Bordet-Gengou Agar Research Articles

Bordet-Gengou agar medium supplemented with albumin-containing biologics for cultivation of bordetellae.

Bordetella pertussis, B. parapertussis, and B. bronchiseptica cause respiratory infections in mammals, including humans, and are generally cultivated on Bordet-Gengou (BG) agar plates in laboratories. The medium requires animal blood as a supplement for better bacterial growth. However, using blood is problematic, as its constant supply is occasionally difficult because of the limited shelf-life. This study proposes modified BG agar plates supplemented with bovine serum albumin and fetal bovine serum as a simple and convenient medium that confers sufficient growth of bordetellae.

Aboratory Diagnosis of Pertussis

Background: The resurgence of pertussis highlights the importance of standardized, sensitive, and specific laboratory diagnoses, the lack of which is responsible for the large differences in pertussis notifications between countries. Accurate laboratory diagnosis is also important for distinguishing between the several etiologic agents of pertussis-like diseases, which involve both viruses and bacteria. If pertussis is diagnosed in a timely manner, antibiotic treatment of the patient can mitigate the symptoms and prevent transmission. The aim was to evaluate the relationship between reported and confirmed cases in patients treated at Zona Norte Children's Hospital, to analyze the methods used for the diagnosis of pertussis and to study the susceptibility of the isolates to erythromycin as the drug of choice to treatment of pertussis. Methods & Materials: A retrospective study of pertussis notifications and confirmations in patients treated from 1 January 2007 until 30 september 2017 was carried. All nasopharyngeal aspirate specimens for diagnosis of pertussis were assay by amplification of 481 insertion sequence and pertussis toxin by PCR and culture on Regan Lowe and Bordet Gengou agar. To all those strains that remained viable MICs to erythromycin were performed using the Etest method. Results: It was observed that of 1670 notifications only 6.8% (114) were confirmed. It is noted that in 2007, 2008,2011 and 2012 there was a marked increase in notifications and confirmations. The same was in the order of 8,1%; 14,6%, 16,1% and 7% respectively of the reported cases. 57.0% (65) of the diagnoses were performed by both methods (PCR + Culture); 37.7% (43) of the samples were diagnosed only by PCR and 5.3% (6) were positive only by culture. Erythromycin resistance was tested in 81.6% (58) of the recovered strains, all of which were sensitive. Conclusion: The increase in notifications and confirmations in 2007; 2008; 2011 and 2012 coincides with the detected epidemic situation in our province .PCR is most sensitive and should always be included to complement culture. This observation confirms the need for maintaining the ability to culture to analyze the antimicrobial drug resistance. Erythromycin can be a good drug for empirical treatment.

Trends in the Minimum Inhibitory Concentrations of Erythromycin, Clarithromycin, Azithromycin, Ciprofloxacin, and Trimethoprim/Sulfamethoxazole for Strains of Bordetella pertussis isolated in the Czech Republic in 1967-2015.

The aim of the study was to determine trends in the minimum inhibitory concentrations (MICs) of erythromycin used as first-line therapy and alternative antibiotics against Bordetella pertussis (B. pertussis) strains isolated from patients with whooping cough in the Czech Republic (CR) in three periods from 1967 to 2015. In total, 135 isolates from the years 1967–2015 were analysed. The strains were divided into three groups by the year of isolation: 1967–1999 (42 strains), 2004–2010 (43 strains), and 2011–2015 (50 strains). MIC of selected antibiotics (erythromycin, clarithromycin, azithromycin, ciprofloxacin, and trimethoprim/sulfamethoxazole) were obtained by the reference agar dilution method on Bordet Gengou Agar with 15% defibrinated sheep blood. The study set included 70 strains previously tested for MICs of erythromycin and four other antibiotics. In the three study periods, the MICs of the tested antibiotics for B. pertussis were nearly identical. All but a single strain, inhibited by erythromycin at a concentration of 0.03 mg/l, were inhibited by two concentrations of erythromycin and azithromycin (0.06 and 0.125 mg/l). Clarithromycin inhibited the strains from all three study periods at the following concentrations: 0.03, 0.06, and 0.125 mg/l. Any of the 135 strains was inhibited by ciprofloxacin at a single concentration of 0.06 mg/l and by trimethoprim/sulfamethoxazole at three concentrations (0.125, 0.25, and 0.5 mg/l). The study set of 135 Czech strains of B. pertussis isolated in 1967–2015 appears to be homogeneous in terms of the MICs for five antimicrobials. The MICs remained in a narrow range of two to three low concentrations; the unimodal distribution of the MICs suggests the absence of resistance mechanisms. The highest MICs of erythromycin, clarithromycin, and azithromycin were equally 0.125 mg/l, that of ciprofloxacin was 0.06 mg/l, and that of trimethoprim/sulfamethoxazole was 0.5 mg/l. Over the study period of 55 years, the MICs of the study antibiotics remained in the same ranges.

