Penicillin-binding Protein Genes Research Articles

Contributions of PBP 5 and DD-carboxypeptidase penicillin binding proteins to maintenance of cell shape in Escherichia coli.

Escherichia coli has 12 recognized penicillin binding proteins (PBPs), four of which (PBPs 4, 5, and 6 and DacD) have DD-carboxypeptidase activity. Although the enzymology of the DD-carboxypeptidases has been studied extensively, the in vivo functions of these proteins are poorly understood. To explain why E. coli maintains four independent loci encoding enzymes of considerable sequence identity and comparable in vitro activity, it has been proposed that the DD-carboxypeptidases may substitute for one another in vivo. We tested the validity of this equivalent substitution hypothesis by investigating the effects of these proteins on the aberrant morphology of DeltadacA mutants, which produce no PBP 5. Although cloned PBP 5 complemented the morphological phenotype of a DeltadacA mutant lacking a total of seven PBPs, controlled expression of PBP 4, PBP 6, or DacD did not. Also, a truncated PBP 5 protein lacking its amphipathic C-terminal membrane binding sequence did not reverse the morphological defects and was lethal at low levels of expression, implying that membrane anchoring is essential for the proper functioning of PBP 5. By examining a set of mutants from which multiple PBP genes were deleted, we found that significant morphological aberrations required the absence of at least three different PBPs. The greatest defects were observed in cells lacking, at minimum, PBPs 5 and 6 and one of the endopeptidases (either PBP 4 or PBP 7). The results further differentiate the roles of the low-molecular-weight PBPs, suggest a functional significance for the amphipathic membrane anchor of PBP 5 and, when combined with the recently determined crystal structure of PBP 5, suggest possible mechanisms by which these PBPs may contribute to maintenance of a uniform cell shape in E. coli.

Penicillin-binding proteins in Leptospira interrogans.

The Leptospira interrogans ponA and pbpB genes were isolated and characterized. ponA and pbpB encode the penicillin-binding proteins (PBPs) 1 and 3, respectively. There is little sequence variation between the PBP genes from two L. interrogans strains (serovar icterohaemorrhagiae strain Verdun and serovar pomona strain RZ11). The deduced L. interrogans PBP 1 and PBP 3 protein sequences from the two strains shared over 50% similarity to homologous proteins from Escherichia coli. It was demonstrated for strain Verdun that ponA and pbpB are transcribed individually from their own promoter. The ponA and pbpB genes from both strains are separated by 8 to 10 kb and oriented such that their transcription is convergent. The L. interrogans PBP 1 and PBP 3 proteins were synthesized in E. coli and were modified with ampicillin using a digoxigenin-ampicillin conjugate. These data show that both genes encode functional PBPs.

Cloning and characterization of the gene encoding penicillin-binding protein A of Streptomyces griseus

An internal segment of the penicillin-binding protein gene, pbpA, of Streptomyces griseus was amplified from genomic DNA using the polymerase chain reaction and used as a hybridization probe to isolate the complete gene from a cosmid library. pbpA encodes a 485 amino acid sequence that conserves three motifs of PBPs, SXXK, SXN, and KTG. The pbpA gene was located downstream of a gene homologous to the Bacillus subtilis spoVE gene. The pbpA gene was disrupted by replacing an ApaI fragment of the pbpA gene in S. griseus chromosome with an apramycin resistance gene cassette or directly inserting this apramycin resistance gene cassette at the NcoI site of pbpA penicillin-binding domain. No obvious defects in growth, sporulation, or spore sonication resistance were observed in the constructed pbpA mutants, suggesting that PBPA is not essential for growth and sporulation under normal laboratory conditions in S. griseus.

Cloning and characterization of the gene encoding penicillin-binding protein A of Streptomyces griseus.

