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Mucoid bacteria, predominately Pseudomonas aeruginosa, are commonly associated with decline in pulmonary function in children with cystic fibrosis (CF), and are thought to persist at least in part due to a greater propensity toward forming biofilms. We isolated a higher frequency of mucoid Streptococcus pneumoniae (Sp) expressing high levels of capsular polysaccharides from sputa from children with CF, compared to those without CF. We compared biofilm formation and maturation by mucoid and non-mucoid isolates of Sp collected from children with and without CF. Non-mucoid Sp serotype 19A and 19F isolates had significantly higher levels of biofilm initiation and adherence to CF epithelial cells than did serotype 3 isolates. However, strains expressing high levels of capsule had significantly greater biofilm maturation, as evidenced by increased density and thickness in static and continuous flow assays via confocal microscopy. Finally, using a serotype 3 Sp strain, we showed that highly encapsulated mucoid phase variants predominate during late adherence and better colonize CFTR–/– as compared to wild-type mice in respiratory infection studies. These findings indicate that overexpression of capsule can enhance the development of mature pneumococcal biofilms in vitro, and may contribute to pneumococcal colonization in CF lung disease.

The 13-valent pneumococcal conjugate vaccine (PCV13) was introduced in the United States in 2010 for the prevention of invasive pneumococcal disease (IPD) and otitis media. While many studies have reported its potential efficacy for IPD, not much is known about the epidemiology of noninvasive disease following its introduction. We characterized the capsular types and surface protein genes of noninvasive pediatric pneumococcal isolates collected between 2002 and 2010 ( n = 1,058) at Children's of Alabama following the introduction of PCV7 and tested a subset of noninvasive and previously characterized IPD isolates for the presence of the pspA , pspC , and rrgC genes, which encode protection-eliciting proteins. PCV7 serotypes had dramatically decreased by 2010 ( P < 0.0001), and only 50% of all noninvasive infections were caused by the PCV13 capsular serotypes. Serotype 19A accounted for 32% of the noninvasive isolates, followed by serotypes 35B (9%), 19F (7%), and 6C (6%). After 7 years of PCV7 usage, there were no changes in the frequencies of the pspA or pspC genes; 96% of the strains were positive for family 1 or 2 pspA genes, and 81% were also positive for pspC . Unexpectedly, more noninvasive than invasive strains were positive for rrgC ( P < 0.0001), and the proportion of rrgC -positive strains in 2008 to 2010 was greater than that in 2002 to 2008 (IPD, P < 0.02; noninvasive, P < 0.001). Serotypes 19F, 19A, and 35B were more frequently rrgC positive ( P < 0.005) than other serotypes. A vaccine containing antigens, such as PspA, PspC, and/or RrgC, can provide coverage against most non-PCV13-type pneumococci. Continued surveillance is critical for optimal future vaccine development.

Recent studies have reported the isolation of highly mucoid serotype 3 Streptococcus pneumoniae (Sp) from the respiratory tracts of children with cystic fibrosis (CF). Whether these highly mucoid Sp contribute to, or are associated with, respiratory failure among patients with CF remains unknown. Other mucoid bacteria, predominately Pseudomonas aeruginosa, are associated with CF respiratory decline. We used a mouse model of CF to study pneumococcal pneumonia with highly mucoid serotype 3 and non-mucoid serotype 19A Sp isolates. We investigated susceptibility to infection, survival, and bacterial counts from bronchoaviolar lavage samples and lung homogenates, as well as associated inflammatory cytokines at the site of infection, and lung pathology. Congenic CFTR-/- mice and wild-type (WT)-mice were infected intranasally with CHB756, CHB1126, and WU2 (highly mucoid capsular serotype 3, intermediately mucoid serotype 3, and less mucoid serotype 3, respectively), or CHB1058 (non-mucoid serotype 19A). BAL, lung homogenates, and blood were collected from mice 5 days post-infection. Higher CFU recovery and shorter survival were observed following infection of CFTR-/- mice with CHB756 compared to infection with CHB1126, WU2, or CHB1058 (P≤0.001). Additionally, CFTR-/- mice infected with CHB756 and CHB1126 were more susceptible to infection than WT-mice (P≤0.05). Between CFTR-/- mice and WT-mice, no significant differences in TNF-α, CXCL1/KC concentrations, or lung histopathology were observed. Our results indicate that highly mucoid type 3 Sp causes more severe lung disease than non-mucoid Sp, and does so more readily in the lungs of CFTR-/- than WT-mice.

