|Home | About | Journals | Submit | Contact Us | Français|
We have developed a modified cyclodextrin solid (MCS) medium using the selective antibiotic cefdinir. MCS medium exhibited higher sensitivity (95.6%; any culture-positive sample as reference) and greater inhibition of nasopharyngeal flora than did Bordet-Gengou agar (65.2%, P = 0.009) or cyclodextrin solid medium (39.1%, P < 0.001).
Pertussis is an acute respiratory infection caused by Bordetella pertussis. Since the introduction of the acellular pertussis vaccine, the number of reported pertussis cases has drastically decreased. However, occasional outbreaks have still been reported (2-4, 7), and adult pertussis has emerged (2, 3).
Culturing B. pertussis from clinical specimens is the “gold standard” for diagnosis of pertussis, although this remains an insensitive method (12, 13). In addition, isolation of clinical strains is required for epidemiological analysis (including phenotypic and genotypic characterization). It is also required for the determination of the appropriate vaccine strain and the antimicrobial susceptibility of isolates in order to control the spread of pertussis (12, 13). Bordet-Gengou agar (BG agar) was the standard culture medium for isolation of B. pertussis but has the problem of low selectivity. More-selective media, such as BG agar with 40 μg/ml of cephalexin (cefalexin) (BG agar) and Regan-Lowe agar with 40 μg/ml of cephalexin (RL agar; charcoal agar based), have been described; however, these media have a short shelf life because they contain blood (6, 15). Cyclodextrin solid medium with 5 μg/ml of cephalexin (CS medium) does not contain blood products and is reported to have improved selectivity and a long shelf life (1). However, the rate of detection of B. pertussis by this medium was lower than that achieved with other conventional media (10). Moreover, β-lactam (especially narrow-spectrum cephalosporins like cephalexin)-resistant bacteria, such as Haemophilus influenzae, Streptococcus pneumoniae, Staphylococcus aureus, and Moraxella catarrhalis, have recently emerged (8, 11). Therefore, cephalexin may no longer be a suitable selective reagent for isolation of B. pertussis from nasopharyngeal specimens. To address this problem, we have attempted to improve the selective isolation of B. pertussis when performing direct plating of clinical specimens. Modified CS medium (MCS medium) was prepared by replacing cephalexin with cefdinir (Asteras Pharma Inc., Tokyo, Japan), which inhibits the growth of M. catarrhalis (9, 16); vancomycin; and amphotericin B at final concentrations of 4 μg/ml, 8 μg/ml, and 4 μg/ml, respectively. The concentration of cefdinir was set by the MIC data from 50 clinical strains of M. catarrhalis and 40 clinical strains of B. pertussis. MCS medium was composed of basic medium and supplement. The basic medium contained the following: 10.7 g of sodium glutamate, 0.24 g of l-proline, 2.5 g of NaCl, 0.5 g of KH2PO4, 0.2 g of KCl, 0.1 g of MgCl2·6H2O, 0.02 g of CaCl2, 6.1 g of Tris, 2.5 g of Casamino Acids (Difco Laboratories, Detroit, MI), 2.0 g of dimethyl-β-cyclodextrin (Teijin Ltd., Osaka, Japan), and 17.0 g of Bacto agar (Difco) per liter of distilled water. The supplement preparation for MCS medium containing 40 mg of l-cysteine, 10 mg of FeSO4·7H2O, 20 mg of ascorbic acid, 4 mg niacin, and 150 mg of reduced glutathione per 10 ml was added to each liter of basic medium. All the chemical reagents were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), unless otherwise specified. For making MCS medium, all of the ingredients except antibiotics were autoclaved for 15 min at 121°C. After the mixture was cooled down to 50°C, the sterile stock solution (100×) of each antibiotic was added.
To perform the recovery test of Bordetella species on three media, one colony of each strain was streaked on BG agar, CS medium, and MCS medium plates and incubated at 35°C for 5 days. On the 5th day, the growth of each bacterial strain was observed and categorized as no growth, poor growth (<0.1-mm colony size), or positive growth (≥0.1-mm colony size). A total of 56 strains of Bordetella species (43 of B. pertussis including B. pertussis CCUG 30837T, three of Bordetella parapertussis including B. parapertussis CCUG 413T, two of Bordetella bronchiseptica including B. bronchiseptica CCUG 219T, four of Bordetella holmesii including B. holmesii CCUG 34073T, and the four strains Bordetella avium CCUG 13726T, Bordetella hinzii CCUG 33847T, Bordetella petrii CCUG 43448T, and Bordetella termatum CCUG 32381T) could be grown on the MCS medium. However, BG agar could not support the growth of B. avium, B. hinzii, B. holmesii, and B. termatum, and B. termatum grew poorly on CS medium (data not shown).
