

7. pumps of the resistance-nodulation-division (RND) superfamily can confer resistance to
antibiotics, transport hydrophobic proteins involved in cell division [12].
All antibiotic use, especially irrational use like overuse is since long one of the major concern for
development and spread of ABR in humans [13]. There have however been a limited number of
studies aiming to identify context specific barriers and facilitators for appropriate clinical
management of infectious diseases in lower middle[ income countries like India. Identifying
barriers and facilitators is a major challenge in designing and implementing successful
interventions in general and forchild health in particular [14]. There is need to document the current
prescribing patterns along with knowledge and attitudes in relation to treatment of common
infectiousetiologies by health care practitioners on one hand and also to understand the community
health seeking behaviour on the other hand in order to limit the spread of ABR [15]. Apart from
fundamental applications in preventing and treating infections in humans, antibiotics are used e.g.
in agriculture [16]. Due to incomplete metabolism of ingested antibiotics or disposal of unused
antibiotics, antibiotics enter the environment [17]. In environment, antibiotic residues might
induce the development of ABR genes in the bacteria. There is worldwide concern about
emergence of ABR in bacteria carried by healthy individuals and in individuals in the community
treated with antibiotics [18]. E. coli of animal, humans and environmental origin including that
from water sources serve as natural habitats for ABR genes [19]. As mentioned above, antibiotic
residues in the environment not only alter the ecology of the environment but also give rise to
selection of ABR. It has also been found that the biophysical and socio-behavioral environment
example prescriber behavior and attitudes modifies antibiotic use and ABR [20]. The extensive
use of antibiotics in clinical therapy of human infectious diseases and in animal husbandry during
the past 50 years has resulted in the emergence and rapid global spread of antibiotic resistance
determinants [21]. Tetracycline inhibits bacterial growth by interfering with protein synthesis
when the antibiotic binds to the 30S ribosomal subunit thereby preventing aminoacyl-t-RNA
binding to the ribosomal-A site and preventing synthesis of polypeptides [22]. Once resistance
genes are introduced into the environment, they are also exposed to selective pressure, such as

8. antibiotics produced by indigenous antibiotic producers in soil. However, selection can occur in
the environment without antibiotic selective pressure [23].
There are about 50 heavy metals that are of special concern for their toxicological importance to
human health and many of them, like Zn, Cu, Ni, Fe and Mn are also essential trace elements for
living organisms. However, if these metals accumulate at high level or are ingested in greater
amounts than the required concentration, then they can cause serious health problem [24]. The
micro-organisms respond to these heavy metals by several processes including bio-sorption to the
cell walls and entrapment in extracellular capsules, transport across the cell membrane,
precipitation, oxidation-reduction reactions and complications [25]. Some industrial processes
results in the release of heavy metals into aquatic systems. This has led to increasing concern about
the effect of toxic heavy metals as environmental pollutants. This kind of contamination presents
a challenge, as the presence of heavy metals in soils and aqueous effluents leads to serious
problems because they cannot be biodegraded. Unlike many other pollutants, heavy metals are
difficult to remove from the environment [26].

9. CHAPTER-2
AIM AND OBJECTIVES
AIM: Isolation and characterization of bacteria from soil brass industries.

10. 1.Sample collection from soil
2.Serial dilution
3.Isolation of bacterial strain from soil
4.Characterization
5.Isolation of DNA

11. CHAPTER -3
REVIEW OF LITERATURE
REVIEW OF LITREATURE

12. Despite an exceptional number of bacterial cells and species in soils, bacterial diversity
seems to have little effect on soil processes, such as respiration or nitrification that can be affected
by interactions between bacterial cells. The aim of this study is to understand how bacterial cells
are distributed in soil to better understand the scaling between cell-to-cell interactions and what
can be measured in a few milligrams, or more, of soil [27]. Water constitutes not only a way of
dissemination of antibiotic-resistant organisms among human and animal populations, as drinking
water is produced from surface water, but also the route by which resistance genes are introduced
in natural bacterial ecosystems. In such systems, nonpathogenic bacteria could serve as a reservoir
of resistance genes and platforms. Moreover, the introduction (and progressive accumulation) in
the environment of antimicrobial agents, detergents, disinfectants, and residues from industrial
pollution, as heavy metals, contributes to the evolution and spread of such resistant organisms in
the water environment. The heavy use of prophylactic antibiotics in aquaculture [28] can be
particularly relevant. On the contrary, environmental bacteria act as an unlimited source of genes
that might act as resistance genes when entering in pathogenic organisms [29], the set of genes
able to be converted in antibiotic-resistance genes. Human health risk assessment protocols for
antibiotic and resistant bacteria in water are starting to be discussed [30]. Antibiotic resistance
evolves in bacteria because of the effect of industrially produced antimicrobial agents on bacterial
populations and communities. Genetic reactors are places in which the occasion occurs for genetic
evolution, particularly because of high biological connectivity, generation of variation, and
presence of specific selection. Beyond mutational events, significant genetic variation occurs as a
consequence of re-combinatorial events, frequently resulting from genetic exchanges among
organisms inside populations and communities. There are four main genetic reactors in which
antibiotic resistance evolves. The primary reactor is constituted by the human and animal micro-
biota, with more than 500 bacterial species involved, in which therapeutic or preventive antibiotics
exert their actions. The secondary reactor involves the hospitals, long-term care facilities, farms,
or any other place in which susceptible individuals are crowded and exposed to bacterial exchange.
The tertiary reactor corresponds to the wastewater and any type of biological residues originated
in the secondary reactor, including for instance lagoons, sewage treatment plants, or compost
toilets, in which bacterial organisms from many different individuals have the opportunity to mix
and genetically react. The fourth reactor is the soil and the surface or ground water environments,

13. where the bacterial organisms originated in the previous reactors mix and counteract with
environmental organisms. Water is involved as a crucial agent in all four genetic reactors, but
particularly in the last ones. The possibility of reducing the evolvability of antibiotic resistance
depends onthe ability of humans to control the flow of active antimicrobial agents, bacterial clones,
and genetically based biological information along these genetic reactors.Binding to soil particles
(and sediments) delays its biodegradation and explains longterm permanence of the drugs in the
environment. Of course, soil particles also remove antibiotics from water, so that a kind of water–
soil pharmacokinetics might be considered. Antimicrobial agents are retained in soil by its
association with soil chemicals. For instance, Elliot soil humic acids produce complexation of
antibiotics [31]. Human and animal pathogenic and potentially pathogenic bacteria are constantly
released with wastewater into the water environment. Many of these organisms harbor antibiotic-
resistance genes, eventually inserted into genetic mobile platforms (plasmids, transposons,
integrons) able to spread among water and soil bacterial communities [32].
All these applications made antibiotics to be released in large amounts in natural ecosystems. Little
is known on the overall effects of antibiotics on the population dynamics of the microbiosphere.
However, the effect of antibiotics used for treating infections or for farming purposes in the
selection of antibiotic-resistant microorganisms, which can impact human health has been studied
in more detail [33]. Antibiotics at much higher concentrations that usually found in natural
ecosystems can be found in soils (e.g. soils treated with manure and farm soils). However, these
high concentrations are usually concentrated to areas of human activity, whereas pristine
environments usually have low concentrations of antibiotics [34]. Risk assessments might thus
take into consideration mainly those areas with high antibiotic load and containing human-
associated microorganisms (reactors for evolution of resistance see for analyzing the effect of
antibiotic pollution on natural ecosystems [35]. Since antibiotics are efficient inhibitors of bacterial
growth produced by environmental microorganisms, it has been widely accepted that their role in
nature will be to inhibit microbial competitors. Conversely, antibiotic resistance determinants
should serve to avoid the activity of antibiotics, in such a way that they would be a good example
of the Darwinian struggle for life. Although this can be true in some occasions, an alternative
hypothesis stating that antibiotics could be signal molecules that shape the structure of microbial
communities has been proposed. Similarly, it has been stated that some elements that serve to resist

