New Aspects of the Interplay between Penicillin Binding Proteins, murM, and the Two-Component System CiaRH of Penicillin-Resistant Streptococcus pneumoniae Serotype 19A Isolates from Hungary

ABSTRACT The Streptococcus pneumoniae clone Hungary19A-6 expresses unusually high levels of β-lactam resistance, which is in part due to mutations in the MurM gene, encoding a transferase involved in the synthesis of branched peptidoglycan. Moreover, it contains the allele ciaH232, encoding the histidine kinase CiaH (M. Müller, P. Marx, R. Hakenbeck, and R. Brückner, Microbiology 157:3104–3112, 2011, https://doi.org/10.1099/mic.0.053157-0 ). High-level penicillin resistance primarily requires the presence of low-affinity (mosaic) penicillin binding protein (PBP) genes, as, for example, in strain Hu17, a closely related member of the Hungary19A-6 lineage. Interestingly, strain Hu15 is β-lactam sensitive due to the absence of mosaic PBPs. This unique situation prompted us to investigate the development of cefotaxime resistance in transformation experiments with genes known to play a role in this phenotype, pbp2x, pbp1a, murM, and ciaH, and penicillin-sensitive recipient strains R6 and Hu15. Characterization of phenotypes, peptidoglycan composition, and CiaR-mediated gene expression revealed several novel aspects of penicillin resistance. The murM gene of strain Hu17 (murM Hu17), which is highly similar to murM of Streptococcus mitis, induced morphological changes which were partly reversed by ciaH232. murM Hu17 conferred cefotaxime resistance only in the presence of the pbp2x of strain Hu17 (pbp2x Hu17). The ciaH232 allele contributed to a remarkable increase in cefotaxime resistance in combination with pbp2x Hu17 and pbp1a of strain Hu17 (pbp1a Hu17), accompanied by higher levels of expression of CiaR-regulated genes, documenting that ciaH232 responds to PBP1aHu17-mediated changes in cell wall synthesis. Most importantly, the proportion of branched peptides relative to the proportion of linear muropeptides increased in cells containing mosaic PBPs, suggesting an altered enzymatic activity of these proteins.

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Identification and Characterization of the Penicillin-Binding Protein 2a of Streptococcus pneumoniae and Its Possible Role in Resistance to β-Lactam Antibiotics

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.44.6.1745-1748.2000 ◽

2000 ◽

Vol 44 (6) ◽

pp. 1745-1748 ◽

Cited By ~ 20

Author(s):

Genshi Zhao ◽

Timothy I. Meier ◽

Joann Hoskins ◽

Kelly A. McAllister

Keyword(s):

Streptococcus Pneumoniae ◽

Binding Protein ◽

Penicillin Resistance ◽

Penicillin Binding Protein ◽

Insertional Mutant ◽

Identification And Characterization

ABSTRACT To further understand the role of penicillin-binding protein 2a (PBP 2a) of Streptococcus pneumoniae in penicillin resistance, we confirmed the identity of the protein as PBP 2a. The PBP 2a protein migrated electrophoretically to a position corresponding to that of PBP 2x, PBP 2a, and PBP 2b of S. pneumoniae and was absent in a pbp2ainsertional mutant of S. pneumoniae. We found that the affinities of PBP 2a for penicillins were lower than for cephalosporins and a carbapenem. When compared with other S. pneumoniae PBPs, PBP 2a exhibited lower affinities for β-lactam antibiotics, especially penicillins. Therefore, PBP 2a is a low-affinity PBP for β-lactam antibiotics in S. pneumoniae.

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Methylation of 23S rRNA Nucleotide G748 by RlmAIIMethyltransferase Renders Streptococcus pneumoniae Telithromycin Susceptible

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00164-13 ◽

2013 ◽

Vol 57 (8) ◽

pp. 3789-3796 ◽

Cited By ~ 11

Author(s):

Akiko Takaya ◽

Yoshiharu Sato ◽

Tatsuma Shoji ◽

Tomoko Yamamoto

Keyword(s):

