Christopher G. Dowson

Horizontal transfer of multiple penicillin‐binding protein genes, and capsular biosynthetic genes, in natural populations of Streptococcus pneumoniae

Multiply antibiotic‐resistant serotype 23F isolates of Streptococcus pneumoniae are prevalent in Spain and have also been recovered recently in the United Kingdom and the United States. Analysis of populations of these isolates by multilocus enzyme electrophoresis, and restriction endonuclease cleavage electrophoretic profiling of penicillin‐binding protein (PBP) genes, has demonstrated that these isolates are a single clone (Muñoz et al., 1991). Here we report studies of non‐serotype 23F penicillin‐resistant pneumococci isolated in Spain and the United Kingdom. One of the isolates expressed serotype 19 capsule but was otherwise indistinguishable from the serotype 23F clone on the basis of multilocus enzyme electrophoresis, antibiotic resistance profiling, and restriction endonuclease patterns of genes encoding PBP1A, PBP2B and PBP2X, a result which suggests that horizontal transfer of capsular biosynthesis genes had occurred. These same techniques revealed that six other resistant isolates, all expressing serotype 9 polysaccharide capsule, represent a clone. Interestingly, the chromosomal lineage of this clone is not closely related to the 23F clone; however, the serotype 9 and 23F clones harbour apparently identical PBP1 A, ‐2B and ‐2X genes. To explain these data, we favour the interpretation that horizontal gene transfer in natural populations has distributed genes encoding altered forms of PBP1A, ‐2B and ‐2X to distinct evolutionary lineages of S. pneumoniae.

Evolution of penicillin resistance in Streptococcus pneumoniae; the role of Streptococcus mitis in the formation of a low affinity PBP2B in S. pneumoniae.

Penicillin‐resistant strains of Streptococcus pneumoniae possess forms of penicillin‐binding proteins (PBPs) that have a low affinity for penicillin compared to those from penicillin‐sensitive strains. PBP genes from penicillin‐resistant isolates are very variable and have a mosaic structure composed of blocks of nucleotides that are similar to those found in PBP genes from penicillin‐sensitive isolates and blocks that differ by up to 21%. These chromosomally encoded mosaic genes have presumably arisen following transformation and homologous recombination with PBP genes from a number of closely related species. This study shows that PBP2B genes from many penicillin‐resistant isolates of S. pneumoniae contain blocks of nucleotides originating from Streptococcus mitis. In several instances it would appear that this material alone is sufficient to produce a low affinity PBP2B. In other examples PBP2B genes possess blocks of nucleotides from S. mitis and at least one additional unidentified species. Mosaic structure was aiso found in the PBP2B genes of penicillin‐sensitive isolates of S. mitis or S. pneumoniae. These mosaics did not confer penicillin resistance but nevertheless reveal something of the extent to which localized recombination occurs in these naturally transformable streptococci.

Origin and molecular epidemiologY of penicillin-binding-protein-mediated resistance to β-lactam antibiotics

Resistance to beta-lactam antibiotics in some naturally transformable bacterial pathogens has arisen by interspecies recombinational events that have generated hybrid penicillin-binding proteins with reduced affinity for the antibiotics. This type of resistance is of particular concern in pneumococci, in which it is increasing worldwide.

Genetics of resistance to third-generation cephalosporins in clinical isolates of Streptococcus pneumoniae

Resistance to third‐generation cephalosporins in a clinical isolate of Streptococcus pneumoniae was shown to be due to the production of altered forms of penicillin‐binding proteins (PBPs) 2X and 1A. The cloned PBP2X gene from the resistant strain was able to transform a susceptible strain to an intermediate level of resistance. The resulting transformant could be transformed to the full level of resistance of the clinical isolate using the cloned PBP1A gene from the latter strain. Chromosomal DNA from the resistant strain (and from other resistant strains) could readily transform a susceptible strain to the full level of resistance to third‐generation cephalosporins (>250‐fold for cefotaxime; >100‐fold for ceftriaxone) in a single step (transformation frequency of about 10‐5). The resistant transformants obtained with chromosomal DNA were shown by gene fingerprinting to have gained both the PBP1A and PBP2X genes from the DNA donor.

Genetic analysis of clinical isolates of Streptococcus pneumoniae with high-level resistance to expanded-spectrum cephalosporins

