Robert E. Gertz

Population Snapshot of Emergent Streptococcus pneumoniae Serotype 19A in the United States, 2005

BACKGROUND Serotype 19A invasive pneumococcal disease (IPD) increased annually in the United States after the introduction of the 7-valent conjugate vaccine (PCV7). To understand this increase, we characterized serotype 19A isolates recovered during 2005. METHODS IPD cases during 1998-2005 were identified through population-based surveillance. We performed susceptibility testing and multilocus sequence typing on 528 (95%) of 554 serotype 19A isolates reported in 2005. RESULTS The incidence of IPD due to serotype 19A increased from 0.8 to 2.5 cases per 100,000 population between 1998 and 2005 (P < .05), whereas the overall incidence of IPD decreased from 24.4 to 13.8 cases per 100,000 population (P < .05). Simultaneously, the incidence of IPD due to penicillin-resistant 19A isolates increased from 6.7% to 35% (P < .0001). Of 151 penicillin-resistant 19A isolates, 111 (73.5%) belonged to the rapidly emerging clonal complex 320, which is related to multidrug-resistant Taiwan(19F)-14. The remaining penicillin-resistant strains were highly related to other clones of PCV7 serotypes or to isolates within major 19A clonal complex 199 (CC199). In 1999, only CC199 and 3 minor clones were apparent among serotype 19A isolates. During 2005, 11 multiple-isolate clonal sets were detected, including capsular switch variants of a serotype 4 clone. CONCLUSIONS PCV7 ineffectiveness against serotype 19A, antibiotic resistance, clonal expansion and emergence, and capsular switching have contributed to the genetic diversity of 19A and to its emergence as the predominant invasive pneumococcal serotype in the United States.

Sequential Multiplex PCR Approach for Determining Capsular Serotypes of Streptococcus pneumoniae Isolates

ABSTRACT Accurate serotyping is essential to monitor the changes in the seroepidemiology of Streptococcus pneumoniae. We devised a simple and schematic sequence-based system of seven multiplex PCRs, in a sequence order based upon Active Bacterial Core surveillance (ABCs) serotype distribution during 2002 to 2003, to reliably deduce specific pneumococcal serotypes. A total of 421 isolates from ABCs were randomly chosen to evaluate this system. Two hundred twenty-nine of the isolates (54.3%) were specifically assigned 1 of 17 serotypes by the multiplex PCR system, with the results in complete concordance with conventional serotyping. One hundred seventy-two additional isolates (40.9%) were assigned to 11 specific sets of 2 to 4 serotypes that with one exception (serotypes 6A and 6B) consisted of the single frequently occurring targeted serotype and 1 to 3 additional rare serotypes primarily within the same serogroup as the targeted serotype. Only 20 isolates (4.8%) could not be assigned specific serotypes or serotype sets, since they were either of rare serotypes not included in the assay design or were nonserotypeable. Overall, we found this system to be highly reliable, with the potential to greatly reduce our reliance upon conventional serotyping. Especially important is the capability of this system to give serotype-determining potential to any facility that lacks the expensive typing sera and expertise needed for conventional serotyping yet has the modest equipment necessary for DNA amplification and electrophoresis.

Postvaccine Genetic Structure of Streptococcus pneumoniae Serotype 19A from Children in the United States

BACKGROUND The introduction of the 7-valent conjugate pneumococcal vaccine (PCV7) in children may result in serotype replacement. We estimated the rate of increase of invasive pneumococcal disease (IPD) caused by serotype 19A in children <5 years old and determined the genetic composition of these isolates. METHODS Cases of IPD between July 1999 and June 2004 were identified through the Active Bacterial Core Surveillance. Serotype 19A isolates obtained from children <5 years old between January 2003 and June 2004 were characterized by serotyping, antibiotic susceptibility testing, and pulsed-field gel electrophoresis (PFGE). Select isolates representing homologous PFGE clusters were subjected to multilocus sequence typing, and eBURST was used to delineate clonal groups. RESULTS Between July 1999 and June 2004, the overall rate of IPD decreased from 23.3 to 13.1 cases/100,000 population (P<.00001). In children <5 years old, the rate decreased from 88.7 to 22.4 cases/100,000 population (P<.00001), whereas the rate in persons > or =5 years old decreased from 18.4 to 12.4 cases/100,000 population (P<.0001). The rate of serotype 19A IPD in children <5 years old increased significantly from 2.6 cases/100,000 population in 1999-2000 to 6.5 cases/100,000 population in 2003-2004; this was accompanied by significant increases in penicillin nonsusceptibility (P=.008) and multidrug resistance (P=.002) among serotype 19A isolates. As was observed during the pre-PCV7 era, clonal complex (CC) 199 predominated within serotype 19A, representing approximately 70% of invasive serotype 19A isolates from children <5 years old during 2003-2004. New serotype 19A genotypes were observed during 2003-2004, including 6 CCs that were not found among pneumococcal serotype 19A isolates during surveillance in 1999. CONCLUSION Serotype 19A is, at present, the most important cause of IPD by replacement serotypes, and it is increasingly drug resistant. CC199 is the predominant CC among type 19A serotypes in children <5 years old. Our data suggest that some of the increase in rates of infection with serotype 19A may be due to serotype switching within certain vaccine type strains.

