Recovery of Bordetella holmesii from patients with pertussis-like symptoms: use of pulsed-field gel electrophoresis to characterize circulating strains

A 4-year retrospective study showing that we isolated Bordetella holmesii, but not Bordetella pertussis, from patients with pertussis-like symptoms was performed. From 1995 through 1998, we isolated B. holmesii from 32 nasopharyngeal specimens that had been submitted from patients suspected of having pertussis. Previously, B. holmesii had been associated mainly with septicemia and was not thought to be associated with respiratory illness. A study was undertaken to describe the characteristics of the B. holmesii isolates recovered and why we were successful in detecting the organism in nasopharyngeal specimens. B. holmesii isolates were characterized for drug sensitivities and for genetic relatedness by pulsed-field gel electrophoresis (PFGE). These isolates, an additional strain of B. holmesii isolated from a blood culture and previously confirmed by the Centers for Disease Control and Prevention, Atlanta, Ga., and 14 other clinical isolates of Bordetella spp., including 4 of B. bronchiseptica, 5 of B. parapertussis, and 5 of B. pertussis, were studied. They were all separately inoculated on three Bordet Gengou (BG) selective media containing either 0.625 microgram of oxacillin per ml, 40 microgram of cephalexin per ml, or 2.5 microgram of methicillin per ml, on BG agar with no antibiotic (control), and on charcoal agar (CA) with and without 40 microgram of cephalexin per ml. We found that cephalexin, the antibiotic commonly incorporated in both CA and BG agar for the recovery of Bordetella spp., is inhibitory to the growth of B. holmesii. In addition, the genotypic analysis of the 32 B. holmesii isolates by PFGE following restriction with XbaI and SpeI identified the dominant strains circulating during the study period.     

Cephalexin-supplemented Jones-Kendrick charcoal agar for selective isolation of Bordetella pertussis: comparison with previously described media.

Four agar media, Jones-Kendrick (JK) charcoal agar unsupplemented, JK agar supplemented with 0.5 U of penicillin per ml, JK medium supplemented with 2.5 micrograms of methicillin per ml, and JK medium supplemented with 40 micrograms of cephalexin per ml, were evaluated to determine their ability to support growth of Bordetella pertussis, their ability to selectively inhibit normal pharyngeal flora while maintaining growth of B. pertussis, and their stability during storage. Five stock cultures of B. pertussis were plated on each of the media. Penicillin- and cephalexin-supplemented media were more inhibitory for early growth of B. pertussis than was medium supplemented with methicillin. However, after 5 days of incubation at 35 degrees C, all media supported good growth of this organism. When employed to detect B. pertussis in sham specimens, prepared by mixing normal pharyngeal material with each of the five B. pertussis stock cultures, the medium containing cephalexin was judged superior to all other media tested in its combined ability to suppress the growth of normal pharyngeal flora and to allow early detection of Bordetella colonies. All media tested retained their efficacy after 9 weeks of storage at 2 to 8 degrees C.     

Comparison of four charcoal media for the isolation of Bordetella pertussis.

Charcoal-horse blood agar with 40 micrograms of cephalexin per ml, charcoal-horse blood agar with 3 micrograms of lincomycin per ml, charcoal agar with 3 micrograms of lincomycin per ml, and Legionella (buffered charcoal-yeast extract) agar with 3 micrograms of lincomycin per ml were compared for isolation of Bordetella pertussis. Charcoal-horse blood agar with 40 micrograms of cephalexin per ml gave the best results, with a B. pertussis recovery rate of 100%. Growth was most rapid and the mean number of colonies was highest on this agar, and growth of pharyngeal flora was completely suppressed.     

Effects of transport temperature and medium on recovery of Bordetella pertussis from nasopharyngeal swabs.