An internal segment of the penicillin-binding protein gene, pbpA, of Streptomyces griseus was amplified from genomic DNA using the polymerase chain reaction and used as a hybridization probe to isolate the complete gene from a cosmid library. pbpA encodes a 485 amino acid sequence that conserves three motifs of PBPs, SXXK, SXN, and KTG. The pbpA gene was located downstream of a gene homologous to the Bacillus subtilis spoVE gene. The pbpA gene was disrupted by replacing an ApaI fragment of the pbpA gene in S. griseus chromosome with an apramycin resistance gene cassette or directly inserting this apramycin resistance gene cassette at the NcoI site of pbpA penicillin-binding domain. No obvious defects in growth, sporulation, or spore sonication resistance were observed in the constructed pbpA mutants, suggesting that PBPA is not essential for growth and sporulation under normal laboratory conditions in S. griseus.

Use of digoxigenin-labelled ampicillin in the identification of penicillin-binding proteins in Helicobacter pylori.

Amoxycillin is used in current therapeutic regimens to treat the infection caused by the human gastric pathogen, Helicobacter pylori. The penicillin-binding proteins (PBPs) are the primary targets for the beta-lactam antibiotics, such as amoxycillin, and are involved in the terminal stages of peptidoglycan synthesis. They also play active roles in the determination and maintenance of cellular morphology. It was believed that an organism with a complex morphology, such as H. pylori, would have more than the three PBPs previously suggested. Using digoxigenin-labelled ampicillin (DIG-ampicillin), we report the identification of eight PBPs in H. pylori with masses of 72, 62, 54, 50, 44, 33.5, 30.5 and 28 kDa. A smaller (21 kDa) ninth band was also detected, which may represent another PBP. However, the relatively small size of this apparent PBP raises questions as to whether this is a true PBP. In an attempt to identify the PBPs to which amoxycillin preferentially binds, amoxycillin was used in competition assays with DIG-ampicillin. It appeared that amoxycillin inhibited the binding of DIG-ampicillin to only the 72 kDa PBP. The experimental data were also compared with the seven putative PBPs identified in the two published H. pylori genomes, most of which correlate with the experimental data. To investigate further the properties of these PBPs, the seven putative PBP genes identified in the H. pylori genomes were examined. The derived amino acid sequences of the putative PBPs were examined for the three characteristic motifs found in all conventional PBPs, SXXK, SXN and KTG. We were able to determine that all of the putative PBPs had at least one of these motifs, but none possessed all three motifs with the characteristics of conventional PBPs. These findings suggest that the PBPs of H. pylori are unique.

Molecular epidemiology of penicillin-nonsusceptible Streptococcus pneumoniae among children in Greece.

A total of 145 penicillin-nonsusceptible Streptococcus pneumoniae strains were isolated from young carriers in Greece and analyzed by antibiotic susceptibility testing, serotyping, restriction fragment end labeling (RFEL), and penicillin-binding protein (PBP) genotyping. The serotypes 23A and 23F (54%), 19A and 19F (25%), 9V (5%), 15A, 15B, and 15C (4%), 6A and 6B (4%), and 21 (4%) were most prevalent in this collection. Fifty-three distinct RFEL types were identified. Sixteen different RFEL clusters, harboring 2 to 32 strains each, accounted for 82% of all strains. Eight of these genetic clusters representing 60% of the strains were previously identified in other countries. A predominant lineage of 66 strains (46%) harboring five RFEL types and the serotypes 19F and 23F was closely related to the pandemic clone Spain(23F)-1 (genetic relatedness of > or =85%). Another lineage, representing 11 strains, showed close genetic relatedness to the pandemic clone France(9V)-3. Another lineage of 8 serotype 21 strains was Greece specific since the RFEL types were not observed in an international collection of 193 genotypes from 16 different countries. Characterization of the PBP genes pbp1a, pbp2b, and pbp2x revealed 20 distinct PBP genotypes of which PBP type 1-1-1, initially observed in the pandemic clones 23F and 9V, was predominantly present in 11 RFEL types in this Greek collection of penicillin-nonsusceptible strains (55%). Sixteen PBP types covering 52 strains (36%) were Greece specific. This study underlines the strong contribution of penicillin-resistant international clones to the prevalence and spread of penicillin-nonsusceptible pneumococci among young children in Greece.

