Last data update: Apr 22, 2024. (Total: 46599 publications since 2009)
Records 1-21 (of 21 Records) |
Query Trace: Gertz RE Jr [original query] |
---|
Nonpneumococcal Strains Recently Recovered from Carriage Specimens and Expressing Capsular Serotypes Highly Related or Identical to Pneumococcal Serotypes 2, 4, 9A, 13, and 23A
Gertz RE Jr , Pimenta FC , Chochua S , Larson S , Venero AK , Bigogo G , Milucky J , Carvalho MDG , Beall B . mBio 2021 12 (3) The polysaccharide capsule is a key virulence factor of Streptococcus pneumoniae There are numerous epidemiologically important pneumococcal capsular serotypes, and recent findings have demonstrated that several of them are commonly found among nonpathogenic commensal species. Here, we describe 9 nonpneumococcal strains carrying close homologs of pneumococcal capsular biosynthetic (cps) loci that were discovered during recent pneumococcal carriage studies of adults in the United States and Kenya. Two distinct Streptococcus infantis strains cross-reactive with pneumococcal serotype 4 and carrying cps4-like capsular biosynthetic (cps) loci were recovered. Opsonophagocytic killing assays employing rabbit antisera raised against S. infantis US67cps4 revealed serotype 4-specific killing of both pneumococcal and nonpneumococcal strains. An S. infantis strain and two Streptococcus oralis strains, all carrying cps9A-like loci, were cross-reactive with pneumococcal serogroup 9 strains in immunodiffusion assays. Antiserum raised against S. infantis US64cps9A specifically promoted killing of serotype 9A and 9V pneumococcal strains as well as S. oralis serotype 9A strains. Serotype-specific PCR of oropharyngeal specimens from a recent adult carriage study in the United States indicated that such nonpneumococcal strains were much more common in this population than serotype 4 and serogroup 9 pneumococci. We also describe S. oralis and S. infantis strains expressing serotypes identical or highly related to serotypes 2, 13, and 23A. This study has expanded the known overlap of pneumococcal capsular serotypes with related commensal species. The frequent occurrence of nonpneumococcal strains in the upper respiratory tract that share vaccine and nonvaccine capsular serotypes with pneumococci could affect population immunity to circulating pneumococcal strains.IMPORTANCE The distributions and frequencies of individual pneumococcal capsular serotypes among nonpneumococcal strains in the upper respiratory tract are unknown and potentially affect pneumococcal serotype distributions among the population and immunity to circulating pneumococcal strains. Repeated demonstration that these nonpneumococcal strains expressing so-called pneumococcal serotypes are readily recovered from current carriage specimens is likely to be relevant to pneumococcal epidemiology, niche biology, and even to potential strategies of employing commensal live vaccines. Here, we describe multiple distinct nonpneumococcal counterparts for each of the pneumococcal conjugate vaccine (PCV) serotypes 4 and 9V. Additional data from contemporary commensal isolates expressing serotypes 2, 13, and 23A further demonstrate the ubiquity of such strains. Increased focus upon this serological overlap between S. pneumoniae and its close relatives may eventually prove that most, or possibly all, pneumococcal serotypes have counterparts expressed by the common upper respiratory tract commensal species Streptococcus mitis, Streptococcus oralis, and Streptococcus infantis. |
New pneumococcal serotype 15D
Pimenta F , Moiane B , Gertz RE Jr , Chochua S , Snippes Vagnone PM , Lynfield R , Sigaúque B , Carvalho MDG , Beall B . J Clin Microbiol 2021 59 (5) The pneumococcal serogroup 15 comprises 4 capsular polysaccharide serotypes (15A, 15B, 15C, 15F) that collectively account for an impactful disease burden (1,2).…. |
Streptococcus infantis, Streptococcus mitis , and Streptococcus oralis Strains With Highly Similar cps5 Loci and Antigenic Relatedness to Serotype 5 Pneumococci.
Pimenta F , Gertz RE Jr , Park SH , Kim E , Moura I , Milucky J , Rouphael N , Farley MM , Harrison LH , Bennett NM , Bigogo G , Feikin DR , Breiman R , Lessa FC , Whitney CG , Rajam G , Schiffer J , da Gloria Carvalho M , Beall B . Front Microbiol 2018 9 3199 Streptococcus pneumoniae is a highly impactful bacterial pathogen on a global scale. The principal pneumococcal virulence factor and target of effective vaccines is its polysaccharide capsule, of which there are many structurally distinct forms. Here, we describe four distinct strains of three Mitis group commensal species (Streptococcus infantis, Streptococcus mitis, and Streptococcus oralis) recovered from upper respiratory tract specimens from adults in Kenya and the United States that were PCR-positive for the pneumococcal serotype 5 specific gene, wzy5. For each of the four strains, the 15 genes comprising the capsular polysaccharide biosynthetic gene cluster (cps5) shared the same order found in serotype 5 pneumococci, and each of the serotype 5-specific genes from the serotype 5 pneumococcal reference strain shared 76-99% sequence identity with the non-pneumococcal counterparts. Double-diffusion experiments demonstrated specific reactivity of the non-pneumococcal strains with pneumococcal serotype 5 typing sera. Antiserum raised against S. mitis strain KE67013 specifically reacted with serotype 5 pneumococci for a positive Quellung reaction and stimulated serotype 5 specific opsonophagocytic killing of pneumococci. Four additional commensal strains, identified using PCR serotyping assays on pharyngeal specimens, revealed loci highly homologous to those of pneumococci of serotypes 12F, 15A, 18C, and 33F. These data, in particular the species and strain diversity shown for serotype 5, highlight the existence of a broad non-pneumococcal species reservoir in the upper respiratory tract for the expression of capsular polysaccharides that are structurally related or identical to those corresponding to epidemiologically significant serotypes. Very little is known about the genetic and antigenic capsular diversity among the vast array of commensal streptococcal strains that represent multiple diverse species. The discovery of serotype 5 strains within three different commensal species suggests that extensive capsular serologic overlap exists between pneumococci and other members of the diverse Mitis group. These findings may have implications for our current understanding of naturally acquired immunity to S. pneumoniae and pneumococcal serotype distributions in different global regions. Further characterization of commensal strains carrying homologs of serotype-specific genes previously thought to be specific for pneumococci of known serotypes may shed light on the evolution of these important loci. |
A Population-Based Descriptive Atlas of Invasive Pneumococcal Strains Recovered Within the U.S. During 2015-2016.