We compared relative recoveries of Bordetella pertussis from simulated nasopharyngeal (NP) specimens incubated in three separate transport media at different temperatures. Transport media included one-half-strength Regan-Lowe (RL.5), Regan-Lowe with one-half-strength agar (RL.5A), and buffered charcoal-yeast extract agar supplemented with alpha-ketoglutarate, lincomycin, and anisomycin (BCYE alpha LA). For each transport medium, recovery of B. pertussis was least efficient after storage at 25 degrees C. The highest recovery of B. pertussis from a mixed culture was achieved with RL.5 at 4 degrees C. Overall, RL.5 and RL.5A were comparable as transport media whether held at 4 or 25 degrees C, but fewer organisms were recovered from BCYE alpha LA. In addition, Regan-Lowe (RL), Bordet-Gengou, and cyclodextrin media were compared as primary isolation media for recovering B. pertussis from simulated NP swabs held at 4 and 35 degrees C in RL.5 medium. The highest recovery of B. pertussis was obtained on RL primary isolation medium. Bordet-Gengou medium recovered only 80% and cyclodextrin medium recovered less than 60% of the numbers recovered on RL medium. Based on these results, refrigeration (4 degrees C) of NP swabs shipped in RL.5 transport medium and using RL as the primary isolation medium are recommended for recovering B. pertussis from swab specimens.     

Isolation of Capnocytophaga species with a new selective medium.

A selective medium (CAP) composed of a GC agar base supplemented with 1% hemoglobin, 1% Polyvitex, and an antibiotic mixture of polymyxin B (15 U/ml), vancomycin (5 micrograms/ml), trimethoprim (2.5 micrograms/ml), and amphotericin B (2.5 micrograms/ml) was compared with another selective medium (TBBP) and two nonselective media--a blood agar and a chocolate agar--to isolate Capnocytophaga species from 725 clinical specimens. These included sputa (467 specimens), throat swabs (116 specimens), oral ulcerations (35 specimens), and periodontal pockets (107 specimens). The recovery rate of Capnocytophaga species was significantly higher with the CAP medium (96%) than with the selective TBBP medium (52.2%), the nonselective blood agar (6.2%), and the chocolate agar (4.6%). Growth of the normal flora was best inhibited on CAP medium. Colony size and yellow-brown pigment formation were maximally expressed on chocolate agar and CAP medium, but gliding motility was mostly absent. We conclude that the CAP medium is an excellent medium for the recovery of Capnocytophaga species from contaminated clinical specimens.     

Granada medium for detection and identification of group B streptococci

A new starch serum medium, Granada medium, for isolation and identification of group B streptococci (GBS) anaerobically as red colonies is described. The medium contains 3.8% Proteose Peptone no. 3 (Difco), 15% soluble starch 1252 (Merck), 10% coagulated horse serum, 15 micrograms of trimethoprim per ml, and 0.06 M phosphate buffer (pH 7.8; medium pH 7.4). This medium inhibited fecal flora and at the same time supported growth of GBS. A new pigment-enhancing effect of folate inhibitors on GBS is reported and used in the formulation of the medium. The good selective and differential properties of the Granada medium favor quicker and easier detection of GBS in heavily contaminated specimens. Since the medium is convenient to use and requires only 18 h of incubation to detect and identify GBS, it should be useful in any clinical microbiology laboratory and would assist in the early detection of GBS in clinical specimens.     

Comparison of media and culture techniques for detection of Streptococcus pneumoniae in respiratory secretions.

We compared the relative efficacy of three methods for the isolation of Streptococcus pneumoniae in lower respiratory secretions. Based on results from 294 clinical specimens, we found that S. pneumoniae was isolated at a frequency of 65% with 5% sheep blood agar or 5% sheep blood agar containing 5 micrograms of gentamicin per ml, both incubated in 5% CO2. Anaerobic incubation of 5% sheep blood agar enhanced the recovery rate of S. pneumoniae to 93%. The improved efficacy with anaerobic incubation is due to the greater ease of recognition of the larger and more mucoid colonies of S. pneumoniae, and to the suppression of the growth of other oral bacteria present in the respiratory sections.     

In vitro evaluation of combined usage of fosfomycin and 5-fluorouracil for selective isolation of Leptospira species.