Streptococcus Pneumoniae ◽

Stop Codon ◽

Regulatory Region ◽

Dimethyl Sulfate ◽

23S Rrna ◽

Gene Encoding ◽

Content Type ◽

Resistant Mutants ◽

High Level ◽

Domain V

ABSTRACTSeveral posttranscriptional modifications of bacterial rRNAs are important in determining antibiotic resistance or sensitivity. In all Gram-positive bacteria, dimethylation of nucleotide A2058, located in domain V of 23S rRNA, by the dimethyltransferase Erm(B) results in low susceptibility and resistance to telithromycin (TEL). However, this is insufficient to produce high-level resistance to TEL inStreptococcus pneumoniae. Inactivation of the methyltransferase RlmAII, which methylates the N-1 position of nucleotide G748, located in hairpin 35 of domain II of 23S rRNA, results in increased resistance to TEL inerm(B)-carryingS. pneumoniae. Sixteen TEL-resistant mutants (MICs, 16 to 32 μg/ml) were obtained from a clinically isolatedS. pneumoniaestrain showing low TEL susceptibility (MIC, 2 μg/ml), with mutation resulting in constitutive dimethylation of A2058 because of nucleotide differences in the regulatory region oferm(B) mRNA. Primer extension analysis showed that the degree of methylation at G748 in all TEL-resistant mutants was significantly reduced by a mutation in the gene encoding RlmAIIto create a stop codon or change an amino acid residue. Furthermore, RNA footprinting with dimethyl sulfate and a molecular modeling study suggested that methylation of G748 may contribute to the stable interaction of TEL with domain II of 23S rRNA, even after dimethylation of A2058 by Erm(B). This novel finding shows that methylation of G748 by RlmAIIrendersS. pneumoniaeTEL susceptible.

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Genomic Analyses of DNA Transformation and Penicillin Resistance in Streptococcus pneumoniae Clinical Isolates

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01311-13 ◽

2013 ◽

Vol 58 (3) ◽

pp. 1397-1403 ◽

Cited By ~ 12

Author(s):

Fereshteh Fani ◽

Philippe Leprohon ◽

George G. Zhanel ◽

Michel G. Bergeron ◽

Marc Ouellette

Keyword(s):

Streptococcus Pneumoniae ◽

Binding Proteins ◽

Resistance Mechanisms ◽

Clinical Isolates ◽

Penicillin Resistance ◽

Dna Transformation ◽

Content Type ◽

Penicillin Binding Proteins ◽

Genomic Analyses ◽

Genome Scale

ABSTRACTAlterations in penicillin-binding proteins, the target enzymes for β-lactam antibiotics, are recognized as primary penicillin resistance mechanisms inStreptococcus pneumoniae. Few studies have analyzed penicillin resistance at the genome scale, however, and we report the sequencing ofS. pneumoniaeR6 transformants generated while reconstructing the penicillin resistance phenotypes from three penicillin-resistant clinical isolates by serial genome transformation. The genome sequences of the three last-level transformants T2-18209, T5-1983, and T3-55938 revealed that 16.2 kb, 82.7kb, and 137.2 kb of their genomes had been replaced with 5, 20, and 37 recombinant sequence segments derived from their respective parental clinical isolates, documenting the extent of DNA transformation between strains. A role in penicillin resistance was confirmed for some of the mutations identified in the transformants. Several multiple recombination events were also found to have happened at single loci coding for penicillin-binding proteins (PBPs) that increase resistance. Sequencing of the transformants with MICs for penicillin similar to those of the parent clinical strains confirmed the importance of mosaic PBP2x, -2b, and -1a as a driving force in penicillin resistance. A role in resistance for mosaic PBP2a was also observed for two of the resistant clinical isolates.

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Penicillin- and Cephalosporin-Resistant Streptococcus pneumoniae: An Emerging Microbial Threat

PEDIATRICS ◽

10.1542/peds.93.3.500 ◽

1994 ◽

Vol 93 (3) ◽

pp. 500-503 ◽

Cited By ~ 1

Author(s):

Robert J. Leggiadro

Keyword(s):

Streptococcus Pneumoniae ◽

Pneumococcal Disease ◽

Inhibitory Concentration ◽

Penicillin Resistance ◽

Cell Wall Synthesis ◽

Penicillin Binding Proteins ◽

Clinical Challenge ◽

Recent Developments ◽

High Level ◽

Level Resistance

Recent reports from South Africa,1 Spain,2 Hungary,3 Texas,4-6 and Memphis7,8 document an increasing incidence of penicillin-resistant Streptococcus pneumoniae infections in children. The emergence of penicillin-resistant pneumococci that also demonstrate decreased susceptibility to extended-spectrum cephalosporins presents an even greater clinical challenge.6-9 This commentary reviews recent developments in the epidemiology, identification, and management of penicillin- and cephalosporin-resistant pneumococcal disease in children. Pneumococcal susceptibility to penicillin is defined as a minimal inhibitory concentration (MIC) <0.1 µg/mL. Intermediate (relative) penicillin resistance is defined as an MIC from 0.1 to 1.0 µg/mL and high-level resistance as an MIC >1.0 µ/mL. Pneumococcal penicillin resistance is mediated by alterations in penicillin-binding proteins involved in cell wall synthesis.10

