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

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.

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

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.

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.

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

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

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

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

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

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

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

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

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.

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.

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)

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

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).

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.

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.

Here’s an example of an antibiogram.