Penicillin-binding Protein 2B Gene Research Articles

OL-060 In vitro activity of antibiotics on biofilm producing isolates of Pseudomonas aeruginosa

Background: To evaluate the penicillin-binding protein 2B gene (pbp2b) based PCR and sequencing for screening Streptococcus pneumoniae infection and predict its susceptibility in cerebral spinal fluid from paediatric bacterial meningitis patients. Methods: A nested PCR targeting pbp2b and another two S. pneumoniae specific PCR targeting pneumolysin (ply) and autolysin (lytA) were developed for detection of S. pneumoniae in cerebral spinal fluid from bacterial meningitis patients. The PCR results of three different genes and culture were compared. The consistency of penicillin susceptibility PCR (using resistant and susceptible primers respectively), sequencing and culture based phenotypic penicillin resistant results were compared. Result: Of the 161 specimens studied, there were totally 25 S. pneumoniae infection confirmed by different methods (16 by lytA PCR, 14 by ply PCR, 16 by pbp2b PCR and 9 by cerebrospinal fluid culture). Of the 16 pbp2b positive specimens, penicillin sensitive and resistant sequence types account for half respectively. 4 of the 16 pbp2b positive specimens had culture based phenotypic penicillin-resistant result. 3 of 4 were consistent with penicillin susceptibility PCR result. The result of susceptibility PCR targeting pbp2b was consistent with sequencing result. There were no new point mutations but new sequence types were found in these strains when compared with GenBank. Penicillin resistant in pneumococcal meningitis was 66.67% (6/9) by culture phenotype and 50% (8/16) by PCR and sequencing when culture was negative. Conclusion: pbp2b can be served as a good target gene to detect S. pneumoniae and predict its penicillin susceptibility, especially important when culture was negative.

OL-059 Penicillin-binding protein 2B gene (pbp2b) based PCR and sequencing for screening Streptococcus pneumoniae infection and predicting its susceptibility in cerebral spinal fluid from paediatric bacterial meningitis patients

OL-059 Penicillin-binding protein 2B gene (pbp2b) based PCR and sequencing for screening Streptococcus pneumoniae infection and predicting its susceptibility in cerebral spinal fluid from paediatric bacterial meningitis patients

Detection of penicillin-binding protein 2b gene alteration in Streptococcus mitis by polymerase chain reaction.

Three isolates of beta-lactam-resistant streptococci from the saliva of healthy adults were identified as Streptococcus mitis. Minimum inhibitory concentrations (MICs) were 2 to 4 micro g/ml for ampicillin (ABPC) and 64 to more than 128 micro g/ml for cefaclor (CCL). To determine the position of base alterations of the penicillin-binding protein 2b ( pbp2b) gene, upstream primers containing possible mutation points were designed, and used for polymerase chain reaction (PCR), together with a downstream primer. Alterations adjacent to the conserved motifs of the pbp2b gene were apparent. DNA sequencing data indicated replacements in deduced amino acid sequences in all resistant isolates: from threonine to alanine just after the serine-serine-asparagine (SSN) motif, and from alanine to glycine two residues downstream of the lysine-threonine-glycine (KTG) motif. These changes were the same as those in penicillin-resistant Streptococcus pneumoniae (PRSP), suggesting importance for the enzymatic activity of the protein. Thus, Beta-lactam susceptibility of S. mitis may be partially predicted by PCR using our primer set for pbp2b.

Extensive variation in the ddl gene of penicillin-resistant Streptococcus pneumoniae results from a hitchhiking effect driven by the penicillin-binding protein 2b gene.

An internal fragment of the ddl gene, encoding the cytoplasmic enzyme D-alanyl-D-alanine ligase, was sequenced from 566 isolates of Streptococcus pneumoniae and single isolates of Streptococcus mitis and Streptococcus oralis. The 52 alleles found among the S. pneumoniae isolates fell into two groups. Group A alleles were very uniform in sequence and were present in both penicillin-susceptible and penicillin-resistant pneumococci. Group B alleles were much more diverse and were found only in penicillin-resistant isolates. The Streptococcus oralis and Streptococcus mitis alleles were less diverged from group A alleles than some of the group B pneumococcal alleles, suggesting that the latter alleles contain interspecies recombinational replacements. The ddl gene was located 783 bp downstream of the penicillin-binding protein 2b gene (pbp2b). Sequencing of the pbp2b-recR-ddl-murF region of three penicillin-resistant pneumococci that had diverged ddl alleles showed that the whole region from pbp2b to ddl (or beyond) was highly diverged (about 8%) compared with the sequences from three penicillin-susceptible isolates. The high levels of diversity in the group B ddl alleles from penicillin-resistant isolates were ascribed to a hitchhiking effect whereby interspecies recombinational exchanges at pbp2b, selected by penicillin usage, often extend into, or through, the ddl gene. The data allow the average size of the interspecies recombinational replacements to be estimated at about 6 kb.