Beall B , Chochua S , Gertz RE Jr , Li Y , Li Z , McGee L , Metcalf BJ , Ricaldi J , Tran T , Walker H , Pilishvili T . Front Microbiol 2018 9 2670 Invasive pneumococcal disease (IPD) has greatly decreased since implementation in the U.S. of the 7 valent conjugate vaccine (PCV7) in 2000 and 13 valent conjugate vaccine (PCV13) in 2010. We used whole genome sequencing (WGS) to predict phenotypic traits (serotypes, antimicrobial phenotypes, and pilus determinants) and determine multilocus genotypes from 5334 isolates (~90% of cases) recovered during 2015-2016 through Active Bacterial Core surveillance. We identified 44 serotypes; 26 accounted for 98% of the isolates. PCV13 serotypes (inclusive of serotype 6C) accounted for 1503 (28.2%) isolates, with serotype 3 most common (657/5334, 12.3%), while serotypes 1 and 5 were undetected. Of 305 isolates from children <5 yrs, 60 (19.7%) were of PCV13 serotypes 19A, 19F, 3, 6B, and 23F (58/60 were 19A, 19F, or 3). We quantitated MLST-based lineages first detected during the post-PCV era (since 2002) that potentially arose through serotype-switching. The 7 predominant emergent post-PCV strain complexes included 23B/CC338, 15BC/CC3280, 19A/CC244, 4/CC439, 15A/CC156, 35B/CC156, and 15BC/CC156. These strains accounted for 332 isolates (6.2% of total) and were more frequently observed in children <5 yrs (17.7%; 54/305). Fifty-seven categories of recently emerged (in the post PCV7 period) putative serotype-switch variants were identified, accounting for 402 isolates. Many of these putative switch variants represented newly emerged resistant strains. Penicillin-nonsusceptibility (MICs > 0.12 mug/ml) was found among 22.4% (1193/5334) isolates, with higher penicillin MICs (2-8 mug/ml) found in 8.0% (425/5334) of isolates that were primarily (372/425, 87.5%) serotypes 35B and 19A. Most (792/1193, 66.4%) penicillin-nonsusceptible isolates were macrolide-resistant, 410 (34.4%) of which were erm gene positive and clindamycin-resistant. The proportion of macrolide-resistant isolates increased with increasing penicillin MICs; even isolates with reduced penicillin susceptibility (MIC = 0.06 mug/ml) were much more likely to be macrolide-resistant than basally penicillin-susceptible isolates (MIC < 0.03 mug/ml). The contribution of recombination to strain diversification was assessed through quantitating 35B/CC558-specific bioinformatic pipeline features among non-CC558 CCs and determining the sizes of gene replacements. Although IPD has decreased greatly and stabilized in the post-PCV13 era, the species continually generates recombinants that adapt to selective pressures exerted by vaccines and antimicrobials. These data serve as a baseline for monitoring future changes within each invasive serotype. |
Population and Whole Genome Sequence Based Characterization of Invasive Group A Streptococci Recovered in the United States during 2015.
