Rapid Detection of PBP2a in Staphylococci from Shortly Incubated Subcultures of Positive Blood Cultures by an Immunochromatographic Assay.

Staphylococcus aureus, as well as coagulase-negative staphylococci (CoNS), can cause a wide range of human infections both in nosocomial and community settings. Βeta-lactams are the antibiotics of choice for the treatment of bloodstream infections (BSI) caused by these microorganisms. Resistance to virtually all ß-lactams (also referred to as methicillin resistance) primarily results from the production of an alternative penicillin-binding protein (PBP2a) encoded by the mecA gene. While ß-lactams are still used as first-line therapy against BSI caused by S. aureus, BSI with CoNS are usually treated with vancomycin due to the high prevalence of methicillin resistance. Rapid detection of methicillin resistance is thus critical for continuation or adjustment of the empirical therapy and therewith to improve the clinical outcome of the patients. The revised version of the immunochromatographic assay PBP2a SA culture colony test (SACCT) is a rapid, inexpensive, and easy method that enables reliable detection of PBP2a in mecA-positive staphylococcal isolates after18 to 24 h of incubation. Here, we evaluated the diagnostic performance of the SACCT using primary subcultures of spiked blood cultures after short incubation (4 to 6 h) and established a modified procedure with an equal analytical performance to that of longer-grown cultures. With the proposed method the SACCT can be employed for PBP2a detection from shortly incubated subcultures of clinically relevant staphylococcal isolates, thereby allowing more rapid and effective management of BSI caused by these organisms. IMPORTANCE Antibiotic resistance poses a major threat to health and incurs high economic costs worldwide. Rapid detection of resistance mechanisms can contribute to improving patient care and preventing the dissemination of antimicrobial resistance. Here, we describe a rapid method to detect the most important beta-lactam resistance mechanism (the plasmid-encoded alternative transpeptidase PBP2a) in staphylococcal isolates causing BSI. We show that, using a modified procedure, PBP2a can be reliably detected from primary subcultures of spiked blood cultures after short incubation (4 to 6 h) with a rapid, inexpensive, and simple immunochromatographic test (SACCT). We provide an accurate, inexpensive, and rapid method to facilitate appropriate management and control of infections in patients suffering from invasive staphylococcal infections.

Molecular detection of methicillin heat-resistant Staphylococcus aureus strains in pasteurized camel milk in Saudi Arabia.

Antibiotic- and heat-resistant bacteria in camel milk is a potential public health problem. Staphylococcus aureus (S. aureus) is an opportunistic pathogen in humans, dairy cattle and camels. We characterized the phenotype and genotype of methicillin-resistant staphylococcal strains recovered from pasteurized and raw camel milk (as control) distributed in the retail markets of Saudi Arabia. Of the 100 samples assessed between March and May 2016, 20 S. aureus isolates were recovered from pasteurized milk, 10 of which were resistant to cefoxitin, and as such, were methicillin-resistant. However, raw camel milk did not contain methicillin-resistant S. aureus (MRSA). Antimicrobial susceptibility tests showed that the resistance ratio for other antibiotics was 60%. We performed a polymerase chain reaction (PCR) assay using primers for the methicillin-resistant gene mecA and nucleotide sequencing to detect and verify the methicillin-resistant strains. Basic local alignment search tool (BLAST) analysis of the gene sequences showed a 96-100% similarity between the resistant isolates and the S. aureus CS100 strain's mecA gene. Ten of the methicillin-resistant isolates were heat-resistant and were stable at temperatures up to 85°C for 60 s, and three of these were resistant at 90°C for 60 or 90 s. The mean decimal reduction time (D85-value) was 111 s for the ten isolates. Sodium dodecyl sulfate (SDS)/polyacrylamide gel electrophoresis (PAGE) showed that there was no difference in the total protein profiles for the ten methicillin heat-resistant S. aureus (MHRSA) isolates and for S. aureus ATCC 29737. In conclusion, a relatively high percentage of the tested pasteurized camel milk samples contained S. aureus (20%) and MHRSA (10%).

