Infection Prevention & the Microbiology Lab

1. Doramarie Arocha, PhD, MS, MT (ASCP)SM,CIC, FAPIC Director of Infection Prevention UT Southwestern Medical Center Infection Prevention & the Microbiology Lab

2. Overview • Terminology/definitions • Preanalytic: Specimen collection/submission • Analytic: What happens in the Micro lab • Postanalytic: • Reporting/susceptibilities • Interpreting the reports

4. MICROBIOLOGY • The branch of biology focused on microorganisms and the effects they have on other living organisms • Microorganisms • bacteria • viruses • fungi • parasites They are not all bad

5. IP Role: Develop Good Relationships • Microbiology • Reference Laboratory • Health Department

6. Terminology Normal flora: Bacteria and some yeasts present at a variety of sites Skin, mucosal surfaces Do not cause disease under normal circumstances Participate in maintaining health Colonizer: present on mucous membranes, noninvasive, no host response VRE in stool, MRSA in nares Pathogen: causing infection, invasive with host response Normal flora and colonizers can become pathogens

7. Functions of Normal Flora Provide some nutrients (vit. K) Help develop mucosal immunity: stimulate immune system with cross reactivity against some pathogens Prevent colonization by potential pathogens Aid digestion

8. Beneficial Effects of Normal Flora Compete for space and nutrients with pathogens Produce nutrients: Vit. K, B6, B12 Produce antimicrobial substances Occupy receptors and prevent attachment of pathogens

9. Adverse Effects of Antibiotics on Normal Flora Kill normal flora Select resistant mutants

10. Factors Influencing Normal Flora • Local environment • pH, temperature, oxygen levels, nutrients • Diet • Age • Health/Immune status • Antibiotics • Flora changes with eruption of teeth, weaning, onset/cessation of ovarian function

11. Normal Flora by Site • Mouth/oropharynx • Viridans group streptococci • Veillonellasp • Fusobacterium sp. • Treponema sp. • Prevotella/Porphyromonas • Neisseria/ Moraxella • Streptococcus pneumoniae • Beta hemolytic strep (Strep mlleri/anginosus) • Candida • Haemophilus • Corynebacterium/diphtheroids • Actinomyces • HACEK • Staphylococcus aureus • Lactobacillus • Most normal flora is anaerobic • Skin • Coagulase negative staphylococci • Diphtheroids/Corynebacterium sp. • Propionibacterium • Staphylococcus aureus • Viridans group Streptococci • Bacillus sp. • Malassezia furfur • Candida • Nares • Coagulase negative staphylococci • Viridans group streptococci • Staphylococcus aureus • Neisseria/Moraxella • Haemophilus • Streptococcus pneumoniae

12. Normal Flora by Site • Colon • Bacteroides • Fusbacterium • Clostridium • Peptostreptococcus • Enteric GNRs • Enterococcus • Lactobacillus • Viridans streptococci • Candida • Stomach • Lactobacillus • Viridans streptococci • Staphylococci • Peptostreptococcus • Small Intestine • Lactobacillus • Bacteroides • Clostridium • Enterococci • Enteric GNRs

13. Normal Flora by Site • Urethra • Coagulase negative staphylococci • Diphtheroids/ Corynebacterium sp. • Viridans streptococci • Bacteroides • Fusobacterium • Peptostreptococcus • Vagina • Lactobacillus • Peptostreptococcus • Diphtheroids/ Corynebacterium sp. • Viridans streptococci • Candida • Gardnerellavaginalis

14. Common pathogens by site • Skin, subcutaneous tissue • Sinusitis • Pharyngitis • Bronchitis • Pneumonia (CAP) S.aureus, S.pyogenes, Pseudomonas, S. pneumoniae, H. influenzae, S. pyogenes, S. aureus, M. catarrhalis, Gram Negative Bacilli S. pyogenes, respiratory viruses • Respiratory viruses, S. pneumoniae,H. influenzae, RSV, B. pertussis, M. catarrhalis S. pneumoniae, H. influenzae,K. pneumoniae, Mtb, S. aureus, L. pneumophila, P. carinii, Gram Negative Bacilli

15. Pathogens…continued Empyema Healthcare acquired pneumonia Endocarditis Gastroenteritis Peritonitis, abdominal abscess Urinary tract infection • Anaerobes, oral Streptococcus, S. aureus, • S. pyogenes, H. influenzae • Pseudomonas, S. aureus, Legionella, Enterobacteriaceae • S. viridans, S. aureus, Enterococcus, Haemophilus, S. epidermidis, Candida • Salmonella, Shigella, Campylobacter, invasive E. coli (0157:H7), viruses, Giardia, Yersinia, Vibrio • Bacteroides, anaerobic cocci, S. aureus, Enterococcus, Candida, Enterbacteriaceae • E.coli, Klebsiella, Proteus, Enterococcus, Pseudomonas, Candida, S. saprophyticus

16. …A few more PID Osteomyelitis Septic arthritis Meningitis Septicemia Device related • C. trachomatis, N. gonorrhoeae, Enterobacteriaceae • S. aureus, Pseudomonas S. agalactiae • S. aureus, N. gonorrhoeae, S. pyogenes, S. pneumoniae, P. multocida • H. influenzae, N. meningitidis, Mtb, • S. pneumoniae, S. agalactiae • S. aureus, S. pneumoniae, Salmonella, E.coli, Klebsiella, Candida, Clostridium, Listeria • Coagulase Negative Staph, Corynebacterium, Gram Negative Bacilli, organisms listed under septicemia (source: APIC Text of Infection Control and Epidemiology, 2002)

