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Contoh bakteri tanah yang memiliki plasmid resisten antibiotik.

Antibiotic resistance genes in human pathogens such as methicillin-resistant Staphylococcus aureus1 have become
notorious because they confound the tools that are used to treat disease (FIG. 1). In particular, resistance determinants
in pathogens are commonly encountered after the introduction of an antibiotic to clinical use, and treating human
pathogens with anti- biotics directly affects the frequency of resistance to those antibiotics in these pathogens1–4.

The presence of antibiotic resistance elements in pathogenic bacteria is made all the more problematic because of the
prevalence of horizontal gene transfer, the process by which bacteria acquire genes from the environment 5. Many of
the known antibiotic resist- ance genes are found on transposons, integrons or plasmids, which can be mobilized and
transferred to other bacteria of the same or different species. There is evidence of the transfer of resistance elements
to known human commensal bacteria and pathogens6,7, and gene transfer in the human intestinal microbiome is
extensive8.

What are the sources and reservoirs of these transferable genes? A full understanding of the pres- sures and
circumstances that lead to the evolution and dissemination of antibiotic resistance genes in pathogens is impossible
without a detailed examina- tion of the origin and role of resistance genes in natural environments. This Review
discusses the environmen- tal sources of antibiotic resistance, the functions and roles of resistance genes in microbial
ecology and the ways by which those genes may be disseminated in response to human antibiotic use.

Soil. owing to the movement of antibiotics and resist- ance genes on the wind and on feathers, it is unlikely that any
environment can be considered truly pristine. However, despite the movement of soil particles by physical forces, soil
itself is a stationary complex, and some soils are far removed from human influences. Thus, studies of antibiotic
resistance in soil show that environmental bacteria harbour antibiotic resistance genes independently of human
activities.

Culturable bacteria in soil harbour genes encoding enzymes that degrade or otherwise inactivate antibiotics. Bacteria
that grow on antibiotics as the sole carbon and nitrogen sources include: Pseudomonas fluorescens grown on
streptomycin51; P. fluorescens52, Burkholderia cepacia53 and eight unidentified strains54 grown on penicillin; an
unidentified Streptomyces sp. isolate and Streptomyces venezuelae grown on chlorampheni- col55,56; a Flavobacterium
sp. isolate grown on chloram- phenicol57; and various bacteria, mostly of the phylum Proteobacteria, grown on
various antibiotics58. In addi- tion to antibiotic degradation, culturable soil bacteria use many mechanisms of
resistance to antibiotics. In the most comprehensive study to date on antibiotic resist- ance in one soil species, over
400 actinomycetes cultured from forest, agricultural and urban soils were found to have highly varied resistance
profiles; moreover, some exhibited resistotypes that had not been seen before, such as inactivation of telithromycin
by a novel structural modification59.

Allen, H. K., Donato, J., Wang, H. H., Cloud-Hansen, K. A.,Davies, J. & Handelsman, J. 2010. Call of
the wild: antibiotic resistance genes in natural environments. Connecticut : Macmillan Publishers
Limited p 251-254
Pengertian plasmid curing dan jenis-jenis metode (fisika dan kimia) beserta
mekanisme/mode of action-nya yang dapat digunakan untuk mengamati fenomena
tersebut.

plasmid curing is the process of complete removal of plasmids from bacteria in vitro by chemical agents such as
acriflavin or acridine orange.

In situations where plasmid is stable and the loss of its property is difficult to determine, bacteria can be treated
with the various curing agents. These agents may include the chemical or physical agents that mutate DNA,
interfere with their replication, effect on structural com- ponents or enzymes of the bacterial cell. In this regard,
the elimination of plasmid from bacterial culture establishes the relationship between the genetic trait and
carriage of specific plasmid as the phenotypic characteristics associated with plasmid do not express with the
cured strains and the reintroduction of the plasmid is responsible for the reappearance of the particular
phenotypic characteristic. The efficiency of plasmid curing is dependent on the nature of the plasmid as well as
the host system. In most of the instances, cur- ing experiments are performed under conditions similar to the
conditions used for routine bacte- rial culture. If the treatment is for longer period, then the agent should be used
at a lower concen- tration. However, if acridine orange is used, the culture should be maintained at pH 7.6 as
well as should be incubated in the dark.

The subinhibitory concentrations of chemical agents like acridine orange and SDS may lead to plasmid curing
leading to the generation of cured derivatives. Acridine orange and ethidium bromide act as the intercalating
agents are found to be effective against a wide variety of bacte- rial genera. Though most of the agents have
been practised against various genera of bacteria for plasmid curing, their effect is unpredictable in many cases.

