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Both mAbs were purified on Protein A column . | PMC6331205 | -1 | [] |
The mAbs were conjugated with phycoerythrin ( PE ) and purified on Protein A column by Chromaprobe , Inc. ( Maryland Hts ., MO ) . | PMC6331205 | -1 | [] |
Coupling of CP5 and CP8 to Luminex MagPlex microspheres . | PMC6331205 | -1 | [] |
CP5 and CP8 conjugated to poly-lysine ( CP5-pLL and CP8-pLL ) were passively coated individually onto unique Luminex MagPlex carboxylated microspheres . | PMC6331205 | -1 | [] |
CP5-pLL ( 50 ng / mL final ) or CP8-pLL ( 12.5 ng / mL final ) were added to a 1 mL bead suspension ( 1.25 x 10 microspheres ) in DPBS and incubated for 2.5 h at room temperature with mixing . | PMC6331205 | -1 | [] |
After unbound antigen was washed , the microspheres were blocked and stored in DPBS containing 1 % BSA and 0.05 % NaN . | PMC6331205 | -1 | [] |
Analysis of samples by singleplex liquid array system , Luminex 200 . | PMC6331205 | -1 | [] |
CP5 and CP8 detection assays were performed individually using an antigen competitive assay format . | PMC6331205 | -1 | [] |
CP5 or CP8 coated microspheres in DPBS containing 0.05 % Tween-20 and 0.02 % NaN were added to wells ( 2,500 beads / well ) of the assay plate ( Costar 3912 ) . | PMC6331205 | -1 | [] |
CP5 or CP8 reference standards serially diluted 2.5-fold in DPBS containing 0.05 % Tween-20 and 0.02 % NaN were added in triplicate to wells to create a 12-point titration standard curve . | PMC6331205 | -1 | [] |
The concentration range of the reference standards was 0.06 – 1500 ng / mL and 0.01 – 250 ng / mL for CP5 and CP8 respectively . | PMC6331205 | -1 | [] |
A volume of 50 μ L of unknown samples was tested in duplicate . | PMC6331205 | -1 | [] |
Finally , 50 μ L of mAb CP5-5-1-PE ( 600 ng / mL ) or mAb CP8-119-11-PE ( 105 ng / mL ) in DPBS containing 0.05 % Tween-20 and 0.02 % NaN were added to each well of the assay plate . | PMC6331205 | -1 | [] |
The plate was covered and incubated with shaking at 300 rpm at room temperature for 60 min . | PMC6331205 | -1 | [] |
The plates were then washed three times with DPBS containing 0.05 % Tween-20 and 0.02 % NaN . | PMC6331205 | -1 | [] |
After the final wash , 100 μ L of DPBS containing 0.05 % Tween-20 and 0.02 % NaN was added to each well and the plates were read at high reporter setting using a Bio-Plex reader ( Bio-Plex 200 systems ; Bio-Rad Laboratories , Hercules , CA ) . | PMC6331205 | -1 | [] |
The quantity of CP5 or CP8 polysaccharide in test samples was derived from the CP5 or CP8 reference standard curves fitted using 4-Parameter Logistic ( 4PL ) nonlinear regression analysis . | PMC6331205 | -1 | [] |
Bioinformatics analysis of the cap operon and SNP calling . | PMC6331205 | -1 | [] |
Single nucleotide polymorphisms ( SNPs ) in the cap operon were detected by mapping the paired-end reads against cap operons of selected reference isolates of S. aureus , using SMALT version 0.7.4 . | PMC6331205 | -1 | [] |
( https://www.sanger.ac.uk/resources/software/smalt/ ) . | PMC6331205 | -1 | [] |
Different reference sequences were used for CP5 and CP8 isolates . | PMC6331205 | -1 | [] |
Furthermore , for each CP type two different reference genomes were used to verify identified polymorphisms and only the consensus SNPs were further analyzed ( those found by mapping to both reference genomes of the same CP type ) . | PMC6331205 | -1 | [] |
The CP5 isolates were mapped against the cap operon from the ST22 HO 5096 0412 isolate ( GenBank accession HE681097 ) [ 85 ] and the SNPs were verified by mapping against the ST5 N315 isolate ( GenBank accession BA000018 ) . | PMC6331205 | -1 | [] |
The CP8 isolates were mapped against the cap operon of the ST1 MSSA476 isolate ( GenBank accession BX571857 ) and the SNPs were verified by mapping against the M013 isolate ( ST59 , accession number NC_016928 ) . | PMC6331205 | 26,677 | [
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In silico PCR extraction of individual genes for all analyzed isolates was used to confirm the mapping results and analyses listed above.To determine the distribution of all identified cap polymorphisms in the context of S. aureus phylogeny , evolutionary relationships between all isolates were reconstructed . | PMC6331205 | -1 | [] |
