Bacterial resistance mechanisms and new trends for resistance overcoming

Bacterial resistance

By: Mohammed Fawzy

Microbiology lab at NODCAR

Agenda

I. Overview.II. Origin of resistanceIII. Major mechanisms of resistanceIV. Factors that promote bacterial resistanceV. Antibacterial in food and animal industriesVI. Consequence of antibiotics resistanceVII.New trends for overcoming bacterial resistanceVIII.Questions

overview

Worldwide, antibacterial resistance has increased dramatically over the past few years and is currently recognized as a major medical challenge in most healthcare settings.

After the discovery of penicillin in 1928, a number of treatment failures and occurrence of some bacteria such as staphylococci which were no longer sensitive to penicillin started being noticed. This marked the beginning of the error of antimicrobial resistance.

Increasing prevalence of resistance has been reported in many pathogens over the years in different regions of the world including developing countries(Byarugaba, 2005). As MRSA, Pseudomonas aeruginosa.

Overview

Throughout history, there has been a continual battle between humans and the multitude of microorganisms that cause infection and disease.

Examples:

Bubonic plague, tuberculosis, malaria, and more recently, the human immunodeficiency virus/acquired immunodeficiency syndrome pandemic, have affected substantial portions of the human population, causing significant morbidity and mortality.

Definitions

Antimicrobial resistance (AMR):• It’s define as resistance of a microorganism to an antimicrobial

medicine to which it was originally sensitive.• Resistant organisms (they include bacteria, fungi, viruses and some

parasites) are able to withstand attack by antimicrobial medicines, such as antibiotics, antifungals, antivirals, and antimalarial

• so that standard treatments become ineffective and infections persist increasing risk of spread to others. The evolution of resistant strains is a natural phenomenon that happens when microorganisms are exposed to antimicrobial drugs, and resistant traits can be exchanged between certain types of bacteria.(WHO 2013)

(cont.)

Multi-drug resistance (MDR)• Is defined as having acquired non-susceptibility to at least one

agent in three or more antimicrobial categories.

Extensive drug resistance (EDR)• Is defined as non-susceptibility to at least one agent in all but

two or fewer antimicrobial categories (i.e. bacterial isolates remain susceptible to only one or two categories).

Pandrug-resistant (PDR) • Is defined as non-susceptibility to all agents in all antimicrobial

categories.

ORIGIN OF RESISTANCE

Bacterial resistance to antimicrobial agents may be intrinsic or acquired, intrinsic resistance as resistance of Mycoplasma species to B-lactams antibiotic, due to it’s lack of cell wall and pleomorphic characters.

And acquired resistance is arise from de novo mutation of DNA sequence or by horizontal gene transfer by different mechanisms (transformation, transduction and conjugation ).

Origin of resistance

Intrinsic resistance(IR)is that type of resistance which is naturally coded and

expressed by all (or almost all) strains of that particular bacterial species. An example of intrinsic resistance is the natural resistance of anaerobes to aminoglycosides and Gram-negative bacteria against Vancomycin.

the resistant genes are maintained in nature because of the presence of antibiotics producing bacteria in soil. These antibiotics act on other bacterial species other than the producer bacteria, There has to be a mechanism of protection in the host bacteria against the antibiotics that it produces, which could be the source of genes encoding resistance

(cont.) is the innate ability of a bacterial species to resist activity of a

particular antimicrobial agent through its inherent structural or functional characteristics, which allow tolerance of a particular drug or antimicrobial class. This can also be called “insensitivity” since it occurs in organisms that have never been susceptible to that particular drug. Such natural insensitivity can be due to:

I. lack of affinity of the drug for the bacterial target.II. Inaccessibility of the drug into the bacterial cell.III. Extrusion of the drug by chromosomally encoded

active exporters.IV. Innate production of enzymes that inactivate the drug.

