1. Supplemental testing methods
Screening agar
Agar contains known conc. of antibiotic
Growth on agar indicates resistance
Oxacillin screening agar: 6 g/ml oxacillin
Screening of staphylococci
Vancomycin screening agar: 6 g/ml vanco
Screening of enterococci and staphylococci
Certain resistance mechanisms may be difficult to detect

2. This is an example of oxacillin agar
This is an example of oxacillin agar. It is used to verify results from automated testing for methicillin resistance (MRSA) of Staph.aureus. If the plate and the automated results don’t match the specimen must be retested and no results can be given out. There is another plate used to verify vancomycin resistance for enterococcus species.

3. Supplemental testing methods
Predictor drugs
Staphylococci R to Oxacillin =
R to penicillins, cephalosporins, and imipenem
High level gentimicin R in enterococci =
R to all currently available aminoglycosides
Ampicillin R in enterococci =
R to all penicillin derivatives and imipenem

4. Direct detection of resistance mechanisms
Beta-lactamase (phenotypic)
Chromogenic substrate incorporated into disk
- color change in presence of enzyme
Usefulness is limited:
Penicillin R in Neisseria gonorrhea
Ampicillin R in Haemophilus influenzae
Penicillin R in anaerobes
This refers to one of the spot tests that we looked at. The disk looks similar to an antibiotic testing disk but it has the chromogenic substrate impregnated into the disk and the bacterial colony is smeared across it, if positive there is a dark reddish color change.

5. Direct detection of resistance mechanisms
Extended spectrum beta-lactamase
Mutations in plasmid-encoded beta-lactamases
- hydrolyze extended spectrum cephalosporins
and aztreonam
- more than 100 types have been identified
- isolates are often resistant to other classes
Interpretive criteria available for:
- E. coli, K. pneumoniae, K. oxytoca, P. mirabilis
Patients found to have any of the resistance mechanisms for MRSA, VRE, ESBL or other multi-drug resistant strains must be place in contact isolation immediately to help prevent the spread of resistance.

6. Direct detection of resistance mechanisms
Extended spectrum beta-lactamase
Screen with aztreonam or cefpodoxime
R = requires confirmatory testing
Confirmatory testing:
Ceftazidime v. ceftaz + clavulanic acid
Cefotaxime v. cefotax + clavulanic acid
KB: >/= 5 mm increase w/ BLI
MIC: >/= 3-fold decr in MIC w/ BLI

7. With clavulanic acid Without clavulanic acid
                                                                                    
Note the large difference in zone size between the two disks, the zone size has an increase of 5 mm or greater when clavulanic acid is added. This is what we would see if the bacteria was ESBL.
Without clavulanic
acid

8. Direct detection of resistance mechanisms
Oxacillin R due to PBP2a (phenotypic)
Latex agglutination test to detect altered
PBP in staphylococci
Presence confers resistance to Oxacillin
Depends on expression of protein

9. Direct detection of resistance mechanisms
Oxacillin R due to PBP2a (genotypic)
PCR to detect mecA gene in staphylococci
Positive not dependent on expression, a positive
result simply indicates presence

10. Direct detection of resistance mechanisms
Inducible clindamycin resistance (D test)
Resistance to macrolides can occur through:
efflux (msrA)
ribosome alteration (erm)
Erythro R msrA or inducible erm
Clinda S
Erythro R constitutive erm
Clinda R

11. L: Erythro, R: Clinda No resistance Efflux Constitutive erm
Inducible erm

12. Laboratory strategies for testing
Goals of effective strategies include:
Relevance
Accuracy
Communication

13. Laboratory strategies for testing
Criteria used for assessing relevance:
Clinical significance of isolate
Predictability of susceptibility against drugs
of choice
Availability of reliable standardized methods
Selection of appropriate agents

14. Laboratory strategies for testing
Clinical significance
Abundance in direct smear
Ability to cause disease in that body site
Colonizer or pathogen?
Body site of isolation
Again, the microbiologist must know the source of the specimen to reliably report antibiotic susceptibilities. Some drugs are not affective for some body sites.

15. Laboratory strategies for testing
Predictability of susceptibility
Testing not required when susceptibility is
predictable
Pen S in beta-hemolytic streptococci
Ceph S in GC
Clinical requirements can result in exceptions
For example: physicians can treat strep throat without having done a susceptibility panel because Group A strep is considered universally susceptible to the penicillin family.

16. Laboratory strategies for testing
Availability of standardized methods
Testing cannot be performed if standardized
method does not exist
Method and interpretive guidelines required –
CLSI guide
Info available for most pathogenic bacteria
Fungi, Nocardia, AFB (Mycobacterium)

17. Laboratory strategies for testing
Selection of agents
Previously discussed criteria:
Organism ID or group
Acquired resistance patterns
Testing method used
Site of infection
Formulary

18. Laboratory strategies for testing
Communication
Prompt and thorough review of results
Prompt resolution of unusual results
Augment susceptibility reports with messages
that help clarify and explain potential
therapeutic problems not necessarily
evident by data alone
The microbiology department works very closely with the infectious disease doctors and the pharmacy. There is constant (daily) communication about the prevalence and resistance patterns currently being seen at the hospital (or other facility).

19. Antibiograms:
The hospital antibiogram is a periodic summary
of antimicrobial susceptibilities of local bacterial
isolates submitted to the hospital's clinical
microbiology laboratory. Antibiograms are often
used by clinicians to assess local susceptibility
rates, as an aid in selecting empiric antibiotic
therapy, and in monitoring resistance trends over
time within an institution.

20. Antibiograms:
Antibiograms can also used to compare
susceptibility rates across institutions and
track resistance trends. These trends can then
be used to develop the appropriate formulary
for the hospital.

21. Here’s an example of an antibiogram.