Cross-reactive “common” pneumococcal antigens offer an attractive alternative, or complement, to polysaccharides and polysaccharide-protein conjugate vaccines. These common antigens should be protective against strains of a wider range of capsular types than can be achieved with conjugate vaccines. Common protein antigens would be expected to be highly immunogenic in young children and should be able to be manufactured relatively inexpensively using recombinant techniques. It is hoped that these antigens will lead to a vaccine(s) that could have application worldwide, even in the poorest developing countries where the rates of fatal pneumococcal disease in children are the highest.

We characterized 32 levofloxacin-nonsusceptible Streptococcus pneumoniae (LNSP) isolates obtained from a broad geographic region of North America over a 5-year period by using capsular serotypes, antimicrobial susceptibility profiles, BOX-PCR, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). Sixteen international clones identified by the Pneumococcal Molecular Epidemiology Network also were included for comparison. Fifteen serotypes were represented, with serogroups 6, 9, 14, 19, and 23 accounting for 63% of isolates. Among isolates whose quinolone resistance-determining regions were sequenced, all contained gyrA and parC point mutations. Sixty-three percent were penicillin susceptible, and 84% were erythromycin susceptible. BOX-PCR analysis identified 39 different band patterns among 32 LNSP and 16 international clones and grouped 16 isolates, including 2 international clones, into seven unrelated groups of 2 to 4 isolates each. PFGE analysis identified 35 different band patterns among 32 LNSP and 16 international clones and grouped 21 isolates, including 3 international clones, into eight unrelated groups of 2 to 6 isolates each. MLST performed on 10 isolates identified five allelic profiles and separated 9 isolates into four groups of 2 to 3 isolates each. Overall, each typing method indicated that the LNSP were heterogeneous and that resistance to fluoroquinolones was not closely associated with a particular serotype or with coresistance to other antimicrobial classes and suggests that LNSP have likely arisen through independent mutational events as a result of selective pressure. However, seven LNSP were found to be related to three international clones by PFGE.

To study the evolution and virulence of pneumococcal populations, we used multilocus sequence typing to identify the major clones among 212 carriage and invasive isolates expressing capsular serogroup 6 from 39 countries. The global population consisted of 8 major complexes and 6 minor complexes of related clones and 32 clones of diverse origin. Surprisingly, serotype 6A clones evolved by mutation nearly as often as by recombination, whereas serotype 6B clones evolved almost exclusively by recombination ( P = 0.0029). This is the first report of population genetic differences among serotypes of this species. The largest clonal complex was associated with invasive disease ( P = 0.019) and included a common ancestor for five previously identified drug-resistant clones. The putative ancestors of the major clonal complexes were represented by a greater proportion of carriage isolates than were their descendents ( P = 0.001), and the ancestors tended to be less virulent than their descendents in a mouse model of infection. These data suggested that virulent serogroup 6 clones have evolved multiple times from less-virulent ancestral clones.