Detection and characterization of bacterial polysaccharides in drug-resistant enterococci.

Enterococcus faecium (E. faecium) has emerged as one of today's leading causes of health care-associated infections that is difficult to treat with the available antibiotics. These pathogens produce capsular polysaccharides on the cell surface which play a significant role in adhesion, virulence and evasion. Therefore, we aimed at the identification and characterization of bacterial polysaccharide antigens which are central for the development of vaccine-based prophylactic approaches. The crude cell wall-associated polysaccharides from E. faecium, its mutant and complemented strains were purified and analyzed by a primary antibody raised against lipoteichoic acid (LTA) and diheteroglycan (DHG). The resistant E. faecium strains presumably possess novel capsular polysaccharides that allow them to avoid the evasion from opsonic killing. The E. faecium U0317 strain was very well opsonized by anti-U0317 (~95%), an antibody against the whole bacterial cell. The deletion mutant showed a significantly increased susceptibility to opsonophagocytic killing (90-95%) against the penicillin binding protein (anti-PBP-5). By comparison, in a mouse urinary tract and rat endocarditis infection model, respectively, there were no significant differences in virulence. In this study we explored the biological role of the capsule of E. faecium. Our findings showed that the U0317 strain is not only sensitive to anti-LTA but also to antibodies against other enterococcal surface proteins. Our findings demonstrate that polysaccharides capsule mediated-resistance to opsonophagocytosis. We also found that the capsular polysaccharides do not play an important role in bacterial virulence in urinary tract and infective endocarditis in vivo models.

Structural Basis for E. coli Penicillin Binding Protein (PBP) 2 Inhibition, a Platform for Drug Design.

Penicillin-binding proteins (PBPs) are the targets of the ß-lactams, the most successful class of antibiotics ever developed against bacterial infections. Unfortunately, the worldwide and rapid spread of large spectrum ß-lactam resistance genes such as carbapenemases is detrimental to the use of antibiotics in this class. New potent PBP inhibitors are needed, especially compounds that resist ß-lactamase hydrolysis. Here we describe the structure of the E. coli PBP2 in its Apo form and upon its reaction with 2 diazabicyclo derivatives, avibactam and CPD4, a new potent PBP2 inhibitor. Examination of these structures shows that unlike avibactam, CPD4 can perform a hydrophobic stacking on Trp370 in the active site of E. coli PBP2. This result, together with sequence analysis, homology modeling, and SAR, allows us to propose CPD4 as potential starting scaffold to develop molecules active against a broad range of bacterial species at the top of the WHO priority list.

Chemiresistive DNA hybridization sensor with electrospun nanofibers: A method to minimize inter-device variability.

Chemiresistive platforms are best suited for developing DNA hybridization detection systems, owing to their ease of fabrication, simple detection methodology and amenability towards electronics. In this work, we report development of a generic, robust, electrospun nanofiber based interdigitated chemiresistive platform for DNA hybridization detection. The platform comprises of interdigitated metal electrodes decorated with electrospun nanofibers on the top. Two approaches viz., drop casting of graphene doped Mn2O3 nanofibers (GMnO) and direct electrospinning of polyaniline/polyethylene oxide (PANi/PEO) composite nanofibers, have been utilized to decorate these electrodes. In both approaches, inter-device variability, a key challenge for converting this proof-of-concept into a tangible prototype/product, has been addressed using a shadow masking technique. Consequently, the relative standard deviation for multiple PANi/PEO nanofiber based chemiresistors has been brought down from 17.82% (without shadow masking) to 4.41% (with shadow masking). The nanofibers are further modified with single-stranded probe DNAs, to capture a desired hybridization event. To establish the generic nature of the platform, detection of multiple target DNAs has been successfully demonstrated. These targets include dengue virus specific consensus primer (DENVCP) and four DNAs corresponding to Staphylococcus aureus specific genes, namely nuc, mecA, vanA and protein A. The chemiresistive detection of DENVCP has been performed in the concentration range of 10 fM - 1 µM, whereas the detection of the other targets has been carried out in the range of 1 pM - 1 µM. Using a 3σ method, we have estimated the limit of detection for the chemiresistive detection of DENVCP to be 1.9 fM.

