Antibiotic sensitivity and resistance .pptx seminar 2

Antibiotic sensitivity and resistance

1. ANTIBIOTIC SENSITIVITY AND RESISTANCE Presented by: Dr. Mitali Thamke (I MDS)

2. INDEX INTRODUCTION HISTORICAL ASPECT DEFINITION CLASSIFICATION MECHANISMS OF ACTION OF ANTIMICROBIAL DRUGS RESISTANCE TO ANTIMICROBIAL DRUGS PRODUCTION OF ENZYMES PRODUCTION OF ALTERED ENZYMES SYNTHESIS OF MODIFIED TARGETS ALTERATION OF PERMEABILITY OF CELL WALL ALTERATION OF METABOLIC PATHWAY

3. BASIS OF RESISTANCE NONGENETIC BASIS GENETIC BASIS CHROMOSOME- MEDIATED RESISTANCE PLASMID- MEDIATED RESISTANCE TRANSPOSONS- MEDIATED RESISTANCE ANTIBIOTIC SENSITIVY TESTING DISC DIFFUSION TESTS DILUTION TESTS Mechanism of biofilm resistance against antimicrobials Factors promoting resistance Prevention of drug resistance Conclusion

4. INTRODUCTION Antibiotics have not only saved patients lives, they have played a pivotal role in achieving major advances in medicine and surgery. They have successfully prevented or treated infections that can occur in patients who are receiving chemotherapy treatments; who have chronic diseases such as diabetes, end-stage renal disease, or rheumatoid arthritis; or who have had complex surgeries such as organ transplants, joint replacements, or cardiac surgery.

5. Antibiotics have also helped to extend expected life spans by changing the outcome of bacterial infections. In 1920, people in the U.S. were expected to live to be only 56.4 years old; now, however, the average U.S. life span is nearly 80 years. Antibiotics have had similar beneficial effects worldwide. In developing countries where sanitation is still poor, antibiotics decrease the morbidity and mortality caused by food-borne and other poverty-related infections.

6. HISTORICAL ASPECT Paul Ehrlich coined the term chemotherapy in 1913 to mean “injury of invading organism without injury to the host”. Ehrlich’s Magic Bullets

7. Gerhard Domagk - Prontosil The modern era of chemotherapy of infection started by Domagk in 1935 with the demonstration of therapeutic effects of prontosil , a sulfonamide dye, in pyogenic infection.

8. Fleming and Penicillin In 1928 Alexander Fleming observed that a contaminating mould on a staphylococcal culture plate caused the adjacent bacterial colonies to undergo lysis. Fleming named antibacterial principle penicillin.

9. Chain and Florey followed up this observation in 1939 which culminated in the clinical use of penicillin in 1941 . It was not extensively used until the 2nd World War when it was used to treat war wounds

10. Selman Waksman In 1940’s Waksman and his colleagues undertook a systematic search of Actinomycetes as source of antibiotics and discovered streptomycin in 1944.

11. DEFINITIONS ANTIBIOTICS: Antibiotics are the substances produced by microorganism, which selectively suppress the growth of or kill other microorganisms at very low concentrations. CHEMOTHEARPY: Treatment of systemic infections with specific drugs that selectively suppress the infecting micro-organisms without significantly affecting the host.

12. Due to analogy between the malignant cells and the pathologic microbes, treatment of neoplastic diseases with drugs is also called chemotherapy It would be more meaningful to use the term antimicrobial agent to designate synthetic as well as naturally obtained drugs that attenuate microorganisms.

13. CLASSIFICATION OF ANTIBIOTICS WITH RESPECT TO THEIR CHEMICAL STRUCTURE ACCORDING TO SPECTRUM OF ACTIVITY TYPE OF ACTION ACCORDING TO MECHANISM OF ACTION

14. With respect to their chemical structure SULPHONAMIDE AND RELATED STRUCTURES Sulfadiazine and others Sulfones – Dapsone (DDS), Paraaminosalicylic acid DI-AMINO-PYRIMIDINES Trimethoprim pyrimethamine