We studied 198 macrolide-resistant S. pneumoniae isolates obtained from adults and children to evaluate whether 2-μg clindamycin disks can distinguish between isolates manifesting ermB - versus mefE -mediated resistance to clarithromycin and to determine the relative frequency with which each resistance mechanism occurred in these populations. The mefE gene was predominant among 109 isolates from children, occurring in 73.4% versus 50.6% of 89 isolates from adults. Three isolates (1.5%) did not amplify either gene. Among 125 mefE + isolates, the MIC of clarithromycin at which 90% of the isolates tested were inhibited, determined by Etest, was 32 μg/ml versus >256 μg/ml in 70 ermB + isolates. All ermB + isolates were highly resistant to clindamycin (MICs >256 μg/ml), whereas all mefE + isolates were susceptible to clindamycin using the 2-μg disk. Testing S. pneumoniae from the respiratory tract for susceptibility to clindamycin by agar disk diffusion is an easy and inexpensive method to estimate the frequency of resistance mediated by ermB in specific patient populations. Macrolide resistance mediated by ermB is usually of greater magnitude than that due to mefE . Clinical studies are needed to determine the significance of high- versus low-level macrolide resistance in S. pneumoniae .

Background. Children

Pneumococcal conjugate vaccines (PCVs) are recommended for the prevention of invasive pneumococcal disease (IPD) in young children. Since the introduction of the heptavalent pneumococcal vaccine (PCV7) in 2000, IPD caused by serotypes in the vaccine has almost been eliminated, and previously uncommon capsular serotypes now cause most cases of pediatric IPD in the United States. One way to protect against these strains would be to add cross-reactive protein antigens to new vaccines. One such protein is pneumococcal surface protein A (PspA). Prior to 2000, PspA families 1 and 2 were expressed by 94% of isolates. Because PCV7 vaccine pressure has resulted in IPD caused by capsular serotypes that were previously uncommon and unstudied for PspA expression, it was possible that many of the new strains expressed different PspA antigens or even lacked PspA. Of 157 pediatric invasive pneumococcal isolates collected at a large pediatric hospital in Alabama between 2002 and 2010, only 60.5% had capsular serotypes included in PCV13, which came into general use in Alabama after our strains were collected. These isolates included 17 serotypes that were not covered by PCV13. Nonetheless, pneumococcal capsular serotype replacement was not associated with changes in PspA expression; 96% of strains in this collection expressed PspA family 1 or 2. Continued surveillance will be critical to vaccine strategies to further reduce IPD.

This article is part of a series of papers presented at a symposium entitled "Antimicrobial Resistance in Streptococcus pneumoniae" at the 5th European Congress of Clinical Microbiology and Infectious Diseases in Oslo in September 1991., Examination of several hundred penicillin-resistant clinical isolates of Streptococcus pneumoniae has revealed extensive strain-to-strain variation in the number and molecular size of penicillin-binding proteins (PBPs), This polymorphism has been used to classify resistant isolates into groups (PBP families) that share distinct electrophoretic profiles, We describe herein properties of four such PBP families: two from Spain (and/or Ohio) and one each from Hungary and Alaska. We have discovered that representative isolates assigned to each PBP family also share capsular serotype, antibiotic resistance pattern, pneumococcal surface protein A type, and multilocus enzyme genotype. The results demonstrate independent clonal origin for strains assigned to each PBP family. Each resistant clone occurs with uniquely high incidence within specific geographic areas., These investigations were supported by the National Institute of Allergy and Infectious Diseases (RO I Al 16794, RO I AI33119, and PDI HD17812).

The relationships among capsular type, protein type, and penicillin resistance for capsular group 9 Streptococcus pneumoniae collected in northwestern Canada between 1974 and 1987 were examined. The group 9 relatively penicillin-resistant (RPR) isolates were of the rare 9L capsular type. Of 47 penicillin-susceptible (PS) group 9 isolates that were typed for capsule, only 1 was 9L. Among 29 PS group 9 isolates that were protein typed, 9 protein types were observed. Of the 70 RPR isolates, 51 were protein type P23, 1 was P19, and 18 could not be typed (P0). Protein types P23 differed from P0 by a single epitope on pneumococcal surface protein A. These results suggest that the Canadian P23 and P0 capsular group 9 isolates are likely subclones of a primordial 9L RPR strain.