As nasopharyngeal flora (NPF) contains various microorganisms which interfere with the growth of B. pertussis, we evaluated the selectivity of B. pertussis on BG agar, CS medium, RL agar (14), and MCS medium by the use of artificially prepared (“mock”) samples. H. influenzae ATCC 49247, S. pneumoniae ATCC 49619, S. aureus ATCC 43300, M. catarrhalis NK-015 (clinical strain, β-lactamase producer), and Candida albicans ATCC 24433 were used to prepare an artificial mix of NPF. Final concentrations of 104 to 107 CFU/ml of NPF were mixed with 104 CFU/ml of B. pertussis CCUG 30837T. Ten microliters of each aliquot was spread onto each medium. The plates were incubated at 35°C under aerobic conditions. After 5 days, the growth of NPF and B. pertussis was determined (Table (Table1).1). B. pertussis could not be detected on BG agar or CS medium when cultured with any of the NPF concentrations and could be detected on RL agar only with 104 CFU/ml of NPF. However, B. pertussis was easily detected on MCS medium with all concentrations of NPF tested.
To evaluate the advantages of MCS medium in the clinical setting, 120 nasopharyngeal specimens were collected using a rayon swab (Seedswab no. 2; Eiken Chemical, Tokyo, Japan), in a blinded fashion between December 2001 and November 2002. Samples were collected predominantly from children with symptoms of pertussis. The specimens were sent to our laboratory within 2 days of collection from institutions participating in the Japanese pertussis surveillance group. All institutions had ethical approval for participation in this study. Each swab was then suspended in 300 μl of normal sterile saline. One loopful (approximately 2 μl) of this suspension was streaked out onto BG agar, CS medium, and MCS medium. Each medium was then incubated in humidified air at 35°C for 7 days. B. pertussis was identified by Gram staining, oxidase reaction, and agglutination test with polyclonal antisera (Denka Seiken, Tokyo, Japan) (12, 13). Identification of other bacterial species was performed according to the Manual of Clinical Microbiology (13). Crude DNA extracts were then prepared by boiling the remaining 100 μl of each suspension at 100°C for 15 min. Samples were then centrifuged at 18,500 × g for 5 min, and supernatants were used for PCR. To compare the detection of B. pertussis and B. parapertussis, nested duplex PCR targeting IS481 and IS1001 was performed as described by Farrell et al. (5). The chi-square test was used for statistical evaluation, and P values of less than 0.05 were considered significant. Table Table22 shows the results for detection of B. pertussis by culture on each medium or by PCR. Of the 120 samples, 23 (19.1%) were positive on at least one of the three different culture media and 44 (36.6%) were positive by PCR on at least one of the three media. There were no PCR-negative, culture-positive cases. B. parapertussis was not detected from any specimens by culture or PCR. The sensitivity of culture on BG agar, CS medium, and MCS medium was 34.0, 20.4, and 50.0%, respectively, when PCR was used as the gold standard and 65.2, 39.1, and 95.6%, respectively, by any culture-positive sample as a reference. The number of B. pertussis strains isolated on MCS medium was greater than that on CS medium (P = 0.003) and also greater than that on BG agar, but this was not statistically significant. However, when any positive culture was used as a reference, MCS medium was superior to CS medium (P < 0.001) or BG agar (P = 0.009). In addition, eight strains were detected only on MCS medium (P = 0.01 versus BG agar, P = 0.003 versus CS medium). Growth of NPF, except B. pertussis, from clinical specimens on each of the three media is shown in Table Table3.3. Growth of NPF on MCS medium was significantly less than that on BG agar and CS medium. Moreover, the number of samples with complete inhibition of NPF on MCS medium (73; 60.8%) was significantly greater than those on BG agar (24; 20.0%) and on CS medium (32; 26.6%). Table Table44 summarizes the NPF microorganisms from clinical specimens isolated on each medium. Of the three media used, MCS medium yielded the fewest NPF microorganisms other than B. pertussis and successfully inhibited growth of all the α streptococci and Haemophilus, Bacillus, and Staphylococcus strains. MCS medium could inhibit M. catarrhalis more effectively than could CS medium, probably due to the selective inhibition of β-lactamase-producing M. catarrhalis strains by cefdinir (9, 16). More α streptococci and Neisseria, Haemophilus, Staphylococcus, and Corynebacterium strains were grown on BG agar than on MCS medium, and this result was statically significant. Our results suggest that MCS medium not only inhibited indigenous NPF overgrowth but also supported the growth of B. pertussis.
MCS medium was shown to support the growth of B. pertussis Tohama and B. pertussis Yamaguchi strains for up to 6 months and could inhibit S. aureus IFO 13726, S. aureus ATCC 43300, M. catarrhalis NK-015, and C. albicans ATCC 10231 completely for up to 5 months when stored at 4 to 9°C (data not shown). A long shelf life is another benefit of this medium because most clinical microbiology laboratories are infrequently required to culture specimens from pertussis patients (6, 15). The cost of MCS medium is similar to that of BG agar or RL agar.
In conclusion, MCS medium improved the selective isolation of B. pertussis from clinical specimens. This culture method will assist future studies aimed at a better understanding of the pathophysiology of pertussis.
We thank the participants from the Japanese pertussis surveillance group for their invaluable contribution to this study.
Published ahead of print on 21 October 2009.