Jiaji Zhou

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.

Role of interspecies transfer of chromosomal genes in the evolution of penicillin resistance in pathogenic and commensal Neisseria species.

SummaryThe two pathogenic species of Neisseria, N. meningitidis and N. gonorrhoeae, have evolved resistance to penicillin by alterations in chromosomal genes encoding the high molecular weight penicillin-binding proteins, or PBPs. The PBP 2 gene (penA) has been sequenced from over 20 Neisseria isolates, including susceptible and resistant strains of the two pathogenic species, and five human commensal species. The genes from penicillin-susceptible strains of N. meningitidis and N. gonorrhoeae are very uniform, whereas those from penicillin-resistant strains consist of a mosaic of regions resembling those in susceptible strains of the same species, interspersed with regions resembling those in one, or in some cases, two of the commensal species. The mosaic structure is interpreted as having arisen from the horizontal transfer, by genetic transformation, of blocks of DNA, usually of a few hundred base pairs. The commensal species identified as donors in these interspecies recombinational events (N. flavescens and N. cinerea) are intrinsically more resistant to penicillin than typical isolates of the pathogenic species. Transformation has apparently provided N. meningitidis and N. gonorrhoeae with a mechanism by which they can obtain increased resistance to penicillin by replacing their penA genes (or the relevant parts of them) with the penA genes of related species that fortuitously produce forms of PBP 2 that are less susceptible to inhibition by the antibiotic. The ends of the diverged blocks of DNA in the penA genes of different penicillin-resistant strains are located at the same position more often than would be the case if they represent independent crossovers at random points along the gene. Some of these common crossover points may represent common ancestry, but reasons are given for thinking that some may represent independent events occurring at recombinational hotspots.

Sequence diversity within the argF, fbp and recA genes of natural isolates of Neisseria meningitidis: interspecies recombination within the argF gene

Studies of natural populations of Neisserla meningitidis using multilocus enzyme electrophoresis have shown extensive genetic variation within this species, which, it has been proposed, implies a level of sequence diversity within meningococci that is greater than that normally considered as the criterion for species limits in bacteria. To obtain a direct measure of the sequence diversity among meningococci, we obtained the nucleotide sequences of most of the argF, recA and fbp genes of eight meningococci of widely differing electrophoretic type (from the reference collection of Caugant). Sequence variation between the meningococcal strains ranged from 0–0.6% for fbp, 0–1.3% for argF, and 0–3.3% for recA. These levels of diversity are no greater than those found within Escherichia coli‘housekeeping’genes and suggest that multilocus enzyme electrophoresis may overestimate the extent of nucleotide sequence diversity within meningococci. The average sequence divergence between the Neisseria meningitidis strains and N. gonorrhoeae strain FA19 was 1.0% for fbp and 1.6% for recA. The argF gene, although very uniform among the eight meningococcal isolates, had a striking mosaic structure when compared with the gonococcai argF gene: two regions of the gene differed by >13% in nucleotide sequence between meningococci and gonococci, whereas the rest of the gene differed by <1.7%. One of the diverged regions was shown to have been introduced from the argF gene of a commensal Neisseria species that is closely related to Neisseria cinerea. The source of the other region was unclear.

Interspecies recombination, and phylogenetic distortions, within the glutamine synthetase and shikimate dehydrogenase genes of Neisseria meningitidis and commensal Neisseria species

Visual inspection showed clear evidence of a history of intraspecies recombinational exchanges within the neighbouring meningococcal shikimate dehydrogenase (aroE ) and glutamine synthetase (glnA) genes, which was supported by the non‐congruence of the trees constructed from the sequences of these genes from different meningococcal strains, and by statistical tests for mosaic structure. Many examples were also found of highly localized interspecies recombinational exchanges between the meningococcal aroE and glnA genes and those of commensal Neisseria species. These exchanges appear to have inflated the sequence variation at these loci, and have resulted in major distortions of the phylogenetic trees constructed from the sequences of the aroE and glnA genes of human pathogenic and commensal Neisseria species. Statistical tests for sequence mosaicism, and for anomalies within the Neisseria species trees, strongly supported the view that frequent interspecies recombination has occurred within aroE and glnA. The high levels of sequence variation, and intra‐ and interspecies recombination, within aroE and glnA did not appear to be due to a ‘hitch‐hiking’ effect caused by positive selection for variation at a neighbouring gene. Our results suggest that interspecies recombinational exchanges with commensal Neisseria occur frequently in some meningococcal ‘housekeeping’ genes as they can be observed readily even when there appears to be no obvious selection for the recombinant phenotypes.