14. high concentrations of antibiotics, have disparate functional roles (e.g. cell homeostasis, signal
trafficking, metabolic enzymes) in their original hosts.The strong increase of antibiotic
concentrations in natural ecosystems as the consequence of human activities (human therapy,
farming) shifts the original functions of antimicrobials and resistance elements to the
weapon/shield roles they play in hospitals or farms [36]. Besides selecting antibiotic-resistant
mutants and favoring the acquisition of antibiotic resistance determinants by gene-transfer
elements that can spread among the environmental microbiota, antibiotic pollution can enrich the
population of intrinsically resistant microorganisms, and reduce the population of susceptible
microbiota. Cyanobacteria, which are responsible of more than a third of total free oxygen
production and carbon-dioxide fixation, are susceptible to antibiotics. There is not at the moment
any indication that the Cyanobacteria population is suffering the impact of antibiotic pollution,
and the risks for this situation are likely very low. However, the dramatic effect that eliminating
Cyanobacteria as the consequence of antibiotic pollution might have for the biosphere reinforce
the idea that the release of antibiotics in natural environments have relevant consequences not just
in terms of resistance but for the maintenance of the global activity of the microbiosphere also. in
the structure of suspended and attached algae, in the nutrient processing capacity and in the natural
food web of the ecosystems .A similar study has demonstrated that tetracycline have a negative
impact on the functional diversity of soil microbial communities [37,38].
Antibiotic utilization for clinical or farming purposes selects resistant microorganisms .It is thus
predictable that residues from hospitals or farms will contain both types of pollutants: antibiotics
and resistance genes. Nevertheless, the fate of both types of pollutants is likely different. Several
antibiotics are natural compounds that have been in contact with environmental microbiota for
millions of years and are thus biodegradable, an even serve as a food resource for several
microorganisms [39].
The fact that antibiotics are degraded in natural ecosystems does not mean that they are not relevant
pollutants. For instance the degradation process is slow at low temperatures in winter and the
composition and moisture of the soil clearly impact antibiotic degradation [40]. More important,
some ecosystems suffer a constant release of antibiotics (e.g. hospital effluents, farms residues),
so that they are constantly polluted irrespectively of antibiotic degradation. As stated by Lindberg

15. et al. ecotoxicity tests are usually performed using very high concentrations of antibiotics for short
periods of time, whereas in these types of environments, the organisms are continuously exposed
to antibiotics at sub-therapeutic levels .Since sub-inhibitory concentrations of antibiotics trigger
specific transcriptional responses in bacteria .the presence of antibiotics will necessarily modify
the metabolic activity of the microbiota present in these polluted environments. Finally, the impact
on the structure of bacterial populations due to the presence of antibiotics might remain even when
the antimicrobials have been mineralized .In any case, the fate of antibiotics in natural ecosystems
is their degradation [41]. First, utilization on antibiotics can select for antibiotic-resistant bacteria
within the treated host. In the case of antibiotics used for farming purposes, selection of resistance
can be important for both the treatment of animal infections and for human health. Several
evidences support an association between the use of antimicrobial agents in food animals and
antimicrobial resistance among bacteria isolated from humans [42].
The effect of antibiotics used for farming in human health has mainly focused on foodborne
pathogens. These bacteria are present in the animals and can infect humans. Examples of
foodborne pathogens are Campylobacter jejuni, E. coli, SalmonellaorEnterococcus faeciumamong
others[43]. For those pathogens, both mutation-driven antibiotic resistance and the acquisition of
antibiotic resistance genes are important concerns for human health, because the same strain can
colonize both animals and humans, and antibiotic resistance genes can easily spread among
bacterial species (or clones) that are closely phylogenetically related [44].
First, antibiotic selective pressure in natural ecosystems may select the integration and further
dissemination of antibiotic resistance genes in gene-transfer units, which can be then considered
as contaminants. A good example of this situation is the quinolone-resistance gene (qnr), which is
chromosomally encoded in several water-borne bacteria. It has been shown that contamination of
river waters by quinolones favours the integration of the qnr gene into plasmids and its further
dissemination among natural ecosystems that can be geographically distant [45].
Second, residues from hospitals, houses and farms contain bacteria that can carry antibiotic
resistance determinants. Search of specific antibiotic resistance genes in the sediments of Cache

16. La Poudre River, which have high concentrations of antibiotics related to urban and agricultural
activities showed the presence of resistance genes in all sites, although impacted sites presented
higher concentration of those genes than pristine environments. The finding of specific antibiotic
resistance genes, which are already disseminated among human, animals or plants bacterial
pathogens (or commensals) will be an indication of a history of contamination. Differing to the
situation with antibiotics, this contamination is not necessarily local neither dependent on the
constant release of residues, because once those genes are in the environment, they can disseminate
among different bacterial species and distinct habitats. It has been demonstrated that antibiotic
resistance genes can migrate between connected aquatic systems. It is unclear whether the presence
of antibiotic resistance genes is the result of the migration of antibiotic-resistant bacteria or the
transmission of resistance genes by HGT [46]. Worldwide transport and commercial activities are
helping as well the dissemination of bacteria even between different oceans and continents [47].
For this reason, antibiotic resistance genes firstly reported in human pathogens, are found as well
in several different habitats including those in which antibiotic pollution is very low or even null.
The fact that remote human populations with minimal antibiotic exposure carry antibiotic resistant
commensal bacteria further support the worldwide dissemination of resistance genes [48].
Resistant infections are becoming more difficult or even impossible to treat with current
antibiotics, leading to infections causing higher morbidity and mortality, imposing huge costs on
our society [49]. This increasing resistance involves many common human pathogens, including
Enterococcus faecium, Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae,
Acinetobacter baumannii, Pseudomonas aeruginosa, and other Enterobacter species [50,51].
However, many of these bacteria and/or their modes of resistance came from the natural
environment, including bacteria within soils and water. Antibiotic resistance development is not
just a local public health issue but includes broader environmental influences, which are amplified
by international travel and global trade in foodstuffs. The World Health Organization (WHO)
recently announced a suite of policies that, if implemented, should mitigate the emergence and
further dissemination of antibiotic-resistant organisms [52].

17. These initiatives have focused on antibiotic stewardship in the hospital and community settings,
and reducing antibiotic use in livestock production. However, if we are to better manage antibiotic
resistance, it is also vital that we consider the broader environment. Therefore, an improved
understanding of the impacts of human activities on antibiotic resistance development is needed,
such as nonhuman antibiotic use, pharmaceutical manufacturing waste, domestic and agricultural
waste releases into the environment, and the influence of poor sanitation and unsafe water supplies.
There are emerging concerns that anthropogenic impacts are changing environmental reservoirs
of resistance genes, “the resistome” [53], which will increase the probability of recruitment of
resistance genes into clinically relevant pathogens [54]. For example, wastewater treatment, drug
manufacturing, and agricultural effluents release massive quantities of antibiotic residues and
resistant bacteria, selected in the digestive tracts of people or animals by antibiotic use [55].
Exposure of environmental bacteria to antibiotics as well as to large numbers of resistant bacteria
may accelerate the evolution of resistance, increase the abundance and distribution of resistance
genes within the resistome that is critical to the development of clinical resistance, and increase
exchange of antibiotic resistance genes between bacteria [56,57]. People and animals are
connected to each other through the environment, and it is important to consider antibiotic
resistance within the “One Health” concept, which provides a global strategy for expanding
interdisciplinary collaboration and communication.
ANTIBIOTIC RESISTANCE GENES ARE UBIQUITOUS AND ANCIENT
Our world is inhabited by approximately 5 × 1030 bacteria, the vast majority of which are not
pathogenic. Through evolutionary time, microorganisms developed capabilities for the
biosynthesis of chemicals toxic to bacteria, “antibiotics,” which vary widely in chemical structures,
mode of action, and spectrum of activity. This was paralleled by the development of strategies to
defeat antibiotics. Environmental bacteria, which predate the modern antibiotic era by billions of