Whole genome SNPs were detected by mapping the paired-end reads against the S. aureus N315 reference genome using SMALT . | PMC6331205 | -1 | [] |
The generated whole genome sequence alignment was curated to exclude accessory regions ( mobile genetic elements identified from the N315 reference genome ) . | PMC6331205 | -1 | [] |
Based on the core genome SNP alignment an approximately-maximum-likelihood phylogenetic tree was constructed with FastTree software using generalized time reversible ( GTR ) model with GAMMA method of correction for among site rate variation . | PMC6331205 | -1 | [] |
The phylogenetic tree was annotated with the distribution of identified SNPs and indels ( S3 Fig ) . | PMC6331205 | -1 | [] |
Detection of capsule production in vivo . | PMC6331205 | -1 | [] |
Immunofluorescence microscopy assay ( IFA ) for the detection of surface polysaccharides . | PMC6331205 | -1 | [] |
IFA was used to detect in vivo expression in S. aureus isolates . | PMC6331205 | -1 | [] |
S. aureus isolation from infected animals and immunofluorescence staining were performed as previously described [ 64 ] . | PMC6331205 | -1 | [] |
The accuracy of IFA in confirming capsule expression was first demonstrated using three different S. aureus strains ; wild type Reynolds ( CP5 ) , its isogenic CP5-negative mutant , and the CP8-expressing isogenic Reynolds derivative in which cap5H-cap5K was replaced with cap8H-cap8K of the cap8 gene cluster [ 86 ] . | PMC6331205 | -1 | [] |
The strains were tested in a blinded fashion with two independent experiments per strain . | PMC6331205 | -1 | [] |
Bacteremia ( intraperitoneal challenge with 7 × 10 to 2 × 10 CFU S. aureus per animal ) and wound infection ( a 1-cm incision is made in the right thigh muscle of mice then closed with a suture , and 5 μ L of an S. aureus suspension ( 5 × 10 CFU ) was introduced into the muscle incision under the suture ) models were used . | PMC6331205 | -1 | [] |
Female , CD1 mice 8 to 12 weeks old ( Charles River Laboratories ) were used in the in vivo experiments Duplicate samples were tested , one with CP5 specific antibody and the other with CP8 specific antibody . | PMC6331205 | -1 | [] |
After specificity of the CP5 and CP8 antibodies had been confirmed with the S. aureus isogenic derivatives of strain Reynolds , strains from the T.E.S.T collection were tested in duplicate using the same IFA procedure . | PMC6331205 | -1 | [] |
Genetic comparison of challenge inocula with the bacteria recovered from infected animals . | PMC6331205 | -1 | [] |
Mice ( 2 mice per bacterial strain ) were challenged with one of three USA300 strains ( PFESA0119 , PFESA0029 and PFESASA0021 ) or Reynolds . | PMC6331205 | -1 | [] |
Blood samples were collected from animals at two time points ; T0 ( pre-challenge ) and 6 h after infection . | PMC6331205 | -1 | [] |
Challenge stocks used to inoculate mice as well as blood samples were plated on Tryptic Soy Agar plates with 5 % sheep blood . | PMC6331205 | -1 | [] |
No colonies were recovered from the pre-challenge blood samples . | PMC6331205 | -1 | [] |
Five colonies from the respective bacterial inocula and 10 colonies recovered from mice challenged with Reynolds strain ( 5 colonies per animal ) and 20 colonies from mice challenged with each of the USA300 strains ( 10 colonies per strain per animal ) were subjected to whole genome sequencing for comparative analysis of genotypic features and cap operon analysis . | PMC6331205 | -1 | [] |
Detection of capsule transcripts in vivo by qRT-PCR . | PMC6331205 | -1 | [] |
RNA expression of two cap operon genes ( cap5D , cap5E ) and regulators ( mgrA , sarA , agrA , RNA III and ccpA ) was evaluated in animals infected with S. aureus isolates . | PMC6331205 | -1 | [] |
Determination of 16S rRNA expression was included to normalize RNA expression levels of target genes . | PMC6331205 | -1 | [] |
The isolates tested represented lineages that had a mixture of CP-expression positive and negative in vitro phenotypes ; Reynolds , CC8-USA300 ( PFESA0029 , PFESA0119 and PFESA0021 ) and USA500 ( PFESA0065 ) strains . | PMC6331205 | 157,817 | [
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Approximately , 4x10 CFU of mid log phase bacterial cultures grown in TSB were used to infect 10 – 12 week old CD1 mice intraperitoneally ( IP ) . | PMC6331205 | -1 | [] |
