1. Microbiology in Infection Prevention Leslie Teachout MT (ASCP), CIC Riverton Memorial and Lander Regional Hospitals

2. Infection Prevention

3. Objectives Discover and discuss the importance of the infection preventionist and microbiology working as a team. Learn a basic understanding of microbiology and how it helps infection preventionists. Discuss and interpret the antimicrobial susceptibility report and the antibiogram.

4. Specimen Collection Get a good specimen to Get good result! Is very important!

5. The Basics Bugs are small – 2-5 microns or (10 6 meters) – Viruses are even smaller – nanometers (10 9 ) Classification based on three things – Shape – Gram Reaction – Growth requirements

6. Shape

7. Gram stain process There are four basic steps of the Gram stain: 1.Primary stain with crystal violet to a heat- fixed smear of a bacterial culturecrystal violet 2.Followed by the addition of a trapping agent Gram's iodineiodine 3.Rapid decolorization with alcohol or acetonealcoholacetone 4.Counterstaining with safraninsafranin

8. Gram stain

9. Using Gram stain information!! Gives a quick look at the specimen – Presumptive identification Can interpret quality of specimen – Number of “pus” (polymorphonuclear) cells present

10. Using Gram stain information (cont.)!! Number of epithelial cells present – Surface Number of bacteria present – Normal vs. abnormal

11. Why the Gram Stain is important!!!! Can help direct antibiotic therapy – Based on cell wall composition Not so helpful if lots of normal flora present – Throats, stool, decubital ulcers QUITE significant on sterile body sites – CSF, blood, urine and other fluids – Assists in the interpretation of culture results

12. Other Stains Acid fast stain is another process. Tuberculosis can not be seen in a gram stain.

13. Normal Respiratory Flora Oral anaerobes Streptococci species Neisseria species Haemophilus species

14. Respiratory Tract Infections Is this a sterile body site? Streptococcus pneumoniae Klebsiella pneumoniae Staphylococcus aureaus Haemophilus influenzae

15. NORMALLY STERILE SITES IN THE HUMAN BODY: Colonization of one of these sites generally involves a defect or breach in the natural defenses that creates a portal of entry Brain; Central nervous system Blood; Tissues; Organ systems Sinuses; Inner and Middle Ear Lower Respiratory Tract: Larynx; Trachea; Bronchioles (bronchi); Lungs; Alveoli Kidneys; Ureters; Urinary Bladder; Posterior Urethra Uterus; Endometrium (Inner mucous membrane of uterus ); Fallopian Tubes; Cervix and Endocervix

16. Never Normal Flora Mycobacterium tuberculosis Legionella species Brucella species Growth in a sterile body fluid

17. Growth requirements

18. What is Bacterial Growth Media? A growth media is a mixture of nutrients, moisture and other chemicals that bacteria need for growth. Media are used to grow bacterial colonies

19. Using Media to Identify Bacteria Like the differential staining of bacteria, special types of media can be used to provide clues about a microbe’s identity. There are many types of media that are specific about what they grow, or that provide information about the type of microbes presentdifferential staining of bacteria

20. MRSA on Chromagar Typical Appearance of microorganisms Methicillin Resistant Staphylococcus aureus (MRSA) → rose to mauve Methicillin Susceptible Staphylococcus aureus (MSSA) → inhibited Other bacteria → blue, colorless or inhibited

21. Hemolysis: complete lysis of RBCs

22. Other identification requirements Oxygen requirements Ability to ferment or oxidize sugars to produce acid end products Temperature ranges Salt tolerance Chemical tolerance Enzymes Motility

23. Gram Positive or Gram Negative bacteria

24. Catalase Tests the organism’s ability to liberate oxygen from hydrogen peroxide If it bubbles it staphylococci

25. Coagulase The ability of the organism under study to clump, clot or coagulate rabbit plasma. – Can use plasma or latex particles Used as main identification of Staphylococcus aureus, distinguishing it form other Staph. species

26. Coagulase test results Coag positive Staph aureusCoag. Negative Staph epi

27. Basic Growth Times Most human pathogens take 18-24 hours to grow enough on media to be visible and to be able to distinguish single colonies with the naked eye Sensitivity testing from a pure culture can be anywhere from 4-24 hours later. Full identification can take 24 to 48 hours.

28. Pathogens in urine Is urine a sterile fluid? – Escherichia coli or E. coli – Enterococcus faecalis – Proteus species – Klebsiella Pneumoniae – Enterobacter species

29. Other Pathogens: Skin and wound – Steptococcus – Staphylococcus Gastroenteritis – Salmonella – Shigella – Campylobacter species

30. Other Frequently Isolated Organisms (Seldom Pathogens) Diphtheroids Propionibacterium Bacillus species

31. Sensitivity Testing Basically expose organism to antibiotic and see if it kills the bug. – Antibiotic impregnated discs – Micro-wells to which an organism suspension is added – Take 4-24 hours

