Multi-Drug Resistant Organisms

(MDROs)Dr Mostafa Mahmoud, MD, Ph D,

Consultant MicrobiologistAssist. Prof. of Medical Microbiology &

Immunology

Genetic materials controlling bacterial functions are:

• 1- Chromosome• 2- Mobile genetic elements (plasmids, transposons, genetic

islands).

Bacterial cell structure (Prokaryote)

Classification and mechanism of action of antimicrobials

• Antimicrobial agents are substances that kill or inhibit the growth of microorganisms and are suitable for systemic use.

• If this antimicrobial substance is synthesized in the laboratory it is named Chemotherapeutic.

• The term “antibiotic” is a substance produced as a secondary metabolite by a bacterium which inhibits or kills other microorganisms.

Some Naturally Produced AntibioticsSpecies Microorganism Antibiotic produced

Gram positive Bacteria Bacillus subtilis Bacitracin Bacillus polymixa Polymixin

Actinomycetes Micromonospora purpurea Gentamycin Streptomyces Streptomyces erythreus Erythromycin

Streptomyces griseus Streptomycin Streptomyces rimosus Tetracycline Streptomyces orientalis Vancomycin

Fungi Penicillium chrysogenum Penicillin Cephalosporium acremonium Cephalosporins

Miscellaneous Pseudomonas fluorescens pseudomonic acids*

classifications of antimicrobials:

• Several ways according to:• Mechanism of action: (inhibit cell wall, protein, or NA synthesis

etc).• Spectrum of activity: (broad or narrow spectrum). • Killing or inhibitory effect upon microorganism: (Bacteriostatic or

Bactericidal).• The chemical structure.

The classification of AB by the mechanism of action

1- Inhibition of bacterial cell wall synthesisGroup Examples Spectrum of action Penicllins: 1-Natural Penicillin G (injection)

Penicillin V (oral)Gram positive bacteria.

2-Penicillinase-Resistant Penicillins

Cloxacillin - Dicloxacillin - Methicillin, Nafcillin - Oxacillin

Antistaphylococcal actions

3- Aminopenicillins Amoxicillin - AmpicillinAmoxicillin/clavulanate Ampicllin/sulbactambacapicillin

Gram +ve and Gram -ve bacteria.

4- Carboxypenicillins Carbenicillin - Ticracillin,Ticracillin/clavulante

Greater activity against gram negative organisms

5-Ureidopenicillins and Piperazine

Mezlocillin - PiperacillinPiperacillin/tazobactam

They have the broadest-spectrum of all penicillins especially on Pseudomonas aeruginosa

Group Examples Spectrum of action

2-Cephalosporins:1st generation

Cefadroxil - Cefazolin - Cephalexin Cephalothin - Cephradine

Gram (+ve) & few Gram (–ve) e.g. E. coli, Klebsiella

2nd generation Cefaclor – Cefamandole - Cefmetazole, Cefoxitin - Cefoprzil - Cefuroxime

More G (–ve) e.g. Klebsiella, Proteus, less on (G+ve)

3rd generation Cefixime – Cefoprazone - Cefotaxime, Ceftazidime - Ceftriaxone

Pseudomonas & Enterobacter.- TTT of HAIs.

4th generation Cefepime upon G (+ve) & G (-ve) organisms, including P. aeruginosa

Other cell wall inhibitors:

Group Examples Spectrum of action 3- Carbapenems Imipenem - Meropenem

Etrapenem - Doripenembroad-spectrum of activity (MSSA- Pseudomonas).

4- Glycopeptides Vancomycin Bacitracin Teicoplanin

MRSA

5- Monobactams Aztreonam Aerobic Gram-negative microorganisms

N.B. - Beta-Lactam Antimicrobials include: PENICLLINS, CEPHALOSPORINS, CARBAPENEMS, MONOBACTAMS. - All Beta-Lactam Antimicrobials are generally Bactericidal on bacteria.

2- Interference with cell membrane function

• This group includes some antibacterial agents e.g. polymyxin B and colistin, and antifungal agents e.g. amphotricin B, imidazoles and nystatin.