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.

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.

Rapid detection of penicillin-resistant Streptococcus pneumoniae in cerebrospinal fluid by a seminested-PCR strategy.

A seminested-PCR assay, based on the amplification of the pneumococcal penicillin-binding protein 2B gene (pbp2B), was developed for the detection of penicillin-resistant and -susceptible pneumococci in cerebrospinal fluid (CSF) specimens. Species-specific primers (P5 and P6) which amplified a 682-bp conserved region of the transpeptidase-encoding region of the pbp2B gene were used. Four "resistance" primers were designed to bind to altered areas of the pbp2B gene identified in penicillin-resistant South African wild-type strains. Together with the downstream primer P6, the upstream resistance primers amplified fragments which were used to detect the presence of penicillin resistance. This system identified all 35 of the S. pneumoniae isolates evaluated, including strains of 11 different serotypes and a range of penicillin-resistant and -susceptible strains. The specificity of the assay was demonstrated by its inability to amplify DNA from other bacterial species which commonly cause meningitis. It was possible to detect pneumococcal DNA from culture-negative CSF inoculated with 2.5 pg of purified DNA or 18 CFU. Analysis of 285 CSF specimens showed that PCR detected the pneumococcus in 18 samples positive by culture, including the identification of four penicillin-resistant isolates. The positive predictive value and the negative predictive value of the assay were each 100%.

An outbreak of penicillin resistant Streptococcus pneumoniae investigated by a polymerase chain reaction based genotyping method.

AIMS: To characterise the genotypes of penicillin resistant Streptococcus pneumoniae infecting patients in a care of the elderly ward and to study its transmission in a hospital environment. METHODS: Isolates...

Determination of penicillin susceptibility of Streptococcus pneumoniae using the polymerase chain reaction.

AIM: To develop a polymerase chain reaction (PCR) based method to detect penicillin susceptibility in isolates of Streptococcus pneumoniae (SP). METHOD: PCR primers were designed to amplify differential nucleotide sequences...

Directly repeated insertion of 9-nucleotide sequence detected in penicillin-binding protein 2B gene of penicillin-resistant Streptococcus pneumoniae

We investigated the molecular mechanism of 50 penicillin-resistant Streptococcus pneumoniae strains (penicillin: MIC, > or = 0.125 microgram/ml) having neither class A nor class B mutations in the penicillin-binding protein 2B gene (pbp2b). An analysis of the nucleotide sequences of the pbp2b genes from seven strains revealed an unique direct repeat of 9 nucleotides (TGGTATACT) between active-site serine (residue 385) and Ser-X-Asn (residues 442 to 444) motifs. The same insertion was detected in 13 strains.

Combinational detection of autolysin and penicillin-binding protein 2B genes of Streptococcus pneumoniae by PCR.

PCR was used to identify penicillin resistance in 1,062 clinical isolates of Streptococcus pneumoniae. Three sets of primers were designed to amplify (i) a 240-bp fragment of the penicillin-binding protein (PBP) 2B gene (pbp2b) of penicillin-susceptible S. pneumoniae (PSSP), (ii) a 215-bp fragment of the class A mutations of the pbp2b gene present in penicillin-resistant S. pneumoniae, and (iii) a 286-bp fragment of the class B mutation. In addition, a set of primers that amplify 273 bp of the autolysin (lytA) gene was applied in combination with the above to identify S. pneumoniae. Of 621 isolates for which MICs of penicillin were < or = 0.06 mu g/ml, 614 (98.9%) were ascertained as having DNA fragments amplified by the PSSP primers. Of 441 isolates for which MICs of penicillin were > or = 0.125 mu g/ml, a class A mutation was detected in only 8 (1.8%), a class B mutation was detected in 310 (70.3%), and neither class A nor class B mutations were found in the remaining 123 (27.9%). However, when analysis was limited to isolates for which MICs of penicillin were > or = 1.0 mu g/ml, 247 isolates (89.8%) of 275 were found to possess a class B mutation. When PBPs were analyzed in 12 isolates with unclear mutations of the pbp2b gene by using [3H]benzylpenicillin, low affinity to PBP 2B was observed in them all. These findings suggest that a pbp2b mutation other than class A or class B is present in these isolates. These results also indicate that it may be possible to identify PSSP and penicillin-resistant S. pneumoniae by applying PCR using a combination of primers to detect the susceptible pbp2b gene, resistant pbp2b gene mutations, and the lytA gene.

