Todd hewitt broth thb

The virulent S. suis strain ZY05719 of serotype 2 was used in this study. This strain was isolated in Ziyang, China, in 2005 and has been confirmed to form biofilms in vitro. Escherichia coli DH5α was used as the host strain for plasmid construction, replication, and preservation. The plasmid pMD19-T vector was used as the source for rRNA genes (16S rRNA and 23S rRNA), and SCOTS clones were prepared in pMD19-T. S. suis was cultured on Todd-Hewitt broth (THB) (Oxoid Ltd., Basingstoke, UK) or on Todd-Hewitt agar (THA) at 37°C. E. coli DH5α was cultured routinely in Luria-Bertani broth or plates (Oxoid, Basingstoke, UK) at 37°C. Ampicillin (50 μg/mL), IPTG (isopropyl β-d-thiogalactopyranoside) (100 μg/mL), and X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) (200 μg/mL) in LB medium were used as required. All primers used in this study are shown in Table S1. Healthy, S. suis-free, 30-day-old female piglets were obtained from a farm free of relevant swine pathogens. Piglets were randomly divided into two groups: uninfected (n = 1) and infected (n = 5).

A total of 32 GAS isolates, including 27 erythromycin-resistant [minimum inhibitory concentration (MIC) ≥ 1 μg/mL] and 5 erythromycin-susceptible strains isolated throughout Italy from children with pharyngitis (Varaldo et al., 1999 (link)), were examined. All strains had previously been characterized (Facinelli et al., 2001 (link); Spinaci et al., 2004 (link), 2006 (link)) in terms of erythromycin resistance phenotype/genotype [erm(B)/cMLS (n = 6); erm(B)/iMLS (n = 5); erm(TR)/iMLS (n = 6); mef(A)/M (n = 10)]; emm type (12 different emm types); the presence of the prtF1 gene, and cell invasiveness. Each of the 32 strains is a clone identified among Italian GAS isolates.
Blood agar base (BAB) supplemented with 5% sheep blood, Müller-Hinton agar (MHA) supplemented with 5% sheep blood, Müller-Hinton cation-adjusted broth (CAMHB) supplemented with 3% laked sheep blood, brain heart infusion (BHI) agar and broth, Todd-Hewitt broth (THB) and Tryptone Soya Broth (TSB), all from Oxoid (Basingstoke, UK) were used throughout the study. Isolates were maintained in glycerol at –70°C and subcultured twice on BAB before testing.

The bacterial strains and plasmids used in this study are listed in Table 1, strain SC19 was isolated from a diseased pig during an epidemic outbreak in 2005 in Sichuan, China (Li et al., 2009 (link)). The mnmE deletion mutant of SC19 (ΔmnmE) obtained from our previous research were also used in this study (Gao et al., 2019 (link)). The SS2 were grown in Todd-Hewitt broth (THB; Oxoid, Basingstoke, England) or on THB agar (THA; Oxoid, Basingstoke, England) plates supplemented with 5% sheep blood (Maojie, Nanjing, China) at 37°C. The arginine metabolic pathway study was performed using a chemically defined medium (van de Rijn and Kessler, 1980 (link)). Escherichia coli (E. coli) strain DH5α (Vazyme, Nanjing, China) used as host strain for cloning, were grown in LB broth (Difco Laboratories, Franklin Lakes, NJ, USA) or on LB agar plates at 37°C. If necessary, erythromycin (90 μg/ml), spectinomycin (100 μg/ml), and streptomycin (20 μg/ml) were supplemented to promote bacterial selection.

Bacterial strains used in this study are described in Table 2. S. mutans Xc (Koga et al., 1989), a serotype c wild‐type (WT) strain, was a kind gift of Dr. Y. Yamashita (Kyushu University, Japan) and was routinely grown in Todd‐Hewitt Broth (THB, Oxoid) or on THB agar at 37°C with 5% CO₂. When appropriate, S. mutans was cultured with 10 µg ml⁻¹ erythromycin (ERY) or 3 µg/ml chloramphenicol (CHL). GAS 5448, a representative of the epidemic M1T1 clone, was cultured in Todd‐Hewitt Broth (Becton Dickinson) supplemented with 1% yeast extract (THY; Oxoid) or on THY agar at 37°C. E. coli MC1061 was used for cloning purposes and was grown in lysogeny broth (LB, Oxoid) or on LB agar containing 10 µg/ml CHL at 37°C. Mycobacterium tuberculosis laboratory strain H37Rv was routinely cultured in Middlebrook 7H9 broth (Difco) supplemented with 10% (v/v) Albumin Dextrose Catalase (ADC) enrichment (Difco), 0.05% (v/v) tyloxapol and 0.02% (v/v) glycerol. Liquid cultures were grown at 37°C in 50 mL centrifugation tubes with rotation of 40 rpm until mid‐exponential phase (OD₆₀₀ ~ 1). For solid growth drug susceptibility experiments, M. tuberculosis H37Rv was grown in Middlebrook 7H10 (Difco) agar medium supplemented with 10% (v/v) of Oleic acid ADC enrichment (OADC) (Difco) and 0.5% (v/v) glycerol (c7H10) at 37°C incubator.

Streptococcus pneumoniae was grown in Todd-Hewitt broth (THB; Oxoid, Basingstoke, Hampshire, UK) supplemented with 0.5% yeast extract (Oxoid, Basingstoke, Hampshire, UK), brain heart infusion (BHI) broth (Oxoid, Basingstoke, Hampshire, UK), or on blood agar (BA) plates (Isolab, Shah Alam, Selangor, Malaysia) at 37 °C and in 5% CO₂. For storage, S. pneumoniae were grown in BHI broth at 37 °C to an OD₆₀₀ of around 0.5, and 800 μL culture was frozen with the addition of 200 μL 80% glycerol (final concentration of glycerol in the stock media was 20%) and kept at −80 °C.

For infection experiments, S. pneumoniae serotype 6B and 19F were cultured on blood agar at 37 °C for 18 h, followed by a passage onto Todd Hewitt broth (THB, Oxoid, Cambridge, UK) overnight at 37 °C. Pneumococci were harvested 3600 g for 10 min and washed with sterile PBS. The challenge of animals was performed via the nasal route with 10⁴, 10⁵, or 10⁶ CFU of pneumococci per mouse, as described above. S. pneumoniae counts in lung and blood were determined on day 2 or 7 post-infection [11 (link)].
After euthanasia, lungs were excised, weighed, and homogenized in sterile peptone water. The respective homogenates were diluted and plated onto blood agar for bacterial counts (CFU log//g lung). Bacteremia was reported as either negative or positive.

S. aureus, S. capitis, S. carnosus, S. epidermidis, S. lugdunensis, S. pseudintermedius, S. saprophyticus, and S. simulans strains (46 (link)– (link)55 (link)) (see Table S1 in the supplemental material) were grown overnight at 37°C with agitation in 5 ml Todd-Hewitt broth (THB; Oxoid). For S. aureus strains that were plasmid complemented, THB was supplemented with 10 μg/ml chloramphenicol (Sigma-Aldrich). Overnight cultures were subcultured the next day in fresh THB and grown to an optical density at 600 nm (OD₆₀₀) of 0.4 for S. capitis and an OD₆₀₀ of 0.6 to 0.7 for all other bacteria, which correspond to mid-exponential growth phases.