scholarly journals Highly Variable Penicillin Resistance Determinants PBP 2x, PBP 2b, and PBP 1a in Isolates of Two Streptococcus pneumoniae Clonal Groups, Poland23F-16 and Poland6B-20

2007 ◽  
Vol 52 (3) ◽  
pp. 1021-1027 ◽  
Author(s):  
Radosław Izdebski ◽  
Jens Rutschmann ◽  
Janusz Fiett ◽  
Ewa Sadowy ◽  
Marek Gniadkowski ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Reference Strain ◽  
Pattern Analysis ◽  
Profile Analysis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Reactivity Pattern ◽  
Multiple Gene ◽  
Penicillin Binding Proteins ◽  
Resistance Determinants

ABSTRACT Penicillin-binding proteins (PBPs) in representatives of two Streptococcus pneumoniae clonal groups that are prevalent in Poland, Poland23F-16 and Poland6B-20, were investigated by PBP profile analysis, antibody reactivity pattern analysis, and DNA sequence analysis of the transpeptidase (TP) domain-encoding regions of the pbp2x, pbp2b, and pbp1a genes. The isolates differed in their MICs of β-lactam antibiotics. The majority of the 6B isolates were intermediately susceptible to penicillin (penicillin MICs, 0.12 to 0.5 μg/ml), whereas all 23F isolates were penicillin resistant (MICs, ≥2 μg/ml). The 6B isolates investigated had the same sequence type (ST), determined by multilocus sequence typing, as the Poland6B-20 reference strain (ST315), but in the 23F group, isolates with three distinct single-locus variants (SLVs) in the ddl gene (ST173, ST272, and ST1506) were included. None of the isolates showed an identical PBP profile after labeling with Bocillin FL and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and only one pair of 6B isolates and one pair of 23F isolates (ST173 and ST272) each contained an identical combination of PBP 2x, PBP 2b, and PBP 1a TP domains. Some 23F isolates contained PBP 3 with an apparently higher electrophoretic mobility, and this feature also did not correlate with their STs. The data document a highly variable pool of PBP genes as a result of multiple gene transfer and recombination events within and between different clonal groups.

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

2015 ◽  
Vol 59 (6) ◽  
pp. 3548-3555 ◽  
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.

Isolation, Cultivation, and Characterization of Borrelia burgdorferi from Rodents and Ticks in the Charleston Area of South Carolina

2000 ◽  
Vol 38 (1) ◽  
pp. 120-124
Author(s):  
J. H. Oliver ◽  
K. L. Clark ◽  
F. W. Chandler ◽  
L. Tao ◽  
A. M. James ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Borrelia Burgdorferi ◽  
Reference Strain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Western Blots ◽  
Cotton Rat ◽  
Cotton Mouse ◽  
B 31

ABSTRACT Twenty-eight Borrelia burgdorferi isolates from the Charleston, S.C., area are described. This represents the first report and characterization of the Lyme disease spirochete from that state. The isolates were obtained from December 1994 through December 1995 from the tick Ixodes scapularis , collected from vegetation, and from the rodents Peromyscus gossypinus (cotton mouse), Neotoma floridana (eastern wood rat), and Sigmodon hispidus (cotton rat). All isolates were screened immunologically by indirect immunofluorescence with monoclonal antibodies to B. burgdorferi -specific outer surface protein A (OspA) (antibodies H5332 and H3TS) and B. burgdorferi -specific OspB (antibodies H6831 and H614), a Borrelia (genus)-specific antiflagellin antibody (H9724), Borrelia hermsii -specific antibodies (H9826 and H4825), and two polyclonal antibodies (one to Borrelia species and another to B. burgdorferi ). Six of the isolates were analyzed by exposing Western blots to monoclonal antibodies H5332, H3TS, H6831, and H9724. All isolates were also analyzed by PCR with five pairs of primers known to amplify selected DNA target sequences specifically reported to be present in the reference strain, B. burgdorferi B-31. The protein profiles of six of the isolates (two from ticks, one from a cotton mouse, two from wood rats, and one from a cotton rat) also were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We conclude that the 28 Charleston isolates are B. burgdorferi sensu stricto based on their similarities to the B. burgdorferi B-31 reference strain.

scholarly journals Genome analysis of a MDR Streptococcus pneumoniae 23F serotype causing meningoencephalitis in a 10-months refugee infant

2018 ◽  
Vol 12 (03) ◽  
pp. 196-203
Author(s):  
Tamara Salloum ◽  
Elie Tannous ◽  
Samar Merheb-Ghoussoub ◽  
Elie Ghoussoub ◽  
Sima Tokajian
Keyword(s):  
Streptococcus Pneumoniae ◽  
Virulence Factors ◽  
Genome Analysis ◽  
Reference Strain ◽  
Clinical Diagnostics ◽  
Penicillin Binding Proteins ◽  
Multiple Components ◽  
Genes Encoding ◽  
Resistant Strains ◽  
Reference Strains

