The PCR/ESI-MS method enables identification and characterization of Staphylococcus isolates at several levels. At the genus level, primer pair BCT2249 (Table ) hybridizes with a region of the tufB gene (encoding EF-Tu) and amplifies DNA isolated from all known species within the Staphylococcus genus but not from other genera of bacteria (Fig. ). The base composition of the amplicon distinguishes S. aureus from all other species of staphylococci. Representative spectra from S. aureus isolates and several coagulase-negative Staphylococcus species are shown in Fig. . In ESI-MS analysis of PCR amplicons, the forward and reverse strands of the DNA are separated, producing two distinct peaks in the mass spectrum. Almost all unique base compositions are distinguishable by MS, even those separated by single nucleotide substitutions. Thus, this primer pair is capable of detecting all species of staphylococci for which sequence data exist based on bioinformatics analyses of sequence databases and unambiguously distinguishes S. aureus from other species. Analyses of amplicons from more than 1,000 confirmed S. aureus isolates (317 in this study) to date have shown identical base compositions (43 A, 28 G, 19 C, and 35 T residues). The ability of this primer pair to unambiguously identify strains of non-S. aureus staphylococci to the species level has not yet been determined experimentally; however, in specificity testing, 29 coagulase-negative Staphylococcus isolates were correctly categorized in the coagulase-negative Staphylococcus grouping and showed no cross-reactions suggesting S. aureus identification patterns.
FIG. 2. Detection and identification of multiple Staphylococcus spp. by base composition analysis with a genus-level primer pair targeting the tufB gene. (A) Alignment showing the target and amplified region for the Staphylococcus-specific tufB primer pair BCT2249. (more ...)
The nuc primer pair was shown to have 100% sensitivity and specificity for all isolates in this study. Additional primer pairs were used to determine the presence or absence of other genes important in the characterization of S. aureus.
Using the primers shown in Table , we employed PCR/ESI-MS to characterize 30 S. aureus
isolates from the CDC that had been genotyped previously by PFGE, MLST (23 isolates), and multilocus PCR/ESI-MS and characterized by real-time PCR (17
). These isolates represent diverse genotypes corresponding to each of the major clonal complexes. The results of PCR/ESI-MS characterization are shown in Table . As expected, the tufB
- and nuc
-targeting primer pairs generated amplicons consistent with S. aureus
for all isolates except CDC0031, a blind unknown isolate that was nuc
negative and gave a PCR/ESI-MS tufB
signature inconsistent with S. aureus
but consistent with its correct identification, S. schleiferi
PCR/ESI-MS signatures of CDC panel of S. aureus isolatesa
All isolates were either positive for both mecA and mecR1 genes or negative for both. PCR/ESI-MS results were consistent with previous characterization at the CDC by broth microdilution and mecA PCR. The presence or absence of ermA and ermC as detected by PCR/ESI-MS was concordant with in vitro resistance to erythromycin and clindamycin as determined at the CDC. PCR/ESI-MS detection of mupA in CDC006 was consistent with broth microdilution and PCR results; all other isolates were phenotypically mupirocin susceptible and negative for mupA by PCR/ESI-MS. The lukE and lukD genes of this sample set were not characterized independently, so the PCR/ESI-MS results for these genes could not be corroborated.
There was no correlation between the presence or absence of PVL genes or any of the antibiotic resistance genes and PCR/ESI-MS types or clonal complexes, suggesting that the acquisition of these genes happened on a more recent time scale than the evolution of the housekeeping genes used in MLST analysis. However, the presence or absence of lukE-lukD was highly correlated with both PCR/ESI-MS types and clonal complexes (Table ). All isolates belonging to clonal complex 8 (CC8) and CC5 were lukE-lukD positive, and all isolates of CC30, CC45, and CC59 were lukE-lukD negative, suggesting a stable relationship between the housekeeping genes used in MLST and the lukE-lukD operon.
