INFECTION PREVENTION & THE MICROBIOLOGY LAB Doramarie Arocha, MS, MT (ASCP)SM,CIC, FAPIC Director of Infection Prevention UT Southwestern Medical Center
Overview • Terminology/definitions • Preanalytic: Specimen collection/submission • Analytic: What happens in the Micro lab • Postanalytic: – Reporting/susceptibilities – Interpreting the reports
MICROBIOLOGY • The branch of biology focused on microorganisms and the effects they have on other living organisms • Microorganisms – bacteria – viruses – fungi – parasites
IP Role: Develop Good Relationships • Microbiology • Reference Laboratory • Health Department
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
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
Beneficial Effects of Normal Flora
Adverse Effects of Antibiotics on Normal Flora
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
Normal Flora by Site • Mouth/oropharynx • Most normal flora is anaerobic • Skin – – Coagulase negative staphylococci Viridans group streptococci – Veillonella sp – Diphtheroids/Corynebacterium sp. – Fusobacterium sp. – Propionibacterium – Treponema sp. – – Prevotella/Porphyromonas Staphylococcus aureus – Neisseria/ Moraxella – Viridans group Streptococci – Streptococcus pneumoniae – – Bacillus sp. Beta hemolytic strep (Strep mlleri/anginosus) – Malassezia furfur – Candida – Candida – Haemophilus – Corynebacterium /diphtheroids • Nares – Actinomyces – Coagulase negative staphylococci – HACEK – – Staphylococcus aureus Viridans group streptococci – Lactobacillus – Staphylococcus aureus – Neisseria/Moraxella – Haemophilus – Streptococcus pneumoniae
Normal Flora by Site • Colon • Stomach – Bacteroides – Lactobacillus – Fusbacterium – Viridans streptococci – Clostridium – Staphylococci – Peptostreptococcus – Peptostreptococcus – Enteric GNRs • Small Intestine – Enterococcus – Lactobacillus – Lactobacillus – Bacteroides – Viridans streptococci – Clostridium – Candida – Enterococci – Enteric GNRs
Normal Flora by Site • Urethra • Vagina – Coagulase negative – Lactobacillus staphylococci – Peptostreptococcus – Diphtheroids/ – Diphtheroids/ Corynebacterium sp. Corynebacterium sp. – Viridans streptococci – Viridans streptococci – Bacteroides – Candida – Fusobacterium – Gardnerella vaginalis – Peptostreptococcus
S.aureus, S.pyogenes, Pseudomonas, Skin, subcutaneous tissue S. pneumoniae, H. influenzae, S. pyogenes, Sinusitis S. aureus, M. catarrhalis, Gram Negative Bacilli Pharyngitis S. pyogenes , respiratory viruses Bronchitis Respiratory viruses, S. pneumoniae, H. influenzae , RSV, B. pertussis, M. catarrhalis Pneumonia (CAP) S. pneumoniae, H. influenzae,K. pneumoniae , Mtb, S. aureus, L. pneumophila, P. carinii , Gram Negative Bacilli
Pathogens…continued • Anaerobes, oral Streptococcus, S. aureus, Empyema S. pyogenes, H. influenzae • Healthcare acquired Pseudomonas, S. aureus, Legionella, pneumonia Enterobacteriaceae • Endocarditis S. viridans, S. aureus, Enterococcus, Haemophilus, S. epidermidis, Candida • Gastroenteritis Salmonella, Shigella, Campylobacter, invasive E. coli (0157:H7) , viruses, Giardia, Yersinia, Vibrio • Peritonitis, abdominal abscess Bacteroides , anaerobic cocci, S. aureus, Enterococcus, Candida, Enterbacteriaceae • Urinary tract infection E.coli, Klebsiella, Proteus, Enterococcus, Pseudomonas, Candida, S. saprophyticus
…A few more C. trachomatis, N. gonorrhoeae, • PID Enterobacteriaceae • Osteomyelitis S. aureus, Pseudomonas S. agalactiae • Septic arthritis S. aureus, N. gonorrhoeae, S. pyogenes, S. pneumoniae, P. multocida • Meningitis H. influenzae, N. meningitidis, Mtb, S. pneumoniae, S. agalactiae • Septicemia S. aureus, S. pneumoniae, Salmonella, E.coli, Klebsiella, Candida, Clostridium, Listeria Coagulase Negative Staph, Corynebacterium , • Device related Gram Negative Bacilli, organisms listed under septicemia (source: APIC Text of Infection Control and Epidemiology, 2002)
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
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*
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
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
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
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
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
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)
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
BACTERIAL IDENTIFICATION • Presumptive – Gram Stain – Colony morphology and odor – Spot tests: catalase, indole • Definitive – Biochemical tests • Manual • Automated system – Molecular diagnostics
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
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
Sputum
Neisseria/Moraxella
Haemophilus influenzae
Listeria monocytogenes
Staphylococcus
Gram Negative Rods
Staph and Strep
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, …
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