17. Probably normal…usually not identified Coagulase Negative Staphylococcus (CNS) unless present in several blood cultures Yeasts in respiratory cultures, rarely cause pneumonia 3 or more gram negatives in urine, recollect Pseudomonas in stool

18. The Eight Major Classifications for Taxonomic Ranking (Order) Life Domain Kingdom Phylum or Division Class Order Family - the family name always ends in -ae Genus* Species*

19. Taxonomy Classification and Grouping Biochemical phenotype Molecular DNA or RNA Other considerations - rRNA Examples of Classification Family: Enterobacteriaceae Genus: Klebsiella Species: pneumoniae (not capitalized) Note: names are italicized

20. Specimen Collection Avoid contamination from indigenous flora, to ensure a sample representative of the infectious process Select the correct anatomic site from which to obtain the specimen Submit tissue or needle aspirates when possible Collect adequate volumes; insufficient material may yield false negative results

21. Specimen Collection • Try to collect specimens before administering antimicrobials • Request direct smears when appropriate • Label each specimen container with the patient’s name, MR, source, specific site, date, time of collection, and initials of collector • Designations of wound or abscess are acceptable as long as the exact anatomic location is also stated • Transport specimen to lab ASAP

22. Swabs Limited volume Should only be used for specimens from mucous membranes Have no place in the OR Organisms get caught in fibers and die Anaerobes die upon exposure to air but survive in fluids and tissues

23. Blood Cultures Quality of collection affects microbial recovery, contamination rates, and the ability of physicians to interpret test results. Even with good collection technique, 1%-3% of blood cultures are found to be contaminated (rates are higher in teaching hospitals and EDs) Meticulous attention to skin antisepsis is necessary to prevent contamination

24. Blood Cultures 2-3 cultures from different venipuncture sites are recommended A single culture is inappropriate A single draw for multiple sets is inappropriate Volume of blood cultured is the most important variable in recovering a pathogen 20 cc should be drawn from each venipuncture site with 10cc added to each bottle (aerobic and anaerobic)

25. Specimen Rejection • No label or requisition does not match specimen • Prolonged transport • Improper or leaking container • Specimen unsuitable for request • Duplicate specimens on the same day for the same request (except blood and tissue) • Sputum specimens consisting of oropharyngeal secretions • Routine bacterial stool cultures on patients in-house >3 days

26. BACTERIAL IDENTIFICATION Presumptive Gram Stain Colony morphology and odor Spot tests: catalase, indole Definitive Biochemical tests Manual Automated system Molecular diagnostics

28. Gram Stain • Bacteria colorless and usually invisible to light microscopy. Staining allows for early classification • Gram positive microorganisms • Cell wall high peptidoglycans • Stain purple • Cocci or rod shaped • Gram negative microorganisms • Cell wall high lipids • Stain red or pink • Rod or cocci shaped

29. Bacterial Morphology • Bacteria have four major shapes • Cocci: spherical • Bacilli: rods. Short bacilli are called coccobacilli • Spiral Forms: comma-shaped, S-shaped or spiral • Pleomorphic: Lacking a distinct shape

30. Sputum

31. Neisseria/Moraxella

32. Haemophilusinfluenzae

33. Listeriamonocytogenes

34. Staphylococcus

35. Gram Negative Rods

36. Staph and Strep

37. Gram Negative Bacteria • Coccobacilli • Haemophilus • Diplococci • Neisseria and Moraxella • Spiral • Treponema pallidum • Pleomorphic • Campylobacter • Remainder are rods • Escherichia, Salmonella, Shigella and other Enterobacteriaceae, Pseudomonas, Stenotrophomonas, Legionella, Acinetobacter, …

38. Oxygen Requirements • Obligate aerobes • Require 02 (have all of the enzymes) • Pseudomonas, Mtb, Bacillus • Facultative anaerobes • Can grow in the absence of 02 • Staph, E. coli, Listeria, yeast • Microaerophilic • Require around 5% oxygen • Campylobacter, Neisseria • Obligate anaerobes- 02is fatal • Clostridium, Bacteroides

39. MEDIAbroths or agars used to support the growth of microorganisms Routine Blood agar Chocolate agar Selective MacConkey agar Columbia CNA Differential Chomogen agar Hektoen enteric agar Enrichment Broth medium

40. Colony Morphology • Size, color and odor on non-selective media • Growth on media type – chocolate vs blood • Hemolysis on blood agar • Beta (clear zone) - Group A Streptococcus • Alpha (green zone) - Streptococcus pneumoniae • Lactose fermentation on MacConkey agar • Positive - Escherichia coli • Negative - Salmonella, Shigella

41. MEDIA PLATES FOR BACTERIAL GROWTH

46. Identification • Quick tests • Automated systems • MALDI-TOF

47. Susceptibility Testing

48. Susceptibility Report Moderate Growth Escherichia coli Antibiotic M.I.C. Interpretation Ampicillin >16 R Cefotaxime <=8 S Ciprofloxacin <=1 S Gentamicin <=2 S Levofloxacin <=1 S Piperacillin/Tazo 64 I Trim/Sulfa >2/38 R Ticarcillin/Clav >64 R Tobramycin <=4 S

49. Antibiotic Resistance ESBL Extended Spectrum Beta Lactamase Resistant to 3rd generation cephalosporins (cefotaxime, ceftazidime, cefpodoxime) and monobactams (aztreonam) D Test Inducible Clindamycin Resistance

50. Resistant StrainsRare Antimicrobial Exposure x x x x x x x x x x x x Resistant Strains Dominant Selection for Resistant Strains