18. years, carry genes encoding resistance to antibiotics that have become critically important in
medicine [58]. However, because only approximately 1% of environmental strains are culturable
[59], our knowledge of the true diversity and composition of the environmental resistome is
limited.
The ability to quantitatively link the transfer of specific resistance genes from environmental
strains to human pathogens has been difficult and, grossly underappreciated, although the ancient
nature of environmental resistance is clear. For example, viable multidrug-resistant bacteria were
cultured from the Lechuguilla Cave in New Mexico even though it has been totally isolated for >4
million years [60].
These bacteria were resistant to at least 1 antibiotic and often 7–8 antibiotics, including β-lactams,
aminoglycosides, and macrolides, as well as newer drugs such as daptomycin, linezolid,
telithromycin, and tigecycline. Two distinct new macrolide inactivation mechanism swere
identified, suggesting that the utilization of the environmental microbiome could be used to help
combat resistance through the development of novel antibiotics designed not to be inactivated by
these mechanisms. Likewise, DNA extracted from 30 000-year-old Beringian permafrost
contained genes coding for resistance to β-lactams, tetracyclines, and glycopeptides, confirming
that resistance predates antibiotic use in medicine and agriculture. Furthermore, major β-lactamase
classes predate the existence of humans. Class A β-lactamases evolved approximately 2.4 billion
years ago and were horizontally transferred into the gram-positive bacteria about 800 million years
ago[61]. Overall, these studies provide compelling evidence of the breadth of the resistome in
environmental strains and the intrinsic capacity for all bacteria to gain resistance.
One explanation is that bacteria that produce antibiotics must be resistant to them to avoid self-
destruction. In a highly diverse and competitive microbial environment such as soil, antibiotic-
resistant bacteria will have a competitive advantage against susceptible bacteria. In addition,
antibiotics are products of secondary metabolism, and some have important physiological
functions at different concentrations, including the regulation of gene expression and

19. communication between bacteria [62]. Antibiotics at sub-lethal concentrations can promote
genetic exchanges through multiple pathways involving various stress responses [63].Frequency
of transfer of tetracycline-resistance plasmids in S. aureus was increased by up to 1000-fold in the
presence of sub-inhibitory concentrations of β-lactams [64]. Also, antibiotics in animal feed
induced prophages in swine fecal microbiomes and contributed to phage-mediated resistance gene
transfer [65], highlighting multiple environmental vectors for the horizontal transfer of resistance
genes. Finally, many bacteria, while also resistant to multiple antibiotics, can actually use
antibiotics as their sole carbon source [66]. Overall, the ancient origin of resistance genes
highlights the need to take effective measures to control antibiotic usage in people and animals,
the major drivers for the modern emergence of resistance. Indeed, in Australia, low levels of
resistance to fluoroquinolones in key pathogens have resulted from restricted quinolone use in
humans and absent use in food animals [67].
Human activity since the industrialization of antibiotic production after World War II has changed
the distribution and increased the abundance of resistance genes. Genes encoding resistance were
2–15 times more abundant in 2008 compared to the 1970s in DNA extracted from archived soil
samples collected between 1940 and 2008 in the Netherlands [68]. In particular, genes encoding
resistance to β-lactams and tetracyclines were enriched. Worrisomely, an increase in extended
spectrum β-lactamases (ESBLs) of the CTX-M family was observed, which appears to predate any
clinical detection of theseenzymes. Furthermore, since industrialization, millions of tons of
antibiotics have been released into the environment, including via wastewater effluents, land
application of animal wastes, treatment of crop diseases, aquaculture, and many other activities.
For example, 71% of total Danish antibiotic consumption (kg) in 2010 was for animal production
[69]. A similar trend of antibiotic use in human’s versus animals was also observed in Canada
[70].
Public health impacts from antibiotic use in agriculture and aquaculture have already drawn much
attention in the last decade [71]. Importantly, antibiotics used in humans and animals often belong
to the same classes. The WHO has established a list of “critically important” antibiotics in humans
to ensure prudent drug use in both human and veterinary medicine [72]. The third- and fourth-

20. generation cephalosporins, fluoroquinolones, and macrolides are considered the drugs most
urgently requiring risk management of their use in food animals[73,74]. The use of extra-label
third-generation cephalosporins poses an important challenge [75,76]. The relationship between
antibiotic use and resistance is exemplified in a novel manner by recent work on the longterm
exposure of tetracyclines on honeybees, which showed the accumulation of mobile tetracycline
resistance genes closely related to those from human pathogens in the gut microbiota of bees [77].
Antibiotic are used in large-scale industrial agricultural facilities to raise food animals at high-
density, highlight many public health impacts including increased resistance and decreased water
quality [78,79]. Similarly, impacts from largescale and widespread antibiotic use in aquaculture
need to be addressed. Specifically, fish infections are treated through the administration of
antibiotics directly into the water, avoiding any kind of purification processes [80]. Aquaculture is
increasingly important because fish production has increased substantially over the last 50 years
with 52.5 million tons processed in 2008 [81].
Many antibiotics are excreted unchanged, are environmentally persistent, and can be detected
downstream of wastewater treatment plants and adjacent to fields receiving animal manures [82].
In treated effluents and sewage sludge, antibiotic residues of several classes range in
concentrations from nanograms per liter up to low micrograms per liter [83]. Although these are
well below minimum inhibitory concentrations (MICs), even low concentrations provide selective
advantages for certain resistant strains [84].
We also release large numbers of resistant bacteria that have multiplied exponentially in the
gastrointestinal tracts of people and animals treated with antibiotics. These bacteria, in agricultural
and wastewater effluents, harbor resistance genes and genetic elements that promote their
exchange between bacteria [85,86]. Commensals as well as pathogens are important sources of
resistance genes that can be shared, eventually leading to human infections and disease [87].
Indirect selection for antibiotic resistance also needs to be considered. Resistance mechanisms to
biocides or heavy metals may be present on the same genetic elements as those conferring
resistance to antibiotics [88],

21. Antibiotic resistance can be acquired through mutation of existing DNA, uptake of foreign DNA
by means of transformation or phage-mediated transduction, and/or by conjugation (DNA
exchange directly from other bacteria). Transposition of DNA within genomes also plays an
important role in the mobilization of resistance determinants. Horizontal gene transfer is highly
important in the evolution and transmission of resistance genes between species and includes the
movement of resistance genes from fecal bacteria to environmental bacteria, as well as the reverse;
that is, emergence of novel mechanisms of acquired resistance in pathogens, genes that originally
were present in harmless bacteria [89]. Transduction has been identified to be important in the
exchange of these genes with other organisms, particularly in freshwater [90].
There is an interrelationship between humans, animals, and the environment. Both methicillin-
resistant S. aureus and-producing E. coli can be used as indicators to evaluate the movement of
resistant bacteria in the environment. ESBL-producing bacteria cause serious infections around
the world and can be recovered from foods for human consumption as well as in wildlife [91,92].
The rapid emergence of infections associated with multidrug resistance in Acinetobacter species
has been increasingly observed globally. In the 1970s–1980s, Acinetobacter, a gram-negative
organism commonly found in soil and water, was often susceptible to antibiotics. Today,
Acinetobacter is one of the most difficult resistant gram-negative bacteria to control and treat [93].
Outbreaks have been associated with contamination of the hospital environment and equipment
with multidrugresistant strains introduced into hospitals by returning soldiers [94] and earthquake
survivors [95]. Multidrug-resistant A. baumannii possesses almost all typical mechanisms of
resistance (e.g, multiple β-lactamases including carbapenamases, aminoglycoside-modifying
enzymes, and drug efflux pumps) that render the organism resistant to almost all classes of
antibiotics. Resistance islands in the chromosome of A. baumannii have large numbers of
resistance genes and mobile genetic elements which explains the sophisticated mechanisms of
resistance in this species [96].