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 ß-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.

The cell cycle regulator GpsB functions as cytosolic adaptor for multiple cell wall enzymes.

Bacterial growth and cell division requires precise spatiotemporal regulation of the synthesis and remodelling of the peptidoglycan layer that surrounds the cytoplasmic membrane. GpsB is a cytosolic protein that affects cell wall synthesis by binding cytoplasmic mini-domains of peptidoglycan synthases to ensure their correct subcellular localisation. Here, we describe critical structural features for the interaction of GpsB with peptidoglycan synthases from three bacterial species (Bacillus subtilis, Listeria monocytogenes and Streptococcus pneumoniae) and suggest their importance for cell wall growth and viability in L. monocytogenes and S. pneumoniae. We use these structural motifs to identify novel partners of GpsB in B. subtilis and extend the members of the GpsB interactome in all three bacterial species. Our results support that GpsB functions as an adaptor protein that mediates the interaction between membrane proteins, scaffolding proteins, signalling proteins and enzymes to generate larger protein complexes at specific sites in a bacterial cell cycle-dependent manner.

Prevalence of mecA: Genotyping screening of community acquired-MRSA isolates in Karachi, Pakistan.

Among resistant nosocomial and community pathogens, MRSA has become the most serious pathogen, causing life threatening infections worldwide. In S.aureus, quick and exact recognition of methicillin (cefoxitin) resistance has become essential. The benchmark for MRSA identification among S.aureus is the detection of the mecA gene that causes the expression of protein (PBP2a) culpable for classic ß-lactam resistance. However, the utter reliance on amplification of mecA gene as a hallmark in confirmation of methicillin (cefoxitin) resistant S. aureus is the matter of distrust by some investigators. The current investigation designed to analyse the prevalence of mecA gene among phenotypically positive MRSA isolates using molecular method and to correlate its prevalence to conventional techniques. Furthermore, antimicrobial sensitivity of mecA positive staphylococci was determined by Kirby Baeuer method. For this purpose, 201 clinical staphylococcal specimens were recovered from various diagnostic laboratories in Karachi City, Pakistan. Phenotypic existence of methicillin resistance in S. aureus was observed to be 51.7%. In contrast, when organisms were subjected for amplification of mecA gene by PCR, mecA positive isolates were 36/104 (35%) MRSA isolates. Current work raise question towards the usefulness of molecular identification of mecA gene in confirmation of methicillin resistance without correlating with conventional methods. Therefore, it is essential to consider the other possible resistance mechanisms for ß-lactams that may interact with mecA gene in the development of methicillin resistance mechanism in Staphylococcus.

The structures of penicillin-binding protein 4 (PBP4) and PBP5 from Enterococci provide structural insights into ß-lactam resistance.

The final steps of cell-wall biosynthesis in bacteria are carried out by penicillin-binding proteins (PBPs), whose transpeptidase domains form the cross-links in peptidoglycan chains that define the bacterial cell wall. These enzymes are the targets of ß-lactam antibiotics, as their inhibition reduces the structural integrity of the cell wall. Bacterial resistance to antibiotics is a rapidly growing concern; however, the structural underpinnings of PBP-derived antibiotic resistance are poorly understood. PBP4 and PBP5 are low-affinity, class B transpeptidases that confer antibiotic resistance to Enterococcus faecalis and Enterococcus faecium, respectively. Here, we report the crystal structures of PBP4 (1.8 Å) and PBP5 (2.7 Å) in their apo and acyl-enzyme complexes with the ß-lactams benzylpenicillin, imipenem, and ceftaroline. We found that, although these three ß-lactams adopt geometries similar to those observed in other class B PBP structures, there are small, but significant, differences that likely decrease antibiotic efficacy. Further, we also discovered that the N-terminal domain extensions in this class of PBPs undergo large rigid-body rotations without impacting the structure of the catalytic transpeptidase domain. Together, our findings are defining the subtle functional and structural differences in the Enterococcus PBPs that allow them to support transpeptidase activity while also conferring bacterial resistance to antibiotics that function as substrate mimics.

