M45: INFREQUENTLY
ISOLATED OR
FASTIDIOUS BACTERIA
Romney
Humphries,
PhD D(ABMM)
UCLA Clinical
Microbiology
Under
Revision!
ORGANISMS INCLUDED IN M45 VS. M100
M100
M45
Enterobacteriaceae
Abiotrophia / Granulicatella
Pseudomonas aeruginosa
Aeromonas / Pleisiomonas
Acinetobacter spp
Bacillus spp. (not B. anthracis)
Burkholderia cepacia
Campylobacter jejuni / coli
Pediococcus
S. maltophilia
Corynebacterium spp.
Vibrio spp
Other Non-Enterobacteriaceae
Erysipelothrix rhusiopathiae
Agents of Bioterrorism
Staphylococcus spp.
HACEK group
Enterococcus
Helicobacter pylori
H.influenzae / parainfluenzae
Lactobacillus
Streptococcus spp, β hemolytic
Leuconostoc
Streptococcus spp, viridans group
Listeria monocytogenes
Neisseria meningitidis
Moraxella catarrhalis
Anaerobes
Pasteurella spp.
Don’t test
these!!
M100 VS. M45
 Breakpoints
Established from:
 Microbiological data
 Clinical data
 Pharmacodynamic data
 Method described in
M23
 “Standard”
 Many breakpoints
Adapted from M100
breakpoints.
 Breakpoints based on:
 Literature search
 Clinical experience
 Does not follow M23
process
 “Guideline”
FASTIDIOUS ORGANISMS?
Require media supplemented with blood or
blood components
Possibly need an atmosphere other than
ambient air (eg, 5% CO2)
NOTE: CO2 lowers the pH of media:
- aminoglycoside MICs high
- quinolone MICs high
- clindamycin MICs high
- penicillin MICs low
- macrolide MIC low
ORGANISMS THAT USE “STANDARD” TEST
METHODS
Enterobacteriaceae methods (MIC / DD) Aeromonas / Pleisiomonas
Vibrio spp.
Streptococcus methods (MIC only)
Corynebacterium spp
E. rhusiopathiae
HACEK group
Lactobacillus
Leuconostoc
Listeria monocytogenes
Pediococcus
Streptococcus methods
Moraxella catarrhalis
Streptococcus methods (no CO2, if
requires CO2, don’t use DD)
Pasteurella spp.
ORGANISMS THAT USE “SPECIAL” TEST
METHODS
Abiotrophia / Granulicatella*
CAMHB with LHB + 0.0015 pyridoxal
Campylobacter
CAMHB with LHB or BMHA, but
microaerobic conditions / 42C
H. pylori**
Agar dilution with MHA and aged sheep
blood
*These should be sent to a reference laboratory / only performed by those with
experience with AST for these organisms
** Send to a reference laboratory, if testing indicated!
SUMMARY OF TEST CONDITIONS
WHEN DO I TEST THESE ORGANISMS?
 Only if:
 Infection that warrants antimicrobial therapy
 Susceptibility cannot be reliably predicted
 Many of the organisms in M45 are:
 Par t of the normal human microbiota:





Corynebacterium
Abiotrophia
Lactobacillus
Pediococcus
Leuconostoc
 Environmental :
 Bacillus spp.
 Have a controver sial need for AST:




Vibrio spp (from stool)
Aeromonas (from stool)
Campylobacter (from stool)
H. pylori
Don’t test routinely;
Don’t test if isolated from nonsterile /
superficial sites
Don’t test if not in pure culture
Test with ID / physician consultation
CASE 1
 69 year old woman with fever, nausea, vomiting and diarrhea.
 Diarrhea started 5 days ago, vomiting 4 days ago
 Stool Culture:
 No salmonella, shigella or campylobacter isolated.
 Aeromonas hydrophila
Is susceptibility testing warranted for the
A. hydrophila?
CASE 2
 9 year old with otocephalic mandibular syndrome undergoes
reconstructive surgery
 Venous congestion is observed after the surgery
 Leeches are applied + ciprofloxacin prophylaxis
 Presents 3 days later with purulent drainage from surgical site
 Wound cultures collected:
Anaerobe Culture
Prevotella
B. fragilis
Aerobe Culture
Morganella morganii
Aeromonas hydrophila
Is susceptibility testing warranted for the
A. hydrophila? And if so, how do we approach this?
M45 GUIDANCE
 “The need for susceptibility testing of Aeromonas and Vibrio
spp. isolates recovered from feces is controversial. Testing
should only be under taken in consultation with an infectious
disease-trained physician or other exper t clinicians who can
assist in determining if susceptibility testing is needed in the
management of a specific patient, and in the interpretation of
any results generated. Generally, testing of these organisms
should be limited to isolates recovered from normally sterile
sites, [or] serious wound infections …”
TESTING GUIDANCE FOR AEROMONAS
SPP.
1. Perform AST just like for Enterobacteriaceae (DD, MIC)
2. Interpretive criteria are based on those from M100 S20
3. QC strains are the same as in M100 S20
Note: Interpretive breakpoints for cefazolin, carbapenems for the Enterobacteriaceae
were change AFTER publication of M45 S2.
These will likely be harmonized with next M45 edition (2015).
TESTING GUIDANCE FOR AEROMONAS
SPP.
Note: Should test P. aeruginosa
ATCC 27853 for carbapenems to
achieve on-scale endpoints (M100)
FURTHER GUIDANCE FOR AEROMONAS
 “Aeromonas spp. are uniformly resistant to ampicillin;
however, susceptibility to amoxicillin -clavulanic acid and
cefazolin dif fers among species. Aeromonas strains may
possess multiple, distinct inducible beta -lactamases, and like
other genera with inducible beta -lactamases, resistance may
emerge during therapy.”
 i.e. consider more frequent re -testing of isolates if repeatedly
recovered for patient on treatment (like Enterobacter spp.)
AEROMONAS HYDROPHILA FROM CASE 2
Ampicillin-sulbactam
>32
R
Cefepime
≤0.5
S
4
R
≤1
S
Ciprofloxacin
Trimethoprim sulfamethoxazole
Patient treated with cefepime for 3 weeks
Two additional surgeries performed for tissue debridement
Discharged after 2 months in hospital
Doing well!
Note: Leeches were obtained from pharmacy, and from all ciprofloxacin –
resistant A. hydrophila were isolated… change to hospital policy on prophylaxis.
CASE 3
 28 YO male
 Diagnosis:
 HIV (on antiretroviral therapy, viral load = 590,000 copies; CD4 =585)
 Guillain Barre syndrome (lower extremity weakness, sensory
abnormalities, rapid loss of reflexes)
 Blood cultures:
 1 / 4 bottles grow a curved Gram negative rod
 Team discharges patient, with note
 “likely contaminant”
CASE 3 CONTINUED
 Microbiology laboratory calls ID team when it is realized
patient has been discharged
 ID contacts Neurology service and has the patient re -admitted
 Repeat blood cultures also grow Campylobacter
 Patient treated with IV meropenem, which is transitioned to
ciprofloxacin upon receipt of AST results
% S among Campylobacter blood stream isolates
Cipro
Amox-Clav Cefotax
SXT
Imipenem
30%
92%
75%
100%
79%
Fernandez-Cruz 2010 Medicine. 89:319
CAMPYLOBACTER IN BLOOD
 NEVER a contaminant
 Uncommon cause of BSI (0.24%)
 present with history of abdominal pain (35%); diarrhea (34%)
 Untreated, in HIV patients, is associated with mortality in
~50% of cases
 Even treated, in HIV+ patients is associated with high
mortality (33% vs 9.8%, p=0.04)
Patient’s isolate S to ciprofloxacin  de-escalate therapy & discharged
AST TESTING OF CAMPYLOBACTER
 You don’t need to test isolates at both temperatures  some
do not grow well @ 37 ○ C
CASE 4
 5 year old girl
 Diagnosis: Acute Otitis Media
 Ear swab submitted for culture:
 Many M. catarrhalis
Do we perform AST?
SANFORD GUIDE
 Initial empiric therapy: Amoxicillin Clavulanate, oral
cephalosporin, SXT
 Note: up to 90% of patients infected with M. catarrhalis will
have spontaneous resolution
 Might wait to treat / use analgesic if no fever & questionable
exam
M45: “Testing is not recommended routinely. May be useful for
management of patients with prolonged / severe infections”
Most M. catarrhalis are β-lactamase positive:
Resistant to amoxicillin, ampicillin, penicillin
Susceptible to amoxicillin-clavulanate (commonly prescribed)
UCLA APPROACH
 Perform β-lactamase testing using nitrocefin test
 Report β-lactamase positive (to remind physicians of common
resistance to amoxicillin, which is also commonly prescribed
empirically for otitis media)
WHAT WILL BE NEW WITH M45 A3?
 New organisms!




Aerococcus spp.  treated like viridans group streptococci
Lactococcus  treated like Staphylococcus
Micrococcus spp.  treated like Staphylococcus
Rothia mucilaginosa  treated like Staphylococcus
 HACEK group will be split in two:
 Aggregatibacter / Eikenella
 Cardiobacterium and Kingella spp.
CHALLENGES WITH TESTING HACEK
 Recommended media: CAMHB with LHB
 Some isolates require 48 h incubation
 Some isolates just won’t grow in this media!
 Recent study of 174 HACEK isolates
 59.5% failed to grow
 Aggregatibacter spp: 60 – 83% didn’t grow
 Eikenella is also problematic
 Studies at UCLA suggest Brucella broth with hemin and lysed
horse blood (5% v/v) might work better for these
 Further studies underway to confirm this…
AEROCOCCUS SPP.
 5 species of clinically relevant Aerococcus isolated from
humans:





Aerococcus
Aerococcus
Aerococcus
Aerococcus
Aerococcus
urinae
viridans
sanguinicola
urinaehominis
christensenii
very little data, not included in M45
 A. urinae most common
 isolated from the urine of elderly women
 Clinical significance here is uncertain when isolated from urine
(controversial!)
 Numerous reports of invasive infections caused by A. urinae and
other species
KEY THINGS TO KNOW ABOUT
AEROCOCCI
 A. urinae and Trimethoprim-sulfamethoxazole :
When tested in LHB, will test with low MICs
“Report as
When tested on sheep blood, have high MICs (literature)
Thymidine?
resistant to
• Low in human urine, serum ( but depends on diet)
SXT”
• High in sheep blood
• Low in horse blood
So m e s ug g est “ i n h erent re s i st anc e to s ul fona mides l i ke t h e e n te ro c oc c i”
A . u ri n a e g e n ome e n c o de s a h i g h -a f finit y fo l a te t ra n s po r t bi n di n g prote i n Fo l T
( but n ot ot h e r a e ro c o cc i )
•
•
•
•
•
 A. sanguinocola and A. viridans and FQ
 Resistance more common vs. A.urinae
 Related to mutation to gyrA or parC
Levofloxacin Susceptibility
100%
80%
A. sanguinocola
60%
A. urinae
 More information:
 Humphries and Hindler 2014 JCM 52:2177
40%
A. viridans
20%
0%
2
4
8