THEORETICAL PART

Contents

INTRODUCTION...........................................................................................................................................1

CHAPTER 1. ANTIBIOTICS: DEFINITION, CLASSIFICATION, MECHANISMS OF ACTION.................................3

1.Definition..............................................................................................................................................3

1.2 Main classes of antibiotics.................................................................................................................3

Chapter 2. Genetic and biochemical resistance to antibiotics in the main Gram-positive and Gram-

negative bacterial strains of clinical importance.......................................................................................10

2.1 Natural resistance vs acquired resistance........................................................................................16

2.2. Chromosomal versus plasmidial resistance....................................................................................20

2.3. Biochemical mechanisms of antibiotic resistance...........................................................................23

BIBLIOGRAPHY:..........................................................................................................................................26

INTRODUCTION

For half a century, antibiotics are the most important weapon in the fight against

dangerous infection-causing bacteria from the ear and the skin infection to the blood infection.

Infectious diseases causing thousands of deaths and much suffering in the past, such as

tuberculosis, are now under control with antibiotics.1

2

Despite their undeniable importance in modern medicine, these drugs are not always the

best therapeutic solution . There are two main types of germs that cause bacterial infections and

viruses. The most important points to note are:

- Antibiotics have no effect on diseases caused by viruses;

1 Agerso et al., 2005

2 https://www.google.ro/search?q=antibiotice&es_sm=122&source=lnms&tbm=isch&sa=X&ved=0CAcQ_AUoAWoVChMI-o7kj4zHxwIVSEAUCh3DvA_O&biw=1024&bih=475#tbm=isch&q=antibiotice+pentru+raceala

- Only diseases caused by bacteria can be treated with antibiotic;

At first administration of the antibiotic, the initial dose kills weak bacteria at which time the

general condition improves. The strongest bacteria survive, and if treatment is discontinued

before the period recommended by physicians, they multiply and become resistant to the

antibiotic (which can subsequently be transmitted to other family members and the community).

When a person is infected with a bacterium that has gained strength, it requires treatment

administration with stronger antibiotic substances.

Besides the induction of antibiotic-resistant bacteria, abuse of these drugs lead to the appearance

of other side effects such as allergic reactions or serious diarrhea infections caused by

Clostridium difficile . In rare cases, these complications can even lead to death.3

According to American specialists, many infections spread among health care professionals are

caused by bacteria that have become resistant to many antibiotics. These include:

- MRSA (a recent variety of Staphylococcus aureus bacterium );

- Enterococcus (resistant to vancomycin);

- K. pneumonia (resistance to cephalosporins);

- E. coli and Enterobacter spp. (Carbapenem resistant);

- P. aeruginosa (carbapenem resistance);

3 Agerso et al., 2005

CHAPTER 1. ANTIBIOTICS: DEFINITION, CLASSIFICATION,

MECHANISMS OF ACTION

1.Definition

Antibiotics (anti+bios = with adverse effects) = heterogeneous group of chemical

substances produced by microorganisms in the biosynthetic processes that kill or inhibit the

growth of other species of microorganisms.

 Chemotherapeutic agents = chemical synthesis substances used to treat infections caused

by different microorganisms (bacteria, microscopic fungus, and protozoans) (P. Ehrlich -l904).

The present definition = chemical substances obtained by biosynthesis, semi-synthesis,or by

chemical synthesis, which, in low concentrations inhibits the multiplication or kills

microorganisms. = antimicrobial substances

> 500 identified substances having antibiotic properties

~ l00 used in therapy

1.2 Main classes of antibiotics

The large number of currently known antibiotics has questioned the classification of these

products. The following classifications has been proposed :4

1. After the origin of producing microorganism :

• antibiotics produced by bacteria

4 SOCKETT. Et al., 2006

- Gramicidin

- Bacitracin

- Polymyxins

• antibiotics produced by the actinomycete:

- Streptomycin

- Neomicima

- Kanamycin

- Nystatin

• antibiotics produced by fungus

- penicillin

- grizeofulvin

2. After the chemical structure:

- antibiotics with aliphatic, aromatic structure

- heterocyclic

3. After biogenesis:

- antibiotics derived from amino acids

- from acetate units

- from carbohydrates

4. After pharmacological action:

- Antibacterial antibiotics

- Antituberculosis antibiotics

- Antiviral antibiotics

- Anticancer antibiotics

- Antifungal antibiotics

5 https://www.google.ro/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0CAcQjRxqFQoTCIqMpKmMx8cCFcbNFAod06YFGQ&url=http%3A%2F%2Fwww.psmicrographs.co.uk%2Fmould-fungus--penicillium-expansum-%2Fscience-image%2F80200576&ei=mt_dVYrXFcabU9PNlsgB&psig=AFQjCNHVFX9XEQM3JUJ4KTDNu3PCj34vfg&ust=1440690451933819

 The classification of antibiotic after structure

1. lactam: penicillins and cephalosporins

2. aminoglycosides gentamicin, kanamycin, streptomycin

3. Macrolides: erythromycin, clarithromycin

4. Tetracyclines: Tetracycline and Doxycycline

5. Chloramphenicol

6. sulfamides (are 23323k1021x synthetic)

• bactericidal antibiotics: penicillins, cephalosporins, aminoglycosides.

• bacteriostatic antibiotics: Erythromycin, Tetracycline, Chloramphenicol, sulfamides. • broad-

spectrum antibiotics [1] broad-spectrum penicillin (Ampicillin, Amoxicillin), chloramphenicol,

tetracycline.

