RESUMO
BACKGROUND: Antibiotic-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), pose a significant threat to public health. Existing detection methods, like cultivation-based techniques, demand significant time and labor, while molecular diagnostic techniques, such as PCR, necessitate sophisticated instrumentation and skilled personnel. Although previous multiplex loop-mediated isothermal amplification assays based on fluorescent dyes (mfLAMP) offer simplicity and cost-effectiveness, they are prone to false-positive results. Therefore, developing a rapid and efficient multiplex assay for high-sensitivity MRSA is imperative to create a practical diagnostic tool for point-of-care testing. RESULTS: Here, we developed a mfLAMP combined with a lateral flow assay (mfLAMP-LFA) for the visual and simultaneous detection of the mecA (PBP2a-specific marker) and nuc (S. aureus-specific marker) genes in MRSA. We optimized mfLAMP-LFA using graphene oxide (GO)-based purification and specific DNA probes and evaluated its sensitivity, specificity, and stability. Utilizing GO to mitigate false-positive results by acting as a trap for free DNA probes, the mfLAMP-LFA method successfully identified mecAf and nucf-probes, exhibiting distinct red, green, and yellow fluorescence signals. The detection sensitivity of the developed mfLAMP-LFA method (1 CFU mL-1 in phosphate-buffered saline (PBS)) was comparable to other highly sensitive MRSA detection methods (1 CFU mL-1 in PBS). Furthermore, the method demonstrated specificity for MRSA, detecting it in irrigation water samples within the desired range and achieving reliable recovery rates from spiked samples. SIGNIFICANCE: This novel strategy is the first to incorporate GO into mfLAMP-LFA, enabling specific and sensitive MRSA detection and advancing rapid bacterial detection. This assay facilitates MRSA diagnostics, contributing to improved public health and food safety by delivering rapid, cost-effective point-of-care results. It enables the simultaneous detection of multiple bacteria, even in irrigation water samples artificially inoculated with MRSA, which contain aerobic bacteria at 2.7 × 102 CFU mL-1.
Assuntos
Proteínas de Bactérias , Staphylococcus aureus Resistente à Meticilina , Nuclease do Micrococo , Técnicas de Amplificação de Ácido Nucleico , Proteínas de Ligação às Penicilinas , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Staphylococcus aureus Resistente à Meticilina/genética , Proteínas de Ligação às Penicilinas/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Nuclease do Micrococo/genética , Proteínas de Bactérias/genética , Fluorescência , Técnicas de Diagnóstico Molecular/métodos , Corantes Fluorescentes/química , GrafiteRESUMO
The increase in the resistance of mutant strains of Neisseria gonorrhoeae to the antibiotic ceftriaxone is pronounced in the decrease in the second-order acylation rate constant, k2/KS, by penicillin-binding protein 2 (PBP2). These changes can be caused by both the decrease in the acylation rate constant, k2, and the weakening of the binding affinity, i.e., an increase in the substrate constant, KS. A501X mutations in PBP2 affect second-order acylation rate constants. The PBP2A501V variant exhibits a higher k2/KS value, whereas for PBP2A501R and PBP2A501P variants, these values are lower. We performed molecular dynamic simulations with both classical and QM/MM potentials to model both acylation energy profiles and conformational dynamics of four PBP2 variants to explain the origin of k2/KS changes. The acylation reaction occurs in two elementary steps, specifically, a nucleophilic attack by the oxygen atom of the Ser310 residue and C-N bond cleavage in the ß-lactam ring accompanied by the elimination of the leaving group of ceftriaxone. The energy barrier of the first step increases for PBP2 variants with a decrease in the observed k2/KS value. Submicrosecond classic molecular dynamic trajectories with subsequent cluster analysis reveal that the conformation of the ß3-ß4 loop switches from open to closed and its flexibility decreases for PBP2 variants with a lower k2/KS value. Thus, the experimentally observed decrease in the k2/KS in A501X variants of PBP2 occurs due to both the decrease in the acylation rate constant, k2, and the increase in KS.