Chochua S , Metcalf BJ , Li Z , Rivers J , Mathis S , Jackson D , Gertz RE Jr , Srinivasan V , Lynfield R , Van Beneden C , McGee L , Beall B . mBio 2017 8 (5) Group A streptococci (GAS) are genetically diverse. Determination of strain features can reveal associations with disease and resistance and assist in vaccine formulation. We employed whole-genome sequence (WGS)-based characterization of 1,454 invasive GAS isolates recovered in 2015 by Active Bacterial Core Surveillance and performed conventional antimicrobial susceptibility testing. Predictions were made for genotype, GAS carbohydrate, antimicrobial resistance, surface proteins (M family, fibronectin binding, T, R28), secreted virulence proteins (Sda1, Sic, exotoxins), hyaluronate capsule, and an upregulated nga operon (encodes NADase and streptolysin O) promoter (Pnga3). Sixty-four M protein gene (emm) types were identified among 69 clonal complexes (CCs), including one CC of Streptococcus dysgalactiae subsp. equisimilisemm types predicted the presence or absence of active sof determinants and were segregated into sof-positive or sof-negative genetic complexes. Only one "emm type switch" between strains was apparent. sof-negative strains showed a propensity to cause infections in the first quarter of the year, while sof+ strain infections were more likely in summer. Of 1,454 isolates, 808 (55.6%) were Pnga3 positive and 637 (78.9%) were accounted for by types emm1, emm89, and emm12 Theoretical coverage of a 30-valent M vaccine combined with an M-related protein (Mrp) vaccine encompassed 98% of the isolates. WGS data predicted that 15.3, 13.8, 12.7, and 0.6% of the isolates were nonsusceptible to tetracycline, erythromycin plus clindamycin, erythromycin, and fluoroquinolones, respectively, with only 19 discordant phenotypic results. Close phylogenetic clustering of emm59 isolates was consistent with recent regional emergence. This study revealed strain traits informative for GAS disease incidence tracking, outbreak detection, vaccine strategy, and antimicrobial therapy.IMPORTANCE The current population-based WGS data from GAS strains causing invasive disease in the United States provide insights important for prevention and control strategies. Strain distribution data support recently proposed multivalent M type-specific and conserved M-like protein vaccine formulations that could potentially protect against nearly all invasive U.S. strains. The three most prevalent clonal complexes share key polymorphisms in the nga operon encoding two secreted virulence factors (NADase and streptolysin O) that have been previously associated with high strain virulence and transmissibility. We find that Streptococcus pyogenes is phylogenetically subdivided into loosely defined multilocus sequence type-based clusters consisting of solely sof-negative or sof-positive strains; with sof-negative strains demonstrating differential seasonal preference for infection, consistent with the recently demonstrated differential seasonal preference based on phylogenetic clustering of full-length M proteins. This might relate to the differences in GAS strain compositions found in different geographic settings and could further inform prevention strategies. |
Validation of ß-lactam minimum inhibitory concentration predictions for pneumococcal isolates with newly encountered penicillin binding protein (PBP) sequences.
Li Y , Metcalf BJ , Chochua S , Li Z , Gertz RE Jr , Walker H , Hawkins PA , Tran T , McGee L , Beall BW . BMC Genomics 2017 18 (1) 621 BACKGROUND: Genomic sequence-based deduction of antibiotic minimum inhibitory concentration (MIC) has great potential to enhance the speed and sensitivity of antimicrobial susceptibility testing. We previously developed a penicillin-binding protein (PBP) typing system and two methods (Random Forest (RF) and Mode MIC (MM)) that accurately predicted beta-lactam MICs for pneumococcal isolates carrying a characterized PBP sequence type (phenotypic beta-lactam MICs known for at least one isolate of this PBP type). This study evaluates the prediction performance for previously uncharacterized (new) PBP types and the probability of encountering new PBP types, both of which impact the overall prediction accuracy. RESULTS: The MM and RF methods were used to predict MICs of 4309 previously reported pneumococcal isolates in 2 datasets and the results were compared to the known broth microdilution MICs to 6 beta-lactams. Based on a method that specifically evaluated predictions for new PBP types, the RF results were more accurate than MM results for new PBP types and showed percent essential agreement (MICs agree within +/-1 dilution) >97%, percent category agreement (interpretive results agree) >93%, major discrepancy (sensitive isolate predicted as resistant) rate < 1.2%, and very major discrepancy (resistant isolate predicted as sensitive) rate < 1.4% for all 6 beta-lactams. The identification of new PBP types over time was well approximated by a diminishingly increasing curve (Pearson's r = 0.99) and minimally impacted overall MIC prediction performance. CONCLUSIONS: MIC prediction using the RF method could be an accurate alternative of phenotypic susceptibility testing even in the presence of previously uncharacterized PBP types. |
Short-read whole genome sequencing for determination of antimicrobial resistance mechanisms and capsular serotypes of current invasive Streptococcus agalactiae recovered in the United States.
Metcalf BJ , Chochua S , Gertz RE Jr , Hawkins PA , Ricaldi J , Li Z , Walker H , Tran T , Rivers J , Mathis S , Jackson D , Glennen A , Lynfield R , McGee L , Beall B . Clin Microbiol Infect 2017 23 (8) 574 e7-574 e14 OBJECTIVES: Our objective was to evaluate and exploit a whole genome sequence (WGS) bioinformatics pipeline for predicting antimicrobial resistance and capsular serotypes from invasive group B streptococci (iGBS). METHODS: For 1975 iGBS recovered during 2015 from CDC's Active Bacterial Core surveillance, we compared pipeline predictions to broth dilution testing. Fifty-six isolates from earlier surveillance were included for testing beta-lactams.. Conventional serotyping was compared to WGS-based assignments for 302 isolates. RESULTS: All 28 isolates with reduced susceptibility to beta-lactam antibiotics harbored one of 19 rare PBP2x types. Resistances to erythromycin/clindamycin (808/1975 isolates, 41.0%), erythromycin (235/1975, 11.9%), and lincosamide/streptogramin A/pleuromutilins (56/1975, 2.8%) were predicted by presence of erm-methylase, mef, and lsa determinants, respectively (41 of 56 lsa gene positive isolates also contained lnu, erm, and/or mef genes). Presence of both erm and lsa determinants (25 isolates) predicted nonsusceptibility to quinupristin/dalfopristin. Most isolates (1680/1975, 85.1%) were tet gene-positive, although 41/1565 (2.6%) tetM-positive isolates were tetracycline-susceptible. All 53 fluoroquinolone-resistant isolates contained ParC and/or GyrA substitutions. Resistances to rifampin (8 isolates), trimethoprim, chloramphenicol and vancomycin (2 isolates each) were predicted by the pipeline. Resistance to macrolides/lincosamides without pipeline prediction was rare and correlated to divergent resistance genes or rRNA A2062G substitution. A selection of 267 isolates assigned WGS-based serotypes were also conventionally serotyped. Of these, 246 (92.1%) were in agreement, with the remaining 21 (7.8%) conventionally non-serotypeable. For thirty-two of 1975 isolates (1.6%), WGS-based serotypes could not be assigned. CONCLUSION: WGS-based assignment of iGBS resistance features and serotypes is an accurate substitute for phenotypic testing. |
Using whole genome sequencing to identify resistance determinants and predict antimicrobial resistance phenotypes for year 2015 invasive pneumococcal disease isolates recovered in the United States.
