Abstract
Colony morphology on kanamycin esculin azide agar was investigated as a means of selecting different species and strains of enterococci from clinical specimens. Four representative colonies of each morphotype were indistinguishable by pulsed-field gel electrophoresis, biotype, and antibiogram analysis. The optimum time for identification of different colony morphotypes was 72 h.
Over the past 2 decades, enterococci have become important nosocomial pathogens, probably due to inherent resistance to antibiotics (such as cephalosporins), ability to adhere to indwelling medical devices, and ability to survive adverse environmental conditions. Enterococci normally inhabit the human gastrointestinal tract and are also found in the mouth and vagina and on the skin of healthy individuals (12). Although multiple strains of enterococci can be found in human clinical specimens (1, 5, 20), the extent of colonization with different strains is unknown. Epidemiological studies have been hampered by the lack of a simple, reliable method of distinguishing individual strains within a sample. In addition, the optimum period of incubation required to recover enterococci from clinical samples is not known. This study aimed to determine whether variations in colonial morphology (13) could be used to select representative examples of each strain from clinical samples containing mixtures of enterococci and also to identify the optimum incubation time for identification of colony variants.
The local ethics committee approved the study protocol, and 10 inpatients on a hematology ward consented to participate. Fecal samples were processed by suspending a 0.5-g portion in 4.5 ml of sterile saline (0.85%, wt/vol). Aliquots (100 μl) of decimal dilutions of the resulting suspension were spread-plated onto kanamycin esculin azide agar (KAAA; Oxoid, Basingstoke, United Kingdom) and incubated aerobically at 37°C for 3 days and then at room temperature for a further 5 days. Plates were examined daily by eye, and the number of different colony types was recorded. Four single colonies representative of each colony type that exhibited a brown to black halo on KAAA were screened initially by Gram staining and a catalase test. Catalase-negative, gram-positive cocci were subcultured onto horse blood agar (HBA; Oxoid) for further identification. Stock cultures were frozen at −70°C in phosphate-buffered saline with 40% glycerol. The 4 CFU of each distinct colony type in a sample were identified to species level by conventional methods including carbohydrate fermentation, motility, colony pigmentation, pyruvate fermentation, and tellurite tolerance (7, 11, 17). Pyruvate fermentation and tellurite tolerance were determined according to methods described elsewhere (10, 14). Biochemical identification was confirmed using PCR amplification of genus-specific (tuf gene) and species-specific (Enterococcus faecalis, E. faecium, E. casseliflavus, and E. gallinarum) oligonucleotide primers as described previously (3, 9, 15, 16). Genomic DNA for pulsed-field gel electrophoresis (PFGE) was prepared and digested with SmaI as described previously (6, 8), and fingerprints were compared by using GelCompar (version 4.0; Applied Maths, Kortrijk, Belgium).
A breakpoint agar dilution method was used for susceptibility testing according to guidelines M7A-4 and M100-S7 of the National Committee for Clinical Laboratory Standards (18). Pharmaceutical-grade vancomycin (Dumex-Alpharma, Copenhagen, Denmark), teicoplanin (Marion Merrel, Uxbridge, United Kingdom), linezolid (Pharmacia and Upjohn Company, Kalamazoo, Mich.), and rifampin (CIBA Laboratories, Camberley, United Kingdom) were used. Ampicillin, erythromycin, gentamicin, tetracycline, and chloramphenicol were obtained from Sigma-Aldrich Co., Ltd. (Poole, United Kingdom).
Ten fecal samples from 10 patients were examined, each yielding ≥2 different colony types (Table 1). Variations in colony morphology were clearly apparent on KAAA and also on HBA (Fig. 1). Visual recognition of different colony types was possible in some cases on day 1, increased on day 2, and peaked on day 3, with no further new morphotypes appearing thereafter. In 60% (6 of 10) of the samples, new colony types continued to appear after 48 h of incubation. This was also observed in samples that grew vancomycin-resistant enterococci (VRE).
