Characteristic Signatures of the lytA Gene Provide a Basis for Rapid and Reliable Diagnosis of Streptococcus pneumoniae Infections

ABSTRACT The nucleotide sequences of the lytA gene from 29 pneumococcal isolates of various serotypes and 22 additional streptococci of the mitis group (including two Streptococcus pseudopneumoniae strains) have been compared and found to correspond to 19 typical (927-bp-long) and 20 atypical (921-bp-long) alleles. All the Streptococcus pneumoniae strains harbored typical lytA alleles, whereas nonpneumococcal isolates belonging to the mitis group always carried atypical alleles. A sequence alignment showed that the main difference between typical and atypical lytA alleles resided in 102 nucleotide positions (including the 6 bp absent from atypical alleles). These nucleotides were perfectly conserved in all the typical alleles studied, and the corresponding nucleotides of the atypical alleles were also perfectly conserved. The presence in these signatures of distinctive restriction sites (namely, SnaBI, XmnI, and BsaAI) allowed the development of a simple, reliable, and fast method that combines PCR amplification of the lytA gene, digestion with BsaAI, and separation of the products by agarose gel electrophoresis. This assay allows the rapid and consistent identification of true S. pneumoniae strains and represents an improved diagnostic tool for the study of pneumococcal carriage.

[1]  W. Hanage,et al.  Using Multilocus Sequence Data To Define the Pneumococcus , 2005, Journal of bacteriology.

[2]  A. Fenoll,et al.  Analysis of the Genetic Structure of Nontypeable Pneumococcal Strains Isolated from Conjunctiva , 2005, Journal of Clinical Microbiology.

[3]  Ernesto García,et al.  Characterization of LytA-Like N-Acetylmuramoyl-l-Alanine Amidases from Two New Streptococcus mitis Bacteriophages Provides Insights into the Properties of the Major Pneumococcal Autolysin , 2004, Journal of bacteriology.

[4]  Ernesto García,et al.  Recent trends on the molecular biology of pneumococcal capsules, lytic enzymes, and bacteriophage. , 2004, FEMS microbiology reviews.

[5]  A. Steigerwalt,et al.  Accuracy of Phenotypic and Genotypic Testing for Identification of Streptococcus pneumoniae and Description of Streptococcus pseudopneumoniae sp. nov , 2004, Journal of Clinical Microbiology.

[6]  G. Carlone,et al.  Comparison of four polymerase chain reaction assays for specificity in the identification of Streptococcus pneumoniae. , 2004, Diagnostic microbiology and infectious disease.

[7]  Ernesto García,et al.  Genomic organization and molecular analysis of the inducible prophage EJ-1, a mosaic myovirus from an atypical pneumococcus. , 2004, Virology.

[8]  J. García,et al.  Cell Wall Hydrolases , 2004 .

[9]  C. Dowson.  What Is a Pneumococcus , 2004 .

[10]  A. J. Martin-Galiano,et al.  Genetic Characterization of Optochin-Susceptible Viridans Group Streptococci , 2003, Antimicrobial Agents and Chemotherapy.

[11]  J. García,et al.  Genome Organization and Molecular Analysis of the Temperate Bacteriophage MM1 of Streptococcus pneumoniae , 2003, Journal of bacteriology.

[12]  J. García,et al.  VO1, a temperate bacteriophage of the type 19A multiresistant epidemic 8249 strain of Streptococcus pneumoniae: analysis of variability of lytic and putative C5 methyltransferase genes. , 2003, Microbial drug resistance.

[13]  J. García,et al.  Molecular Peculiarities of the lytA Gene Isolated from Clinical Pneumococcal Strains That Are Bile Insoluble , 2002, Journal of Clinical Microbiology.

[14]  K. Lohman,et al.  Sensitive and Specific Method for Rapid Identification of Streptococcus pneumoniae Using Real-Time Fluorescence PCR , 2001, Journal of Clinical Microbiology.

[15]  Elliot J. Lefkowitz,et al.  Genome of the Bacterium Streptococcus pneumoniae Strain R6 , 2001, Journal of bacteriology.

[16]  A. Pikis,et al.  Optochin resistance in Streptococcus pneumoniae: mechanism, significance, and clinical implications. , 2001, The Journal of infectious diseases.

[17]  J. Kellogg,et al.  Identification of Streptococcus pneumoniae Revisited , 2001, Journal of Clinical Microbiology.

[18]  S. Salzberg,et al.  Complete Genome Sequence of a Virulent Isolate of Streptococcus pneumoniae , 2001, Science.

[19]  M. J. Jedrzejas.  Pneumococcal Virulence Factors: Structure and Function , 2001, Microbiology and Molecular Biology Reviews.

