Contents

BACTERIOLOGY

Susceptibility Testing – Susceptibility testing allows us to predict the likelihood of successfully treating an infection with a particular antimicrobial agent, using in vitro methods. However, clinical outcome may depend on a variety of factors, such as host immunity or surgical treatment, which are not reflected in laboratory tests. All methods of susceptibility testing are based on diffusion or dilution.

A. Automated Susceptibility Testing

Automated antimicrobial susceptibility testing is performed using the Microscan system, which is based on broth microdilution. This system allows the laboratory to rapidly perform identification and susceptibility testing on most common pathogens (e.g. Enterobacteriaceae, Staphylococci, Enterococci, and Pseudomonas aeruginosa). The antibiotics tested vary based upon the Microscan panel used and the antibiotics that are currently on The Nebraska Medical Center hospital formulary. However, the microbiology laboratory reports antibiotics (that are on formulary) from most antibiotic classes that are appropriate for the specific organism tested. For instance, if the laboratory recovers an Escherichia coli isolate from urine, the following results are reported: penicillin, penicillin/b-lactamase inhibitor combination(s), first generation cephalosporin, a cephamycin, multiple expanded-spectrum cephalosporins (including cefepime), a carbapenem, one or two fluoroquinolones, at least two aminoglycosides, trimethoprim-sulfamethoxazole, and nitrofurantoin.

The results obtained from the Microscan system are based on the minimum inhibitory concentration (MIC). The MIC is defined as the lowest concentration of antibiotic that completely inhibits growth of the specific organism being tested. For instance, in figure 1, the organism being tested grew in wells containing 0.5, 1.0, 2.0 and 4.0 mg/ml of antibiotic. The lowest concentration of antibiotic (MIC) that completely inhibits growth was 8.0 mg/ml.

Figure 1

The MIC is then interpreted (S=susceptible, I=Intermediate, or R=resistant) using CLSI (formerly NCCLS) standards, which are published each year in January. For example, the MIC interpretive standards for ampicillin against E. coli are <8mg/ml=susceptible, 16 mg/ml=intermediate, and >16 mg/ml=resistant. These interpretive standards are based on many studies, including clinical, pharmacokinetic/pharmacodynamic, and microbiological studies.

B. Disk Diffusion

The Nebraska Medical Center Microbiology laboratory does not routinely perform disk diffusion (commonly referred to as Kirby-Bauer testing) antimicrobial susceptibility testing except for Pseudomonas aeruginosa isolates obtained from cystic fibrosis (CF) patients. The Microscan system is not FDA approved to perform susceptibility testing on CF P. aeruginosa isolates due to the large amount of extracellular material typically produced by these isolates. Disk diffusion allows for measurement of the zone of growth inhibition (Figure 2).

Measure the diameter

of the zone of

inhibition

Figure 2

The CLSI provides interpretive standards for reporting an organism as S, I, or R based on the zone of inhibition. The main difference between disk diffusion testing and MIC testing is that disk diffusion gives clinicians qualitative results, whereas MIC testing gives quantitative results. Knowing the MIC can help clinicians incorporate pharmacodynamic/pharmacokinetic principles into the design of the treatment regimen. For instance, if we want to use ceftriaxone, a time-dependent/concentration-independent antibiotic, to treat meningitis due to Streptococcus pneumoniae, we need to achieve a concentration in the cerebrospinal fluid (CSF) of approximately four times the MIC for about 40% of the dosing interval. Therefore, if the MIC of the S. pneumoniae isolate to ceftriaxone is 0.25 mg/ml, we want a concentration of at least 1 µg/ml in the CSF for 40% of the dosing interval.

C. E-test

The CLSI only interprets MIC results for common pathogens (Enterobacteriaceae, Pseudomonas aeruginosa, Acinetobacter spp., Stenotrophomonas maltophilia, Burkholderia cepacia, Staphylococcus sp., Enterococcus sp., Haemophilus sp., Neisseria gonorrhoeae, Streptococcus pneumoniae, Streptococcus sp., and Vibrio cholerae). However, in many cases, bacterial species are isolated that do not have CLSI standards (i.e. Corynebacterium sp. or certain gram-negative glucose non-fermenting organisms, such as Flavobacterium sp. or Alcaligenes sp.) that need antibacterial susceptibility testing. Many of these bacterial species are also not FDA approved to use with the Microscan system or do not grow well under these conditions. Therefore, the E-test methodology is typically used under these conditions. The E-test is an agar based method that uses antibiotic impregnated strips that contain decreasing concentrations of antibiotic on the strip. The MIC is read at the point where the bacterial ellipse passes the E-test strip (Figure 3). If susceptibility testing is needed for an organism that does not have CLSI standards, please call the microbiology laboratory to discuss the antibiotic regimen to be tested.