Assuntos
Ceftriaxona , Simulação de Dinâmica Molecular , Neisseria gonorrhoeae , Proteínas de Ligação às Penicilinas , Ceftriaxona/farmacologia , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/efeitos dos fármacos , Neisseria gonorrhoeae/metabolismo , Proteínas de Ligação às Penicilinas/genética , Proteínas de Ligação às Penicilinas/química , Proteínas de Ligação às Penicilinas/metabolismo , Antibacterianos/farmacologia , Mutação , Farmacorresistência Bacteriana/genética , Acilação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , D-Ala-D-Ala Carboxipeptidase Tipo SerinaRESUMO
The function of many bacterial processes depends on the formation of functional membrane microdomains (FMMs), which resemble the lipid rafts of eukaryotic cells. However, the mechanism and the biological function of these membrane microdomains remain unclear. Here, we show that FMMs in the pathogen methicillin-resistant Staphylococcus aureus (MRSA) are dedicated to confining and stabilizing proteins unfolded due to cellular stress. The FMM scaffold protein flotillin forms a clamp-shaped oligomer that holds unfolded proteins, stabilizing them and favoring their correct folding. This process does not impose a direct energy cost on the cell and is crucial to survival of ATP-depleted bacteria, and thus to pathogenesis. Consequently, FMM disassembling causes the accumulation of unfolded proteins, which compromise MRSA viability during infection and cause penicillin re-sensitization due to PBP2a unfolding. Thus, our results indicate that FMMs mediate ATP-independent stabilization of unfolded proteins, which is essential for bacterial viability during infection.
Assuntos
Proteínas de Bactérias , Microdomínios da Membrana , Proteínas de Membrana , Staphylococcus aureus Resistente à Meticilina , Proteínas de Membrana/metabolismo , Microdomínios da Membrana/metabolismo , Staphylococcus aureus Resistente à Meticilina/metabolismo , Proteínas de Bactérias/metabolismo , Desdobramento de Proteína , Trifosfato de Adenosina/metabolismo , Proteínas de Ligação às Penicilinas/metabolismo , Proteínas de Ligação às Penicilinas/genética , Proteínas de Ligação às Penicilinas/química , Humanos , Estabilidade Proteica , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/metabolismo , Animais , CamundongosRESUMO
Penicillin-binding protein 2 (PBP2), a vital protein involved in bacterial cell-wall synthesis, serves a target for ß-lactam antibiotics. Acinetobacter baumannii is a pathogen notorious for multidrug resistance; therefore, exploration of PBPs is pivotal in the development of new antimicrobial strategies. In this study, the tertiary structure of PBP2 from A. baumannii (abPBP2) was elucidated using X-ray crystallography. The structural analysis demonstrated notable movement in the head domain, potentially critical for its glycosyltransferase function, suggesting that abPBP2 assumes a fully closed conformation. Our findings offer valuable information for developing novel antimicrobial agents targeting abPBP2 that are applicable in combating multidrug-resistant infections.
Assuntos
Acinetobacter baumannii , Proteínas de Ligação às Penicilinas , Conformação Proteica , Acinetobacter baumannii/metabolismo , Acinetobacter baumannii/química , Proteínas de Ligação às Penicilinas/química , Proteínas de Ligação às Penicilinas/metabolismo , Proteínas de Ligação às Penicilinas/genética , Cristalografia por Raios X , Modelos Moleculares , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Antibacterianos/farmacologia , Antibacterianos/química , Sequência de AminoácidosRESUMO
The high-level resistance to next-generation ß-lactams frequently found in Staphylococcus aureus isolates lacking mec, which encodes the transpeptidase PBP2a traditionally associated with methicillin-resistant Staphylococcus aureus (MRSA), has remained incompletely understood for decades. A new study by Lai et al. found that the co-occurrence of mutations in pbp4 and gdpP, which respectively cause increased PBP4-mediated cell wall crosslinking and elevated cyclic-di-AMP levels, produces synergistic ß-lactam resistance rivaling that of PBP2a-producing MRSA (L.-Y. Lai, N. Satishkumar, S. Cardozo, V. Hemmadi, et al., mBio 15:e02889-23. 2024, https://doi.org/10.1128/mbio.02889-23). The combined mutations are sufficient to explain the high-level ß-lactam resistance of some mec-lacking strains, but the mechanism of synergy remains elusive and an avenue for further research. Importantly, the authors establish that co-occurrence of these mutations leads to antibiotic therapy failure in a Caenorhabditis elegans infection model. These results underscore the need to consider this unique and novel ß-lactam resistance mechanism during the clinical diagnosis of MRSA, rather than relying on mec as a diagnostic.