MECHANISM NATURAL RESISTANCE AGAINST

ORGANISM

Lack of oxidative metabolism to drive uptake of aminoglycosides

Aminoglycoside Anaerobic bacteria

Inability to reduce drug to active form

Metronidazole Aerobic bacteria

Lack of PBPs Aztreonam Gram-positive bacteriaLack of uptake(increase thickness of PG layer)

Vancomycin Gram-negative bacteria

Beta-lactamase Ampicillin Klebsiella spp.Beta-lactamase Imipenem Stenotrophomonas.

maltophila

Lack of appropriate cell wall precursor target

Vancomycin Lactobacilli and Leuconostoc

Lack of uptake resulting Sulfonamides, trimethoprim, tetracycline, or chloramphenicol

Pseudomonas aeruginosa

Lack of sufficient oxidative metabolism to drive uptake of aminoglycosides

Aminoglycosides Enterococci

Lack of PBPs All cephalosporins

(cont.)Acquired resistance(AR)

Acquired resistance is said to occur when a particular microorganism obtains the ability to resist the activity of a particular antimicrobial agent to which it was previously susceptible.By mutation By horizontal gene transfer

1. Mutation It’s define as permanent change in the sequence of DNA nucleotide of gene. This change can take place either by alteration, loss or gain of the nucleotide. Types

1. Spontaneous mutation ( occurs by natural physical agents as HEAT and IRRADIATION , in which energize DNA nucleotide so that subsequent intra-molecular rearrangement of bases lead to incorrect base –pairing and ultimately mutation.

2. Induced mutation(occurs by intentional treatment of the cell with physical or chemical agents that alter base sequences.

(cont.)

Other types of mutation:1. Point mutation → change in single base-pair in the DNA.2. Substitution → replacement of an original base-pair or sequence of base-

pair by another, may be transition (same) or transversion (different).3. Deletion.4. Insertion.5. Silent.6. Reading frame shift mutation.7. Non-sense. 8. Missense.9. Lethal mutation.10. Back mutation. 11. Condition lethal mutation.12. Suppressor mutation.

(cont.)

2- Horizontal gene transfer(HGT) It’s recombination between two genetically different DNA molecules, then the resistance is acquired. Acquisition of foreign genetic elements in prokaryotes may occur by three main mechanisms.

I. TRANSFORMATION → direct passage of free DNA (naked) from one cell to another. The receiving bacteria then simply introduce the free DNA in to their cytoplasm and then incorporate it to their own DNA.

II. TRANSDUCTION → transfer of genetic element by mean of vector (usually virus) called bacteriophage.

III. CONJUGATION→it’s the most important and most common mechanism of gen transfer, this mechanism is mediated by plasmid

(bacteria containing plasmid called F positive. But the other cell is

called F negative.

(cont.) Transposon

It’s a mobile genetic element involved in horizontal gen transfer. Have the ability to move from place to place on the chromosome and in to and out plasmid. Types:

1- Replicative → it's leave a copy of itself at the original site. 2- Non replicative → it's not leave a copy of itself at the original site.

N.B. transposon can enter the functional gene Size about 5 kilobases. Two enzyme are involved in transposition process

1-Transposase2-Resolvase

Transposon contains two inverted repeat, in which the two enzymes are identifying. Mobile genetic element are probably responsible for most of the genetic variability in natural

bacterial population, and the spread of bacterial resistance genes. Some transposons may contain a special, more complex DNA fragment called ‘‘integron’’, a site

capable of integrating different antibiotic resistance genes and thus able to confer multiple antibiotic resistance to a bacteria. Integrons have been identified in both gram-negative and gram-positive bacteria, and they seem to confer high-level multiple drug resistance to the bacteria that carry and express them

MECHANISM INVOLVED RESISTANCE OBSERVED ACQUIRED RESISTANCE THROUGH

Point mutations in the rifampin-binding region of rpoB

Mycobacterium tuberculosis resistance to rifamycins

Mutations

Mutations in the chromosomal gene specifying dihydrofolate reductase

E.coli, Hemophilius influenzae resistance to trimethoprim

Via acquisition of mecA genes which is on a mobile genetic element called “staphylococcal cassette chromosome” (SCCmec) which codes for penicllin binding proteins (PBPs) that are not sensitive to ß-lactam inhibition

Staphylococcus aureus resistance to methicillin (MRSA)

Horizontal gene transfer

Major biological mechanisms of antimicrobial resistance

Whichever way a gene is transferred to a bacterium, the development of antibiotic resistance occurs when the gene is able to express itself and produce a tangible biological effect resulting in the loss of activity of the antibiotic.

Microbes utilize numerous mechanisms of resistance to antimicrobial Drugs they can be summarized as follow:

I. Decreased uptake and increased efflux of drug from the microbial cell.

II. Expression of resistance genes that code for an altered version of the substrate to which the antimicrobial agent binds.

III. Covalent modification of the antimicrobial drug molecule which inactivates its antimicrobial activity.

IV. Increased production of a competitive inhibitor of antibiotic.V. Drug tolerance of metabolically inactive persisters.VI. Biofilms.VII. Swarming.

