Detection Of Penicillin-binding Proteins Research Articles

PBP Isolation and DD-Carboxypeptidase Assay.

Penicillin-binding proteins (PBPs) share the namesake because of their ability to bind penicillin or any beta-lactam antibiotic. In other words, PBPs are the targets of β-lactam antibiotics that hold nearly 60% of the global antibiotic market. These enzymes catalyze the final stages of peptidoglycan (PG) biosynthesis by acting as transglycosylases and transpeptidases. PBPs are also involved in PG remodeling by catalyzing DD-carboxypeptidase (DD-CPase) and endopeptidase reactions. Though the cross-linking abilities of PBPs are well known, the process of remodeling is still unclear, thereby drawing attention toward the DD-CPase enzymes. Here, we describe the step-by-step procedures for isolation of the bacterial cell membrane and detection of PBPs in it, followed by the purification of PBPs (DD-CPases) by both ampicillin-affinity and nickel-nitrilotriacetic acid (Ni-NTA) chromatography. The protocols to determine the enzymatic efficiency are also elucidated. The assays are aimed to determine the kinetic parameters for the interaction of the PBP with BOCILLIN, to evaluate its acylation and deacylation rates, and with its peptide substrates, to assess its DD-CPase activity.

Transcriptional Modulation of Penicillin-Binding Protein 1b, Outer Membrane Protein P2 and Efflux Pump (AcrAB-TolC) during Heat Stress Is Correlated to Enhanced Bactericidal Action of Imipenem on Non-typeable Haemophilus influenzae.

Objective: The purpose of the present study was to investigate the penicillin binding proteins (PBPs), drug influx and efflux modulations during heat stress and their effects on the bactericidal action of imipenem on non-typeable Haemophilus influenzae (NTHi).Methods: The two NTHi clinical isolates (GE47 and GE88, imipenem MICs by E-test > 32 μg/mL) examined in this study were collected at Geneva University Hospitals. The imipenem killing activity was assessed after incubation of the NTHi strains at either 37 or 42°C for 3 h with increasing concentrations of imipenem. The detection of PBPs was carried out by Bocillin-FL. Global transcriptional changes were monitored by RNA-seq after pre-incubation of bacterial cells at either 37 or 42°C, and the expression levels of relevant target genes were confirmed by qRT-PCR.Results: Quantitation of NTHi viable cells after incubation with 0.25 μg/mL of imipenem for 3 h revealed more than a twofold decrease in GE47 and GE88 viable cells at 42°C as compared to 37°C. Transcriptome analysis showed that under heat stress conditions, there were 141 differentially expressed genes with a | log2(fold change)| > 1, including 67 up-regulated and 74 down-regulated genes. The expression levels of ponB (encoding PBP1b) and acrR (regulator of AcrAB-TolC efflux pump) were significantly increased at 42°C. In contrast, the transcript levels of ompP2 (encoding the outer membrane protein P2) and acrB gene (encoding AcrB) were significantly lower under heat stress condition.Conclusion: This study shows that the transcriptional modulation of ponB, ompP2, acrR, and acrB in the heat stress response is correlated to enhanced antimicrobial effects of imipenem on non-typeable H. influenzae.

Phenotypic occurrence of methicillin-resistant <i>Staphylococcus aureus</i> in camels slaughtered at Kano abattoir, Kano, Nigeria