3-Inhibition of bacterial protein synthesisGroup Examples Mechanism of action Effect bacteria

Aminoglycosides Streptomycin - Neomycin, Kanamycin - Tobramycin, Netilimicin - Amkacin

Irreversible binding to 30S ribosomal subunit

Bactericidal

Tetracyclines Tetracycline - Oxytetracycline Demeclocycline – Doxycycline Minocycline

Reversibly bind to 30S subunit

Bacteriostatic (Chlamydia & rickettsia)

Glycocyclines (30S= TAGG)

Tigecycline Binds to 30S ribosomal subunit.

Bacteriostatic (mostly)(for MRSA, GISA, ESBL, and VRE)

Group Examples Mechanism of action Effect bacteriaChloramphenicol Chloramphenicol Binds 50S and inhibits

peptidyl transferaseBacteriostatic

Clindamycin Clindamycin Binds to 50S Bacteriostatic(Anaerobes)

Macrolides Erythromycin, azithromycin, clarithromycin Dirthromycin, Troleandomycin

Bind to the 50S subunit inhibiting RNA-dependent protein synthesis.

Bacteriostatic or Bactericidal

Ketolides Telithromycin Binds to the 50S subunit. Bacteriostatic or Bactericidal

Inhibitors of protein synthesis (continue

Group Examples Mechanism of action Effect bacteriaOxazolinones Linezolid Binds to the 50S subunit Bacteriostatic or

Bactericidal(for VRE, MSSA and MRSA)

Streptagramins Quinupristin-dalfopristin (Synercid)

binding to the 50S ribosomal subunit of gram + bacteria

Bactericidal

Cyclic lipopeptides Daptomycin Not completely understood, but alters cell membrane activity

Bactericidal

Inhibitors of protein synthesis (continue)

4- Inhibition of nucleic acid synthesis

Group Examples Mechanism of action

Effect on bacteria

Fluoroquinolone (1st) Nalidixic acid(2nd)–Ciprofloxacin - Levofloxacin Norfloxacin - Ofloxacin(3rd)–Gatifloxacin(4th)–Trovafloxacin

Inhibit DNA gyrase Bactericidal

Sulfonamides Sulfisoxazole - Sulfamethoxazole, Sulfadiazine - Sulfadoxine,Sulfasalazine - Sulfapyridine

Inhibit folic acid (FA) synthesis.

Bacteriostatic

Group Examples Mechanism of action Effect on bacteriaTrimethoprim Trimethoprim Inhibit dihydrofolate reductase

enzyme BacteriostaticBactericidal if combined with sulfa

Cyclic lipopeptides

Daptomycin Not clearly understood, disruption of DNA, RNA and protein synthesis

Bactericidal Natural For MRSA & VRE

Rifamycins Rifampin,Rifabutin,Rifapentine.

Inhibiting RNA by binding to DNA-dependentRNA polymerase

Bactericidal (Antibiotics)TTT of TB

Inhibitors of nucleic acid synthesis (continue).

Mechanisms of antimicrobial drug resistanceClassification:• The resistance of microorganisms to antimicrobials is classified

as being either natural or acquired.• 2.3.2.1 Natural resistance • An organism is termed as having natural (intrinsic) resistance

when it has an inherent resistance to the action of an antibiotic; this pattern of resistance is common to all isolates of the species, e.g. the resistance of Escherichia coli to macrolides and the resistance of Pseudomonas aeruginosa to most drugs.

• The intrinsic (natural) resistance of a microorganism to an antimicrobial is explained by the absence or inaccessibility of the target of the drug action, e.g. Gram-negative bacteria are naturally resistant to some antibiotics e.g. erythromycin due to the non-permeability of the outer membrane

Acquired resistance• Acquired resistance is developed to an antibiotic to which the

microorganism was previously susceptible; it develops within one or more isolates of the species, i.e. not all strains of a species are resistant.

• For an antimicrobial to produce its intended action, it has to have a target for its action (e.g. in the form of an enzyme or protein) within the bacterial cell, to be able to reach this target, and also to reach the target in its active form, i.e. not having been destroyed.