[20]  C. Dowson,et al.  Genetic Relationships between Clinical Isolates of Streptococcus pneumoniae, Streptococcus oralis, and Streptococcus mitis: Characterization of “Atypical” Pneumococci and Organisms Allied to S. mitis HarboringS. pneumoniae Virulence Factor-Encoding Genes , 2000, Infection and Immunity.

[21]  T. Skopek,et al.  A novel assay for allelic discrimination that combines the fluorogenic 5' nuclease polymerase chain reaction (TaqMan) and mismatch amplification mutation assay. , 1999, Mutation research.

[22]  C. Dowson,et al.  The Autolysin-Encoding Gene (lytA) ofStreptococcus pneumoniae Displays Restricted Allelic Variation despite Localized Recombination Events with Genes of Pneumococcal Bacteriophage Encoding Cell Wall Lytic Enzymes , 1999, Infection and Immunity.

[23]  T Ezaki,et al.  Genetic approaches to the identification of the mitis group within the genus Streptococcus. , 1999, Microbiology.

[24]  A. Tomasz,et al.  A High Incidence of Prophage Carriage among Natural Isolates of Streptococcus pneumoniae , 1999, Journal of bacteriology.

[25]  C. Dowson,et al.  Molecular Characterization of Equine Isolates ofStreptococcus pneumoniae: Natural Disruption of Genes Encoding the Virulence Factors Pneumolysin and Autolysin , 1999, Infection and Immunity.

[26]  R. Yolken,et al.  PCR-Enzyme Immunoassay for Detection ofStreptococcus pneumoniae DNA in Cerebrospinal Fluid Samples from Patients With Culture-Negative Meningitis , 1998, Journal of Clinical Microbiology.

[27]  J. Claverys,et al.  Competence‐specific induction of recA is required for full recombination proficiency during transformation in Streptococcus pneumoniae , 1998, Molecular microbiology.

[28]  C. Whitney,et al.  Use of polymerase chain reaction to identify pneumococcal infection associated with hemorrhage and shock in two previously healthy young children. , 1997, Clinical chemistry.

[29]  K. Ubukata,et al.  Combinational detection of autolysin and penicillin-binding protein 2B genes of Streptococcus pneumoniae by PCR , 1996, Journal of clinical microbiology.

[30]  J. Thompson,et al.  CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. , 1994, Nucleic acids research.

[31]  B. Greenwood,et al.  Detection of Streptococcus pneumoniae DNA in blood cultures by PCR , 1994, Journal of clinical microbiology.

[32]  Ernesto García,et al.  Molecular basis of the optochin‐sensitive phenotype of pneumococcus: characterization of the genes encoding the F0 complex of the Streptococcus pneumoniaeStreptococcus oralis H+‐ATPases , 1994, Molecular microbiology.

[33]  V. Emery,et al.  Detection of Streptococcus pneumoniae in sputum samples by PCR , 1994, Journal of clinical microbiology.

[34]  C. Black,et al.  Evaluation of polymerase chain reaction for diagnosis of pneumococcal pneumonia , 1993, Journal of clinical microbiology.

[35]  R. Carey,et al.  Identification of Streptococcus pneumoniae with a DNA probe , 1992, Journal of clinical microbiology.

[36]  E. Díaz,et al.  EJ-1, a temperate bacteriophage of Streptococcus pneumoniae with a Myoviridae morphotype , 1992, Journal of bacteriology.

[37]  E. Díaz,et al.  Role of the major pneumococcal autolysin in the atypical response of a clinical isolate of Streptococcus pneumoniae , 1992, Journal of bacteriology.

[38]  R. López,et al.  Sequence of the Streptococcus pneumoniae bacteriophage HB-3 amidase reveals high homology with the major host autolysin , 1990, Journal of bacteriology.

[39]  T. Ezaki,et al.  Simple genetic method to identify viridans group streptococci by colorimetric dot hybridization and fluorometric hybridization in microdilution wells , 1988, Journal of clinical microbiology.

[40]  E. Tuomanen,et al.  Microbiological and clinical significance of a new property of defective lysis in clinical strains of pneumococci. , 1988, The Journal of infectious diseases.

[41]  R. Lopez,et al.  Nucleotide sequence and expression of the pneumococcal autolysin gene from its own promoter in Escherichia coli. , 1986, Gene.

[42]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[43]  J. Henrichsen,et al.  Chapter XI Laboratory Diagnosis, Serology and Epidemiology of Streptococcus pneumoniae , 1978 .

[44]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[45]  A. Tomasz,et al.  Choline-containing teichoic acid as a structural component of pneumococcal cell wall and its role in sensitivity to lysis by an autolytic enzyme. , 1970, The Journal of biological chemistry.

[46]  S. Lacks,et al.  A study of the genetic material determining an enzyme activity in Pneumococcus , 1960 .