Thirty Streptococcus pneumoniae clinical isolates resistant to levofloxacin were analyzed for the quinolone resistance-determining DNA sequences to identify point mutations and were tested for in vitro susceptibility to multiple drug classes. Of these isolates, 29 had mutations in both gyrA and parC genes of DNA gyrase and topoisomerase IV, respectively. In GyrA, an amino acid change from Ser-81Phe was detected in 27 isolates and a Glu-85 Lys change was found in the remaining three. Of the 29 isolates for which ParC data were available, Ser-79 Tyr or Phe were the predominant mutations observed. MICs for levofloxacin were 4–16 mg/l, whereas those for moxifloxacin were 1–2 mg/l. Twenty-four (80%) isolates were susceptible to erythromycin, 25 (83%) to azithromycin, 26 (87%) to clarithromycin, 27 (90%) to clindamycin, 20 (67%) to penicillin, 21 (70%) to ceftriaxone and 30 (100%) to amoxycillin/clavulanate. These results confirm the presence of double mutations among clinical isolates of S. pneumoniae from diverse geographical regions of North America and also suggest that quinolone resistance may develop independently of resistance to other drug classes. © 2001 Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved.

To determine whether nasopharyngeal carriage isolates of Streptococcus pneumoniae are of the same genetic background as isolates that caused invasive disease in one community, IS1167 and boxA genotypes were obtained for 182 pneumococcal isolates from children living in central Tennessee. The isolates represented 70 combined IS1167-boxA genotypes. The genotypic diversity of the invasive isolates was significantly less than that of the total population ( ). Most of the carriage isolates belonged to genotypes unique to carriage, whereas P p .003 most of the invasive isolates belonged to genotypes common to carriage and disease (P p ). Monte Carlo simulations showed a greater number of genotypes unique to carriage than .02 can be explained by chance ( in all cases). Two genotypes were identified by multilocus P ! .05 sequence typing as members of globally disseminated clones, and one such genotype that was strictly carriage in this sample caused disease in other studies. Thus, clones can have different propensities for carriage and invasion. Streptococcus pneumoniae can be carried asymptomatically in the human nasopharynx, but little is known about the clones involved in carriage and their genetic relationships to clones associated with invasive disease. Invasion is correlated with at least transient carriage [1, 2]. Several studies have reported a link between carriage of pneumococci and the occurrence of acute otitis media [3–5]. Moreover, carriage isolates can exhibit the same capsule serotypes and genetic backgrounds as invasive isolates. For example, in a study by Takala et al. [6], the serotype 7 and 14 pneumococci were mostly clonal and invasive; however, there were 2 carriage isolates each of serotypes 7 and 14, and these belonged to the same genotypes as the isolates from disease. In a

Antimicrobial susceptibility, pneumococcal surface protein A (PspA) serotyping, and pulsed-field gel electrophoresis (PFGE) were used to evaluate clonal relatedness among 66 invasive isolates of Streptococcus pneumoniae serotype 6B collected during 1982-1996 from patients in Alaska. Thirty-seven (56%) of the isolates had penicillin minimal inhibitory concentration values >/=0.125 microgram/mL and were resistant to at least 1 other antibiotic. Fourteen PspA serotypes were observed; PspA 16 was the most common (35%). Forty-five (68%) of the 66 isolates shared common and highly related PFGE patterns using 3 enzymes. Twenty-six (58%) of the isolates with common PFGE patterns were from Native Alaskan children

These studies have identified a major genetic lineage of capsule serotype 12F Streptococcus pneumoniae, which has maintained two different types of the pneumococcal surface protein A (PspA) virulence factor and caused invasive disease in geographically disjoint locations. Twenty outbreak strains from a Texas jail and Maryland day care center and 16 reference strains from Texas, Maryland, Washington, Michigan, Oklahoma, Missouri, Alaska, and Australia were examined. Although the Texas and Maryland outbreak strains were indistinguishable by IS1167 and boxA genotyping procedures, all strains examined were members of a genetically similar lineage. The microevolutionary history of pspA differed from that of the overall genetic background of the strains. Taken together, these findings suggested that the Texas and Maryland outbreaks were caused by different clones of a major genetic lineage of serotype 12F pneumococci, within which at least one PspA has been acquired via localized genetic recombination.