The functional mechanisms of acquired resistance

What bacteria can do to combat antimicrobials?• 1) they may destroy or inactivate the antibiotic;• 2) bacteria can use an efflux system to exclude the drug from its

interior ; • 3) bacteria can produce alterations in the target site used by

antimicrobials to act or they may completely prevent this binding; • 4) bacteria can reduce their cell surface permeability or even

completely block the entrance of the antimicrobial to the cell, so that the antimicrobial can no longer act; and

• 5) bacteria can produce a bypass mechanism by using alternative pathways which are different from those inhibited by the antibiotic

Different mechanisms of antimicrobial resistance used by bacteria

Mechanism Examples of affected antimicrobials 1- Destruction, modification, or inactivation of the antimicrobial.

- β-lactam antibiotics - Chloramphenicol- Aminoglycosides

2- Multidrug efflux pumps. -Tetracycline

3- Target site alteration. -β-lactam antibiotics - Chloramphenicol - Streptomycin - Quinolones - Fusidic acid - Erythromycin – Glycopeptides - Rifampicin

Different mechanisms of antimicrobial resistance used by bacteria

Mechanism Examples of affected antimicrobials 4- Reduction in the cell surface permeability or access of the antimicrobial to the cell interior.

Tetracyclines - Quinolonesβ-lactam antibioticsAminoglycosides - Chloramphenicol

5- New metabolic bypass mechanism.

Trimethoprim - Sulphonamides

The basis (mechanism) of antimicrobial resistance

• A-Passive (intrinsic or phenotypic) drug resistance:• bacteria stop multiplying they are not affected by the

antimicrobial; Mycobacterium tuberculosis, where “persister” • L-form bacteria, which lack the cell wall which is the target for

action by some antimicrobials like penicillins and cephalosporins; this is sometimes termed “phenotypic tolerance” rather than resistance

B-Active drug resistance:

• Attributed to the emergence or acquisition of a new gene(s) which controls the process of resistance either following mutation, which may be spontaneous or induced, or by the transfer of a gene from another bacterium or another locus within the same bacterium by the process of transposition

1- Mutation• Mutation is a heritable change in the structure of the genes which

may arise spontaneously as an error of replication. • Due to UV light, radiation, or alkylating agents • Termed chromosomal resistance, as it usually originates in a

chromosome as a spontaneous mutation in a locus responsible for the antimicrobial drug action.

• May arise by insertion or deletion of nucleotide(s).

Examples of Mutations: 1-Mutation producing a single amino acid change in the PBPs

gives low level resistance to penicillins and cephalosporins.2-Mutations in the 23S ribosomal RNA gene also lead to linezolid-

resistant strains of VRE and MRSA,

Mobile Genetic Elements (carriers of antimicrobial resistance genes (ARGs)) from one species to another??)

• Spread of antimicrobial resistance genes (ARGs) among, human, animal, and environmental bacteria is mediated through this mobile genetic elements (MGEs).

• The MGEs include: conjugative plasmids, gene cassettes within the integrons, plasmids, transposons, and Insertion Sequence (IS) elements

2- Gene Transfer

1- Conjugative plasmids • Conjugative plasmids are those having the ability to transfer

themselves and other plasmids from one bacterial cell to another carrying ARGs present in both Gram positive and Gram negative bacteria.

• Spread of conjugative plasmids can occur at narrow or wide range of species.

2- Integrons• Integrons, which are present naturally as gene expression elements as

they contain open reading frames (ORFs) enabling them to express the genes it contains, are formed from two conserved flanking regions which incorporate one or more resistance gene(s) in-between.

3-Gene cassettes• The mobile genetic elements (MGEs) within the integrons are known

as the genetic cassettes. Many identified genetic cassettes are known and have been identified that mediate resistance to several antimicrobials e.g. penicillins, cephalosporins, aminoglycosides, chloramphenicol, and trimethoprim.

4- Insertion sequences and transposons• The simplest transposable DNA sequences are known as the

insertion sequence (IS) elements. They are a heterogeneous class of MGEs in bacteria, having the ability to promote their own translocation and do not carry antimicrobial resistance genes (ARGs).

• Transposons are mobile genetic elements which contain self-transmissible elements, including transposase and recombination DNA segments (e.g. ARGs).