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

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).

A Novel Secondary Acyl Chain in the Lipopolysaccharide of Bordetella pertussis Required for Efficient Infection of Human Macrophages

Lipopolysaccharide is one of the major constituents of the Gram-negative bacterial outer membrane and is a potent stimulator of the host innate immune response. The biosynthesis of the lipid A moiety of lipopolysaccharide is a complex process in which multiple gene products are involved. Two late lipid A acyl transferases, LpxL and LpxM, were first identified in Escherichia coli and shown to be responsible for the addition of secondary acyl chains to the 2' and 3' positions of lipid A, respectively. Here, we describe the identification of two lpxL homologues in the genome of Bordetella pertussis. We show that one of them, LpxL2, is responsible for the addition of the secondary myristate group that is normally present at the 2' position of B. pertussis lipid A, whereas the other one, LpxL1, mediates the addition of a previously unrecognized secondary 2-hydroxy laurate at the 2 position. Increased expression of lpxL1 results in the appearance of a hexa-acylated lipopolysaccharide form with strongly increased endotoxic activity. In addition, we show that an lpxL1-deficient mutant of B. pertussis displays a defect in the infection of human macrophages.

The reemergence of pertussis in immunized populations: a case study.

To present a case of classical pertussis occurring in previously vaccinated male siblings, 11 and 13 years of age, living in El Paso TX; also to present an overview and update of the changing epidemiology of pertussis including pathophysiology, diagnosis, and treatment. Nasopharyngeal swabs and blood samples were collected from two male siblings, 11 and 13 years of age, presenting with cold-like symptoms and persistent cough during the second week of infection. Nasopharyngeal swabs were plated onto Bordet-Gengou agar plates and incubated for 48 hours. Blood samples were analyzed for the presence of antibodies (IgM and IgA) against Bordetella pertussis antigens using indirect enzyme linked immunosorbent assay (ELISA). Cultures and serological analysis was conducted at the University of Texas at El Paso, Clinical Laboratory Science Program Research facility. Bacterial cultures of both children were positive for Bordetella pertussis and the sera revealed positive IgM and IgA antibodies (> 11 PANBIO UNITS) against a mixture of antigens including: pertussis toxin, filamentous hemagglutinin, pertactin, and fimbriae. Pertussis immunity wanes overtime, leaving most adolescents and adults susceptible to infection. Physicians must be prepared to diagnose and treat pertussis in any age group regardless of vaccination status.

Growth phase influences complement resistance of Bordetella pertussis.

The BrkA protein of Bordetella pertussis inhibits killing by the antibody-dependent classical pathway of complement; however, susceptibility to complement can be highly variable. Log-phase bacteria grown in Stainer-Scholte (SS) broth plated on Bordet-Gengou (BG) agar were about 500 times more sensitive to killing by complement than stationary-phase SS-BG cultures. While always more susceptible to complement than the wild-type strain, a BrkA mutant displayed a similar growth phase variation in susceptibility to complement. Growth phase susceptibility to complement was also observed for a mutant constitutive for Bvg activation of BrkA, suggesting that modulation of virulence factor expression was not responsible for sensitivity to complement. Susceptibility was not due to differential antigenic expression, since serum adsorbed with complement-resistant, stationary-phase SS-BG cultures lacked bactericidal activity against B. pertussis harvested at all times during the growth cycle. These results suggest that log-phase susceptibility to complement is not due to variable expression of BrkA or antigenic differences and may be an inherent property of rapidly growing cultures. Implications for vaccine development are discussed.

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.

