Trypticase soy agar

The CA-MRSA USA300 isolate LAC³⁸ (generously provided by Dr. Frank DeLeo, Rocky Mountain National Laboratories, National Institutes of Health, Hamilton, MT) was used for infection studies. Early exponential-phase bacteria were prepared as previously described^{119 (link)} and stored at −80 °C for no more than two weeks prior to use. For infection studies, bacteria were diluted in USP-grade saline (B. Braun Medical, Irvine, CA) to yield 4 × 10⁷ CFU per 50 µL. The number of CFU was verified by plating ten-fold serial dilutions onto Trypticase soy agar containing 5% sheep blood (Becton, Dickinson and Company; Franklin Lakes, NJ).

Bacterial strains and plasmids used in this study are listed in Supplementary Table 1. Strain MGAS10870 is a previously described invasive serotype M3 isolate whose genome has been fully sequenced^{67 (link)}. MGAS10870 is representative of serotype M3 strains that cause invasive infections and has wild-type sequences for all known major regulatory genes^{67 (link)}. Escherichia coli DH5α strain was used as the host for plasmid constructions and BL21(DE3) strain was used for recombinant protein overexpression. GAS was grown routinely on Trypticase Soy agar containing 5% sheep blood (BSA; Becton Dickinson) or in Todd–Hewitt broth containing 0.2% (w/v) yeast extract (THY; DIFCO). When required, kanamycin or ampicillin was added to a final concentration of 50 or 100 µg/ml, respectively. Chloramphenicol was used at a final concentration of 15 µg/ml. All GAS growth experiments were done in triplicate on three separate occasions for a total of nine replicates. Overnight cultures were inoculated into fresh media to achieve an initial absorption at 600 nm (A₆₀₀) of 0.03. Bacterial growth was monitored by measuring the absorption at 600 nm (A₆₀₀). The E. coli strain used for protein overexpression was grown in Lysogeny broth (LB broth; Fisher).

For in vitro growth, GAS strains were grown in Todd-Hewitt broth (Becton, Dickinson [BD]) supplemented with 0.2% yeast extract (THY medium). This medium was used (i) because it is a standard medium for GAS growth used by many investigators and (ii) because there is no medium that adequately mimics in vivo conditions. THY medium was supplemented with spectinomycin (150 μg ml⁻¹) as needed. Escherichia coli strains were grown in Luria-Bertani (LB) medium at 37˚C. LB medium was supplemented with spectinomycin (50 μg ml⁻¹), as needed. Trypticase soy agar supplemented with 5% sheep red blood cells (Becton, Dickinson and Co.) was also used for growth of GAS strains. Spectinomycin dihydrochloride pentahydrate was purchased from Sigma. Gap electroporation cuvettes (2-mm gap size) were purchased from BTX Harvard apparatus. Growth experiments were performed as described previously (93 (link)). Locus tag equivalence between strains MGAS5005 and MGAS2221 is shown in Table S1J in the supplemental material.

At varied days of gestation in the first trimester (between days 36 to 50) or third trimester (between days 110 to 135 [full term is day 165]), monkeys were sedated, the uterus was examined by ultrasound to confirm a viable pregnancy, and a single dose between 10⁶ and 10⁸ colony forming units (CFUs) log-phase cells of strain LM2203 (serotype 4b, derived from an outbreak of listeriosis among pregnant women in Winston-Salem, North Carolina in the year 2000 MacDonald et al., 2005 (link)), were administered in 10 ml of whipping cream via a soft intragastric feeding tube as previously described (Smith et al., 2003 (link)). For each inoculation, Lm was cultured at 37°C in Tryptic Soy Broth (Becton Dickinson, Sparks, MD). 500 ul of the inoculum was diluted in phosphate-buffered saline (PBS; Catalog #P5368, Sigma-Aldrich, St. Louis, MO), plated on Trypticase soy agar with 5% sheep blood (Becton Dickinson, Sparks, MD), and incubated at 37°C to confirm the dose given to each animal as CFUs of Lm per milliliter. Six monkeys were given a whipping cream inoculum without Lm in an identical manner to be included as uninfected controls.

Sample preparation followed the protocol described previously [11 (link),24 (link)]. Each isolated sample was collected into a sterile tube or container (except blood culture samples, which were inoculated immediately in blood culture-specific bottles (BD BACTEC^TM; Becton Dickinson, Franklin Lakes, NJ, USA), sent to the laboratory within two to three hours of collection, and maintained at room temperature. Samples that were not inoculated immediately during a non-working period were stored at room temperature, except urine and CSF, which were maintained at 4 °C and 35 °C, respectively. All bacterial culture samples were inoculated within 24 h of isolation. For sputum cultures, initial Gram stains for quality scores (<10 squamous epithelial cells per low power field) were checked before reporting final results. For urine samples, quantitative cultures using sterile loops were established, and the reporting criteria were >10⁵ and >10³ CFU for midstream and indwelling catheters, respectively. All specimens retrieved from patients were streaked across Trypticase soy agar with 5% sheep blood (TSA II)/Levine eosin methylene blue EMB agar (Becton Dickinson) and incubated at 37 °C. Bacterial isolates were identified as ACB and P. aeruginosa by use of an automated microbiologic analysis system (BD Phoenix; Becton Dickinson) [10 (link)].

A total of nine clinical Enterobacterales isolates were obtained from the Centers for Disease Control and Food and Drug Administration Antimicrobial Resistance Isolate Bank (n = 1), the Antibacterial Research Leadership Group isolate bank (n = 5), the American Type Culture Collection (n = 2), and the Center for Anti-Infective Research and Development (CAIRD) isolate library (n=1). Escherichia coli (n = 5) and Klebsiella pneumoniae (n = 4) isolates were selected for their ESBL-harboring and wild-type genotypes. The ceftibuten broth microdilution MICs ranged from 0.03–4 mg/L. The individual isolate genotype and ceftibuten MICs are available in Table 4. Notably, one K. pneumoniae isolate (KP 956) was characterized as SHV-harboring, but displayed the phenotypic profile (ceftibuten MIC: 0.03 mg/L) of a wild-type isolate and thus was included in the wild-type isolate analysis. All of the isolates were stored frozen at −80 °C in skim milk. Prior to examination, each isolate was sub-cultured twice on trypticase soy agar with 5% sheep blood (Becton Dickinson and Co., Sparks, MD, USA) and incubated at 37 °C for 18–24 h.