Metcalf BJ , Chochua S , Gertz RE Jr , Li Z , Walker H , Tran T , Hawkins PA , Glennen A , Lynfield R , Li Y , McGee L , Beall B . Clin Microbiol Infect 2016 22 (12) 1002 e1-1002 e8 OBJECTIVES: We assessed our whole genome sequence (WGS) pipeline for accurate prediction of current antimicrobial phenotypes. METHODS: For 2316 invasive pneumococcal isolates (IPD) recovered during 2015 we compared WGS pipeline data to broth dilution testing (BDT) for 18 antimicrobials. RESULTS: For 11 antimicrobials categorical discrepancies were assigned when WGS-predicted minimum inhibitory concentrations (MICs) and BDT MICs resulted in different predictions for susceptibility, intermediate-resistance or resistance, ranging from 0.9% (tetracycline) to 2.9% (amoxicillin). For beta-lactam antibiotics, the occurrence of >4-fold MIC differences ranged from 0.2% (meropenem) to 1.0 % (penicillin), although phenotypic retesting resolved 25 - 78% of these discrepancies. Nonsusceptibility to penicillin, predicted by penicillin binding protein types, was 2.7% (non-meningitis criteria) and 23.8% (meningitis criteria). Other common resistance determinants included mef (475 isolates), ermB (191 isolates), ermB + mef (48 isolates), tetM (261 isolates) and cat (51 isolates). Additional accessory resistance genes (tetS, tet32, aphA-3, sat4) were rarely detected (in 1- 3 isolates). Rare core genome mutations conferring erythromycin-resistance included a 2 codon rplD insertion (rplD69-KG-70) and the 23S rRNA A2061G substitution (6 total isolates). Intermediate cotrimoxazole-resistance was associated with 1-2 codon insertions within folP (238 isolates) or the folA I100L substitution (38 isolates), while full cotrimoxazole-resistance was attributed to alterations in both genes (172 isolates). Levofloxacin-resistance (2 isolates) was associated with parC and/or gyrA mutations. Of 11 remaining isolates with moderately elevated MICs to both ciprofloxacin and levofloxacin, 7 contained parC or gyrA mutations. The two rifampin-resistant isolates contained rpoB mutations. CONCLUSIONS: WGS-based MIC prediction was an informative alternative to BDT for current IPD isolates. |
Penicillin-Binding Protein Transpeptidase Signatures for Tracking and Predicting ß-Lactam Resistance Levels in Streptococcus pneumoniae.
Li Y , Metcalf BJ , Chochua S , Li Z , Gertz RE Jr , Walker H , Hawkins PA , Tran T , Whitney CG , McGee L , Beall BW . mBio 2016 7 (3) beta-Lactam antibiotics are the drugs of choice to treat pneumococcal infections. The spread of beta-lactam-resistant pneumococci is a major concern in choosing an effective therapy for patients. Systematically tracking beta-lactam resistance could benefit disease surveillance. Here we developed a classification system in which a pneumococcal isolate is assigned to a "PBP type" based on sequence signatures in the transpeptidase domains (TPDs) of the three critical penicillin-binding proteins (PBPs), PBP1a, PBP2b, and PBP2x. We identified 307 unique PBP types from 2,528 invasive pneumococcal isolates, which had known MICs to six beta-lactams based on broth microdilution. We found that increased beta-lactam MICs strongly correlated with PBP types containing divergent TPD sequences. The PBP type explained 94 to 99% of variation in MICs both before and after accounting for genomic backgrounds defined by multilocus sequence typing, indicating that genomic backgrounds made little independent contribution to beta-lactam MICs at the population level. We further developed and evaluated predictive models of MICs based on PBP type. Compared to microdilution MICs, MICs predicted by PBP type showed essential agreement (MICs agree within 1 dilution) of >98%, category agreement (interpretive results agree) of >94%, a major discrepancy (sensitive isolate predicted as resistant) rate of <3%, and a very major discrepancy (resistant isolate predicted as sensitive) rate of <2% for all six beta-lactams. Thus, the PBP transpeptidase signatures are robust indicators of MICs to different beta-lactam antibiotics in clinical pneumococcal isolates and serve as an accurate alternative to phenotypic susceptibility testing. IMPORTANCE: The human pathogen Streptococcus pneumoniae is a leading cause of morbidity and mortality worldwide. beta-Lactam antibiotics such as penicillin and ceftriaxone are the drugs of choice to treat pneumococcal infections. Some pneumococcal strains have developed beta-lactam resistance through altering their penicillin-binding proteins (PBPs) and have become a major concern in choosing effective patient therapy. To systematically track and predict beta-lactam resistance, we obtained the sequence signatures of PBPs from a large collection of clinical pneumococcal isolates using whole-genome sequencing data and found that these "PBP types" were predictive of resistance levels. Our findings can benefit the current era of strain surveillance when whole-genome sequencing data often lacks detailed resistance information. Using PBP positions that we found are always substituted within highly resistant strains may lead to further refinements. Sequence-based predictions are accurate and may lead to the ability to extract critical resistance information from nonculturable clinical specimens. |
Strain features and distributions in pneumococci from children with invasive disease before and after 13 valent conjugate vaccine implementation in the United States.
