Do it quick! Update on CLSI M35A2 - SWACM

<strong>Do</strong> <strong>it</strong> <strong>quick</strong>!
<strong>Update</strong> on CLSI M35A2 –
Rapid Methods for Identification
Mary K. York, Ph.D.
Marykyork@gmail.com

Changes in second ed<strong>it</strong>ion
More organisms
Includes bioterrorist
agents
New safety precautions
defined
User friendly tables
added
New biochemicals
Revised biochem
section

References Prove Value :
<strong>Do</strong>ern, G., R. Vautour, M. Gaudet, and B. Levy.
1994. Clinical impact of rapid in v<strong>it</strong>ro
susceptibil<strong>it</strong>y testing and bacterial
identification.
MIC 9.6 h from colonies vs. 25.9 h- showed
less mortal<strong>it</strong>y, length of stay and orders of
laboratory tests
Barenfanger, J., C. Drake, and G. Kacich.
1999. Clinical and financial benef<strong>it</strong>s of rapid
bacterial identification and antimicrobial
susceptibil<strong>it</strong>y testing.
evaluated evening vs. next day - 5 h
difference - length of stay and cost was
significantly less

Goals
Rapid (same day) identification and
reporting of >90% of pathogens
Decrease costs of testing
Increase safety of personnel
Make use of microbiologists abil<strong>it</strong>y to
recognize organisms
Reduce need to confirm - lim<strong>it</strong>ations
stated

Grows on MAC
Spreading Proteus

M35-A2 Nov. 2009
M35A GRAM-NEGATIVE M35A2 GRAM-NEGATIVE
Escherichia coli Brucella species
Haemophilus influenzae Campylobacter jejuni/coli
Moraxella catarrhalis Cardiobacterium hominis
Proteus species Eikenella corrodens
Pseudomonas aeruginosa Francisella tularensis
Kingella kingae
Neisseria gonorrhoeae
Neisseria mening<strong>it</strong>idis
Neisseria lactamica

M35-A2 Nov. 2009
M35A GRAM-POSITIVE M35A2 GRAM-POSITIVE
Enterococcus Listeria monocytogenes
Staphylococcus aureus Staphylococcus lugdunensis
Streptococcus agalactiae
(Gr. B)
Aerococcus viridans
S. pneumoniae Streptococcus anginosus
group (S. milleri)
S. pyogenes (Gr. A) Viridans group streptococci

Example of new tables

“Is Use of Odor Safe”
“Sniffing” plates is dangerous
Opening plates on bench is safe
(once dangerous ones & mold are ruled out)
Odor is recognized when opening if
strong and distinctive
Can be used in identification

Technologist must….
Begin w<strong>it</strong>h pure colony
Recognize what <strong>it</strong> could be from
typical colony morphology
Perform rapid tests accurately and
read them correctly
<strong>Do</strong>n’t cut corners…

Supervisor must…..
Validate the competency of the staff
doing tests
Check to see that all tests are done
Be sure that procedures are wr<strong>it</strong>ten and
QC is done usually by lot

Perform a Gram stain
unless growing on
MacConkey.
Exceptions will be noted
as we proceed.

Not “Presumptive”
>95% accuracy of rapid methods
If cr<strong>it</strong>eria are met, no further testing is
required for defin<strong>it</strong>ive identification
Exception: will further testing provide
information clinically useful for patient
outcome (e.g. confirm a bioterorist
identification)

Start w<strong>it</strong>h Indole and Oxidase Test
Cinnamaldehyde is blue
Required for anaerobes
Benzaldehyde (Kovacs) in acid alcohol, not amyl, is red
Can be used for tube test

Many rapid indole tests….