Assuntos
Antibacterianos , Caenorhabditis elegans , Staphylococcus aureus Resistente à Meticilina , Proteínas de Ligação às Penicilinas , Infecções Estafilocócicas , beta-Lactamas , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/metabolismo , beta-Lactamas/farmacologia , Antibacterianos/farmacologia , Proteínas de Ligação às Penicilinas/genética , Proteínas de Ligação às Penicilinas/metabolismo , Caenorhabditis elegans/microbiologia , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/tratamento farmacológico , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Resistência beta-Lactâmica/genética , Mutação , Testes de Sensibilidade Microbiana , Parede Celular/metabolismo , Parede Celular/efeitos dos fármacos , Humanos , AMP Cíclico/metabolismo , Antibióticos beta LactamRESUMO
Staphylococcus aureus has the ability to invade cortical bone osteocyte lacuno-canalicular networks (OLCNs) and cause osteomyelitis. It was recently established that the cell wall transpeptidase, penicillin-binding protein 4 (PBP4), is crucial for this function, with pbp4 deletion strains unable to invade OLCNs and cause bone pathogenesis in a murine model of S. aureus osteomyelitis. Moreover, PBP4 has recently been found to modulate S. aureus resistance to ß-lactam antibiotics. As such, small molecule inhibitors of S. aureus PBP4 may represent dual functional antimicrobial agents that limit osteomyelitis and/or reverse antibiotic resistance. A high throughput screen recently revealed that the phenyl-urea 1 targets PBP4. Herein, we describe a structure-activity relationship (SAR) study on 1. Leveraging in silico docking and modeling, a set of analogs was synthesized and assessed for PBP4 inhibitory activities. Results revealed a preliminary SAR and identified lead compounds with enhanced binding to PBP4, more potent antibiotic resistance reversal, and diminished PBP4 cell wall transpeptidase activity in comparison to 1.
Assuntos
Antibacterianos , Simulação de Acoplamento Molecular , Proteínas de Ligação às Penicilinas , Staphylococcus aureus , Proteínas de Ligação às Penicilinas/metabolismo , Proteínas de Ligação às Penicilinas/antagonistas & inibidores , Antibacterianos/farmacologia , Antibacterianos/química , Relação Estrutura-Atividade , Staphylococcus aureus/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Ureia/química , Ureia/farmacologia , Ureia/análogos & derivados , Animais , Camundongos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/antagonistas & inibidoresRESUMO
Penicillin binding proteins (PBPs) are involved in biosynthesis, remodeling and recycling of peptidoglycan (PG) in bacteria. PBP-A from Thermosynechococcus elongatus belongs to a cyanobacterial family of enzymes sharing close structural and phylogenetic proximity to class A ß-lactamases. With the long-term aim of converting PBP-A into a ß-lactamase by directed evolution, we simulated what may happen when an organism like Escherichia coli acquires such a new PBP and observed growth defect associated with the enzyme activity. To further explore the molecular origins of this harmful effect, we decided to characterize deeper the activity of PBP-A both in vitro and in vivo. We found that PBP-A is an enzyme endowed with DD-carboxypeptidase and DD-endopeptidase activities, featuring high specificity towards muropeptides amidated on the D-iso-glutamyl residue. We also show that a low promiscuous activity on non-amidated peptidoglycan deteriorates E. coli's envelope, which is much higher under acidic conditions where substrate discrimination is mitigated. Besides expanding our knowledge of the biochemical activity of PBP-A, this work also highlights that promiscuity may depend on environmental conditions and how it may hinder rather than promote enzyme evolution in nature or in the laboratory.
Assuntos
Escherichia coli , Proteínas de Ligação às Penicilinas , Peptidoglicano , Escherichia coli/genética , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Proteínas de Ligação às Penicilinas/metabolismo , Proteínas de Ligação às Penicilinas/genética , Proteínas de Ligação às Penicilinas/química , Peptidoglicano/metabolismo , Especificidade por Substrato , Cianobactérias/metabolismo , Cianobactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , SynechococcusRESUMO
Most rod-shaped bacteria elongate by inserting new cell wall material into the inner surface of the cell sidewall. This is performed by class A penicillin binding proteins (PBPs) and a highly conserved protein complex, the elongasome, which moves processively around the cell circumference and inserts long glycan strands that act as barrel-hoop-like reinforcing structures, thereby giving rise to a rod-shaped cell. However, it remains unclear how elongasome synthesis dynamics and termination events are regulated to determine the length of these critical cell-reinforcing structures. To address this, we developed a method to track individual elongasome complexes around the entire circumference of Bacillus subtilis cells for minutes-long periods using single-molecule fluorescence microscopy. We found that the B. subtilis elongasome is highly processive and that processive synthesis events are frequently terminated by rapid reversal or extended pauses. We found that cellular levels of RodA regulate elongasome processivity, reversal and pausing. Our single-molecule data, together with stochastic simulations, show that elongasome dynamics and processivity are regulated by molecular motor tug-of-war competition between several, likely two, oppositely oriented peptidoglycan synthesis complexes associated with the MreB filament. Altogether these results demonstrate that molecular motor tug-of-war is a key regulator of elongasome dynamics in B. subtilis, which likely also regulates the cell shape via modulation of elongasome processivity.