Metcalf BJ , Gertz RE Jr , Gladstone RA , Walker H , Sherwood LK , Jackson D , Li Z , Law C , Hawkins PA , Chochua S , Sheth M , Rayamajhi N , Bentley SD , Kim L , Whitney CG , McGee L , Beall B . Clin Microbiol Infect 2015 22 (1) 60 e9-60 e29 The effect of second generation pneumococcal conjugate vaccines on invasive pneumococcal disease (IPD) strain distributions have not yet been well described. We analyzed IPD isolates recovered from children <5 years of age through Active Bacterial Core surveillance before (2008-2009; n=828) and after (2011-2013; n=600) 13-valent vaccine (PCV13) implementation. We employed conventional testing, PCR/electrospray ionization mass spectrometry, and whole genome sequence (WGS) analysis to identify serotypes, resistance features, genotypes, and pilus types. PCV13, licensed in February of 2010, effectively targeted all major 19A and 7F genotypes and decreased antimicrobial resistance primarily due to removal of the 19A/ST320 complex. The strain complex contributing most to remaining beta-lactam resistance during 2011-2013 was 35B/ST558. Significant emergence of non-vaccine clonal complexes was not evident. Due to the removal of vaccine serotype strains, positivity for one or both pilus types (PI-1 and PI-2) decreased in the post PCV13 years 2011-2013 relative to 2008-2009 (decreases of 32-55% for PI-1, > 95% for PI-2 and combined PI-1 + PI-2). beta-lactam susceptibility phenotypes correlated consistently with transpeptidase region sequence combinations of the three major penicillin binding proteins (PBPs) determined through WGS. Other major resistance features were predictable by DNA signatures from WGS. Multilocus sequence data combined with PBP combinations identified progeny, serotype donors, and recipient strains in serotype switch events. PCV13 decreased all PCV13 serotype clones and concurrently decreased strain subsets with resistance and/or adherence features conducive for successful carriage. Our results serve as a reference describing key features of current pediatric IPD strains in the United States after PCV13 implementation. |
Streptococcus equi subsp. zooepidemicus infections associated with guinea pigs
Gruszynski K , Young A , Levine SJ , Garvin JP , Brown S , Turner L , Fritzinger A , Gertz RE Jr , Murphy JM , Vogt M , Beall B . Emerg Infect Dis 2015 21 (1) 156-8 Streptococcus equi subsp. zooepidemicus is a known zoonotic pathogen. In this public health investigation conducted in Virginia, USA, in 2013, we identified a probable family cluster of S. zooepidemicus cases linked epidemiologically and genetically to infected guinea pigs. S. zooepidemicus infections should be considered in patients who have severe clinical illness and report guinea pig exposure. |
Non-pneumococcal mitis-group streptococci confound detection of pneumococcal capsular serotype-specific loci in upper respiratory tract
Carvalho Mda G , Pimenta FC , Moura I , Roundtree A , Gertz RE Jr , Li Z , Jagero G , Bigogo G , Junghae M , Conklin L , Feikin DR , Breiman RF , Whitney CG , Beall BW . PeerJ 2013 1 e97 We performed culture-based and PCR-based tests for pneumococcal identification and serotyping from carriage specimens collected in rural and urban Kenya. Nasopharyngeal specimens from 237 healthy children <5 years old (C-NPs) and combined nasopharyngeal/oropharyngeal specimens from 158 adults (A-NP/OPs, 118 HIV-positive) were assessed using pneumococcal isolation (following broth culture enrichment) with Quellung-based serotyping, real-time lytA-PCR, and conventional multiplexed PCR-serotyping (cmPCR). Culture-based testing from C-NPs, HIV-positive A-NP/OPs, and HIV-negative A-NP/OPs revealed 85.2%, 40.7%, and 12.5% pneumococcal carriage, respectively. In contrast, cmPCR serotypes were found in 93.2%, 98.3%, and 95.0% of these sets, respectively. Two of 16 lytA-negative C-NPs and 26 of 28 lytA-negative A-NP/OPs were cmPCR-positive for 1-10 serotypes (sts) or serogroups (sgs). A-NP/OPs averaged 5.5 cmPCR serotypes/serogroups (5.2 in HIV-positive, 7.1 in HIV-negative) and C-NPs averaged 1.5 cmPCR serotypes/serogroups. cmPCR serotypes/serogroups from lytA-negative A-NP/OPs included st2, st4, sg7F/7A, sg9N/9L, st10A, sg10F/10C/33C, st13, st17F, sg18C/18A/18B/18F, sg22F/22A, and st39. Nine strains of three non-pneumococcal species (S. oralis, S. mitis, and S. parasanguinis) (7 from A-OP, 1 from both A-NP and A-OP, and 1 from C-NP) were each cmPCR-positive for one of 7 serotypes/serogroups (st5, st13, sg15A/15F, sg10F/10C/33C, sg33F/33A/37, sg18C/18A/18B/18F, sg12F/12A/12B/ 44/46) with amplicons revealing 83.6-99.7% sequence identity to pneumococcal references. In total, 150 cmPCR amplicons from carriage specimens were sequenced, including 25 from lytA-negative specimens. Amplicon sequences derived from specimens yielding a pneumococcal isolate with the corresponding serotype were identical or highly conserved (>98.7%) with the reference cmPCR amplicon for the st, while cmPCR amplicons from lytA-negative specimens were generally more divergent. Separate testing of 56 A-OPs and 56 A-NPs revealed that approximately 94% of the positive cmPCR results from A-NP/OPs were from OP microbiota. In contrast, A-NPs yielded >2-fold more pneumococcal isolates than A-OPs. Verified and suspected non-pneumococcal cmPCR serotypes/serogroups appeared to be relatively rare in C-NPs and A-NPs compared to A-OPs. Our findings indicate that non-pneumococcal species can confound serotype-specific PCR and other sequence-based assays due to evolutionarily conserved genes most likely involved in biosynthesis of surface polysaccharide structures. |
Concurrent serotyping and genotyping of pneumococci by use of PCR and electrospray ionization mass spectrometry.