Rapid Identification of E.coli
•Indole +
•Oxidase -
•Gram-negative rod
•Lactose pos<strong>it</strong>ive
•Beta-hemolytic or PYR
negative
= E. coli

PYR negative = E. coli
1. Wet disk slightly
2. Add reagent after 2 minutes
3. <strong>Do</strong> not use selective media

PYR reactions
Genus Indole Lactose PYR
C<strong>it</strong>robacter Some + V +
E. coli + V -
Klebsiella Some + + +
Yersinia Some + - +
+
Enterobacter - + -
+
Serratia - + -
Morganella + - -
Proteus Some + - -
Providencia + - -
Edwardsiella + - -
Hafnia - + -
Shigella Some + - -
Salmonella - - +
-
+

Brucella
blood, lymph nodes, body fluids
• <strong>Do</strong>es not grow on MacConkey
• Non- hemolytic, slow-growing colonies
• Tiny cocco-bacilli
• Catalase pos<strong>it</strong>ive
• Oxidase pos<strong>it</strong>ive
• Indole negative
• Rapid pos<strong>it</strong>ive urease (2 hours)
• Phenylalanine deaminase negative

This is Brucella unless
proven otherwise
Work in a BSC
Confirm w<strong>it</strong>h urease
Reportable disease
Bioterrorism

Urease rapid test-incubate 2 h
-
+
+ w<strong>it</strong>h Fe Cl
Phenylalanine deaminase test

Brucella Diagnosis
Symptoms are non-specific
Onset is insidious
Risks are eating raw dairy products or
working in a microbiology laboratory
W<strong>it</strong>hout treatment there is no cure :
<strong>Do</strong>xycycline plus rifampin

Other Oxidase + catalase + urease +
Paracoccus yeei (vacuoles and round cells)
Psychrobacter immobilis (rose odor, likes
20°C)
Methylobacterium spp. (pink at 72 hours)
Psychrobacter phenylpyruvicus (PDA +)
Oligella ureolytica (PDA +, motile)

Paracoccus yeei

Campylobacter in stools
Distinctive colony on
selective media at
42°C
Oxidase pos<strong>it</strong>ive
Catalase pos<strong>it</strong>ive
Gull wing gram stain
Darting motil<strong>it</strong>y

Pos<strong>it</strong>ive
hippurate
Is Campylobacter
jejuni

Pos<strong>it</strong>ive Indoxyl Acetate
is C. jejuni/coli
5-10 minute test for blue color

Indoxyl acetate
C. lari is negative
Can also be used
for identification of
Moraxella
catarrhalis

Colonies move when pushed

Identification of M. catarrhalis
Gram negative
diplococci
Indoxyl acetate
or butyrate
pos<strong>it</strong>ive
Actually all
Moraxella and
Acinetobacter are
butyrate + but they
are coccobacilli

HACEK bacteria
BAP & MAC
Pos<strong>it</strong>ive blood culture

Cardiobacterium hominis

Cardiobacterium hominis
Indole-pos<strong>it</strong>ive in blood culture
Oxidase-pos<strong>it</strong>ive
Catalase-negative
Non-hemolytic
Chronic endocard<strong>it</strong>is
Slow growing
Fatal if not treated-beta-lactam surgery
N<strong>it</strong>rate-negative if needed to confirm
<strong>Do</strong>n’t let the gen<strong>it</strong>al isolate Pasteurella bettyae or
rare isolate Suttonella indologenes confuse you

Oxidase +, catalase -, indole -
Eikenella corrodens
Non-hemolytic-sticks in agar
Odor of bleach
Slight yellow

Blood agar hemolysis-
Found in joint and
blood infections in
children
Kingella

Kingella kingae
Oxidase + Catalase - Indole -

Francisella tularensis
If growth takes longer than 24 h, may be
Francisella which does not satell<strong>it</strong>e
• Grows on CHOC but not on BAP.
• Oxidase-negative; catalase weak.
• No k<strong>it</strong> will identify
• <strong>Do</strong> cefinase-result is pos<strong>it</strong>ive.
• Send to health department for confirmation
• Safety concerns

No growth on BAP
Tiny rod in smear

Rapid identification
Oxidase - Catalase V Indole -

Francisella is
beta
lactamase
pos<strong>it</strong>ive

Francisella tularensis - BSL III

Identification of
Haemophilus influenzae
Small gram-negative
coccobacilli
Growth of large
colonies only on
CHOC in 24 h or
around
staphylococci
Satell<strong>it</strong>e separates <strong>it</strong>
from Francisella