22. Bacteria do not live in isolation, but are readily dispersed through the world by humans, animals,
plants, soil, water, and air. An underappreciated exposure route for the dissemination of antibiotic
resistance is water, and multidrug-resistant bacteria have been detected from various water
sources, including drinking water. This is a major concern in developing countries and has been a
major route for the transmission of pathogenic bacteria to people in developed countries in the past
[97]. Consumption or handling of water, whether treated or not, can lead to the colonization of the
gastrointestinal tract in humans and animals with bacteria containing resistance genes. This in turn,
can result in exchange of genes with bacteria (commensal or pathogenic) already present in the
human/ animal gut. In addition, water is used for the irrigation of plants for animal and human
consumption, contaminating products that could also lead to human/animal colonization with
antibiotic-resistant organisms [98].
CHAPTER-4
MATERIALS AND METHODS

23. MATERIALS AND METHOD
4.1.Collection of samples:
Soil sample was collected from the near brass industry in Moradabad U.P. The soil sample was
sieved to extract fine soil particle which were then serially diluted for isolation of bacteria.
4.2 Nutrient agar media:-

24. Nutrient agar is used as a general purpose medium for the growth of a wide variety of non-
fastidious microorganisms. It consists of peptone, beef extract and agar. This relatively simple
formulation provides the nutrients necessary for the replication of a large number of non-fastidious
microorganisms. Nutrient Agar/broth is used for the cultivation and maintenance of non-fastidious
organisms as well as enumeration of organisms in water, sewage, dairy products, feces and other
materials.
Composition of nutrient agar:-
Beef extract is an aqueous extract of lean beef tissues. It contains water-soluble substances of
animal tissue, which include carbohydrates, organic nitrogen compounds, water soluble vitamins,
and salts.
Peptone is made by digesting proteinaceous materials e.g., meat, casein, gelatin, using acids or
enzymes. Peptone is the principal source of organic nitrogen and may contain carbohydrates or
vitamins. Depending up on the nature of protein and method of digestion, peptones differ in their
constituents, differing in their ability to support the growth of bacteria.
Agar is a complex carbohydrate obtained from certain marine algae. It is used as a solidifying
agent for media and does not have any nutritive value. Agar gels when the temperature of media
reaches 45°C and melts when the temperature reaches 95 °C.
4.3 Serial Dilution Method:
1 gram of soil was weighed and mixed in 10 ml of distilled water to get 1:10 dilution, then
thoroughly mixed by vigorousshaking after allowing the sediment to settles. Supernatant was used
for subsequent dilution. Dilutions were prepared by taking one ml of stock solution (having 1:10
dilution) and transferring in to 9 ml sterile distilled water in another test tube to give 1:100. 0.1ml
Peptone 0.5g
Beef extract 0.3g
Agar 1.5g
NaCl 0.5g

25. of soil inoculum from each dilution was taken and inoculated by separately on to Petridishes with
nutrient agar media of pH 7-7.2 plates were incubated at room temperature for two days in inverted
position.
Material required:
1. Test tubes
2. Test tubes stands
3. Distilled water
4. Micropipette
Procedure:
1.Suspend one gram soil in the test tube containing 9ml steriledistilled water and shake the tube
on a shaker.
2.Perform serial dilution technique up 10-1
10-2 10-3 10-4 10-5 and 10-6 dilution.
3.Vigrously shake the dilution on a rotary shaker to obtain uniform suspension of microorganisms.
4.Transfer aliquots of 0.1ml suspension form 10-5 dilution blank on sterilized Petridishes.
4.4 Pour plate method:
Pour plate method is usually the method of choice for counting the number of colony-forming
bacteria present in a liquid specimen. In this method, fixed amount of inoculums (generally 1 ml)
from a broth/sample is placed in the center of sterile Petri dish using a sterile pipette. Molten cooled
agar (approx. 15mL) is then poured into the Petri dish containing the inoculums and mixed
well. After the solidification of the agar, the plate is inverted and incubated at 37°C for 24-48
hours.
Procedure:

26. 1. Inoculate labeled empty petri dish with specified mL (0.1 or 1.0 mL) of diluted specimen
2. Collect one bottle of sterile molten agar (containing 15 mL of melted Plate Count Agar or any
other standard culture media) from the water bath (45°C).
3. Pouring the molten agar medium.
4. Hold the bottle in the right hand; remove the cap with the little finger of the left hand.
5. Flame the neck of the bottle.
6. Lift the lid of the Petri dish slightly with the left hand and pour the sterile molten
agar into the Petri dish and replace the lid.
7. Flame the neck of the bottle and replace the cap.
8. Gently rotate the dish to mix the culture and the medium thoroughly and to ensure that the
medium covers the plate evenly.
9. Do not slip the agar over the edge of the petri dish.
10. Allow the agar to completely Seal and incubate the plate in an inverted position at 37°C for
24-48 hours gel without disturbing it, it will take approximately 10 minutes.
4.5. Spread Plate Technique
The spread plate technique involves using a sterilized spreader with a smooth surface made of
metal or glass to apply a small amount of bacteria suspended in a solution over a plate. The plate
needs to be dry and at room temperature so that the agar can absorb the bacteria more readily. A
successful spread plate will have a countable number of isolated bacterial colonies evenly
distributed on the plate.
Material required:
1. Spreader L shape
2. Bunsen burner
3.Micropipette
Procedure:

27. 1. Make a dilution series from a sample.
2. Pipette out 0.1 ml from the appropriate desired dilution series onto the center of the surface of
an agar plate.
3. Dip the L-shaped glass spreader into alcohol.
4. Flame the glass spreader (hockey stick) over a Bunsen burner.
5. Spread the sample evenly over the surface of agar using the sterile glass spreader, carefully
rotating the Petridish underneath at the same time.
6. Incubate the plate at 37°C for 24 hours.
7. Calculate the CFU value of the sample. Once you count the colonies, multiply by the
appropriate dilution factor to determine the number of CFU/mL in the original sample.
4.6 Streak plate method:
The streak plate method is a rapid qualitative isolation method. The techniques commonly used
for isolation of discrete colonies initially require that the number of organisms in the inoculums be
reduced. It is essentially a dilution technique that involves spreading a loopful of culture over the
surface of an agar plate. The resulting diminution of the population size ensures that, following
inoculation, individual cells will be sufficiently far apart on the surface of the agar medium to
effect a separation of the different species present. Although many type of procedures are
performed, the four ways or quadrant streak is mostly done.
Materials required:
1. Inoculation loop,
2. A striker/lighter
3. Bunsen burner,
4. Agar plate (Nutrient agar or any other agar medium)
Procedure:

28. 1. Sterilize the inoculating loop in the bunsen burner by putting the loop into the flame until it is
red hot. Allow it to cool.
2. Pick an isolated colony from the agar plate culture and spread it over the first quadrant
(approximately 1/4 of the plate) using close parallel streaks or Insert your loop into the
tube/culture bottle and remove some inoculum. You don’t need a huge chunk.
3. Immediately streak the inoculating loop very gently over a quarter of the plate using a back
and forth motion.
4. Flame the loop again and allow it to cool. Going back to the edge of area 1 that you just streaked,
extend the streaks into the second quarter of the plate.
5. Flame the loop again and allow it to cool. Going back to the area that you just streaked (area
2), extend the streaks into the third quarter of the plate.
6. Flame the loop again and allow it to cool. Going back to the area that you just streaked (area
3), extend the streaks into the center fourth of the plate.
7. Flame your loop once more.
4.7.Gram Staining:
Gram staining differentiates bacteria by the chemical and physical properties of their cell walls by
detecting peptidoglycan, which is present in the cell wall of Gram-positive bacteria. Gram-positive
bacteria retain the crystal violet dye, and thus are stained violet, while the Gram-negative bacteria
do not; after washing, a counterstain is added (commonly safranin or fuchsine) that will stain these
Gram-negative bacteria a pink color. Both Gram-positive bacteria and Gram-negative bacteria pick
up the counterstain. The counterstain, however, is unseen on Gram-positive bacteria because of
the darker crystal violet stain.
The Gram stain is almost always the first step in the preliminary identification of a bacterial
organism. While Gram staining is a valuable diagnostic tool in both clinical and research settings,
not all bacteria can be definitively classified by this technique. This gives rise to gram-
variable and gram-indeterminate groups.
Gram staining is a bacteriological laboratory technique[
used to differentiate bacterial species into
two large groups (gram-positive and gram-negative) based on the physical properties of their cell

29. walls.Gram staining is not used to classify archaea, formerly archaeabacteria, since these
microorganisms yield widely varying responses that do not follow their phylogenetic groups.
The Gram stain is not an infallible tool for diagnosis, identification, or phylogeny, and it is of
extremely limited use in environmental microbiology. It is used mainly to make a preliminary
morphologic identification or to establish that there are significant numbers of bacteria in a clinical
specimen. It cannot identify bacteria to the species level, and for most medical conditions, it should
not be used as the sole method of bacterial identification. In clinical microbiology laboratories, it
is used in combination with other traditional and molecular techniques to identify bacteria. Some
organisms are gram-variable (meaning they may stain either negative or positive); some are not
stained with either dye used in the Gram technique and are not seen. In a modern environmental
or molecular microbiology lab, most identification is done using genetic sequences and other
molecular techniques, which are far more specific and informative than differential staining.
4.7.1 Gram-positive bacteria:
Gram-positive bacteriaarebacteriathatgive apositiveresult inthe Gram staintest. Gram-positivebacteriatakeup
thecrystalvioletstainusedinthetest,andthenappeartobepurple-colouredwhenseenthroughamicroscope.This
isbecausethethickpeptidoglycan layerinthebacterialcellwallretainsthestainafteritiswashedawayfromtherest
ofthesample,inthedecolorizationstageofthetest.
4.7.2 Gram negative bacteria
Gram-negativebacteriacannotretainthevioletstainafterthedecolorizationstep;alcoholusedinthisstagedegrades
theoutermembraneofgram-negativecellsmakingthecellwallmoreporousandincapableofretainingthecrystal
violet stain. Their peptidoglycan layer is much thinner and sandwiched between an inner cell membrane and
abacterialoutermembrane,causingthemtotakeupthe counterstain(safraninorfuchsine)andappearredorpink..
Materials Required:

30. 1.Clean glass slides
2. Inoculating loop
3. Bunsen burner
4. Bibulous paper
5. Microscope
6. Lens paper and lens cleaner
7. Immersion oil
8.Distilled water
9.18 to 24 hour cultures of organisms
Reagents:
1.Primary Stain - Crystal Violet
2.Mordant - Grams Iodine
3.Decolourizer - Ethyl Alcohol
4. Secondary Stain - Safranin
Procedure :
1.Place slide with heat fixed smear on staining tray.
2. Gently flood smear with crystal violet and let stand for 1 minute.
3. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle.
4.Gently flood the smear with Gram’s iodine and let stand for 1 minute.
5. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle.
The smear will appear as a purple circle on the slide.

31. 6.Decolorize using 95% ethyl alcohol or acetone. Tilt the slide slightly and apply the alcohol
drop by drop for 5 to 10 seconds until the alcohol runs almost clear. Be careful not to over-
decolorize.
7. Immediately rinse with water.
8.Gently flood with safranin to counter-stain and let stand for 45 seconds.
9.Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle.
10. Blot dry the slide with bibulous paper.
11.View the smear using a light-microscope under oil-immersion.
4.8 Antibiotic sensitivity test:
The introduction of various antimicrobials for treating variety of infections showed the necessity
of performing antimicrobial susceptibility testing as a routine procedure in all microbiology
laboratories. In laboratories it can be made available by using antibiotic disk which will diffuse
slowly into the medium where the suspected organism is grown. The basic principle of the
antibiotic susceptibility testing has been used in microbiology laboratories over 80 years. Various
chemical agents such as antiseptics, disinfectants, and antibiotics are employed to combat with the
microbial growth. Among these, antibiotics are generally defined as the substances produced by
the microorganism such as Penicillium, which has the ability to kill or inhibit the growth of other
microorganisms, mainly bacteria. Antimicrobial susceptibility tests (ASTs) basically measures the
ability of an antibiotic or other antimicrobial agent to inhibit the invitro microbial growth.
There are many different procedures that microbiologists use to study the effects of various
antimicrobial agents in treating an infection caused by different microorganisms. Mueller Hinton
Agar is considered as best for the routine susceptibility testing since it is has batch-to-batch
reproducibility, low concentration of inhibitors of sulphonamide, trimethoprim and tetracyclines
and produce satisfactory results for most of the non-fastidious pathogens. Fastidious organisms
which require specific growth supplements need different media to grow for studying the
susceptibility patterns.The Kirby Bauer test is a qualitative assay whereby disks of filter paper are

32. impregnated with a single concentration of different antibiotics or any chemicals that will diffuse
from the disk into the agar. The selected antibiotic disks are placed on the surface of an agar plate
which has already been inoculated with test bacteria. During the incubation period, the
antibiotics/chemicals diffuse outward from the disks into the agar. This will create a concentration
gradient in the agar which depends on the solubility of the chemical and its molecular size. The
absence of growth of the organism around the antibiotic disks indicates that, the respected
organism is susceptible to that antibiotic and the presence of growth around the antibiotic disk
indicates the organism is resistant to that particular antibiotic. This area of no growth around the
disk is known as a zone of inhibition, which is uniformly circular with a confluent lawn of growth
in the media.
4.9 Endospore staining:
Endospore staining is a technique used in bacteriology to identify the presence of endospores in a
bacterial sample, which can be useful for classifying bacteria. Within bacteria, endospores are
quite protective structures used to survive extreme conditions, but this protective nature makes
them difficult to stain using normal techniques. Special techniques for endospore staining include
the Schaeffer–Fulton stain and the Moeller stain. A good stain to use for spore staining is malachite
green. It takes a long time for the spores to stain due to their density, so time acts as the mordant
when doing this differential stain; the slide with the bacterium should be soaked in malachite green
for at least 30 minutes. Water acts as the decolorizer. A counterstain to differentiate the vegetative
cells is 0.5% safranin. Types of endospores that could be identified are free endospores, central
endospores, central and swollen endospores, and subterminal endospores.
Materials Required:
1. Clean glass slides
2. Inoculating loop
3. Bunsen burner
4. Bibulous paper
5. Microscope

33. 6. Lens paper and lens cleaner
7. Immersion oil
8.Distilled water
Reagents:
1. Malachite green (dye)
2.Safranin stain
Procedure:
1.Prepare smears of organisms to be tested for presence of endospores on a clean microscope slide
and air dry it.
2.Heat fix the smear.
3.Place a small piece of blotting paper (absorbent paper) over the smear and place the slide (smear
side up) on a wire gauze on a ring stand.
4.Heat the slide gently till it starts to evaporate (either by putting the slide on a staining rack that
has been placed over a boiling water bath or via bunsen burner).
5.Remove the heat and reheat the slide as needed to keep the slide steaming for about 3-
5 minutes. As the paper begins to dry add a drop or two of malachite green to keep it moist, but
don’t add so much at one time that the temperature is appreciably reduced.
6.After 5 minutes carefully remove the slide from the rack using a clothespin.
7.Remove the blotting paper and allow the slide to cool to room temperature for 2 minutes.
8.Rinse the slide thoroughly with tap water (to wash the malachite green from both sides of the
microscope slide).
9.Stain the smear with safranin for 2 minutes.
10.Rinse both side of the slide to remove the secondary stain and blot the slide/ air dry.