Resistance to Aminoglycosides of Methicillin-Resistant Strains of Staphylococcus aureus, Originating in the Surgical and Transplantation Wards of the Warsaw Clinical Center-A Retrospective Analysis.

INTRODUCTION: Aminoglycoside resistance (AR) is common in health care-associated methicillin-resistant Staphylococcus aureus (HA-MRSA). AR is most often associated with the production of antibiotic modifying enzymes: bidomain AAC(6')-Ie/APH(2″)-Ia acetyltransferase and phosphotransferase, ANT(4')-Ia nucleotidyltransferase, and APH(3″)-IIIa phosphotransferase. AIM: Determination of aminoglycoside sensitivity, presence of genes encoding enzymes, and molecular typing of HA-MRSA strains derived from patients hospitalized in surgical and transplantation wards. MATERIALS AND METHODS: Fifty-four HA-MRSA strains, isolated from various materials from patients in the surgical and transplantation wards of Warsaw's clinical hospital, hospitalized between 1991 and 2007. The MIC values of gentamicin-GEN/tobramycin-TOB/amikacin-AK/netilmicin-NET were determined by the E-test (CLSI/EUCAST). Genes mecA/aacA-aphD/aadD/aph(3″)-IIIa were detected using PCR. SCCmec types were determined according to the Oliveira method and the sequence type (ST)/clonal complex (CC) by the MLST method. RESULTS: Of the isolates tested, 36 (66.7%) showed resistance to at least one aminoglycoside: TOB (57.4%), GEN (53.7%), AK (55.6%), NET (24.1%). The aacA-aphD gene was present in 29 MRSA-GEN-R (most often in combination with aadD, 15/29 or aph(3″)-IIIa, 10/29); the aacA-aphD gene was the only determinant of resistance in 1 isolate. The AR variants mainly belonged to the CC8 clonal complex (ST239/247/241/254/8) and most frequently contained SCCmec type III (3A) cassettes. CONCLUSIONS: Resistance to at least one aminoglycoside was present in 66.7% of HA-MRSA and in more than 22% to all of them. The presence of the aacA-aphD gene was sufficient to express the resistance phenotype to GEN/TOB/AK/NET. Resistant isolates were closely related to each other.

Aptamer-based fluorometric assay for direct identification of methicillin-resistant Staphylococcus aureus from clinical samples.

Accurate and rapid identification of methicillin-resistant Staphylococcus aureus (MRSA) is of important clinical significance. In this study, a novel aptamer-based fluorometric assay was developed for detection of MRSA in clinical samples by coupling with immunomagnetic separation. The S. aureus cells in clinical specimens were enriched by magnetic separation. Following lysis by staphylococcal lysin, the PBP2a proteins were released from S. aureus cells and detected by the aptamer-based fluorometric assay. Without lengthy period of bacteria cultivation in the traditional susceptibility testing, this test has an overall testing time of only 2 h with the detection limit of 2.63 × 103 and 1.38 × 103 CFU/mL in PBS and spiked nasal swab, respectively. Since it is simple, rapid and sensitive, this method could be used for the detection of MRSA in various clinical samples.

Detection of the mecA gene and identification of Staphylococcus directly from blood culture bottles by multiplex polymerase chain reaction.

