Bacterial strains and human clinical specimens.
Throat swab specimens from the posterior pharynx were collected from 18 pediatric patients presenting with signs and symptoms consistent with acute pharyngitis at a clinic in Houston, Tex. The swabs were immediately cultured to confirm GAS pharyngitis and frozen on dry ice. The GAS strains and throat swabs were shipped on dry ice by commercial courier to Rocky Mountain Laboratories, Hamilton, Mont. The emm
type of each GAS strain was determined by methods described previously (35
). The 18 strains included emm2
= 1 strain), emm4
= 2 strains), emm6
= 2), emm12
= 3), emm28
= 2), emm75
= 3), emm77
= 2), and emm89
= 3). The study protocol was approved by the Human Subjects Institutional Review Board at Baylor College of Medicine and Affiliated Hospitals. Written informed consent was obtained from all human subjects.
Strain MGAS5005 (serotype M1) was used for the nonhuman primate infection studies. This organism is genetically representative of serotype M1 isolates obtained from patients with pharyngitis and invasive infections in the United States, Canada, and western Europe (22
). It has been characterized extensively genetically and used in mouse models of infections and in vitro studies (16
Nonhuman primates and experimental inoculation.
The study protocol was approved by the Animal Care and Use Committee, Rocky Mountain Laboratories. Three cynomolgus macaques were used, including a juvenile female (15 months, 2.2 kg), an adult female (6 years, 8 months; 3.3 kg), and an adult male (9 years, 6 months; 7.1 kg). Two throat swabs and one venous blood specimen were collected from each animal on days 0, 2, 4, 7, 9, and 11 of the study. Day 0 samples were obtained immediately before inoculation with GAS. One swab was used to determine the level of GAS CFU by colony count measurement after overnight growth on blood agar plates, and a second swab was used to extract GAS RNA. Bacterial colonies with a morphology consistent with GAS were verified as such by sequencing of the emm gene. Plasma was separated from whole blood by centrifugation at 200 × g for 10 min.
Preinoculation throat swab specimens were collected by swabbing the tonsils vigorously with a sterile cotton applicator and culturing overnight on sheep blood agar plates. None of these specimens grew GAS. Strain MGAS5005 (serotype M1) used for experimental inoculation was cultured overnight on sheep blood agar plates at 37°C in 5% CO2, seeded into 11-ml of prewarmed Todd-Hewitt broth supplemented with 0.2% yeast extract (THY), and grown overnight at 37°C in 5% CO2. The overnight growth was subcultured to prewarmed THY broth and incubated for 5 h to late exponential phase (i.e., an optical density at 600 nm of 0.5). The culture was centrifuged, and the bacteria were suspended in pyrogen-free sterile phosphate-buffered saline (PBS) to a concentration of 107 CFU/ml. The viable bacterial cell count was verified by plating on sheep blood agar. Each monkey was anesthetized with ketamine and inoculated by dribbling 1 ml of the bacterial suspension slowly into the nares. GAS colony counts were obtained by culturing throat swabs taken on days 0, 2, 4, 7, 9, and 11. The swabs were immersed in 300 μl of sterile PBS, diluted serially in sterile PBS, plated onto sheep blood agar, and cultured overnight at 37°C in 5% CO2.
Clinical observations of nonhuman primates inoculated with GAS.
To investigate the cynomolgus macaque as a model of acute pharyngitis, clinical observations were made by one attending veterinarian. Pharyngeal erythema, tonsil size, presence of cervical lymphadenopathy, skin condition, weight, and eating behavior were evaluated. A grading scheme was used to estimate pharyngitis severity and tonsil size. Pharyngitis was scored as follows: mild erythema with hyperemic blood vessels (+1), more intense erythema and palatal petechiae (+2), and intense erythema with palatal petechiae and exudative tonsillitis (+3). Feinstein and Levitt (9
) have established criteria for scoring tonsil enlargement during human GAS pharyngitis (0 to +4). The same criteria were used except cynomolgus macaque tonsils were scored from +1 to +4 since healthy macaque tonsils resemble slightly enlarged tonsils (+1) in humans.
Extraction of GAS RNA from throat swabs.