Genotypic survey of recent beta-lactam-resistant pneumococcal nasopharyngeal isolates from asymptomatic children in Chile.

To assess pneumococcal strain variability among young asymptomatic carriers in Chile, we used serotyping, antibiotic susceptibility testing, and genotyping to analyze 68 multidrug-resistant pneumococcal isolates recovered from 54 asymptomatic children 6 to 48 months of age. The isolates represented capsular serotypes 19F (43 isolates), 14 (14 isolates), 23F (7 isolates), 6B (3 isolates), and 6A (1 isolate). Genotypic analysis, which included pulsed-field gel electrophoresis (PFGE) of chromosomal digests, penicillin binding protein (PBP) gene fingerprinting, and dhf gene fingerprinting, revealed that the isolates represented six different genetic lineages. Clear circumstantial evidence of capsular switching was seen within each of four of the genetically related sets. The majority of the isolates, consisting of the 43 19F isolates and 2 type 6B isolates, appeared to represent a genetically highly related set distinct from previously characterized pneumococcal strains. Each of three other genetically defined lineages was closely related to one of the previously characterized clones Spain(6B)-2, France(9V)-3, or Spain(23F)-1. A fifth lineage was comprised of four type 23F isolates that, by the techniques used for this study, were genetically indistinguishable from three recent type 19F sterile-site isolates from the United States. Finally, a sixth lineage was represented by a single type 23F isolate which had a unique PFGE type and unique PBP and dhf gene fingerprints.

Escherichia coli mutants lacking all possible combinations of eight penicillin binding proteins: viability, characteristics, and implications for peptidoglycan synthesis.

The penicillin binding proteins (PBPs) synthesize and remodel peptidoglycan, the structural component of the bacterial cell wall. Much is known about the biochemistry of these proteins, but little is known about their biological roles. To better understand the contributions these proteins make to the physiology of Escherichia coli, we constructed 192 mutants from which eight PBP genes were deleted in every possible combination. The genes encoding PBPs 1a, 1b, 4, 5, 6, and 7, AmpC, and AmpH were cloned, and from each gene an internal coding sequence was removed and replaced with a kanamycin resistance cassette flanked by two res sites from plasmid RP4. Deletion of individual genes was accomplished by transferring each interrupted gene onto the chromosome of E. coli via lambda phage transduction and selecting for kanamycin-resistant recombinants. Afterwards, the kanamycin resistance cassette was removed from each mutant strain by supplying ParA resolvase in trans, yielding a strain in which a long segment of the original PBP gene was deleted and replaced by an 8-bp res site. These kanamycin-sensitive mutants were used as recipients in further rounds of replacement mutagenesis, resulting in a set of strains lacking from one to seven PBPs. In addition, the dacD gene was deleted from two septuple mutants, creating strains lacking eight genes. The only deletion combinations not produced were those lacking both PBPs 1a and 1b because such a combination is lethal. Surprisingly, all other deletion mutants were viable even though, at the extreme, 8 of the 12 known PBPs had been eliminated. Furthermore, when both PBPs 2 and 3 were inactivated by the beta-lactams mecillinam and aztreonam, respectively, several mutants did not lyse but continued to grow as enlarged spheres, so that one mutant synthesized osmotically resistant peptidoglycan when only 2 of 12 PBPs (PBPs 1b and 1c) remained active. These results have important implications for current models of peptidoglycan biosynthesis, for understanding the evolution of the bacterial sacculus, and for interpreting results derived by mutating unknown open reading frames in genome projects. In addition, members of the set of PBP mutants will provide excellent starting points for answering fundamental questions about other aspects of cell wall metabolism.