Assuntos
Bacillus subtilis , Proteínas de Bactérias , Parede Celular , Proteínas de Ligação às Penicilinas , Bacillus subtilis/metabolismo , Bacillus subtilis/genética , Parede Celular/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Ligação às Penicilinas/metabolismo , Proteínas de Ligação às Penicilinas/genética , Peptidoglicano/metabolismo , Peptidoglicano/biossíntese , Microscopia de Fluorescência , Imagem Individual de Molécula , Proteínas Motores Moleculares/metabolismo , Proteínas Motores Moleculares/genéticaRESUMO
BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) poses a great health threat to humans. Looking for compounds that could reduce the resistance of S. aureus towards methicillin is an effective way to alleviate the antimicrobial resistance crisis. METHODS AND RESULTS: Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), Time-killing growth curve, staphyloxanthin and penicillin-binding protein 2a (PBP2a) were detected. A quantitative polymerase chain reaction was used to measure the effect of BBH on the gene transcription profiles of MRSA. The MIC of MRSA-ST59-t437 towards oxacillin was 8 µg/ml, and MBC was 128 µg/ml. After adding a sub-inhibitory concentration of BBH, the MIC and MBC of MRSA-ST59-t478 towards oxacillin went down to 0.125 and 32 µg/ml respectively. The amount of PBP2a and staphyloxanthin were reduced after treatment with BBH. Moreover, the transcription levels of sarA, mecA and fni genes were downregulated. CONCLUSIONS: It is for the first time reported that BBH could inhibit staphyloxanthin synthesis by inhibiting fni gene. Moreover, fni might be the target gene of sarA, and there might be another regulatory pathway to inhibit staphyloxanthin biosynthesis. BBH could effectively reduce the methicillin resistance of MRSA-ST59-t437 by downregulating fni, sarA and mecA genes.
Assuntos
Antibacterianos , Proteínas de Bactérias , Berberina , Staphylococcus aureus Resistente à Meticilina , Testes de Sensibilidade Microbiana , Xantofilas , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/genética , Xantofilas/farmacologia , Berberina/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Antibacterianos/farmacologia , Proteínas de Ligação às Penicilinas/genética , Proteínas de Ligação às Penicilinas/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Oxacilina/farmacologiaRESUMO
Ovoid-shaped bacteria, such as Streptococcus pneumoniae (pneumococcus), have two spatially separated peptidoglycan (PG) synthase nanomachines that locate zonally to the midcell of dividing cells. The septal PG synthase bPBP2x:FtsW closes the septum of dividing pneumococcal cells, whereas the elongasome located on the outer edge of the septal annulus synthesizes peripheral PG outward. We showed previously by sm-TIRFm that the septal PG synthase moves circumferentially at midcell, driven by PG synthesis and not by FtsZ treadmilling. The pneumococcal elongasome consists of the PG synthase bPBP2b:RodA, regulators MreC, MreD, and RodZ, but not MreB, and genetically associated proteins Class A aPBP1a and muramidase MpgA. Given its zonal location separate from FtsZ, it was of considerable interest to determine the dynamics of proteins in the pneumococcal elongasome. We found that bPBP2b, RodA, and MreC move circumferentially with the same velocities and durations at midcell, driven by PG synthesis. However, outside of the midcell zone, the majority of these elongasome proteins move diffusively over the entire surface of cells. Depletion of MreC resulted in loss of circumferential movement of bPBP2b, and bPBP2b and RodA require each other for localization and circumferential movement. Notably, a fraction of aPBP1a molecules also moved circumferentially at midcell with velocities similar to those of components of the core elongasome, but for shorter durations. Other aPBP1a molecules were static at midcell or diffusing over cell bodies. Last, MpgA displayed nonprocessive, subdiffusive motion that was largely confined to the midcell region and less frequently detected over the cell body.
Assuntos
Proteínas de Bactérias , Proteínas de Ligação às Penicilinas , Streptococcus pneumoniae , Streptococcus pneumoniae/metabolismo , Streptococcus pneumoniae/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Ligação às Penicilinas/metabolismo , Proteínas de Ligação às Penicilinas/genética , Peptidoglicano/metabolismo , Peptidoglicano Glicosiltransferase/metabolismo , Peptidoglicano Glicosiltransferase/genéticaRESUMO
In Algeria, the issue of antibiotic resistance is on the rise, being the Staphylococcus aureus infection as a significant concern of hospital-acquired infections. The emergence of antibiotic resistance in this bacterium poses a worldwide challenge. The aim of this study aims to establish the incidence of S aureus strains in Algeria as well as identify phenotypic and genotypic resistance based on the "mecA" and "nuc" genes. From 2014 to 2017, a total of 185 S aureus strains were isolated from patients at a hospital in the city of Rouïba, Algiers the number of isolates was slightly higher in males at 58.06% compared to females at 41.94%, resulting in a sex ratio of 1.38. the Oxacillin and Cefoxitin DD test (1 µg oxacillin disk and 30 µg cefoxitin disk) identified 42 strains as resistant. The results indicated high resistance to lactam antibiotics, with penicillin having a 100% resistance rate. There was also significant resistance to oxacillin (51.25%) and cefoxitin (50%). This resistance was frequently associated with resistance to other antibiotic classes, such as aminoglycosides (50%) and Macrolides (28.29%). To confirm methicillin-resistant characteristics, a polymerase chain reaction (PCR) multiplex was conducted on 10 isolates (6 SARM; 4 MSSA) on a phenotypic level. Three isolates tested positive for "mecA," while 7 were negative. All strains carry the nuc gene, which is specific to S aureus. In Algeria, the incidence of S aureus resistance is slightly lower compared to other countries, but it is increasing over time. It is now more crucial than ever to restrict the proliferation of multidrug-resistant strains and reduce undue antibiotic prescriptions. To achieve this, it is vital to keep updated on the epidemiology of this bacterium and its antibiotic susceptibility. This will enable the formulation of appropriate preventive control measures to manage its progression.