34. 4.10.Catalase test:
The enzyme catalase mediates the breakdown of hydrogen peroxide into oxygen and water. The
presence of the enzyme in a bacterial isolate is evident when a small inoculum is introduced into
hydrogen peroxide, and the rapid elaboration of oxygen bubbles occurs. The lack of catalase is
evident by a lack of or weak bubble production. The culture should not be more than 24 hours old.
H2O2H2O + O2
Catalase
Bacteria thereby protect themselves from the lethal effect of Hydrogen peroxide which is
accumulated as an end product of aerobic carbohydrate metabolism.
Materials Required:
1. Clean glass slides
2. Inoculating loop
3. Bunsen burner
Reagents:
1. Hydrogen peroxide
Procedure of catalase test (Slide Test):
1.Transfer a small amount of bacterial colony to a surface of clean, dry glass slide using a loop or
sterile wooden stick
2.Place a drop of 3% hydrogen peroxide on to the slide and mix.
3.A positive result is the rapid evolution of oxygen (within 5-10 sec.) as evidenced by bubbling.
4.A negative result is no bubbles or only a few scattered bubbles.
5.Dispose of your slide in the biohazard glass disposal container.

35. CHAPTER –5
RESULTS AND DISCUSSION

36. RESULTS AND DISCUSSION
5.1.Streaking:
Streaked plate are incubated at37°Cfor 24 hours. Examine the colonies grown in the plate carefully. All colonies
should have the same general appearance. If there is more than one type of colony, each type should be streaked
againonaseparateplatetoobtainapureculture.
5.2 . Gram Staining:
Gram-positive bacteria will stain violet/purple.

37. 5.2.1. Gram-positive bacteria:
Gram-positive bacteriaarebacteriathatgive apositiveresult inthe Gram staintest. Gram-positivebacteriatakeup
thecrystalvioletstainusedinthetest,andthenappeartobepurple-colouredwhenseenthroughamicroscope.This
isbecausethethickpeptidoglycan layerinthebacterialcellwallretainsthestainafteritiswashedawayfromtherest
ofthesample,inthedecolorizationstageofthetest.
Figure.5.2.Showinggrampositivebacteria
5.3 Antibiotic sensitivity test:

38. Figure5.2.[A] Bacteria’s sensitive to TEI antibiotic and shows zone of clearance.
Figure5.3 [B] Bacteria’s resistant to TCC antibiotic and they do not show zone of clearance.
5.4 Endospore staining:
Observedthebacteriaunder40X(oilimmersion) totalmagnification.
Thesporesappearedgreenincolor.

39. Fig- 5.4 [A] Bacteria showsspores [green colored]
5.6.Catalase test:
Catalase Positive reactions: Evident by immediate effervescence (bubble formation).Catalase
Negative reaction: No bubble formation (no catalase enzyme to hydrolyze the hydrogen peroxide)

40. Figure.5.6.Showing aerobic bacteria
CHAPTER-6
CONCLUSION

41. CONCLUSION
Weisolated bacteria present from the soil sample and characterized it by using the gram staining
method. From which we observed that the bacteria we isolated was gram positive bacteria since it
showed purple color. Antibiotic sensitivity test was done to determine whether the
bacteria’spresent in the soil sample was sensitive or resistance. So for that we used TCC and TEI
antibiotic disc. We found thatthe bacteria were sensitive to TEI (zone of clearance was formed)

42. and resistant to TCC. Endospore staining test showed that endospores were present in whichthe
spores appeared green. Catalase test was performed to check whether the bacteria were aerobic or
anaerobic. Immediate effervescence (bubble formation) was observed thus indicating that the
bacteria are aerobic.

43. CHAPTER-7
REFERENCES
REFERENCES
1. Sharpe M. High on pollution: drugs as environmental contaminants. J Environ Monit.
2003;5:43–46. [PubMed]

44. 2. Pathak SP, Gaur A, Bhattacherjee JW. Distribution and antibiotic resistance among aerobic
heterotrophic bacteria from rivers in relation to pollution. J Environ Sci Health A. 1993;28:73–87.
3. Kruse H. Indirect transfer of antibiotic resistance genes to man. Acta Vet Scand. 1999;92:59–
65. [PubMed]
4. Bush K, Courvalin P, Dantas G, Davies J, Eisenstein B, et al. (2011) Tackling antibiotic
resistance. Nat Rev Microbiol 9: 894–896. doi: 10.1038/nrmicro2693 PMID: 22048738
5 . Rokem JS, Lantz AE, Nielsen J (2007) Systems biology of antibiotic production by
microorganisms. Nat Prod Rep 24: 1262–1287. PMID: 18033579
6 . Yim G, Wang HH, Davies J (2007) Antibiotics as signalling molecules. Philos Trans R Soc
Lond B Biol Sci 362: 1195–1200. PMID: 17360275
7. Michael CA, Dominey-Howes D, Labbate M causes, consequences, and management. Front
Public Health. 2014;2:145. The antimicrobial resistance crisis.
8. Min B, Allen-Scott LK, Buntain B. Transdisciplinary research for complex One Health issues:
a scoping review of key concepts. Prev Vet Med. 2013;112(3-4):222–9.
9. Aminov RI, Mackie RI (2007) Evolution and ecology of antibiotic resistance genes. FEMS
Microbiol Lett271: 147–161. PMID: 17490428
10. Steven B, Leveille R, Pollard WH, Whyte LG (2006) Microbial ecology and biodiversity in
permafrost. Extremophiles 10: 259–267. PMID: 16550305
11. Juck DF, Whissell G, Steven B, Pollard W, McKay CP, et al. (2005) Utilization of fluorescent
microspheres and a green fluorescent protein-marked strain for assessment of microbiological
contamination of permafrost and ground ice core samples from the Canadian High Arctic. Appl
Environ Microbiol 711035–1041. PMID: 15691963

45. 12. Su CC, Long F, Zimmermann MT, Rajashankar KR, Jernigan RL, et al. (2011) Crystal
structure of the CusBA heavy-metal efflux complex of Escherichia coli. Nature 470: 558–562. doi:
10.1038/ nature09743 PMID: 21350490
13. World Health Organisation Improving the containment of antimicrobial resistance WHA;
2005. [http://www.searo.who.int/entity/medicines/topics/ wha_58_27.pdf]. Accessed 23 Dec
2015.
14. Lassi ZS, Mallick D, Das JK, Mal L, Salam RA, Bhutta ZA. Essential interventions for child
health. Reprod Health. 2014;11 Suppl 1:S4.
15. Bebell LM, Muiru AN. Antibiotic use and emerging resistance: how can resource-limited
countries turn the tide? Global Heart. 2014;9(3):347–58.
16. Gilchrist MJ, Greko C, Wallinga DB, Beran GW, Riley DG, Thorne PS. The potential role of
concentrated animal feeding operations in infectious disease epidemics and antibiotic resistance.
Environ Health Perspect. 2007;115(2):313–6.
17. Economou V, Gousia P. Agriculture and food animals as a source of antimicrobial-resistant
bacteria. Infect Drug Resist. 2015;8:49–61.
18. Macfarlane S. Antibiotic treatments and microbes in the gut. Environ Microbiol.
2014;16(4):919–24
19. Zhang SH, Lv X, Han B, Gu X, Wang PF, Wang C, et al. Prevalence of antibiotic resistance
genes in antibiotic-resistant Escherichia coli isolates in surface water of Taihu Lake Basin, China.
Environ Sci Pollut Res Int. 2015
20. Stålsby Lundborg C, Tamhankar AJ. Understanding and changing human behaviour–antibiotic
mainstreaming as an approach to facilitate modification of provider and consumer behaviour. Ups
J Med Sci. 2014; 119(2):125–33.