(cont.)

I. Decreased uptake(impermeability) and increased efflux of drug from the microbial cell.• Decreased uptake of antimicrobial drugs and/or use of transmembrane efflux pumps

prevents the concentration of antimicrobial agent from increasing to toxic levels within the microbial cell (↓uptake↓conc↓effect).

• Gram negative bacteria have an outer membrane surrounding a periplasmic space (which contains a peptidoglycan cell wall),which surrounds an innermembrane, whereas Gram positive bacteria have a peptidoglycan cell wall surrounding only a single plasma membrane .

• This outer membrane may provide an extra barrier against drug uptake (especially hydrophobic drugs) in Gram negative bacteria, which is not present in Gram positive bacteria. This is one explanation why Gram negative bacteria are less susceptible than Gram positive bacteria to many antibiotics, including beta-lactams and macrolides.

(cont.)• E.g. P. aeruginosa and E.coli are containing proton-dependant

efflux pump which expel the drug outside the cell.• Exampls

Tetracyclin resistance byTetA,B and k gen mediated efflux pump.Fluroquinolon resistance by decreas uptak Vancomycin resistance By increas thickness of bacterial cell wall,

so decreas uptak.

EFFLUX AND IMMPERMEABILITY

(cont.)

II. Expression of resistance genes that code for an altered version of the substrate to which the antimicrobial agent binds

GENE mutation → translated to altered protein( substrate) → low binding affinity→ reduced antibacterial activity → resistance developed.

E.g.

• MacA resistance gene codding for PBP2A (altered form than wild-type), represent resistance of MRSA against B-lactams.

• VanA resistance gene codding for altered binding substrate (D-alanine–D-lactate ligase, Vancomycin has 1000 times lower affinity for D-alanine–D-lactate than D-alanine–D-alanine, so the VanA gene confers resistance to vancomycin. Both vancomycin resistant Enterococcus (VRE) and vancomycin-resistant S. aureus (VRSA) express VanA.

(cont).• Expression of altered DIHYDROFOLATE PETROATE represent sulfonamide

resistance, Bacteria using this resistance mechanism include S. pneumoniae, S. pyogenes, Neisseria meningitidis, and E. coli.

• Altered gyrA and gyrB, represent resistance of Gm-ve against Quinolones.• Altered Topoisomerase IV, represent resistance of Gm+ve against

Quinolones.

(cont.)

III. Covalent modification of the antimicrobial drug molecule which inactivates its antimicrobial activity.

Microbes can also express drug resistance genes that code for enzymes that covalently modify the antimicrobial drug, thereby reducing its antimicrobial activity.

E.g.i. beta-lactamases hydrolyze the beta-ring of betalactams, thereby

inactivating the antibiotic activity of the beta-lactam molecule and conferring beta-lactam resistance.

ii. ACT N-acetyltransferse, which acetylates an NH2 group of the aminoglycoside molecule.

iii. APH O-phosphotransferase, which phosphorylatesan OH group of the aminoglycoside molecule.

iv. and the ANT O-adenyltransferase, which adenylates an OH group of the aminoglycoside molecule.

v. Acetyltransferases, which acetylate and thereby inactivate chloramphenicol.

(cont.)Aminoglycoside inactivating

enzymes Penicillin inactivating enzyme

(cont.)

IV. Increase production of competitive inhibitors.

Bacteria can also achieve antibiotic resistance by synthesizing a molecule that is a competitive inhibitor of the antibiotic(Enzyme Substrate).

Example Mechanism of sulfonamide resistance is increased

synthesis by bacteria of para-aminobenzoic acid (PABA), which competes with the sulfonamide drug for the binding site of bacterial dihydropteroate synthetase.

This mechanism of sulfonamide resistance is used by S. aureus and N. meningitidis.

(cont).

V. Drug tolerance of metabolically inactive persisters.

The presence of metabolically inactive persisters at the site of infection in close to actively bacterial population, results in antibacterial tolerance.

Recurrence of infection after treatment is usually occur. This mechanism occur due to expression of gene called

Toxin-Antitoxin, which cause their metabolic activity to slow or stop.

After the hos exposed to antibacterial agent, the actively metabolic bacterial of population eradicated.

And the persisters are turn to metabolically active and cause recurrence of infection.

(cont).

VI. Biofilm.Biofilm formation can result in tolerance of bacteria to very high concentrations of multiple antibiotics, resulting in chronic infections despite antibiotic treatment.