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Long Persistence of a Streptococcus pneumoniae 23F Clone in a Cystic Fibrosis Patient

mSphere ◽

10.1128/msphere.00201-17 ◽

2017 ◽

Vol 2 (3) ◽

Cited By ~ 2

Author(s):

Martin Rieger ◽

Harald Mauch ◽

Regine Hakenbeck

Keyword(s):

Cystic Fibrosis ◽

Streptococcus Pneumoniae ◽

Gene Transfer ◽

Cystic Fibrosis Patient ◽

Penicillin Resistance ◽

Specific Gene ◽

Gene Encoding ◽

Content Type ◽

Long Period ◽

Genes Encoding

ABSTRACT Streptococcus pneumoniae is a common resident in the human nasopharynx. However, carriage can result in severe diseases due to a unique repertoire of pathogenicity factors that are rare in closely related commensal streptococci. We investigated a penicillin-resistant S. pneumoniae clone of serotype 23F isolated from a cystic fibrosis patient on multiple occasions over an unusually long period of over 3 years that was present without causing disease. Genome comparisons revealed an apparent nonfunctional pneumococcus-specific gene encoding a hyaluronidase, supporting the view that this enzyme adds to the virulence potential of the bacterium. The 23F clone harbored unique mosaic genes encoding penicillin resistance determinants, the product of horizontal gene transfer involving the commensal S. mitis as donor species. Sequences identical to one such mosaic gene were identified in an S. mitis strain from the same patient, suggesting that in this case S. pneumoniae played the role of donor. Streptococcus pneumoniae isolates of serotype 23F with intermediate penicillin resistance were recovered on seven occasions over a period of 37 months from a cystic fibrosis patient in Berlin. All isolates expressed the same multilocus sequence type (ST), ST10523. The genome sequences of the first and last isolates, D122 and D141, revealed the absence of two phage-related gene clusters compared to the genome of another ST10523 strain, D219, isolated earlier at a different place in Germany. Genomes of all three strains carried the same novel mosaic penicillin-binding protein (PBP) genes, pbp2x, pbp2b, and pbp1a; these genes were distinct from those of other penicillin-resistant S. pneumoniae strains except for pbp1a of a Romanian S. pneumoniae isolate. All PBPs contained mutations that have been associated with the penicillin resistance phenotype. Most interestingly, a mosaic block identical to an internal pbp2x sequence of ST10523 was present in pbp2x of Streptococcus mitis strain B93-4, which was isolated from the same patient. This suggests interspecies gene transfer from S. pneumoniae to S. mitis within the host. Nearly all genes expressing surface proteins, which represent major virulence factors of S. pneumoniae and are typical for this species, were present in the genome of ST10523. One exception was the hyaluronidase gene hlyA, which contained a 12-nucleotide deletion within the promoter region and an internal stop codon. The lack of a functional hyaluronidase might contribute to the ability to persist in the host for an unusually long period of time. IMPORTANCE Streptococcus pneumoniae is a common resident in the human nasopharynx. However, carriage can result in severe diseases due to a unique repertoire of pathogenicity factors that are rare in closely related commensal streptococci. We investigated a penicillin-resistant S. pneumoniae clone of serotype 23F isolated from a cystic fibrosis patient on multiple occasions over an unusually long period of over 3 years that was present without causing disease. Genome comparisons revealed an apparent nonfunctional pneumococcus-specific gene encoding a hyaluronidase, supporting the view that this enzyme adds to the virulence potential of the bacterium. The 23F clone harbored unique mosaic genes encoding penicillin resistance determinants, the product of horizontal gene transfer involving the commensal S. mitis as donor species. Sequences identical to one such mosaic gene were identified in an S. mitis strain from the same patient, suggesting that in this case S. pneumoniae played the role of donor.

