Analysis of Morphologically Similar Staphylococcus aureus Colonies for Assessment of Phenotypic and Genotypic Correlation

LETTER

In an effort to track and control the transmission of Staphylococcus aureus, patient isolates are saved for epidemiological studies (1–3). Study investigators often assume that colonies with the same morphology on the original culture plate represent the same clone. However, there is very limited literature to support this assumption. Many studies do not address how isolates are selected from a culture (3–5) and state that one colony is chosen as a representative sample (6). Currently, the Treating Parents to Reduce NICU Transmission of Staphylococcus aureus (TREAT PARENTS) trial (registration no. NCT02223520) is assessing the concordance of S. aureus strains colonizing parents and their neonates (7). To test the aforementioned assumption, multiple S. aureus colonies were saved from a single culture plate and tested to determine their genotypes and susceptibility profiles.

Once parents or guardians consented to the TREAT PARENTS trial, swab samples were collected from the nares, throat, groin, and perianal region to screen for the presence of S. aureus. The samples were collected with the Copan Eswab transport system (Copan, Murrieta, CA). For each sample, 10 μl was aliquoted onto a quarter of one S. aureus selective chromogenic agar (SASelect; Bio-Rad, Hercules, CA) plate and one 5% sheep blood agar (SBA; Remel, Lenexa, KS) plate and incubated at 37°C for 16 to 24 h. At the same time, 100 μl of each sample was aliquoted into tryptic soy broth containing 6.5% sodium chloride (Bio-Rad, Hercules, CA) and incubated at 37°C for 16 to 24 h. After incubation, 10 μl of each broth was plated on SASelect medium and 5% SBA, streaked for isolation, and incubated at 37°C for 16 to 24 h. The colonies had to have the same color, size, consistency, and entirety to be called identical. Two medical technologists independently read the plates, and there were no inconsistencies between them in the determination of identical versus different morphologies on the basis of the criteria described. For each S. aureus morphology on every positive plate, five separate colonies were individually subcultured on 5% SBA and then frozen. These isolates were analyzed by pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility testing (AST) with standard antistaphylococcal agents.

PFGE was performed in accordance with standard methods by using SmaI as the restriction enzyme (8). Restriction digestion patterns were analyzed with the Fingerprinting 2 software (Bio-Rad). PFGE results were interpreted by using modified Tenover criteria, and isolates were considered related if their patterns had three or fewer band differences (9).

Isolate susceptibility was determined with the BD Phoenix 100 instrument (BD Diagnostics Inc., Sparks, MD) with Phoenix PMIC/ID-105 panels in accordance with the manufacturer's instructions.

A combination of 14 neonates and adult participants had positive S. aureus cultures that yielded 205 isolates (5 isolates from 41 morphologies observed). Of the isolates tested, 99.5% (204 of 205) had PFGE patterns that were identical to those of the other isolates of a particular morphology. One isolate in one group had a one-band difference from the other four isolates, and these are considered epidemiologically related.

AST indicated that 95.1% (195 of 205) of the isolates tested had the same profile of susceptibility to all 16 antibiotics tested (data not shown). All isolates showed categorical agreement (CA; agreement of susceptible/intermediate/resistant results) for all antibiotics except erythromycin (90% CA). When it could be determined, essential agreement (EA; MICs that match exactly or are within ±1 2-fold dilution) ranged from 90 to 100%. The 95% confidence interval for the proportion of morphologies with exact agreement is 0.91 to 1 when using an exact binomial confidence interval (10).

Because of its high colonization frequency and its role in health care-associated infections (1, 6, 8, 11), S. aureus will remain a commonly archived pathogen for epidemiological studies. A lack of literature specifying S. aureus colony selection seems to suggest that a fundamental assumption in these studies is that a single colony of the same morphotype is representative of the same strain. The results of our study show that this assumption is appropriate for morphologically similar colonies from a single culture. An interesting, but not fully unexpected, result was differing susceptibility profiles for 5% of the isolates that had identical banding patterns.