Assuntos
Antibacterianos , Testes de Sensibilidade Microbiana , Infecções Estafilocócicas , Staphylococcus aureus , Humanos , Antibacterianos/farmacologia , Feminino , Masculino , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificação , Infecções Estafilocócicas/epidemiologia , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/tratamento farmacológico , Argélia/epidemiologia , Prevalência , Proteínas de Bactérias/genética , Oxacilina/farmacologia , Adulto , Proteínas de Ligação às Penicilinas/genética , Cefoxitina/farmacologia , Pessoa de Meia-Idade , Nuclease do Micrococo/genética , Farmacorresistência Bacteriana/genética , Farmacorresistência Bacteriana Múltipla/genética , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/isolamento & purificaçãoRESUMO
The accurate and rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) holds significant clinical importance. This work presents a new method for detecting methicillin-resistant Staphylococcus aureus (S. aureus) in clinical samples. The method uses an aptamer-based colorimetric assay that combines a recognizing probe to identify the target and split DNAzyme to amplify the signal, resulting in a highly sensitive and direct analysis of methicillin-resistance. The identification of the PBP2a protein on the membrane of S. aureus in clinical samples leads to the allosterism of the recognizing probe, and thus provides a template for the proximity ligation of split DNAzyme. The proximity ligation of split DNAzyme forms an intact DNAzyme to identify the loop section in the L probe and generates a nicking site to release the loop sequence ("3" and "4" fragments). The "3" and "4" fragments forms an intact sequence to induce the catalytic hairpin assembly, exposing the G-rich section. The released the G-rich sequence of LR probe induces the formation of G-quadruplex-hemin DNAzyme as a colorimetric signal readout. The absorption intensity demonstrated a strong linear association with the logarithm of the S. aureus concentration across a wide range of 5 orders of magnitude dynamic range under the optimized experimental parameters. The limit of detection was calculated to be 23 CFU/ml and the method showed high selectivity for MRSA.
Assuntos
Aptâmeros de Nucleotídeos , Colorimetria , DNA Catalítico , Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , DNA Catalítico/metabolismo , Colorimetria/métodos , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/diagnóstico , Humanos , Técnicas Biossensoriais/métodos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificação , Sensibilidade e Especificidade , Resistência a Meticilina , Proteínas de Ligação às Penicilinas/metabolismo , Proteínas de Ligação às Penicilinas/genéticaRESUMO
BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen that can cause many community and hospital-acquired infections. This study was conducted to investigate the SCCmec gene types responsible for methicillin resistance in MRSA isolates isolated from hospitalised patients. MATERIAL AND METHODS: MRSA isolates isolated from samples sent from various clinics to Gaziantep University Hospital Microbiology Laboratory between March 2021-January 2022 were included in the study. Bacteria were identified using by VITEK 2 automated system. Cefoxitin (FOX) resistance was determined by the disc diffusion method according to EUCAST standards. Cefoxitin resistance was confirmed by the Penicillin Binding Protein 2' latex agglutination test. Types of mecA, mecC, coa, nuc, Panton Valentin Leukocidin (PVL), ccrC2, class A mec, SCCmec types in isolates detected as MRSA were investigated by real-time PCR. RESULTS: In this study, 116 isolates meeting the study criteria were examined. By detecting the nuc and coa genes in all isolates by PCR, the phenotypic identification of S.aureus was confirmed. While the mecA gene was detected in all MRSA isolates, no mecC gene was detected in any isolates. Detected SCCmec types were as follows; SCCmec Type 1 (2.6%), Type II (28.4%), Type III (12.9%), Type IVa (11.2%), Type IVb (3.4%), Type IVc (3.4%), Type IVg (12.1%), Type V (0.9%), Type VII (4.3%), Type VIII (18.1%), Type IX (0.9%), Type XII (1.7%). On the other hand, SCCmec Type VI, X, XI and XIII were not found in any isolate. It was determined that four of the MRSA isolates (3.4%) carried the PVL gene that two (50%) of these were found in SCCmec Type VIII. CONCLUSION: Monitoring of FOX resistance is an effective and safe method for determination of MRSA isolates. The change in the mec gene causes resistance, which should be monitored regularly with molecular methods. Our study is the first study in Turkey.
