enoriega/keyword_pubmed · Datasets at Hugging Face

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For example , CC5 isolates were largely implicated in HA-SA invasive and respiratory infections , whereas CC8 isolates accounted for the majority of HA-SA wound infections ( Fig 1B ) .	PMC6331205	-1	[]
The distribution of S. aureus isolates collected from inpatient wards at different census regions was reflective of the total S. aureus population with high prevalence of CC5 and CC8 isolates ( S1 Fig ) .	PMC6331205	-1	[]
The distribution of clonal complexes among disease isolates collected during 2004 ( n = 117 ) , 2009 ( n = 162 ) and 2010 ( n = 237 ) is illustrated in Fig 1C .	PMC6331205	-1	[]
In 2004 , 13 CC were associated with disease , whereas 20 CC were associated with disease in 2009 – 10 .	PMC6331205	-1	[]
For all time periods , CC8 and CC5 constituted the two most prevalent lineages followed by CC30 and CC45 .	PMC6331205	5,703	[ 18 ]
While there was a trend in emergence of CC59 over time , the variation in the distribution of CC among 2004 , 2009 and 2010 isolates was not statistically significant ( P = 0.138 ) .	PMC6331205	-1	[]
spa type distribution .	PMC6331205	-1	[]
A total of 124 spa types were identified in 97 % ( n = 501 of 516 ) of isolates where a spa type was defined ( S2 Table ) .	PMC6331205	-1	[]
The distribution of prevalent spa types across the population in association with different infections reflected the CC distribution ( S2 Fig ) .	PMC6331205	-1	[]
The most common spa types t008 ( 28 % , 146/516 ) and t002 ( 20 % , 105/516 ) were associated with the two dominant CC in the population , CC8 and CC5 , respectively .	PMC6331205	-1	[]
CC30 was mostly associated with spa type t012 , while CC45 had greater spa type diversity compared to other prominent CC where a single spa type dominated each lineage .	PMC6331205	5,703	[ 10 ]
CC8-USA300 lineage .	PMC6331205	-1	[]
USA300 isolates belong to CC8 , contain the SCCmec type IV element , and carry signature toxin genes such as Panton-Valentine Leukocidin ( PVL ) [ 87 , 88 ] .	PMC6331205	-1	[]
The molecular epidemiology of CC8-USA300 isolates analyzed here was described previously [ 89 ] .	PMC6331205	-1	[]
Briefly , of the 191 CC8 isolates in the S. aureus population , 154 isolates ( 81 % , 154/191 ) were typed as the USA300 clone .	PMC6331205	-1	[]
The majority of USA300 isolates ( 86 % , 132/154 ) were genotyped as spa t008 .	PMC6331205	-1	[]
The majority of these isolates ( 84 % , 129/154 ) were MRSA .	PMC6331205	-1	[]
Most infections caused by USA300 isolates were associated with skin and wound infections ( 41 % , 63/154 ) .	PMC6331205	-1	[]
A relatively small number of USA300 isolates were obtained from invasive infections ( 7.8 % , 12/154 ) .	PMC6331205	-1	[]
The CC8 lineage also contained four representatives of the USA500 clone and six additional isolates closely related to USA500 but descending from a distinct node ( USA500-like ) [ 89 ] .	PMC6331205	157,817	[ 9, 18 ]
All USA500 and USA500-like isolates belonged to spa type t064 .	PMC6331205	157,817	[ 1 ]
Capsular polysaccharide genotype distribution .	PMC6331205	-1	[]
Genotyping revealed that all study isolates possessed either cap5 or cap8 specific genes to direct the biosynthesis of CP5 or CP8 , respectively ( 72 % CP5 and 28 % CP8 ) .	PMC6331205	-1	[]
As previously reported [ 20 ] , capsule genotypes were highly correlated with isolate lineage ( Fig 2 ) .	PMC6331205	-1	[]
Most MRSA isolates were CP5 ( 96 % , 254/265 ) , whereas MSSA isolates had a nearly equal distribution between CP5 and CP8 ( S3 Fig ) .	PMC6331205	-1	[]
A CP5 genotype was significantly more prevalent among isolates collected in either time period from both inpatient and outpatient wards ( P < 0.001 ) ( Table 1 and Fig 2 ) .	PMC6331205	-1	[]
The prevalence of CP5 isolates was higher among invasive isolates ( 67 % , 70/105 ) .	PMC6331205	-1	[]
The same trend was observed for other infection types where CP5 was the dominant genotype amongst clinical isolates .	PMC6331205	-1	[]
No association was observed between CP genotype distribution and either the type of infection ( P = 0.724 ) or the ward source ( P = 0.208 ) .	PMC6331205	-1	[]
Opsonophagocytic killing of diverse clinical S. aureus isolates with immune sera .	PMC6331205	-1	[]
