Lesley McGee

Rapid Pneumococcal Evolution in Response to Clinical Interventions

Streptococcus pneumonia evades vaccines and drugs by high levels of recombination and rapid adaptation. Epidemiological studies of the naturally transformable bacterial pathogen Streptococcus pneumoniae have previously been confounded by high rates of recombination. Sequencing 240 isolates of the PMEN1 (Spain23F-1) multidrug-resistant lineage enabled base substitutions to be distinguished from polymorphisms arising through horizontal sequence transfer. More than 700 recombinations were detected, with genes encoding major antigens frequently affected. Among these were 10 capsule-switching events, one of which accompanied a population shift as vaccine-escape serotype 19A isolates emerged in the USA after the introduction of the conjugate polysaccharide vaccine. The evolution of resistance to fluoroquinolones, rifampicin, and macrolides was observed to occur on multiple occasions. This study details how genomic plasticity within lineages of recombinogenic bacteria can permit adaptation to clinical interventions over remarkably short time scales.

Nomenclature of major antimicrobial-resistant clones of Streptococcus pneumoniae defined by the pneumococcal molecular epidemiology network

ABSTRACT The emergence of disease caused by penicillin-resistant and multidrug-resistant pneumococci has become a global concern, necessitating the identification of the epidemiological spread of such strains. The Pneumococcal Molecular Epidemiology Network was established in 1997 under the auspices of the International Union of Microbiological Societies with the aim of characterizing, standardizing, naming, and classifying antimicrobial agent-resistant pneumococcal clones. Here we describe the nomenclature for 16 pneumococcal clones that have contributed to the increase in antimicrobial resistance worldwide. Guidelines for the recognition of these clones using molecular typing procedures (pulsed-field gel electrophoresis, BOX-PCR, and multilocus sequence typing) are presented, as are the penicillin-binding profiles and macrolide resistance determinants for the 16 clones. This network can serve as a prototype for the collaboration of scientists in identifying clones of important human pathogens and as a model for the development of other networks.

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.

Evaluation and improvement of real-time PCR assays targeting lytA, ply, and psaA genes for detection of pneumococcal DNA.

ABSTRACT The accurate diagnosis of pneumococcal disease has frequently been hampered not only by the difficulties in obtaining isolates of the organism from patient specimens but also by the misidentification of pneumococcus-like viridans group streptococci (P-LVS) as Streptococcus pneumoniae. This is especially critical when the specimen comes from the respiratory tract. In this study, three novel real-time PCR assays designed for the detection of specific sequence regions of the lytA, ply, and psaA genes were developed (lytA-CDC, ply-CDC, and psaA, respectively). These assays showed high sensitivity (<10 copies for lytA-CDC and ply-CDC and an approximately twofold less sensitivity for psaA). Two additional real-time PCR assays for lytA and ply described previously for pneumococcal DNA detection were also evaluated. A panel of isolates consisting of 67 S. pneumoniae isolates (44 different serotypes and 3 nonencapsulated S. pneumoniae isolates from conjunctivitis outbreaks) and 104 nonpneumococcal isolates was used. The 67 S. pneumoniae isolates were reactive in all five assays. The new real-time detection assays targeting the lytA and psaA genes were the most specific for the detection of isolates confirmed to be S. pneumoniae, with lytA-CDC showing the greatest specificity. Both ply PCRs were positive for all isolates of S. pseudopneumoniae, along with 13 other isolates of other P-LVS isolates confirmed to be non-S. pneumoniae by DNA-DNA reassociation. Thus, the use of the ply gene for the detection of pneumococci can lead to false-positive reactions in the presence of P-LVS. The five assays were applied to 15 culture-positive cerebrospinal fluid specimens with 100% sensitivity; and serum and ear fluid specimens were also evaluated. Both the lytA-CDC and psaA assays, particularly the lytA-CDC assay, have improved specificities compared with those of currently available assays and should therefore be considered the assays of choice for the detection of pneumococcal DNA, particularly when upper respiratory P-LVS might be present in the clinical specimen.