INTRODUCTION: Staphylococcus spp. - both S. aureus, including methicillin-resistant strains (MRSA) and coagulase negative staphylococci (CoNS) - are relevant agents of healthcare-associated infections. Therefore, the rapid recognition of MRSA and methicillin-resistant CoNS from blood stream infections is critically important for patient management. It is worth noting that inappropriate empiric therapy has been associated with higher in-hospital mortality. MATERIAL AND METHODS: In this study we evaluated a multiplex polymerase chain reaction (multiplex PCR) standardized to detect Staphylococcus spp., S. aureus, and mecA gene-encoded oxacillin resistance directly from blood culture bottles. A total of 371 blood cultures with Gram-positive microorganisms confirmed by Gram-stain were analyzed. Results from multiplex PCR were compared to phenotypic characterization of isolates. RESULTS: Staphylococcus aureus was detected in 85 (23.0%) blood cultures and CoNS in 286 (77.0%). There was 100% agreement between phenotypic and multiplex PCR identification. Forty-three (50.6%) of the 85 S. aureus carried the mecA gene and among the 286 CoNS, 225 (78.7%) were positive for the mecA gene. CONCLUSIONS: The multiplex PCR assay developed here was found to be sensitive, specific, rapid, and showed good agreement with the phenotypic results besides being less expensive. This PCR method could be used in clinical laboratories for rapid identification and initiation of specific and effective treatment, reducing patient mortality and morbidity. Furthermore, this method may reduce misuse of antimicrobial classes that are more expensive and toxic, thus contributing to the selection of antibiotic-resistant Staphylococcus spp.

Detection of the mecA gene and identification of Staphylococcus directly from blood culture bottles by multiplex polymerase chain reaction

ABSTRACT Introduction: Staphylococcus spp. - both S. aureus, including methicillin-resistant strains (MRSA) and coagulase negative staphylococci (CoNS) - are relevant agents of healthcare-associated infections. Therefore, the rapid recognition of MRSA and methicillin-resistant CoNS from blood stream infections is critically important for patient management. It is worth noting that inappropriate empiric therapy has been associated with higher in-hospital mortality. Material and methods: In this study we evaluated a multiplex polymerase chain reaction (multiplex PCR) standardized to detect Staphylococcus spp., S. aureus, and mecA gene-encoded oxacillin resistance directly from blood culture bottles. A total of 371 blood cultures with Gram-positive microorganisms confirmed by Gram-stain were analyzed. Results from multiplex PCR were compared to phenotypic characterization of isolates. Results: Staphylococcus aureus was detected in 85 (23.0%) blood cultures and CoNS in 286 (77.0%). There was 100% agreement between phenotypic and multiplex PCR identification. Forty-three (50.6%) of the 85 S. aureus carried the mecA gene and among the 286 CoNS, 225 (78.7%) were positive for the mecA gene. Conclusions: The multiplex PCR assay developed here was found to be sensitive, specific, rapid, and showed good agreement with the phenotypic results besides being less expensive. This PCR method could be used in clinical laboratories for rapid identification and initiation of specific and effective treatment, reducing patient mortality and morbidity. Furthermore, this method may reduce misuse of antimicrobial classes that are more expensive and toxic, thus contributing to the selection of antibiotic-resistant Staphylococcus spp.

An ultrasensitive electrochemical biosensor for the detection of mecA gene in methicillin-resistant Staphylococcus aureus.

