Improved Selective Isolation of Bordetella pertussis by Use of Modified Cyclodextrin Solid Medium

Abstract

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 g/ml of cephalexin (cefalexin) (BG agar) and Regan-Lowe agar with 40 g/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 g/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, ␤-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 g/ml, 8 g/ml, and 4 g/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 KH 2 PO 4 , 0.2 g of KCl, 0

Tracheal colonization factor A (TcfA) is a biomarker for rapid and specific detection of Bordetella pertussis

“…Candidates that were known virulence determinants or cytotoxic factors 9 were prioritized to increase the probability of a diagnostic's long-term utility due to selective pressure against loss of expression. Candidates were also required to have multiple, spatially separated regions with high predicted antigenicity, high conservation across B. pertussis strains, and low homology to other microorganisms reported in nasopharyngeal specimens [25][26][27][28][29][30][31] . Ultimately, we selected five proteins that met the above criteria: tracheal colonization factor A (TcfA), adenylate cyclase toxin (ACT), filamentous hemagglutinin (FHA), pertussis toxin subunit 1 (PTXS1), and autotransporter (Vag8).…”

Development of vaccines against pertussis caused by Bordetella holmesii using a mouse intranasal challenge model

“…Individual lungs were homogenized in 10 mL/lung DPBS in an ULTRA‐TURRAX tube dispenser and DT‐20M sterile tube (IKA, Staufen im Breisgau, Germany). After appropriate dilution, in general 10 1 ‐, 10 3 ‐, and 10 4 ‐fold, each homogenate was spread on cyclodextrin agar and incubated for 5 days at 36.5°C. Number of viable cells of B. holmesii was calculated from the number of CFU in each lung.…”

Untitled

“…modified cyclodextrin agar or liquid medium; the cyclodextrin medium was supplemented with ammonium sulfate (4.00 g/L), D-glucose (18.2 g/L) and sodium aspartate (7.15 g/L) 8 . Leclercia adecarboxylata was grown for 3 days at 25°C or 37°C on a modified cyclodextrin agar medium; the bacterial cells were harvested, suspended in modified cyclodextrin broth lacking or containing mizoribine added at 0.63 or 1.25 mmol/L, inoculated at 1× 10 8 colony-forming units (CFU)/mL into 3 mL of modified cyclodextrin broth in 25cm 2 flasks (Thermo Fisher Scientific, Waltham, MA, USA) and cultured for 3 days at 25°C or 37°C under static conditions.…”