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 (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 heatfixed smear of a bacterial culture
2. Followed by the addition of a trapping agent
Gram's iodine
3. Rapid decolorization with alcohol or acetone
4. 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
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•
•
•
Oral anaerobes
Streptococci species
Neisseria species
Haemophilus species
Respiratory Tract Infections
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•
•
•
•
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 present
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 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
Escherichia coli
Pseudomonas aeruginosa
Enterobacter spp.
Klebsiella spp.
1131
Staphylococcus aureus (incl MRSA)
Methcillin Resistant S.aureus
Staphylococcus coag neg
585
72
92 51
176
29
75
82
68
87 47
Enterococcus faecalis
Streptococcus pneumoniae
meningeal susceptible
non-meningeal susceptible
Streptococcus agalactiae (Grp B)
"viridans" Streptococcus ssp.
166
64
74
97
92
110
62
8
217
0
87
97
97
92
93
97
88
92
86
95
100 86
82
89
95
96
85
99
96
96
95
99
84
98
17 100 97
96
8
49 100
100
100 100
50
96
98
100 99
97
87
99
90
92
98
100 98
64
0
96
97
99
94
98
98
78
62
98
8
99
79
73 100 99
70
10
30 100 100
0
0
72 100 100 64
35
81
96 100
100
Vancomycin
Tobramycin
79
Trimethoprim/Sulfa
Tetracycline
Rifampin
Streptomycin (synergy)
Quinupristin/Dalfopristin
Piperacillin/Tazobactam
Piperacillin
Penicillin G
Oxacillin
Nitrofurantoin
Linezolid
Levofloxacin
Imipenem
Gentamicin (synergy)
Gentamicin
Clindamycin
Erythromycin
Ciprofloxacin
Cefuroxime-parenteral
Cefuroxime-oral
Ceftriaxone
Ceftazidime
Cefepime
Cefazolin
Aztreonam
Ampicillin/Sulbactam
Ampicillin
# of isolates tested
Antibiograms
92
99
95
99 100
99
96
98 100
100
71
93 100
98
82 26
100
88 56
97
59
77
73 100
44 44
100
100
100
67
100
98
87
33
46
100
70
79 57
- 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
Beta-lactam/Beta-lactamase inhibitor combination
monobactam
cephem
fluoroquinolone
lincosamide
macrolide
aminoglycoside
carbapenem
oxazolidinone
nitrofurantoin
streptogramin
ansamycin
tetracycline
folate pathway inhibitor
glycopeptide
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 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
Sporulation
Host Exit
Spore state
Disease
Host Entry
The life
cycle of
Clostridium
difficile
Toxin
Production
Germination
Vegetative
state
Reproduction
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
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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??