Assuntos
Proteínas de Bactérias , Cefoxitina , Staphylococcus aureus Resistente à Meticilina , Reação em Cadeia da Polimerase em Tempo Real , Infecções Estafilocócicas , Humanos , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Staphylococcus aureus Resistente à Meticilina/classificação , Cefoxitina/farmacologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Infecções Estafilocócicas/microbiologia , Proteínas de Bactérias/genética , Antibacterianos/farmacologia , Proteínas de Ligação às Penicilinas/genética , Testes de Sensibilidade Microbiana , Leucocidinas/genética , Exotoxinas/genética , Toxinas Bacterianas/genéticaRESUMO
BACKGROUND: Staphylococcus aureus (S. aureus) associated with COVID-19 has not been well documented. This cross-sectional study evaluated the association between nasal S. aureus carriage and COVID-19. METHODS AND RESULTS: Nasopharyngeal samples were collected from 391 participants presenting for COVID-19 test in Lagos, Nigeria, and S. aureus was isolated from the samples. Antimicrobial susceptibility test was done by disc diffusion method. All S. aureus isolates were screened for the presence of mecA, panton-valentine leucocidin (PVL) and toxic shock syndrome toxin (TSST) virulence genes by polymerase chain reaction. Staphylococcal protein A (spa) typing was conducted for all the isolates. Participants with COVID-19 had double the prevalence of S. aureus (42.86%) compared to those who tested negative (20.54%). A significant association was seen between S. aureus nasal carriage and COVID-19 (p = 0.004). Antimicrobial sensitivity results showed resistance to oxacillin (100%), cefoxitin (53%), and vancomycin (98.7%). However, only 41% of the isolates harbored the mecA gene, with SCCmecV being the most common SCCmec type. There was no association between the carriage of virulence genes and COVID-19. A total of 23 Spa types were detected, with t13249 and t095 being the two most common spa types. CONCLUSION: This study examined the association between nasal S. aureus carriage and SARS-COV-2 infection. Further research is required to fully explore the implications of S. aureus co-infection with COVID-19.
Assuntos
COVID-19 , SARS-CoV-2 , Infecções Estafilocócicas , Staphylococcus aureus , Humanos , COVID-19/microbiologia , COVID-19/epidemiologia , COVID-19/virologia , Infecções Estafilocócicas/epidemiologia , Infecções Estafilocócicas/microbiologia , Estudos Transversais , Masculino , Feminino , Staphylococcus aureus/genética , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/patogenicidade , Staphylococcus aureus/isolamento & purificação , Adulto , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Pessoa de Meia-Idade , Toxinas Bacterianas/genética , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Comorbidade , Proteínas de Bactérias/genética , Virulência/genética , Nigéria/epidemiologia , Farmacorresistência Bacteriana Múltipla/genética , Antibacterianos/farmacologia , Portador Sadio/epidemiologia , Portador Sadio/microbiologia , Testes de Sensibilidade Microbiana , Proteínas de Ligação às Penicilinas/genética , Leucocidinas/genética , Exotoxinas/genética , Fatores de Virulência/genética , Adulto JovemRESUMO
Heterogeneity of Helicobacter pylori communities contributes to its pathogenicity and diverse clinical outcomes. We conducted drug-susceptibility tests using four antibiotics, clarithromycin (CLR), amoxicillin (AMX), metronidazole and sitafloxacin, to examine H. pylori population diversity. We also analyzed genes associated with resistance to CLR and AMX. We examined multiple isolates from 42 Japanese patients, including 28 patients in whom primary eradication with CLR and AMX had failed, and 14 treatment-naïve patients. We identified some patients with coexistence of drug resistant- and sensitive-isolates (drug-heteroR/S-patients). More than 60% of patients were drug-heteroR/S to all four drugs, indicating extensive heterogeneity. For the four drugs except AMX, the rates of drug-heteroR/S-patients were higher in treatment-naïve patients than in primary eradication-failure patients. In primary eradication-failure patients, isolates multi-resistant to all four drugs existed among other isolates. In primary eradication-failure drug-heteroR/S-patients, CLR- and AMX-resistant isolates were preferentially distributed to the corpus and antrum with different minimum inhibitory concentrations, respectively. We found two mutations in PBP1A, G591K and A480V, and analyzed these in recombinants to directly demonstrate their association with AMX resistance. Assessment of multiple isolates from different stomach regions will improve accurate assessment of H. pylori colonization status in the stomach.
