Nonpneumococcal Strains Recently Recovered from Carriage Specimens and Expressing Capsular Serotypes Highly Related or Identical to Pneumococcal Serotypes 2, 4, 9A, 13, and 23A.

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.

A Population-Based Descriptive Atlas of Invasive Pneumococcal Strains Recovered Within the U.S. During 2015-2016.

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 µg/ml) was found among 22.4% (1193/5334) isolates, with higher penicillin MICs (2-8 µg/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 µg/ml) were much more likely to be macrolide-resistant than basally penicillin-susceptible isolates (MIC < 0.03 µg/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.

Streptococcus mitis Expressing Pneumococcal Serotype 1 Capsule.

Streptococcus pneumoniae's polysaccharide capsule is an important virulence factor; vaccine-induced immunity to specific capsular polysaccharide effectively prevents disease. Serotype 1 S. pneumoniae is rarely found in healthy persons, but is highly invasive and a common cause of meningitis outbreaks and invasive disease outside of the United States. Here we show that genes for polysaccharide capsule similar to those expressed by pneumococci were commonly detected by polymerase chain reaction among upper respiratory tract samples from older US adults not carrying pneumococci. Serotype 1-specific genes were predominantly detected. In five oropharyngeal samples tested, serotype 1 gene belonging to S. mitis expressed capsules immunologically indistinct from pneumococcal capsules. Whole genome sequencing revealed three distinct S. mitis clones, each representing a cps1 operon highly similar to the pneumococcal cps1 reference operon. These findings raise important questions about the contribution of commensal streptococci to natural immunity against pneumococci, a leading cause of mortality worldwide.

Streptococcus infantis, Streptococcus mitis, and Streptococcus oralis Strains With Highly Similar cps5 Loci and Antigenic Relatedness to Serotype 5 Pneumococci.

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.

Population and Whole Genome Sequence Based Characterization of Invasive Group A Streptococci Recovered in the United States during 2015.

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.

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 ß-lactam MICs for pneumococcal isolates carrying a characterized PBP sequence type (phenotypic ß-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 ß-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 ß-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.

Penicillin-Binding Protein Transpeptidase Signatures for Tracking and Predicting ß-Lactam Resistance Levels in Streptococcus pneumoniae.

UNLABELLED: ß-Lactam antibiotics are the drugs of choice to treat pneumococcal infections. The spread of ß-lactam-resistant pneumococci is a major concern in choosing an effective therapy for patients. Systematically tracking ß-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 ß-lactams based on broth microdilution. We found that increased ß-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 ß-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 ß-lactams. Thus, the PBP transpeptidase signatures are robust indicators of MICs to different ß-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. ß-Lactam antibiotics such as penicillin and ceftriaxone are the drugs of choice to treat pneumococcal infections. Some pneumococcal strains have developed ß-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 ß-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.

Streptococcus equi subsp. zooepidemicus infections associated with guinea pigs.

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.

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 ∼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.

Outbreak of bacterial meningitis among patients undergoing myelography at an outpatient radiology clinic.

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.

Concurrent serotyping and genotyping of pneumococci by use of PCR and electrospray ionization mass spectrometry.

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.

Using PCR-based detection and genotyping to trace Streptococcus salivarius meningitis outbreak strain to oral flora of radiology physician assistant.

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.

Pneumococcal genome sequencing tracks a vaccine escape variant formed through a multi-fragment recombination event.

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(1). The highly effective introduction of the PCV7 pneumococcal vaccine in 2000 in the United States(2,3) 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(4), 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.

Characterization of highly antimicrobial-resistant clinical pneumococcal isolates recovered in a Chinese hospital during 2009-2010.

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 ß-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.

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.

Shifting genetic structure of invasive serotype 19A pneumococci in the United States.

BACKGROUND: Following 7-valent conjugate vaccine introduction in the United States in 2000, invasive serotype (sero19A) pneumococcal disease (IPD) emerged rapidly. Sero19A IPD incidence increased slightly during 2005-2008 (from 2.3 cases to 2.5 cases per 100,000 population), whereas sero19A penicillin resistance (defined as a minimum inhibitor concentration [MIC] ≥2 µg/mL) increased significantly (from 28.7% to 43.7%). To better understand changes, we characterized sero19A isolates recovered during 2004-2008. METHODS: We performed antimicrobial susceptibility testing on all 2767 sero19A IPD isolates identified through the Centers for Disease Control Active Bacterial Core surveillance during 2004-2008. We genotyped 1804 (96.3%) of 1874 sero19A isolates recovered during 2005-2007 and all 148 year 2008 sero19A isolates from children <5 years of age. RESULTS: Resistant clonal complex (CC) 320/271(19A) increased from 20.9% (115 of 550) to 32.9% (208 of 633; P < .001) of IPD isolates during 2005-2007, which paralleled increased sero19A penicillin resistance (from 28.7% [163 of 567 isolates] to 39.5% [261 of 661 isolates]; P < .001). Total IPD due to 320/271(19A) increased during 2005-2007 and increased from 2.1 to 3.6 cases per 100,000 population during 2005-2008 in children <5 years of age. The penicillin-susceptible/intermediate, putative vaccine-escape CC695(19A) increased from 7.5% (41 of 550) to 13.6% (85 of 633) of sero19A isolates during 2005-2007 (P = .002). CONCLUSIONS: Sero19A rates may have plateaued; however, clonal shifts are increasing resistance. Increased IPD caused by CC320/271(19A) and CC695(19A) could reflect additional selective advantages in addition to resistance.

Streptococcus salivarius meningitis case strain traced to oral flora of anesthesiologist.

Two women in labor received intrapartum spinal anesthesia from the same anesthesiologist approximately 1 h apart. Within 15 h, both patients developed Streptococcus salivarius meningitis and one patient died. Blood and cerebrospinal fluid (CSF) samples from both patients and tongue swab specimens from the anesthesiologist yielded isolates of an indistinguishable S. salivarius strain.

Increased penicillin nonsusceptibility of nonvaccine-serotype invasive pneumococci other than serotypes 19A and 6A in post-7-valent conjugate vaccine era.

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.