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

Infection Prevention

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.

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

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

Shape

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

Gram stain

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

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

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

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

Normal Respiratory Flora Oral anaerobes Streptococci species Neisseria species Haemophilus species

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

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

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

Growth requirements

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

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

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

Hemolysis: complete lysis of RBCs

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

Gram Positive or Gram Negative bacteria

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

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

Coagulase test results Coag positive Staph aureusCoag. Negative Staph epi

Basic Growth Times Most human pathogens take 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.

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

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

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

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

National excepted criteria for zone size

Sensitivity Example

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 Pseudomonas aeruginosa Enterobacter spp Klebsiella spp Staphylococcus aureus (incl MRSA) Methcillin Resistant S.aureus Staphylococcus coag neg Enterococcus faecalis Streptococcus pneumoniae meningeal susceptible 98 non-meningeal susceptible 87 Streptococcus agalactiae (Grp B) "viridans" Streptococcus ssp 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

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

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

Positive Blood culture, Is follow up needed?

Wound culture to follow up on:

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?

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

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

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

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

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

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

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,

Pathogen factors Strain type Antibiotic resistance Sporulation rates Toxin regulation

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

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

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.

Thank You Any questions??