Purpose: Streptococcus pneumoniae is an important human pathogen causing invasive pneumococcal diseases (IPD). The re-emergence of eradicated S. pneumoniae-associated meningoencephalitis in Lebanon is a major point of concern. Methods: We aimed at conducting a comparative genome analysis of a multi-drug resistant S. pneumoniae, LAU-23F, linked to meningoencephalitis and fatality in a 10-months Syrian refugee infant in Lebanon, and 24 related publically available genome sequences. Serotype, capsular genes, MLST, SNPs, phylogenetic relatedness and repertoire of resistance genes were investigated. Genes encoding penicillin binding proteins (PBPs) were examined for mosaicity. Virulence factors were screened for SNPs as compared to reference strains. Results: The isolate belonged to ST-277 and was of serotype 23F. It showed an intermediate resistance to ciprofloxacin, cefuroxime and penicillin and carried multiple components of different efflux pumps. Gene mosaicity was observed in pbp2x, it was also distinct from other penicillin-resistant strains; pbp1a and pbp2b appeared to be conserved between LAU-23F and the reference strain SP49. The arrangement of capsular gene loci was similar to ATCC 700669 though polymorphism was detected in the cpsABCD region, believed to be conserved among different Streptococcus species. Amplitude of virulence factors was detected showing varying degrees of conservation compared to reference strains. Observed zones of high heterogeneity were associated with phage encoded regions. Conclusions: The fine levels of diversity throughout the genome could account for the pronounced invasiveness of this isolate. The genomics-based methods used support the importance of implementing WGS in routine clinical diagnostics and surveillance of streptococcal diseases.

scholarly journals Numerical analysis of SDS–PAGE protein patterns ofSerratia marcescens: a comparison with other typing methods

1990 ◽  
Vol 105 (1) ◽  
pp. 107-117 ◽  
Author(s):  
B. Holmes ◽  
M. Costas ◽  
L. L. Sloss
Keyword(s):  
Numerical Analysis ◽  
Reference Strain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Clinical Isolates ◽  
Protein Patterns ◽  
Sds Page ◽  
Typing Methods ◽  
Effective Adjunct ◽  
Reference Strains

SUMMARYTwenty-five cultures comprising 18 clinical isolates ofSerratia marcescensfrom two hospitals, the type strain ofS. marcescens, two reference strains ofS. marinorubra, the type or a reference strain of three other Serratia species and a reference strain of undetermined species, were characterized by one-dimensional sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) of whole-cell proteins. The protein patterns were highly reproducible and were used as the basis of a numerical analysis which divided the clinical isolates into eight protein types. Comparison with O-serotyping indicated that the level of discrimination by SDS–PAGE was similar. As with O-serotyping, a secondary scheme, such as phage typing, is necessary to differentiate strains of the same protein type. We conclude that high-resolution SDS–PAGE of proteins provides an effective adjunct to other methods for typing isolates ofS. marcescens.

scholarly journals Mutation of luxS of Streptococcus pneumoniae Affects Virulence in a Mouse Model

2003 ◽  
Vol 71 (6) ◽  
pp. 3206-3212 ◽  
Author(s):  
Uwe H. Stroeher ◽  
Adrienne W. Paton ◽  
A. David Ogunniyi ◽  
James C. Paton
Keyword(s):  
Streptococcus Pneumoniae ◽  
Mouse Model ◽  
Bacterial Species ◽  
Systemic Disease ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Wild Type ◽  
Pneumococcal Infections ◽  
Wide Range

ABSTRACT The LuxS protein is required for the biosynthesis of the type 2 autoinducer (AI-2), which is involved in quorum sensing in a wide range of bacterial species. We have determined the effects of a defined luxS mutation on the virulence of Streptococcus pneumoniae. Although the luxS mutant displayed reduced virulence relative to its wild-type parent, the type 2 strain D39, it was by no means avirulent in a mouse model. After intranasal administration, the luxS mutant was able to colonize the nasopharynx of the mouse as efficiently as the wild type. However, it was less able to spread from the nasopharynx to the lungs or the blood. Intraperitoneal coadministration studies indicated that the luxS mutant was less fit and was readily outcompeted by wild-type D39. However, when administered on its own by this route, the mutant was able to proliferate and cause fatal systemic disease, albeit at a lower rate than the wild type. Western blot analysis of whole-cell lysates of the mutant and its parent did not reveal any differences in the levels of several well-characterized virulence proteins. However, analysis of Coomassie blue-stained protein profiles after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that mutation of luxS had pleiotropic effects on protein expression in all cellular compartments. This is consistent with the product of luxS having a regulatory role in S. pneumoniae. This is the first report of a direct role for luxS (and by inference, AI-2) in the virulence of a gram-positive pathogen. However, the fact that mutagenesis of luxS does not completely attenuate S. pneumoniae has implications for the possible use of AI-2 antagonists for treatment of pneumococcal infections.