• penicillins are antibiotics = drugs that destroy bacteria . There are several types:

Characteristics of some commonly used antibiotics

1. Beta-lactams (so called because of their chemical structure)

1.1. Peniciline

1.1.1. Narrow-spectrum penicillins

Benzylpenicillin (Penicillin G) is a natural penicillin. It is active against certain types of

bacteria: streptococcus ( that causes streptococcal angina, scarlet fever, erysipelas),

pneumococcal (lobar franca pneumonia ), meningococcal (meningitis), Treponema pallidum

(syphilis), clostridia (gas gangrene), the bacillus tetanus (tetanus) bacillus coal (anthrax).

It may not be administered orally because it is inactivated by stomach acid. It is usually given

intramuscularly, intravenously in severe cases. PenicilinaG can be potassium or sodium. A

patient with heart failure will prefer PenicilinaG potassic, because sodium attracts water and

overweight self circulation. A patient with kidney failure will prefer PenicilinaG sodium

because sodium is eliminated through the kidneys easier.

Phenoxymethylpenicillin (Penicillin V, Ospen) is resistant to gastric acid therefore is

administered orally. The absorption from the digestive tract is incomplete and performed

blood concentration of antibiotics is low, because of this drug is administered in case of low

gravity infections: For instance streptococcal angina. penicilinaG is administered in more

severe infections .

Benzathine benzylpenicillin (Moldamin) is a deposit penicillin. It injects deep

intramuscular where it performs a deposit of active substance (benzylpenicillin) which is

released slowly into the blood (for several weeks). The blood concentration is sufficient to

protect the body from a possible strep infection. It is used to prevent re-streptococcal

infections in persons with a history of poststreptococice disease (rheumatic fever)

Procainpeniclina (Efitard) is a combination of procaine and penicilinaG which is slowly

absorbed into the blood from the injection site. It is effective for 12 hours. It has increased

risk of allergic reactions due to the presence of procaine.

1.1.2. Staphylococcal penicillins: Oxacillin, Cloxacillin, Nafcillin

Synthetic penicillins can not be destroyed by penicillinase, so resistant germs are active on

narrow-spectrum penicillins. The only indication for these antibiotics are staphylococcal

infections (furunculosis, osteomyelitis, etc.)

1.1.3. Broad-spectrum penicillins: ampicillin, amoxicillin

Are active on the germs that are narrow-spectrum penicillins active and in addition to Gram-

negative: Haemophilus influenza E.Colli, Salmonella, Shigella. Not active against

staphylococci that are inactivated by penicillinase.

Ampicillin can be taken internally or injected. It should be administered on an empty

stomach, because it decreases the absorption of food. It focuses much in the bile and urine, as

indicated in infections at these levels.

Amoxicillin has the advantage that it absorbs better digestive,the absorption is not affected

by food.

Amoxicillin may be associated with clavulanic acid which is an inhibitor of penicillinase,

resulting antibiotic called Augmentin (amoxiclav). Augmentin has a very broad spectrum of

action: it is active on all germs that are narrow-spectrum penicillins active, on staphylococci

and gram negative.

·        Penicillins have degenerative bactericidal effect (kills bacteria multiply). Prevents bacterial

wall synthesis.

1.2. Cephalosporins

Broad-spectrum antibiotics are indicated in serious infections that do not respond to other

antibiotics. Some are taken orally: cephalexin, cephalothin, cefuroxime (Zinnat)), others only by

injection (parenteral): Latamoxef, cefepime, cefpirome. If a person had a serious allergic reaction

to penicillin we recommend to avoid cephalosporin because of the risk of cross-allergy .

2. Aminglicozide: Streptomycin, gentamicin, kanamycin, neomycin.

These antibiotics are not used alone, because germs are quickly becoming resistant to them.

Always use together with other antibiotics.

Streptomycin is used to treat tuberculosis TB together with other chemotherapeutic agents.

Gentamicin and Kanamycin is associated with penicilinaG.

If taken digestive, these antibiotics are not absorbed. However sometimes it is administered

digestive to treat intestinal infections (enteritis, enterocolitis). For other infections

(tuberculosis, urinary tract infections, genital infections, bronchitis etc) aminoglycosides are

administered by injection. Neomycin is so toxic that is administered only digestive or

external (skin or mucosa).

·        Erythromycin is an antibiotic that is given to patients allergic to penicillin. It is

bacteriostatic active, gram positive. It inhibits bacterial protein synthesis.

·        Co-trimoxazole (Biseptol) is a combination of two chemotherapy agents Sulfamethoxazole

and trimethoprim. It has bactericidal effect. It manages internal respiratory infections,

digestive and urinary.

·        Tetracycline is a broad spectrum antibiotic. It has bacteriostatic effect (stops the

multiplication of bacteria). It is indicated in periodontal infections (periodontal abscess). Do

not use in pregnant women or children younger than 12 years because it causes

malformations of teeth. If taken long, oral candidiasis occurs. Is NOT administered with milk

or antacids that decreases the absorption of tetracycline from the digestive tube.

·        Chloramphenicol is used in Salmonella infections. It has bacteriostatic effect. It can have

serious reactions: gray syndrome in infants and inhibiting hematopoiesis [2] in adults\

Antibiotic Resistence mechanisms of bacteria

Betalactamine

(penicillins, cephalosporins,etc.)