A comparison of the nucleotide sequences of theadk andrecA genes of pathogenic and commensalNeisseria species: Evidence for extensive interspecies recombination withinadk

The sequences of the adenylate kinase gene (adk) and the RecA gene (recA) were determined from the same isolates ofNeisseria gonorrhoeae, N. meningitidis, N. lactamica, N. polysaccharea, N. cinerea, N. mucosa, N. pharyngis var.flava, N. flavescens, andN. animalis. The patterns of sequence divergence observed atadk andrecA were very different. Dendrograms constructed from therecA data using two different algorithms were statistically robust and were congruent with each other and with the relationships between the species previously proposed using other data. In contrast, the dendrograms derived from theadk data were noncongruent with each other, and with those from therecA data, and were statistically poorly supported. These results, along with the uniform distribution of pairwise sequence divergences between the species atadk, suggest there has been a history of interspecies recombination within theadk gene of the humanNeisseria species which has obscured the phylogenetic relationships between the species. This view was supported by Sawyers runs test, and the Index of Association (IA) between codons, which provided significant evidence for interspecies recombination between theadk genes from the humanNeisseria species, but no evidence of interspecies recombination between therecA sequences.

Monofunctional biosynthetic peptidoglycan transglycosylases

The high-molecular-weight penicillin-binding proteins (highMr PBPs) catalyse the final stages of bacterial peptidoglycan synthesis and are the physiological targets of the blactam group of antibiotics (Spratt and Cromie, 1988, Rev Inf Dis 10: 699–711; Ghuysen, 1991, Annu Rev Microbiol 45: 33–67). The carboxy-terminal domains of these enzymes catalyse the penicillin-sensitive peptidoglycan transpeptidation (TPase) reaction. The aminoterminal domains of the class A high-Mr PBPs (those that are homologous to Escherichia coli PBP1A and PBP1B) catalyse a penicillin-insensitive peptidoglycan transglycosylase (TGase) reaction; the function of this domain in the class B high-Mr PBPs (those that are homologous to E. coli PBP2 and PBP3) is uncertain (Ghuysen, 1991, ibid). It has been proposed that several species, including Staphylococcus aureus and E. coli, possess a peptidoglycan TGase that is not associated with a TPase domain and which is therefore not a PBP (Hara and Suzuki, 1984, FEBS Lett 168: 155–160; Park and Matsuhashi, 1984, J Bacteriol 157: 538–544). The best-documented report is that of Hara and Suzuki (1984, ibid.), who described a protein of Mr = 34 000 from E. coli that could synthesise linear glycan strands from lipid-linked precursors. This type of TGase activity appears to have been largely ignored since the initial reports. We have recently identified open reading frames (ORFs) in Neisseria gonorrhoeae (and Neisseria meningitidis), Klebsiella pneumoniae, E. coli and Haemophilus influenzae that appear to encode biosynthetic peptidoglycan TGases that are not PBPs, and which may correspond to this activity. The K. pneumoniae and N. gonorrhoeae putative TGase genes were identified within regions that were being sequenced for other reasons in our laboratories. In both cases, comparisons of the products of translation, in all reading frames, against the protein databases, using BLASTX, gave very highly significant matches against the amino-terminal regions of class A high-Mr PBPs (using the K. pneumoniae TGase, the number of matches expected by chance to be equal to, or greater than, those observed ranged from 2.6610 to 4.4610). No significant matches to any other classes of proteins (including class B high-Mr PBPs or lytic transglycosylases) were obtained. The K. pneumoniae protein is encoded by an ORF that is downstream of the rpoN operon. A similar gene (unassigned, ORF242; EMBL /GenBank/DDJB Nucelotide Sequence Data Libraries accession number U18997) is found downstream of the E. coli rpoN operon. Although, whereas in K. pneumoniae ORF242 is immediately adjacent to the last gene of the operon (ptsO; accession number Z50803), in E. coli another ORF (ORF210) of unknown function separates ptsO and ORF242. In both species the putative TGase gene is transcribed in the opposite direction to the rpoN operon and appears to be translationally coupled to an upstream gene described in E. coli as ‘sigma cross-reacting protein 27A’ (accession number D13188). The N. gonorrhoeae (and N. meningitidis) protein is encoded by an ORF immediately downstream of the shikimate dehydrogenase gene (aroE ). These genes are transcribed in the same direction with only two base pairs between the end of aroE and the start of the ORF. A search of the H. influenzae genome sequence (accession number L42023) revealed a homologous TGase gene (unassigned ORF, HI0831). The products of all of these genes were of similar size (about 240 residues) and none of them were associated with a TPase domain. We propose the genetic nomenclature mtgA (monofunctional TGase) for this class of gene. The sequences of the K. pneumoniae and N. gonorrhoeae mtgA genes have been submitted to the EMBL database as Z54198 and L47159, respectively. Figure 1 shows the alignment of the putative TGases with themselves and with the TGase domain of PBP1A of E. coli. Of the 19 residues that are completely conserved in all class A high-Mr PBPs (Popham and Setlow, 1995, J Bacteriol 177: 327–335), 15 are conserved in the four monofunctional TGases, and two are conservative replacements (lysine for arginine and serine for threonine). The carboxy-terminus of these proteins corresponds to a position in PBP1A that is approx. 115 residues before the active-site serine (the residue in the TPase domain that is acylated by penicillin) and presumably marks the boundary between the TGase and TPase domains of this bifunctional high-Mr PBP. High-Mr PBPs are translocated across, and retained in, the cytoplasmic membrane by a hydrophobic aminoterminal, non-cleaved, signal-like sequence (Edelman et al., 1987, Mol Microbiol 1: 101–106). This feature is present in the putative TGases, suggesting they are also translocated across the cytoplasmic membrane. The TGase from H. influenzae appears to lack this feature, Molecular Microbiology (1996) 19(3), 639–647