Detection of Streptococcus pneumoniae in whole blood by PCR

Streptococcus pneumoniae is a major cause of bacteremia in both children and adults. Currently, the diagnosis of pneumococcal bacteremia relies on the isolation and identification of the bacteria from blood cultures. We have developed a sensitive assay for the detection of S. pneumoniae in whole blood by the PCR. A specific primer-probe set (JM201 and JM202 primers with JM204 probe) designed from the penicillin-binding protein 2B gene was demonstrated to reproducibly detect between 10 and 100 fg of input purified S. pneumoniae DNA. This assay system was shown to be inclusive for all strains of S. pneumoniae evaluated, including 15 different serotypes and a battery of penicillin-resistant and -sensitive strains. The specificity of this PCR-based assay was demonstrated by its inability to support amplification from a series of human, bacterial, and yeast genomic DNAs. A general specimen preparation method which should be suitable for the purification of DNA from any pathogens in whole blood was developed. With this protocol it was possible to detect S. pneumoniae-specific DNA from whole blood specimens inoculated with as little as 4 CFU/ml. Copurified human blood DNA, ranging from 0 to 4.5 micrograms per PCR, did not affect the sensitivity of S. pneumoniae detection by PCR. A blinded clinical trial was used to compare the PCR-based assay with standard microbiological blood culture for the detection of S. pneumoniae bacteremia in 36 specimens obtained from pediatric patients seen in the emergency room of Children's Hospital of Pittsburgh. With culture as the "gold standard," the PCR-based assay had a sensitivity of 80% (4 of 5 culture-positive specimens were PCR positive) and a specificity of 84% (26 of 31 culture-negative specimens were PCR negative). However, three patients whose specimens were PCR positive and culture negative had histories suggestive of bacteremia, including recent positive blood cultures, treatment with antibiotics, cellulitis, and multiple emergency room visits for fever within a 24-h period. These data suggest that PCR-based assays for S. pneumoniae may prove useful to augment current methods of detection for S. pneumoniae bacteremia.

Relatedness among penicillin-binding protein 2b genes of Streptococcus mitis, Streptococcus oralis, and Streptococcus pneumoniae.

Penicillin-binding protein (PBP) 2b similarities among Streptococcus mitis, S. oralis, and S. pneumoniae using DNA fingerprinting and sequencing were investigated. The polymerase chain reaction (PCR) was performed on 41 penicillin-susceptible and -resistant clinical isolates of S. mitis and S. oralis using the susceptible S. pneumoniae R6 PBP 2b primers. PCR products were then analyzed using Hinf I and Sty I restriction enzymes. Of 41 S. mitis/S. oralis isolates studied 15 strains produced a PCR product of a similar size to that of S. pneumoniae R6. On fingerprinting these 15 strains, 11 different patterns were seen using Sty I restriction enzyme and 12 different patterns with Hinf I. The PBP 2b genes of the S. mitis and S. oralis isolates studied were found to be very heterogenous. The PBP 2b genes of two S. mitis isolates, MICs 0.5 and 2 micrograms/ml, were sequenced. These PBP 2b genes were found to possess a mosaic structure when compared to those of other S. pneumoniae and viridans streptococcal species. Analysis of these mosaic blocks indicates that both S. mitis strains contain areas that originated from S. pneumoniae as well as regions of unknown origin. PBP 2b sequence comparisons of a susceptible S. oralis with reported sequences of S. pneumoniae R6 and S. mitis NCTC 10712 revealed what appears at this stage to be nucleotide regions unique to S. oralis. A penicillin-resistant S. oralis strain contained a pneumococcal region of 272 bp that was flanked by S. oralis sequences. These specific S. oralis regions have been located in PBP 2b genes of penicillin-resistant S. oralis and S. pneumoniae isolates described from Europe and South Africa.