Inactivating enzymes (beta-lactamases), (C, P)

Change PBP (C, P)

Reduction in cellular permeability (C)

Aminoglycosides Enzymatic inactivation (aminoglycosides modifying enzymes)

(C, P)

The alteration of the ribosomal target protein (C)

Changing active transport systems through bacterial shells (C)

Quinolones The affinity reduction of action target (changing DNA gyrase)

(C)

Reducing the permeability of the outer membrane (C)

Active efflux of antibiotics in bacterial cell (C, P)

Macrolides Changing target share (C, P)

Active efflux of antibiotics in bacterial cell

ATB modifying enzymes bacteria acquisition (P)

Lincosamides Changing target share (C, P)

ATB modifying enzymes bacteria acquisition (P)

Vancomycin Changing target share

Tetracilcine Active efflux of antibiotics in bacterial cell (P)

Changing ribosomal protein (P)

Chloramphenicol Enzymatic inactivation (chloramphenicol acetyltransferase)

Reducing the bacterial wall permeability (C)

Sulphamides Reducing the bacterial permeability (C, P)

Modification of enzymes (C, P)

Overproduction of APAB (C)

Trimethoprim Reducing the bacterial permeability (C, P)

Nitrofurans Reducing the bacterial permeability

Deficiency in nitrofuran reductase activity (C)

Nitroimidazoles Reducing the bacterial permeability

Insufficient nitrofuran reductase activity6

6 http://europroxima.com/products/antibiotics/nitrofurans/

Chapter 2. Genetic and biochemical resistance to

antibiotics in the main Gram-positive and Gram-negative

bacterial strains of clinical importance

Resistance to antibiotics

Antibiotic resistance is the naturally or acquired ability of a microorganism to resist the

effects of one or more antibiotics. Although this concept is also used relating to natural resistance

- intrinsic ability of bacteria to resist certain antibiotics - a greater scientific and clinical

importance is the acquired resistance.

Resistance to antibiotics can occur through natural selection or by mutation (in the

mutagenic effect of environmental factors or uncorrected errors in the DNA replication process).

With emerging antibiotic resistance, gene coding for this character may spread to other cells

through transfer of plasmids. A bacterium can use multiple antibiotic resistance gene, in this case

being called multi-resistant.7

Antibiotic resistance of a microorganism can also be artificially induced , by

transformation techniques (introduction of foreign genetic material into the cell and expression

of encoded characters). This method is useful in genetic engineering and biotechnology for the

selection of bacteria bearing a specific additional character, transmitted along with the gene of

resistance.

The emergence of antibiotic resistance is a evolutionary phenomenon, caused by selective

pressure of environmental factors. Antibiotics are a selection factor and the bacteria that suffers a

benefical mutation (the emergence of resistance to antibiotic) will survive and will possibly send

these characters to descendants. The increasingly frequent use of antibiotics to treat infections

resulted in emergence of a growing number of bacterial strains resistant to a growing number of

antibiotics.

There are several mechanisms by which this phenomenon may occur:

7 SOCKETT. Et al., 2006

o Antibiotic inactivation or destruction - for example Penicillin inactivation by producing

enzyme (beta-lactamase) that break a beta-lactam bond in the molecule.

o Changing a target (antibiotic binding site) so antibiotic molecule no longer can react with

cellular components - for example, ribosomes and enzymes involved in the synthesis of

the bacterial wall

o Change in the metabolic pathways in which antibiotics act - the case of bacteria resistant

to sulphonamides, which, instead of synthesizing folic acid starting from para-

aminobenzoic acid, they use preformant folic acid, like mammal cells.

o inhibition of penetration of the antibiotic into the cell - for example the resistance

mechanism of E. coli to macrolide

o Eliminating antibiotic (active efflux) - seen in some species of E. coli and S. aureus.

8

8

? Conform http://www.britannica.com/science

The natural result of the discovery of this phenomenon was the continuous search of new

antibiotics capable of inactivating microorganisms resistant to antibiotics already available.

However, it was found that after a certain time from the introduction of a new semi-synthetic

antibiotic, or a new class of antibiotic, the existance of resistant bacterial strains is, due to the

fact that antibiotic resistance is an evolutionary phenomenon. 9

Among the examples of continual evolution and adaptations of pathogenic bacteria to new

antibiotics, are increasingly frequent cases of multi-resistant tuberculosis and infections with

methicillin-resistant Staphylococcus aureus.

The concept of microbial resistance defines the ability of pathogenic germs to survive

and multiply in the presence of antibiotics. The germs are resistant or become "indifferent"

("tolerant") to antibiotics, thereby avoiding desired antibacterial effect through various ways ,

following multiple usual non-toxic doses . When exposed to an antibiotic, the bacteria is the

selected for resistance . This natural biological process leads to the survival of the resistant

strains (FEDES, 2000).

After HEINEMANN (1999), antimicrobials are approaching the end of their

effectiveness. Although evolution of resistance to these drugs was expected, mechanisms by

which genes conferring resistance will spread were not intuited . Therefore antimicrobial agents

used in the future must avoid effects caused by pathogens.

Types of antibiotic resistance:

Factors influencing bacterial antibiotic resistance. The term strength can be used in two

ways, as microbiological resistance and as clinical resistance.

Microbial resistance is an absolute concept and it must be distinguished from pseudo or

false strength, relative notion of practice clinical treatments.