The combined usage of fosfomycin (FOM) and 5-fluorouracil (5-FU) as selective agents for the isolation of leptospires from contaminated materials was investigated. Additive or synergistic antibacterial activity was apparent with the combination compared with each agent used separately. Of 54 bacterial strains tested, 52 were inhibited, while all 5 Leptospira strains tested were unaffected by the combined addition of FOM (400 micrograms/ml) and 5-FU (100 micrograms/ml) to Korthof medium. Furthermore, this combination successfully supported the selective growth of Leptospira interrogans serovar copenhageni in experimentally contaminated specimens. This FOM-5-FU combination is surmised to be useful for the selective isolation of leptospires from contaminated clinical, pathological, or environmental materials.     

Selective medium for isolation of Actinobacillus actinomycetemcomitans.

A selective medium, TSBV (tryptic soy-serum-bacitracin-vancomycin) agar, was developed for the isolation of Actinobacillus actinomycetemcomitans, TSBV agar contained (per liter) 40 g of tryptic soy agar, 1 g of yeast extract, 100 ml of horse serum. 75 mg of bacitracin, and 5 mg of vancomycin. The TSBV medium suppressed most oral species and permitted significantly higher recovery of A. actinomycetemcomitans than nonselective blood agar medium. The distinct colonial morphology and positive catalase reaction of A. actinomycetemcomitans easily distinguished this bacterium from Haemophilus aphrophilus, Capnocytophaga species, and a few other contaminating organisms. With the TSBV medium, even modestly equipped laboratories will be able to isolate and identify A. actinomycetemcomitans from clinical specimens.     

Evaluation of novel vancomycin-containing medium for primary isolation of Kingella kingae from upper respiratory tract specimens.

A new selective medium (BAV), consisting of trypticase agar with 5% sheep hemoglobin and 2 micrograms of vancomycin per ml, was compared with the routine blood-agar medium for the primary isolation of Kingella kingae from upper respiratory specimens from a population of young children. Infection was detected by the BAV medium in 43 of 44 (98%) cultures positive for K. kingae, and detection of the organism was facilitated by inhibition of gram-positive flora. Infection was detected in only 10 of 44 (23%) positive cultures by the blood-agar medium, and plates were usually covered by abundant normal flora, making the recognition of K. kingae much more difficult. Challenge of the medium with different organisms of respiratory origin showed that the BAV medium was inhibitory for gram-positive cocci and Haemophilus influenzae but that it supported growth of eight K. kingae strains isolated from patients with invasive infections. The new medium appears to be a useful epidemiological tool for studying the respiratory carriage of K. kingae.     

Selective recovery of oral Capnocytophaga spp. with sheep blood agar containing bacitracin and polymyxin B.

On the basis of in vitro susceptibility testing of antibiotics, dyes, and other antimicrobial agents, we developed and evaluated a medium, TBBP, for the selective isolation of oral Capnocytophaga spp. TBBP medium consists of 4% Trypticase soy agar (BBL Microbiology Systems, Cockeysville, Md.), 5% sheep blood, 0.1% yeast extract, 50 micrograms of bacitracin per ml, and 100 micrograms of polymyxin B per ml. A total of 34 Capnocytophaga stock cultures grew well on TBBP medium. Except for some streptococcal strains, TBBP medium inhibited growth of all test stock culture isolates of common oral gram-positive and gram-negative species. In a clinical study of 15 deep periodontal pockets, TBBP medium demonstrated Capnocytophaga recoverability that was similar to or higher than that shown by a nonselective blood agar medium. Typical Capnocytophaga colonial morphology enabled us to readily distinguish this organism from the few other bacteria which could grow on TBBP medium.     

Isolation medium for the recovery of Pseudomonas cepacia from respiratory secretions of patients with cystic fibrosis.

A new medium for the isolation of Pseudomonas cepacia from respiratory tract secretions of patients with cystic fibrosis (CF) is described. This medium consists of inorganic salts, 0.5% pyruvate, and 0.1% proteose peptone as nutritive components and 0.0001% crystal violet, 0.15% bile salts, 100 micrograms of ticarcillin per ml, and 300 U of polymyxin B per ml as selective agents. The medium, designated PC medium, supported superior growth of 38 of 50 stock isolates of P. cepacia after 48 h of incubation when compared with MacConkey agar (0 of 50). The medium completely inhibited the growth of 112 of 124 stock isolates of organisms commonly found in respiratory secretions of CF patients. Cultures were made on PC medium with respiratory secretions of 169 CF patients. P. cepacia was recovered from 35 patients with isolates on PC medium but from only 21 patients with isolates on MacConkey agar. Of 221 other potentially pathogenic isolates found in these specimens, only six (two Pseudomonas aeruginosa isolates, two molds, one yeast, and one Serratia marcescens isolate) grew on PC medium. PC medium should facilitate the recovery of P. cepacia from CF patients.     