Two hundred forty isolates of S. aureus obtained from JHH were characterized by PCR/ESI-MS. These isolates, previously genotyped by PFGE at JHH, were grouped by PCR/ESI-MS into 23 unique genotypes (Fig. ), primarily from six major clonal complexes, with the largest numbers of isolates from CC8 (82 of 240; 34%) and CC5 (70 of 240; 29%). Upon analysis of these same isolates for the presence of the lukE-lukD operon, PVL genes, mecA, ermA, ermC, and mupA, the number of unique PCR/ESI-MS genotypes increased from 23 to 59. A visualization of the occurrence of these genes in conjunction with the PCR/ESI-MS genotype and clonal complex assignment is shown in a radial plot in Fig. . The presence of the lukE-lukD operon, PVL genes, the mec gene, and either ermA or ermC is depicted by the stippled segments, and the absence of these genes is shown using the background color for the clonal complex. Thirty-four of the 240 isolates (14%) were PVL positive, and the majority of these (29 isolates) were of PCR/ESI-MS type 1 (corresponding to CC8, which contains USA300 isolates). All but three of these were also mecA positive. Overall, 95 of the 240 isolates (40%) were mecA positive, 69 (29%) were ermA positive, 44 (18%) were ermC positive, and only 4 (<2%) were mupA positive. Only three of the erm-positive isolates were both ermA and ermC positive. The distribution of the antibiotic resistance genes was less correlated with PCR/ESI-MS types and clonal complexes than was that of the leukotoxin operons (see the two outer rings of Fig. ). The results obtained from PCR/ESI-MS compared favorably with the results of phenotypic methods to detect resistance to oxacillin, erythromycin, and mupirocin and with PCR and PFGE typing data (Table ). Sensitivities and specificities ranged from 90 to 100%, except for erythromycin resistance. The presence of either ermA or ermC was highly predictive of erythromycin resistance detected by culture (specificity, 98%). The absence of an ermA or ermC gene did not ensure erythromycin susceptibility (70%), however, implying resistance by other mechanisms, such as msr(A).
FIG. 3. PCR/ESI-MS analysis of S. aureus isolates obtained from geographically distinct hospital collections. (A) Radial plot of results from genotyping and characterization of 240 S. aureus isolates from JHH. The area of each wedge corresponds to the number (more ...)
Sensitivity and specificity of PCR/ESI-MS with respect to phenotypes for Johns Hopkins isolates
S. aureus isolates obtained from the University of Arizona were also characterized by PCR/ESI-MS, and the data compared to results from conventional culture methods for species identification and antimicrobial resistance as shown in Fig. . PCR/ESI-MS spectra from the tufB and nuc gene-targeting primers showed the characteristic spectral signatures for S. aureus, with no variation in base composition from the canonical signatures. The 47 S. aureus isolates comprised six PCR/ESI-MS types and three clonal complexes, predominantly CC5 and CC8. All but one of the isolates contained the lukE-lukD operon. Isolates belonging to CC5 (e.g., USA100) were PVL negative, and all but one of the isolates belonging to CC8 (e.g., USA300 and USA500) were PVL positive. The mecA gene was present in 42 of 47 isolates (89%), the ermA gene was present in 36 (77%), and the ermC gene was present in 4 isolates (9%); 3 of the 4 were both ermA and ermC positive. The presence of the mecA gene correlated with in vitro cefoxitin resistance for all 47 isolates. Of the 37 isolates that contained either an ermA or ermC gene, all but 2 were erythromycin resistant; however, as was observed with the CDC and JHH isolates, the absence of ermA or ermC did not ensure erythromycin susceptibility. Of the 10 ermA- and ermC-negative isolates, only 2 were susceptible to erythromycin. Of the 47 isolates, only 1 had the mupA gene; this isolate was confirmed to be mupirocin resistant in vitro. The other isolates were mupirocin susceptible.