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Microtiter Plate-Based Assay for Inhibitors of Penicillin-Binding Protein 2a from Methicillin-Resistant Staphylococcus aureus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01327-10 ◽

2011 ◽

Vol 55 (6) ◽

pp. 2783-2787 ◽

Cited By ~ 8

Author(s):

Sudheer Bobba ◽

V. K. Chaithanya Ponnaluri ◽

Mridul Mukherji ◽

William G. Gutheil

Keyword(s):

Staphylococcus Aureus ◽

Binding Protein ◽

Binding Constants ◽

Public Health Problem ◽

Microtiter Plate ◽

Penicillin Binding Protein ◽

Methicillin Resistant ◽

Content Type ◽

High Level

ABSTRACTPenicillin-binding protein 2a (PBP2a), the molecular determinant for high-level β-lactam resistance in methicillin-resistantStaphylococcus aureus(MRSA), is intrinsically resistant to most β-lactam antibiotics. The development and characterization of new inhibitors targeting PBP2a would benefit from an effective and convenient assay for inhibitor binding. This study was directed toward the development of a fluorescently detected β-lactam binding assay for PBP2a from MRSA. Biotinylated ampicillin and biotinylated cephalexin were tested as tagging reagents for fluorescence detection by using a streptavidin-horseradish peroxidase conjugate. Both bound surprisingly well to PBP2a, with binding constants of 1.6 ± 0.4 μM and 13.6 ± 0.8 μM, respectively. Two forms of the assay were developed, a one-step direct competition form of the assay and a two-step indirect competition form of the assay, and both forms of the assay gave comparable results. This assay was then used to characterize PBP2a binding to ceftobiprole, which gave results consistent with previous studies of ceftobiprole-PBP2a binding. This assay was also demonstrated for screening for PBP2a inhibitors by screening a set of 13 randomly selected β-lactams for PBP2a inhibition at 750 μM. Meropenem was observed to give substantial inhibition in this screen, and a follow-up titration experiment determined its apparentKito be 480 ± 70 μM. The availability of convenient and sensitive microtiter-plate based assays for the screening and characterization of PBP2a inhibitors is expected to facilitate the discovery and development of new PBP2a inhibitors for use in combating the serious public health problem posed by MRSA.

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Identification, Purification, and Characterization of Transpeptidase and Glycosyltransferase Domains of Streptococcus pneumoniae Penicillin-Binding Protein 1a

Journal of Bacteriology ◽

10.1128/jb.180.21.5652-5659.1998 ◽

1998 ◽

Vol 180 (21) ◽

pp. 5652-5659 ◽

Cited By ~ 39

Author(s):

Anne Marie Di Guilmi ◽

Nicolas Mouz ◽

Jean-Pierre Andrieu ◽

JoAnn Hoskins ◽

S. Richard Jaskunas ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Fusion Protein ◽

Limited Proteolysis ◽

Penicillin Binding Protein ◽

Penicillin Binding Proteins ◽

Substrate Analogues ◽

Extracellular Region ◽

Parent Protein ◽

Gst Fusion Protein

ABSTRACT Resistance to β-lactam antibiotics in Streptococcus pneumoniae is due to alteration of penicillin-binding proteins (PBPs). S. pneumoniae PBP 1a belongs to the class A high-molecular-mass PBPs, which harbor transpeptidase (TP) and glycosyltransferase (GT) activities. The GT active site represents a new potential target for the generation of novel nonpenicillin antibiotics. The 683-amino-acid extracellular region of PBP 1a (PBP 1a*) was expressed in Escherichia coli as a GST fusion protein. The GST-PBP 1a* soluble protein was purified, and its domain organization was revealed by limited proteolysis. A protease-resistant fragment spanning Ser 264 to Arg 653 exhibited a reactivity profile against both β-lactams and substrate analogues similar to that of the parent protein. This protein fragment represents the TP domain. The GT domain (Ser 37 to Lys 263) was expressed as a recombinant GST fusion protein. Protection by moenomycin of the GT domain against trypsin degradation was interpreted as an interaction between the GT domain and the moenomycin.

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Profiling of β-Lactam Selectivity for Penicillin-Binding Proteins in Streptococcus pneumoniae D39

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05142-14 ◽

2015 ◽

Vol 59 (6) ◽

pp. 3548-3555 ◽

Cited By ~ 44

Author(s):

Ozden Kocaoglu ◽

Ho-Ching T. Tsui ◽

Malcolm E. Winkler ◽

Erin E. Carlson

Keyword(s):

Streptococcus Pneumoniae ◽

Binding Proteins ◽

Morphological Changes ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Sublethal Concentration ◽