Microbiological resistance

9 Agerso et al., 2005

Absolute microbial resistance occurs when a microorganism is resistant in vitro, the

antibiotic used proving to be unable to stop the reproduction of them or when stopping the

multiplication is carried out only by very high concentrations of the antibiotics, which would

require toxic doses in vivo practically unusable in the patient. From a microbiological point of

view, resistant organisms are those that possess any type of mechanism of resistance or

resistance genes. The minimum inhibitory concentration (MIC) of an antibiotic gives quantitative

information about bacterial susceptibility. Usually an organism is considered susceptible when

MIC is less than the limit indicated by various laboratories authorized for such standards

(SOCKETT. Et al., 2006).

Clinical resistance

Clinical resistance is more complex than microbiological resistance, since it is linked to

the likelihood of response to antimicrobial therapy. Prior to treatment, information concerning

the susceptibility of the bacteria involved in the infection are received from specialized

laboratories, although not fully reflects the antibacterial activity of antibiotics in clinical

conditions (Swartz, 2000).

Pseudo-resistance or false resistance

It represents the failure of antibiotic treatment due to characteristics of the host organism

and of the infection and especially incorrect use of antibiotics. Pseudo-resistance is a temporary

phenomenon, reversible, which shall be exercised only in vivo. In clinical terms, the

development of resistance is facilitated by pharmacokinetic characteristics of different classes of

antibiotics and an incorrect treatment (insufficient dose , too short duration of treatment or long-

term use of antibiotics), as well as inefficient active concentration of the antibiotic at the place of

infection (hard sterilizable outbreaks,serous barriers, avascular tissue, inactivation of antibiotics,

unfavorable pH etc.). Another cause of treatment failure is relative resistance to

pathogens,sensitive in vitro, but became insensitive, usually temporarily, in vivo (persisters,

germs with reduced metabolism).

Moreover, other factors such as the immune status of the patient may affect the

therapeutic response. In most patients, antibiotics do not totally destroy pathogens, but assists the

immune system in an attempt to eliminate the infection. An enormous selection force is the large

amount of antibiotics used in agriculture as well as the therapy and prophylaxis of human and

veterinary medicine (HARDY, 2002; CROMWELL, 2002; DIBNER et al., 2005).

Microbial resistance to antibiotics can be natural and acquired: natural resistance is the

resistance of all members of a bacterial species to one or more antibiotics present in the

maximum dose tolerated by the treated body without risk, doses that can inhibit growth or

destroy other bacterial species . After ENGELKIRK Burton (2002), natural resistance is the

intrinsic property of the species thus total, genetically fixed. For instance, the resistance to

penicillin G of the strains of Salmonella;

Pseudomonas aeruginosa is naturally resistant to chloramphenicol; Proteus genus species

are resistant to tetracycline; anaerobes (Bacteroides, Clostridium, some streptococci) and Serratia

genus are resistant to aminoglycosides. Acquired resistance refers to the state arising from

naturally sensitive species after the entering of an antibiotic into therapy and consists in

decreasing or cancellation of antibiotic sensitivity.10

This is a phenotypic character correlated with an altered genetic material. In practice, the

bacteria is considered resistant when there is an subunitary ratio between medium serum levels

supported by the patient and minimum inhibitory concentrations of the antibiotic or antibacterial

(TODAR, 2002). Until now several acquired resistance types are known, and are classified by

various criteria:

a. The moment infection is installed

The primary resistance, the state of resistance of the bacteria at the initiation of the

infection. Secondary resistance, state acquired by infected strain during treatment.

b. The number of antibiotics to which resistance is installed. Mono-resistance - microbial

resistance to a single antibiotic. Multi-resistance - microbial resistance to several antibiotics.

10 Agerso et al., 2005

c. The rate of installing resistance to antibiotics. Streptomycin fast resistance type ,

consists of a single stage (single mutant, interesting only one gene). Penicillin-type progressive

resistance constituted in several steps (successive mutations, interesting multiple processes)

d. The presence of antimicrobial factor. Inducible resistance - expressed only in the

presence of antibiotic resistance. Constitutive resistance - the ability of a gene to continuously

express their resistance independent of the presence or absence of the antibiotic.

e. Other types of resistance. Cross-resistance - the ability of some strains to manifest

resistance to some antibiotics related by chemical structure. Adaptive resistance - a condition

which, without interesting the genome is sometimes transmitted to successive generations and

which appears under the influence of subinhibitory doses of antibiotic, bacteria returning to its

previous state after several generations from the disappearance of inducing factor

(ANGELESCU, 1998; Swartz, 2000).

Prevention of antibiotic resistance

Antibiotic resistance is defined as a natural or acquired ability of a microorganism to

survive the effects of one or more antibiotics. This phenomenon is evolutionary caused by

selective pressure of environmental factors. Following frequent use of antibiotics in treating

infections , a growing number of bacterial strains resistant to more and more types of antibiotics,

has appeared.11

Both patients and health professionals must collaborate to stagnate the phenomenon of

antibiotic resistance development. Experts recommend a series of measures by which the danger

of impossibility of treating serious infections does not become a harsh reality for mankind.

Medical experts recommend you

- carry out a specialist medical inspection to establish the exact nature of the infection (viral or

bacterial);

11 Swartz, 2000

- Require access to antibiogram, so to make sure that your doctor has prescribed antibiotic in

removing bacteria or right contacted bacteria ;

- Follow antiobiotic treatment according to precise physician recommendations (dose, time,

etc.).

- Do not skip doses even if you feel better after only 1-2 days of treatment.