TABLE 1.
Molecular typing, selected biochemical results, and antibiotic susceptibility patterns of 100 enterococcal colonies representing 25 morphotypes isolated from 10 fecal samples
| Fecal sample no. | Morphotypea | PFGE type | Resultb of the indicated biochemicalc or antibiotic susceptibilityd test
|
Species | |||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Pig | Tell | Pyr | Ara | Sor | Xyle | Mgp | AMP | CHL | TEC | VAN | ERY | RIF | GEN | TET | LZD | ||||
| 1 | 1a | A | − | − | − | + | − | − | − | R | R | S | R | R | R | S | R | S | E. faecium |
| 1b | B | − | + | + | − | + | − | − | S | S | S | S | S | S | S | S | S | E. faecalis | |
| 1c | C | − | − | − | + | − | − | − | R | R | S | R | R | S | R | S | S | E. faecium | |
| 2 | 2a | D | − | − | − | + | − | − | − | R | S | S | S | S | R | S | S | S | E. faecium |
| 2b | E | − | + | + | − | + | − | − | S | S | S | S | S | R | S | S | S | E. faecalis | |
| 3 | 3a | F | − | − | − | + | − | − | − | S | S | S | S | R | R | S | R | S | E. faecium |
| 3b | G | − | + | + | + | + | + | + | S | S | S | I | S | S | S | S | S | E. gallinarum | |
| 3c | H | + | + | + | + | − | − | + | S | R | S | I | R | S | S | S | S | E. casseliflavus | |
| 4 | 4a | I | − | − | − | + | − | − | − | R | S | S | S | R | R | R | R | S | E. faecium |
| 4b | J | − | − | − | + | − | − | − | S | S | S | S | S | S | S | S | S | E. faecium | |
| 4c | K | − | − | − | + | + | − | − | S | S | S | S | R | S | R | R | S | E. raffinosus | |
| 5 | 5a | L | − | − | − | + | − | − | − | S | S | S | S | S | S | S | S | S | E. faecium |
| 5b | M | + | − | − | + | − | − | + | S | S | S | I | S | S | S | S | S | E. casseliflavus | |
| 6 | 6a | N | + | − | − | + | − | − | + | S | S | S | I | S | S | S | S | S | E. casseliflavus |
| 6b | O | − | + | + | − | + | − | − | S | S | S | S | R | R | S | S | S | E. faecalis | |
| 6c | P | − | + | + | + | + | − | − | S | S | S | S | R | S | S | S | S | E. faecalis | |
| 7 | 7a | Q | − | − | − | + | − | − | − | R | S | S | S | S | S | S | S | S | E. faecium |
| 7b | R | − | + | + | − | + | − | − | S | R | S | S | R | R | R | R | S | E. faecalis | |
| 8 | 8a | S | − | − | − | + | − | − | − | S | S | S | S | R | R | S | R | S | E. faecium |
| 8b | T | − | − | − | + | + | + | + | S | S | S | I | S | S | S | S | S | E. gallinarum | |
| 9 | 9a | U | − | + | + | + | + | + | + | S | S | S | I | S | S | S | S | S | E. gallinarum |
| 9b | V | − | − | − | + | − | − | − | R | S | S | S | S | S | S | S | S | E. faecium | |
| 10 | 10a | W | − | − | − | + | − | − | − | R | R | S | S | R | R | R | R | S | E. faecium |
| 10b | X | − | − | − | + | + | + | + | R | R | S | I | R | R | R | R | S | E. gallinarum | |
| 10c | Y | + | − | − | + | − | − | + | S | S | S | R | R | S | S | R | S | E. casseliflavus | |
Four colonies of each morphotype were tested.
+, positive; −, negative; S, sensitive; R, resistant; I, intermediate.
Pig, pigment; Tell, tellurite; Pyr, pyruvate; Ara, arabinose; Sor, sorbitol; Mgp, methyl-α-d-glucopyranoside; Xyl, xylose.