Clinical sera from human subjects immunized with an investigational vaccine ( SA3Ag ) containing CP5 - and CP8-CRM197 conjugates [ 82 ] were evaluated for the ability to kill encapsulated S. aureus isolates in opsonophagocytic assays ( OPA ) .	PMC6331205	-1	[]
OPA analyses of representative isolates from the four prominent CC ( 5 , 8, 30 and 45 ) , were conducted to evaluate the breadth of coverage of this investigational vaccine .	PMC6331205	-1	[]
An OPA titer was defined as the reciprocal of the sample serum dilution required to kill 50 % of the bacteria in the test .	PMC6331205	-1	[]
Geometric mean OPA titers and 95 % confidence intervals ( CI ) were calculated for samples ( n = 10 ) tested in two independent experiments ( Fig 3 ) .	PMC6331205	-1	[]
OPA titers in sera from SA3Ag immunized subjects increased substantially compared to preimmune sera and for all isolates tested ( Fig 3 ) .	PMC6331205	-1	[]
Preabsorption of test sera with homologous capsule blocked the majority of vaccine elicited opsonic killing , illustrating the functional activity of antibody directed to the CP antigens .	PMC6331205	-1	[]
Detection of capsule expression in vitro by Luminex based assays .	PMC6331205	-1	[]
OPA demonstrated that antibodies elicited to the CP conjugate antigens contained in SA3Ag were able to kill diverse S. aureus clinical isolates .	PMC6331205	-1	[]
Since OPA are complex functional assays that are time consuming to develop for a large number of strains , CP5 / 8 detection assays were developed using a competitive antigen binding assay format to evaluate capsule expression in vitro for a larger panel of S. aureus clinical isolates .	PMC6331205	-1	[]
Initially , five randomly selected representatives from each of the 13 prominent CC among US disease causing isolates were tested .	PMC6331205	-1	[]
To account for the spa diversity within a given CC , additional isolates within each lineage were also evaluated ( Table 2 ) .	PMC6331205	-1	[]
All CP5 isolates tested were negative in the CP8 assay and vice versa .	PMC6331205	-1	[]
Among the representative CC 9 , 15 , 25 , 30 , 45 , 59 , 72 and 121 isolates tested , all expressed capsule in vitro ( Table 2 ) .	PMC6331205	-1	[]
However , CC5 , 8, 12 , 97 and 188 contained some isolates for which capsule production in vitro was below detectable levels ( approximately 35 % of the total isolates tested ) ( Table 2 ) .	PMC6331205	-1	[]
Genetic analysis of the cap5 / 8 biosynthetic operon .	PMC6331205	-1	[]
To investigate the potential genetic basis for the lack of in vitro capsule expression in some isolates , whole genome sequencing data corresponding to the cap biosynthetic operon was analyzed for each of the 516 S. aureus isolates .	PMC6331205	-1	[]
Nucleotide polymorphisms were identified relative to the S. aureus reference isolates HO 5096 0412 ( ST22 ) for CP5 isolates and MSSA476 ( ST1 ) for CP8 isolates .	PMC6331205	-1	[]
Substantial single nucleotide polymorphism ( SNP ) diversity was detected within both CP5 and CP8 operons ( S4 Fig ) .	PMC6331205	-1	[]
A small number of insertion / deletion ( indel ) mutations were also observed ( S3 Fig ) .	PMC6331205	-1	[]
Most of the SNPs were detected in isolates that expressed capsule in vitro , suggesting that they do not negatively impact capsule production in vitro .	PMC6331205	-1	[]
For the SNP analysis of CC8 isolates , we focused on previously identified mutations that were linked to the CP-negative phenotype ; whereas for the other CC , we screened the entire cap operon to correlate the occurrence of SNPs and the observed CP-negative phenotype .	PMC6331205	-1	[]
When compared with the respective reference isolates , a total of 8 different mutation types were identified among the isolates that lacked detectable capsule expression in vitro ( Table 3 ) .	PMC6331205	-1	[]
For CC8 isolates , four different mutations in the cap5 operon were identified and in vitro negative CP phenotypes were associated with isolates that carried combinations of two or more of these mutations .	PMC6331205	-1	[]
These mutations have been previously described [ 55 ] .	PMC6331205	-1	[]
The cap5 promoter had a transition mutation ( T → C ) positioned 73 nucleotides upstream of the ATG translation start codon of cap5A and was identified in 186 of 191 ( 97 % ) CC8 isolates .	PMC6331205	-1	[]
