Regine Hakenbeck

Interspecies recombinational events during the evolution of altered PBP 2x genes in penicillin-resistant clinical isolates of Streptococcus pneumoniae

Penicillin resistance in pneumococci is due to the appearance of high molecular‐weight penicillin‐binding proteins (PBPs) that have reduced affinity for the antibiotic. We have compared the PBX2x genes (pbpX) of one penicillin‐susceptible and five penicillin‐resistant clinical isolates of Streptococcus pneumoniae isolated from various parts of the world. All of the resistant isolates contained a low‐affinity form of PBP 2x. The 2kb region of the two penicillin‐susceptible isolates differed at only eight nucleotide sites (0.4%) and resulted in one single amino acid difference in PBP 2x. In contrast, the sequences of the PBP 2x genes from the resistant isolates differed overall from those of the susceptible isolates at between 7 and 18% of nucleotide sites and resulted in between 27 and 86 amino acid substitutions in PBP 2x. The altered PBP 2x genes consisted of regions that were similar to those of susceptible strains (<3% diverged), alternating with regions that were very different (18–23% diverged). The presence of highly diverged regions within the PBP 2x genes of the resistant isolates contrasts with the uniformity of the sequences of the amylomaltase genes from the same isolates, and with the uniformity of the PBP 2x genes in the two susceptible isolates. It suggests that the altered PBP 2x genes have arisen by localized interspecies recombinational events involving the PBP 2x genes of closely related streptococci, as has been suggested to occur for altered PBP 2b genes (Dowson etal., 1989b). The PBP 2x genes from the resistant isolates could transform the susceptible strain R6 to increased levels of resistance to β‐lactam antibiotics. Indicating that the altered forms of PBP 2x in the resistant isolates contribute to their resistance to penicillin.

Mosaic Genes and Mosaic Chromosomes: Intra- and Interspecies Genomic Variation of Streptococcus pneumoniae

ABSTRACT Streptococcus pneumoniae remains a major causative agent of serious human diseases. The worldwide increase of antibiotic resistant strains revealed the importance of horizontal gene transfer in this pathogen, a scenario that results in the modulation of the species-specific gene pool. We investigated genomic variation in 20S. pneumoniae isolates representing major antibiotic-resistant clones and 10 different capsular serotypes. Variation was scored as decreased hybridization signals visualized on a high-density oligonucleotide array representing 1,968 genes of the type 4 reference strain KNR.7/87. Up to 10% of the genes appeared altered between individual isolates and the reference strain; variability within clones was below 2.1%. Ten gene clusters covering 160 kb account for half of the variable genes. Most of them are associated with transposases and are assumed to be part of a flexible gene pool within the bacterial population; other variable loci include mosaic genes encoding antibiotic resistance determinants and gene clusters related to bacteriocin production. Genomic comparison between S. pneumoniae and commensal Streptococcus mitis andStreptococcus oralis strains indicates distinct antigenic profiles and suggests a smooth transition between these species, supporting the validity of the microarray system as an epidemiological and diagnostic tool.

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.

A two‐component signal‐transducing system is involved in competence and penicillin susceptibility in laboratory mutants of Streptococcus pneumoniae

Penicillin resistance in Streptococcus pneumoniae has been attributed so far to the production of penicillin‐binding protein (PBP) variants with decreased affinities for β‐lactam antibiotics. Cefotaxime‐resistant laboratory mutants, selected after several steps on increasing concentrations of this β‐lactam, become deficient in transformation as well. A DNA fragment conferring both cefotaxime resistance and transformation deficiency was isolated and cloned from the mutant C306. The cefotaxime resistance associated with this resistance determinant was not accompanied with apparent changes in PBP properties, and it mapped on the chromosome distinct from the known resistance determinants, genes encoding PBP2x, PBP1a or PBP2b. Determination of a 2265 bp DNA sequence of the resistance determinant revealed two open reading frames, claR and claH, whose deduced amino acid sequence identified the corresponding proteins as the response regulator and histidine kinase receptor, respectively (members of the two families of bacterial signal‐transducing proteins). Two hydrophobic peptide regions divided the histidine kinase ClaH into two putative domains: an N‐terminal extracelluiar sensor part, and an intracelluiar C‐terminal domain with the conserved His‐226 residue, the presumed phosphorylation site. The single point mutations responsible for cefotaxime‐resistance and transformation deficiency of C306 and of another two independently isolated cefotaxime‐resistant mutants were each located in the C‐terminal half of ClaH. A small extracellular protein, the competence factor, is required for induction of competence. Neither C306 nor the transformants obtained with the mutated claH gene produced competence factor, and exogenous competence factor could not complement the transformation deficiency, indicating that the signal‐transducing system cia is involved in early steps of competence regulation.