15. QUINOLONES Nalidixic acid Norofloxacin Ciprofloxacin etc. TETRACYCLINE Oxytetracycline Doxycycline NITROBENZENE DERIVATIVE CHLORAMPHENICOL LACTAM ANTIBIOTICS Penicillin Cephalosporin Monobactum Cabapenems

16. AMINOGLYCOSIDES Streptomycin Gentamycin MACROLIDE ANTIBIOTICS Erythromycin Roxithromycin Azithromycin POLYPEPTIDE ANTIBIOTIC Polymyxin -B Colistin Bacitracin

17. Glycopeptides Vancomycin Teicoplanin Oxazolidinone Linezolid Nitrofuran derivatives Nitrofurantoin Furazolidone Nitroimidozoles Metronidozole Tinidazole

18. Nicotinic acid derivatives Isoniazid Pyrazinamide Ethionamide Polyene antibiotics Nystatin Amphotericin-B Azole derivatives Miconazole Clotrimazole Ketoconazole Others Rifampin Lincomycin Clindamycin

19. According to mechanism of action Inhibit cell wall synthesis Penicillins Cephalosporin Vancomycin Bacitracin Damage to the cytoplasmic membrane: Polypeptides Polyene 3) Inhibit DNA gyrase Fluoroquinolones Ciprofloxacin 4 ) Interfere with DNA function Rifampicin Metronidazole

20. Classification D) Spectrum of activity: Narrow spectrum: Penicillin G Streptomycin Erythromycin Broad spectrum: Tetracyclines Chloramphenicol Intermediate spectrum Aminopenicillin Cephalosporins Fluoroquinolones

21. Classification E) Type of action: Primarily bacteriostatic Sulfonamides Tetracyclines Chloramphenicol Macrolide Ethambutol Primarily bactericidal Penicillins Aminoglycosides Rifampin Cotrimoxazole Cephalosporins Vancomycin Quinolones

22. Mechanism of action of antimicrobial drug The antibiotics act against bacteria by following mechanism: Inhibition of cell wall synthesis Inhibition of protein synthesis Inhibition of nucleic acid synthesis Alteration of cell membrane function

23. Inhibition by cell wall synthesis: Penicillin , cephalosporin, vancomycin and caspofungin acts against bacteria by interfering cell wall synthesis. Penicillin and cephalosporins are called -lactum antibiotics because they posses an intact -lactam ring, essential for antimicrobial activity.  

24. Antibiotic Mechanism of action Penicillin inhibiting penicillin –binding proteins(PBPs)also known as transpeptidases that link the cross-bridges between NAMs, thereby, greatly weakening the cell wall meshwork Cephalosporin Similar to penicillin Vancomycin Similar to penicillin Caspofungin It kills bacteria by preventing synthesis of -glucan, a polysaccharide component of bacterial cell wall. Antibiotic Mechanism of action Penicillin inhibiting penicillin –binding proteins(PBPs)also known as transpeptidases that link the cross-bridges between NAMs, thereby, greatly weakening the cell wall meshwork Cephalosporin Similar to penicillin Vancomycin Similar to penicillin Caspofungin

25. Inhibition of protein synthesis : Bacteria have 30S and 50S ribosomal units, whereas mammalian cell have 80S ribosomes. The subunit of each type of ribosome, their chemical composition, and their functional speficities are sufficiently different, which explain why these antimicrobial drugs can inhibit protein synthesis in bacterial ribosomes without having major effect on ribosomal ribosomes

26. Aminoglycosides and tetracyclines act at the level of 30S ribosomal subunits, whereas erythromycins,chloramphenical and clindamycin act at the level of 50S ribosomal subunits.

27. Antibiotic Mechanism of action Aminoglycosides binding to 30S subunit ribosome which block the initiation complex, leading to no formation of peptide bonds or polysomes Tetacycline acts by inhibiting protein synthesis of bacteria by blocking the binding of aminoacyl t-RNA to 30S ribosomal subunits Macrolides inhibiting protein synthesis of bacteria by blocking the release of t-RNA after it has transferred its amino acid to the growing peptide. Chloramphenocol binds to 50S subunit of the ribosome and block peptidyl transferase, the enzyme that delivers the amino acid to growing polypeptide, resulting in the inhibition of bacterial protein synthesis Clindamycin It inhibits bacterial protein synthesis by blocking the release of t-RNA

28. Inhibition of nucleic acid synthesis: Sulphonamide ,trimethoprim,quinolones and rifampicin are examples of drugs that act by inhibition of nucleic aid synthesis.