CC8 isolates with just this single mutation in the cap5 promoter did express detectable CP5 in vitro as shown in Table 2 .	PMC6331205	-1	[]
The majority of CC8 isolates ( 97 % , 185/191 ) carried a single nucleotide insertion in cap5D ( poly A heptamer occurring after nucleotide position 992 ) resulting in a translation frameshift and the introduction of a premature stop codon .	PMC6331205	-1	[]
This mutation was absent in the CC8 isolates where capsule was detected when cultured in vitro .	PMC6331205	-1	[]
The G → T transversion mutation at nucleotide position 223 of cap5E resulted in the non synonymous substitution , Asp75Tyr , and was detected in all USA300 isolates ( 81 % of the total CC8 isolates , 154/191 ) , but not in other CC8 isolates .	PMC6331205	-1	[]
A T → C transition mutation at nucleotide position 478 in cap5G was detected in 89 % ( 170/191 ) of CC8 isolates ( including spa types t008 , t024 , t121 and t681 ) and resulted in the non synonymous substitution , Phe160Leu .	PMC6331205	98,913	[ 31 ]
USA500 and USA500-like isolates ( spa type t064 ) harbored the cap5 promoter and the cap5D frameshift mutations , but the cap5E and cap5G mutations were not detected in these sub-lineages of S. aureus CC8 isolates.The presence of capsule operon mutations was not restricted to the CC8 lineage of S. aureus isolates .	PMC6331205	157,817	[ 0 ]
In a single CC5-t062 isolate with a CP-negative in vitro phenotype , a premature stop codon was detected in cap5E ( C → T resulting in a nonsense mutation ) .	PMC6331205	-1	[]
A different CC5 isolate of the same spa type that lacked this mutation was CP-positive in vitro .	PMC6331205	-1	[]
A similar genotype to phenotype correlation was observed for the CC188-t189 isolates .	PMC6331205	-1	[]
One isolate that did not express capsule in vitro had a unique SNP in cap8O resulting in a Gly251Val substitution .	PMC6331205	-1	[]
This SNP was not detected in four CC188-t189 isolates with a positive CP in vitro phenotype .	PMC6331205	-1	[]
Four of the five CC12 isolates did not express capsule in vitro and three of these shared a single nucleotide deletion in cap8D ( position 671 ) , resulting in a frameshift and premature stop codon .	PMC6331205	-1	[]
The same three isolates ( two with spa type t160 and one with spa type t771 ) carried a G → T transversion mutation at nucleotide position 683 of cap8E resulting in a non synonymous substitution , Ser156Ile .	PMC6331205	-1	[]
Interestingly , no specific mutations were detected in the fourth CC12 isolate ( spa type t5318 ) with the CP-negative in vitro phenotype .	PMC6331205	-1	[]
A single CC97 isolate that did not express capsule in vitro harbored a 1448 nucleotide deletion involving the cap5D and cap5E genes .	PMC6331205	-1	[]
Detection of capsule expression in vivo .	PMC6331205	-1	[]
Detection of surface polysaccharides in the murine model by immunofluorescence assay ( IFA ) .	PMC6331205	-1	[]
To assess whether isolates with undetectable capsule expression in vitro may express capsule in vivo , IFA was used to test the majority of S. aureus isolates that were CP-negative in vitro [ 21 , 64 ] .	PMC6331205	-1	[]
Blood was harvested from mice 6 h following infection with S. aureus strains , and evaluated by immunohistochemistry using anti-CP5 or anti-CP8 specific antibodies .	PMC6331205	-1	[]
The specificity of the anti-capsular polysaccharide antibodies used to detect capsule production by IFA was confirmed using three isogenic S. aureus Reynolds strains that expressed CP5 , CP8 , or no capsule ( S5 Fig ) .	PMC6331205	-1	[]
The CP5 mAb only detected wild type S. aureus Reynolds , the prototype serotype 5 strain .	PMC6331205	-1	[]
Likewise the CP8 mAb only recognized the isogenic CP8 expressing Reynolds strain ; neither antibody recognized the isogenic acapsular mutant strain .	PMC6331205	-1	[]
Of the five CC12 isolates tested , four ( spa types t160 , t5318 and t771 ) were CP-negative in vitro while one isolate ( CC12-t156 ) was CP-positive .	PMC6331205	-1	[]
As discussed previously , the in vitro CP-negative CC12 isolates either had two SNPs in their capsule operon ( spa types t160 and t771 ) , or none ( spa type t5318 ) .	PMC6331205	-1	[]