Steps of biofilmI. Formation of conditioning biofilm.II. initial attachment.III. Irreversible attachment and synthesis and secretion of a matrix

consisting of extracellular polymeric substance (EPS). This EPS matrix accumulates and eventually surrounds the population of bacterial cells

IV. Biofilm growing.V. Detachment.VI. Formation of a new conditioning biofilm in other site in host.

Biofilm steps

Role of Extracellular polymeric substance in resistanse

I. Act as barrier to diffusion of oxygen and nutrients. In turn the deeply located bacteria to metabolically in active and tolerate antibacterial agent rather than superficially located bacteria.

II. Decrease diffusion of antibacterial agent to bacterial population, so concentration not reach to MIC due to:

Small pores of EPS. The negative charge of the EPS matrix also traps antibiotic molecules

before they can affect the bacterial cells Third, enzymes within the EPS matrix also covalently modify

antibiotic molecules, thereby inactivating their antimicrobial activity.

(cont.)

VII.Swarming. type of multicellularity in bacteria and operates by the

following mechanism:I. Planktonic bacterial cells differentiate into elongated cells

with multiple flagella (swarm cell).II. More swarm cell adhere together and act as single unit.

These swarm cells are also tolerant to antimicrobial agent.III. Subculturing of swarm cell in a liquid media, reverse back

to planktonic bacteria which no longer have tolerance to antibacterial agent.

E.g. Bacillus subtilis, Serratia marcescens, E. coli, Salmonella typhimurium and P. aeruginosa.

Planktonic form: are single-cells that may float or swim in a liquid medium.

Factors that promote bacterial resistance

suboptimal use of antimicrobials for prophylaxis and treatment of infection.

noncompliance with infection-control practices. prolonged hospitalization, increased number and duration of

intensive care-unit stays, multiple comorbidities in hospitalized patient.

increased use of invasive devices and catheters. ineffective infection-control practices, transfer of colonized patients

from hospital to hospital grouping of colonized patients in long-term-care facilities. antibiotic use in agriculture and household chores. increasing national and international travel.Lack of education and poverty.

Consequence of antibacterial resistance

Antibacterial in food and animal industries

Veterinary antibiotics (VAs) are widely used in many countries worldwide to treat disease and protect the health of animals.

They are also incorporated into animal feed to improve growth rate and feed efficiency. As antibiotics are poorly adsorbed in the gut of the animals, is excreted unchanged in faeces

and urine. Given that land application of animal waste as a supplement to fertilizer. there is a growing international concern about the potential impact of antibiotic residues on

the environment. E.g. tetracycline, chloramphenicol, triclosan and bacitracin.

New trends for overcoming bacterial resistance

Due to global emergence of antibacterial resistance, scientists are introduce a new strategies to overcome resistance.

Many of this strategies are

I. Plant compounds with resistance modifying activities.II. Nanotechnology as a therapeutic tool to combat microbial

resistance.

I. Some antibiotic resistance modifying compounds from plants

REFERANCE ANTIBIOTIC POTENTIATED

PLANT SOURCE COMPOUND

Smith et al. (2007) Oxacillin, Tetracycline,NorfloxacinTetracycline

Chamaecyparis lawsoniana

Ferruginol5-Epipisiferol

Marquez et al. (2005) Ciprofloxacin, Norfloxacin,Pefloxacin, Acriflavine and Ethidium bromide

Jatropha elliptica 2,6-dimethyl-4-phenylpyridine-3,5-dicarboxylicacid diethyl ester

Oluwatuyi et al. (2004)

Erythromycin Rosmarinus officinalis Carnosic acid carnosol

Shibata et al. (2005) B-lactams Caesalpinia spinosa Ethyl gallate

Gibbons et al. (2004)Hu et al. (2002)Zhao et al. (2001)

NorfloxacinImipenemPanipenemB-Lactams

Camellia sinensis Epicatechin gallateEpigallocatechin gallate

II. Nanotechnology as a therapeutic tool to combat microbial resistance.

Use of nanoparticles is among the most promising strategies to overcome microbial drug resistance.

Example

Nanoparticles with multiple simultaneous mechanisms of action against microbes

Nitric oxide-releasing nanoparticles (NO NPs).Chitosan-containing nanoparticles (chitosan NPs).Metal-containing nanoparticles.Nanoparticles that target antimicrobial agents to the

site of infection.Liposomes nano-particles.Dendrimers.

Finally

So………..