Multidrug Resistant Oraganisms (MDRO) infection control

Description of the major classes of antimicrobial drug, resistant mechanisms developed by bacteria to combat the action of antimicrobials, and the control measures needed to limit this horizontal gene transfer.

1. Multi-Drug Resistant Organisms (MDROs) Dr Mostafa Mahmoud , MD, Ph D, Consultant Microbiologist Assist . Prof. of Medical Microbiology & Immunology

2. Genetic materials controlling bacterial functions are: 1- Chromosome 2- Mobile genetic elements (plasmids, transposons, genetic islands).

3. Bacterial cell structure (Prokaryote)

4. 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.

5. Some N aturally Produced Antibiotics Species 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*

6. 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 .

7. The classification of AB by the mechanism of action

8. 1- Inhibition of bacterial cell wall synthesis Group 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 - Ampicillin Amoxicillin/ clavulanate Ampicllin / sulbactam bacapicillin Gram + ve and Gram - ve bacteria. 4- Carboxypenicillins Carbenicillin - Ticracillin , Ticracillin / clavulante Greater activity against gram negative organisms 5-Ureidopenicillins and Piperazine Mezlocillin - Piperacillin Piperacillin / tazobactam They have the broadest-spectrum of all penicillins especially on Pseudomonas aeruginosa

9. Group Examples Spectrum of action 2- Cephalosporins : 1 st generation Cefadroxil - Cefazolin - Cephalexin Cephalothin - Cephradine Gram (+ ve ) & few Gram (– ve ) e.g. E . coli, Klebsiella 2 nd generation Cefaclor – Cefamandole - Cefmetazole , Cefoxitin - Cefoprzil - Cefuroxime More G (– ve ) e.g. Klebsiella , Proteus , less on ( G+ve ) 3 rd generation Cefixime – Cefoprazone - Cefotaxime , Ceftazidime - Ceftriaxone Pseudomonas & Enterobacter . - TTT of HAIs . 4 th generation Cefepime upon G (+ ve ) & G (- ve ) organisms, including P. aeruginosa

10. Other cell wall inhibitors: Group Examples Spectrum of action 3- Carbapenems Imipenem - Meropenem Etrapenem - Doripenem broad-spectrum of activity (MSSA- Pseudomonas). 4- G lycopeptides 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 .

11. 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.

12. 3-Inhibition of bacterial protein synthesis Group 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 )

13. Group Examples Mechanism of action Effect bacteria Chloramphenicol Chloramphenicol Binds 50S and inhibits peptidyl transferase Bacteriostatic 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

14. Group Examples Mechanism of action Effect bacteria Oxazolinones 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 )

15. 4- Inhibition of nucleic acid synthesis Group Examples Mechanism of action Effect on bacteria Fluoroquinolone (1 st ) Nalidixic acid (2 nd )–Ciprofloxacin - Levofloxacin Norfloxacin - Ofloxacin (3 rd ) – Gatifloxacin (4 th )– Trovafloxacin Inhibit DNA gyrase Bactericidal Sulfonamides Sulfisoxazole - Sulfamethoxazole , Sulfadiazine - Sulfadoxine , Sulfasalazine - Sulfapyridine Inhibit folic acid (FA) synthesis. Bacteriostatic

16. Group Examples Mechanism of action Effect on bacteria Trimethoprim Trimethoprim Inhibit dihydrofolate reductase enzyme Bacteriostatic Bactericidal 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-dependent RNA polymerase Bactericidal (Antibiotics) TTT of TB Inhibitors of nucleic acid synthesis (continue).

17. Mechanisms of antimicrobial drug resistance Classification: 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.

18. 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

19. 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

20. 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

21. 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

22. 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 - Quinolones β-lactam antibiotics Aminoglycosides - Chloramphenicol 5- New metabolic bypass mechanism. Trimethoprim - Sulphonamides

23. 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

24. 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

25. 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).

26. 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,

27. 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

28. 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.

29. 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.

30. 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 ).

31. 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

32. 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 ).

33. 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

34. 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.

35. 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.

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

37. 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.

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

39. How complex the spread of ARGs In the community & Environment

40. 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 )

41. 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 ).

42. 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 .

43. 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

44. 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.

45. MDRO Prevention and Control 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).

46. 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 ).

47. Methods for obtaining ASC/AST : MRSA : Studies suggest that cultures of the nare s . 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).

48. 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).

49. 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).

50. Stop the Abuse of Antimicrobials Follow antimicrobial Policy and stewardship program in hospitals.

51. Stop the misuse of antibiotics by the community

52. Control of antibiotic usage for non-therapeutic purposes

53. 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.

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

55. Thank You