Selective medium for Branhamella catarrhalis with acetazolamide as a specific inhibitor of Neisseria spp.

Several semiselective media for Branhamella catarrhalis have been proposed. These media allow growth of all members of the family Neisseriaceae, and further differentiation is necessary. By addition of 10 micrograms of acetazolamide, a carbonic anhydrase inhibitor, per ml and incubation in air, a medium was created which reduced growth of Neisseria spp. When saliva samples from 178 healthy schoolchildren were screened for the presence of B. catarrhalis, the carrier rate for this organism was estimated to be 48.9% with the selective medium compared with 12.4% when a semiselective medium, which contains only 10 micrograms of vancomycin, 5 micrograms of trimethoprim, and 2 micrograms of amphotericin B per ml, was used and 6.2% when a nonselective blood agar plate was used. The number of Neisseria spp. isolated dropped from 297 on the semiselective agar to 55 on the selective agar.     

New selective medium for Pseudomonas aeruginosa with phenanthroline and 9-chloro-9-[4-(diethylamino)phenyl]-9,10-dihydro-10- phenylacridine hydrochloride (C-390).

A new selective medium (PC agar) for the isolation of Pseudomonas aeruginosa was developed, consisting of 30 micrograms of 9-chloro-9-[4-(diethylamino)phenyl]-9,10-dihydro-10-phenylacridine hydrochloride (C-390) per ml and 30 micrograms of phenanthroline per ml in Columbia agar. PC agar was superior to phenanthroline, C-390, acetamide, and cetrimide agars for the selective growth of P. aeruginosa.     

Semiselective medium for isolation of Moraxella bovis from cattle with infectious keratoconjunctivitis.

The incorporation of 2.5 micrograms/ml of cloxacillin into 5% bovine blood agar provided an inexpensive, easily prepared culture medium for the primary isolation of Moraxella bovis from bovine lacrimal and nasal secretions. With this medium, the time required to identify and isolate M. bovis from large numbers of field specimens was substantially reduced, whereas the sensitivity of isolation was increased by 60%.     

Selective diagnostic medium for pathogenic Listeria spp

Pathogenic Listeria serovars produced hemolysis on agars containing 5% rabbit erythrocytes and 10 micrograms of acriflavin, 40 micrograms of nalidixic acid, and 7.5 activity units of equi factor per ml. Apathogenic Listeria innocua was nonhemolytic on this medium.     

Heptakis(2,6-O-dimethyl)beta-cyclodextrin: a novel growth stimulant for Bordetella pertussis phase I.

The effect of cyclodextrins on the growth of Bordetella pertussis Tohama phase I in synthetic medium was evaluated. The addition of cyclodextrins, especially heptakis(2,6-O-dimethyl)beta-cyclodextrin (Me beta CD), to a complete synthetic medium such as Stainer-Scholte medium gave the same number of individual colonies and growth rates as those on Bordet-Gengou medium. Furthermore, with the addition of Me beta CD, growth inhibition by fatty acids such as oleic or palmitic acid was overcome and normal cell growth was observed. This modified Stainer-Scholte medium, designated as cyclodextrin solid medium (CSM), supported excellent growth of 20 lyophilized clinical isolates. Serotypes of the organisms after 10 passages on this CSM plate were not changed. These results suggest that Me beta CD is a significant growth stimulant and CSM is one of the most suitable synthetic media for culture of B. pertussis phase I.     