Total RNA was extracted from throat swabs with the FastPrep FP 120 kit (Qbiogene, Carlsbad, Calif.) by immersing the swab tips directly into the FastPrep Blue tubes containing 300 μl of 5 mM ammonium aurintricarboxylate, 500 μl of CRSR-Blue (Qbiogene), and 500 μl of acid phenol-chloroform (5:1, pH 4.5; Ambion, Austin, Tex.). The mixture was homogenized (80 s at speed 5), heated at 65°C for 20 min, and centrifuged at 16,000 × g for 15 min. The aqueous phase was collected, 250 μg of glycogen (Roche Applied Science, Indianapolis, Ind.) was added, and the mixture was concentrated to 100 μl with a vacuum concentrator (Eppendorf). The concentrate was purified by using the Qiagen RNeasy kit (Qiagen, Valencia, Calif.) according to the manufacturer's protocol (Qiagen). Contaminating DNA was removed by DNase I treatment (DNA-free; Ambion). To ensure that contaminating DNA was absent, an aliquot of RNA from each sample was subjected to 40 cycles TaqMan real-time PCR for the proS gene. All swabs analyzed yielded a PCR product for the proS gene when the proS primers were tested against cDNA synthesized from the purified swab RNAs, indicating that the proS gene primer sites were conserved in all GAS strains. The RNA was treated with DNase I (Ambion) until no signal was detected by TaqMan real-time reverse transcription-PCR (RT-PCR) by using the conserved proS gene as a target. None of the GAS primers (Table ) cross-hybridized to cDNAs synthesized from RNA made from the oral flora present in the monkeys (data not shown).
TaqMan real-time RT-PCR primers, probes, and their sequences
TaqMan real-time PCR assay.
The sequences of the primers and probes used in the present study are listed in Table . The Superscript II Choice system (Invitrogen, Carlsbad, Calif.) was used for cDNA synthesis as described by the manufacturer. RNA purified from each swab was divided into four aliquots, to which 0.8 μg of bacteriophage MS2 carrier RNA (Roche) was added to each aliquot, and reverse transcribed with 1.5 μg of random hexamer primers at 42°C for 1 h. The cDNA samples were treated with RNase H−
(Invitrogen) for 1 h and diluted with water to 100 μl. TaqMan 5′ nuclease real-time PCR assays (Applied Biosystems, Foster City, Calif.) were carried out in a 384-well format with an 7900HT instrument (Applied Biosystems) in 10-μl reactions containing 1× universal master mix, 100 nM 6-carboxy-4′5′-dichloro-2′7′-dimethylfluorescein (JOE) and QSY-7 labeled putative prolyl-tRNA synthetase (proS
, Spy1962; Table ), 200 nM concentrations of target forward and reverse primers, and 100 nM concentrations of the target TaqMan oligonucleotide for 50°C for 2 min and 95°C for 10 min and then for 40 cycles of 95°C for 15 s and 60°C for 1 min. All TaqMan oligonucleotide probes were labeled with 6-carboxyfluorescein (6-FAM) at the 5′ end and the quencher carboxytetramethylrhodamine (TAMRA) at the 3′ end. The comparative CT
method was used to determine the ratio of target and endogenous control (Applied Biosystems) as described previously (16
Characterized or putative GAS regulatory and virulence genes analyzed in vivo
Measurement of antibody response by the nonhuman primates to GAS protein antigens.
Antibody titers to streptococcal inhibitor of complement (Sic; Spy2016), a GAS homolog of Listeria monocytogenes internalin A (InlA; Spy1361), and streptolysin O (SLO; Spy0167) were measured by enzyme-linked immunosorbent assay (ELISA). Sic and InlA were purified at Rocky Mountain Laboratories, and SLO was purchased from Sigma (St. Louis, Mo.). Sic and InlA were diluted to 2.5 μg/ml in buffer (0.05 M Tris, pH 7.5; 0.15 M NaCl), and SLO was diluted to 10 μg/ml. Microtiter plates (Immulon-2; Dynex Technologies, Inc., Chantilly, Va.) were coated (100 μl) overnight at 4°C, washed with washing buffer (0.05 M Tris, pH 7.5; 0.15 M NaCl; 0.5% Tween 20), and blocked with 2% bovine serum albumin in washing buffer for 2 h at 37°C. After the washing step, 100 μl of plasma was serially diluted in washing buffer with 2% bovine serum albumin and added to the wells, and the mixtures were incubated at 37°C for 1 h. Plates were washed again, and a 1:500 dilution of 100 μl of alkaline phosphatase-labeled goat anti-monkey immunoglobulin G (Rockland, Gilbertsville, Pa.) was added to the wells, followed by incubation for 1 h at 37°C, and 100 μl of substrate (p-nitrophenyl phosphate; Zymed, South San Francisco, Calif.) was added. The absorbance at 405 nm was measured with an ELISA reader (Dynex Technologies, Inc.). ELISA titers are expressed as the reciprocal of plasma dilutions giving an absorbance threshold value of ≥0.3. Preinoculation plasma was used as a negative control, and the ELISA absorbance value of this sample was <0.1.