Microbiology in Infection Prevention

Leslie TeachoutMT (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 toGet good result!

• Is very important!

The Basics

• Bugs are small – 2-5 microns or (106 meters)– Viruses are even smaller – nanometers (109)

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 culture2. Followed by the addition of a trapping agent

Gram's iodine3. Rapid decolorization with alcohol or acetone4. Counterstaining with safranin

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 BacteriaLike 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 present

MRSA on ChromagarTypical Appearance of microorganismsMethicillin Resistant Staphylococcus aureus (MRSA)→ rose to mauveMethicillin Susceptible Staphylococcus aureus (MSSA) → inhibitedOther 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 aureus Coag. Negative Staph epi

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.

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 isolate

s tested

Ampicillin

Ampicillin/Sulbactam

Aztreonam

Cefazolin

Cefepime

Ceftazidim

e

Ceftriaxon

e

Cefuroxime-

oral

Cefuroxime-

parenteral

Ciprofloxacin

Clindamycin

Erythromycin

Gentamicin

Gentamicin (synergy)

Imipenem

Levofloxacin

Linezolid

Nitrofurantoin

Oxacillin

Penicillin G

Piperacillin

Piperacillin/

Tazobactam

Quinupristin/

Dalfopristin

Rifampi

n

Streptomycin

(synergy)

Tetracycline

Tobramycin

Trimethoprim/Sulfa

Vancomycin

Escherichia coli 1131 64 74 97 92 97 97 97 88 92 86     95   100 86   96     79 99         96 84  

Pseudomonas aeruginosa 110         92 93       82     89   95 85         96 95         99    

Enterobacter spp. 62 8   98 17 100 97 96 8 49 100     100   100 100   50     96 98         100 99  

Klebsiella spp. 217 0 87 97 87 99 94 99 90 92 98     98   100 98   64     0 96         97 92  

                                                             

Staphylococcus aureus (incl MRSA) 585                   72 92 51 98     73 100 99 70 10       99   95   99 100

Methcillin Resistant S.aureus 176                   29 75 8       30 100 100 0 0     100 99   96   98 100

Staphylococcus coag neg 82                   68 87 47 99     72 100 100 64 35       100   71   93 100

                                                             

Enterococcus faecalis 166 98                 78       79   81 96 100   98         82 26     100

Streptococcus pneumoniae 62                     88 56       97       59           77   73 100

meningeal susceptible               98                                            

non-meningeal susceptible               87                                            

Streptococcus agalactiae (Grp B) 33 100                   44 44       100       100                 100

"viridans" Streptococcus ssp. 46             70       79 57               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

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?

Yes

Yes

MycobacteriumAFB 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 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, 2241-2250.

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 toGet 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??