Use of 9-chloro-9-(4-diethylaminophenyl)-10-phenylacridan as a primary medium for recovery of Pseudomonas aeruginosa from clinical specimens

Several concentrations of 9-chloro-9-(4-diethylaminophenyl)-10-phenylacridan (C-390), ranging from 5 to 100 micrograms/ml, were incorporated in brain heart infusion agar, MacConkey agar, and xylose-lysine-deoxycholate agar to evaluate the recovery of Pseudomonas aeruginosa from 538 sputum, 174 urine, and 22 stool samples. Seventy-six sputum samples containing P. aeruginosa grew this bacterium alone on brain heart infusion and MacConkey agars with a C-390 concentration of 25 micrograms/ml or greater. Other microorganisms present in these specimens grew only on media without C-390, and significantly less growth was observed on media with less than 20 micrograms of C-390 per ml. In few samples containing Klebsiella pneumoniae (3 of 30) and Serratia spp. (3 of 10), these organisms grew on all C-390 media and concentrations tested. The remaining sputum samples grew other bacteria and yeasts only on media without C-390. Brain heart infusion and MacConkey agars with C-390 were equally effective in recovering P. aeruginosa and suppressing the growth of a wide range of bacteria and yeasts from urine and stool samples. Xylose-lysine-deoxycholate agar with C-390 did not show a selective or suppressive advantage over xylose-lysine-deoxycholate agar alone for recovering P. aeruginosa from stool specimens. These results indicate that use of the correct medium and C-390 concentration would provide a suitable primary medium for inhibiting a wide range of bacteria and yeasts and would select the growth of P. aeruginosa from clinical specimens.     

Comparison of six media, including a semisolid agar, for the isolation of various Campylobacter species from stool specimens.

A recently described semisolid blood-free selective motility medium (SSM) (J. Goossens, L. Vlaes, I. Galand, C. Van den Borre, and J. P. Butzler, J. Clin. Microbiol. 27:1077-1080, 1989) was compared with two charcoal-based selective media (charcoal-based selective medium [CSM] and modified charcoal cefoperazone deoxycholate agar [CCDA]), two blood-based media (Skirrow medium [SKM] and CampyBAP), and a passive, 0.65-microns-pore-size cellulose acetate membrane filter technique for the recovery of campylobacters from stools of patients with diarrhea. A total of 1,980 specimens were tested, 161 of which were found to be positive for campylobacters. Campylobacter jejuni was isolated in 148 specimens (91.9%), C. coli was isolated in 27 (7.5%), and "C. upsaliensis" was isolated in 1 (0.6%). After 72 h of incubation with a single medium, the cumulative percentages of Campylobacter-positive specimens isolated on CSM, CCDA, SKM, and SSM were 87, 83, 80, and 72%, respectively. The filter method alone enabled us to recover 61% of all campylobacters. The "C. upsaliensis" strain was isolated by this method only. The highest isolation rates were observed when two media, including CSM, were combined. The combination of CSM and SSM yielded the highest rates (96%), but these were not statistically different from the rates observed with combinations of CSM and SKM (94%) or of CSM and the filter method (91%). Extending the incubation time from 48 to 72 h led to an increase in the isolation rate regardless of the medium used (P less than 0.001). CSM and CCDA were the most selective media. SKM and CampyBAP appeared to be the most inhibitory media for the isolation of C. coli.     

Isolation of mycobacteria from clinical specimens by use of selective 7H11 medium.

7H11 agar containing carbenicillin, amphotericin B, polymyxin B and trimethoprim lactate (selective 7H11 or S7H11) was used for the selective isolation of mycobacteria from clinical specimens. This medium as previously described contained 100 mug. carbenicillin per ml. It was found that reducing the concentration of carbenicillin to 50 mug. per ml. made S7H11 less inhibitory to certain strains of mycobacteria. However, some strains of M. kansaii, M. intracellulare, and M. gordonae still do not grow well on S7H11. Of 3,134 clinical specimens (mostly sputum) received, processed, and plated on 7H11 agar and the S7H11 medium, 508 positive specimens were isolated. Of these, 402 were positive on both types of media, 30 were positive on 7H11 only, 19 were positive on S7H11 only, 52 were contaminated on 7H11 but positive on S7H11, while only 5 were contaminated on S7H11 and positive on the plain medium. Thus, the total positive specimens on 7H11 was 437 and total positive specimens on the selective medium was 473.Used in conjunction with nonselective media, S7H11 agar appears to be a valuable culture medium for used in diagnostic mycobacteriology laboratories.