AMP, ampicillin; CHL, chloramphenicol; TEC, teicoplanin; VAN, vancomycin; ERY, erythromycin; RIF, rifampin; GEN, gentamicin; TET, tetracycline; LZD, linezolid.
Positive within 2 h of incubation.
FIG. 1.
Variations in enterococcal colony morphology on HBA plates at 24 (A), 48 (B), and 72 (C) h of incubation in air at 37°C. Arrow indicates appearance of a new colony. Boxes highlight the area on the plate in which the new colony is seen after 72 h of incubation.
In all, 25 morphotypes (a total of 100 CFU) were studied. In every case, the four representative colonies of each morphotype were shown to be indistinguishable by antibiogram, biochemical profile, and PFGE. Some biochemical tests, in particular arabinose, mannitol, and sorbitol fermentation, produced equivocal results, but variations were consistent among identical morphotypes. Identical morphotypes were, therefore, always found to be the same strain. Distinct morphotypes in a single sample were not necessarily found to be different strains; on three occasions, they were found to be indistinguishable by PFGE, antibiogram, and biochemical profile, highlighting the presence of colonial variation among some enterococcal strains.
Simultaneous fecal colonization with ≥1 species of Enterococcus, such as E. faecalis and either E. gallinarum or E. casseliflavus, has been reported (1, 5). The extent of mixed colonization with different enterococcal species and strains is unknown but is of importance to clinical epidemiological investigation and research. A range of incubation times has been used in studies of the epidemiology of VRE infection (usually 48 to 72 h), but neither current guidelines for the isolation of enterococci (2) nor the Manual of Clinical Microbiology (12) specifies the optimum incubation period. A previous study in which selective plates were incubated for as long as 72 h found that only 85% of VRE were recovered after 48 h of incubation (19). In the present study, 72 h of incubation was the optimum time for detection of distinct enterococcal colony types, especially in samples containing E. gallinarum and E. raffinosus and in the 60% of samples that were positive for VRE. This finding is important, not only for epidemiological research, but also for the detection of VRE in the diagnostic laboratory. Forty-eight hours of incubation would appear to be insufficient time for the recovery of VRE from clinical samples, but increasing incubation times would delay reporting for a further 24 h.
Colony morphology was found to be a reliable method of screening for different enterococcal strains in the clinical samples tested. In every case, the four representative colonies of each colony type were shown to be indistinguishable by antibiogram, biochemical profile, and PFGE. In practical terms, this means that testing of a single colony of each different morphotype in a mixed culture could reliably recover different species and strains, avoiding the need to sample and test multiple colonies. On three occasions, samples contained colonies with different morphotypes that subsequently turned out to be the same strain by PFGE. This highlights the importance of molecular typing to confirm results obtained by colony morphotyping. Morphotyping was investigated using KAAA and may not be applicable to other media. Although HBA appears to yield similar results (data not shown), it is insufficiently selective to use for the initial isolation of putative enterococcal isolates from clinical material likely to contain mixed bacterial populations, such as fecal specimens.
In this study, biochemical profiles and antibiograms were unreliable methods of screening for different strains, since examples of genotypically unrelated isolates of E. faecalis, E. faecium, and E. casseliflavus that shared the same biochemical reactions and antibiotic susceptibilities were found. Furthermore, the problem of equivocal results with some biochemical tests, noted in a previous report (4), serves to highlight the limited applicability of this technique.
In conclusion, differences in colony morphology on KAAA after 72 h of incubation at 37°C appear to be reliable for the identification of different enterococcal strains in a mixed culture. It would be interesting to undertake work on clinical samples from other centers in order to establish the reproducibility of this finding in different geographical locations. In addition, we recommend that laboratories which screen fecal samples for VRE should investigate the sensitivity of their recovery method after 48 h of incubation compared with 72 h.
Acknowledgments
This work was supported by a grant from Wyeth Ayerst Research.
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