Characterization, Distribution, and Expression of Novel Genes among Eight Clinical Isolates of Streptococcuspneumoniae

Abstract

Eight low-passage-number Streptococcus pneumoniae clinical isolates, each of a different serotype and a different multilocus sequence type, were obtained from pediatric participants in a pneumococcal vaccine trial. Comparative genomic analyses were performed with these strains and two S. pneumoniae reference strains. Individual genomic libraries were constructed for each of the eight clinical isolates, with an average insert size of ϳ1 kb. A total of 73,728 clones were picked for arraying, providing more than four times genomic coverage per strain. A subset of 4,793 clones were sequenced, for which homology searches revealed that 750 (15.6%) of the sequences were unique with respect to the TIGR4 reference genome and 263 (5.5%) clones were unrelated to any available streptococcal sequence. Hypothetical translations of the open reading frames identified within these novel sequences showed homologies to a variety of proteins, including bacterial virulence factors not previously identified in S. pneumoniae. The distribution and expression patterns of 58 of these novel sequences among the eight clinical isolates were analyzed by PCR-and reverse transcriptase PCR-based analyses, respectively. These unique sequences were nonuniformly distributed among the eight isolates, and transcription of these genes in planktonic cultures was detected in 81% (172/212) of their genic occurrences. All 58 novel sequences were transcribed in one or more of the clinical strains, suggesting that they all correspond to functional genes. Sixty-five percent (38/58) of these sequences were found in 50% or less of the clinical strains, indicating a significant degree of genomic plasticity among natural isolates.Streptococcus pneumoniae is a gram-positive cocci that is etiologically associated with meningitis as well as numerous infections of the respiratory mucosa, including pneumonia, otitis media (OM), and sinusitis. On a worldwide basis, it is estimated that there are over 10,000 pneumococcus-related deaths per day. S. pneumoniae possesses an inducible system (38) for the uptake of DNA from its environment and has served as the model organism for the study of bacterial transformation for nearly 8 decades (4,23,28). These autocompetence and autotransformation mechanisms, together with its role as a major human pathogen, make S. pneumoniae an ideal model organism for studying the effects of in vivo bacterial strain evolution on pathogenicity and persistence during chronic infection.Genomic diversity among multiple strains within a pathogenic bacterial species has been proposed to play a key role in virulence by the continual evolution of new strains via horizontal gene transfer (HGT) mechanisms during polyclonal infections (16,17,31,32,45). There are over 90 catalogued serotypes of S. pneumoniae, each of which is distinguished by a unique capsular gene cluster which exists as a cassette at a common site within the genome. Moreover, comparative genomic studies (11) of the sequenced S. pneumoniae reference strains, TIGR4 (49) and R6 (27), toge...

Construction and characterization of a highly redundant Pseudomonas aeruginosa genomic library prepared from 12 clinical isolates: Application to studies of gene distribution among populations

“…Apparently the novel nucleotide sequences that we detected are widely distributed among the constituent clinical strains of the library and no clustering within a strain (or strains) was observed for the eight unique clones tested. These findings of interstrain genomic plasticity for P. aeruginosa are similar to those for the human pathogens Haemophilus influenzae and Streptococcus pneumoniae [32,33] indicating that high degrees of genomic plasticity may be a common feature of bacterial pathogens.…”

“…If bacteria are able to re-assort distributed contingency genes from a supra-genome during polyclonal chronic infectious processes this would provide a major survival advantage to pathogens at the population level [9,10,32,33,34]. Recent observations using random amplification of polymorphic DNA (RAPD) [35] and amplified fragment length polymorphism (AFLP) [36] have established that a substantial number of P. aeruginosa infections (27 %), [30] are polyclonal.…”

Phenotypic Characterization ofStreptococcus pneumoniaeBiofilm Development

“…All strains used in this study are listed in Table 1. Eight pneumococcal strains, designated BS68 to BS75, were obtained as nasal washes from symptomatic pediatric participants at Children's Hospital of Pittsburgh who were enrolled in a pneumococcal vaccine trial (60). Three additional pneumococcal strains were obtained from the middle ear and one from sputum.…”

“…S. pneumoniae is characterized by extensive interstrain phenotypic and genotypic diversity as demonstrated by the ability of different isolates to produce 90 serologically distinct types of capsular polysaccharide (33,45) and the finding that each strain varies in genomic content from every other strain by 5 to 10% (60). To determine whether the biofilm growth conditions were suitable for various other S. pneumoniae serotypes and/or clinical isolates, we analyzed the three-dimensional structure of biofilms of various S. pneumoniae clinical isolates (see Table 1) by CLSM after 6 days of biofilm growth.…”

Role of Conjugative Elements in the Evolution of the Multidrug-Resistant Pandemic CloneStreptococcus pneumoniaeSpain23FST81

“…However, the functional genetic variation to which this differing ability to cause disease is attributable remains largely unknown. Genome sequencing efforts have mainly focused on clinical pneumococcal isolates; the complete genomes of two highly invasive strains, TIGR4 (70) and D39 (38) (and the laboratory-adapted D39 derivative R6) (34), have been published, along with draft sequences for serotype 19F strain G54 (26) and eight clinical isolates from a hospital in Pittsburgh (65). However, in order to understand the bacterial population structure, and the reasons underlying the variation in pathogenicity, genomic studies of strains that only rarely invade past the mucosal surfaces are required.…”