Effect of use of 13-valent pneumococcal conjugate vaccine in children on invasive pneumococcal disease in children and adults in the USA: analysis of multisite, population-based surveillance

BACKGROUND In 2000, seven-valent pneumococcal conjugate vaccine (PCV7) was introduced in the USA and resulted in dramatic reductions in invasive pneumococcal disease (IPD) and moderate increases in non-PCV7 type IPD. In 2010, PCV13 replaced PCV7 in the US immunisation schedule. We aimed to assess the effect of use of PCV13 in children on IPD in children and adults in the USA. METHODS We used laboratory-based and population-based data on incidence of IPD from the Active Bacterial Core surveillance (part of the Centers for Disease Control and Preventions Emerging Infections Program) in a time-series model to compare rates of IPD before and after the introduction of PCV13. Cases of IPD between July 1, 2004, and June 30, 2013, were classified as being caused by the PCV13 serotypes against which PCV7 has no effect (PCV13 minus PCV7). In a time-series model, we used an expected outcomes approach to compare the reported incidence of IPD to that which would have been expected if PCV13 had not replaced PCV7. FINDINGS Compared with incidence expected among children younger than 5 years if PCV7 alone had been continued, incidence of IPD overall declined by 64% (95% interval estimate [95% IE] 59-68) and IPD caused by PCV13 minus PCV7 serotypes declined by 93% (91-94), by July, 2012, to June, 2013. Among adults, incidence of IPD overall also declined by 12-32% and IPD caused by PCV13 minus PCV7 type IPD declined by 58-72%, depending on age. We estimated that over 30 000 cases of IPD and 3000 deaths were averted in the first 3 years after the introduction of PCV13. INTERPRETATION PCV13 reduced IPD across all age groups when used routinely in children in the USA. These findings provide reassurance that, similar to PCV7, PCVs with additional serotypes can also prevent transmission to unvaccinated populations. FUNDING Centers for Disease Control and Prevention.

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.

Acquisition, Carriage, and Transmission of Pneumococci with Decreased Antibiotic Susceptibility in Young Children Attending a Day Care Facility in Southern Israel

The prevalence and transmission of antimicrobial drug-resistant pneumococci was studied in 48 children attending a day care facility in southern Israel. Nasopharyngeal cultures were obtained every 2 weeks for 10 months, and antibiotic susceptibility of isolates was determined by disk diffusion and E-test. Relatedness of isolates was investigated by capsular typing, ribotyping, and arbitrarily primed polymerase chain reaction. Pneumococci were recovered during 362 (63%) of 573 fortnights, and 219 (60%) of these isolates showed decreased susceptibility to at least one drug; 154 (43%) were intermediately susceptible to penicillin and 51 (14%) were multiresistant. Combining the different typing methods showed that a limited number of clones circulated in the facility. Clones exhibiting decreased antibiotic susceptibility (especially 23F, intermediately susceptible to penicillin and resistant to trimethoprim-sulfamethoxazole, and multiresistant 6B) were more frequently isolated and persisted longer than did fully susceptible clones. By multivariate analysis, carriage of organisms with decreased antibiotic susceptibility was associated with young age, female sex, winter season, and exposure to antimicrobial drugs during the previous month.

Fitness Costs of Fluoroquinolone Resistance in Streptococcus pneumoniae

ABSTRACT The fitness cost of the genes responsible for resistance to fluoroquinolones in clinical isolates of Streptococcus pneumoniae were estimated in vitro in a common genetic background. Naturally occurring parC, parE, and gyrA loci containing mutations in the quinolone-resistance-determining regions were introduced by transformation into S. pneumoniae strain R6 individually and in combinations. The fitness of these transformants was estimated by pairwise competition experiments with a common R6 strain. On average, single par and gyr mutants responsible for low-level MIC resistance (first-step resistance) impose a fitness burden of approximately 8%. Some of these mutants engender no measurable cost, while one, a parE mutant, reduces the fitness of these bacteria by more than 40%. Most interestingly, the addition of the second par or gyr mutations required for clinically significant, high-MIC fluoroquinolone resistance does not increase the fitness burden imposed by these single genes and can even reduce it. We discuss the implications of these results for the epidemiology of fluoroquinolone resistance and the evolution of acquired resistance in treated patients.