Figure 3

Interpretation of these MIC results is based upon clinical, pharmacokinetic and pharmacodynamic experience (i.e. the MIC =2 and it is known that one can achieve 16mg/ml trough levels of a particular antibiotic at a particular site) as well as published reports of clinical success/failure. If susceptibility testing is performed in these situations, the following statement will be added to the final report “National Standards for antimicrobial susceptibility testing for this isolate have not been established and results may not predict clinical response. The Infectious Disease Service may be contacted for specific treatment and recommendations.”

D. Special susceptibility testing issues

Extended-spectrum ß -lactamases (ESBLs)

ESBLs are ß-lactamases that are capable of hydrolyzing expanded-spectrum cephalosporins (ceftriaxone, cefotaxime, and ceftazidime) as well as cefepime and aztreonam. ESBLs can be isolated from many different Enterobacteriaceae species, but are most commonly isolated from Klebsiella pneumonia, K. oxytoca, E. coli, or Proteus mirabilis. Using in vitro testing systems such as Microscan, isolates that carry ESBLs can initially be intermediate or resistant to one or all of the expanded-spectrum cephalosporins, cefepime or aztreonam. This is due to the fact that there are many different ESBLs with different substrate specificities. If a particular Klebsiella pneumonia, K. oxytoca, E. coli, or Proteus mirabilis isolate is resistant or intermediate to any of the expanded-spectrum cephalosporins, cefepime or aztreonam, the following statement will be included in the preliminary report: “Suspected Extended Spectrum Beta-Lactamase (ESBL), confirmation to follow.” An ESBL test will then be performed which is based upon the fact that clavulanic acid will inhibit ESBLs (Figure 4) The test is performed using disk diffusion disks that contain either cefotaxime or ceftazidime and corresponding disks containing cefotaxime/clavulanic acid or

Figure 4

ceftazidime/clavulanic acid. If the disk containing cefotaxime (or ceftazidime)/clavulanic acid is 5mm in diameter greater than either cefotaxime (or ceftazidime) alone, it is considered a positive test. Note that in figure 4, the zone of inhibition surrounding ceftazidime/clavulanate (22 mm) and cefotaxime/clavulanate (26 mm) is at least 5 mm greater than the zone of inhibition surrounding ceftazidime (13 mm) or cefotaxime (21 mm) alone, demonstrating that this isolate is producing an ESBL. If the isolate is positive for an ESBL, the following statement is added to the final report “Positive for Extended-Spectrum Beta-lactamase (ESBL). ESBL-producing strains may be clinically resistant to all cephalosporins, cefepime, and aztreonam.” In addition, all β-lactams excluding the cephamycins, piperacillin/tazobactam and the carbapenems, are changed to resistant (if they were initially reported as susceptible or intermediate).

Inducible clindamycin-resistance in Staphylococcus aureus

Erythromycin resistance within staphylococci is typically mediated through two distinct mechanisms. The first mechanism entails protection of the ribosome from erythromycin (and clindamycin) through methylation (also referred to as MLS_B resistance). This mechanism may be constitutive (conferring resistance to both erythromycin and clindamycin) or inducible (conferring resistance only to erythromycin). The second resistance mechanism is conferred through efflux of erythromycin out of the cell through specific pumps (encoded by the msrA gene). Staphylococcal isolates carrying the MsrA efflux pump are resistant only to erythromycin and not clindamycin. If a S. aureus isolate is resistant to erythromycin and susceptible to clindamycin and the clinician would like to use clindamycin for therapy, a D-test should be performed (the laboratory needs to be called for this test to be performed). Published clinical reports have demonstrated that S. aureus isolates carrying an inducible MLS_B resistance gene should be considered resistant to clindamycin even if the in vitro result considers the isolate susceptible to clindamycin. To detect whether the isolate has an inducible MLS_B gene or msrA, a D-test is used. The D-test is set up by placing an erythromycin disk 15 mm away from a clindamycin disk. Organisms that demonstrate flattening of the clindamycin zone adjacent to the erythromycin disk are considered positive for inducible MLS_B resistance and clindamycin should not be used during therapy (Figure 5). The following statement will be added to the final report if the isolate carries an MLS_B resistance gene “This isolate demonstrates inducible clindamycin resistance. Use of clindamycin may result in clinical failure.”

Figure 5