Bacterial strains, culture media, and antibiotics

Spn reference strains and mutants used in the present study were: TIGR4 [21], SPJV41 [22, 23], D39 [24], EF3030 [25], S19F 4924 [26] and S6B 8655 [27]. Strains were routinely cultured on blood agar plates (BAP), or grown in Todd Hewitt broth containing 0.5% (w/v) yeast extract (THY), at 37 °C with a 5% CO2 atmosphere. Where indicated, streptomycin (Str, 200 µg/ml), trimethoprim (Tmp, 10 µg/ml), tetracycline (Tet, 1 µg/ml), or/and erythromycin (Ery, 1 µg/ml) was added to BAP. All antibiotics were purchased from Millipore-Sigma Sigma-Aldrich.

Preparation of inocula

The inoculum was prepared as previously described [28]. Briefly, bacteria were inoculated on blood agar plates (BAP) and incubated overnight at 37 °C in a 5% CO2 atmosphere. Bacteria were then harvested from plates by PBS washes and this suspension was used to inoculate THY or cell cultures, that was brought to a final OD600 of ∼0.1. This suspension contained ∼5.15 × 108 cfu/ml as verified by serial dilution and platting of aliquots of the suspension.

Cell cultures

Human pharyngeal Detroit 562 cells (ATCC CCL-138) were cultured in Eagle’s minimum essential medium (MEM) (Gibco) supplemented with non-heat-inactivated 10% fetal bovine serum (FBS) from R&D systems, 1% nonessential amino acids (Gibco), 1% glutamine (Gibco), penicillin (100 U/ml), and streptomycin (100 µg/ml), and the pH was buffered with HEPES (10 mM) (Gibco). Cells were harvested with 0.25% trypsin (Gibco), resuspended in the cell culture medium, and incubated at 37 °C in a 5% CO2 humidified atmosphere.

Fluorescent labeling of anti-capsule antibodies

Antibodies were purchased from the Staten Serum Institute (Denmark). The following antibodies were used throughout this study: Omni antiserum (recognizes 92 serotypes), type serum 2, type serum 4, group serum 6, and group serum 19. Serum was depleted of proteins less than 100 kDa using an Amicon Ultra-100 K centrifugal filter (Millipore-Sigma), washed three times with PBS. Proteins were quantified using Bradford reagent, and 1 mg of the antibodies was labeled with either Alexa-488 or Alexa-555 following the manufacturer’s recommendations (Molecular Probes). After purification by size exclusion chromatography (≥ 40 kDa), collected fractions were assessed for reactivity against the specific serotype, serogroup, or several serotypes for the Omni antiserum. Proteins were quantified from reactive fractions using the Bradford assay, and antisera were stored at 4 °C for short-term or -20 °C for long-term storage. Labeled antibodies stored at 4 °C remained reactive for up to three months, while those frozen have been effective in labeling bacteria for over three years. For the sake of simplicity, the fluorescently labeled Omni antiserum is referred to as Spn-FLUO throughout this manuscript.

In vitro imaging of pneumococci

Pneumococci were inoculated into 8-well glass slides (Lab-Tek®) and incubated for 4 h at 37 °C in a 5% CO2 atmosphere. Some slides contained cultures of Detroit 562 cells (ATCC CCL-198) grown to confluence, typically 7 days post seeding. Pneumococci attached to the glass substratum or human cells were then washed twice with PBS and fixed with 2% PFA for 15 min at room temperature. After removing the 2% PFA, the bacteria were washed with PBS and blocked with 2% bovine serum albumin (BSA) for 1 h at 37 °C. The pneumococci were then incubated with the specified labeled antibodies (∼20 µg/ml) for 1 h at room temperature. The stained preparations were subsequently washed twice with PBS and mounted with ProLong Diamond Antifade Mountant with DAPI (Molecular Probes). In some preparations, DAPI was replaced with TO-PRO-3 (1 µM), a carbocyanine monomer nucleic acid stain (Molecular Probes). Fluorescence images were acquired using an upright, epi-fluorescence Nikon Ni-U Research Microscope equipped with a Nikon DS-Qi2 sCMOS Camera system. Confocal images were obtained using a Nikon C2 laser scanning confocal microscopy system, and the micrographs were analyzed with ImageJ version 1.49k (National Institutes of Health, USA) or Imaris software (Bitplane).