SUMMARY Streptococcus pneumoniae is a problematic infectious agent, whose seriousness to human health has been underscored by the recent rise in the frequency of isolation of multidrug-resistant strains. Pneumococcal pneumonia in the elderly is common and often fatal. Young children in the developing world are at significant risk for fatal pneumococcal respiratory disease, while in the developed world otitis media in children results in substantial economic costs. Immunocompromised patients are extremely susceptible to pneumococcal infection. With 90 different capsular types thus far described, the diversity of pneumococci contributes to the challenges of preventing and treating S. pneumoniae infections. The current capsular polysaccharide vaccine is not recommended for use in children younger than 2 years and is not fully effective in the elderly. Therefore, innovative vaccine strategies to protect against this agent are needed. Given the immunogenic nature of S. pneumoniae proteins, these molecules are being investigated as potential vaccine candidates. Pneumococcal surface protein A (PspA) has been evaluated for its ability to elicit protection against S. pneumoniae infection in mouse models of systemic and local disease. This review focuses on immune system responsiveness to PspA and the ability of PspA to elicit cross-protection against heterologous strains. These parameters will be critical to the design of broadly protective pneumococcal vaccines.

Pneumococcal surface protein A (PspA) has been shown to be a serologically variable virulence factor of Streptococcus pneumoniae. In mice, PspA can elicit antibodies capable of protecting them against otherwise fatal infections with encapsulated pneumococci. In previous studies it has been reported that almost all isolates have two apparently unlinked genomic sequences that are highly homologous to the 5' and 3' halves of Rx1 pspA, although out MAbs to PspA have not detected more than one PspA in any given isolate of S. Pneumoniae. Recently, we have identified four isolates from a clone of capsular serotype 6B pneumococci (MC25-28) that simultaneously express two distinct PspAs. Each of the isolates (MC25-28) exhibited the same two Kpn I fragments (each containing a Hind III site) that hybridized with Rx1 pspA. MAbs specific for PspA detected two PspAs characterized by different molecular weights and different serologic patterns of reactivity (PspA type 6 detected by MAbs XiR278 and 2A4, and PspA type 34 detected only by MAb 7D2) in each of the four isolates. In previous studies XiR278 and 2A4 frequently have been observed to react with PspA epitopes of the same strain. Based on molecular weight data both epitopes were always present on the same molecule. Our present findings raise the possibility that pneumococci make a second serologically variable PspA which is generally not detected by currently available MAbs to PspA.

Previous studies have suggested that relatively penicillin-resistant (RPR) capsular group 9L strains in western Canada may be clonally related. To test this hypothesis, restriction fragment length polymorphisms (RFLPs) were examined using DNA probes for pspA and a newly recognized pneumococcal genetic element, IS1167. Penicillin-binding proteins (PBPs) and PBP genes from representative strains were also studied. All RPR type 9L strains demonstrated an identical RFLP when probed with IS1167, and 12 of 14 RPR strains had the same RFLP when examined with pspA. Amplification of pspA by polymerase chain reaction and restriction endonuclease digestion showed that the 9L strains had common DNA fragments not identified in any of the penicillin-susceptible strains. The 9L strains apparently have a low-affinity PBP 2B distinct from those of other capsular types. These data derived from new genetic markers and PBP analysis strongly support a clonal origin of RPR type 9L pneumococci of western Canada.