In vitro activity of macrolides against Bordetella pertussis strains isolated in 1968 and 30 years later in Poland

Bordetella pertussis causes whooping cough, a severe respiratorytract infection. The disease affects all age groups of people, but its severity is greatest in young children. In spite of vaccination programmes, whooping cough epidemics continue to occur [1,2]. Erythromycin (main component, erythromycin A) is regarded as the antibiotic of choice for treatment and postexposure prophylaxis of B. pertussis infection. Widespread use of macrolides may lead to changes in susceptibility patterns and the emergence of resistance in earlier susceptible bacteria. Resistance of B. pertussis to erythromycin had not previously been reported until recently,when two B. pertussis isolates that were resistant to this antibiotic were found in the United States [3,4]. The data may indicate a necessity to evaluate the activity of new antimicrobial agents against B. pertussis. The aim of this study was to compare the susceptibility to macrolides of B. pertussis strains isolated in 1968 and 30 years later in Poland. The analysed strains of B. pertussis were isolated from the nasopharynx of children with whooping cough in 1968 (n = 21) and in the years 1997–98 (n = 34). The B. pertussis isolates from 1968 were collected at the Pertussis Laboratory, National Institute of Hygiene and the strains isolated in the years 1997– 98 were collected at Warsaw Sanitary and Epidemiological Station. Macrolide standards of known potency were applied as follows: erythromycin (USP, Rockville, DC, USA, 945 U/mg), azithromycin (Pliva, Zagreb, Croatia, 96.9%), clarithromycin (Abbott Laboratories, Rungis Cedex, France, 992 U/mg), dirithromycin (Eli Lilly, Indiana, IN, USA, 96.9%), oleandomycin (Sigma, St. Louis, MI, USA, 95%), roxithromycin (Hoechst Marion Roussel, Paris La Defense Cedex, France, 100%), spiramycin (Rhone Poulenc Rorer, Antony Cedex, France, 4375 U/mg) and josamycin (Virbac, Carros, France, 999 U/mg). All B. pertussis isolates were stored in a freeze-dried form. The antibiotic agar dilution method was used. Before analysis the strains were subcultured twice on Bordet-Gengou agar supplemented with 30% sheep blood and

Clinical and laboratory diagnostic features of Bordetella spp. — pertussis and beyond

B. pertussis and B. parapertussis remain important human pathogens capable of causing endemic or epidemic pertussis in the community and nosocomial infection in the hospital setting. Infections due to the former organism can be particularly severe and life-threatening in the infant or young child. When encountered in the clinical laboratory, they should always be regarded as pathogenic. The availability of acellular vaccines with greater immunogenicity and fewer adverse effects than traditional whole-cell vaccines should contribute to higher immunization rates in children and fewer cases of disease. The use of these newer vaccines for periodic booster immunization in adults may further control pertussis. Newer amplification-based, nucleic acid detection methods will improve diagnosis, but these procedures will likely be available only in some pediatric reference centers. Culture isolation using Regan-Lowe, Bordet-Gengou, or newer agar media, coupled with serologic identification, will continue to be more commonly used. Notwithstanding the report of at least one erythromycin-resistant isolate of B. pertussis , antibiotics of the erythromycin class remain the drugs of choice for pertussis, and in vitro susceptibility testing for these organisms is neither standardized nor necessary. The remaining Bordetella spp. are infrequent agents of human disease. When encountered from human sources other than sterile body sites, the possibility that they are colonizers must be considered. As a group, they are readily recovered on general purpose microbiologic media, but their biochemical identification, with the exception of B. bronchiseptica , will present a challange. Again, except for B. bronchiseptica , there is little information available regarding the antimicrobial susceptibility of these organisms. Therefore, it is appropriate to attempt to perform dilution MIC testing when these organisms are involved in infection.

Influence of culture conditions on expression of antigens in Bordetella bronchiseptica.