Massire C , Gertz RE Jr , Svoboda P , Levert K , Reed MS , Pohl J , Kreft R , Li F , White N , Ranken R , Blyn LB , Ecker DJ , Sampath R , Beall B . J Clin Microbiol 2012 50 (6) 2018-25 A pneumococcal serotyping/genotyping system (PSGS) was developed based upon targeted PCR, followed by electrospray ionization mass spectrometry and amplicon base composition analysis. Eight multiplex PCRs, 32 targeting serotype-determining capsular biosynthetic loci, and 8 targeting multilocus sequence typing (MLST) loci were employed for each of 229 highly diverse Streptococcus pneumoniae isolates. The most powerful aspect of the PSGS system was the identification of capsular serotypes accounting for the majority of invasive and carried pneumococcal strains. Altogether, 45 different serotypes or serogroups were correctly predicted among the 196 resolvable isolates, with only 2 unexpected negative results. All 33 isolates that represented 23 serotypes not included in the PSGS yielded negative serotyping results. A genotyping database was constructed using the base compositions of 65- to 100-bp sections of MLST alleles compiled within http://www.mlst.net. From this database, one or more MLST sequence types (STs) that comprised a PSGS genotype were identified. The end result of more PSGS genotypes (163) than conventional STs actually tested (155) was primarily due to amplification failures of 1 to 3 targets. In many instances, the PSGS genotype could provide resolution of single- and double-locus variants. This molecular serotyping/genotyping scheme is well suited to rapid characterization of large sets of pneumococcal isolates. |
Pneumococcal genome sequencing tracks a vaccine escape variant formed through a multi-fragment recombination event.
Golubchik T , Brueggemann AB , Street T , Gertz RE Jr , Spencer CC , Ho T , Giannoulatou E , Link-Gelles R , Harding RM , Beall B , Peto TE , Moore MR , Donnelly P , Crook DW , Bowden R . Nat Genet 2012 44 (3) 352-5 Streptococcus pneumoniae ('pneumococcus') causes an estimated 14.5 million cases of serious disease and 826,000 deaths annually in children under 5 years of age. The highly effective introduction of the PCV7 pneumococcal vaccine in 2000 in the United States provided an unprecedented opportunity to investigate the response of an important pathogen to widespread, vaccine-induced selective pressure. Here, we use array-based sequencing of 62 isolates from a US national monitoring program to study five independent instances of vaccine escape recombination, showing the simultaneous transfer of multiple and often large (up to at least 44 kb) DNA fragments. We show that one such new strain quickly became established, spreading from east to west across the United States. These observations clarify the roles of recombination and selection in the population genomics of pneumococcus and provide proof of principle of the considerable value of combining genomic and epidemiological information in the surveillance and enhanced understanding of infectious diseases. |
Using PCR-based detection and genotyping to trace Streptococcus salivarius meningitis outbreak strain to oral flora of radiology physician assistant.