Pre- and Postvaccination Clonal Compositions of Invasive Pneumococcal Serotypes for Isolates Collected in the United States in 1999, 2001, and 2002

ABSTRACT Monitoring of serotypes and their clonal associations is critical as pneumococci adapt to the selective pressures exerted by the pneumococcal seven-valent conjugate vaccine (PCV7). We genotyped 1,476 invasive isolates from the Active Bacterial Core surveillance (705 [89.8%] of the isolates were obtained from children <5 years of age, and 771 [18.4%] of the isolates were obtained from individuals >5 years of age) in 2001 and 2002 (after the introduction of PCV7). The data were compared to the results for 1,168 invasive isolates (855 [83.9%] of the isolates were from children <5 years of age) collected in 1999. Among children <5 years of age, the incidence of invasive disease due to non-PCV7 serogroups together with serogroup 19A increased (P < 0.001). Eighty-three clonal sets, representing 177 multilocus sequence types (STs), were compiled from the 3-year isolate set. Among the non-PCV7 serogroups, newly emerging clones were uncommon; and a significant expansion of already established clones occurred for serotypes 3 (ST180), 7F (ST191), 15BCF (ST199), 19A (ST199), 22F (ST433), 33F (ST662), and 38 (ST393). However, additional minor clonal types within serotypes 1, 6A, 6B, 7C, 9N, 10A, 12F, 14, 15B/C, 17F, 19A, 19F, 20, 22F, and 33F that were absent in 1999 were found during 2001 and 2002. Although 23 clonal sets exhibited multiple serotypes, for most serotypes there were either no changes or modest changes in clonal compositions since the introduction of PCV7. The only example of an identical ST shared between non-PCV7 and PCV7 or PCV7-related serotypes was ST199; however, ST199 was prevalent within serotypes 15B/C and 19A before and after PCV7 introduction. Continued genotypic surveillance is warranted, since certain clones not targeted by PCV7 are expanding, and their emergence as significant pathogens could occur with maintained vaccine pressure.

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.

Point Mutation in the Group B Streptococcal pbp2x Gene Conferring Decreased Susceptibility to β-Lactam Antibiotics

ABSTRACT Beta-lactam antibiotics (BLAs) are the first-line agents used against group B streptococci (GBS) infection. A clonal set of four independent, invasive GBS isolates with elevated MICs to BLAs were identified that shared a pbp2x mutation (Q557E) corresponding to a resistance-conferring pneumococcal mutation. BLA sensitivity was restored through allelic replacement or complementation with the wild-type pbp2x.

PCR-Based Quantitation and Clonal Diversity of the Current Prevalent Invasive Serogroup 6 Pneumococcal Serotype, 6C, in the United States in 1999 and 2006 to 2007

Following introduction of the 7-valent pneumococcal conjugate vaccine to the United States, rates of invasive pneumococcal disease (IPD) caused by serotype 6A declined among all age groups, while rates of IPD caused by newly identified serotype 6C increased slightly among persons 5 years of age and older. Conventionally serotyped 6A isolates (CS6As) from active population-based surveillance during 1999 and 2006 to 2007 were classified as serotypes 6A and 6C by an expedient and highly accurate serotype 6C-specific PCR assay developed during this study. PCR testing of 636 year 1999, 2006, and 2007 CS6As revealed 6C proportions of 35/214 (16.4%), 141/218 (64.7%), and 141/204 (69.1%), respectively. These results agreed with those from a previously devised monoclonal antibody-based serotyping system (346 CS6As compared). Type 6C IPD incidence significantly increased during 2006 and 2007 compared to during 1999 (0.57 to 0.58 cases per 100,000 and 0.22 cases per 100,000, respectively; 164% increase from 1999 to 2007 [95% confidence interval, 87 to 270%]), while rates of IPD due to types 6A and 6B markedly decreased. In 2007, 31.2% of 6C isolates were not susceptible to penicillin. Serotype 6C is now the predominant serotype associated with serogroup 6 IPD in the United States and is often penicillin nonsusceptible. We performed multilocus sequence typing (MLST) on a limited sampling of 6C isolates with different antimicrobial susceptibility profiles. MLST of 42 6C isolates revealed 12 genotypes distributed among six distinct genetic groups. Fifteen 6C isolates shared one of four different MLST types with 6C-negative CS6As. MLST results suggest 6C strains arose from independent recombination events involving only serotype 6A and 6C parental strains.

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.

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

ABSTRACT 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.

Emergence of a Novel Penicillin-Nonsusceptible, Invasive Serotype 35B Clone of Streptococcus pneumoniae within the United States

Monitoring antibiotic-resistant pneumococcal strains not covered by the 7-valent conjugate vaccine is an important priority. The Centers for Disease Control and Preventions Active Bacterial Core Surveillance identified 68 invasive penicillin-nonsusceptible serotype 35B (PN35B) isolates recovered from 1995 to 2001 from patients residing in the states of California, Colorado, Connecticut, Georgia, Maryland, Minnesota, New York, Oregon, Tennessee, and Texas. Nonsusceptible isolates accounted for 69% of all serotype 35B isolates recovered during this time. Twelve (18%) of the 68 PN35B isolates recovered since 1995 were obtained from pediatric patients. These 68 isolates exhibited penicillin MICs of 0.25-2 microg/mL and reduced susceptibility to cefotaxime. Representative PN35B isolates exhibited a common chromosomal macrorestriction profile and identical penicillin-binding-protein gene restriction profiles characteristic of penicillin-resistant strains, and they shared a unique 7-locus sequence type that included 3 new alleles. The mosaic pbp2b and divergent ddl sequences were suggestive of interspecies recombination at the ddl-pbp2b chromosomal region.