Cloning and Characterization of a Gene, pbpF , Encoding a New Penicillin-Binding Protein, PBP2B, in Staphylococcus aureus

A previously unrecognized penicillin binding protein (PBP) gene, pbpF, was identified in Staphylococcus aureus. This gene encodes a protein of 691 amino acid residues with an estimated molecular mass of 78 kDa. The molecular mass is very close to that of S. aureus PBP2 (81 kDa), and the protein is tentatively named PBP2B. PBP2B has three motifs, SSVK, SSN, and KTG, that can be found in PBPs and beta-lactamases. Recombinant PBP2B (rPBP2B), which lacks a putative signal peptide at the N terminus and has a histidine tag at the C terminus, was expressed in Escherichia coli. The purified rPBP2B was shown to have penicillin binding activity. A protein band was detected from S. aureus membrane fraction by immunoblotting with anti-rPBP2B serum. Also, penicillin binding activity of the protein immunoprecipitated with anti-rPBP2B serum was detected. These results suggest the presence of PBP2B in S. aureus cell membrane that covalently binds penicillin. The internal region of pbpF and PBP2B protein were found in all 12 S. aureus strains tested by PCR and immunoblotting.

Emerging issues in antibiotic resistant infections in long-term care facilities.

Managing patients infected with antibiotic resistant bacteria is becoming one of the major clinical obstacles facing physicians who treat patients in long-term care facilities (LTCFs). Penicillin-resistant pneumococci (PRP), vancomycin-resistant enterococci (VRE), gram-negative bacteria that produce extended-spectrum and ampC-type beta-lactamase enzymes, and quinolone-resistant gram-positive and gram-negative bacteria are the major resistant pathogens that are emerging in these settings. The mechanisms responsible for the evolution of these antibiotic resistant organisms (molecular rearrangement of penicillin binding protein genes, acquisition of a mobile genetic element, and point mutation that alter the active site) are reviewed. Vancomycin intermediate Staphylococcus aureus (VISA) and multidrug efflux pumps in gram-negative bacteria are also threatening our most potent antimicrobials. Aggressive screening, education, antibiotic-control measures, and immunization are advocated as important preventive measures. The combined efforts of the medical directors, infection-control personnel, and administrators are needed to stem this problem.

Spread of drug-resistant Streptococcus pneumoniae in Asian countries: Asian Network for Surveillance of Resistant Pathogens (ANSORP) Study.

Antimicrobial susceptibility of 996 isolates of Streptococcus pneumoniae from clinical specimens was investigated in 11 Asian countries from September 1996 to June 1997. Korea had the greatest frequency of nonsusceptible strains to penicillin with 79.7%, followed by Japan (65.3%), Vietnam (60.8%), Thailand (57.9%), Sri Lanka (41.2%), Taiwan (38.7%), Singapore (23.1%), Indonesia (21.0%), China (9.8%), Malaysia (9.0%), and India (3.8%). Serotypes 23F and 19F were the most common. Pulsed-field gel electrophoresis (PFGE) of 154 isolates from Asian countries showed several major PFGE patterns. The serotype 23F Spanish clone shared the same PFGE pattern with strains from Korea, Japan, Singapore, Taiwan, Thailand, and Malaysia. Fingerprinting analysis of pbp1a, pbp2x, and pbp2b genes of 12 strains from six countries also showed identical fingerprints of penicillin-binding protein genes in most strains. These data suggest the possible introduction and spread of international epidemic clones into Asian countries and the increasing problems of pneumococcal drug resistance in Asian countries for the first time.

Molecular epidemiological survey of penicillin-resistant Streptococcus pneumoniae from Asia, Europe, and North America

One hundred penicillin-resistant Streptococcus pneumoniae (PRSP) strains from Asia, Europe, and North America were analyzed using pulsed-field gel electrophoresis; fingerprinting of penicillin binding protein ( pbp) genes; and BOX PCR. Results show that six PFGE patterns (three patterns comprising ≥ 2 serotypes) were found widespread and accounted for 64 of the 100 PRSP strains.