Slide comment for the figure:• Transposable DNA elements: a) the insertion sequence (IS) elements which are the

simplest transposable DNA sequences, having reversed identical sequences (inverted repeats: IR) of 10-40 nucleotides at both ends flanking the transposase (tnp) gene. The direct repeats formed from 5-9 bp at the extremities of the structure are the target for the enzyme transposase during the integration process; b) the simple Tn3 transposons containing the transposase gene tnpA, regulator sequence (tnpR) and the (res) site to which the resolvase enzyme binds; c) the composite transposons which are formed from two IS elements making a frame for a region which is not essential for transposition e.g. tetracycline resistance gene (tetB); d) represents the conjugative transposons which have certain segments encoding factors used in the control of the transfer (Tra) and transposition (Tn) processes

Horizontal (lateral) gene transfer (HGT)

• Gene transfer between different bacteria occurs through one of three mechanisms: conjugation, transformation, or transduction. Conjugation is the most frequent mechanism mediated in HGT.

1- Conjugation • Conjugation is the process of gene transfer between bacteria from

the donor to the recipient via intimate contact, termed “mating through a channel”.

• The transposable DNA elements include the insertion sequences (IS) and the different forms of tranposons (simple, composite, and conjugative transposons).

Transfer of a conjugative plasmid by the process of conjugation between two bacterial cells via the sex pili: a) formation of the conjugation channel, b) start of the transfer of a single strand of the plasmid cleaved by the endonuclease enzyme at a specific point, c) the cleaved strand entering the recipient cell where d) a complementary strand is synthesized

2- Transformation• Bacteria can take exogenous DNA (genes) from the surrounding

environment. This ability to take exogenous genes is called competence and it is encoded by chromosomal genes within the bacterium, and it is mostly a time-limited process and occurs in a wide variety of bacteria e.g. Hemophilus, Helicobacter, Campylobacter, Niesseria, Staphylococci, and Pseudomonas species.

• Transformation can occur as natural process or can by induced by certain factors e.g. nutrient access, altered growth conditions, or starvation.

• Antimicrobial resistant gene (ARG) transfer is the natural one which can transmit resistance gene among different bacterial strains. The DNA is up taken as double stranded one which then converted to one strand while passing the inner membrane.

3- Transduction• In transduction, the bacterial genes are transferred between different

bacterial strains by the means of bacteriophage, which is a bacteria infecting virus.

• The phage for transfer must be the temperate (Lysogenic) one and not the lytic phage.

N.B. Transposition• In transposition, a DNA segment (mobile genetic element) can move

either to another locus in the same molecule (chromosome or plasmid) or transfer between them i.e. inside the cell not involved in HGT.

Schematic representation of different gene transfer mechanisms in bacteria: A= transformation, B= Transduction, and C= Conjugation

Source of Resistant Bacteria• Large amounts of antibiotics are used for human therapy, as well as

for farm animals and even for fish in aquaculture, resulted in the selection of bacteria resistant to multiple drugs.

• These environmental resistant bacteria carry great harm to human beings when genes are transferred to clinical isolates by HGT.

• MDROs are those resistant to more than 2 classes of antimicrobials!!.• Examples of MDROs include: MRSA, ESBLs, VRE, • Pan-Resistant organisms are isolated nowadays resistant to all

available antimicrobials.• Going to the pre-antibiotic era is the actual threat.

Various routes for antimicrobial resistance gene spread from human activity origins to the environment

How complex the spread of ARGsIn the community & Environment

Example of MDROs

Resistant to: Treatment

MRSA Penicillins, Cephalosporins, Monobactams, Carbapenems. (additionally to aminoglycosides, macrolides, tetracycline, chloramphenicol, and lincosamides).

Vancomycin, Linezolid,

ESBLs Penicillins, cephalsporins, Monobactams. Synercid, Tigecyclines,

VRE, VRSA & VISA

Penicillins, Cephalosporins, Monobactams, Carbapenems, Vancomycin.

Linezolid

PDROs All available antimicrobials Colistin ??CRE imipenem, meropenem, doripenem, or ertapenem

(Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, or Enterobacter)

CDC definitions of MDROs• MRSA: Includes S. aureus cultured from any specimen that tests

oxacillin-resistant, cefoxitin-resistant, or methicillin-resistant by standard susceptibility testing methods (AST) OR, positive FDA approved direct testing from sampling (e.g. PCR) for MecA gene.