Neisseria species
Gram-negative diplococci
Oxidase pos<strong>it</strong>ive
Species identification by rapid
test and growth on various media

Neisseria mening<strong>it</strong>idis
Colony grows on BAP as glistening and nonhemolytic
and not yellow pigmented
Delta -glutamyl amino peptidase – pos<strong>it</strong>ive
• May be carried as normal flora in the throat
• Isolated from the cervix or urethra of men or women
• Sporadic cases or epidemics of mening<strong>it</strong>is.
• 13 serogroups
• Use caution BSC - vaccine available for lab staff.

Neisseria lactamica
Colony grows on BAP non-hemolytic and not
yellow pigmented
Beta galactosidase– pos<strong>it</strong>ive
• Associated w<strong>it</strong>h ocular infections

NET test 30 min
•Blue - Pos<strong>it</strong>ive for betagalactosidase:
Neisseria lactamica
•Yellow – Pos<strong>it</strong>ive for
gammaglutamylaminopeptidase:
Neisseria mening<strong>it</strong>idis

Next step if not e<strong>it</strong>her of these species or
rule out Moraxella
Moraxella catarrhalis is negative for both
but pos<strong>it</strong>ive for e<strong>it</strong>her butyrate or indoxyl
acetate
confirm lack of growth on nutrient agar.
• Nutrient agar
• TSA
• MH
Or does <strong>it</strong> grow on Thayer Martin =
vancomycin and colistin resistant

Neisseria
gonorrhoeae
Gram negative diplococci
Oxidase-pos<strong>it</strong>ive
Catalase-pos<strong>it</strong>ive -
strong w<strong>it</strong>h 30% peroxide
No growth on nutrient agar and no pigment
…..Or Colistin/ polymyxin B resistant by growth
on selective medium
K<strong>it</strong> identification not other Neisseria
Usually proline pos<strong>it</strong>ive

Neisseria Table
All are catalase + and oxidase +;
GC is 4+ catalase
MH Bgal Glut Pro
N. mening<strong>it</strong>idis + + v
N. lactamica + + +
N. gonorrhoeae - +
N. cineria + +
Neisseria (other)
Moraxella
+ v

Staphylococcus aureus - BAP

Staphylococcus Latex tests
Staph Latex
Fibrinogen
IgG to attach to Protein A

Staphylococcus saprophyticus
Latex test can be
pos<strong>it</strong>ive
Slide coagulase is
negative
Gram-pos<strong>it</strong>ive cocci in urine

S. lugdunensis can be Latex + or -
Significant
cause of
bacteremia
and wound
infections
Recognize
colony
Catalase +
Can be slide
coagulase + or
latex +

Staphylococcus lugdunensis
Clumpy slide test
Negative tube test
PYR Magenta

Algor<strong>it</strong>hm for Staphylococcus ID

Aerococcus viridans
Alpha colony
Tetrads
PYR pos<strong>it</strong>ive
PYR +

PYR-pos<strong>it</strong>ive α hemolytic
catalase negative colony

Streptococcus PYR - LAP +
Enterococcus PYR + LAP +
Aerococcus viridans PYR + LAP -
Courtesy of Color Atlas of Medical Bacteriology, ASM Press, 2004

•Streptococcus anginosus (“milleri” grp)
• Alpha or Beta-hemolytic on BAP
• Pinpoint colonies

Group A, C, G Streptococcus
vs. Streptococcus anginosus
Odor of vanilla
Tiny colonies
PYR not pos<strong>it</strong>ive if A

Listeria monocytogenes
(blood, CSF, products of conception)
Pos<strong>it</strong>ive rapid hippurate
Tumbling motil<strong>it</strong>y
Catalase pos<strong>it</strong>ive

Bach Mai
Hanoi,Vietnam

Rapid testing does make a
difference
Questions?