Molecular epidemiology of penicillin-resistant pneumococci isolated in Nairobi, Kenya.

A total of 26% of the pneumococci isolated from an outpatient clinic in Nairobi, Kenya, during 1991 to 1992 had intermediate levels of penicillin resistance. Gene fingerprinting and DNA sequencing were used to distinguish the penicillin-binding protein (PBP) 1A, 2B, and 2X genes in 23 resistant isolates. Isolates were grouped into those that had identical forms of each of the three PBP genes (fingerprint groups) and those that had identical rRNA gene restriction patterns (ribotypes). Both methods divided the isolates into 11 groups. In a few cases, horizontal gene transfer appeared to have distributed an identical altered PBP gene into different pneumococcal lineages. Eight isolates were indistinguishable by ribotyping or multilocus enzyme electrophoresis and contained identical PBP 1A genes. Although these isolates were therefore members of the same clone, they were divided into two fingerprint groups which contained different PBP 2X and 2B genes. Presumably, members of this clone have acquired different altered PBP 2X and 2B genes on two separate occasions. One of these fingerprint groups contained isolates of serotype 14, whereas the other contained isolates of both serotypes 14 and 7. The identification of isolates in the latter group that are identical by all criteria, except serotype, implies the occurrence of a change in serotype. The predominant serotypes of the penicillin-resistant pneumococci from Nairobi were serotypes 14 and 19. In both cases, isolates of the same serotype which required the same MIC of penicillin were not members of a single clone, indicating that identity of serotype and MIC are not sufficient criteria for defining clones of resistant pneumococci even when the bacteria are isolated from a single clinic.

Genetic diversity of penicillin-binding protein 2B and 2X genes from Streptococcus pneumoniae in South Africa

Streptococcus pneumoniae (the pneumococcus) is believed to have developed resistance to penicillin by the production of altered forms of penicillin-binding proteins (PBPs) that have decreased affinity for penicillin. Sixty-eight clinical isolates of serogroup 6 and 19 pneumococci (MICs, < 0.015 to 8 micrograms/ml) were randomly selected from hospitals across South Africa which are at substantial geographic distance from each other. The polymerase chain reaction was used to isolate the penicillin-binding domain of PBPs 2B and 2X from the chromosomal DNAs of the bacteria; the purified PBP DNA was digested with restriction enzymes, the fragments were end-labelled and separated on polyacrylamide gels, and the DNA fingerprints were visualized following autoradiography. Fingerprint analysis revealed that at least 19 PBP 2B gene variants occur in the serogroup 6 and 19 pneumococci. The PBP 2B gene revealed a uniform profile among penicillin-susceptible isolates, with variation from this profile occurring only in isolates for which MICs were > or = 0.06 micrograms/ml. Analysis of the PBP 2X gene revealed a greater diversity in the population with 26 variant genes, including some diversity among susceptible isolates. Discrete profiles of both genes were found only within narrow bands of the penicillin MIC, so that the gene pattern predicted the MIC. PBP 2X gene variation and the lack of variability among PBP 2B genes in pneumococci inhibited at low MICs confirm that PBP 2X alteration may be responsible for low-level penicillin resistance, while alterations in both PBP 2B and PBP 2X are required for high-level resistance. The extensive diversity of PBP genes in South African serogroup 6 and 19 strains suggests that altered PBP genes have arisen frequently in this population.

Horizontal spread of an altered penicillin-binding protein 2B gene between Streptococcus pneumoniae and Streptococcus oralis

Horizontal spread of an altered penicillin-binding protein 2B gene between Streptococcus pneumoniae and Streptococcus oralis

Deletion analysis of the essentiality of penicillin-binding proteins 1A, 2B and 2X of Streptococcus pneumoniae.