Electrochemical DNA biosensor has unique advantages for on-site pathogenic microorganism detection, yet the detection of long DNA towards genome DNA (gDNA) analysis remains challenge. In this work, we report a novel electrochemical biosensor for the ultrasensitive analysis of mecA DNA on methicillin-resistant Staphylococcus aureus (MRSA) genome, using a multi-signal probes (MSP) system. The MSP consists of 7 biotin-labelled signal probes that will combine to the target DNA in a prehybridization step, and then the complex will be captured by a DNA tetrahedron structure probe (TSP) on the electrode surface. Then, after the introduction of the streptavidin-labelled HRP enzyme, a catalysis current signal is detected that is found to be corresponding to the concentration of the target DNA. MSP in this work plays a critical role not only for the signal amplification through bringing 7 biotins, but also dramatically improves the accessibility of the target sequence embedded in the double-strand DNA molecules and complex second structures. The 3-D DNA TSP here provides steady support and optimized surface density for the very "large" complex of MSP system and gDNA, as a base of the capture probe. Finally, as low as 10fM synthetic target DNA was successfully detected, which is at least 3 magnitudes lower than that using single signal probe. Most importantly, we demonstrated the practicability of our analysis method by analyzing a 57fM MRSA gDNA sample showing excellent selectivity, and the reliability of the analysis was also demonstrated by digital PCR.

Prevalence and characterisation of Staphylococcus aureus causing community-acquired skin and soft tissue infections on Java and Bali, Indonesia.

OBJECTIVES: To define the role of Staphylococcus aureus in community settings among patients with skin and soft tissue infections (SSTI) in Indonesia. METHODS: Staphylococcus aureus were cultured from anterior nares, throat and wounds of 567 ambulatory patients presenting with SSTI. The mecA gene and genes encoding Panton-Valentine leukocidin (PVL; lukF-PV and lukS-PV) and exfoliative toxin (ET; eta and etb) were determined by PCR. Clonal relatedness among methicillin-resistant S. aureus (MRSA) and PVL-positive S. aureus was analysed using multilocus variable-number tandem-repeat analysis (MLVA) typing, and multilocus sequence typing (MLST) for a subset of isolates. Staphylococcal cassette chromosome mec (SCCmec) was determined for all MRSA isolates. Moreover, determinants for S. aureus SSTI, and PVL/ET-positive vs PVL/ET-negative S. aureus were assessed. RESULTS: Staphylococcus aureus were isolated from SSTI wounds of 257 (45.3%) patients, eight (3.1%) of these were MRSA. Genes encoding PVL and ETs were detected in 21.8% and 17.5% of methicillin-susceptible S. aureus (MSSA), respectively. PVL-positive MRSA was not detected. Nasopharyngeal S. aureus carriage was an independent determinant for S. aureus SSTI (odds ratio [OR] 1.8). Primary skin infection (OR 5.4) and previous antibiotic therapy (OR 3.5) were associated with PVL-positive MSSA. Primary skin infection (OR 2.2) was the only factor associated with ET-positive MSSA. MLVA typing revealed two more prevalent MSSA clusters. One ST1-MRSA-SCCmec type IV isolate and a cluster of ST239-MRSA-SCCmec type III were found. CONCLUSIONS: Community-acquired SSTI in Indonesia was frequently caused by PVL-positive MSSA, and the hospital-associated ST239-MRSA may have spread from the hospital into the community.

Comparison of Oxacillin and Cefoxitin for the Detection of mecAGene to Determine Methicillin Resistance in Coagulase Negative Staphylococci(CoNs).

The aim of the study was to compare the effectiveness of Cefoxitin with that of Methicillin/Oxacillin in the determination of mecAgene in Methicillin resistant Coagulase-negative staphylococci(CoNS). We assessed 57 CoNS isolates for mecA gene via PCR, which were subsequently subjected to Methicillin/Oxacillin and Cefoxitin disc diffusion test. These methods are simple, inexpensive and easily available compared to PCR despite less specificity. Out of 41 mecApositive species, 33 (80.5%) were resistant to Methicillin/Oxacillin. Cefoxitin-resistance was seen in all 41 (100%) mecApositive samples. Two (12.5%) mecAnegative isolates of S.saprophyticuswere Methicillin/Oxacillin resistant, but were Cefoxitin sensitive. Four (9.7%) isolates of S.saprophyticus, three (7.3%) of S.epidermidisspecies, and one (2.4%) S.haemolyticusthat were mecApositive were sensitive to Methicillin/Oxacillin but resistant to Cefoxitin. Cefoxitin resistance provides a more accurate picture of mecAgene positivity as compared to Methicillin and Oxacillin.