Mouse models of pneumococcal nasopharyngeal carriage and disease

Inbred 6–7 week-old C57BL/6 mice -pneumococcal carriage-, or 4–5 week-old Balb/c mice -pneumococcal pneumonia- model (Charles River Laboratories) were anesthetized with 5% isoflurane (vol/vol) in oxygen (2 L/min) administered via an RC2 calibrated vaporizer (VetEquip Inc) and then infected with ∼1 × 105 CFU of strain Spn EF3030 (carriage model) or ∼1 × 108 cfu of TIGR4 or SPJV41 (pneumonia model). The animals were weighed daily, and their behavior and appearance were monitored twice daily for up to four days. Mice in the pneumonia model were euthanized when they lost ≥ 20% of their body weight, compared to their body weight before infection, or when mice were non-responsive to manual stimulation, and/or if they show signs of illness such as ruffled fur, intermittent hunching, and exhibiting respiratory distress. Mice were then euthanized, and the upper airways, including nasopharyngeal tissue or the lungs, were aseptically collected, placed in THY with 10% glycerol and immediately homogenized. For fluorescence staining (explained below), the nasopharynx from some mice was collected and fixed with 10% PFA. The specimens were then stored at -80 °C. This aliquot was used for bacterial counts and detection with labeled antibodies. To obtain bacterial counts, the homogenized tissue was serially diluted in PBS and plated onto BAP with gentamicin. The Institutional Animal Care and Use Committee (IACUC) at the University of Mississippi Medical Center approved the protocol used in this study (1584), overseeing the welfare, well-being, and proper care of all mice utilized in this study. All mouse experiments followed the guidelines outlined in the National Science Foundation Animal Welfare Act (AWA).

Fluorescence staining of nasopharyngeal tissue

The PFA-fixed nasopharynxes were paraffin-embedded, sectioned (∼5 μm), mounted on a slide and deparaffinized. Tissues were washed three times with PBS and the preparations were blocked with 2% bovine serum albumin (BSA) for 1 h at room temperature. These preparations were then incubated for 1 h with serogroup-specific (S19) polyclonal antibody (Statens Serum Institute, Denmark) (20 µg/ml) that had been previously labeled with Alexa-555 (Molecular Probes). Stained tissues were finally washed three times with PBS, air dried and then mounted with ProLong Diamond Antifade mounting medium containing DAPI (Molecular Probes). Confocal micrographs were obtained using a Nikon C2 laser scanning confocal microscopy system and analyzed using the Imaris software (Bitplane).

Specimens from humans with pneumococcal pneumonia

Human specimens, including sputum and bronchoalveolar lavages from de-identified patients with microbiologically and radiologically confirmed pneumococcal pneumonia, were utilized in this study. Institutional Review Board (IRB) exemption was obtained for the use of these de-identified specimens. When available, the age of the patients ranged from 19 months through 60 years. The specimens were collected according to institutional guidelines by the University of Mississippi Medical Center’s clinical laboratory and stored at -80 °C until processed. As part of the microbiological diagnostics, the pathology laboratory at UMMC isolated a Spn strain from each specimen. Additionally, the Spn etiology was confirmed by lytA-based real-time PCR, as detailed [29, 30] and briefly described below.

Human nasopharyngeal specimens

Human nasopharyngeal specimens (N = 50) utilized in the current study had been collected from Peruvian children and processed for pneumococcal detection and quantification by qPCR and culture in previous studies [31, 32]. Children enrolled in the mentioned studies were aged 0–3 years of age; details on the study population have been published elsewhere [32,33,34]. De-identified specimens were processed at Emory University, obtaining IRB exemption. Briefly, these nasopharyngeal samples were collected following WHO recommendations [35] with a deep NP swab, using rayon polyester swabs and were immediately placed in 2.0 ml cryogenic vials with 1 ml of transport medium, a mixture containing skim milk-tryptone-glucose-glycerin (STGG) and then stored at -80 °C [36].

Identification of pneumococcus using Spn-FLUO in mouse tissue or human specimens

Specimens were thawed on ice and an aliquot (10 µl) of each specimen was immediately mixed with Spn-FLUO (50 µg/ml) and incubated for 10 min at room temperature in the dark. Five µl of this suspension was dropped onto a microscope slide and covered with a coverslip. The stained sample was analyzed within five minutes using an upright, epi-fluorescence Nikon Ni-U Research Microscope equipped with a Nikon DS-Qi2 sCMOS Camera system. We first situated the specimen in the correct optical plane (i.e., visualizing cells, tissue, and/or bacteria) with the 60x objective and a bright light source. The slides were then analyzed and scored using epifluorescence in the green channel, following a semiquantitative algorithm: 1–5 pneumococci per field in at least three fields corresponded to “+”, 5–10 pneumococci per field to “++”, and more than 10 pneumococci per field was scored as “+++”. Negative samples had no pneumococci observed. Specimens were analyzed in a blinded fashion by personnel with varying levels of expertise (e.g., laboratory technicians, PhDs, MDs, MD-PhDs). The assessments consistently yielded similar outcomes, regardless of the analyzer’s qualifications, for both positive and negative samples. Micrographs from positive specimens were obtained using the 60x and 100x objectives.