46. 21.Davies J (1994) Inactivation of antibiotics and the dissemination of resistance genes. Science
264: 375±381
22. Bryskier A. Tetracyclines. In: Bryskier A, editor. Antimicrobial agents: antibacterials and
antifungals. Washington: ASM Press; 2005. p. 642–51.
23. Alonso A. Sa´nchez P, Martı´nez JL: Environmental selection of antibiotic resistance genes.
Environ Microbiol. 2001;3:1–9.
24. Vallee BL, Ulmer DD. Biochemical effects of mercury, cadmium and lead. Ann Rev Biochem,
1972; 41: 91-128.
25. Rai LC, Gaur JP, Kumar HD. Phycology and heavy metal pollution. Biol Rev, 1981; 56: 99-
151.
26. Ren WX, Li PJ, Geng Y, Li XJ. Biological leaching of heavy metals from a contaminated soil
by Aspergillus niger. J. Hazardous materials, 2009; 167: 164-169. 3–42
27.Alonso, Ana, Patricia Sanchez, and Jose L. Martinez. "Environmental selection of antibiotic
resistance genes." Environmental microbiology 3.1 (2001): 1-9.
28.Cabello, Felipe C. "Heavy use of prophylactic antibiotics in aquaculture: a growing problem
for human and animal health and for the environment." Environmental microbiology 8.7 (2006):
1137-1144.
29.D'costa, Vanessa M., et al. "Sampling the antibiotic resistome." Science 311.5759 (2006): 374-
377.
30.Kim, Sungpyo, and Diana S. Aga. "Potential ecological and human health impacts of antibiotics
and antibiotic-resistant bacteria from wastewater treatment plants." Journal of Toxicology and
Environmental Health, Part B 10.8 (2007): 559-573.
31.Gu, Cheng, and K. G. Karthikeyan. "Sorption of the antibiotic tetracycline to humic-mineral
complexes." Journal of environmental quality 37.2 (2008): 704-711.
32.Cabello, Felipe C. "Heavy use of prophylactic antibiotics in aquaculture: a growing problem
for human and animal health and for the environment." Environmental microbiology 8.7 (2006):
1137-1144.

47. 33.Ferber, Dan. "WHO advises kicking the livestock antibiotic habit." Science 301.5636 (2003):
1027-1027.
34.Witte, Wolfgang. "Medical consequences of antibiotic use in agriculture." Science 279.5353
(1998): 996-997.
35.Baquero, Fernando, José-Luis Martínez, and Rafael Cantón. "Antibiotics and antibiotic
resistance in water environments." Current opinion in biotechnology 19.3 (2008): 260-265.
36.Linares, Juan Francisco, et al. "Antibiotics as intermicrobialsignaling agents instead of
weapons." Proceedings of the National Academy of Sciences 103.51 (2006): 19484-19489.
37.Kong, W-D., et al. "The veterinary antibiotic oxytetracycline and Cu influence functional
diversity of the soil microbial community." Environmental Pollution 143.1 (2006): 129-137.
38.Halling-Sørensen, Bent, et al. "Occurrence, fate and effects of pharmaceutical substances in the
environment-A review." Chemosphere 36.2 (1998): 357-393.
39.Dantas, Gautam, et al. "Bacteria subsisting on antibiotics." Science 320.5872 (2008): 100-103.
40.Sukul, Premasis, and Michael Spiteller. "Fluoroquinolone antibiotics in the
environment." Reviews of environmental contamination and toxicology.Springer New York,
2007.131-162.
41.Pei, Ruoting, et al. "Effect of river landscape on the sediment concentrations of antibiotics and
corresponding antibiotic resistance genes (ARG)." Water research 40.12 (2006): 2427-2435.
42.Angulo, F. J., V. N. Nargund, and T. C. Chiller. "Evidence of an association between use of
anti‐microbial agents in food animals and anti‐microbial resistance among bacteria isolated from
humans and the human health consequences of such resistance." Journal of Veterinary Medicine,
Series B 51.8‐9 (2004): 374-379.
43.Garofalo, Cristiana, et al. "Direct detection of antibiotic resistance genes in specimens of
chicken and pork meat." International journal of food microbiology 113.1 (2007): 75-83.
44.Sundsfjord, A., G. SkovSimonsen, and P. Courvalin. "Human infections caused by
glycopeptide‐resistant Enterococcus spp: are they a zoonosis?." Clinical Microbiology and
Infection 7.s4 (2001): 16-33.
45.Cattoir, Vincent, et al. "Unexpected occurrence of plasmid-mediated quinolone resistance
determinants in environmental Aeromonas spp." Emerging infectious diseases 14.2 (2008): 231.

48. 46.Cattoir, Vincent, et al. "Unexpected occurrence of plasmid-mediated quinolone resistance
determinants in environmental Aeromonas spp." Emerging infectious diseases 14.2 (2008): 231.
47.Ruiz, Gregory M., et al. "Global spread of microorganisms by ships." Nature 408.6808 (2000):
49-50.
48.Grenet, Karine. "Antibacterial Resistance, Wayampis Amerindians, French Guyana-Volume
10, Number 6—June 2004-Emerging Infectious Disease journal-CDC." (2004).
49.De Kraker, Marlieke EA, et al. "Mortality and hospital stay associated with resistant
Staphylococcus aureus and Escherichia coli bacteremia: estimating the burden of antibiotic
resistance in Europe." PLoS Med 8.10 (2011): e1001104.
50.Carlet, Jean, et al. "Society's failure to protect a precious resource: antibiotics." The
Lancet 378.9788 (2011): 369-371.
51.Boucher, Helen W., et al. "Bad bugs, no drugs: no ESKAPE! An update from the Infectious
Diseases Society of America." Clinical Infectious Diseases 48.1 (2009): 1-12.
52.Leung, Emily, et al. "The WHO policy package to combat antimicrobial resistance." Bulletin
of the World Health Organization 89.5 (2011): 390-392.
53.D'costa, Vanessa M., et al. "Sampling the antibiotic resistome." Science 311.5759 (2006): 374-
377.
54.Knapp, Charles W., et al. "Antibiotic resistance gene abundances correlate with metal and
geochemical conditions in archived Scottish soils." PLoS One 6.11 (2011): e27300.
55.Peak, Nicholas, et al. "Abundance of six tetracycline resistance genes in wastewater lagoons at
cattle feedlots with different antibiotic use strategies." Environmental microbiology 9.1 (2007):
143-151.
56.Gillings, Michael R., and H. W. Stokes. "Are humans increasing bacterial
evolvability?." Trends in ecology & evolution 27.6 (2012): 346-352.
57.Wright, Gerard D. "Antibiotic resistance in the environment: a link to the clinic?." Current
opinion in microbiology 13.5 (2010): 589-594.
58.D’Costa, Vanessa M., et al. "Antibiotic resistance is ancient." Nature 477.7365 (2011): 457-
461.
59.Monier, Jean-Michel, et al. "Metagenomic exploration of antibiotic resistance in soil." Current
opinion in microbiology 14.3 (2011): 229-235.