In vivo functional and molecular characterization of the Penicillin-Binding Protein 4 (DacB) of Pseudomonas aeruginosa.

BACKGROUND: Community and nosocomial infections by Pseudomonas aeruginosa still create a major therapeutic challenge. The resistance of this opportunist pathogen to ß-lactam antibiotics is determined mainly by production of the inactivating enzyme AmpC, a class C cephalosporinase with a regulation system more complex than those found in members of the Enterobacteriaceae family. This regulatory system also participates directly in peptidoglycan turnover and recycling. One of the regulatory mechanisms for AmpC expression, recently identified in clinical isolates, is the inactivation of LMM-PBP4 (Low-Molecular-Mass Penicillin-Binding Protein 4), a protein whose catalytic activity on natural substrates has remained uncharacterized until now. RESULTS: We carried out in vivo activity trials for LMM-PBP4 of Pseudomonas aeruginosa on macromolecular peptidoglycan of Escherichia coli and Pseudomonas aeruginosa. The results showed a decrease in the relative quantity of dimeric, trimeric and anhydrous units, and a smaller reduction in monomer disaccharide pentapeptide (M5) levels, validating the occurrence of D,D-carboxypeptidase and D,D-endopeptidase activities. Under conditions of induction for this protein and cefoxitin treatment, the reduction in M5 is not fully efficient, implying that LMM-PBP4 of Pseudomonas aeruginosa presents better behaviour as a D,D-endopeptidase. Kinetic evaluation of the direct D,D-peptidase activity of this protein on natural muropeptides M5 and D45 confirmed this bifunctionality and the greater affinity of LMM-PBP4 for its dimeric substrate. A three-dimensional model for the monomeric unit of LMM-PBP4 provided structural information which supports its catalytic performance. CONCLUSIONS: LMM-PBP4 of Pseudomonas aeruginosa is a bifunctional enzyme presenting both D,D-carboxypeptidase and D,D-endopeptidase activities; the D,D-endopeptidase function is predominant. Our study provides unprecedented functional and structural information which supports the proposal of this protein as a potential hydrolase-autolysin associated with peptidoglycan maturation and recycling. The fact that mutant PBP4 induces AmpC, may indicate that a putative muropeptide-subunit product of the DD-EPase activity of PBP4 could be a negative regulator of the pathway. This data contributes to understanding of the regulatory aspects of resistance to ß-lactam antibiotics in this bacterial model.

A Graphene Oxide-Based Sensing Platform for the Determination of Methicillin-Resistant Staphylococcus aureus Based on Strand-Displacement Polymerization Recycling and Synchronous Fluorescent Signal Amplification.

To develop new technology for detecting methicillin-resistant Staphylococcus aureus (MRSA), a novel fluorescent biosensor based on Klenow fragment (KF)-assisted target recycling amplification and synchronous fluorescence analysis was created. Carboxy-fluorescein (FAM)-labeled single-stranded DNA (ssDNA) containing a capture probe and a signal probe was adsorbed onto the surface of graphene oxide (GO) via π-stacking interactions, resulting in the fluorescence quenching of the dye. When target and primer were introduced, the fluorescence was restored due to P0 being completely released from the surface of the GO. Meanwhile, by using the KF and exploiting the synergistic effect of FAM and the double-stranded DNA (dsDNA)-SYBR Green I duplex structure, the fluorescence in this detection system was considerably amplified and the sensitivity was improved. The proposed strategy for mecA gene analysis showed a good linear range from 1 to 40 nmol/L, with a lower limit of detection of 0.5 nmol/L. In addition, a bacterial sample harboring the mecA gene was also detected, and its lower detection limit was up to 300 colony-forming units (CFU)/mL. Accordingly, this biosensor exhibits high sensitivity and selectivity and has great potential for early clinical diagnosis and treatment.