Assuntos
Amoxicilina , Antibacterianos , Farmacorresistência Bacteriana , Infecções por Helicobacter , Helicobacter pylori , Testes de Sensibilidade Microbiana , Mutação , Humanos , Helicobacter pylori/genética , Helicobacter pylori/efeitos dos fármacos , Helicobacter pylori/isolamento & purificação , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/tratamento farmacológico , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Farmacorresistência Bacteriana/genética , Amoxicilina/farmacologia , Amoxicilina/uso terapêutico , Masculino , Feminino , Metronidazol/farmacologia , Estômago/microbiologia , Claritromicina/farmacologia , Pessoa de Meia-Idade , Idoso , Adulto , Proteínas de Bactérias/genética , Proteínas de Ligação às Penicilinas/genética , Fluoroquinolonas/farmacologia , Fluoroquinolonas/uso terapêuticoRESUMO
In this review, we explore the regulation of septal peptidoglycan (sPG) synthesis in bacterial cell division, a critical process for cell viability and proper morphology. Recent single-molecule imaging studies have revealed the processive movement of the FtsW:bPBP synthase complex along the septum, shedding light on the spatiotemporal dynamics of sPG synthases and their regulators. In diderm bacteria (E. coli and C. crescentus), the movement occurs at two distinct speeds, reflecting active synthesis or inactivity driven by FtsZ-treadmilling. In monoderm bacteria (B. subtilis, S. pneumoniae, and S. aureus), however, these enzymes exhibit only the active sPG-track-coupled processive movement. By comparing the dynamics of sPG synthases in these organisms and that of class-A penicillin-binding proteins in vivo and in vitro, we propose a unifying model for septal cell wall synthesis regulation across species, highlighting the roles of the sPG- and Z-tracks in orchestrating a robust bacterial cell wall constriction process.
Assuntos
Bactérias , Proteínas de Bactérias , Parede Celular , Peptidoglicano , Imagem Individual de Molécula , Parede Celular/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Peptidoglicano/metabolismo , Peptidoglicano/biossíntese , Bactérias/metabolismo , Bactérias/enzimologia , Bactérias/genética , Divisão Celular , Proteínas de Ligação às Penicilinas/metabolismo , Proteínas de Ligação às Penicilinas/genéticaRESUMO
Macrocyclization of peptides reduces conformational flexibilities, potentially leading to improved drug-like properties, such as target specificities and metabolic stabilities. As chemical methodologies often allow side reactions like epimerization and oligomerization, keen attention has been directed toward enzymatic peptide cyclization using peptide cyclases from specialized metabolic pathways. Penicillin-binding protein-type thioesterases (PBP-type TEs) are a recently identified family of peptide cyclases involved in the biosynthesis of non-ribosomal peptides in actinobacteria. PBP-type TEs have undergone intensive investigation due to their outstanding potential as biocatalysts. This review summarizes the rapidly growing knowledge on PBP-type TEs, with special emphasis on their functions, scopes, and structures, and efforts towards their biocatalytic applications.
Assuntos
Biocatálise , Proteínas de Ligação às Penicilinas , Proteínas de Ligação às Penicilinas/metabolismo , Proteínas de Ligação às Penicilinas/química , Tioléster Hidrolases/metabolismo , Tioléster Hidrolases/química , Tioléster Hidrolases/genética , CiclizaçãoRESUMO
Antimicrobial resistance has recently been considered an emerging catastrophe globally. The public health and environmental threats were aggravated by the injudicious use of antibiotics in animal farming, aquaculture, and croup fields, etc. Consequently, failure of antibiotic therapies is common because of the emergence of multidrug-resistant (MDR) bacteria in the environment. Thus, the reduction in antibiotic spillage in the environment could be an important step for overcoming this situation. Bear in mind, this research was focused on the green synthesis of chitosan nanoparticles (ChiNPs) using Citrus lemon (Assam lemon) extract as a cross-linker and application in controlling MDR bacteria to reduce the antibiotic spillage in that sector. For evaluating antibacterial activity, Staphylococcus aureus and Escherichia coli were isolated from environmental specimens, and their multidrug-resistant pattern were identified both phenotypically by disk diffusion and genotypically by detecting methicillin- (mecA), penicillin- (blaZ), and streptomycin (aadA1)-resistance encoding genes. The inhibitory zone's diameter was employed as a parameter for determining the antibacterial effect against MDR bacteria revealing 30 ± 0.4 mm, 34 ± 0.2 mm, and 36 ± 0.8 mm zones of inhibition against methicillin- (mecA) and penicillin (blaZ)-resistant S. aureus, and streptomycin (aadA1)-resistant E. coli, respectively. The minimum inhibitory concentration at 0.31 mg/mL and minimum bactericidal concentration at 0.62 mg/mL of yielded ChiNPs were used as the broad-spectrum application against MDR bacteria. Finally, the biocompatibility of ChiNPs was confirmed by showing a negligible decrease in BHK-21 cell viability at doses less than 2 MIC, suggesting their potential for future application in antibiotic-free farming practices.