T0 represents the bacterial challenge sample . | PMC6331205 | -1 | [] |
Bacteria were harvested from ~ 1.5 – 2 mL of blood at 1 and 4 hr post challenge ( 2 mice per time point per isolate ) and mixed with 2 volumes of RNAProtect Bacteria Reagent ( Qiagen , Valencia , CA ) ; the samples were processed immediately for RNA extraction where samples were mixed thoroughly by vortexing and incubated at room temperature for 10 – 30 min . | PMC6331205 | -1 | [] |
Mixtures were centrifuged at 5,000 x g for 10 min and supernatants removed . | PMC6331205 | -1 | [] |
Pellets were suspended in 500 uL of RNA Protect and homogenized with an Omni tissue homogenizer ( TH ) ( Omni International , GA ) assembled with disposable Soft Tissue Omni Tip plastic homogenizing probes . | PMC6331205 | -1 | [] |
Homogenates were centrifuged at 5,000 x g for 10 min and supernatants removed . | PMC6331205 | -1 | [] |
Pellets were suspended in 1 mL of RLT buffer / β - mercaptoethanol buffer ( QIAgen RNeasy kit ) , homogenized and centrifuged as above . | PMC6331205 | -1 | [] |
Pellets were suspended in 110 μ L of 2X TE buffer and transferred to 2 mL tubes harboring 50mg of glass beads ( Sigma ) . | PMC6331205 | -1 | [] |
A volume of 385 μ L of RLT / β - mercaptoethanol buffer were used to rinse the 15 mL tubes and combined with the 110 uL 2x TE . | PMC6331205 | -1 | [] |
Bacterial lysates were generated by beating the tubes at 1/30 s for 10 min in a Tissue Lyser ( Qiagen ) and cleared by centrifugation at 14,000 rpm for 2 min . | PMC6331205 | -1 | [] |
A volume of 450 μ L of lysate were mixed with 250 μ L100 % ethanol and applied to an RNeasy column ( QIAgen ) . | PMC6331205 | -1 | [] |
RNA was purified according to the QIAgen RNeasy Kit instructions with on-column DNase treatment to remove genomic DNA . | PMC6331205 | -1 | [] |
RNA was eluted twice in 40 μ L RNase-free water , quantified on a Nanodrop and stored -80 ° C. RNA integrity was evaluated by Bioanalyzer 2100 ( Agilent , Santa Clara , CA ) . | PMC6331205 | -1 | [] |
For in vivo transcript profiling , 10 μ L of RNA was reverse transcribed using SuperScript Vilo Master Mix ( Invitrogen , Carlsbad , CA ) and the resulting cDNA was diluted 5 fold with RNase-free water . | PMC6331205 | -1 | [] |
Real-Time PCR reactions were assembled by adding 4 μ L of diluted cDNA , 400 nM forward and reverse primers , 200 nM fluorescent-labeled probe ( Applied Biosystems , Foster City , CA ) and 5 μ L of Taqman Fast Advanced Master Mix ( Applied Biosystems ) in a total volume of 10 μ L. Reactions were performed using the 7900HT Fast Real-Time PCR System ( Applied Biosystems ) under the following conditions : 30 s at 95 ° C and 40 cycles of 3 s at 95 ° C and 30 s at 60 ° C. | PMC6331205 | -1 | [] |
In some instances , transcripts were measured in one-step real-time PCR reactions assembled with 4 μ L of RNA , 400 nM forward and Reverse primer , 200 nM Fluorescently labeled probe , 9.5 μ L of EXPRESS One-Step qRT-PCR SuperMix ( Applied Biosystems ) in a total volume of 15 μ L. | PMC6331205 | -1 | [] |
The AB 7900HT cycling conditions consisted of 15 min at 55 ° C for cDNA synthesis , 2 min at 95 ° C and 40 cycles of 15 s at 95 ° C and then followed by 1 min at 60 ° C. For each transcript , the number of copies was determined from standard curves generated using 10 to 10 copies of plasmid harboring the corresponding gene . | PMC6331205 | -1 | [] |
Normalized expression of transcripts was then expressed as copies per 10 copies of 16S rRNA . | PMC6331205 | -1 | [] |
Competitive Luminex immunoassay ( cLIA ) . | PMC6331205 | -1 | [] |
cLIA was used to measure the level of serum antibody titers against CP5 and CP8 antigens in animals challenged with USA300 isolates ( PFESA0029 [ CDC3 ] , PFESA0119 and PFESA0119 Δ cap5HIJK ermC ) . | PMC6331205 | 1,884 | [
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CD1 mice ( 7 – 9 weeks of age , 10 per group ) were challenged with three intraperitoneal injections of ~ 2x10 CFU / animal in 0.5 mL at 0 , 6 and 14 weeks and bled before vaccination and two weeks after the final inoculation . | PMC6331205 | -1 | [] |
CP5 antibody responses were measured using a CP5 specific cLIA assay . | PMC6331205 | -1 | [] |
CP5 and CP8 antigens were coupled to a distinct fluorescent Luminex microsphere . | PMC6331205 | -1 | [] |