Srinivasan V , Gertz RE Jr , Shewmaker PL , Patrick S , Chitnis AS , O'Connell H , Benowitz I , Patel P , Guh AY , Noble-Wang J , Turabelidze G , Beall B . PLoS One 2012 7 (2) e32169 We recently investigated three cases of bacterial meningitis that were reported from a midwestern radiology clinic where facemasks were not worn during spinal injection of contrast agent during myelography procedures. Using pulsed field gel electrophoresis we linked a case strain of S. salivarius to an oral specimen of a radiology physician assistant (RPA). We also used a real-time PCR assay to detect S. salivarius DNA within a culture-negative cerebrospinal fluid (CSF) specimen. Here we extend this investigation through using a nested PCR/sequencing strategy to link the culture-negative CSF specimen to the case strain. We also provide validation of the real-time PCR assay used, demonstrating that it is not solely specific for Streptococcus salivarius, but is also highly sensitive for detection of the closely related oral species Streptococcus vestibularis. Through using multilocus sequence typing and 16S rDNA sequencing we further strengthen the link between the CSF case isolate and the RPA carriage isolate. We also demonstrate that the newly characterized strains from this study are distinct from previously characterized S. salivarius strains associated with carriage and meningitis. |
Outbreak of bacterial meningitis among patients undergoing myelography at an outpatient radiology clinic
Chitnis AS , Guh AY , Benowitz I , Srinivasan V , Gertz RE Jr , Shewmaker PL , Beall BW , O'Connell H , Noble-Wang J , Gornet MF , Van Beneden C , Patrick SL , Turabelidze G , Patel PR . J Am Coll Radiol 2012 9 (3) 185-90 PURPOSE: To investigate an outbreak of bacterial meningitis at an outpatient radiology clinic (clinic A) and to determine the source and implement measures to prevent additional infections. METHODS: A case was defined as bacterial meningitis in a patient undergoing myelography at clinic A from October 11 to 25, 2010. Patients who underwent myelography and other procedures at clinic A during that period were interviewed, medical records were reviewed, and infection prevention practices were assessed. Case-patient cerebrospinal fluid (CSF) specimens, oral specimens from health care personnel (HCP), and opened iohexol vials were tested for bacteria. Bacterial isolates were compared using pulsed-field gel electrophoresis. A culture-negative CSF specimen was tested using a real-time polymerase chain reaction assay. RESULTS: Three cases were identified among 35 clinic A patients who underwent procedures from October 11 to 25, 2010. All case-patients required hospitalization, 2 in an intensive care unit. Case-patients had myelography performed by the same radiology physician assistant and technician on October 25; all patients who underwent myelography on October 25 were affected. HCP did not wear facemasks and reused single-dose iohexol vials for multiple patients. Streptococcus salivarius (a bacteria commonly found in oral flora) was detected in the CSF of 2 case-patients (1 by culture, 1 using real-time polymerase chain reaction) and in HCP oral specimens; 1 opened iohexol vial contained Staphylococcus epidermidis. Pulsed-field gel electrophoresis profiles from the case-patient S salivarius and the radiology physician assistant were indistinguishable. CONCLUSIONS: Bacterial meningitis likely occurred because HCP performing myelography did not wear facemasks; lapses in injection practices may have contributed to transmission. Targeted education regarding mask use and safe injection practices is needed among radiology HCP. |
Characterization of highly antimicrobial-resistant clinical pneumococcal isolates recovered in a Chinese hospital during 2009-2010
Zhang B , Gertz RE Jr , Liu Z , Li Z , Fu W , Beall B . J Med Microbiol 2012 61 42-8 Ninety-one consecutive pneumococcal isolates (primarily from sputum), recovered in Chongqing Southwest Hospital during a 12 month period in 2009-2010 from individuals of all ages with suspected cases of pneumococcal disease, were subjected to PCR-serotyping, Quellung reaction serotyping, antimicrobial-susceptibility testing and multilocus sequence typing (MLST). Although 20 different serotypes were observed, most isolates (69, 75.8 %) were of serotypes included in the pneumococcal 13-valent conjugate vaccine (PCV13), including 33 of the 46 (71.7 %) isolates recovered from individuals less than 5 years of age. The prevalent serotypes were 19F (34 %), 19A (9.9 %), 6B (9.9 %), 23F (7.7 %), 14 (6.6 %) and 6A (4.4 %). PCR-determined serotypes were in agreement with Quellung testing, with the exception of two serotype 33C isolates. Most or all isolates within each PCV13 serotype were represented by one genotype, with the globally disseminated MLST sequence types (STs) ST271, ST320, ST90 and ST81 each accounting for the highly resistant isolates within serotypes 19F, 19A, 6B and 23F, respectively. Sixty-six (72.5 %) isolates were resistant to combinations of beta-lactam antibiotics (BLAs). A total of 63 of these 66 (95.5 %) BLA-resistant isolates were of serotypes included in PCV13; however, 3 serogroup 15 isolates were also BLA-resistant. Most isolates (88/91 = 96.7 %) were resistant to erythromycin and clindamycin. The majority of isolates were also resistant to tetracycline (76, 84 %) and to cotrimoxazole (67, 74 %). This work revealed that the majority of antimicrobial-resistant isolates (50/91 = 54.9 %) recovered in this Chinese hospital were represented by four global clones. Serotypes for these as well as more obscure strains were readily determined by using PCR. |
Serotype and genotype distributions of pneumococcal carriage isolates recovered from Brazilian children attending day-care centres.