- Take only prescribed antibiotics personally do not share with anyone and do not use those

remaining from previous treatments (different types of antibiotics treat specific types of

infections

- the administration will postpone wrong substance healing and stimulate bacterial growth).

- Do not save antibiotics for possible future diseases and do not recommend them to others just

because they worked in your treatment.

- No doctor urges an antibiotic prescription if he does not deem it necessary. Remember always

that these drugs have side effects!

- Prevent infection by washing hands and correct immunization (vaccination).

For children with symptoms like fever, fatigue, muscle pain or irritated throat, do not quickly

conclude that they contacted bacteria . Most often, it is a common cold, against which antibiotics

have no effect. Carry out a pediatric inspection and help children fight viral infection by

- Prolonged bed rest;

- Additional hydration by water, fruit juices, soups and tea;

- Anti-inflammatory drugs based on ibuprofen;

- Nasal sprays with saline solution and cough syrup;

- Steam baths;

Prevention of antibiotic resistance depends largely on healthcare professionals, who are

the only ones authorized to issue prescriptions on which patients can purchase antibiotics. They

must:12

12 Ibidem

- Prescribe antibiotics correctly (crop sampling, performing sensitivity testing, establishing the

required dosage and administration period, issuing the prescription after the patient has been

tested properly).

- To document on the dose, timing and indications on the prospectus of antibiotics sold in

pharmacies.

- constantly worrying about the high degree of risks in the preading of antibiotic resistant

bacteria in the medical unit in which they work.

- Attend and support all measures taken by hospital practices and authorities, to discourage

improper antibiotic prescriptions for patients.

- Respect all rules of hygiene and other infection control measures with each patient treated.

- Inform patients about the dangers they face when abusing antibiotics.13

13 Swartz, 2000

14 http://www.google.ro/imgres?imgurl=http://evolution.berkeley.edu/evolibrary/images/interviews/resistance.gif&imgrefurl=http://evolution.berkeley.edu/evolibrary/article/bergstrom_03&h=238&w=484&tbnid=2SYhLvqfuxO4hM:&docid=N6590bDHHJrnIM&ei=V-HdVe26L4HwUvidqPAB&tbm=isch&ved=0CB8QMygBMAFqFQoTCK3C1v2Nx8cCFQG4FAod-A4KHg

2.1 Natural resistance vs acquired resistance15

Efflux phenomena

Besides impermeability, there is another mechanism that explains accumulation in

bacteria; excretion or active efflux. The antibiotic enters in the bacteria, but before it could fix

on target, it is taken by membrane proteinsoutside and excreted outside the bacteria. This system

works with a cytoplasmic membrane protein that is the carrier (pump), a protein the outer

membrane that forms the excretion channel and a protein periplasmic responsible for the link

between the previous ones.16

Natural resistance

This system occur naturally in certain bacteria. Also in P. aeruginosa, such a system

largely explains the high natural resistance against a large number of antibiotic molecules. This

system exists in staphylococci against quinolones.

Gained resistance

First resistance efflux acquired by E. coli was reported in 1980. It was the tetracycline excretion.

Since then, this resistance mechanism has been revealed to:

- E coli, P. aeruginosa and Staphylococcus aureus against quinolones;

- staphylococci against macro-LIDE ;

- P. aeruginosa against beta lactams;

This may involve acquiring resistance genes as a result of introduction of a plasmid or, most

often, a mutation engaging existing chromosomal gene overexpression but little or no casted.

15 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3257838/

16 Swartz, 2000

The lack of affinity

After cell penetration of the antibiotic, there is a stage of target recognition.This type of

resistance occurs at the remembered level. It's either a natural resistance with a weak affinity of

certain antibiotics for targets, or a resistance acquired with the modification of targets and the

affinity loss to antibiotics for these targets.

Natural resistance

Various molecules in the family does not have all the same affinity for the bacterial targets.

Aztreonam (monobactams) has weak affinity for the PLP of Gram-positive and rigorous

anaerobic bacteria , which explains their natural resistance against this antibiotic.17

Likewise, first-generation quinolones (nalidixic acid and pipemidic) have only weak affinity for

DNA gyrase Staphylococcus.

Acquired resistance

This type of mechanism is responsible for a large number of acquired resistance. We

quote Staphylococcus resistance against beta-lactams. It is about the acquisition of genetic

material (mecA gene). This gene encodes the synthesis of a new PLP: PLP 2a or 2 'which

possesses a weak affinity for beta-lactams, whatever the molecule, also explaining cross-

resistance against the whole family.

Similarly, Streptococcus pneumoniae, sensitive to penicillin G is no longer sensitive

(only in 40% of the cases in France). The acquisition of foreign genes by transformation from

other streptococci or pneumococcus is responsible for this resistance.

17 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3257838/

Another way of acquiring this type of resistance is met in bacteria: a chromosome

mutation touches the gene ruling the target . DNA gyrase gene mutations train modified enzyme

synthesizing possessing low affinity against quinolones molecules.18

Resistance by enzymatic modification

Quantitatively and qualitatively, this type of mechanism is certainly the most important.

Many classes of antibiotics and virtually all bacterial species are concerned. To be active, the

antibiotic must reach its target intact. If there is modification of the antibiotic by enzymes present

in bacteria, at any level, modified form of the antibiotics molecule is most often inactivate.

These enzymes occurr naturally or acquired at bacteria of clinical interest .

