Recovery of Bordetella holmesii from Patients with Pertussis-Like Symptoms: Use of Pulsed-Field Gel Electrophoresis To Characterize Circulating Strains

Strains.Thirty-two B. holmesii isolates from the nasopharynx and one isolate (95R0375) from blood which had been previously confirmed as B. holmesii at the CDC were included in the study. In addition, four isolates of B. bronchiseptica, five isolates of B. parapertussis, and five isolates of B. pertussis previously recovered and identified at the Massachusetts State Laboratory Institute (SLI) were used. Acinetobacter transformation strain KC1842 was obtained from R. Weyant, Special Bacteriology Reference Laboratory, Division of Bacterial and Mycotic Diseases, CDC, and the positive control strain used for the transformation assay wasAcinetobacter sp. ATCC 19606.

Method for isolating strains used.Our laboratory provides a pertussis culture kit to the healthcare community. Three of the items included in the kit are a sterile nasopharyngeal calgiswab, a tube containing 0.5 ml of 1% casein hydrolysate (CAS) holding medium, and a charcoal agar with cephalexin (CA-LEX) transport agar slant. The clinician is directed to obtain a nasopharyngeal specimen from the patient by using the sterile swab provided and, after it has been taken, to immediately place the swab in the 1% CAS holding medium, in which it may remain for up to 30 min. The clinician is then directed to remove the swab from the 1% CAS, to roll it over the CA-LEX transport agar slant provided, and to return the CA-LEX transport slant to the SLI for testing. It is recommended that the culture kit be transported by same-day courier, although delivery by an overnight transport service on cold packs is acceptable. Cultures that cannot be processed on the day of receipt at the SLI are refrigerated until they are processed. When received at the laboratory, each nasopharyngeal specimen previously streaked onto CA-LEX slants by the health care providers is further subcultured onto a Bordet-Gengou agar plate containing methicillin (BG-MET plate). To do this, a sterile swab is saturated with fresh sterile 1% CAS and swabbed over the slant of the CA-LEX transport agar. It is then streaked onto a BG-MET plate and cross-hatched to obtain isolated colonies. (A wet swab is used to transfer the growth from the slanted culture in order to replenish moisture that may have been lost in transit.) Both the CA-LEX slant and the BG-MET plate are incubated and examined for Bordetellaspp.

Media and biochemical tests.CA-LEX (charcoal agar with 10% horse blood plus 40 μg of cephalexin per ml) was prepared full strength according to the manufacturer's instructions. Ten milliliters of the sterile molten medium was dispensed into wide-mouthed screw-cap bottles (3.0 by 6.5 cm) and allowed to cool in a slanted position. BG agar plates with 20% sheep blood (Difco Laboratories, Detroit, Mich.) with and without antibiotics were prepared according to the manufacturer's directions and dispensed at 30 ml/plate. One percent CAS (Difco Laboratories) was dispensed at 0.5 ml per tube and sterilized. B. holmesii isolates were identified according to the conventional tests and methods of the Special Bacteriology Reference Laboratory, CDC (10). The inoculated selective media were observed after 3 days of incubation and then daily up to 7 days. A final reading was taken after a total incubation of 12 days. Media and biochemical tests were incubated aerobically at 35 to 36°C. The oxidase test was performed on 3-day-old growth obtained from nonselective BG agar plates. Smears were prepared and stained withB. pertussis and B. parapertussis conjugates (Difco Laboratories). All smears prepared for fluorescent-antibody staining consisted of cells taken from colonies growing on BG selective or nonselective agar plates, which consistently gave satisfactory results at a 1:5 or 1:4 dilution, respectively. It was found that smears prepared from cells growing on CA-LEX gave unsatisfactory results when they were stained with Difco conjugates. The transformation assay procedure as described by Juni (3) was used to determine if the test strains were genetically related toAcinetobacter. The transformation assay involves the mixing of crude DNA extracts of the test strain with the viable cellular mass of a specific strain of A. calcoaceticus that is an amino acid auxotroph. The mixture is incubated on a heart infusion agar plate for 4 to 6 h and then subcultured to a plate with a minimal medium (lactate mineral agar) upon which only the prototrophic transformants will grow. Conversion of genetically related strains from auxotroph to prototroph is evidenced by the subsequent growth of colonies after a 24-h incubation on the minimal medium (10).

Testing of Bordetella spp. on selective media.All isolates were subcultured two consecutive times onto antibiotic-free BG agar plates. After 3 days, a sweep of colonies from the final culture plates was suspended in phosphate-buffered saline (pH 7.2). The cell suspensions were adjusted to a density equivalent to a 0.5 McFarland standard, and subsequent working dilutions were made to yield a final concentration equivalent to 1.5 × 10⁴CFU/ml. A 10-μl aliquot of the working dilution (1.5 × 10² CFU) of each cell suspension was inoculated onto four culture plates of BG agar under different antibiotic conditions: (i) without antibiotic (BG agar alone), (ii) with 0.625 μg of oxacillin per ml (BG-OXA), (iii) with 40 μg of cephalexin (BG-LEX), and (iv) with 2.5 μg of methicillin per ml (BG-MET). In addition, CA and CA-LEX were inoculated. The plates were incubated aerobically at 35 to 36°C and observed for seven consecutive days. The partial inhibition or absence of bacterial growth was qualitatively observed for each plate. Partial inhibition was defined as a reduction in the colony count on the experimental plate relative to the bacteria's growth on the antibiotic-free BG agar plate.

PFGE of B. holmesii.All B. holmesiiisolates were incubated for 3 days at 37°C on antibiotic-free BG agar. The colonies were harvested with cotton swabs and suspended in 1.5 ml of pH 8.0 cell suspension buffer (CSB) (10 mM Tris, 100 mM EDTA) to a density approximately equal to a 3.5 McFarland standard. The cell suspensions were washed twice with CSB at room temperature, followed by the embedding of the cells in agarose plugs, and processed as previously described by de Moissac et al. (2). Two segments were cut from each plug, and PFGE was performed following restriction with XbaI on one segment and SpeI on the other. The gels were run using contour-clamped homogeneous electric field MAPPER (Bio-Rad) at 14°C using TBE running buffer (10.9 g of Tris, 6.0 g of boric acid, 14 ml of 0.5 M EDTA, and distilled water to make 1.0 liter [pH 8.0]). PFGE was run for 18 h with initial and final switch times of 2.16 and 35.07 s, respectively.