Antigen-coated microspheres were incubated overnight at 4 ° C with appropriately diluted serum samples , controls , or reference standard serum . | PMC6331205 | -1 | [] |
Antigen-specific functional mouse mAbs were then added to the microsphere / serum mixture and bound mAbs were detected with R-Phycoerythrin ( PE ) labeled rat anti-mouse IgG1 secondary antibody ( Southern Biotech ) using a BioPlex reader ( BioRad ) to measure fluorescence . | PMC6331205 | -1 | [] |
In this competitive assay , the magnitude of the fluorescent PE signal is inversely proportional to the amount of antigen-specific antibody in the sample.CP8 immune responses were also measured in animals challenged with CP8 S. aureus isolates including five CC12 isolates ( PFESA1194 , PFESA1305 , PFESA1405 , PFESA1502 , PFESA2455 ) and two CC188 isolates ( PFESA2058 and PFESA2089 ) . | PMC6331205 | -1 | [] |
CD1 mice ( 7 – 9 weeks of age , 10 per group ) were administered with three intraperitoneal injections of ~ 2x10 CFU / animal in 0.5 mL at 0 , 4 and 12 weeks and bled at week 27 . | PMC6331205 | -1 | [] |
CP8 antibody responses were measured using a CP8 specific cLIA assay as described above for CP5 cLIA assay . | PMC6331205 | -1 | [] |
Statistical analyses . | PMC6331205 | -1 | [] |
The categorical variables in the molecular epidemiology analyses were compared using either the χ or likehood ratio test . | PMC6331205 | -1 | [] |
A P value of < 0.05 was considered significant . | PMC6331205 | -1 | [] |
The cLIA serology data were described with geometric mean titer ( GMT ) and corresponding 95 % confidence intervals ( CIs ) . | PMC6331205 | -1 | [] |
The CIs were constructed by back transformation of the confidence limits computed for the mean of the logarithmically transformed assay data based on Student t distribution . | PMC6331205 | -1 | [] |
P-values based on 2-sample t-test or Wilcoxon test were presented to identify potential differences in immunoglobulin concentration . | PMC6331205 | -1 | [] |
Results . | PMC6331205 | -1 | [] |
Patient demographics . | PMC6331205 | -1 | [] |
The demographics of the patients with S. aureus infections in 2004 and 2009 – 10 are shown in Table 1 . | PMC6331205 | -1 | [] |
The differences in % males ( P = 0.462 ) and median age ( P = 0.717 ) were not significant among patients from the two time periods . | PMC6331205 | -1 | [] |
Clinical characteristics and sources of isolates . | PMC6331205 | -1 | [] |
In total , 516 S. aureus isolates , collected through the T.E.S.T program from 12 hospitals in 6 US census regions in 2004 ( n = 117 ) and 2009 – 10 ( n = 399 ) , were analyzed . | PMC6331205 | -1 | [] |
A list of the 516 isolates is shown in S1 Table . | PMC6331205 | -1 | [] |
No ward information was available for 10 isolates collected in 2009 – 10 . | PMC6331205 | -1 | [] |
The epidemiological characteristics of the isolates are described in Table 1 . | PMC6331205 | -1 | [] |
The proportion of MRSA infections among all S. aureus infections in inpatient and outpatient wards combined was significantly higher in 2004 ( 74 % , 86/117 ) compared to 2009 – 10 ( 45 % , 179/399 ) ( P < 0.001 ) . | PMC6331205 | -1 | [] |
The majority of S. aureus isolates evaluated were obtained from healthcare-associated S. aureus ( HA-SA ) infections ( 72 % , 374/516 ) and approximately 25 % of these isolates were derived from invasive infections . | PMC6331205 | -1 | [] |
Population structure of S. aureus isolates . | PMC6331205 | -1 | [] |
Clonal complexes . | PMC6331205 | -1 | [] |
The S. aureus population in the US , over the time period surveyed , included 20 clonal complexes ( CC ) comprised of 69 sequence types ( ST , S2 Table ) . | PMC6331205 | -1 | [] |
Overall , 13 CC represented 98 % of disease causing isolates ( Fig 1 ) . | PMC6331205 | -1 | [] |
Four of these CC represented >80 % of isolates ( Fig 1 ) : CC8 ( 37 % ) , CC5 ( 29 % ) , CC30 ( 8 % ) and CC45 ( 7 % ) . | PMC6331205 | 5,703 | [
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No clear distinction was observed between disease-causing CC in the healthcare setting compared to the community ; four major lineages caused disease in both settings including CC5 , 8, 30 and 45 ( Fig 1A ) . | PMC6331205 | -1 | [] |
There was a significant association between the isolate genotype ( CC ) and the type of clinical infection in inpatient wards ( P = 0.036 ) . | PMC6331205 | -1 | [] |