Staining and confocal analysis of human sputum

An aliquot of sputum sample (10 µl) was dropped onto a slide and air dry for ∼20 min at room temperature. The sputum was then stained for 1 h with Spn-FLUO (20 µg/ml) and wheat germ agglutinin conjugated to Alexa-555 [(WGA), 5 µg/ml]. The stained specimen was washed three times with PBS, air-dried, and mounted with ProLong Diamond Antifade mounting medium containing DAPI (Molecular Probes). Confocal micrographs were obtained using a Nikon C2 laser scanning confocal microscopy system and z-stacks micrographs were analyzed using the NIS Elements Basic Research software, version 4.30.01 build 1021. For 3D visualization, creation of animations, and quantification purposes, images were processed using the Imaris software 64x Version 10.1.0 (Oxford Instruments).

DNA extraction and quantitative PCR (qPCR)

DNA was extracted as described earlier using a QIAamp DNA mini kit (Qiagen) [31, 37]. Bacterial density was quantified by qPCR reactions in a total 25 µl volume. Reactions contained 1x Platinum qPCR superMix (Invitrogen), 200 nm each of primers and probe, and 2.5 µl of purified DNA. The nucleotide sequence of primers and probe were published elsewhere [13]. No template controls (NTC) were run with each set of samples. qPCR reactions were carried out using a CFX96 Real-Time PCR Detection System (Bio-Rad). The following amplification parameters were utilized, 95 °C for 2 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. qPCR standards using genomic DNA from reference strain TIGR4 (ATCC BAA-334) were run in parallel to construct a standard curve utilized to calculate genome equivalents (GenEq)/ml using the software Bio-Rad CFX manager. Standards DNA Preparation: Purified DNA was adjusted to a concentration of 1 ng/µl in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and immediately stored at -80 °C until use. Standards for quantification were prepared within an hour before reactions by serially diluting a 1 ng/µl aliquot in TE buffer to final concentrations of 100 pg/µl, 10 pg/µl, 1 pg/µl, 100 fg/µl, 50 fg/µl, and 5 fg/µl of pneumococcal DNA. Given the 2.1608 Mb genome size of TIGR421, these standards corresponded to 4.29 × 105, 4.29 × 104, 4.29 × 103, 4.29 × 102, 4.29 × 101, 2.14 × 101, or 2.14 GenEq, respectively. Standards prepared using this protocol consistently achieved an efficiency greater than 90% throughout the study (data not shown).

Isolation and identification of S. pneumoniae strains

Nasopharyngeal specimens were thawed, vortexed for 15 s and 200 µl transferred to a 5 ml Todd-Hewitt broth (THY) containing 0.5% of yeast extract and 1 ml of rabbit serum (Gibco® by Life Technologies) [38]. This enriched culture was incubated for 6 h at 37 °C in a 5% CO2 atmosphere and then inoculated onto BAP and incubated for 18–24 h at 37 °C in a 5% CO2 atmosphere. Spn strains were isolated and identified using the optochin test (Remel) and bile solubility test as previously described [38].

Spn strains were isolated and identified from lower respiratory tract specimens including sputum, endotracheal aspirates, bronchoalveolar lavage fluids, bronchial washings, and lung aspirates, using standard methodologies [39]. Briefly, after Gram staining, the primary media including 5% Columbia sheep blood, Chocolate, and MacConkey agars (BD BBL™) were inoculated. Thioglycolate broth was employed for bronchial brush samples. The media were incubated for 18 to 24 h at 35–37 °C in 5% CO2, and cultures that remained negative after this period were re-incubated for another 24 h.

Pathogenic organisms were identified and susceptibility testing was performed only on significant growth, characterized by moderate to abundant colonies in the second or further quadrants of the plate, small numbers of a pathogen consistent with the predominant Gram-stain morphotype, and associated with inflammatory white blood cells, or colonies in the first quadrant if minimal or no other normal flora was present. Alpha-hemolytic, dry, or mucoid colonies resembling Spn strains were identified using Matrix Assisted Laser Desorption Ionization Time-of-Flight (VITEK® MS) or VITEK® 2 Gram-Positive identification card (GP), and purity was confirmed with the optochin disc.

All Spn isolates were serotyped using latex agglutination followed by the Quellung reaction using Neufeld reagents (Statens Serum Institute, Copenhagen, Denmark) [29, 40] and further confirmed by PCR assays [41, 42].

Statistical analysis

We performed one-way analysis of variance (ANOVA) followed by Dunnett’s multiple-comparison test when more than two groups were compared or Student’s t test to compare two groups, as indicated. All statistical analysis was performed using the software GraphPad Prism (version 8.3.1).