To assess the occurrence of MRSA among camels in Kano abattoir, a total of 300 nasal swabs were collected from camels at the lairage in Kano abattoir, Kano state, Nigeria to isolate and biochemically characterize Staphylococcus aureus and confirm methicillin-resistant Staphylococcus aureus among isolates using oxacillin resistance screening agar basal medium, disc diffusion method, PCR and also through detection of penicillin binding protein 2’. Samples were collected into universal sample bottles containing trypticase soy broth with 6.5% Nacl. amples ere incubated at 37 o C for 24 hrs and sub-cultured on Baird Parker agar (Oxoid Ltd, Basingstoke, UK). Suspect Staphylococcus spp isolates were confirmed using coagulase, DNAse, haemolysis and sugar fermentation tests (mannitol, sucrose, lactose, mannose and xylose). Fifteen of the 42 isolated Staphylococcus aureus were confirmed to be MRSA on oxacillin resistance screening agar basal medium of which 12 were also resistant to oxacillin using disc diffusion method. Five (33.3%) of the 15 isolates were confirmed to be MRSA using the PBP2 latex agglutination test kit. The isolates were however negative for mec A by PCR. The prevalence of Staphylococcus aureus and MRSA was higher in males than in females, but this difference was found to be statistically insignificant (p > 0.05). Multidrug resistance was displayed by all Staphylococcus aureus isolates with 100% resistance to ampicillin and penicillin, but 97.6% of the isolates were susceptible to amikacin and 90% to ciprofloxacin and gentamicin. There was no statistically significant difference in antibiotic resistance between Staphylococcus aureus and MRSA to amikacin, ciprofloxacin, chloramphenicol, cloxacillin, erythromycin, gentamicin, penicillin, tetracycline, sulphamethoxazole, vancomycin ( p-value > 0.0), but there was statistical significance to oxacillin (p = 0.0001; OR = 0.7143). MRSA strains were found in 5% of camels and thus may play a potential role in disseminating the pathogen between animals and humans as well as within the community. Keywords: Camels, Kano abattoir, Occurrence, Methicillin resistant Staphylococcus aureus , Multidrug resistance

Titania nanotube-modified screen printed carbon electrodes enhance the sensitivity in the electrochemical detection of proteins

The use of titania nanotubes (TiO2-NT) as the working electrode provides a substantial improvement in the electrochemical detection of proteins. A biosensor designed using this strategy provided a robust method to detect protein samples at very low concentrations (Cprotein ca 1ng/μl). Reproducible measurements on protein samples at this concentration (Ip,a of 80+1.2μA) could be achieved using a sample volume of ca 30μl. We demonstrate the feasibility of this strategy for the accurate detection of penicillin binding protein, PBP2a, a marker for methicillin resistant Staphylococcus aureus (MRSA). The selectivity and efficiency of this sensor were also validated using other diverse protein preparations such as a recombinant protein tyrosine phosphatase (PTP10D) and bovine serum albumin (BSA). This electrochemical method also presents a substantial improvement in the time taken (few minutes) when compared to conventional enzyme-linked immunosorbent assay (ELISA) protocols. It is envisaged that this sensor could substantially aid in the rapid diagnosis of bacterial infections in resource strapped environments.

Discrimination of methicillin-resistant Staphylococcus aureus from methicillin-susceptible Staphylococcus aureus or coagulase-negative staphylococci by detection of penicillin-binding protein 2 and penicillin-binding protein 2′ using a bioluminescent enzyme immunoassay

For the discrimination of methicillin-resistant S. aureus (MRSA) from methicillin-susceptible S. aureus (MSSA) or coagulase-negative staphylococci (CNS), we developed a bioluminescent enzyme immunoassay (BLEIA) for detecting penicillin-binding protein 2 (PBP2) and penicillin-binding protein 2′ (PBP2′) using biotinylated firefly luciferase. The BLEIA was able to detect recombinant PBP2 at 50pg/ml and recombinant PBP2′ at 500pg/ml. PBP2 and PBP2′ present in the membranes of S. aureus were extracted by acid and detergent treatment. The method was able to detect PBP2 or PBP2′ extracted from 106 colony forming units of S. aureus because of efficient extraction and the high sensitivity of luciferase. In a study of clinical isolates previously characterized as either MRSA or MSSA by antibiotic susceptibility testing, all 34 specimens identified as MRSA were both PBP2 and PBP2′ positive. The 34 MSSA specimens were PBP2 positive and PBP2′ negative. Moreover, the BLEIA could detect PBP2′ extracted from four species of methicillin-resistant CNS, but not PBP2 extracted from four species of methicillin-resistant and methicillin-susceptible CNS. This result suggested that PBP2 could be a unique marker for discrimination of S. aureus from CNS. A BLEIA that is able to detect PBP2 and PBP2′ may be useful in clinical diagnostics.

Fluorescent Bocillins: synthesis and application in the detection of penicillin-binding proteins.