29. Antibiotic Mechanism of action Sulphonamide antibiotic inhibit the synthesis of tetrahydrofolic acid, the main donor of the methyl groups that are essential to synthesis adenine, guanine and cytosine Trimethoprim enzyme reduce dihydrofolic to tetrahydrofolic acid, leading to decreased synthesis of purines and ultimately DNA Quinolones act by inhibiting bacterial DNA synthesis by blocking DNA gyrase. Rifampicin Rifampicin inhibits bacterial growth by binding strongly to the DNA-dependent RNA polymerase of bacteria

30. Alteration of cell membrane function The cytoplasm of all living cells is surrounded by the cytoplasmic membrane, which serves as a selective permeability barrier. The cytoplasmic membrane carries out active transport functions, and thus controls the internal composition of cell. If the functional integrity of cytoplasmic membrane is disrupted, macromolecules and ions escape from cell, and cell damage or death ensues.

31. Antifungal drugs act by altering the cell membrane function of fungi. They show selective toxicity because cell membrane of fungi contain ergosterol whereas human cell membrane has cholesterol. Bacteria with exception of mycoplasm do not have sterol in their cell membranes, hence resistant to action of these drugs

32. Polymix b which is an antibacterial binds to lipopolysaccharide in outer membrane of Gram negative bacteria and disrupt both outer and inner membrane

33. Amphotericin B and azoles are frequently used antifungal drugs. Amphotericin B acts against the fungi by disrupting the cell membrane by binding at the site of ergosterol in the membrane. Azoles such as ketoconazole inhibits synthesis of ergosterol, hence are toxic to fungi.

34. Resistance to antimicrobial drugs As early as 1945, Sir Alexander Fleming raised the alarm regarding antibiotic overuse when he warned that the “public will demand [the drug and] ... then will begin an era ... of abuses . The overuse of antibiotics clearly drives the evolution of resistance. Epidemiological studies have demonstrated a direct relationship between antibiotic consumption and the emergence and dissemination of resistant bacteria strains.

35. Mechanism of antibiotic resistance Production of enzymes Production of altered enzymes Synthesis of modified targets Alteration of permeability of cell wall Alteration of metabolic pathway

36. Basis of resistance The resistance by bacteria against antibiotic may be classified as: Nongenetic basis Genetic basis

37. Nongenetic basis Nongenetic basis of resistance plays less important role in development of drug resistance: Certain bacteria under ordinary circumstances are usually killed by penicillin. But these bacteria , if they loss their cell wall and become protoplast, become nonsusceptible to the action of cell wall-acting drug such as penicillins.

38. 2. In certain condition as in abscess cavity, bacteria can be walled off which prevent drug to penetrate effectively into bacteria. Surgical drainage of the pus, however, makes these bacteria again susceptible to action of antibiotics

39. 3. The presence of foreign bodies such as surgical implants and catheters and penetration injury caused by splinters and sharpeners make successful antibiotic treatment more difficult.

40. 4. Nonreplicating bacteria in their resting stage are less sensitive to the action of cell wall inhibitors such as penicillin and cephalosporin. This is particularly true for certain bacteria such as Mycobacterium tuberculosis that remains in resting stage in tissues for many years, during which it is insensitive to drugs. However when these bacteria begin to multiply, they become susceptible to antibiotic

41. Genetic basis of Drug Resistance The genetic basis of drug resistance , mediated by genetic change in the bacteria, is most important in development of drug resistance in bacteria. This is of 3 types as follows: Chromosomes-mediated resistance. Plasmid –mediated resistance. Transposons-mediated resistance.