Emergence in Vietnam of Streptococcus pneumoniae Resistant to Multiple Antimicrobial Agents as a Result of Dissemination of the Multiresistant Spain23F-1 Clone

ABSTRACT Surveillance for Streptococcus pneumoniae resistant to penicillin and other antimicrobial agents is necessary to define the optimal empirical antibiotic therapy for meningitis in resource-poor countries such as Vietnam. The clinical and microbiological features of 100 patients admitted to the Centre for Tropical Diseases in Ho Chi Minh City, Vietnam, between 1993 and 2002 with invasive pneumococcal disease were studied. A penicillin-nonsusceptible pneumococcus (MIC, ≥0.1 μg/ml) was isolated from the blood or cerebrospinal fluid of 8% of patients (2 of 24) between 1993 and 1995 but 56% (20 of 36) during 1999 to 2002 (P < 0.0001). Pneumococcal isolates resistant to penicillin (MIC, ≥2.0 μg/ml) increased from 0% (0 of 24) to 28% (10 of 36) (P = 0.002). Only one isolate was ceftriaxone resistant (MIC, 2.0 μg/ml). Penicillin-nonsusceptible pneumococci were isolated from 78% of children younger than 15 years (28 of 36) compared with 25% of adults (16 of 64) (P = 0.0001). Isolation of a penicillin-nonsusceptible pneumococcus in adults with meningitis was independently associated with referral from another hospital (P = 0.005) and previous antibiotic therapy (P = 0.025). Multilocus sequence typing showed that 86% of the invasive penicillin-resistant pneumococcus isolates tested (12 of 14) were of the Spain23F-1 clone. The serotypes of >95% of the penicillin-nonsusceptible pneumococci were included in the currently available pneumococcal vaccines. Our findings point to the recent introduction and spread of the Spain23F-1 clone of penicillin-resistant pneumococci in Vietnam. Simple clinical predictors can be used to guide empirical antibiotic therapy of meningitis. Pneumococcal vaccination may help to control this problem.

Identification of multidrug-resistant Streptococcus pneumoniae strains isolated in Poland by multilocus sequence typing.

Multilocus sequence typing (MLST) of 35 isolates of multidrug-resistant Streptococcus pneumoniae recovered in Poland during 1995-1996 distinguished 10 different sequence types (ST). The majority of the isolates were assigned to two Polish clones of serotypes 6B and 23F, although the international clones, Spain23F-1 and Spain9V-3, were also identified. Similar results were obtained using pulsed-field gel electrophoresis (PFGE), providing a direct comparison of these two typing methods.