Streptococcus pneumoniae CS109 and CS111 were isolated in the United States in 1991 and have high levels of resistance to expanded-spectrum cephalosporins (MICs of 8 and 32 micrograms of cefotaxime per ml, respectively). CS109, but not CS111, also showed high-level resistance to penicillin. As both strains expressed the serotype 23F capsule, were very closely related in overall genotype, and possessed identical or closely related mosaic pbp1a, pbp2x, and pbp2b genes, it is likely that they have arisen from a recent common ancestor. High-level resistance to expanded-spectrum cephalosporins was entirely due to alterations of penicillin-binding proteins (PBPs) 1a and 2x, since a mixture of the cloned pbp1a and pbp2x genes from the resistant strains could transform the susceptible strain R6 to the full level of cephalosporin resistance of the clinical isolates. Both PBP1a and PBP2x of these strains were more resistant to inhibition by cephalosporins than those of typical highly penicillin-resistant isolates. The pbp1a genes of CS109 and CS111 were identical in sequence, and the fourfold difference in their levels of resistance to cephalosporins was due to a Thr-550-->Ala substitution at the residue following the conserved Lys-Ser-Gly motif of PBP2x. This substitution was also the major cause of the 16-fold-lower resistance of CS111 to penicillin. The pbp2x gene of CS111, in an appropriate genetic background, could provide resistance to 16 micrograms of cefotaxime per ml but only to 0.12 microgram of benzylpenicillin per ml. Removal of the codon 550 mutation resulted in a pbp2x gene that provided resistance to 4 microgram of cefotaxime per ml and 4 microgram of benzylpenicillin per ml. The Thr-550-->Ala substitution in CS111 therefore appears to provide increased resistance to expanded-spectrum cephalosporins but a loss of resistance to penicillin.

Extensive re-modelling of the transpeptidase domain of penicillin-binding protein 2B of a penicillin-resistant South African isolate of Streptococcus pneumoniae

Clinical isolates of Streptococcus pneumoniae that have greatly increased levels of resistance to penicillin (>1000‐fold) have been reported from South Africa during the last ten years. Penicillin resistance in these strains is entirely due to the development of penicillin‐binding proteins (PBPs) with decreased affinity for penicillin. We have cloned and sequenced the coding region for the transpeptidase domain of penicillin‐binding protein 2B from three penicillin‐sensitive strains of S. pneumoniae and from a penicillin‐resistant South African strain. The amino acid sequences of the transpeptidase domains of PBP2B of the three penicillin‐sensitive strains were identical and there were only between one and four differences in the nucleotide sequences of their coding regions. The corresponding region of the PBP2B gene from the penicillin‐resistant strain differed by 74 nucleotide substitutions which resulted in 17 alterations in the amino acid sequence of PBP2B. The most remarkable alteration that has occurred during the development of the‘penicillin‐resistant’form of PBP2B is the substitution of seven consecutive residues in a region that is predicted to form a loop at the bottom of the penicillin‐binding site.

Insertion of an extra amino acid is the main cause of the low affinity of penicillin-binding protein 2 in penicillin-resistant strains of Neisseria gonorrhoeae

Non‐β‐lactamase‐ producing, penicillin‐resistant strains of Neisseria gonorrhoeae (CMRNG strains) produce altered forms of penicillin‐binding protein 2 (PBP2) that have decreased affinity for penicillin. A feature of PBP2 from all CMRNG strains is the presence of an additional residue (Asp‐345A) that is absent from PBP2 of penicillin‐sensitive strains. The role of the additional aspartic acid residue in the decreased affinity of PBP2 is unclear as PBP2 of all previously examined CMRNG strains possess several other amino acid sequence alterations, in addition to the insertion of Asp‐345A, compared to PBP2 of penicillin‐sensitive strains. Site‐directed mutagenesis has been used to insert the Asp‐345A codon into the penA gene from a penicillin‐sensitive gonococcus. The resulting penA gene expressed an altered form of PBP2 that had a decreased affinity for benzylpenicillin and was able to transform a pencillin‐sensitive strain of N. gonorrhoeae to an increased level of resistance to benzylpenicillin. Insertion of amino acids other than aspartic acid did not produce forms of PBP2 that provided increased resistance to penicillin. Removal of the Asp‐345A codon from the penA gene of a CMRNG strain reduced its ability to transform a penicillin‐sensitive strain to an increased level of penicillin resistance. The reduction in the affinity of PBP2 in CMRNG strains is therefore largely, although not exclusively, due to the insertion of Asp‐345A. Clinical isolates that produce altered forms of PBP2 that differ from that of penicillin‐sensitive strains only in the insertion of Asp‐345A have been identified.

Penicillin‐binding protein 2 genes of non‐β‐lactamase‐producing, penicillin‐resistant strains of Neisseria gonorrhoeae

Oligonucleotides that correspond to regions of the penicillin‐binding protein 2 gene (penA) that differ between penicillin‐sensitive and penicillin‐resistant strains have been used as probes to classify the penA genes in a collection of penicillin‐resistant gonococci isolated in Britain. 44/47 of those gonococcal strains that had minimal inhibitory concentrations of ≥0.25 μg benzylpenicillin per ml contained extensively altered penA genes which appeared to be very similar (or identical) to one or other of the two classes of altered penA genes that have been described previously. Since these two classes of altered penA genes are related, it appears that the great majority of the altered penA genes of non‐β‐lactamase‐producing penicillin‐resistant gonococci have a clonal origin. The other three penicillin‐resistant strains had altered penA genes that were different to those described previously. A crucial step in the development of the altered forms of PBP2 with decreased affinity for penicillin appears to have been the insertion of an extra codon within the transpeptidase domain of the penA gene. This insertion was found in the penA gene of all gonococci with minimal inhibitory concentrations of >0.016μg benzylpenicillin per ml but was not found in any strains with minimal inhibitory concentrations of ≤0.016μg per ml.