• VRE: Enterococcus faecalis, Enterococcus faecium, or Enterococcus species unspecified that is resistant to vancomycin, by standard susceptibility testing methods (AST) or by direct testing by FDA approved test e.g. PCR for genes (VanA, VanB, VanC).

• CRE (Carbapenem-Resistant Enterobacteria): Any Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, or Enterobacter spp. testing resistant to imipenem, meropenem, doripenem, or ertapenem by standard susceptibility testing methods (AST). OR by production of a carbapenemase (i.e., KPC, NDM, VIM, IMP, OXA-48) genes by PCR.

• MDR-Acinetobacter: Any Acinetobacter spp. testing non-susceptible (i.e., resistant or intermediate) to at least one agent in at least 3 antimicrobial classes of the following 6 antimicrobial classes:

Antimicrobial class

β-lactam/β-lactam β-lactamase inhibitor combination

Aminoglycosides Carbapenems

Representatives Piperacillin Piperacillin/tazobactam

Amikacin Gentamicin Tobramycin

Imipenem Meropenem Doripenem

Antimicrobial class

Fluoroquinolones Cephalosporins Sulbactam

Representatives Ciprofloxacin Levofloxacin

Cefepime Ceftazidime

Ampicillin/sulbactam

Burdens of MDROs Problem

1- Antimicrobial resistance kills (no treatment).2- Antimicrobial resistance hampers the control of infectious

diseases.3- Antimicrobial resistance increases the costs of health care.4- Antimicrobial resistance jeopardizes health care gains to

society.

MDRO Prevention and Control1. Administrative support: (Financial and HR, communication system,

HH facilities, staff levels, adherence to IPC recommendations).

2. Education: Facility-wide, unit-targeted, and informal, educational interventions.

3. Judicious use of antimicrobial agents: (antimicrobial stewardship program). Use narrow spectrum, treat only infections not contaminant or colonizers, duration limited, restricted Abs validation).

Control of MDROs Spread:4. MDRO surveillance: (new pathogen, trends, effective

interventions, by either reviewing micro lab results or by Active Surveillance Culture/Testing (ASC/AST) to detect colonization.

• Antibiograms (simplest for of MDROs surveillance).• MDRO Infection Rates reviews• Molecular typing of MDRO isolates.• Surveillance for MDROs by Detecting Asymptomatic Colonization

(great impact up to 65% reduction of spread ).

Methods for obtaining ASC/AST:

• MRSA: Studies suggest that cultures of the nares.• VRE: Stool, rectal, or perirectal swabs.• MDR-GNBs: peri-rectal or rectal swabs + oro-pharyngeal,

endotracheal, inguinal, or wound cultures.• Rapid detection methods: (media containing chromogenic enzyme

substrates – real-time PCR-based tests for MRSA from swabs, vanA and vanB genes (VRE or VRSA) from rectal swabs).

5. Infection Control Precautions: (Standard and Contact isolation Precautions for MDROs, Hand hygiene) .

• Cohorting and other MDRO control strategies: (cohorting of patients, cohorting of staff, use of designated beds or units, unit closure are necessary for control of transmission.

• Duration of Contact Precautions: controversial. 3 negative swabs, better for all period of stay in facility.

• Barriers used for contact with patients infected or colonized with MDROs: (gloves with or without gowns, .

• Impact of Contact Precautions on patient care and well-being: (adverse effects).

6. Environmental measures e.g. surfaces and medical equipment, environmental cultures are not recommended routinely, .

• Stick to proper environmental , surfaces, and equipment cleaning).

7. Decolonization: (treat colonized persons with MRDOs to eradicate them e.g. MRSA, little success in VRE).

Stop the Abuse of Antimicrobials

Follow antimicrobial Policy and stewardship program in

hospitals.

Stop the misuse of antibiotics by the community

Control of antibiotic usage for non-therapeutic purposes

If the world does not cooperate together fighting microbial

resistance, then all these drugs will have no value.Eventually we will go to the pre-antibiotic era.

References:

• http://www.who.int/mediacentre/factsheets/fs194/en/.• http://www.cdc.gov/hicpac/mdro/mdro_4.html.• GCC Infection Control Manual 2013.

Thank You