Bordetella (B.) bronchiseptica isolates from the respiratory tract of rat and pig in their virulent phase-I and their spontaneously developed avirulent phase-III were investigated. The strains were cultured on Bordet-Gengou agar, on nutrition agar with 10% horse blood, and with 5% sheep blood, on yeast agar, on minimal nutrition agar (MM) and Simmon's citrate agar with glycerol, starch, and nicotinic acid. Antigenic modulation was induced by MgSO4 on Bordet-Gengou agar. The B. bronchiseptica strains were cultivated at 37 degrees C for 48 h. The influence of different MgSO4 concentrations after five passages (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100 mM/ml and low (20 degrees C) and high (42 degrees C) temperatures on antigenic modulation was investigated on Bordet-Gengou (B-G) agar. B. bronchiseptica colonies were characterized in relation to morphology, haemolysis, slide agglutination with B. bronchiseptica phase-I polyclonal antibodies, haemagglutination with horse and calf erythrocytes and polypeptide and LPS patterns in SDS-PAGE. Cultivation of B. bronchiseptica phase-I on B-G agar and ACMM agar at 37 degrees C for 48 h resulted in strong phase-I antigenic patterns, and, on peptone-rich media, in antigenic modulation. The morphology of B. bronchiseptica phase-I-strain colonies on peptone media was different from that of B-G and ACMM agar. The LPS pattern of both strains resembled that of phase-III strains. MgSO4 concentrations of 1 mM/ml (strain 1636 I) and 3 mM/ml (Ratte I) were able to induce LPS-pattern-like phase III.

Shelf life of prepared Bordet-Gengou and Regan-Lowe agar plates for isolation ofBordetella pertussis

The shelf life of prepared agar plates used for the isolation of Bordetella pertussis was studied. They contained Bordet-Gengou agar, Bordet-Gengou agar with cephalexin, Regan-Lowe agar, Regan-Lowe agar with cephalexin, or Regan-Lowe agar with both cephalexin and amphotericin B. Plates stored were compared to freshly prepared control plates for up to a maximum of 18 weeks. They were inoculated with clinical isolates of Bordetella pertussis, either in pure culture, or mixed with a defined oropharyngeal flora. Bordet-Gengou agar plates may be used, with proper storage at 4 degrees C in airtight-sealed plastic bags, for up to 10 weeks, Regan-Lowe agar plates for up to 14 weeks. Field studies are needed to substantiate our findings.

Rapid detection of Bordetella pertussis by a monoclonal antibody-based colony blot assay

Monoclonal antibodies to Bordetella pertussis filamentous hemagglutinin (FHA) and lipopolysaccharide (LPS) were used in a colony blot enzyme-linked immunosorbent assay designed for rapid detection of B. pertussis. Bacterial colonies from Bordet-Gengou agar plates were blotted onto nitrocellulose filter disks, lysed by immersion in chloroform, and reacted with monoclonal antibodies. Following reaction with peroxidase-conjugated rabbit anti-mouse immunoglobulin antisera and 4-chloro-1-naphthol, blue dots representing single colonies appeared on the filters. Blotting of single B. pertussis colonies could be performed after incubation for 40 h, i.e., before the colonies were visible by eye on the agar surface. Ten of ten B. pertussis strains showed positive blotting reactions with antibodies specific for B. pertussis FHA and LPS. Fourteen of fourteen B. parapertussis strains reacted with two of the FHA-specific antibodies but not with two of the LPS-specific antibodies. Strains of B. bronchiseptica showed a variable reaction pattern. No cross-reactions were observed with strains of Streptococcus mitis, S. pyogenes, S. pneumoniae, Staphylococcus aureus, Branhamella catarrhalis, or Klebsiella pneumoniae. This assay may be useful for identification of B. pertussis and B. parapertussis in suspected cases of whooping cough.

Isolation and characterization of mutant strains of Bordetella bronchiseptica lacking dermonecrotic toxin-producing ability.

Mutant strains of Bordetella bronchiseptica, named B-42, B-76, B-84, and B-119, were obtained after serial passages of a parent strain, L3, on Bordet-Gengou agar plates containing 20% horse blood and 200 micrograms of nalidixic acid per ml (BGN-20 agar plates) at 42 degrees C. Mutant strains completely lacked dermonecrotic toxin-producing ability, and lethal activity of the strains for mice was apparently reduced compared with that of strain L3. Mutant strains were able to grow at 42 degrees C, and the strains were nalidixic acid resistant. The mutant strains showed domed (Dom+) colony morphology with smooth texture (Scs+) and no production of zone of hemolysis (Hly-), but the agglutinability of these strains to antiserum prepared with Dom+ Scs+ Hly+ organisms of strain L3 was the same as that of strain L3. When strain B-42 was inoculated intramuscularly or intranasally into guinea pigs, all the animals survived without manifesting clinical signs and produced a high-level of serum agglutination antibodies against strain L3. These inoculated animals were protected against intranasal challenge with strain L3. These properties of mutant strains are hereditarily stable after 50 subcultures on BGN-20 agar plates or 20 passages in mice. These data suggest that the mutant strains lacking dermonecrotic toxin-producing ability can be used as a live attenuated vaccine against swine atrophic rhinitis.