CP8 expression was detected in blood following infection with three of the four in vitro CP-negative CC12 isolates ( IFA data is shown for a representative isolate in Fig 4A ) .	PMC6331205	-1	[]
The CC12-t160 isolate that was CP-negative in vivo had mutations in cap8D and cap8E ( mutation types 6 and 7 , Table 3 ) .	PMC6331205	-1	[]
The second CC12-t160 isolate with the same two mutations was positive for CP8 expression in vivo , despite having no detectable capsule expression in vitro .	PMC6331205	-1	[]
The single CC188-t189 isolate that was phenotypically CP-negative in vitro and harbored the non synonymous Gly251Val substitution in cap8O ( mutation type 8, Table 3 ) did express capsule in vivo ( Fig 4A ) .	PMC6331205	-1	[]
The CC5-t062 isolate that was phenotypically CP-negative in vitro and contained the CP5 mutation ( cap5E ) resulting in a premature termination codon was not tested in the bacteremia model.To more thoroughly investigate in vivo expression of CP5 in S. aureus CC8 isolates in light of contradictory conclusions in recent reports [ 21 , 55 , 64 ] , additional CC8 S. aureus isolates from a contemporary US collection obtained from the CDC were included to supplement the CC8 isolates from the T.E.S.T collection .	PMC6331205	-1	[]
A total of 26 CC8 isolates were tested for capsule expression in mice , including 20 t008 isolates ( 17 of which are USA300 isolates ) , five t064 isolates ( four of which are USA500 isolates ) , and one t024 USA300 isolate ( Table 2 ) .	PMC6331205	157,817	[ 35 ]
Three of the 26 CC8 S. aureus isolates in this subset expressed capsule in vitro ( 12 % ) .	PMC6331205	-1	[]
As was seen with isolates from other lineages ( e.g .	PMC6331205	-1	[]
CC12 ) , no consistent association was found between the presence of mutations in the cap operon and the ability to express capsule in vivo ( Table 2 ) .	PMC6331205	-1	[]
Twenty of the 26 CC8 isolates tested expressed capsule in vivo ( 77 % ) .	PMC6331205	-1	[]
Detection of capsule in vivo expression is shown for representative CC8-t008 ( USA300 ) and CC8-t064 ( USA500 ) isolates in Fig 4A.To assess whether the observed in vivo expression was due to either mutations reverting in vivo or contamination by other S. aureus that are capsule producers , mice ( two mice per bacterial strain tested ) were challenged individually with three USA300 strains ( PFESA0119 , PFESA0029 and PFESASA0021 ) and Reynolds strain .	PMC6331205	157,817	[ 17 ]
For each of these strains , ST , spa type and cap operon sequences were identical in the challenge bacteria and the bacteria that were recovered from the animals after infection ( S3 Table ) .	PMC6331205	-1	[]
There was no evidence of genetic changes or strain contamination.Capsule polysaccharide expression in vivo was further explored by evaluating whether RNA transcripts from the cap operon could be detected in mouse blood following infection with a representative panel of USA300 ( two in vivo CP-positive isolates , PFESA0119 and PFESA0029 [ CDC3 ] and an in vivo CP-negative isolate , PFESA0021 ) and USA500 ( in vivo CP-positive isolate PFESA0065 ) isolates .	PMC6331205	1,884	[ 51, 63 ]
RNA transcripts corresponding to cap5D and cap5E were detected in vivo for each of the four isolates tested ( Fig 4B ) .	PMC6331205	-1	[]
Detection of CP5 immune responses to USA300 isolates .	PMC6331205	-1	[]
To further evaluate whether USA300 isolates express capsule in vivo , mice were infected with two in vivo CP-positive USA300 MRSA isolates PFESA0029 [ CDC3 ] and PFESA0119 as well as the isogenic capsule knockout mutant ( PFESA0119 Δ cap5HIJK ermC ) .	PMC6331205	1,884	[ 24 ]
S. aureus ( MSSA ) strain Reynolds was used as a CP5 positive control .	PMC6331205	-1	[]
Sera were collected from mice before infection and 2-weeks after the final inoculation and were evaluated for the presence of CP specific antibodies .	PMC6331205	-1	[]
Antibody responses to CP5 were measured using a CP5-specific competitive luminex immunoassay ( cLIA ) .	PMC6331205	-1	[]
Anti-CP5 antibodies were not detected in cohort mice prior to infection .	PMC6331205	-1	[]
However , a CP5 immune response was detected in mice infected with either of the USA300 isolates indirectly indicating CP5 capsule expression in vivo .	PMC6331205	-1	[]