An internal fragment from each of the penicillin-binding protein (PBP) 1A, 2B and 2X genes of Streptococcus pneumoniae, which included the region encoding the active-site serine residue, was replaced by a fragment encoding spectinomycin resistance. The resulting constructs were tested for their ability to transform S. pneumoniae strain R6 to spectinomycin resistance. Spectinomycin-resistant transformants could not be obtained using either the inactivated PBP 2X or 2B genes, suggesting that deletion of either of these genes was a lethal event, but they were readily obtained using the inactivated PBP 1A gene. Analysis using the polymerase chain reaction confirmed that the latter transformants had replaced their chromosomal copy of the PBP 1A gene with the inactivated copy of the gene. Deletion of the PBP 1A gene was therefore tolerated under laboratory conditions and appeared to have little effect on growth or susceptibility to benzylpenicillin.

Antimicrobial resistance in Streptococcus pneumoniae: a South African perspective.

Resistance to penicillin among South African strains of Streptococcus pneumoniae increased from 4.9% in 1979 to 14.4% in 1990. Except for resistance to co-trimoxazole (44%), resistance to other antimicrobial agents remained relatively low. Multiply resistant strains belonged mainly to serovars 6B, 19A, 14, and, more recently, 23F. Use of chloramphenicol to treat meningitis caused by strains relatively resistant to penicillin proved to be unsatisfactory, probably because of the inadequate bactericidal activity of chloramphenicol against these strains. Spread of penicillin-resistant nasopharyngeal strains in pediatric wards was most common among children who received antimicrobial therapy. Penicillin-binding protein (PBP) patterns were shown to vary in resistant clinical strains. Interspecies transfer of penicillin resistance between Streptococcus mitis and S. pneumoniae was demonstrated and antigenic homology was found in PBPs 1A and 2B of strains belonging to these species. Restriction enzyme mapping following DNA amplification of the PBP 2B gene revealed six arrangements among South African strains within serogroup 19. Despite extensive studies in South Africa and several other countries, many questions with regard to the global problem of antimicrobial resistance among S. pneumoniae strains remain unanswered, especially those that relate to prevalence in developing regions of the world.

Relatedness between Streptococcus pneumoniae and viridans streptococci: transfer of penicillin resistance determinants and immunological similarities of penicillin-binding proteins

The occurrence of highly variable penicillin-binding proteins (PBPs) in penicillin-resistant Streptococcus pneumoniae suggested that transfer of homologous genes from related species may be involved in resistance development. Antiserum and monoclonal antibodies raised against PBPs 1a and 2b from the susceptible S. pneumoniae R6 strain were used to identify related PBPs in 41 S. mitis, S. sanguis I and S. sanguis II strains mostly isolated in South Africa with MIC values ranging from < 0.15 to 16 mg/ ml . Furthermore, the possibility of genetic exchange was examined with 30 penicillin-resistant strains of this collection (MIC ⩾ 0.06 mg/ ml ) as donors using S. pneumoniae R6 as recipient in transformation experiments. The majority of S. mitis and S. sanguis II strains but none of the S. sanguis I strains could transform penicillin resistance genes into S. pneumoniae R6. All positive donor strains and all susceptible isolates of S. mitis and S. sanguis II strains contained PBPs which cross-reacted with the anti-PBP 1a and/or anti-PBP 2b antibodies. On the other hand, only five of the 14 S. sanguis I strains contained a PBP that reacted with one of the antibodies. This strongly suggested the presence of genes homologous to the pneumococcal PBP 1a and 2b genes in viridans streptococci and documents that penicillin resistance determinants can be transformed from viridans streptococci into the pneumococcus.

Intercontinental Spread of a Multiresistant Clone of Serotype 23F Streptococcus pneumoniae

Isolates of serotype 23F Streptococcus pneumoniae with high levels of resistance of penicillin have been commonly recovered in Spain for more than a decade. Recently penicillin-resistant serotype 23F S. pneumoniae strains were also isolated from children attending a day-care center in Cleveland. A number of Spanish and Cleveland isolates were compared by electrophoretic analysis of penicillin-binding protein (PBP) profiles and DNA restriction endonuclease cleavage profiles of the PBP 2X and 2B genes amplified with the polymerase chain reaction and by multilocus enzyme electrophoresis. All strains were identical by these criteria. The findings demonstrate that the Spanish and Cleveland isolates are clonally related and suggest that this antibiotic resistant clone of serotype 23F S. pneumoniae has spread intercontinentally from Spain to the United States.