Genetic Analysis of Serotype 23F Streptococcus pneumoniae Isolates from Several Countries by Penicillin-Binding Protein Gene Fingerprinting and Pulsed-Field Gel Electrophoresis

We characterized 21 strains of serotype 23F Streptococcus pneumoniae isolated in various countries with various levels of penicillin susceptibility by penicillin-binding protein (PBP) gene fingerprinting and pulsed-field gel electrophoresis (PFGE). Pneumococci isolated in Israel, Hungary, Bulgaria, Slovakia, Rumania, France, the United States, Spain and Japan were included. These strains were classified into 12 and 18 groups by PBP gene fingerprinting and PFGE, respectively. Some of the pneumococci isolated in Spain, the United States and France appeared to be genetically related by PFGE, showed the same PBP gene pattern and had similar antimicrobial susceptibility patterns. One penicillin-susceptible Bulgarian strain, with a similar PFGE pattern but a different fingerprinting pattern, may be an ancestral recipient strain that became transformed into the resistant variants. Rumanian and Israeli strains were also genetically related by PFGE. These results indicate the existence of widely spread but related pneumococci in the world. PBP 2X gene profiles of pneumococci with MICs of 0.25 µg/ml were different from each other and from penicillin-susceptible pneumococci (PSP). PBP 2B gene profiles of these resistant strains were identical. PBP 2B gene profiles of pneumococci (penicillin MICs ≥0.5 µg/ml) were different from PSP. PBP gene profiles may not only be useful for genetic analysis but also for presumed penicillin susceptibility.

Phylogenetic analysis of macrorestriction fragments as a measure of genetic relatedness in Staphylococcus aureus: the epidemiological impact of methicillin resistance.

The present study was undertaken for the purpose of defining the epidemiology and genetic relatedness of Staphylococcus aureus strains in a region of Italy by investigating the molecular background for which resistance to methicillin, mediated by the acquisition of another penicillin-binding protein gene, is embedded. Restriction fragment length polymorphism (RFLP) data were used for phylogenetic analyses, since genetic distance values can be used as a general measure of the number of events generating distinct clones. The percentages of methicillin-resistant S. aureus (MRSA) isolates in a six-month period from inpatients and outpatients were, respectively, 12% (22 out of 180) and 0.4% (1 out of 257). On the basis of RFLP obtained after pulsed field gel electrophoresis (PFGE), it was possible to designate isolates as indistinguishable, closely related, possibly related and unrelated. We were able to demonstrate the occurrence of at least five distinct MRSA cross-infection episodes in two hospitals, four involving two patients each and one involving four patients. Phylogenetic analyses overcame the simple pairwise comparison of common bands between strains, and provided a comprehensive epidemiological scenario, identifying three major clusters of MRSA, including different levels of genetic relatedness, while excluding the circulation of a single clone in Italy. Moreover, multidimensional scaling analysis of the obtained genetic distance confirmed that MRSA strains belong to a restricted set of clones, thus demonstrating the relatedness of broad evolutionary lineages within the species S. aureus.

Molecular epidemiology of penicillin-resistant Streptococcus pneumoniae isolates recovered in Italy from 1993 to 1996.

Thirty-nine penicillin-resistant Streptococcus pneumoniae isolates recovered among the approximately 700 pneumococcal strains collected from 1993 to 1996 in central and northern Italy were analyzed for several characteristics, including serotype, antibiotic susceptibility profile, chromosomal relatedness (by using pulsed-field gel electrophoresis [PFGE]), restriction fragment length polymorphism (RFLP) of the penicillin-binding protein (PBP) genes 1A, 2X, and 2B, and the presence of a variety of antibiotic resistance genes (determined by hybridization with appropriate DNA probes). The MICs of penicillin for most of the isolates (30 of 39) were high, in the range of 1 microgram/ml or higher, and these 30 isolates carried additional resistance traits to two or more drugs (erythromycin, chloramphenicol, co-trimoxazole, and tetracycline) and expressed serotypes 9, 19, and 23 and three distinct PFGE patterns. More than half (22 of 30) of the isolates for which MICs were high were identified as representatives of two widespread international epidemic clones of S. pneumoniae. The first one of these clones (seven isolates) expressed serotype 23F and possessed all properties characteristic of the widespread Spanish/USA international clone. Seven additional strains with serotype 19 also had the same PFGE pattern, PBP gene, and RFLP polymorphisms, and other properties typical of the serotype 23 Spanish/USA clone, suggesting that these strains were the products of a capsular transformation event (from serotype 23F to serotype 19) in which the Spanish/USA clone was the recipient. The second international clone was represented by eight serotype 9 isolates which were resistant to penicillin and trimethoprim-sulfamethoxazole and had the molecular properties of the French/Spanish epidemic clone. The remaining eight isolates for which penicillin MICs were high appeared to represent a hitherto-undescribed "Italian" clone; they had a novel PFGE type, unique RFLPs for the PBP genes, and resistance to tetracycline, trimethoprim-sulfamethoxazole, and erythromycin, and the penicillin MICs for these isolates were 2 to 4 microgram/ml.