The natural resistance

The most important enzymes are enzymes directed clinically against beta-lactam beta-

lactamases. These enzymes destroy beta-lactam ring, making the antibiotic inactive. Within this

family, the molecule choice is obviously based on the certain or supposed bacterium . The first

criterion is the presence of a natural resistance, more often the presence of beta-lactamases . For

this family, it is essential to have a good knowledge of wild phenotypes, so natural resistances.

Many species naturally produce these inactivating enzymes . This is the case of

Enterobacteriaceae, Pseudomonas and acinar-tobacter. Similarly, the Group II of

Enterobacteriaceae possess beta-lactamase that inhibits penicillin (amino, carboxy,

ureidopeniciline). Enterobacteriaceae from Groups III and IV, like Pseudomonas and

Acinetobacter produce another enzyme: a cephalosporinase explaining the natural resistance of

these bacteria. This type of resistance is not only beta-lactam reserved. In its natural state,

18 Ibidem

certain bacteria produce enzymes that inactivate aminozide.This is the case of Serratia marces-

census , that inactivates tobramycin, netilmicin and amikacin, as the Providencia stuartii that

inactivates gentamicin, tobramycin and netilmicin.19

Accquired resistance

Following the beta-lactams described, it is clear that it is important to detect MECA-resistant

organisms through the acquisition of enzymes. Geneticaly , beta-lactamases should distinguish

the plasmid origin from the chromosomal origin. In most cases, it is a genetic material

acquisition through plasmids. We are talking about plasmid resistance . Depending on their

spectrum of activity, we organize:in penicillinases, as staphylococci; in broad spectrum senior

penicillinases and beta-lactamases, as the enterobacteria.

If we talk about aminozide, there arevnumerous inactivating enzymes of plasmid origin .

Depending on their type, they are responsible for the inactivation of one or more molecules used

in clinical gentamicin, tobramycin, netilmicin or amikacin. They are frequent in

Enterobacteriaceae, Pseudomonas and, more frequently, in staphylococci.

Another mechanism of acquiring resistance is often seen in Gram negative bacteria. This is beta-

lactams. This is no longer foreign gene, but mutations in genes that govern the synthesis of

natural cephalosporinase. This is no longer synthesized at low levels, but at a very high level,

responsible for inhibiting a greater number of molecules of beta-lactams.

Mechanisms to acquire antibiotic resistance. The interaction of the microbial resistance

and anti-bacterial agents appears in a direct and indirect manner (RUBINSTEIN, 1999):

• directly - by the development of resistance to the antibiotics used or to the antimicrobial

agents belonging to the same class: for example, betalactamase induction by both gram-positive

and gram-negative; as well as through the development of resistance to the components of the

different classes of antibiotics used to treat for example, loss of susceptibility to penicillin of

Streptococcus pneumoniae, together with a simultaneous loss of susceptibility to erythromycin

and tetracycline.

19 Ibidem

• indirectly - microbial resistance can develop by selection of resistant organisms when

the patient is treated with antibiotics, when the environment is contaminated with antibiotics

(intensive care unit) or the antibacterial agents used in agriculture or as animal growth promoters

2.2. Chromosomal versus plasmidial resistance

Genetic mechanisms

Antibiotic resistance in bacteria can be induced by mechanisms of genetic variation both

endogenous (mutations and translocations) and exogenous (chromosomal recombination,

plasmid transfer resistant, etc.). 21

The chromosomal resistance. This type of resistance occurs due to mutations in the

bacterial chromosome nucleotide sequences that determine the synthesis of proteins and other

macromolecules that differ to a great extent from original chemical entities that interfere with

antibiotic activity. Chromosome mutations can be spontaneous or induced by mutagen agents

(especially antibiotics) (Henderson, 1999; Kuhn et al., 2003), are genotyped, with vertical

transmission (performed without contact with the antibiotic), appear suddenly in one medicine 20 https://www.google.ro/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0CAcQjRxqFQoTCNqhn5WOx8cCFcVcFAodxksO3Q&url=http%3A%2F%2Flyonlive.com%2Fsupport%2Fext1731.html&ei=ieHdVZq5DMW5UcaXuegN&psig=AFQjCNFZyG666dmesjr5qT2Qae2l0LR5OQ&ust=1440690949701197

21 Ibidem

and are final. However, the number of resistant mutants will decrease after the end of exposure to

the antibiotic (Swartz, 2000).

In a population exposed to an antibiotic, development of chromosome resistance is

usually a gradual process, careful monitored, carried out through several consecutive mutations;

However, for some antibiotics a single mutation may determin resistance resulting in an intense

increase of MIC. The emergence of resistant mutants is much less frequent in vivo than in vitro,

probably because mutations leading to resistance are usually associated with other cellular

changes that may be disadvantageous for bacteria. For this reason, some scientists see the

development of resistance determined by mutations on chromosome as a small problem

compared with transferable resistance. However, in practice, these mutations have little

significance due to the fact that rarely occur (10%) (Jackson et al., 2004).

Transferable resistance.

Bacteria have extremely efficient genetic transfer systems capable of exchanging and

accumulating resistance genes. Some genes, including genes which encode resistance, can move

between the elements of chromosomal and extrachromosomal DNA of the bacteria.

Extrachromosomal mutations are more frequent (90%), so of great practical importance. Genes

can be transmitted between bacteria of the same species or different species or different genres of

bacteria (horizontal transfer), that have common habitat in the body. Interspecies transfer means

that, once transferable resistance gene have occurred, the bacteria carrying the gene will remain

a potential donor for other bacterial genes (Agerso et al., 2005).