42. Chromosome-mediated resistance Chromosome-mediated resistance occurs as a result of spontaneous mutation. This is cause by mutation in gene that codes for either the target of drugs or the transport system in the membrane of cell wall that controls the entry of drugs into cells The frequency of chromosomal mutation is much less than the plasmid-mediated resistance

43. Plasmid-mediated resistance Plasmid-mediated drug resistance in bacteria occurs by transfer of plasmid and genetic materials. It is mediated by resistance plasmid, otherwise known as R-factor

44. R-factors They are circular, double stranded DNA molecules that carry the genes responsible for resistance against variety of antibiotics. These factors may carry one or even two or more resistant genes. These genes encode for a variety of enzymes that destroy the antibiotic by degrading antibiotics or modify membrane transport.

45. Plasmid-mediated antibiotic resistance Antibiotic Mechanism of resistance lactams -lactamases break down the lactam ring to an inactive form Aminoglycosides Aminoglycosides modifying enzymes: acetyltransferases, phosphotransferases and nucleotidyltransferases Erythromycin and clindamycin Induced enzymatic activity due to methylating ribosomal RNA Chloramphenicol Acetylation of the antibiotic to an inactive form Tetracycline Alteration of cell membrane, decreases permeability to antibiotic Antibiotic Mechanism of resistance Aminoglycosides Aminoglycosides modifying enzymes: acetyltransferases, phosphotransferases and nucleotidyltransferases Erythromycin and clindamycin Induced enzymatic activity due to methylating ribosomal RNA Chloramphenicol Acetylation of the antibiotic to an inactive form Tetracycline Alteration of cell membrane, decreases permeability to antibiotic

46. Plasmid mediated resistance plays a very important role in antibiotics usage in clinical practice. This is because A high rate of transfer of plasmids from one bacterium to another bacterium takes place by conjugation Plasmid mediated resistance to multiple antibiotics Plasmid mediated resistance occurs mostly in Gram-negative bacteria.

47. Transposons-mediated drug resistance Drug resistance is also mediated by transposons that often carry the drug resistance genes Transposons are small pieces of DNA that move from one site of bacterial chromosome to another and from bacterial chromosome to plasmid DNA. Many R factors carry one or more transposons.

48. Difference between mutational and transferable drug resistance Mutational drug resistance Transferable drug resistance Chromosome mediated Plasmid mediated Resistance to one drug Resistance to multiple drug Resistance is nontransferable Resistance is transferable Virulence of organism lowered Virulence of organism not lowered Low-degree of resistance High-degree of resistance Due to decreased permeability ,development of alternative metabolic pathway or inactivation of drug Due to production of many degrading enzymes.

49. Specific mechanism of resistance Penicillins Resistance to penicillin is mainly mediated by 3 mechanism: 1. Production of penicillin-destroying enzymes( -lactamases) 2. Mutation in genes coding for PBP 3. Reduced permeability to drug  

50. 1. Production of penicillin destroying enzymes( lactamases):   Resistance to penicillins may be determined by organism’s production of penicillin-destroying enzymes( -lactamases). - lactamases such as penicillinases and cephalosporinases open the - lactam ring of penicillin and cephalosporin and abolish their antimicrobial activity. - lactamases have been described for many species of Gram-positive and Gram-negative bacteria.  

51. Some -lactamases are plasmid-mediated(eg,penicillinase of S.aureus),while other are chromosomally mediated(eg,many species of Gram-negative bacteria such as Enterobacter spp.,Citrobacter spp.,Pseudomonas spp.,etc) There is one group of -lactamases that is occasionally found in certain species of Gram-negative bacilli, usually Klebsiella pneumoniae and E.coli  

52. These enzymes are termed extended-spectrum -lactamases because they confer upon the bacteria the additional ability to hydrolyze the - lactam rings of cefotaxime,ceftazidime,or azetreonam.  

53. 2. Mutation in genes coding for PBP: This form of resistance occur due to absence of some penicillin receptors(PBP) and occurs as a result of chromosomal mutation The mechanism is responsible for both low-level and high-level resistance seen in S.pneumonia to penicillin G and in S.aureus to nafcillin.

54. 3.Reduced permeability to drug: Low-level resistance of Neisseria gonorrhoeae to penicillin is caused by poor permeability of drug. However, high-level resistance is mediated by plasmid coding for penicillinase.

55. Vancomycin Resistance to vancomycin is mediated by change in D-ALA-D-ALA part of peptide in the peptidoglycan to D-ALA-D-lactate. This result in the inability of vancomycin to bind to a bacteria. Vancomycin resistance in Enterococcus is been increasingly documented in different clinical conditions.