For example , CC5 isolates were largely implicated in HA-SA invasive and respiratory infections , whereas CC8 isolates accounted for the majority of HA-SA wound infections ( Fig 1B ) .

PMC6331205

-1

[]

The distribution of S. aureus isolates collected from inpatient wards at different census regions was reflective of the total S. aureus population with high prevalence of CC5 and CC8 isolates ( S1 Fig ) .

PMC6331205

-1

[]

The distribution of clonal complexes among disease isolates collected during 2004 ( n = 117 ) , 2009 ( n = 162 ) and 2010 ( n = 237 ) is illustrated in Fig 1C .

PMC6331205

-1

[]

In 2004 , 13 CC were associated with disease , whereas 20 CC were associated with disease in 2009 – 10 .

PMC6331205

-1

[]

For all time periods , CC8 and CC5 constituted the two most prevalent lineages followed by CC30 and CC45 .

PMC6331205

5,703

[ 18 ]

While there was a trend in emergence of CC59 over time , the variation in the distribution of CC among 2004 , 2009 and 2010 isolates was not statistically significant ( P = 0.138 ) .

PMC6331205

-1

[]

spa type distribution .

PMC6331205

-1

[]

A total of 124 spa types were identified in 97 % ( n = 501 of 516 ) of isolates where a spa type was defined ( S2 Table ) .

PMC6331205

-1

[]

The distribution of prevalent spa types across the population in association with different infections reflected the CC distribution ( S2 Fig ) .

PMC6331205

-1

[]

The most common spa types t008 ( 28 % , 146/516 ) and t002 ( 20 % , 105/516 ) were associated with the two dominant CC in the population , CC8 and CC5 , respectively .

PMC6331205

-1

[]

CC30 was mostly associated with spa type t012 , while CC45 had greater spa type diversity compared to other prominent CC where a single spa type dominated each lineage .

PMC6331205

5,703

[ 10 ]

CC8-USA300 lineage .

PMC6331205

-1

[]

USA300 isolates belong to CC8 , contain the SCCmec type IV element , and carry signature toxin genes such as Panton-Valentine Leukocidin ( PVL ) [ 87 , 88 ] .

PMC6331205

-1

[]

The molecular epidemiology of CC8-USA300 isolates analyzed here was described previously [ 89 ] .

PMC6331205

-1

[]

Briefly , of the 191 CC8 isolates in the S. aureus population , 154 isolates ( 81 % , 154/191 ) were typed as the USA300 clone .

PMC6331205

-1

[]

The majority of USA300 isolates ( 86 % , 132/154 ) were genotyped as spa t008 .

PMC6331205

-1

[]

The majority of these isolates ( 84 % , 129/154 ) were MRSA .

PMC6331205

-1

[]

Most infections caused by USA300 isolates were associated with skin and wound infections ( 41 % , 63/154 ) .

PMC6331205

-1

[]

A relatively small number of USA300 isolates were obtained from invasive infections ( 7.8 % , 12/154 ) .

PMC6331205

-1

[]

The CC8 lineage also contained four representatives of the USA500 clone and six additional isolates closely related to USA500 but descending from a distinct node ( USA500-like ) [ 89 ] .

PMC6331205

157,817

[ 9, 18 ]

All USA500 and USA500-like isolates belonged to spa type t064 .

PMC6331205

157,817

[ 1 ]

Capsular polysaccharide genotype distribution .

PMC6331205

-1

[]

Genotyping revealed that all study isolates possessed either cap5 or cap8 specific genes to direct the biosynthesis of CP5 or CP8 , respectively ( 72 % CP5 and 28 % CP8 ) .

PMC6331205

-1

[]

As previously reported [ 20 ] , capsule genotypes were highly correlated with isolate lineage ( Fig 2 ) .

PMC6331205

-1

[]

Most MRSA isolates were CP5 ( 96 % , 254/265 ) , whereas MSSA isolates had a nearly equal distribution between CP5 and CP8 ( S3 Fig ) .