The most important carrier for the transfer of resistance genes in bacteria are plasmids,

transposons and integrons (Swartz, 2000). As specified by McDermott (2002), in recent years a

number of resistance genes have been associated with the massive transfer of extrachromosomal

DNA elements called plasmids, which can be other mobile DNA elements such as transposons

and integrons.

It has been shown that these mobile elements transmit genetic determinants of

antimicrobial resistance mechanisms and they matter in the dissemination of resistance genes

between different bacteria. Plasmids are extrachromosomal DNA molecules, replicable, which

may contain resistance genes (MARBLE, 1999). Replication is independent of chromosomal

DNA.22

They are important in the development of bacterial plasmids, since they affect the

replication, metabolism, bacteria fertility, as well as resistance to bacterial toxins (bacteriocins),

antibiotics and bacteriophages, thus ensuring a better chance for survival and propagation.

However, the general plasmids are not strictly necessary for the survival of the bacteria. They

have been identified in the majority of bacterial species, having the ability to be transferred

(conjugative), or co-transferred (non-conjugative) from a bacterium to another, thereby leading

to a wide dissemination of the plasmid-encoded characteristics within an ecosystem.

The genes encoded by the plasmid is inherently more mobile than chromosomal genes

because plasmids can be transferred within a bacterial species or between different species

(Swartz, 2000). R-plasmids are plasmids containing the resistance gene (AOKI, 1993, KIM. Et

al., 1996, Diaz et al., 2006). The purchase of new resistance determinants may occur more

rapidly than the R-plasmid genetic mutation.

One R-plasmid may encode resistance simultaneously to more than 10 different

antibiotics. There have been found many different r-plasmids. A single bacterial cell may contain

several different plasmids and each plasmid may have more resistance genes. Plasmids isolates

from human and veterinary seem to be very similar, suggesting even their transmission from

animals to humans (WRAY C. et al., 1986) or from humans to animals (Sannes et al., 2004).

Dissemination of plasmids can occur through clonal distribution and intra- and inter-species

transfer, leading to a gradual increase in the proportion of microorganisms in a bacterial

community transporting one or more factors R.23

Transmission of plasmids from one bacteria to another is done in several ways: -

conjugation (recombinant) - by sexual pili; - Transducing phage - the plasmid is taken from a

bacteriophage, and transferred to a plasmid-free bacteria; - Transformation - the plasmid is taken

22 Ibidem

23 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3257838/

directly from another organism, after a bacteria destruction (KEHRENBERG, 2000;

FURUSHITA, 2003).

The resistance plasmid is dependent on the genotype, having the transmision both

horizontally and vertically as possible from the one or more antibiotics (poly-resistant) (Johnson

et al., 1994). Transposons ("jumping genes") are short sequences of DNA that can move from

plasmids, between the plasmid and the bacterial chromosome or between a plasmid and a

bacteriophage (bacterial virus) (Henderson, 2000). In contrast to plasmids, transposons are not

able to replicate independently and must be maintained in a functional replicon (eg. Plasmid or

chromosome).

Transposons at gram-negative bacteria are non-conjugative, while at gram-positive

bacteria Bacteriodes spp., may be either conjugative or non-conjugative. However, if a gram-

negative bacteria of the transposon DNA is part of a conjugative plasmid, horizontal transfer is

possible. Transposons, including those carrying resistance genes (KEHRENBERG et al., 1998;

RIBERA et al., 2003),are easily purchased by the plasmids and then incorporated into bacterial

DNA. Transposons are often more crowded on the same plasmid, causing the transfer of several

resistance determinants in a single conjugations. Also, plasmids from different backgrounds can

carry multiple sets of identical genes of resistance.

The intracellular transfer of the transposon from plasmids between bacterial

chromosomes and plasmids also interbacterian transfer of conjugative plasmids and transposons

can lead to a rapid development of resistance in bacterial populations. Expression of the

resistance gene located on transposons (eg. The production of enzymes) may require the

presence of the antibiotic. Moreover, the presence of antibiotic resistance will promote the

transfer.

Antibiotics create an environment in which the resistance determinants possession is

advantageous and, in addition, will increase the rate of transfer of resistance genes. Integrons.

Integrons are natural components of gene expression. They are composed of two conserved

regions and a variable interposed region, containing cassettes of genes for resistance to

antibiotics (Hall and Collis 1995).

Gene cassettes are elements that include a single gene and a place ("Site") of

recombination. More than 40 cassettes have been identified, , and all but 5 of them contain the

resistance gene (Agerso et al., 2005). One of the conserved regions of integrons contains

integrase gene, which is responsible for specific insertion at the place ("Site") in the boxes

(Swartz, 2000). Integrons can be located in chromosomal DNA, but more often are located in

plasmids (Diaz et al., 2006) or transposons and are therefore mobile. For example, the

chromosome resistance characteristic pattern of Salmonella typhimurium DT104 is associated

with the presence of integrons (Swartz, 2000).

Antibiotic resistance 1940-2000

2.3. Biochemical mechanisms of antibiotic resistance

Bacterial resistance to antibiotics, both the natural and acquired resistance, is achieved

through a variety of mechanisms (ANGELESCU, 1998):

1.Enzymatic inactivation of antibiotics, resistant plasmids and, more rarely, the

chromosomes, encodes enzymes of antibiotics inactivation . We know several categories of

inactivating enzymes: beta-lactam antibiotics, aminoglycoside antibiotics, chloramphenicol.