PspA is anchored to the surface of all pneumococci by the C-terminal end of the molecule. The N-terminal half of PspA is known to be serologically variable and to be able to elicit protective immune responses. Molecular analysis with DNA probes spanning different regions of pspA was carried out to identify homologous sequences among pneumococcal isolates. At high stringency, DNA probes derived from the 3'-half of pspA (encoding the C-terminal half of PspA) hybridized to all of 37 pneumococcal isolates tested, representing 20 capsular serotypes and 12 PspA serotypes. Most strains had two sequences highly homologous to this region of pspA. Using derivatives of strain Rx1, with insertion mutations in pspA, it was possible to identify the functional pspA sequence. At 50% stringency, the 3' pspA probes also detected lytA and additional sequences. lytA encodes autolysin and shares homology with the 3' portion of pspA. A probe derived from the 5'-half of pspA (encoding the N-terminal half of PspA) hybridized with only 75% of strains and generally detected only one of the two sequences recognized by the 3' probes. Thus, the 3'-half of pspA appears to contain more highly conserved sequences than the 5'-half of pspA and shares homology with several additional sequences, suggesting that the pneumococcus might make several proteins that interact with the surface by the same mechanism as PspA.

Previous studies have demonstrated that mouse antibodies to phosphocholine (PC) can protect mice against fatal infection caused by several, but not all, mouse-virulent laboratory strains of Streptococcus pneumoniae. Because the pneumococcal strains used in previous studies had been mouse passed and were propagated for many years outside of humans, it was not known whether antibody to PC would be able to protect mice against S. pneumoniae freshly isolated from humans. In the present study, we examined the ability of an immunoglobulin G (IgG) monoclonal antibody (MAb) to PC to protect against infections in mice caused by 14 pneumococcal strains of capsular types 3, 4, 6A, and 6B. Nine of these strains were selected as the most virulent strains for mice from a group of 69 fresh clinical isolates. Five were mouse-passed laboratory strains. Mouse IgG3 MAb to PC was able to exhibit protective effects (survival or increased time to death) against infection with virtually all of the strains injected intravenously and against infection with 70% of the strains injected intraperitoneally. The protective effects of antibody to PC appeared to be partially dependent on capsular type. MAb to PC was most effective against capsular type 3 strains and least effective against type 4 strains. With type 3 and type 4 strains, MAb to PC could frequently protect against larger numbers of CFU injected intravenously than intraperitoneally. For capsular type 6A and 6B strains the reverse was true.

The relationship between capsular type and virulence for mice was examined with 69 fresh human isolates of Streptococcus pneumoniae. These isolates represented eight capsular types or groups. Serologic and molecular weight differences in PspA (pneumococcal surface protein A) indicated that the strains were clonally distinct. Mice were infected intravenously with washed bacteria of all 69 isolates in sterile salt solutions. Twenty-eight of the isolates were also injected intraperitoneally to permit comparisons between the intravenous and intraperitoneal routes. With a few exceptions, there was concordance between the ability of strains to cause fatal infections by the two routes. About 30% of the 69 isolates were virulent for mice. The abilities of the isolates to kill mice and the length of time between inoculation and death were strongly associated with capsular type. All type 4 isolates, 40% of type 3 isolates, and 60% of group 6 isolates were virulent for mice; type 1 isolates were marginally virulent; and all type or group 14, 19, and 23 isolates were avirulent. Times to death were generally longer for mice infected with group 6 or type 1 than for those infected with type 3 or 4 pneumococci. There was no relationship between clinical diagnosis or tissue source of the isolates and virulence for mice.

Naturally occurring antibodies reactive with the phosphocholine (PC) determinant of pneumococcal teichoic acids may be useful for evaluating the potential of patients to make antibodies to polysaccharides. Antibodies to PC are present in most adults under the age of 60 years, are absent in very young children, and are present at low levels in Wiscott-Aldrich patients and in IgG2-deficient adults. These last three groups respond very poorly to polysaccharide antigens. Antibodies to PC are also found at low levels in the elderly, a group that has previously been shown to have low levels of antibody to blood groups A and B carbohydrates. The levels of antibody to PC over time were constant in most individuals and, in adults, seemed to show slightly less variation than did titers of antibody to blood group B. Our findings suggest that titers of antibody to PC may be superior to titers of antibody to blood group A or B for monitoring responsiveness to carbohydrate antigens.