49. 60.Bhullar, Kirandeep, et al. "Antibiotic resistance is prevalent in an isolated cave
microbiome." PloS one 7.4 (2012): e34953.
61.Hall, Barry G., and Miriam Barlow. "Evolution of the serine β-lactamases: past, present and
future." Drug Resistance Updates 7.2 (2004): 111-123.
62.Aminov, Rustam I. "The role of antibiotics and antibiotic resistance in nature." Environmental
microbiology 11.12 (2009): 2970-2988.
63.Blázquez, Jesús, et al. "Antimicrobials as promoters of genetic variation." Current opinion in
microbiology 15.5 (2012): 561-569.
64.Barr, V., et al. "β-Lactam antibiotics increase the frequency of plasmid transfer in
Staphylococcus aureus." Journal of Antimicrobial Chemotherapy 17.4 (1986): 409-413.
65.Allen, Heather K., et al. "Antibiotics in feed induce prophages in swine
fecalmicrobiomes." MBio 2.6 (2011): e00260-11.
66.Dantas, Gautam, et al. "Bacteria subsisting on antibiotics." Science 320.5872 (2008): 100-103.
67.Finley, Rita L., et al. "The scourge of antibiotic resistance: the important role of the
environment." Clinical Infectious Diseases (2013): cit355.
68. Knapp, Charles W., et al. "Evidence of increasing antibiotic resistance gene abundances in
archived soils since 1940." Environmental science & technology 44.2 (2009): 580-587.
69. Gagliotti, C., et al. "Escherichia coli and Staphylococcus aureus: bad news and good news
from the European Antimicrobial Resistance Surveillance Network (EARS-Net, formerly
EARSS), 2002 to 2009." Eurosurveillance (2011).
70. Nesbitt, A., et al. "Integrated surveillance and potential sources of Salmonella Enteritidis in
human cases in Canada from 2003 to 2009." Epidemiology and infection 140.10 (2012): 1757.
71. Heuer, Ole E., et al. "Human health consequences of use of antimicrobial agents in
aquaculture." Clinical Infectious Diseases 49.8 (2009): 1248-1253.
72. World Health Organization. Critically important antimicrobials for human medicine. World
Health Organization, 2012.

50. 73. Dutil, Lucie, et al. "Ceftiofur resistance in Salmonella enterica serovar Heidelberg from
chicken meat and humans, Canada." Emerging infectious diseases 16.1 (2010): 48.
74. Wittum, Thomas E. "The challenge of regulating agricultural ceftiofur use to slow the
emergence of resistance to extended-spectrum cephalosporins." Applied and environmental
microbiology 78.22 (2012): 7819-7821.
75. Kluytmans, Jan AJW, et al. "Extended-Spectrum β-lactamase–producing Escherichia coli from
retail chicken meat and humans: comparison of strains, plasmids, resistance genes, and virulence
factors." Clinical Infectious Diseases (2012): cis929.
76. Overdevest, Ilse. "Extended-Spectrum β-Lactamase Genes of Escherichia coli in Chicken Meat
and Humans, the Netherlands-Volume 17, Number 7—July 2011-Emerging Infectious Disease
journal-CDC." (2011).
77. Tian, Baoyu, et al. "Long-term exposure to antibiotics has caused accumulation of resistance
determinants in the gut microbiota of honeybees." MBio 3.6 (2012): e00377-12.
78. Burkholder, JoAnn, et al. "Impacts of waste from concentrated animal feeding operations on
water quality." Environmental health perspectives (2007): 308-312.
79. Greger, Michael, and Gowri Koneswaran. "The public health impacts of concentrated animal
feeding operations on local communities." Family & community health 33.1 (2010): 11-20.
80. Kümmerer, Klaus. "Antibiotics in the aquatic environment–a review–part
II." Chemosphere 75.4 (2009): 435-441.
81. Costello, Christopher, et al. "Status and solutions for the world’s unassessed
fisheries." Science 338.6106 (2012): 517-520.

51. 82. Zhang, Tong, and Bing Li. "Occurrence, transformation, and fate of antibiotics in municipal
wastewater treatment plants." Critical reviews in environmental science and technology 41.11
(2011): 951-998.
83. Kümmerer, Klaus. "Pharmaceuticals in the environment." Annual review of environment and
resources 35 (2010): 57-75.
84. Andersson, Dan I., and Diarmaid Hughes. "Evolution of antibiotic resistance at non-lethal drug
concentrations." Drug Resistance Updates 15.3 (2012): 162-172.
85. Gaze, William H., et al. "Impacts of anthropogenic activity on the ecology of class 1 integrons
and integron-associated genes in the environment." The ISME journal 5.8 (2011): 1253-1261.
86. Czekalski, Nadine, et al. "Increased levels of multiresistant bacteria and resistance genes after
wastewater treatment and their dissemination into Lake Geneva, Switzerland." (2012): 18.
87. Hawkey, Peter M., and Annie M. Jones. "The changing epidemiology of resistance." Journal
of Antimicrobial Chemotherapy 64.suppl 1 (2009): i3-i10.
88. Martinez, Jose Luis. "Environmental pollution by antibiotics and by antibiotic resistance
determinants." Environmental pollution 157.11 (2009): 2893-2902.
89. Baquero, Fernando, José-Luis Martínez, and Rafael Cantón. "Antibiotics and antibiotic
resistance in water environments." Current opinion in biotechnology 19.3 (2008): 260-265.
90. Lupo, Agnese, Sébastien Coyne, and Thomas Ulrich Berendonk. "Origin and evolution of
antibiotic resistance: the common mechanisms of emergence and spread in water
bodies." Analyzing possible intersections in the resistome among human, animal and environment
matrices (2012): 116.

52. 91. Guenther, Sebastian, Christa Ewers, and Lothar H. Wieler. "Extended-spectrum beta-
lactamases producing E. coli in wildlife, yet another form of environmental pollution?." Analyzing
possible intersections in the resistome among human, animal and environment matrices (2011):
103.
92. Smet, Annemieke, et al. "Broad-spectrum β-lactamases among Enterobacteriaceae of animal
origin: molecular aspects, mobility and impact on public health." FEMS microbiology
reviews 34.3 (2010): 295-316.
93. Eliopoulos, George M., Lisa L. Maragakis, and Trish M. Perl. "Acinetobacter baumannii:
epidemiology, antimicrobial resistance, and treatment options." Clinical infectious diseases 46.8
(2008): 1254-1263.
94. Whitman, Timothy J., et al. "Occupational transmission of Acinetobacter baumannii from a
United States serviceman wounded in Iraq to a health care worker." Clinical Infectious
Diseases 47.4 (2008): 439-443.
95. Tao, Chuanmin, et al. "Microbiologic study of the pathogens isolated from wound culture
among Wenchuan earthquake survivors." Diagnostic microbiology and infectious disease 63.3
(2009): 268-270.
96. Fournier, Pierre-Edouard, et al. "Comparative genomics of multidrug resistance in
Acinetobacter baumannii." PLoS Genet 2.1 (2006): e7.
97. Koczura, Ryszard, et al. "Antimicrobial resistance of integron-harboring Escherichia coli
isolates from clinical samples, wastewater treatment plant and river water." Science of the Total
Environment 414 (2012): 680-685.
98. Coleman, B. L., et al. "The role of drinking water in the transmission of antimicrobial-resistant
E. coli." Epidemiology and infection 140.04 (2012): 633-642.

53. LIST OF ABBREVIATIONS
Zn Zinc
Cu Copper
Ni Nickel
Fe Iron
Mn Manganese
DNA deoxyribonucleic acid
ABR Antibiotic resistance
RND resistance-nodulation-division
e.g. example

54. qnr quinolone-resistance gene
% percentage
MICs minimum inhibitory concentrations