PMC6331205

-1

[]

A CP5 genotype was significantly more prevalent among isolates collected in either time period from both inpatient and outpatient wards ( P < 0.001 ) ( Table 1 and Fig 2 ) .

PMC6331205

-1

[]

The prevalence of CP5 isolates was higher among invasive isolates ( 67 % , 70/105 ) .

PMC6331205

-1

[]

The same trend was observed for other infection types where CP5 was the dominant genotype amongst clinical isolates .

PMC6331205

-1

[]

No association was observed between CP genotype distribution and either the type of infection ( P = 0.724 ) or the ward source ( P = 0.208 ) .

PMC6331205

-1

[]

Opsonophagocytic killing of diverse clinical S. aureus isolates with immune sera .

PMC6331205

-1

[]

Clinical sera from human subjects immunized with an investigational vaccine ( SA3Ag ) containing CP5 - and CP8-CRM197 conjugates [ 82 ] were evaluated for the ability to kill encapsulated S. aureus isolates in opsonophagocytic assays ( OPA ) .

PMC6331205

-1

[]

OPA analyses of representative isolates from the four prominent CC ( 5 , 8, 30 and 45 ) , were conducted to evaluate the breadth of coverage of this investigational vaccine .

PMC6331205

-1

[]

An OPA titer was defined as the reciprocal of the sample serum dilution required to kill 50 % of the bacteria in the test .

PMC6331205

-1

[]

Geometric mean OPA titers and 95 % confidence intervals ( CI ) were calculated for samples ( n = 10 ) tested in two independent experiments ( Fig 3 ) .

PMC6331205

-1

[]

OPA titers in sera from SA3Ag immunized subjects increased substantially compared to preimmune sera and for all isolates tested ( Fig 3 ) .

PMC6331205

-1

[]

Preabsorption of test sera with homologous capsule blocked the majority of vaccine elicited opsonic killing , illustrating the functional activity of antibody directed to the CP antigens .

PMC6331205

-1

[]

Detection of capsule expression in vitro by Luminex based assays .

PMC6331205

-1

[]

OPA demonstrated that antibodies elicited to the CP conjugate antigens contained in SA3Ag were able to kill diverse S. aureus clinical isolates .

PMC6331205

-1

[]

Since OPA are complex functional assays that are time consuming to develop for a large number of strains , CP5 / 8 detection assays were developed using a competitive antigen binding assay format to evaluate capsule expression in vitro for a larger panel of S. aureus clinical isolates .

PMC6331205

-1

[]

Initially , five randomly selected representatives from each of the 13 prominent CC among US disease causing isolates were tested .

PMC6331205

-1

[]

To account for the spa diversity within a given CC , additional isolates within each lineage were also evaluated ( Table 2 ) .

PMC6331205

-1

[]

All CP5 isolates tested were negative in the CP8 assay and vice versa .

PMC6331205

-1

[]

Among the representative CC 9 , 15 , 25 , 30 , 45 , 59 , 72 and 121 isolates tested , all expressed capsule in vitro ( Table 2 ) .

PMC6331205

-1

[]

However , CC5 , 8, 12 , 97 and 188 contained some isolates for which capsule production in vitro was below detectable levels ( approximately 35 % of the total isolates tested ) ( Table 2 ) .

PMC6331205

-1

[]

Genetic analysis of the cap5 / 8 biosynthetic operon .

PMC6331205

-1

[]

To investigate the potential genetic basis for the lack of in vitro capsule expression in some isolates , whole genome sequencing data corresponding to the cap biosynthetic operon was analyzed for each of the 516 S. aureus isolates .

PMC6331205

-1

[]

Nucleotide polymorphisms were identified relative to the S. aureus reference isolates HO 5096 0412 ( ST22 ) for CP5 isolates and MSSA476 ( ST1 ) for CP8 isolates .

PMC6331205

-1

[]

Substantial single nucleotide polymorphism ( SNP ) diversity was detected within both CP5 and CP8 operons ( S4 Fig ) .

PMC6331205

-1

[]

A small number of insertion / deletion ( indel ) mutations were also observed ( S3 Fig ) .

PMC6331205

-1

[]

Most of the SNPs were detected in isolates that expressed capsule in vitro , suggesting that they do not negatively impact capsule production in vitro .

PMC6331205

-1

[]

For the SNP analysis of CC8 isolates , we focused on previously identified mutations that were linked to the CP-negative phenotype ; whereas for the other CC , we screened the entire cap operon to correlate the occurrence of SNPs and the observed CP-negative phenotype .