Association of Serotypes of Streptococcus pneumoniae with Disease Severity and Outcome in Adults: An International Study

BACKGROUND The introduction of conjugate pneumococcal vaccination for children has reduced the burden of invasive disease due to pneumococcal conjugate vaccine (PCV) types (i.e., serotypes 9V, 14, 6B, 18C, 23F, 19F, and 4) in adults. As nonvaccine serotypes become predominant causes of invasive disease among adults, it is necessary to evaluate the disease severity and mortality associated with infection due to nonvaccine serotypes, compared with PCV serotypes, in adults. METHODS The association of pneumococcal serotype and host-related variables with disease severity and mortality was statistically examined (with multivariable analysis) in 796 prospectively enrolled, hospitalized adult patients with bacteremia due to Streptococcus pneumoniae. RESULTS In multivariate analyses of risk in patients with invasive pneumococcal disease, older age (age, > or = 65 years; P = .004), underlying chronic disease (P = .025), immunosuppression (P = .035), and severity of disease (P < .001) were significantly associated with mortality; no association was found between nosocomial infection with invasive serotypes 1, 5, and 7 and mortality. The risk factors meningitis (P = .001), suppurative lung complications (P < or = .001), and preexisting lung disease (P = .051) were significantly associated with disease severity, independent of infecting serotype. No differences were seen in disease severity or associated mortality among patients infected with PCV serotypes, compared with patients infected with nonvaccine serotypes. CONCLUSIONS Our data support the notion that host factors are more important than isolate serotype in determining the severity and outcome of invasive pneumococcal disease and that these outcomes are unlikely to change in association with nonvaccine serotype infection in the post-conjugate vaccine era.

Levofloxacin-Resistant Invasive Streptococcus pneumoniae in the United States: Evidence for Clonal Spread and the Impact of Conjugate Pneumococcal Vaccine

ABSTRACT The emergence of fluoroquinolone resistance in sterile-site isolates of Streptococcus pneumoniae is documented in this study characterizing all invasive levofloxacin-resistant (MIC, ≥8 mg/liter) S. pneumoniae isolates (n = 50) obtained from the Centers for Disease Control and Prevention Active Bacterial Core Surveillance from 1998 to 2002. Resistance among all isolates increased from 0.1% in 1998 to 0.6% in 2001 (P = 0.008) but decreased to 0.4% in 2002, while resistance among vaccine serotypes continued to increase from 0.3% in 1998 to 1.0% in 2002, suggesting that fluoroquinolones continue to exert selective pressure on these vaccine serotypes. Only 22% of resistant isolates were not covered by the conjugate vaccine serogroups. Multilocus sequence typing revealed that 58% of resistant strains were related to five international clones identified by the Pneumococcal Molecular Epidemiology Network, with the Spain23F-1 clone being most frequent (16% of all isolates). Thirty-six percent of the isolates were coresistant to penicillin, 44% were coresistant to macrolides, and 28% were multiresistant to penicillin, macrolides, and fluoroquinolones. Fifty percent of the isolates were resistant to any three drug classes. Ninety-four percent of the isolates had multiple mutations in the quinolone resistance-determining regions of the gyrA, gyrB, parC, and parE genes. In 16% of the isolates, there was evidence of an active efflux mechanism. An unusual isolate was found that showed only a single parE mutation and for which the ciprofloxacin MIC was lower (2 mg/liter) than that of levofloxacin (8 mg/liter). Our results suggest that invasive pneumococcal isolates resistant to levofloxacin in the United States show considerable evidence of multiple resistance and of clonal spread.