56. AMINOGLYCOSIDES Resistance to aminoglycoside is mediated by three important mechanism as follows: 1. Plasmid-dependent resistance to aminoglycosides enzymes is most important mechanism. It depends on production of plasmid-mediated phosphorylating, and acetylating enzymes that destroy the drug

57. 2. Chromosomal resistance of microbes to aminoglycosides in second mechanism. The chromosomal mutation in the genes results in lack of a specific protein receptor on the 30S subunit of ribosome, essential for binding of the drug.

58. 3. A ‘permeability defect’ is the third mechanism. This lead to an outer membrane change that reduces active transport of aminoglycosides into cell so that the drug cannot reach the ribosome. Often this is plasmid-mediated

59. Tetracycline Resistance to tetracycline occurs by 3 mechanisms: Efflux Ribosomal protection, and Chemical modification The first two are the most important

60. Efflux pumps, located in bacterial cell cytoplasmic membrane, are responsible for pumping the drug out of the cell. Tet gene products are responsible for protecting the ribosome, possibly through mechanism that induce conformational changes. These conformational changes either prevent binding of the teracyclines or causes their dissociation from the ribosome. This is often plasmid-controlled.

61. Antibiotic sensitivity testing Antibiotic sensitivity testing is carried out to determine the appropriate antibiotic agents to be used for a particular bacterial strain isolated from clinical specimen qualitative quantitative

62. Disc diffusion tests Most commonly used In this method as name suggests, disc is impregnated with known concentrations of antibiotics are placed on agar plate that has been inoculated with a culture of bacterium to be tested. The plate is incubated at 37 for 18- 24 hours  

63. After diffusion the concentration of antibiotic usually remain higher near the site of antibiotic disc but decreases with distance. Susceptibility to the particular antibiotic is determined by measuring the zone of inhibition of bacterial growth around the disc. Zone of inhibition

64. Selection of media Media that support both test and control strain is selected for carrying out AST of the bacteria. For e.g., Mueller Hinton agar is used for testing Gram-negative bacilli and Staphylococcus spp; Blood agar for Streptococcus spp. And Enterococcus spp Chocolate agar for Haemophilus influenza Wellcotest medium for sulphonamide and cotrimoxazole .

65. Medium is prepared by pouring onto the flat horizontal surface of petri dishes of 100mm to a depth of 4mm. pH is maintained at 7.2-7.4 More alkaline pH increases activity of tetracyclines ,novobiocin and fusidic acid Acidic pH reduced the activity of aminoglycosides and macrolides such as erythromycin. Plates are stored at 4 for upto 1 week.  

66. Type of disc diffusion tests Disc diffusion tests are of following types: Kirby-Bauer disc diffusion method Strokes disc diffusion method Primary disc diffusion test

67. Kirby-Bauer disc diffusion method Most common method used Both test strain and control strain are tested in separate plates. Test organism is inoculated in suitable broth solution, followed by incubation at 37 for 2-4 hours. 0.1 ml of broth is inoculated on surface of agar medium by streaking with a sterile swab  

68. THE TEST BACTERIA IS ISOLATED FROM ITS CULTURE PLATE A LIQUID CULTURE OF THE TEST BACTERIUM IN A SUITABLE BROTH IS PREPARED IN A TEST TUBE

69. THIS IS THEN POURED ON TO A SUITABLE SOLID AGAR MEDIUM(NUTRIENT/MUELLER-HINTON) IN A PETRI DISH WHICH IS TILTED TO ENSURE UNIFORM SPREADING EXCESS BROTH IS PIPETTED OFF.

70. Alternately,a sterilized cotton swab may be dipped in the liquid bacterial suspension and streaked across the solid agar medium in different angle to ensure uniformity. The plate is dried at 37 degree Celsius for 30 minutes The antibiotic filter paper(4/5 per 10cm dish)are placed with sterile forceps and incubated overnight A ’ lawn’culture develops with zone of in hibition

71. If the bacterium is susceptible to the drug growth is inhibited,but if it is resistant no zone of inhibition will be seen.