Novel fluorescent analogs of penicillin V were synthesized and evaluated for efficacy in the detection of penicillin binding proteins (PBPs). These molecules include the full structure of penicillin V, with the potent Bodipy fluorophore attached to the para-position of the penicillin V phenyl group. The green fluorescent Bocillin FL and the near-infrared (IR) fluorescent Bocillin 650/665 probes were shown to bind to PBPs, both purified and from membrane preparations, with high affinity and specificity. These reagents allow for facile detection of 2-4 ng of purified PBP with the aid of a fluorescent scanner.

Detection of Penicillin-Binding Proteins in Bacillus Cereus by Using Biotinylated β-Lactams

A !lew rnethod for detection or penicillin hinding proteim: from bacterial membranes has been developed in t.his study. The method, that ernployed biotin (llllpicillin conjugate (Bin-peA), is v('z;,' rapid and can be a significant alternative of hazardous and lillie commrnirnng convenLional radiometric method for detection of PBPs in cell membranes. PRPs from R cereus were !examined by this LCcIUli411C. In vegetative and sporulating cells, 8 major PBI's were rietertr'd. Comparing \villl standard marker proteiIls, these PBPs were estimated for their molecular \veight as 106, 83, 75, 72, ();3, 4G, 40, (I.llel :32 kDa. Affinity of cephnlcxin, ecfoxitin, and cefotaxime to PBPs ,vas measured indirectly by competition for subsequent binding orRio-PCA. PI3Ps 2, :3, 4, and 7 ' .... ere decreased or disappeared in the elect.rophoregnlTll by prpbimiillg \\'ith these ;3 -lactams. TwCl PEPs, PDP ;3 and PUP 4, ... vhich Viere predominant in vegetat.ive and sporularing cells, r(>sl'ect.iv(>ly, showed strong rrffinily for ceplmlexin.

Characterisation of a monoclonal antibody and its use in the immunoaffinity purification of penicillin-binding protein 2' of methicillin-resistant Staphylococcus aureus.

The additional penicillin-binding protein (PBP) 2' that is important in determining intrinsic resistance in methicillin-resistant strains of Staphylococcus aureus (MRSA) has been purified by affinity chromatography using monoclonal antibodies. Monoclonal antibody 1/423.10.351 reacted in ELISA with detergent extracts of membranes from resistant organisms, but not in immunoblots with PBP 2' separated by SDS-PAGE. Immunoprecipitation experiments showed that antibody 1/423.10.351 reacted with PBP 2' in detergent extracts. The latter antibody, covalently coupled to protein A-Sepharose through the Fc region, served as an affinity matrix to purify PBP 2'. The PBP was detected in immunoblots using a second monoclonal antibody, 2/401.43. Conjugation of this antibody with alkaline phosphatase afforded more rapid detection of PBP 2' for the immunological detection of PBP 2' both in affinity-purified fractions and in resistant strains.

Immunological detection of penicillin-binding protein 2′ in clinical isolates of methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis

Immunological detection of penicillin-binding protein 2′ in clinical isolates of methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis

Immunological detection of penicillin-binding protein 2′ in clinical isolates of methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis

The additional penicillin-binding protein (PBP 2') that is important in determining intrinsic resistance in methicillin-resistant strains of Staphylococcus aureus (MRSA) has been detected immunologically in strains from a variety of world-wide locations. This additional protein has also been definitively identified both immunologically and as a PBP in methicillin-resistant strains of S. epidermidis (MRSE). The assay described is rapid, specific and sensitive and has been used to detect PBP 2' in S. haemolyticus but not in beta-lactam resistant Streptococci.

Fractionation of the Providencia stuartii cell envelope.

Cell envelopes (i.e. unfractionated inner and outer membranes) were obtained from Providencia stuartii by following procedures previously applied to the isolation of envelopes from Escherichia coli. The P. stuartii envelopes contained known inner membrane enzymes that included a variety of dehydrogenases and ATPase. The catalytic activity of the ATPase depended upon the concentration of magnesium ions, the substrate (ATP) level and the ratio of magnesium ions to ATP. Cell envelopes from P. stuartii were further fractionated to recover inner and outer membrane polypeptides by treatment with the detergent Sarkosyl. Proteins from the periplasmic region were recovered by a simple osmotic shock procedure also previously applied to E. coli. The purity of the various P. stuartii cell envelope fractions was assessed by a combination of techniques that included one- and two-dimensional gel electrophoresis of proteins, enzyme assays and detection of penicillin-binding proteins.