Alterations in PBP 1A essential-for high-level penicillin resistance in Streptococcus pneumoniae.

High-level penicillin resistance in pneumococci is due to alterations in penicillin-binding proteins (PBPs) 2X, 2B, and 1A. We have sequenced the penicillin-binding domain of PBP 1A from penicillin-resistant South African pneumococcal isolates and have identified amino acid substitutions which are common to all the resistant isolates analyzed. Site-directed mutagenesis was then used to determine whether particular amino acid substitutions at specific positions in PBP 1A mediate penicillin resistance. PCR was used to isolate PBP 2X, 2B, and 1A genes from clinical isolate 8303 (penicillin MIC, 4 micrograms/ml). These wild-type PBP genes were cloned into pGEM-3Zf and were used as the transforming DNA. Susceptible strain R6 (MIC, 0.015 microgram/ml) was first transformed with PBP 2X and 2B DNA, resulting in PBP 2X/2B-R6 transformants for which MICs were 0.25 microgram/ml. When further transformed with PBP 1A DNA, 2X/2B/1A-R6 transformants for which MICs were 1.5 micrograms/ml were obtained. Site-directed mutagenesis of the PBP 1A gene from isolate 8303 was then used to reverse particular amino acid substitutions, followed by transformation of PBP 2X/2B-R6 transformants with the mutagenized PBP 1A DNA. For PBP 2X/2B/1A-R6 transformants, the introduction of the reversal of Thr-371 by Ser or Ala in PBP 1A decreased the MIC from 1.5 to 0.5 micrograms/ml, whereas the reversal of four consecutive amino acid substitutions (Thr-574 by Asn, Ser-575 by Thr, Gln-576 by Gly, and Phe-577 by Tyr) decreased the MIC from 1.5 to 0.375 micrograms/ml. These data reveal that amino acid residue 371 and residues 574 to 577 of PBP 1A are important positions in PBP 1A with respect to the interaction with penicillin and the development of resistance.

Application of molecular typing to the epidemiology of Streptococcus pneumoniae.

The spread of antibiotic resistance and the development of new vaccines have focused attention on the epidemiology of Streptococcus pneumoniae over recent years. While serotyping and the determination of antibiotic...

Capsular transformation of a multidrug-resistant Streptococcus pneumoniae in vivo.

Several multidrug-resistant (MDR) isolates of Streptococcus pneumoniae recovered from a variety of clinical sources and expressing serotypes 9N, 14, 19F, and 3 could not be distinguished from the capsular type 23 Spanish/USA epidemic clone in antibiotype, pulsed-field gel electrophoretic pattern, and restriction fragment length polymorphism types of their penicillin binding protein genes 1A, 2X, and 2B. When tested in a mouse model of virulence, isolates expressing capsular type 3 were lethal for 100% of infected animals within 2 days after intraperitoneal injection of 10(2) cfu/mouse. In contrast, several capsular type 23F isolates belonging to the MDR Spanish/USA clone and recovered from the same site were not lethal, even when injected at the dose of 10(7) cfu/mouse. These data suggest that the pneumococcal isolates expressing serotypes 9N, 14, 19F, and 3 represent products of in vivo capsular transformation events in which the MDR epidemic capsular type 23F Spanish/USA clone was the recipient.