72. Stroke’s method Similar to Kirby- bauer method,however control organism is used. The control organism used are Staph.Aureus E.coli Ps.aeruginosa A standard sesitive control organism is inoculated on one side of the plate. The test bacterium is inoculated on the other side of the plate Antibiotic disc are placed at junction of two layers. Comparison of zone of inhibition indicates the susceptibility with respect to Std.bacterium .

73. In modified Strokes method, control strain is inoculated in the central part but test strains are inoculated on the upper and lower third of plate. Reporting of result is carried out by comparing the zone of inhibition of test and control bacteria.

74. The zone is measured from the edge of the disc to edge of zone. It is interpreted as follows: Sensitive (S): the zone of test bacterium is equal to or more than that of control strain. The difference between the zone sizes of control and test strain should not be more than 3mm if the zone size of test bacterium is smaller than that of control. 2. Intermediate sensitive(I): The zone size of the test bacterium should be at least 2 mm, and the differences between the zone of test. 3. Resistant (R): The zone size of test bacterium is 2mm or less. Antibiotic disc Inner zone: resistant strain Black zone: intermediate susceptibility Outer zone: suceptible strain

75. Interpretation of disc diffusion tests: Results of disc diffusion tests such as Kirby-Bauer and Strokes method are interpreted as follows: Sensitive(S) : infection treatable by the normal dosage of the antibiotic Intermediate(I) : Infection may respond to higher dosage Resistant(R) : Unlikely to respond to usual dosage of the antibiotics

76. Primary disc diffusion test: It is carried out directly on clinical specimens unlike Kirby-Bauer or Strokes diffusion method which are performed on pure cultures of bacteria isolates from clinical specimens. In this method the clinical specimen is inoculated uniformly on surfaces of the agar to which antibiotic are applied directly. Plate is incubated overnight at 37 for demonstration of zone of inhibition.  

77. Method is used to know antibiotic sensitivity result urgently but these results should always be confirmed by testing isolates subsequently by Kirby-Bauer or Strokes diffusion method.

78. Dilution tests Dilution test is performed to determine the minimum inhibitory concentration of (MIC)antimicrobial agent. MIC is defined as the lowest concentration of the antimicrobial agent that inhibits the growth of organisms.

79. Estimation of the MIC is useful to: Regulate the therapeutic dose of antibiotic accurately Test antimicrobial sensitivity patterns of slow growing bacteria such as M.tuberculosis.

80. Following method are carried out to determine the MIC: Broth dilution method Agar dilution method Epsilometer test

81. Broth dilution method Quantitative method for determining MIC. The antimicrobial agent is serially diluted in Mueller-Hinton broth by doubling dilution in tubes, and then a standard suspension of broth culture of test organism is added to each of the antibiotic dilution and control tube. This is mixed gently and incubated for 16-18 hours at 37  

82. An organism of known susceptibility is included as control. MIC is recorded by noting the lowest concentration of the drug at which there is no visible growth as demonstrated by the lack of turbidity in tube. Main advantage is that this is a simple procedure for testing a small number of isolates.

83. Added advantage is that by using same tube ,the minimum bactericidal concentration (MBC) of bacteria an be determined. MBC is determined by subculture from each tube, showing no growth on a nutrient agar without antibiotics

84. The plates are examined for the growth, if any, after incubation overnight at 37 The tube containing the lowest concentration of drug that fail to show any growth on subculture plate is considered as the MBC of the antibiotic for that strain.  

85. Agar dilution method It is quantitative method for determining the MIC of antimicrobial agent against test organism. It is useful: To test organism from serious infection like bacterial endocarditis To verify equivocal results of disc diffusion test

86. Mueller-Hinton agar is used Serial dilution of antibiotic are made in agar and poured into petri dishes. Dilution are made in distilled water and added to the agar which has been melted and cooled to more that 60 One control plate without antibiotic Organism to test is inoculated and incubated overnight at 37  

87. Plates examined for presence or absence of bacteria. The concentration at which bacterial growth is completely inhibited is considered as the MIC of antibiotic. Organisms is reported sensitive, intermediate, or resistance by comparing test MIC value Main advantage is that number of organisms can be tested simultaneously on each plate containing an antibiotic solution.