PMC6331205

-1

[]

When compared with the respective reference isolates , a total of 8 different mutation types were identified among the isolates that lacked detectable capsule expression in vitro ( Table 3 ) .

PMC6331205

-1

[]

For CC8 isolates , four different mutations in the cap5 operon were identified and in vitro negative CP phenotypes were associated with isolates that carried combinations of two or more of these mutations .

PMC6331205

-1

[]

These mutations have been previously described [ 55 ] .

PMC6331205

-1

[]

The cap5 promoter had a transition mutation ( T → C ) positioned 73 nucleotides upstream of the ATG translation start codon of cap5A and was identified in 186 of 191 ( 97 % ) CC8 isolates .

PMC6331205

-1

[]

CC8 isolates with just this single mutation in the cap5 promoter did express detectable CP5 in vitro as shown in Table 2 .

PMC6331205

-1

[]

The majority of CC8 isolates ( 97 % , 185/191 ) carried a single nucleotide insertion in cap5D ( poly A heptamer occurring after nucleotide position 992 ) resulting in a translation frameshift and the introduction of a premature stop codon .

PMC6331205

-1

[]

This mutation was absent in the CC8 isolates where capsule was detected when cultured in vitro .

PMC6331205

-1

[]

The G → T transversion mutation at nucleotide position 223 of cap5E resulted in the non synonymous substitution , Asp75Tyr , and was detected in all USA300 isolates ( 81 % of the total CC8 isolates , 154/191 ) , but not in other CC8 isolates .

PMC6331205

-1

[]

A T → C transition mutation at nucleotide position 478 in cap5G was detected in 89 % ( 170/191 ) of CC8 isolates ( including spa types t008 , t024 , t121 and t681 ) and resulted in the non synonymous substitution , Phe160Leu .

PMC6331205

98,913

[ 31 ]

USA500 and USA500-like isolates ( spa type t064 ) harbored the cap5 promoter and the cap5D frameshift mutations , but the cap5E and cap5G mutations were not detected in these sub-lineages of S. aureus CC8 isolates.The presence of capsule operon mutations was not restricted to the CC8 lineage of S. aureus isolates .

PMC6331205

157,817

[ 0 ]

In a single CC5-t062 isolate with a CP-negative in vitro phenotype , a premature stop codon was detected in cap5E ( C → T resulting in a nonsense mutation ) .

PMC6331205

-1

[]

A different CC5 isolate of the same spa type that lacked this mutation was CP-positive in vitro .

PMC6331205

-1

[]

A similar genotype to phenotype correlation was observed for the CC188-t189 isolates .

PMC6331205

-1

[]

One isolate that did not express capsule in vitro had a unique SNP in cap8O resulting in a Gly251Val substitution .

PMC6331205

-1

[]

This SNP was not detected in four CC188-t189 isolates with a positive CP in vitro phenotype .

PMC6331205

-1

[]

Four of the five CC12 isolates did not express capsule in vitro and three of these shared a single nucleotide deletion in cap8D ( position 671 ) , resulting in a frameshift and premature stop codon .

PMC6331205

-1

[]

The same three isolates ( two with spa type t160 and one with spa type t771 ) carried a G → T transversion mutation at nucleotide position 683 of cap8E resulting in a non synonymous substitution , Ser156Ile .

PMC6331205

-1

[]

Interestingly , no specific mutations were detected in the fourth CC12 isolate ( spa type t5318 ) with the CP-negative in vitro phenotype .

PMC6331205

-1

[]

A single CC97 isolate that did not express capsule in vitro harbored a 1448 nucleotide deletion involving the cap5D and cap5E genes .

PMC6331205

-1

[]

Detection of capsule expression in vivo .

PMC6331205

-1

[]

Detection of surface polysaccharides in the murine model by immunofluorescence assay ( IFA ) .

PMC6331205

-1

[]

To assess whether isolates with undetectable capsule expression in vitro may express capsule in vivo , IFA was used to test the majority of S. aureus isolates that were CP-negative in vitro [ 21 , 64 ] .

PMC6331205

-1

[]

Blood was harvested from mice 6 h following infection with S. aureus strains , and evaluated by immunohistochemistry using anti-CP5 or anti-CP8 specific antibodies .

PMC6331205

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The specificity of the anti-capsular polysaccharide antibodies used to detect capsule production by IFA was confirmed using three isogenic S. aureus Reynolds strains that expressed CP5 , CP8 , or no capsule ( S5 Fig ) .