Live Cells ◽

Bacterial Cell Division ◽

Content Type ◽

Penicillin Binding Proteins

ABSTRACTSelective fluorescent β-lactam chemical probes enable the visualization of the transpeptidase activity of penicillin-binding proteins (PBPs) at different stages of bacterial cell division. To facilitate the development of new fluorescent probes for PBP imaging, we evaluated 20 commercially available β-lactams for selective PBP inhibition in an unencapsulated derivative of the D39 strain ofStreptococcus pneumoniae. Live cells were treated with β-lactam antibiotics at different concentrations and subsequently incubated with Bocillin FL (Boc-FL; fluorescent penicillin) to saturate uninhibited PBPs. Fluorophore-labeled PBPs were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorescence scanning. Among 20 compounds tested, carbapenems (doripenem and meropenem) were coselective for PBP1a, PBP2x, and PBP3, while six of the nine penicillin compounds were coselective for PBP2x and PBP3. In contrast, the seven cephalosporin compounds tested display variability in their PBP-binding profiles. Three cephalosporin compounds (cefoxitin, cephalexin, and cefsulodin) and the monobactam aztreonam exhibited selectivity for PBP3, while only cefuroxime (a cephalosporin) was selective for PBP2x. Treatment ofS. pneumoniaecultures with a sublethal concentration of cefuroxime that inhibited 60% of PBP2x activity and less than 20% of the activity of other PBPs resulted in formation of elongated cells. In contrast, treatment ofS. pneumoniaecultures with concentrations of aztreonam and cefoxitin that inhibited up to 70% of PBP3 activity and less than 30% of other PBPs resulted in no discernible morphological changes. Additionally, correlation of the MIC and IC50s for each PBP, with the exception of faropenem, amdinocillin (mecillinam), and 6-APA, suggests that pneumococcal growth inhibition is primarily due to the inhibition of PBP2x.

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Quintuplex PCR to Detect Antibiotic Resistance Genes in Streptococcus pneumoniae

Journal of Clinical and Health Sciences ◽

10.24191/jchs.v2i1.5861 ◽

2016 ◽

Vol 1 (2) ◽

pp. 22 ◽

Cited By ~ 2

Author(s):

Navindra Kumari Palanisamy ◽

Parasakthi Navaratnam ◽

Shamala Devi Sekaran

Keyword(s):

Antibiotic Resistance ◽

Streptococcus Pneumoniae ◽

Resistance Genes ◽

Bacterial Pathogen ◽

Antibiotic Resistance Genes ◽

Pcr Amplification ◽

Penicillin Resistance ◽

Penicillin Binding Proteins ◽

Efflux Mechanism ◽

Mic Value

Introduction: Streptococcus pneumoniae is an important bacterial pathogen, causing respiratory infection. Penicillin resistance in S. pneumoniae is associated with alterations in the penicillin binding proteins, while resistance to macrolides is conferred either by the modification of the ribosomal target site or efflux mechanism. This study aimed to characterize S. pneumoniae and its antibiotic resistance genes using 2 sets of multiplex PCRs. Methods: A quintuplex and triplex PCR was used to characterize the pbp1A, ermB, gyrA, ply, and the mefE genes. Fifty-eight penicillin sensitive strains (PSSP), 36 penicillin intermediate strains (PISP) and 26 penicillin resistance strains (PRSP) were used. Results: Alteration in pbp1A was only observed in PISP and PRSP strains, while PCR amplification of the ermB or mefE was observed only in strains with reduced susceptibility to erythromycin. The assay was found to be sensitive as simulated blood cultures showed the lowest level of detection to be 10cfu. Conclusions: As predicted, the assay was able to differentiate penicillin susceptible from the non-susceptible strains based on the detection of the pbp1A gene, which correlated with the MIC value of the strains.

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Increasing Ceftriaxone Resistance and Multiple Alterations of Penicillin-Binding Proteins among Penicillin-Resistant Streptococcus pneumoniae Isolates in Taiwan

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01563-06 ◽

2007 ◽

Vol 51 (9) ◽

pp. 3404-3406 ◽

Cited By ~ 20

Author(s):

Cheng-Hsun Chiu ◽

Lin-Hui Su ◽

Yhu-Chering Huang ◽

Jui-Chia Lai ◽

Hsiu-Ling Chen ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Amino Acid ◽

Binding Proteins ◽

Binding Protein ◽

Penicillin Binding Protein ◽

Penicillin Binding Proteins

ABSTRACT The rate of nonsusceptibility of penicillin-resistant Streptococcus pneumoniae strains to ceftriaxone increased significantly in Taiwan in 2005. Approximately 90% of the ceftriaxone-nonsusceptible isolates were found to be of four major serotypes (serotypes 6B, 14, 19F, and 23F). Seven amino acid alterations in the penicillin-binding protein 2B transpeptidase-encoding region specifically contributed to the resistance.

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