Betalactamazele. They are enzymes found in most bacterial species, which have the ability to

hydrolyze the beta-lactam ring of beta-lactam antibiotics, which leads to complete inactivation of

the antibiotic. Betalactamase may have chromosomal or plasmid-determinism. 24

The genes for the beta-lactamases are located on chromosome or on plasmid, and can be

translocated in the chromosome or in another plasmid by the transposon. Transfer within and

between species or genres successfully explains the spread of resistance mediated by these

ennzime. Betalactamazese classification can be based on molecular characteristics and / or

functional characteristics: Staphylococcus aureus beta-lactamase - there are four serologically

distinct forms that are closely related at the molecular level. Its production can be mediated

plasmidic or chromosomial.

They are particularly active in the penicillin. Chromosomal cephalosporinases of gram-

negative bacteria - virtually all gram-negative bacteria produce enzymes mediated

chromosomyal. Most of these preferential beta-lactamases hydrolyze cephalosporins. Some of

these chromosomal beta-lactamases are able to hydrolyse betalactmine newer such as

cefotaxime, cefuroxime, moxolactam, aztreonam, imipenem.

Plasmidic-mediated betalactamase - are more common gram-negative bacteria and can be

divided in their turn into three main groups: broad spectrum penicillinases (TEM), which

hydrolyze penicillins and cephalosporins in similar proportions, the most widespread in

Enterobacteriaceae; oxacilinaze's (OXA), which rapidly hydrolyze oxacillin and cloxacillin;

carbenicilinaze's (CARB), which hydrolyze preferentially carbenicillin. Metallo-beta-lactamases

- perhaps the most formidable beta-lactamases known. Rapidly hydrolyze most beta-lactam

agents, including carbapenems.It is resistant to Beta-lactamase inhibitors. These enzymes have

been limited to a few strains originally isolated from B. cereus and Bacteroides fragilis, as

chromosomal enzymes, but were subsequently identified in Japan on plasmids carried by

Bacteriodes fragilis, Serratia marcescens, Klebsiella pneumoniae and Pseudomonas aeruginosa.

Such strains appear to be confined to localized areas, but have potential for wide

dissemination. Enzymes amending aminoglycosides. Nucleotidiltransferase, acetyltransferase

24 Hall and Collis 1995

genes encoded plasmid phosphotransferase or transposable elements that often gives them a high

level of resistance. Aminoglycosides are modified by these enzymes at the level of amino and

hydroxyl groups especially through mechanisms of phosphorylation, acetylation or adenilare,

and these changes lead to inactivation of antibiotics.

Inactivating enzymes of chloramphenicol. Resistance is likely a plasmid and is due to the

production of an enzyme by the bacterium, acetyltransferase, causing antibiotic acetylation,

resulting its inactivation.

2. Antibiotic resistance through bacteria changes a. modifying the permeability of bacterial

coatings

• Structural alterations of bacterial coatings - outer membrane permeability reduction by

reducing the synthesis of porins so as decreasing the number of functional porins → applicable

gram-negative bacteria which are in this manner impermeable to betalactamine and quinolones -

development of impermeable cell walls due to extremely narrow porins → for example

Pseudomonas aeruginosa compared to multiple antibiotics - strengthening the barrier function of

the outer membrane proteins by producing additional intercalated protein in its complex

structure. → thus producing E. coli tetracycline resistance - reducing bacterial wall permeability

through the loss of a outer membrane protein . → resistance of bacteria compared to

chloramphenicol - reducing bacterial permeability deficient in intracellular penetration of

antibiotics → is performed compared to sulfonamides, trimethoprim, nitrofurans, nitroimidazoles

• modification of active antibiotics transport systems through bacterial coatings → for example

Pseudomonas aeruginosa compared to aminoglycosides

b. alteration of headquarters - the target of inhibitory activity of the antibiotic - modification of

PBP (penicillin binding proteins), inserted proteins on the external membrane of the cell, as

action target: betalactamine - ribosomal protein modification target: aminoglycosides,

tetracyclines - macrolides, lincosamides, glycopeptides also determined the modifying of the

target of action - reducing the affinity of action for quinolones is caused by a structural change in

DNA gyrase. 25

25 Hall and Collis 1995

c. Active efflux of antibiotics in the bacterial cell - impedes the effective concentrations of

antibioticsin the cell : tetracycline, fluoroquinolones (Staphylococcus aureus), erythromycin

d. modifying enzymes: dihidropterat synthase (DHPS) for sulfonamides and dihydrofolate

reductase (DHFR) to trimethoprim, so that the chemotherapeutic agents can not achieve

competition with the enzymes involved in folate synthesis

e. lack of action of some enzymes: nitrofuran-reductase in the case of nitrofurans and nitro-

reductase in the case of nitroimidazoles

f. the acquisition by the bacteria of some enzymes - esterase, nucleotidiltransferases, enzymes of

plasmid origin, changes macrolides and lincosamides .

g. metabolite increased production of the antimicrobial agent is in competition - increasing

paraaminobenzoic acid synthesis (APAB) cancels competitive inhibitory action of sulfonamides.

Therefore, given the complexity of resistance to antibiotics and continued growth of this

phenomenon, constant surveillance is needed, involving medical- veterinary laboratories and the

empowered authorities worldwide.

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