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The CP5 mAb only detected wild type S. aureus Reynolds , the prototype serotype 5 strain .

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Likewise the CP8 mAb only recognized the isogenic CP8 expressing Reynolds strain ; neither antibody recognized the isogenic acapsular mutant strain .

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Of the five CC12 isolates tested , four ( spa types t160 , t5318 and t771 ) were CP-negative in vitro while one isolate ( CC12-t156 ) was CP-positive .

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As discussed previously , the in vitro CP-negative CC12 isolates either had two SNPs in their capsule operon ( spa types t160 and t771 ) , or none ( spa type t5318 ) .

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CP8 expression was detected in blood following infection with three of the four in vitro CP-negative CC12 isolates ( IFA data is shown for a representative isolate in Fig 4A ) .

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The CC12-t160 isolate that was CP-negative in vivo had mutations in cap8D and cap8E ( mutation types 6 and 7 , Table 3 ) .

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The second CC12-t160 isolate with the same two mutations was positive for CP8 expression in vivo , despite having no detectable capsule expression in vitro .

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The single CC188-t189 isolate that was phenotypically CP-negative in vitro and harbored the non synonymous Gly251Val substitution in cap8O ( mutation type 8, Table 3 ) did express capsule in vivo ( Fig 4A ) .

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The CC5-t062 isolate that was phenotypically CP-negative in vitro and contained the CP5 mutation ( cap5E ) resulting in a premature termination codon was not tested in the bacteremia model.To more thoroughly investigate in vivo expression of CP5 in S. aureus CC8 isolates in light of contradictory conclusions in recent reports [ 21 , 55 , 64 ] , additional CC8 S. aureus isolates from a contemporary US collection obtained from the CDC were included to supplement the CC8 isolates from the T.E.S.T collection .

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A total of 26 CC8 isolates were tested for capsule expression in mice , including 20 t008 isolates ( 17 of which are USA300 isolates ) , five t064 isolates ( four of which are USA500 isolates ) , and one t024 USA300 isolate ( Table 2 ) .

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Three of the 26 CC8 S. aureus isolates in this subset expressed capsule in vitro ( 12 % ) .

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As was seen with isolates from other lineages ( e.g .

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CC12 ) , no consistent association was found between the presence of mutations in the cap operon and the ability to express capsule in vivo ( Table 2 ) .

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Twenty of the 26 CC8 isolates tested expressed capsule in vivo ( 77 % ) .

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Detection of capsule in vivo expression is shown for representative CC8-t008 ( USA300 ) and CC8-t064 ( USA500 ) isolates in Fig 4A.To assess whether the observed in vivo expression was due to either mutations reverting in vivo or contamination by other S. aureus that are capsule producers , mice ( two mice per bacterial strain tested ) were challenged individually with three USA300 strains ( PFESA0119 , PFESA0029 and PFESASA0021 ) and Reynolds strain .

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For each of these strains , ST , spa type and cap operon sequences were identical in the challenge bacteria and the bacteria that were recovered from the animals after infection ( S3 Table ) .

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There was no evidence of genetic changes or strain contamination.Capsule polysaccharide expression in vivo was further explored by evaluating whether RNA transcripts from the cap operon could be detected in mouse blood following infection with a representative panel of USA300 ( two in vivo CP-positive isolates , PFESA0119 and PFESA0029 [ CDC3 ] and an in vivo CP-negative isolate , PFESA0021 ) and USA500 ( in vivo CP-positive isolate PFESA0065 ) isolates .

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RNA transcripts corresponding to cap5D and cap5E were detected in vivo for each of the four isolates tested ( Fig 4B ) .

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Detection of CP5 immune responses to USA300 isolates .

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To further evaluate whether USA300 isolates express capsule in vivo , mice were infected with two in vivo CP-positive USA300 MRSA isolates PFESA0029 [ CDC3 ] and PFESA0119 as well as the isogenic capsule knockout mutant ( PFESA0119 Δ cap5HIJK ermC ) .

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S. aureus ( MSSA ) strain Reynolds was used as a CP5 positive control .

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Sera were collected from mice before infection and 2-weeks after the final inoculation and were evaluated for the presence of CP specific antibodies .

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Antibody responses to CP5 were measured using a CP5-specific competitive luminex immunoassay ( cLIA ) .

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Anti-CP5 antibodies were not detected in cohort mice prior to infection .

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However , a CP5 immune response was detected in mice infected with either of the USA300 isolates indirectly indicating CP5 capsule expression in vivo .

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