Microbiological aspects

By the 1930s several studies had shown that Streptococcus pneumoniae was an important pathogen in Nairobi (Kenya) and various risk factors for infection were recognized, including seasonally cold conditions, overcrowding and recent arrival in the city. Research into pneumococcal disease declined with the arrival of penicillin but recently interest has been rekindled by recogńition of the pneumococcus as a human immunodeficiency virus (HIV)-associated pathogen and by the emergence of antibiotic resistance. The pneumococcus and its association with HIV were studied during the course of the Wellcome Trust/Kenya Medical Research Institute HIV Programme in Nairobi (1988–1993). There were generally high rates of pneumococcal disease. The pneumococcus (with tuberculosis and salmonellosis) was a major HTV-related pathogen. One study showed HIV seropositivity to confer a relative risk of 17·8 for pneumococcal infection. Recurrent infection accounted for a large proportion (25%) of disease episodes in a longitudinally studied cohort of HIV patients. There were higher pneumococcal carriage rates in HIV-positive than in HIV-negative patients (28% vs. 16%, P = 0.003). High rates of resistance were found to penicillin (25%). Molecular characterization of penicillin-resistant strains identified 11 separate clones, showing great genetic diversity in a small sample of isolates, and there was evidence of horizontal spread of penicillin-binding protein genes between separate lineages. Molecular characterization of isolates from patients with recurrent disease suggested that both relapse and reinfection might occur. There was molecular evidence of transfer of capsular genes between clones (serotype switching). The overall spectrum of serotypes resembled those reported elsewhere, most serotypes being included in the 23-valent vaccine. Higher numbered serotypes were associated with respiratory tract source and antibiotic resistance. Various methods were used to show 82% concordance between pernasal and blood isolates in pneumonia cases. HIV-seropositive patients were more prone to infection with penicillin- and tetracycline-resistant organisms than seronegative patients (penicillin, 27% vs. 7%; tetracycline, 40% vs. 17%), a difference reflected by different serotype profiles in the 2 groups. These studies highlight the importance of the pneumococcus as an HIV-related pathogen in one part of Africa. The high rates of antibiotic resistance are a cause of concern. There should be continued monitoring of resistance patterns, and assessments of the significance of penumococcal disease made elsewhere in Africa are to be encouraged.

Stability of clonally related DNA fingerprints and cell-wall peptide patterns in geographic isolates of multiresistant epidemic clones of Streptococcus pneumoniae

Objective: To determine the stability of multidrug-resistant epidemic clones of Streptococcus pneumonia. Methods: Polymerase chain reaction (PCR) was used to amplify the penicillin-binding protein (PBP) genes 1A, 2X, and 2B, followed by fingerprinting after restriction digestion of the amplified molecules. Cell walls were isolated, digested by the autolytic amidase of pneumococcus, and the family of stem peptides was separated by high-pressure liquid chromatography. Results: Twenty-eight isolates of multidrug-resistant capsular type 6B S. pneumoniae recovered in Iceland between 1990 and 1992 and ten isolates of the multidrug-resistant capsular type 23F S. pneumoniae recovered in the United States, Portugal, Croatia, and South Korea between 1989 and 1996 were compared for restriction fragmentation length polymorphism (RFLP) of their PBP genes 1A, 2X, and 2B, using the PCR for amplification. Five isolates each of the multidrug-resistant capsular type 6B pneumococci from Iceland and the capsular type 23F isolates from Cleveland, Ohio, were also compared for their muropeptide profiles, using a high-performance liquid chromatography (HPLC) system to separate the family of stem peptides generated by enzymatic hydrolysis. The isolates representing the two major and internationally spread epidemic clones have retained the clone-specific RFLP fingerprints of their PBP genes and also reproduced distinct cell-wall stem peptide patterns characteristic of the two pneumococcal clones. Conclusions: The data further document the considerable stability of clonal types of the multiresistant epidemic clones of S. pneumoniae over time and over a wide range of geographic isolation sites.