Assuntos
Antibacterianos , Quitosana , Farmacorresistência Bacteriana Múltipla , Escherichia coli , Nanopartículas , Staphylococcus aureus , Antibacterianos/farmacologia , Antibacterianos/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Quitosana/farmacologia , Quitosana/química , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Química Verde , Testes de Sensibilidade Microbiana , Nanopartículas/química , Proteínas de Ligação às Penicilinas/genética , Proteínas de Ligação às Penicilinas/metabolismo , Proteínas de Ligação às Penicilinas/antagonistas & inibidores , Staphylococcus aureus/efeitos dos fármacosRESUMO
Introduction. Resistance towards amoxicillin in Helicobacter pylori causes significant therapeutic impasse in healthcare settings worldwide. In Malaysia, the standard H. pylori treatment regimen includes a 14-day course of high-dose proton-pump inhibitor (rabeprazole, 20 mg) with amoxicillin (1000 mg) dual therapy.Hypothesis/Gap Statement. The high eradication rate with amoxicillin-based treatment could be attributed to the primary resistance rates of amoxicillin being relatively low at 0%, however, a low rate of secondary resistance has been documented in Malaysia recently.Aim. This study aims to investigate the amino acid mutations and related genetic variants in PBP1A of H. pylori, correlating with amoxicillin resistance in the Malaysian population.Methodology. The full-length pbp1A gene was amplified via PCR from 50 genomic DNA extracted from gastric biopsy samples of H. pylori-positive treatment-naïve Malaysian patients. The sequences were then compared with reference H. pylori strain ATCC 26695 for mutation and variant detection. A phylogenetic analysis of 50 sequences along with 43 additional sequences from the NCBI database was performed. These additional sequences included both amoxicillin-resistant strains (n=20) and amoxicillin-sensitive strains (n=23).Results. There was a total of 21 variants of amino acids, with three of them located in or near the PBP-motif (SKN402-404). The percentages of these three variants are as follows: K403X, 2%; S405I, 2% and E406K, 16%. Based on the genetic markers identified, the resistance rate for amoxicillin in our sample remained at 0%. The phylogenetic examination suggested that H. pylori might exhibit unique conserved pbp1A sequences within the Malaysian context.Conclusions. Overall, the molecular analysis of PBP1A supported the therapeutic superiority of amoxicillin-based regimens. Therefore, it is crucial to continue monitoring the amoxicillin resistance background of H. pylori with a larger sample size to ensure the sustained effectiveness of amoxicillin-based treatments in Malaysia.
Assuntos
Amoxicilina , Antibacterianos , Variação Genética , Infecções por Helicobacter , Helicobacter pylori , Proteínas de Ligação às Penicilinas , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Amoxicilina/farmacologia , Amoxicilina/uso terapêutico , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana/genética , Quimioterapia Combinada , Infecções por Helicobacter/tratamento farmacológico , Infecções por Helicobacter/microbiologia , Helicobacter pylori/genética , Helicobacter pylori/efeitos dos fármacos , Malásia , Testes de Sensibilidade Microbiana , Mutação , Proteínas de Ligação às Penicilinas/genética , Filogenia , Inibidores da Bomba de Prótons/uso terapêuticoRESUMO
Penicillin-binding protein 2 (PBP2) plays a key role in the formation of peptidoglycans in bacterial cell walls by crosslinking glycan chains through transpeptidase activity. PBP2 is also found in Campylobacter jejuni, a pathogenic bacterium that causes food-borne enteritis in humans. To elucidate the essential structural features of C. jejuni PBP2 (cjPBP2) that mediate its biological function, we determined the crystal structure of cjPBP2 and assessed its protein stability under various conditions. cjPBP2 adopts an elongated two-domain structure, consisting of a transpeptidase domain and a pedestal domain, and contains typical active site residues necessary for transpeptidase activity, as observed in other PBP2 proteins. Moreover, cjPBP2 responds to ß-lactam antibiotics, including ampicillin, cefaclor, and cefmetazole, suggesting that ß-lactam antibiotics inactivate cjPBP2. In contrast to typical PBP2 proteins, cjPBP2 is a rare example of a Zn2+-binding PBP2 protein, as the terminal structure of its transpeptidase domain accommodates a Zn2+ ion via three cysteine residues and one histidine residue. Zn2+ binding helps improve the protein stability of cjPBP2, providing opportunities to develop new C. jejuni-specific antibacterial drugs that counteract the Zn2+-binding ability of cjPBP2.