88. Epsilometer test Combines the principle of disc diffusion and agar dilution Similar to Kriby-Baurer method but uses a thin inert plastic strip impregnated with a known gradient of varying drug concentration along its length. Here, a symmetrical inhibition ellipse is produced. The intersection of inhibitory zone of edge and the calibrated carrier strip indicates the MIC value. E test is a very useful test for easy interpretation of the MIC of an antibiotic. MIC

89. Mechanisms of Biofilm Resistance Against Antimicrobials

90. Mechanisms responsible for a resistance in biofilms are as following Biofilm Impermeability to Antimicrobial Agents Antimicrobial molecules must reach their target in order to inactivate the enmeshed bacteria. The biofilm glycocalyx protects infecting cells from humoral and cellular host defence systems as well as the diffusion of the antimicrobial molecules to the target, acting as a barrier by influencing the rate of transport of molecules into the biofilm interior.

91. Physical (coaggregation and coadhesion), metabolic, and physiological (gene expression and cell–cell signalling) interactions yield a positive cooperation among different species within the biofilm. A key role is played by F. nucleatum , able to form the needed “bridge” between early and late colonizers. The presence of F. nucleatum , enables anaerobes to grow, even in the aerated environment of the oral cavity. In the absence of F. nucleatum , P. gingivalis cannot aggregate with the microbiota

92. Because subgingival bacteria are organized in biofilms, in principle, they are less susceptible to antimicrobials. Therefore, The oral plaque biofilm needs to be mechanically debrided or disturbed in order for antimicrobials to be effective

93. 2) Altered Growth Rates in Biofilm Organisms the growth rate of the organisms is significantly slower than the growth of planktonic (biofilm free) cells; therefore, the uptake of the antimicrobial molecules is diminished. 3) The Biofilm Microenvironment Antagonizing The Antimicrobial Activity Relatively large amounts of antibiotic-inactivating enzymes such as b-lactamases which accumulate within the glycocalyx produce concentration gradients that can protect underlying cells.

94. The most prominent b-lactamase-producing organisms belonged to the anaerobic genus Prevotella . Other enzyme-producing anaerobic strains were F. nucleatum, and Peptostreptococcus sp.

95. 4) The Role of Horizontal Dissemination in the Biofilm Horizontal transmission of genetic information between bacteria can occur by three gene transfer mechanisms: conjugation, transduction, and transformation

96. Conjugation requires that the donor cell have a conjugative element, usually a plasmid or a transposon, and that physical contact be made between donor and recipient cells to initiate transfer of the DNA molecule. Transduction process involves transducing bacteriophage particles that harbour the foreign DNA. Gene transfer by transformation , does not require a living donor cell, since free DNA released during cell death and lysis is the principal source of the donor DNA.

97. These all above mentioned 3 mechanisms of horizontal gene transfer require basic components i.e. plasmids, conjugative transposons (CTn), and bacteriophages. Plasmids Usually exist as independently replicating units. Plasmids are common in both gram-positive and gram-negative organisms isolated from the oral cavity. Among the most important plasmids in mediating broad host-range gene transfer are those of the IncP group .

98. Conjugative Transposons The gram-negative oral bacterium A. actinomycetemcomitans and E.corrodens has been implicated as a causative agent of several forms of periodontal diseases. The conjugative tetracycline resistance transposon Tn916 was transduced to their recipients as a unit.

99. Bacteriophage Bacteriophage can contribute to horizontal gene transfer by transduction or lysogenic conversion. A range of bacteriophages specific for species of Veillonella spp, Actinomyces spp, S. mutans , Enterococcus faecalis , and A. actinomycetemcomitans have been described in dental plaque samples or in saliva.

100. Communications Systems (Quorum Sensing) Gram-positive bacteria generally communicate via small diffusible peptides, while many gram-negative bacteria secrete acyl homoserine lactones (AHLs). AHLs are involved in quorum sensing whereby cells are able to modulate gene expression in response to increases in cell density. Several strains of P. intermedia , F. nucleatum , and P. gingivalis were found to produce such activities.