Mosaic pbpX genes of major clones of penicillin-resistant Streptococcus pneumoniae have evolved from pbpX genes of a penicillin-sensitive Streptococcus oralis

Penicillin‐resistant clinical isolates of Streptococcus pneumoniae contain mosaic penicillin‐binding protein (PBP) genes that encode PBPs with decreased affinity for β‐lactam antibiotics. The mosaic blocks are believed to be the result of gene transfer of homologous PBP genes from related penicillin‐resistant species. We have now identified a gene homologous to the pneumococcal PBP2x gene (pbpX) in a penicillin‐sensitive Streptococcus oralis isolate M3 from South Africa that diverged by almost 20% from pbpX of penicillin‐sensitive pneumococci, and a central sequence block of a mosaic pbpX gene of Streptococcus mitis strain NCTC 10712. In contrast, it differed by only 2‐4% of the 1 to 1.5 kb mosaic block in pbpX genes of three genetically unrelated penicillin‐resistant S. pneumoniae isolates, two of them representing clones of serotype 6B and 23F, which are prevalent in Spain and are also already found in other countries. With low concentrations of cefotaxime, transformants of the sensitive S. pneumoniae R6 strain could be selected containing pbpX genes from either S. mitis NCTC 10712 or S. oralis M3, demonstrating that genetic exchange can already occur between β‐lactam‐sensitive species. These data are in agreement with the assumption that PBPs as penicillin‐resistance determinants have evolved by the accumulation of point mutations in genes of sensitive commensal species.

beta-lactam resistance in Streptococcus pneumoniae: penicillin-binding proteins and non-penicillin-binding proteins.

The β‐lactams are by far the most widely used and efficacious of all antibiotics. Over the past few decades, however, widespread resistance has evolved among most common pathogens. Streptococcus pneumoniae has become a paradigm for understanding the evolution of resistance mechanisms, the simplest of which, by far, is the production of β‐lactamases. As these enzymes are frequently plasmid encoded, resistance can readily be transmitted between bacteria. Despite the fact that pneumococci are naturally transformable organisms, no β‐lactamase‐producing strain has yet been described. A much more complex resistance mechanism has evolved in S. pneumoniae that is mediated by a sophisticated restructuring of the targets of the β‐lactams, the penicillin‐binding proteins (PBPs); however, this may not be the whole story. Recently, a third level of resistance mechanisms has been identified in laboratory mutants, wherein non‐PBP genes are mutated and resistance development is accompanied by deficiency in genetic transformation. Two such non‐PBP genes have been described: a putative glycosyltransferase, CpoA, and a histidine protein kinase, CiaH. We propose that these non‐PBP genes are involved in the biosynthesis of cell wall components at a step prior to the biosynthetic functions of PBPs, and that the mutations selected during β‐lactam treatment counteract the effects caused by the inhibition of penicillin‐binding proteins.

Anthrax kills wild chimpanzees in a tropical rainforest

Infectious disease has joined habitat loss and hunting as threats to the survival of the remaining wild populations of great apes. Nevertheless, relatively little is known about the causative agents. We investigated an unusually high number of sudden deaths observed over nine months in three communities of wild chimpanzees (Pan troglodytes verus) in the Taï National Park, Ivory Coast. Here we report combined pathological, cytological and molecular investigations that identified Bacillus anthracis as the cause of death for at least six individuals. We show that anthrax can be found in wild non-human primates living in a tropical rainforest, a habitat not previously known to harbour B. anthracis. Anthrax is an acute disease that infects ruminants, but other mammals, including humans, can be infected through contacting or inhaling high doses of spores or by consuming meat from infected animals. Respiratory and gastrointestinal anthrax are characterized by rapid onset, fever, septicaemia and a high fatality rate without early antibiotic treatment. Our results suggest that epidemic diseases represent substantial threats to wild ape populations, and through bushmeat consumption also pose a hazard to human health.