32. National excepted criteria for zone size

33. Sensitivity Example

34. Antibiograms # of isolates tested Ampicillin Ampicillin/Sulbactam Aztreonam Cefazolin Cefepime Ceftazidime Ceftriaxone Cefuroxime-oral Cefuroxime-parenteral Ciprofloxacin Clindamycin Erythromycin Gentamicin Gentamicin (synergy) Imipenem Levofloxacin Linezolid Nitrofurantoin Oxacillin Penicillin G Piperacillin Piperacillin/Tazobactam Quinupristin/Dalfopristin Rifampin Streptomycin (synergy) Tetracycline Tobramycin Trimethoprim/Sulfa Vancomycin Escherichia coli 11316474979297 889286 95 10086 96 7999 9684 Pseudomonas aeruginosa 110 9293 82 89 9585 9695 99 Enterobacter spp. 628 98171009796849100 50 9698 10099 Klebsiella spp. 2170879787999499909298 10098 64 096 9792 Staphylococcus aureus (incl MRSA) 585 72925198 73100997010 99 95 99100 Methcillin Resistant S.aureus 176 29758 30100 00 99 96 98100 Staphylococcus coag neg 82 68874799 72100 6435 100 71 93100 Enterococcus faecalis 16698 78 79 8196100 98 8226 100 Streptococcus pneumoniae 62 8856 97 59 77 73100 meningeal susceptible 98 non-meningeal susceptible 87 Streptococcus agalactiae (Grp B) 33100 44 100 "viridans" Streptococcus ssp. 46 70 7957 67 100 - Values are expressed in % susceptible - Shaded areas indicate that the antimicrobial was not tested against the organism, is not appropriate to report, or is a limitation of the test methods used. - % susceptible results for clindamycin on staphylococcus, Group B strep, and beta-hemolytic strep have been corrected to reflect isolates that demonstrated inducible clindamycin resistance - H.influenzae is only tested for beta-lactamase production; 89% of the isolates tested were beta-lactamase negative (ampicillin susceptible) penicillin macrolide streptogramin Beta-lactam/Beta-lactamase inhibitor combination aminoglycoside ansamycin monobactam carbapenem tetracycline cephem oxazolidinone folate pathway inhibitor fluoroquinolone nitrofurantoin glycopeptide lincosamide

35. Antimicrobial Resistance Prevention and Control: New drug development Management of antimicrobial use Surveillance Periodic preparation and dissemination of institutional resistance patterns P&T Committee team work

37. Daily Micro Review Culture source – Wounds - check previous admissions – Throats and vaginal cultures tend not to be hospital acquired (check admission date) Location of the patient Admission date Culture date is this more than 24 hours from admission

38. Positive Blood culture, Is follow up needed?

39. Wound culture to follow up on:

41. How to handle this information? Spinal fluid with gram negative cocci – Is this a sterile body site? – Is this organism at pathogen or potential pathogen?

42. Mycobacterium AFB stain Does not stain with Gram’s Stain Staining process uses carbol fushsin, slide is heated, then decolorize with HCI and alcohol for 5 minutes – Acid fast (AFB-bacillus) – Retain red color

43. Mycobacterium M. Tuberculosis (MTb) is a human pathogen M. avium-intracellularae (MAI)in HIV patient Divide once every 24 hours – 2-8 weeks for visible colonies Some environmental species – M. gondonae – M. marinum

44. What is a virus? Viruses are not like bacteria! Viruses DO NOT “grow” or divide Viruses make copies of themselves using: – Tools like enzymes or proteins they code – Using cell machinery – May target specific cells like the liver

45. What is a Virus? Obligate intracellular parasite NOT a cellular organism – No organelles or – ribosome, energy-less Not Free-living – Completely dependent on host cells

46. Viruses Enveloped – Easiest to kill, less hardy Non-enveloped – Hardy, resistant to lower concentration of alcohol Both DNA and RNA viruses Test is generally sent to a reference lab

47. Yeasts Single cell organisms Numerous species – Candida albicans Opportunistic – Can be normal respiratory flora

48. The life cycle of Clostridium difficile Host Entry Germination Vegetative state Reproduction Toxin Production Disease Host Exit Sporulation Spore state Adapted from description in Paredes-Sabja, D., Bond, C., Carman, R. J., Setlow, P. & Sarker, M.R. (2008). Germination of spores of Clostridium difficile strains, including isolates from a hospital outbreak of Clostridium difficile-associated disease (CDAD). Microbiology, 154, 2241-2250.

49. Pathogen factors Strain type Antibiotic resistance Sporulation rates Toxin regulation

50. Hypervirulent NAP1 “This is a specific strain of C. difficile that emerged first in North America, in Pennsylvania. This NAP1 strain has a genetic change that results in literally 16 to 23 times more toxin production in vitro,” explains William Jarvis, MD

51. Last Thoughts The names may change but the bugs stay the same Get a good specimen to Get good result!

52. Objectives Discover and discuss the importance of the infection preventionist and microbiology working as a team. Learn a basic understanding of microbiology and how it helps infection preventionists. Discuss and interpret the antimicrobial susceptibility report and the antibiogram.

53. Thank You Any questions??