scholarly journals Protein and Antigenic Profile among Mycoplasma bovis Field Strains Isolated in Bosnia and Herzegovina

2009 ◽  
Vol 78 (1) ◽  
pp. 151-154 ◽  
Author(s):  
Maid Rifatbegović ◽  
Patrícia Assunção ◽  
Šemso Pašić ◽  
Christian de la Fe ◽  
Jose B. Poveda
Keyword(s):  
Bosnia And Herzegovina ◽  
Gel Electrophoresis ◽  
Reference Strain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Geographic Region ◽  
Mycoplasma Bovis ◽  
Nasal Swabs ◽  
Geographic Regions ◽  
Antigenic Profile

Mycoplasma bovis is a serious, worldwide-spread but often overlooked pathogen causing respiratory disease, mastitis, and arthritis in cattle. In this study we characterize the protein and antigenic profiles of M. bovis field strains isolated in Bosnia and Herzegovina by sodium dodecyl sulphate polyacrylamide gel electrophoresis and immunoblotting, and analyze possible variations among these strains. Greater differences occurred when comparing field strains with the reference strain PG45. One field strain isolated from lung samples of a heifer was markedly different from strains isolated from nasal swabs taken from cattle raised in another geographic region. A possible correlation may exist between protein and antigen profiles of M. bovis field strains, geographic regions and anatomical sites of isolation.

scholarly journals Profiling of β-Lactam Selectivity for Penicillin-Binding Proteins in Escherichia coli Strain DC2

2015 ◽  
Vol 59 (5) ◽  
pp. 2785-2790 ◽  
Author(s):  
Ozden Kocaoglu ◽  
Erin E. Carlson
Keyword(s):  
Escherichia Coli ◽  
Binding Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Coli Strain ◽  
Bacterial Cell Division ◽  
Content Type ◽  
Penicillin Binding Proteins ◽  
Whole Cells ◽  
E Coli

ABSTRACTPenicillin-binding proteins (PBPs) are integral players in bacterial cell division, and their catalytic activities can be monitored with β-lactam-containing chemical probes. Compounds that target a single PBP could provide important information about the specific role(s) of each enzyme, making identification of such molecules important. We evaluated 22 commercially available β-lactams for inhibition of the PBPs in liveEscherichia colistrain DC2. Whole cells were titrated with β-lactam antibiotics and subsequently incubated with a fluorescent penicillin derivative, Bocillin-FL (Boc-FL), to label uninhibited PBPs. Protein visualization was accomplished by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation and fluorescent scanning. The examined β-lactams exhibited diverse PBP selectivities, with amdinocillin (mecillinam) showing selectivity for PBP2, aztreonam, piperacillin, cefuroxime, cefotaxime, and ceftriaxone for PBP3, and amoxicillin and cephalexin for PBP4. The remaining β-lactams did not block any PBPs in the DC2 strain ofE. colior inhibited more than one PBP at all examined concentrations in this Gram-negative organism.

Electrophoretic analysis of Geoffroea (Leguminosae, Papilionoideae): taxonomic inferences in Argentinean populations

2009 ◽  
Vol 22 (2) ◽  
pp. 137
Author(s):  
Alicia L. Lamarque ◽  
Diana O. Labuckas ◽  
Julián Greppi ◽  
Renée H. Fortunato
Keyword(s):  
Profile Analysis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Taxonomic Status ◽  
Electrophoretic Analysis ◽  
Dry Forest ◽  
Protein Patterns ◽  
Phenotypic Differences ◽  
Forest Flora ◽  
Sds Page

The genus Geoffroea Jacq. was circumscribed to two species without varietal division. Nevertheless, in the taxonomic treatment of Argentinean dry-forest flora, on the basis of habit and foliar and floral features, it was possible to distinguish G. decorticans (Hook. & Arn.) Burkart var. decorticans from var. subtropicalis (Lillo) Burkart and to recognise intraspecific variation among the populations of G. spinosa. The purposes of the present study were to provide seed-protein data of Geoffroea species, and to analyse the relationships among them. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) was carried out on mature seeds harvested at different locations. Electrophoretic profile analysis showed outstanding differences between G. decorticans var. decorticans and var. subtropicalis and supported the view that these two taxa are less closely related than previously assumed, warranting their recognition at the varietal level. Moreover, attentive to the differences in the protein patterns from the analysed population of G. spinosa (Argentina: Chaco and Salta provinces), in addition to phenotypic differences, materials from other disjunct areas where this species grows need to be studied to verify their taxonomic status.

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