The Streptococcus pneumoniae cia Regulon: CiaR Target Sites and Transcription Profile Analysis

The ciaR-ciaH system is one of 13 two-component signal-transducing systems of the human pathogen Streptococcus pneumoniae. Mutations in the histidine protein kinase CiaH confer increased resistance to beta-lactam antibiotics and interfere with the development of genetic competence. In order to identify the genes controlled by the cia system, the cia regulon, DNA fragments targeted by the response regulator CiaR were isolated from restricted chromosomal DNA using the solid-phase DNA binding assay and analyzed by hybridization to an oligonucleotide microarray representing the S. pneumoniae genome. A set of 18 chromosomal regions containing 26 CiaR target sites were detected and proposed to represent the minimal cia regulon. The putative CiaR target loci included genes important for the synthesis and modification of cell wall polymers, peptide pheromone and bacteriocin production, and the htrA-spo0J region. In addition, the transcription profile of cia loss-of-function mutants and those with an apparent activated cia system representing the off and on states of the regulatory system were analyzed. The transcript analysis confirmed the cia-dependent expression of seven putative target loci and revealed three additional cia-regulated loci. Five putative target regions were silent under all conditions, and for the remaining three regions, no cia-dependent expression could be detected. Furthermore, the competence regulon, including the comCDE operon required for induction of competence, was completely repressed by the cia system.

Nucleotide sequences of the pbpX genes encoding the penicillin-binding proteins 2x from Streptococcus pneumoniae R6 and a cefotaxime-resistant mutant, C506.

Development of penicillin resistance in Streptococcus pneumoniae is due to successive mutations in penicillin‐binding proteins (PBPs) which reduce their affinity for β‐lactam antibiotics. PBP2x is one of the high‐Mr PBPs which appears to be altered both in resistant clinical isolates, and in cefotaxime‐resistant laboratory mutants. In this study, we have sequenced a 2564 base‐pair chromosomal fragment from the penicillin‐sensitive S. pneumoniae strain R6, which contains the PBP2x gene. Within this fragment, a 2250 base‐pair open reading frame was found which coded for a protein having an Mr of 82.35 kD, a value which is in good agreement with the Mr of 80–85kD measured by SDS‐gel electrophoresis of the PBP2x protein itself. The N‐terminal region resembled an unprocessed signal peptide and was followed by a hydrophobic sequence that may be responsible for membrane attachment of PBP2x. The corresponding nucleotide sequence of the PBP2x gene from C504, a cefotaxime‐resistant laboratory mutant obtained after five selection steps, contained three nucleotide substitutions, causing three amino acid alterations within the β‐lactam binding domain of the PBP2x protein. Alterations affecting similar regions of Escherichia coli PBP3 and Neisseria gonorrhoeae PBP2 from β‐lactam‐resistant strains are known. The penicillin‐binding domain of PBP2x shows highest homology with these two PBPs and S. pneumoniae PBP2b. In contrast, the N‐terminal extension of PBP2x has the highest homology with E. coli PBP2 and methicillin‐resistant Staphylococcus aureus PBP2′. No significant homology was detected with PBP1a or PBP1b of Escherichia coli, or with the low‐Mr PBPs.

Identification of the genes directly controlled by the response regulator CiaR in Streptococcus pneumoniae: five out of 15 promoters drive expression of small non-coding RNAs.

The two‐component regulatory system CiaRH of Streptococcus pneumoniae has been implicated in β‐lactam resistance, maintenance of cell integrity, competence and virulence, but the genes that are regulated directly by the system have not been defined. Using transcriptional mapping, in vitro CiaR binding, and in vivo analysis of CiaR‐mediated regulation, 15 promoters were identified to be directly controlled by the response regulator CiaR. A direct repeat, TTTAAG‐N5‐TTTAAG, was found to be essential for CiaR binding and regulation. It is present, either completely or with subtle changes, in all promoter regions. Fourteen promoters of the regulon are activated by CiaR, and one was found to be controlled negatively. The genes that are transcribed from these promoters included ciaRH, loci that are predicted to be involved in the modification of teichoic acids (lic), in sugar metabolism (mal, man), stress response (htrA), chromosome segregation (parB), protease maturation (ppmA) and unknown functions. Remarkably, the five strongest promoters of the CiaR regulon drive expression of small RNAs. These small RNAs, designated csRNAs for cia‐dependent small RNAs, are non‐coding, between 87 and 151 nt in size, and show a high degree of similarity to each other. The analysis of deletion mutants in the csRNA genes revealed that csRNA4 and csRNA5 affect stationary‐phase autolysis. The identification of five small non‐coding regulatory RNAs opens new perspectives to approach the physiological role of the CiaRH two‐component regulatory system.