101. van Winkelhoff et al and Slots revealed that systemically administered antibiotics provided a clear clinical benefit in terms of mean periodontal attachment level “gain” post therapy when compared with groups not receiving these agents. Meta-analyses performed by Herrera et al and Haffajee et al indicated that adjunctive systemically administered antibiotics can provide a clinical benefit in the treatment of periodontal infections.

102. The tetracyclines, metronidazole, and b-lactams are among the most widely used agents for treating periodontal conditions. Mechanisms of bacterial resistance to these antibiotics have been extensively described and attributed to resistance genes . Many genes for bacterial resistance to tetracycline have been identified and characterized. These include tet A , B , C , D , E , G , H , I , K , L , M , O , Q , and X associated with gram-negative bacteria, and tet K , L , M , O , P , Q , S , Otr A , B , and C (oxytetracycline resistance determinants) associated with gram-positive bacteria.

103. Kuriyama et al. found that bacteria of the genus Porphyromonas and of the genus Fusobacterium showed susceptibility to cephalosporins Fosse et al. found resistance to tetracycline frequently associated with β-lactamase production, with 50% of Gram-negative oral anaerobes isolated resistant both to tetracycline and to penicillins . Andres et al. were able to link erythromycin and tetracycline resistance and β-lactamase production in Gram-negative anaerobes, concluding that they were associated with conjugative elements in oral Prevotella species. The co-transfer of resistance determinants to these three antibiotic classes occurs frequently within this genus

104. Antibiotics promote resistance If a patient taking a course of antibiotic treatment does not complete it Or forgets to take the doses regularly, Then resistant strains get a chance to build up The antibiotics also kill innocent bystanders bacteria which are non-pathogens © 2008 Paul Billiet ODWS

105. This reduces the competition for the resistant pathogens The use of antibiotics also promotes antibiotic resistance in non-pathogens too These non-pathogens may later pass their resistance genes on to pathogens

106. Resistance gets around When antibiotics are used on a person, the numbers of antibiotic resistant bacteria increase in other members of the family In places where antibiotics are used extensively e.g. hospitals and farms antibiotic resistant strains increase in numbers © 2008 Paul Billiet ODWS

107. Prevention of drug resistance It is utmost clinical importance to curb development of drug resistance. Measures are: No indiscriminate and inadequate or unduly prolonged use of antimicrobial agents(AMA’s) should be made. This would minimize the selection pressure and resistance strain will get less chance to preferentially propagate.

108. 2. Prefer rapidly acting and selective(narrow spectrum) AMA’s whenever possible; broad-spectrum drug should be used only when a specific one cannot be determined or is not suitable.

109. 3. Use combination of AMA’s whenever prolonged therapy is undertaken,eg, Tuberculosis ,SABE 4. Infection by organism notorious for developing resistance e.g., staph. Aureus, E.coli,M.tberculosis,Proteus,etc must be treated intensively.

110. For acute localized infections in otherwise healthy patient ,symptom determined shorter courses of AMA’s are being advocated now

111. Conclusion Antibiotic resistance is rising to dangerously high levels in all parts of the world. New resistance mechanisms are emerging and spreading globally, threatening our ability to treat common infectious diseases. A growing list of infections – such as pneumonia, tuberculosis, blood poisoning and gonorrhea – are becoming harder, and sometimes impossible, to treat as antibiotics become less effective.

112. Where antibiotics can be bought for human or animal use without a prescription, the emergence and spread of resistance is made worse. Similarly , in countries without standard treatment guidelines, antibiotics are often over-prescribed by health workers and veterinarians and over-used by the public.

113. Without urgent action, we are heading for a post-antibiotic era, in which common infections and minor injuries can once again kill.

114. References Essentials of medical pharmacology,6 th edition ,K.D Tripathi Textbook of Microbiology and Immunology ; Subhash Chandra Parija Carranza’s Clinical periodontology,11 th edition Rajiv Saini Biofilm: A dental microbial infection. J Natural science,biology and medicine 2011;2: 71-75 Basics & clinical pharmacology,9 th edition,Bretram G . Katzung L.C .Sweeny et.al . Antibiotic resistance in general dental practice J.Antimicrob.chemotherapy 2004;53: 567-76

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