Bordetella bronchiseptica phase variation induced by crystal violet.

A method for effective induction of phase variation in Bordetella bronchiseptica by treatment with crystal violet (CV) is presented. When grown in CV-broth, phase I cells dissociated into three serial phases. Appearance of variant cells was observed simultaneously with the beginning of cell multiplication. The maximum effect of CV was obtained at a concentration of 8 micrograms/ml, when the proportion of variants in the population reached 100%. The main factors which affected phase variation were concentration of CV, culture age, and temperature of treatment. The phase variants obtained were phenotypically stable upon serial passages on Bordet-Gengou agar plates. By this treatment, no reversion of phase descendants to former phases was observed.

Isolation of Bordetella pertussis: benefits of using both Bordet-Gengou and charcoal agar media.

Isolation of Bordetella pertussis: benefits of using both Bordet-Gengou and charcoal agar media.

Studies on culture conditions of Bordetella pertussis and relationship to immunogenicity of vaccines

Conditions for growing Bordetella pertussis bacteria in fermentors were studied for the purpose of producing highly protective vaccines. Bacteria with high protective potency were obtained in fermentors when the inoculum consisted of a dense suspension of young B. pertussis cells grown on Bordet-Gengou agar plates. When the time of cultivation in fermentors exceeded 30 h, bacteria harvested showed lower protective potency activity and lower levels of histamine sensitizing factor (HSF) and leukocytosis promoting factor (LPF), indicating that the optimal time for collection is at the end of the logarithmic growth phase. In fermentors, the ATP concentration in the bacterial cells increased from the time of inoculation until the end of the logarithmic growth phase. At the late logarithmic growth phase, the amounts of ATP, HSF, LPF and protective antigen declined rapidly. ATP kinetics thus provides a way of predicting the immunogenicity of vaccines. These results may be applicable to the production of vaccines exhibiting high protective activity at low bacterial concentrations, and to the preparation of soluble vaccines resulting from high-yield extraction of protective antigens. A correlation between the potency and LPF activity of vaccines was established for use in the rapid screening of vaccines for protective activity.

Phenotypic variation and modulation in Bordetella bronchiseptica.

Most of the isolates of Bordetella bronchiseptica obtained by this laboratory possessed a characteristic colonial morphology when grown on Bordet- Gengou agar (BGA) at 37 degrees C. The colonies appeared domed (Dom+) with a smooth colonial surface (Scs+) and a clear zone of hemolysis ( Hly +). From these Dom+ Scs+ Hly + BGA colony types arose flat (Dom-), smooth colonial surface (Scs+) and nonhemolytic ( Hly -) variants at frequencies of 10(-2) to 10(-3). Isogenic pairs of Dom+ Scs+ Hly + and Dom- Scs+ Hly - BGA phenotype variants (BGA-PVs) were picked from 11 strains of B. bronchiseptica, and their whole cell lysates were compared with each other by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Characteristic SDS-PAGE profiles were observed for each of the Dom+ Scs+ Hly + and Dom- Scs+ Hly - BGA-PVs with regard to (i) surface-exposed proteins, based on autoradiographs of 125I- Iodogen -labeled organisms, (ii) polypeptide differences, based on gels stained with Coomassie brilliant blue R-250, and (iii) lipopolysaccharide differences based on gels stained with silver after oxidation with periodic acid. SDS-PAGE profiles were then used to monitor the phenotypes expressed by Dom+ Scs+ Hly + and Dom- Scs+ Hly - BGA-PVs transferred and grown on brucella agar, Trypticase soy agar, and nutrient agar. When grown on non-BGA media, the Dom+ Scs+ Hly + BGA-PVs from six of eight strains showed SDS-PAGE profiles identical to those of Dom- Scs+ Hly - BGA-PVs. This phenotypic change was reversible even after 15 subcultures on the non-BGA media, since Dom+ Scs+ Hly + organisms passed back onto BGA expressed both Dom+ Scs+ Hly + colonial morphology and Dom+ Scs+ Hly + SDS-PAGE profiles. The influence of cultural conditions on maintenance of virulence is discussed.