Pimenta FC , Carvalho MG , Gertz RE Jr , Bastos-Rocha CG , Oliveira LS , Lacerda Pigosso L , Lima JA , Marquez Franco C , Andrade AL , Beall BW . J Med Microbiol 2011 60 1455-9 Pneumococcal nasopharyngeal carriage isolates recovered from Brazilian children attending day-care centres in 2005 were assessed for serotype, genotype and penicillin susceptibility phenotype. As 124 of the 253 isolates (49 %) were characterized previously with respect to serotype and penicillin susceptibility, the primary objectives were to examine clonal associations and penicillin susceptibility within major serotypes and to assess the suitability of conventional multiplex PCR for deducing carriage serotypes within this population. Using a combination of PCR-based serotyping and the Quellung reaction, serotypes were identified for 81 % (205/253) of the isolates, with serogroups or types 14, 6, 23F, 19F and 18 being predominant. Included within the 205 isolates successfully serotyped by PCR were 28 isolates that had become non-viable. Forty-eight isolates were non-typable using both the PCR method and the Quellung reaction. Penicillin non-susceptibility was observed within 16 of the 18 multilocus sequence types detected. Thus, this study provides further evidence from a diverse collection of pneumococcal clones that PCR-based serotype deduction is useful for providing supportive evidence for pneumococcal conjugate vaccine implementation. |
Increased penicillin nonsusceptibility of nonvaccine-serotype invasive pneumococci other than serotypes 19A and 6A in post-7-valent conjugate vaccine era
Gertz RE Jr , Li Z , Pimenta FC , Jackson D , Juni BA , Lynfield R , Jorgensen JH , Carvalho MG , Beall BW . J Infect Dis 2010 201 (5) 770-5 According to population-based invasive pneumococcal surveillance in the United States during 2007, 898 (26%) of 3,511 isolates were penicillin nonsusceptible. Non-7-valent pneumococcal conjugate vaccine (PCV7) serotypes other than 19A accounted for 40% of these penicillin-nonsusceptible isolates; of these, serotypes 15A (11%), 23A (8%), 35B (8%), and 6C (5%) were most common (cumulatively 32% of penicillin-nonsusceptible isolates). Each except 6C represented a single serotype and clonal complex combination that predated the introduction of PCV7. We evaluated the genetic characteristics and nonsusceptibility to penicillin of non- PCV7 serotypes, and we found increased proportions of specific penicillin-nonsusceptible clones in serotypes 15A, 23A, 35B, and 6C, which potentially indicates a basic change of population structure within these individual serotypes. |
Genetic relationships deduced from emm and multilocus sequence typing of invasive Streptococcus dysgalactiae subsp. equisimilis and S. canis recovered from isolates collected in the United States
Ahmad Y , Gertz RE Jr , Li Z , Sakota V , Broyles LN , Van Beneden C , Facklam R , Shewmaker PL , Reingold A , Farley MM , Beall BW . J Clin Microbiol 2009 47 (7) 2046-54 Beta-hemolytic group C and G streptococci cause a considerable invasive disease burden and sometimes cause disease outbreaks. Little is known about the critical epidemiologic parameter of genetic relatedness between isolates. We determined the emm types of 334 Streptococcus dysgalactiae subsp. equisimilis isolates, and attempted emm typing of 5 Streptococcus canis isolates from a recent population-based surveillance for invasive isolates. Thirty-four emm types were observed, including one from S. canis. We formulated multilocus sequence typing (MLST) primers with six of the seven loci corresponding to the Streptococcus pyogenes MLST scheme. We performed MLST with 65 of the 334 surveillance isolates (61 S. dysgalactiae subsp. equisimilis isolates, 4 S. canis isolates) to represent each emm type identified, including 2 to 3 isolates for each of the 25 redundantly represented emm types. Forty-one MLST sequence types (STs) were observed. Isolates within 16 redundantly represented S. dysgalactiae subsp. equisimilis emm types shared identical or nearly identical STs, demonstrating concordance between the emm type and genetic relatedness. However, seven STs were each represented by two to four different emm types, and 7 of the 10 S. dysgalactiae subsp. equisimilis eBURST groups represented up to six different emm types. Thus, S. dysgalactiae subsp. equisimilis isolates were similar to S. pyogenes isolates, in that strains of the same emm type were often highly related, but they differed from S. pyogenes, in that S. dysgalactiae subsp. equisimilis strains with identical or closely similar STs often exhibited multiple unrelated emm types. The phylogenetic relationships between S. dysgalactiae subsp. equisimilis and S. pyogenes alleles revealed a history of interspecies recombination, with either species often serving as genetic donors. The four S. canis isolates shared highly homologous alleles but were unrelated clones without evidence of past recombination with S. dysgalactiae subsp. equisimilis or S. pyogenes. |
Rarely occurring 19A-like cps locus from a serotype 19F pneumococcal isolate indicates continued need of serology-based quality control for PCR-based serotype determinations
Pimenta FC , Gertz RE Jr , Roundtree A , Yu J , Nahm MH , McDonald RR , Carvalho Mda G , Beall BW . J Clin Microbiol 2009 47 (7) 2353-4 Determination of the DNA sequences corresponding to all 91 pneumococcal capsular biosynthetic (cps) loci (1, 3, 6, 9) has allowed serotype determinations from pneumococcal isolates and clinical specimens using conventional PCR assays (2, 4, 7, 8, 10). We developed sequential, multiplexed PCR schemes that resolve 33 serologic specificities, including 20 serotypes and 13 serotype subsets (4, 7, 8). We recently expanded the scheme to 40 specificities (www.cdc.gov/ncidod/biotech/strep/pcr.htm). | PCR primers targeted serotype-specific open reading frames within central portions of known cps loci (4, 7, 8). Subsequent information (1, 3, 6) revealed that we primarily targeted serotype-specific oligosaccharide repeat unit polymerases (wzy encoded), glycosyl transferases, and flippases (wzx encoded). Our serotype 19A primers, however, targeted the mnaA gene that encodes UDP-N-acetylglucosamine-2-epimerase (8). Although the mnaA sequence differs markedly among the 15 cps loci that harbor it (serotypes 19A, -B, -C, -F; 12A, -B, -F; 9A, -N, -L, -V; 4; 36; 44; 46), due to potential exchanges of heterologous mnaA genes, we recently changed the 19A PCR assay to target wzy (our unpublished data). The wzy19A and wzy19F genes putatively provide the basis of the structural difference between the related 19A and 19F capsules (1). |
- Page last reviewed:Feb 1, 2024
- Page last updated:Apr 22, 2024
- Content source:
- Powered by CDC PHGKB Infrastructure