Moreover, the elimination (curing) of a genetic trait by treating the bac- terial population with chemical or physical curing
agents such as acridine dyes, ethidium bromide, sodium dodecyl sulfate (SDS),antibiotics, high temperature, or electropora-
tion (63, 95) indicates that the expression of that genetic trait is linked to the presence of a plasmid. Most curing agents are
also mutagenic agents. In most cases, however, it is essential to document that a plasmid is present and un- equivocally
associated with the genetic trait in question. Clinically, the plasmid content of a cell can also be a useful epidemiological
marker (62, 103).If possible, it is best to transfer a plasmid by physical or genetic means into a bac- terial host that is known
to be devoid of plasmids.
Metode

2.1. Intercalating dyes

Intercalating dyes such as acriflavine, acridine orange, ethidium bromide and quinacrine have been successfully used in
curing bacteria of plas- raids.

2.2. Coumermycin and novobiocin

Both coumermycin and novobiocin are inhibi- tors of DNA gyrase in intact cells of E. coil [22]. DNA gyrase
catalyzes negative superhelical turns into double-stranded, closed, circular DNA [21].

2.3. Rifampicin

Rifampicin binds directly to the RNA poly- merase molecule in both E. coil and S. aureus [25].

2.4. Mitomycin C

Mitomycin C owes its inhibitory activity to metabolic activation via reduction to a corre- sponding
hydroquinone [28]. The hydroquinone undergoes further changes and produces a reactive intermediate which
can react with purine bases [28]. Because of this type of nucleophilic attack, it can insert alkylating cross-links
between the two strands of double-stranded DNA. Any cross-lin- kage of DNA is sufficient to block
transcription by RNA polymerase [28]

2.5. Sodium dodecyl sulfate

Sodium dodecyl sulfate (SDS) is capable of curing certain plasmids. Some plasmid-containing cells are
presumed to be more sensitive to SDS because of plasmid-specific pili on their cell surface

2.6. Elevated growth temperature

Elevated incubation temperature (5-7°C above the normal or optimal growth temperature) can be employed as a
curing method. For example, strains that normally have an optimum growth temper- ature of 37°C can be
incubated at 43°C [11].

2.7. Thymine limitation

Thymine limitation can be used to cure thymine-requiring auxotrophs. Caro et al. [4] re- ported that is has only
been employed with E. coli, and has summarized a protocol for use with this organism. A minimal medium
containing 50 #g/ml thymine is inoculated with the auxotrophic strain, and incubated overnight (12 h). The cells
are aseptically diluted into a series of tubes containing the same minimal medium and thymidine con-
centrations between 0-25 #g/ml. After overnight incubation, serial dilutions are prepared and plated onto
thymine-supplemented nutrient agar [4]. In- dividual colonies can then be tested for plasmid lost.

2.8. Protoplast formation and regeneration


It has been reported that the elimination of certain plasmids in 80% of S. aureus cells can occur during the
formation and regeneration of lysostaphin-produced protoplasts [36]. It was sug- gested that plasmid curing may
occur during the several protoplast divisions that take place prior to cell wall regeneration. Curing was
postulated to be due to a disruption of the plasmid partioning mechanism when the cell wall was removed. This
curing procedure may prove to be especially suita- ble for Gram-positive bacteria that are difficult to cure using
other procedures, yet are easily made into protoplasts via lysozyme treatment.

Das, S. (2015). Microbial Biotechonology - A Laboratory Manual for Bacterial Systems (1st
ed.). New Delhi.

Acridine orange is a nucleic acid binding dye which is used mostly for the determination of cell cycle
information. It is permeable to the cell wall and readily interacts with nucleic acids by intercalation or
electrostatic attractions. During epifluorescence microscopy the role of this dye is of worth use. DNA and RNA
can also be distin- guished by the use of acridine orange as it emits green colour when forms complex with
DNA whereas, emits orange colour upon binding with the RNA molecule.

Das, S. (2015). Microbial Biotechonology - A Laboratory Manual for Bacterial Systems (1st
ed.). New Delhi.

Penjelasan mengenai acridine orange dan mode of action dalam plasmid curing (sertakan
gambar).

Spengler, G., Molnár, A., Schelz, Z., Amaral, L., Sharples, D., & Molnár, J. (2006). The
Mechanism of Plasmid Curing in Bacteria. Journal of Physical Chemistry Chemical Physics,
823–841.

Komposisi dan fungsi medium Luria Bertani (LB).

LB Medium

Luria Bertani (LB) broth is a rich medium that permits the fast growth and good growth yields for many species
including E. coli. It is the most commonly used medium used in molecular biol- ogy studies for E. coli cell
culture. Easy to make, fast growth of most E. coli strains, readily avail- able and simple compositions contribute
the popularity of LB broth. LB can support E. coli growth OD600 2–3 under normal shaking incuba- tion
conditions.

Das, S. (2015). Microbial Biotechonology - A Laboratory Manual for Bacterial Systems (1st
ed.). New Delhi.

Spengler, G., Molnár, A., Schelz, Z., Amaral, L., Sharples, D., & Molnár, J. (2006). The
Mechanism of Plasmid Curing in Bacteria, Journal of Current Drug Targets, 823-841(19)