Isolation and characterization of Bordetella pertussis phenotype variants capable of growing on nutrient agar: comparison with phases III and IV.

Unsupplemented nutrient agar (NA) was used to select spontaneous phenotype variants (PVs) of Bordetella pertussis Tohama I and 3779 which, by their growth on NA, could possibly be considered equivalent to phase IV in the system of Leslie and Gardner (P.H. Leslie and A.D. Gardner, J. Hyg. 31:423-434, 1931) or phase III in the system of Kasuga et al. (T. Kasuga, Y. Nakase, K. Ukishima, and K. Takatsu, Kitasato Arch. Exp. Med. 26:121-134, 1953). NA growers (Gna+) were selected from the flat, nonhemolytic, non-NA grower (Dom- Hly- Gna-) PV of both strains at a rate of between 10(-7) to 10(-8) per cell per generation. When cultured on Bordet-Gengou agar (BGA), more than one colony type was observed in strain 3779; these all retained the Gna+ characteristic during 10 to 30 passages on BGA. Analysis of 125I-surface-labeled whole cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed no major changes between the Dom- Hly- Gna+ PV and their Dom- Hly- Gna- PV parents in polypeptide profile (by Coomassie stain), in surface exposure of proteins (by autoradiography), or in lipopolysaccharide profile (by silver stain). Increased resistance to oleic acid, tetracycline, erythromycin, rifampin, and penicillin G, however, was characteristic for the Dom- Hly- Gna+ PV. Five phase IV strains and a phase III B. pertussis strain had similar antibiotic and oleic acid sensitivity profiles as the Dom- Hly- Gna+ isolates and plated with similar efficiency on NA, despite heterogeneity in BGA colonial morphology and lipopolysaccharide profile.

Isolation and characterization of isogenic pairs of domed hemolytic and flat nonhemolytic colony types of Bordetella pertussis.

Four different serotype strains of Bordetella pertussis, 3779BL(2)S(4), Tohama I, 353/Z, and 2753, were plated on Bordet-Gengou agar, where they grew as domed, hemolytic (D(+)H(+)) wild-type colonies. Cloned D(+)H(+) colony types of all four strains were passed onto modified Stainer-Scholte medium solidified with 1% Noble Agar. Colonies were selected from Stainer-Scholte agar, and these subsequently grew as flat, nonhemolytic (D(-)H(-)) colonies when transferred back onto Bordet-Gengou agar. The frequency of D(-)H(-) organisms within a population of cloned D(+)H(+) was determined to be between 5 x 10(-5) and 5 x 10(-6). The D(-)H(-) colony types maintained their flat, nonhemolytic characteristics for over 80 single-colony passages on Bordet-Gengou agar. The isogenic pairs of D(+)H(+) and D(-)H(-) colony types from the four strains were compared for hemagglutination titer, lymphocytosis-promoting activity, adenylate cyclase activity, and presence of agglutinogens by agglutination. In all cases the D(-)H(-) colony types showed reduced activities or amounts of antigen compared with their D(+)H(+) parents. Freely diffusible antigens were markedly different between the two phenotypes as noted by double diffusion of antisera added to plates on which colonies of the variants were growing. Antigens solubilized from the two colony types by Triton X-100 were also markedly different as judged by radial immunodiffusion with antifimbrial hemagglutinin, antilymphocytosis-promoting factor, and anti-353/Z adsorbed with autoclaved 353/Z. In addition, autoradiographs of (125)I-surface-labeled whole cells separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed unique banding patterns for each colony type. Since all organisms, regardless of colony type, were grown on Bordet-Gengou agar, the differences reported could not be due to medium composition. Differences between phenotypes were also independent of passage number on Bordet-Gengou agar. By analogy to previous studies, the D(-)H(-) organisms appear to fulfill the criteria for phase III or phase IV in the system of Leslie and Gardner (P. H. Leslie and A. D. Gardner, J. Hyg. 31:423-434, 1931) or phase III in the system of Kasuga et al. (T. Kasuga, Y. Nakase, K. Ukishima, and K. Takatsu, Kitasato Arch. Exp. Med. 26:121-134, 1954).